advances in processing and quality assurance of dairy foods-2011.pdf
TRANSCRIPT
Compendium of Lectures
Advanced Course in Faculty Training
on
ADVANCES IN PROCESSING AND QUALITY ASSURANCE
OF DAIRY FOODS
22nd March – 11th April, 2011
Editing and Compilation
Dr. Chand Ram, Senior Scientist
Dr. V.K. Gupta, Principal Scientist
Dr. (Mrs.) Shilpa Vij, Senior Scientist
Dr. Naresh Kumar, Senior Scientist
Mr. Manju G.
DAIRY MICROBIOLOGY DIVISION
CENTRE OF ADVANCED FACULTY TRAINING IN DAIRY
PROCESSING
NATIONAL DAIRY RESEARCH INSTITUTE
(Deemed University)
Indian Council of Agricultural Research
Karnal-132001 (HARYANA)
Course Director:
Dr. Chand Ram
Senior Scientist (DM)
Co-Directors:
Dr. V.K. Gupta, Principal Scientist (DT)
Dr. (Mrs.) Shilpa Vij, Senior Scientist (DM)
Dr. Naresh Kumar, Senior Scientist (DM)
Course Co-ordinator:
Dr. Rameshwar Singh
Head, Dairy Microbiology Division & Registrar (Academic)
Course Advisor:
Dr. A. A. Patel
Director, Centre of Advanced Faculty Training in Dairy Processing
& Head, Dairy Technology Division
ALL RIGHTS RESERVED
No part of the lecture compendium may be reproduced or transmitted in any form or by
any means, electronic or mechanical, including photocopy, recording or any information
storage & retrieval system without the permission of the Director, NDRI, Karnal.
FOREWORDDairy food systems in developing countries are awaiting good headway but yet to achieve the
standards as prevalent in the industrialised world. Milk in India is largely produced by small farmers. This has its own socio-economic advantages but at the same time posses serious challenges to processing and quality assurance. Quality and safety aspects of dairy products are of utmost importance. Food Safety and Standard Authority of India (FSSAI) has been established, which is actively involved in developing a suitable system for ensuring quality & safe food to consumer as per international standards.
Recently, there have been major advances in processing, preservation and quality assurance technologies to save energy and time while delivering wholesome, safe and shelf stable dairy products to the consumers. Milk is considered as an ideal vehicle for developing value added products, as it already contains a number of beneficial major and minor micronutrients and bioactive health promoting attributes. New technologies and R& D efforts are focussing on harnessing these bioactive molecules to develop functional dairy foods.
In this advanced course in faculty training, the course curriculum has been designed to provide information on latest development in dairy processing sector and quality assurance. Apart from classroom lectures, the participants will also be provided hands on practical knowledge of various techniques. The lectures include design of new equipment for mechanized continuous production of traditional dairy foods, alternative process technologies like membrane and hydrostatic high pressure technology, utilization of by-products to produce value added composite dairy foods, molecular characterization and typing of probiotics, low cholesterol dairy products and rapid methods for detection of adulterants and contaminants in dairy products, and use of statistical tools in dairy industry etc.
I am sure that the deliberations during the 21 day Advanced Course in Faculty Training on “Advances in Processing and Quality Assurance of Dairy Foods” will be highly useful for the participants in further developing their concepts in the area of processed dairy foods. The information compiled by the organizers in the form of compendium of lectures will also benefit not only trainees but also serve as reference material for scientists and students of NDRI.
Wish Advanced Course in Faculty Training a great success.
(A.K. Srivastava)
LIST OF LECTURES
ADVANCES IN PROCESS TECHNOLOGIES
1. Application of Nanotechnology in Food Industry Gautam Kaul
2
2. Applications of Wireless Sensor Network for Animal Management T.K. Mohanty and A.P. Ruhil
8
3. Designer Dairy Foods Latha Sabikhi
18
4. Dietary Food Formulation D. K. Thompkinson
23
5. Innovations in Packaging for Perishable Food Supply Chain for Quality and Safety P.S. Minz
27
6. Mechanization of Traditional Dairy Products P.S. Minz and A.K. Dodeja
31
7. Application of Membrane Processing in the Production of Indian Dairy Products Vijay Kumar Gupta
35
8. Application of Membrane Processing for Production of Quality Dairy Products Vijay Kumar Gupta
42
9. Recent Developments in the Manufacture of Low-Calorie Milk Products P. Narender Raju and Ashish Kumar Singh
49
10. Technology of Fresh Cheeses with Enhanced Health Attributes S.K. Kanawjia, Y. Khetra and A. Chatterjee
60
11. Technologies to Reduce Cholesterol in Milk and Milk Products Vivek Sharma, Darshan Lal and Raman Seth
70
12. Application of Bacteriocin Based Formulation in Bio-preservation of Dairy Foods R. K. Malik, Arun Bhardwaj, Gurpreet Kaur and Naresh Kumar
80
13. Statistical Analysis Using SAS Enterprise Guide Ravinder Malhotra & Vipul Sharma
94
14. Opportunities for Small Scale Milk Processing for Entrepreneurs Surinder Kumar
115
15. Application of High Hydrostatic Pressure (HHP) Technology in Processing of Milk & Milk Products Ashish Kumar Singh, Prateek Sharma and P. N. Raju
120
16. Technological Aspects of Composite Dairy Products Ashish Kumar Singh and P. Narender Raju
S
ADVANCES IN QUALITY ASSURANCE 17. Biosensors for Heavy Metal Ions
Neelam Verma 129
18. Health Hazards Associated with Engineered Nanomaterials Gautam Kaul
131
19. Food Allergens: Their Detection and Prevention Rajeev Kapila & Suman Kapila
140
20. ISO 22000 Food Safety Management System Bimlesh Mann
147
21. Rancimat (Accelerated and Automated) Method for Evaluation of Oxidative Stability of Fats and Oils Sumit Arora
154
22. Lateral Flow Assay – Principle and its Application in Analytical Food Science Rajan Sharma and Y. S. Rajput and Priyanka Singh Rao
162
23. Microbiological Risk Assessment: A Global Management Approach to Dairy Food Safety Naresh Kumar and Raghu H. V
169
24. Safety Aspects of Food Additives Sathish Kumar M.H. and Ameeta Salaria
181
25. Spore Based Biosensors and Their Role in Monitoring Potential Environmental Contaminants in Dairy Foods Naresh Kumar, Raghu. H. V. Avinash Yadav, Gurpreet and Geetika Thakur
191
26. Quality Management System and its Application in Dairy Industry Naresh Kumar and Raghu H V
204
27. Application of HACCP in Dairy Industry Vaishali, Rajeev Patel and Naresh Kumar
216
28. Conventional and Advanced Technique for Enumeration of Spoilage and Pathogenic Bacteria in Milk Raghu, H. V, Naresh Kumar, Mandeep, B., Ramakant, L, V. K. Singh
222
29. Preparation and Characterization of Gold Nanoparticles, their Conjugation with Antibodies and Construction of Lateral Flow Devices Priyanka Singh Rao, Swapnil Sonar, Y.S. Rajput and Rajan Sharma
232
30. Detection of Adulterants in Milk by Rapid Methods Rajan Sharma and Amit K. Barui
236
31. Estimation of Cholesterol Content in Ghee Using a Cholesterol Estimation Kit Vivek Sharma, Darshan Lal, Manvesh Sihag and Karuna Meghwal
244
32. Production and Quality Evaluation of Direct Vat Starters Rameshwar Singh, Surajit Mandal and R.P. Singh
246
33. Pathogen Monitoring in Food Systems S.G.Kulkarni
249
34. Medical Diagnostics and Clinical Microbiology for Detection of Pathogens Bhagat Singh, Chand Ram and Renu Singh
251
35. Concept of Laboratory Accreditation and its Implementation Rajan Sharma
257
ADVANCES IN FUNCTIONAL FOODS 36. Antimicrobial Factors of Colostrum: Application and its Health Benefits
Raman Seth and Anamika Das 265
37. Biofunctional Dairy Beverages Shilpa Vij, Deepika Yadav, Subrota Hati,
272
38. Role of Laboratory Animals Studies for Assessment of Safety and 280
Bioavailability of Nutraceuticals Ayyasamy Manimaran and Chand Ram
39. Microencapsulation of Lactobacillus Spp. in Calcium Alginate Surajit Mandal, Sandip Basu, R.P. Singh, Chand Ram and Rameshwar Singh
287
40. Electron Microscopy as a Tool for Study of Functional Attributes of Probiotics Sudhir Kumar Tomar
281
41. Emerging Trends in Molecular Techniques for Identification, Characterization and Typing of Novel Probiotics V. K. Batish, Ashwani Kumar, Rahul Rathore and Sunita Grover
303
42. LAB-Cell Factories for Novel Dairy Ingredients Shilpa Vij, Subrota Hati, Deepika Yadav
306
43. Technological Advances in the Manufacture of Value Added Traditional Dairy Products P. Narender Raju and Ashsih Kumar Singh
312
44. Probiotics as Biotherapeutics for Management of Inflammatory Metabolic Disorders Sunita Grover, Aparna, V, Harsh Panwar, Rashmi, H.M, Ritu Chauhan, and V.K.Batish
324
45. Diabetes Management through Enzymes Inhibitory Potential of Lactobacilli Priti Mudgil, Sumit Singh Dagar, Dinesh Dahiya and Anil Kumar Puniya
326
46. Direct Vat Starters: Concentrated Cultures for Fermented Milks
Rameshwar Singh, Surajit Mandal and R. P. Singh
332
47. Microencapsulation – an Efficient Delivery System for Functional Food Ingredients Surajit Mandal, Sandip Basu, R. P. Singh, Chand Ram and Rameshwar Singh
341
48. Milk Bioactive Peptides and Their Immunomodulatory Role Suman Kapila and Rajeev Kapila
350
49. Evaluation of Immunomodulatory Property of Milk Protein Suman Kapila and Rajeev Kapila
355
50. Concepts and Skills in Technical and Scientific Writing Meena Malik
359
51. Novel Health Promoting Poly-functional Bioactive Peptide from Bovine Milk Fermented with Lactobacillus helveticus Bhagat Singh, Chand Ram and Renu Singh
365
52. Gene Expression Microarrays in Livestock Genomics M Mukesh and Monika Sodhi
374
53. Evaluation Methods for Quality of Milk and Dairy Products Purshotam Kaushik
379
54. Application of Enzymes in Gluten Free Rice Bread Hardeep Singh Gujral
S
i
CENTRE OF ADVANCED FACULTY TRAINING IN DAIRY PROCESSING DAIRY MICROBIOLOGY DIVISION
NDRI, KARNAL
Advanced Course in Faculty Training on
Advances in Processing and Quality Assurance of Dairy Foods (22.03.2011 – 11.04.2011)
PROGRAMME
22nd March, 2011 (Tuesday)
10.00 AM- 11.00 AM Registration Surajit Mandal
11.00 AM-11.15 AM Tea
11.30 AM-12.15 PM Formal Inauguration
12.15 PM-1.15 PM Evaluation Methods for Quality of Milk and Milk Products
Prof. Purshotam Kaushik
1.15 PM- 2.00 PM Lunch
2.00 PM-3.00 PM Diabetes Management Through Enzymes Inhibitory Potential of Lactobacilli ( Theory)
A. K. Puniya
3.00 PM-3.15 PM Tea
3.15 PM-5.00 PM Digestive Enzyme Inhibition Assay Using Lactobacilli (Practical)
A. K. Puniya
23rd March, 2011 (Wednesday)
10:00 AM-11.15 AM Innovations in Packaging for Perishable Food Supply Chain for Quality and Safety
P.S. Minz
11.15 AM-11.30 AM Tea
11.30 AM- 1.00 PM Application of High Hydrostatic Pressure (HHP) Technology in Processing of Milk & Milk Products
A.K. Singh
1.00 PM – 2.00 PM Lunch
2.00 PM- 5.00 PM
Spore Based Biosensors and Their Role in Monitoring Potential Environmental Contaminants in Dairy Foods (Theory & Practical)
Naresh Kumar
24th March, 2011 (Thursday)
9.30 AM – 1.00PM Conventional and Advanced Technique for Enumeration of Spoilage and Pathogenic Bacteria in Milk (Theory & Practical)
Raghu, H. V.
1.00 PM- 2.00 PM Lunch
2.00 PM- 5.00 PM Technology of Whey Based Drinks (Practical) A. K. Singh
ii
25th March, 2011 (Friday)
10:00 AM- 12.00 PM Detection of Adulterants in Milk by Rapid Methods (Theory & Practical)
Rajan Sharma
12.00 PM-1.00 PM Mechanization of Traditional Dairy Products A. K. Dodeja
1.00 PM – 2.00 PM Lunch
2.00PM – 3.00 PM Technologies to Reduce Cholesterol in Milk and Milk Products
Vivek Sharma
3.00 PM-4.00PM DLS Assay-Devices for Protein Applications Rahul Sharma, Wyatt Technology
4.00PM -6.00PM Estimation of Cholesterol Content in Ghee using Cholesterol Estimation Kit (Practical)
Vivek Sharma
26th March, 2011 (Saturday)
9.30 AM- 11.00 AM Food Allergens: Their Detection and Prevention Rajeev Kapila
11.00 AM-11.15 AM Tea
11.15 AM-12.00 PM Role of Laboratory Animals Studies for Assessment of Safety and Bioavailability of Nutraceuticals
A. Manimaran
12.00 PM-1.00PM Concepts and Skills in Technical and Scientific Writing
Meena Malik
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00 PM Technology of Fresh Cheeses with Enhanced Health Attributes (Theory)
S. K. Kanawjia
3.00 PM- 5.30 PM Manufacture of Functional Soft Cheese (Practical) S. K. Kanawjia
27th March, 2011 (Sunday)
28th March, 2011 (Monday)
10.00 AM-11.00 AM Application of Bacteriocin Based Formulation in Bio-preservation of Dairy Foods
R. K. Malik
11.00 AM-11.15 AM Tea
11.15 AM- 1.00 PM Comparative Antimicrobial Activities of Different Bacteriocins (Practical)
R. K. Malik
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00PM
Antimicrobial Factors of Colostrum: Application and its Health Benefits
Raman Seth
3.00 PM-3.15 PM Tea
3.15 PM- 5.00 PM Applications of Wireless Sensor Network for Animal Management ( Theory & Practical)
T. K. Mohanty
29th March, (Tuesday)
10.00AM –11.15 AM ISO 22000 Food Safety Management System Bimlesh Mann
11.15 AM-11.30 AM Tea
11.30 AM – 1.00 PM Direct Vat Starters: Concentrated Cultures for Fermented Milks
Rameshwar Singh
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00 PM Application of HACCP in Dairy Industry Vaishali and Rajeev Patel 3.00 PM- 5.00 PM Visit to Model Dairy Plant
iii
30th March, 2011 (Wednesday)
10.00 AM –11.00AM Microbiological Risk Assessment: A Global Management Approach to Dairy Food Safety
Raghu H.V.
11.00AM-12.00PM Opportunities for Small Scale Milk Processing for Entrepreneurs
Surinder Kumar KVK, Karnal
12.00PM-1.00PM Medical Diagnostics and Clinical Microbiology for Detection of Pathogens
Bhagat Singh
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00 PM Lateral Flow Assay – Principle and its Application in Analytical Food Science
Y.S. Rajput
3.00 PM – 5.00 PM Preparation and Characterization of Gold Nanoparticles, their Conjugation with Antibodies and Construction of Lateral Flow Devices (Practical)
Y.S. Rajput
31st March, 2011 (Thursday)
9.30 AM -10.30 AM Microencapsulation – An Efficient Delivery System for Functional Food Ingredients (Theory
Surajit Mandal
10.30 AM-1.30PM Microencapsulation of Lactobacillus Spp. in Calcium Alginate (Practical)
Surajit Mandal
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00 PM Milk Bioactive Peptides and Their Immunomodulatory Role (Theory)
Suman Kapila
3.00 PM- 5.30 PM Evaluation of Immunomodulatory Property of Milk Protein (Practical)
Suman Kapila
1st April, 2011 (Friday)
10.00 AM-11.15 AM Application of Membrane Processing in the Production of Indian Dairy Products (Theory)
V. K. Gupta
11.15 AM – 1.00 PM Production of Skim Milk Retentate Using UF Process ( Practical)
V. K. Gupta
1.00 PM – 2.00 PM Lunch
2.00 PM- 5.00 PM Production and Quality Evaluation of Direct Vat Starters (Practical)
R.P. Singh
2nd April, 2011 (Saturday)
10.00 – 11.00 AM Biofunctional Dairy Beverages Shilpa Vij
11.00 AM-11.15 AM Tea
11.15 AM-1.00 PM Rancimat (Accelerated & Automated) Method for Evaluation of Oxidative Stability of Fats and Oils (Theory & Practical)
Sumit Arora
1.00 PM – 2.00 PM Lunch
2.00 PM- 4.30 PM Pathogen Monitoring in Food Systems S. G. Kulkarni, Nestle India Ltd
4.30 PM-5.30PM Novel Health Promoting Poly-functional Bioactive Peptide from Bovine Milk Fermented with Lactobacillus helveticus
Bhagat Singh
iv
3rd April, 2011 (Sunday)
4th April, 2011 (Monday)
9.30 AM– 11.00 AM Technological Advances in the Manufacture of Value Added Traditional Dairy Products
P. N. Raju
11.00 AM-12.00 AM Application of Membrane Processing for Production of Quality Dairy Products
V. K. Gupta
12.00 PM-1.00 PM Quality Management Systems and its Application in Dairy Industry
Naresh Kumar
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00 PM Designer Dairy Foods Latha Sabikhi
3.00 PM- 5.30 PM Recent Developments in the Manufacture of Low-Calorie Milk Products (Theory & Practical)
P. N. Raju
5th April, 2011 (Tuesday)
10.00 AM- 1.00 PM Statistical Analysis Using SAS Enterprise Guide (Theory & Practical)
Ravinder Malhotra & Vipul Sharma
1.00 PM – 2.00 PM Lunch
2.00 PM- 3. 30 PM Biosensors for Heavy Metal Ions Neelam Verma
3.30PM-5.00 PM Gene Expression Microarrays in Livestock Genomics
Manishi Mukesh, NBAGR, Karnal
6th April, 2011 (Wednesday)
9.30 AM -10.30 AM Technological Aspect of Composite Dairy Products
A. K. Singh
10.30 PM-11.30 PM Concept of Laboratory Accreditation and its Implementation
Rajan Sharma
11.30 AM -1.00 PM Emerging Trends in Molecular Techniques for Identification, Characterization and Typing of Novel Probiotics
V. K. Batish
1.00 PM – 2.00 PM Lunch
2.00 PM- 5.00 PM
Identification and Typing of Probiotic Lactobacilli by PCR and RAPD (Practical)
Sunita Grover
7th April, 2011 (Thursday)
9.45 AM-11.00 AM Probiotics as Biotherapeutics for Management of Inflammatory Metabolic Disorders
Sunita Grover
11.00 AM -12.00 AM Application of Nanotechnology in Food Industry Gautam Kaul
12.00 PM-1.00 PM Health Hazards Associated with Engineered Nanomaterials
Gautam Kaul
1.00 PM – 2.00 PM Lunch
2.00 PM- 3.00 PM Dietary Food Formulation D. K. Thompkinson
3.00 PM-5.30 PM Application of Statistical Tools in Dairy Research using MS Excel (Theory & Practical)
Ravinder Malhotra & Vipul Sharma
8th April 2011 (Friday)
9.30 AM -10.30 AM Electron Microscopy as a Tool for Study of Functional Attributes of Probiotics
S. K. Tomar
10.30 AM-11.30 AM LAB-Cell Factories for Novel Dairy Ingredients Shilpa Vij
11.30 AM-1.00PM Safety Aspects of Food Additives Sathish M. H.
1.00 PM – 2.00 PM Lunch
v
2.00 PM- 5.00 PM
Application of Extrusion Technology in Manufacture of Dairy Products (Practical)
A. K. Singh
9th April, 2011 ( Second Saturday) - Visit to Dairy Plant
10th April,2011 (Sunday)
11th April,2011 (Monday)
10.00AM- 11.00 AM Application of Enzymes in Gluten Free Rice Bread
H. S. Gujral GNDU, Amritsar
11.00AM-12.00PM Course evaluation
12.00 PM – 1.00 PM Discussion and Interaction with faculty
1.00 PM – 2.00 PM Lunch
2.30 PM- 3.30 PM Valedictory function
1
SECTION I
Advances in Processing Technologies
2
Application of Nanotechnology in Food Industry
Gautam Kaul
Animal Biochemistry Division, National Dairy Research Institute Karnal-132001, India
Introduction
Atoms and molecules combine to form dynamic structures and systems that are the building
blocks of every organism’s existence. For humans, cell membranes, hormones, and DNA are
examples of vital structures that measure in the nanometer range. In fact, every living organism
on earth exists because of the presence and interaction of various nanostructures.
Nanotechnology deals with the capability to image, measure, model, control, and manipulate
matter at dimensions of roughly 1–100 nanometers, where novel interfacial phenomena
introduce new functionalities. This exceptional capability has led to a vast array of new
technologies that have an impact on virtually every aspect of science and technology, industry,
economy, the environment and human lives. All organisms represent a consolidation of various
nanoscale-size objects. Even food molecules such as carbohydrates, proteins and fats are the
results of nano scale-level mergers between sugars, amino acids, and fatty acids. The electron
microscope and, more recently, the development of tools such as probe microscopes, has
provided unparalleled opportunities for understanding heterogeneous food structure at the
sub-molecular level. This has provided new solutions to previously intractable problems in food
science and offers new approaches to the rational selection of raw materials, or the processing
of such materials to enhance the quality of food products. As it applies to the food industry,
nanotechnology involves using biological molecules such as sugars or proteins as target-
recognition groups for nanostructures that could be used, for example, as biosensors on foods.
Such biosensors could serve as detectors of food pathogens and other contaminants and as
devices to track food products. Nanotechnology may also be useful in encapsulation systems
for protection against environmental factors. In addition, it can be used in the design of food
ingredients such as flavors and antioxidants. The goal is to improve the functionality of such
ingredients while minimizing their concentration.
The recent explosion in the general availability of nanoproducts makes it almost certain
that nanotechnology will have both direct and indirect impacts on the food industry. Some
nanoscale phenomena have been utilized in nutraceutical and functional food formulation,
manufacturing, and processes. New concepts based on nanotechnology are being explored to
improve product functionality and delivery efficiency. Some of these nano-based technologies
are outlined below.
Nanoparticulate Delivery Systems for Foods
Systems containing large interfacial areas such as emulsion, dispersion, and bi-
continuous structured fluid are a rich source of new knowledge. Newly developed capabilities in
3
nanoscale characterization offer a better visualization of these structures in nanometer
resolution, and further a better understanding of their functionality. When amphiphilic
molecules like surfactants, lipids, and copolymers that have both polar and nonpolar
characteristics are dispersed in a polar solvent, hydrophobic interactions cause them to
spontaneously self-assemble into a rich array of thermodynamically stable, lyotropic, liquid
crystalline phases with characteristic length scales in the nanometers. These include micelles,
hexagonal (tubular) structures, lamellar structures, and cubosomes, which possess a high
degree of molecular orientation order despite the fact that they exist in a liquid state.
Micelles: These are submicron spherical particles, typically 5–100 nm in diameter, that are
formed spontaneously upon dissolution of surfactants in water at concentrations that exceed a
critical level, known as the “critical micelle concentration” (CMC). This self-assembly process is
thermodynamically driven; i.e., interactions of the hydrophobic tail group of surfactants with
water are minimized, while interactions of the hydrophilic surfactant head groups with water
are maximized. Because of this, micelle integrity under a given set of environmental conditions
(pH, temperature, salt concentration) is often maintained for many years. A remarkable
property of micelles is that they have the ability to encapsulate nonpolar molecules such as
lipids, flavorants, antimicrobials, antioxidants, and vitamins. Compounds that ordinarily are not
water soluble or are only sparingly soluble can, with the help of micelles, be made water
soluble. Micelles containing solubilized materials are referred to as microemulsions or swollen
micelles. While micelles have been used as a delivery system for pharmaceutical compounds for
quite a long time, their use as carrier systems for functional food components has only recently
attracted increased attention. Reports of successful application of microemulsions include
encapsulation of limonene, lycopene, lutein, and omega-3 fatty acids using a variety of food-
grade emulsifiers, although in some cases addition of ethanol as a co-surfactant was required.
Patent applications have been filed for the use of microemulsions to incorporate essential oils
in flavoured carbonated beverages and to encapsulate alpha-tocopherol to reduce lipid
oxidation in fish oil.
Liposomes: Liposomes or lipid vesicles are formed from polar lipids that are available in
abundance in nature, mainly phospholipids from soya and egg. Like micelles, liposomes can
incorporate a wide variety of functional components in their interior. However, in contrast to
micelles, they can be used to encapsulate both water and lipid-soluble compounds. Liposomes
are spherical, polymolecular aggregates with a bilayer shell configuration. Depending on the
method of preparation, lipid vesicles can be unilamellar or multilamellar, containing one or
many bilayer shells, respectively. Liposomes typically vary in size between 20 nm and a few
hundred micrometers. Their core is aqueous in nature, its chemical composition corresponding
to that of the aqueous solution in which the vesicles are prepared. Because of the charge of the
polar lipids used in the preparation of liposomes, charged but water-soluble ionic species can
be trapped inside the liposomes. The pH and ionic strength of the liposomal core can thus differ
4
from those of the continuous phase in which the liposomes are later dispersed. Liposomes have
been successfully used to encapsulate proteins and provide a microenvironment in which
proteins can continue to function regardless of external environmental conditions. On the other
hand, the interior of the bilayer has properties resembling those of an organic solvent.
Consequently, lipid compounds can be encapsulated inside the bilayer, a process known as
adsolubilization. Liposomes have been shown to increase shelf life of dairy products by
encapsulating lactoferrin, a bacteriostatic glycoprotein as well as nisin Z, an antimicrobial
polypeptide. Antimicrobial efficiency of other ingredients in the encapsulated form has also
been reported. Liposomal entrapped phosvitin was used to inhibit lipid oxidation in a variety of
dairy products and ground pork. Recent research has demonstrated that, liposome-
encapsulated vitamin C retained 50% activity after 50 days of refrigerated storage, whereas
free ascorbic acid lost all activity after 19 days.
Nanoemulsions: These are simply very fine oil-in-water (o/w) emulsions with mean droplet
diameter of 50–200 nm. An emulsion is defined as a mixture of two completely or partially
immiscible liquids, such as oil and water, with one liquid being dispersed in the other in the
form of droplets. Examples of emulsified food products are mayonnaise, milk, sauces, and salad
dressings. In contrast to these well-known o/w emulsions, nanoemulsions are small enough not
to scatter light in the visible region of the spectra; thus, they appear clear instead of being
optically opaque. Because of their small size, they also do not cream within an appreciable
time. Creaming is the process whereby oil droplets move to the top of the emulsion to form a
concentrated oil-droplet layer. This is often followed by a complete breakdown of the emulsion,
yielding a clearly visible oil layer on top of the emulsion. Nanoemulsions and macroemulsions
can be manufactured in a similar fashion using high-pressure homogenizers, or membrane and
microfluidic channels. It should be noted that the proper choice of surfactants and/or polymers
is critical in the production of nanoemulsions. Because of their small size, nanoparticles have
excellent penetration properties to ensure rapid delivery of high concentrations of active
ingredients to cell membranes. Bioavailability of lipophilic active ingredients can be
substantially improved by delivery in Nanoemulsions. For example, nanoemulsions have been
used in parenteral nutrition for quite some time. Also because of their small size, they may also
exhibit some interesting textural properties that differ from those of an emulsion containing
larger droplets. For example, they may behave like a viscous cream even at low oil droplet
concentrations, a fact that has attracted attention in the development of low-fat products.
Biopolymeric nanoparticles: These consist of a matrix of biopolymers that may be linked
through intermolecular attractive forces or through chemical covalent bonds to form solid
particles. Nanoparticles may consist of a single biopolymer or may have a core-shell structure.
Because of the versatility in terms of compounds that can be encapsulated and the degree to
which these particles can be engineered and surface properties can be tailored, they have
rapidly become the most promising nanoscale delivery systems in the pharmaceutical and
5
cosmetics industries. Food-grade biopolymers such as proteins or polysaccharides can be used
to produce nanometer-sized particles. Using aggregative (net attraction) or segregative (net
repulsion) interactions, a single biopolymer separates into smaller nanoparticles. The
nanoparticles can then be used to encapsulate functional ingredients and release them in
response to distinct environmental triggers. One of the most common components of many
biodegradable biopolymeric nanoparticle is polylactic acid. But its high cost and susceptibility to
hydrolytic breakdown were believed to make it unsuitable for use in biomedical or agricultural
applications or sparingly used in research. However, the use of this polymer as an ideal material
for sutures was discovered in the 1970s, and a process was developed in the 1980s to produce
the polymer via bacterial fermentation, greatly reducing costs and increasing production rates.
Today, a wide variety of natural and synthetic polymers have been used to encapsulate and
deliver compounds. Among these are chitosan, a natural antimicrobial and anti-oxidative
polymer obtained from crustacean shells and the synthetic polymers L-, D-, and D,L-polylactic
acid (PLA), polyglycolic acid (PGA), and polycaprolactic acid (PCL). Copolymers created using
combinations of the monomers lactide, galactide, and caprolactone are also increasingly used.
Cubosomes: These are bicontinuous cubic phases which consist of two separate, continuous,
but non-intersecting hydrophilic regions divided by a lipid layer that is contorted into a periodic
minimal surface with zero average curvature. The continuous and periodic structure results in a
very high viscosity of the bulk cubic phase. However, cubosomes prepared in dispersion
maintain a nanometer structure identical to that of the bulk cubic phase but yield a much
lower, water-like viscosity. Its tortuosity can be useful for slowing diffusion in controlled
transport applications. Its isotropic optical property permits uses in many different products.
Compared to liposomes, cubosomes have much higher bilayer area-to-particle volume ratios.
The cubosome structure can be changed by modifying the environmental conditions, such as
pH, ionic strength, or temperature, thus achieving controlled release of the carried compound.
Cubosomes may be used in controlled release of solubilized bioactives in food matrices as a
result of their nanoporous structure (approximately 5–10 nm); their ability to solubilize
hydrophobic, hydrophilic, and amphiphilic molecules; and their biodegradability and
digestibility by simple enzyme action. The cubic phase is strongly bioadhesive, so it may find
applications in flavor release via its mucosal deposition and delivery of effective compounds.
Yet, its tortuous structure may lead to applications where masking unpleasant taste or flavor is
desirable, because of the slow effective diffusivity. The rate of release appears tunable through
system optimization or ideal formulation of products for specific purposes.
Nanolaminates: Besides nanodispersions and nanocapsules, another nanoscale technique that is
commercially viable for the food industry is nanolaminates. Consisting of two or more layers of material
with nanometer dimensions, a nanolaminate is an extremely thin food-grade film (1–100 nm/ layer) that
has physically bonded or chemically bonded dimensions. Because of its advantages in the preparation of
edible films, a nanolaminate has a number of important food-industry applications. Edible films are
present on a wide variety of foods: fruits, vegetables, meats, chocolate, candies, baked goods, and
6
French fries. Such films protect foods from moisture, lipids, and gases, or they can improve the textural
properties of foods and serve as carriers of colors, flavors, antioxidants, nutrients, and antimicrobials.
Currently, edible nanolaminates are constructed from polysaccharides, proteins, and lipids. Although
polysaccharide- and protein-based films are good barriers against oxygen and carbon dioxide, they are
poor at protecting against moisture. On the other hand, lipid-based nanolaminates are good at
protecting food from moisture, but they offer limited resistance to gases and have poor mechanical
strength. Because neither polysaccharides and proteins, nor lipids provide all of the desired properties in
an edible coating, researchers are trying to identify additives that can improve them, such as polyols. For
now, coating foods with nanolaminates involves either dipping them into a series of solutions containing
substances that would adsorb to a food’s surface or spraying substances onto the food surface. While
there are various methods that can cause adsorption, it is commonly a result of an electrostatic
attraction between substances that have opposite charges. The degree of a substance’s adsorption
depends on the nature of the food’s surface as well as the nature of the adsorbing substance. Different
adsorbing substances can constitute different layers of a nanolaminate like polyelectrolytes (proteins
and polysaccharides), charged lipids, and colloidal particles. Consequently, different nanolaminates
could include various functional agents such as antimicrobials, anti-browning agents, antioxidants,
enzymes, flavors, and colours.
Conclusion
Undoubtedly nanotechnology is having potential applications in all areas of food production and
processing, however many of the methods are either too expensive or too impractical to implement on
a commercial scale. There is an urgent need for nanoscale techniques that are most cost-effective in
development of new functional materials, food formulations, food processing at microscale and
nanoscale levels, product development, and storage. Although the products of nanotechnology
intended for food consumption are likely to be classified as novel products, they require testing and
clearance, and there are concerns, particularly in the area of food contact materials, that there could be
inadvertent release and ingestion of nanoparticles of undetermined toxicity. Such concerns need to be
addressed because the ultimate success of products based on nanotechnology will depend on consumer
acceptance. Consideration should be given to the consequences of the use of nanotechnology to
enhance the bioavailability of nutrients. This should consider the safety of the products, the
consequences of enhanced or altered metabolism, and also the need for labelling, regulation and testing
of health claims for such food supplements.
References:
1. Chen, H, Weiss, J and Shahidi, F., Nanotechnology in nutraceuticals and functional foods. Food Technol.,
2006. 60 (3): 30-36.
2. Flanagan, J. and Singh, H., Microemulsions: A potential delivery system for bioactives in food. Crit. Rev.
Food Sci. Nutr., 2006. 46: 221-237.
3. Moraru, C.I., Panchapakesan, C.P., Huang, Q., Takhistove, P., Liu, S., and Kokini, J.L., Nanotechnology: A
new frontier in food science. Food Technol., 2003. 57(12): 24-29.
7
4. McClements, D.J., Decker, E.A., and Weiss, J., inventors; University of Massachussetts, assignee. Novel
procedure for creating nanolaminated edible films and coatings, U.S. patent application. 2005.
5. Nakajima, M., Development of Nanotechnology and Materials for Innovative Utilization of Biological
Functions, Proceedings of the 34th
United States and Japan Natural Resources (UJNR) Food and
Agriculture Panel, Susono, Japan. 2005.
6. Scott, N. and Chen, H., Nanoscale science and engineering for agriculture and food systems, Report
submitted to Cooperative State Research, Education and Extension Service (CSREES), U.S. Dept. of
Agriculture. 2003.
7. Taylor, T.M., Davidson, P.M., Bruce, B.D., and Weiss, J., Liposomal nanocapsules in food science and
agriculture. Crit. Rev. Food Sci. Nutr., 2005. 45: 1-19.
8. Worldnutra., 6th
International Conference and Exhibition on Nutraceuticals and Functional Foods.
Anaheim, Calif., October 2005.
8
Applications of Wireless Sensor Network for Animal Management
T.K. Mohanty and A.P. Ruhil
Computer Centre, NDRI, Karnal
The paper discusses the need and benefit of wireless sensor networks in farm animal
management. It presents a brief overview of wireless sensor technologies and standards for
wireless communications as applied to wireless sensors networks. Examples of wireless sensors
networks applied in farm management, agriculture and food production for environmental
monitoring, precision agriculture is given. The paper also discusses advantages and limitations
of wireless sensors for adoption in field conditions.
Introduction
Indian dairy sector is suffering from non availability of qualitative and quantitative data and
decision support system for animal management at field level due to which planners and
managers face difficulties in formulating policies and making effective decisions in time. Major
problems prevailing in the field both at organized dairy farms as well as unorganized sector are
identification of animals, heat detection, monitoring health and comfort level of animals,
automation of milking process, segregation of animals based on health and production, shelter
management etc. which is causing great economic losses to dairy farmers.
A lot of qualitative data is required on various parameters to draw any conclusion for
better animal selection and management of animals. For example, heat detection, the animal
has to be observed continuously at regular interval about its position, movement, activities,
body temperature etc. To acquire such huge and complex data flawlessly is very difficult (if not
impossible) besides being a costly affair through visual observation.
For precision animal husbandry reliable information is required continuously without
human intervention for better decision making, so that the corrective measures can be taken
immediately as and when required. With the advances in wireless communication and digital
computing it is now possible to produce of small, low cost sensors which integrate sensing,
processing and communication capabilities and form an autonomous entity. These sensors can
be deployed in the field and sensor independently senses the environmental parameters and
collaboratively achieves complex information gathering and dissemination tasks like intrusion
detection, target tracking, environmental monitoring, etc.
In this endeavor, sensor network based technology may be customized to meet specific
needs of dairy farm management for bringing efficiency and profitability of dairy farms. Dairy
animals mounted with sensor belt serve as dynamic nodes (active) and a sink (receiver) with
9
computational capabilities are provided, animal behavior in large dairy can be monitored and
regularized since animals in groups behave in certain patterns. Optimum temperature and
humidity levels can be regularized through on-off type of misting solution can be placed in grid
on the dairy house floor controlled by sensors and actuators. The flow of water to mist can be
regularized as the environment of animal housing /changes. Activity of animals can be
correlated with animal in heat and sickness. Feeding behavior will also help in the decision
making for better management. Decision support system placed at sink to process the acquired
data will increase the efficiency of dairy farm management.
Wireless Sensor Network (WSN)
A wireless sensor network generally consists of spatially distributed sensor nodes and
base station(s) (or “gateway”) that can communicate with a number of wireless sensor nodes
scattered in a region via a radio link to cooperatively monitor physical or environmental
conditions, such as temperature, humidity, sound, vibration, pressure, and motion or air
pollutants. Data is collected at the wireless sensor node, compressed, and transmitted to the
gateway directly or, if required, uses other wireless sensor nodes to forward data to the
gateway. The transmitted data is then presented to the system (end user) by the gateway
connection which has the capability of communicating with other computers via other
networks, such as a LAN, a WLAN, a WPAN and the Internet. In other words spatially distributed
sensor nodes in a region constitute a wireless ad-hoc network, where each sensor node
cooperate in routing data packets to base station using multi-hop routing algorithm as shown in
figure given below (Fig. 1).
Fig.1: Wireless Sensor Network Architecture
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Sensor node is just like a small computing device, extremely basic in terms of their interfaces
and their components. They usually consist of a processing unit with limited computational
power and limited memory, sensors, a communication device (usually radio transceivers or
alternatively optical), and a power source usually in the form of a battery. The size of a sensor
node may vary from matchbox to the size of a dust particle. A tiny sensor node (dust/ sand
particle size – smartdust) is also known as “motes”. The base station(s) are component of the
WSN with more computational, energy and communication resources. They act as a gateway
between sensor nodes and the end user.
Characteristics of WSN
Unique characteristics of a WSN include:
Limited power they can harvest or store
Ability to withstand harsh environmental conditions
Ability to cope with node failures
Mobility of nodes
Dynamic network topology
Communication failures
Heterogeneity of nodes
Large scale of deployment
Unattended operation
Node capacity is scalable, only limited by bandwidth of gateway node.
Wireless sensor networks are self-organizing, self-configuring, self-diagnosing and self-
healing.
Wireless Sensors, “Smart Transducers” and Actuators
The words 'sensor' and 'smart transducer' are both widely used in the description of
measurement systems. However there is minor difference between 'sensor' and 'transducer'. A
sensor; is a device that measures a physical quantity and converts it into a signal which can be
read by an observer or by an instrument. Sensors are now being commonly used to detect or
quantitatively determine physical parameters such as pressure, temperature, humidity,
position, light colour and intensity, magnetic field, displacement, speed, chemical composition
or velocity over a measuring range.
A transducer is a device that converts one type of energy from one system to another in the
same or in the different form. In general, a sensor needs a transducer that transforms the
measured magnitude in another one that is easier to interpret or visualize. A sensible
11
distinction is to use 'sensor' for the sensing element itself and 'transducer' for the sensing
element plus any associated circuitry. All transducers would thus contain a sensor and most
(though not all) sensors would also be transducers. “Smart transducers” are equipped with
microcontrollers to provide local “intelligence” and network capability.
Actuator is an electromechanical device that converts energy into linear or rotary motion for
controlling a system. It takes energy, usually created by air, electricity, or liquid, and converts
that into some kind of motion. That motion can be anything from blocking to clamping to
ejecting. Actuators are typically used in manufacturing or industrial applications and may be
used in things like motors, pumps, switches, relays and valves. Computer uses sensor data to
control different systems through the use of actuators.
Hardware and Software Requirements
Hardware requirements for deploying WSN include:
Robust radio technology
Low cost, energy-efficient processor
Flexible i/o for various sensors
Long-lifetime energy source
Flexible, open source development platform
Software requirements for wireless sensors include:
Small footprint to run on small processors
Efficient energy use
Capability of fine grained concurrency
High modularity
Robust ad hoc mesh networking that requires low power
Event driven programming
Operating systems for wireless sensor network nodes are generally less complex in comparison
to general-purpose operating systems both because of the special requirements of sensor
network applications and because of the resource constraints in sensor network hardware
platforms. For example, sensor network applications are usually not interactive in the same way
as applications for PCs. Because of this, the operating system does not need to include support
for user interfaces. Furthermore, the resource constraints in terms of memory and memory
mapping hardware support make mechanisms such as virtual memory either unnecessary or
impossible to implement.
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The “TinyOS” operating system is specifically designed for wireless sensor networks. It is an
event driven operating system composed of event handler and tasks modules. Other operating
systems are Contiki, MANTIS, Nano-RK, SOS, LiteOS, ERIKA Enterprise etc.
Types of Sensor Nodes
A number of wireless sensor nodes are available in the market, a few of them are mentioned
below:
Accelerometers
Barometric pressure sensors
Light sensors
GPS modules
Temperature sensors
Humidity sensors
Acoustic sensors
Magnetic RPM sensors
Magnetometers
Solar radiation sensors
Soil moisture sensors
Soil temperature sensors
Wind speed sensors
Rainfall meters
Seismic sensors
Load sensors
Wireless Standards and Sensor Technologies
Various wireless standards have been established for developing WSN. Among them, the
following standards are used more widely for measurement and automation applications. All
these standards use the instrumentation, scientific and medical (ISM) radio bands, which
include band of 2.400–2.4835 GHz. The 2.4 GHz band has a wider bandwidth that allows more
channels and frequency hopping and permits compact antennas.
Data Visualization and Fusion
The data gathered from wireless sensor networks is saved in the form of numerical data in a
central base station. Open Geospatial Consortium (OGC) is specifying standards for
interoperability interfaces and metadata encodings that enable real time integration of
13
heterogeneous sensor webs into the Internet, allowing any individual to monitor or control
Wireless Sensor Networks through a Web Browser.
Data fusion, also called information fusion, is a technique for processing sensor data by
filtering, aggregating, and making inferences about the gathered data from many
heterogeneous sensors, on many platforms, into a single composite picture of the environment.
Information fusion deals with the combination of multiple sources to obtain improved
information: cheaper, greater quality or greater relevance. Within the wireless sensor networks
domain, simple aggregation techniques such as maximum, minimum, and average, have been
developed for reducing the overall data traffic to save energy., Data fusion might be viewed as
a set reduction technique with improved confidence whereas data integration is set
combination wherein the larger set is retained.
Applications in Agriculture and Animal Management
The wireless sensor networks were developed for monitoring, tracking, or controlling
applications. Such networks are being used extensively in military applications such as
battlefield surveillance, industrial process monitoring and control, machine health monitoring,
environment and habitat monitoring, healthcare applications, home automation, object
tracking, fire detection, land slide detection, traffic control etc.
WSN has potential applications in agriculture sector where it can be used effectively to increase
the productivity by minimizing the input requirements. However, deployment of wireless
sensors and sensor networks in agriculture is still at the beginning stage and new applications
are emerging day by day. Wireless sensors have been used in precision agriculture to assist in
spatial data collection, soil health data (temperature, moisture etc.), precision irrigation for
efficient water usage, variable-rate technology to regulate the fertilizer application rate and
supplying data to farmers. Gravity fed water systems can be monitored using pressure
transmitters to monitor water tank levels, pumps can be controlled using wireless I/O devices,
and water use can be measured and wirelessly transmitted back to a central control center for
billing. Tata Consultancy Services (TCS) has recently launched a pilot project in few villages of
Uttar Pradesh and Punjab to detect late blight disease in potato crop based on the information
collected on soil and weather (humidity, temperature and rainfall) parameters from a wireless
sensor network spread across the farms.
A number of cases for the deployment of wireless sensor networks for monitoring animals and
other related purposes have been reported in the literature. Heterogeneous sensor networks
have been installed on a large scale working dairy farm for studying and monitoring the animal
behavior with respect to environmental changes [8]. Sensor networks have also been deployed
for studying the effect of micro-climate factors in habitat selection by sea birds [2-4]. The
14
networks are used for tracking the movements of wild animals such as Zebras [5]. Another
application of wireless sensor network describes the monitoring of cattle health. The purpose
of their experiment was two fold, to test the capabilities of sensors and wireless sensor
networks for monitoring animal health, as well as to provide a preliminary investigation into
movement in cow’s rumen by putting the sensors inside a standard drug release capsule and
finally placing this capsule in the rumen. A number of health parameters like internal
temperature, pressure, pH level, conductivity etc. were measured [6]. Another paper discusses
the design of a remote health monitoring system for cattle that hosts a suite of sensors and
communicates through a wireless link with a base station via Bluetooth telemetry [7]. Another
application describes the creation of “Moving Virtual Fence” for herding cows to keep them
within the boundaries of pasteurized land using WLAN, GPS and sound amplifier [9]. Recently a
paper has shown that native animals living in a forest, with sensors as mobile biological sensors,
can be used in early detection of forest fire through animal behavior classification and/or
thermal detection [12]. Kansas State University is working to develop a sensor based system to
monitor the health and activity of individual animals in a herd [13]. Some articles have
published the utility of sensor in testing of a three-dimensional acceleration measuring system
with wireless data transfer (WAS) for behavior analysis on free moving cows and horses; and
contact less measurement of cow behavior in a milking robot [14,15].
In India, few groups are doing research independently on wireless sensor networks (WSN) for
home security, industrial surveillance etc. in research organizations like IITs, IISc, C-DAC etc. No
instance has been found for developing and deploying WSN and sensor products for dairy farm
automation. However few (mostly foreign) MNCs are marketing their products like electronic
identification, automatic milking parlors, activity meters, automatic feeding stations for farm
management in India. Most of these products are based on active/ passive sensors using radio
frequency identification (RFID) mechanism. Chitle dairy farm, Maharashtra, has deployed RFID
network for electronic identification of animals, linking of RFID with computerizing dairy farm
operations, feeding schedule, breeding data and milking operations by Delaval India Pvt. Ltd.
Parag milk and milk facilities, Pune, has automated milking and animal management process by
installing 50 station rotary parlor with RFID network by Westfalia Separator India Pvt. Ltd.
Farmers dairy, Chandigarh, has installed 40 station parallel parlor to automate milking and
animal management system with the help of Westfalia Separator India Pvt. Ltd.
NDRI efforts in Developing WSN for Animal Management
Efforts are being made at NDRI to improve the farm animal management practices by using the
state of the art technologies. A project on “Development of wireless sensor network for animal
management” in collaboration with Indian Institute of Technology, Delhi has been sanctioned
and funded by NAIP at this institute. The project aims to improve the accuracy in data
15
acquisition about animals in unorganized and organized to increase efficiency of dairy farming
and to improve management practices to manage dairy animals. The project also address the
issues of tracing of nomadic herds for disease surveillance and migratory route Deployment of
wireless sensor network is proposed to acquire data on impossible measurements about
animals to enhance the quality and quantity of data for management of production, disease,
comfort and traceability.
It is proposed to design and develop indigenous low cost sensors which can be mounted on
animals for recording temporal and spatial data about activities, movement, position, feeding
patterns, and other behavioral parameters of animals. The following sensor based automatic
devices are proposed to develop for the benefit of small farmers as well as large dairy farmers:
Smart bucket with provision for measuring/ recording milk weight, pH, and conductivity
for mastitis detection.
Smart belt for monitoring vital health parameters i.e. respiration, heartbeat, body
temperature for monitoring health of animal continuously without human intervention.
Online body weight and allometric measurements device for monitoring growth of
animals.
Micro-climate controlling device for shelter management.
Feeding machine for providing optimum feed to animal based productivity of animal
Water trough to minimize wastage of natural resources.
Tracking of nomadic herds in desert etc.
A decision support system will be developed to analyze the temporal and spatial data along
with production data using computational intelligence techniques for heat detection, illness
and lameness identification, behavioral analysis, shelter management, tracking of nomadic
herds for disease surveillance and other undiscovered problems.
These data will help in breeding strategies for improving productivity through selective
breeding for desired traits. The accurate data collection is a major problem in the field
conditions where 80% of dairy animals reside. Collecting data from these animals will further
help in increasing the selection intensity in larger population for faster increase in productivity.
These data will also help in diagnosing diseases like mastitis, lameness, illness, and reproductive
problems with minimum intervention of animal owner.
Advantages of WSN
Wireless sensor network has number of advantages a few are mentioned as below:
Automatic recording and transmission of data without human intervention.
Significant reduction and simplification in wiring and harness
16
Wireless technology reduces maintenance complexity and costs
Suitable to monitor dangerous, hazardous, unwired or remote areas and locations
Faster deployment and installation
Flexible extension of the network
Wireless sensors can be mounted on moving objects i.e. mobility of sensors
Provide ubiquitous (anywhere everywhere) computing environment
Limitations of WSN
Despite the fact that WSN has great potentials as recognized and supported by many
enthusiastic industry alliances and users, adoption of wireless sensor technology has not been
as fast as one would imagine. Main obstacles are as follows:
Standardization is not yet completed
Compatibility with legacy systems is not addressed
Security issues need to be resolved
Power supply is always a great concern for wireless systems
The reliability of wireless sensor system remains unproven
Lack of experienced staff for troubleshooting.
Conclusion
Wireless sensor networks are enabling applications that previously were not practical. It
provides massive qualitative data which can be used for finding hidden patterns and developing
decision support systems. WSN has proven it utility in studying the behavior of animals which
was otherwise difficult in manual system. As new standards-based networks are released and
low power systems are continually developed, we will start to see the widespread deployment
of wireless sensor networks.
Important References Cited
1. P. Sikka, P. Corke, P. Valencia, C. Crossman, D. Swain, G. Bishop-Hurley, Wireless Adhoc Sensor and
Actuator Networks on the Farm, In Proceeding of 5th international; conference on Information
Processing in Sensor Networks (IPSN 2006), April 19-21, Nashville, TN, USA., pages 492-499, ACM Press
2006
2. R. Szewczyk, A. Mainwaring, J. Polastre, J. Anderson, and D. Culler. An analysis of a large scale habitat
monitoring application. In Proceedings of the 2nd international conference on Embedded networked
sensor systems, pages 214-226. ACM Press, 2004.
3. A. Mainwaring, J. Polastre, R. Szewczyk, D. Culler and J. Anderson. Wireless Sensor Networks for Habitat
Monitoring, In Proceeding of the 1st ACM Workshop on Wireless Sensor Networks and Application,
September 28, 2002. Atlanta, GA, USA.
17
4. G. Tolle, J. Polastre, R. Szewczyk, D. Culler, N. Turner, K. Tu, S. Burgess, and T. Dawson. A macroscope in
the redwoods. In Proceedings of the Third International Conference on Embedded Networked Sensor
Systems, pages 51-63. ACM Press, 2005.
5. P. Zhang, C. M. Sadler, S. Lyon, and M. Martonosi. Hardware design experiences in zebranet. In
Proceedings of the 2nd international conference on Embedded networked sensor systems. ACM Press,
2004.
6. K. Mayer, K. Taylor, and K. Ellis. Cattle health monitoring using wireless sensor networks, 2nd IASTED
International Conference on Communication and Computer Networks, Cambridge, Massachusetts, USA,
Nov. 2004.
7. Nagl, L., Warren, S., Yao, J. & Schmitz, R. 'Wearable Sensor System for Wireless State-of-Health
Determination in Cattle', Engineering in Medicine and Biology Society - Proceedings of the 25th Annual
International Conference of the IEEE, Volume 4, pp. 3012 - 3015. 2003,
8. Trevarthen, A. "The Importance of Utilizing Electronic Identification for Total Farm Management: A Case
Study of Dairy Farms on the South Coast of NSW", Ph.D. thesis submitted to University of Wollongong,
2005.
9. Butler, Z., Corke, P., Peterson, R., Rus, D., “Virtual fence for controlling cows”, Proceedings of the IEEE
International conference on Robotics and Automation, New Orleans, LA, USA, PP. 4429-4436, April 26-
May 1, 2004.
10. Wang, N., Zhang, N., Wang, M., “Wireless Sensors in Agriculture and Food Industry- Recent
Development and Future Perspective”, Computers and Electronics in Agriculture, 50, pp. 1-14, 2006
11. P. Juang, H. Oki, Y. Wang, M. Martonosi, L.-S. Peh, and D. Rubenstein, “Energy-efficient computing for
wildlife tracking: Design tradeoffs and early experiences with zebranet”, In Tenth International
Conference on Architectural Support for Programming Languages and Operating Systems (ASPLOS-X).
ACM Press, 2002.
12. Yasar Guneri Sahin, “Animals a Mobile Biological Sensors for Forest Fire Detection”, Sensors, 7, 3084-
3099, 2007
13. Kansas State University project site accessed on 10/01/08 http://www.k-
state.edu/media/WEB/News/Webzine/safetyandsecurity/livestockmonitor.html
14. Schiebe, Klaus Manfred, Gromann, Cora, “Application testing of a new three-dimensional acceleration
measuring system with wireless data transfer (WAS) for behaviour analysis”, Behavior Research
Methods, Vol.38, Number 3, PP. 427-433, 2006
15. Pastell, M., Aisla, A.-M., Hautala, M., Ahokas, J., Poikalainen, V., Praks, J. “Contactless measurement of
cow behaviour in a milking robot”, Behavior Research Methods, Vol.38, Number 3, PP. 479-486, 2006
16. http://en.wikipedia.org/wiki/Wireless_sensor_network
17. Crossbow Technology Inc., 2004. Smart Dust/Mote Training Seminar. Crossbow Technology, Inc., San
Francisco, California, July 22–23.
18
Designer Dairy Foods
Latha Sabikhi
Dairy Technology Division, NDRI, Karnal
Introduction
Altering milk composition for processing and/or animal and human health by employing
nutritional and genetic approaches is a novel area of Dairy biotechnology. Newer value-added
products can be derived from milk and milk products by these interventions. Recent attempts
are directed toward enhancing the value of milk and studying its health implications although
earlier, the emphasis was on breeding policies for producing more milk. Milk composition can
be altered by nutritional management or through the exploitation of naturally occuring genetic
variation among cattle. Researchers are now hoping to develop 'designer milk' tailored to
consumer preferences or rich in specific milk components that have implications in health as
well as processing by combining the two strategies.
Opportunities in ‘milk designing’
To realise the full potential of the healthful and therapeutic benefits of milk, it would be
desirable to have the opportunity to alter its composition in several ways. For example, a
greater proportion of unsaturated fatty acids in milk fat, reduced lactose content in milk for
lactose-intolerant people, and/or milk free from -lactoglobulin (-LG) would benefit human
diet and health. Processing aids that would be useful would be alteration of primary structure
of casein to improve technological properties of milk, production of high protein milk,
engineering milk meant for cheese manufacturing that leads to accelerated curd clotting time,
increased yield and/or more protein recovery, milk containing nutraceuticals and replacement
for infant formula.
Alteration in carbohydrate
Lactose, the major milk sugar, regulates the osmotic process of lactation, thus causing the
movement of water into milk. This carbohydrate is synthesized in the secretory vesicles of the
mammary glands by the lactose synthase complex. As lactose cannot diffuse out of the vesicles,
it draws water into the vesicles by osmosis. Thus, the volume of milk produced is directly
dependent on the amount of lactose synthesized.
Lactose cannot be transported to the bloodstream directly and can be absorbed only after its
enzymatic hydrolysis to the monosaccharides glucose and galactose by intestinal lactase (-
galactosidase). As milk is a major component in the human diet, lactose intolerance (caused by
the absence of this enzyme) limits the use of a valuable nutritional source for many people. In
19
addition, since milk can provide much of the calcium we require to maintain bone health,
lactose intolerance can also be associated with osteopenia in later life.
Lactose intolerance can also be tackled through the use of -galactosidase-replacement
(preharvest) or hydrolyzed low-lactose (postharvest) products. Besides the nutritional
advantage, a reduction in milk lactose content could also benefit the industry with less volume
to transport, better milk coagulation, and less effluent production. The complete removal of
lactose from milk creates milk that is extremely viscous, containing very little water. It is
extremely difficult to extract this milk from the mammary gland, making the milking process
difficult and painful for the animal. However, research has shown that with controlled
reduction in the lactose content of milk, it is possible to decrease the water, increase the
percentage of total solids, and reduce the lactose yield of the milk while keeping fluidity intact.
The pre-harvest methodologies of reducing lactose either by the removal of α-lactalbumin (α -
LA) and gene 'knock-out' methodologies or by introducing the lactase enzyme into milk via
mammary gland specific expression have not been successful, as the former resulted in highly
viscous milk (Karatzas and Turner, 1997) and the latter, in milk with high osmotic pressure due
to the mono-saccharides produced (Bremel et al., 1989).
Jost et al. (1999) explained an in vivo technique for low-lactose milk production by generating
transgenic mice that selectively produce a biologically active lactase in their milk. In contrast to
previous results, the lactose content reduced while retaining most of the monosaccharides of
the milk. In addition, transgene expression did not affect the milk protein levels, thus helping to
maintain a balanced nutrient supply.
Alteration in fat
Manipulation of composition of milk fat is possible through feeding practices for dairy cows.
Feeding of unsaturated fats in an encapsulated or protected form results in a prompt rise in the
degree of unsaturation of the serum lipids, tissue fat and milk fat. Trials conducted at the
University of Alberta (US) have demonstrated that adding a blend of canola oil and linseed oil to
the cow's diet enhances the nutritional quality of milk fat and improves the spreadability of
butter. A 'designer cow' called Daisy which can produce semi-skimmed (half-fat) milk (diet:
dehusked oats) and soft-spreading butter that spreads straight from the refrigerator (diet:
rapeseed oil) has been bred in Britain. Similarly, restricted quantities of fish oil, fish meal or
plankton added to the cow's diet of grass or silage can produce milk rich in omega-3 fatty acids.
Dietary fats such as corn oil fed to cows in the protected form results in the production of milk
with substantially increased levels of conjugated linoleic acid (CLA), which reportedly
suppresses carcinogens, inhibits proliferation of leukemia and colon, prostate, ovarian, and
breast cancers.
20
In changing the fat composition, targeting enzymes that influence the synthesis of fat is
important. As an example, reduction of acetyl CoA carboxylase that regulates the rate of fat
synthesis within the mammary gland would translate to a drastic reduction in the fat content of
milk and reduce the energy required by the animal to produce milk. Similarly, genetic variants
of stearoyl-CoA desaturase has an influence on degree of unsaturation and on concentration of
conjugated linolenic acid in the milk fat. The type of fatty acids present in milk fat can influence
the flavor and physical properties of dairy products. There are reports that butter produced
from cows fed high oleic sunflower seeds and regular sunflower seeds were similar in flavour
and texture to the control butter. Extruded soybean and sunflower diets yielded a Cheddar
cheese that had higher concentrations of unsaturated fatty acids while maintaining flavor,
manufacturing, and storage characteristics similar to that of control cheese. It is also beneficial
from a safety point of view, as the accumulation of fatty acids, namely C12, C14, C18:1 and C18:2
enhanced the safety of cheeses against Listeria monocytogenes and Salmonella typhimurium.
Alteration in protein
One of the major products of the mammary glands being protein, exciting possibilities in
research and technology extends the frontiers for better protein supplementation. Improved
amino acid profile by the addition of L-taurine, L-leucine and L-phenylalanine offers additional
nutritional benefits. Active whey peptides such as glyco-macro-peptide (GMP) is valuable in diet
preparations for children with phenylketonurea (PKU) disorder, a condition that can lead to
mental retardation if not treated early. Those with this rare metabolic disorder have an
impaired ability to metabolise phenylalanine, a component of most foods. Transgenic animals
can also secrete in their milk, proteins such as blood clotting factors needed by patients of
haemophilia.
Caseins, being easily digestible are quite sensitive to plasmin, a serine protease occurring
naturally in milk and also plasminogen. Thus, -casein the most abundant casein in ruminant
milk undergoes limited proteolysis by plasmin. This can be disadvantageous as casein
proteolysis decreases the curd yield in cheese and can induce organoleptic defects and gelation
of UHT milk. A milk enriched with specific inhibitor of either plasmin or plaminogen activator
would therefore be alternative for the process industry.
Several human proteins that are of high value, low volume and therapeutic have been
expressed in milk of domestic animals with success. The major advantage of transgenic
technology is that these proteins can be produced at a very low cost. Economic comparison of
production costs of human tissue plasminogen activator (htPA) through bacterial fermentation,
mammalian cell culture and cow transgenic technology estimates the cost per gram of htPA to
be 20000, 10000 and 10 US dollars respectively (Karatzas and Turner, 1997). Two proteins,
human antitrypsin and human antithrombin II have been purified from milk of transgenic
21
ruminants. Human antithrombin III, a plasma protein that helps prevent harmful blood clotting
is also being tested.
Bovine milk to resemble human milk
Mother's breast milk is the ultimate designer food for babies. However, due to varying reasons,
a number of infants are fed formulas based on bovine milk. The composition of these formulas
could be greatly improved to suit the needs of the infant by incorporating ingredients that
resemble those of human milk, thereby 'humanising' the bovine milk.
Lactoferrin (LF), the iron-binding protein has antimicrobial properties and may also mediate
some effects of inflammation and have a role in regulating various components of the immune
system. Its level in human milk is about one gram per litre (in human colostrum about seven
gram per litre). As the levels of LF in cow milk is only about one tenth of that in human milk, this
has caught the attention of those involved in designing human milk replacement formulas. The
human LF (hLF) gene has already been expressed at low levels (0.1 to 36 mg/ml) in the milk of
transgenic mice and a transgenic bull that carries the gene for hLF has been produced.
Human milk contains 0.4 g/L of lysozyme (LZ), an enzyme that provides antibacterial activity in
human milk. Active human LZ (hLZ) has been produced in the milk of transgenic mice at the
concentrations of 0.78 g/L (Maga and Anderson, 1995). On the processing front, the expression
of LZ in milk results in the reduction of rennet clotting time and greater gel strength in the clot.
A double transgenic cow that co-expresses both hLF and hLZ in milk may also reduce the
incidence of intra-mammary infection or mastitis.
Yet another application of transgenic technology could be to produce the human lipase, which
is stimulated by bile salt in the milk of bovines. The lipase thus produced could be used as a
constituent of formulas to increase the digestibility of lipids especially in premature infants who
have low lipase activity (Lonnerdal, 1996).
Several children are allergic to cow’s milk, owing to the presence of -lg, which is not found in
human milk. Elimination of this protein by knocking out -lg gene from cow’s milk is unlikely to
have any detrimental effects, on either cow or human formula, and might actually overcome
many of the major allergy problems associated with cow’s milk.
Other advantages
Mice that produce milk with 33% more total solids (40-50% TS) and 17% less lactose than
normal control mice have been generated by transgene. Due to a decrease in lactose
synthetase activity, less lactose is being produced and less water is being transferred into milk
causing a reduction in milk volume. So it appears that the same amounts of total milk fat and
protein are being produced in a lesser total milk volume. If this technology could be translated
to dairy animals, milk that contains 6.5% protein, 7% fat, 2.5% lactose and 50% less water is not
22
an improbable accomplishment. This would mean a direct economic benefit in terms of 50%
reduction in the cost of shipping milk. In addition, since the cow would be producing one half
her normal volume of milk there would be less stress on the cow and on her udder.
From the processing point of view, after removal of fat from this type of milk, a skim milk
having twice protein content and have half the lactose content of normal milk could be
produced. This type of milk would also make it easier to produce low lactose or lactose free
dairy products. The concentrated milk should lead to better product yields from the same
amount of initial input. The lowering of milk volume and lactose content will reduce the total
whey output produced during processing. The reduction of stress on the mammary gland of
the cow and the more viscous milk may also decrease the susceptibility to obtaining mastitis
infection. Organisms that cause mastitis use lactose as their energy source and since lactose
would be reduced in the system there would be a decrease in the available food source for
these bacteria.
Challenges
There is a tendency among human beings to resist change, especially those that trouble their
inner instincts. As all changes that arise as a consequence of biological research would fall into
this category, there is bound to be tremendous resistance to topics such as transgenics. The
future of biotechnologically derived foods is, therefore, at crossroads even after two decades of
positive results. Acceptability will depend ultimately on the four key factors of animal welfare,
demonstrable safety of the product, enhanced health properties of the product and increased
profitability as compared with conventional practices. Various ethical, legal and social aspects
of biotechnological research need to be addressed before we would see designer transgenic
herds similar to the organic herds that thrive in the current economic and social climate. Hi-
tech milk processing may be more acceptable to consumers than transgenesis for altering milk
composition.
Selected References
Bremel, R.D., Yom, H.C. and Bleck, G.T. 1989. Alteration of milk composition using molecular genetics. J.
Dairy Sci. 72:2826.
Jost, B., Vilotte, J-Luc., Duluc, I., Rodeau, J-Luc. and Freund, J-Noel. 1999. Production of low-lactose milk
by ectopic expression of intestinal lactase in the mouse mammary gland. Nature Biotechnol. 17(2):160.
Karatzas, C.N. and Turner, J.D. 1997. Toward altering milk composition by genetic manipulation: Current
status and challenges. J. Dairy Sci. 80:2225.
Lonnerdal, B. 1996. Recombinant human milk proteins- an opportunity and a challenge. Am. J. CL. Nutr.
63:622S.
Maga, E.A. and Anderson, G.B. 1995. The effect of mammary gland expression of human lysozyme on the
properties of milk from transgenic mice. J. Dairy Sci. 78:2645.
23
Dietary Food Formulation
D. K. Thompkinson
Dairy Technology Division, NDRI, Karnal.
Introduction - In the past century, increase in population, urbanization and life span etc. have
drove the food industry to be large scale, health conscious and convenience of foods being the
harbinger of technological transformation. With the next twenty five years the world
population is expected to grow by two millions people leading to total greater demand for food.
Convenience has always been the strongest drive for change. Along with the concept of eating
healthy food, there emerged a need for food that nourish, heal and fortify. This introduces as a
basis of food design for body requirements as well as therapeutic adjuncts that can serve as
health aids.
Formulated foods serve as important vehicle to meet nutritional requirement of normal
individuals and those in need of special diet. Processed food industry produces a vide array of
value added foods based on available raw materials in the country. Formula for processed
foods targeted for ameliorated diet related metabolic disorders are indeed possible in the light
of recent nutritional knowledge. Growing awareness towards beneficial role of specially
formulated products has led to new range of functional and dietetic foods and therapeutic
adjunct has opened immense opportunity for manufacturers of food with specific nutritional
merits.
Formulated foods – Formulated or fabricated foods are foods designed and built according to
plan from individual components, to yield a product that has specific physical, chemical and
functional properties. The earliest known formulated food is bread, which does not exist per se
in nature. Historically such foods were developed to use available ingredients in a convenient
and utilitarian manner. There are two basic types – one are those that are designed to simulate
natural counterparts and other which have no counter parts, but are prepared to give variety to
the diet. Under these two types falls products that are – fortified, enriched, nutritionally
modified, simulated, imitation and convenience foods.
Principles of food formulation – Fabricated foods are made by combining three basic building
blocks of all food products- fat, protein and carbohydrates – in a way that provides
convenience, texture, flavour and other desirable characteristics. It involves manipulation of
these basic components along with water, vitamins, flavours etc. to design a product of
predictable composition, texture, flavour and storage properties. The final aim is to achieve
uniformity in product attributes which are dependent upon complex inter-relationship to
various aspects like- multi-component solubility behaviour, crystal growth and assemblies,
wetting, emulsification, stability and mechanical properties. In the case of dispersed system like
24
emulsions, the stabilizing effect is achieved by raising the viscosity of continuous phase or by
adding proteinaceous material which acts as barrier to coalescence. Generally polysaccharides
are used as viscosity enhancer, where the polymer chain forms the ordered structure and give
rise to gel formation of different strength and stability. It is therefore, necessary to take into
consideration the complex structures and interactions involved among food components. The
ingredients used must be readily available, economical, safe and must serve a useful function.
Dietetic foods – Increasing consumer awareness of the importance of diet in health and as
therapeutic adjunct in control of many diseases has opened vistas for manufacturers to provide
foods with specific nutritional merits. Dietetic foods include products for dietary management
of people suffering with specific metabolic disorders. Under this category comes infant
formulae, weaning foods, slimming foods, energy rich foods and beverages, low sodium foods,
food supplements for management of cardio-vascular and diabetic health.
Dietary supplement - Dietary supplements, also known as “food supplements or nutritional
supplements”, are preparation intended to supplement the diet and provide nutrients that
may be missing or may not be consumed in sufficient quantity in a person's diet. Nutraceuticals
are also gaining importance as a dietary adjunct for preventing various diseases. They may be
considered as a food or part of food that provides medical or health benefits, including the
prevention of disease. Dietary patterns of consumers have changed and they are going in for
foods with functional ingredients beneficial for health. This has resulted in the growth of
functional foods market by about 60 percent.
Cardiovascular disease (CVD) - It is the major cause of premature deaths in the most affluent
societies all over the world (Bahl et al. 2001). It is responsible for 51% of human deaths in the
world. The prevalence of cardiovascular disease in India has increased from 4% in 1960 to 11%
in 2001. Specifically, every 9th individual in India can be confidently suspected of having CVD
(Krishnaswami, 2002). Coronary heart disease (CHD) is a condition in which the main coronary
arteries supplying blood to the heart are no more capable of supplying sufficient oxygenated
blood to the heart muscle. The main cause of reduced flow is an accumulation of plaques,
manly in the intima of arteries, a disease called “Atherosclerosis” . A number of risk factors
known to predispose an individual to CVD. High levels of Low-density-lipoprotein (LDL)
cholesterol and low levels of High-density-lipoprotein cholesterol (HDL) are regarded as major
indicators of CVD risk.
Dietary intervention - An increasing number of potential nutritional products with medical and
health benefits, so called “functional foods” have gained an important place in the world
market. Functional foods are derived from naturally occurring ingredients and should be
consumed as part of the daily diet. When ingested, they are expected to perform particular
functions such as enhancement of the biological defense mechanisms, prevention/recovery
25
from a specific disease. Foods can be modified by the addition of phytochemicals, bioactive
peptides, omega-3 PUFA and probiotics and/or prebiotics to become functional. Diet is believed
to influence the risk of CHD through its effects on certain risk factors mentioned above. In
recent years, the possible hypocholsterolemic effects of several dietary components such as
prebiotics, dietary fiber (beta-glucan), omega-3 fatty acids and dietary antioxidants
(tocopherols, tocotrienols) have attracted much interest. Recent research indicates that foods
rich in omega-3 fatty acids, antioxidant vitamins and dietary fiber may provide some heart-
health benefits. These dietary strategies are all aimed at improving cardiac health.
Role of fat intake - The present recommendation is to decrease saturated fatty acids and
increase the intake of monounsaturated acids. First line of treatment for individuals with
moderately raised cholesterol and/or TAG is to modify their diet by reducing the percentage of
dietary energy derived from fat to approximately 30%, of which not more than 10% of energy
should come from saturated fat. There are three major types of omega-3 fatty acids that are
ingested in foods and used by the body: (a) alpha-linolenic acid (ALA), (b) Eicosapentaenoic acid
(EPA), (c) Docosahexaenoic acid (DHA). Once eaten, the body converts ALA to EPA and DHA,
which are more readily used by the body. The benefits of the increased intake of n-3 PUFA lie in
their ability to reduce thrombosis and decrease plasma TAG levels (Lovegrove and Jackson,
2000). The ratio of dietary ALA to linoleic acid to 1:4 is important in prevention of secondary
CHD (Allman, 1995).
Role of dietary fiber - Dietary fiber is a mixture of many complex organic substances, each
having unique physical and chemical properties. Results of various human studies indicate that
a variety of different soluble fibers, including guar, psyllium, pectin and oat bran have
hypocholesterolemic properties. The products of bacterial fermentation of dietary fiber may
also play a role in lipid metabolism. The physico-chemical changes in the gastrointestinal tract
(i.e. increased viscosity) interfere with micelle formation and lipid absorption, thus resulting in
reduction of serum cholesterol. Oat bran in particular has received a great deal of attention as a
fiber source with an appreciable level of soluble fiber that has been shown to reduce plasma
cholesterol levels under controlled conditions. The ability of oats to reduce plasma cholesterol
and in particular, LDL-cholesterol is because of the soluble beta-glucan gum, which is the major
hypocholesterolemic component (Welch, 1998).
Anti-oxidant vitamins - These include Vitamin E, beta- carotene and Vitamin C. Increasing the
Vitamin E content of the LDL by dietary supplementation of volunteers also inhibited oxidation
of LDL to the atherogenic form. Vit.E’ is often used to denote a mixture of biologically active
tocopherols which are potent inhibitors of lipid peroxidation. The epidemiological and
biochemical studies indicate that protection of high-risk groups from CVD could require an
intake of 36-100mg/day. Beta-carotene is particularly effective at scavenging peroxy radicals
26
under physiological conditions and is also a potent scavenger of singlet oxygen. Beta-carotene
and lycopene inhibit the oxidation of LDL to its atherogenic form. Vitamin C is a strong, water-
soluble antioxidant and is the first line of defense against oxidative stress in plasma. It serves as
an intercellular and extra cellular quencher of free radicals. It thus protect biomembranes and
LDL from peroxidative damage.
Conclusion - Formulating food products with combination of nutrients may provide health
benefits. Recent research indicates that foods rich in omega-3 and omega-6 fatty acids,
antioxidant vitamins and fibers may be beneficial for cardio-vascular health. Certain dietary
supplements claiming to lower down the serum cholesterol levels or helpful in maintaining
cardiac health are available in foreign market. Presently few dietary supplements for diabetic,
arthritic, renal patients, sports people are available in India. However, no supplement for CHD
patients has yet been introduced. Current projections suggest that in the next 20 years India
will have the largest CVD burden in the world. An insight on the occurrence of CHD suggests
that cardiac health needs protection.
References:
Allman M.A.1995 .Plant sources of n-3 fatty acids. Supplement to Food Australia. 47(3): S14-S17
Bahl V.K.; Prabhakaran, D. and Karthikeyen,G. 2001. Coronary artery disease in Indians. Indian Heart
Journal.53(6): 701-713.
Bhavana Vashishtha 2005 Formulation of dietary supplement for cardio-vascular health. Ph.D.Thesis,
National Dairy Research Institute,Deemed University,Karnal.
Krishnaswami, S. 2002.Prevalence of coronary artery disease in India. Indian Heart Journal.54: 103.
Lovegrove J.A. and Jackson, K.G. 2000. Coronary Heart Disease. In: Functional Foods: Concept to
Product.ed.Glenn R Gibson and Christine M William. Woodhead Publishing Limited, Cambridge, England.
Welch R.W. 1998. Oats – a multifunctional food. In: Functional Foods – the consumer, the products and
the evidence. eds. Michele.J.Sadler and Michael Saltmarsh. The Royal Society of Cambridge, U.K. pp99-
105.
27
Innovations in Packaging for Perishable Food Supply Chain
for Quality and Safety
P. S. Minz
Dairy Engineering Division, NDRI, Karnal-132001
Introduction
In today’s highly competitive marketplace, packaging is as vital to success as actual product.
Selecting and developing the right container to effectively market product requires an
understanding of packaging materials - advantages and disadvantages - and how materials can
be used as innovative tools for creating distinction. A better protection is a key to lengthening
product’s shelf-life, a desire by many marketers that is driven by the economics of today’s
expanding marketplace. This paper covers packaging technologies which have already replaced
conventional packaging and have a tremendous scope in near future.
Bioplastics
Because of this growing problem of waste disposal and because petroleum is a non-renewable
resource with diminishing quantities, renewed interest in packaging research is underway to
develop and promote the use of “bioplastics.” Bioplastics is a term used for packaging materials
derived from renewable resources, and which are considered safe to be used in food
applications. These new materials include starch, cellulose, and those derived from processes
involving microbial fermentation. Bioplastic development efforts have focused predominantly
upon starch, which is a renewable and widely available raw material. Starch is economically
competitive with petroleum and has been used in several methods for preparing compostable
plastics. Starch alone cannot form films with satisfactory mechanical properties (high
percentage elongation, tensile and flexural strength) unless it is plasticized, blended with other
materials, chemically modified, or modified with a combination of these treatments. Starch-
based thermoplastic materials have been commercialized over the last several years and
currently dominate the market of bio-based, compostable materials. Food-related applications
include films for food wrapping and thermoplastics for food packaging and other food
containers such as bowls, plates, cups and egg trays (Liu, 2006).
Active packaging
Active food contact materials are intended to extend the shelf life to maintain and improve the
condition of packaged food. They are designed to deliberately incorporate components that
would release or absorb substances into or from the packaged food or the environment
surrounding the food. Polymers are appropriate materials for the development of active
structures thanks to their mass transport characteristics: permeation, sorption and migration.
The active components can be incorporated into the package walls by diverse procedures. From
there, the active agent can be released into the food or headspace to make their beneficial
28
action, can remove food or headspace components which are absorbed into the polymer matrix
or act by food contact.
Edible films and coatings
Edible films and coatings enhance the quality of food products by protecting them from
physical, chemical, and biological deterioration (Kester and Fennema, 1986). The application of
edible films and coatings is an easy way to improve the physical strength of the food products,
reduce particle clustering, and enhance the visual and tactile features of food product surfaces
(Cuq et al, 1995). They can also protect food products from oxidation, moisture
absorption/desorption, microbial growth, and other chemical reactions (Kester and Fennema,
1986). The most common functions of edible films and coatings are that they are barriers
against oils, gas or vapours, and that they are carriers of active substances such as antioxidants,
antimicrobials, colors and flavours (Guilbert and Gontard, 1995; Krochta and De Mulder-
Johnston, 1997). Thus edible films and coatings enhance the quality of food products, which
results in an extended shelf life and improved safety.
Antimicrobial packaging
Antimicrobials in food packaging are used to enhance quality and safety by reducing surface
contamination of processed food; they are not a substitute for good sanitation practices (Brody
et al., 2001; Cooksey 2005). Antimicrobials reduce the growth rate and maximum population of
microorganisms (spoilage and pathogenic) by extending the lag phase of microbes or
inactivating them (Quintavalla and Vicini 2002). Antimicrobial agents may be incorporated
directly into packaging materials for slow release to the food surface or may be used in vapour
form.
Intelligent packaging
Intelligent packaging system contains an external or internal indicator to provide information
about aspects of the history of the package and/or quality of the food. Intelligent packaging
devices are capable of sensing and providing information about the function and properties of
packaged food and can provide assurances of pack integrity, tamper evidence, product safety
and quality, as well as being utilised in applications such as product authenticity, anti-theft and
product traceability. Intelligent packaging devices include time–temperature indicators, shelf
life indications, ripeness indicators, biosensors, gas sensing dyes, microwave doneness
indicators, microbial growth indicators, physical shock indicators, radio frequency identification
and numerous examples of tamper proof and anti-counterfeiting technologies. The benefits
include increased food safety, quality and consumer confidence. Such features may be
appreciated and increasingly demanded by consumers in the light of issues such as product
29
recalls, food poisoning cases and food scares. The use of intelligent packaging technologies
comes at a cost and an application of such a technology must be justified by a benefit analysis.
RFID systems for packaged foods
Radio frequency identification (RFID) is a system that uses radio waves to track items wirelessly.
RFID makes use of tags or transponders (data carriers), readers (receivers), and computer
systems (software, hardware, networking, and database). The tags consist of an integrated
circuit, a tag antenna, and a battery if the tag is passive (most active tags do not require battery
power). The integrated circuit contains a non-volatile memory microchip for data storage, an
AC/DC converter, encode/decode modulators, a logic control, and antenna connectors. The
wireless data transfer between a transponder/tag and a reader makes RFID technology far
more flexible than other contact identifications, such as the barcode system (Finkenzeller 2003;
RFID Journal Inc. 2005), and thus makes it ideal for food packaging. The working principles of an
RFID system are as follows:
1. Data stored in tags are activated by readers when the objects with embedded tags enter the
electromagnetic zone of a reader;
2. Data are transmitted to a reader for decoding; and
3. Decoded data are transferred to a computer system for further processing.
Conclusion
Food packaging has developed strongly during recent years, mainly due to increased demands
on product safety, shelf-life extension, cost-efficiency, environmental issues, and consumer
convenience. In order to improve the performance of packaging in meeting these varied
demands, innovative packagings such as bio-plastic, active, intelligent packaging etc are being
developed, tested and optimised in laboratories around the world. All these novel packaging
technologies have great commercial potential to ensure the quality and safety of dairy food
with fewer or no additives and preservatives, thus reducing wastage, food poisoning and
allergic reactions. Intelligent packaging can also monitor product quality and trace a product’s
history through the critical points in the food supply chain. An intelligent product quality
control system thus enables more efficient production, higher product quality and a reduced
number of complaints from retailers and consumers.
References
Brody A, Strupinsky ER, Kline LR. 2001. Odor removers. In: Brody A, Strupinsky ER, Kline LR, editors. Active
packaging for food applications. Lancaster, Pa.: Technomic Publishing Company, Inc. p 107–17.
Cooksey K. 2005. Effectiveness of antimicrobial food packaging materials. Food Addit Contam 22(10):980–
7.
Cuq, B., Gontard, N. and Guilbert, S. (1995). Edible films and coatings as active layers. In: Active Food
Packaging (M. Rooney, ed.), pp. 111-142. Blackie Academic & Professional, Glasgow, UK.
30
Finkenzeller K. 2003. RFID handbook: fundamentals and applications. 2nd ed. West from:
http://www.rfidjournal.com/article/articleview/1339/1/129/.
Guilbert, S. and Gontard, N. (1995). Edible and biodegradable food packaging. In: Foods and Packaging
Materials - Chemical Interactions (P. Ackermann, M. Jagerstad and T. Ohlsson, eds), pp. 159-168. The
Royal Society of Chemistry, Cambridge, UK.
Kester, J. J. and Fennema, O. R. (1986). Edible films and coatings: a review. Food Technol. 48(12), 47-59.
Krochta, J. M. and De Mulder-Johnston, C. (1997). Edible and biodegradable polymer films: challenges and
opportunities. Food Technol. 51(2), 61-74.
Liu, L. 2006. Bioplastics in Food Packaging: Innovative Technologies for Biodegradable Packaging.
www.iopp.org/files/public/SanJoseLiuCompetitionFeb06.pdf
Quintavalla S, Vicini l. 2002. Antimicrobial food packaging in meat industry. Meat Sci 62:373–80.
RFID Journal Inc. 2005. What is RFID? RFID Journal [Internet magazine]. Available
Sussex, U.K.: JohnWiley & Sons Ltd. 452 p.
31
Mechanization of Traditional Dairy Products
P. S. Minz* and A.K. Dodeja**
*Scientist, Head and Principal Scientist**
Dairy Engineering Division, NDRI, Karnal-132001
Introduction
Traditional dairy products in our country are made manually in small-scale sector with variable
quality depending on the skills of "halwais". Operations employing equipments have poor
hygiene, and inefficient energy use besides being labour intensive resulting in poor and non-
uniform product quality. For manufacture of indigenous dairy products we require units, which
should have flexibilities in their designs features such as: convenient and hygienic handling of
raw materials and products, control on product quality by facilitating inspection during all
stages of processing and provision for multi-process capability. Serious efforts have been made
by R&D institutes, engineering department of various colleges, industries etc in last three
decades to mechanize the production of Indian dairy products. This paper will give an overview
of different equipments for semi-automatic to fully automatic production of traditional dairy
products like khoa, peda, panner, rabri, basundi etc.
1. Single stage scraped surface heat exchanger (SSHE)
It has a rotor with two or four hinged blades with rotor drive operated at different rpm. This
single stage system had less operational flexibility. The capacity of system (kg of milk processed
per hour per unit heat transfer area) depends upon the mass flow rate of milk, steam
temperature, rotor speed and number of blades (Dodeja, 2008).
Application: Milk concentration, Doda Burfi, Sandesh (Bhadania et al., 2005)
2. Two stage thin film scraped surface heat exchanger (TFSSHE)
In this, two thin film SSHEs were arranged in cascade fashion. Milk enters into first SSHE where
it is concentrated to around 30% T.S. The rotor of first SSHE is provided with four variable
clearance blades and operated at 200rpm. This concentrated product flows by gravity into
second SSHE which had a different kind of rotor arrangement .It had two variable clearance
blades and two skewed blades to provide conveying force to khoa towards outlet. Further it is
operated at a lower speed of 2.5 rps. The system was tested for its industrial potential (Dodeja
et al., 1992). The product so made was compared with the product made from conventional
method in terms of its sensory attributes and found it comparable. But due to the problem of
pastiness of final product and since small change in feed rate affected the product consistency,
the idea of three stage system was conceived .
Application: Khoa
32
3. Three stage scraped surface heat exchanger (SSHE)
This equipment consisted of three identical thin film SSHEs with similar rotor in first two heat
exchanger and different unique rotor design in third stage thin film SSHE. All rotors are having
independent mechanism of varying rotor speed to alter the texture of the final product. A
precise feed control mechanism was incorporated to keep known feed rate during trials. A
screw conveying mechanism was provided at the inlet of third stage SSHE for blending sugar in
khoa. Various steam pressures controllers having sensing and transmitting signals are provided
in each steam inlet to avoid any steam pressure fluctuations (Dodeja, 2008).
Application: Khoa, burfi, basundi, rabri
4. Inclined stage scraped surface heat exchanger (SSHE)
At National Dairy Development Board an inclined scraped surface heat exchanger (ISSHE) for
continuous manufacture of khoa has been developed by Punjrath et al. (1990). In this
machine, concentrated milk of 42 to 45 per cent total solids is used as feed. The inclination of
ISSHE permits formation of a pool of boiling milk similar to traditional karahi method and is
critical to development of typical flavour and texture in khoa. This unit has received a wide
acceptance in the dairy industry.
Application: Khoa
5. Conical process vat
This equipment consists of a stainless steel conical vat with cone angle 60° and steam jacket
partitioned into 3-segments for efficient use of thermal energy and less heat loss. The
mechanism is consisting of 3-equidistant arms supported at two points in the shaft and each
arm having three independent spring-loaded blades for scraping the surface. Positive
displacement screw pump is connected to the outlet at the bottom of the vat for recirculation
and spreading of the product over heat transfer surface (Agrawala et al., 1987). The equipment
has been improvised for discharge of viscous dairy products.
Application: Khoa, burfi, rabri, ghee, basundi
6. Rheon shaping and forming machine
Industrial method of manufacture of peda has been adopted by Sugam Dairy, Baroda. Khoa
made in ISSHE is transferred to a planetary mixer and sugar @ 30% of khoa,
flavouring/colouring ingredients, additives etc is properly mixed. The peda mass is cooled to 4oC
and forming/shaping of peda ball is done by Rheon shaping and forming machine. The capacity
of the machine is 6000 pieces/hr and average weight of peda is 20 gms.
Application: Peda
33
7. Channa making device
This product is popular in the eastern part of India. A patent is obtained on the mechanized
production of chhana by IIT, Kharagpur. In this device milk is boiled and cooled down to 80°C,
through regeneration and mixed with 2 to 3% coagulant to reach pH 5.5 to 5.6 and the whey is
strained instantaneously on the perforated cone. Chhana, an unmatted mass, is collected and
bagged for sweets manufacture.
8. Equipment for continuous chhana ball forming and cooking of rasogolla
Equipment has been developed do knead the chhana and make it into balls for preparation of
rasogolla in continuous manner. In this equipment the raw chhana mass is pushed axially by the
screw and with the shearing action the desired kneading is obtained. This kneaded mass comes
out through a die in the form of cylindrical pieces. These cylindrical pieces roll through a
cylinder gyrating in an eccentric mode and in the process get modified in to spherical shape of a
ball. This equipment is capable of making 800 balls per hour each weighing about 10 gm and
can be scaled-up or down to the desired capacity.
Rasogolla Cooker: A small capacity continuous rasogolla cooker system has been developed for
hooking up with the above unit. The unit consists of a steam-jacketed cooker with a product
conveying system which is filled up with sugar syrup kept at boiling temperature for cooking of
rasogolla balls @ 2000-3000 balls per hr. (Choudhary et al., 2005).
9. Portioning and ball rolling machine
These equipments are presently used in Sugam dairy, Vadodara for mechanized production of
gulabjamun. The capacity of the portioning machine is 60 kg/hr and it makes portion of 8 gms
from the dough. The ball rolling machine then gives a spherical shape to the cut portions. The
capacity of the ball rolling machine is 3000 balls/hr.
Application: Gulabjamun, Rasogulla
Conclusion
Innovation and development of new equipments is the key to the success of dairy industry.
Since the specific processing requirements for equipment development for traditional Indian
milk products are diversified, it is therefore very important to pay close attention to R&D. An
innovative gap also exists on development of appropriate packaging machinery together with
the suiting packaging materials for traditional milk products, for integration with the
mechanized processing line.
34
References:
Dodeja, A.K. 2008. Success story of continuous khoa making machine. Proceedings of 5th
Convention of
Indian Dairy Engineers Association and National Seminar on "Dairy Engineering for the Cause of Rural
India" held at IGKV, Raipur. Pg. 159-164
Bhadania, A.G., Patel, J.S., and Shah, B.P. 2005. Sandesh Making – An Innovative Approach. Proceedings of
3rd
Convention of Indian Dairy Engineers Association held at NDRI, Karnal. Pg. 35
Agrawala, S. P., Sawhney, I. K andBikram Kumar (1987) Mechanized conical process vat. Patent No.
165440.
Punjrath, J. S., Veeranjanyalu, B., Mathunni, M. I, Samal,P.K and Aneja, R.P (1990) Inclined scraped surface
heat exchanger for continuous khoa making. Indian Dairy Sci., 43(2): 225-230.
Choudhary, R.L., Jha, S..N., Makker, S.K. and Narsaiah, K.N. 2005. A mechanized system for continuous
production for chhana ball. Annual Report - NDRI Kamal pp-32.
35
Application of Membrane Processing in the Production of Indian Dairy Products
Vijay Kumar Gupta
Dairy Technology Division, NDRI, Karnal-132 001
1.0 INTRODUCTION
The pressure driven membrane processes are based on the ability of semi-permeable
membranes of appropriate physical and chemical nature to discriminate between molecules-
primarily on the basis of size and to a lesser extent on shape and chemical composition. The
main membrane systems in ascending order of pore size are: reverse osmosis (RO),
nonofiltration (NF), ultrafiltration (UF) and microfiltration (MF). The distinction between RO,
NF, UF and MF is somewhat arbitrary and has evolved with time and usage. In a broader sense,
RO is essentially a dewatering technique, NF a demineralization process, UF a method for
fractionation and MF a clarification process.
Membrane processes have many applications in the dairy industry and are increasingly
being used because of several inherent advantages. Membrane processes can be carried out at
ambient temperature. Thus, thermal degradation problems common to evaporation processes
can be avoided resulting in better nutritional and functional properties of milk constituents.
Further, these are continuous molecular separation processes that do not involve either a
phase change or inter-phase mass transfer. Therefore, energy requirements of membranes
processes are very low compared with other processes such as evaporation, freeze
concentration, and freeze-drying. Further, easy, simple and economical operation, improved
recovery of constituents and better yield of products are other advantages for which
membrane processes are valued.
2.0 APPLICATION OF REVERSE OSMOSIS
RO is the most energy efficient dewatering process. Fluid milks and buttermilk can be
partially concentrated economically using RO, particularly for the preparation of concentrated
and dried products including indigenous dairy products like khoa, chakka, shrikhand, rabri,
basundi and kheer. The economical levels of RO concentration for whole milk is up to 30% TS
and for skim milk, 22% TS.
2.1 Khoa from RO concentrates
Khoa, an important indigenous Indian milk product, is presently manufactured on a
small scale by continuous boiling of whole milk until a desirable solids concentration (65-70%
total solids) is obtained. In recent years, several attempts have been made to develop new
methods including the use of scraped surface heat kettles or heat exchangers for commercial
production of khoa. The use of concentrated milk having up to 30% TS has produced khoa of
36
highly satisfactory quality. The reverse osmosis, being energy effective process for pre-
concentration of milk prior to the manufacture of khoa, has great potential in India. Khoa has
been prepared from cow milk as well as buffalo milk by atmospheric boiling of RO retentates in
a steam kettle (Gupta and Pal, 1994; Pal and Cheryan, 1987). The most important difference in
control khoa and RO khoa was the higher moisture retention and lower free fat content in the
later. Use of highly concentrated milk adversely affects the flavour quality. The process is
conveniently amenable to continuous production of khoa from RO milk retentate using SSHE.
Such process offers attractive energy saving in the initial concentration of milk. The energy
consumption in RO concentration was estimated to be about 80 kcal/kg of milk for batch
process and 25 kcal for continuous process, which brings about a net saving of 335 to 430
kcal/kg of milk.
2.2 Chakka from RO concentrates
Sachdeva et al. (1994) reported manufacture of ‘Chakka’ from milk concentrated by
reverse osomosis (RO). Cow milk, standardised to fat : SNF ratio of 1 : 2.2 (12.5% TS), was
pasteurised and concentrated (2.5 fold) using an RO plant The concentrate was subjected to
heat treatment of 90°C/5 min, cooled to 22°C, cultured at the rate of 2% with a mixed strain
lactic culture and incubated for 18 hours. The coagulum thus obtained was filtered and a
minimal amount of whey (4.5 lit./40 lit. of coagulum) having 18% TS was removed from it to get
the chakka. Good quality shrikhand could be produced from RO chakka.
The RO chakka had 32.7% TS, fat 10.3%, 8.8% protein, 11.7% Lactose and 1.9% ash
against the respective values for conventional chakka of 28.0%, 11.5%, 12.6%, 2.6% and 1.3%.
The yield of RO Chakka was 35.5% as compared to 28.3% in case of conventional chakka.
Increased yield, higher solids recovery, reduced processing time, increased throughput, access
to mechanisation and alleviation of whey disposal problem are claimed as major advantages of
this process.
3.0 Application of nanofiltration
Pal et al. (2002) and Sudhir (2002) reported that the inherent problem of salty taste and
sandy texture in khoa could be overcome by nanofiltration of cow milk to 1.5 fold conentration
before khoa manufacture. Dahi prepared from nanofiltered cow milk was also found to be
superior to that of normal cow milk dahi.
4.0 Application of ultrafiltration
Ultrafiltration has a wide range of applications in the dairy industry. From milk, UF
produces a permeate containing water, lactose, soluble minerals, non-protein nitrogen and
water-soluble vitamins and a retentate in which proteins, fat and colloidal salts content
increase in proportion to the amount of permeate removed. The process has also been used for
37
the manufacture of several fermented dairy products like Yoghurt and Srikhand. UF retentate
seems to be a highly promising base for chhana, rasogolla mix powder, long-life paneer. UF
technology has also been applied to upgrade khoa maufacture from cow and buffalo milks.
4.1 Chhana
Preparation of good quality chhana using skim milk ultrafiltered-diafiltered retentate
and plastic cream has been reported (Sharma and Reuter, 1991). Skim milk, heated to 95°C for
5 min., is ultrafiltered (26% TS). The retentate is diafiltered (23% TS) with equal amount of
water to reduce lactose. For preparation of chhana, the retentate is mixed with plastic cream
to a protein/fat ratio of 0.722. The mixture is heated to 85-90°C/5 min. and coagulated with
dilute lactic acid to develop the characteristic grain. The granular mass is subsequently pressed
to remove free moisture, yielding chhana. The process is reported to yield about 18-19 percent
extra product and also no significant difference in flavour, body and texture and appearance
compared to traditional method. High yield, easy automation and flexibility in operation are
emphasized as advantages of this method for adoption for large-scale production.
Kumar et al. (2005) reported improved quality of UF chhana from cow milk. Cow skim
milk was ultrafiltered and diafiltered to an optimum 23.88% TS. The required quantity of 63-
65% fat fresh cream was then added to the UF retentate for standardization of fat. An
innovative new approach i.e. addition of coagulant to UF retentate mixture at room
temperature and then heating to coagulation temperature, optimum being 60°C, resulted in
production of desired softer chhana with higher moisture content, suitable for making sweets
(rasogolla and sandesh), along with higher yield (12.92%) and higher total solid recovery
(10.89%) than in traditional chhana and lesser total solid losses in whey compared to when UF
chhana was prepared using traditional approach. Slow stirring (60-80 rpm) during heating and
coagulation of UF retentate mixture yielded lower moisture (54.53%) content in chhana,
compared to 56.93% moisture with rapid stirring (130-150 rpm). Standardized UF chhana met
PFA standards and was comparable to traditional chhana organoleptically. Rasogolla and
sandesh, prepared with modified process from UF chhana, scored ”liked moderately” to “liked
very much” on sensory evaluation.
Kumar (2006) standardized the manufacturing process of good quality chhana from a
mixture of buffalo milk and sweet cream buttermilk by employing UF process. The standardized
process gave higher yield (13.03%) and higher total solid recovery (11.49%) in UF chhana
compared to the traditional process. The standardized UF chhana had 57.6% moisture and
scored 7.5 for body and texture on 9-point Hedonic scale. The manufacturing process of
optimum quality rasogolla and sandesh produced from UF chhana were also standardized. UF
rasogolla & sandesh scored 7.7 & 8.17, respectively, for overall sensory acceptability on 9-point
Hedonic scale.
38
4.2 Rasogolla Mix Powder
Manufacture of rasogolla is probably most difficult amongst all the milk-based
delicacies. It requires lot of art and experience in addition to the right type of raw materials.
The use of ultrafiltration process has been made in our endeavour to produce base for the
rasogolla mix powder (Pal et al., 1994). Cow skim milk is ultrafiltered to about 3-fold
concentration to achieve a product containing all the milk proteins and part of the minerals and
lactose. To reduce the mineral and lactose level to almost the same level as in chhana, UF
retentate has to be diafiltered. The pasteurised cream is added to diafiltered retentate
followed by spray drying adopting standard conditions. The dried retentate is blended with
selected additives to produce desired flavour and texture. The dried rasogolla mix has about 5
months at 30oC. Production of rasogolla mix powder offers following benefits:
Offers economic use of seasonal and regional milk surpluses.
Produce sweets of consistent quality at the convenience of users.
Adaptable to medium and industrial scale dairy processing operations.
Allows product diversification with manageable investments for improved productivity
of the dairy industry.
The products offer good export potential.
4.2.1 Rasogolla making from dried mix
Equal quantities of water is added to the mix powder and kept for about 5 min for
rehydration of proteins. Circular balls of about 7g size are rolled out in a manner that no cracks
appear on the surface. Balls are cooked in the boiling sugar syrup, (maintained at 60%
consistency) for 15 min with plenty of foam around the balls. The cooked balls are transferred
into another hot sugar syrup of about 40% consistency. The yield is almost 20% higher than that
obtained by traditional method.
4.3 Paneer
Production of good quality paneer using ultrafiltration (UF) has been reported by
Sachdeva et al. (1993). The process offers advantages like access to mechanisation, uniform
quality, improved shelf life, increased yield and nutritionally better product. The method
involves standardisation and heating of milk followed by UF, whereby lactose, water and some
minerals are removed. The concentrated mass, which has about 40 percent total solids, is cold
acidified to get the desired pH. Till this point, the product is flowable and can be easily
dispensed into containers with automatic dispensing machines. The filled containers are then
subjected to texturisation by microwave heating. The resulting product has typical
characteristics of normal paneer. The yield increases by about 25 percent due to the retention
of good quality whey proteins and the slightly increased moisture content.
39
Table 1 Compositional comparison between various types of paneers made by the traditional
processes and Long life paneer made by the texturizing process.
Chemical attribute Traditional paneers Concentrated
milk paneer
UF paneer
Full fat
(5.8%)
Low fat
(1.5%)
Skim milk
(0.05%)
Fat 23.41
(50.84)
8.60
(22.47)
0.20
(0.56)
5.39
(17.42)
7.20
(23.51
Protein 18. 33
(39.81)
21.56
(56.32)
25.83
(72.92)
13.50
(43.63)
15.92
(51.98)
Lactose 2.40
(5.22)
* * 10.13
(32.74)
5.30
(17.30)
Ash 1.90
(4.13)
* * 1.92
(6.21)
2.21
(7.22)
Total Solids 46.04 38.28 35.42 30.94 30.63
Yield 20.00 16.30 14.10 40.00 25.00
Figures in parentheses indicate the values on moisture free basis
In another approach, a fully sterilization process has been developed which yields a long
shelf life paneer like product (Rao, 1991). Standardised buffalo milk is concentrated partly by
vacuum concentration process and partly by employing UF to a level of total solids desired in
the fnal product. After packing in metallised polyester pouches, product is formed by a
texturising process at 1150C, which permits concomitant sterilization. The process permits
greater product yield due to retention of whey solids, being 35 per cent as compared to 15 per
cent obtained by conventional batch process.
4.4 Shrikhand
The traditional technology allows the whey proteins to drain along with whey during the
process of chakka making. These proteins, having high biological value could be recovered in
chakka by the application of ultrafltration to make, so called UF-chakka (Sharma and Reuter,
1992). Chakka and Shrikhand of good sensory quality and meeting PFA standards could be
successfully prepared using ultrafiltration technology (Shukla, 2004). In standardized
ultrafiltration process, skim milk coagulum obtained by fermentation of skim milk with yoghurt
culture was heated to 600C for 5 minute with continuous agitation and ultrafiitered up to
around 16.60% TS concentration. Whey was then removed from this concentrated coagulum by
hanging it in a muslin cloth (eight layered) at room temperature followed by mild pressing to
get chakka. Chakka was then kneaded in a planetary mixer with 70% fat cream and sugar to
40
prepare Shrikhand of smooth consistency. UF process resulted in nil fat loss in whey and 20.70%
extra recovery of total solids in chakka. The protein content in skim milk chakka through UF
process and in shrikhand prepared from it was higher than in traditional process.
4.5 Khoa
Khoa from cow milk has been reported to be salty in taste, sticky/pasty in body and
texture and slight yellowish in colour. WPC addition has shown to improve the flavour, body
and texture, colour and appearance and thereby overall sensory attributes of cow milk khoa.
Addition of 5% WPC solids to cow milk improved the flavour, body and texture and colour of
khoa prepared (Patel et al., 1993). WPC incorporated cow milk khoa compared well with the
traditional buffalo milk khoa.
Though the flavour score for 12% WPC added khoa were higher than other WPC added
khoa samples, the improvement was not statistically significant between 8%, 10% and 12%
WPC added khoa (Sudhir, 2002). Increased level of WPC increased the grain size of khoa and
decreased stickiness/pastiness, however, it also resulted in reduced cohesiveness and increased
dryness in the product. Hence, the selection of level of WPC is subject to the requirement of
type of khoa intended for further use e.g. Khoa prepared by addition of higher level can be
suitable for kalakand like product.
Sudhir (2002) reported that the khoa with added WPC (80) from nanofiltered cow milk
scored higher for flavour and overall scores (47 and 91.29, respectively) than khoa from
nanofiltered cow milk (45.71 and 90.43, respectively). A definite increase in grain size for WPC
added khoa from nanofiltered cow milk was observed. Khoa with added 12% WPC from
nanofiltered cow milk scored more in flavour, body and texture (30.86), colour and appearance
(13.42) and overall sensory scores than 12% WPC added khoa from cow milk (44.57, 30.36,
12.71 and 87.64, respectively). The scores of khoa from nanofiltered cow milk with added WPC
were also comparable to commercial buffalo milk khoa, which scored 47.07, 31.5, 13.79 and
92.35 for flavour, body and texture and colour and appearance, respectively. However, the
product obtained from use of nanofiltered cow milk tended to be sticky, which could be
because of homogenization effect on cow milk. Nanofiltration of skimmed cow milk followed by
standardization to fat : TS ratio of 0.38-0.4 and subsequent khoa making by WPC addition might
probably obliviate this problem.
Reuter et al. (1990) incorporated 10 and 18% WPC (27.41% TS) solids in buffalo milk for
the manufacture of khoa, Greater amount of WPC produced bigger grains in khoa, which is a
desirable property for preparing Kalakand - a popular khoa based Indian sweet.
41
5. References
Gupta, S. K and Pal, D. (1994) Production of khoa from buffalo milk concentrated by Reverse Osmosis.
Indian J. Dairy Sci., 47 (3) 211-214.
Pal, D. and Cheryan, M. (1987) Application of reverse osmosis in the manufacture of khoa: Process
optimization and product quality. J. Fd. Sci. & Tech. 24, 233.
Pal, D., Rajorhia, G.S., Garg, F.C. and Verma, B.B. (1993) Development of technology for dried rasogolla
mix. NDRI Annual Report 1992-93, pp 90.
Pal, D., Rajorhia, G.S., Garg, F.C. and Verma, B.B. (1994) Production of dried rasogolla mix from
ultrafiltered milk retentate. 24th Int. Dairy Congr., Melbourne, Australia 18-22 Sept, pp 424.
Kumar, J. (2006) Admixing of buttermilk to buffalo milk for production of chhana and chhana based
sweets - Ph.D. Dissertation submitted to NDRI (Deemed University), Karnal.
Kumar, J., Gupta, V.K. and Patil, G.R. (2005) Studies on improvement of chhana using ultrafiltration
process. Indian J. Dairy Sci.58 (3), 162-168.
Rao, K. V. S. S. (1991) A mechanized process for manufacture of paneer - Ph.D. Dissertation submitted to
NDRI (Deemed University), Karnal.
Sachdeva, S., Patel, R.S., Kanawijia, S.K., Singh, S. and Gupta, V.K. 1993. Paneer manufacture employing
ultrafiltration, 3rd
Int. Food Conv., IFCON-93, Mysore.
Sachdeva, S., Patel, R.S., Tiwary, B.D. and Singh, S. 1994. Manufacture of chakka from milk concentrated
by Reverse osmosis. 24th
International dairy congr. Melbourne, Australia, Jb. 36 : 415.
Sharma, D.K. and Reuter, H. 1991. A method of chhana making by ultrafiltration technique. Indian J.
Dairy Sci., 44 (1) : 89.
Sharma, D.K. and Reuter, H. 1992. Ultrafiltration technique for shrikhand manufacture. Indian J. Dairy
Sci., 45 (4) : 209.
Shukla, K.K. (2003) Studies on the production of shrikhand using ultrafiltration process- M.Sc. Thesis
submitted to Institute of Food Technology, Bundelkhand University, Jhansi.
Sudhir, V.K (2002) Studies on improvement of quality of khoa using ultrafiltration technique- M.Sc. Thesis
submitted to NDRI Deemed University, Karnal.
42
Application of Membrane Processing for Production of Quality Dairy Products
Vijay Kumar Gupta
Dairy Technology Division, NDRI, Karnal-132 001
Introduction
The main membrane systems in ascending order of pore size are: reverse osmosis (RO),
nonofiltration (NF), ultrafiltration (UF) and microfiltration (MF). The distinction between RO,
NF, UF and MF is somewhat arbitrary and has evolved with time and usage. In a broader sense
RO is essentially a dewatering technique, NF a demineralization process, UF a method for
fractionation and MF a clarification process.
Membrane processes have many applications in the dairy industry and are increasingly
being used because of several inherent advantages. Membrane processes can be carried out at
ambient temperature. Thus, thermal degradation problems common to evaporation processes,
can be avoided resulting in better nutritional and functional properties of milk constituents.
Further, these are continuous molecular separation processes that do not involve either a
phase change or inter-phase mass transfer. Therefore, energy requirements of membranes
processes are very low compared with other processes such as evaporation, freeze
concentration, and freeze-drying. Further, easy, simple and economical operation, improved
recovery of constituents and better yield of products are other advantages for which
membrane processes are valued.
Application of nanofiltration
Acid whey is particularly very rich in mineral contents. Whey can be partially
demineralized (about 40%), particularly with respect to monovalent ions, and concentrated
simultaneously to approximately 25 % TS using nanofiltration process. Pal et al. (2002) and
Sudhir (2002) reported that the inherent problem of salty taste and sandy texture in khoa could
be overcome by nanofiltration of cow milk to 1.5 fold. Dahi prepared from NF cow milk was
also found to be superior to that of normal cow milk dahi.
Application of ultrafiltration
Ultrafiltration has a wide range of applications in the dairy industry. UF produces from
milk a permeate containing water, lactose, soluble minerals, non-protein nitrogen and water-
soluble vitamins and a retentate in which proteins, fat and colloidal salts content increase in
proportion to the amount of permeate removed. The UF process has been used for milk protein
standardisation, preparation of protein rich milk, low lactose powder etc. The process has also
been used for the manufacture of several fermented dairy products like Yoghurt, Srikhand and
Ymer and various types of soft and semi soft varieties of cheese. The development of
43
ultrafiltration processes has proved a boon for cheese makers in the treatment of whey. The
use of ultrafiltration to fractionate and concentrate the whey proteins, followed by evaporation
and drying is now a commercial process for the manufacture of whey protein concentrate for
edible and other applications. Other industrial applications include enzyme recovery. More
recently, membrane processes have been utilised for the preparation of enzymatic derivatives
of milk proteins having pharmacological significance.
Milk protein standardisation
Standardisation of protein content of milk and milk products has become an
international issue and receiving attention of the planners and research workers alike. Views
on protein standardisation of fluid milk for drinking, other fluid milk products and cream are
receiving considerable attention in context with the economic implications of protein
standardisation. From processing point of view this issue can be attempted through the use of
UF technology.
UF milk retentate
UF milk retentate has widely been used for the manufacture of cheese and other
fermented short shelf-life products where protein increase is desirable, but lactose and ash
increase is not desirable (Darghn and Savello, 1990; Green, 1990, Singh et al., 1994). In the
Indian context UF retentate seems to be a highly promising base for long-life paneer (Rao,
1991; Singh et al., 1994). UF technology has also been applied to produce milk protein
concentrates, low lactose powder, non-dairy whitener, rasogolla mix powder, cheese base etc.
1. High protein/high calcium diet
The UF process offered dairy technologists a powerful and versatile tool for the
fractionation and concentration of milk constituents that inspired their efforts to develop new
dairy dietary products and to tailor the properties according to market need and need of the
patients. A wide range of novel in container sterilised milk concentrates have also been
developed from ultrafiltered skim milk with a shelf life above one year (Muir et al 1984,
Sweetsur and Muir 1985) and can be used for sports persons and old people.
2. Manufacture of milk protein concentrates
Typically with a protein purity of 50-85 %, Milk protein concentrates can be considered
as a functional ingredient to be used in the manufacture of other foodstuffs. To obtain milk
protein concentrates with 85 % protein/TS, it is necessary to employ diafiltration treatment.
Dried milk protein concentrates can be used for the production of many dietetic foods.
44
3. Low lactose powder
Lactose intolerance is a global problem. UF technology is employed for the manufacture
of low-lactose powder. Additional diafiltration treatment is employed to further reduce lactose.
During the ultrafiltration process, some of the soluble salts like calcium, sodium and potassium
are bound to go in the permeate. These salts are important for giving milk its natural taste. To
maintain the salt level and thereby revive the original taste of milk on reconstitution, a salt
mixture of kcl and -citrate in the ratio of 1:0.77 is added to the to the formulation before spray
drying. Further, for better reconstitution properties, malto-dextrin is added in the formulation
4. Non-dairy whitener
Non-dairy whiteners are widely used as a substitute for fresh milk, cream or evaporated
milk in coffee, tea, cocoa or drinking chocolate and are also suitable for adding to foods like
soups, sauces, puddings and cereal dishes. The replacement of sodium caseinate, the
conventionally used protein source in the non-dairy whiteners, by UF skim milk retentate has
many advantages like reduction in product cost, process simplification and presence of
nutritious whey proteins. The suitability of using UF skim milk retentate as whitener has been
reported by Jimenez-Florez and Kosikowski (1986). Mukherjee (1996) standardised the
manufacture of non-dairy whitener using UF skim milk retentate as a base.
5. Cheese
The major use of UF technology is in the manufacture of soft cheeses, defined as those
containing more than 45% moisture. Extending the use of UF technology to all the cheeses may
not be simple as UF retentate contains appreciable quantities of whey protein. Undenatured
whey proteins retained in the cheese are resistant to proteolysis. High buttering capacity of the
cheese curd, due to the increased concentration of calcium in the retentate, retards the rate of
lactic starter autolysis and consequently hydrolysis of casein network. Continuous efforts are
being made to manufacture good quality hard cheese from UF milk employing certain process
modifications and using modified starters.
Advantage of cheese making by UF
It increases the yield of cheese up to 10-30% due to entrapment of whey proteins and
possibly additional bound water associated with the whey proteins. The yield depends
on the level of concentration achieved during ultrafiltration and the type of cheese
made. About 8% higher yield for hard cheese e.g. Cheddar cheese and up to 30% for
semi-hard and soft varieties of cheeses are commercially obtainable.
Requirements for starter culture and rennet are reduced.
It reduces the energy requirement during heating and cooking steps.
Whey disposal problem is substantially reduced because of lesser whey production.
45
Process is amenable to mechanization and automation in cheese making.
Manufacture of cheese base and processed cheese
Cheese base is a paste of the same composition and ph as Cheddar cheese but without
the Cheddar flavour and structure. It is used to replace the young cheese component for the
manufacture of processed cheese. For the production of cheese base, milk is pasteurised and
standardised to 3.8% fat, cooled to 50°C, ultrafiltered to 30% TS, diafiltered to reduce lactose to
desired level, further ultrafiltered to 40% TS, re-pasteurised, cooled and 1% Cheddar starter
culture added and evaporated to 60% TS. Processed cheese is made by blending cheese base
(30%) with 70 % normal aged Cheddar cheese.
6. Chhana
Preparation of good quality chhana using skim milk ultrafiltered-diafiltered retentate
and plastic cream has been reported. Skim milk, heated to 95°C for 5 min., is ultrafiltered (26%
TS). The retentate is diafiltered (23% TS) with equal amount of water to reduce lactose. For
preparation of chhana the retentate is mixed with plastic cream to a protein/fat ratio of 0.722.
The mixture is heated to 85-90°C/5 min. And coagulated with dilute lactic acid to develop the
characteristic grain. The granular mass is subsequently pressed to remove free moisture,
yielding chhana. The process is reported to yield about 18-19 percent extra product and also
no significant difference in flavour, body and texture and appearance compared to traditional
method. High yield, easy automation and flexibility in operation are emphasized as advantages
of this method for adoption for large-scale production.
7. Rasogolla Mix Powder
Manufacture of rasogolla is probably most difficult amongst all the milk-based
delicacies. It requires lot of art and experience in addition to the right type of raw materials.
The use of ultrafiltration process has been made in our endeavour to produce base for the
rasogolla mix powder (Pal et al., 1993). Cow skim milk is ultrafiltered to about 3-fold
concentration to achieve a product containing all the milk proteins and part of the minerals and
lactose. To reduce the mineral and lactose level to almost the same level as in chhana, UF
retentate has to be diafiltered. The pasteurised cream is added to diafiltered retentate
followed by spray drying adopting standard conditions. The dried retentate is blended with
selected additives to produce desired flavour and texture.
8. Paneer
Production of good quality paneer using ultrafiltration (UF) has been reported by
Sachdeva et al. (1993). The process offers advantages like access to mechanisation, uniform
quality, improved shelf life, increased yield and nutritionally better product. The method
46
involves standardisation and heating of milk followed by UF, whereby lactose, water and some
minerals are removed. The concentrated mass, which has about 40 percent total solids, is cold
acidified to get the desired ph. Till this point, the product is flowable and can be easily
dispensed into containers with automatic dispensing machines. The filled containers are then
subjected to texturisation by microwave heating. The resulting product has typical
characteristics of normal paneer. The yield increases by about 25 percent.
Whey protein concentrates
UF process is now a major means of WPC production throughout most of the dairy
countries of the world. WPC with 35% protein is perceived to be a universal substitute for
NFDM, because of the similarity in gross composition and its dairy character. WPC can also be
seen competing with casein, egg albumin and soya proteins within the existing markets.
Commercially, dried WPC products produced by UF may contain 30 to 80% protein. In order to
achieve higher protein values (up to 90% of dry matter), one or more diafiltration steps may
follow.
Lactose
Ultrafiltration technology offers distinct advantages over the conventional technology
for the manufacture of lactose. The protein and mineral contents of whey are the limiting
factors for the crystallization of lactose and hence permeate obtained on ultrafiltration is
considered as a better substrate for lactose production.
APPLICATIONS OF MICROFILTRATION
The potential applications of MF in dairy industry include separation of bacteria and
spores, fractionation of milk proteins and clarification of whey.
Improving microbiological quality of milk
The emergence of MF as a means of bacteria and spore removal from milk has
generated much interest about the alternative technologies for the manufacture of quality
dairy products. Removal of spores using MF is 10 times better compared to bactofugation,
regardless of initial count. The feasibility of removing microorganisms by MF appeared remote
until the development of Membralox multichannel 1.4 µm membrane, which proved to be
suitable for debacterisation of skim milk with only minor losses in solids-not fat (Jost and Jelen,
1997). Somatic cells, which can induce detectable defects in dairy products made from milk
having a content higher than 4 x 105 SCC/ml, are absent in the MF treated milk. This leads to
increased technical advantages and hygienic safety in dairy processing. Alfa-Laval has patented
a process called 'Bacto Catch' for removing microorganims from skim milk using Alumina
membrane of 1.4 µ (Larsen, 1996). The microbial count was reduced by 99.91%. The retentate
(having bacteria and some milk solids) was heat treated (130°C) and recombined with
47
microfilterate and whole lot was pasteurized. The keeping quality of milk is enhanced further
to 3-4 days. For cheese manufacture, the microfiltrate retentate is mixed with the cream,
heated to 130°C for 4 sec and mixed with the microfilterate (Kessler, 1997). The number of
anaerobic spores in cheese milk is reduced significantly (Samuelsson et al., 1997).
Clarification of whey
Microfiltration can be used to remove casein fines, microorganisms, fat globules,
somatic cells etc. From whey. Pearce et al., (1992) reported 30 to 80 percent residual lipids
removal from cheddar cheese whey using an Alfa-Laval MFS-7 fitted with Ceraver ceramic
membranes of 1.4 and 0.8 µ porosity, respectively. There is a 1.8 fold increase in the rate of UF
of whey proteins when the lipids had been removed by MF ( Karleskind et al., 1995).
Pretreatment of whey by MF has emerged as a necessary step in producing high purity
whey protein concentrates. A control pretreatment consists of a physico-chemical process
comprising increased ionic calcium and ph accompanied by heat (50°C, 15 min.) To cause
aggregation of complex lipid-calcium phosphate particles, which are then separated by MF
(Gesan et al., 1995). In another study (Pierre et al., 1994), physico-chemical pretreatment of
whey was carried out combining calcium addition, ph increase to 7.3 and a heat treatment
(60°C, 10 min.). Studies have shown that when MF is performed on sweet whey as an
intermediate step within the UF process, a fat content below 0.4 percent in 85 percent WPC
powder can be achieved (Jensen et al., 1992).
References
Bird, J. (1996) The application of membrane systems in the dairy industry. J. Soc. Dairy Technol., 49: 16.
Darghn, R.A. and Savello, P.A. (1990) Yoghurt with improved physical properties from ultrafiltered and
UHT treated skim milk. J. Dairy Sci., 73 (Suppl.): 1, 94.
Famelart, M.H., Lepesant, F., Gaucheron, F., Le Graet, Y. And Schuck, P. 1996 ph-Induced physicochemical
modifications of native phosphocaseinate suspensions : Influence of aqueous phase. Lait 76 : 445-460.
Jensen, M., Jensen, J., Larsen, P.H. and Pannetier, E. (1992) Defatted high functional 85% WPC. Alfa-Laval
Publication, 9.
Jost, R. And Jelen, P. (1997) Crossflow microfiltration-an extension of membrane processing of milk and
whey. IDF Bull. 320 : 9-15.
Karleskind, D., Laye, I., Mei, F.I. and Morr, C.V. (1995) Chemical pretreatment and microfiltration for
making delipidised whey protein concentrate. J. Food Sci. 60 (2) : 221.
Kessler, H.G. (1997) Engineering aspects of currently available technological processes. IDF Bull. 320 : 16-
25.
Larsen, P.H. (1996) Microfiltration for pasteurised milk. IDF Special Issue 9602 : 232-239.
Maubois, J.L. (1997) Current uses and future perspectives of MF technology in the dairy industry. IDF Bull,
320 : 37-40.
Muir, D.D. and Banks, J.M. (1985) J. Soc. Dairy Technol.38, 116-119.
Mukherjee, M. (1996) Studies on UF skim milk retentate as a base for non-dairy whitener. M.Sc. Thesis
submitted to NDRI Deemed University, Karnal.
48
Pal, D., Rajorhia, G.S., Garg, F.C. and Verma, B.B. (1993) Development of technology for dried rasogolla
mix NDRI Annual Report, pp 90.
Rao, K. V. S. S. (1991) A mechanized process for manufacture of paneer, Ph.D. Dissertation submitted to
NDRI (Deemed University), Karnal.
Sachdeva, S., Patel, R.S., Kanawijia, S.K., Singh, S. And Gupta, V.K. (1993) Paneer manufacture employing
ultrafiltration, 3rd
Int. Food Conv., IFCON-93, Mysore.
Singh, S., Kanawjia, S.K., Patel, R.S., Sachdeva, S. And Rai, T. (1994) Development of fresh/short ripened
cheese from UF cow and buffalo milk. Annual Rept., NDRI, Karnal, PP. 85.
Samuelsson, G., Dejmek, P., Tragarth, G. And Paulsson, M. (1997) Rennet coagulation of heat-treated
retentate from crossflow microfiltration of skim milk. Milchwissenschaft 52 : 187-192.
Sudhir, V.K (2002) Studies on improvement of quality of khoa using ultrafiltration technique. M.Sc. Thesis
submitted to NDRI Deemed University, Karnal.
49
Recent Developments in the Manufacture of Low-Calorie Milk Products
P. Narender Raju and Ashish Kumar Singh
Dairy Technology Division, NDRI, Karnal-132001, India
Introduction
Worldwide non-communicable diseases such as obesity, diabetes, cardiovascular
diseases and cancer have become major health problems due to changing lifestyle and dietary
patterns among people. The World Health Organization indicated that worldwide
approximately 1.6 billion adults (age 15+) and 20 million children under the age of 5 years were
overweight and at least 400 million adults were obese in 2005 and projected that
approximately 2.3 billion adults will be overweight and more than 700 million will be obese by
the year 2015 (WHO, 2006). Further, recent estimations revealed that worldwide more than
220 million people have diabetes (WHO, 2009). In 2005, an estimated 1.1 million people died
from diabetes, with the number likely to be doubled by the year 2030 (WHO, 2009). India has
the largest diabetic population with one of the highest diabetes prevalence rates in the world
(King et al., 1998; Bjrok et al., 2003). It is predicted that the Indian diabetic population would
rise to more than 80.9 million by the year 2030 (King, et al., 1998). An Indian National Urban
Diabetes Survey reported the average diabetes prevalence rate as 12.1% (Ramachandran, et al.,
2001). However, there was a large regional variation and the prevalence rates varied from 9.3%
in Mumbai to 16.6% in Hyderabad. Type-2 diabetes is a chronic progressive disease that
requires lifestyle changes (Knowler et al., 2002), the key lifestyle interventions being physical
activity and a nutritional plan with reduced caloric intake (Franz, 1997). In India, non-
communicable diseases caused 5.10 million deaths in the year 2002, of which cardiovascular
diseases were responsible for 2.78 million deaths (Beaglehole and Yach, 2003). However, there
are large disparities in cardiovascular disease mortality in different Indian states. The dietary
factors such as high intake of fats, sugars, milk and its products and low intake of fruits and
vegetables were ascribed for the role in the cardiovascular disease mortality (Gupta et al.,
2006). Being aware of the impact of high fat and high sugar on health, today’s health conscious
consumer is looking for the low-fat, low-sugar or sugar-free dairy products. Successful efforts of
chemists and food technologists worldwide led to the development of novel food additives that
impart low- or zero-calories. With the continuous invention of fat replacers and low-calorie and
high-intensity sweeteners it has been possible to develop dietetic dairy products for the benefit
of health conscious consumers in general and calorie conscious consumers in particular. In the
present paper, technological developments in the manufacture of low-calorie dairy products
have been presented.
50
Additives for low-calorie products
Fat is a crucial contributor to several texture attributes, such as creaminess, softness,
melting in the mouth, juiciness and thickness while sweeteners elicit pleasurable sensations
with or without energy and contribute to bulk and characteristic colour. Most of these
attributes are desired attributes and hence positively regarded qualities in food products. But,
calorie conscious people need to achieve a negative energy balance to maintain ideal body
weights by cutting down their caloric intake. Hence, low-fat, low-sugar or sugar-free products
are formulated or designed so as to meet the dietary requirements of obese, persons at risk of
cardiovascular diseases, diabetics and persons on weight management diets. Most dairy
products including Indian traditional dairy products contain high fat and high sugar and it is well
known that these macro nutrients provide about 9 and 4 kcal of energy per gram, respectively.
Hence, it is imperative to choose food additives or ingredients that contribute to few or no
calories in the development of low-calorie dairy and food products without compromising the
sensory and overall quality. In this context, sweeteners and fat replacers are the vital additives
for the development of such products.
Sweeteners are be classified, based on their contribution towards energy, as nutritive
and non-nutritive sweeteners. Nutritive sweeteners are those substances, which when
consumed, not only provide sweet taste but also contribute 4 kcal per gram of substance. It
includes sugar, honey, D-glucose, invert sugar, caramel, maltodextrin, high-fructose corn syrup
and dextrose syrup. Low-calorie sweeteners are nutritive sweeteners that are relatively less
sweet than sucrose and provide energy between 1 to 3 kcal per gram. Polyols are low-calorie
sweeteners (about 2 kcal per gram) that occur naturally in a number of fruits, all vegetables,
cereals, algae, mushrooms, seaweeds, etc. e.g. sorbitol, maltitol, lactitol and mannitol. Polyols
are industrially obtained under high temperature by catalytic hydrogenation of the relevant
saccharides. Non-nutritive sweeteners are those sweeteners that offer no energy such as
aspartame, acesulfame-K, sucralose etc. The intensity of the sweetness of a given substance in
relation to sucrose is made on a weight basis (Table-1).
51
Table-1. Relative Sweetness of Sweeteners
Sweetener Approximate
Sweetness
Sucrose 1.0
Crystalline fructose 1.2 - 1.7
HFCS, 55% 1.0
HFCS, 90% 1.0
Hydrogenated
starch hydrolysates 0.4-0.9
Lactitol 0.4
Trehalose 0.45
Isomalt 0.45-0.65
Sorbitol 0.6
Mannitol 0.7
Maltitol 0.9
Xylitol 1.0
Aspartame 180
Acesulfame
potassium 200
Saccharin 300
Sucralose 600
Stevioside 300
Alitame 2000
Neotame 8000
Table-2. Examples of Fat Replacers
Source Example
Carbohydrate based
Corn
maltodextrin
Maltrin®
Sta-Slim®
Resistant starch Crystalean®
Modified starch FirmTex®
Fantesk®
Tapioca dextrins N-Oil®
Potato dextrins Paselli®
β-Glucans Oatrim®
MCC Avicel®
Inulin (FOS) Raftiline®
Raftilose®
Polydextrose Litesse®
Guar gum Novagel®
Fat based
Structured lipids Caprenin®
Salatrim®
Sucrose fatty acid
polyesters (SPEs) Olestra (Olean®)
Synthetic fats
Dialkyl
dihexadecylmalonate
(DDM)
Trialkoxytricarballylat
e (TATCA)
Protein based
Whey protein,
partially
denatured
Dairy-Lo®
Whey protein,
microparticulate
d
Simplesse®
52
Fat may be replaced in foods by reformulating the foods with food additives called as fat
replacers which represent a variety of chemical types with diverse functional and sensory
properties and physiological effects. Mostly they are characterized into two groups – fat
substitutes and fat mimetics. Fat substitutes are macromolecules that physically and chemically
resemble triglycerides and which can theoretically replace the fat on a one-to-one, gram-for-
gram basis (fat- or lipid-based fat replacers). Examples of fat substitutes are sucrose fatty acid
polyesters (SPEs), sucrose fatty acid esters (SFEs), structured lipids, etc (Table-2). Structured
lipids are developed for specific purposes, such as reducing the amount of fat available for
metabolism and potentially, caloric value. Fat mimetics are substances that imitate
organoleptic or physical properties of triglycerides but which cannot replace fat on a one-to-
one, gram-for-gram basis (protein- or carbohydrate-based fat replacers). Fat mimetics generally
adsorb substantial amount of water and are not suitable for frying. They are generally less
flavourful than the fats the mimetics are intended to replace as they carry water-soluble
flavours but not lipid-soluble flavour compounds. Examples of fat mimetics include
carbohydrate- and protein-based fat replacers.
LOW-CALORIE MILK PRODUCTS
The dairy industry has responded to the growing needs of health conscious consumers
for low-calorie foods. Consequently, a large number of dairy products made with low-calorie
and/or non-nutritive sweeteners and fat replacers have been developed and some were
witnessed in the super market shelves. Some of the R&D efforts in this area are discussed here.
Ice-cream and Frozen desserts
Frozen desserts are delicate, delicious and nutritious food liked by all age groups
throughout the world. In its broadest sense the term ‘ice cream’ covers a wide range of
different types of frozen desserts. It includes dairy ice cream, non-dairy ice cream, gelato,
frozen yoghurt, milk ice, sherbet, fruit ice, etc. What these all have in common is that they are
sweet, flavoured, contain ice and unlike any other frozen food, are normally eaten in the frozen
state. In India, as per PFA Act, ice cream shall contain not less than 10 per cent milk fat. Olsen
(1989) suggested an ice cream formulation with low fat and low sugar content having 3% fat,
0% sugar, 4% glucose syrup, 3% bulking agent, 0.05% aspartame and 0.7% stabilizer/emulsifier.
Palumbo, et al. (1995) developed aspartame sweetened ice cream and ice milk bulked with
lactitol and/or polydextrose. Mingione and Kohlmann (1995) developed formulation of a low
fat, low cholesterol and lactose free dairy dessert. It was reported that the dessert formulation
may contain non-dairy milk, a sweetener (7-45% sucrose or a sugar substitute such as
aspartame or dextrose), filler (whey, whey protein concentrate or maltodextrin), stabilizer and
flavourings. Olinger and Pepper (1996) described a process for frozen dessert sweetened with
acesulfame-K in combination with lactitol and hydrogenated starch hydrolysate was used as the
53
bulk sweeteners. Taste, texture, hardness, melting and overrun properties of the frozen dessert
were reported to be comparable to those in conventional products sweetened with sucrose
and corn syrup. Verma (2002) had developed frozen dessert using artificial sweeteners and
reported that amongst the various sweeteners attempted, aspartame produced the most
acceptable product. Further, it was reported that such frozen dessert contained 5.5% fat, 12.5%
MSNF, 9.9% maltodextrin, 9.3% sorbitol, 1.5% WPC, 0.38% stabilizer and emulsifier and 400
ppm aspartame. Basyigit, et al. (2006) developed a human-derived probiotic ice cream using
sucrose and aspartame and reported that the probiotic cultures remained unchanged in ice
cream stored for 6 months regardless of the sweeteners used.
Fermented dairy products
Cheese and yoghurt represents very significant fermented dairy products around the
world. Cheese is a generic name for a group of fermented milk-based products, produced in a
wide range of flavours and forms throughout the world. Although the primary objective of
cheesemaking is to conserve the principal constituents of milk, cheese has evolved to become a
food of high-quality with epicurean qualities, as well as being highly nutritious. Processed
cheese, in most generic terms, is a blend of one or more natural cheeses of different ages,
emulsifying salts, water and other dairy and non-dairy ingredients. As per a compilation done at
University of Wisconsin, there are about 1400 varieties of cheeses in the world. Cheddar cheese
one of the most common semi-hard and ripened variety, on an average, contains about 35g of
fat and 25g protein per 100g and contributes to about 400 kcal of energy. Yoghurt is a semisolid
fermented product made from a heat-treated standardized milk mix by the activity of a
synbiotic blend of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
The popularity of yoghurt has increased due to its variety of flavours, variety of textures,
packaging innovations, convenience and perceived health benefits. Due to wide consumption
and popularity of these products and increasing awareness of role of fat and cane sugar in
human health, lot of work has been done for the development of low-fat and/or low calorie
cheeses and yoghurts.
Removing all or part of the fat from cheese can adversely affect its taste and texture and
its functionality. Many low-fat cheeses tend to have a flat and noncharacteristic taste, more
translucency, poorer melting and baking properties, and more rubbery and gummy texture and
mouthfeel (Johnson et al., 2009). Functionality of modified tapioca starch and lecithin as fat
mimetic in Feta cheese was studied by Sipahioglu et al. (1999). Cheeses were made with
modified tapioca starch (1%), lecithin (0.2%), and a combination of tapioca starch (0.5%) and
lecithin (0.1%). Feta cheese containing no fat mimetic was considered as control. It was
reported that levels of fat and fat mimetic significantly affected moisture, protein, yield, and
hardness of cheese. Reduced-fat cheeses with modified tapioca starch had the highest moisture
(67.6%) and lowest protein (13.5%) content and their hardness was higher. The combination of
54
modified tapioca starch and lecithin improved flavor, texture and overall acceptability of
reduced-fat and low-fat Feta cheeses. Koca and Metin (2003) studied the textural, melting and
sensory properties of semi-hard Turkish traditional cheese, known as Kashar cheese containing
5% w/w Raftiline®HP, 1% w/w Simplesse®D-100 and 1% w/w Dairy-Lo™ as fat replacers. The
results were compared with the control samples of low-fat cheese without fat replacer and the
full-fat cheese. The changes in cheese characteristics were examined during storage for 90
days. It was found that deterioration of hardness, springiness, gumminess and chewiness
occurred due to the usage of fat replacers while cheese cohesiveness was increased. The use of
carbohydrate-base fat replacer, Raftiline®HP had slightly increased the cheese meltability. These
results indicated that Simplesse®D-100 and Raftiline®HP can improve the texture and sensory
properties of low-fat fresh kashar cheese. Fat replacers affected the microstructure of low-fat
cheddar cheese aged for 6 months at 5°C. It was reported that Simplesse and DairyLo showed
rippled surface while Novagel and Stellar resulted in cheese with undulated and rough surface
microstructure. It was reported that Simplesse and Novagel softened low-fat Cheddar cheese
by imparting discontinuity to the casein matrix (Aryana and Haque, 2001).
Fat solids reduction in yoghurt has been associated with poor texture, where commonly
the fat removed is substituted by skim milk powder, sodium caseinate or whey protein
concentrate (WPC). Sandoval-Castilla et al., (2004) studied the effect of three commercial fat
replacers consisting of WPC, microparticulated whey protein (MWP) and modified tapioca
starch (MTS) on the texture and microstructure of seven reduced-fat yogurts prepared from
reconstituted milk. They found that yogurts with WPC and blends of WPC and MWP possessed
textural characteristics that resemble those of full-fat yogurt (FFY), whereas yogurt with MWP
showed lower tension and firmness but higher cohesiveness. Yogurt with MTS showed higher
firmness than FFY. Blends of carbohydrate–protein-based fat replacer resulted in yogurts that
are less dense, firm and adhesive, but more cohesive than FFY. Further, scanning electron
micrographs showed that the protein matric of the reduced-fat yoghurts made with and
without fat replacers showed differing structures, which in general terms were more open and
less dense than that of FFY. Pinheiro, et al. (2005) reviewed the effect of different sweeteners
in low-calorie yogurts. Keller, et al. (1991) had formulated an aspartame-sweetened frozen
dairy dessert with increased MSNF but without bulking agents by treating it with lactase. It was
reported that there were no significant differences in the scores of lactase-treated and
artificially sweetened frozen desserts. Malone and Miles (1984) was granted a patent by the US
patents organization for the development of a gelled, artificially sweetened yogurt prepared by
mixing a stabilizer solution containing high methoxyl pectin (2-7%), low methoxyl pectin (3-8%)
and an aspartic acid-based sweetener (0.1-0.75%). Farooq and Haque (1992) developed a non-
fat low-calorie yogurt using aspartame and sugar esters and reported that sugar esters had
improved the overall quality of non-fat low calorie yoghurt. It was reported that yoghurt with
sugar esters, mainly stearate-type yoghurt with an HLB range of 5 to 9, had firmer body,
55
texture, and mouth feel than yoghurts without sugar esters. Further it was reported that skim
milk yoghurts sweetened with aspartame had 50% fewer calories per serving than regular
yoghurt containing 3.25% fat and 4% sucrose. Keating and White (1990) had developed plain
and fruit-flavoured yogurts using 9 different alternative sweeteners including aspartame,
sodium and calcium saccharins, and acesulfame-K. It was reported that among all the plain and
fruit flavoured yoghurts, yoghurts sweetened with sorbitol and aspartame received highest
sensory flavour scores. Fellows, et al. (1991) developed a sundae-style yogurt using aspartame
and reported that during the manufacture, aspartame has excellent stability in fruit
preparation. Fernandez-Gracia, et al. (1998) has developed a reduced-calorie, fiber fortified
yogurt using natural alternative sweeteners.
Traditional dairy products
Burfi
Burfi, the most popular khoa based confection among Indian traditional dairy products,
has its own distinguished niche in Indian diets during festive season as well as day-to-day life. It
contains high amounts of fat (20%) and sugar (30%). Successful attempts were made by Prabha
and Pal (2006) in developing a technology for the production of dietetic burfi for a target group
of obese, diabetic and those prone to heart related problems. Studies were conducted for
screening of the suitable fat replacers and bulking agents. The necessary process modifications
were made for use of these fat replacers and sugar replacers. The critical compositional
variables of dietetic burfi including levels of milk fat, fat replacers and bulking agents were
optimized using RSM. Aspartame and neotame showed poor stability in dietetic burfi. Sucralose
was selected as a high potency sweetener on the basis of its most preferred sweetness profile
and excellent stability in the product. Shelf life studies reveled that vacuum packaged dietetic
burfi can be stored without spoilage for 12 days at 30C and 40 days at 5C. Arora et al. (2007)
reported that use of artificial sweeteners viz. saccharin, acesulafem-K, sucralose and aspartame
in burfi resulted in low instrumental hardness, adhesiveness, springiness, gumminess and
chewiness with a decreased compactness of the network as revealed by the scanning electron
microscopy. Recently, Arora et al. (2010) studied the stability of aspartame in burfi and
reported that aspartame sweetened (0.065%) burfi resembled control burfi in sweetness with
94% recovery of aspartame when stored at 6-8°C for 7 days.
Rasogolla
Rasogolla is the most popular chhana based Indian sweetmeat. Because of its pleasant
and delightful taste, the fame of this sweet has not only spread throughout India but is
becoming popular abroad as well. Quite a considerable quantity of this sweet is now being
exported to Middle East and European countries from Bikaner and West Bengal. Because of its
high sugar content (32-55%) the people who are suffering from diabetes are not able to relish
56
this delicious product. Technology has been developed for the manufacture of sugar free
rasogolla using artificial sweeteners for such a large group of people. The levels of aspartame
and sorbitol were optimized on the basis of sensory quality of the product using D6 Hokes
design (RSM). The use of 40% sorbitol and 0.08% aspartame was found to be optimum for
cooking of rasogolla balls. The higher sorbitol level resulted in hard body and unacceptable
flavour where as lower level caused flattening of rasogolla balls with surface cracks. Aspartame
did not much affect the sensory quality of the product except for its sweetness. No signs of
deterioration in terms of flavour body and texture, color and appearance and sweetness of the
product were observed up to 20 days at refrigeration temperature and up to 15 days at
ambient temperature.
Kulfi
Kulfi is a popular frozen dessert of Indian origin that occupies a privileged position
amongst the traditional Indian dairy products and contains high sugar (13-20%) in it.
Technology for the production of artificially sweetened kulfi using combination of bulking
agents mainly maltodextrin, sorbitol and artificial sweeteners such as aspartame, acesulfame-K
and sucralose has been developed. Aspartame was found to be a suitable sweetener with
maltodextrin and sorbitol as bulking agents. Kulfi mix was flavored with cardamom, filled in
mould and frozen in ice and salt mixture. The levels of maltodextrin, sorbitol and aspartame
were optimized on the basis of sensory quality and melting rate using CCRD. The level of
aspartame had a major impact on sweetness of the product. The body and texture were mainly
affected by levels of maltodextrin and sorbitol.
Gulabjamun
Gulabjamun is a khoa based sweet popular in India. The traditional method of
preparation involves blending of khoa, refined wheat flour and baking powder into a
homogeneous mass so as to obtain smooth dough along with small amount of water. The balls
of the dough are deep fat fried in ghee or refined vegetable oil to a golden brown colour and
subsequently transferred to sugar syrup. Chetna et al (2004) optimized the critical variable of
gulabjamun preparation using sugar substitutes i.e. concentration of syrup, soaking
temperature and duration of soaking using response surface methodology. Based on the
optimized conditions gulabjamun without sugar could be prepared without affecting the quality
of product. Soaking of fried gulabjamun balls in sorbitol syrup of 54B strength added with
aspartame @ 0.25% maintained at 65C for 3 hrs yielded the good quality product.
Misti dahi
In eastern India, the traditional fermented dairy product, dahi, has been elevated to a
dessert by sweetening it. The sweetened variety of dahi is popularly known as misti dahi or
misthi doi. Misti dahi has creamish to light brown color, firm consistency, smooth texture and
57
pleasant aroma. Various market survey reports on the quality of misti dahi sold in different
parts of the country revealed wide variations in the fat (1-12%) and cane sugar (6-25%)
contents. High fat and sugar contents in misti dahi may pose a hurdle for its successful
marketing in other parts of the country in the present health foods regime. With an aim to
develop reduced fat misti dahi, Raju and Pal (2009) studied the effect of reduction of milk fat,
by keeping the total milk solids constant, and reported that highly acceptable reduced fat misti
dahi can be produced with 3.0% fat and 15.0% milk solids-not-fat (MSNF). Further, studies were
carried out to replace cane sugar in misti dahi with a blend of sweeteners along with bulking
agents and it was reported that maltodextrin was found to be the most suitable bulking agent
in the preparation of artificially sweetened misti dahi using a binary blend of aspartame and
acesulfame-K (Raju and Pal, 2011).
Shrikhand
Shrikhand an acid coagulated indigenous and sweetish-sour, fermented milk product is a
popular delicacy in Gujarat, Maharashtra and part of Karnataka. It is consumed as a dessert.
This indigenous dairy product is prepared by lactic acid coagulation of milk, separation of whey
form curd followed by blending with grounded sugar, flavour, colour and selected spices. It has
very high content of sugar (40). The effect of sugar replacers on sensory attributes and storage
stability of shrikhand was studied by Singh and Jha (2005). Among various combinations of
sugar and raftilose tired, shrikhand prepared with raftilose (4%) and sugar (12.5%) was rated as
most acceptable by the sensory panelists. Sugar and raftilose exhibited significant effect
(p<0.01) on flavour, body and texture and overall acceptability no significant effect was
observed on color and appearance.
Dairy-based beverages
Lassi is a traditional South Asian beverage, originated in Punjab (India, Pakistan) and
made by blending dahi with water, salt and spices until frothy. It is a healthy dairy beverage, the
thickness of which depends on the ratio of dahi to water. The product is relished sweet in the
northern parts of the country, whereas the salt variety is preferred in the south. Kumar (2000)
developed a low calorie lassi, a traditional fermented refreshing beverage, by using aspartame
and reported that aspartame at a level of 0.08% was required to replace 15% of cane sugar in
lassi. Recently, George et al. (2010) studied the stability of multiple sweeteners in lassi and
reported that binary blend of aspartame and acesulfame-K was found to be the best as it
resembled control sample in all the sensory attributes up to 5 days of storage. Beukema and
Jelen (1990) studied the suitability of developing whey-based drinks using high potency
sweeteners and reported that both aspartame and acesulfame-K may be suitable sweetening
agents in cottage cheese whey based fruit drinks. It was further reported that, in such drinks,
the total calories were reduced to almost 50%. Yau, et al. (1989) studied the effects of
aspartame on flavour properties of still or carbonated blueberry flavoured milks and found no
58
significant effect on overall flavour intensity, sweetness or blue berry flavour. Bharadwaj (2003)
replaced sugar with artificial sweeteners in the preparation of flavoured milks. Based on
sensory scores a combination of saccharin and aspartame (33 mg/L and 368 mg/L) was found to
have equisweetness to that of control samples containing 7% sugar. Physicochemical,
microbiological and sensory qualities of the three types of flavoured milks viz. toned, double
toned and skimmed milk did not differ greatly with that of their counterparts with sucrose.
CONCLUSION
With growing evidence of the role of diet and dietary components especially fat and
sugar in non-communicable diseases such as obesity, diabetes, cardiovascular diseases etc.
worldwide people are cautious of what they eat. With the continuous invention of food
additives such as fat replacers and low-calorie and high-intensity sweeteners it has been
possible to develop dietetic dairy products that suit the palate of local consumers. R&D
institutes in India such as NDRI, Karnal and other academia too has contributed for the
development of low-calorie dairy products such as dietetic rasogolla, burfi, misti dahi, kulfi, etc.
for the benefit of health conscious consumers in general and calorie conscious consumers in
particular. It is not far off for the Indian dairy industry to exploit and reap the benefits of such
inventions.
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59, 161-165.
Arora, S., Gawande, H., Sharma, V., Wadhwa, B. K., George, V., Sharma, G. S. & Singh, A. K. (2010). The
development of burfi sweetened with aspartame. International Journal of Dairy Technology, 63, 1, 127-
135.
Arora, S., Singh, V.P., Yarrakula, S., Gawande, H., Narendra, K., Sharma, V., Wadhwa, B.K., Tomer, S.K. &
Sharma, G.S. (2007). Textural and microstructural properties of burfi made with various sweeteners.
Journal of Texture Studies, 38(6), 684-697.
Aryana, K. J. & Haque, Z. U. (2001). Effect of commercial replacers on the microstructure of low-fat
Cheddar cheese. International Journal of Food Science and Technology, 36, 169-177.
Beaglehole, R. & Yach, D. (2003). Globalization and the prevention and control of non-communicable
disease: the neglected chronic disease of adults. Lancet, 362, 903-908.
Bjrok, S., Kapur, A., King, H., Nair, J. & Ramachandran, A. (2003). Global policy: aspects of diabetes in India.
Health Policy. 66, 61-72.
Chetana, R., Manohar, B. & Reddy, S. R. Y. (2004). Process optimization of gulab jamun, an Indian
traditional sweet, using sugar substitutes. European Food Research and Technology, 219, 386-392.
Franz, M. J. (1997). Lifestyle modifications for diabetes management. Endocrinology and Metabolism
Clinics of North America, 26(3), 499-510.
George, V., Arora, S., Sharma, V., Wadhwa, B. K. & Singh, A. K. (2010). Stability, physico-chemical,
microbial and sensory properties of sweetener/sweetener blends in lassi during storage. Food and
Bioprocess Technology, DOI 10.1007/s11947-009-0315-7.
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Gupta, R., Misra, A., Pais, P., Rastogi, P. & Gupta, V. P. (2006). Correlation of regional cardiovascular
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Keller, S. E., Fellows, J. W., Nash, T. C. & Shazer, W. H. (1991). Formulation of aspartame-sweetened
frozen dairy desserts without bulking agents. Food Technology, 45, 102-106.
King, H., Aubert, R. E. & Herman, W. H. (1998). Global burden of diabetes, 1995-2025: prevalence,
numerical estimates, and projections. Diabetes Care, 21(9), 1414-1431.
Knowler, W. C., Barrett-Connor, E., Fowler, S. E., Hamman, R. F., Lachin, J. M., Walker, E. A., Nathan, D. M.
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England Journal of Medicine, 346(6), 393-403.
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International Conference on Traditional Dairy Foods (pp. 1-27). National Dairy Research Institute, Karnal
(Haryana), Nov 14-17.
Pinheiro, M. V. S., Oliveira, M. N., Penna, A. L. B. & Tamime, A. Y. (2005). The effects of different
sweeteners in low-calorie yogurts – a review. International Journal of Dairy Technology, 58 (4), 193-199.
Raju, P. N. & Pal, D. (2009). The physico-chemical, sensory and textural properties of misti dahi prepared
from reduced fat buffalo milk. Food and Bioprocess Technology, 2, 101-108.
Raju, P. N. & Pal, D. (2011). Effect of bulking agents on the quality of artificially sweetened misti dahi
(caramel colored sweetened yoghurt) prepared from reduced fat buffalo milk. LWT-Food Science and
Technology (In Press).
Sandoval-Castilla, O., Lobato-Calleros, C., Aguirre-Mandujano, E. & Vernon-Carter, E. J. (2004).
Microstructure and texture of yogurt as influenced by fat replacers. International Dairy Joournal, 14, 151-
159.
Sandrou, D. K. & Arvanitoyannis, I. S. (2003). Low-fat/calorie foods: current state and perspectives. Critical
Reviews in Food Science and Nutrition, 40, 427-447.
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properties of feta cheese made with tapioca starch and lecithin as fat mimetics. International Dairy
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Switzerland s(http://www.who.int).
60
Technology of Fresh Cheeses with Enhanced Health Attributes
S.K. Kanawjia, Y. Khetra and A. Chatterjee
Division of Dairy Technology, NDRI, Karnal
1.0 INTRODUCTION:
Milk and milk products make a significant contribution to the supply of nutrients to
human beings. Among the various dairy products, within recent years, consumption of cheese
has increased dramatically, impart because of its versatility, high nutrient content and
convenience. Cheese, as delightful fermented food contributing to a variety in our diets, has
been recognized to provide important nutrients and considered superior over non-fermented
dairy products in terms of nutritional attributes as the micro flora present produce simple
compounds like lactic acid, amino acids and free fatty acids that are easily assimilable. In
addition, cheese is also a good source of vitamins, riboflavin and minerals. Further, fermented
foods are reported to be anticarcinogenic, anticholesterolemic, anticariogenic, and
antihypertensive. Some of the cheese flora has been reported to inhibit the growth of certain
toxin-producing bacteria in the intestine. Cheese has, therefore, been truly classified as a value
added product and is consumed in various other forms like dietetic foods, snacks fast foods and
spreads. Cheese contains a high concentrated form of essential nutrients, particularly good
quality protein, several vitamins and minerals. This is an excellent food for the people of all
ages and suitable for individuals who are lactose intolerants. Cheeses have been reported to
have hypocoholesterolemic properties. Recently, cheeses have been developed with the
probiotic bifidobacteria to cater the mass with healthy way. Fresh acid-curd cheeses refer to
those varieties produced by coagulation of milk, cream or whey via acidification or a
combination of acid and heat, and which are ready for consumption once the manufacturing
operations are complete (Guinee et al., 1993). Quarg, cottage, cream, fromage-frais and Ricotta
are commercially the most important types under this category of cheese. Most fresh cheese is
very versatile and particularly suitable for processing into fresh cheese preparations (Cheese
cakes and sauces, desserts).
2.0 Major Fresh Acid-curd Cheese Varieties
Approximate Composition of Varies Fresh Cheeses
Variety Dry
matter
Fat Protein % w/w
lactose
Salt Ca (mg/
1 00g)
pH
Cream cheese
Double 40 30 8-10 2-3 0.75 80 4.6
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Single 30 14 12 3-5 0.75 100 4.6
Neufchetal 35 20 10-12 2-3 0.75 75 4.6
Labneh 25 11.6 8-4 4.3 -- -- 4.2
Quarg
Skim milk 18 0.5 13 3-4 -- 120 4.5
Full fat 27 12 10 2-3 -- 100 4.6
Cottage cheese
Low fat 21 2 14 -- -- 90 4.8
Creamed 21 5 13 -- -- 60 4.8
Fromage frais
Skim milk 14 1 8 3.5 -- 0.15 4.4
Queso
blanco
49 15 23 1.8 3.9 -- 5.4
Ricotta
Whole milk 28 13 11.5 3.0 -- 200 5.8
Part skim 25 8 12 3.6 -- 280 5.8
Ricottone 18 0.5 11 5.2 -- 400 5.3
Mozzarella Cheese
Standardized 46 18 22 2-3 1.2 150 5.3
3.0 Cottage Cheese:
Cottage cheese, designated as slim cheese with low calorific value (96 Kcal/100g), and
low fat with reduced cholesterol content is very much suitable for the people suffering from the
metabolic and physical mayhems like lactose intolerance, atherosclerosis, obesity etc. So its
incorporation as ethnic food in the diet list of modern consumers rummaging around newer
taste everyday may wake up the dormant opportunity to the Indian dairy industry to brace our
national economy, which requires extensive studies to make it accuser to the consumers in
terms of quality and palatability as well as to the industry in terms of technological accessibility.
Cottage cheese has a pleasant mild flavour which is attuned to the olfaction of Indian
people with its widespread consumer appeal both as a savoury and dessert product, and its
potential as low cost, good quality high protein and low fat product, the consumption of
Cottage cheese seems to increase significantly, as because of the consciousness of a large group
of Indian population concerning over-weight and cardiovascular as well as other metabolic
ailments. This is also the main reason for a drastic boost in production of Cottage cheese in the
USA and many other countries. Popularising Cottage in India will not only help to increase the
nutritional status and provides a substitute for ripened varieties of cheese, a luxurious item on
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account of its high cost but also satisfy the crave of modern patrons for tasting cheese, who
have grown phobia over obesity and cardiovascular as well as metabolic disorders.
Probiotic Cottage cheese: The technology of Probiotic Cottage cheese developed intended to
amalgamate some health benefits of probiotic culture to the product with special emphasis
given on the hypocholesterolemic effect of probiotic to reduce the risks of atherosclerotic
CVDs. Animal study using rat model revealed that feeding probiotic cheese considerably reduce
plasma cholesterol and plasma LDL levels.
The technology of manufacture this cheese is as follows:
Receiving (Pasteurized) skim milk
Adding Calcium chloride
Adding starter
Adding rennet
Setting (32oC/ 5h)
Cutting (pH 4.8)
Cooking (1-2 h – 46oC)
Drainage of whey
Washing and draining the curd
Salting (@ 1% of curd, or 15% of milk)
Creaming (@20%)
Packaging and storage
4.0 Cream Cheese
Cream cheese is a soft, unripened cheese made from cream, coagulated either by
microbial development of lactic acid (aided by milk-coagulating enzymes) or by direct
acidification. This is followed by collection of the formed soft curd by centrifugation or pressing
63
in cloth bags. This cheese is creamy-white in colour, has a fine, smooth, spreadable texture, and
a full rich cream-like flavour with a slight acidic taste. The product has a shelf-life of 3 months at
8oC and most popular in North America.
Cream cheese is generally made from a cream base that contains 12 to 20% milk fat; the
fat content of the finished cheese may vary from the typical minimum of 30% to as high as 40%.
The moisture content will vary in inverse proportion.
Neufchatel is a similar cheese made from whole milk of high fat content and hence has a
correspondingly lower milk fat content (20-25%) in the final product.
In recent years, a US manufacturer has developed a direct acidification process for
converting cream or milk base into cream cheese or Neufchatel cheese. Glucono-delta-lactone
(GDL) and phosphoric acid are the acidulants used for coagulating the milk protein. Gluconic
acid in formed, when GDL is added to an aqueous system such as the cream or milk base; the
resulting pH decrease induces the clotting of casein. Milk or cream is usually preacidified with
phosphoric acid. The GDL requires heating of the milk for its conversion to gluconate.
5.0 Quarg Cheese
Quarg is a natural, unripened, soft fresh cheese. It is essentially a milk protein paste,
manufactured by acid coagulation of milk by proper bacterial cultures with a small rennet
addition for better separation of the protein coagulum from the whey and thus better yields. It
is produced in a variety of fat contents, ranging from an essentially fat-free type to a variant
with as much as 40 per cent fat in the dry matter. Quarg cheese is milky white in color, may be
even faintly yellowish. Body and texture are homogeneously soft, smooth and mildly supple or
elastic. Spreadability must be good. There should be no appearance of water or whey, dryness
or graininess, bacteriological deterioration, over-acidification or bitter flavour during storage.
Odour and taste, i.e. the flavour, must be clean and may be mildly acidic. It is sometimes
loosely referred to as chakka in India. Also it is referred to as Tvorog in some European
countries. In the manufacture of quarg, several processing (hydrocolloid, addition, heating,
homogenization and/ or aeration) and addition of various materials (species, herbs, fruit,
cream, sugar, other fresh fermented milk products of different fat levels) to quarg give rise to a
range of quarg-based products such as half-fat (20% FDM) and full fat (40% FDM) quarg and
savoury quarg’s, shrikhand dairy desserts and fresh cheese preparations (Patel et al., 1986;
Guinee, 1990). Ultrafiltration is now being used on a large scale for the commercial production
of quarg .
Enrichment with Dietary Fibers:
The human gut micro-biota can play a major role in host health, thus there is currently a
dynamic interest in the manipulation of the gut flora toward a potentially remedial community
64
with the application of prebiotics. The concept of prebiotics has become very popular since its
introduction in 1995. Prebiotics are “non-digestible dietary components that pass through to
the colon and selectively stimulate the proliferation and/or activity of populations of desirable
bacteria in-situ”. Food ingredients classified as prebiotics must not be hydrolyzed or absorbed
in the upper GIT, need to be a selective substrate for one or a limited number of beneficial
colonic bacteria, must alter the microbiota in the colon to a healthier composition and should
induce luminal or systematic effects that are beneficial to host health. Dietary fiber, especially
soluble fibers are associated with carbohydrate and lipid metabolism has shown to have
hypercholesterolemic properties. Keeping in view the reported beneficial effect of dietary fiber
on cardiac disease, inulin (Raftiline), oat (Vitacel) fiber and soy fiber hyave been assessed for
their suitability.
Optimization of level of incorporation of plant sterol esters
Phytosterols are important structural components of plant membranes, and they play a
key role in plant cell membrane function just as cholesterol does in animal cell membranes
(Quílez et al., 2003). Phytosterols are found in significant amounts in seeds, nuts, fruits and
vegetables; however, the most concentrated source is vegetable oils (Ostlund, 2002). Since
humans are not able to synthesize phytosterols, all phytosterols in the human body originate
from dietary intake. As part of a normal healthy diet, most people eat 100-500 mg of
phytosterol each day (Ostlund, 2002). Most of the phytosterols or phytostanols currently
incorporated into foods are esterified to unsaturated sterol/stanol esters to increase lipid
solubility, thus allowing maximal incorporation into a limited amount of lipid. Phytosterol or
phytostanol intake from functional foods (e.g. bread spreads) is usually 1.5-3g/day. Phytosterol
and phytostanol products reduce the serum concentration of total cholesterol by up to 15%
and that of LDL cholesterol by up to 22% (Ostlund, 2002; Christiansen et al., 2001). Although
many studies have been conducted to resolve the mechanisms of action by which phytosterols
lower serum cholesterol, the molecular actions are not fully understood. The main physiological
response to ingestion of phytosterols is known to be reduced intestinal absorption of both
dietary and endogenously produced cholesterol without, however, any decrease in the levels of
high-density lipoprotein (HDL)-cholesterol or triglycerides (Moreau et al., 2002, Ostlund et al.,
2002). This interference with absorption is probably related to the similarity in the chemical
structures of phytosterols, stanols, and cholesterol (Salo et al., 2002; Plat and Mensink, 2005).
At this institute studies have been conducted to incorporate different levels of plant
sterol esters in fiber enriched quarg cheese with the aspiration to explore the enrichment of
quarg cheese with plant sterol esters. The study demonstrated that adding plant sterol ester
had no significant change in sensory quality of fiber enriched quarg cheese.
65
Extension of shelf life:
Quarg cheese has a shelf life of about 2 week under refrigeration storage.
Commercialization of any technology depends on the ability to be preserved in its fresh form
for longer time at retail outlets. The use of MicroGARDTM 100 or Nisin could be successfully
practiced to extend the shelf life of the Quarg cheese over 6 weeks without adversely affecting
the quality of Quarg cheese.
Enrichment of Quarg cheese with prebiotic and probiotic attributes
Probiotic bacteria are defined as ‘living microorganisms, which upon ingestion in certain
numbers exert health benefits beyond inherent basic nutrition’ (Ross et al., 2002). A number of
health benefits for product containing live probiotic bacteria have been claimed including
alleviation of symptoms of lactose intolerance, treatment of diarrhea, anticarcinogenic
properties, reduction of blood cholesterol and improvement in immunity. High levels of daily
consumption of probiotic bacteria, however, are required to confer health benefits. Probiotic
Quarg cheese manufactured at this institute and evaluated for the sensory, textural, physico-
chemical and survivability of probiotic in fresh cheese sample as well as during storage also. It
was also observed that Quarg manufactured using probiotic L. casei (NCDC 298) possessed
good overall acceptability and survivability during storage of 30 days.
Flow diagram of method of manufacture of quarg
Milk
Cream Separator
Skim Milk
Pasteurize (74oC/ 15 s)
Addition of st. culture → Preacidification (23oC, 2h)
Addition of Rennet → Souring/ Renneting (23oC/ 15 hr)
Stirring (15 min)
Curd Separator - Whey
66
Quarg
Cool (5oC)
Packing
6.0 Ricotta Cheese:
Ricotta is a soft, cream coloured, unripened cheese, with a sweet-cream and somewhat
nutty-caramel flavour and a delicate aerated-like texture. The cheese, which was traditionally
produced in Italy from chese whey of ewe’s milk, now enjoys more widespread popularity, in
particular in North America, where it is produced mainly from whole or partly skimmed bovine
milk or whey/ skim milk. Ricotta cheese because of its relatively high pH, high moisture and
open manner of moulding and cooling is very susceptible to spoilage by yeasts, mold and
bacteria, and hence a relatively short-life of 1-3 weeks at 4oC. Excellent quality Ricotta cheese
produced by using ultrafiltration. Ricotta cheese, while being a very acceptable product
itself, has many applications, including a base for whipping dairy dessert, use in confectionary
fillings and cheese cakes and as a base for products such as cream cheese and processed cheese
(Kosikowski, 1982).
7.0 Queso Blanco:
Queso Blanco is the generic name for white, semi-soft cheeses, produced in central and
south America and which can be consumed fresh: however, some cheers may be held for
period of 2 weeks to 2 months before consumption.
In latin America, Queso blanco covers many white cheese varieties which differ from
each other by the method of production (i.e. acid/ heat or rennet coagulated), composition,
size, shape and region of production like Queso del Cincho, Queso del Pais and Queso Llanero
(acid/ heat coagulated), and Queso de Matera and Queso Pasteurizado (Rennet coagulated).
In general, Queso blanco-type cheeses are creamy, high salted and acid in flavour, the
texture and body resembles those of young high-moisture cheddar and the cheese has good
slicing properties. One of the properties of acid-coagulated Queso blanco is its melt
resistance (due to inclusion of whey protein); this makes the cheese suitable for use in deep-
fried snack foods such as cheese sticks in batter.
The texture and hence the sliceability, of Queso Blanco is influenced by the moisture
content and the age of the cheese. Major volatile compound contributing to the flavour and
aroma of this type of cheese include acetaldehyde, acetone, ethyl, iso-propyl and butyl alcohols
and formic, acetic, propionic and butyric acids.
67
7.0 Mozzarella Cheese
Mozzarella cheese was originally manufactured from high fat buffalo milk in the Battipaglia
region of Italy, but it is now made all over Italy, in other European countries and USA from cow
milk. It belongs to the cheese classified as”pasta filata” which involves the principle of skillfully
stretching the curd in hot water to get a smooth texture and grain in cheese. It is a soft, white
un-ripened cheese which may be consumed shortly after manufacture. Its melting and
stretching characteristics are highly appreciated in the manufacture of Pizza where it is a key
ingredient.
The method of manufacture of Mozzarella cheese, irrespective of the milk system from
which it is made involves (1) optimum addition of starter culture or proper acidification of milk,
(2) renneting of milk, (3) cutting the curd at the right firmness, (4) stirring and cooking the curd
particles to the correct consistency and (5) proper cheddaring, stretching and salting of curd for
optimum plasticity and elasticity.
Mozzarella Cheese Types
S.No. Type of Mozzarella Moisture % FDM%
1 Mozzarella 52-60 45
2 Low moisture 45-52 45
3 Part Skim 52-60 >30 <45
4 Low moisture Part skim 45-52 >30 <45
Manufacturing steps (Traditional method)
Milk
Filtration/ Clarification
Standardization (3-4% fat)
Pasteurization(63°C/30min.)
Cooling (31°C)
68
Starter addition
Streptococcus salivarius subsp. thermophillus and Lactobacillus delbrueckii subsp. bulgaricus
(1:1) @ 1-2%)
Rennet addition (1.0- 1.5 g/100 l. milk)
Cutting
Cooking (42-44°C)
Pitching
Draining
Cheddaring (0.70% acidity)
Milling
Plasticizing / stretching under hot water (80-85°C)
Molding
Brining (20-22% chilled brine)
Packaging
Storage
8.0 Conclusion
The scenario of cheese production in India is quite bright because of the fact that cheese
has all the beneficial attributes of an ideal dairy product including therapeutic,
anticholesterolemic, anticarcinogenic and anticariogenic, etc. beyond their basic nutritive value.
There are many cheeses which are less liked by the consumers because of their strong flavour
and high cost. This could be surmounted to a great extent by introducing fresh cheese like
quarg type from low fat milk. The current trend is of functional foods to enhance the health
attributes by fortification with functional ingredient. The processes developed for manufacture
of fresh type cheeses with and without dietary fibers, phytosterols and also with probiotics
appear to have great industrial potential. The shelf life of these products has been extended
considerably using biopreservatives for commercial exploitation and marketing.
69
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development of probiotic foods. J Appl. Microbiol. 98 (6): 1410-17.
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F.D. Gunstone (ed.), 183-224. The Oily Press, Bridgw
Sorensen, H.H. (2001). The world market for cheese. Bulletin 359. International Dairy Federation, Burssels.
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Technologies to Reduce Cholesterol in Milk and Milk Products
Vivek Sharma, Darshan Lal and Raman Seth
Dairy Chemistry Division, NDRI, Karnal
Introduction
The importance of milk and milk products, in India, has been recognized since Vedic times. Milk
is considered to be a complete food as it contains almost all essential nutrients required for
human health and growth. Lipids, the most important constituent of milk, play significant role
in the nutrition, flavour and physico-chemical properties of milk and milk products. They are
also rich source of fat-soluble vitamins (A, D, E & K) and essential fatty acids, apart from having
pleasant sensory attributes. Milk fat is easily digestible than other oils and fats. It contains
number of components which show anticarcinogenic activity, e.g. sphingomyeline, conjugated
linoleic acid, -carotene etc. So one (especially vegetarians) cannot avoid it in one’s diet. But
recent trend, in the society, is against fat-rich dairy products due to the presence of saturated
fat & cholesterol as these are known to increase the incidence of coronary heart disease (CHD).
CHD is one of the common causes of heart attack. Through a period of time, many researchers
have shown that dietary cholesterol, serum cholesterol and occurrence of coronary heart
disease (CHD) have positive correlation. Milk fat contains about 0.25 to 0.40% cholesterol.
Consumption of ghee and other fat-rich dairy products makes appreciable contribution to
cholesterol intake. Furthermore, some cholesterol oxidation products (COPs) have been
reported to be more harmful than cholesterol itself as they are cytotoxic, atherogenic,
mutagenic and carcinogenic. Recent wave against cholesterol-containing foods has damaged
the image and market growth of fat-rich dairy products. The educated and urban society, in
particular, is more conscious about the presence of cholesterol in their diet. This segment of
the society is the major consumer of dairy and other food items manufactured by the organized
sector. In recent years, demand of cholesterol-free foods has increased tremendously. This has
led to increase in market of margarine, vegetable fat filled dairy products, milk fat replaced
dairy products, etc.
Owing to the adverse affects of cholesterol on human health, various physical, chemical and
biological methods have been developed for reducing cholesterol in foods. These include
blending of milk fat with vegetable oils, extraction with organic solvent, adsorption with
activated charcoal and saponin, vacuum distillation, molecular distillation, degradation of
cholesterol by enzyme (cholesterol oxidase) and removal of cholesterol by supercritical carbon
dioxide. Recently, - cyclodextrin (a starch hydrolysed product) has been effectively used for
cholesterol removal from milk, cream, cheese, lard and egg-yolk. Beta cyclodextrin is reported
to be non-toxic, non-hygroscopic, chemically stable and edible.
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Cholesterol
Cholesterol is a waxy material found in all cells of the body and is a necessary part of cell
membranes, some hormones and other body components. In particular, it participates in the
formation of myelin sheaths in the brain and peripheral nerves, and modulates the absorption
of dietary fats in the intestine. It also acts as a precursor in the biosynthesis of bile acids, steroid
hormones and vitamin D. The body makes all the cholesterol it needs; it is not necessary to get
any cholesterol from the diet. A high level of cholesterol in the blood is a major risk factor for
CHD and heart attack.
Structure and Properties of Cholesterol
The term cholesterol was derived from the
Greek words chole and stear, which mean
"bile" and "hard fat," respectively. The origin
of the term is a reflection of the fact that the
substance was first identified as a hard &
white solid in gallstones. Though discovered
by Poulletier de la Salle in 1769, cholesterol
was not named until 1818, when Michel
Chevreul rediscovered it and dubbed it as
cholesterine, believing that the material was
like a fat (Sabine, 1977). Cholesterol is a
hydrophobic sterol consisting of a four-ring structure (Figure A) with molecular weight 386.66
and molecular formula: C27H46O.
Cholesterol is insoluble in water, sparingly soluble in cold alcohol or petroleum ether, and
soluble in hot alcohol and most other organic solvents. Cholesterol melts at 148.50C. It can be
sublimed and distilled under high vacuum. The polar hydroxyl group, which gives cholesterol a
slightly hydrophilic nature, can be esterified to a fatty acid, producing cholesterol ester. Both
cholesterol and cholesterol ester are important structural components of cell membranes.
Cholesterol is also a major determinant of membrane fluidity due to its hydrophobic and
hydrophilic regions (Webb et al, 1987).
Sources of Cholesterol in Body
In the body, cholesterol appears through endogenous synthesis and from the diet. Cholesterol
synthesis in the body is most active in the liver and intestine and averages 11 mg per kg body
weight per day. This equals 770 mg for a 70 kg man on a low (less than 300 mg per day)
cholesterol diet (McNamara, 1987). Normally, liver makes 80% of the total blood cholesterol
Fig: A
72
and only 20% comes from the diet (Renner and Gurr, 1991; Allred, 1993). Cholesterol is not
considered as an essential dietary nutrient because of its endogenous synthesis. On the other
hand, Thomas and Holub (1994) reported that if less dietary cholesterol is consumed, the body
compensates by making more cholesterol.
Digestion, Absorption and Transportation of Cholesterol in the Blood
Digestion and absorption of cholesterol occurs in the small intestine (Grundy, 1983).
Cholesterol ester is broken down by a pancreatic cholesterol esterase into free cholesterol,
which, absorbed into the cells lining of the intestine. The absorption of endogenous cholesterol
(as bile acids) is more efficient than dietary cholesterol absorption.
Fat, including cholesterol, absorbed from the diet, is insoluble in the aqueous medium of the
blood. To enable transport through blood system, the various fat components are incorporated
into particles called lipoproteins (Grundy, 1983; Mahley and Innerarity, 1983). Lipoproteins
consist of a lipid core of triglyceride and cholesterol ester with a surface of mainly
phospholipids, protein and some free cholesterol. The four major lipoprotein fractions found in
the blood are chylomicrons, very-low density lipoprotein (VLDL), low-density lipoprotein (LDL)
and high-density lipoprotein (HDL).
Chylomicrons are very rich in triglycerides (about 85%) but also contain absorbed cholesterol in
the free or esterified form. VLDL is also rich in triglyceride (about 50%) and contains a
substantial portion of cholesterol mainly as cholesterol ester. VLDL transport about 15% of the
total cholesterol found in the blood.
LDL is enriched in cholesterol and accounts for about 60% of the total blood cholesterol level. It
is deposited in artery walls, increasing the buildup of plaque and hence also known as bad
cholesterol. HDL carries as much as 20% of the total blood cholesterol level. HDL is thought to
be antiatherogenic since it picks up cholesterol from peripheral tissues for delivery to the liver
and excretion. Consequently, HDL is called good cholesterol. A better indicator of risk for CHD is
the LDL/HDL cholesterol ratio (Thomas and Holub, 1994; Gurr, 1995).
Synergistic effect of cholesterol with saturated fatty acids on plasma cholesterol level
Some saturated fatty acids are reported to affect total plasma cholesterol concentration. While,
stearic acid has little effect on plasma cholesterol concentration, myristic and palmitic acids
have been reported to have the greatest cholesterol raising potential (Hegsted et al., 1965).
Some evidence suggests that the effect of myristic and palmitic acids depends on the
concomitant intake of dietary cholesterol (National Academy of Sciences, 1989). Such an
interaction is clear in several experimental mammals (Spady et al., 1993) and has also been
found in some human studies (Fielding et al., 1995). The above reports suggesting an
73
interaction between cholesterol and saturated fat intake; provide a further reason to limit
dietary cholesterol.
Coronary heart disease and atherosclerosis
Coronary heart disease (CHD) is a condition in which the main coronary arteries which supply
blood to the heart are no longer able to supply sufficient blood and oxygen to the heart muscle.
CHD, the common cause of heart attack, is one of the most frequent causes of death in the
developed and developing countries (AHA, 1989). The rates of mortality due to CHD throughout
the world vary. For example, in one study among men aged 40-59 years, the annual incidence
rate varied from 15 per 100,000 in Japan to 198 per 100,000 in Finland (Lovegrove and Jackson,
2003). According to Chopra (1997), 2.5 million Indians become victims of heart disease every
year, and Indian women are the fastest rising group of coronary patients in the world. He
further observed that 33 per 1000 Indians have a greater chance of requiring treatment and
intervention for heart disease than either European or Americans. Atherosclerosis is a silent,
painless process and the main cause of CHD characterized by build up of cholesterol-rich fatty
deposits on the inner lining of the coronary arteries, which decrease blood flow to the heart
muscle by narrowing the arteries substantially (Tabas, 2002). The atherosclerosis plaques
usually develop at a point of minor injury in the arterial wall.
Cholesterol in milk and milk products
Animal food products like milk and milk products, meat and meat products and eggs are the
major sources of cholesterol in our diet. Among these, chicken egg contains highest amount
(about 215 mg/egg) of cholesterol. Normally, most of the dieticians believe milk fat as a main
source of dietary cholesterol and the main culprit for CHD disease. Cholesterol accounts for
0.25-0.45% of the total lipids in milk. Cholesterol concentrates in the milk fat globule
membrane (MFGM). In milk, 80% of the cholesterol is associated with the milk fat globules and
the remaining 20% is partitioned into the skim milk phase where it is associated with fragments
of cell membrane (Patton & Jensen, 1975). However, any event disrupting the membrane
structure, e.g. churning of cream will result in the partial passing of cholesterol alongwith
ruptured membrane material to the aqueous phase. Arul et. al., (1987) studied the distribution
of cholesterol in various milk fat fractions viz., solid fraction (m. pt. 39oC), semisolid fraction
(m. pt. 21oC) and liquid fraction (m. pt. 12oC) and reported that 80% of the total cholesterol
content was present in the liquid fraction of the milk fat. 80-90% of the cholesterol is present in
milk in the free form, while 10-20% is esterified (Bindal and Jain, 1973; Wood and Bitman, 1986;
Jensen, 1987; Schlimme & Kiel, 1989).
Pantulu and Murthy (1982) observed 8-10 times higher content of cholesterol in whey than in
whole milk. Srinivasan (1984) reported the average cholesterol content of cow and buffalo milk
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as 2.8 and 1.9 mg/g fat, respectively. However, Prasad and Pandita (1990) showed that buffalo
milk (20 mg %) contained more cholesterol than cow milk (15.5 mg %). Similarly, they found
that dahi from buffalo milk contained more cholesterol as compared to dahi from milk of
different breed of cows. In general, dahi had lower cholesterol values than the fresh milk (Ismail
and Ahmad, 1978; Prasad and Pandita, 1990). Cholesterol in channa samples exhibited a highly
significant variation, being minimum in buffalo, while such species variations were not observed
in case of khoa calculated on dry weight basis (Prasad and Pandita, 1990).
Cheese was found to contain 52.3-76.6 (av. 69.3) mg of cholesterol/100 g of cheese and 198-
298 (av. 273) mg/100g fat in cheese (Fuke and Matsuoka, 1974). Tylkin et al., (1975) reported 9
times higher cholesterol/g fat in butter milk than butter. Aristova and Bekhova (1976) observed
cholesterol content in unsalted butter as 244 mg/100 g. Vyshemirskii et al., (1977) reported
that 80-90 % cholesterol initially present in cream passed into butter and 10-20% to butter milk.
Masson and Martinez (1984) reported cholesterol content as 177–208 mg/100 g fat in butter.
Bindal and Jain (1972) estimated free and esterified cholesterol in Desi ghee, using TLC method
and reported their contents as 0.288 and 0.038% and 0.214 and 0.056 % in cow and buffalo
ghee, respectively. Prasad and Pandita (1987) observed cholesterol content of ghee prepared
from milk of Haryana, Sahiwal and Sahiwal X Friesian cows and from Murrah buffaloes, to be
303, 310, 328 and 240 mg/100 g fat, respectively.
Factors Affecting Level of Cholesterol in Milk and Milk Products
Effect of Species/Breeds
Bindal and Jain (1973) reported that cow ghee (0.31%) contained higher cholesterol than
buffalo ghee (0.267%). Bernolak (1979) observed that cow milk, with 2.8% fat, contained 237
mg total sterols/100 g fat (92.8% cholesterol of total sterols). Prasad and Pandita (1987, 1990)
also reported higher cholesterol content in cow ghee compared to that in buffalo ghee. Singh
and Gupta (1982) observed that goat ghee contain higher cholesterol (0.236 g/100 g fat) than
cow (0.230 g/100 g fat) and buffalo (0.196 g/100 g fat) ghee.
Effect of Season/Stage of lactation
Season has also been reported to affect the cholesterol content of milk fat. Treiger (1979)
reported that total cholesterol content of cow milk fat ranged from 0.24-0.29 g/100 g fat in
spring and 0.18-0.25 g/100 g fat in summer season. Prasad and Pandita (1987, 1990) observed
that cholesterol content of ghee was higher in winter than in summer (301 vs 291 mg/100g fat).
Krzyzewski et al. (2003) also observed a significantly lower (by about 16%) concentration of
cholesterol in milk during winter season. Ghee prepared from milk of old animals (Lal, 1982)
and late lactation milk (Nigam, 1989) was found to contain highest level of cholesterol.
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Effect of Heat
Bector and Narayanan (1975) observed that when cow and buffalo ghee were heated at 225°C
for 2 h, respectively 26.1 and 27.3 % of cholesterol was lost. Similarly, Rai and Narayanan (1984)
also reported 28.2 and 49% loss of cholesterol after 12 h of intermittent frying in aluminium
and iron container.
Methods of cholesterol removal from milk fat
Since dairy products contain significant amounts of cholesterol, a number of processes for
removal of cholesterol have been developed to produce low-cholesterol dairy products. These
include steam stripping, molecular distillation, solvent or super-critical extraction, reaction with
cyclic anhydride, enzymatic method and treatments with adsorbents like saponin, activated
charcoal and cyclodextrin. These are briefly discussed below.
1. Steam Stripping
This process is similar to that used in the deodorization of vegetable oils and removal of
unsaponifiable matter. To remove cholesterol by steam stripping, the fat is first deairated
under vacuum after which it is heated with steam upto 2320C and then subjected to steam at
low pressure in cylindrical tall chamber. The anhydrous milk fat (AMF) passing over a series of
plates is spread in many thin layers, which increases the stripping efficiency. The steam rises
and carries with it the evaporated cholesterol to be condensed and collected with other
volatiles. This process can remove upto 93% of cholesterol though with 5% fat losses. The
major disadvantage to the process is that it removes flavouring compounds also (Schlimme &
Kiel, 1989).
2. Molecular Distillation
In this process, AMF is molecularly distilled at temperature 190 and 210oC at a vacuum of 10-4
Torr. Fractions distilled at 190 and 210oC represented 3.43 and 3.99 % of the initial mass and
contained more than 93 % of the total cholesterol (Lanzani et al, 1994 and Sharma et al.,
1999). Arul et al. (1988) fractionated AMF into four fractions at temperatures of 245 and
2650C and pressure of 220 and 100 mm Hg. Two low melting point fractions were blended
together to yield a total of three fractions (liquid, intermediate and solid). About 78% of the
total cholesterol was found in the liquid fraction while the remaining was found in the
intermediate (18%) and solid (4%) fractions in the esterified form. But, because of the high
heat used in the process, the quality of the end product is adversely affected.
3. Solvent Extraction
In this process butter oil is mixed with propane and ethanol in the mixing vessel. The low
viscous mixture of butter fat, ethanol and propane is fed into the extraction column. A
mixture of ethanol and water, containing a small amount of propane is used as extractant.
76
The extract, a solution of cholesterol and butter fat in a mixture of ethanol, water and some
propane is withdrawn at the bottom of the extraction column, which is splitted into two
phases. The upper phase consists of fat and cholesterol, which are subsequently separated, in
a further processing step. Around 90 to 95% of the cholesterol is extracted in this counter-
current procedure operated at 30oC and 10 bar (Czech et al., 1993).
4. Supercritical Carbon Dioxide Extraction:
Some studies have shown that supercritical carbon dioxide (SC-CO2) can be used to fractionate
AMF with evidence that cholesterol can be concentrated into selected fractions. Kaufmann et
al., (1982) obtained two fractions of milk fat by SC-CO2 extraction at a pressure of 200 bars
and temperature of 80oC. In this process, the liquid fractions were enriched in total
cholesterol. However, Huber et al. (1996) observed that direct supercritical extraction of
cholesterol from AMF is not feasible because of the low selectivity of cholesterol and poor
solubility of AMF. Moreover, under these conditions, important milk flavours also get
separated with the cholesterol. Therefore, they proposed another process for cholesterol
removal from AMF, dissolved in SC-CO2 under high solubility conditions for AMF (40 MPa at
70oC) to achieve rapid extraction. In this process, the dissolved AMF in SC-CO2 is passed
isobarically and isothermally through a high-pressure column, filled with a suitable adsorbent
(e.g. silica gel) to eliminate cholesterol. Finally, the supercritical mixture is fractionated by
either descending or ascending temperature profile in separators connected in series. Karkare
and Alkio (1993) found that over 99% of cholesterol from milk fat could be removed using an
SC-CO2 extraction system equipped with a silica gel column.
5. Reaction with Cyclic Anhydride:
Gu et al. (1994) developed a method for cholesterol removal from milk fat based on the
reaction between the hydroxyl group of cholesterol and a cyclic anhydride such as succinic
anhydride. The conversion of cholesterol into an acid derivative makes it possible to remove
these from fats by extraction with aqueous alkali. Addition of acetic acid increases the rate of
reaction and prevents the distillation of cyclic anhydride from reaction mixture. They removed
50% cholesterol from animal fats but alongwith it - tocopherol (50%), - and - lactones also
get removed.
6. Enzymatic Method:
McDonald et al. (1983) have described an enzymatic process using cholesterol reductase for
conversion of cholesterol to biologically inactive, e.g., non-toxic, non-absorbable products like
coprosterol, which is either not or is only poorly adsorbed by the body. This approach, which
is theoretically suitable for reducing the cholesterol content of milk fat, has been verified
77
biologically at least in part, by the finding that a portion of the intestinal cholesterol is reduced
to coprosterol by intestinal bacteria and subsequently eliminated.
7. Adsorption Methods
Cholesterol can be removed by its adsorption on certain material. Adsorbents, which are used
to remove cholesterol, are activated charcoal, saponins and -cyclodextrin.
(A) Activated charcoal
Bindal et al., (1994) could remove half of the cholesterol present in milk fat through treatment
of liquid fat with activated charcoal. Another activated charcoal method claimed 95% of
cholesterol removal from AMF but many other compounds including yellow pigments were
also removed simultaneously (Sharma et al., 1999).
(B) Saponins
Saponins are naturally occurring plant compounds that can be used to selectively bind to
cholesterol and precipitate it out. 80% and 90% cholesterol reduction in cream and anhydrous
milk fat was obtained by using this method (Riccomini et al., 1990). Oh et al. (1998) found
70.5% of the cholesterol removal when milk was treated with 1.5% saponin at 45oC for 30
min. Further, addition of 0.25% celite increased cholesterol removal to 72%. However, the
methods using activated charcoal or saponins are relatively non-selective and remove flavour
and nutritional components also when cholesterol is removed (Lee et al., 1999; Sharma et al.,
1999).
(C) -cyclodextrin
Beta cyclodextrin, one of the well known members of cyclodextrin family, is a cyclic
oligosaccharide of seven glucose units joined ‘head to tail’ by -1, 4 linkage and is produced
by the action of enzyme cyclodextrin glycosyl transferase (CGT) on hydrolyzed starch syrup.
Beta cyclodextrin has torus like structure. The central cavity is hydrophobic, giving the
molecule its affinity for non-polar molecules such as cholesterol (Szejtli, 2004). The radius of
the cavity can accommodate a cholesterol molecule almost exactly, explaining the highly
specific nature of -cyclodextrin’s ability to form an inclusion complex with cholesterol
(Hettinga, 1996).
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Application of Bacteriocin based Formulation in Bio-preservation of Dairy Foods
R. K. Malik, Arun Bhardwaj, Gurpreet Kaur and Naresh Kumar
Dairy Microbiology Division, NDRI, Karnal
Introduction
In India agricultural and dairy sectors have achieved remarkable successes over the last three
and a half decades. Besides being one of the world’s largest producers of food-grains, India
ranks second in the world in the production of fruits and vegetables and first in milk
production–providing much needed food security to the nation. The accomplishments of the
green and white revolutions have, however, not been matched by concurrent developments in
supply chain management, and in new technologies for better processing, preservation, and
storage of food. There is a quest for safe food. The public is willing to accept levels of risk in
other aspects of their life but not in food. This is a consequence of the special role food plays
in the society. In spite of modern advances in technology, the preservation of foods is still a
debated issue, not only for developing countries (where implementation of food preservation
technologies are clearly needed) but also for the industrialized world. Amelioration of
economic losses due to food spoilage, lowering the food processing costs and avoiding
transmission of microbial pathogens through the food chain while satisfying the growing
consumers’ demands for foods that are ready to eat, fresh-tasting, nutrient and vitamin rich,
and minimally-processed and preserved, are major challenges for the food industry. The
extent of microbiological problems in food safety was clearly reflected in the WHO food
strategic planning meeting (WHO, 2002):
The emergence of new pathogens and pathogens not previously associated with food
consumption is a major concern;
Microorganisms have the ability to adapt and change, and changing modes of food
production, preservation and packaging have, therefore, resulted in altered food safety
hazards.
The empirical use of microorganisms and/or their natural products for the preservation of
foods (biopreservation) has been a common practice in the history of mankind (Ross et al.,
2002). Lactic Acid Bacteria (LAB) have contributed in the increased volume of fermented foods
world wide especially in foods containing probiotics or health promoting bacteria. LAB play an
important role in the food industry, because they significantly contribute to the flavour,
texture, and in many cases to the nutritional value of the food products. The interest in the
application of microorganisms and their metabolites in the prevention of the food spoilage
and the extension of the shelf life of foods have been increased during the last decade. The
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application of antimicrobial peptides from lactic acid bacteria (LAB) that target food spoilage
and pathogenic organisms without toxic or other adverse effects has received great attention.
Food Processing and Challenges Ahead
The single most important development permitting the formation of civilization was the ability
to produce and store large quantities of food. Food processing is a highly complex multi-
disciplinary activity involving the application from the wide range of fields. Food processing, to
a large extent, embraces techniques of food preservation, as in addition to producing modified
products, spoilage is also reduced. The main distinction between preservation and processing
lies in the fact that processing may be carried out solely for the purpose of extending product
lines and variety and not necessarily to extend shelf life as in preservation. The challenges in
processing lie in retaining the nutritional value, flavor, aroma, and texture of foods, and
presenting them in near natural form with added conveniences. The challenges for the food
preservation, distribution and processing sectors are diverse and demanding, and need to be
addressed on several fronts to derive maximum market benefits.
In the developed world and now in the developing countries the abundant supply of
food, in combination with changes in the social economic and demographic scenario besides
the liberalization of global trade under WTO, the changes in the consumers’ concepts of
nutrition preference for different types of food, food selection patterns, along with
technological innovations and competition among the food processors, have created several
unique problems in the area of food preservation. Moreover, changes in the demographic
patterns have created a huge demand for convenience foods that can be eaten with varied
preparations
Despite the widespread popularity and acceptability of traditional milk products such
as Srikhand, Gulab jamun, Burfi, Peda, Paneer etc. in the Indian market, the organized sector
has so far not been able to tap into this market potential for many reasons such as lack of
published literature on their technology, inadequacy of appropriate technologies for their
commercial production, inadequacy of packaging materials and labeling to take care of new
pattern in consumer demand, low keeping quality and lack of quality assurance systems.
Although 46 per cent of the milk produced in the country is consumed as liquid milk, an
estimated 50 to 55 per cent of the milk produced in India is converted into a variety of
traditional Indian dairy products. The short shelf life of traditional dairy products is the major
limitation in organized marketing of these products. The conventional preservation techniques
such as sterilization, freezing etc can not be used for traditional dairy products due to their
adverse effects on sensory and textural quality. This calls for application of newer concepts of
food preservation technologies.
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Preservation: Past and Present
Traditionally, the most popular preservation technologies for the reduction of microbial
contamination of food, and pathogens in particular, have been the manipulation of the water
activity and/or pH, heat treatments, the addition of chemical preservatives, and the control of
storage temperature of foods. Lately, and mainly as a result of consumer demand for “fresher
products,” other technologies are emerging as alternatives for extension of product shelf life
(for better quality products) and reduction of pathogenic organisms (for safer products).
Inactivation of microorganisms is influenced by a number of microorganism-related
factors that are generally independent of the technology itself. These include the type and
form of target microorganism; the genus, species, and strain of microorganisms; growth stage;
environmental stress selection mechanisms; and sub-lethal injury. Each factor influences the
bacterial resistance independently of the apparent inactivation capacity of that particular
process. Thermal food preservation is a well known and old technique for reducing the
microbial count of foods. This technique is adapted to the difficult balance between
overheating (reducing the food's organoleptic properties) and underheating (leading to unsafe
and low-quality food products). For heat sensitive food products, however, thermal
pasteurization can impart undesirable organoleptic changes in addition to some detrimental
affects to the nutritional quality of the food. Preservation of food by chemical preservatives is
quite common but there are several health and questionable safety issues related with these
preservatives that render them unsafe for consumption. They are, therefore, of least choice in
food industry for preserving and extending the shelf life of different food products.
With an increased consumer demand for nutritious, fresh-like food products having
high organoleptical quality and an extended shelf life, non thermal processing alternatives
have been proposed. Among these non-thermal inactivation technologies, high hydrostatic
pressure (HHP) and pulsed electrical fields (PEF) are the most investigated ones (Devlieghere
et al., 2004). Especially, HHP is envisaged as a promising processing alternative to improve the
microbial safety of food products, while preserving nutritional and sensory characteristics.
Although HHP offers some great opportunities for food preservation, it also has some serious
limitations, such as i) the occurrence of pressure resistant vegetative bacteria after successive
pressure treatments, ii) the large investment costs (due to the high pressures involved), iii) at
present non continuous nature of the process, and iv) regulatory and product safety related
issues which need to be further clarified (Devlieghere et al., 2004; Estrada-Girón et al., 2005).
These drawbacks are hampering widespread implementation of HHP preservation by the food
industry.
Another preservation methodology is the antimicrobial effect of high pressure CO2
treatment that can be exploited at room temperature. It has been widely demonstrated in last
years (Erkmen & Karaman, 2001; Spilimbergo & Bertucco, 2003). At moderate temperature
and pressure CO2 is able to significantly inactivate bacterial vegetative cells, moulds and yeasts
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and, at suitable conditions, CO2 can also inactivate intracellular and proteolytic enzymes. So
far, no industrial applications are exploiting the high pressure CO2 technology, mainly due to
two reasons: the lack of knowledge on high pressure installations for food processing and the
poor understanding of the inactivation mechanism induced by pressure of CO2 over
microorganisms. The absence of an adequate plant design and operation procedure represents
a big obstacle to the development of this technology in view of any industrial applications.
Specific applications of radiation treatments are now permitted in more than 35
countries. These include the treatment of meat, poultry, eggs and shrimps to remove parasites
and Salmonella and the decontamination of food ingredients such as spices and herbs.
Parasites are more sensitive to irradiation than bacteria and doses as low as 0.3 kGy can
render them non-infective. Concerns over the potential safety of food irradiation have been
extensively investigated and found to be without foundation. Despite its undoubted potential
to contribute to food safety, commercial uptake of irradiation has been limited because of
consumer resistance to the concept of irradiated foods. Ultraviolet light can kill
microorganisms but, unlike ionizing radiation, its penetrating power is very limited. Its use is
restricted to disinfecting surfaces and also reducing the population of airborne fungal spores in
areas where they would pose a threat to a product’s shelf life.
Biopreservation & Bacteriocins as Biopreservatives
Bacterial fermentation of perishable raw materials has been used for centuries to
preserve the nutritive value of food and beverages over an extended period. According to
Steinkraus (1995), the traditional fermented foods contain high nutritive value and develop a
diversity of flavors, aromas, and textures in food substrates. Food fermentations are important
in developing countries where the lack of resources limits the use of techniques such as
vitamin enrichment of foods, and the use of energy and capital intensive processes for food
preservation. In a number of food fermentations, the key event is the conversion of sugars to
lactic acid by lactic acid bacteria (LAB) which includes the genera Lactococcus, Streptococcus,
Lactobacillus, Leuconostoc and Pediococcus. Lactic acid and other end products of LAB
metabolism, including hydrogen peroxide, diacetyl, acetoin and other organic acids act as
biopreservatives by altering the intrinsic properties of the food to such an extent as to actually
inhibit spoilage microorganisms. While the role of these metabolic end products has long been
appreciated, the contribution of LAB-derived bacteriocins may frequently have been
overlooked. The wide spread ability of LAB to produce bacteriocins implies an important
biological role maintained over many generations and the precise nature of this role has been
the subject of intensive research in recent times. Bacteriocin production could be considered
advantageous to producer organism as in sufficient amount these peptides can kill or inhibit
bacteria competing for the same ecological niche or the same nutrient pool. Although
bacteriocins are produced by many Gram positive and Gram negative species, those produced
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by LAB are of much commercial importance. Although various methods other than bacteriocin
production are employed for the preservation of food, an increasingly, health conscious public
may seek to avoid foods that have undergone extensive processing or which contain chemical
preservatives. Therefore, the production of bacteriocins by LAB is not only advantageous to
the bacteria themselves but could also be exploited by the food industry as a tool to control
undesirable bacteria in a food grade and natural manner, which is likely to be more acceptable
to consumers
Bacteriocins of Lactic Acid Bacteria
Bacteriocins are antimicrobial peptides or small proteins which inhibit, by a bactericidal
or bacteriostatic mode of action, micro-organisms that are usually closely related to the
producer strain (Schillinger and Holzapfel 1996). Generally, bacteriocins are low molecular
weight, cationic, amphiphilic, peptides which tend to aggregate and are benign to the
producing organism. In cases where the mode of action is known, the cell membrane is usually
the site of action. These agents are generally heat-stable, yet are apparently hypoallergenic
and are readily degraded by proteolytic enzymes in the human gastro intestinal tract. The
bacteriocins produced by LAB are of particular interest to the food industry (Nettles and
barefoot, 1993), since these bacteria have generally been regarded as safe (GRAS status).
Moreover, majority of bacteriocin producing LAB are natural food isolates, they are ideally
suited for food biopreservation.
Classification of Bacteriocins of Lactic Acid Bacteria
Most LAB bacteriocins are small (< 6 kDa), cationic, heat-stable, amphiphilic,
membrane-permeabilizing peptides that may be divided into three main groups: the modified
bacteriocins, known as lantibiotics (Class I), the heat-stable unmodified bacteriocins (class II),
and the larger heat-labile bacteriocins (Class III) as proposed by Klaenhammer (1993). A fourth
group (Class IV) with complex bacteriocins carrying lipid or carbohydrate moieties is often
included in bacteriocins classifications. Recently a fifth class of cyclic bacteriocins has been
included in the classification scheme with lesser amount of modified amino acids.
Biopreservation by Bacteriocins of Lactic Acid Bacteria
The term “biopreservative” includes the antimicrobial compounds that are of plant,
animal and microbial origin and have been used in human food for long time, without any
adverse effect on human health. Lactic acid bacteria have a major potential for use in
biopreservation because they are safe to consume and during storage they naturally dominate
the microflora of many foods. Dairy products are nutrient-dense foods that are important to
good health. At the same time, they are highly perishable commodities and attention is
required for their preservation. Bacteriocins are examples of metabolites that have
considerable potential in the realm of bio-preservation. As broad spectrum bacteriocins inhibit
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a large number of food borne pathogens, it is particularly suitable to use them as bio-
preservatives in foods. Strategies for incorporating bio-preservatives into foods include the use
of LAB with proven anti-microbial activity as starter cultures or starter adjuncts, the use of a
bio-preservative preparation in the form of a previously fermented product, and / or the use
semi-purified or purified bacteriocins. Bacteriocins can be incorporated into foods as a
concentrated, though not purified, preparation made with food-grade techniques. The use of
purified bacteriocins is not always attractive to the food industry, as in this form they may
have to be labeled as additives and require regulatory approval.
To date, the only commercially produced bacteriocins are nisin (or group N inhibitory
substance), produced by Lactoccocus lactis, and pediocin PA-1, produced by Pediococcus
acidilactici, marketed as Nisaplin® (product description-PD45003-7EN; Danisco, Copenhagen,
Denmark) and ALTATM 2431 (Kerry Bioscience, Carrigaline, Co. Cork, Ireland), respectively.
Nisin has been shown to be effective in a number of food systems, inhibiting the growth of a
wide range of Gram positive bacteria, including many important food borne pathogens such as
Listeria monocytogenes (Tagg et al., 1976). It is used predominantly in canned foods and dairy
products and is especially effective when utilized in the production of processed cheese and
cheese spreads where it protects against heat-resistant spore-forming organisms such as those
belonging to the genera Bacillus and Clostridium. This has particular significance in the case of
preventing contamination with Clostridium botulinum as there can be serious repercussions
resulting from toxin formation by this species. The two other alternatives (fermented
ingredient/starter culture) do not require regulatory approval or preservative label
declarations. These options are frequently regarded as more attractive routes through which
bacteriocins can be incorporated into a food.
Nisin
In 1969, nisin was approved for use as an antimicrobial in food by the Joint FAO/WHO
Expert Committee on Food Additives. Nisin is utilised as an additive and was assigned the
number E234 (EEC, 1983 EEC commission directive 83/463/EEC) and is permitted currently for
use in over 50 countries. In Australia and New Zealand it is allowed in cream products
(flavoured, whipped, thickened, and sour cream) at a maximum of 10 mg/kg; in crumpets,
flapjacks and pikelets (hot plate flour products) at a maximum of 250 mg/kg; and in cheese
and cheese products, oil emulsions (<80% oil), tomato products pH 4.5, beer and related
products, liquid egg products, dairy and fat based desserts, dips and snacks, sauces, toppings,
mayonnaises and salad dressings at levels compliant with good manufacturing practice. Nisin
has been sold under the trade name of Nisaplin®. Nisaplin® contains approximately 2.5% nisin,
the balance consisting of milk and milk solids derived from the fermentation of a modified milk
medium by nisin producing strains of L. lactis. The product is standardized to an activity of one
million international units per gram.
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Dairy Products
Nisin is used in pasteurized, processed cheese products to prevent outgrowth of spores such
as those of Clostridium tyrobutyricum that may survive heat treatments as high as 85–105°C.
Use of nisin allows these products to be formulated with high moisture levels and low NaCl
and phosphate contents, and also allows them to be stored outside chill cabinets without risk
of spoilage. The level of nisin used depends on food composition, likely spore load, required
shelf life and temperatures likely to be encountered during storage. (Hirsch et al., 1951). Nisin
is also used to extend the shelf life of dairy desserts which cannot be fully sterilized without
damaging appearance, taste or texture. Nisin can significantly increase the limited shelf life of
such pasteurized products.
Other pasteurised dairy products
Other pasteurised dairy products, such as dairy desserts, cream, clotted cream and
mascarpone cheese, often cannot be subjected to full sterilization without damaging quality
and are thus sometimes preserved with nisin. Tests on a chocolate dairy dessert resulted in a
20 day increase in shelf life at 7C with 3.75 mg/kg nisin while the same nisin level gave a 30
day increase in shelf life at 12C for a crème caramel dessert. The addition of nisin to
pasteurised milk is permitted in some countries. In trials at Reading University, UK, nisin added
at 1 mg/L before pasteurisation at 72C/15s, 90C/15s or 115C/2s resulted in significant shelf-life
extension of the milk at 10oC.
Yoghurt
The addition of nisin to stirred yoghurt post-production has an inhibitory effect on the starter
culture (a mixture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus
thermophilus strains), thereby preventing subsequent over-acidification of the yogurt. Thus an
increase in shelf life is obtained by maintaining the flavour of the yoghurt (less sour) and
preventing syneresis. Typical addition levels for this application are 0.5–1.25 mg/kg. Kalra et al
(1973) studied the effect of nisin (100IU/gm) on the preservation of khoa at 10, 22 and 30C. At
10C, the nisin treated khoa could be preserved for up to 90 days; at 22C for 42 days and at 30C
for 28 days. Similarly De et al (1976) studied the shelf life of control, sterilized and nisaplin
added cans of kheer at 37C and 4C. Nisin was added at the rate of 2 gm/10kg kheer (200
IU/gm) it was observed that at 37C, the control sample has a shelf life of 2-3 days, sterilized 3-
4 days and nisaplin added 8-10 days. However, at 4C the control sample has a shelf life of 10 -
15 days, sterilized 60-70 days and nisin added has 100-150 days. It was concluded that while
sterilization treatment of canned product shows considerable increase in shelf life under
refrigeration storage, addition of nisaplin showed remarkable increase under similar
conditions of storage.
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Gupta and Prasad (1989) evaluated the effect of various levels of nisin incorporated in stirred
yoghurt on the biochemical and growth characteristics of the natural and contaminating
microflora and recommend the optimum level of nisin required to enhance the shelf life of the
product. The overall assessment of the product revealed that an addition of 50IU nisin/g to
yoghurt after preparation gave an acceptable product with increased shelf-life upto 10 days at
refrigeration temperature without any change in flavor, body & texture, and consistency. In
another attempt, to increase the shelf life of Lassi by the incorporation of nisin, Kumar and
Prasad (1996) observed that the preservative effect of nisin on lassi enhanced considerably
with the decrease of storage temperature from 30 to 20C. The shelf life of lassi containing 100
to 200 IU nisin/ml increased by two folds and the product was acceptable up to 24 hrs. Shelf
life of lassi was also carried out at refrigeration temperature. Preliminary trials revealed that
the product containing 500 IU nisin/ml could be kept up to 8 to 10 days without much change
in acceptability (Kumar and Prasad, 1996).
Pediocin
Pediocin is produced by Pediococcus acidilactici, generally recognized as a safe (GRAS)
organism is commonly found and used in fermented sausage production. Most pediocins are
thermostable proteins and function under a wide range of pH (Rodriguez et al., 2002).
Pediocin AcH has been proven to be effective against both spoilage and pathogenic organisms,
including L. monocytogenes, Enterococcus faecalis, Staphylococcuc aureus, and Clostridium
perfringens (Bhunia et al., 1988). Pediocin PA-1 has been observed to inhibit Listeria in dairy
products such as cottage cheese, ice cream, and reconstituted dry milk. In situ production in
dry fermented sausage inhibits L. monocytogenes throughout fermentation and drying,
possibly owing to a combination of the reduction in pH and bacteriocin production.
Pediococcus acidilactici is also used as a low-level inoculum in reduced-nitrite bacon to prevent
the outgrowth of Clostridium botulinum spores and subsequent toxin production. A broad
spectrum bacteriocin pediocin 34 produced by Pediococcus pentosaceus 34 isolated from
Cheddar Cheese has been studied for its application in different dairy products (Malik et al.,
2005).
Improvement of Shelf-Life of Dairy Products Using Pediocin 34
Pilot scale shelf-life studies on the treatment of the paneer samples with pediocin along with
EDTA/Na-citrate and Potassium sorbate were carried out at refrigeration temperature (5-7C).
Though after 15 days, the treated samples had a higher total viable count than the 0 day
control, their keeping and organoleptic qualities remained comparable to that of 0 day control.
A prolonged shelf-life of up to 59-60 days was obtained in the samples treated with the
bacteriocin based biopreservative.
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Effect of pediocin preparation on the shelf-life of paneer was studied by first dipping of paneer
samples in the bacteriocin preparation for 30, 60 and 120 minutes (treatment 1) and by first
dipping in water followed by dipping in bacteriocin preparation for 30, 60 and 120 min.
(treatment 2). Paneer samples given treatment 1 exhibited longer shelf-life (> 75 days) as
compared to paneer samples subjected to treatment 2. This was also reflected by bacterial
counts at different time intervals during storage at refrigeration temperatures. The bacteriocin
preparations – pediocin, nisin and pediocin + nisin (50: 50) in combination with NaCl, K-
Sorbate and EDTA/Na Citrate were found to be quite effective in enhancing the shelf-life of
Paneer to about 75 days (Malik et al., 2005). Attempts have been made in author’s lab to
improve the shelf life of khoa and peda through the use of bacteriocin based bipreservative
formulation. Similarly, Vij et al (2007)(personal communication)studied the effect of antifungal
substance (AFS) produced by Lactobacillus species for the biopreservation of paneer along
with lactoferrin, and pediocin 34.
Microgard™ and ALTA™ 2341
Apart from nisin (Nisaplin®), two other commercial compounds that have been licensed for
addition to foods, MicrogardTM and ALTATM 2341, are ferments of food grade bacteria that
impart antibacterial properties to the foods. It is commonly stated that, except for nisin,
applied studies on bacteriocins are lacking. This is understandable because no other
bacteriocin have been licensed for addition to foods. Convincing evidence of inhibition of
pathogens and spoilage bacteria is required to stimulate commercial interest in bacteriocins as
agents for biopreservation. Unfortunately, except for a few bacteriocins, they have a narrow
antibacterial spectrum and they are not active against Gram negative bacteria. Use of nisin
with a chelating agent expands the antibacterial spectrum of nisin to include Gram-negative
bacteria. MicrogardTM (DANISCO, Denmark) is commercially produced from grade A skim milk
fermented by a strain of Propionibacterium shermanii, and has a wide antimicrobial spectrum
including some Gram-negative bacteria, yeasts and fungi. This product is added to 30% of the
cottage cheese produced in the USA as an inhibitor against psychrotrophic spoilage bacteria. It
is available as a liquid concentrate, spray-dried or freeze-dried preparation. It is added to a
variety of dairy products such as cottage cheese and yoghurt and a nondairy version is also
available for use in meat and bakery goods. The inhibitory activity almost certainly depends
primarily on the presence of propionic acid, but there has also been a role proposed for a
bacteriocin-like protein produced during the fermentation. Makhal and Kanawjia
(2005)observed that the coomercial biopreservative MicrogardTM 100 , MicrogardTM 200 and
MicrogardTM 400 at the level of 0.5% successfully enhance the shelf life of direct acidified
cottage cheese from 12- 20, 24 and 26 days, respectively without hampering the quality of the
product.
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ALTA™ 2341 is produced from Pediococcus acidilactici fermentation and has to rely on the
inhibitory effects of pediocin PA-1/AcH. It is added to Mexican soft cheese which is susceptible
to listerial contamination (Glass et al., 1995). Pediocin in the form of ALTA™ 2341 has been
used in combination with sodium diacetate (SD) and sodium lactate (SL) as dipping solutions. It
is very effective in controlling L. monocytogenes in vacuum packed beef franks stored at 4oC.
Another product, BIOPROFIT™, a combination of specific lactic strains, is used in normal starter
cultures to inhibit the growth of yeasts, moulds, Bacillus spp. Clostridium spp. and
heterofermentative lactobacilli during dairy fermentations. (Mayra-Makinen and Suomalainen,
1995).
Lacticin 3147
Lacticin 3147 produced by Lc. lactis DPC3147 was used to ferment reconstituted demineralized
whey (10% solids), which was pasteurized, concentrated and spray dried to produce a
bioactive lacticin 3147 powder (Morgan et al., 1999). This powder was subsequently found to
be effective in inhibiting L. monocytogenes Scott A and Bacillus cereus in natural yoghurt,
cottage cheese and soups showing the potential of lacticin 3147 as an aid to eliminate
pathogenic organisms. Recently, a food-grade strain has been developed to produce both
lacticin 3147 and lacticin 481. This strain addresses both the food safety and food
improvement aspects. Significantly, the killing effect of this double producer was more
pronounced, when tested against Lb. fermentum and L. monocytogenes, than either
bacteriocin producer alone (O’Sullivan et al., 2003). The use of strains that produce multiple
bacteriocins could be advantageous to limit the potential emergence of bacteriocin-resistant
populations.
Bacteriocin Producing Protective Cultures
The use of cultures to produce bacteriocins in situ as a means of bio-preservation has received
a great deal of interest in recent times. The system of incorporating a bacteriocin-producing
culture into a food gives it its own built in biopreservation, thereby returning to a more natural
method of shelf-life extension and improving the safety of food (O’Sullivan et al., 2002). The
food service sector has yet to apply protective cultures despite the availability of commercial
protective cultures preparations. These include nisin-producing BS-10® (L. lactis spp. lactic)
from Chr Hansen, BIOPROFIT™ (L. rhamnosus LC705) from BioGaia, the Bovamine Meat
CulturesTM from Texas Tech University (Taxes, U.S.) active against Salmonella and Escherichia
coli in meat, and HOLDBAC™ series (L. plantarum, L. rhamnosus, L. sakei, L. paracasei and
Propionibacterium freundenreichii spp. shermanii) from DANISCO (Denmark) active against
Listeria. The companies claim shelf-life extension and a reduction in distribution costs as an
additional benefit to the food safety improvements.
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Furthermore, some protective cultures have already been tested in a range of non-fermented
products: raw produce (ground beef, raw chicken meat, pasteurized liquid eggs and seafood)
as well as ready-to-eat meals including salads. Applications inhibiting L. monocytogenes are of
particular interest to cook-chill operators. This pathogen was reduced to the level of <10 cfu/g
by 106 cfu/g of lactocin S-producing L. sakei 148 at 7C during 27 days (Vermeiren et al., 2006).
The U.S. Food and Drug Administration recently approved bio-control of fresh-cut produce
with the lytic bacteriophages. This is a novel way of bio-control with viruses against L.
monocytogenes and Salmonella (Levernetz et al., 2003).
Bacteriocins And Hurdle Technology
Hurdle technology refers to the manipulation of multiple factors (intrinsic and extrinsic)
designed to prevent bacterial contamination or control growth and survival in food. A
combination of preservation methods may work synergistically or at least provide greater
protection than a single method alone, thus improving the safety and quality of a food. While
in certain foods intrinsic properties such as high salt may provide adequate protection, the
conscious addition of an extra hurdle(s) can ensure safety. (Leistner, 2000)
The benefits of combining nisin with HHP has been demonstrated extensively (Stewart
et al., 2000; Lopez-Pedemonte et al., 2003) and to a lesser extent PEF (Terebiznik et al., 2000)
has been demonstrated extensively. Lacticin 3147 was also used in concert with HHP at
pressures of 150–275MPa to investigate the effects on Staphylococcus aureus and L. innocua.
Using 10,000 AUmL-1 and the lower pressure of 150MPa resulted in a 2.1 log kill of S. aureus
relative to a control lacking lacticin where a <0.5 log kill was observed. A more pronounced > 6
log kill was observed when lacticin 3147 was combined with a higher pressure of 275MPa.
Similar, although less marked, trends were seen for L. innocua (Morgan et al., 2000). The first
ever successful application of hurdle technology in India was made in NDRI for preservation of
ready to eat paneer curry (Rao and Patil, 1999). It involved optimization of water activity, pH,
extent of heat treatment and level of preservatives to obtain shelf stable product. The product
has a shelf life of one month. Application of hurdle technology in preservation of paneer
(Yadav and Sanyal, 1999) and heat coagulated colostrums milk (Premaralli et al., 1999) has also
been reported. The work on preservation of burfi and milk cake using hurdle technology is in
progress in NDRI.
Several researchers have also examined the synergistic action of nisin and other antibacterial
products/processes on various microorganisms—nisin and sodium lactate (Nykanen et al.,
2000), nisin and sodium chloride (Pawar et al., 2000), nisin and carvacrol (Pol and Smid, 1999),
and Sorbate and nisin (Avery and Buncic, 1997).
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Incorporation of bacteriocins into packaging films to control food spoilage and pathogenic
organisms has been an area of active research for the last decade. Antimicrobial packaging film
prevents microbial growth on food surface by direct contact of the package with the surface of
foods, such as meats and cheese. Coating of solutions containing nisin, citric acid, EDTA, and
Tween 80 onto polyvinyl chloride, linear low density polyethylene, and nylon films reduced the
counts of Salmonella typhimurium in fresh broiler drumstick skin by 0.4- to 2.1-log10 cycles
after incubation at 4 °C for 24 h (Natrajan and Sheldon, 2000). This incorporation of
bacteriocins in packaging films can also be helpful in the preservation of traditional dairy foods
that could check the post processing contamination of foods.
Conclusions
The potential for use of bacteriocins in the food industry has spurred research in the area of
food preservation. Bacteriocins have been envisaged as an effective means of aiding in the
preservation of foods by controlling fermentation, and by preventing or reducing food spoilage
while extending the shelf life and stability of the product with regards to microbial activity. It
would be naive to believe that bacteriocins represent the ultimate solution to food safety
problems. However, given the effectiveness of bacteriocins, the existence of economically
viable means through which they can be incorporated and a consumer desire for minimally
processed food, they may represent an excellent alternative for chemical preservatives. There
are also a number of yet-to-be commercialized bacteriocins reported in the scientific literature
such as pediocin 34, lacticin 3147 and lacticin 481, which have shown the potential for
exploitation as natural food bio-preservatives and flavor enhancers. Intensive studies to
elucidate the fundamental structural and functional properties of bacteriocins have been
valuable. However, applied research carried out with a view to determining the impact of food
components and processing methods on the structure, solubility and activity of bacteriocins is
of extreme importance when considering potential food applications. In addition to the
ongoing study of existing bacteriocins, the discovery of new bacteriocins, combined with
imaginative developments regarding their application, can only be beneficial and will increase
the likelihood that the use of these peptides can be optimized to fulfill their potential in food
applications. Further research into the synergistic reactions of these compounds and other
natural preservatives in combination with advanced technologies could result in developing
novel strategies for food preservation or could allow less severe processing treatments, while
still maintaining adequate microbiological safety and quality in foods. Bacteriocins of lactic
acid bacteria and bacteriocin-producing cultures are attractive options in the realm of
biopreservation and will be promising future preservatives to extend the shelf life of
indigenous and exotic traditional foods globally.
92
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packaged beef at refrigeration temperature. Journal of Food Protection. 60: 1075–1080.
Bhunia, A. K., Johnson, M. C. and Ray, B. 1988. Purification, characterization and antimicrobial spectrum
of a bacteriocin produced by Pediococcus acidilactici. J. App. Bacteriol., 65: 261–268.
De S, Thompkinson DK, Gahlot DP and Mathur ON. 1976. Studies on method of preparation and
preservation of kheer. Indian Journal of Dairy Science. 29 (4). 316-318
Devlieghere, F.,Vermeiren, L.,Debevere, J., 2004.New preservation technologies: possibilities and
limitations. International Dairy Journal 14, 273–285.
Erkmen, O., and Karaman, H. 2001. Kinetic studies on the high pressure carbon dioxide inactivation of
Salmonella typhimurium. Journal of Food Engineering, 50, 25–28.
Estrada-Girón, Y., Swanson, B.G., Barbosa-Cánovas, G.V., 2005. Advances in the use of high hydrostatic
pressure for processing cereal grains and legumes. Trends in Food Science and Technology 16, 194–203.
Garneau S., Martin N., and Vederas JC. 2002. Two-peptide bacteriocins produced by lactic acid bacteria.
Biochimie, 84, 577– 92.
Glass, K.A., B. Bhanu Prasad, J.H. Schlyter, H.E. Uljas, N.Y. Farkye and J.B.Luchansky, 1995. Effects of acid
type and ALTATM
2431 on Listeria monocytogenes in a Queso Blanco type of cheese. Journal of Food
Protection, 58: 737-741.
Hirsch A., Grimstead E., Chapman HR., and Mattic ATR. 1951. A note on the inhibition of an anaerobic
sporeformer in Swiss cheese by a nisin producing streptococcus, Journal of Dairy Science, 18, 205-206
Kalra MS., Laxminarayana H. and Dudani AT. 1973. Journal of Food Science and Technology. 10 (3). 92-94
Klaenhammer TR. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiology
Reviews, 12, 39–86.
Leistner L. 2000. Basic aspects of food preservation by hurdle technology. International Journal of Food
Microbiology, 55, 181–186.
Leverentz, B., Conway, W.S., Mary, J.C., Wojciech, J.J., Abuladze, T., Yang, M., Saftner, R., Sulakvelidze,
A., 2003. Biocontrol of Listeria monocytogenes on freshcut produce by treatment with lytic
bacteriophages and a bacteriocin. Applied and Environmental Microbiology 69 (8), 4519-4526.
Lopez-Pedemonte T J., Roig-Sagues AX., Trujillo AJ., Capellas M., and Guamis B. 2003. Inactivation of
spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme.
Journal of Dairy Science, 86, 3075–3081.
Makhal S. and Kanawjia. 2005. Shelf life extension of direct acidified cottage cheese using Microgard. In:
Souvenir of National Seminar on Value Added Dairy Products. Dec 21-22. 2005. National Dairy Research
Institute, Karnal, Pp 177
Malik RK., Rao KN., Bandhopadhyay P., and Kumar N. 2005. Bacteriocins: natural and safe anti microbial
peptides for food preservation. Indian Food Industry; 24 (1), 69-70
Mayra-Makinen, A. and T. Suomalainen, 1995. Lactobacillus casei spp. rhamnosus, bacterial preparations
comprising said strain and use of said strain and preparations for the controlling of yeast and moulds.
United States Patent US., 5: 378-458.
Morgan, S. M., Galvin, M., Kelly, J., Ross, R. P. and Hill, C. 1999. Development of a lacticin 3147-enriched
whey powder with inhibitory activity against foodborne pathogens. Journal of Food Protection. 62:
1011–1016.
Morgan SM., Ross RP., Beresford T., and Hill C. 2000. Combination of hydrostatic pressure and lacticin
3147 causes increased killing of Staphylococcus and Listeria. Journal of Applied Microbiology, 88, 414–
420.
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Natrajan, N., and Sheldon, B. 2000. Efficacy of nisin-coated polymer films to inactivate Salmonella
typhimurium on fresh broiler skin. Journal of Food Protection. 63: 1189-1196.
Nettles CG., and Barefoot SF. 1993 Biochemical and genetic characteristics of bacteriocins of food-
associated lactic acid bacteria. Journal of Food Protection, 56, 338-346.
Nykänen, A., Weckman, K. and Lapveteläinen, A. 2000. Synergistic inhibition of Listeria monocytogenes
on cold-smoked rainbow trout by nisin and sodium lactate, Inernational Journal of Food Microbiology.
61: 63-72.
O'Sullivan L., Ross RP., and Hill C. 2002. Potential of bacteriocin-producing lactic acid bacteria for
improvements in food safety and quality. Biochimie, 84, 593–604.
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which produce the lantibiotics lacticin 3147 and lacticin 481. Appl. Environ. Microbiol., 69: 3681–3685.
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combination with sodium chloride on the survival of Listeria monocytogenes added to raw buffalo meat
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monocytogenes. Letters in Applied Microbiology. 29:166–170.
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traditional cereal products. In: Proceedings of National Seminar on Food Preservation by Hurdle
Technology and Related Areas. 29- 30 Dec. 1999. Defence Food Laboratory,Mysore, pp 156-62
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Food Science and Technology. 36, 37-41
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lactic acid bacteria. Crit. Rev. Food Sci. Nutr., 42: 91–121.
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94
Statistical Analysis Using SAS Enterprise Guide
Ravinder Malhotra* & Vipul Sharma**
DES & M Division, National Dairy Research Institute, Karnal – 132001
[email protected]; [email protected]
1. Introduction
SAS Enterprise Guide (or SAS EG) is a windows application that provides a point-and-click
interface to the SAS System. SAS EG does not itself analyze data, instead it generates SAS
program. Every time we run a task in SAS EG, it writes a SAS program.SAS EG can be used to
connect to SAS server on remote system or on the local system also. SAS Enterprise Guide
communicates with the SAS System to access data, perform analysis, and generate results.
From SAS Enterprise Guide one can access and analyze many types of data, such as SAS data
sets, Excel spreadsheets, and third-party databases. One can either use a set of task dialog
boxes or write its own SAS code for performing the analysis.
SAS Enterprise Guide provides following features
a. access to much of the functionality of SAS
b. ready-to-use tasks for analysis and reporting
c. easy ways to export data and results to other applications
d. transparent access to data
e. a code editing facility
2. Start SAS Enterprise Guide
To open SAS Enterprise Guide click the Start → SAS → Enterprise Guide 4.2 (or the version
available on your system) from menu bar, otherwise double click the shortcut icon Enterprise
Guide 4.2 on the desktop of your system. Every time you open SAS EG, it brings up SAS EG
window in the background, with welcome screen (shown in Fig 2.1) in the foreground. It allows
one to choose options like open previous saved project, new project, new SAS program etc.
Fig 2.1
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The first time you start SAS EG, the windows are arranged in the default application layout. This
layout consists of the Project Tree, Resource Pane, and the Workspace window.
The Project Tree window displays tree structure of the project.
Resource Pane window shows Server List, Task List, SAS folder etc.
The Workspace window is the container for the process flow, results, data grids, SAS code
etc.
At first, the process flow (shown in Fig 2.2) is the only window that is open in the workspace
area. When you generate reports or open data, other windows open in the workspace with a
tabbed interface. You can also use the recently viewed items menu in the upper-left corner of
the workspace to navigate between the windows.
Fig 2.2
If one wants to customize layout by changing the position of any window or by closing the
window, then it gets automatically saved for the current session of SAS EG. If you close any of
the application then click on Menu option View and select the window name you want to
reopen. If one wants to restore the default layout then click on Tools → Options from menu
bar then click on Restore Window Layout button.
Creating new SAS Data set or Entering Data
To get Data grid click File → New → Data from menu bar. A New Data wizard opens (as shown
in Fig 2.3). This is the first step for entering the data in which mention the name of Data Set or
(SAS data table) in the first text box. Then select library where you want to save the data set. By
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default WORK library is selected but this is a temporary storage location so better to select
SASUSER library or any User defined library. Click Next to proceed to next step. In second
window (Fig 2.4) one can assign column or variable name and there properties. By default there
are six columns one can add more or delete as per requirement. Set properties like Name,
Label, Type of variable Numeric or Character etc.
Fig 2.3 Fig 2.4
After making necessary entries click on Finish Button. A new data table appears in the data grid
form in workspace window (shown in Fig 2.5). A shortcut icon of Data Set name is also there in
the Project tree under process flow.
Fig 2.5
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After completing data entry you need to protect data before start working on it. To protect
data click on Edit → Protect Data from Menu bar. If you forget to protect data and start
working then SAS EG automatically ask to protect data. One can insert or delete Row and
Column from data set by selecting row or column where you want to perform action and by
clicking right mouse button and choosing insert rows or delete rows and insert column or
delete.
Importing Files other than SAS Data set
Click on File → Import Data from menu bar. A window appears where you mention name of the
file you need to open for eg. Lactation.xls. Then clicks on open button. An Import Data wizard
will open having 4 steps in which one can found various option if you want no change in the
data then simply click on Finish button to create SAS Data Set of the file. A SAS Data Set will
open in workspace area with shortcut icon in Project tree.
3. Creating New Project
In this step one create a new project to store the data and results. Select File→ New→ Project.
If you already had a project open in SAS Enterprise Guide, you might be prompted to save the
project. Select the appropriate response. The new project opens with an empty Process Flow
window. There is also a New button on the toolbar to accomplish the same function.
4. Save the Project
One can save the project in a single file at any desired location on local computer as well as
server also (if you are connected with server). Select File → Save Project As… from menu bar. A
Save window opens in which you can select the location where you want to save the project file
(as shown in Fig 4.1). Enter filename in the textbox; file will be saved with .egp extension. Click
on save button.
Fig 4.1
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To open saved projects select File → Open → Project. An open window appears, one can select
location either local or server and then select the project you need to open.
5. The Project Tree
Project tree is hierarchal representation of active project (Fig 5.1). It shows related data, results
file, tasks, and programs. One can manage items in the project from project tree. You can
rename, rearrange and delete objects from the project.
Fig 5.1
6. The Workspace and Process flow
When one creates a new project, an empty Process Flow window opens (as shown in Fig 6.1).
As you add data, run tasks, and generate output, an icon for each object is added to the process
flow. The process flow displays the objects in a project, any relationships that exist between the
objects, and the order in which the objects will run when one run the process flow.
1. Resource Pane
This window consists of four sub options Task List, SAS Folder, Server List, and Prompt
Manager.
Task List
One can use task to do manipulation in data, execute analytical procedures, create graphs and
generate reports etc. One can select any task from task list or can have Tasks as an option in
menu bar. You can view listing of Task list based on Category, Name or Task template (as shown
in Fig 7.2).
SAS Folder
It displays list of all of your stored processes, information maps, and projects. You can select an
item from this list and open it.
Server List
It displays a list of all the available SAS servers.
Prompt Manager It displays a list of all the available prompts (as shown in Fig 7.2).
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Fig 6.1
Fig 7.1 Fig 7.2
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2. SAS EG Help
SAS EG provides us a comprehensive help for our ease of access. Select Help → SAS Enterprise
Guide Help. In Help window you can browse through the table of content and index or you can
use search feature (Fig 8.1).
Fig 8.1
3. Menu Bar
SAS EG has following list of Menu. While clicking on any menu option sub-menu items appears
in drop down format.
Menu Functions
File Open and save project, data, code, report, and process flow.
Import and export data. Print process flow.
Edit Modify or copy text, search and replace data. Expand or collapse
data.
View Customize the look of the SAS Enterprise Guide window by
selecting to view the tool bars for Project Flow, Task List, and Task
Status.
Tasks Perform statistical procedures to manage data, create graphs, and
produce descriptive and inferential statistics.
Program Open new or existing program (where one type SAS code to
perform analyses), run or stop current program.
Tools Combine multiple reports into one. Set style of report. Set options
such as window layout and enabling particular features.
Help Get help on SAS Enterprise Guide tasks. Getting Started.
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4. Working with Tasks
In SAS EG, one can use Tasks to do statistical analysis procedures as well as for creating reports.
One way to select tasks is from Menu Bar (as shown in Fig 10.1) and other way by using the
Task List (as shown in Fig 7.1). As you scroll down the Task List you see tasks in the Statistical
Analysis, Graph etc. categories. In each task, there are certain steps that you must complete
before running the task. For example, you must specify which variables you want to analyze,
how to analyze them, format in which one can save its results, mentioning analysis title etc.
Once you have specified the necessary information to run the task, the Run button becomes
available and one can run the task and get the results.
Fig 10.1
Exploring Task Window
Every task has two lists of variables, (as shown in Fig 10.2) Variables to assign and Task roles.
The Variables to Assign list displays all the variables from the data that you have selected. In
Task Roles list you assign variables to roles in the task. This is how you tell SAS EG how you
want to analyze your data. The Task Roles list displays all the ways that variables can be used in
a task.
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Fig 10.2
To assign a variable to a task role, one can select the variable and drag it to the role. You can
also select the variable by clicking the right arrow, and select the role from the menu that
appears. On the left hand side of window there is a Selection Pane having options like Results,
Titles, and Options etc. You can set these values according to your need. You can see the code
and modify it also, if required by clicking the Preview code button in the lower-left of each task
window.
How to Analyze Data
During this analysis we will be using Body_weight SAS Data set which consist of following
variables WT_FC (Weight at First Calving), AFC (Age at First Calving), FLY (Milk Yield at First
Lactation), FLL (First Lactation Length), FCI (First Calving Interval) and FSP (First Service Period).
Fig 10.3
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Descriptive Statistics
Select Tasks→ Describe→ Summary Statistics. On the Summary Statistics Wizard
select the AFC, FLL, FLY variable from Variable to assign list and drag it to Analysis
Variable under Task Role window.
One can select desired Basic Statistics like Mean, Standard deviation, Standard Error
etc. from Selection Pane
After selecting desired variables you can click on Run.
Result will be displayed in the result window (Fig 10.4)
Fig 10.4
t Test
t Test (one Sample)
Select Tasks → ANOVA → t Test.
In the wizard select One Sample option.
Click on Data option and select the AFC, FLL, FLY variable from Variable to assign list
and drag it to Analysis Variable under Task Role window.
Click on Run and result will be displayed in the result window (Fig 10.5)
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Fig 10.5
Two Sample
During this analysis we will be using following SAS Data set which consist of following
variables Trt (Treatments), Score (Acceptance Score).
Select Tasks → ANOVA → t Test.
In the wizard select Two Sample option.
Click on Data option and select the Score variable from Variable to assign list and
drag it to Analysis Variable under Task Role window.
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Select the Trt variable from Variable to assign list and drag it to Classification
Variable under Task Role window.
Click on Run and result will be displayed in the result window (Fig 10.6)
Fig 10.6
Correlation and Regression
The task that generates results for correlation is Correlations.
In the Process Flow window, select the data set on which you want to perform
analysis. Then select Task → Multivariate → Correlations.
Select the FLY variable on which you want to perform analysis and drag it to Analysis
Variables under Task Role window.
Select the variable AFC to which you want to view correlation and drag it to
Correlation with under Task Role window.
Select Option from Selection Pane, by default type of correlation is selected as
Pearson. One can select any correlation type by clicking on the check box. By
checking Fisher Options, one can select level of significance, one can also select type
of alternative hypothesis as lower, upper or two sided from the drop down list.
Select Results in Selection Pane, here one can select show statistics for each variable
check box or show significance probabilities associated with correlations
After selecting desired variables you can click on Run.
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Result window will be opened in the process flow area showing results of your
analysis. From there you can get options to create report, export report in any
defined format, and modify the task etc. (as shown in Fig 10.7).
Fig 10.7
Partial Correlation
In the Process Flow window, select the data set on which you want to perform
analysis. Then select Task → Multivariate → Correlations.
Select the FLY variable on which you want to perform analysis and drag it to Analysis
Variables under Task Role window.
Select the variable FLL to which you want to view correlation and drag it to
Correlation with under Task Role window.
You need to select a variable under Partial Variables option to perform partial
correlation. So, select the FCI variable and drag it to Partial Variables under Task
Role window.
Select Option from Selection Pane, by default type of correlation is selected as
Pearson. One can select any correlation type by clicking on the check box. By
checking Fisher Options, one can select level of significance, one can also select type
of alternative hypothesis as lower, upper or two sided from the drop down list.
107
Select Results in Selection Pane, here one can select show statistics for each variable
check box or show significance probabilities associated with correlations
After selecting desired variables you can click on Run.
Result will be displayed in the result window (Fig 10.8)
Fig 10.8
Multiple Regression
In the Process Flow window, select the data set on which you want to perform
analysis. Then select Task → Regression → Linear Regression.
Select the FLY variable and drag it to Dependent Variable under Task Role window.
Select the variable AFC & FLL and drag it to Explanatory Variable under Task Role
window.
You can also select any variable and drag it to Group Analysis by under Task Role
window.
After selecting desired variables you can click on Run.
Result will be displayed in the result window (Fig 10.9)
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Fig 10.9
One-Way ANOVA
During this analysis we will be using following SAS Data set which consist of following variables
Trt (Treatments), Rep (Replication), Score (Acceptance Score).
In the Process Flow window, select the data set on which you want to perform
analysis. Then select Task → ANOVA → One-Way ANOVA.
Select the Trt variable from Variable to assign list and drag it to Dependent
Variables under Task Role window.
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Select the Score variable from Variable to assign list and drag it to Independent
Variables under Task Role window.
Under Means in the selection pane, select Comparison.
If you reject the null hypothesis of equality of treatment effects, then use multiple
comparison procedure for all possible pair wise treatment comparisons to
determine which of the mean are different. A desired Multiple Comparison
Procedure from the available options, say Tukey’s studentized range test (HSD).
For this example one can select Tukey’s studentized range test (HSD). Tukey’s
method examines the difference between all possible combinations of two
treatment means.
Click Run to run the One-Way ANOVA.
Result will be displayed in the result window (Fig 10.10)
Fig 10.10
Two-Way ANOVA
During this analysis we will be using following SAS Data set which consist of following variables
Trt (Treatments), Mth (Methods), Mois Cont (Moisture Content).
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In the Process Flow window, select the data set on which you want to perform
analysis. Then select Task → ANOVA → Linear Models.
Select the Mois Cont variable from Variable to assign list and drag it to Dependent
Variables under Task Role window.
Select the Trt and Mth variable from Variable to assign list and drag it to
Classification Variables under Task Role window.
In the Selection Pane, select Model option.
In the Class and Quantitative variables list, select Trt and Mth and click on main
button.
For desired type of Sum of Squares select from Model Options.
For multiple comparison procedure, select from Post Hoc Tests, Least squares and
then select the trt under the options for means tests.
Click Run to run the Two-Way ANOVA.
Result will be displayed in the result window (Fig 10.11)
Fig 10.11
Factorial Randomized Complete Block Design
During this analysis we will be using following SAS Data set which consist of following variables
Stain (First Factor), Time (Second Factor) , Rep (Replication), Fat Content.
111
In the Process Flow window, select the data set on which you want to perform
analysis. Then select Task → ANOVA → Linear Models.
Select the Fat Cont variable from Variable to assign list and drag it to Dependent
Variables under Task Role window.
Select the Stain, Time and Rep variable from Variable to assign list and drag it to
Classification Variables under Task Role window.
In the Selection Pane, select Model option.
In the Class and Quantitative variables list, select Stain, Time and Rep and click on
main button, then press ctrl and select Stain and Time and click on Cross button.
For desired type of Sum of Squares select from Model Options.
Click Run to run the Two-Way ANOVA.
Result will be displayed in the result window (Fig 10.12)
Fig 10.12
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5. Export a SAS Report
Suppose you would like to export Linear Regression Report as a step in a project, and you would
like it to be an HTML file. For this purpose you need to follow following steps.
Click on Export → Export SAS Report → Linear Regression1 As A Step In Project.
The first page of the Export wizard enables you to select the file that you want to
export. In this case, select SAS Report → Linear Regression1. Click Next.
Fig 11.1
The second page of the Export wizard enables you to select the file type of the
exported file. To save the report as an HTML file, select HTML documents.
Fig 11.2
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The third page of the Export wizard allows you to specify a location for the exported
file. If you would like to change the name of the file, simply click on Browse button
and mention new filename and destination path. No need to change file extension
(.html). Click Next.
Fig 11.3
The fourth page of the Export wizard enables you to review the selections that you
have made. Click Finish.
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Fig 11.4
References:
Susan J. Slaughter & Lora D. Delwiche. The Little SAS Book for Enterprise Guide 4.2.
http://support.sas.com/
http://web.iasri.res.in/sscnars/sas_manual
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Opportunities for Small Scale Milk Processing for Entrepreneurs
Surinder Kumar
SMS, KVK, NDRI, Karnal
Indian dairy sector has shown an impressive growth during the last three decades. Our country
which was producing about 17 million tonnes of milk during 1950-51 is presently the world
leader with milk production of about 112 million tonnes. The per capita availability of milk also
increased from 124 gm/day to 265 gm/day during the similar period indicating that growth in
milk production surpassed the population growth in our country. The contribution of different
species in milk production in the country also makes it unique. Livestock census 2003 indicates
a cattle population of 185 million and buffalo population of 98 million in our country (Table 1).
Table 1: Livestock population in the country (census 2003)
Species Number (million) Rank in world
Cattle 185 2nd ( Brazil is First now )
Buffalo 98 1st
Goat 124 2nd (China is first now)
Sheep 61 3rd
Pig 13.5 6th
Poultry 489 7th
Source: Dept. of A.H.D.F. GOI
As per the latest figures of the cross bred population in the country has increased from 24.69
million in 2003 to 27.57 million in 2007. The preparation of bovines bred through AI is about
20% of the breed able animals indicating huge scope for genetic improvement of domestic
animals. Even though there has been deceleration in growth of rate of livestock output per se
after mid 1990s, over the years the growth in livestock sector has been faster than in crop
sector. The contribution of livestock in agriculture in terms of output which was 17.3 percent
during 1980-81, increased to 26.9 percent in 2007-08. Similarly the contribution of the sector to
the national GDP has been around 5.5 percent over the years despite pronounced variations
observed in contribution of crop sector to national GDP, indicating the stability of the livestock
sector. About 70 percent of our milk producers are small and marginal farmers, with limited
resources. Of the total milk produced buffaloes, cows and goats contribute about 50, 41 and 4%
respectively.
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Table 2: Production and availability of milk
Year Milk production
(million tones)
Per Capita Availability
(gm/day)
1950-51 17.00 124
2000-01 80.60 220
2006-07 100.90 246
2007-08 104.80 252
2008-09 108.53 260
2009-10
(anticipated)
112.03 265
Demand for milk and milk products
Most economists agree that demand for milk, pulses, vegetables, fruits and eggs will grow at a
rate much faster than that of cereals and that there is adequate evidence to show that per
capita consumption of milk, in particular, increased during the last one decade. A recent survey
carried out by 64th round of NSSO has shown that an average Indian family allocates an average
17 percent of the expenditure incurred for food products on milk and milk products, with rural
families allocating 15 percent while families in the urban area allocating 18 percent. With
increasing income the demand for milk is going to rise faster now than seen in the previous
decade. The higher GDP growth rate and enhanced income of rural households through
programmes such as NREGA are influencing the demand for milk both in rural and urban areas.
With the GDP growth of 9-10% it is expected that consumption of milk and milk products will
continue to grow at about 7%.
Milk Processing
Of the total milk produced in the country, about 50% is retained in rural areas while remaining
comes for marketing. Of the total marketable surplus, organized sector comprising private and
cooperative dairies handles about 30% milk. Of the total milk produced about 50% is used as
liquid milk while 45% is converted into traditional milk products and remaining into western
milk products. Current level of processing in organized sector is about 46 million Kg milk per
day. In our country any establishment handling more than 10,000 lit of milk per day needs to
get itself registered. The units handling between 10,000 - 2.00 lakh lit per day need to be
registered with state authorities, while handling more than that and units with multistate
activities need to be registered with central registration authority. At present as per the figures
maintained by central registrar there are about 925 milk processing units as on 31.03.2010 as
detailed in the following table.
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Table 3: Milk processing plants in the country (31.03.2010)
Centrally Registered State Registered
Units Registered Processing
Capacity (lakh lit per day)
Units Registered Processing
Capacity (lakh lit per day)
Coop 124 318.07 141 59.50
Private 94 353.44 531 265.41
Govt. 16 32.00 19 7.21
Total 234 703.51 691 332.12
Opportunities for Entrepreneurs in Dairy Sector
Dairy sector comprise four different but interlinked and important activities i.e. production,
procurement, processing and marketing. Milk production in our country is scattered throughout
the country and rural based. Individual farmers keep the animals and produce milk. About 40%
of the country’s rural population own milch animals. However, now the trend for commercial
dairy farming is picking up. Many large industrial houses are planning to put up dairy farms, to
ensure the regular supply of quality milk and to preserve good quality germplasm.
Due to scattered milk production, its procurement is another opportunity in the dairy sector for
the entrepreneurs. The entrepreneur can procure milk through well established network and
transport that milk to processing plants. Due to tropical climate the preservation of milk has
always been a hard task. Of late use of bulk milk coolers has increased in the milk procurement
network. By putting up bulk milk cooler, near the production cluster, the quality of milk is
maintained thereby fetching premium on its sale to milk processers.
Milk processing offers an attractive scope in tiny and small scale industry sector for
manufacturing products like cream, butter, ghee, dahi, paneer, khoa, ice cream etc. The
production of milk products like casein and milk powders are not only capital intensive but also
require large volumes of milk (about one lakh lit) to handle per day to make the operations
viable. Moreover casein industry is totally dependent upon exports therefore the viability of the
unit producing it gets affected by fluctuations in the International market.
Milk processing offers various avenues for the small entrepreneurs by using fat, unit for
production of cream, butter and ghee can be set up, while SNF offers an opportunity to put up
unit for production of Skimmed Milk Powder (SMP), casein and dahi. However, small units for
processing of whole milk into packaged milk, paneer, chhana, khoa, dahi, lassi and ice cream
etc. Milk processing units needs to be defined clearly and according equipment need to be
purchased as it requires product specific equipment. It means equipment for making paneer
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cannot be used for khoa making similarly with other products. Different units which may be set
up by small entrepreneur are:
Cream-butter-ghee
Paneer-chhana
Liquid milk packaging
Ice cream
Cheese
Khoa
Steps for Project Planning
• Feasibility study to work out its viability
• Planning in terms of market and design of dairy plant etc. decide the capacity.
• Quantifying the product mix – availability of raw material
• Formation of dairy plant specifications
• Plant construction
• Marketing plan
• Arrange for sanctions/approvals
• Coordination of civil construction and installation of equipment
• Appointments of staff
• Placing the products in market
Export Potential of Milk and Milk Products
Prior to last decade, our country was exporting very few indigenous milk products to the
Indians settled abroad. However, now with the improvement in the quality of milk products
produced in the country, we have a significant influence in the international market, despite
the fact that the total exports of milk and milk from India constitute about 0.3% of the total
milk produced in the country. Main products being exported from India are Skimmed Milk
Powder (SMP), casein, indigenous milk products etc. to not only Asian countries like
Bangladesh, Nepal, Afghanistan, Saudi Arabia but also to other countries like USA, Singapore,
South Korea and France. Exports during the last three years from India had been tabulated in
the following Table.
Table 4: Exports of milk and milk products from India
Year Amount (Rs. Crores)
2007-08 866.56
2008-09 980.86
2009-10 402.68
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Schemes for Promotion of Milk Processing
For promotion of milk processing in the country, the state and central Governments are
implementing various schemes. The state sponsored schemes are state specific. The central
government schemes are implemented throughout the country, the brief of such two schemes
is given further.
Ministry of Food Processing Industries, GOI
Under the plan scheme of Technology Upgradation/ Establishment/ Expansion/
Modernization of Food Processing Industries, Ministry of Food Processing Industries extends
the financial assistance in the form of grant-in-aid @25% of the cost of plant & machinery and
technical civil works subject to a maximum of Rs.50 lakhs in general areas or 33.33% subject to
a maximum of Rs.75 lakhs in difficult areas under the scheme.
Department of Animal Husbandry, Dairying & Fisheries Min. of Agri., GOI
The Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture is also
implementing a central sector Plan Scheme “Dairy Entrepreneurship Development Scheme”
where a subsidy for setting up of dairy units is provided. The components are covered under
the scheme and the maximum unit cost covered under the scheme is as follows:
Small dairy farm (5 lakhs), Rearing of heifer (4.8 lakhs), Vermicompost (0.20 lakhs), Milk
machines/ Milkotesters/BMC (18 lakhs), Dairy processing equipment (12 lakhs), cold chain &
transportation (24 lakhs), cold storages (30 lakhs), private veterinary clinics (2.4/1.8 lakhs),
dairy marketing outlet/ dairy parlour (0.56 lakhs)
The Pattern of Assistance
Entrepreneurs contribution (margin)-10% of outlay (Minimum)
Back ended capital subsidy 25% of the outlay for general category and 33% for SC/ST farmers
subject to component wise ceiling which will be adjusted against the last few installments of
repayment of bank loan. Effective Bank Loan – Balance portion, minimum of 40% of outlay.
Implementing agency
The scheme is being implemented through NABARD who is the nodal agency for the scheme.
For further details, the entrepreneurs may contact National Dairy Research Institute, Karnal
(Haryana) and attend the training programmes on milk processing.
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Application of High Hydrostatic Pressure (HHP) Technology in Processing of Milk
& Milk Products
Ashish Kumar Singh, Prateek Sharma and P. N. Raju
Dairy Technology Division, NDRI, Karnal
Introduction
Conventional processing treatments aimed at enhancing the shelf-life and ensuring the
consumer safety invariably suffer with other quality defects. Thermal treatments lower the
nutritional quality of foods and also impair the sensory characteric. Also, energy optimization
and heat recovery in the food industry has been a focus in the past decades for conventional
processes, but their replacement by novel techniques for food preservation or modification
may still provide a potential to reduce energy consumption and costs of operation, as well as to
improve sustainability of production (Toepfl et al, 2006).. Several non- thermal food processing
technologies have emerged to overcome such problems including irradiation, Ohmic heating,
microwave processing, Pulse electric field (PEF), Ultrasound and high hydrostatic pressure
technology (HHP). Among these HHP is gaining acceptance, owing to its ability to inactivate
spoilage as well as pathogenic micro-organisms with minimal heat treatment, along with almost
complete retention of nutritional and sensory characteristics of fresh food. High pressure
technology is increasingly being used in the food industry particularly to produce high-value-
added products.
Hite (1899) is among the pioneer workers who initiated the investigations on effect of
HHP on food borne micro-organisms by subjecting milk to pressure of 650 MPa and reported a
significant reduction in the viable number of microbes. Later on despite the ability of high
pressure treatment in inactivating the microbes the technology has not gained attention from
researchers mainly due to the non-availability of processing equipments. The first commercial
product was introduced in Japanese market in early 1990’s and now several HHP processed
products like jams, fruit juices, (, meat, oysters, ham, fruit jellies and pourable salad dressings,
salsa, poultry available on shelf across the world (Mohácsi-Farkas et al, 2005).
Working Mechanism:
In high pressure processing food either in packaged or as such is subjected to
pressures in the range of 300-700 MPa & is effective in inactivating most of the vegetative
bacteria at pressure above 400 MPa. The most attractive feature which has made the process
worldwide acceptable is uniform processing ability as the pressure is applied uniformly
throughout the food material, independent of its mass and time. The time required to
pressurize the vessel is influenced by the compressibility of the pressure medium and the
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nature of the food material. Generally in almost all the cases, water is used as the pressure –
transmitting medium. Presence of air in the food increases the pressurization time; since air is
considerably more compressible than water. The pressure is applied isostatically. Therefore,
pressure remains uniform in the product and thus the entire product undergoes the same
treatment. High pressure is non-thermal in principle, even though the pressure increase in itself
causes a small adiabatic rise in temperature (Ohlsson & Bengtsson, 2002).
Process can be broadly classified into three main categories and are:
Batch process
Semi continuous process
Continuous process
Microbial Inactivation using HP Treatment
Milk being a perishable commodity, is usually thermally processed to provide acceptable safety
and shelf life. However, HP treatment has potential to destroy the pathogenic swell as spoilage
microorganisms thus enhance the shelf-life. HP causes a number of morphological and
biochemical changes apart from affecting the cell membrane and genetic material. The lethal
effect of HP is mainly attributed to its effect of cell membrane permeability and also on the
activity of membrane bound ATPase. The resistance of microorganisms to pressure in food is
variable depending on HP processing conditions (pressure, time, temperature, cycles, etc.),
food constituents, its properties and the physiological state of the microorganism (Smelt, 1998).
Cells at their exponential growing stage are more sensitive to pressure than cells in the
stationary phase. The bacterial spores are always more resistant than vegetative cells and they
can survive at pressure of 1000 MPa (Cheftel, 1992). However, it has been found that the
pressurization along with mild heat treatment triggers spores to germinate and after
germination, microorganisms lose their resistance towards pressure & heat, and gets killed
(Gould and Sale, 1970; Knorr, 1995; Gould, 2000). Gram-positive microorganisms are more
resistant to pressure than gram-negative. Gram-positive microorganisms need an application of
500–600 MPa at 25° C for 10 min to achieve inactivation, while gram-negative microorganisms
can be inactivated with treatments of 300–400 MPa with same time temperature combination.
Vegetative forms of yeasts and moulds are most pressure sensitive compared to spores
produces thereof (Smelt, 1998). Many studies have been conducted on raw milk using this
technology and has been proved that HPP treatment gives raw milk quality (pressurized at 400–
600 MPa) comparable to that of pasteurized milk (but not that of sterilized milk due to
presence of HP resistant spores), as it is equally effective in destroying pathogenic and spoilage
microorganisms For example, to achieve a shelf life of 10 days at a storage temperature of 10°
C, a pressure treatment of 400 MPa for 15 min or 600 MPa for 3 min at 20° C is necessary
(Rademacher & Kessler, 1997). In order to get quality as comparable to that of sterilized milk,
the combined treatments like HP along with heat is an important consideration in this regard.
Effect of HHP on Milk Constituents
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Normally any changes which are associated with volume reduction are favoured by high
pressure. High pressure influences the properties of milk and milk components; however the
effect may depends on native structure of macromolecules and the extent of pressurization.
HP treatment do not affect covalent bonds in the temperature range of 0-400C, hence primary
structure of protein remains intact on pressurization, however it influence electrostatic an ionic
interactions responsible for the maintenance of secondary structure. At pressure above 200
MPa significant changes in tertiary structure is observed. The casein micelles are disintegrated
into smaller particles resulting in an increase of caseins and calcium phosphate levels in the
serum phase of milk and a decrease in the both non-casein nitrogen and serum nitrogen
fractions (Law et al., 1998). Pressure treatment in the range of 1000-3000atmosphere generally
tends to be reversible but above 3000 atmosphere it led to irreversible denaturation (Jaenicke,
1981). Pressurization of milk causes conformational changes in milk proteins and on applying
HP treatment the size and number of casein micelles increases, because the spherical particles
join together to form chains or clusters of sub-micelles. However, the effect of pressure
treatment on casein moiety is temperature dependent as well. Among the whey proteins β-
lactoglobulin denaturation initiates above 150 MPa but complete denaturation occurs above
500 MPa at 25 °C. Immunoglobulin, α-Lactalbumin and bovine serum albumins are more
resistant and their denaturation occurs at the highest pressures and at temperature above 50°
C. It could be a strategy for preserving the colostrum Immunoglobulins which are heat labile
(Felipe et al., (1997). The variation in HP induced denaturation among whey proteins may be
attributed to the presence and number of disulphide bonds and lack of free sulfhyfryl groups in
case of α-Lactalbumin.
High pressure treatment has been observed to induce crystallization in milk fat and an
increase in total solid content in cream. Studies carried out by Gervilla et al. (2001) on Free
fatty acids (FFA) content in ewe's milk have showed that HP treatments between 100–500 MPa
at 4, 25 and 50° C did not increase FFA content. HP treatment at higher processing temperature
resulted lower FFA values in the milk. The phenomenon is of great interest to avoid production
of off flavours in milk and milk products, often encountered due to lipolytic rancidity in milk.
Hydrostatic pressure up to 500 MPa affects changes in size and distribution of milk fat globules
of ewe's milk. HP treatments at 25 and 50° C showed an increase in the number of small
globules in the range 1–2 μm, whereas at 4°C the tendency was reverse (Gervilla et al., 2001).
These changes on distribution of milk fat globules could be due to phenomenon of aggregation
and disaggregation /disintegration & offers certain advantages for HP-treated milk. HP
treatment increases the stability of milk treated at 25 and 50° C, whereas at 4° C increases the
creaming-off, which could improve cream separation during butter manufacture.
During heating of milk lactose may isomerise to lactulose and then degrade to form
acids and other sugars. No changes in these compounds have been observed after
pressurization in the range of 100–400 MPa for 10–60 min at 25 °C, suggesting that lactose
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isomerization or maillard reaction occurs in milk after pressure treatment (López Fandiño et al.,
1996). Contrary to thermal treatments, where covalent as well as non-covalent bonds are
affected, HP treatment at room and mild temperatures only disrupts relatively weak chemical
bonds (hydrogen bonds, hydrophobic bonds, ionic bonds). Thus, small molecules such as
vitamins, amino acids, simple sugars and flavour compounds remain unaffected by the HP
treatment. HP treatment of milk at 400 MPa (@ 2.5 MPa/s for 30 min at 25 °C) results in non-
significant loss of Vitamin B1 and B6 (Sierra et al., 2000). Inactivation of native enzymes by HP
treatment has been attempted by several workers and quite variables results obtained. Alkaline
phosphatase remains resistant to pressurization of 400 MPa but at higher pressure (600-800
MPa) and elevated temperature inactivation increases. Several other enzymes like
lactoperoxidase, phosphohexoseisomerase, γ-glutamyltransferase and plasmin have also been
reported to resistant for HP treatment. García Risco et al. (2000) found that HP treatments at
400 MPa for 15 min at 40–60° C reduces the proteolytic activity, and at 25–60° C improves the
organoleptical properties of milk, suggesting that these combined treatments could be used to
produce milk of good sensory properties with an increased shelf life.
HHP Induced Effects on Functional Characteristics of Milk
Micelle disintegration induced by HP treatment also affects the milk Colour. Treatment of 200
MPa at lower temperature had little effect on L value (whiteness) of the milk but a lower L
value is reported for milk treated at 250-450 MPa mainly due to disintegration of casein
micelle. A study was carried out by Harte et al. (2003) to observe the series of changes during
combine treatment of thermal and HHP for yogurt manufacture and it was observed that milk
subjected to HHP treatment and thermal treatment followed by HHP, loses its white colour and
turns into yellowish colour and might be due to reduction in size of casein micelles (Needs et
al., 2000), whereas milk when first subjected to HP followed by thermal treatment regained its
whitish colour and is attributed to reversible nature of casein micelles (or reaggregation of
disrupted micelles) towards HHP treatment when applied in the range of 300-676 MPa followed
by thermal treatment.
Liu et al. (2005) investigated the effect of HHP treatments on hydrophobicity of whey
protein and observed enhanced yields of Whey Protein Concentrate (WPC) with an increase in
the number of binding sites which leads to certain modifications of proteins. It also indicated
that high pressure can be applied to improve the functional properties of food proteins. Similar
observations for improved hardness, surface hydrophobicity, solubility, gelation and
emulsifying properties were observed in whey proteins (Lee et al., 2006)
Liu et al. (2005) studied the effect of HHP on flavor binding properties using whey protein
concentrate and observed that treatment of 600 MPa at 50° C resulted in an increase in
number of binding sites of WPC from 0.23 to 0.39 per molecule of protein for heptanone and
from 0.21 to 0.40 for octanone. Conformational changes in casein moiety reduced the Rennet
Coagulation Time (RCT) as the area of casein micelle get increased resulting in better access to
124
enzymes for action. However reduction in RCT is reported for treatment in the range of 200-
600 MPa. However, higher pressurization and longer duration for treatment did not cause any
substantial change in RCT.
HP Treatment for Cheese Manufacture
Milk pasteurization destroys pathogenic and almost all, but not all, spoilage microorganisms,
and it is the most important heat treatment applied to cheese milk to provide acceptable safety
and quality. However, milk pasteurization is known for its adverse affects with respect to many
sensory characteristics of cheese, leading to alterations in texture and often delayed
maturation (Grappin & Beuvier, 1997). HP technology can be used to increase the
microbiological safety and quality of milk to produce high quality cheeses. As it has been
mentioned above, HP processing of milk at room temperature causes several protein
modifications, such as whey protein denaturation and micelle fragmentation, and alters mineral
equilibrium. It has been observed that denaturation of whey proteins is due to applied
pressure, and results in interaction between denatured whey protein and casein, which in turn
increases the retention of former within casein matrix of cheese. Thus, these changes results in
modifying the technological aptitude of milk to make cheese, improving the rennet coagulation
properties and yield of cheese milk (Gonzalez et al., 2004; Trujillo et al., 1999). Microbiological
quality of cheeses from HP-treated milk (500 MPa for 15 min at 20° C) was comparable to
pasteurized milk (72° C for 15 sec) cheeses (Buffa et al., 2001). However, the application of HP
technology to cheese milk causes differences in cheese composition and ripening in comparison
to pasteurized milk cheese. The HP-treated milk cheeses retain higher moisture, salt and total
free amino acids contents than raw or pasteurized milk cheeses. On the other hand, cheeses
made from HP-treated milk showed a similar level of lipolysis in cheeses made from raw milk,
whereas the level of lipolysis in cheese made from pasteurized milk was lower and this
behaviour was explained by heat-sensitive but partial pressure-resistant characteristics of the
indigenous milk lipase. Also pressure treated cheese shows more viscoelastic texture and poses
less resistance to flow.
Cheese ripening always received a special attention and importance in cheese making
industries owing to its expensiveness and thus accelerated ripening is highly desirable. Most of
the work in this field has been done using elevation of ripening temperature, addition of cheese
slurries or exogenous enzymes or by the use of adjunct starters, either as such or in modified
form. The potential use of HP to accelerate cheese ripening was first elucidated in a patent by
Yokoyama, Sawamura and Motobayashi (1992). Experimental Cheddar cheese samples were
exposed to pressure from 0.1 to 300 MPa for 3 days at 25°C after cheese making and best
results were obtained at 50 MPa, where cheese had free amino acid content and taste
comparable to that of a 6 month old commercial cheese. However, similar studies in Cheddar
cheese and in other cheese varieties have shown notable differences respect to the level of
proteolysis as claimed in the Yokoyama's (1992) patent. It should be noted that the method of
125
Cheddar cheese making reported by these authors was substantially different from
conventional procedure. In particular, the kind of starter bacteria added to the cheese milk was
highly proteolytic and added at least 10-fold higher rates than conventional inoculation rates. In
certain cheese varieties such as Mozzarella and Gouda, pressurization increases rate of
proteolysis on exposure to pressure treatment of 400-600 MPa for 5-15 min.
Many HP conditions have been tested for accelerating cheese ripening which involves
‘high’ HP treatments (400–600 MPa) short times (5–15 min) or an initial ‘high’ HP treatment
(400–600) short times (5–15 min) followed by a ‘low’ HP treatment (50 MPa) long times (72 h)
for different cheese varieties While long treatments at moderate pressure produce an increase
in proteolysis whereas short and intense treatments produce a permanent effect on proteolysis
rates. The enhancement effect is assumed to be caused by the release of starter enzymes. An
increase in free amino acid amount was found on cheese stored for two weeks after pressure
treatment at 400 MPa for 5 min.
Quality Improvement in Yoghurt and Ice Cream Through HPP
Yoghurt, a popular dairy product suffers from common defect of syneresis and low
viscosity. Quality of yoghurt can be improved in terms of its preservation and improved
rheological properties by pressurization treatment. Skim milk treated with combined
treatments of high hydrostatic pressure (400-500 MPa) and thermal treatment (85° C for 30
min) shows increased yield stress, resistance to normal penetration, elastic modulus and
reduced syneresis (Harte et al., 2003). Similarly, Needs et al. (2000) recorded lower values of
fracture stress in set yoghurts made from milk pressure treated at 60 MPa for 15 min compared
to heat treated milk.
Reps et al. (1999) investigated the effect of pressurization on inactivation of microflora present
in yogurt and found that HP treatment of 400 MPa completely inactivates Lactobacillus
bulgaricus but Streptococcus thermophilus was found more resistance towards pressure but the
resistance varies from strain to strain with varied destruction in the range of 35.3 to 99.9 %
which gives an idea that shelf life of yogurt can be enhanced by HHP treatment. Penna et al.
(2007) observed the effect of HPP (676 MPa for 5min) along with heat treatment (85° C for
30min) on microstructure of low- fat yoghurt and found dense aggregated protein structure
with smooth surface; compact gel with improved gel texture and improved viscosity as
compared to fewer interconnected chains in untreated yogurt.
HPP treatment induces fat crystallization, shortens the time required to achieve a
desirable solid fat content, & thereby thus reduces the ageing time of Ice-Cream, and also
enhances the physical ripening of cream for butter making (Buchheim and Frede, 1996). In a
study it has been observed that pressurization treatment improves whipping ability of cream
when treated for 2 minutes at 600 MPa and is possibly due to better crystallization properties
of milk fat (Eberhard et al., 1999). Looking at the potential of modifying the conformational and
functional characteristics of milk molecules the HP technology has generated considerable
126
interests for improving the quality characteric of various value added products from milk.
Conclusion
HPP products are becoming choice of a modern consumer in terms of health and safety
aspects. Being one of the emerging technologies high pressure technology offers the food
technologists an opportunity to develop novel products with enhanced shelf life and higher
safety with better sensory and nutritional aspects of food intact within and being applicable to
a wide range of products, this technology offers food processors to manufacture minimally
processed shelf stable products. Also the application of these modern non- thermal
technologies provides a potential to reduce energy requirements in food processing industries.
Thus, in coming future, the day is not far off when we will have these products available in local
markets and presence of such products will eradicate the products made by the obsolescing
technologies available that only help in preserving the food but destroys its nutritive value.
References:
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emulsified fats. DMZ Lebensmittel industrie and Milchwirtschaft 117(5): 228–237.
Buffa M.;. Trujillo A.J and Guamis B. (2001). Changes in textural, microstructure, and colour characteristics
during ripening of cheeses made from raw, pasteurised or high-pressure-treated goats’ milk. International
Dairy Journal 11 (12): 927–934.
Cheftel J.C. (1992). Effect of high hydrostatic pressure on food constituents- An overview. In: High
Pressure and Biotechnology (Ed. By: Balny R Hayashi; Heremans H.; and Masson K ), Colloque INSERM,
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Eberhard P.; Strahm W. and Eyer H. (1999). High pressure treatment of whipped cream. Agrarforschung 6
(9): 352–354
Felipe X.; Capellas M. and Law A. R. (1997). Comparison of the effects of high-pressure treatments and
heat pasteurisation on the whey proteins in goat's milk. J. Agricultural and Food Chemistry. 45(3): 627–
631.
García Risco M.R.; Olano A.; Ramos M. and López Fandiño R. (2000). Micellar changes induced by high
pressure. Influence in the proteolytic activity and organoleptic properties of milk. J. Dairy Sci. 83 (10):
2184–2198.
Gervilla R.; Ferragut V. and Guamis B. (2001). High hydrostatic pressure effects on colour and milk-fat
globule of ewe's milk. J. Food Sci. 66(6): 880–885.
Gonzalez-Martin M. F. San; Chanes- Welti J. S. and Barbosa- Canovas G. V. (2004). Cheese manufacturing
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Gould W. Grahame (2000). Preservation: Past, Present and Future. British Medical Bulletin. 56 (1): 84-96.
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Grappin R. and Beuvier E. (1997). Possible implications of milk pasteurisation on the manufacture and
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Harte F; Luedecke L; Swanson B and Barbosa-Canovas .G.V. (2003) Low fat set yogurt made from milk
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SECTION II
Advances in Quality Assurance
129
Biosensors for Heavy Metal Ions
Neelam Verma
Biosensor Technology Lab, Department of Biotechnology,
Punjabi University, Patiala.
A biosensor is an analytical tool that consists of an immobilized component in close proximity
or in conjunction with a transducer that represents a synergic combination of biotechnology
and microelectronics. The use of biosensors for detection and quantification of heavy metal
ions is of great concern as contamination of heavy metal ions leads to deteriorating health
problems since these substances are non-biodegradable and retained by the ecological system.
Conventional analytical techniques( like atomic absorption spectrometry and inductively
coupled plasma mass spectrometry) are although highly precise but suffer from disadvantage of
high cost, the need for trained personnel and the fact that these are mostly laboratory bound.
Biosensors have the advantages of low cost, ease of use, specificity, portability and the ability
to furnish real time signals. The analysis of heavy metal ions can be carried out with biosensors
by using both protein and whole cell based approaches and DNA based biosensors (Verma and
Singh, 2005). A variety of enzymes have been used in the analysis of heavy metal ions based on
activation ( alkaline phosphatase apoenzyme for zinc ions). The more common situation of
heavy metal inhibition of enzymes is based on the interaction of metal ions with exposed thiols
or methy thiol groups of protein amino acids. Non-enzymatic proteins, ranging from naturally
occurring metal binding proteins to various engineered proteins that are constructed to bind
specific metal ions, have been utilized in biosensor development. Antibody based biosensors,
DNA based biosensors, naturally occurring whole cell based biosensors( Verma et al. 2010) and
genetically engineered microorganism based biosensors are also used for monitoring heavy
metal ions in the industrial effluents and food samples.( Figure 1).
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Figure 1: Classification of the types of biosensors used for the analysis of heavy metal ions
References:
Verma, N. and Singh, M.( 2005). Biosensors for heavy metals. Biometals 18: 121-129.
Verma, N. Singh,M. and Kumar V.( 2005) Development of enzyme based biosensor for monitoring copper
ions in industrial effluents and food samples. CHEM.ENVRON.RES. 14: 53-58.
Verma, N. and Singh, M. (2006). A Bacillus sphaericus based biosensor for monitoring nickel ions in
industrial effluents and food. Journal of automated methods & management in chemistry. 1-4
Verma N, Kumar S, Kaur H (2010) Fiber Optic Biosensor for the Detection of Cd in Milk. J Biosens
Bioelectron 1:102. doi:10.4172/2155-6210.1000102
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Health Hazards Associated with Engineered Nanomaterials
Gautam Kaul
Animal Biochemistry Division, NDRI, Karnal-132001, India
Nanotechnology has been defined by the U.S. National Nanotechnology Initiative (NNI) as
“understanding and control of mater at dimensions of roughly 1 to 100nm (nanomaterials)
where unique phenomena enable novel applications” (NNI, 2007). Nanomaterials – used to
describe materials with one or more components that have at least one dimension in the range
of 1 to 100 nm and include nanoparticles, nanofibres and nanotubes, composite materials and
nano-structured surfaces. Examples are Gold NPs, Carbon NPs, Europium oxide NPs, Titanium
NPs, Magnetic NPs, Biodegradable NPs (PLGA), Nanotubes (singled-walled and multi-walled),
Nanowires, Fullerene derivatives, Quantum dots etc. Research on toxicologically relevant
properties of these engineered nanomaterials has increased tremendously during the last few
years. Nanomaterials may have different properties like chemical, optical, magnetic, and
structural; and hence consequently they are having differential toxicity profiles (Lanone and
Boczkowski., 2006; Studart et al., 2007). ‘Engineered nanomaterials’’ (ENMs) are nanomaterials
with specific physico-chemical characteristics manufactured intentionally by humans.
Nanomaterials hold great promise in a range of biomedical applications, including medical
imaging and diagnostics and for targeted delivery of therapeutic compounds, or the
simultaneous monitoring of disease processes and therapeutics (theranostics). Engineered
nanoparticles are intentionally designed, which have application in nanomedicine are
monodispersed and in solid form, where as unintentional nanosized particles that are
polydispersed and chemically complex (Oberdorster et al., 2005; Moghimi et al., 2005).
However, the same toxicological principles apply to unintentionally and intentionally designed
nanoparticles (Oberdorster et al., 2005).
Nanomaterials being a potent toxin it affects almost all the tissues which come in contact with
it as shown in the Figure 1. Nanotoxicology refers to the study of the interactions of
nanostructures with biological systems with an emphasis on elucidating the relationship
between the physical and chemical properties of nanostructures with induction of toxic
biological responses (Oberdorster et al., 2005). Mammal’s skin, lungs and the gastro-intestinal
tract are in constant contact with the environment. The lung and gastro-intestinal tract are
more susceptible compared to the skin because it has effective barrier to foreign substances.
These three ways are the most critical points of entry for natural or anthropogenic
nanoparticles. Injections and implants are other minor possible routes of exposure, primarily
limited to engineered materials.
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Fig 1: An interdisciplinary-science: Nanotoxicology. An overview of the potential toxic effects
associated with nanomaterials, in vivo and in vitro. Figure showing the different toxicity due to
nanomaterials like Genotoxicity, Neurotoxicty, Pulmonary toxicity, Cardiovascular toxicity, GIT
toxicity, Nephrotoxicity, Spermatotoxicty and Dermal toxicity. (Modified from A. El-Ansary and
S. Al-Daihan 2009).
Entry of nanoparticles into living system:
Possible routes of entry into the body include inhalation, absorption through the skin or
digestive tract, injection, and absorption or implantation for drug delivery systems. In
particular, nanoparticles uptake by inhalation and ingestion are likely to be the major routes in
terrestrial organisms Brigger et al., (2002).
Respiratory tract: The respiratory tract can be divided into three regions: nasopharyngeal,
tracheobronchial, and alveolar regions. Significant amounts of certain particle size ranges can
deposit in each region for example, about 50% of nanoparticles of 20nm in diameter deposit in
the alveolar region and remaining 15% in the nasopharyngeal region, 15% in the
tracheobronchial region. In comparison, nanoparticle of 1nm size does not reach the alveolar
region and about 90% deposit in nasopharyngeal region, 10% in the tracheobronchial region
(Moghimi et al., 2005). Inhalation nanoparticles are deposited in all regions of the respiratory
tract, but only smaller particles reach distal airways and larger particles may be filtered out in
the upper airways (Curtis et al., 2006; Hagens et al., 2007). The nanoparticels are absorbed
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across the lung epithelium and enter into the blood and lymph to reach cells in the bone
marrow, lymph nodes, spleen, and heart . Diesel exhaust (DE) and DE particles (DEP) are one of
the major compounds responsible for air pollution. These compounds consist of nanopaticles
which induce adverse health effects. Several studies reported that the effects of nanoparticles
on the human body (mammals) have shown that nanoparticles exacerbate lung injury . When
the nanoparticles are administered through the nasal, they accumulate in the brain via the
olfactory nerve and exacerbated inflammatory reactions (Elder et al., 2006), and that
nanoparticles affect the circulatory system by altering heart rate (Chalupa et al., 2004).
Nanomaterial toxicity: Mechanism of Action
Nanomaterials have unique properties and characteristics of high surface area to volume ratio,
hence results into a unique mechanism of toxicity. In particular, toxicity has been thought to
originate from nanomaterial size, surface area, composition, and shape as reviewed by Lanone
and Boczkowski (2006). Size of the particle can also affect the mode of endocytosis, cellular
uptake, and the efficiency of particle processing in the endocytic pathway (Lanone and
Boczkowski, 2006; BeruBe et al., 2007). As the particles size decreases then it leads to an
exponential increase in surface area relative to volume, which makes the nanomaterial surface
more reactive on itself (aggregation) and to its surrounding environment (biological
components). This activity includes a potential for inflammatory and pro-oxidant, which explain
early findings showing mixed results in terms of toxicity of NSPs (Nano Sized Particles) to
environmentally relevant species. When the nanomaterial uptake is increased into certain
tissues then it may lead to accumulation, where they may interfere with critical biological
functions (Lanone and Boczkowski, 2006; Sayes et al., 2007). The chemical interaction of the
nanomaterial at the surface is largely defined by the chemical composition, since the surface is
in direct contact with the body whereas the limited bulk volume is hidden.
The main molecular mechanism of in vivo nanotoxicity is the induction of oxidative stress by
free radical formation and these free radicals will also cause damage to biological components
through oxidation of lipids, proteins and DNA. This leads to more oxidative stress on the body
which have a role in the induction or the enhancement of inflammation through up-regulation
of redox sensitive transcription factors (e.g.NF-κB), activator protein-1 and kinases involved in
inflammation. Interactions of nanomaterials with the mitochondria and cell nucleus are being
considered as main sources of toxicity. The organs like liver and spleen are the main targets of
oxidative stress because of slow clearance and accumulation (storage) of potential free radical
producing nanomaterials as well as prevalence of numerous phagocytic cells in the organs of
the reticuloendothelial system (RES). Additionally, organs of high blood flow that are exposed
to nanomaterials, such as the kidneys and lungs, can also be affected.
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Carbon nanotubes: Three types of SWCNTs (single walled carbon nanotubes) were investigated
in an intratracheal instillation (study in Mice) (Lam et al., 2004). The results showed that
regardless of the amount of metal impurities, dose-dependent lung lesions were characterized
chiefly by interstitial granulomas and SWCNTs was taken up by alveolar macrophages. In
macrophages SWCNTs clustered to form granulomas in centrilobular locations. Muller et al.
(2005) compared the pulmonary toxicity of ground and unground MWCNTs in rats, using
asbestos (Rhodesian chrysotile) and carbon black as references. They found that after 60 days
there were indications of a higher degree of pulmonary inflammation with ground MWCNTs
than that with intact MWCNT-treated animals. They also noticed that the adverse effects of
MWCNTs depend on the length of the material used in vivo. Scanning electron microscope
(SEM) images of multi-walled carbon nanotubes (MWNTs) scaffold prepared in our lab on
polyethyleneimine-coated glass surface at different magnifications and different views is shown
in Fig. 2 (a) and (b). The topological features of nano-network assembly and the surface
modification by protein adsorption served to convert CNTs into a bioactive material with
pronounced cell growth and functional activities (Rafeeqi, Kaul. 2010a & 2010b).
Fig.2 (a & b): Scanning electron microscopy images of MWNTs scaffold. When observed by SEM
at different magnifications and different views, these scaffolds with compact structure were
composed of many thousands of highly entangled nanotubes with diameters ranging from nm
to several micrometers in length. SEM micrographs show MWNTs distributed all over the
surface. Scale bars represent (a) 5 μm (b) 1 μm. (Our lab: Rafeeqi, Kaul. 2010a)
Zinc, iron and selenium nanoparticles :
Cha et al. (2007) exposed zinc (300 nm), iron (100 nm), selenium (10-20, 40-50, 90-110 nm;
0.24–2400 _ 1029 g ml21) nanoparticles to glioma cell line. Results showed that the
nanoparticles did not alter the membrane permeability and the cytotoxicity in vitro was low.
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Moreover, it was not dependent on the types and the sizes of nanoparticles and thus here the
toxicity was inferred to be due to material chemistry rather than size (Cha and Myung, 2007).
Fe2O3 magnetic nanoparticles: The temporary exposure to Fe2O3 magnetic nanoparticles
(MNPs) results in a dose-dependent reduced ability of rat pheochromocytoma (growing neuron
cell line PC12) to respond to nerve growth factor (NGF). PC12 cells exposed to different doses of
Fe2O3 MNPs show reduced viabilities, increased cytoskeletal disruption, decreased intracellular
contact, and diminished ability to form mature neuritis in response to NGF exposure as
compared to control cells (Pisanic II et al., 2007).
Magnetic nanoparticles: The effect of magnetic nanoparticles on the adhesion and cell viability
concerned to astrocytes was assessed by Au et al. (2007). They observed that nanoparticles
impede the attachment of astrocytes to the substratum. However, once astrocytes attach to
the substratum and grow to confluence, nanoparticles may cause mitochondrial stress. Due to
lack of a significant difference between the control and nanoparticle-treated group strongly
suggests that the addition of nanoparticles to astrocytes does not disturb membrane integrity.
When SWCNT exposed to chicken embryonic spinal cord or dorsal root ganglia, the DNA
content is significantly decreased. This effect was more pronounced when cells were exposed
to highly agglomerated SWCNTs than when they were exposed to better dispersed SWCNT
bundles (Belyanskaya et al., 2009).
Gold nanoparticles: Wiwanitkit et al. (2008) evaluated the effect of gold nanoparticles on RBC
in vitro. Mixture of gold nanoparticle solution and blood sample was analyzed. And observed
that accumulation of gold nanoparticles in the red blood cell but showed no significant
destruction of the red blood cell.
Carbon nanotubes, zinc oxide and iron oxide nanoparticles: Loeb et al. investigated the toxic
effect of multi walled carbon nanotubes (MWCNT), zinc (II) oxide (ZnO) and iron(III) oxide
(Fe2O3) nanomaterials on human red blood cells (RBC). As hemolysis of erythrocytes is a useful
method to examine the effects of particles on the cell membrane. The interaction of RBC and
nanoparticles were studied with the help of ultra high resolution imaging systems. This unveiled
attachment of nanoparticles to RBC and their cross linking effects. And MWCNT were able to
induce only hemolysis where as Fe2O3 displayed only hemagglutination, and ZnO nanorods
showed both hemolysis as well as hemagglutination. It showed that the MWCNT, ZnO and
Fe2O3 are toxic to human red blood cells, irrespective of the blood group.
Carbon black nanoparticles (CB): The in-utero effect of CB on the reproductive function of male
offspring was investigated by Yoshida et al. (2010). They administered CB in-utero and observed
that, the DSP was significantly reduced in male offspring. Even when CB was administered to
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adult mice, DSP decreased significantly (Yoshida et al., 2009). When adult mice were exposed to
CB, the incidence of seminiferous tubule damage was high (vacuolation of the seminiferous
tubules); however, its severity was mild (Yoshida et al., 2009). The intercellular adhesions of
seminiferous epithelia and seminiferous tubules damage were observed in testis of male
offspring and thus inhibited the spermatogenesis. Fig-3 (a) and (b) shows the spermatogonial
stem cells cultured on multi-walled carbon nanotube and functional multi-walled carbon
nanotube scaffold, pre-prepared on polyethyleneimine-coated glass surface. The SEM images
showed that the spematogonial stem cells had adhered properly and extensions of the cell
were seen in all directions on carbon nanotube scaffolds. The results provided that the degree
of biocompatibility between spermatogonial cells and CNTs, and the real possibility for CNTs to
be used as an alternative nano-material for in vitro growth of these cells (Rafeeqi, Kaul. 2010c).
Fig.3 (a & b): Higher magnification SEM images of cells during in vitro culture on MWNTs and
fMWNTS. Note the cell body maintaining its shape and adhering properly with substratum.
Scale bars represent (a) 1 μm and (b) 2 μm. (Our lab: Rafeeqi, Kaul. 2010c).
Titanium dioxide and zinc oxide nanoparticles
Gopalan et al. (2009) assessed the effects of ZnO and TiO2 nanoparticles (40-70 nm range) in
the presence and absence of Ultra-Violet (UV) light in human sperm and human lymphocytes in
the dark (D), after pre-irradiation with UV (PI) and simultaneous irradiation with UV (SI). The
effect of TiO2 nanoparticles showed that the percentage reduction in head DNA was greater for
PI and SI samples compared with samples treated in the dark. However with regard to
photogenotoxicity, sperm exhibited no significant differences when the results for PI and SI and
the dark were compared, except at the lowest concentration for SI samples in the case of ZnO
and the lowest concentration for PI in the case of TiO2. The effect of Diesel Exhaust Particle
(DEP), carbon black (CB) and TiO2 on mouse Leydig TM3 cells, (the testosterone-producing cells
of the testis). They assessed that, TiO2 was more cytotoxic to Leydig cells than other
nanoparticles. The proliferation of Leydig cells was suppressed transiently by treatment with
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TiO2 or DEP. When mouse Leydig TM3 cells treated with DEP then the expression of heme
oxygenease-1 (HO-1) a sensitive marker for oxidative stress, was induced remarkably. The gene
expression of the steroidogenic acute regulatory (StAR) protein, the factor that controls
mitochondrial cholesterol transfer was slightly increased when exposed to CB and DEP. Hence
overall results were found that DEPs, TiO2 and CB nanoparticles were taken up by Leydig cells,
and affected the viability, proliferation and gene expression (Komatsu et al., 2008). Liu et al.
(2010) investigated the effect of calcium phosphate nanoparticles on both steroid hormone
production and apoptosis in human ovarian granulosa cells. Results showed that calcium
phosphate nanoparticles could enter into granulosa cells, and distributed in the membranate
compartments, including lysosome, mitochondria and intracellular vesicles. Treatment with
calcium phosphate nanoparticles at concentrations of 10-100 mM didn't significantly change
either the progesterone or estradiol level in culture fluid, and the expression levels of mRNAs.
Liu et al. concluded that the calcium phosphate nanoparticles interfered with cell cycle of
cultured human ovarian granulosa cells thus increasing cell apoptosis.
Carbon nanotubes
The effect of single-walled carbon nanotubes (SWCNTs) on primary immune cells in vitro was
investigated by Zhang et al. (2008). The results showed that SWCNTs (25 and 50 mg/mL) could
promote the proliferation of spleen cells but not at concentrations of 1 and 10 mg/mL.
Interestingly they can inhibit T-lymphocyte proliferation at higher concentrations but no effect
on T-lymphocyte proliferation stimulated by concanavalin-A (ConA) at lower concentrations.
They also observed that SWCNTs inhibited the B-lymphocyte proliferation stimulated by
lipopolysaccharides (LPS) at concentrations of 1, 10, 25 and 50 mg/mL. Authors concluded that
SWCNTs have possibly negative effects on immune cells in vitro.
Conclusion:
Several researches were carried out with different nanoparticles causing abiotic stress on the
animal and human health. This shows us that engineered nanoparticles must be handled with
care and workers exposure must be minimized, since these effects are extremely variable from
one product to another. Although studies are conflicting regarding the magnitude and
mechanisms of nanomaterial toxicity, it is evident that some nanomaterials that were
previously considered biocompatible due to safety of the bulk material may indeed be toxic.
Still the pharmaco-kinetic behaviour of different types of nanoparticles requires detailed
investigation and a database of health risks associated with different nanoparticles (e.g. target
organs, tissue or cells) should be created. Existing research on nanotoxicity has concentrated on
empirical evaluation of the toxicity of various nanoparticles, with less regard given to the
relationship between nanoparticle properties (exact composition, crystallinity, size, size
dispersion, aggregation, ageing, etc) and their toxicity in the mammals. This approach gives very
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limited information, and should not be considered adequate for developing predictions of
toxicity of seemingly similar nanoparticle materials. The studies must include, research on
nanoparticles translocation pathways, accumulation, short- and long-term toxicity, their
interactions with cells, the receptors and signalling pathways involved, cytotoxicity, and their
surface functionalization for an effective phagocytosis in the mammals. Hence there is a serious
lack of information concerning the human health, animal health and environmental
implications of manufactured nanomaterials. Understanding the interactions of these “new age
materials” with biological systems is key to the safe usage of these materials in novel
biomedical fields like diagnostics and therapeutics. Since these are relatively new particles, it
requires thoughtful environmental, human health, animal health and safety research,
meaningful, and an open discussion of broader societal impacts, and urgent toxicological
oversight action.
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BeruBe K, Balharry D, Sexton K, Koshy L, Jones T. 2007. Combustion derived nanoparticles: mechanisms of
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Cha KE, Myung H. 2007. Cytotoxic effects of nanoparticles assessed in vitro and in vivo. J. Microbial Biotechnol.
17: 1573–1578.
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crystal phases. 2008. Toxicol. Lett. 183: 72–80. (doi:10. 1016/j.toxlet.2008.10.001).
Chalupa DC, Morrow PE, Oberd€orster G, Utell MJ, Frampton MW. 2004. Ultrafine particle deposition in
subjects with asthma. Environ Health Perspect. 112: 879–82.
Curtis J, Greenberg M, Kester J, Phillips S, Krieger G. 2006. Nanotechnology and nanotoxicology: a primer for
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El-Ansary A, Al-Daihan S. 2009. On the Toxicity of Therapeutically Used Nanoparticles. Journal of Toxicology.
Article ID 754810, 9 pages. (doi:10.1155/2009/754810)
Elder A, Gelein R, Silva V, Feikert T, Opanashuk L, Carter J, Potter R, Maynard A, Ito Y, Finkelstein J,
Oberdörster G. 2006. Translocation of inhaled ultrafine manganese oxide particles to the central nervous
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Food Allergens: Their Detection and Prevention
Rajeev Kapila & Suman Kapila
Animal Biochemistry Division, NDRI, Karnal
Adverse food reaction is a broad term representing any abnormal clinical response
associated with ingestion of a food. They are further classified as food intolerance or food
allergy based on the pathophysiological mechanism of the reaction. Food intolerance refers to
an adverse physiologic response to a food and may be due to inherent properties of the food
(i.e. toxic contaminant, pharmacologic active component) or to characteristics of the host (i.e.
metabolic disorders, idiosyncratic responses, psychological disorder), they may not be
reproducible, and they are often dose dependent. However, food allergy refers to an abnormal
immunologic response to a food that occurs in a susceptible host. These reactions are
reproducible each time the food is ingested and they are often not dose dependent. Based on
the immunological mechanism involved, food allergies may be further classified in a) IgE-
mediated, which are mediated by antibodies belonging to the Immunoglobulin E (IgE) and are
the best-characterized food allergy reactions; b) cell mediated when the cell component of the
immune system is responsible of the food allergy and mostly involve the gastrointestinal tract;
c) mixed IgE mediated-cell mediated when both IgE and immune cells are involved in the
reaction.
Immunological Mechanisms in Food Allergy
IgE-Mediated (Immediate Hypersensitivity):
IgE-mediated allergy is the best-understood allergy mechanism and, in comparison to
non-IgE-mediated reactions, is relatively easily diagnosed. Since the onset of symptoms is rapid,
occurring within minutes to an hour after allergen exposure, IgE-mediated allergy is often
referred to as "immediate hypersensitivity". In healthy immune systems, this type of
inflammatory response has evolved to target multicellular parasites such as worms. Allergic
responses occur when benign environmental antigens, such as food proteins, are incorrectly
targeted. The development of IgE-mediated occurs in two stages. The first, "sensitization",
occurs when the immune system is aberrantly programmed to produce IgE antibodies to food
proteins. These antibodies attach to the surface of mast cells and basophils, arming them with
an allergen-specific trigger. Subsequent exposure to food proteins leads to "activation" when
the cell-associated IgE binds the allergenic epitopes on food and triggers the rapid release of
powerful inflammatory mediators leading to allergy symptoms. The symptoms associated with
IgE-mediated CMA include one or more of cutaneous , gastrointestinal or respiratory
manifestations .
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Non-IgE-Mediated (Delayed Hypersensitivity)
A significant proportion of infants and the majority of adults with food allergy do not
have circulating milk protein-specific IgE and show negative results in skin prick tests and RAST.
These non-IgE-mediated reactions tend to be delayed, with the onset of symptoms occurring
from 1 hour to several days after ingestion food. Hence, they are often referred to as "delayed
hypersensitivity". As with IgE-mediated reactions, a range of symptoms can occur, but are most
commonly gastrointestinal and/or respiratory in nature. The gastrointestinal symptoms, such as
nausea, bloating, intestinal discomfort and diarrhoea, mirror many of those that are
symptomatic of lactose intolerance, complicating self-diagnosis. Adults with non-IgE-mediated
allergy to milk tend to suffer ongoing allergy without the development of milk tolerance. A
number of mechanisms have been implicated, including type-1 T helper cell (Th1) mediated
reactions, the formation of immune complexes leading to the activation of Complement, or T-
cell/mast cell/neuron interactions inducing functional changes in smooth muscle action and
intestinal motility.
Dysfunctional Tolerance
Food antigens contact the immune system throughout the intestinal tract via the gut
associated lymphoid system (GALT), where interactions between antigen presenting cells and T
cells direct the type of immune response mounted. Unresponsiveness of the immune system to
dietary antigens is termed "oral tolerance" and is believed to involve the deletion or switching
off (anergy) of reactive antigen-specific T cells and the production of regulatory T cells (T reg)
that quell inflammatory responses to benign antigens.Food allergy such as from cow milk is
believed to result from the failure to develop these tolerogenic processes or from their later
breakdown. In the case of IgE-mediated allergy, a deficiency in regulation and a polarisation of
food-specific effector T cells towards type-2 T helper cells (Th2) lead to signalling of B-cells to
produce food protein-specific IgE.. Non-IgE-mediated reactions may be due to Th1 mediated
inflammation. Dysfunctional T reg cell activity has been identified as a factor in both allergy
mechanisms (Tiemessen et al.,2004). Additionally, the induction of tolerance in children who
have outgrown their cow milk allergy has been shown to be associated with the development of
T reg cells.
Dominant Food Allergens
The most common food allergies are:
Dairy allergy
Egg allergy
Peanut allergy
Tree nut allergy
Seafood allergy
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Shellfish allergy
Soy allergy
Wheat allergy
These are often referred to as "the big eight." They account for over 90% of the food allergies.
The top allergens vary somewhat from country to country but milk, eggs, peanuts, tree-nuts,
fish, shellfish, soy, wheat and sesame tend to be in the top 10 in many countries. Allergies to
seeds - especially sesame - seem to be increasing in many countries.
Milk proteins as allergens
Hypersensitivity to milk proteins is one of the main food allergies and affects mostly but
not exclusively infants, while it may also persist through adulthood and can be very severe. Cow
milk contains more than 20 proteins allergens that can cause allergic reactions. Casein fractions
and β-lactoglobulins (β-lg) are the most common cow milk allergens. Human milk is free of β-lg,
similar to camel milk (El-Agamy, 2007). On the contrary, β-lg is a major whey protein in cow,
buffalo, sheep, goat, mare and donkey milk. Caseins in milk of the different species differ in
fraction number, amino acid composition, and their peptide mappings. β -Casein is the major
fraction in goat casein, which is similar to human casein and different from cow casein. The
peptide mappings of goat α-la and β-Ig are completely different from those of cow milk.
Allergies to milk proteins of non bovine mammals have documented due to cross reactivity
between cow milk proteins and their counterparts in other species and even between goat and
sheep caseins. Genetic polymorphism of milk proteins play an important role in eliciting
different degree of allergic reactions (Bell et al, 2006). Goat’s milk may contain only trace
amounts of the allergenic casein protein, αS1-CN. Several studies have reported real and
dramatic benefits from using goat, camel, mare and even soy milk as alternatives in cases of
cow milk allergy and they can be considered hypoallergenic (Monaci et al.,2006).
Diagnosis
The best method for diagnosing food allergy is to be assessed by an allergist. The
allergist will review the patient's history and the symptoms or reactions that have been noted
after food ingestion. If the allergist feels the symptoms or reactions are consistent with food
allergy, he/she will perform following allergy tests.
Skin Prick Test
Skin prick testing is easy to do and results are available in minutes. In these tests, a tiny
amount of the suspected allergen is put onto the skin or into a testing device, and the device is
placed on the skin to prick, or break through, the top layer of skin. This puts a small amount of
the allergen under the skin. A hive will form at any spot where the person is allergic. This test
generally yields a positive or negative result. It is good for quickly learning if a person is allergic
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to a particular food or not, because it detects allergic antibodies known as IgE. Skin tests cannot
predict if a reaction would occur or what kind of reaction might occur if a person ingests that
particular allergen. They can however confirm an allergy in light of a patient's history of
reactions to a particular food. Non-IgE mediated allergies cannot be detected by this method.
Immunoassay based methods
Blood tests are another useful diagnostic tool for evaluating IgE-mediated food allergies.
For example, the RAST (RadioAllergo Sorbent Test) detects the presence of IgE antibodies to a
particular allergen. The RAST test is a specific type of test with greater specificity: it can show
the amount of IgE present to each allergen. Researchers have been able to determine
"predictive values" for certain foods. These predictive values can be compared to the RAST
blood test results. If a person’s RAST score is higher than the predictive value for that food,
then there is over a 95% chance the person will have an allergic reaction (limited to rash and
anaphylaxis reactions) if they ingest that food. Currently, predictive values are available for the
following foods: milk, egg, peanut, fish, soy, and wheat. Blood tests allow for hundreds of
allergens to be screened from a single sample, and cover food allergies as well as inhalants.
Antibodies play a major role in most allergen detection methods. The specific binding between
antibodies and their recognized antigens has been exploited to create very sensitive and
specific systems for the detection of proteins. The majority of immunoassay methods use the
ELISA format for the detection of food allergens. Enzyme immunoassay (EIA) and enzyme-linked
immunosorbent assay (ELISA) have become household names for medical laboratories,
manufacturers of in vitro diagnostic products, regulatory bodies, and external quality
assessment and proficiency-testing organizations. ELISAs are rapid, sensitive, cost effective and
can be performed in a high-throughput manner. In contrast, techniques like
immunofluorescence and RIA are tedious, time consuming, having short shelf-life of the
reagents, requiring sophisticated expensive equipments and the strict regulatory controls on
the use of isotope. Though, this technique is relatively less sensitive as compared to radio-
immuno-assay (RIA) but efforts are continuing to increase its sensitivity.
SDS-PAGE immunoblotting
IgE binding capacity of individual proteins in a food extract can be analyzed if they are
physically separated. The standard procedure is one dimensional sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting, which includes the
electrotransfer of separated proteins onto a membrane, incubation of such membrane with
serum from allergic patients and detection with radio or enzyme labeled anti-IgE antibodies.
The method allowed the identification of new allergens from conventional foods as well as
from biotechnological novel food sources.
Bioinformatic tools
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The crucial step in the assessment of the potential allergenicity of proteins is sequence
similarity search against known allergens. In this sense a protein which shares more than 35%
sequence identity (over an 80 amino acids window) or at least six identical contiguous amino
acids with a known allergen is considered as likely allergenic. It is supposed that the two rules
account for the presence of T-(cells) or B- (IgE) epitopes in the query protein. Although only a
limited number of epitopes are known to date, it seems that T- epitopes are linear or
continuous motifs of about 8-24 amino acid residues , whereas B epitopes can also be
conformational.
Preventive measures
Effects of processing on allergen stability
The portion of a food protein that may cause an allergic reaction may be a simple
stretch of a few amino acids along the primary structure or it may be a unique three
dimensional motif of the protein structure, respectively referred to as linear and
conformational epitopes. An allergenic protein may contain a single epitope that is repeating or
may have several different epitopes. In order to have IgE cross-linking, there must be more than
one epitope on the allergen. The understanding relationships between the nature of the
allergenic epitopes and the corresponding clinical symptoms is crucial in designing ways to
reduce/eliminate allergenicity of the targeted allergens. Since foods/food ingredients are often
subjected to a variety of processing conditions, alteration in immunodominant epitopes may
potentially affect protein allergenic properties. Processing may destroy existing epitopes on a
protein or may generate new ones (neoallergen formation) as a result of change in protein
conformation. More commonly, processing methods have been associated with decreased
allergenicity (e.g., pollen-related fresh fruit and vegetable food allergens upon heating) or with
no significant effect (e.g., heat-stable allergens from shrimp upon heating).Conformational
epitopes are typically expected to be more susceptible to processing induced destruction than
the linear epitopes on the same allergen. Linear epitopes are more likely to be altered if the
linear epitopes are hydrolyzed. Alternatively, linear epitopes may be chemically modified during
food processing or be intentionally changed by introducing mutations through genetic
engineering. Since food processing involves thermal as well as non-thermal treatments and
each type of treatment may differ in its effect on epitopes, individual treatments must be
considered carefully when evaluating allergen stability. Thermal processing may be
accomplished by dry heat (e.g., oven roasting, oil roasting, infrared heating, and ohmic heating)
or may use wet heating conditions such as the ones encountered in cooking in aqueous media,
microwave cooking, pressure cooking (autoclaving), extrusion, blanching, boiling, and steaming.
Non-thermal processing methods include irradiation (e.g., γ-irradiation), soaking, germination,
milling, fermentation, high-pressure processing, dehulling and dehusking, and grinding.
Processing may alter food in a manner that may permit masking or unmasking of allergenic
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epitopes thereby reducing or enhancing allergen recognition and therefore potentially altering
allergenicity of the offending food. Alteration in protein structure (by food processing) can lead
to epitope destruction, modification, masking, or unmasking thereby decreasing, increasing or
having no effect on allergenicity (Sathea et al., 2005).
Extensively hydrolysed formula (eHF)
Manufacturers of hypoallergenic infant milk formulas have approached the problem by
destroying allergenic epitopes through extensive hydrolysis of milk proteins to peptides
typically smaller than 1500 Da. These extensively hydrolyzed formulas (eHF) successfully
prevent the triggering of allergy symptoms in the majority of allergic infants and are evidently
effective for both IgEmediated and non-IgE-mediated reactions. In a small percentage of cases,
even eHF trigger symptoms in highly sensitive infants and amino acid-based formulas are
required (Walker-Smith, 2003). While extensive hydrolysis eliminates allergenicity, it also
destroys the physical and biological functionalities of milk proteins, and the search for
alternative methods to produce hypoallergenic milks continues (Crittenden and Bennett, 2005).
Partially hydrolysed formula (pHF)
The proteins in hypoallergenic cow’s milk infant formulas are extensively hydrolyzed in
order to destroy allergenic epitopes. While these extensively hydrolyzed formulas (eHF) remove
allergenicity, the loss of immunogenicity also prevents the immune system from developing
tolerance to milk proteins. Partially hydrolyzed cow’s milk formulas (pHF) have been developed
with the aim of minimizing the number of sensitizing epitopes within milk proteins, while at the
same time retaining peptides with sufficient size and immunogenicity to stimulate the induction
of oral tolerance. Since they contain larger peptides than eHF, pHF trigger activation of
symptoms in a relatively large percentage of already sensitized infants and are therefore not
recommended where there is a risk of severe milk allergy symptoms. Human intervention
studies in at-risk infants have shown that pHF reduce the incidence of atopic dermatitis in the
first 2 years compared to intact cow’s milk protein formulas. However, despite animal studies
indicating that pHF have an increased capacity to induce tolerance, there remains no clear
evidence from human studies that they are better than eHF in preventing CMA. Further
prospective human feeding studies are required to establish if they can play a useful role in
preventing CMA.
Probiotics
Epidemiological evidence shows that allergy is more common in industrialized countries
than in developing nations and more frequent in urban compared to rural communities. This
has lead to the development of the “hygiene hypothesis”, which speculates that a decline in
Th1-inducing exposure to pathogens and parasites contributes to the Th2- skewed immunity
seen in IgE-mediated allergies. Providing a microbial challenge in the form of dietary probiotic
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bacteria (live Lactobacillus and Bifidobacterium cultures used in fermented dairy products) has
redressed Th1/Th2 imbalances and induced regulatory T cell activity in animal studies .
Interestingly, controlled feeding studies using probiotics in human infants have produced
clinically significant ameliorations of atopic dermatitis that have been maintained up to the age
of 4 years. Probiotics are now included in some infant formulas, together with oligosaccharides
(prebiotics), which can induce the development of a Bifidobacterium- dominated intestinal
microbiota, replicating the effect of human breast milk. Although still in its infancy, the use of
probiotics, prebiotics and components of intestinal parasites in the prevention of allergy is an
exciting and burgeoning area of research.
Immune Factors in Milk
Regulatory cytokines in human milk, such as transforming growth factor-beta (TGF), play
an important role in promoting appropriate responses to food antigens during early infancy
when the gut immune system is still developing. However, cow’s milk-based infant formulas are
generally deficient in regulatory cytokines. Using a rodent model, Penttila et al. (2001) reported
that supplementing infant formulas with cow’s milk fractions rich in immunoregulatory factors
enhanced the development of oral tolerance to food antigens. In the future, replicating the
immunoregulatory capacity of human breast-milk may prove a valuable strategy to promote
the tolerogenicity of cow’s milk formulas. Food processing may inactivate certain
conformational epitopes, but not all allergens. Enzymatic hydrolysis may help eliminate certain
epitopes. However, from a food product quality and acceptability viewpoint, protein hydrolysis
may result in undesirable and or unacceptable changes in food structure and sensory attributes.
When food allergens are present in trace quantities avoidance of the offending agent requires
the foreknowledge of their presence. Therefore, there is a critical need to develop robust,
reliable, sensitive, and accurate allergen detection methods.
References:
Bell, S.J., Grochoski, G.T., Clarke, A.J. (2006). Health implications of milk containing β-casein with the A2
variant. Crit. Rev. Food Sci. Nut.46: 93-100.
Crittenden, R.G and. Bennett L. E. (2005). Cow’s Milk Allergy: A Complex Disorder. J.Am. College Nut., 24: 582S–591S.
El-Agamy,E.I. (2007). The challenge of cow milk protein allergy. Small Rumin. Res. 68: 64-72.
Monaci, L., Tregoat, V. ,van Hengel, A.J. and Elke Anklam (2006). Milk allergens, their characteristics and their detection in food: A review. Eur Food Res Technol 223: 149–179.
Penttila IA, Zhang MF, Bates E, Regester G, Read LC, Zola H(2001). Immune modulation in suckling rat pups by a growth factor extract derived from milk whey. J Dairy Res 68:587–599.
Sathea, S.K., Teuberb, S.S. and Roux , K. H. (2005). Effects of food processing on the stability of food allergens. Biotech. Adv. 23: 423–429.
Walker-Smith J (2003). Hypoallergenic formulas: are they really hypoallergenic? Annal Allergy Asthma Immunol 90:112–114.
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ISO 22000 Food safety Management System
Bimlesh Mann
Dairy Chemistry Division, NDRI, Karnal
Every player in the food supply chain or food sector is ever more eager to ensure that
appropriate systems are in place to manage their food supply channels. Many companies are
looking for appropriate certification standards to give their products a sound seal of approval. It
is now generally accepted by legislators and food professionals that a formal, structured food
safety management system is a must for effectively managing and controlling food safety
hazards in the preparation and handling of food and food products. Food safety means taking
care with all aspects of food production and preparation to make sure that the final product is
safe without any contamination. Food safety is linked to the presence of food-borne hazards in
food at the point of consumption. Food safety is an integral part of food quality assurance.
Since food safety hazards can occur at any stage in the food chain it is essential that adequate
control be in place. Therefore, a combined effort of all parties through the food chain is
required. The increased demand for safe food by health conscious consumers is result of media
exposure, globalization and international trade.
ISO 22000:2005
ISO 22000, published on 1 September 2005, is a new International Standard designed to
ensure safe food supply chains worldwide and the first of a family on food safety management
systems. ISO 22000 is an international standard designed to ensure worldwide safe food supply
chains and provide a framework of internationally harmonized requirements for the global
approach that is needed. ISO 22000:2005, Food safety management systems – Requirements
for any organization in the food chain, provides a framework of internationally harmonized
requirements for the global approach that is needed. The standard has been developed within
ISO by experts from the food industry, along with representatives of specialized international
organizations and in close cooperation with the Codex Alimentarius Commission, the body
jointly established by the United Nations’ Food and Agriculture Organization (FAO) and World
Health Organization (WHO) to develop food standards. A major resulting benefit is that ISO
22000 will make it easier for organizations worldwide to implement the Codex HACCP (Hazard
Analysis and Critical Control Point) system for food hygiene in a harmonized manner, which
does not vary with the country or food product concerned. Food reaches consumers via supply
chains that may link many different types of organization and that may stretch across multiple
borders. One weak link can result in unsafe food that is dangerous to health – and when this
happens, the hazards to consumers can be serious and the cost to food chain suppliers
considerable. As food safety hazards can enter the food chain at any stage, adequate control
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and communication throughout is essential. Food safety is a joint responsibility of all the actors
in the food chain and requires their combined efforts.
ISO 22000 is therefore designed to allow all types of organization within the food chain
to implement a food safety management system. These range from feed producers, primary
producers, food manufacturers, transport and storage operators and subcontractors to retail
and food service outlets – together with related organizations such as producers of equipment,
packaging material, cleaning agents, additives and ingredients. The standard has become
necessary because of the significant increase of illnesses caused by infected food in both
developed and developing countries. In addition to the health hazards, food borne illnesses can
give rise to considerable economic costs covering medical treatment, absence from work,
insurance payments and legal compensation. As a result, a number of countries have developed
national standards for the supply of safe food and individual companies and groupings in the
food sector have developed their own standards or programmes for auditing their suppliers
which generated risks of uneven levels of food safety, confusion over requirements, and
increased cost and complication for suppliers that find themselves obliged to conform to
multiple programmes. Consequently, there was a perceived need for international
harmonization of such global standards a role for which the ISO22000 has been created. ISO
22000, backed by international consensus, harmonizes the requirements for systematically
managing safety in food supply chains and offers a unique solution for good practice on a
worldwide basis. In addition, food safety management systems that conform to ISO 22000 can
be certified – which answers the growing demand in the food sector for the certification of
suppliers – although the standard can be implemented without certification of conformity,
solely for the benefits it provides.
Key elements of ISO 22000:
This international standard specifies the requirements for a food safety management
system that combines the recognized generally recognized key elements to ensure food safety
along the food chain, up to the point of final consumption:
Interactive communication
Clear communication along the food chain is essential to ensure that all relevant food
safety hazards are identified and adequately controlled at each step with in the food chain. This
implies communication of the needs of the organization to organizations both upstream and
downstream in the food chain. Communication with customers and suppliers, based on the
information generated through systematic hazard analysis, will also assist in establishing
customer and supplier requirements in terms of feasibility, need and impact on the end
product. Recognition of the organization’s role and position within the food chain is essential to
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ensure effective interactive communication throughout the chain in order to deliver safe food
products to the consumers.
System management
The most effective food safety systems are designed, operated and updated within the
framework of a structured management system and incorporated into the overall management
activities of the organization. This provides maximum benefit for the organization and
interested parties. ISO 22000 is aligned with the requirements of ISO 9001:2000 in order to
enhance the compatibility of the two standards and to ease their joint or integrated
implementation. This international standard can be aligned or integrated with existing related
management system requirements, while organizations may utilize existing management
systems to establish a food safety management system that complies with the requirements of
this international standard.
Prerequisite programmes (PRPs)
Basic conditions and activities those are necessary to maintain a hygienic environment
throughout the food chain suitable for the production, handling and provision of safe end
products and safe food for human consumption. The prerequisite programmes needed depend
on the segment of the food chain in which the organization operates and the types of
organization. Examples of equivalent terms are: Good Agricultural Practice (GAP), Good
Veterinarian Practice (GVP), Good Manufacturing Practice (GMP), Good Hygienic Practice
(GHP), Good Production Practice(GPP), Good Distribution Practice (GDP) and Good Trading
Practice (GTP).
Hazard control
This international Standard integrates the principles of the Hazard Analysis and Critical
Control Point (HACCP) system and application steps developed by Codex Alimentarius
Commission.
Principle of HACCP
It is important to identify the possible hazards that can occur at every stage of the food
business from growth, processing, manufacturing, storage and distribution, until the point
where it is sold to the customer. As far as possible manufacturer should consider how the
customer might handle it too. The HACCP system consists of seven principles:
Principle 1: Identify hazards
Principle 2: Determine critical control points
Principle 3: Establish critical limits
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Principle 4: Establish a monitoring system
Principle 5: Establish corrective action
Principle 6: Establish verification procedures
Principle 7: Establish record keeping and documentation requirements
During hazard analysis, the organization determines the strategy to be used to ensure
hazard control by combining the prerequisite programmes (PRPs) and the HACCP plan. Hazard
analysis is the key to an effective food safety management system, since conducting a hazard
analysis assists in organizing the knowledge required to establish an effective combination of
control measures. ISO 22000 requires that all hazards that may be reasonably expected to
occur in the food chain, including hazards that may be associated with the type of process and
facilities used, are identified and assessed. Thus it provides the means to determine and
document why certain identified hazards need to be controlled by a particular organization and
why others need not. During hazard analysis, the organization determines the strategy to be
used to ensure hazard control by combining the PRPs, operational PRPs and the HACCP plan.
Sections of ISO 22000:
Organizations implementing ISO 22000, which includes the principles of the Codex HACCP
system, can now cover the key requirements of the various global standards by using a single
document. Since ISO22000 is designed to be fully compatible with ISO 9001, a food supply
company with an established quality management system will find it easy to extend their
system to include this new standard. The sections of ISO 22000 have been deliberately made
similar to those of ISO9000, to simplify and streamline the work for those integrating both food
safety and quality, and to retain the usefulness of the familiar, proven structure. A brief
overview of the eight sections of ISO22000 has been given below:
1. Scope:
Define how and where standard can be applied across any part of the chain.
2. Normative References
Outlines how the standard was developed through the defined way of developing
consensus among stakeholders.
3. Terms and Definitions
Defines necessary terms and ensures proper use of such terms in documents to ensure clear
communication and understanding.
4. Food Safety Management System, including
General requirements (such as overall statements which will be presented in more
detail elsewhere in a company’s ISO documentation).
Documentation requirements, including processes for document approval, changes,
retention and control.
5. Management Responsibly
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Management commitment and evidence of this commitment through its business
objectives, its communication within the organization, its food safety policy and
beyond.
Food safety policy
Food safety management system planning
Responsibility and authority, each to be clearly defined
Food safety team leader
Communication, both within and outside the company
Emergency preparedness and response
Management review at planned intervals
6. Resource Management
Competency, awareness and training for personnel to ensure they are capable of
carrying out the requirements of adhering to the ISO22000 standards
Infrastructure, to ensure the suitability of the environment, equipment, ventilation,
process equipment and supporting services.
Work environment to ensure safe flow patterns to prevent cross contamination, and
appropriate facilities for employees
7. Planning and Realization
Prerequisite programs, meaning those standards to which a particular company
must comply
Hazard analysis and critical control point
Operational programs, such as SOPs, which are process specific
HACCP plan covers all the principles establishing critical limits, monitoring,
corrective action, verification and record
Updates, verification, traceability and control of nonconformity
8. Validation, verification and improvement of the food safety management system
Validation of control measures, to ensure that prerequisite programs and CCPs are
still working
Control of measurement devices, including calibration and corrective action for food
safety devices
Verification through internal audits and other activities and measurements
Improvement through reviews, updates and input from management
An ISO technical specification (ISO/TS 22004) gives details about guidance on the
implementation of the standard, with a particular emphasis on small and medium sized
enterprises. Another technical specification (ISO/TS 22003) explains certification requirements
applicable when third party certification is used.
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Bureau of Indian standards(BIS),being a certification body, does not provide any form of
consultancy body, does not provide any form of consultancy services for implementation of the
requirement of IS/ISO 22000 under its Food Safety Management Systems Certification Scheme.
However, BIS will be the guiding instrument for any organization who is interested to obtain
license for FSMS. There are number of certifying bodies for standards related to food.
Benefits for users
Organizations implementing the standard will benefit from:
• Organized and targeted communication among trade partners
• Optimization of resources (internally and along the food chain)
• Improved documentation;
• Better planning, less post process verification
• More efficient and dynamic control of food safety hazards
• All control measures subjected to hazard analysis
• Systematic management of prerequisite programmes
• Wide application because it is focused on end results
• Valid basis for taking decisions
• increased due diligence
• Control focused on what is necessary
• Saving resources by reducing overlapping system audits
Benefit for other stakeholders
Other stakeholders will benefit from
• Confidence that the organizations which are implementing ISO 22000 have the
ability to identify and control food safety hazards.
Value-adding features
• It is an auditable standard with clear requirements;
• It is internationally accepted
• It integrates and harmonizes various existing national and industry-based
certification schemes
• Food processing industries are waiting for this standard
• It is aligned with both ISO 9001:2000 and HACCP
• It contributes to a better understanding and further development of HACCP.
The food safety has become a prime concern in recent time however different quality
management systems and standards have evolved through past few decades. ISO 22000 gives
top importance to the safety of the consumer based on the strong and healthy management
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practices of the revolutionized standard. The future success of the standard will depend on its
take-up across the food industry by key customers, notable retailers and major purchasers.
References:
ISO 22000:2005 Standard http://www.iso.org./ iso
ISO management Systems, www. Iso.org./ims
SOF Institute website, Safe Quality Food, www.sqti.com
Surak, John G. "A Recipe for Safe Food: ISO 22000 and HACCP". Quality Progress. October 2007. pp. 21-27.
World food safety organization website www.worldfoodsafety.org
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Rancimat (Accelerated and Automated) Method for Evaluation of Oxidative
Stability of Fats and Oils
Sumit Arora
Dairy Chemistry Division, NDRI, Karnal.
Rancimat is a modern, computer controlled analytical instrument for the comfortable
determination of oxidative stability index to predict oxidation stability of oils and fats, and
hence, their shelf life. It was developed by Hador and Zurcher in 1974 to replace the time
consuming active oxygen method (AOM) and other such methods. Oxidative stability is an
important criterion for evaluating the quality of oils, fats and fatty acid methyl esters
(biodiesel). Lipid oxidation in foodstuffs is one of the most important critical factors affecting
major quality parameters such as colour, flavour, aroma and nutritive value, which reduces
their shelf life and influence its suitability for consumption. Therefore, it has great importance
in food industry to predict the shelf life of foods especially fatty foods. Determining oxidative
stability is a tedious and time-consuming process when performed at room temperature, thus it
is necessary to use accelerated methods to obtain the oxidative stability in a shorter time. For
this reason, several accelerated methods have been developed such as Schaal oven test, Active
Oxygen Method (AOM) and Rancimat Method. AOM and Schaal oven test are non-reproducible
and time-consuming methods, however, Rancimat method is comparatively more popular
because of its ease of handling and reproducibility of results. The unique temperature
extrapolation allows an approximate estimation of the storage stability of a product, thus
saving both time and money.
Advantages:
Automated computer-controlled instrument, therefore, is easy to operate
Conversion of induction time to other temperatures i.e. extrapolation to predict the
shelf life of samples
Excellent data security and reproducibility
Time and money saving
Evaluation can be done at two different temperatures simultaneously
Independent heating blocks having individual start of each position
Principle:
Oil or fat sample is heated at higher temperature in a sealed reaction vessel. Stream of air is
passed through the oil or fat sample which results in oxidation of lipid molecules. The volatile
products formed upon oxidation are transported through the stream of air to a second vessel
containing distilled water, whose conductivity increases with increase in content of oxidation
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products. A graph is plotted between conductivity and time which can be used to estimate the
induction time or oxidative stability index of oil or fat, thus predicting the shelf life of sample.
Standards
The Rancimat method is included in various national and internationals standards, such as:
a. AOCS Cd 12b-92 (Sampling and analysis of commercial fats and oils: Oil Stability Index)
b. ISO 6886 (Animal and vegetable fats and oils– Determination of oxidation stability by
accelerated oxidation test)
c. 2.4.28.2-93 (Fat stability test on Autoxidation. CDM, Japan)
d. Swiss Food Manual (Schweizerisches Lebensmittelbuch), section 7.5.4
Determination of Oxidation Stability:
Prepare fat/oil sample
Switch on 743 Rancimat as suggested by the manufacturer
Select method
Start heating
Insert and connect reaction vessels when temperature is reached
Start determination
Determination finished when stop criteria* is reached
Result display
Clean vessels and accessories
Figure 1: Flow diagram showing working of 743 Rancimat
*Stop criteria may be induction time, conductivity or end point (point at which conductivity
starts increasing abruptly)
RANCIMAT:
A. INSTRUMENTATION:
a. Heating Blocks:
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The 743 Rancimat has two independent heating blocks that allow up to eight samples to
be analyzed at one or two temperatures. Up to four Rancimats can be connected to one
computer, so that the maximum number of samples that can be analyzed in parallel can
be increased to 32. Each measuring position can be started individually. As soon as the
measurement has been completed the measuring position is immediately ready for a
new sample, which means that the instrument can be used to its full capacity.
b. Reaction Vessel:
Weighing out the sample and assembling the reaction vessel are extremely simple and
safe. Reaction vessel does not need to be expensively cleaned at the end of the
measurement, thus reducing the analysis costs.
Figure 2: Reaction Vessel
c. Measuring Vessel:
Easy-to-clean polycarbonate beakers are used for the automatic conductivity
measurement. Glass beakers are available as an alternative.
Figure 3: Measuring Vessel
d. Cover with built-in conductivity cell:
The conductivity cell is incorporated in the measuring vessel cover. When the cover is
placed in position the cell is immersed in the water. At the same time electrical contact
is made to the electronics in the instrument. The use of fragile glass conductivity
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electrodes with lengthy connecting cables went out of fashion a long time ago. The new
conductivity cell is also very easy to clean.
Figure 4: Conductivity cell
e. Connections:
In order to make operation as simple as possible, there are no controls at all on the
instrument. All its functions are controlled from the computer. Apart from the power
switch, the only features you will find on the instrument are the RS-232 socket for
connection to a computer and a socket for connecting the Pt-100 temperature sensor.
Figure 5: Connections
f. Air inlet filter and molecular sieve:
The air used for the measurement is aspirated through a filter that prevents particles
from entering the instrument. The molecular sieve removes water vapour from the
aspirated air; as water contributes to the hydrolytic decomposition of the fat molecules,
it could interfere with the measurement.
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Figure 6: Air inlet filter and molecular sieve
g. Air supply line:
The amount of air that passes through the sample is automatically controlled via the
rotation rate of the built-in pump according to the method settings. A separate supply
of compressed air is not necessary.
B. VALIDATION WITH THE GLP SET:
The optionally available GLP Set facilitates the validation of 743 Rancimat. It contains a
certified Pt-100 temperature sensor with accessories that can be used for testing the
temperature regulation of the heating block. A test plug for checking the conductivity
measurement inputs is also supplied.
Figure 7: GLP Set
C. SOFTWARE FUNCTIONS:
All functions of the 743 Rancimat are controlled by the Rancimat software, which excels by
its user-friendliness. All the functions are clearly arranged in just a few windows, the
operation is intuitive.
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D. RANCIMAT CONTROL:
This is where the measuring parameters can be called up and edited. The instrument
functions are controlled directly from here; the measurements are also started and shown in
the live display field. The arrangement of this window corresponds to a view of the
instrument from above. This means that the assignment of sample information and
measuring position is perfectly clear. The timer function can be used to automatically switch
on the heating blocks before the start of work, so that it is no longer necessary to wait while
they warm up.
The functions in a nutshell:
• Individual start/stop for each position
• Live display
• Temperature display
• Method definition
• Instrument controls
• Calculation formulas, automatic result transformation to other temperatures
• Timer function
E. RESULTS:
At the end of each determination, the measured data is stored in a database and can be
viewed by the user in the results window. Sample information and results are shown in
tabular form and can be exported in various formats. The measured curves can be shown
individually or in groups. It is also possible to edit the automatic evaluation and recalculate
the results. The temperature extrapolation function for estimating the storage stability is
available in this section of the software. All the displayed data can be sorted or filtered and
display can be adapted to meet our requirements. Results can be obtained in the following
forms:
• Overview table
• Curve display: individual or multiple plots
• Re-evaluation: induction time, stability time and manual tangent method
• Report printout
• Temperature extrapolation (estimation of storage stability)
• Database functions: filtering, sorting
• Data export
F. APPLICATIONS:
Determination of oxidation stability of foods:
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Just like the pure substances, the oils and fats contained in foods are also subject to
oxidation, which contributes to their spoilage. In such cases the Rancimat can be used to
determine the oxidation stability of foods containing oils and fats. Meltable foods with a high
fat content, such as ghee, butter, margarine, lard or tallow, can be analyzed directly without
any further sample preparation. For liquid or semi-liquid foods, such as salad dressings or
mayonnaise, it is better to split the emulsion and analyze the separated fat phase. For solid,
non-meltable foods it is also necessary to separate off the fat phase. In this case the fat is
normally cold-extracted with petroleum ether and the isolated fat is then analyzed.
Following food samples can be analysed: butter, margarine, ghee, vegetable oils, baby foods,
ice-cream, cereals, chocolate, nuts and biscuits.
G. Technical specifications:
1. Heating blocks:
Two aluminium heating blocks; electrically heated; can be set to different temperatures
2. Number of samples:
Eight samples (4 measuring positions per heating block)
3. Temperature control and measurement
Temperature range : 50 to 220 °C, adjustable in 10C steps
Temperature correction : -9.9 to +9.9 °C, adjustable in 0.10C
steps
Reproducibility of set temperature : Typically better than ±0.2 °C*
Temperature variation : Typically <0.1 °C*
Temperature difference between : Typically <0.3 °C*
different measuring positions
Instrument heating-up time from 20 °C : 45min (to ±0.1°C temp. stability)
To 1200C
Instrument heating-up time from 20 °C : 60min (to ±0.1°C temp. stability)
to 220 °C
Outer temperature of instrument : <50 °C (at an operating temp. 220 °C)
Response temperature of thermal : 2600C
protection device
*When operating temperature has been reached, with inserted reaction vessels with an
identical filling and 20 L/h air throughput.
4. Air throughput:
Pump : Diaphragm pump
Output range : 7 to 25 L/h
161
5. Conductivity measurement:
Measurement range : 0 to 400 μS/cm
Electrodes : 6.0913.130 conductivity cell with double steel pin electrode
built into vessel cover
6. Temperature:
Nominal working range : +50C to +400C (at 20 to 80% relative humidity)
Storage : -200C to +700C
Transport : -400C to +700C
7. Line power
Voltage : 2.743.0014/2.743.0114: 230 V (220...240 V ±10%)
2.743.0015/2.743.0115: 115 V (100...120 V ±10%)
Frequency : 50 to 60 Hz
Power consumption : <450 VA (depending on heating power)
8. Dimensions
Width : 405 mm
Height : 268 mm (without accessories)
353 mm (with accessories)
Depth : 466 mm
9. Weight 27.6 kg (with accessories)
10. PC requirements
Processor : Pentium III with 700 MHz or higher
Operating system : Windows TM NT, Windows TM 2000 or Windows TM XP
Memory : 20 MB for program files, 200 MB recommended for
measuring data storage
RAM : Working memory 128 MB, recommended 256 MB or higher
(particularly for Windows TM XP)
Graphics resolution : min. 800 x 600, recommended 1024 x 768 or higher
Interface : 1 free RS-232C interface (COM)
Printer : All printers supported by WindowsTMt advantage
162
Lateral Flow Assay – Principle and its Application in Analytical Food Science
Rajan Sharma1 and Y. S. Rajput2, and Priyanka Singh Rao1
1Dairy Chemistry Division; 2Animal Biochemistry Division, NDRI, Karnal
Introduction
The lateral flow assay (LFA), also called the immunochromatographic assay or the strip
assay is a simple device intended to detect the presence (or absence) of a target analyte in
sample (matrix). This technique is based on an immunochromatographic procedure that utilizes
antigen–antibody properties and enables rapid detection of the analyte. It includes several
benefits, such as a user-friendly format, rapid results, long-term stability over a wide range of
weather conditions, and relatively low manufacturing costs. These characteristics render it
ideally suited for onsite testing by untrained personnel. The main application of this technology
had been the human pregnancy test which came in picture in the 1970s. However, to fully
develop the lateral flow test platform, a variety of other enabling technologies were also
required. These include technologies as diverse as nitrocellulose membrane manufacturing,
antibody generation, fluid dispensing and processing equipment, as well as the evolution of a
bank of knowledge in development and manufacturing methodologies. Many of these
facilitative technologies had evolved throughout the early 1990s, the first lateral flow products
were introduced to the market in the late 1980s. Since then, as of 2010, over 200 companies
worldwide are producing a range of testing formats. The world market for LF-based tests
(Rosen, 2009) is estimated at $2,270 million in 2005 and, with a compounded annual growth
rate (CAGR) of 10%, it will reach $3,652 million in 2012. This estimate includes LF-based tests
used in human and veterinary medicine, food and beverage manufacturing, pharmaceutical,
medical biologics and personal care product production, environmental remediation, and water
utilities.
Architecture and Working of a Lateral Flow Immunoassay
Figure 1 shows the typical configuration of a LFA which is composed of a variety of
materials, each serving one or more purposes. The parts overlap onto one another and are
mounted on a backing card using a pressure-sensitive adhesive. The assay consists of several
zones, typically constituted by segments made of different materials. When a test is run,
sample is added to the proximal end of the strip, the sample pad. Here, the sample is treated to
make it compatible with the rest of the test. The treated sample migrates through this region to
the conjugate pad, where a particulate conjugate has been immobilized. The particle can
typically be colloidal gold, or a colored, fluorescent, or paramagnetic monodisperse latex
particle. This particle has been conjugated to one of the specific biological components of the
assay, either antigen or antibody depending on the assay format. The sample re-mobilizes the
163
dried conjugate, and the analyte in the sample interacts with the conjugate as both migrate
into the next section of the strip, which is the reaction matrix. This reaction matrix is a porous
membrane, onto which the other specific biological component of the assay has been
immobilized. These are typically proteins, either antibody or antigen, which have been laid
down in bands in specific areas of the membrane where they serve to capture the analyte and
the conjugate as they migrate by the capture lines. Excess reagents move past the capture lines
and are entrapped in the wick or absorbent pad. Results are interpreted on the reaction matrix
as the presence or absence of lines of captured conjugate, read either by eye or using a reader.
Figure 1.: Typical configuration of a lateral flow immunoassay test strip
Lateral Flow Assay Formats
This test can be performed on two platforms, either direct (sandwich) or competitive
(inhibition) and also can be used to accommodate qualitative, semi quantitative and in limited
cases, fully quantitative determination.
Direct assay format: Direct assays (Figure 2) are typically used when testing for larger analyte
with multiple antigenic sites i.e. analyte presenting several epitopes. This system (equivalent to
sandwich ELISA) employs two different antibodies (polyclonal and monoclonal) that bound
distinct epitopes of the analyte: a labelled polyclonal antibody is placed in a dehydrated state
onto a glass-fiber membrane (conjugate pad) to serve as detector reagent and a monoclonal
antibody specific to the analyte is sprayed at the test line of the nitrocellulose membrane to
serve as capture reagent. An additional antibody specific to the detection antibody (species
specific) could be used to produce a control signal at control line.
When a sample extract is applied to sample pad, the liquid migrates up by capillary force
and the detector reagent is then released. Some of the analyte bind to the detection antibody
and some remain free in the solution. Subsequently, the mixture passes through the capture
164
zone (test line) and both unbound analyte and bound analyte bind to the capture antibody. The
response in the capture zone (test line) is directly proportional to the amount of analyte in the
sample.
Competitive assay
Competitive assay formats are typically used when testing for small molecules with
single antigenic determinants, which cannot bind to two antibodies simultaneously. In this
format, an analyte-protein conjugate coated on the test zone of a nitrocellulose membrane
captures a labelled anti-analyte monoclonal antibody complex, allowing colour particle (e.g.
colloidal gold) to concentrate and form a visible line on the test zone. Another specific antibody
coated on the control line allows the capture of the excess antibody complex. One band colour
will therefore be visible in the control zone regardless of the presence of target analytes,
confirming correct test development. Conversely, a negative sample will result in the formation
of two visible colour bands (test line and control line).
Figure 3. Competitive Lateral Flow Assay
Materials and Processes in Lateral Flow immunoassay Development and Construction
A typical test strip consists of the following components:
a. Membrane/Analytical Region: The purpose of the analytical region in a lateral flow
immunoassay is to bind proteins at the test and control areas and to maintain their stability and
activity over the shelf-life of the product. The membrane material is typically a hydrophobic
nitrocellulose or cellulose acetate membrane onto which anti-target analyte antibodies are
immobilised in a line across the membrane as a capture zone or test line. A control zone may
also be present, containing antibodies specific for the conjugated antibodies. Nitrocellulose,
while extremely functional, the only material that has been successfully and widely applied for
LIFA because of it’s relatively low cost, true capillary flow characteristics, high protein-binding
capacity, relative ease of handling.
165
b. Conjugate Pad or reagent pad: This contains antibodies specific to the target analyte
conjugated to coloured particles (usually colloidal gold particles, or latex microspheres). The
role of the conjugate pad in a lateral flow immunoassay is to accept the conjugate, hold it
stable over its entire shelf life, and release it efficiently and reproducibly when the assay is run.
The materials of choice are glass fibers, polyesters, or rayons.
c. Sample Pad: Sample pad is an absorbent pad onto which the test sample is applied. One of
the major advantages of the lateral flow concept is that these assays can be run in a single step
with many different sample types in a variety of application areas. The role of the sample pad is
to accept the sample, treat it such that it is compatible with the assay, and release the analyte
with high efficiency.The materials used for the sample pad depend on the requirements of the
application. Examples of such materials are cellulose, glass fiber, rayon, and other filtration
media.
d. Wick or waste reservoir: The wick is the engine of the strip. It is designed to pull all of the
fluid added to the strip into this region and to hold it for the duration of the assay. It should not
release this fluid back into the assay or false positives can occur. The material is typically a high-
density cellulose.
e. Backing Materials: All components of the lateral flow assay are laminated to the backing
material to provide rigidity and easy handling of the strip. The backing material is coated with a
pressure-sensitive adhesive to hold the various components in place. The backing materials are
typically polystyrene or other plastic materials coated with a medium to high tack adhesive.
f. Labels for Detection: The most commonly used particulate detector reagents in lateral flow
systems are colloidal gold and monodisperse latex. Latex particles coupled with a variety of
detector reagents, such as colored dyes, fluorescent dyes, and magnetic or paramagnetic
components, are available commercially.
Applications of Lateral flow assays in Food Quality Assurance
In the past 3–5 years, food safety issues and concerns for public health have led to more
stringent legislation in food safety requirements. Legislation has produced increased demand
for pathogen and toxin tests in just about every segment of the food production industry –
processed food, meats, poultry, beverages, and dairy; and by all major food producers
worldwide. For monitoring residue contaminants such as veterinary, pesticide and antibiotic
residues, an analytical strategy has been recommended using two different methods. This
strategy comprises: (i) screening with a first method optimized to prevent false negative results,
with a high sample throughput (e.g. ELISA), an acceptable percentage of false positive results
and low cost, and (ii) confirmation with an independent second method optimized to prevent
false positive results. Confirmatory methods are generally separative techniques coupled with
various detectors such as HPLC and GC–MS. Chromatography methods are sensitive and
specific, but suffer from being time consuming, laborious and multi-complex. In addition, these
166
technologies are unaffordable to the farmers and some laboratories in the developing
countries. Therefore there have been emergent needs for developing highly accurate, rapid and
cheap analytical tools. Lateral flow tests provide advantages in simplicity and rapidity when
compared to the conventional detection methods. LFT has also been confirmed to be a rapid
and sensitive method in the detection of food borne pathogens such as Salmonella, Listeria,
Campylobacter, Clostridium and Escheriachia coli. Apart form these pathogens, LFA also has
been employed for the detection of bacterial toxins and zoonotic viruses such as Avian
Influenza (AIV). LFA have also been used for the detection of potentially allergenic peanut and
hazelnut in raw cookie dough and chocolate. The Table summarizes the published reports on
LFT applications in this field. A driver in the demand for rapid and LF tests in food production is
the adoption of Hazard Analysis and Critical Control Points (HAACP) regulations that prescribe
test procedures throughout the manufacturing process. A number of manufacturers have come
with LF type tests. No one company dominates the market for LF food tests. The leaders are
Strategic Diagnostics, Inc. (Newark, DE), Neogen, Idexx Labs and Biocontrol Systems
(Brownsville, CA). Other companies include Celsis International PLC (Chicago, IL), Medical Wire
& Equipment Co. (Wiltshire, United Kingdom), Merck KgaA (Dermstadt, Germany), and M-Tech
Diagnostics Ltd. (Cheshire, United Kingdom).
Conclusions:
A variety of analytical methods available for detecting pathogen organisms or hazardous
chemicals related to food safety, human health and environment suffers from being time-
consuming, too expensive and too complicated to use. Major advantages found on LFT are low-
cost, speed, portable; do not require complicated equipment and technical expertise, which are
critical components during testing in the field. Since its initial development in the 1980s, the
technology of Lateral Flow Immunoassay has gained wide acceptance. The main reason for its
popularity is the simplicity of the test design. The lateral flow immunoassay devices are
compact and easily portable. Most of them do not require external reagent for results. Results
are quick and easy to interpret, usually without the help of an instrument. The technology is
also powerful. Multiple analytes can be tested simultaneously with a single device. It can also
be easily combined with other technology to provide a comprehensive analysis like
simultaneous drug and alcohol determinations by the police force in a roadside testing
situation. Manufacturing of the test is relatively easy and inexpensive. Advancement in the
detection moieties, improvement in material components, availability of better processing
equipment, and increased attention to quality manufacturing all these factors contribute to
increase in the reliability, accuracy, and applications of the technology. However, the
continuing demand for quantitative result and sensitivity has presented great challenge for
assay developer.
167
Table :- Applications of Lateral Flow Assays in Food Analysis
Analyte Assay format Labels Sample Sensitivities Referenc
e
Detection of pathogen bacteria and related toxins
Staphylococcus
aureus
Sandwich Colloidal gold Pork, Beef, Fried
Chicken
<25 CFU/g
(93.0–100%)
[4]
Escherichia coli Sandwich Colloidal gold Milk Powder, Flour,
Starch, Etc
105CFU/ml [18]
Listeria
monocytogenes
Sandwich Carbon black Dairy Products 10 CFU/25 mL [1]
Salmonella
enteritidis
Sandwich Colloidal gold Raw Eggs 107CFU/ mL [15]
Staphylococcus
aureus
enterotoxin B
Sandwich Fluorescent
immunoliposome
s
Water, Apple Juice,
Ham , Milk, Cheese
0.02–0.6 pg/
ml
[5]
Detection of veterinary drug residues mycotoxins and pesticides
Veterinary Drug Residue
Progesterone Competitive Colloidal gold Bovine Milk 0.6–1.2 μg/L [3]
Mycotoxins
Deoxynivanelol and
Zearalenone
Competitive Colloidal gold Wheat 100–1500
μg/kg
[6]
Deoxynivanelol Competitive Colloidal gold Wheat and Maize 50 ng./mL [22]
Aflatoxin B1 Competitive Colloidal gold Rice, Corn ,Wheat 0.05–2.5 ng/
ml
[23]
Aflatoxin B2 Competitive Magnetic
nanogold
microsphere
Peanut, Hazelnut,
Pistacia, Almond
0.9 ng/ ml [17]
Total B fumonisins
(B1, B2 and B3)
Competitive Colloidal gold Maize 4,000 μg/ kg [8]
Ochratoxin Competitive Colloidal gold Coffee 5 ng/ml [21]
Ochratoxin Competitive Colloidal gold Barley, Wheat, Oat,
Corn, Rice etc
1 ng/ ml [19]
Pesticides
Methamidophos Competitive Colloidal gold Green Vegetables 1.0 μg/ ml [2]
Thiabendazole and
Methiocarb
Competitive Carbon black Fruit Juices 0.005–0.5
mg/kg
[16]
Carbaryl Competitive Colloidal gold Rice And Barley 50–10 μg/L [20]
Detection of Allergens
Hazelnut Protein Competitive Unknown Chocolates 3.5 mg/kg [13]
168
Doughs 2.6 mg/kg
Allergenic Peanut
Protein Ara H1
Competitive Unknown Chocolates 0.8 mg/kg
Doughs 0.6 mg/kg
Detection of Adulteration
Rennet whey in
milk& milk powder
Sandwich Latex beads Milk and milk powder 15 ng/ml [7]
Thermal-stable
ruminant-specific
muscle protein,
troponin I
Competitive Coloured
particles
Beef
in
chicken
Raw 0.50
(%, w/w)
[12]
Cooked
Sterilized
Lamb-in-
pork
Raw 0.05
(%, w/w) Cooked
Sterilized
Beef-in-
turkey
Raw 0.10
(%, w/w) Cooked
Sterilized
References : 1. Blaskoza M, Koet M, Rauch P, Amerogen AV (2009) Eur Food Res Technol 229:867–874 2. Chenggang S, Suqing Z, Kun Z, Guobao H, Zhenyu Z (2008) J Environ Sci 20:1392–1397 3. Geertruida A, Posthuma T, Jakob K, Amerogen AV (2008) Anal Bioanal Chem 392:1215-1223 4. Huang S-H, Wei H-C, Lee Y-C (2007) Food Control 18:893–897 5. Khreich N, Lamourette P, Boutal H, Deveilliers K, Creminon C, Vollad H (2008) Anal Biochem 377:182–188 6. Kolosova AY, De Saeger S, Sibanda L, Verheijen R, Peteghem CV (2007) Anal Bioanal Chem 389:2103–2107 7. Martı´n-Herna´ndez, C.; Mun˜oz, M.; Daury, C.; Weymuth, H.; Kemmers-Voncken, A.E.M,; Corbato´n, T.;
Toribio, T. and Bremer, M.G.E.G. (2009). Int Dairy J. 19:205–208. 8. Molinelli A, Grossalber K, Krska R (2009) Anal Bioanal Chem 395:1309–1316 9. Ngom, B., Guo, Y. Wang, X and Bi, D. (2010) Anal. Bioanal. Chem. 397: 1113-1135. 10. O’Farrell, B. (2009) Evolution in lateral flow-based systems. In: Lateral flow immunoassay. (Ed. R.C. Wong
and H.Y. Tse). Springer, NY, USA. 11. Ponti, J.S. (2009) Material platform for the assembly of lateral flow immunoassay test strips. In: Lateral
flow immunoassay. (Ed. R.C. Wong and H.Y. Tse). Springer, NY, USA. 12. Q. Rao, Y.-H. Peggy Hsieh. (2007) Meat Science. 76: 489–494 13. Röder M , Vieths S, Holzhauser T (2009) Anal Bioanal Chem 395:103–109 14. Rosen, S. (2009) Market Trends in Lateral Flow Immunoassays. In: Lateral flow immunoassay. (Ed. R.C.
Wong and H.Y. Tse). Springer, NY, USA. 15. Seo K-H, Holt PS, Gast RK, Stone HD (2003) Int J Food Microbiol 87:139–144 16. Smidova Z, Blazkova M, Fukal L, Rauch P (2009) Czech J Food Sci 27:S414–S416 17. Tang D, Sauceda JC, Lin Z, Basova SOE, Goryacheva I, Biselli S, Lin J, Niessner R, Knopp D (2009) Biosens
Bioelectron 25:514 18. Wang J, Chen WN, Hu KX, Li W (2006) Chinese J 35:439–441 19. Wang XH, Liu T, Xu N, Zhang Y, Wang S (2007) Anal Bioanal Chem 389:903–911 20. Wang S, Zhang C, Wang J, Zhang Y (2005) Anal Chim Acta 546:161–166 21. Wang J, Yu F-Y (2008) Anal Chem 80:7029–7035 22. Xu Y, Huang Z-B, He QH, Deng SZ, Li LS, Li YP (2010) J Food Chem 119:834–839 23. Xiulan S, Xiaolian Z, Jian T, Xiaohong G, Jun Z, Chu FS (2006) Food Control 17:256–262
169
Microbiological Risk Assessment: A Global Management Approach to Dairy Food
Safety
Naresh Kumar and Raghu H. V
Dairy Microbiology Division, NDRI, Karnal
The significance of milk in human nutrition in now well established as it is considered as
the best, ideal and complete food for all age groups. Milk can also serve not only as a potential
vehicle for transmission of some pathogens but also allows these organisms to grow, multiply
and produce toxins. A variety of pathogenic organisms may gain access in milk and milk products
from different sources and cause different types of food born illnesses which includes food
infection, intoxication and toxio-infection (Aneja et al., 2002). Recent development regarding
Quality and safety management systems such as ISO and Hazard Analysis Critical Control Point
(HACCP) has reduced such incidences. The safety of milk and milk products has been extensively
reviewed by regulatory agencies in India and internationally. A large number of risk assessments
and risk profiles have been undertaken, examining the risks across the entire dairy supply chain
and conducting in-depth evaluations of specific pathogen-product combinations. This risk
assessment will summarise the major body of relevant work undertaken to date.
Setting public health goals – The concept of Appropriate Level of Protection (ALOP):
During the past decade, there has been increased interest and effort in developing tools
to more effectively link the requirements of food safety programs with their expected public
health impact. This document introduces two such tools, the "Food Safety Objective" (FSO) and
the “Performance Objective” (PO). These can be used to communicate food safety requirements
to industry, trade partners, consumers and other countries. Good practices and HACCP remain
essential food safety management systems to achieve FSOs or POs. Setting goals for public
health are the right and responsibility of governments. These goals may specify the maximum
number of harmful bacteria that may be present in a food. Where possible, the determination of
this number should be based on scientific and societal factors. The level of risk can be expressed
in a qualitative way (e.g., high, medium or low risk), or when possible, as the number of cases of
foodborne disease per number of people per year. The ICMSF ranking scheme categorizes
hazards by the severity of the threat they pose to human health, taking into consideration the:
likely duration of illness; likelihood of death; and potential for ongoing adverse health effects.
The severity of adverse health effects caused by a hazard is ranked as moderate, serious or
severe according to the following definitions:
170
Severity Description
Moderate Not usually life threatening; no sequelae; normally short duration; symptoms
are self-limiting; can be severe discomfort
Serious Incapacitating but not life threatening; sequelae infrequent; moderate
duration
Severe Life threatening, or substantial sequelae, or long duration
Under the ICMSF ranking, severe hazards are further divided into those applying to the general
population and those applying to specific sub-populations, that is, susceptible individuals (for
example, the very young and old, the immunocompromised, and pregnant women and their
unborn children). This takes into account those situations where a hazard considered to be of
moderate or serious to the general population may cause a severe illness in certain susceptible
sub-populations. The estimates of the risk level have to be based on clinical information
available (e.g., how many stool samples have been found to contain salmonellae) in combination
with results from microbiological surveys of foods, evaluations of the types of foods that are
produced, how they are produced and how they are stored, prepared and used. A few countries
may use scientific techniques such as Quantitative Microbiological Risk Assessment (QMRA) to
estimate the risk of illnesses using detailed knowledge of the relationship between the number
of microorganisms in foods and the occurrence of foodborne diseases. Whatever method is used
to estimate the risk of foodborne illness, the next step is to decide whether this risk can be
tolerated or needs to be reduced. The level of risk a society is willing to accept is referred to as
the "Appropriate Level Of Protection" (ALOP). Importing countries with more strict requirements
for a particular hazard (e.g., harmful bacteria) may be asked to determine a value for the ALOP
according to the SPS agreement. When a country is willing to accept the current risk of illnesses,
that level is the ALOP. However, most countries will wish to lower the incidence of foodborne
disease and may set targets for future ALOPs. For instance, the current level of listeriosis could
be 6 per million people per year and a country may wish to reduce this to 3 per million people
per year.
A Food Safety Objective (FSO): When a government expresses public health goals relative to the
incidence of disease, this does not provide food processors, producers, handlers, retailers or
trade partners with information about what they need to do to reach this lower level of illness.
To be meaningful, the targets for food safety set by governments need to be translated into
parameters that can be assessed by government’s agencies and used by food producers to
process foods. The concepts of food safety objectives (FSOs) and performence objectives (POs)
have been proposed to serve this purpose. The position of these concepts appearing in the food
chain can be seen in Figure 1. An FSO is “The maximum frequency and/or concentration of a
hazard in a food at the time of consumption that provides or contributes to the appropriate level
171
of protection (ALOP)” It transforms a public health goal to a concentration and/or frequency
(level) of a hazard in a food. The FSO sets a target for the food chain to reach, but does not
specify how the target is to be achieved. Hence, the FSO gives flexibility to the food chain to use
different operations and processing techniques that best suit their situation, as long as the
maximum hazard level specified at consumption is not exceeded.
FSO and Product/pathogen/Pathway Analysis:
The ICMSF has introduced a simple equation that summarises the fate of a hazard along the
food chain as follows:
Ho - SR + SI = FSO
Where:
FSO = Food Safety Objective
Ho =Initial level of the hazard
SR = the cumulative (total) decrease in level
SI = the cumulative (total) increase in level
≤ = preferably less than, but at least equal to FSO, Ho, R, and I are expressed in
log10 units
I (increase) is determined by growth (G) as well as by recontamination (RC). Since the FSO is the
level of a hazard at the moment of consumption, another term is needed to describe the level at
another point in the food chain. The term Performance Criterion has been proposed by the
ICMSF, but this term is also used to describe the outcome of a processing step (for example a 6
decimal reduction of a pathogen). For this reason the term Performance Standard is used in this
document to reflect the level of a hazard and Performance Criterion to describe the impact of a
process on the level of a hazard.
As a consequence of this, the following equation is proposed:
Where:
FSO = Food Safety Objective
172
PS = Performance Standard
Ho = Initial level of the hazard
SR = the cumulative (total) decrease of the hazard
SIRC = the cumulative (total) recontamination with the hazard
SIG = the cumulative (total) growth of the hazard
≤ = preferably less than, but at least equal to
Note that the PS of one point of the food chain may be the Ho of the following one.
This equation is helpful to determine the effect of control measures necessary to meet a FSO. It
is important to recognise that data used in PPP analyses that can be used to determine the
various values of Ho, R, IRC, IG and PS, may differ according to their source and use.
A Performance Objective (PO): For some food hazards, the FSO is likely to be very low,
sometimes referred to as "absent in a serving of food at the time of consumption". For a
processor that makes ingredients or foods that require cooking prior to consumption, this level
may be very difficult to use as a guideline in the factory. Therefore, it is often required to set a
level that must be met at earlier steps in the food chain. This level is called a performance
objective (PO). A PO may be obtained from an FSO, as will be explained below, but this is not
necessarily always the case. Foods that need to be cooked before consumption may contain
harmful bacteria that can contaminate other foods in a kitchen. Reducing the likelihood of cross-
contamination from these products could be important in achieving a public health goal. The
level of contamination that should not be exceeded in such a situation is a PO. For example, raw
chicken may be contaminated with Salmonella. Although thorough cooking will make the
chicken safe (absence of Salmonella in a serving), the raw chicken may contaminate other foods
during preparation of a meal. A PO of “no more than a specified percentage of raw chicken
carcasses may contain Salmonella” may reduce the likelihood that Salmonella will contaminate
other foods. In products, such as ready-to-eat foods, the POs can be calculated from the FSO by
subtracting expected bacterial contamination and/or growth between the two points.
The difference between an FSO, PO and Microbiological Criteria (MC): Microbiological criteria
need to be accompanied by information such as the food product, the sampling plan, methods
of examination and the microbiological limits to be met. Traditional MC are designed to be used
for testing a shipment or lot of food for acceptance or rejection, especially in situations where
no prior knowledge of the processing conditions is available. In contrast, the FSO or the PO are
maximum levels and do not specify the details needed for testing. However, MC can be based on
Pos in certain instances where testing of foods for a specific microorganism can be an effective
means for their verification. There are several approaches to sampling (e.g., lot testing, process
control testing) but they all compare the results obtained against a predetermined limit, i.e. a
number of microorganisms.
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Responsibility for compliance with the FSO: The marketing of food that is not harmful to
consumers when used in the intended way is the responsibility of the various food businesses
along the food production chain. This responsibility will not change with the introduction of the
FSO and PO concepts. In fact, the use of FSOs and POs will make food professionals involved in
the various parts of the food chain more aware of the fact that they share this responsibility.
Government or third parties can assess programs, such as the good practices and HACCP, to
confirm the likelihood that the products will meet the FSOs. This can and will be extended across
national boundaries, as some countries will ask that imported products are produced under food
safety management programmes based on GHP and HACCP.
Meeting the FSO: Since the FSO is the maximum level of a hazard at the point of consumption,
this level will frequently be very low. Because of this, measuring this level is impossible in most
cases. Compliance with POs set at earlier steps in the food chain can sometimes be checked by
microbiological testing. However, in most cases, validation of control measures, verification of
the results of monitoring critical control points, as well as auditing good practices and HACCP
systems, will provide the reliable evidence that POs and thus the FSO will be met.
Microbiological criteria can be derived from FSOs and POs, if such levels are available. If such
levels are not stated, microbiological criteria can be develop, if appropriate. The ICMSF (2002)
has provided guidance on the establishment of microbiological criteria.
Figure 1. Model food chain indicating the position of a food safety objective and derived
performance objectives
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Figure 2. FSOs and POs are means of communicating public health goals to be met by food
processors by good practices and HACCP. Also, industry can set POs to ensure that FSOs are met.
Microbiological Risk Profile: Microbiological risk assessment is an essential element of risk
analysis, as it describes food-borne risks related to the occurrence of pathogenic microorganisms
in the whole food chain. The novelty of this concept is that risks are assessed throughout the
food chain on the basis of sound science, combining qualitative and quantitative data in the
areas of epidemiology and pathogenicity of microorganisms with data from monitoring of food
and food animals, food production and handling. According to the Codex definition, risk
assessment should be commissioned, scoped and targeted by risk managers through a risk
assessment policy. It is, however, necessary to remember that different types of problem
warrant different types of assessment. In principle, some risk assessments can be done over a
very limited time span, whereas others (typically) need a full MRA, including all four components
and a significant input of specialized man-hours.
The 4 phases or steps of microbiological risk assessment can be outlined as follows:
1. Hazard Identification: The qualitative indication that a substance may cause adverse health
effects. The identification of agents capable of causing adverse health effects which may be
present in a particular food or group of foods. The purpose of hazard identification in MRA is
to identify the microorganism(s) or microbial toxin of concern and to collect evidence that it
is indeed a potential hazard when present in the particular food.
2. Hazard characterization (dose-response assessment): the qualitative and quantitative
evaluation of the nature of the adverse health effects; the relationship between the
magnitude of the exposure and the probability of occurrence of adverse health effects.
The description of the relationship between different doses and their relative effect (the
dose-response relationship), the impact of the composition of food, the virulence of strains
and the sensitivity of (sub-populations of) consumers at risk.
3. Exposure characterization: the qualitative and quantitative evaluation of the degree of
exposure likely to occur. The determination of the numbers/quantities of pathogens or
toxins ingested by the consumer and the prevalence of such ingestion(s). This is the part of
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MRA where food companies can make important contributions through the provision of
data. Most (theoretical) data should be developed taking into account not only processing
during manufacture but rather the whole distribution chain from primary production of raw
materials to the preparation and use by the consumer. This aspect of MRA involves
determination of the probability and extent of exposure to a population, possibly including
consideration of sub-populations exposed to varying quantities of the microorganism.
4. Risk characterization: Integration of the above steps into an estimation of the adverse
effects likely to occur in a population, to be used in decision-making (risk management).
The quantitative and/or qualitative estimation (with attendant uncertainties) of the
probability of occurrence and severity of known or potential adverse health effects in a
given population. It may consist of different estimates, based on different scenarios or
assumptions, which may help the risk managers to evaluate the effectiveness of various
control options. Risk characterization is the last step in risk assessment, on the basis of
which a risk management strategy can be formulated. Bringing together the information of
the previous stages, it provides an estimate of risk to a given population or sub-population.
The WTO/SPS agreement (WHO, 1997) describes the rules for the international trade in safe
food and has introduced the term "appropriate level of protection" (ALOP) to express what is
mentioned in the first bullet point above. This ALOP has also been called "acceptable level of
risk". This term is similar to the expression "tolerable level of risk" (TLR) preferred by the ICMSF,
because it recognises that risks related to the consumption of food are seldom accepted, but at
best tolerated. Also implied is that for a number of food safety hazards, “zero risk” does not
exists and/or too costly (financial, societal) to achieve. One of the tasks of governmental Risk
Managers is thus to decide upon what is adequate, appropriate or tolerable in terms of food
safety or health risk. How they have to do this is not described in detail by the WTO or the
Codex. However, the determination of ALOP/TLR should be science based, should include
economic and societal factors and should minimise negative trade effects. Integral to the
agreement is that imported food should not compromise the ALOP. An exporting country can
contest an importing country's judgement that a food is not meeting the ALOP, by using
scientific methods such as risk assessment. Codex standards, codes and guidelines are
mentioned as reference documents. A country cannot demand that imported foods are "safer"
than similar domestically produced foods. Figure 3 Illustrates how Microbiological Risk
Assessment (MRA) could be used in acceptance procedures of internationally traded food
products. Under the heading of transparency it is mentioned that member states shall ensure
that "reasonable questions can be answered concerning SPS measures and that relevant
documents can be provided such as: risk assessment procedures, factors taken into
consideration, as well as the determination of the ALOP". Changes in regulations should be
notified. Although it is not specified how an ALOP should be expressed, it is commonly seen as
the number of illnesses per annum that should not be exceeded.
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Uncertainty and variability in exposure assessment: One has to deal with uncertainty and
variability when conducting an exposure assessment. Uncertainty is the (quantitative)
expression of our lack of knowledge. Variability is the heterogeneity of the subjects modelled
and includes both stochastic variability (randomness) and inter-individual variability. Uncertainty
can be reduced by additional measurement or information, while variability can not (Vose,
2000).
Variability: Variability is always present in biological systems. It is important to realize variability
occurs at many levels. Thus, there may be variability in genotype, strain type, time, place,
experimental conditions, etc. It is crucial to define the denominator of the variability (like
variability per year and variability per flock), and variability from different sources should not be
mixed without careful consideration.
Uncertainty: There are many types of uncertainty in exposure assessment, including process
uncertainty, model uncertainty, parameter uncertainty, statistical uncertainty, and even
uncertainty in Variability.
1. Process uncertainty refers to the uncertainty about the relationship between the food chain
as documented in the exposure assessment and the processes that take place in reality. For
example, rare, undocumented events in food production or consumer behavior may have a
relevant impact on the exposure without being fully considered in the model.
2. Model uncertainty comprises both the correctness of the way the complexity of the food
chain is simplified, and the correctness of all the sub models that are used in the exposure
assessment. To enable the construction of the food chain model, process simplification may
be inevitable, but the level to which this is appropriate is subjective, and should be reviewed
by experts. Sub models used to describe processes, such as growth during storage at a
particular stage, are the choice of the assessor and may be based on the availability of both
data and models. As different models may yield different predictions, there will be
uncertainty about the appropriateness of a given model.
3. Parameter uncertainty incorporates uncertainties dealing with errors resulting from the
methods
Risk Characterization in Dairy Products: In preparing the Dairy Risk Profile, previous risk
assessments conducted by other scientific agencies were reviewed and evaluated in this
document. There have been few assessments undertaken for dairy products, and typically they
address specific pathogen: commodity pairs. This profile considers the entire dairy supply chain,
including the wide range of milk and milk products. Dairy products likely to support the growth
of pathogens and prone to contamination after pasteurization may be categorised as higher risk
than other dairy products. Alternatively, dairy products that do not support the growth of
pathogens, if correctly formulated, can be classified as low risk.
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Figure below illustrates how Microbiological Risk Assessment (MRA) could be used in acceptance
procedures of internationally traded food products
The initial RA estimated the likelihood of exceeding microbiological limit value set by law in the
final products taking into account the influence of the manufacturing process on the hazards
considered (Salmonella spp., Listeria monocytogenes, Aflatoxin M1, Bacillus cereus spores and
toxin, and Staphylococcus aureus toxin). The risk-based monitoring targeted each year different
combinations of hazards and products according to the RA and the results of previous years.
The actual ranking of the dairy products is quite variable. Once a shelf-stable UHT product is
opened, it may become contaminated and when subjected to temperature abuse it could
become a high-risk food. In contrast, the low pH and low water activity of extra hard cheese
means its will be very robust and unlikely to support the growth of any pathogen that
adventitiously contaminates the surface. Dried milk powders and infant formulae are inherently
stable products due to their low water activity, however these products may be prone to
contamination, and upon reconstitution become higher risk, especially if improperly
reconstituted and stored. Following criteria in food matrix may be considered while
characterizing the risk:
Intrinsic properties of the product (i. e. the impact of aw, pH, salt concentration, and their
effect on the growth of contaminating microorganism)
Extent to which food is exposed to factory environment or handling after heat treatment
Hygiene and control during distribution and retail sale
Degree of reheating or cooking before consumption (many dairy products are RTE, so this is
rarely a factor).
Attribution of Food-borne Illness to Dairy Products: While there is enhanced quantitative data
on the incidence of illness due to specific pathogens, there is often not the ability or capacity to
identify or distinguish specific food vehicles. The causative agent of an illness is usually
determined through epidemiological studies, but confirming the identity of a key ingredient or
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the original source of product contamination, or critical factors contributing to their occurrence
is problematic. This inability to attribute cases of food-borne illness to causal vehicles is a major
issue internationally, and is especially difficult where illness is linked to foods with multiple
ingredients. Critical in this process is the capacity to link epidemiological data to animal and
food monitoring data. The development of public health interventions requires accurate data
defining the source from which humans are acquiring pathogens and how specific foods
contribute to the total burden of food-borne illness. However, outbreak data represents only a
small component of actual cases of food-borne illness, as many outbreaks go unrecognized.
People do not always seek medical attention for mild forms of gastroenteritis, and not all food-
borne illnesses require notification to health authorities.
Risk Management Issues and Control Strategies for Dairy foods: The critical factors having the
most significant impact on the safety of processed dairy products are as follows:
The quality of raw materials
Correct formulation
Effective processing
The prevention of recontamination of product
Maintenance of temperature control through the dairy supply chain.
While pathogenic microorganisms may contaminate raw milk supplies, pasteurization is a very
effective Critical Control Point (CCP) in eliminating pathogens; good manufacturing practices
must also be employed to ensure that post-pasteurization contamination does not occur. The
effectiveness of pasteurization is dependent upon the microbiological status of the incoming raw
milk. Control measures at the primary production level involve minimizing the likelihood of
microbiological hazards contaminating the raw milk. This is achieved through the
implementation of a food safety program incorporating good agricultural practices (GAP). These
measures are effective in reducing the microbial load of milk being sent for processing.
However, should microbial contamination of raw milk occur, it is critical that milk is
stored at a temperature that minimizes the opportunity for the bacteria to multiply.
Temperature abuse of the milk may allow growth of pathogenic bacteria to the extent where the
pasteurization process may not eliminate all pathogenic bacteria and/or toxins. The Aflatoxins
can be formed and ingested by dairy cattle during feeding, eventually contaminating the milk.
Aflatoxin contamination of milk is more common where intensive supplementary feeding of
dairy herds is conducted.
Correct formulation: Ingredients used in the manufacture of dairy products that are added post
pasteurization must be of a high microbiological standard. Many non-dairy ingredients added to
ice-cream mix after heat treatment include fruits (canned, fresh, or frozen and usually in
concentrated sugar syrups), nuts, chocolate, pieces of toffee and biscuit, colors and flavors.
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These ingredients and those added to other dairy products such as yoghurt, dairy desserts, dairy
dips and cheese may introduce pathogens into the product (ICMSF, 1998).
The addition of ingredients added after pasteurization was identified as a high risk factor
by Jansson et al (1999) who recommended that dairy products with these additions (eg ice-
cream and cheeses) be moved into the high risk category and the finished product be subject to
additional end product microbiological analysis. The microbial quality of dry-blended ingredients
into infant formula was identified as a significant source of contamination, as there is no heat
treatment to destroy bacteria in the final product.
Effective processing (pasteurization): Dairy processing facilities primarily use High Temperature
Short Time (HTST) pasteurization (minimum 72°C for 15 seconds) or batch pasteurization
(minimum 65°C for 30 minutes) to eliminate the pathogens of concern in milk. However, most
factories actually heat the milk to higher temperatures and hold it for a longer time period as an
in-built safety margin. In most cases, milk and dairy products are consumed as RTE foods and will
readily support the growth of any contaminating microorganism. In the past, the dairy industry
has been subjected to a high level of food safety regulation; ensuring high levels of hygiene and
sanitation are maintained. The pasteurization process eliminates all pathogenic bacteria found in
raw milk, with the exception of the spore forming bacteria B. cereus and C. perfringens.
The prevention of recontamination of product: Post-pasteurization contamination can pose a
major problem where good manufacturing practices are not employed (Zottola & Smith, 1991).
Pathogenic microorganisms can be introduced into a dairy processing environment with raw
milk. Once these organisms gain access to the processing plant, the presence of nutrients and
moisture can allow not only for survival, but multiplication of these organisms. The application
of food safety programs including elements of Good manufacturing practice (GMP) and Good
hygienic practice (GHP) are critical to limit the potential for pathogens to contaminate dairy
products after pasteurization. The primary organisms of concern are Listeria monocytogenes for
most dairy products and Salmonella in dried milk products.
Maintenance of temperature control through the dairy supply chain: The intrinsic nature of
many dairy products means they will support the growth of pathogenic bacteria that may
contaminate the product. This categorizes these products as ‘potentially hazardous foods’. The
exception to this are products such as yoghurt and hard cheeses (low pH) and ice- cream (stored
frozen). As potentially hazardous foods, maintenance of temperature control through the dairy
supply chain is critical to ensure these foods remain safe and suitable.
Concluding Remarks: FSOs and POs are new concepts that have been introduced to further
assist government and industry in communicating and complying with public health goals. These
tools are additional to the existing programmes of GAPs, GHPs and HACCP which are the means
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by which the levels of POs and FSOs will be met. Hence FSOs and POs build on, rather than
replace, existing food safety practices and concepts.
References:
Aneja, R. P., B. N. Mathur, R. C. Chandan, A. K. Banerjee, 2002. Technology of Indian Milk Products. A Dairy India Publication, Delhi.
Codex Committee on Food Hygiene (CCFH, CX/FH 04/5/6), 2004. Proposed draft process by which the committee on food hygiene could undertake its work in microbiological risk assessment/risk management, Alinorm 04/27/13.
Havelaar, A. H., Nauta, M. J., Jansen, J. T., 2004. Fine-tuning food safety objectives and risk assessment. International Journal of Food Microbiology 93, 11–29
ICMSF, 1998, Principles for the establishment of microbiological food safety objectives and related control measures. Food Control 9, 379-384.
ICMSF, 2002. Microorganisms in Foods 7. Microbiological testing in food safety management. Kluwer Academic / Plenum Publishers, New York, USA.
Jansson, E., Moir, C., Richardson, K., 1999. Final Report Review of Food Safety Systems developed by the NSW Dairy Corporation. Food Science Australia Report.
WHO, 1997. Food Safety and Globalization of Trade in Food, a challenge to the public health sector. WHO/FSF/FOS/97.8 Rev. 1, WHO, Geneva.
Vose, D. 2000. Risk Analysis: A quantitative guide. 2nd ed. John Wiley & Sons, UK.
Zottola, E.A., Smith, L.B., 1991. Pathogens in cheese. Food Microbiology 8, 171-182.
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Safety Aspects of Food Additives
Sathish Kumar M.H. and Ameeta Salaria
Dairy Technology Division, NDRI, Karnal
Food Additives
Food additives have been used for centuries. Food preservation began when man first
learned to safeguard food from one harvest to the next and by the salting and smoking of meat
and fish. The Egyptians used colours and flavourings, and the Romans used saltpetre (potassium
nitrate), spices and colours for preservation and to improve the appearance of foods. Over the
last 50 years, developments in food science and technology have led to the discovery of many
new substances that can fulfil numerous functions in foods. These food additives are now
readily available and include emulsifiers in margarine, sweeteners in low calorie products and
wider range of preservatives and antioxidants which not only slow down product spoilage rate
and rancidity but also maintain taste. Food additives afford us the convenience and enjoyment
of a wide variety of appetizing, nutritious, fresh, and palatable foods. Their quantities in food
are small, yet their impact is great. Without additives, it is practically impossible to relish many
number of food items available in the modern day world. Some food additives are derived from
natural sources while others are made synthetically.
A food additive can be defined as ‘any substance that becomes part of a food product
either directly or indirectly during some phase of processing, storage or packaging’. According
to FDA, food additives are substances added to foods for specific physical or technical effects.
They may not be used to disguise poor quality but may aid in preservation and processing or
improve the quality factors of appearance, flavour, nutritional value and texture.
Need for Additives
The primary aim of the food-manufacturing industry is to provide a wide range of safe,
wholesome, nutritious and attractive products at affordable prices all year round in order to
meet consumer requirements for quality, convenience and novelty. It would be impossible to
do this without the use of food additives. They are essential in the battery of tools used by the
food manufacturer to convert agricultural raw materials into products that are safe, stable, of
consistent quality and readily prepared and consumed.
Food Additives – Ingredients with a Purpose
Modern food processing technologies include the use of a variety of food additives proven
effective and safe through long use and rigorous testing. Additives carry out a variety of useful
functions which we often take for granted. Foods are subjected to many environmental
conditions, such as temperature changes, oxidation and exposure to microbes, which can
change their original composition. Food additives play a key role in maintaining consistent
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quality of food, keeping food safe, wholesome, appealing and characteristics that consumer
demand.
Different types of additive are used for different purposes, though many individual
additives perform more than one function. Food additives are very carefully regulated and the
general criteria for their use is that they perform a useful purpose, are safe to the consumer.
For the purposes of both classification and regulation, they are grouped according to their
primary function. The main groupings, or classes, of additives are explained below, together
with their functions and some examples of their use.
Classes of Food Additives
Preservatives
Colours
Flavours and flavouring agents
Emulsifying, Stabilizing, Anticaking and Antifoaming agents
Antioxidants
Sequestering and Buffering agents/ Acidulants
High intensity / low calorie sweeteners
Vitamins and minerals
Nutraceuticals
Probiotics/Prebiotics
Functional additives
Some specific examples of food additives and their functions include
Anti-caking agents that keep powders running freely (for example, magnesium
carbonate in icing sugar)
Colours (natural and synthetic) that give food an appetizing appearance (for example,
carotene in butter and cheese)
Enzymes that are involved in desired chemical reactions in foods (for example, rennet in
cheese making)
Preservatives that inhibit the growth of moulds, yeast, or bacteria (for example, sodium
benzoate in carbonated soft drinks)
Texture-modifying agents that provide a desired consistency in foods (for example,
diglycerides in ice cream)
Safety Concerns
The potential hazards to health presented by the use of food additives
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This is not to say that the chemicals used as food additives are never hazardous. Even sodium
chloride, perhaps the most common of food additives, has caused death when misused. The
possible adverse effects on health of each use of an additive will, in general, have been
evaluated before the use is permitted and judged to be insignificant. Potential direct
toxicological effects of food additives are of greater concern with respect to food safety. Few
additives are used at levels that will cause a direct toxicological impact. Of particular concern
are the hypersensitivity reactions to some additives that can have a direct and severe impact on
sensitive individuals even when the chemicals are used at legally acceptable levels. The
reactions to sulfites and other additives are examples of such a problem.
Toxicological problems resulting from the long-term consumption of additives are not
well documented. Cancer and reproductive problems are of primary concern, although there is
no direct evidence linking additive consumption with their occurrence in humans. There are,
however, animal studies that have indicated potential problems with some additives. Although
most of these additives have been banned, some continue to be used, the most notable being
saccharin.
Food additives in general can lead to:
Genotoxicity (cause changes in the DNA of cells)
Carcinogenicity
Changes in behaviour e.g. hyperactivity in children
Can cause allergies
Temporarily inhibit digestive enzyme function.
May cause bronchial problems
Lower oxygen carrying capacity of blood
May combine with other substances to form nitrosamines that are carcinogens
Growth retardation and severe weight loss in animals
Food Colours
Food colourings, in particular, have been blamed long time for behaviour problems in
children. It has been over 30 years since Feingold suggested that artificial food colours and
preservatives had a detrimental effect on the behaviour of children. In 2007, a study on the
effect of two mixtures of certain artificial food colours together with the preservative sodium
benzoate showed an adverse effect on the hyperactive behaviour of children. Also, a very small
number of individuals, one or two of every ten thousand, are sensitive to FD&C Yellow #
5(tartrazine), used as a food coloring, causing itching and hives.
In addition, the use of many colours has been discontinued during the last decade, on the basis
of results of animal studies. One of the first colours to be discontinued was butter yellow, which
was used in margarine until 1940 when it was shown to induce hepatomas in the rat.
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Artificial Sweeteners
Cyclamates were classified as GRAS in 1958, marketed in the form of tabletop
sweetener for diabetics; also have synergistic sweetening properties and improved taste with
saccharin. The banning of cyclamates raised significant questions about the validity of testing
methods and the interpretation of results. Cyclamates, it was claimed, when fed to rats in the
ratio 10:1 cyclamate: saccharin caused an increase in bladder tumours in rats. Thus in 1970
cyclamate was removed from GRAS status.
A toxicological study on saccharin reveals its potential carcinogenic properties. At a dose
of 5% or greater an increase frequency of urinary bladder cancer was found in male rats. The
carcinogenic potential of aspartame was also revealed by Soffritti and co-workers in 2007. Their
research, using Sprague Dawley fetal rats, has demonstrated a significant increase of malignant
tumours in males, an increase in the incidence of lymphomas and leukemias in males and
females, and an increase in the incidence of mammary cancer in females.
People with a rare genetic disease known as phenylketouria (PKU) should avoid foods
sweetened with aspartame (Equal). Aspartame is made from two amino acids, one being
phenylalanine. Individuals with PKU cannot metabolize this amino acid, and if consumed can
cause serious side effects including tissue damage.
Food Preservatives
Sub acute toxicity studies of benzoic acid in mice indicates that ingestion of benzoic acid
or its sodium salt caused weight loss, diarrhoea, irritation of internal membranes, internal
bleeding, enlargement of liver and kidney, hypersensitivity and paralysis followed by death. In
rare occasions some individuals can experience adverse reactions to sulphites. A small
percentage of asthmatics can react to sulphites, substances used to prevent certain foods from
browning.
Preformed exogenous nitrosamines are found mainly in cured meat products, smoked
preserved foods, foods subjected to drying by additives such as malt in the production of beer
and whiskey, pickled and salty preserved foods. On the other hand, nitrosamines are formed
endogenously from nitrate and nitrite. Two important nitrosamines, namely N-
nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA), are classified as probably
carcinogenic to humans by International Agency for Research on Cancer (IARC).The
carcinogenic effects of nitrosamines have been very well documented in recent years. Earlier
nitrates were also used as preservatives in cheese found to be potential source for formation of
nitrosamines.
Food Flavours and Flavour Enhancer
Lung disorders were observed in workers handling diacetyl flavour in microwave
processed popcorn manufacturing units and flavour manufacturing companies. European Food
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Safety Authority (EFSA) recently declared one of the ingredient used to prepare smoke flavour
is toxic to humans.
Monosodium glutamate (MSG), commonly found in Chinese foods, can also cause
adverse reactions in small groups of people. The symptoms, usually mild, include body tingling
or warmth, and chest pain. However, these symptoms are usually mild and often last less than
an hour, but in sensitive individuals these may prove fatal. In a recent study on MSG reveals its
deleterious effects on the fallopian tubes of adult female Wistar rats at 0.08mg/kg dose and
continuous exposure further may causes of female infertility.
Adverse reactions to food additives are caused by several mechanisms. Food additives
are ingested irregularly and in small doses. Additives are usually low molecular weight
chemicals, unlike many high molecular weight proteins which are potent allergens. There is
very little evidence of an immunological basis in reactions caused by food additives. Adverse
effects due to various pharmacological or other mechanisms are much more common. The best
advice to any individual that has adverse reactions to any food additives is to read labels
carefully and avoid these products whenever possible. If an adverse reaction does occur, be
sure to contact your physician immediately
Finally, studies of populations exposed to food additive chemicals during manufacture
and use (or otherwise) might reveal long-term hazards not demonstrable in the laboratory, and
such studies should be, and frequently are, part of the continuing evaluation of safety of these
chemicals.Generally, additives have to be tested extensively before they can be permitted to be
used in food of human consumption. They are tested to see how they react within the body and
whether the additive has any toxic effects. This also includes tests to see if the additive poses
any genetic risk and whether it can be seen to cause cancer.
Food Safety
Food safety in India is ensured by Government of India’s Ministry of Health under the
provisions of Prevention of Food Adulteration Act & Rules. They are responsible for Food Laws
and the rules there in. State government, Food & Drug Administration (FDA), which carries out
surveillance using food inspectors, does the enforcement. There are food analysis labs, both
state and central, which verify the authenticity of food products.
Any food safety legislation or standard requires involvement of several aspects including
Research & Development, Information & Documentation, Education & Training, Quality
Assurance Program, Codex & International Norms, Advisory System, Planning, Enforcement and
Surveillance. Various activities take place at different places such as education & research
institutions, government laboratories, data bases including international & national, industry
production and quality evaluation centres, and finally state level enforcement and surveillance
departments. Due to the complex nature, any change is standards and enforcement has to be
properly planned and executed after careful consideration of all these factors.
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Food Additives: Approval Process
Any new additive before approving must undergo rigorous toxicity studies, including
acute and chronic studies involving biochemical evaluation, teratogenic studies, and
reproductive studies besides the LD50 tests. Exposure assessment is very important in
determining the risk involving any additive under the modern practice of determining safety.
The Risk Analysis, adopted nowadays involves, risk assessment, wherein the Hazard is identified
& characterized, Exposure is assessed and thus risk is characterized. Once the Risk is assessed, it
must then be managed so hazardous conditions do not arise. Finally the risk must then be
communicated.
Food safety policy has a long history of using risk analysis to guide public decisions. A
study by U.S. Food and Drug Administration (FDA) toxicologists in the mid-1950s introduced
safety factors to establish acceptable daily intake of food additives on the basis of acute
toxicity, an approach still applied today (Lehman and Fitzhugh 1954).Much of Codex’s effort has
gone into producing model standards. These include commodity standards aimed at preventing
consumer fraud, quantitative standards for food additives, and quantitative tolerances for
contaminants such as pesticides and veterinary drugs.
From the start of the process to the end it can take up to 10 years for an additive to be passed
as safe for use in food. This consists of 5 years for the actual safety testing, two year of
assessment by the European Food Safety Authority and at least another three years for EU
approval.
JECFA: The Joint FAO/WHO Expert Committee on Food Additives (JECFA) is an international
scientific expert committee that is administered jointly by the Food and Agriculture
Organization of the United Nations FAO and the World Health Organization WHO. It has been
meeting since 1956, initially to evaluate the safety of food additives. Its work now also includes
the evaluation of contaminants, naturally occurring toxicants and residues of veterinary drugs
in food.
To date, JECFA has evaluated more than 1500 food additives, approximately 40
contaminants and naturally occurring toxicants, and residues of approximately 90 veterinary
drugs. The Committee has also developed principles for the safety assessment of chemicals in
food that are consistent with current thinking on risk assessment and take account of recent
developments in toxicology and other relevant sciences.
JECFA and JMPR (Joint FAO/WHO Meeting on Pesticide Residues) and role in Risk Assessment
policy:
The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has repeatedly noted
the importance of reviewing substances previously evaluated when new data on those
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substances become available and in light of further developments in science and risk
assessment methodologies. The fortieth session of CCFA in 2008 requested an evaluation by
JECFA of the impact on dietary exposures to cyclamates of different maximum levels of use of
cyclamates in the Codex GSFA Food Category 14.1.4 Use of same HPLC method for
quantification of residual octenyl succinic acid utilizing as a standard octenyl succinic acid
anhydride, which is commercially available has also been recommended. The Committee
recommended that the specifications and toxicity of hexanes should be reconsidered at a
future meeting in view of new data on the toxicity of n-hexane. The Committee decided to
update the General Specifications and Considerations for Enzymes Used in Food Processing.
In these policies, animal models are relied upon with certain assumptions to establish
potential human effects. A 100-fold safety factor is used for many assumptions and variations
between species. The policy does not assign any ADI (average daily intake) to additives, drugs
and pesticides that are found to be genotoxic carcinogens. This permits some of these
contaminants to be at levels “as low as reasonably achievable” (ALARA).
Food Additives and Regulations
There are different sets of regulations everywhere. Each country has its own set of rules
for regulating food additives for example; US FDA Guidelines & Regulations gives the American
regulations for food additives. Thus anyone producing and marketing food products in the US
must abide by them. India has its own set of regulations under Prevention of Food Adulteration
(PFA) Act & Rules. Each country has a set of regulations. When an Indian company wants to
export to US, then it will have to follow the US regulations. When it wants to export to Australia
their rules have to be followed. So there might be difficulties trying to follow many sets of
regulations.
A group of countries may have a common regulation for example, European Union Directives,
which give regulations for countries affiliated to it. This allows free exchange of food products
across those EU countries. It avoids confusion because of many different regulations being
followed for different countries. For international trade we have Codex, SPS, and TBT
regulations. Under the WTO agreements, common regulations have been arrived at for those
countries signatories to the agreement and this allows the international trade without much
problems. FAO/WHO has come up with Codex rules, which are accepted by these countries.
Purpose of Law
The Food Laws or Regulations are made in order to protect people consuming these
foods from undue risks, which may arise from processing, transport, retailing and consumption
of food products which may undergo contamination, spoilage, inclusion of harmful
chemicals/microbes or at harmful levels. Besides safety, consumers are also protected by these
laws with respect to quality, quantity and substance that the food products are supposed to
188
represent. Finally, laws also aim to protect the consumers from Nutrition and Health
considerations with special consideration being given to vulnerable group such as infants,
pregnant women etc.
Regulatory Environment: India
The Indian law, Prevention of Food Adulteration Act, 1954 & Rules, 1955, address consumer
and safety concerns. There is legislation, which created authority and infrastructure to govern
and enforce the law and also provisions for Rules and standards for various products in which
even the type and amounts of various additives allowed are given. This is the major law, but
there are many others like Fruit Products Order (FPO), MFPO (Meat Food Products Order), Milk
& Milk Products Order (MMPO), Edible Oils Order etc. which deal with specific group of
products. There are weights and measures regulations, which not only include food products
but also the other non-food products.
All the above are the mandatory food laws. Besides there are some optional food laws
or standards referred to as quality standards, e.g. those of Bureau of Indian Standards (BIS) and
Agmark, which are used when products are said to be of those qualities. Thus India has a
number of laws, which may govern the same food products.
Limitations of current Food Laws
While the market scenario is not very friendly but highly competitive although there are
many opportunities, any lack of support due to limitation in food laws is bound to make a
negative impact on the industry. At present, there are many laws for same food products are at
times they overlap or contradict one another. Many of the laws and standards are quite rigid
and inflexible. There are rules, which lay down standards for hundreds of products with very
little scope for innovation. This denies consumers the choice, which then will be provided by
imported products and the market will be lost for Indian industry. There is also weak
enforcement and at times the implementation is ad-hoc and not uniform. This creates
uncertainty among the industry about compliance, which may result in unjust harassment and
threat of prosecution. This is not a healthy state for good growth of industry.
Need for Integrated Food Law
Integrated Food Law is imperative for just and focused enforcement as well as for
healthy growth of industry. Many countries have unified food laws including USA, Malaysia,
Thailand, Indonesia and Pakistan. There are examples of groups of countries that have come
together and formulated unified common food laws. Examples are Australia and New Zealand
as well as European Union countries. They not only have integrated laws but also laws
conducive for healthy industry producing safe and high quality products. In order to overcome
189
the shortcomings of the existing food laws in India there was a much needed demand to amend
the existing laws and give them a unified form.
Food Safety and Standards Act 2006
The Food Safety and Standards Act 2006 was introduced to overcome the shortcomings
of existing food laws and to give more importance to safety standards. This Act consolidates the
laws relating to food and establishes the Food Safety and Standards Authority of India (FSSA)
for laying down science-based standards for articles of food and to regulate their manufacture,
storage, distribution, sale and import, to ensure availability of safe and wholesome food for
human consumption. There is focus on in-process quality control rather than product testing.
Food Additives as regulated by FSSA (Food Safety and Standards Act):
Food additives / processing aids are to be added only in accordance with provisions /
regulations under the Act; Food additives under this unified act falls under Regulations 6.1.1.
For the purpose of this regulation “good manufacturing practices (GMP) for use of food
additives” means the food additives used under the following conditions namely
(i) the quantity of the additive added to food shall be limited to the lowest possible level necessary to accomplish its desired effect;
(ii) the quantity of the additive becomes a component of food as a result of its uses in the manufacturing, processing or packaging of a food and which is not intended to accomplish any physical or other technical effect in the food itself; is reduced to the extent reasonably possible; and
(iii) the additive is prepared and handled in the same way as a food ingredient.
Conclusion:
Thus there should be criteria for testing and evaluation of these food additives, which
includes estimating exposure and predicting toxicity from chemical structure. Test procedures
should be evaluated. In experimental toxicity studies effects with functional manifestations,
neoplasmic lesions with morphological manifestations and reproductive/ developmental
toxicity should be evaluated. Certain metabolic and pharmacokinetic studies in safety
assessment involving identification of relevant animal species, determining the mechanisms of
toxicity, metabolism into normal body constituents and influence of gut microflora i.e., either
effects of gut microflora on chemicals or chemical on gut microflora must be done. The
Influence of age, nutritional status, and health status on the design and interpretation of
studies must be assessed. Then further the use of human studies like epidemiological and food
intolerance in safety evaluation can be a valuable tool in deciding the limits and use of these
additives. Also setting the ADI is necessary for safety evaluation of additives. It is important that
statistical analyses are correctly selected and applied to the results available. In most food
applications more than one food additive is available that could fulfil a required role. But in
190
cases where this is not so, and a possible hazard to human health is indicated, the evaluation
must weigh up the risks of its consumption versus the benefits it gives the human population by
virtue of its use and at the same time, industry should aim to develop a safer, suitable
alternative.
The evaluation of food additives would be facilitated by good lines of communication
and active collaboration between government and industry. Where possible the design for
carcinogenicity tests should be standardized to enable comparison of results from all studies
performed. The safety-in-use of food additives is closely monitored in many countries
worldwide, and with the present laws the consumer is well protected against any deleterious
effects of food additives. With the improvements in testing methodology the consumer can
only benefit to an even greater extent in the future.
"All things are poisons; nothing is without poison; only the dose determines whether there is
a harmful effect". Paracelsus (16th Century Philosopher)
It must be remembered that all substances (chemicals) are poisons. There is none that
may not act as poison; only the right dose differentiates a poison and a remedy. Some of the
known toxins are at times given as remedy and some of the nutrients and medicines at very
high levels are toxic. No food substance is unequivocally safe or unsafe. The safety depends
both on the amount in the diet and on level of its exposure. It is also important to know that
both natural and synthetic additives must be considered from safety aspects.
References:
Bateman B. The effects of a double blind, placebo controlled artificial food colourings and benzoate preservative challenge on hyperactivity in a general population sample of preschool children. Archives of Disease in Childhood, 2004, 89, 506-11.
Bran, A. L., Davidson P. M., Salminen S. Food Additives. 2nd
edition. Marcel Ilekker, Inc. 2002.
Christina R. Whitehouse et al. The Potential Toxicity of Artificial Sweeteners. AAOHN Journal vol. 56, (6) June
2008, 251-259.
Feingold B.F. Hyperkinesis and learning disabilities linked to artificial food flavors and colors. American Journal of Nursing, 1975, 75,797–803.
Food Safety and Standards Act 2006 of India Ministry of Health and Family Welfare, New Delhi, Government of India www.mohfw.nic.in/pfa.htm .
191
Spore Based Biosensors and Their Role in Monitoring Potential
Environmental Contaminants in Dairy Foods
Naresh Kumar, Raghu. H. V. Avinash Yadav, Gurpreet and Geetika Thakur
Dairy Microbiology Division, NDRI, Karnal
Introduction:
The development of sensors for detecting foodborne pathogens has been motivated by the need to
produce safe foods and to provide better healthcare (Irudayaraj, 2009). Improving food and water safety
and security depends on the ability to detect, identify, and trace food and water pathogens. As milk is a
compulsory part of daily diet and being nutritious food for human beings, also serves as a good medium
for the growth of many microorganisms which cause spoilage of milk and milk products Earlier for
detection of pathogens conventional methods were used, and are considered a gold-standard for
foodborne pathogen detection which rely on specific media to enumerate and isolate viable bacterial
cells in food. These methods are very sensitive, inexpensive and can give both qualitative and
quantitative information and involve the basic steps: pre-enrichment, selective enrichment, selective
plating, biochemical screening and serological confirmation. Hence, a complete series of tests is often
required before any identification can be confirmed (Mandal et al., 2010/11) Although methods are
powerful, error-proof, and dependable but are lengthy, cumbersome and are often ineffective because
they are not compatible with the speed at which the products are manufactured and the short shelf life
of products. To overcome these challenging criteria of time and sensitivity rapid methods which include
nucleic acid, fluorescent antibody or immuno-based techniques have been developed which gives
instant or real time results but requiring additional expensive devices and equipments (Ivnitski et al.,
1999). Biosensor based tools offer the most promising solutions and address some of the modern-day
needs for fast and sensitive detection of pathogens in real time. Biosensors are defined as analytical
devices integrating biological elements and signal transducers. The biological elements interact
specifically with an analyte, producing a signal that the transducer recognizes and converts into
measurable parameters as shown in fig.1 (Rasooly and Herold, 2006). Currently biosensor is defined as a
sensor that integrates a biological element with a physiochemical transducer to produce an electronic
signal proportional to a single analyte which is then conveyed to a detector.
Fig 1 Diagrammatic representation of Biosensor and its working principle
192
(E. Eltzov, R.S. Marks., 2010)
Biosensor achievements have revolutionized the detection method and provide us with simple to use
device, cost-effective, rapid and appropriate detection method that give immediate and accurate results
comparable to or better than the conventional analytical systems in terms of performance i.e. reliability,
sensitivity, selectivity, specificity and robustness and can identify the contaminants much faster, more
efficient and can give effective real time monitoring of pathogens and most importantly ensuring
customer safety (Scott, 1998).
History of biosensor: In 1956, Leland C Clark Jr., who is known as the father of Biosensors and he
published his definitive paper on the oxygen electrode. In 1962, he described "how to make
electrochemical sensors more intelligent" by adding "enzyme transducers as membrane enclosed
sandwiches”. The year wise development in the field of biosensor is as follows:
193
Types of Biosensors (bioreceptors): A Bioreceptor is a biological molecular species or a living biological
system that utilizes a biochemical mechanism for recognition (Tantilipikara, 2005). Depending upon the
mechanism of biochemical interaction between the receptor and the analyte the biosensor can
categorised as follows.
Type of biological recognition elements Name of the biosensors
194
Enzymes, Protein, Antibodies, NA,
Organelles, Microbial cells, Plant and
animal tissues
Enzyme electrodes, Immunosensor, DNA sensors,
Microbial Sensors
Types of Biosensors (Detection mode): The transducing element of a biosensor is used to convert the
biological recognition step into the measurable signal that can be detected and displayed. It is further
classified into following types:
Type of transducers Measured property
Electrochemical Potentiometric, Amperometric, Voltametric
Electrical Surface conductivity, Electrolyte conductivity
Optical Fluorescence, Adsorption, Reflection
DNA sensors
Mass Sensitive Thermal Rezonans frequency of piezocrystals, Heat of reaction, Heat of
adsorption
Spores based biosensor: Bacterial spores appears to have great potential for their application as bio-
sensor as they have the ability to sense environmental changes and to respond using explosive
molecular mechanisms that transform dormant spores into rapid growing cells. There are a great
number of bacterial species which produce spores for example; genus Bacillus (widely dispersed in soil,
plant matter, and air) may be readily grown in the laboratory to form spores: B. cereus, B. licheniformis,
B. megaterium, B. sphaericus, B. stearothermophilus, B. subtilis, and B. thuringiensis. They can also
survive in a very harsh condition. For the development of bacterial spore as a biosensor, it is a
prerequisite to have a complete or descriptive knowledge regarding their germinants (carbohydrates,
nucleotides, amino acids etc.) which by their action on the dormant spores convert them into vegetative
cells. The germination process of a whole population of spore may be completed in a very short duration
of time (15-30min) followed by a sequence of metabolic reactions and synthesis of enzymes resulting in
outgrowth of vegetative cells. After germination de novo acetyl esterase is released from the core of the
spore which act upon DAF and its hydrolysis results in flouroscence and the signal can be captured using
optical device to quantify the presence of target analyte (Rotman, 2001).
Characteristic features of spores: Bacillus species have inherent characteristics to produce endospore.
These are the dormant form of life having no metabolic activity. They are resistant to environmental
stress like heat, desiccation, irradiation and chemical compounds and can be stored in a medium for long
time even in the absence of nutrients. Spores are resistant due to the Calcium-dipicolinate present in the
spores that stabilize and protects the DNA from denaturation. DNA-binding proteins helps in protecting
the DNA from heat, drying, chemicals, and radiations. While dehydration process that is the loss of water
195
provides them resistance toward heat and radiation. And finally during the germination process
damaged DNA they get repaired by DNA repair enzymes (Setlow, 2003).
Sporulation: Spore formation, sporogenesis or sporulation, normally commences when growth ceases
due to lack of nutrients. It is a complex process and may be divided into seven stages .An axial filament
of nuclear material forms (stage-I), followed by an inward folding of the cell membrane to enclose part
of the DNA and produce the forespore septum (stage-II). The membrane continues to grow and engulfs
the immature spore in a second membrane (stage-III). Next, cortex is laid down in the space between
the two membranes, and both calcium and dipicolinic acid is accumulated (stage-IV). Protein coats then
are formed around the cortex (stage-V), and maturation of the spore occurs (stage-VI). Finally, lytic
enzymes destroy the sporangium releasing the spore (stage-VII). Diagram representing the different
stages of sporulation (Prescott et al., 2002).
Germination: In the presence of favorable growth conditions spores get germinated. The germination
process is essentially a biophysical and degradative one– the spore’s inner membrane increases in
fluidity and ion fluxes resume; monovalent cations, potassium and sodium, move across the spore
membrane, and calcium ions and dipicolinate are excreted. The peptidoglycan of the spore cortex is
degraded, and the coat layers are partially degraded. ATP synthesis and oxidative metabolism resume,
DNA damage is repaired and the DNA-complexing small acid-soluble proteins (SASPs) are degraded by a
specific protease, providing a source of amino acids for outgrowth. As germination events precede any
de novo synthesis of macromolecules, the apparatus required for spore germination must be already
present in the mature spore (Moir et al., 2002).
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(Setlow, P, 2003.)
Application of spores as biosensor: Bacterial spores are suitable for use as biosensor because they have
the ability to sense environmental changes in response to specific “germinant” and transform into rapid
growing cells. The spores are heat resistant and can remain in non metabolic state for many years. This
characteristic can effectively be used as a biosensor for tracking these residues in milk and milk products
and the details of biosensor developed are as follows:
Development of analytical process for detection of antibiotic residues in milk using bacterial spores
as biosensor.(Patent no Reg# IPR /4.9.1/05074/2006)(Kumar et al., 2006)
A kit for detection of β-lactam antibiotic group in milk using bacterial spore as biosensor (Patent Reg
# IPR/ 4.14.1/08073/del/2009) (Kumar et al., 2009)
Development of Spore Inhibition Based -Enzyme Substrate Assay (SIB-ESA) for monitoring Aflatoxin
M1 in milk
Development of Enzyme Substrate Assay (ESA) for Monitoring Enterococci in Milk
Development of Analytical Process for Detection of Antibiotic Residues in Milk Using Bacterial Spores
as Biosensor
Outer
membrane Core
Inner
membrane
Corte
Germ cell
wall Coat
Germination trigger
1) Cation release
2) ca2+
dipicolinic acid release
3) Partial core hydration
• Cortex hydrolysis
• Further core hydration
• Start of metabolic activity
• Core expansion
• Further loss of resistance Increase of metabolic activity
RNA synthesis
Protein synthesis
Escape from the spore coat Coat
a
197
Introduction & Prior Art: This invention relates to application of dormant bacterial spores as biosensor
.The bacterial spores have unique ability to sense environmental changes in response to specific
“germinant” and transform rapidly into growing vegetative cells. The spores are heat resistant and can
remain in non metabolic state for many years. This characteristic can effectively be used as a biosensor
for tracking these residues in milk and milk products. In the present invention, an analytical process of
transformation of dormant spore of Bacillus stearothermophilus into active vegetative cell through
activation, germination and outgrowth has been developed .This analytical process can track major
groups of antibiotic residues in milk within 2.30-3.0 hours at MRL / or above levels recommended by
codex .
Brief of Invention: An analytical process which involves sporulation & activation of dormant spores of
B.stearothermophilus in newly developed medium & their germination/ outgrowth in presence of
selective germinant mixture has been developed ( Patent Reg # IPR/ 4.9.1.4/ 05074/ 1479 /DEL /2006).
The validated process is in line with AOAC approved charm 6602 system & can be used effectively for
semi-quantitative detection of antibiotic residues in different types of milk system within 2.30-3.0 hrs at
MRL/ or above levels as recommended by the codex /EU . This cost effective process can also find
applications in targeting spoilage and pathogenic organisms in dairy and non dairy foods.
Novel features of process:
1. Cost effective
2. Better sensitivity
3. Semi-quantitative detection
4. No false positive /negative results
5. Insensitivity towards detergents / sanitizers
6. Consistency in color development with in 3.0 hrs
7. Validated with AOAC approved charm 6602 system
8. Wide spectrum of application for different types of milk
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A Kit for Detection Of Β-Lactam Antibiotic Group in Milk Using Bacterial Spore as Biosensor: This
invention relates to application of dormant bacterial spores as biosensor .The study is based on the
resistance mechanism of some β-lactamase generating Bacillus spp. Some spore forming bacteria such
as B. cereus and B. licheniformis produce β-lactamase enzyme due to induction by β-lactam antibiotics
and the enzyme production is proportional to the concentration of inducer present in milk. A real time
microbial assay based on β-lactamase enzyme using starch iodine as color indicator has been developed.
The microbial assay is working on principle of non competitive enzyme action on inducer (β-Lactam)
resulting in indirect reduction of starch iodine mixture through penicilloic acid. A comparison of the
intensity of the test reaction with that of a control was taken as criteria to determines whether the
sample is positive or negative (Kumar et al., 2009) (Patent Regd. #IPR / 4.14 .1/08073). The assay can
detect specifically β- lactam groups in spiked milk with in 15-20 min at regulatory codex limits with
negligible sensitivity towards non β- lactam groups. The presence of Inhibitors other than antibiotic
residues in milk did not interfere with the working principle of microbial assays. A significant correlation
between microbial assay & receptor based assay (charm 6202) was established in survey work with raw,
pasteurized milk and dried products with no false positive/ negative results. Spore suspension was found
stable up to 5 months when stored under refrigeration conditions. The microbial assay (Rs 20.54/- test)
is cost effective can find immense application in dairy industry as “ON FARM” milk screening test for β-
lactam group (Kumar et al.,2009).
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The impact of innovation on life of Rural India: The invention was carried out to test drug residues at
farm level. These drug residues have immense public health and processing implications. The kit/ test
can be executed at farm levels with minimal cost of Rs 16/ - test. The field level testing will be of public
heath and processing value to dairy farmers and entrepreneurs who are involved in dairy small business.
Development of Spore Inhibition Based –Enzyme Substrate Assay (SIB-ESA) for Monitoring Aflatoxin
M1
Brief about Innovation: Aflatoxins are toxic, carcinogenic, mutagenic immuno-suppressive agents
produced as secondary metabolites by the fungi Aspergillus flavus & A. parasiticus. Four major Aflatoxins
B1, B2, G1, and G2 have been isolated from feeds. Aflatoxin M1 is hydroxylated derivative of aflatoxin
B1.The bacterial spores as nano-molecules have unique ability to sense environmental changes in
response to specific “germinant” and transform rapidly into growing vegetative cells. This characteristic
can be effectively used as biosensor for tracking microbial and non–microbial contaminants (Kumar et
al.,2005; Rotman 2001 & 2003).The present hypothesis is based on the specific spore germination
inhibition principle in presence of specific analyte i.e. aflatoxin M1. In case where analyte is absent in
milk system, specific indicator enzyme (s) are produced by active bio-sensing molecules which will act
specifically on Chromogenic/or fluorogenic substrate resulting in colored reaction/or fluorescence as
end product which is measured semi-quantitatively by either visually/or using optical system at specific
excitation/emission spectra (Kumar et al., 2010).
200
Patent on development of spore inhibition based–enzyme substrate assay (SIB-ESA) for monitoring
Aflatoxin M1 in milk has been filed at NDRI and is under processing: The end product response is
significantly different in case of buffer/ or milk system containing specific analyte i. e. Aflatoxin M1. The
developed test assay was validated by analyzing 25 samples of each raw and pasteurized milk procured
from different organized/or private own dairies and other reputed brands using AOAC approved RIA and
ELISA based system and a significant correlation with ELISA at Codex MRL Limit (0.5 ppb) of Aflatoxin M1
was established.
Development of Enzyme Substrate Assay (ESA) For Monitoring Enterococci in Milk: An Enzyme
Substrate Assay (ESA) based on β-D-glucosidase activity was attempted for specific detection of
Enterococci to meet the emerging demand of dairy industry. Four enrichment broths commercially
available in the market were screened for selective recovery of Enterococci based on β-D-glucosidase
activity. One of these broths namely Chromocult Enterococcus Broth (CEB) showed better performance
in terms of selectivity and enzyme activity with partial inhibition of contaminants other than Enterococci.
The selected medium was further improved for desired features by increasing the concentration of
Sodium Azide from 0.06 to 0.15g/100ml resulting in significant inhibitory effect on growth pattern of L.
lactis, L. casei, Leuconostoc mesenteroides and L. monocytogenes.
Development of bacterial spore based bio-chip for on line monitoring of
Aflatoxin M1 in milk (Patent Reg #3064/DEL/2010 )
Chromogenic Assay Fluorogenic Assay
Residual enzyme activity for positive sample (0.25 -0.5ppb) = ≤ 0.45
Residual enzyme activity for negative sample = ≥ 0.45
Novel Features:
Real time cost effective (Rs. 25 per test) Chromogenic assay for Aflatoxin M1working within 45min
Real time fluorogenic assay working for Aflatoxin M1within 25min
Semi quantitative detection at 0.25-0.5 ppb level and validated with AOAC approved microbial
receptor assay and ELISA test
Test can be applied at Dairy farm as well as reception dock for monitoring Aflatoxin M1 in raw, heat
treated & dried milk
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Other media components and supplements were also optimized for enhanced sensitivity and selectivity
of Enterococcus sp. The optimized selective enrichment medium i. e. Esculin Based Sodium Azide
Medium (EBSAM) demonstrated superior features in terms of sensitivity, selectivity, fastness, accuracy
etc. and may be a suitable substitute for existing media used for routine monitoring of Enterococci in
R&D institutions. Developed assay was screened for Enterococci count with 32 samples of raw milk and it
could detect 2.67, 3.50, 4.25 and 4.8 log counts within incubation period of 12, 7½, 6½ and 5 hr
respectively. ESA could also detect Enterococci log counts of 2.84 in pasteurized milk within 12 hrs of
incubation; however, assay was insensitive at very low level of 1.13 and 0.915 log counts. As such ESA
developed in current investigation may find industrial application as Hygiene Indicator test for detection
of Enterococci in raw milk & pasteurized milk with in 5-12 hrs as against 36-48hrs required in
conventional method (Thakur et al., 2010).
Germination Signals
(Dextrose)
SPORE
Signal
Receptor
Measurement of fluorescent signal using plate reader/EMCCD
camera
Transduced Fluorescent Signals
After DAF hydrolysis
Spores on microplate/biochip get germinated in
presence of dextrose and release marker enzyme
Enterococci
(ß-D-glucosidase)
Selective
Enrichment
202
Concluding remarks: Biosensors are making a great impact on the development of rapid, sensitive assays
for the detection of microbial and non – microbial contaminants in food system. Kits are now available
for several organisms such as E. coli O157:H7 and Salmonella typhimurium and it is hoped that more will
become available shortly. The most viable openings in the food industry will arise where a biosensor can
rapidly detect total microbial contamination. The largest area of application for the environment lies in
the development of biosensors for monitoring bacteria in drinking and waste water, rivers, reservoirs
and supplies. Spores have a great potential to be used as a biosensor and the bioassay are cost effective,
rapid, easy to perform and require almost negligible infra-structural facilities.
References
Belluzo, M. S., Ribone, M. E., Lagier, C. M., 2008. Assembling Amperometric Biosensors for Clinical Diagnostics.
Sensors 8, 1366-1399.
Chauhan, S., Rai, V., Singh, H. B., 2004. Biosensors. Resonance. 33-44.
E. Eltzov, R.S. Marks, 2010. Whole-cell aquatic biosensors. Anal Bioanal Chem. published online 11th
September 2010
in Springer.
Irudayaraj, J., 2009. Pathogen Sensors. Sensors 9, 8610-8612.
Ivnitski, D., Abdel-Hamid, I., Atanasov, P., Wilkins, E., 1999. Biosensors for detection of pathogenic bacteria.
Biosensors & Bioelectronics 14, 599–624.
Kumar, N., Das, S., Manju, G., 2009. A kit for detection of β-lactam antibiotic group in milk using bacterial spore as
biosensor (Patent Reg # IPR/115/del/2009).
Kumar, N., Sawant, S., Malik, R.K., Patil, G.R., 2005. Development of analytical process for detection of antibiotic
residues in milk using bacterial spores as biosensor (Patent Reg # IPR/4.9.1.4/05074/1479/del/2006).
Kumar, N., Singh, N., Singh, V.K., Bhand, S., Malik, R.K., 2010. Development of spore inhibition based–enzyme
substrate assay (SIB-ESA) for monitoring Aflatoxin M1 in milk (Patent Reg #3064/DEL/2010).
203
Mandal, P. K., Biswas, A. K., Choi, K., Pal, U. K., 2010/11. Methods for Rapid Detection of Foodborne Pathogens: An
Overview. American Journal of Food Technology 6(2), 87-102
Moir, A., Corfe, B. M., Behravan, J., 2002. Spore germination. Cell Mol Life Sci 59, 403–409.
Prescott, L. M., Harley, Klein., 2002. Microbiology. 5th Edition. The McGraw-Hill companies,. (Chapter 3)
Rasooly and Herold, 2006. Biosensors for the Analysis of Food- and Waterborne Pathogens and their Toxins. Journal
of AOAC international 89(3), 873-883.
Rogers, K. R., 2000. Principles of affinity-based biosensors. Molecular Biotechnology 14(2), 109-129.
Rotman, B., 2001. Using living spores for real-time biosensing. Gen. Eng. News 21, 65.
Rotman, B., Cote, M. A., 2003. Application of a real-time biosensor to detect bacteria in platelet concentrates.
Biochem. Biophys. Res. Comm 300, 197-200.
Scott, A. O., 1998. Biosensor for food analysis. Published by Royal Society of chemistry, Cambridge, UK. (Chapter 1)
Setlow, P., 2003. Spore germination. Current Opinion in Microbiology 6, 550–556.
Tantilipikara, P., 2005. Optical biosensor for microalbumin determination. A thesis submitted in partial fulfilment of
the requirement for the degree of Master of Science. Mahidol University.
Thakur, G., Kumar, N., Raghu, H. V., Malik, R. K., (2010). Development of Off-Line Enzyme Substrate Based Assay for
Monitoring Enterococci in Milk. NDRI Newsletter Apr – June 2010. Pp 2-3.
Thevenot, D. R., Toth, K., Durst, R. A., Wilson, G. S., 2001. Electrochemical biosensors: recommended definitions and
classification. Biosensors & Bioelectronics 16(1), 121-131.
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Quality Management System and its application in dairy industry
Naresh Kumar and Raghu H V
Dairy Microbiology Division, NDRI, Karnal.
The concept of quality has existed for many years, though it’s meaning has changed and
evolved over time. In the early twentieth century, quality management meant inspecting
products to ensure that they met specifications. In the 1940s, during World War II, quality
became more statistical in nature. Statistical sampling techniques were used to evaluate quality,
and quality control charts were used to monitor the production process. In the 1960s, with the
help of so-called “quality gurus,” the concept took on a broader meaning. Quality began to be
viewed as something that encompassed the entire organization, not only the production process.
Since all functions were responsible for product quality and all shared the costs of poor quality,
quality was seen as a concept that affected the entire organization. The meaning of quality for
businesses changed dramatically in the late 1970s. Before then quality was still viewed as
something that needed to be inspected and corrected. Today, successful companies understand
that quality provides a competitive advantage. They put the customer first and define quality as
meeting or exceeding customer expectations. Since the 1970s, competition based on quality has
grown in importance and has generated tremendous interest, concern, and enthusiasm.
Companies in every line of business are focusing on improving quality in order to be more
competitive. In many industries quality excellence has become a standard for doing business.
Companies that do not meet this standard simply will not survive. The term used for today’s new
concept of quality is total quality management or TQM. Fig. 1 presents a timeline of the old and
new concepts of quality. You can see that the old concept is reactive, designed to correct quality
problems after they occur. The new concept is proactive, designed to build quality into the
product and process de-sign. Next, we look at the individuals who have shaped our
understanding of quality.
Fig. 1 presents a timeline of the old and new concepts of quality
205
QMS definition: “A set of co-ordinated activities to direct and control an organisation in order to
continually improve the effectiveness and efficiency of its performance.” Collective policies, plans,
practices, and the supporting infrastructure by which an organization aims to reduce and eventually
eliminate non-conformance to specifications, standards, and customer expectations in the most cost
effective and efficient manner. These activities interact and are affected by being in the system, so
the isolation and study of each one in detail will not necessarily lead to an understanding of the
system as a whole. The main thrust of a QMS is in defining the processes, which will result in the
production of quality products and services, rather than in detecting defective products or
services after they have been produced.
Companies perform many activities besides manufacturing of APIs, such as development,
marketing, purchasing, warehousing and distribution. All these activities are processes which are
required to be managed in a systematic manner. Therefore, the company shall establish,
document and implement within its organization a Quality Management System that is designed
to continually improve its effectiveness. Top management is called to establish a customer
oriented organization:
By defining the systems and processes that can be managed and improved in effectiveness
and efficiency,
Acquiring and using process data and information on a continuing basis,
Directing progress towards continual improvement,
Using suitable methods to evaluate process improvement.
ISO 9000:2000 standard defines process as the "system of activities that uses resources to
transform inputs into outputs". This definition has a strong point in two major rules: (1) Inputs
of one process are mainly outputs of another and (2) processes are managed in order to
create new values that correspond to requirements and expectations of customers. So,
cybernetic approach to management is at use today, an approach that establishes connection
between inputs and outputs, during which process outputs must be verified according to input
requirements in order to satisfy customer requirements and requirements of other interested
sides. Also, process inputs must be defined and recorded in order to provide a base for demand
formulation, that is to be used for output validation and verification. Input requirements that are
crucial for product or process must be identified in order to assign proper responsibilities and
resources (ISO 9000:2000) Production process represents a flow that begins with external
requirements of buyers and ends with the product that is used by buyers. Buyer makes
judgment about realization or non-realization of his requirements. ISO 9000:2000 standards
recommend that: (1) desired results can be more efficiently achieved if proper resources and
activities are managed as processes and (2) System approach to management: identification,
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understanding and system management of related processes to achieve goal that are set. Make
the efficient organization.
Process Network - Network Architecture: Considering the definition that the process is " a
system of activities...", then, every process can be structured as the unity of activities or chain
of activities, and any activity can be structured as the chain of elementary tasks. For both
definitions, for activities and tasks, the second part of the definition is the same " ....that
uses resources in order to transform inputs into outputs". ISO 9000:2000 standards explain the
consistency of such structure as follows: "Any activity that transforms inputs into outputs can
be considered as the process ". In order for it's efficient functioning the organization should
identify and manage inter related process. Process model of the standard ISO 9000:2000 is
shown in figure. 2
Fig. 2 Process module of the standard ISO 9000:2000
ISO 9000 family of standards: ISO 9000 consists of a set of standards and a certification process
for companies. By receiving ISO 9000 certification, companies demonstrate that they have met
the standards specified by the ISO. The standards are applicable to all types of companies and
have gained global acceptance. In many industries ISO certification has become a requirement
for doing business. Also, ISO 9000 standards have been adopted by the European Community as
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a standard for companies doing business in Europe. In December 2000 the first major changes
to ISO 9000 were made, introducing the following three new standards:
ISO 9000:2000–Quality Management Systems–Fundamentals and Standards: Provides the
terminology and definitions used in the standards. It is the starting point for understanding
the system of standards. This standard describes the concepts of a quality management
system (QMS) and defines the fundamental terms used in the ISO 9000 family. The standard
also includes the eight quality management principles which were used to develop ISO 9001
and ISO 9004. This standard replaces ISO 8402:1994 and ISO 9000-1:1994.
ISO 9001:2000–Quality Management Systems–Requirements: This is the standard used for
the certification of a firm’s quality management system. It is used to demonstrate the
conformity of quality management systems to meet customer requirements. This standard
specifies the requirements for a QMS, whereby an organization needs to assess and
demonstrate its ability to provide products that meet customer and applicable regulatory
requirements, and thereby enhance customer satisfaction. This standard replaces ISO
9001:1994, ISO 9002:1994 and ISO 9003:1994.
ISO 9004:2000–Quality Management Systems–Guidelines for Performance: Provides
guidelines for establishing a quality management system. It focuses not only on meeting
customer requirements but also on improving performance. This standard provides
guidance for continual improvement and can be used for performance improvement of an
organization. While ISO 9001 aims to give quality assurance to the manufacturing processes
for products and to enhance customer satisfaction, ISO 9004 takes in a broader perspective
of quality management and gives guidance for future improvement. This standard replaces
ISO 9004-1:1994. Guidelines for self-assessment have been included in Annex A of ISO
9004:2000. This annex provides a simple, easy-to-use approach to determine the relative
degree of maturity of an organization’s QMS and to identify the main areas for
improvement.
Major changes between the 1994 and 2000 versions of the ISO 9001standard: The new
standard is less biased towards the manufacturing sector and thus more generic. It can be used
by all organizations, regardless of type, size and product category.
All the requirements of this new standard may not be applicable to all organizations. As the
distinction between ISO 9001, ISO 9002 and ISO 9003 has been removed, an “application
clause” (clause 1.2) in the new standard allows companies to exclude certain requirements of
section 7 (Product realization) that are not relevant to them. For example, an organization that
was certified to ISO 9002:1994 and does not carry out design activities may seek exclusion for
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clause 7.3 of ISO 9001:2000, relating to “design and development”, so long as it states the
reasons for exclusion in its Quality Manual.
A new “process-oriented” structure and more logical sequence of the contents differentiate the
new standard from the 1994 version, which was “clause-oriented”. The standard retains a large
part of ISO 9001:1994, but the 20 requirements have been grouped in five sections: quality
management system; management responsibility; resource management; product realization;
and measurement, analysis and improvement. The new standard has also reduced significantly
the amount of documentation required. Documented procedures have been reduced from
eighteen to six, although the organization, if required, may document other procedures,
instructions, etc.
Management Principles: The eight quality management principles on which the quality
management system standards of the ISO 9000:2000 and ISO 9000:2008 series are based.
These principles can be used by senior management as a framework to guide their
organizations towards improved performance. The principles are derived from the collective
experience and knowledge of the international experts who participate in ISO Technical
Committee ISO/TC 176, Quality management and quality assurance, which is responsible for
developing and maintaining the ISO 9000 standards. The eight quality management principles
are defined in ISO 9000:2005, Quality management systems Fundamentals and vocabulary, and
in ISO 9004:2000, Quality management systems Guidelines for performance improvements.
This document gives the standardized descriptions of the principles as they appear in ISO
9000:2005 and ISO 9004:2000. In addition, it provides examples of the benefits derived from
their use and of actions that managers typically take in applying the principles to improve their
organizations' performance.
Principle 1: Customer focus
Principle 2: Leadership
Principle 3: Involvement of people
Principle 4: Process approach
Principle 5: System approach to management
Principle 6: Continual improvement
Principle 7: Factual approach to decision making
Principle 8: Mutually beneficial supplier relationships
Principle 1: Customer focus: Organizations Depend On Their Customers And Therefore Should
Understand Current And Future Customer Needs, Should Meet Customer Requirements And
Strive To Exceed Customer Expectations.
Applying the principle of leadership typically leads to:
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Increased revenue and market share obtained through flexible and fast responses to market opportunities.
Increased effectiveness in the use of the organization's resources to enhance customer satisfaction.
Improved customer loyalty leading to repeat business.
Applying the principle of customer focus typically leads to:
Researching and understanding customer needs and expectations.
Ensuring that the objectives of the organization are linked to customer needs and expectations.
Communicating customer needs and expectations throughout the organization.
Measuring customer satisfaction and acting on the results.
Systematically managing customer relationships.
Ensuring a balanced approach between satisfying customers and other interested parties (such as owners, employees, suppliers, financiers, local communities and society as a whole).
Principle 2: Leadership: Leaders establish unity of purpose and direction of the organization.
They should create and maintain the internal environment in which people can become fully
involved in achieving the organization's objectives.
Applying the principle of leadership typically leads to:
Considering the needs of all interested parties including customers, owners, employees, suppliers, financiers, local communities and society as a whole.
Establishing a clear vision of the organization's future.
Setting challenging goals and targets.
Creating and sustaining shared values, fairness and ethical role models at all levels of the organization.
Establishing trust and eliminating fear.
Providing people with the required resources, training and freedom to act with responsibility and accountability.
Inspiring, encouraging and recognizing people's contributions.
Principle 3: Involvement of people: People at all levels are the essence of an organization and
their full involvement enables their abilities to be used for the organization's benefit.
Applying the principle of involvement of people typically leads to:
People understanding the importance of their contribution and role in the organization.
People identifying constraints to their performance.
People accepting ownership of problems and their responsibility for solving them.
People evaluating their performance against their personal goals and objectives.
People actively seeking opportunities to enhance their competence, knowledge and experience.
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People freely sharing knowledge and experience.
People openly discussing problems and issues.
Principle 4: Process approach: A desired result is achieved more efficiently when activities and
related resources are managed as a process.
Applying the principle of process approach typically leads to:
Systematically defining the activities necessary to obtain a desired result.
Establishing clear responsibility and accountability for managing key activities.
Analysing and measuring of the capability of key activities.
Identifying the interfaces of key activities within and between the functions of the organization.
Focusing on the factors such as resources, methods, and materials that will improve key activities of the organization.
Evaluating risks, consequences and impacts of activities on customers, suppliers and other interested parties.
Principle 5: System approach to management: Identifying, understanding and managing
interrelated processes as a system contributes to the organization's effectiveness and efficiency
in achieving its objectives.
Applying the principle of system approach to management typically leads to:
Structuring a system to achieve the organization's objectives in the most effective and efficient way.
Understanding the interdependencies between the processes of the system.
Structured approaches that harmonize and integrate processes.
Providing a better understanding of the roles and responsibilities necessary for achieving common objectives and thereby reducing cross-functional barriers.
Understanding organizational capabilities and establishing resource constraints prior to action.
Targeting and defining how specific activities within a system should operate.
Continually improving the system through measurement and evaluation.
Principle 6: Continual improvement: Continual improvement of the organization's overall
performance should be a permanent objective of the organization.
Applying the principle of continual improvement typically leads to:
Employing a consistent organization-wide approach to continual improvement of the organization's performance.
Providing people with training in the methods and tools of continual improvement.
Making continual improvement of products, processes and systems an objective for every individual in the organization.
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Establishing goals to guide, and measures to track, continual improvement.
Recognizing and acknowledging improvements.
Principle 7: Factual approach to decision making: Effective decisions are based on the analysis
of data and information
Applying the principle of factual approach to decision making typically leads to:
Ensuring that data and information are sufficiently accurate and reliable.
Making data accessible to those who need it.
Analysing data and information using valid methods.
Making decisions and taking action based on factual analysis, balanced with experience and intuition.
Principle 8: Mutually beneficial supplier relationships: An organization and its suppliers are
interdependent and a mutually beneficial relationship enhances the ability of both to create
value
Applying the principles of mutually beneficial supplier relationships typically leads to:
Establishing relationships that balance short-term gains with long-term considerations.
Pooling of expertise and resources with partners.
Identifying and selecting key suppliers.
Clear and open communication.
Sharing information and future plans.
Establishing joint development and improvement activities. Inspiring, encouraging and recognizing improvements and achievements by suppliers.
Requirement of ISO 9001: 2000:
The 14 essential steps are to be followed through in order to implement ISO 9000 quality
management system successfully.
Step 1: Top Management Commitment
The top management (managing director or chief executive) should demonstrate a
commitment and a determination to implement an ISO 9000 quality management system in the
organization. Without top management commitment, no quality initiative can succeed. Top
management must be convinced that registration and certification will enable the organization
to demonstrate to its customers a visible commitment to quality. It should realize that a quality
management system would improve overall business efficiency by elimination of wasteful
duplication in management system.
Step 2: Establish Implementation Team
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ISO 9000 is implemented by people. The first phase of implementation calls for the
commitment of top management - the CEO and perhaps a handful of other key people. The
next step is to establish implementation team and appoint a Management Representative (MR)
as its coordinator to plan and oversee implementation. Its members should include
representatives of all functions of the organization -Marketing, Design and development,
Planning, Production, Quality control, etc.
Step 3: Start ISO 9000 Awareness Programs
ISO 9000 awareness programs should be conducted to communicate to the employees the aim
of the ISO 9000 quality management system; the advantage it offers to employees, customers
and the organization; how it will work; and their roles and responsibilities within the system.
Suppliers of materials and components should also participate in these programs.
Step 4: Provide Training
Since the ISO 9000 quality management system affects all the areas and all personnel in the
organization, training programs should be structured for different categories of employees -
senior managers, middle-level managers, supervisors and workers. The ISO 9000
implementation plan should make provision for this training. The training should cover the
basic concepts of quality management systems and the standard and their overall impact on
the strategic goals of the organization, the changed processes, and the likely work culture
implications of the system. In addition, initial training may also be necessary on writing quality
manuals, procedures and work instruction; auditing principles; techniques of laboratory
management; calibration; testing procedures, etc.
Step 5: Conduct Initial Status Survey
ISO 9000 does not require duplication of effort or redundant system. The goal of ISO 9000 is to
create a quality management system that conforms to the standard. This does not preclude
incorporating, adapting, and adding onto quality programs already in place. So the next step in
the implementation process is to compare the organization’s existing quality management
system, if there is one -- with the requirements of the standard (ISO 9001:2000). For this
purpose, an organization flow chart showing how information actually flows (not what should
be done) from order placement by the customer to delivery to this customer should be drawn
up. From this over-all flow chart, a flow chart of activities in each department should be
prepared. With the aid of the flow charts, a record of existing quality management system
should be established. A significant number of written procedures may already be in place.
Unless they are very much out of date, these documents should not be discarded. Rather, they
should be incorporated into the new quality management system.
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Step 6: Create a Documented Implementation Plan
Once the organization has obtained a clear picture of how its quality management system
compares with the ISO 9001:2000 standard, all non-conformances must be addressed with a
documented implementation plan. Usually, the plan calls for identifying and describing
processes to make the organization’s quality management system fully in compliance with the
standard.
Step 7: Develop Quality Management System Documentation
Documentation of the quality management system should include:
Documented statements of a quality policy and quality objectives, A quality manual, Documented procedures and records required by the standard ISO 9001:2000, and Documents needed by the organization to ensure the effective planning, operation and
control of its processes.
Step 8: Document Control
Once the necessary quality management system documentation has been generated, a
documented system must be created to control it. Control is simply a means of managing the
creation, approval, distribution, revision, storage, and disposal of the various types of
documentation. Document control systems should be as simple and as easy to operate as
possible -- sufficient to meet ISO 9001:2000 requirements and that is all.
Document control should include: ƒ
Approval for adequacy by authorized person (s) before issue, Review, updating and re-approval of documents by authorized person (s), Identification of changes and of the revision status of documents, Availability of relevant versions of documents at points of use, Identification and control of documents of external origin, Assurance of legibility and identifability of documents, and Prevention of unintended use of obsolete documents.
The principle of ISO 9000 document control is that employees should have access to the
documentation and records needed to fulfil their responsibilities.
Step 9: Implementation
It is good practice to implement the quality management system being documented as the
documentation is developed, although this may be more effective in larger firms. In smaller
companies, the quality management system is often implemented all at once throughout the
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organization. Where phased implementation takes place, the effectiveness of the system in
selected areas can be evaluated.
Step 10: Internal Quality Audit
As the system is being installed, its effectiveness should be checked by regular internal quality
audits. Internal quality audits are conducted to verify that the installed quality management
system:
Conforms to the planned arrangements, to the requirements of the standard (ISO 9001:2000) and to the quality management system requirements established by your organization, and
Is effectively implemented and maintained.
Step 11: Management Review
When the installed quality management system has been operating for three to six months, an
internal audit and management review should be conducted and corrective actions
implemented. The management reviews are conducted to ensure the continuing suitability,
adequacy and effectiveness of the quality management system.
Step 12: Pre-assessment Audit
When system deficiencies are no longer visible, it is normally time to apply for certification.
However, before doing so, a pre-assessment audit should be arranged with an independent and
qualified auditor. Sometimes certification bodies provide this service for a nominal charge. The
pre-assessment audit would provide a degree of confidence for formally going ahead with an
application for certification.
Step 13: Certification and Registration
Once the quality management system has been in operation for a few months and has
stabilized, a formal application for certification could be made to a selected certification
agency. The certification agency first carries out an audit of the documents (referred to as an
"adequacy audit"). If the documents conform to the requirements of the quality standard, then
on-site audit is carried out. If the certification body finds the system to be working
satisfactorily, it awards the organization a certificate, generally for a period of three years.
During this three-year period, it will carry out periodic surveillance audits to ensure that the
system is continuing to operate satisfactorily.
Step 14: Continual Improvement
Certification to ISO 9000 should not be an end. You should continually seek to improve the
effectiveness and suitability of the quality management system through the use of:
Quality policy
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Quality objectives Audit results Analysis of data Corrective and preventive actions Management review ISO 9004:2000 provides a methodology for continual improvement.
Conclusion: While the world witnesses technological break through and emerging global markets,
global standardization and certification systems encapsulate these developments and provide
tools to facilitate international transactions of goods and services. Thus standardization and
quality systems have become indispensable for development of the national economy all over
the world. The approach to progressive development has reckoned with unbecoming ease that
one has to work better within the limitation of resources and attempt capturing larger markets,
with interplay of various non price factors which have come to have a bearing on international
trade. It is in this contest that ISO 9000 Quality Management System Standards brought out by
International Organization for Standardization (ISO) has assumed greater importance for
achieving the objective of facilitating global trade for safe and wholesome foods. This is the
reasons why ISO 9000 has unanimously received a worldwide acceptance.
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APPLICATION OF HACCP IN DAIRY INDUSTRY
Vaishali, Rajeev Patel and Naresh Kumar*
Model Dairy Plant, N.D.R.I, Karnal-132001
*Dairy Microbiology division, N.D.R.I.,Karnal-132001
Introduction: Food safety is a global concern. Not only because of the continuing importance
for public health, but also because of its impact on international trade. Effective Food Safety
Systems shall therefore manage and ensure the safety and suitability of foodstuffs. In many
countries world-wide, legislation on the safety and suitability of foodstuffs requires HACCP to
be put in place by any food business or organization, whether profit-making or not and whether
public or private, carrying out any or all of the following activities: preparation, processing,
manufacturing, packaging, storage, transportation, distribution, handling or offering for sale or
supply of foodstuffs.
HACCP was developed in the 1960s by the US food industry and National Aeronautics and Space
Administration (NASA) as a ‘zero-defect’ approach to feed astronauts. The bases of HACCP are
that it is a process control rather than a product control and that it focuses control on steps in
the processing systems that are critical to consumer health. HACCP has won wide acceptance as
a voluntary control programme in the food industry. There can hardly be HACCP without Good
Manufacturing or Management Practices (GMP). Briefly, GMP is a description of all the steps
(which should represent good practice) in a processing facility, while HACCP is a documentation
that the steps important to consumer health are under control.
Application of HACCP: The application of HACCP involves three distinct but interrelated steps
namely prerequisite compliance, preliminary steps and steps based on HACCP principles.
Prerequisite Compliance: The Implementation of HACCP system requires compliance to food
hygiene and good manufacturing practices (GMP).Without implementing prerequisite
programmes food safety management system based on HACCP cannot be effectively
implemented. This programme is designed in the “Codex Standard CAC/RCP-1(1969)
Recommended International Code of Practice General Principles of Food Hygiene”.The code
follows the food chain from primary production through to final consumption, highlighting the
key hygiene controls at each stage. The controls described in these general principles are
internationally recognized as essential to ensure the safety and suitability of food for
consumption.
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Preliminary steps:
Constitution of HACCP team: A multi-disciplinary HACCP team is constituted with specialization
involved in processing, quality assurance and maintenance. The team needs to have been
exposed to HACCP principles and practices.
Description of the Product: The HACCP team keeping in view the raw material/ingredients
used, processes of manufacture and distribution channel, has to describe the product. It should
include following:
Common or Usual Name?
Raw, Ready-to-Eat or must it be cooked before consumption?
Preservation Method?
Type of package?
Method of Distribution?
Is product distributed frozen, Refrigerated, or is it shelf stable?
Length of shelf-life?
Temperature?
Label instructions?
Are special distribution controls needed?
End Use of the Product: The description of end use of the product should include following:
What is the normal use of the food by intended consumers?
Who will consume the food?
Is the food intended for High-Risk populations (Infants, Elderly, Immuno- compromised?)
Is food intended for retail or food service?
Is food held refrigerated, frozen or hot before consumption?
Construction of Process Flow Diagram: The HACCP team, after careful study of all process
stages, has to prepare the process flow diagrams. It should include on-site verification of the
flow diagrams to ensure that all stages and conditions are covered.
Validation of Process Flow Diagram: The process flow diagram prepared by the HACCP team is
to be validated on the shop floor by the team for its correct description of the processes in the
correct sequence of operations. During internal audits the audit teams have to revalidate the
process flow diagram.
Steps Based On HACCP Principles:
Conducting Hazard Analysis (HACCP Principle 1): After the process flow diagrams are
completed and verified, the team has to list all possible biological, chemical and physical
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hazards that can reasonably be expected to occur at each step and have to describe the
preventative measures by which these hazards can be controlled. Included in the list are
hazards of such nature that their elimination or reduction to acceptable levels is essential to the
production and distribution of safe products. The team have to consider applicable
preventative measures, for each hazard. Preventive measures are actions and activities
required for eliminating hazards or reducing their impact or occurrence to acceptable levels.
More than one preventive measure may be required to control a specific hazard(s) and more
than one hazard may be controlled by a specified preventative measure.
Identification Of Critical Control Points (HACCP Principle 2): Applying the decision tree given in
HACCP Standard (IS :15000:1995) facilitates the identification of a CCP in the HACCP system.
Team members should be trained in the application of the decision tree. All hazards that may
reasonably be expected to occur, or be introduced at each step, have to be considered. If a
hazard was identified at a step where control is necessary for safety and no preventative
measure exists at that or another step, then the product or process is to be modified suitably to
include a preventative measure. Decision tree is to be applied to determine whether the step is
a CCP for an identified hazard.
Establishing Control Limits (HACCP Principle 3): CCPs define the boundaries between safe and
unsafe products. Hence it is vital to set these correctly for each criterion. The team has to,
therefore, use a criteria governing safety at each CCP in order to set the appropriate critical
limits. Critical limits are specified for each preventative measure .Where required more than
one critical limit, is to be elaborated at a particular step. Criteria to be used may include
measurements of temperature, time, adulterants, microbial load and sensory parameters such
as taste and flavor and visual appearance.
Establishing CCP Monitoring Mechanism (HACCP Principle 4): Monitoring, one of the most
important aspects of HACCP system, is the scheduled measurement or observation of a CCP
relative to its critical limit. Monitoring procedures are designed to enable detection of loss of
control at the CCP and provide timely information for corrective action to regain process
control before a stage of product rejection is reached. Monitoring procedures decided for CCPs
are done rapidly as they relate to online processes and lengthy analytical testing is not
practicable. Physical and chemical measurements are preferred to microbiological testing
because they can be done rapidly (or rapid microbiological tests may be carried out).All records
and documents associated with monitoring of CCPs are signed by the person monitoring the
CCP and by a responsible reviewing official.
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Establish Corrective Action (HACCP Principle 5): Specific corrective actions developed for each
CCP in the HACCP system have to deal with deviations that may occur. Actions are taken ensure
that the CCP has been brought under control and also include proper disposition of affected
products. Deviation and product disposition procedures are documented in the HACCP record
keeping. Corrective actions are also taken when monitoring indicates a trend towards loss of
control at a CCP. Action is taken to bring the process back into control before deviation leads to
safety hazards.
Establishing Procedure For Verification (HACCP Principle 6): HACCP system includes verification
procedures for assuring that the system is being complied with on day-to-day basis. Internal
quality audit methods are used to ensure most effectively that the HACCP system is working
correctly. Monitoring and auditing methods, procedures and tests, including random sampling
and analysis, too are used to determine if the HACCP system is working correctly. Appropriate
frequency of audit and verification is used to validate the HACCP system.
Examples of verification activities include:
Review of the HACCP system and its records.
Review of deviations and product dispositions.
Operations to determine if CCPs are under control.
Validation of established critical limits.
Establishing Effective Record Keeping (HACCP Principle 7): Efficient and accurate record
keeping is essential for application of the HACCP system. Records of all areas critical to product
safety are kept to demonstrate that the HACCP system is compliant with the documented
system. The quality manual and procedures include the documentation of all steps of HACCP
procedures. Records form a base for analysis of trends and investigation of any food safety
incidents that might have occurred. A unique reference number has to be allocated to each
HACCP record.
Food Safety Management System (IS/ISO:22000:2005): Food safety Management system
IS/ISO:22000:2005 has been developed by ISO in close cooperation with Codex alimentarius
commission..A major benefit of ISO:22000 is that it make it easier for organizations worldwide
to implement the codex HACCP system for food hygiene in a harmonized manner, which
doesn’t vary with the country of origin of food product. The ISO 22000:2005 standard was
published in Sept. ’2005 with the aim to unify principles of the quality systems used in the food
industry. It is an optional standard because it goes beyond the framework of the GHP/GMP and
HACCP requirements. Its range encompasses:
The PRP,GMP,GHP and other good practices
The HACCP system
The identification system(Traceability system)
The Quality management system (ISO 9001:2008)
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Comparison Of ISO 22000 with HACCP: The main changes of ISO 22000 compared with HACCP
are the following:
1. Extension of the scope to include all the food businesses from feed and primary production as well as the organizations indirectly involved in the food chain, such as suppliers of equipment, food packaging, insecticides, veterinary drugs, detergents ⁄ disinfectants, which could introduce possible dangers in the food chain either with the supply of raw materials or their services.
2. The hazards that require control are those managed not only by CCPs (either with continuous monitoring or with an adequate frequency for an immediate implementation of corrective actions), but also through prerequisite programmes (PRPs) and operational pre-requisite programmes (OPRPs).
3. In this new standard there is provision of crisis management procedures in the case that external dangers turn up, dangers which are not included in hazard analysis, such as natural destruction, environmental pollution.
4. Additional requirements for external communication exist between the food organizations and the relevant authorities involved in food safety beyond the internal communication requirements.
The incorporation of PRPs and oPRPs in the ISO 22000 has made the system more flexible since
a smaller number of CCPs is introduced.
Advantages of ISO 22000
1. Optimum distribution of resources inside the food chain/ organization. 2. Effective communications of suppliers, clients, authorities and other authorities
involved. 3. Focus on the PRPs, conditions and hygiene measures, planning of preventive actions
with the aim of eliminating any possible failures. 4. Better documentation. 5. More efficient and dynamic control of Food safety hazards 6. It is aligned with both ISO 9001:2008 and HACCP
Conclusion: HACCP is a management system to assess hazards and establish control systems
throughout the food chain from primary production to final consumption that focus on
preventive measures rather than relying mainly on end-product testing. It enhances food
safety besides better utilization of resources and timely response to problems in the system. It
is now widely embraced by the food industries and by the government regulatory agencies
around the world as a most cost effective means of minimizing the occurrence of identifiable
food borne biological, chemical and physical hazards and maximizing product safety. The new
standard ISO:22000 ‘Food Safety Management Systems – Requirements for Food Chain
Organizations’ aims at the proper implementation worldwide of the internationally well-known
principles of HACCP from the food chain organizations to provide safe food to the consumers.
ISO Secretary–General Alan Bryden commented “Notably FAO/WHO‘s Codex Alimentarius
commission, is responsible for well known HACCP system for food hygiene. Thanks to the
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strong partnership between ISO and Codex, ISO 22000 will facilitate the implementation of
HACCP and the food hygiene principles developed by this pre-eminent body in this field”.
References:
CAC/RCP-1(1969) Rev.3 (1997) Recommended International Code of Practice General Principles of Food Hygiene. Codex Alimentarius Commission Rome.
JOINT FAO/WHO Food Standards Programme Codex Committee on Food Hygiene (2002) Proposed draft code of hygienic practices for milk and milk products - 35
th Session Washington DC, USA Oct - 2002
SAREEN,S (2002) Meeting global food safety requirements - challenges for India. Presented in Asian Seminar on Safe and high quality food for international trade held at New Delhi on 4-5
th April 2002
IS/ISO 22000:2005 Food Safety Management Systems –requirements for any organization in food chain, (reprint June 2007),BIS,New Delhi.
IS:15000:1998 Food Hygiene-Hazard Analysis and Critical Control Point (HACCP)-System and guidelines for its application
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Conventional and Advanced Technique for Enumeration of Spoilage and Pathogenic
Bacteria in Milk
Raghu, H. V, Naresh Kumar, Mandeep, B., Ramakant, L, V. K. Singh
Dairy Microbiology Division, NDRI, Karnal
Introduction: Food spoilage is an enormous economic problem worldwide. Through microbial
activity alone, approximately one-fourth of the world’s food supply is lost. Milk is a highly
nutritious food that serves as an excellent growth medium for a wide range of Microorganisms.
The microbiological quality of milk and dairy products is influenced by the initial flora of raw milk,
the processing conditions, and post-heat treatment contamination. Undesirable microbes that can
cause spoilage of dairy products include Gram-negative psychrotrophs, coliforms, lactic acid
bacteria, yeasts, and molds. Pathogens are virtually present everywhere, reaching every aspect of
life. Potentially threatening bacteria in foods, soil and in water has historically outrun any detection
efforts resulting in unwarranted deaths and illness. Current trends in nutrition and foods
technology are increasing the demand on food microbiologist to ensure a safe food supply.
Bacterial pathogens encountered to human’s illness in the last decades are through consumption
of undercooked or minimally processed dairy products such as soft cheeses made with
unpasteurized milk, ice cream, butter, etc.). However, the presence of pathogens in these products
is a serious concern since these products do not receive any further treatment before
consumption. The dairy products are important reservoir for many of the food borne pathogens
such as Salmonella spp., Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica,
pathogenic strains of Escherichia coli and Enterotoxigenic strains of Staphylococcus aureus may
also be found in milk and dairy products. The infectious doses of many of these pathogens are very
low (10-1000 cfu/ml). Further, consumers have become much more aware of food safety issues as
a result of publicity given to food borne diseases in the media. Hence, we are in urgent need to
implement programmes such as HACCP as a part of Good Manufacturing Practices (GMP) and
Sanitary and Phytosanitary measures (SPS) to monitor the quality of the products produced for the
presence of spoilage and pathogens (APHA,1987). This is an ideal situation wherein rapid methods
such as online monitoring system can be useful to quickly screen large number of samples and
thereby enhancing processing efficiency. The analysis of food for the presence of spoilage and
pathogenic bacteria is a standard practices for ensuring safety and quality. However the advent of
technology has greatly altered food testing methods and there are numerous companies that are
actively developing assay that are specific faster and often more sensitive than conventional
methods in testing for microbial contaminants in food.
A rapid method can be an assay that gives instant or real time results, but on the other hand it can
also be a simple modification of a procedure that reduces the assay time. These rapid methods not
only deals with the early detection and enumeration of microorganisms, but also with the
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characterisation of isolates by use of microbiological, biochemical, biophysical, molecular and
immunological methods.
Conventional Methods: conventional bacterial testing methods rely on specific media to
enumerate and isolate viable bacterial cells in dairy food. These methods are very sensitive,
inexpensive and can give both qualitative and quantitative information on the number and the
nature of microorganism present in the dairy food sample. Traditional methods for the
detection of bacterial involve the following basic steps: pre-enrichment, selective enrichment,
selective plating, biochemical screening and serological confirmation. Hence, a complete series
of tests is often required before any identification can be confirmed. These conventional
methods require several days to give results because they rely on the ability of the organisms to
multiply to visible colonies. Moreover, culture medium preparation, inoculation of plates and
colony counting makes these methods labour intensive. Conventional methods generally
regarded as the golden standard often takes days to complete the identification of viable
pathogens. Any modification that reduces the analysis time that can technically be called rapid
methods.
Detection and Enumeration of Microorganisms: There are several methods for detection and
enumeration of microorganisms in food. The method that is used depends on the purpose of the
testing.
1. Direct Enumeration: Using direct microscopic counts (DMC), Coulter counter etc. allows a
rapid estimation of all viable and nonviable cells. Identification through staining and
observation of morphology also possible with DMC.
2. Viable Enumeration: The use of standard plate counts, most probable number (MPN),
membrane filtration, plate loop methods, spiral plating etc., allows the estimation of only
viable cells. As with direct enumeration, these methods can be used in the food industry to
enumerate fermentation, spoilage, pathogenic, and indicator organisms.
3. Metabolic Activity Measurement: An estimation of metabolic activity of the total cell
population is possible using Resazurin, Methylene blue dye reduction, acid production,
electrical impedance etc. The level of bacterial activity can be used to assess the keeping
quality and freshness of milk. Toxin levels can also be measured, indicating the presence of
toxin producing pathogens.
4. Cellular Constituents Measurement: Using the Luciferase test to measure ATP is one
example of the rapid and sensitive tests available that will indicate the presence of even
one pathogenic bacterial cell.
Isolation of microorganisms is an important preliminary step in the identification of most food
spoilage and pathogenic organisms. This can be done using a simple streak plate method.
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Constraints in food analysis: Microbiological analysis of food especially for particular pathogenic
species remains a challenging task for virtually all assay and technologies. The problems may be
due to the fact that:
Bacteria are not uniformly distributed in the food
Heterogeneity of food matrices
a. Ingredients such as proteins, carbohydrates, fats, oil, chemicals, preservatives
b. Physical form of food (powder, liquid, gel, semisolid or other forms)
c. Difference in viscosity due to fats and oils, which may interfere in proper mixing
Presence of indigenous microbes which do not cause health risk but their presence often
interferes with the selective identification and isolation of specific pathogens, which are
usually found in low numbers
Need for Rapid methods: Since traditional enumeration procedures often require rather long
incubation times, there is a need for rapid methods to detect food borne pathogens, indicator and
spoilage organisms. In most food legislation, microbiological criteria are stated for food borne
pathogens, but to a lesser extent for indicator organisms. The laboratory needs to choose whether
to use traditional or rapid methods. However, due to the very low numbers of some food borne
pathogens present in a product (e.g. Salmonella in milk powder or Cronobacter in infant formula),
time-consuming enrichment procedures are necessary (the time varying from 1-2 days, depending
on the type of enrichment). Many rapid methods, mainly immunological and/or DNA-based, are
commercially available for the detection of food borne pathogens. However, traditional methods
are still first choice for the enumeration of indicator and spoilage organisms. The effective testing
of bacteria requires methods of analysis that can meet a number challenging criterions. Time and
sensitivity of analysis (Table 1) are the most important limitation related to the usefulness of
microbial testing. The food industry is in need of more rapid methods which are sensitive for the
following reasons:
To provide immediate information on the possible presence of pathogen in raw material
and finished products
Low numbers of pathogenic bacteria are often present in complex biological environment
along with many other non –pathogenic organism
The presence of even a single pathogenic organisms in the food may be a infectious dose
For monitoring of process control, cleaning and hygienic practices during manufacture
To reduce human errors and to save time and labour cost
Table.1 Characteristics of some alternative and rapid methods
Method Detection limit
(cfu/mL or g)
Time before results Specificity
Plating technique 1 1-3days Good
Bioluminescence 104 ½ h No
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Flow cytometry 102- 103 ½ h Good
DEFT 103- 104 ½ h No
Impedance 1 6-24h Moderate/good
Immunological methods 104 1-2h Moderate/good
Nucleic acid based assay 103 6-12h excellent
Rapids methods can be classified into the following categories:
1. Modified and automated conventional methods
2. Biosensor’s
a. Bioluminescence biosensor
b. Impedometric (electrical impedance)
c. Piezoelectric biosensors
i. Flow cytometry
ii. Solid phase cytometry
iii. Electronic nose
3. Immunological methods
4. Nucleic acid based assays
a. DNA hybridisation
b. Polymerase chain reaction (PCR)
c. DNA micro assays (Gene Chip technology)
Modified and automated conventional methods: Many attempts have been made to improve
laboratory efficiency by making the procedures for traditional agar based methods more
convenient, user friendly and to reduce the cost material and labour. Several modification in
sample preparation, plating technique, counting and identification system have made these
conventional methods faster and easier.
Sample Preparation: Gravimetric diluters-automatically adds the correct amount of diluents to
the test sample before homogenization.
Stomacher: massages sample in a sterile disposable bag eliminating need to sterilize and to use
blender cups.
Pulsifier: this apparatus beats the outside of a sterile disposal bag at high frequency (3500rpm)
producing a combination of shock waves and intense stirring which drives the microbes into
suspension.
Plating technique: there are several methods of adding sample homogenate to the agar plates.
Spiral plater-this deposits a small volume onto the surface of the agar in spiral fashion such that
there is a dilution ratio of 104 from the centre to edge of the plate. The colonies appearing
along the spiral pathway can be counted manually or electronically. As the volume dispensed at
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any volume at any point is known, this technique eliminates the need fro serial dilution before
plating and less time required fro colony counting.
Use of fluorogenic and Chromogenic substrate: in selective media detection, enumeration and
identification. This eliminates the use of subculture media and further biochemical tests. These
compound yield bright color fluorescent products when reacting with specific bacterial
enzymes or metabolites. Fluorogenic enzyme substrates are derived from coumarin, such as 4-
methylumbelliferrone, while Chromogenic enzymes compound are mainly phenol derivatives.
Petrifilm: Petrifilm plates are designed to be as accurate as conventional plating methods.
Ingredients usually vary from plate to plate depending on what micro-organism is being
cultured, but generally a Petrifilm plates are a thin film, sample ready, dehydrated, version of
the conventional Petri dish agar plate. Petrifilm comprises a cold-water-soluble gelling agent,
nutrients, and indicators for activity and enumeration. A typical Petrifilm plate has a 10 cm(H) ×
7.5 cm(W) bottom film which contains a foam barrier accommodating the plating surface, the
plating surface itself (a circular area of about 20 cm2), and a top film which encloses the sample
within the Petrifilm. A 1 cm × 1 cm yellow grid is printed on the back of the plate to assist
enumeration. A plastic “spreader” is also used to spread the inoculum evenly. Petrifilm plates
have International recognition such as AOAC and AFNOR, and are widely used in industry in
Australia and Internationally. One millilitre of liquid sample is placed on the centre of film
system and the dehydrated growth of microorganisms. After incubation, the colonies can be
counted directly from the film system is in conventional plates. These Petrifilm can be used fro
aerobic plate count, coliform, yeast and mould, Enterobacteriacea count, Staphylococcus
aureus, Listeria monocytogenes etc.
Biosensors: Biosensors are defined as indicators of biological compounds that can be as
simple as temperature-sensitive paint or as complex as DNA-RNA probes. Food
microbiologists are constantly seeking rapid and reliable automated systems for the detection
of biological activity. Bio- sensors provide sensitive, miniaturized systems that can be used
to detect un- wanted microbial activity or the presence of a biologically active compound,
such as glucose or a pesticide. Immunodiagnostics and enzyme biosensors are two of the
leading technologies that have had the greatest impact on the food industry.
The use of these two systems has reduced the time for detection of pathogens such as
Salmonella to 24 h and has provided detection of biological compounds such as cholesterol or
chymotrypsin. The continued development of biosensor technology will soon make available
“on-line quality control” of food production, which will not only reduce cost of food
production but will also provide greater safety and increased food quality.
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Bioluminescence: Chemiluminescence is the measurement of light emitted from a chemical
reaction. When caused by biological enzymatically catalyzed reaction, this chemical reaction is
often referred to as bioluminescence. One example of a biosensor system utilizing
Chemiluminescence is the luciferase system. In this system, ATP from viable microorganisms is
detected and quantified by addition of the sample to the cofactor luciferin and the enzyme
Luciferase:
Luciferin + Luciferase + ATP 3 AMP + light + C02
This biosensor is capable of detecting bacteria in the range of 104 ceIls/ml in only a few minutes.
ATP Bioluminescence: All living cells contain the molecule ATP. This molecule may be analysed
simply using an enzyme and coenzyme complex (Luciferase-luciferrin) found in the tail of firefly
(Photinus pyralis). The total light output of the sample is directly proportional to the amount of
ATP present and can be quantified by luminometers. At least 104 cells/mL are required to
produce a signal. This system lacks specificity, but because of rapid response time for obtaining
results, this system is very suitable for on-line monitoring of HACCP programme. This technique
has a detection limit of 1 pg ATP which is equivalent to 1000bacterial cells. ATP is present in
both non-microbial and microbial cells. To determine microbial ATP selective extraction is used.
First, non-microbial ATP is extracted with non-ionic detergent and then destroyed with high
levels of potato ATPase for 5 minutes. Subsequently microbial ATP is extracted using either
trichloro-acetioc acid (5%) or an organic solvent (ethanol, acetone or chloroform).
Bacterial Bioluminescence: The genes responsible for bacterial bioluminescence (lux gene) has
been identified and cloned. The DNA carrying this gene can be introduced into host specific
phages. These phages do not posses the intracellular biochemistry necessary to express this
gene, hence they remain dark. However, on transfer of lux gene to the host bacterium during
infection results in light emission that can be easily detected by luminometers. This technique
can be detect 1X 102 cells fro 60min. the specificity of this assay depends on phage specificity
e.g. bacteriophage p22 is specific for Salmonella typhimurium.
Fibre Optic Biosensors: Fibre optic biosensor is one of the first commercially available
biosensors for the detection of foodborne pathogens. The basic principle of the fibre optic
sensor is that when light propagates through the core of the optic fibre i.e. wave guide, it
generates an evanescent filed outside the surface of the wave guide. The wave guides are
generally made up of polystyrene fibres or glass slides. When fluorescent labelles analytes such
as pathogens or toxins bound to the surface of the waveguide are excited by the evanescent
wave generated by a laser (635nm) and emit fluorescent signal, the signal travels back through
the waveguide in high order mode to be detected by a fluorescent detector in real time.
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Surface plasmon Resonance (SPR) Sensor: SPR is a phenomenon that occurs during optical
illumination of a metal surface and it can be used for bimolecular interaction analysis.
Receptors or antibodies immobilized on the surface of thin film of a precious metal (gold)
deposited on the reflecting surface is located above or below a high index resonant layer and
low index coupling layer. When a visible or near-infrared radiation (IR) is passed through the
wave guide in such a way, it causing an internal total reflection on the surface of plasma or
cloud of electrons on the high index metal surface and the resonance effect causes a strong
absorbance. The exact wavelength of this absorption depends on the angle of incidence, the
metal, the amount of capture molecule immobilized on the surface and the surrounding
material. The presence of ligands or antigens interacting with the receptor or antibody causes
shift in the resonance to longer wavelengths and the amount of shift can be related to the
concentration of the bound molecules. SPR based sensors are governed by two basic principles:
wavelength interrogation and angle interrogation. Wavelength interrogation uses a fixed angle
of incidence but measures spectral changes, while in angle interrogation, a fixed wavelength, a
fixed wavelength is sued but the angle of reflectance is monitored. Most of the commercial SPR
systems are operated based on the angle interrogation mode. SPR based sensor allows real
time or near real-time detection of binding events between two molecules. The detection
system is label free, this eliminating the need for additional reagents, assay steps and time. The
sensor can be reused for the same analyte repeatedly. it is highly sensitive and it can detect
molecule in the fentomolar range.
Electrical Impedance Biosensor: Impedance microbiology detects microbes either directly due
to production of ions from metabolic end products or indirectly from liberation of Co2.
Microbial metabolism usually results in an increase in both conductance and capacitance,
causing a decrease in impedance. A bridge circuit usually measures impedance. This method is
well suited for detection of bacteria in milk samples and to monitor quality and detect specific
food pathogens.
In this method, a population of microbes is provided with nutrients (non-electrolyte) like
lactose and microbes may utilize that nutrients and convert it to lactic acid (ionic form) thus
changing the impedance. This impedance is measured over a period of 20h after inoculation in
specific media. Since this does not involve serial dilution, this technique is simple to perform
and faster than agar plate count. This system is capable of analysing hundreds of sample at the
same time since the instrument (Bactometer) is computer driven and automated to enable
continuous monitoring. Typically most impedance analysis of food samples can be completed in
24h. This technique is not suited for testing samples with low number of microorganism and
that the food matrix may interfere with the analysis.
Impedance-based Biochip sensor: though the concept of this detection method is old, now
getting wider popularity. Impedance is based on the changes in conductance in a medium due
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to the microbial breakdown of inert substrates into electrically charged ionic compounds and
acidic by-products. The principle of all impedance-based system is that they measure the
relative or absolute changes in conductance, impedance, or capacitance at regular intervals. So
threshold value for the detection of target pathogens is mainly depends on initial inoculums
and the physiological state of the cells. In media-based impedance methods, bacterial
metabolism results in increased conductance and capacitance, with decreased impedance. The
major advantage of this system is that it allows the detection of only the viable cells, which is
the major concern in food safety.
Piezoelectric biosensors: This system is very attractive and offers a real time output, simplicity
of use and cost effectiveness. The general principle is based on coating the surface of
piezoelectric sensor with a selective binding substances for example antibodies to bacteria and
then placing it in a solution containing bacteria. The bacteria will bind to the antibodies and the
mass of the crystal will increase while the resonance frequency of oscillation will decrease
proportionally.
Flow cytometry: this may be considered as the form of automated fluorescence microscopy in
which instead of sample being fixed to slide, it is injected into a fluid (dye), which passes
through a sensing medium of flow cell. In flow cytometer the cells are carried by laminar flow of
water through a focus of light the wavelength of which matches the absorption spectrum of the
dye with which the cells have been stained. On passing through the focus each cell emits a
pulse of fluorescence and the scattered light is collected by lenses and directed on to selective
detectors (Photomultiplier tubes). These edetctors transform the light pulses into an equivalent
electrical signal. The light scattering of the cells gives information on their size, shape and
structure. This system is highly effective means fro rapid analysis of individual cells at the rate
of thousand cells per second.
Immunological methods: Immunological methods rely on the specific binding of an antibody to
an antigen. Immunoassay refers to the qualitative and quantitative determination of antigen
and antibody in a specimen by immunological reaction. The increased use of immunosensor for
rapid detection of microbes is due to:
Development of new and highly sensitive assays
Mechanical devices to automate tedious steps
Techniques to construct predetermined antibodies of specificity (Hybridoma
technology)
Polyclonal antibodies contain a collection of antibodies having different cellular origin and
therefore somewhat different specificity. The development of monoclonal antibodies greatly
enhanced the filed of immunology by providing a constant and reliable source of characterised
antibodies.
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Nucleic Acid based assay: advances in biotechnology have lead to the development of a
diverse array of assays for detection of food pathogens. Rapid analysis that used nucleic acid
hybridisation and nucleic acid amplification technique offer more sensitive and specificity than
culture based method as well as dramatic reduction in the time to get results. The essential
principle of nucleic acid based assay is the specific formation of double stranded nucleic acid
molecules from two complementary single stranded under defined physical and chemical
conditions. There are many nucleic acid based assays only DNA probe and PCR has been
developed commercially for detecting food pathogens. Recently number of DNA based
molecular typing methods, including pulsed filed gel Electroporesis (PFGE). Restriction fragment
length Polymorphisms (RFLP) and ribotyping have also been developed.
Requirement of Alternative and Rapid methods: there are several factors which must be
considered before adapting new alternative or rapid methods:
Accuracy: false- positive and false-negative results must be minimal or preferably zero.
The method must be sensitive as possible and the detection limit as low as possible.
Validation: Test should be validated against standard test and evaluated by
collaborative studies. In these studies, preferences should be given to naturally
contaminated food specimens; the tests are then performed under conditions in which
users will apply them. Results obtained with samples containing a low level
contamination should be emphasized, since there is sufficient evidence that in most
cases high number of target cells will lead top positive test results.
Speed: rapid tests for the detection of pathogens or toxins should give accurate results
within hours or at the utmost one day. However, many detection need an overnight
enrichment for resuscitation and amplification of the target pathogens, are they rely on
the presence of at lest 104-105 organisms/mL for the result should be reliable.
Automation and computerization: The ability to test many sample at the same time.
Many systems utilizing the microtitre plate format can handle 96 samples at one time.
However, for smaller laboratories, the availability of single unit test is also very
important.
Sample matrix: New systems should give a good performance of the matrices to be
labelled. Baseline extinction values may depend on the type of food being tested.
Background flora natural substances or debris can interfere with the test method and
invalidated the test result.
Cost
Simplicity: methods should be user friendly
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Conclusion: The food borne pathogens are growing concern for human illness and death. There
is an increasing demand to ensure safe food supply. Current methods for the rapid detection of
spoilage in meats are inadequate and all have the same recurring theme in that they are time
consuming, labour intensive and, therefore, give retrospective information. Despite this
knowledge the ability to correlate biochemical change with microbial biomass is a complex
problem, and perhaps only very recently surmountable. There is continuous development of
methods for the rapid and reliable detection of food borne pathogens. Improvements in the
filed of biosensors, immunology, molecular biology, automation and computer technology
continue to have a positive effect on the development of faster, more sensitive and more
convenient methods in microbiological analysis of milk and milk products. With continuous
advances in analytical instrumentation coupled with the realization that miniaturization
instrumentation is assuming increasing importance, as computers processing speeds get more
powerful, as our understanding of complex multivariate spectroscopic data and their machine
learning interpretation deepens, it will not be long before the so-called ‘rapid’ detection
methods used at present are replaced by those which are truly rapid and detect quantitatively
microbial spoilage in milk and milk products within seconds as opposed to hours.
References:
P. K. Mandal, A.K. Biswas, K. Choi and U K. Pal (2011). Methods for rapid detection of food borne pathogens:
An overview. American Journal of Food Technology 6(2): 87-102.
David I. Ellis and Royston Goodacre (2001). Rapid and quantitative detection of the microbial spoilage of
muscle foods: current status and future trends. Trends in Food Science & Technology 12 (2001) 414–424.
Invitski, D., L.A. Harnid, P. Atanasov and E. Wilkins (1999). Biosensors for detection of Pathogenic Bacteria.
Biosensors and Bioelectronics, 14: 599-624.
Ritcher, E. R. (1993). Biosensors: Application for dairy Industry. J. Dairy Sci., 76:3114-3117
232
Preparation and Characterization of Gold Nanoparticles, their Conjugation with
Antibodies and Construction of Lateral Flow Devices
Priyanka Singh Rao1, Swapnil Sonar2, Y.S. Rajput2 and Rajan Sharma1
Dairy Chemistry Division1; Animal Biochemistry Division2 , NDRI, Karnal
Lateral Flow Assays also known as Immunochromatographic assays are a simple device
intended to detect the presence (or absence) of a target analyte in sample (matrix).
Traditionally designed assays are composed of a variety of materials, each serving one or more
purposes. Colloidal gold is the most widely used label today in commercial lateral flow
immunoassays for many reasons. It is fairly easy and inexpensive to prepare in the laboratory.
The color is intense, and no development process is needed for visualization. A large body of
protocols exist in the literature for its conjugation and application. Gold colloids are formed by
the reduction of gold tetrachloric acid through a “nucleation” process. The size and shape of
the colloids depend on the type and amount of reducer used. The label is very stable in liquid or
dried form and is non-bleaching after staining on membranes. An accurate and reproducible
lateral-flow assay requires the use of high-quality gold conjugates. Gold particles can be
produced that range in size from 5 to 100 nm in diameter. The most common size of colloidal
gold particle used is 40 nm. In addition, colloidal gold in unconjugated forms (which are ready
for labeling) and conjugated forms (conjugated with biologicals) are now readily available from
many commercial sources. In addition to the dry parts of a lateral-flow assay, there are also the
biological components that allow the visualization of the results. By virtue of their high levels of
specificity and binding affinities, antibodies are the ideal choice of agent for detection. In
Lateral Flow Assay an antibody molecule is conjugated to a colloidal gold particle. Antibodies
can be polyclonal or monoclonal. Once the antibody has been conjugated, the quality of the
gold conjugate must be assessed before incorporation into the rapid-test assay. Usually,
electron microscopy is employed as a quality-control measure. Conjugation of colloidal gold
particles and antibodies depends on the availability and accessibility of three amino acid
residues—lysine, tryptophan, and cysteine. Once a high-quality antibody–gold conjugate is
formed, it can be applied to the conjugate pad either by soaking or by spraying. The drying
process that follows is essential. The lateral flow immunoassay devices are compact and easily
portable. A test strip typically consists of a plastic backing holding together a sample pad for
deposition of sample fluids, a conjugate pad pre treated with sample detection particles, a
microporous membrane containing sample capturing reagents, and an absorbent pad at the
distal end serving to collect excess fluids.
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A. Preparation of Gold Nano Particle
Material: All the chemicals required for the preparation for gold nanoparticles can be procured
from Sigma-Aldrich Ltd.
Reagents:
1. Stock gold chloride (tetrachloroauric acid trihydrate, Mol.Wt.393.83; 200 mM) solution- 787.6 mg of HAuCl4.3H2O is dissolved in Millipore water and volume is made up to 10 ml. The stock solution is stored at room temperature.
2. Working gold chloride solution (50 mM) - Stock gold chloride solution is diluted four times with Millipore water.
3. Trisodium citrate dihydrate (38.8 mM; M.W. 294) - 114 mg of trisodium citrate dihydrate is dissolved in 10 ml Millipore water.
Procedure:
1. Prepare aqua regia by mixing 3:1 concentrated HCl:HNO3 in a large beaker in a fume hood. Be
extremely careful when preparing and working with aqua regia. Wear goggles and gloves,
and perform the experiment in a fume hood. Aqua regia should be freshly prepared and
should never be stored in a closed vessel. The capped aqua regia bottle may explode. Render
it safe by dilution and neutralization.
2. Soak the 200 ml two-neck flask, magnetic stir bar, stopper and condenser in aqua regia for at
least 15 min. Rinse the glassware with copious amount of deionized water and then
Millipore-filtered water. Obtaining high-quality nanoparticles is the first important step
towards the success of the experiment. Care should be taken to make sure that no
contamination is introduced during nanoparticle synthesis.
3. Load 98 ml of Millipore water into the two-neck flask. Add 2 ml of 50 mM HAuCl4 solution so
that the final HAuCl4 concentration is 1 mM.
4. Connect the condenser to one neck of the flask, and place the stopper in the other neck. Put
the flask on the hot plate to reflux while stirring.
5. When the solution begins to reflux, remove the stopper. Quickly add 10 ml of 38.8 mM
sodium citrate, and replace the stopper. The color should change from pale yellow to deep
red in 1 min. Allow the system to reflux for another 20 min.
6. Turn off heating and allow the system to cool to room temperature (23–25 °C) under stirring.
B. Characterization of gold nanoparticles
The diameter of such prepared nanoparticles is ~13 nm. The extinction value of the 520 nm
plasmon peak is 3.8, and the nanoparticle concentration is ~13 nM. The colour should be
burgundy red, and the nanoparticle shape should be spherical under transmission electron
microscopy (TEM). All gold sols display a single absorption peak in the visible range between
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510 and 550 nm, and the absorption maximum shifts to a longer wavelength with increasing
particle size. The relative uniformity of the particles or the range of particles can be gauged by
the width of the absorption spectra: the sharper the band, the more uniform the particles. The
relative concentration of each batch of colloidal gold can be determined by absorbance at 520
nm. Various batches can be brought to the same relative concentration by the addition of de-
ionized water.
C. Labelling of gold nanoparticles with antibody
Reagents
1. NaOH (0.2 N) 2. Carbonate Buffer (5 mM)
3. Tris-HCl buffer (pH 8.2) containing 1% BSA and 0.1 sodium azide
Procedure: Adjust the pH of Gold nanoparticle using 0.2 N NaOH to 8.5. 6 µl (20 µg) of affinity
purified antibody (against glycomacropeptide) is diluted to 160 µl with carbonate buffer and
added to nanoparticles. The mixture is incubated overnight at 4:C. Centrifuge the contents at
7,000 rpm at 15ºC for 15 minutes. Suspend the pellet in carbonate buffer and again centrifuge
at 7,000 rpm at 15ºC for 15 minutes. Decant the supernatant and dissolve the pellet in Tris-HCl
buffer. Store the antibody labelled nanoparticles at 4°C till further use.
D. Construction and working of lateral flow strip
Materials: All the material required for the construction of lateral flow strip, can be purchased
from Milipore India Pvt. Ltd. Bangalore.
Procedure: Lateral flow assays are composed of a variety of materials, each serving one or
more purposes. The parts overlap onto one another and are mounted on a backing card using a
pressure-sensitive adhesive. Each component of the test, including membrane, backing
substrate, and each of the pad materials, has a defined dimension. On conjugation pad Gold
nanoparticles are coated with antigen specific antibodies.Test line is also coated with the
antigen specific antibody and Control line with species specific anti-antibody for the antibody in
the particulate conjugate.
Side view of test-strip construction
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The sample is treated to make it compatible with the rest of the test. The treated sample
migrates through this region to the conjugate pad, where a particulate conjugate has been
immobilized. The sample interacts with the conjugate as both migrate into the next section of
the strip, which is the membrane. Excess reagents move past the capture lines and are
entrapped in the wick or absorbent pad. The control line indicates that the test developed
properly and test line indicates that the test is positive.
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Detection of Adulterants in Milk by Rapid Methods
Rajan Sharma and Amit K. Barui
Dairy Chemistry Division, NDRI, Karnal
Introduction
Adulteration in milk has become a common feature for fulfilling the milk demands of over
populated country. For milk vendors and shop-keepers, adulteration of milk with water to
increase the quantity in order to supply milk in large number of house-holds also has become a
common practice. The lack of timely action against the adulterators by the Public Health
Departments and lack of easier and rapid methods for detection of adulteration further
encouraged this menace. Common man i.e. consumers are not aware of the methods and
chemicals used in the methods. Now in NDRI Karnal, the procedures for the detection of
various adulterants and neutralizers have been simplified to be easily adopted by the house-
holds. The prepared reagents as well as a KIT for the detection of adulterants and neutralizers
are available in the Dairy Chemistry Division of NDRI, Karnal.
Preservatives
A. Test for formaldehyde
Formalin (40% water solution of formaldehyde) is generally used by Public Health
Departments to preserve the milk samples for chemical analysis purpose. Formaldehyde is very
poisonous chemical. Though, it can preserve the milk for very long time, it should never be
added to milk meant for processing due to its poisonous property. Moreover, it affects the
quality of the milk products. If milk kept at room temperature (25 to 35ºC) for longer time, did
not sour, then that milk must be tested for formaldehyde by the following simple method:
Method I: Leach test
1. Take about 5 ml of milk in a test tube.
2. Add to it equal volume of Conc. HCl containing 1 ml of 10% ferric chloride solution to each
500 ml of the acid.
3. Keep the tube in boiling water bath for about 3-4 min.
4. Observe the colour of the solution in the tube. The tube containing pure sample will turns
yellowish. The positive sample (i.e. containing HCHO) will turn violet to brown black.
Method II: Chromotropic acid test
Reagent: Saturated solution of 1,8-dihydroxynaphthalene-3,6-disulphonic acid in about 72%
sulphuric acid (about 500 mg/100 ml). Light straw-coloured solution should result.
1. Take one ml of milk sample in a test tube. Add 1 ml of the Chromotropic acid reagent and
mix well.
2. Appearance of yellow colour confirms the presence of formalin in the sample, whereas;
control sample will remain colourless.
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B. Test for hydrogen peroxide
Hydrogen Peroxide is a preservative, but as per PFA rule it is not permitted to be added
in milk. Hence if it is found, then milk is said to be adulterated.
Method I
Reagent: Para-phenylenediamine solution (2%, Aq, w/v).
Procedure:
1. Add to about 5 ml of milk in a test tube, an equal volume of raw milk, followed by five drops
of a 2 % of para-phenylenediamine.
2. A blue colour is developed in the presence of hydrogen peroxide.
Note: It is unlikely that the addition of less than 0.1% of H2O2 to milk could be detected after 24
h, owing to the action of peroxidase and catalase which stimulate its conversion into water. If
more than 0.2% H2O2 is added, some will persist for considerable long time. Owing to the fact
that larger amount of H2O2 are known to destroy peroxidase, it is always advisable to add to the
sample an equal volume of raw unpreserved milk and to follow with a few drops of a 0.2%
solution of para-phenylenediamine. Under these circumstances a blue colour will develop
immediately if H2O2 is added.
Method II
A method using potassium iodide and starch was standardized for the detection of
hydrogen peroxide in milk.
Procedure: Take one ml milk sample in a test tube. Add one ml of potassium iodide-starch
reagent (mix equal volumes of 20% potassium iodide solution and 1% starch solution) to the
test tube. Appearance of blue colour indicates the presence of hydrogen peroxide in the milk
sample whereas control samples remain colorless.
C. Detection of Neutralizers
Alkali in various forms like sodium carbonate, sodium bicarbonate, sodium hydroxide
and lime are used to neutralize developed acidity in milk. Detection of such neutralizers can be
made by the following two tests.
Method I. Rosalic Acid Test:
Reagents: Ethanol (95%), Rosalic acid solution (1% in alcohol).
Procedure:
1. Take in test tube about 5 ml milk and mix with 5-ml ethanol followed by 2-3 drops of rosalic
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acid solution.
2. Formation of rose red colour indicates the presence of alkali as neutralizer. Pure milk
produces brownish or brownish yellow colour only.
Rosalic acid is an organic dye, which is used as an indicator-changing colour at pH 7.0 to 8.0.
Hence, milk made even faintly alkaline by addition of neutralizers can be detected due to
formation of rose red colour with rosalic acid solution.
Method II. Ash alkalinity test
Neutralization of milk whether with lime, soda, or caustic soda, invariably increases the
ash content and the total alkalinity of the ash from a fixed quantity.
Reagent: HCl (standard, 0.1 N), Phenolphthalein indicator.
Procedure:
1. Pipette 20 ml of milk into a porcelain basin and evaporate to dryness on boiling water bath.
2. Remove the basin, cool to room temperature and ignite the residue by heating over Bunsen
flame until gray-white ash is obtained.
3. Cool the basin to room temperature. Add to the residue 10-ml of water and disperse the
ash in water by stirring with a glass rod.
4. Titrate the ash dispersed by standard HCl using phenolphthalein indicator. If the volume of
0.1 N HCl required to neutralize the ash dispersate exceeds 1.20 ml; the milk is suspected to
contain neutralizers.
D. Detection of starch or cereal flours
Reagent: Iodine solution (1%), Dissolve 2.5 g potassium iodide in 100 ml water, add to it 1 g
pure iodine crystal, shake well to give a clear solution.
Procedure:
1. Take one ml of well-mixed milk sample in a test tube.
2. Heat the milk to just boiling by holding the tube over flame, and thereafter cool to room
temperature.
3. Add 1-2 drops of 1% iodine solution.
4. Observe the development of colour. Formation of blue-violet colour indicates presence
of starch cereal flours.
E. Detection of cane sugar
Sugar or cane-sugar, is generally added to milk in order to raise the lactometer reading
of the milk which was diluted with water, so that by lactometer reading, the detection of added
water is prevented. In suspected samples, sugar can be easily detected by following method:
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Reagent: Resorcinol, conc. HCl. (or prepare sucrose detecting reagent by dissolving 0.5 g of
resorcinol in about 40 ml of distilled water. Then add 35 ml of 12 N conc. HCl. Make up the
volume to 100 ml using distilled water.)
Procedure:
1. To about 5 ml of milk in a test tube, add 1 ml of conc. HCl and 0.1 g of resorcinol and mix.
2. Place the tube in boiling water bath for 5 min.
3. In the presence of cane sugar, red colour is produced.
Note: The test can be simplified by taking 1 ml of suspected sample of milk is a test tube
followed by the addition 1 ml of sucrose detecting reagent. In the presence of cane sugar, red
colour is produced.
F. Detection of glucose
Glucose being a reducing sugar poses many problems in its detection. Moreover, it is
easily available in commercial form as concentrated syrup. These days adulteration of milk with
glucose is increasing. Now it has become possible to detect Glucose in milk by the following
method:
Reagents:
2. Barfoed’s reagent: Dissolve 24 g cupric acetate in 450 ml boiling water and immediately add
25 ml of 8.5% lactic acid to the hot solution. Shake to dissolve almost all precipitate, cool
and dilute with water to 500 ml. If necessary decant of filter to get a clear solution.
3. Phosphomolybdic acid reagent: Take 35 g ammonium molybdate and 5 g sodium tungstate
in a large beaker; add 200 ml of 10% NaOH solution and 200 ml water. Boil vigorously (20-
60 min) so as to remove nearly whole of ammonia. Cool, dilute with water to about 350 ml.
Add 125 ml conc. H3PO
4 (85%) and dilute further to 500 ml.
Procedure:
1. Take 1 ml of milk sample in a test tube. Add 1 ml of modified Barefoed’s reagent.
2. Heat the mixture for exact 3 min in a boiling water bath and then rapidly cool under tap
water.
3. Add one ml of phosphomolybdic acid reagent to the turbid solution and observe the colour.
4. Immediate formation of deep blue colour indicates the presence of added glucose. In case
of pure milk only faint bluish colour is formed due to the dilution of Barefoed’s reagent.
G. Detection of nitrates (pond water)
Pond water is heavier than the tap water; some unscrupulous persons for adulteration
in milk usually prefer it. However, it can be easily detected by the following method. This
method actually detects nitrates present in the pond water. In the pond water nitrates may
come from fertilizers used in the fields.
Reagent: Diphenylamine: Prepare 2% solution of diphenylamine in conc. sulfuric acid.
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Procedure:
Take 2 ml of milk in a test tube. Rinse the tube with the milk and drain the milk from the test
tube. Add two-three drops of the reagent along the side of the test tube. Deep blue colour will
be formed in presence of nitrate.
H. Detection of Urea in milk
Urea is a natural constituent of milk and it forms a major part of the non-protein
nitrogen of milk. Urea concentration in milk is variable within herd. Urea is one of the
ingredients of synthetic milk along with caustic soda, detergent, sugar and foreign fats.
Adulteration of natural milk with synthetic milk increases the level of urea to such an extent
that on consumption of this adulterated milk causes toxicological hazards. Estimation of urea
concentration in milk may serve as a tool for checking the menace of adulteration of natural
milk with synthetic milk. The average urea content in milk of Karan Swiss, Karan Fries and
Sahiwal cows was reported to be 28.57, 28.79 and 25.39 mg/100 ml (range 20 to 35 mg/100
ml). In buffalo milk, the average urea content was found to be
35.10 mg (range 25 to 40 mg/100 ml). The addition of urea to milk can be detected by using
DMAB method. This method is based on the principle that urea forms a yellow complex with p-
dimethyl aminobenzaldehyde (DMAB) in a low acidic solution at room temperature. The
intensity of yellow colour is measured at 425 nm.
Method I:
Reagent:
1.6% DMAB reagent: Dissolve 1.6 g DMAB in 100-ml ethyl alcohol and add 10-ml conc. HCl.
Procedure:
1. Take equal quantity of milk and equal quantity of 24% TCA in a glass stoppered test tube.
Mix and filter it.
2. Take 3 ml of filtrate in a test tube and add 3 ml of 1.6% DMAB reagent in ethyl alcohol and
HCl. Note the colour obtained.
3. The occurrence of distinct yellow colour indicates the presence of added urea in milk.
Method II:
Reagents
1. 1.6% p-dimethyl aminobenzaldehyde (DMAB): Dissolve 1.6 g DMAB in 50 ml ethanol (95%,
v/v) and add 10 ml concentrated hydrochloric acid (sp. gr. 1.16). Make the final volume to
100 ml with ethanol.
Urea + DMAB
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2. Trichloroacetic acid (TCA): 24% (w/v, aq.).
3. Phosphate buffer (pH 7.0): Dissolve 3.403 g anhydrous potassium dihydrogen
orthophosphate (KH2PO4) and 4.355 g anhydrous di-potassium monohydrogen
orthophosphate (K2HPO4) in distilled water and make the volume to one litre with distilled
water.
4. Diluting reagent: Equal volumes of 24% (w/v) TCA and phosphate buffer (pH 7.0) are mixed
to make the diluting reagent.
5. Standard urea solution (1 mg/ml): Weigh 100 mg of AR Grade quality urea and dissolve in
phosphate buffer (pH 7.0). Make up the volume to 100 ml with the above phosphate buffer.
Procedure:
1. Take 10 ml of milk sample and add 10 ml of 24% TCA in 50 ml glass stoppered test tube. Mix
the content and filter through Whatman filter paper grade 42.
2. Take 5 ml of the above filtrate in a test tube and add 5 ml of 1.6% DMAB reagent.
3. Take the absorbance of the yellow colour so obtained at 425 nm in a spectrophotometer
against reagent blank.
4. For reagent blank take 5 ml of diluting reagent and add 5 ml of 1.6% DMAB reagent.
5. For standard curve preparation, take different concentration of urea solution (0.1, 0.2, 0.4,
0.6, 0.8, 1.0, 1.2, 1.4, 1.6 mg) separately in different test tubes and make the total volume
to 5 ml in each case with diluting reagent. Then add 5 ml of 1.6% DMAB reagent to each
test tube to develop yellow colour. Take the absorbance at 425 nm against the reagent
blank.
6. Draw a standard curve by plotting the absorbance along Y-axis and urea concentration
along X-axis.
Calculation
Read from the graph the concentration of urea (mg) corresponding to absorbance of the
sample.
Say the absorbance for the sample be X and the corresponding concentration from the
standard curve for urea is = Y mg
Therefore, 5 ml of filtrate from sample has = Y mg urea
20 ml of filtrate from sample has = (Y/5) X 20 mg urea
i.e. 10 ml milk has = (Y/5) X 20 mg urea
100 ml of milk sample has = ((Y/5) X 20 X 100)/10 mg urea
= 40 Y mg urea/100 ml of milk
Note: The control (milk sample containing no added urea) showed a slight yellow colour due to
the presence of natural urea in milk.
I. Maltodextrin
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To 5 ml milk sample in a test tube, 2 ml of dilute iodine solution (0.05 N) is added. Appearance
of chocolate red brown colour developed indicates the presence of maltodextrin.
J. Sodium chloride
Take 5 ml of milk and 1 ml of silver nitrate solution (0.1 N). Mix well and add two drops of a
solution of 10% potassium chromate. Yellow colour indicates the presence of added salt.
Otherwise, red colour will appear.
K. Ammonium salts
The added ammonium salts e.g ammonium chloride, ammonium sulfate, ammonium nitrate
and ammonium dihydrogen orthophosphate can be detected in milk by two methods i.e
Nessler’s reagent method and turmeric paper method.
Method I: Nessler’s reagent method
Reagent : Nessler’s reagent: Dissolve the following chemicals separately.
1. 8.0 g of mercuric chloride in 150 ml distilled water.
2. 60.0 g of sodium hydroxide in 150 ml distilled water.
3. 16.0 g of potassium iodide in 150 ml distilled water. Add reagent a to reagent b and mix
well. To this mixture, add reagent c, mix and dilute the contents to 500 ml. Leave this
solution undisturbed and decant the clear upper layer of the solution and store in a
stoppered glass bottle.
Procedure:
Pipette 5 ml of suspected milk sample into a test tube and add 1 ml of Nessler’s reagent.
Mix the contents of the tube thoroughly. Appearance of yellowish or grey colour confirms the
presence of added ammonium salts in milk
Method II. Turmeric paper method
This method is based on the principle that ammonium salts on addition of strong alkali
liberate ammonia and the liberated ammonia turns turmeric paper to pinkish red.
Reagents:
1. Turmeric paper: Dissolve 10 g of pure turmeric powder in 100 ml distilled water and dip
Whatman filter paper Grade 1 strips into it for 2 min. Dry the paper at room temperature.
The dried filter paper is wetted with distilled water before use.
2. Sodium hydroxide solution: 10% (aq.)
Procedure:
Pipette 5 ml of suspected milk sample in a test tube and add 1 ml of 10% sodium
hydroxide solution in such a manner that should not touch the rim of the test tube while
adding. Mix the contents of the tube. Place a piece of wet turmeric paper on the rim of the test
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tube and keep the test tube undisturbed. Observe the change in the colour of the turmeric
paper. Appearance of pinkish red colour confirms the presence of ammonium salt in milk.
L. Sulfate salts
Presence of sulfates in milk can be detected by using barium chloride.
Reagents:
a. Barium chloride (BaCl2.2H2O) solution: 5% (w/v, aq.)
b. Trichloroacetic acid (TCA): 24% (w/v, aq.). Procedure: Take 10 ml of milk in a 50 ml stoppered
test tube and add 10 ml of TCA solution. Filter the coagulated milk through Whatman filter
paper Grade 42. Take 5 ml of clear filtrate and add few drops of barium chloride solution.
Formation of milky-white precipitates indicates the presence of added sulfates like ammonium
sulfate, sodium sulfate, zinc sulfate and magnesium sulfate etc. to milk
M. Detection of refined oil in milk
This method is based on the principle that BR reading of milk fat is comparatively lower
than that of most of the foreign fats/oils. Its adulteration with vegetable and/or animal body
fats/oils significantly increases the BR reading. For taking BR reading of the milk fat the milk fat
is isolated from the specially designed butyrometer which has both ends open. Milk fat after
centrifugation is taken with the help of a capillary and BR reading is noted at 40°C. A correction
factor is added to the observed BR reading. This is done to eliminate the inherent hydrolytic
effect of H2SO
4.
Actual BR at 40°C = Observed BR at 40°C + (0.08 X observed BR at 40°C)
References:
Bector BS, Moti R & Singhal OP 1998 Rapid platform test for the detection/determination of added urea in
milk. Indian Dairyman 50 59-62
IS:1479 (Part II) – 1961 Methods of test for Dairy Industry-Part II Chemical analysis of milk.
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Estimation of Cholesterol Content in Ghee Using a Cholesterol Estimation Kit
Vivek Sharma, Darshan Lal, Manvesh Sihag and Karuna Meghwal
Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana – 132 001.
Principle: Cholesterol is extracted in unsaponifiable matter as free cholesterol. The aliquot of
unsaponifiable matter is made to react with the reagents of the cholesterol estimation kit and the color
developed is measured at 505nm. The absorbance values in the sample and control are used to
calculate the cholesterol content in a given sample of ghee.
Materials: Ghee, Enzymatic Diagnostic kit, Methanol, Potassium hydroxide, Hexane, Teflon line screw
capped tubes.
Equipment: Water bath, Spectrophotometer, Cuvettes.
Protocol for cholesterol estimation in milk fat after saponification using enzymatic diagnostic kit:
Milk fat (0.1-0.15 g) in test tube with teflon lined screw cap
Add 5 ml 5% methanolic KOH and mix
Incubate capped tubes in water bath for 90 °C/ 20 min with shaking every 5 min.
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Cool contents by tap water
Add 1 ml distilled water
Add 5 ml hexane
Vortex the contents for 1 min
Centrifuge at 2000 rpm/ 2 min
Pipette out upper hexane layer
Take 0.2 ml of aliquot in dry test tube
Evaporate solvent under nitrogen at 60-70°C
Add 10 µl of absolute ethanol to dissolve dried residue
Add 1.0 ml cholesterol reagent provided in kit and incubate at 37°C/10min
Cool to room temp (28 – 30°C).
Measure colour (pink) intensity at 505 nm
Calculation: Cholesterol (mg/100 g) = 0.02 × OD of sample × ml of hexane (5 ml) × 100
OD of standard × ml of hexane aliquot (0.2 ml) × Weight of sample (g)
Where, 0.02 is the concentration (mg) of cholesterol in 10 µl of standard solution provided in the kit.
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Production and Quality Evaluation of Direct Vat Starters
Rameshwar Singh, Surajit Mandal, Chand Ram and R.P. Singh
National Collection of Dairy Cultures, NDRI, Karnal – 132001
Introduction
Commercial starter cultures currently available for direct addition to production vats contain
billions of viable bacteria per gram, preserved in a form that could be readily and rapidly
activated in the product mix to perform the functions necessary to transform the product mix
to the desired cultured product. To attain that, the selected starter bacteria need to be grown
in a suitable menstrum to high numbers and to concentrate the cells. The composition of the
media used to grow various bacteria differs. Usually, the materials used in the growth media
consist of food grade, agricultural by-products and their derivatives. The generally used
ingredients in media formulations include nonfat milk, whey, hydrolysates of milk and whey
proteins, soy isolates, soy protein hydrolysates, meat hydrolysates and extracts, egg proteins,
com steep liquor, malt extracts, potato infusions, yeast extracts/yeast autolysates, sugars such
as lactose, glucose, high-fructose com syrup, com sugar, sucrose, and minerals such as
magnesium, manganese, calcium, iron, phosphates, salt, etc. For some fastidious bacteria,
amino acids and vitamins may be included. The phosphates are added to provide mineral
requirements as well as for buffering. For some bacteria, which need unsaturated fatty acids to
protect cell membranes, trace quantities of polysorbates (Tweens) are added. To control
foaming, foodgrade anti foam ingredients may be incorporated.
The medium is then either sterilized by heating at 121°C for a minimum of 15 minutes or heat-
treated at 85-95°C for 45 minutes or subjected to ultrahigh temperature treatment (UHT) for a
few seconds. After heat treatment, the medium is cooled to the incubation temperature. After
the addition of the inoculum, the medium is incubated until the predetermined endpoint is
reached. During incubation, the pH is maintained at a predetermined level (constant neu-
tralization to maintain pH). Generally, the endpoint coincides with the exhaustion of sugar
reflected by the trace of the neutralization curve. The frequency of neutralization reflects the
activity of the culture in the fermenter, and when the frequency decreases, it indicates the near
depletion of the sugar. Samples are usually taken to microscopically examine the fermentate
for cell morphology, for any gross contamination, for a rough estimation of cell numbers, and
for quantitative measurement of sugar content. After ascertaining these, the fermentor is
cooled. The cells are harvested either by centrifugation or by ultrafiltration. The cell
concentrate is obtained in the form of a thick liquid of the consistency of cream and is weighed
and rapidly cooled. Sterile preparations of cryoprotectants (glycerol, nonfat milk, monosodium
glutamate, sugars, etc.) are added, and uniformly mixed with the cell concentrate. The
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concentrate may be filled as such into cans and frozen or frozen in droplet form in liquid
nitrogen (pellets), retrieved, and packaged. The concentrate as such or in pellet form may also
be lyophilized in industrial scale freeze dryers or spray dried.
Protocol
a) Activation of freeze dried cultures/ preserved stock cultures
1) Sterilize the surface of the glass ampoules with alcohol
2) Break the ampoule above the cotton ball plug inside the ampoule
3) Remove the cotton with sterile forcep
4) Transfer a small amount of litmus milk into ampoule with Pasteur's pipette and mix the
contents thoroughly.
5) Transfer the mixture of the ampoule into the tubes with chalk litmus milk
6) Incubate at optimum temperature/ time combination (mesophilic cultures at 25-30°C and
thermophilic cultures at 37-42°C for 18-24 h)
7) After coagulation of milk and appearance of pink colour, store the tubes at refrigerated
condition and sub-culture at 3 months interval
8) Subsequently re-activate in suitable culture medium before use
b) Steps for bulk starter preparation
1. Prepare suitable food grade media – e.g. whey based media
2. Heat the milk to 85-90°C, and holding at that temperature for 30-45 minutes to destroy
contaminants including bacteriophages.
3. Cool to the required incubation temperature.
4. Transfer the media in a fermenter.
5. Inoculate with the culture using aseptic precautions with agitation to uniformly mix in the
inoculums (@ 1-2%).
6. Incubate at thermostatically at optimum growth temperature (30°C for mesophilic or
37/42°C for thermophilic cultures) up to stationary phase of growth (18h) with continuous
neutralization by external supply of base to optimum growth pH.
7. Cool to the desired temperature - preferably 5-7°C, if needed to be held longer before use.
8. Harvest the cell biomass at low temperature by centrifugation (80000-10000 rpm for 15
min) and wash the cells using sterile saline.
9. Suspend the cell biomass in suitable suspending medium e.g. cryoprotective medium (5%
glutamate in skim milk) to get high cell densities (1010 to 1011 cfu per gram).
10. Preserved the cell:
i. Freeze drying
ii. As frozen concentrate
iii. Spray drying
11. Pack the dry material with concentrated cells in suitable pouches under vacuum and
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stored at 5°C or low temperature.
Quality control tests
1) Viable cell numbers
2) Absence of contaminants, pathogens, and extraneous matter
3) Acid-producing and other functional activities
4) Package integrity, accuracy of label information on the package
5) Shelf life of the product according to specification
Starter cultures are produced as commercial starter cultures as DVS/ DVI. Most cultured dairy
products commercial concentrated direct-to-vat-set (DVS) cultures are used. For probiotic
supplementation of cultured dairy products and non-fermented dairy and food products, DVS
cultures of probiotics are used.
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Pathogen Monitoring in Food Systems
S.G.Kulkarni NQMS Project Manager, Nestle India Ltd , Email - [email protected]
The objective of “pathogen monitoring” is to ensure:
Food Safety
Validate, monitor and verify the effectiveness of the pre-requisite programs.
Demonstrate Regulatory compliance Pathogens are one of the major causes of food poisoning and their prevention is moral responsibility of any food manufacture. The consequences of pathogen contamination in food not only include loss of the consumer trust but bring down the reputation and may lead to very huge financial loss. The 7 mandatory elements of pathogen monitoring program are given below
Element Description
1 It must include raw materials, factory environment, production lines, and finished products.
2 It must utilize hygiene monitoring (i.e. "EB", coliforms) to assist in managing the hygienic conditions within the factory.
3 It must be designed to assure that effective source detection strategies for target pathogen(s) are consistently performed.
4 It must have a documentation system that allows for trend analysis of the data.
5 The results obtained from the monitoring activities must be reviewed regularly so that appropriate action can be taken in a timely manner.
6 It must allow for dynamic adaptation depending upon the results and their evaluation.
To be effective the pathogen monitoring plan should include four different types samples as given in the illustration below :-
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The level of sampling will be dependent on various factors as mentioned below:
• Product Category? • Control level: e.g. Minimum, Medium, Maximum? • Hygienic status and size of factory? • Manufacturing environment? • Regulatory requirements? • Past results? • Emerging issues? • Factory events e.g. water leak? • Maintenance on line? • Building work?
Most important is the implementation of pre-requisite programs before pathogen monitoring is put in place and these pre-requisite programs mainly include:
• Hygienic design of factory • Zoning of factory to prevent entry of pathogens • Application of effective cleaning and sanitation practices • Well designed RM selection and monitoring program • Microbiological surveillance • Pest Management • Good House keeping • Personnel training to assure the prerequisites are applied correctly
The pathogen monitoring plan should include the component of investigative samples and remember Quality of samples is more important than Quantity of samples! Application of a sound Pathogen monitoring plan is must for a food manufacturing unit to ensure food safety.
Environmental Monitoring
Line Testing Raw Material Management
Finished Products Testing
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Medical Diagnostics and Clinical Microbiology for Detection of Pathogens
Bhagat Singh1, Chand Ram2 and Renu Singh1
1Microbiology, Institute of Applied Medicines and Research, Duhai, Ghaziabad (Utter Pradesh) E-mail: [email protected], [email protected]
2National Dairy Research Institute, Karnal- 132 001 (Haryana)
Introduction
Extreme care must be taken by those involved in collecting, handling and processing specimens
that are to be examined for the presence of microorganisms. High quality specimens are
requested to achieve accurate, clinically relevant results. The three components of specimen
quality are (1) Proper specimens selection (i.e. the correct type of specimen must be submitted)
(2) Proper specimen collection and (3) Proper transport of the specimen to the laboratory.
Whenever possible, specimens must be collected in a manner that will eliminate or minimize
contamination of the specimen with indigenous micro flora. Certain types of specimens must
be rushed to the laboratory. Some require transit on ice, whereas others must never be placed
on ice. The laboratory must provide written guidelines regarding specimen selection, collection
and transport. Copies of this “Floor Manual” must be available in every ward in every clinic.
Furthermore, the laboratory is responsible for ensuring that proper specimen collection and
transport devices are available.
When specimens are improperly collected and handled, (1) the etiologic (causative) agent may
be destroyed, (2) overgrowth by indigenous micro flora may mask the pathogen and (3)
contaminations may interfere with the identification of pathogens and the diagnosis of the
patient’s infectious disease.
A close working relationship among the members of the healthcare team is essential for the
proper identification of pathogens. When the attending physician recognizes the clinical
symptoms of a possible infectious disease, certain specimens and clinical test may be
requested. The clinical microbiologist who performs the laboratory microbial analysis must
provide adequate collection materials and instructions for their proper use. The doctor, nurse,
medical technologist, or other qualified healthcare professional must perform the collection
procedure properly and then the specimen must be transmitted properly to the laboratory
where it is cultured, stained, and analyzed. Laboratory findings must then be conveyed to the
attending physicians as quickly as possible to facilitate the prompt diagnosis and treatment of
the infectious disease.
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Guidance to users
The laboratory should issue guidance to potential users of the service in a leaflet or booklet
distributed to hospital units, medical staff, family doctors and environmental health officers.
This leaflet should give the address and telephone number of the laboratory, the arrangements
for the emergency ‘call out’ of staff out of hours and the supply of specimen containers and
request forms. It should also outline the range of examinations undertaken in the laboratory
each kind of specimen from the patients and for sending specimens to the laboratory, including
the safety precautions to be observed with specimens likely to contain especially dangerous
pathogens.
Delivery of specimens
There must be clearly defined arrangements for the collection of specimens from users of the
service and their safe delivery to the laboratory. Collection and delivery may be done by the
portering service within the hospital in which the laboratory is located and by a special van
service from other hospitals, clinics and general-practice health centers. Suitable trays or boxes
should be provided for safe transport of the specimen containers. If specimens are to be
delivered to the laboratory by mail or courier, the postal regulations specifying the types of
container and packaging must be observed.
Proper collection of specimens
When collecting specimens, these general precautions should be taken:
(1) All specimens should be placed or collected into a sterile container to prevent
contamination of the specimen by indigenous micro flora and airborne microbes.
(2) The material should be collected from a site where the suspected pathogen is most likely to
be found and where the least contamination is likely to occur.
(3) Whenever possible, specimens should be obtained before antimicrobial therapy has begun.
If this is not possible, the laboratory should be informed as to which antimicrobial agents
(s) the patient is receiving.
(4) The acute stage of the disease (when the patient is experiencing the signs and symptoms of
the disease) is the appropriate time to collect most specimens. Some viruses, however, are
more easily isolated during the onset stage of disease.
(5) Specimen collection should be performed with care and tact to avoid harming the patient,
causing discomfort, or causing undue embarrassment. If the patient is to collect the
specimen, such as sputum or urine, the patient must be given clear and detailed collection
instructions.
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(6) A sufficient quantity of specimen must be obtained to provide enough material for all
required diagnostic test. The amount of specimen to collect should be specified in the
“floor manual.”
(7) Specimens should be protected from heat and cold and promptly delivered to the laboratory
so that the results of the analyses will validly represent the number and types of organisms
present at the time of collection. If delivery to the laboratory is delayed, some delicate
pathogens might die, e.g., obligate anaerobes die when exposed to air. Any indigenous
micro flora in the specimen may overgrow, inhibit, or kill pathogens. Delay of delivery
considerably decreases the chances of isolating pathogens. Certain types of specimens
should never be refrigerated or placed on ice due to the fragile nature of the pathogens.
Specimen transport instruction should be contained in the “Floor Manual.”
(8) Hazardous specimens must be handled with even greater care to avoid contamination of the
courier, patients, and healthcare professionals. Such specimens must be placed in a sealed
plastic bag immediate and careful transport to the laboratory.
(9) Whenever possible, sterile, disposable specimen containers should be used. If reusable it
should be properly sterilized. Person collecting the specimen should contain request slip
containing adequate instructions. At minimum, labels must contain the patient’s name and
identification number, specific source of specimen, the date and time of collection, and the
collector’s initials. The laboratory should always be given sufficient clinical information to
aid in performing appropriate analyses. The request slip that accompanies a wound
specimen, for e.g., should state the specific type of wound (e.g., burn wound, dog bite
wound, post surgical wound infection, etc.)
(10) Specimens should be collected and delivered to the laboratory as early in the day as
possible to give the technologist sufficient time to process the material, especially when
the hospital or clinical do not have 24 h laboratory service.
Types of specimens usually required
Different types of specimen are required for different disease. Special techniques in collection
and handling are required to obtain specific types of specimens.
Blood
Blood from healthy individual is almost sterile. Bacteria in the bloodstream (bacterea-mia) may
indicate a disease, although temporary or transient bacteremias may occur following oral
surgery, tooth extraction, etc. to prevent contamination of the blood specimen with indigenous
skin flora, extreme care must be taken to use sterile technique when collecting blood for
culture. After locating a suitable vein, disinfect the skin with 70% isopropyl alcohol and then
with an iodophor. When disinfecting the site, use a concentric swabbing motion, starting at the
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point you intend to insert the needle, and working outward from that point. Allow the iodophor
to dry. Apply a tourniquet and withdraw 10ml to 20ml of blood with a 21-gauge needle into
sterile blood culture bottle, containing an anticoagulant. After venipuncture, remove the
iodophor from the skin with alcohol. The blood culture bottle(s) should be transported
promptly to the laboratory for incubation at 370C.
Bactereamia may occur during certain stages of many infectious diseases. These diseases
include bacterial meningitis, typhoid fever and other salmonella infections, pneumococcal
pneumonia, urinary infections, endocarditis, brucellosis, tularemia, plague, anthrax, syphilis,
and wound infections caused by -hemolytic streptococci, staphylococci, and other invasive
bacteria. Septicemia is a serious disease characterized by chills, fever, prostration, and the
presence of bacteria and/or their toxins in the bloodstreams. The most severe type of
septicemia is those caused by Grams-negative bacilli, due to the endotoxin that is released from
their cell walls. Endotoxin can induce the fever and septic shock, which can be fatal. To
diagnose either bactereamia or septicemia, it is recommended that at least three blood
cultures be collected over a 24h period.
Urine
Urine is ordinarily sterile while it is in the urinary bladder. However, during urination, it
becomes contaminated by indigenous micro flora of the distal urethra (the section of the
urethra furthest from the bladder). Contamination can be reduced by collecting a “clean-catch,
midstream urine” (CCMS urine). Clean-catch refers to the fact that the area around the external
opening of the urethra is cleansed by washing with soap and rinsing with water before
urination. This cleansing removes the indigenous micro flora that lives in the area. “Midstream”
refers to the fact that the initial portion of the urine stream is directed into a toilet or bedpan,
and then the urine stream directed into a sterile container. Thus, the microorganisms that live
in the distal urethra are flushed out of the urethra by the initial portion of the urine stream,
into the toilet or bedpan rather than into the specimen container. In some circumstances, the
physician may prefer to collect a catheterized specimen or to use the suprapubic needle
aspiration technique to obtain a sterile sample of urine. In the latter technique, a needle is
inserted through the abdominal wall into the urinary bladder and a syringe is used to withdraw
urine from the bladder. To prevent continued bacterial growth, all urine specimens must be
processed within an hour or refrigerated at 4C until they can be analyzed (within 5 h).
A urinary tract infection (UTI) is indicated if the number of bacteria in CCMS urine equals or
exceeds 1x105 CFU/ ml]. A CCMS urine collected from someone who does not have a UTI
usually (but not always) contains fewer than 10,000 CFU/ml. The presence of two or more
bacteria per x1000 microscopic field of a Gram-stained urine smear indicates bacteriuria
(bacteria in urine) with 100,000 or more CFU/ ml.
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Sputum
Sputum (pus that accumulates in the lungs) may be collected by allowing the patient to spit the
coughed-up specimen into a sterile wide-mouthed bottle with a lid, after warning the patient
not to contaminate the sputum with saliva. If proper mouth hygiene is maintained, the sputum
will not be severely contaminated with oral flora. If tuberculosis is suspected, extreme care in
collecting and handling the specimen should be exercised because one could easily be infected
with the pathogens. Usually, sputum specimens may be refrigerated for several hours without
loss of the pathogens. The physician may wish to obtain a better quality specimen by bronchial
aspiration through a bronchoscope or by a process known as Transtracheal aspiration. Needle
biopsy of the lungs may be necessary for diagnosis of Pneumocystis carinii pneumonia (as in
AIDS patients) and for certain other pathogens. Although once classified as a protozoan, P.
carinii is currently considered to be a fungus.
Feces
Ideally, fecal specimen should be collected at the laboratory and processed immediately to
prevent a decrease in temperature, which allow the pH to drop, causing the death of many
Shigella and Salmonella species; or the specimen may be placed in a container with a
preservative that maintains a pH of 7. Because the colon is anaerobic, fecal bacteria are
obligate, aerotolerant, and facultative anaerobes. However, fecal specimens are cultured
anaerobically only when Clostridium difficile associated diseases is suspected or for diagnosing
clostridial food poisoning. In intestinal infections, the pathogens frequently overwhelm the
microflora so that they are the predominant seen in smears and cultures. A combination of
culture, direct microscopic examination, and immunological tests may be performed to identify
bacteria (e.g., enteropathogenic E. coli, Salmonella spp., Clostridium perfringens, Clostridium
difficile, Vibrio cholerae, Campylobacter spp. and Staphylococcus spp.), fungi (Candida),
intestinal protozoa (Giardia, Entamoeba), and intestinal helminths. Sterile container is used to
collect feces, having spoon fitted in lid of container.
Mucous membrane swabs
Sterile polyester swabs are used to collect specimens of exudates and secretions of the throat,
nose, ear, eye, urethra, rectum, wounds, operative sites, and ulcerations. Cotton swabs are no
longer used because fatty acids in the cotton inhibit the growth of some microorganisms.
Handy, sterile, disposable collection units can be obtained from many medical supply
companies. Each unit contains a sterile polyester swab and transport medium in sterile tube. By
using this set-up, pathogens are kept alive and protected during transportation to the
laboratory. When attempting to diagnose gonorrhea, vaginal, cervical, and urethral swabs
should be inoculated immediately onto Thayer- martin chocolate agar plates and incubated in a
CO2 environment. Alternatively, they should be inoculated in a tube or bottle (e.g. Transgrow)
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that contains an appropriate culture medium and CO2, while the bottle is healed in an upright
position to prevent loss of the CO2. These cultures should be incubated at 370C overnight, and
then shipped to a public health diagnostic facility for positive identification of gonococci.
Cerebrospinal fluid
Meningitis, encephalitis, and meningoencephalitis are rapidly fatal diseases that can be caused
by a verity of microbes, including bacteria, fungi, protozoa, and viruses. To diagnose these
diseases, spinal fluid specimens must be collected into a sterile tube by a lumbar puncture
(“spinal tap”) under surgically aseptic conditions (fig.10-3). This difficult procedure is performed
by physician. Because Neisseria meningitides (meningococci) are susceptible to cold
temperatures, the specimen must be cultured immediately and not refrigerated. Specimens to
be further examined for viruses may be kept frozen at -20C.
Conclusion
The laboratory should have a carefully considered and clearly formulated policy for the
selection of stains or special microscopy, culture procedures, biochemical tests, serological
tests, and antibiotics for sensitivity test to be used in the examination of struck be each kind of
specimen and microbial isolate. A balance must be struck between the extra precision and
reliability of results to be gained from the multiplication of isolation methods and identification
tests, and the need for economy in labor and materials. The greatest effort should be made to
diagnose the more serious infections with epidemic potential, but in most infections the use of
more than two or three methods of isolation is hardly justified by the small increase in the
probability of detecting the pathogen.
Reference:
Yamauchi, K., et al., Infect. Immune. 61: 719-728. (1993). Antimicrobial activity of lactoferrin and a pepsin derived lactoferrin peptide fragment.
Tomita, M., et al., J. Dairy Sci. 74: 4137-4142. (1991). Potent antimicrobial peptides generated by pepsin digestion of bovine lactoferrin.
Leffineur, N.E., Genetet and Leonin, J. 1996. Immunomodulatory activity of β-casein permeate medium fermented by Lactic Acid Bacteria. J. Dairy Sci., 79: 2112-2120.
Lahov, E. and Regelson, W. 1996. Antibacterial and immunostimulating casein derived substances from milk: casecidin, isracidin peptides. Food Chem. Toxicol. 34: 131-145.
Loukas, S., Panetsos, F., Donga, E., Zioudrou, C. 1990. Selective δ- antagonist peptides, analoges of α-casein exorphin, as probes for the opioid receptor. In: β-casorphins and related peptides (Eds F. Nyberg and N. Brand) pp143-149. Fyris Tryck AB, Uppsala.
McCarron D. A, Morris C. D, Henry H. J, Stanton J. L. Blood pressure and nutrient intake in the United States. Science 1984; 224:1392-1398.
Meisel, H. 1986. Chemical characterization and opioid activity of an exorphin isolated from in vivo digestion to casein. FEBS Lett. 196: 223-227.
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Concept of Laboratory Accreditation and its Implementation
Rajan Sharma
Senior Scientist, Dairy Chemistry Division, NDRI, Karnal.
Accreditation is a procedure by which the accrediting body gives formal recognition that
a body or organization is competent to carry out specific tasks. The concept of laboratory
accreditation was developed to provide a means for third-party certification of the competence
of laboratories to perform specific type(s) of testing and calibration. In the past, experience in
many interlaboratory studies at national and international level has demonstrated that beside
standardized and validated methods, analytical quality assurance plays a key role for the
reliability of laboratory results. Introduction of systematic quality assurance procedures (such
as laboratory accreditation and GLP in some cases) of the analytical work itself is now a
requirement for confidence in laboratories and for the acceptance of results. The globalization
of Indian economy and the liberalization policies initiated by the Government in reducing trade
barriers and providing greater thrust to exports makes it imperative for laboratories to be at
international level of competence.
Laboratory accreditation provides formal recognition to competent laboratories, thus
providing a ready means for customers to identify and select reliable testing, measurement and
calibration services. To maintain this recognition, laboratories are re-evaluated periodically by
the accreditation body to ensure their continued compliance with requirements, and to check
that their standard of operation is being maintained. The laboratory may also be required to
participate in relevant proficiency testing programs between reassessments, as a further
demonstration of technical competence.
Accredited laboratories usually issue test or calibration reports bearing the accreditation
body’s logo or endorsement, as an indication of their accreditation. Clients are encouraged to
check with the laboratory as to what specific tests or measurements they are accredited for,
and for what ranges or uncertainties. This information is usually specified in the laboratory’s
scope of accreditation, issued by the accreditation body. The description in the scope of
accreditation also has advantages for the customers of laboratories in enabling them to find the
appropriate laboratory or testing service.
Laboratory accreditation uses criteria and procedures specifically developed to
determine technical competence. Specialist technical assessors conduct a thorough evaluation
of all factors in a laboratory that affect the production of test or calibration data. The criteria
are based on an international standard called ISO/IEC 17025, which is used for evaluating
laboratories throughout the world. Laboratory accreditation bodies use this standard
specifically to assess factors relevant to a laboratory’s ability to produce precise, accurate test
and calibration data, including the:
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- technical competency of staff - validity and appropriateness of test methods - traceability of measurements and calibrations to national standards - suitability, calibration and maintenance of test equipment - testing environment - sampling, handling and transportation of test items - quality assurance of test and calibration data
Manufacturing organizations may also use laboratory accreditation to ensure the testing of
their products by their own in-house laboratories is being done correctly.
A marketing advantage
Laboratory accreditation is highly regarded both nationally and internationally as a
reliable indicator of technical competence. Many industries, such as the construction materials
industry, routinely specify laboratory accreditation for suppliers of testing services. Unlike
certification to ISO 9001, laboratory accreditation uses criteria and procedures specifically
developed to determine technical competence, thus assuring customers that the test,
calibration or measurement data supplied by the laboratory or inspection service are accurate
and reliable. Many accreditation bodies also publish a directory of their accredited laboratories,
which includes the laboratories’ contact details plus information on their testing capabilities.
This is another means of promoting a laboratory’s accredited services to potential clients.
A benchmark for performance
Laboratory accreditation benefits laboratories by allowing them to determine whether
they are performing their work correctly and to appropriate standards, and provides them with
a benchmark for maintaining that competence. Many such laboratories operate in isolation to
their peers, and rarely, if ever, receive any independent technical evaluation as a measure of
their performance. A regular assessment by an accreditation body checks all aspects of a
facility’s operations related to consistently producing accurate and dependable data. Areas for
improvement are identified and discussed, and a detailed report provided at the end of each
visit. Where necessary, follow-up action is monitored by the accreditation body so the facility is
confident that it has taken the appropriate corrective action.
International recognition for your laboratory
Many countries around the world have one or more organizations responsible for the
accreditation of their nation’s laboratories. Most of these accreditation bodies have now
adopted ISO/IEC 17025 as the basis for accrediting their country’s testing and calibration
laboratories. This has helped countries employ a uniform approach to determining laboratory
competence. It has also encouraged laboratories to adopt internationally accepted testing and
measurement practices, where possible. This uniform approach allows countries to establish
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agreements among themselves, based on mutual evaluation and acceptance of each other’s
accreditation systems. Such international agreements, called mutual recognition arrangements
(MRAs), are crucial in enabling test and calibration data to be accepted between these
countries. In effect, each partner in such an MRA recognizes the other partner’s accredited
laboratories as if they themselves had undertaken the accreditation of the other partner’s
laboratories.
ILAC (International Laboratory Accreditation Co-operation) is the peak international
authority on laboratory accreditation, with a membership consisting of accreditation bodies
and affiliated organizations throughout the world. It is involved with the development of
laboratory accreditation practices and procedures, the promotion of laboratory accreditation as
a trade facilitation tool, the assistance of developing accreditation systems, and the recognition
of competent test and calibration facilities around the globe. In 1996, 44 national bodies signed
a Memorandum of Understanding in Amsterdam that provided the basis for the development
of the co-operation and the eventual establishment of a recognition agreement between ILAC
member bodies. As part of its global approach, ILAC also provides advice and assistance to
countries that are in the process of developing their own laboratory accreditation systems. In
conjunction with ILAC, specific regions have also established their own accreditation co-
operations, notably in Europe (EAL) and the Asia-Pacific (APLAC). These regional co-operations
work in harmony with ILAC and are represented on ILAC’s board of management. India is also a
signatory to ILAC Arrangements as well as APLAC MRAs.
The developing system of international MRAs among accreditation bodies has enabled
accredited laboratories to achieve a form of international recognition, and allowed data
accompanying exported goods to be more readily accepted on overseas markets and thus a
step towards elimination of technical barrier to trade. This effectively reduces costs for both
the manufacturer and the importer, as it reduces or eliminates the need for products to be
retested in another country.
How does using an accredited laboratory benefit government and regulators?
Government bodies and regulators are constantly called upon to make decisions related to:
• Protecting the health and welfare of consumers and the public
• Protecting the environment
• Developing new regulations and requirements
• Measuring compliance with regulatory and legal requirements
• Allocating resources, both technical and financial
Government bodies and regulators must have confidence in the data generated by
laboratories in order to make these decisions. Using an accredited laboratory can help establish
and assure this confidence. If a laboratory is accredited, it means that the laboratory has
achieved a prescribed level of technical competence to perform specific types of testing,
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measurement and calibration activities. The result is assurance that the laboratory is capable of
producing data that are accurate, traceable and reproducible - critical components in
governmental decision-making.
Using an accredited laboratory benefits government and regulators by:
Increasing confidence in data that are used to establish baselines for key analyses and decisions
Reducing uncertainties associated with decisions that affect the protection of human health and the environment
Increasing public confidence, because accreditation is a recognizable mark of approval Eliminating redundant reviews and improving the efficiency of the assessment process
(which may reduce costs) Purchases received from suppliers are safe and reliable Costs associated with laboratory problems, including re-testing, resampling, and lost time
are minimized False positives and negatives, which can directly affect compliance with regulations, are
minimized Using accredited laboratories also facilitates trade and economic growth because data
generated by an accredited laboratory may lead to the more ready acceptance of exported
goods in overseas markets. This reduces costs and eases exports and imports, as it reduces or
eliminates the need for retesting in another country.
Why is a laboratory’s technical competence as critical to you as a manufacturer, supplier,
exporter or customer?
Minimized Risk: Throughout the world today, customers seek reassurance that the products,
materials or services they produce or purchase meet their expectations or conform to specific
requirements. This often means that the product is sent to a laboratory to determine its
characteristics against a standard or a specification. For the manufacturer or supplier, choosing
a technically competent laboratory minimizes the risk of producing or supplying a faulty
product.
Avoid Expensive Retesting: Testing of products and materials can be expensive and time
consuming, even when they are done correctly the first time. If not done correctly, then the
cost and time involved in re-testing can be even higher if the product has failed to meet
specifications or expectations. Not only costs go up, but your reputation as a supplier or
manufacturer can go down. You can also be held liable for any failure of your product,
particularly if it involves public safety or financial loss to a client. Choosing a technically
competent laboratory minimizes the chance of retesting being required.
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Enhance Your Customers’ Confidence: Confidence in your product is enhanced if clients know it
has been thoroughly evaluated by an independent, competent testing facility. This is
particularly so if you can demonstrate to them that the laboratory itself has been evaluated by
a third party. Increasingly customers are relying on independent evidence, rather than simply
accepting a supplier’s word that the product is “fit for purpose”.
Reduce Costs and Improve acceptance of Your our Goods Overseas: Through a system of
international agreements technically competent, accredited laboratories receive a form of
international recognition, which allows their data to be more readily accepted on overseas
markets. This recognition helps to reduce costs for manufacturers and exporters that have their
products or materials tested in accredited laboratories, by reducing or eliminating the need for
retesting in the importing country.
What types of laboratories can seek accreditation?
Most national accreditation bodies can provide comprehensive accreditation for:
facilities undertaking any sort of testing, product or material evaluation, calibration or
measurement; private or government laboratories; one-person operations or large multi-
disciplinary organizations; remote field operations and temporary laboratories.
Accreditation of Food Laboratories
A food laboratory may be accredited for the following classes of tests:
Food Products - Chemical Testing Food Products - Microbiological Testing Food Products - Micronutrients Food Products - Residues Food Products - Sensory Evaluation Microbiological Condition of Food Processing Factories Packaging tests Shelf Life testing Laboratories seeking accreditation for chemical, microbiological and sensory food analyses
must be able to demonstrate that they can competently use the methods included in the scope
of the accreditation. If a method is to be used for the official control of foods there are
extensive requirements on internal verification, i.e. that the laboratory is able to demonstrate
that it can use the method in a way, which enables the analytical task to be solved. The
following requirements are examples of factors which laboratories seeking accreditation should
pay attention to, since they often are included in a competence assessment:
the laboratory must have information on the method: is it based on a standard or reference method, or has it been internally developed?;
any deviation in a method as compared to a reference method is fully described and the effects of the deviation have been investigated;
the method has been verified, e.g. by analysing spiked samples of relevant matrices;
the laboratory's own written method text is available;
the method has been in use in the laboratory for a time period of a minimum of three
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months during which a number of 'real' samples of relevant types have been analyzed;
quality control procedures are in place, e.g. analysis of reference or control materials, or control strains;
if possible, the laboratory participates in proficiency testing schemes and evaluates, on a continuous basis, the results;
where relevant, the measurement uncertainty has been estimated and
if a sensory laboratory, it monitors the performance of individual sensory assessors and of panels.
Documentation showing that the laboratory complies with the requirements presented
above must normally be available to the accreditation body and their technical assessors three
to four weeks before the assessment. This information is a useful tool for the assessors when
they select which parts of an analytical chain are to be assessed. The evaluation of a
laboratory's results on the basis of the elements listed above is carried out in order to assess
the analytical activities and capabilities of a laboratory to obtain an overall impression of the
laboratory. The result should demonstrate whether the laboratory is competent and proficient
in the use of the methods for which accreditation is sought.
The standardization and accreditation of sensory quality evaluation methods is a pressing
need for the certification of food products, particularly for foods and beverages with specific
sensory characteristics, such as those with a protected designation of origin (Lea et al., 1995). A
training and qualification process for expert panelists is required. In cheese, panelists score
quality of overall sensory parameters (shape, rind, paste colour, eyes, odour, texture, flavour
and aftertaste) on a scale, based on how close the product lies to a specific quality standard.
Panelists justify the quality scores given on the basis of the absence/presence of specific
characteristics in the product and/or the presence of defects. Training requires the prior
establishment of references for both characteristics and defects. Qualification trials determine
whether or not the expert panelists (both individually and as a panel) are appropriately
qualified to carry out the sensory evaluation. This work also shows the quality control
maintenance of qualifications for the expert panellist. This approach could be generalized to
any type of food and beverage as a reference for the accreditation of sensory quality evaluation
methods according to ISO 17025. In this way, each product manufacturer would be able to
define its quality standard and, on the basis of this standard, carry out the sensory evaluation
using a panel specifically trained for this purpose (Elortondo et al., 2007).
Laboratory accreditation in India
Government of India has authorized National Accreditation Board for Testing and
Calibration Laboratories (NABL) as the sole accreditation body for Testing and Calibration
laboratories. NABL is an autonomous body under the aegis of Department of Science &
Technology, Government of India, and is registered under the Societies Act. NABL has been
established with the objective to provide Government, Industry Associations and Industry in
general with a scheme for third-party assessment of the quality and technical competence of
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testing and calibration laboratories. NABL is a full member of both ILAC and APLAC. NABL had
undergone the first peer evaluation by a 4 member team of APLAC in July 2000, based on which
NABL qualified as an APLAC MRA Partner as well as a Signatory to ILAC Arrangements. NABL
was reassessed in July 2004 & July 2008 and as stated earlier the signatory status of NABL
within APLAC MRA has been confirmed for further four years i.e. October 2012. NABL provides
laboratory accreditation services to laboratories that are performing tests / calibrations in
accordance with NABL criteria, which is based on internationally accepted standards and
guidelines, such as ISO / IEC Guide 25, ISO / IEC 17025 and EN 45001. These services are offered
in a non-discriminatory manner and are accessible to all testing and calibration laboratories in
India and abroad, regardless of their ownership, legal status, size and degree of independence.
NABL has established its Accreditation System in accordance with ISO/IEC 17011:2004, which is
followed internationally. A list of NABL accredited laboratories involved in food testing is
available at http://www.nabl-india.org.
Conclusion
It is apparent that laboratory accreditation has an important role to play in establishing
the credibility of laboratories. Customers of the providers of analytical data need to be assured
about the quality of the data that is being given to them. Experience in many laboratory studies
at national and international level in the past has demonstrated that besides standardized and
validated methods (although these are key factors); analytical quality assurance plays a key role
for the reliability of laboratory results. Introduction of systematic quality assurance procedures
of the analytical work itself is now expected to become a requirement for confidence in
laboratories and for the acceptance of the results. In this regard laboratory accreditations play
an important role in establishing the credibility of analytical laboratories.
References:
Elortondo FJP, Ojeda M, Albisu M, Salmerón J, Etayo I and Molina M (2007). Food quality certification: An approach for the development of accredited sensory evaluation methods. Food Quality and Preference, 18: 425-439.
ISO (2005) General requirements for the competence of testing and calibration laboratories. IS/ISO/IEC 17025: 2005. Bureau of Indian Standards, Manak Bhavan, 9 Bahadur Shah Zafar Marg, New Delhi.
Lea P, Rodbotten M and Naes, T (1995) Measuring validity in sensory analysis. Food Quality and Preference, 6: 321-326.
Lopez-Fandino, R (2003). Accreditation and quality assurance in dairy laboratories following ISO 17025. Bulletin of the International Dairy Federation; 380: 33-36.
Williams, A. (1993). The evident (and urgent) need for analytical quality assurance. In: Analytical quality assurance and good laboratory practice in dairy laboratories. International Dairy Federation Special Issue., No. 9302: 13-19
Wilson, DW (1999). General application, utilization of accreditation. Bulletin of the International Dairy Federation; 344: 4-7.
Wood R, Nilsson A and Walin, H (1998). Quality in the food analysis laboratory. RSC Food Analysis Monograph. The Royal Society of Chemistry, Cambridge.
http://www.ilac.org
http://www.nabl-india.org
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SECTION III
Advances in Functional foods
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Antimicrobial Factors of Colostrum: Application and its Health Benefits
Raman Seth and Anamika Das
Dairy Chemistry Division, N.D.R.I. KARNAL
Introduction
Colostrum is the first mammary secretion produced during the first 72 hours after
parturition, provides nourishment for the newborn. Colostrum is the first natural food
produced by female mammals during the first 24–36h directly after giving birth. Chemically,
colostrum is a very complex fluid rich in nutrients, antimicrobial factors and growth factors. In
cows the antibodies present in colostrums provide passive immunity to the new born calf,
whereas the growth factors especially stimulate the growth of the gut. Bovine colostrum has
also been used as a raw material for immunonoglubulin-rich commercial products (immune
milk preparations). These products can be given orally to patients who are suffering infections
of the gastrointestial tract or in order to prevent these infections. The other antimicrobial
components of colostrum include lactoferrin, lysozyme and lactoperoxidase. Usually, the cows
have to be hyperimmunized against microorganisms, if specific antibodies are required.
Antimicrobial factors of colostrum may be used as potential components in clinical nutrition in
the future.
Antimicrobial components in colostrum
Immunoglobulins
Immunoglobulins (Igs) are present in the whey component of milk but the highest natural
concentrations occur in colostrum.Immunoglobulins (Igs) are glycoproteins that form an
important part of the immune system. They are special immune cells (activated by B
lymphocytes) produced by the body in response to the host being exposed to foreign
substances (antigens) such as infectious microbes. Bovine colostrum contains large amounts of
sIgA, which protects against viruses (e.g. poliovirus, influenza virus and herpes simplex virus)
and bacteria such as E. coli, salmonella and streptococcus. The orally administered bovine
immunoglobulin concentrate from colostrum protects from Shigella flexneri infection, a
bacteria that causes dysentery epidemics. The two predominant Igs in colostrum are: IgG and
IgA.Immunoglobulins from bovine colostrum act as anti-infective agents against a wide range of
bacteria, viruses and protozoa as well as various bacterial toxins. Igs may exert their beneficial
effects by several different mechanisms of action. These actions can vary depending upon the
type of pathogens.
In general, immunoglobulins work to:
neutralise toxins or viruses
prevent adhesion of pathogens to host cell surfaces e.g. intestinal epithelium
bind the bacteria
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enhance the ability of host immune cells to remove pathogens (opsonisation)
damage the micro-organisms themselves e.g. in conjunction with complement
cells.
Table1.Concentration of immunoglobulin in colostrums and milk(Mach and Pahud,1971)
Lactoferrin
Lactoferrin is an iron binding glycoprotein. The concentration of lactoferrin in bovine colostrum
and mature milk is about 1.5-5 mg/mL and 0.1 mg/mL, respectively. It is a natural antioxidant
with antibacterial, antiviral and immune-stimulating properties. Lactoferrin also plays a role in
the activation of phagocytes and immune response. Some of the biological roles of lactoferrin
may be dependent on its iron-binding activity. It is thought that lactoferrin competes with
pathogenic bacteria for iron, so that its ability to bind iron tightly renders the iron unavailable
for bacterial growth. Lactoferrin has been shown to inhibit the growth in vitro of a range of
micro-organisms, including Escherichia coli, Salmonella typhimurium, Listeria monocytogenes,
Shigella dysenteria, Bacillus subtilis, Bacillus stearothermophilus and Streptococcus mutans.
Bovine lactoferrin and their N-terminal peptides were giardicidal against Giardia lamblia in
vitro. Lactoferrin has been shown to bind lipid A of lipopolysaccharides (LPS) and cause the
release of LPS from cell walls of bacteria. In addition, lactoferrin binds to porin molecules in the
outer membrane of Escherichia coli and Salmonella typhimurium resulting probably in
permeability changes. In addition to its antibacterial activity, lactoferrin has antiviral effects
against herpes simples virus type-l (HSV-1)human immunodeficiency virus-l (HIV-l) and human
cytomegalovirus in vitro.
Lysozyme
Lysozyme is an antibacterial enzyme found in high concentrations in colostrum. Lysozyme in
colostrum may be effective against some bacterial infections in humans. The natural substrate
of the enzyme is the peptidoglycan layer of the bacterial cell wall and its degradation results in
lysis of the bacteria.The antibacterial activity of lysozyme is not only due to its enzymatic
activity, but also because of its cationic and hydrophobic properties. The concentration of
lysozyme in colostrum and in normal milk is about 0.14-0.7 and 0.07-0.6 mg/L, respectively.
Milk lysozyme is active against a number of Gram-positive and some Gram-negative bacteria,
which are completely resistant to egg white lysozyme. The antibacterial activity of lysozyme
267
against Escherichia coli can be enhanced by the presence of lactoferrin which also supports the
hypothesis that lactoferrin damages the outer membrane of Gram-negative bacteria.
Lactoperoxidase
Lactoperoxidase is a major antibacterial enzyme in colostrum. It is a basic glycoprotein
containing a heme-group with Fe3+ and catalyzes the oxidation of thiocyanate (SCN-) in the
presence of hydrogen peroxide (H202), producing a toxic intermediary oxidation product. This
product inhibits bacterial metabolism via the oxidation of essential sulphydryl groups in
proteins. The lactoperoxidase system is also toxic to other Gram-positive and Gram-negative
bacteria such as Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes,
Streptococcus mutans, Staphylococcus aureus and psychrotrophic bacteria in milk. In addition,
the lactoperoxidase system inactivates polio virus and human immunodeficiency virus type 1 in
vitro. Bovine colostrum and milk contain about 11-45mg/L and 13-30mg/L lactoperoxidase,
respectively. The lactoperoxidase system and lactoferrin have been shown to have an additive
antibacterial effect against Streptococcus mutans.
Applications of antimicrobial factors of colostrum
Milk production by the modern dairy cow is far in excess of the nutrient requirements of its calf,
and milk has become a significant economic commodity. Traditionally, milk has been used by
humans for direct consumption or as a raw ingredient for manufacturing cheese, butter,
fermented dairy products, or milk powder. However, recent proteomic studies of bovine
colostrum have revealed a large number of minor components, many of which have an immune
function. Such immune components hold great potential to add significant value to milk, with
applications in infant food, cosmetics, personal care, and health promotion.
Small cationic peptides and proteins of colostrum are increasingly valued for their potential as
antibacterial, antifungal, or antiviral products. They may even hold potential as natural
alternatives to traditional antibiotics, because the development of resistance towards these
components may be less.Several antimicrobial molecules are currently at various stages of
development by several biotechnology companies. Colostrum-derived lactoferrin was one of
the first immune components to be commercially extracted for its antimicrobial and antiviral
properties. It can be found as a valuable ingredient in infant formula and other foods for both
human and pet consumption, in skin care products (e.g., cosmetics), and in oral care products
such as toothpaste, mouthwash, and chewing gum. Lactoperoxidase, another antimicrobial
protein commercially extracted from milk and colostrum, is used for food preservation, oral
care, and even as a plant fungicide. The lytic enzyme lysozyme, which is present in milk and
colostrum, also has application as a food preservative in the food industry.
Immune milk
268
Bovine colostrum is a rich source of natural immunoglobulins. The immunoglobulins from
bovine colostrum at least partially retain biological activity in the human gastrointestinal tract
(GI tract) and a lot of work has been done to prepare purified immunoglobulin fractions from
colostrum for pharmaceutical use. If high amounts of specific antibodies are required, cows
have to be hyperimmunized against specific microorganisms. The hyperimmunization protocols
usually include repeated subcutaneous, intramuscular and/or intravenous injections of
vaccines. Immunoglobulin-rich fractions are usually prepared by removing fat and casein
followed by concentration, sterilization and sometimes lyophilization or spray-drying. The
resulting preparations (sometimes called immune milk preparations) contain high amounts of
specific antibodies against the microorganisms in the vaccines. These preparations can then be
given orally to patients suffering infections of the GI tract or in order to prevent these
infections. Orally administered anti-rotavirus immunoglobulins reduce the duration of rotavirus
excretion and diarrhea and protect children against virus infection. Hyperimmune milk prevents
illness after a Shigella challenge and showed that anti-E.coIi immune milk is effective in
eliminating enteropathogenic E.coli from the intestine. Hyperimmune bovine colostrum
antibodies against Cryptosporidium have been shown to inhibit effectively the parasite infection
in vitro. Immune milk has also been used successfully against Cryptosporidium-associated
diarrhea in acquired immunodeticiency syndrome (AIDS) patients.Immune milk prepared
against Helicobacter pylori has been shown to reduce the colonization of Helicobacter pylori in
piglets and there is some evidence that the anti-bacterial effect of anti-Helicobacter pylori
immune milk may be mediated by complement. Colostral antibodies raised against Clostridium
difficile toxins A and B protect against Clostridium difficile disease. The production costs and
availability of immune milk products limit their use in food formulae, since in most cases
immune milk must be administered daily. Thus, immune milk products may be valuable in
special cases, e.g. in passive protection of hospitalized infants or AIDS patients against rotavirus
and Cryptosporidium infection, when no other efficient treatment is available. If the production
costs could be reduced, immune milk preparations may be used more widely as antimicrobial
supplements in food formulae. In addition to immunoglobulins, non-specilic factors (lysozyme,
lactoperoxidase, etc.) in hyperimmune milk also have positive synergistic antibacterial effec.
Skim milk from hyperimmunized cows has been demonstrated to have cholesterol and blood
pressure lowering effects but the mechanisms of these effects remain unknown and require
further studies. It is suggested that increased amounts of IgG might result in changes in the
human gut microflora, which might enhance the excretion of bile acids, leading to increased
hepatic conversion of cholesterol into newly synthesized bile acids.
Commercial immune milk products
269
Several commercial immune-milk products are available in the market. Some of them are
Gastrogard (Northfield Laboratories, Oakden, Australia), a product used to prevent diarrhea
caused by rotavirus in young children and PRO-IMMUNE 99 (GalaGen Inc., Minnesota, USA), a
product used on young calves to prevent scours caused by E. coli. Furthermore, Biotest Pharm
GmbH (Frankfurt, Germany) produces Lactimmunoglobulin Biotest, a product for human
subjects, which contains immunoglobulins from colostrum of non-immunized cows. It has been
tested in the treatment of severe diarrhea in AIDS patients. Viable Bioproducts Ltd. (Turku,
Finland) produces Bioenervi, a sterile-filtered colostrum-based product, which is designed to
provide growth and antimicrobial factors during strenuous physical activity, e.g. training of
athletes.
HEALTH BENEFITS
Gastrointestinal Function
Gut microflora play a vital role in digestion, nutrient absorption and immune function. If there is
an imbalance in the intestinal microflora this may upset the digestive process and impact on the
immune system. Bovine colostrum has been shown to inhibit the growth or to kill various
gastrointestinal pathogens, e.g. Escherichia coli, Campylobacter jejuni, Helicobacter pylori,
Shigella flexneri, Vibrio cholerae, Cryptosporidium parvum and rotavirus. Bovine colostrum has
also been shown to diminish frequency of E.Coli associated diarrhoea. To generate a
preventative or prophylactic benefit, the bioactive components must act by preventing the
pathogen from adhering to the host cell surface.To ensure that colostrum is therapeutically
active, an oral preparation must survive passage through the intestinal tract. Bovine IgG1 has
been shown to be resistant to proteolytic digestion. Passive immunisation through ingestion of
dietary immunoglobulin source could provide options for an oral treatment against enteric
infections in humans. Several human clinical trials provide some evidence that oral
administration of milk immunoglobulin concentrates from bovine origin could be effective in
preventing an/or treating gastrointestinal tract infections.
Necrotizing enterocolitis
Necrotizing enterocolitis (NEC) is a severe life-threatening illness of young children that causes
severe ulceration of the small and large bowel. Its etiology is unclear, although there are many
possible risk factors, including prematurity, enteric infections, intestinal ischemia and abnormal
immune responses. Although many proinflammatory molecules are likely to be involved in the
etiology of NEC, there is currently interest in the role of the phospholipid-mediator platelet
activating factor (PAF), which is produced by intestinal flora and inflammatory cells during the
development of NEC. Current treatment consists of general supportive measures consisting of
fluid-replacement and antibiotic therapy, although intestinal resection is often required. There
270
is therefore a need for novel therapeutic approaches, eg, the use of peptides to stimulate the
repair process.
Colostrum and eye infections
One study assessed the antimicrobial capacity of human colostrum against Chlamydia
trachomatis, a common agent of ophthalmia neonatorum. Results indicated that topically
applied colostrum was effective in the prophylaxis of ophthalmia neonatorum of chlamydial
etiology. Another investigation revealed that topically applied colostrum alleviates severe eye
dryness and problematic eye lesion.
Growth of bifidobacteria
Whey proteins isolated from buffalo colostrum were investigated for the presence of acidic
glycoproteins and their influence on growth of bifidobacteria. Some of the isolated fractions
were able to significantly promote the growth of Bifidobacterium bifidus at low concentration.
B.bifidus produces acetic and lactic acids, which inhibit the growth of many gram-negative
bacilli and fungal species.
Conclusion:
Bovine colostrum is a rich source of immune components that are contributed by both the
acquired and innate immune systems. Increasingly, immune components from colostrum and
milk are being exploited commercially as antimicrobial agents. Although the commercial
application of some colostrum-derived immune components are increasing, the potential of the
majority of immune-active components identified in colostrum remains untapped. It is
anticipated that colostrum based preparations may have remarkable potential to contribute to
human health care as part of health promoting diet and as an alternative or a supplement to
the medical treatment of specified human diseases.
References
Stelwagen K., Carpenter E., Haigh B., Hodgkinson A. and Wheeler T. T. 2009. Immune components of
bovine colostrum and milk. Journal of Animal Science. 87:3-9.
Jansen A., Nava S., Brussow H., Mahalanbis D. and Hammarstrom L. 1994. Titre of Specific Antibodies in
Immunized and Non-immunized Cow Colostrum Implications for their use in the treatment of patients
with gastro-intestinal infections. Indigenous Antimicrobacterial Agents in Milk–Recent Developments.
Proceedings of the IDF Seminar. IDF, Belgium, 1994.
Korhonen H., Syvaoja E. L., Ahola-Luttilia H., Sivela S., Kopola S., Husu J. and Kosunen T. 1994. Helicobactor
pylori – Specific antibodies and bacterial activity in serum, colostrum and milk of immunised and non-
immunised cows. In Indigenous Anitimicrobial Agents in Milk – Recent Developments. Proceedings of the
IDF Seminar. IDF, Belguim, 1994.
Pakkanen R. and Aalto J. 1997. Growth Factor and Anitmicrobial Factors of Bovine Colostrum.
International Dairy Journal, 7, 285-297.
271
Rona Z. 1998. Clinical Applications: Bovine Colostrum as Immune System Regulator. American Journal of
Natural Medicine 5:19-2.
Rump J.A., Arndt R., Arnold A. 1992. Treatement of Diarrhoea in Human Immunodeficiency Virus-infected
Patients with Immunoglobulins from Bovine Colostrum. The Clinical Investigator 70, 588-594.
Uruakpa F.O., Ismond M. A. H., Akobundu E. N. T. 2002. Colostrum and its benefits: A review. Nutrition
Research 22, 755-767.
Mach J.P. and Pahud J.J. (1971). Secretory IgA, a major immunoglogulin in most bovine external
secretions. Journal of Immunology 106, 552-563.
272
Biofunctional Dairy Beverages
Shilpa Vij, Deepika Yadav, Subrota Hati,
Dairy Microbiology Division, N. D. R. I., Karnal-132001
Lactic acid bacteria (LAB) have been widely used as starter culture for the manufacturing of
various fermented foods such as dairies, beverages, vegetables etc.LAB and their food products
are thought to confer a variety of important nutritional and therapeutic benefits and have
many documented health promoting or probiotic effects in human such as inhibition of
pathogenic organism, antimutagenic and reduction of blood cholesterol. Those, LAB with
scientifically supported health claims define as probiotic and have an increasingly high market
potential. Fermented foods are of great significance since they provide and preserve vast
quantities of nutritious foods in a wide diversity of flavors, aromas and textures, which enrich
the human diet. Over 3500 traditional, fermented foods exist worldwide. Fermented foods
have been with us since humans arrived on earth and of these fermented milks have long been
an important component of nutrition and diet. Originally fermented milks were developed as a
means of preserving nutrients.
Fermented milk products: Fermented milks are manufactured throughout the world and
approximately 400 generic names are applied to traditional and industrialized products but in
actual essence the list may only include a few varieties.
Lactic fermentations that include -
a) mesophilic type, e.g., cultured buttermilk, filmjolk, tatmjolk and langofil;
b) thermophilic type, e.g., yoghurt, Bulgarian buttermilk, zabadi, dahi and
c) therapeutic or probiotic type, e.g., acidophilus milk, Yakult, Onka, Vifit; products within
this group constitute by far the largest number known worldwide;
d) Yeast – lactic fermentations (kefir, koumiss, acidophilus yeast milk); and
e) Mould – lactic fermentations (villi).
The increasing demand from consumers for dairy products with 'functional' properties is a key
factor driving value sales growth in developed markets. This led to the promotion of added-
value products such as probiotic and other functional yoghurts, reduced-fat and enriched milk
products and fermented dairy drinks and organic cheese. There are several principal reasons for
the success of fermented dairy products, which relate to nutrition and health, versatility and
marketing. The consumption of milk drinks and fermented products has been recently reviewed
by the International Dairy Federation, shown briefly in Table 1. It is quite clear from the data
that the consumption of fermented milks has generally increased around the globe over a
period from 2001 to 2004.
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Table 1 Broad classification of fermented milk and beverages
Name Country of origin Microflora
Acidophilus
milk
Australia L.acidophilus
Yoghurt (Bio-
ghurt)
Middle Asia,
Balkans
S.salivarius ssp.thermophilus, L.delbruckii
ssp.bulgaricus, Micrococcus and other lactic acid
bacteria, cocci, yeasts, molds
Kefir Caucasus L.lactis ssp.lactis, Leuconostoc spp.L.delbruckii
ssp.caucasius, Saccharomyces kefir,Torula kefir,
micrococci, spore forming bacilli
Kumiss Asiatic steppes L.delbruckii ssp.bulgaricus, L.acidophilus, Torulla
kumiss, Saccharomyces lactis, micrococci, spore
forming bacilli
Dahi (dadhi) India, Persia L.lactis ssp.lactis, S.salivarius ssp.thermophilus,
L.delbruckii ssp.bulgaricus, lactose fermenting yeasts,
mixed cultures
Srikhand
(chakka)
India S.salivarius ssp.thermophilus, L.delbruckii
ssp.bulgaricus
Lassi India L.lactis ssp.lactis, S.salivarius ssp.thermophilus,
L.delbruckii ssp.bulgaricus
Cultured
buttermilk
Scandinavian and
European countries
L.lactis ssp.lactis, L.lactis ssp.diacetylactis,
Leuconostoc dextranicum ssp citrovorum
Leben, Labneh Lebanon, Arab
countries
L.lactis ssp.lactis, S.salivarius ssp.thermophilus,
L.delbruckii ssp.bulgaricus,Lactose fermenting yeasts
Biofunctional properties of fermented beverages are related to
The digestive system are:Bio-active peptides (satiation, bombesin),Absorption power (anti-
acid),Chelating power (anti-poison), Bioavailability of certain minerals (Ca, Fe, Zn…), Inhibition
of pathogen bacteria (acid, enzymes, bacterin,Non-putrefying fermentation (lactic acid), Source
of short chain fatty acid (energy source for enterocytes, Enzymatic activity (ß-galactosidase)
The effects are: Relief of "irritable bowl",Prevention of pouchitis,Attenuation of the symptoms
of nflammatory diseases, Better tolerence of lactose,Attenuation of food intolerences and
allergies
The immune system are: Immunoglobulin (milk, colostrums),Serum protein (lacto globulin,
lactalbumins), Modulation of the immune response,Microbial cells (oral vaccine), Translocation,
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Mucus secretion, Lectin, Cytokine ,Bio-active peptides. The effects are: Attenuation of diarrhea,
Attenuation of allergic reactions Attenuation of inflammatory reactions,Decline in dental
cavaties,Decline of recurring otitis,Regression of tumors
Cardio vascular system: Fermented product (L. helveticus),Fermented products (L. casei, L.
acidophilus…),Anti-hypertension peptides (enzymnatic hydrolysis),Anti-thrombosis peptides
(caseino-macropeptides) and their effects are: Reduces blood cholesterol levels, Reduces
arterial hypertension (in rats and humans), Reduces blood coagulation activity
Nervous system: Opiate-derived peptides: ß-casein (ß-casomorphin),α-casein , α-lactalbumin ,
ß-lactoglobulin , serumal albumin and works on the intestinal motility,Works on the central
nervous system,Analgesic activities
Cancer: Fermented products, Anti-carcinogenic (ALC, butyric acid, peptides, …),
Sphingomyelin ,Vitamins A, D, beta-carotene,Selenium and effects are :Inhibits the enzymatic
activities associated with cancer,Regression of tumors, Attenuation of the mutagen power of
certain molecules.
The occurrence of various bioactive peptides in fermented milks, e.g., yoghurt, sour milk
and ‘‘Dahi’’, has been reported in many studies. ACE-inhibitory, immunomodulatory and opioid
peptides, e.g., have been found in yoghurt and in milk fermented with a probiotic Lb. casei ssp.
rhamnosus strain. Also, ACE-inhibitory peptides have been detected in yoghurt made from
ovine milk and in kefir made from caprine milk.
Table 2 Commercial dairy products and ingredients with health or function claims based on
bioactive peptides
Brand name Type of product Claimed functional bioactive
peptides
Health/function claims
Calpis Sour milk Val-Pro-Pro, Ile-Pro-Pro, derived
from casein and k-casein
Reduction of blood pressure
Evolus Calcium enriched
fermented milk
Val-Pro-Pro, Ile-Pro-Pro, derived
from casein and k-casein
Reduction of blood pressure
BioZate Hydrolysed whey protein
isolate
b-Lactoglobulin fragments Reduction of blood pressure
BioPURE-
GMP
Whey protein isolate k-casein f(106-169)
(Glycomacropeptide)
Prevention of dental carries,
influence the clotting of blood,
protection against viruses and
bacteria
PRODIET
F200/
Lactium
Flavoured milk, drink,
confectionery, capsules
as1-casein f(91-100) (Tyr-Leu-Gly-
Tyr-Leu-Glu-Gln-Leu-Leu-Arg)
Reduction of stress effects
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Festivo Fermented low-fat hard
cheese
as1-casein f(1-6), (1-7), (1-9) No health claim
Cysteine
peptide
Ingredient/hydrolysate Milk protein derived peptide Aids to raise energy level and
sleep
C12 Ingredient/hydrolysate Casein derived peptide Reduction of blood pressure
Capolac Ingedient Caseinphosphopeptide Helps mineral absorption
PeptoPro Ingredient/hydrolysate Casein derived peptide Improves atheletic performance
and muscle recovery
Recaldent Chewing gum Calcium casein peptone-calcium
phosphate
Anticarogenic
Probiotic dairy beverages
A probiotic is defined as a ‘living organism which when administered in certain numbers exerts
health benefits in the host’ (FAO, 2001). Owing to this property, incorporation of probiotic
micro-organisms in dairy foods has increased rapidly during the last two decades. Consumption
of probiotic bacteria via food products is an ideal way to reestablish the balance of intestinal
microbiota. These include alleviation of lactose intolerance symptoms, lowering cholesterol,
curing antibotic-associated diarrhoea, prevention of intestinal tract infections, prevention of
colon cancer, control of rotavirus, prevention of ulcers related to Helicobacter pylori,
improvement of immune system, irritable bowel syndrome and antihypertensive effects. In
order to produce therapeutic benefits, a suggested range for the minimum level for probiotic
bacteria in probiotic milk is from 106 to 107 colony-forming units (cfu) ⁄mL (IDF 1992). In recent
years, probiotic beverages based on fruit juice, cereal products and daily dose dairy drinks have
also become popular commercially. Today, a wide range of probiotic products is available for
consumers in the market.
Table 3 Probiotic fermented products and beverages
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Many fermented beverages are still produced around the world using natural microflora in
empirical processes based on the spontaneous fermentation of different raw materials.
Ayran is a yoghurt drink produced in Turkey. Ayran is traditionally manufactured by
adding water to yoghurt at a level of 30–50% and salt at a maximum level of 1%. In the
industrial manufacture of Ayran, milk with adjusted dry matter content is fermented using
exopolysaccharide-producing cultures; fermentation continues until a pH of 4.4 – 4.6 is
obtained, and the viscous curd obtained is further diluted with salt-containing water. Ayran is
distinct from other fermented milk beverages, being a yoghurt drink with salt and without any
fruit flavouring. Cooling after manufacture is important in stopping fermentation and
preventing further acidity development.
Matsony is the traditional Georgian dairy product and belongs to the lactate type of
beverages, in which microflora is mainly made up of thermophilic streptococci with different
277
species of lactobacilli. After pasteurizing at 85°C, the milk is cooled to 38-40°C, and inoculated
with 3-5% of ferment in the form of thermophilic streptococci and lactobacilli. As a rule, the
fermentation process is lengthened up to 5-7 hours. The dairy acid of Matsony, apart from
fermentation products also contains products of alcoholic fermentation such as carbon dioxide.
The shelf-life of Matsony is 5-7 days at refrigeration temperatures. In medicine, Matsony is
used as a dietary ingredient. In particular, Matsony is actively used for treating gastro-intestinal
diseases such as gastritis, enteritis, etc.
Fermented soy beverage was developed by fermentation by
Lactobacillus, Streptococcus and Bifidobacterium genera as starters and obtain tasteful
fermented drinks. The drinks contained even portions of L(+) and D(-) lactate, they retained
though well perceived sensory profile and high numbers of beneficial bacterial populations on
storage. These beverages as “bio-drinks” could be taken by adult population and people
revealing allergy on cow milk components (37 % of adult population in Poland).
Non-Probiotic dairy beverages with added bioactive components
Two potent ACE-inhibitory peptides, Valine-Proline-Proline (VPP) and Isoleucine-Proline-Proline
(IPP), derived from caseins during milk fermentation with Lactobacillus helveticus and
Saccharomyces cerevisiae, are responsible for the anti-hypertensive activity shown by Calpis_
sour milk (Calpis Co. Ltd, Tokyo, Japan). Other examples of commercial dairy-based beverages
with added bioactive peptides are Evolus.. The former product is manufactured by
incorporating two tripeptides, Val-Pro-Pro and Ile-Pro-Pro, and is claimed to reduce blood
pressure upon regular consumption. The latter product contains the same tripeptides added to
Evolus plus plant sterols which help to reduce blood cholesterol levels.
Conjugated linoleic acid is found almost exclusively in animal products, with a natural level of
approximaetly 6 mg⁄ g of fat. Normal daily intake of CLA in the diet is 150–400 mg⁄ g. addition
of linoleic acid at a level of 0.1% increased cis9-trans11-CLA content of nonfat yogurt
significantly without affecting the sensory properties of the final product. CLA level in
fermented milk made with the standard yogurt culture (0.57 mg⁄g lipid).
Whey based fermented beverages
Whey, a by-product of cheese, paneer, chhana and coagulated dairy products. It is an important
source of lactose, calcium, milk proteins and soluble vitamins, which make this product to be
considered as a functional food and a source of valuable nutrients. Usually dumped because it
had no value, a practice increasingly frowned upon by environmentalists. In India, there has
been a substantial increase in the production of paneer, resulting in an increased accessibility of
whey. India's annual production is estimated at 1, 50,000 tones of paneer and concerning 2
million tones of whey, containing about 1, 30, 000 tones of valuable milk nutrients are
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produced per annum. Growing environmentalist concern have made dumping expensive while
the development of technology has opened up new and cost effective ways of utilizing the
whey constituents which has helped to find a wide range of new applications and the
development of dairy industry.
Through new technologies, whey and its fractions become versatile ingredients and also have
high economic value. Whey products improve textural properties, extend shelf-life,
emulsification and stability, improve flow properties, enhance color and taste and have been
shown to provide beneficial functionality. Whey products have certain essential amino acids,
good digestibility, and protein efficiency index higher than 3.0. Vitamins such as thiamin,
riboflavin, pantothenic acid, vitamin B6 and B12 are also present. Functional properties of whey
proteins, such as emulsifying, water/fat holding, foaming, thickening and gelling properties,
also make them interesting to be used as a food ingredient. Due to their functional properties,
whey solids/ whey as such could be used in conjunction with fermented milks. Several studies
have focused on the use of milk whey in yoghurt making and use of whey powder or whey–milk
powder mixtures. This process leads to the increase of milk total solid content in order to
provide better consistency, texture and creaminess to the product. Yoghurts prepared with
MPC and SMP, exhibit higher values of viscosity and more syneresis than yoghurts prepared
with WPC. Regarding these results, WPC may be useful for drinking yoghurt production. Lassi
like cultured milk-whey beverages have been developed using paneer whey and cheese whey.
So far the whey is considered to be a waste product in the dairy industry but process has been
developed to produce a healthy drink from this waste material. This beverage unlike the other
carbonated beverages which are of little usefulness has following advantages:
i) It has a good nutritional value
ii) It has therapeutic values namely
a. Protection against gastro-intestinal disorders
b. Bio- availability of vitamins
iii) It has three weeks shelf life under refrigeration.
iv) It is much cheaper in cost compared to the other known and available beverages or,
carbonated drinks.
The microorganisms used in these beverages include certain selected species of probiotic and
non-probiotic lactic acid bacteria (single or mixed) and yeast cultures.
Whey-Based Beverages
a) Lactic fermented Acido whey soft drink
b) Biofunctional strawberry probiotic whey drink
c) Alcoholic fermented Wine and Beer
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d) Low alcoholic whey beverages fortification with grape juice.
In the present scenario of consumption of fermented whey drinks such as Molke in West
Germany, Rivella in Switzerland etc and these products are showing increasing trends in most
of the countries around the world. Keeping in view increased demand of soft drinks and juices
these days in India, there is a tremendous scope and need to exploit commercial production of
these fermented whey beverages since it is the best proposition to convert largest by-product
(whey) of dairy industries into value added product by simple and indigenous processes.
Benefits of whey based fermented drinks:
Whey is an excellent growth medium for Lactic Acid Bacteria to ferment lactose in whey
to form lactic acid.
Whey is a genuine thirst quencher unlike most of the soft drinks.
Whey as a drink can replace much of the lost organic and inorganic salts to the
extracellular fluid.
Whey is rapidly adsorbed due to absence of fat emulsion.
Whey has been used to treat various ailments such as arthritis, liver complaints and
dyspepsia.
It also possesses almost all the electrolytes of Oral Rehydration Solution (ORS) which is
invariably used to control dehydration.
On fermentation with LAB, it becomes a suitable drink for lactose-intolerant people.
Fermentation of whey with LAB also masks the effect of curdy flavor of whey.
At industrial scale, large volumes of whey can be used directly from paneer/cheese vats,
thus eliminating transportation and disposal problems.
Conversion of whey into beverages involves very simple processes.
Utilization of whey generates additional revenue to the dairy plant.
Above all, its utilization also solves the problems of environmental pollution.
References:
LeBlanc, A.M., C. Matar, N. LeBlanc and G. Perdigón, 2005. Effects of milk fermented by Lactobacillus
helveticus R389 on a murine breast cancer model. Breast Cancer Research, 7: 477-486.
Khamrui, K. and G.S. Rajorhia, 1998. Making profits from whey. Indian Dairyman, 50: 13-18.
Spreer, E., 1998. Whey and whey utilization. In Milk and Dairy Technology, Chapter 10.Marcel Dikker INC, New
York.
Adwan L. Fermented dairy drinks under pressure (online). Euromonitor international archive; 2003 Jul 25
(cited 2003 Jul 25). Available from: http://www.euromonitor.com/article.asp?id=1371.
Ozer B and Kirmaci H 2009. Functional Milks and Dairy Beverages . International J Dairy Technology, 63 : 1-15
IDF. The World Dairy Situation. Bulletin of IDF 2005; 399: 81.
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Role of Laboratory Animals Studies for Assessment of Safety and
Bioavailability of Nutraceuticals
Ayyasamy Manimaran and Chand Ram
National Dairy Research Institute, Karnal-132 001
Introduction
Research and awareness about nutraceuticals has increased in last few decades and
expected to continue. The word “nutraceutical” was first coined by DeFelice (2007) who
defined it as “a substance that is a food or a part of food and provides medical and health
benefits, including prevention and treatment of disease.” Basic research using laboratory
animals is critical to furthering understanding of the impact of nutraceuticals on health
promotion and disease management, apart from regulatory prerequisite for conducting further
human clinical trials. A variety of laboratory animals are used for evaluation of functional food
and nutraceutical efficacy/metabolic evaluation. These trials can be conducted in healthy
immune competent- or immunocompromised-animals (ex. nude mice which lack cell mediated
immune response). Although significant evidence exist that functional foods and nutraceuticals
can play key roles in disease prevention and health promotion, as in decreasing the risk of
certain chronic diseases, safety considerations must not be ignored. Despite species variation
animal studies at least give theoretical basis for the assurance of safety due to the following
two reasons (1) there is a direct relationship between the concentration of the chemical and
the biological effect for most of the chemical agents and almost every biological effect, and (2)
there is a similar time-response relationship. Most uncertain aspect of safety evaluation is the
relevance of animal data to human beings. The differential species susceptibility could be due
to the effect of the animal on the substance or the effect of the substance on the animal. For
instances, epinephrine, salicylates, certain antibiotics, and insulin are all known to cause
malformations in laboratory animals but not in man. In contrast, only certain strains of rabbit,
mice and rats have been shown to give teratogenic responses to thalidomide with higher doses
than level that resulted in teratogenesis in humans. The only known human teratogens to have
been identified as teratogenic first in animals were aminopterin, androgenic sex hormones, and
possibly X-radiation. The species differences in drug effects can be due to different rates and
patterns of metabolism or inherent differences in susceptibility due to species specific
receptors. Collectively it implies that no single experimental animal can serve as a model for
humans in every possible situation. Safety pharmacology studies can be performed as
independent studies or can be incorporated as a part of toxicological studies thus reducing the
number of animals used in accordance with the 3R’s principles. Incorporation into toxicological
studies may offer the additional advantage that the effect of the nutraceuticals can be
evaluated not only after a single administration but also after repeated administration for a
given period of time. However, one objection is that the dose levels involved can be much
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higher than the therapeutic dose. In safety pharmacology studies, the low dose should be equal
to or slightly higher than the therapeutic dose. The purpose of toxicity testing of animals is to
know the biological effects of substances, so that precautions can be taken to protect humans,
animals and the environment. Mice are the most widely used species accounting for more than
50% of animals use. Mice biology is well known than any other laboratory animal and this is
widely used by immunologist, oncologist and geneticist. Rats are second most widely used a
laboratory animal species and they are generally preferred over mice by toxicologist and
pharmacologists due to convenient size and they do not have many virally-induced tumors as
mice. Rabbits have been widely used for antisera production, pyrogen testing and reproductive
studies particularly for teratogenicity.
Pharmacological characterization
The pharmacological characterization of a nutraceutical is simply the determination of
its efficacy and safety. Since many nutraceuticals are considers as food items (Dietary
Supplement Health and Education Act 1994), currently, many nutraceuticals (e.g., botanicals)
do not require efficacy and safety testing before marketing. However, Morrow et al. (2005)
reported that there is a concern that many nutraceuticals have pharmacological activity that
can endanger the public health and that certain nutraceuticals (e.g., botanicals) should be
regulated similarly to prescription nutraceuticals. Therefore, future marketing of nutraceuticals
may require more rigorous testing of safety and efficacy before marketing. In fact, the FDA
(2007) developed a current good manufacturing practice requirement for dietary supplements
that obligates manufacturers to evaluate the composition, identity, quality, and strength of
their marketed products. With future increased regulation of nutraceuticals, pharmacological
characterization of nutraceuticals will be useful.
Drug development, testing and review process
The drug review process is roughly divided into preclinical and clinical testing. The
preclinical test is primarily in vitro and animal studies, whereas clinical are human studies.
Preclinical testing in animal model (one rodent, one non-rodent) is useful to evaluate acute and
short term toxicity. Doses will be at normal levels for short and long term or increasingly high
levels to induce toxicity. It is useful to determine lethal dose. Pre-clinical studies will be useful
to assess how drug/chemicals is absorbed, distributed, metabolized, and excreted (ADME) in
animals. Further, clinical studies will be conducted in human being in order to verify the
mechanism and efficacy. Clinical studies in human include the following phases (FDA, 2002;
Berkowitz 2007).
Phase I: 20–80 human subjects, safety, pharmacokinetics
Phase II: 36–300 human subjects, efficacy
Phase III: 300–3,000 human subjects, efficacy, double-blind studies
Phase IV: post-marketing surveillance
Safety studies required by the FDA:
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1. Pharmacology studies: determine ED50
2. Acute toxicity studies: determine LD50
3. Multi-dose toxicity studies
a. Subchronic toxicity: duration of one to three months
b. Chronic toxicity: duration of six months
c. Carcinogenicity: duration of two years
4. Special toxicity studies: route of administration
5. Reproduction studies: birth defects
6. Mutagenicity studies: Ames test
7. Pharmacokinetics studies: absorption, distribution, metabolism and excretion (ADME)
Acute Toxicity
Acute toxicity tests are generally provide data on the relative toxicity likely to arise from
a single or fractionated doses up to 24 hrs for oral and dermal studies, while 4-hr exposure for
inhalation studies. Rats are preferred for oral and inhalation tests where as rabbits preferred
for dermal tests. Young adults of 5 of each sex per dose level with minimum three dose levels
were recommended. Animals should be monitored for 14 days for any clinical symptoms.
Subacute study (Repeated dose exposure)
It is performed to obtain dose for subchronic studies typical protocol is to give 3-4
dosages, and 10 animals for each sex per dose are often used. For non-rodents species, usually
dogs (3-4 of each sex per dose).
Subchronic Toxicity
Subchronic toxicity tests are employed one month to three months (90 days are
common). Detailed clinical observations and pathology examinations should be conducted. Two
species are recommended (rodents and non-rodents). Young adult rodents’ (10-20 animals for
each sex per dose) and non-rodents species, usually dogs (4 of each sex per dose) should be
used for experimentation. At least 3 dose levels, in which high dose produce toxicity but not
more than 10 per cent mortality, low dose not produce toxicity and intermediate dose. The
principal goal of this test is development of No Observable Adverse Effects Level (NOAEL) and
sometimes these protocols can be used for further like chronic and developmental toxicity
studies.
Chronic Toxicity
Long-term or chronic toxicity tests determine toxicity from exposure for a substantial
portion of a subject's life. They are similar to the subchronic tests except that they extend over
a longer period of time, which is depend upon intended period (short or long) of exposure to
human and involve larger groups of animals. In rodents, chronic exposures are usually for 6
months to 2 yrs and in non rodents 1 yr or more. It is useful to assess the cumulative toxicity of
chemicals particularly carcinogencity. Dose selection for the chronic study is generally based on
the results of a series of subchronic (90 day) toxicity studies. Result of weight gain, survival
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information, pharmacokinetic, metabolism data, and histopathology from these experiments
that are used for the dose selection. Highest dose employed should be the maximum tolerated
dose (MTD) which is defined as “the highest dose of the test agent during the chronic study
that can be predicted not to alter the animals' normal longevity from effects other than
carcinogenicity”. Carcinogenicity tests are similar to chronic toxicity tests. Testing in two rodent
species (mice and rats), 50 of each sex per dose level are preferred due to short life span. The
exposure period is at least 18 months for mice and 24-30 month for rats. They should be
observed for 18-24 months for mice and 24-30 month for rats.
Developmental and reproductive toxicity
Developmental toxicity testing detects the potential for substances to produce
embryotoxicity and birth defects. Developmental toxicity is the study of adverse effects on the
developing organism occurring at any time during life span form before conception, during
prenatal development or postnatally until puperty. Teratology study involves from conception
to birth. Reproductive toxicity testing is intended to determine the effects of substances on
gonadal function, conception, birth, and the growth and development of the offspring. The oral
route is preferred.
Pharmacological studies
Apart from safety and efficacy of nutraceuticals, the bioavailability studies are
important. Bioavailability is the measurement of the rate and extent of the active ingredient
that reaches the systemic circulation. This can be determined by measuring the active
ingredient of nutraceuticals or its metabolites from the blood. Active ingredient can be
accurately quantitated pharmacokinetically in the plasma (tmax, Cmax and AUC) or urine (rate of
drug excretion) gives the most objective data on bioavailability. Pharmacokinetic studies are
preferred over pharmacodynamic (deals about mechanism of action) studies. When both
pharmacokinetic and pharmacodynamical studies are not possible, then a clinical study can be
used in human or suitable animals model with assumption of therapeutic success occurred
because there was enough bioavailability when the nutraceuticals was administered. However,
various factors such as diet, disease, or genetics, which can make it difficult to understand the
success or failure (Shargel 1993; FDA, 2003). Whenever potentially active metabolites found
during human cell culture studies, these metabolites can be studied in laboratory animals to
determine their safety and efficacy, which can help determine future in vitro or in vivo human
studies. Moreover, animal studies can be used to examine nutraceuticals-drug interactions with
regard to parent drug and its metabolites. Since several nutraceuticals (example, Grapefruit
and St. John’s Wort) are inhibit or induce a cytochrome P450 which could affect subsequently
administrated drug concentrations.
Assays for ACE-inhibitory and antihypertensive activity
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Determination of the ACE inhibitory activity is the most common strategy followed in
the selection of antihypertensive peptides derived from milk proteins. In order to facilitate the
characterisation of ACE inhibitory peptides, the establishment of a simple, sensitive and reliable
in vitro ACE inhibition assay like, spectrophotometric, fluorimetric, radiochemical, HPLC and
capillary electrophoresis methods can be used to measure ACE activity. This is usually
expressed as the IC50, i.e. concentration needed to inhibit 50% of the enzyme activity. The
spectrophotometric method of Cushman and Cheung (1971) is most commonly utilized. The in
vivo effects are tested in spontaneously hypertensive rats (SHR), which constitute an accepted
model for human essential hypertension. In addition, in many in vivo studies it is also checked
that antihypertensive peptides from milk proteins do not modify the arterial blood pressure of
Wistar-Kyoto (WKY) rats that are the normotensive control of the SHR. The results of
hypotensive effects caused by the short-term administration to SHR of milk protein
hydrolysates, fermented products and isolated milk-derived peptides have been shown to lack
of correlation between the in vitro ACE inhibitory activity and the in vivo action. This poses
doubts on the use of the in vitro ACE inhibitory activity as the exclusive selection criteria for
potential antihypertensive substances, as it does not take into consideration of the
bioavailability of the peptides or other mechanism like antioxidant effects. On other hand, long-
term intake of milk products on blood pressure of SHR was shown that dose dependent
attenuation of the development of hypertension in SHR during 14 weeks of treatment with milk
containing the potent ACE inhibitory peptides (Nakamura et al., 1995; Sipola et al., 2002).
Hypertension animal models
Rats are the most popular species in hypertension. The rat models of hypertension thus
provide ample opportunity not only to investigate the mechanisms involved in the pathogenesis
of hypertension, but also to learn about the critical balance between stress and coping. Among
rats spontaneously hypertensive rat (SHR) is most widely used rat model, although it reflects
only a rare subtype of primary human hypertension, which is due to genetic inheritance. SHR
stroke prone (SHR-SP) is a further developed sub-strain, with even higher levels of blood
pressure, and a strong tendency to die from stroke. Other rat models of hypertension are Dahl
(due to genetic inheritance like SHR), deoxycorticosterone acetate (DOCA)-salt, cause
hypertension due to hormonal alterations (Contreras et al., 2009).
Type 2 diabetic animal models
Chemical induced diabetes model can be produced by administrating drugs like alloxan
in rat (40-200 mg/kg, iv or ip), mice (50-200 mg/kg, iv or ip), rabbit (100-150 mg/kg, iv), dog (50-
75 mg/kg, iv) can cause diabetes. Administration of streptozotocin to rat (35-65 mg/kg, iv or ip),
mice (100-200 mg/kg, iv or ip), hamster (50 mg/kg, ip), dog (20-30 mg/kg, iv) can induce
diabetus in these animals. Selective loss of pancreatic beta cells, residual insulin secretion and
ketosis makes less mortality. Comparatively cheaper, easier to make and maintenance of
animals. Disadvantages are, direct cytotoxic action on the beta cells and insulin deficiency
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rather than consequence of insulin resistance, less stable and reversible. Further, toxic actions
on other body organs are constraints in long-term experiments. Though spontaneous type 2
diabetic models are resemble to human being and minimum variability of results with minimum
sample size, they are limited availability, costly and required sophisticated maintenance. In
dietary or nutrition induced type 2 diabetic models as a result of overnutrition, toxicity of other
vital organ can be avoid. However, long period is required to create diabetes and no frank
hyperglycaemia develops upon simple dietary treatment. Surgical, transgenic and knock out
models of diabetes animals are need cumbersome technical procedure and costly procedure
(Srinivasan and Ramarao, 2007).
Conclusion
Despite the advancement in vitro study strategies, in vivo studies remain very important
component of food chemical or contaminant for human risk assessment. In fact, in vivo animal
studies present the great advantage of providing information on a whole organism, including all
organs and their metabolic functions. Experiments using laboratory animals should be well
designed, efficiently executed, correctly analyzed, clearly presented, and correctly interpreted if
they are to be ethically acceptable. Laboratory animals are nearly always used as models or
surrogates of humans or other species. Animals should be used only if the scientific objectives
are valid (i.e. high probability of meeting the stated objectives and reasonable contributing to
human or animal welfare, possibly in the long term), no other alternative, and the cost to the
animals is not excessive. The reason for choosing the particular animal model and the species,
strain, source, and type of animal used should be clear. The “3Rs” rules (replacement,
refinement and reduction) should be followed to humane use of animals. However, it is
important to recognizing biological effects with sufficient numbers animals in experiments. The
number of animals to be used in an experiment depends on a variety of factors, including
experiment objectives, degree of precision required, expected difference between the effects
of treatments and structure and methods of analysis. Development and application of the
biomarkers to clarify functionality and risk are important to understand the fundamental
molecular mechanism concerning health care and disease prevention of nutraceuticals.
Through use of advanced technologies to study the relationship between nutrition intake and
health associated with genes can be useful for better understanding of nutraceuticals.
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immunoglobulin G in animal milks by new immunosensors. Sensors, 9: 2202-2221.
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effective. http://www.fda.gov/fdac/features/2002/402_drug.html.
Food and Drug Administration. (2003). Guidance for industry. Bioavailability and bioequivalence for orally
administered drug products: General considerations. http://www.fda. gov/cder/guidance/5356fnl.pdf.
Food and Drug Administration. (2007). Final rule promotes safe use of dietary supplements.
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Meltretter, J., Schmidt, A., Humeny, A., Becker, C.M. and Pischetsrieder, M. (2008). Analysis of the peptide
profile of milk and its changes during thermal treatment and storage, J. Agric. Food Chem., 56: 2899-2906.
Morrow, J., T. Edeki, M. El Mouelhi, R. Galinsky, R. Kovelesky, and C. Preuss. (2005). American Society for
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Microencapsulation of Lactobacillus sp. in Calcium Alginate
Surajit Mandal, Sandip Basu, R.P. Singh, Chand Ram and Rameshwar Singh
Dairy Microbiology Division, National Dairy Research Institute, Karnal- 132 001.
Email: [email protected]
Introduction
Probiotics, live micro-organisms which when administered in adequate numbers confer a health
benefit on the host (FAO/WHO 2001), are commonly included in fermented milks, yoghurts and
cheese, but are also available in the form of dietary supplements where the probiotic is in the
form of a dried product. Certain LAB, often members of Lactobacillus genus, have positive
influence on the health of the consumers for successful delivery in foods, probiotics must
survive food processing and storage during product maturation and shelf-life. In addition, the
probiotic food product should be regularly consumed in sufficient quantities to deliver the
relevant dose of live bacteria to the gut, keeping in mind the losses in cell viability typically
encountered during gastric transit. Approaches for selection of an ‘ideal’ strain are difficult and
indeed require considerable resources. Encapsulation, as a means of protecting live cells from
extremes of heat or moisture, such as those experienced during drying and storage is a
technique that is increasingly used in the probiotic food industry. It was found that
encapsulating lactobacilli in calcium-alginate beads improved their heat tolerance.
Encapsulation or immobilization could potentially promote the survival of probiotic bacteria in
food systems and improve the extent of application. Encapsulation also helps to segregate the
bacterial cells from the adverse environment, for example of the product, thus potentially
reducing cell loss. The studies have demonstrated that cultures can be significantly protected
by encapsulation in a variety of carriers, which include milk proteins, food hydrocolloids and
complex carbohydrates (prebiotics).
Microencapsulation of lactobacilli
Culture: Lactobacillus casei NCDC 298 (National Collection of Dairy Cultures, Karnal, India,
as freeze-dried ampoule)
Method of microencapsulation:
o Emulsion technique (phase separation and coacervation)
o Extrusion method
Encapsulating material: Sodium alginate (2, 3 and 4 %)
Continuous phase: Soybean oil + 0.2% Tween–80
Hardening phase: 0.1 M CaCl2 solution
Protocol
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a) Activate the culture in chalk litmus milk (37°C for 24 h) and maintain in refrigerator (7±1°C)
and sub-culture monthly.
b) Re-activate the culture by 2 to 3 transfers in MRS broth (37°C for 15-18 h).
c) Grow the culture in 80 ml MRS broth (37°C for 24 h).
d) Harvested the cell by centrifugation (10000 rpm for 10 min at 4°C) and wash twice, and re-
suspend in 5 ml normal saline.
e) Adjust the cell concentration to1.0x1011 cfu/ ml.
f) Prepare sodium alginate aqueous solution (4%).
g) Sterilize (121°C for15 min) and cool to 38-40°C.
h) Mix 20 ml of the alginate solution and 4 ml of cell suspension in a centrifuge tube (40 ml) by
vortexing for uniform distribution of cells in alginate matrix.
Emulsion technique (phase separation and coacervation)
a) Sterilize soybean oil (100 ml) containing Tween 80 (0.2%).
b) Take 100 ml oil in a beaker (500 ml) and add the alginate-cell mix drop wise into the oil
(continuous phase) while magnetically stirring for 5 min to get uniformly turbid emulsion
c) Add 100 ml 0.1M calcium chloride (chilled) to break the emulsion and hardening of the
alginate microcapsules.
d) Harvest the capsules by centrifugation (1000 rpm for 10 min at 4°C) and wash with sterile
distilled water.
e) Filter through filter paper (Whatman No. 1) to separate the microcapsules and store in
refrigerator (7±1°C) until use.
Extrusion method
a) Take 200 ml 0.1M calcium chloride (chilled) in a beaker (500 ml) and stir magnetically.
b) Add the alginate-cell mix drop wise using hypodermic needle syringe into the calcium
chloride solution (hardening phase) while magnetically stirring.
c) After, complete addition of cell alginate, keep the mixture for 10-15 min for proper
hardening of the alginate microcapsules.
d) Harvest the capsules by filtering through Whatman No. 1filter paper to separate the
microcapsules and store in refrigerator (7±1°C) until use.
Evaluation of encapsulated lactobacilli
1.1. Viability of lactobacilli after encapsulation
1. Check the viability of microencapsulated lactobacilli after depolymerization of the capsules
followed by plating on MRS agar.
2. Transfer microcapsules (1 g) in 10 ml of sterilized phosphate buffer solution (0.1 M, pH
7.1±0.2) and incubate at 37°C for 30 min.
3. Depolymerize by vortexing to obtain a uniform cloudy solution and prepare serial dilution
using normal saline (9ml) tubes.
4. Enumerate the viable lactobacilli using MRS agar (37°C for 48 h).
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Microscopic observation
Examination using phase contrast microscopy
a) Take few microcapsules on glass microscopic slide and disperse in small amount distilled
water.
b) Uniformly spread the capsules on the slide.
c) Observed under 10, 20 or 40 x objective of phase contrast microscopy.
Examination using scanning electron microscopy
a) Dehydrate the beads containing the immobilized cells using routine methods of graded
ethanol series and freeze-drying.
b) Coat the dried beads with gold-platinum alloy and observe at 20 KV with a Hitachi-405A
Scanning Electron Microscope.
Survival of microencapsulated cells on heat treatments
a) Test the tolerances to heat treatment (63°C for 30 min) in sterile distilled water (pH 6.4±0.2)
or sterile skim milk in a water bath.
b) Transfer microcapsules (1 g) or 1m1 of the free cell suspension (1010 cells/ml) in 10 ml of
sterile distilled water or 10 sterile skim milk in test tube
c) Placed the tubes in the water bath maintained at 63°C and record the mixture temperature
by putting thermometer inside on tube.
d) After reaching the content temperature at 63°C, heat the content for 30 min.
e) Take the test tube at initial, 0 min and 30 min at 63°C for enumeration of viable lactobacilli.
f) Cool the content to ambient temperature by placing in tap water.
g) Depolarize the beads in phosphate buffer (0.1 M, pH 7.1±0.2) and prepare serial dilution
using normal saline (9ml) tubes.
h) Enumerate the viable lactobacilli using MRS agar (37°C for 48 h).
Survival of microencapsulated cells in simulated gastric juice
a) Test the tolerance to simulated pH of human stomach.
b) Prepare the simulated gastric juice without pepsin (pH 1.5) containing 0.2% NaCl.
c) Transfer approximately 1 g of microcapsules or 1m1 of the free cell suspension (1010
cells/ml) into test tubes containing 10 ml of simulated gastric juice and incubate at 37°C.
d) At the end of 1 and 3 h, take the tubes and harvest the beads, wash with physiological
saline, and immediately enumerate the viable cells.
e) Depolarize the beads in phosphate buffer (0.1 M, pH 7.1±0.2) and prepare serial dilution
using normal saline (9ml) tubes.
f) Enumerate the viable lactobacilli using MRS agar (37°C for 48 h).
Survival of microencapsulated cells in bile salt solution
a) Test the tolerance to simulated bile concentration of human small intestine.
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b) Transfer approximately 1 g of microcapsules or 1m1 of the free cell suspension (1010
cells/ml) into test tubes containing 10 ml of sterilized 0 and 2% bile salt solution and
incubate at 37°C.
c) At the end of 3 and 12 h, take the tubes and harvest the beads, wash with physiological
saline, and immediately enumerate the viable cells.
d) Depolarize the beads in phosphate buffer (0.1 M, pH 7.1±0.2) and prepare serial dilution
using normal saline (9ml) tubes.
e) Enumerate the viable lactobacilli using MRS agar (37°C for 48 h).
In-vitro release of encapsulated cells in simulated colonic pH solution
a) Study the in-vitro release of microencapsulated L. casei NCDC 298 in simulated colonic pH
solution.
b) Transfer approximately 1 g of microcapsules into the test tubes containing 10 ml simulated
colonic pH solution (0.1 M KH2PO4, pH 7.4±0.2) and mix thoroughly by gentle shaking and
incubate at 37°C.
c) At the end of 0, 0.5, 1.0, 2.0 and 3.0 h, take 1 ml aliquots enumerate the lactobacilli by MRS
agar plating method (37°C for 48 h).
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Electron Microscopy as a Tool for Study of Functional Attributes of Probiotics
Sudhir Kumar Tomar
DM Division, NDRI, Karnal -132 001
INTRODUCTION
Electron microscope provides a markedly higher magnification at a considerable better
resolution than light microscope. Electron microscopy takes advantage of the wave nature of
rapidly moving electrons. Where visible light has wavelengths from 4,000 to 7,000 Angstroms,
electrons accelerated to 10,000 KeV have a wavelength of 0.12 Angstroms. Optical microscopes
have their resolution limited by the diffraction of light to about 1000 diameters magnification.
Electron microscopes, so far, are limited to magnifications of around 1,000,000 diameters,
primarily because of spherical and chromatic aberrations.
Probiotic micro-organisms are often incorporated in foods in the form of yoghurts and
yoghurt-type fermented milk. Recently, there are probiotic ice cream, cheese, infant formulas,
breakfast cereals, sausages, luncheon meats, chocolates, and puddings, probiotic products in
capsules containing freeze-dried cell powders (Figure 1) and in tablet form. However, there are
a number of problems in determining the efficacy of probiotics as a whole. Firstly, although
there are a wide range of species and strains used, the efficacy of some of them remains in
doubt or has not been fully proved. Added to this are the problems of variation in viability or
activity of the cells in the various preparations, the use of mixtures of organisms and their
differential survival, and ensuring that probiotic cells have a long shelf-life and reach their site
of action.
Electron microscope can be used as an effective sophisticated tool to study the
functionality of probiotics especially in terms of their microencapsulation for effective delivery,
digestive stress tolerance and adhesion to human intestine cells.
1.0 TYPES OF ELECTRON MICROSCOPY
1.1 Conventional Electron Microscopy
There are two major electron microscopy modes- Scanning electron microscopy (SEM) and
Transmission Electron microscopy(TEM). The electron beam is focused using magnetic lenses in
both kinds of microscope. The specimen is placed into the path of the electron beam in the
TEM but in the SEM, it is placed at the end of the focused electron beam path. The image is
produced in the form of a shadow on a fluorescent screen in TEM whereas in SEM reflected and
secondary electrons are processed by an electron detector to form a three dimensional image
on a monitor screen.
Since the electrons would be easily absorbed by air, the microscopic examination is
carried out in vacuum. To ensure that the electrons will penetrate a thin section of the
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specimen or its replica, the electron beam is accelerated in the microscope. An anode with an
orifice in its centre is positively charged. The negative electrons rush toward it and those which
are in the centre fly, accelerated, through the orifice toward the specimen. Accelerating voltage
of 3 to 20 kV has been used to do SEM and 60 to 80 kV have been used in TEM of foods.
Traditional electron microscopy requires that the specimen must not release any gas or vapour
when inserted into the transmission or scanning electron microscope. Except for powdered
foods such as flour, sugar, or milk powder, most foods contain water. Drying or freezing at a
very low temperature of -100°C ensure that the condition of not releasing gas or vapour is met.
1.1.1 Scanning Electron Microscopy
The conventional method of sample preparation for SEM includes chemical fixation
(Glutaraldehyde, Osmic Acid), dehydration with a graded series of ethanol or acetone and
subsequently drying by air drying , freeze-drying or critical point drying. The specimen is
mounted on an aluminum stub and coated with heavy metal to make it electrically conductive.
It has been demonstrated that simple air-drying of the specimen yields collapsed micelles even
after proper fixation due to the strong interfacial forces created as a result of passage of
receding water surfaces over the particles. Better results have been obtained with freeze-drying
and critical point drying.
In scanning electron microscopy (SEM), the specimen is examined by a focused electron
beam. An electron gun is the source for this beam. Electrons are emitted from a cathode,
accelerated by passage through electrical fields and focused to a first optical image of the
source. The gun consists of tungsten or lanthanum hexaboride electrode surrounded bya shield
with a circular aperture. Electrons in the gun are accelerated across a potential difference of
the order of 10,000 volts between the cathode (at high negative potential0 and anode (at
ground potential). Some of these electrons are reflected and others generate secondary
electron from the gold coating. (A great variety of other interactions also take place). Secondary
electrons (or, in other applications, backscattered electrons) are used to form an enlarged
image of the specimen surface. The incident electrons carry a negative charge and in order to
be 'neutralized' after they have completed the examination, the specimen should be electrically
conductive. As mentioned earlier this is achieved either by chemical procedures which
impregnate the specimen with osmium or, more frequently, by physically coating its with gold,
a gold-palladium, platinum, or iridium - occasionally both procedures are combined. Metal
coating provides a path for the electrons. It this path is interrupted (by incomplete metal
coating or by cracks), the electrons sit in the area thus isolated and repel any electrons in the
incidental beam in accordance with the rule that electrically charged particles of the same
charge repel each other. Thus the area occupied by the stationary negative charge is by-passed
and cannot be examined. White spots or lines develop in such places and the image is
characterized as suffering from charging artifacts.
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Investigation of water-containing dairy products by SEM requires adequate reinforcement of
fragile structures and also careful selection of drying procedures. Structural stabilization can be
achieved by fixation with glutaraldehyde and dehydration is performed mainly by critical-point
drying. The SEM exhibits only the details of surfaces, internal structure may be studied by
fracturing the sample and examining the surface thus formed. Conventional SEM is used to
study chemically fixed and subsequently dried samples at ambient temperature. Samples fixed
by rapid freezing are examined in the frozen-hydrated state at temperature below –100 C using
Cryo-SEM.
1.1.2 Transmission Electron Microscopy
TEMs are patterned essentially after TLM and yield information on the size, shape and
arrangement of particles which make up the specimen as well as their relationship to each
other on the scale of atomic diameters. The electromagnetic lenses (first & second) determine
the spot size of the electron beam generated by electron gun and also alters the spot to a
pinpoint beam. Further condensor lens restricts the beam by knocking out high angles electrons
and beam strikes the specimen and parts of it are transmitted. The transmitted portion is
focused by the objective lens into an image which is passed down the column through the
intermediate and projector lenses, being enlarged all the way.
Transmission electron microscopy can be performed using various techniques as discussed in
the following sections.
2.1.2.1 Ultrathin Sectioning
One of the most widespread techniques of specimen preparation for electron microscopy is
thin sectioning of plastic-embedded samples. This technique comprises a fixation, dehydration
and finally impregnation in some suitable plastic monomer such as araldite or epon. After
hardening thin sections (15 to 90 nm thick) are cut with ultramicrotome and picked up on an
electron opaque metal grid of 200-400 mesh (lines/in) to provide mechanical support. The
fragile material and viscous foods such as yoghurt, cream, mayonnaise are encapsulated in agar
gel using a thin (0.3-0.5 mm capillary) after the samples have been fixed, post fixed and
dehydrated. These tiny agar capsules can be handled like the pieces of tissue and can be further
processed as described above.
2.1.2.2 Negative Staining
It is a relatively simple procedure whereby minute particles such as casein micelles, dietary
fibre, bacteriophage, or bacteria are mixed with a heavy metal salt solution such as potassium
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tungstate, uranyl acetate or ammonium molybdate and applied on a thin electron-transparent
film.. The specimen and the salt solution are dried and placed into the microscope. The heavy
metal, which surrounds the organic specimen, absorbs electrons but the electrons which pass
through the organic specimen form a 'negatively stained' image. This method can be used to
study dispersion of casein, heat denaturation of whey proteins and of the interaction between
carrageenan and kappa-casein.
2.1.2.3 Metal Shadowing
Metal shadowing or Shadow casting is another simple technique whereby the specimen is dried
on a translucent film and then is 'shadowed' with platinum vapour in vacuo. The thickness of
the coating (less than 0.005 micrometer) depends on the angle of the surface shadowed. The
image is formed by the electron beam which passes through the different thickness of the
coating which depends on the topography of the specimen's surface. This technique has been
successfully used for the study of colloidal protein particles such as casein micelles and their
subunits and of isolated membranes of milk-fat globules.
2.1.2.4 Freeze- fracturing and Freeze-etching
Freeze-fracturing and freeze-etching techniques are the most laborious. They make it possible
to examine the specimen without altering it chemically (fixation) or physically (dehydration,
impregnation with a resin, drying). The specimen is rapidly frozen, then is freeze-fractured at a
temperature below -110°C and the fracture plane is replicated with platinum and carbon either
immediately or after a certain period of freeze-etching, during which a thin layer of ice in the
specimen sublimes off and reveals underlying structures. The specimen is thawed and the
replica is separated from the specimen, cleaned from specimen residues, and examined in the
microscope.
2.2 Cryo-Electron Microscopy
With the recent introduction of suitable cryo-stages for both TEM and SEM, Cryo-electron
microscopy has become a practical tool for the examination of biological nmaterials close to
their native state. A thin film of a dispersion of small particles can be rapidly quench-frozen so
that ni ice crystallization occurs. It is often examined in the electron mic4oscope at –150 c in
the frozen state. In this way, artifacts due to drying, staining, shadowing, fracturing or
sectioning can be avoided.
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2.3 Immuno-Electron Microscopy
Immunolocalization in electron microscopy is a technique which makes it possible to identify a
particular protein or polysaccharide among other proteins and polysaccharides by marking it
with minute gold granules. Examples may include the presence of beta-lactoglobulin in curd
which consists mostly of casein or, in case of a fictitious adulteration, bovine milk proteins in
sheep cheese. Any substance which evokes immunological response as a 'foreign' body may be
detected. Often the chemical composition of the minor substance to be identified is very close
to the major substance, so immunolocalization using colloidal gold is used even in optical
microscopy. These techniques are common in medicine and biology and are increasingly useful
in food science.
2.0 ASSESSMENT OF FUNCTIONALITY OF PROBIOTICS BY ELECTRON MICROSCOPY
2.1 Microencapsulation of Probiotics
Probiotic micro-organisms are often incorporated in foods in the form of yoghurts and yoghurt-
type fermented milk. Recently, there are probiotic ice cream, cheese, infant formulas, breakfast
cereals, sausages, luncheon meats, chocolates, and puddings, probiotic products in capsules
containing freeze-dried cell powders (Figure 1) and in tablet form. However, there are a
number of problems in determining the efficacy of probiotics as a whole. Firstly, although there
are a wide range of species and strains used, the efficacy of some of them remains in doubt or
has not been fully proved. Added to this are the problems of variation in viability or activity of
the cells in the various preparations, the use of mixtures of organisms and their differential
survival, and ensuring that probiotic cells have a long shelf-life and reach their site of action. A
key factor in the development of microencapsulated probiotics is the choice of encapsulation
material, which is dependant on the desired chemical and physical properties, and the process
of microcapsule formation. The microcapsule should be stable and retain its integrity during
passage through the digestive tract until it reaches its target destination, where the capsule
should break down and liberate its contents. The encapsulation material should retain the
bacteria, and should also restrict the movement of the acid wave and digestive enzymes
through the microcapsule. Carbohydrate polymers such as alginate have been used in various
food applications. For alginate capsules, a number of factors determine the internal structure,
including intramolecular distribution and proportion of the guluronic and mannuronic acid
residues, concentration and distribution of mono- and divalent cations and pH. Alginates high in
guluronic acid form stronger and more compact gels in the presence of Ca2+, but is at the same
time more sensitive to fluctuations in calcium concentration than the weaker but more stable
high-mannuronic acid alginate gels . The alginate matrix stays structurally stable in low acid
environments, however, as pH is lowered below the pka values of mannuronic and guluronic
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acid (3.6 and 3.7, respectively) alginate is converted to alginic acid with release of calcium ions
and the formation of a more dense gel.
Microscopy is a useful tool to monitor development and production of microcapsules. It
is vital to characterize the microstructure of polymer microcapsules, since the degradation
kinetics of the microcapsules (the release of entrapped bacteria) are determined by the internal
structure of the microcapsules and the arrangement of the organisms within. In the study of
microencapsulation, Electron microscopy can aid in the determination of the encapsulation
ability of the polymers which comprise the matrix material, thereby assessing the functionality
of the system. EM may contribute information about the size range of empty and bacteria-
loaded microcapsules, matrix microstructure and any matrix changes caused by the entrapped
bacteria. A number of microscopy techniques should be used for a complete microstructural
description of the bacterial loaded microcapsule. Rosenberg et al. (1985) successfully applied
SEM to the morphological study of various microcapsule systems. In addition to using standard
preparation techniques for the examination of the outer structure of microcapsules, they
developed a new embedding and microtoming technique to allow the study of the inner
structure of fractured capsules. The technique uses a new nonpolar embedding resin, Lowicryl
HM-20, which is compatible with the microcapsule shell material, and does not introduce
artifacts associated with the use of epoxy resins. They demonstrated the potential of SEM
techniques as a tool for selection of wall materials, for the study of core materials distribution
in microcapsules, and for elucidating the mechanisms of capsule formation and the effects of
water-vapor uptake on microcapsule properties. Further, Sheu and Rosenberg (1998) studied
microstructure of spray-dried microcapsules with wall systems consisting of mixtures of whey
protein isolate and maltodextrins or corn syrup solidsand observed that structure of
microcapsules was appreciably affected by type of carbohydrate and by the WPI-to-
carbohydrate ratio.
Recently, Wojtas et al (2008) studied Calcium alginate microcapsules, with or without
probiotic bacteria using conventional CSEM, cryo-SEM, and TEM. Each type of microscopy
provided unique microstructural information about the microcapsules and entrapped bacteria.
Microcapsule integrity was not preserved using conventional preparation techniques with
ambient temperature SEM and TEM. Only free bacteria and remnants of capsular material
remained. Cryo-SEM stabilized microcapsule microstructure. It was possible to determine the
size distribution of the microcapsules, to differentiate bacteria-loaded from unloaded
microcapsules, and to describe characteristics of the microcapsule material. Cryo-fracturing
revealed details about the microcapsule matrix, interactions of the bacteria and the
microcapsule, and void spaces around the bacteria. Details of capsule microstructure and
interactions with bacteria could be observed in samples prepared using an anhydrous
procedure followed by TEM. What appeared to be porosity differences existed between
bacteria-loaded and non-loaded microcapsules which could affect viability when exposed to
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gastric conditions. Such microstructural information may be important in designing
microcapsules for food use as well as carriers of other substances for delivery in the body.
Prebiotics are nondigestible food ingredients that beneficially affect the host by
selectively stimulating the growth and/or activity of 1 or a limited number of bacteria in the
colon. Incorporation of prebiotics and calcium alginate as coating materials may provide better
protection for probiotics in food and eventually the intestinal tract because of the potential for
synergy between probiotics and prebiotics. In a study by Chen et al. (2005), prebiotics
(fructooligosaccharides or isomaltooligosaccharides), growth promoter (peptide), and sodium
alginate were incorporated as coating materials to microencapsulate 4 probiotics (Lactobacillus
acidophilus, Lactobacillus casei, Bifidobacterium bifidum, and Bifidobacterium longum). The
proportion of the prebiotics, peptide, and sodium alginate was optimized using response
surface methodology (RSM) to 1st construct a surface model, with sequential quadratic
programming (SQP) subsequently adopted to optimize the model and evaluate the survival of
microencapsulated probiotics under simulated gastric fluid test. Optimization results indicated
that 1% sodium alginate mixed with 1% peptide and 3% fructooligosaccharides as coating
materials would produce the highest survival in terms of probiotic count. The storage results
also demonstrated that addition of prebiotics in the walls of probiotic microcapsules provided
improved protection for the active organisms. These probiotic counts remained at 106 to 107
colony-forming units (CFU)/g for microcapsules stored for 1 mo and then treated in simulated
gastric fluid test and bile salt test.
2.2 Acid Tolerance
Acid stress is of particular importance for bacteria used in food technology. Indeed, a variety of
food products are acidi fied during fermentation by lactic acid bacteria. Probiotic
microorganisms, in particular, are usually provided in the form of fermented milk and suffer
lactic acid stress. Consequently, probiotics, including Bifidobacterium and Lactobacillus strains,
undergo severe mortality during the processing and storage of such products. For this reason,
less acidified products such as cheeses were proposed as carriers of these bacteria. Probiotics
are further challenged by extreme acid stress when reaching the stomach lumen where
hydrochloric acid is present. It is thus clear that the ability to efficiently adapt to acid stress is a
sine qua non condition for a probiotic microorganism in order to reach the intestine and exert
the expected beneficial effects.
Propionibacteria as Porbiotics: Propionibacteria are used both as cheese starters and as
probiotics in human alimentation. Traditionally used as cheese starters, dairy propionibacteria,
including Propionibacterium freudenreichii, display a number of probiotic effects, such as
increased levels of fecal bifidobacteria in humans, inhibition of undesirable flora, beneficial
modification of enzymatic activities within the gut , and treatment of lactose intolerance.
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During the cheese making process, P. freudenreichii resists harsh physical and chemical
stresses, including significant heat and salt stresses. To exert beneficial effects in the intestine,
it also needs to survive digestive stresses. Tolerance to digestive stresses is one of the main
factors limiting the use of microorganisms as live probiotic agents. Adaptation to low pH thus
constitutes a limit to their efficacy. In a study by Jan et al (2001), acid tolerance response (ATR)
was evidenced in a chemically defined medium as an acid stress adaptation in the probiotic SI41
strain of Propionibacterium freudenreichii. Transient exposure to pH 5 afforded protection
toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR,
since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic
expression were observed with two-dimensional electrophoresis during adaptation. Among the
up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA
synthesis and repair were identified during the early stress response, while the universal
chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR
was evident at both the physiological and morphological levels. This study constitutes a first
step toward understanding the very efficient ATR described in P. freudenreichii.
3.3 Bile Salt Tolerance
The major application of probiotics is in the treatment of intestinal disorders which are
destined to be subjected to various physical and chemical stresses before ingestion by the
human host. Bile salts are synthesized from cholesterol in the liver, stored in the gallbladder,
and released into the duodenum. Susceptibility to bile salts and tolerance acquisition in the
probiotic strain P. freudenreichii SI41 were characterized in a study characterized by Leverrier
et al. (2003). They showed that pretreatment with a moderate concentration of bile salts (0.2
g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile
salts challenge led to drastic morphological changes, consistent with intracellular material
leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of
the cells during lethal treatment played an important role in the response to bile salts, as
stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either
thermal or detergent pretreatment conferred significantly increased protection toward bile
salts challenge. In contrast, some other heterologous pretreatments (hypothermic and
hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have
sensitized the cells. These results provided new insights into the tolerance of P. freudenreichii
to bile salts, which must be taken into consideration for the use of probiotic strains and the
improvement of technological processes.
3.4. Adhesion to Intestinal Cells
Several health-related effects associated with the intake of probiotics have been reported in
different animal models as well as in human studies. This bacterial community plays a pivotal
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role in human nutrition and health by promoting the supply of nutrients, preventing pathogen
colonization and shaping and maintaining normal mucosal immunity. While the precise
mechanistic basis of the beneficial effects of probiotics is still obscure and will most likely vary
depending on the strain and species used, a number of mechanisms have been suggested.
Protecting the host from enteropathogen colonization (barrier effects) and immunomodulatory
effects toward host immune response.have been demonstrated in humans and laboratory
animals. For the protection against enteropathogen infections, the possibility of using food
supplements containing probiotic bacteria has been recently considered.
Adhesion and colonization are important for selection and use of probiotic strains. SEM has
been introduced to study the density and survival of probiotics in chicken intestine after
feeding chicken with the probiotic supplements. Chichlowski et al. (2007) reviewed the
metabolic and physiological impact of probiotics on Poultry and on the basis of observations
endorsed by electron microscopy have concluded that beneficial effects of probiotics are the
result of the summation of a complex, multi-variate series of alterations in gut microbial and
whole body metabolism. Those alterations might include whole body and immune function,
feed consumption, absorption of nutrients and beneficial changes in intestinal architecture. To
study the immune response of probiotics in mice TEM was used by Galdeano and
Perdigon(2004) to determine interaction of Lactobacillus casei with the gut. They compared
the influence of viable and nonviable lactic acid bacteria on the intestinal mucosal immune
system (IMIS) and their persistence in the gut of mice. TEM showed whole Lact. casei adhered
to the villi; the bacterial antigen was found in the cytoplasm of the enterocytes. Viable bacteria
stimulated the IMIS to a greater extent than nonviable bacteria with the exception of Lact.
delbrueckii subsp. bulgaricus.
The intestinal tract acts as a reservoir for the intestinal microbiota that exerts both harmful and
beneficial effects on human health. Intestinal microbiota contains an extraordinarily complex
variety of metabolically active bacteria and fungi which interact with the host’s epithelial cells
and provide constant antigenic stimulation to the mucosal immune system. The intestinal
epithelium presents the first line of defense against invading or attaching bacteria. In addition
to serving as a physical barrier to microbial penetration, the intestinal mucosa is the main
interface between the immune system and the luminal environment. Intestinal epithelial cells
(IECs) appear as an essential link in communicating with the immune cells in the underlying
mucosa and the microflora in the lumen via the expression of many mediators. The final
outcome is a considerable infiltration of neutrophils that may perpetuate inflammation and
eventually lead to cell damage, epithelial barrier dysfunction and pathophysiologic change of
diarrhea. The interaction between probiotic strains and the intestinal epithelium is a key
determinant for cytokine production by enterocytes, and probably the initiating event in
probiotic immunomodulatory activity, as it occurs prior to the encounter with the immune
300
system cells. It has been reported that several strains of probiotics belonging to Bifidobacterium
and Lactobacillus are highly relevant to the prevention of the invasion of tissues by
enteropathogens. Moreover, by inhibiting the production of IL-8 in enterocytes, these strains
are also found to be effective in modulating the proinflammatory response in IECs challenged
by enteropathogens such as Salmonella typhimurium (S. typhimurium); such induction is species
and strain specific Since the immunomodulatory properties are strainspecific[ for each
probiotic strain, profiles of the cytokines secreted by lymphocytes, enterocytes and/or DCs that
come into contact with the strain should be established. Shannon et al.(2002) developed an
infant rhesus monkey model to study enteropathogenic Escherichia coli (EPEC) induced
gastroenteritis and employed SEM to investigate effect of L. reuteri-supplementation of infant
formula on growth, nutritional status, and mineral absorption.
Liu et al. (2010) studied Adhesion and immunomodulatory effects of Bifidobacterium lactis
HN019 on intestinal epithelial cells to elucidate the adherence and immunomodulatory
properties of this strain. Adhesion assays of B. lactis HN019 and Salmonella typhimurium (S.
typhimurium) ATCC 14028 to INT-407 cells were carried out by detecting copies of species-
specific genes with real-time polymerase chain reaction. Morphological study was further
conducted by transmission electron microscopy. Interleukin-1β (IL-1β ), interleukin-8 , and
tumor necrosis factor-α (TNF-α ) gene expression were assessed while enzyme linked
immunosorbent assay was used to detect IL-8 protein secretion. The attachment of S.
typhimurium ATCC 14028 to INT407 intestinal epithelial cells was found to be inhibited
significantly by this strain which could be internalized into the INT-407 cells and attenuated IL-
8 mRNA level at both baseline and S. typhimurium induced pro-inflammatory responses. IL-8
secretion was reduced while IL-1β and TNF-α mRNA expression level remained unchanged at
baseline after treated with it. The researchers concluded that B. lactis HN019 does not up-
regulate the intestinal epithelium expressed pro-inflammatory cytokine, it showed the potential
to protect enterocytes from an acute inflammatory response induced by enteropathogen.
Adhesion to Human Intestinal Cell: Adhesion of probiotic to human epithelium cell has been
suggested as an important prerequisite for probiotic action. Adhesions of probiotic are likely to
persist longer in the intestinal tract this to showing the ability to metabolic, immunmodulatory,
stabilize the intestinal mucosal barrier, and provide competitive exclusion of pathogen bacteria.
Appropriate for different human intestinal cell culture models simulating the human situation
has been used widely to study the specific functions of the human intestinal cell (Servin and
Coconnier, 2003). Many studies were done as in vitro model system adhesion of probiotic, such
as the human colon carcinoma cell line HT-29, Caco2, and HT29-MTX are important in the
assessment of adhesion properties (Saarela et. al., 2000). HT-29 used as model for small
intestine and large intestine colon. The location of probiotic adhesion provided with interaction
with the intestinal mucosal surface and contact with gut associate lymphoid tissue (GALT) to
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stimulate immune system. The theoretical benefits of probiotic bifidobacteria in the intestinal,
mediated by modulation the functionality of the intestinal microbial, the gut barrier, and
immune system of the host, and the both therapeutic and prophylactic roles have been
proposed and trailed in animal and human, in recent years, studies of the probiotic effects of
ifidobaeria have been focused in these areas: adherence properties, resistance to infection
diseases, and prevention of colon cancer]. From the identification of a possible probiotic strain,
lead to its production and marketing, through its growth in laboratory, summarizing the whole
process existing behind its development, microencapsulation technologies, safety tests, and the
studies performed to test its resistance to human secretions and stability.
The adhesion ability of two Bifidobacteriums strains Bifidobacterium longum BB536 and
Bifidobacterium psudocatenulatum G4 was done by Ali et al(2008) using HT-29 human
epithelium cell line as in vitro study. Four different level of pH were used 5.6, 5.7, 6.6, and 6.8
with four different times 15, 30, 60, and 120 min. Adhesion was quantified by counting the
adhering bacteria after Gram staining. The adhesion of B. longum BB536 was higher than B.
psudocatenulatum G4. Both species showed significant different in the adhesion properties at
the factors tested. The highest adhesion for both Bifidobacterium was observed at 120 min and
the low adhesion was in 15 min. The findings of this study will contribute to the introduction of
new effective probiotic strain for future utilization.
3.0 CONCLUSION
Electron microscopy both TEM and SEM can be efficiently used in conjunction with other
microscopic techniques(Atomic force microscopy, Confocal laser microscopy), molecular
biological techniques such as Real Time PCR and immunological methods to visualize, assess,
validate and document the functional attributes available and novel probiotics.
Refernces
Ali QS, Farid AJ, Kabeir BM, Zamberi S, Shuhaimi M, Ghazali HM, Yazid AM (2008). adhesion properties of
Bifidobacterium pseudocatenulatum G4 and Bifidobacterium longum BB536 on HT-29 human epithelium
cell line at different times and pH. International Journal of Biological and Medical Sciences, 3-4:267-71.
Allan-Wojtas P, Truelstrup L, Hansen and Paulson AT (2008). Microstructural studies of probiotic bacteria-
loaded alginate microcapsules using standard electron microscopy techniques and anhydrous fixation,
LWT 41:101–108.
Chen KN, Chen MJ, Liu JR, Lin CW and Chiu HY (2005). Optimization of incorporated prebiotics as coating
materials for probiotic microencapsulation. Journal of food science, 70:M 260-M 266.
Chichlowski M, Croom J , McBride BW, Havenstein GB and Koci MD (2007). Metabolic and Physiological
Impact of Probiotics or Direct-Fed-Microbials on Poultry: A Brief Review of Current Knowledge.
International Journal of Poultry Science 6 (10): 694-704.Galdeano CM and Perdigon G (2004). Role of
viability of probiotic strains in their persistence in the gut and in mucosal immune stimulation. J. Appl.
Microbiol., 97:673–68.
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Jan G, Leverrier P, Pichereau V, and Boyaval P (2001). Changes in protein synthesis and morphology during
acid adaptation of Propionibacterium freudenreichii. Applied and environmental microbiology, 67: 2029–
2036.
Leverrier P, Dimova D, Pichereau V, Auffray Y, Boyaval P and Jan G (2003). Susceptibility and Adaptive
Response to Bile Salts in Propionibacterium freudenreichii: Physiological and Proteomic Analysis. Applied
and Environmental Microbiology. 69: 3809–3818.
Liu C, Zhang ZZ, Dong K and Guo XK (2010). Adhesion and immunomodulatory effects of
Bifidobacterium lactis HN019 on intestinal epithelial cells INT-407. World J Gastroenterol., 14;
16(18):2283-2290.
Rosenberg M, Kopelman IJ and Talmon Y (1985). A scanning electron microscopy study of
microencapsulation. Journal of food science, 50:139–144
Saarela M, Mogensen G, Fonden R, Matto J,and Mattila-Sandholm T (2000). Probiotic bacteria: safety,
functional and technological properties. Journal of Biotechnology, vol. 84, pp.197-215.
Servin A, and Coconnier M (2003). Adhesion of probiotic strains to the intestinal mucosa and interaction
with pathogens. Best Practice and Research Clinical Gastroenterology, vol.5, pp. 741–754.
Shannon L, Kelleher, Casas I, Carbajal N and Lonnerdal B (2002). Supplementation of Infant Formula With
the Probiotic Lactobacillus reuteri and Zinc: Impact on Enteric Infection and Nutrition in Infant Rhesus
Monkeys. Journal of Pediatric Gastroenterology and Nutrition, 35:162–168.
Sheu TY and Rosenberg M (1998). Microstructure of microcapsules consisting of whey proteins and
carbohydrates. J. Food Sci., 63: 491-93.
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Emerging Trends in Molecular Techniques for Identification, Characterization
and Typing of Novel Probiotics
V. K. Batish, Ashwani Kumar, Rahul Rathore and Sunita Grover
Molecular Biology Unit, Dairy Microbiology Division, NDRI, Karnal-132001
Probiotics and probiotic based functional/health foods have recently become the focus
of attention across the world including India in view of their multifunctional role in human
health and nutrition. The interest in these magic bugs has grown enormously during the last
decade as they are now fast emerging as possible biotherapeutics for management of gut
related inflammatory metabolic disorders and other chronic diseases both infectious and non-
infectious. With the growing awareness, health conscious consumers are now looking for safe,
cost effective and traditional dietary /food based natural ingredients having novel bioactive
functions for health care as an alternative to medicine/drug based therapy due to possible
adverse side effects of the later. In this context, probiotics are now recognized as the most ideal
candidate for filling this gap and the consumers are now reposing their faith in probiotics for
addressing their health related problems and hence are now keen to use more and more of
probiotics in their daily dietary regimen as prophylactics for boosting their health and mucosal
immunity, by keeping the gut in good healthy status and protecting it against the pathogen
invasion. Because of their novel health promoting physiological functions, and their application
in functional and health foods, probiotics have become a hot commodity with extraordinary
high commercial stakes at the global level Since the properties related to probiotic functions
are highly strain specific, proper identification and authentication of strains is extremely vital to
the success of probiotic applications in the development of novel functional foods for
promoting human health and well being. Molecular techniques that look at the variation
amongst the strains at DNA/RNA level are widely used for genotyping of variety of microbes.
Most of these have also found applications in molecular typing of probiotic microorganisms. In
view of high stakes involved in exploration of the commercial value of probiotics particularly in
the booming functional / health food market, the correct identification of Probiotic cultures has
become extremely important to rule out the possibility of false claims and to resolve disputes
concerning their identity in Probiotic preparations.
Phenotypic approaches
Traditionally, probiotic bacteria have been classified on the basis of phenotypic
properties such as morphology, mode of glucose fermentation, growth at different
temperatures, lactic acid configuration, fermentation of various carbohydrates, methyl esters of
fatty acids and pattern of proteins in the cell wall or entire cell Some of the phenotypic
fingerprinting techniques based on phenotypic and genotypic characteristics are
Polyacrylamide Gel Electrophoresis of soluble proteins, fatty acid analysis, bacteriophage typing
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and sero typing. The phenotypic fingerprints obtained, are usually less sensitive and changes in
the fingerprint may not necessarily mean a different organism, but rather could be attributed to
a change in expression of the particular phenotypic trait.
Some experiments are well documented to compare the phenotypic and genotypic
studies. In a study to assess the methods like carbohydrate fermentation, partial 16S rDNA
sequencing and cellular fatty acid methyl ester methods were used to determine the taxonomic
relationship of the probiotic lactobacilli and Bifidobacteria. The variability among replicates of
FAME analysis was so high that it was concluded that this approach was not useful for
speciation of probiotic lactobacilli and also variation in fermentation profiles were observed in
L. johnsonii strains and this might lead to inaccurate speciation However, the 16S rDNA
sequencing results were highly reliable. The results suggest that the use of the first 500 bp of
the 16S rDNA is effective for species identification.
Drawbacks of conventional methods are lack of reproducibility, type ability and
discriminating power while analyzing the phenotype, since whole information potential of a
genome is never expressed i.e. gene expression is directly related to the environmental
conditions. Also, plate culturing techniques may not always reveal the true microbial
populations because most of the GIT organisms are difficult to cultivate. It has been estimated
that only less than 50% of species present in the gut microflora have been cultured on existing
microbial growth media. All these drawbacks adversely affect the reliability of phenotypic
based methods for culture identification at genus and species level. Hence, polyphasic
approaches combining biochemical, molecular and morphological data are important for
accurate classification of LAB.
Molecular approaches
The phylogenetic information encoded by 16S rDNA has enabled the development of molecular biology techniques, which allow the characterization of the whole human gut microbiota. These techniques have been used in monitoring the specific strains as they have high discriminating power. Molecular techniques have been found to be quite useful and effective in characterization of microbial community, composition, enumeration and monitoring of microbial population and tracking of specific strains of bacteria in the gut microflora. Classification of organisms and evaluation of their evolutionary relatedness by 16SrRNA analysis was first developed as a gold standard. This molecular approach has allowed meaningful phylogenetic relationships between microbes in natural ecosystems to be discerned. The internal transcribed spacer (ITS) has been explored in the genetic characterization of lactobacilli, Bifidobacteria and LAB. RAPD profiling has been successfully applied to distinguish between strains of Bifidobacterium and strains of L. acidophilus group. A multiplex RAPD -PCR using a combination of two 10-mer primers in a single PCR reaction enabled differentiation of Lactobacillus strains from the gastrointestinal tract of mice. RAPD-PCR has also been used for the detection of Lactobacillus rhamnosus and L. fermentum in the human vagina in order to assess probiotic persistence at this site. The group-specific PCR and
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RAPD-PCR have also been explored to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts. Ribo-typing is another potential molecular tool that has been extensively tried for discriminating related strains using four different restriction enzymes which had different recognition sites in the spacer region. However, no different digestion patterns were observed which showed that sequence variation in the spacer region among Lactobacillus strains had not been sufficient for specific identification of L. plantarum strains. Therefore, PCR Ribotyping was determined as an inefficient method for identification of L. plantarum at strain level. DNA finger printing techniques like RFLP have also been and developed new primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene of human faecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI enzymes. Operational Taxonomic Units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. This new T-RFLP method made easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP. Moreover, it also made possible to identify the bacterial species that an OTU represents by cloning and sequencing. PFGE has also been also used to identify strains to assess the accuracy of labeling with regard to genus and species and found the method to be convenient for identifying probiotic lactobacilli in probiotic food and animal feed. Strain typing has been successfully achieved by PFGE for the Lactobacillus acidophilus complex, L. casei, L. delbrueckii and its three subspecies (bulgaricus, delbrueckii and lactis), L. fermentum, L. helveticus, L. plantarum, L. rhamnosus and L. sakei. Fluorescent in situ Hybridization (FISH) has been explored by several investigators in determining the load of viable organisms in the feces and gut. By applying this technique, the number of bacteria in human faecal samples was shown to be approximately ten-fold higher than number estimated through standard culture techniques, when non-specific probes to the 16S rRNA for FISH were used. Amplification rDNA Restriction Analysis (ARDRA) has successfully differentiated various species or strains within the Lactobacillus acidophilus complex, L. casei, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L. reuteri, L. rhamnosus and L. sakeiARDRA has been used to differentiate a variety of lactobacilli at species level, including L. delbrueckii and its three subspecies (bulgaricus, delbrueckii and lactis), L. acidophilus and L. helveticus. Amplified Fragment Length Polymorphism (AFLP) has been found to be a very useful fingerprinting technique for bacteria, affording both species resolution and strain differentiation. Species-level discrimination has been shown for the phylogenetically closely related species L. pentosus, L. plantarum and L. pseudoplantarum using this method. DNA chip/ array is going to be the method of choice for identification of dairy organism in the near future because of its high degree of reliability in terms of specificity and sensitivity. In this presentation, some of the advanced and sophisticated molecular techniques that can be explored for reliable identification of novel probiotic cultures particularly belonging to lactobacilli and bifidobacteria at genus, species and strain level will be highlighted to discriminate their genetic diversity and phylogenetic relatedness.
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LAB-Cell Factories for Novel Dairy Ingredients
Shilpa Vij, Subrota Hati, Deepika Yadav
Dairy Microbiology Division, N. D. R. I., Karnal-132001
Lactic acid bacteria (LAB) are the main group of micro-organisms that has been used
successfully for decades for the production of fermented milks as they are producing different
biofunctional components such as organic acids, exopolysaccharides, bioactive peptides, folate,
oligosaccharides, dietary sugars, vitamins etc. These organisms belong to the genera of
Lactococcus, Leuconostoc, Pediococcus, Streptococcus and Lactobacillus. LAB are industrially
important microbes that are used all over the world in a wide variety of industrial food
fermentations. They are excellent ambassadors for an often maligned microbial world. The
micro-organisms which are employed in fermented milks (including probiotic products,
alcoholic/lactic beverages, cultured cream, and products containing moulds) and the cheese
industry are used singly, or in pairs or multiples, or in a mixture, thus giving the industry the
opportunity to manufacture different products. Beyond the horizons of their conventional role
in acid, flavour and texture development, they are being looked up on as burgeoning “cell
factories” for production of host of functional biomolecules and food ingredients such as
biothickeners, bacteriocins, vitamins, bioactive peptides and amino acids.
Bioactive Peptides
Bioactive peptides are described as ‘food-derived components (genuine or generated)
that, in addition to their nutritional value, exert a physiological effect in the body’. Biological
activities associated with such peptides include immunomodulatory, antibacterial, anti-
hypertensive and opioid-like properties (Meisel, 2005). Milk proteins are recognized as a
primary source of bioactive peptides, which can be encrypted within the amino acid sequence
of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins
using the proteolytic systems of lactic acid bacteria (LAB) is an attractive approach for
generation of functional foods enriched in bioactive peptides given the low cost and positive
nutritional image associated with fermented milk drinks and yoghurt. Thus, fermentation of
milk and milk proteins by proteolytic lactic acid bacteria can lead to development of functional
foods conferring specific health benefits to the consumer beyond basic nutrition. The starter
culture applied in the manufacture of `Festivo’ cheese, a novel bioactive cheese is a mixture of
commercial starter cultures containing 12 different strains of the following genera or species:
Lactococcus sp. and Leuconostoc sp. (BD type cultures), Propionibacterium sp., Lactobacillus sp.
as well as Lactobacillus acidophilus and Bifidobacterium.
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Microorganisms Precursors
proteins
Peptide Sequence Bioavailability
Lactobacillus
helveticus,
Saccharomyces
cerevisiae
β-cn, k-cn Val-Pro-Pro,Ile-Pro-Pro ACE inhibitory,
antihypertensive
Lactobacillus GG
enzymes+pepsin &
trypsin
β -cn, as1-cn Tyr-Pro-Phe-Pro, Ala-Val-Pro-
Tyr-Pro-Gln-Arg, Thr-Thr-Met-
Pro-Leu-Trp
Opioid, ACE inhibitory,
immunostimulatory
L. helveticus CP90
proteinase
β-cn Lys-Val-Leu-Pro-Val-Pro-(Glu) ACE inhibitory
L. helveticus CPN 4 Whey proteins Tyr-Pro ACE inhibitory
L. delbrueckii subsp.
bulgaricus SS1
β -cn, k-cn Many fragments ACE inhibitory
L. delbrueckii subsp.
bulgaricus IFO13953
k-cn Ala-Arg-His-Pro-His-Pro-His-
Leu-Ser-Phe-Met
Antioxidative
L. rhamnosus
+digestion with
pepsin and Corolase
PP
β-cn Asp-Lys-Ile-His-Pro-Phe, Tyr-
Gln-Glu-Pro-
Val-Leu
ACE inhibitory
L. delbrueckii subsp.
bulgaricus
β-cn Ser-Lys-Val-Tyr-Pro-Phe-Pro-
Gly Pro-Ile
ACE inhibitory
Streptococcus
thermophilus+Lc. lactis
subsp.
lactis biovar.
diacetylactis
β-cn Ser-Lys-Val-Tyr-Pro ACE inhibitory
Exopolysaccharides (EPS)
LAB produce exopolysaccharides (EPS), which are homopolysaccharide consisting of α-D-
glucans such as dextrans mainly composed of α-1,6-linked residues with variable (strain
specific) degrees of branching and alternans composed of α-1,3 and α-1,6 linkages. The
biosynthesis process is external and requires sucrose. Specific glycosyltransferase and dextran
or levan sucrase enzymes are involved in the biosynthesis process. Eight glucansucrase-
encoding genes from L. mesenteroides are cloned. The gene encoding the dextransucrase DsrD
can be efficiently expressed and secreted in a heterologous host (i.e. Lc. lactis MG1363) and is
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able to drive dextran synthesis. In dairy technology, dextrans, as with other EPS, are used as
food additives and act as texturizers by increasing viscosity and as stabilizers through
strengthening the rigidity of the casein network by binding hydration water and interacting with
milk constituents. As a consequence, EPS decreases syneresis and improves product stability
(Ricciardi, 2000). They play a recognized role in the manufacturing of fermented milk, cultured
cream; milk based dessert and flavoured milk. EPS contribute to the texture, mouth-feel, taste
perception and stability of the final product. They play a major role in the production of
fermented dairy products in Northern Europe, Eastern Europe and Asia. The
homopolysaccharides dextran and levan are synthesized by secreted or anchored
glycosyltransferases, dextran-sucrase and levan-sucrase, respectively. Once produced, they
convert extracellular sucrose into EPS and monosaccharides. The energy used for the
elongation of the dextran or levan chains is provided by the hydrolysis of sucrose. Conversion of
glucose-6-phosphate to glucose-1-phosphate by phosphoglucomutase is central to generating
the activated sugars. Glucose-1-P reacts with UTP to generate UDP-glucose, which can be
incorporated into the nascent EPS repeating unit or can be converted to UDP-galactose or
dTDP-rhamnose.
EPS Strain Linkage
Dextran L. mesenteroides ssp. mesenteroides
L. mesenteroides ssp. dextranicum
α-D-glc (1-6)
Mutan S. mutans, L. mesenteroides α-D-glc (1-3)
Alternan L. mesenteroides α-D-glc (1-3) (1-6)
β-D-glucan Pediococcus sp. Streptococcus sp. β-D-glc (1-3)
Fructan S. mutans, S. salivarius β-D-Fruc (2-6)
Polygalactan L. lactis ubsp. Lactis H414 α-D-Glc/ β-D-Gal
Mannitol
(D-)Mannitol is a naturally occurring six-carbon sugar alcohol or polyol. Mannitol is a
low-calorie sugar that could replace sucrose, lactose, glucose or fructose in food products. The
mannitol production in lactic acid bacteria is strongly dependent on the pathway of
carbohydrate fermentation: LAB possess either a homofermentative or a heterofermentative
pathway. It is metabolized independently of insulin and is also applicable in diabetic food
products. L. pseudomesenteroides and L. mesenteroides are known for their ability to produce
mannitol in the fermentation of fructose. Mannitol is a valuable nutritive sweetener because it
is non-toxic, non-hygroscopic in its crystalline form and has no teeth decaying effects. It has a
sweet, cool taste and it is about half as sweet as sucrose. Mannitol is only partially metabolized
by humans and it does not induce hyperglycemia, which makes it useful for diabetics. Mannitol
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is applied as a food additive (E421) as a sweet tasting bodying and texturing agent and it is used
as a sweet builder in ‘‘sugar free’’ chewing gum and in pharmaceutical preparations. Mannitol
has some laxative properties and the daily intake of mannitol should therefore not exceed 20 g.
LAB are found to produce small amounts of mannitol intracellularly e.g. Streptococcus mutans,
L. leichmanii; Lactate dehydrogenase-negative mutant of L. plantarum and Lactate
Dehydrogenase deficient mutant of L. lactis., Leuconostoc pseudomesenteroides.
Sorbitol
Sorbitol also referred to as D-glucitol, is mainly found in many fruits and is sweet tasting,
forms a viscous solution, stabilizes moisture, possesses bacteriostatic property and is generally
chemically inert. A recombinant strain of L. casei is constructed, cells of which when pre-grown
on lactose, are able to synthesize sorbitol from glucose. Inactivation of the L-lactate
dehydrogenase gene led to an increase in sorbitol production. It is used as humectant,
sweetener, bodying and viscosity agent, vehicle, anti-caplocking and texture improvement.
Sorbitol is useful to promote the absorption of such as Cs, Sr, F and vitamins B12 (Hugenholtz J.,
2008.).
Galacto-oligosaccharides
Galacto-oligosaccharides (GOS) are thus formed in a kinetically controlled reaction. GOS
produced from lactose through enzymatic transgalactosylation. These are hydrolyzed polymers
of monosaccharides that contain 3-10 linked molecules of simple sugars. Certain other
compounds like lactulose and galactobiose also exhibit similar functional characteristics and are
widely regarded as oligosaccharides. Oligosaccharides can be synthesized by chemical reactions
or by controlled enzymatic hydrolysis of complex polysaccharides or enzyme assisted
transglycolation reactions. Human studies have shown an increase in Bifidobacterium resulting
from OSs ingestion and a reduction in detrimental bacteria such as Cl. perfringens). They found
that ingestion of 210mg /day for several weeks effectively increased bifidobacterial population
in intestine (an average of 7.5 times) and decreased Cl. perifringens (an average of 81%). GOS
has several health beneficial attributes such as mineral absorption, cancer prevention, coronary
heart diseases (CHDs) and in improving the intestinal diseases.
Folate
Folate, an important B-group vitamin, participates in many metabolic pathway such as
DNA / RNA biosynthesis and amino acid inter-conversions. Folic acid or pteroyl glutamic acid
(PGA) is comprised of p-aminobenzoic acid (PABA) linked at one end to a pteridine ring and at
the other end to L-glutamic acid. The naturally occurring folates include 5-
methyltetrahydrofolate (5-MTHF), 5-formyltetrahydrofolate, 10-formyltetrahydrofolate. Most
naturally occurring folates are pteroylpolyglutamates, containing two to seven glutamates
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joined in amide (peptide) linkages to the α-carboxyl of glutamate. Human cannot synthesize
folate; it is necessary to assimilate this vitamin exogenously. Folate deficiency in humans is
associated with several problems, such as cancer, cardiovascular diseases as well as neural tube
defects in newborns. The daily recommended intake (DRI) is set at 200 and 400 µg/day for
adults and women in the periconceptional period, respectively (Forssen, 2000). There ways to
increase the folate levels of food products: i) fotification of food products ii) selection of special
plant cultivars, or fruits with increased folate pools, iii) fermentation fortification.
Folate concentration in dairy products and its contribution to the reference daily intake (RDI)
Product Folate (µg/L) Folate per serving
(µg/240 mL)
% RDI (3 Serving)
Milk 40±10 10±2 6-8
Butter Milk 90±20 22±5 13-20
Yoghurt 80±20 19±5 11-18
Kefir 50±10 12±2 8-11
Ropy-Milk 110±20 26±5 16-23
Sour Buttermilk 75±15 18±4 11-17
Acidophilus milk 75±15 18±4 8-11
S. thermophilus has a strain specific ability of folate production and has been reported to
produce higher quantity of folate in comparison to other LAB; majority of which is excreted into
milk.
Trehalose
Trehalose, also known as mycose, is a natural alpha-linked disaccharide formed by an α,
α-1, 1-glucoside bond between two α-glucose units. Trehalose is found naturally in insects,
plants, fungi and bacteria. Trehalose is a naturally occurring reducer of cell stress, protecting
these organisms from extremes in heat shock and osmotic stress. Trehalose has been accepted
as a novel food ingredient under the GRAS terms in the U.S. and the European Union (Cardoso,
2004). Trehalose is wide spread within the genus Propionibacteruim. Trehalose accumulation in
Propinibacterium e.g. P. acidipropionici and P. freudenreichii subsp. shermanii has also been
observed to occur in response to stress conditions. In this organism, trehalose results from the
conversion of glucose 6-P and ADP glucose via trehalose 6-P synthase to trehalose 6-P and its
subsequent dephosphorylation by trehalose 6-P phosphatase. Alternatively, trehalose can be
fromed from maltose through the action of trehalose synthase.
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Biosurfactant
Biosurfactants are a structurally diverse group of surface active molecules synthesized by large
variety of microoganisms, which vary in their chemical propeties and molecular size. They are
produced on living surfaces, mostly microbial cell surfaces, or excreted extracellularly and
contain hydrophobic and hydrophilic moieties that reduce surface tension and interfacial
tensions between individual molecules at the surface and interface, respectively. Dairy
Streptococcus thermophilus strains is reported to produce biosurfactants which cause their own
desorption and oral Streptococcus mitis strains produce biosurfactants that inhibit adhesion of
Streptococcus mutans (Nitschke, 2007). Major biosurfactants are trehalose lipids, sophorolipids,
rhamnolipids, glycolipids, cellobiose lipids, polyol lipids, phospholipids, sulfonylipids, viscosin,
diglycosyl diglycerides. Biosurfactants by LAB are used more often for medical purpose as
ingredients of therapeutic agents playing a key role in the prevention and control of infections
caused by pathogens representing various groups of microorganisms.
References:
Cardoso, F.S., Gaspar, P., Hughenholtz, J., Ramos, A., Santos, H. 2004. Enhancement of trehalose
production in dairy propionibacteria through manipulation of environment condition. Int. J. Food
Microbiol. 91: 195-204.
Efiuvwevwere, B. J. O., Gorris, L. G. M., Smid, E. J., & Kets, E. P. W. 1999. Mannitol-enhanced survival of
Lactococcus lactis subjected to drying. Applied Microbiology and Biotechnology. 51. 100–104.
Forseen, M. 2000. Folates and dairy product: A critical update.Journal of the American College of
Nutrition. 19.100-110.
Hughenholtz, J. 2008. The lactic acid bacterium as a cell factory for food ingredients production.
International Dairy Journal. 18: 447-466.
Meisel, H. and Bocklmann W. (1999). Bioactive pepitdes encrypted in milk proteins; proteolytic activation
and thropho-funtional properties. Antonie van Leeuwenhoek, 76: 207-215.
Nitschke, M. & Coast, S.G. 2007. Biosurfactants in Food Industry. Trends in Food Sci. Technol. 18. 252-259.
Ricciardi, A., & Clementi, F. 2000. Exopolysaccharides from lactic acidbacteria: Structure, production
andtechnologic al applications. Italian Journal of Food Science.1. 23–45.
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Technological Advances in the Manufacture of Value Added Traditional Dairy
Products
P. Narender Raju* and Ashsih Kumar Singh**
Division of Dairy Technology, National Dairy Research Institute, Karnal, Haryana
1.0 INTRODUCTION
The Operation Flood programme, one of the world’s largest and most successful integrated
dairy development programs initiated in 1970, has led India to emerge as the largest milk
producer in the world. It is estimated that milk production in India reached a record level of
112 MT in 2010 accounting for more than 16% of the world’s total production (697 MT) of
which buffalo milk constitutes more than 50% (FAO, 2010). Historically, surplus milk in the
rural areas where it is produced has been converted into a variety of traditional products
primarily as a means of preservation. The increased availability of milk during the flush
season coupled with lack of facilities to keep liquid milk fresh during transit from rural
production areas to urban market makes conversion of milk into traditional products
particularly attractive. These products include curd, ghee, khoa, chhana, paneer, shrikhand
and a variety of milk sweets, some of which are now increasingly produced even by the
organized sector milk plants. Traditional dairy products and sweets are an integral part of
Indian heritage. These products have great social, religious, cultural, medicinal and
economic importance and have been developed over a long period with the culinary skills of
homemakers and halwais. In addition to preservation of milk solids for longer time at room
temperature, manufacture of traditional dairy products add value to milk and also provide
considerable employment opportunity. It is estimated that about 50% of total milk
produced in India is converted into traditional milk products. Traditional dairy products not
only have established market in India but also great export potential because of strong
presence of Indian diaspora in many parts of the world (Pal and Raju, 2007). In the present
paper, some of the value added Indian traditional dairy products especially ghee, khoa- and
chhana-based sweets are discussed.
2.0 GHEE
Ghee is heat clarified fat derived solely from milk or curd or from desi (cooking) butter
or from cream to which no colouring matter or preservative has been added. It is usually
prepared from cow’s milk, buffalo’s milk or mixed milks. Ghee manufacture has great
significance and relevance to Indian masses and the dairy industry. There is sufficient recorded
evidence to prove that the manufacture of ghee originated in India and it has been used
extensively for dietary and religious purposes since Vedic times (3000-2000 B.C). At present,
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about 28% of the total milk production is utilized for the manufacture of about 1 million tonne
of ghee per annum. Besides, it’s being a product of tradition with an established market,
several other factors favour the production of ghee at different levels i.e. house hold, ghee
trading centres and organized dairies, in India. Some of the factors that goes in favour of ghee
production are its simple technology with relatively low cost, longer shelf life, no required of
refrigeration for storage, several uses, such as direct dressing of food preparations, cooking and
frying medium, and for religious rites. This is probably the only dairy product produced at all
scales starting from household level to very large organized dairies like AMUL. The principle of
manufacturing ghee basically involves following three steps:
i) Concentration of lipid phase: Butterfat in milk is present in form of fat globules, which are
properly emulsified by fat globule membrane and dispersed in serum phase. For efficient
separation of butterfat from the continuous phase (serum), it has to be concentrated
inform of cream or malai. Further concentration of butter fat is possible by converting it
into a continuous phase as in case of butter. The purpose of concentrating butterfat in a
discontinuous (cream) or continuous phase (butter) is to reduce the amount of water and
SNF contents in the raw material and facilitate ghee preparation. Sometimes, some
intermediate operations such as fermentation of milk prior to concentration of lipid phase
or of cream to emanate desired acidic flavor similar to desi ghee is also adopted.
ii) Heat clarification of cream or butter with a view to remove practically all the moisture and
to generate typical flavour and granulation (the final temperature should be normally in
range of 105-110°C to avoid cooked flavor), and
iii) Removal of residue from the heat clarified butter fat with a view to meet the legal
requirements and also to improve the storageability.
Adopting the above principle, different methods are used for the preparation of ghee.
The adoption of a particular method is mainly dependent on the scale of production. The flow
diagram for the manufacture of ghee based on these methods is given in fig.1.
2.1 Indigenous Method
The indigenous methods of ghee making usually involve (i) direct churning of raw milk,
(ii) lactic acid fermentation of heat-treated milk followed by churning of curd or (iii) removal of
thick clotted-cream layers (malai). The lactic acid fermentation method ii) is the most popular
method used in rural areas. Hand-driven wooden beaters are usually employed for separating
what is called ‘makkhan’ (butter). After accumulating sufficient quantity it is heated until
almost all the moisture has been removed. After heating, the contents are left undisturbed.
When the curd particles have settled at the bottom of the pan, the clear fat is carefully
decanted off into ghee storage vessels. The traditional gheemaking practice contributes about
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90% of the total ghee production in India. This method leaves behind a large quantity of
buttermilk and also leads to low fat recoveries (75-85%). This is why modern dairies do not use
the indigenous method.
2.2 Direct Cream Method
The small dairies use a technologically improved method for ghee making. This method
involves separation of cream from milk and directly heating the cream thus obtained to 110-
115°C in a stainless steel, jacketed ghee kettle fitted with agitator, steam control valve,
pressure and temperature gauges. Heating is discontinued as soon as the colour of the ghee
residue turns to golden yellow or light brown. High serum solids content of in the cream lead to
about 4-6% of fat loss in the ghee residue. Use of plastic cream or washed cream, with about
75-80% fat, is recommended for both higher fat recovery and lower steam consumption.
2.3 Creamery Butter Method
This is the standard method adopted in most of the organized dairies where unsalted
creamery butter or white-butter is used as a raw material for ghee-making. A typical plant
assembly for the creamery butter method comprises the following: (i) a cream separator, (ii)
butter churn, (iii) butter melting outfits, (iv) steam-jacketed, stainless steel ghee kettle with
agitator and process controls, (v) ghee filtration devices, such as disc filters or oil clarifier, (vi)
storage tanks for cream, butter and ghee, (vii) pumps and pipelines interconnecting these
facilities. (viii) crystallization tanks, and (ix) product filling and packaging lines. First, the butter
mass is melted at 60°C. The molten butter is pumped into the jacketed ghee boiler and steam
is opened to raise the temperature to boiling. The temperature gradually rises and the heating
at the last stage is carefully controlled. The-point shows the disappearance of effervescence,
appearance of finer air bubbles on the surface of fat, and browning of the curd particles (ghee
residue). At this stage, the typical ghee aroma is also produced. The ghee is then pumped, via
an oil filter or clarifier, into settling tanks which are cooled by re-circulating water at 60°C.
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Fig 1. Flow diagram of manufacturing ghee by different methods
2.4 Prestratification Method
The prestratification method consists of keeping molten butter undisturbed in a ghee
boiler at a temperature of 80-85°C for 30 min for stratifying the mass into three distinct layers.
The top layer is composed of floating, denatured protein particles and impurities, the middle
layer of almost clear fat, and the bottom layers of buttermilk serum. This division helps in
mechanical removal of the bottom layer of buttermilk, carrying about 80% of the moisture and
70% of the SNF contained in butter. Removal of the buttermilk eliminates the need for
prolonged heating and thereby energy saving and significantly low quantity of ghee residue.
The middle layer of fat is heated, usually to 110°C along with some denatured curd particles
floating on the top. This process is essential to promote development of a more
characteristicghee aroma. This method offers the advantages of economy in fuel consumption
up to 35-50 %, saving in time and labour up to 45% and production of ghee with lower free fatty
acid (FFA) levels and acidity. Stratification also helps in the production of ghee with a milder
flavour. Its application is limited to batch-scale operation.
2.5 Efficiency of different methods
The efficiency of different methods of ghee making differs in terms of fat recovery and
energy requirement. The fat recovery in indigenous method is lowest in range of 80-85%, in
creamery butter method it ranges from 88-92% and highest in direct cream method ranging
from 90-95%. The energy requirements in indigenous, direct cream and creamery butter
methods have been reported 1710, 1325 and 414 Kcal/kg of ghee respectively (Pandya et al.,
1987 a & b). Energy requirement are lowest in prestarification method.
3.0 KHOA BASED CONFECTIONS
Khoa is prepared by continuous boiling of milk until desired concentration (65 to 72% TS)
and texture is achieved. According to Prevention of Food Adulteration (PFA) (1955) rules,
khoa sold by whatever variety or name such as Pindi, Danedar, Dhap, Mawa, or Kava means
the product obtained from cow or buffalo (or goat or sheep) milk or milk solids or a
combination thereof by rapid desiccation and having not less than 30 per cent milk fat on
dry weight basis. To achieve the PFA standard a minimum fat level of 5.5 in buffalo milk is
essential. The quality of khoa is better when made from buffalo milk because khoa from
cow milk is inferior due to its moist surface, salty taste and sticky and sandy texture which is
not considered suitable for the preparation of sweetmeats. Also, buffalo milk results in
higher yield of khoa. Khoa is used as a base material for the manufacture of a wider range
of sweetmeats such as burfi, peda, gulabjamun, milk-cake, kalakand and kunda.
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3.1 Burfi
Burfi is the most popular milk-based confection essentially made from khoa. Sugar and
other ingredients are added in different proportions to khoa according to the demand of
consumers. Several varieties of burfi are sold in the market depending on the additives
present, viz., plain mawa, pista, nut, chocolate, coconut and rava burfi. Good quality burfi is
characterized by moderately sweet taste, soft and slightly greasy body and smooth texture with
very fine grains which is attained from buffalo milk khoa. Colour, unless it is chocolate burfi,
should be white or slightly yellowish. Traditionally burfi is prepared by adding sugar to hot khoa
and vigorous blending in a shallow kettle till a homogenous, smooth and fine grains mass is
achieved. In hot condition it is spread in shallow trays for setting. Kumar and Dodeja (2003)
developed a continuous method of making burfi using three-stage TSSHE. It consists of a
continuous khoa-making system (2-stage SSHE) and a burfi-making unit. Sugar was fed into the
burfi-making unit using a sugar dosing mechanism developed for the purpose. Palit and Pal
(2005) adopted TSSHE and Stephan processing kettle for the large scale production of burfi.
They standardized buffalo milk to SNF and fat ratio of 1.5:1 and prepared khoa on a continuous
khoa making machine (TSSHE). Khoa having 38-40% moisture was transferred to a Stephan
process kettle which was reduced to about 30–32% under vacuum. This was followed by sugar
addition @ 30% and kneading and working at 60°C. Burfi, thus obtained was hot filled into
polystyrene tubs and kept at room temperature for setting. Thereafter it was vacuum
packaged. A shelf life of about 60 days at 30°C has been reported by the workers.
3.2 Peda and Brown Peda
Peda, another khoa-based sweet, is granular in texture having dry body because of
comparatvely lower moisture content. Although the method of manufacture of peda vary from
region to region, it is identical to that of burfi preparation wherein a mixture of khoa and sugar
is heated at low-fire till desired texture is attained. Several types of pedas, viz. plain, kesar and
brown are available in the market. Plain peda is made into round balls of about 20–25 g size,
normally by rolling between the palms (Pal, 2000). The product may also be formed into
different shapes and sizes using different dies/moulds. Peda is usually packed in paper board /
boxes having a parchment paper liner or grease-proof paper liner (Reddy, 1985). Dewani and
Jayaprakasha (2002) reported that replacement of milk solids-not-fat (MSNF) up to 40% with
WPC improved all the sensory attributes of plain peda. An industrial method of converting khoa
into kesar peda had been developed at NDDB, Anand (Banerjee, 1997). Dewani and
Jayaprakasha (2004) also applied RO process for pre-concentration of milk as an intermediate
step in the production of plain peda. It was reported that such product was nutritionally better
than the conventionally made peda. Brown peda, another type of peda that is characterized by
caramelized color and highly cooked flavor, is popular in many parts of the country. Some of
the popular brands are Mathura peda, Dharwad peda and Mishra peda. As per an estimate the
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annual production of Dharwad peda varies from 3-6 tonnes per day (Kulkarni and Unnikrishnan,
2006). In almost all of these types, khoa is first cooked to brown colour in ghee and then peda is
prepared from it by blending sugar and other additives. The analysis of the market samples
from different parts of the country revealed significant variation in the quality of brown peda
(Londhe, 2006). Among the various samples analyzed, Mathura peda was reported to be
superior in quality than other types. Londhe (2006) also standardized the method of
manufacture of brown peda and attempted to enhance its shelf life by using different
packaging techniques.
3.3 Gulabjamun
Gulabjamun, is also a khoa-based sweet characterized by brown colour, smooth and spherical
shape, soft and slightly spongy body free from both lumps and hard central core, uniform
granular texture, mildly cooked and oily flavour, free from doughy feel and fully succulent with
sugar syrup. The gross chemical composition of gulabjamun vary widely depending on
numerous factors, such as composition and quality of khoa, proportion of ingredients, sugar
syrup concentration etc. The traditional method of gulabjamun making from dhap khoa has
been standardized by Ghosh et al., (1986). It involves proper blending of khoa, refined wheat
flour, baking powder and water (optional) to make homogenous and smooth dough. The small
balls formed from the dough are deep dried in ghee to golden brown colour and subsequently
transferred to 60% sugar syrup maintained at about 60°C. It takes about 2 hours for the balls to
completely absorb the sugar syrup. Dewani and Jayaprakasha (2002) reported that replacement
of MSNF up to 30% with WPC resulted in increased overall acceptability scores of gulabjamun.
A mechanized semi-continuous system has been developed for the manufacture of gulabjamun
from khoa at commercial scale (Banerjee, 1997). Deep-fat frying is a key operation in
gulabjamun preparation. This process induces typical brown colour and texture required to
produce good quality product. Recently, Kumar et al. (2006) studied the kinetics of colour and
texture changes that take place during deep-fat frying of gulabjamun and reported that the
browning-induced changes in colour parameter L* (lightness or brightness) followed zero-order
reaction, while the ratio of b* (yellowness) and a* (redness) values followed first-order kinetics.
Further, reported that the increase in the texture parameters hardness and firmness followed
zero-order reaction kinetics whereas stiffness rise followed a first-order reaction.
3.4 Kunda
Kunda is defined as a desiccated product prepared by the continuous heating of milk or
high moisture khoa with sugar. It is characterized by semi-brown to brown colour, soft body
and grainy texture, and characteristic sweet, nutty and pleasant flavour. The khoa generally
used for kunda making has high moisture content. If the khoa used has low moisture, then
about 10% milk is added. After the addition of calculated amount of sugar (25–30%), khoa is
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subjected to slow desiccation on direct fire. At the end, a brown mass with granular texture is
obtained which has about 25% moisture (Kulkarni et al., 2001). The shelf life of kunda is
reported to be about 15–28 days at 30°C (Rao et al., 2000). Attempts were made to enhance
the shelf life of kunda by Navajeevan and Rao (2005) using retort pouch processing technology.
However, it was reported that the shelf life of retort processed kunda was limited by chemical
changes during storage and was only 2 weeks at 37°C and 1 week at 55°C.
4.0 CHHANA-BASED CONFECTIONS
Chhana, an important heat and acid coagulated product, serves as a base material for a large
variety of Indian sweetmeats such as rasogolla, sandesh, chum-chum, chhana murki, chhana
podo and rasomalai. Cow milk is better suited to produce chhana as it yields soft and smooth
texture with velvety body, desirable for making chhana based sweetmeats particularly
rasogolla. Chhana produced from buffalo milk is reported to be hard and greasy because of
inherent differences in qualitative and quantitative aspects of buffalo milk. However, attempts
have been made by several workers to overcome these defects. Some of the suggested
measures include addition of sodium citrates, dilution of buffalo milk with 20-30% water,
coagulation at low temperature and homogenization (Rajorhia and Sen, 1988).
4.1 Rasogolla
Rasogolla, a chhana-based delicacy, is stored and served in sugar syrup. For the production of
rasogolla, chhana is thoroughly kneaded and made into small balls, which are subsequently
boiled in clarified sugar syrup followed by slow cooling in comparatively low concentration
sugar syrup. Snow-white in colour, rasogolla possesses a spongy and chewy body and smooth
texture. It is best prepared from soft and freshly made cow milk chhana. Buffalo milk usually
yields hard chhana that lacks sponginess, as well as desired body and texture. Verma and
Rajorhia (1995) made successful attempts in developing rasogolla from buffalo milk. The
method consists of standardizing buffalo milk to 5.0% fat (and 9.8% SNF) and heating to boil
followed by addition of 0.05% sodium alginate (w/w) with constant stirring so as to dissolve it
completely and subsequently cooling to 40°C. Coagulation of milk was achieved by adding 1.0%
citric acid solution (40°C) at pH 5.1. Chhana was obtained, after the coagulum was filtered,
pressed and added with arrowroot, semolina and baking powder. The mixture, after thorough
kneading to a smooth paste and rolled into uniform balls was cooked vigorously in boiling sugar
syrup. Cooked rasogolla balls were then transferred into warm sugar syrup for soaking and
allowed to cool to room temperature. To enhance the shelf life, provide convenience and make
suitable for export, rasogolla is often canned.
4.2 Sandesh
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Sandesh, another popular chhana-based sweet, can be classified broadly into three
types, viz. karapak (low moisture), narampak (medium moisture) and kachhagolla (high
moisture). Among these narampak is the most popular variety. Sandesh is preferably prepared
from chhana obtained from cow milk because it yields soft body and texture with fine and
uniform grains (Sen and Rajorhia, 1990). Buffalo milk chhana on the other hand leads to a
product with a hard body and coarse texture, both undesirable characteristics. However,
successful attempts were made in developing a method for the production of narampak
sandesh using buffalo milk by Sen and Rajorhia (1991). It involved standardization of buffalo
milk to 4.0% fat, heating to boil, dilution with water (30%, the volume of milk) followed by
coagulation of diluted milk to obtain chhana, which was converted into smooth paste and
divided into two equal lots. Ground sugar at the rate of 30% of the total weight of chhana was
mixed with one lot of the chhana and mixture slowly cooked at 75°C with continuous stirring
and scraping. When patting stage had reached the second lot of chhana also mixed to it.
Heating and scraping was continued till a final temperature of 60°C reached. The mix was then
cooled to 37°C and moulded in desired shape and size and packaged in suitable packages.
Kumar and Das (2003) optimized the processing parameters viz. mixing, kneading and cooking
of chhana and sugar mixture for the mechanized production of sandesh from cow milk. But, it
was observed that the desired homogeneity after the initial mixing was lacking in the product.
With a view to overcome this, Kumar and Das (2007) subsequently developed a single-screw
vented extruder for cooking of chhana and sugar mixture that can be integrated with the
mechanized method for the continuous production of sandesh from cow milk. With necessary
modifications, this technology may also be adapted to continuous production of sandesh from
buffalo milk.
4.3 Chhana Podo
Chhana podo is unique as it is the only milk based indigenous dairy product prepared by baking
chhana. It is characterized by a brown crust with a white or light brown inner body. It has a
typical cooked flavour and rich taste. The product is sweetish due to the addition of sugar. It
has a moderately spongy cake-like texture and soft body. Estimated annual production of
chhana podo is approximately 1000 tonnes (Ghosh et al. 2002). The method of production of
chhana podo was standardized by Ghosh et al. (1998). It involved kneading of chhana with
sugar and refined wheat flour (madia) / semolina (suji), spreading of kneaded chhana mix on a
flat, dry, clean pan smeared with ghee and baking in an oven at 200°C for 65 min to obtain a
puffed, brown spongy textured product. Kumar et al., (2002) optimized the commercial method
of chhana podo and reported that the most acceptable product can be made from milk with
4.5% fat, suit 5%, sugar 35%, and water 30% (of china) and baking at 200 + 5°C for 50 min. The
shelf life of chhana pod is only 3 days at 30°C while it is 35 days when vacuum packaged and
stored at 6+1°C (Kumar et al.,, 2002).
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5.0 FERMENTED DAIRY PRODUCTS
5.1 Misti Dahi
Misti dahi, also called as mishti doi or lal dahi or payodhi is a sweetened variety of dahi popular
in Eastern India. It is characterized by a creamish to light brown color, firm consistency, smooth
texture and pleasant aroma. Traditionally, misti dahi is prepared from cow or buffalo or mixed
milk. It is first boiled with a required amount of sugar and partially concentrated over a low
heat during which milk develops a distinctive light cream to light brown caramel color and
flavor. This is then cooled to ambient temperature and cultured with sour milk or previous
day’s dahi (culture). It is then poured into consumer- or bulk-size earthen vessels and left
undisturbed overnight for fermentation. When a firm body curd has set, it is shifted to a cooler
place or preferably refrigerated. Till recently, misti dahi preparation was mainly confined to
domestic or cottage scale operations. However, the technology for the manufacture of misti
dahi in an organized manner was developed by Ghosh and Rajorhia (1990). The process
involves standardization of buffalo milk (5% fat and 13% SNF) followed by homogenization at
5.49 MPa pressure at 65°C, sweetening with cane sugar (14%) and heating mix to 85°C for 10
min. Then cooling the mix to incubation temperature and inoculating with suitable starter
culture and incubating the mix to obtain a firm curd. The firm curd is transferred to cold storage
(4°C) and served chilled. Now, the organized dairies for example, Mother Dairy, Delhi is
manufacturing and marketing misti dahi at large scale.
5.2 Shrikhand
Shrikhand is an indigenous fermented and sweetened milk product having a typical pleasant
sweet-sour taste. It is prepared by blending chakka, a semi-solid mass obtained after draining
whey from dahi, with sugar, cream and other ingredients like fruit pulp, nut, flavor, spices and
color to achieve the finished product of desired composition, consistency and sensory
attributes. Shrikhand has a typical semi-solid consistency with a characteristic smoothness,
firmness and pliability that makes it suitable for consumption directly after meal or with poori
(made of a dough of whole-meal wheat, rolled out and deep-fried) or bread. Although largely
produced on small scale adopting age-old traditional methods, shrikhand is now commercially
manufactured in organized dairy sector to cater to the growing demand. The traditional
method of making shrikhand involves the preparation of curd or dahi by culturing milk
(preferably buffalo milk) with a natural starter (curd of the previous batch). After a firm curd is
formed, it is transferred in a muslin cloth and hung for 12–18 h to remove free whey. The
chakka obtained is mixed with required amount of sugar, color, flavoring materials and spices
and blended to smooth and homogenous consistency (Upadhyay and Dave, 1977). Shrikhand is
stored and served in chilled form. The batch-to-batch large variation in the quality and poor
shelf life of shrikhand are the serious drawbacks of the traditional method. Generally the
recovery of solids in chakka is also low. With a view to overcome the limitations of the
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traditional method, Aneja et al. (1977) developed an industrial process for the manufacture of
shrikhand. Normally skim milk is used for making dahi for the manufacture of shrikhand in this
method. By using skim milk, not only fat losses are eliminated, but also faster moisture
expulsion and less moisture retention in the curd are achieved (Patel, 1982). Sharma and
Reuter (1992) attempted to adopt UF technology for making chakka, the base material for
shrikhand. The objective was to recover all the whey proteins and increase the yield of the final
product while automating the process. It was reported that there was practically no difference
between traditional and UF-shrikhand. Recently, Md-Ansari et al. (2006) also developed
shrikhand using UF pre-concentrated skim milk. Several attempts have been made to
incorporate different additives into shrikhand to address the growing interest in the
diversification of food products to attract a wider range of consumers. The pulp of fruits such as
apple, mango, papaya, banana, guava and sapota (Bardale et al., 1986; Dadarwal et al., 2005),
cocoa powder with and without papaya pulp (Vagdalkar et al., 2002) and incorporation of
probiotic organisms (Geetha et al., 2003) have been tried in shrikhand. However, in case of
post-fermentation addition of pulps, it is essential from the food safety angle, that the fruit
pulp intended for addition must be subjected to heat treatment equivalent to pasteurization.
5.3 Lassi
Lassi is made by blending dahi with water, sugar or salt and spices until frothy. The consistency
of lassi depends on the ratio of dahi to water. Thick lassi is made with four parts dahi to one
part water and/or crushed ice. It can be flavored in various ways with salt, mint, cumin, sugar,
fruit or fruit juice and even spicy additions such as ground chilies, fresh ginger or garlic. The
ingredients are all placed in a blender and processed until the mixture is light and frothy.
Sometimes a little milk is used to reduce the acid tinge and is topped with a thin layer of malai
or clotted cream. Lassi is chilled and served as a refreshing beverage during extreme summers
(Sabikhi, 2006). While sweetened lassi is popular mainly in North India, its salted version is
widely relished in the southern parts of the country. Various varieties of salted lassi include
buttermilk, chhach and mattha. Ancient Indian literature reports that regular use of buttermilk
has therapeutic advantages, being beneficial in haemarrhoids (piles), swelling and duodenal
disorders. Buttermilk warmed with curry and/or coriander leaves, turmeric, ginger and salt, is a
therapy for obesity and indigestion as per the Indian medicinal science of Ayurveda (Sabikhi and
Mathur, 2004). The keeping quality of lassi is extended considerably under refrigeration.
Although, further extension of shelf life of lassi is achieved by ultra high temperature (UHT)
processing of product after fermentation and packaging it aseptically, the sensory quality is
adversely affected due to wheying off. To overcome this problem, Aneja et al. (1989) developed
a method for manufacture of long-life lassi that does not settle down over extended storage in
aseptic packs. Now, UHT-processed lassi and spiced buttermilk are commercially manufactured
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and marketed by different dairies in India. Recently, Khurana (2006) developed suitable
technologies for the manufacture of mango, banana and pineapple lassi.
6.0 CEREAL BASED DAIRY PRODUCTS
6.1 Kheer
Kheer is a heat-desiccated, cereal-based sweetened and concentrated milk confection. Kheer
prepared from buffalo milk is whiter and thick bodied and is, therefore, preferred over that
obtained from cow milk. In addition to milk, kheer also contains substantial amount of non-
dairy ingredients such as rice, sugar, semolina, cardamom, almonds, pistachio, etc. It is
characterized by sweet, nutty and pleasant flavour (Aneja et al., 2002). De et al., (1976)
standardized the method of manufacture of kheer. The suitability of several types of rice viz.
basmati, parmal and parboiled for kheer making were studied by Jha (2000) who reported that
basmati brokens were most suitable for kheer making. Kheer has a limited shelf-life of about
one day at ambient temperature. Hence, a process has been developed with an objective to
enhance its shelf-life by adopting in-package cooking and sterilization of kheer in retort pouches
(Jha et al., 2000).
6.2 Payasam
Payasam, a milk-based delicacy popular in the southern parts of India, forms an integral part of
the cultural ethos of South India. There are several varieties of payasam with distinct
characteristics that may be attributed to the area of their origin and traditional methods of
preparation. These include vermicelli payasam, khus-khus or gasa-gase (poppy seed) payasam,
palada payasam etc. The popularity of different varieties also differs from state to state
(Unnikrishnan et al., 2000). Based on the use of ingredients other than milk and sugar, payasam
is classified as pulse-based, cereal-based, tuber product-based, fruit-based and seed-based. In
general, payasam is thinner in consistency than kheer, although its varieties range from free-
flowing to solid. The colour of payasam varies from white, light cream, cream and light brown
to brown. However, it is distinctly brown when jaggery is used as the sweetening ingredient.
The methods of manufacture of different varieties of payasam and their dry mixes have been
standardized (Venkateshwarlu and Dave, 2003; Nath et al., 2004).
CONCLUSION
Traditional dairy products, apart from being an integral part of Indian heritage, have great
social, religious, cultural, medicinal and economic importance. In addition to preservation of
milk solids for a longer time at room temperature, manufacture of traditional dairy products
add value to milk and also provide tremendous employment opportunity. Owing to the
inherent qualitative and quantitative differences, most of these products, particularly ghee,
khoa, paneer and dahi have higher yield and better quality when they are made from buffalo
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milk. On the other hand, some of these products such as chhana and rasogolla are of superior
quality when they are made from cow milk. Most of these traditional dairy products are well
characterized and the method of manufacture has been standardized using mechanized or
semi-mechanized systems.
References:
Pal, D. (2000). Technological advances in the manufacture of heat desiccated traditional dairy products-An
overview. Indian dairyman 52:27.
Pal, D. and Raju, P. N. (2007) Indian Traditional Dairy Products – An Overview. Theme paper. International
Conference on Traditional Dairy Foods, NDRI, Karnal. Pp: I – XXVI.
Rajorhia, G. S. (2003). Ghee. In “Encyclopedia of Food Sciences and Nutrition”, Second Edn. Eds. Caballero,
B., Trugo, L. C. and Finglas, P. M. Academic Press, UK. Pp: 2883-2889.
Rajorhia, G. S. and Sen, D. C. (1988) Technology of chhana- a review. Indian J. Dairy Sci 41: 141.
324
Probiotics as Biotherapeutics for Management of Inflammatory Metabolic
Disorders
Sunita Grover, Aparna, V, Harsh Panwar, Rashmi, H.M, Ritu Chauhan, and V.K.Batish
Molecular Biology Unit, National Dairy Research Institute, Karnal-132001
The interest in probiotics has been growing enormously during the last few years in view
of their multiple health promoting physiological functions. They are currently being explored as
biotherapeutics for human health applications to manage chronic diseases. Functional foods
with probiotics have emerged as a newer approach to improve human nutrition and well-being
in an environment where metabolic inflammatory disorders due to sedentary lifestyle and
ageing population are considered as a threat to the wellbeing of the society worldwide
including Asia as dietary habits are changing rapidly. Diet-related chronic diseases such as
metabolic inflammatory disorders such as obesity, cardiovascular diseases (CVD) and type two
diabetes (DM-2) have dramatically increased leading to concomitant increase of healthcare and
other societal costs. The advent of health-promoting functional foods has been facilitated by
fast accumulating scientific knowledge about the metabolic and genomic effects of diet and
specific dietary components on human health. As a result of this, opportunities have arisen to
formulate food products which deliver specific health benefits, in addition to their basic
nutritional value. Probiotics are now being intensively investigated as an integral component of
functional foods to act as therapeutic armamentarium of inflammatory metabolic disorders as
an adjunct to the traditional anti-inflammatory and immune-suppressive agents. Action of
probiotics on the host immune system has entered a new and fascinating phase of research in
search for anti-inflammatory agents. It is likely to offer novel and useful means to modulate
host immunity for protection from or treatment of a wide variety of human diseases including
metabolic disorders like obesity, DM-2 and cardio-vascular diseases. The immune system is
extremely complex and amazingly important for maintaining perfect health.
Inflammation is one of the most important defensive methods employed by the
immune system to fight against infections and tissue damage, thereby, preventing the spread
of infection and pathological changes to the rest of the body. Although, inflammation is a
natural defense mechanism against toxic components such as oxidized proteins and lipids, it
has become one of the hottest areas of medical research due to the fact that ‘Inflammation
acts as a secret killer’. It presents a major hazard to individuals inflicted by several of
inflammatory diseases such as IBD, CD, RA, metabolic syndrome including CVD. It is now well
recognized that inflammation plays a central role in the pathogenesis of metabolic diseases.
Evidence linking inflammation to insulin resistance derives from both epidemiological studies
and experimental data in humans and animal models. It is well known that the prevalence of
diabetes, obesity, and Metabolic syndrome all increase with age. Inflammation disturbs the
homeostasis existing between anti and pro-inflammatory cytokines. The increased level of pro-
325
inflammatory cytokines like IL-6 and TNF-α increases the hepatic synthesis of acute phase
proteins like fibrinogen, C reactive protein etc. and at the same time, they decrease the
synthesis of high density lipoprotein (HDL). Few studies have shown that the pro-inflammatory
mediators, particularly TNF-α, can induce a procoagulant state by eliciting tissue factor
production on the surface of vascular endothelium and monocytes, down regulating the protein
C anticoagulant pathway and stimulating thrombin and fibrin formation. Therapeutic
approaches that reduce the levels of pro-inflammatory biomarkers and address traditional risk
factors are specifically important in preventing cardiovascular disease and, potentially
metabolic disorders. It has been shown that some probiotic organisms can modulate the in vitro
expression of pro and anti-inflammatory molecules in a strain-dependent manner. Many
probiotic effects are mediated through immune regulation, particularly through balance control
of pro-inflammatory and anti-inflammatory cytokines. These data show that probiotics can be
used as innovative tools to alleviate intestinal inflammation, normalize gut mucosal
dysfunction, and down-regulate hypersensitivity reactions. Probiotics exhibit adequate fitness
to survive and replenish physiological microflora, suppress pathological microflora and
modulate host immune system. The consumption of probiotics helps to decrease the level of
pro-inflammatory biomarkers which in turn helps to reduce the fibrinogen level in the blood.
The key issue of understanding the functionality of probiotic stains is the identification
of appropriate biomarkers for their health benefits both under in vitro and in vivo conditions.
Quantification of genes at transcriptional levels is an important criteria to know gene
functionality and abnormal alterations in regulation that may result in a disease state since
cellular functions are regulated by changes in gene expressions. Genomics- based studies
reveals numerous bacterial cell-surface-associated proteins with intestinal cell and mucus
binding functions. Relative expression of probiotic marker genes using one of the genomic
approaches like Real Time PCR, Microarray etc. forms an important parameter to select
potential probiotic strain which could be finally used in human clinical trials to ascertain its
probiosis before its exploitation in functional foods. Similarly, specific activity can also be
analysed using proteomics and a more promising metaproteomic approach. Combination of
Genomics, Metagenomcis and Proteomics will enable us to unravel the role of probiotics for gut
health. The strategies based on of all these approaches towards understanding the functionality
of indigenous probiotic strains will be discussed in this presentation.
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Diabetes Management through Enzymes Inhibitory Potential of Lactobacilli
Priti Mudgil, Sumit Singh Dagar, Dinesh Dahiya and Anil Kumar Puniya
Dairy Microbiology Division, National Dairy Research Institute, Karnal
Introduction
With changing lifestyle and eating habits modern society has captured a number of
lifestyle related disease like obesity, hypertension, hypercholestremia and diabetes. Among
these disorders diabetes, a silent assassin is tickling very fast like a time bomb is affecting
millions of people worldwide and affects their quality of life. Global projection of diabetes
clearly demonstrates that this sugar is not sweet in nature. As a silent assassin, diabetes is
affecting nearly 6.6% of world’s adult population cost world economy very dearly both in term
of life and money loss i.e. $376 billion (11.6% of total world healthcare expenditure). India with
nearly one fifth of the total diabetic population is an unchallenged diabetic capital. Diabetes
mellitus is a hyperglycemic syndrome with several characteristic features. It continues to rise
unabatedly in all pockets of world, parallel with affluence and can be controlled not cured.
In the present scenario, diabetes and its associated metabolic syndromes are emerging
as one of the most important public health problems. World over 285 million people have
diabetes whereas, in India it is amounting to 50.8 million, this number is estimated to rise to
435 million in world and 87 million in India by 2030 (International Diabetes Federation Atlas 4th
Edition). It is the sixth most common cause of death in world and significantly affect other more
common cause of death like obesity and will lead to 3.96 million deaths worldwide while one
third of these will occur in India thus making it an unhealthy country in terms of diabetes and
has emerged as world’s unchallenged diabetic capital having largest subject of diabetics to the
tune of 17.8% of total diabetic population. Urban population has a higher incidence of diabetes
with 113 million people as compared to 78 million in rural areas. Treatment cost of diabetes
accounts for more than 11% of total healthcare expenditure while India spends only one
percent of these total diabetic spending. It is a multi-factorial disease with many unknown risk
factors. One of these risk factors is postprandial hyperglycemia (PPG). A person destined to
develop diabetes remains in a postprandial hyperglycaemic state for 10-12 years before the
onset of diabetes and is regarded as an independent risk factor for CVD in diabetics. Control of
postprandial hyperglycemia in early stages has the potential for the treatment of diabetes.
Many drugs are use to control PPG but insulin and digestive enzyme inhibitor are the only
specific drugs available in market that specifically target postprandial hyperglycemia. Alpha
glucosidase and alpha amylase inhibitors have recently taken foremost attention because of
their unambiguous action.
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Enzyme inhibitors and diabetes
Therapeutic approaches for the treatment of type 2 diabetes, such as sulphonylureas,
metformin and insulin therapy are effective in decreasing fasting glucose levels but except for
insulin therapy they have little effect on postprandial hyperglycemia. Increasing importance of
postprandial hyperglycemia and little effectiveness of these oral antihyperglycemic agents have
created a challenge to the researchers for development of new drugs for postprandial
hyperglycemia (Baron, 1998). Among these new agents inhibitors of enzymes involved in
digestion process has gained much importance in past few years. α-Glucosidase inhibitors are
one of these inhibitors and due to their unique mechanism of action there has been an
increased interest in identifying α-glucosidase inhibitors that can be used as an important tool
for understanding biochemical processes and as prospective therapeutic agents for
postprandial hyperglycemia (Markad et. al., 2006; Liu et. al., 2007).
α-Glucosidase inhibitors do not target a specific pathophysiologic aspect of diabetes but
competitively inhibit enzymes in the small intestinal brush border that are responsible for the
breakdown of oligosaccharides and disaccharides into monosaccharides (Lebovitz, 1997). It
works primarily on α-glucosidase (EC 3.2.1.20, 3.2.1.10, 3.2.1.48 and 3.2.1.106), which are
predominant in the proximal half of the small intestine and catalyzes the release of α-D-
glucopyranose from the non-reducing ends of various carbohydrate substrates (Frandsen &
Svensson, 1998). These enzymes also play an important role in the biochemical processes of
glycoproteins and glycolipids. Presence of α-glucosidase inhibitor for example: Acarbose
(Precose®) and Miglitol (Glyset®) in diets inhibit the activity of α-glucosidase thus delays
intestinal absorption of carbohydrates shifted to more distal parts of the small intestine and
colon. This retards glucose entry into the systemic circulation and lowers postprandial glucose
levels.
α-Glucosidase inhibitors act locally at the intestinal brush border and are not absorbed
in intestine but get excreted in faeces. Efficiency of α-glucosidase inhibitor to improve HbA1c
concentrations is by 0.5%- 1.0%. They also have beneficial effects on insulin resistance. α-
Glucosidase inhibitors are generally synthesized chemically. However they can be isolated
naturally from plants, food products, or by microorganisms. Several chemical synthetic
compounds, such as sulfonamide, xanthone derivatives, and deoxy salacious, have been
reported to exhibit inhibitory effects against α-glucosidase activity (Liu et. al., 2007). In
addition, the salacinol from Salacia reticulata (Yoshikawa et. al., 2002), Punica granatum flower
extract (Li et. al., 2005) and the water extract of douchi (Chinese traditional food) also exhibits
α-glucosidase inhibitory activity. Nevertheless, these natural α-glucosidase inhibitors are not
easily produced at large scale (Fujita et. al., 2003) while chemically synthesized α-glucosidase
inhibitors normally cause hepatic disorders. Other negative gastrointestinal symptoms are
bloating, diarrhoea and flatulence. In addition, there are reports for an increased incidence of
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renal tumors and serious hepatic injury and
acute hepatitis by Acarbose (Cheng &
Fantus, 2005).
On contrary α-glucosidase inhibitors
synthesis by microorganisms could be an
effective strategy to produce cost-effective
and productive α -glucosidase inhibitors. It
has been reported that some
microorganisms, including species of
Actinoplanes, Streptomyces and
Flavobacterium saccharophilium, Bacillus
subtilis were able to synthesize α-
glucosidase inhibitors. Due to fast-growing
characteristic of these microbes, there has
been increased interest in identifying α -glucosidase inhibitors producing microorganisms but
they also have been reported to exert some negative health effects. So either enzyme inhibition
through some food sources or by the help of some food grade microorganism can have a dual
advantage of combining nutritional approach with the pharmacological approach. One of these
food grade organisms are Probiotics i.e. live microorganisms which when administered in
adequate amounts (107/ml) confer a health benefit on the host like increased absorbability,
alleviation of lactose intolerance, immuno-stimulation, pathogen exclusion, production of
bioactive compounds, anti-carcinogenic activity and de-conjugation of bile acids to lower blood
cholesterol and other lipids etc.
Probiotics in treatment of diabetes
Various neutraceuticals and probiotics preparations have been recommended for the
treatment of diabetes and its complications as
described earlier (Roberfroid, 2000). Due to the
following properties probiotics can be
considered as an alternative therapeutic regimen
for diabetes:
• Antidiabetic effects
• Antioxidant properties
• Antihypercholesterolemic
• Antiatherogenic properties
• Antihypertensive effects
329
Various research groups in the last decade has demonstrated the beneficial effect of probiotics
on diabetes and its associated complications: Matsuzaki (Matsuzaki, et al., 1997) showed that
oral feeding of 0.1- 0.05% heat killed probiotic Lactobacillus casei to insulin dependent diabetic
NOD mice significantly reduces the incidence of diabetes development along with strong
inhibition of β-cell disappearance from pancreas, reduction in CD8+ T-cells and increase in
CD45R+ B-cells. It also lowered interferon-γ and accelerated IL-2, IL-4, IL-5, IL-6, IL-10 titer thus
indicating increased host immune response. Similarly by feeding of 0.1- 0.05% Lactobacillus
casei to Non Insulin Dependent Diabetic Mice model(NIDDM) KK-Ay mice there was significant
reduction of plasma glucose, plasma insulin & body weight at 8-10 week of age in experimental
group than control group, though no change in food intake, reduction in CD4+ T-cells, IL-2 IFN- γ
was reported (Matsuzaki, et al., 1997). However feeding of 0.1- 0.05% Lactobacillus casei to
alloxan (a type of toxic glucose analogue that destroy β-cell on consumption) induced diabetic
rat at 7-week of age shows decrease in incidence of diabetes, blood glucose, inhibition of
disappearance of islet β-cell, maintenance of serum nitric oxide level and increase in body
weight (Matsuzaki, et al., 1997). Arunachalam (Arunachalam, Gill, & Chandra, 2000) reported
that administration of Bifidobacterium lactis (HN019) reduced the release of inflammatory
cytokines thus preventing the systemic inflammatory induced diabetes.
Feeding of Lactobacillus GG to 9-18 weeks of age in streptozotocin induced diabetic rat
decreases the HbA1c level, increases serum insulin level after 30 min of glucose load and also
improve glucose tolerance (Tabuchi, et al., 2003). Similarly Calcinaro (Calcinaro, et al., 2005)
investigated that oral administration of VSL#3 to NOD mice showed reduced insulitis and β-cell
destruction by increasing the production of IL-10 from Peyer’s patches and prevents
autoimmune diabetes. Lactobacillus johnsonii strain La1 (LJLa1) oral administration for 2 weeks
in NIDDM-KK-Ay mouse model inhibited hyperglycemia induced by 2-deoxy-D-glucose (2DG). In
addition its administration also lowers the elevation of blood glucose and glucagon levels in
streptozotocin-diabetic rats by changing autonomic nerve activity (Yamano, et al., 2006).
Feeding of probiotic dahi containing Lactobacillus casei NCDC 19 & Lactobacillus acidophilus
NCDC 1 to high fructose-induced diabetic rat for eight weeks slows down biochemical changes
that improves insulin resistance (Yadav et. al., 2007) (Cani et al., 2007) positively relates the
concentration of Bifidobacterium spp. in the gut with improved glucose tolerance and insulin
secretion.
High level of Bifidobacterium spp. also decreases endotoxemia & inflammatory
cytokines. Probiotics treatment is also reported to increase the affectivity of other antidiabetic
drugs as reported by Al-Salami et. al., 2008). As probiotic pretreatment increases permeability
of Gliclazide (sulfonylurea) in diabetic rat but decrease its flux in healthy rats. These results
suggest a possible role of Probiotics in treatment of diabetes in synergism with other
antidiabetic drugs. Similar suppressing results on blood glucose were reported by oral
330
administration of Lactobacillus gasseri BNR17 (Yun et. al., 2009). Currently Lactobacillus species
has also been documented to produce digestive enzyme inhibitors raising the hopes that they
can also be used for the management of postprandial hyperglycemia.
Conclusion
Treatment of diabetes requires combined efforts from both medical practitioners as
well as patients. Modification of sedentary lifestyle, exercise and inclusion of healthy diet can
help to manage diabetes and its complication. Many natural therapies without any side effects
are being used from ancient times for its prevention and the recent concept in this is the
inclusion of functional foods containing probiotics. This chapter summarizes the potential of
probiotics in management of diabetes and its related complication like hypertension,
hypercholestremia, oxidative stress etc. via positive modulation of several different
physiological systems, apart from its conventional benefits for gastrointestinal health.
Probiotics have exhibited antidiabetic action via their antihypertensive, antioxidative potential,
improvement of lipid profiles and insulin resistance. These positive findings suggested the
potential use of dietary alternatives such as probiotics, to alleviate the occurrence of diseases
via a fundamental and safe approach as compared to drugs or hormone therapy.
Probiotics could also serve as a complementary supplement to enhance the well-being
for those already suffering the diseases and taking drugs or hormonal therapy to medicate the
condition. Further revelation on the potential of probiotics in future research could lead to a
boost in probiotic-fermented food industries–dairy and non–dairy. Nevertheless, more studies
are needed to better understand the exact mechanisms, in vivo target sites, stability and safety,
prior to using probiotics as an antidiabetic alternative treatment.
References:
Al-Salami, H., Butt, G., Fawcett, J. P., Tucker, I. G., Golocorbin-Kon, S., & Mikov, M. (2008). Probiotic
treatment reduces blood glucose levels and increases systemic absorption of gliclazide in diabetic rats. Eur
J Drug Metab Pharmacokinet, 33(2), 101-106.
Arunachalam, K., Gill, H. S., & Chandra, R. K. (2000). Enhancement of natural immune function by dietary
consumption of Bifidobacterium lactis (HN019). Eur J Clin Nutr, 54(3), 263-267.
Baron, A. D. (1998). Postprandial hyperglycaemia and alpha-glucosidase inhibitors. Diabetes Res Clin
Pract, 40 Suppl, S51-55.
Calcinaro, F., Dionisi, S., Marinaro, M., Candeloro, P., Bonato, V., Marzotti, S., Corneli, R. B., Ferretti, E.,
Gulino, A., Grasso, F., De Simone, C., Di Mario, U., Falorni, A., Boirivant, M., & Dotta, F. (2005). Oral
probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune
diabetes in the non-obese diabetic mouse. Diabetologia, 48(8), 1565-1575.
Cani, P. D., Neyrinck, A. M., Fava, F., Knauf, C., Burcelin, R. G., Tuohy, K. M., Gibson, G. R., & Delzenne, N.
M. (2007). Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in
mice through a mechanism associated with endotoxaemia. Diabetologia, 50(11), 2374-2383.
Cheng, A. Y., & Fantus, I. G. (2005). Oral antihyperglycemic therapy for type 2 diabetes mellitus. CMAJ,
172(2), 213-226.
331
Frandsen, T. P., & Svensson, B. (1998). Plant alpha-glucosidases of the glycoside hydrolase family 31.
Molecular properties, substrate specificity, reaction mechanism, and comparison with family members of
different origin. Plant Mol Biol, 37(1), 1-13.
Fujita, H., Yamagami, T., & Ohshima, K. (2003). Long-term ingestion of Touchi-extract, an [alpha]-
glucosidase inhibitor, by borderline and mild type-2 diabetic subjects is safe and significantly reduces
blood glucose levels. Nutrition Research, 23(6), 713-722.
Lebovitz, H. E. (1997). alpha-Glucosidase inhibitors. Endocrinol Metab Clin North Am, 26(3), 539-551.
Li, Y., Wen, S., Kota, B., Peng, G., Li, G., Yamahara, J., & Roufogalis, B. (2005). flower extract, a potent α-
glucosidase inhibitor, improves postprandial hyperglycemia in Zucker diabetic fatty rats. Journal of
Ethnopharmacology, 99(2), 239-244.
Liu, Y., Ma, L., Chen, W. H., Wang, B., & Xu, Z. L. (2007). Synthesis of xanthone derivatives with extended
pi-systems as alpha-glucosidase inhibitors: insight into the probable binding mode. Bioorg Med Chem,
15(8), 2810-2814.
Markad, S. D., Karanjule, N. S., Sharma, T., Sabharwal, S. G., & Dhavale, D. D. (2006). Synthesis and
evaluation of glycosidase inhibitory activity of N-butyl 1-deoxy-d-gluco-homonojirimycin and N-butyl 1-
deoxy-l-ido-homonojirimycin. Bioorganic & Medicinal Chemistry, 14(16), 5535-5539.
Matsuzaki, T., Nagata, Y., Kado, S., Uchida, K., Hashimoto, S., & Yokokura, T. (1997). Effect of oral
administration of Lactobacillus casei on alloxan-induced diabetes in mice. APMIS, 105(8), 637-642.
Matsuzaki, T., Nagata, Y., Kado, S., Uchida, K., Kato, I., Hashimoto, S., & Yokokura, T. (1997). Prevention of
onset in an insulin-dependent diabetes mellitus model, NOD mice, by oral feeding of Lactobacillus casei.
APMIS, 105(8), 643-649.
Matsuzaki, T., Yamazaki, R., Hashimoto, S., & Yokokura, T. (1997). Antidiabetic effects of an oral
administration of Lactobacillus casei in a non-insulin-dependent diabetes mellitus (NIDDM) model using
KK-Ay mice. Endocr J, 44(3), 357-365.
Roberfroid, M. B. (2000). Prebiotics and probiotics: are they functional foods? The American Journal of
Clinical Nutrition, 71(6), 1682S-1687S.
Tabuchi, M., Ozaki, M., Tamura, A., Yamada, N., Ishida, T., Hosoda, M., & Hosono, A. (2003). Antidiabetic
effect of Lactobacillus GG in streptozotocin-induced diabetic rats. Biosci Biotechnol Biochem, 67(6), 1421-
1424.
Yadav, H., Jain, S., & Sinha, P. (2007). Antidiabetic effect of probiotic dahi containing Lactobacillus
acidophilus and Lactobacillus casei in high fructose fed rats. Nutrition, 23(1), 62-68.
Yamano, T., Tanida, M., Niijima, A., Maeda, K., Okumura, N., Fukushima, Y., & Nagai, K. (2006). Effects of
the probiotic strain Lactobacillus johnsonii strain La1 on autonomic nerves and blood glucose in rats. Life
Sciences, 79(20), 1963-1967.
Yoshikawa, M., Morikawa, T., Matsuda, H., Tanabe, G., & Muraoka, O. (2002). Absolute stereostructure of
potent alpha-glucosidase inhibitor, Salacinol, with unique thiosugar sulfonium sulfate inner salt structure
from Salacia reticulata. Bioorg Med Chem, 10(5), 1547-1554.
Yun, S. I., Park, H. O., & Kang, J. H. (2009). Effect of Lactobacillus gasseri BNR17 on blood glucose levels
and body weight in a mouse model of type 2 diabetes. Journal of Applied Microbiology, 107(5), 1681-
1686.
332
Direct Vat Starters: Concentrated Cultures for Fermented Milks
Rameshwar Singh, Surajit Mandal and R. P. Singh
National Collection of Dairy Cultures, DM Division, N.D.R.I., Karnal
Introduction
Starter cultures are those microorganisms (bacteria, yeasts, and molds or their
combinations) that initiate and carry out the desired fermentation essential in manufacturing
cheese and fermented dairy products such as Dahi, Lassi, Yogurt, Sour cream, Kefir, Koumiss,
etc. Starter cultures have a multifunctional role in dairy fermentations. The production of lactic
acid by fermenting lactose is the major role of dairy starters. The acid is responsible for
development of characteristic body and texture of the fermented milk products, contributes to
the overall flavour of the products, and enhances preservation. Diacetyl, acetaldehyde, acetic
acid, also produced by the lactic starter cultures, contribute to flavor and aroma of the final
product. Carbon-di-oxide produced by some hetero-fermentative lactic acid bacteria involves in
very characteristics texturization in some fermented dairy products, viz. “eye” formation in
cheeses. In cheese making, starters are selected strains of microorganisms that are intentionally
added to milk or cream or a mixture of both, during the manufacturing process and that by
growing in milk and curd cause specific changes in the appearance, body, flavor, and texture
desired in the final end product.
Lactic starter cultures are generally available from commercial manufacturers in spray-dried,
freeze-dried (lyophilized), or frozen form. Spray-dried and lyophilized cultures need to be
inoculated into milk or other suitable medium and propagated to the bulk volumes required for
inoculating a cheese vat as follows:
Stock
culture
(Freze
dried,
frozen,
spray
dried)
Mother
culture
Intermediate
culture
Bulk
culture
Process Milk
(Multipurpose
vat/Cheese
Vat/Lassi tank)
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However, the repeated sub-culturing of certain strains of starter bacteria may loss the plasmids
and consequently can affect the characteristics (i.e., phage-resistant becomes phagesensitive,
lack of lactose utilization etc). The yogurt starter cultures (S. thermophilus and L. delbrueckii
subsp. bulgaricus) are normally used the ratio of cocci : rods as 1 : 1. Starter culture activity is
affected by the rate of cooling after incubation, level of acidity at the end of the incubation
period, and the temperature and duration of storage. Many larger dairy plants develop their
own cultures. However, preparing and maintaining bulk cultures requires specialized facilities
and equipment. Much research and development in the starter culture technology has been
aimed at designing specialized growth media for starters, protecting the starter cultures from
sub-lethal stress and injury during freezing, and minimizing the threat of bacteriophage during
starter culture preparations. Therefore, the use of concentrated direct vat starters is gaining
much importance in preparation of fermented milks. The Direct Vat Starters (DVS) cultures are
highly concentrated cultures that are made of mixtures of defined strains in predetermined
proportions. The advantages of DVS are imporved quality, high yield, less rejection of batches,
ease of use and reliability.
Types of Starter Cultures
In the dairy industry, single or multiple strains of cultures of one or more microorganism are
used as starter cultures. These are belongs to genus Lactococcus (Lactococcus lactis subsp.
cremoris, L. lactis subsp. lactis, L. lactis subsp. lactis biovar diacetylactis), Lactobacillus (L.
delbrueckii subsp. lactis, Lactobaillus acidophilus, Lactobacillus casei), Streptococcus (S.
thermophilus), Leuconostoc, Pediococcus etc. There are two main types of lactic starters:
1) Mesophilic lactic starters(optimum growth temperature: 30°C)
2) Thermophilic lactic starters (optimum growth temperature: 45°C)
Mesophilic cultures usually contain L. cremoris and L. lactis as acid producers and L.
diacetylactis and Leuconostocs as aroma and CO2 producers. Thermophilic starters include
strains of S. thermophilus, and, depending on the product, Lactobacillus bulgaricus, L.
helveticus, or L. lactis. Often, some fermented milks made with thermophilic starters also
contain Lactobacillus acidophilus, L. bulgaricus, and bifidobacteria for their healthful and
therapeutic properties. Table 1 lists the common starter cultures and their applications in
cheese and fermented dairy products.
The lactic starter cultures are also subdivided into two groups:
1) Defined cultures
2) Mixed cultures.
Defined cultures constitute starters in which the number of strains is known. The application of
defined cultures did control the open texture problem, however, and they were prone to slow
acid production due to their susceptibility to bacteriophage. The use of pairs of phage-
unrelated strains and culture rotation to prevent build up of phage in the cheese factory was
334
practiced to minimize the potential for phage problems. Eventually, the use of multiple strain
starter and factory-derived phage-resistant strains was made to control the phage problem.
Lactic starter cultures are also categorized based on flavour or gas production characteristics as
follows:
B or L cultures (Betacoccus or Leuconostoc) contain flavor and aroma producing organisms,
for example, Leuconostoc spp.
D cultures contain Lactococcus diacetylactis
BD or DL cultures contain mixtures of both Leoconostoc and S. diacetylactis strains
O cultures do not contain any flavor/aroma producers but contain L. lactis and L. cremoris
strain.
Table 1: Lactic starter cultures, associated microorganisms and their applications in the dairy
industry
Lactic Acid Bacteria Associated Microorganisms Products
Mesophilic
Lactococcus lactis,
Lactococcus cremosis,
Lactococcus lactis var.
diacetylactis,
Leuconostoc cremosis
Lactococcus lactis var.
diacetylactis,
Penicillium camemberti,
P. roqueforti, P. caseicolum,
Brevibacterium linens
Cheddar, Colby Cottage
cheese, Cream cheese,
Neufachatel, Camembert,
Brie, Roquefort, Blue,
Gorgonzola, Limburger
Thermophilic
Streptococcus thermophilus,
Lactobacillus bulgaricus,
L. lactis, L. casei, L. helveticus,
L. plantarum, Enterococcus
faecium
Candida kefyr, Torulopsis,
spp., L. brevis,
Bifidobacterium bifidum,
Propionibacterium
fureudenreichii, P. shermanii
Parmesan, Romano, Grana
Kefir, Koumiss yogurt,
Yakult, Therapeutic
cultured
milks, Swiss, Emmenthal,
Gruyere
Mixed starters
Lactococcus lactis, S.
thermophilus,
E. faecium, L. helveticus,
L. bulgaricus
… Modified Cheddar, Italian,
Mozzarella, Pasta Filata,
Pizza cheese
What are starter concentrates?
Traditionally 'bulk starter' in liquid form was used to inoculate the milk used in the manufacture
of cheese, yoghurt, buttermilk and other fermented products. Over the past 10-15 years, the
use of starter cell concentrates designated as either Direct Vat Set (DVS) or Direct Vat
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Inoculation (DVI) cultures have increasing being used, particularly in small plants, to replace
bulk starter in fermented dairy product manufacture. (Note that the terms DVI and DVS are
used interchangeably although particular culture suppliers will tend to use only one term)
Starter concentrates used in DVI cultures are concentrated cell preparations containing in the
order of 1011-1013 CFU/g. They are available as frozen pellets (fig. 1) or in freeze-dried granular
form (fig. 2).
Commercial DVS frozen culture in pellet form
Under normal conditions starter growth in milk results in a cell concentration of about 109
cfu/ml. Growth of starters in milk is limited by a number of factors including the accumulation
of lactic acid. Concentrates can be produced by neutralisation (traditional fermentation
technology) or removal of the lactic acid (using diffusion culture), recovering the cells by
centrifugation, and by maintaining starter activity by freeze drying or freezing. Freeze-dried
concentrates can be stored for some months at 4° C. Frozen concentrates are usually stored at -
45°C or lower. Some suppliers recommend that their frozen DVI cultures are stored at -18°C.
Production of starter concentrates
Commercial starter cultures currently available for direct addition to production vats contain
billions of viable bacteria per gram, preserved in a form that could be readily and rapidly
activated in the product mix to perform the functions necessary to transform the product mix
to the desired cultured product. To attain that, the selected starter bacteria need to be grown
in a suitable medium to high numbers and to concentrate the cells. The composition of the me-
dia used to grow various bacteria differs. Usually, the materials used in the growth media
consist of food grade, agricultural by-products and their derivatives. The trade has special
requirements for the raw materials that go into media formulations and for the way they are
mixed and processed.
The generally used ingredients in media formulations include non-fat milk, whey, hydrolysates
of milk and whey proteins, soy isolates, soy protein hydrolysates, meat hydrolysates and
extracts, egg proteins, com steep liquor, malt extracts, potato infusions, yeast extracts/yeast
autolysates, sugars such as lactose, glucose, high-fructose corn syrup, com sugar, sucrose, and
minerals such as magnesium, manganese, calcium, iron, phosphates, salt, etc. For some
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fastidious bacteria, amino acids and vitamins may be included. The phosphates are added to
provide mineral requirements as well as for buffering. For some bacteria, which need
unsaturated fatty acids to protect cell membranes, trace quantities of polysorbates (Tweens)
are added. To control foaming, food grade anti foam ingredients may be incorporated.
The medium is then either sterilized by heating at 121°C for a minimum of 15 minutes or heat-
treated at 85-95°C for 45 minutes or subjected to ultrahigh temperature treatment (UHT) for a
few seconds. After heat treatment, the medium is cooled to the incubation temperature. After
the addition of the inoculum, the medium is incubated until the predetermined endpoint is
reached. During incubation, the pH is maintained at a predetermined level (constant neu-
tralization to maintain pH). Generally, the endpoint coincides with the exhaustion of sugar
reflected by the trace of the neutralization curve. The frequency of neutralization reflects the
activity of the culture in the fermenter, and when the frequency decreases, it indicates the near
depletion of the sugar. Samples are usually taken to microscopically examine the fermentate
for cell morphology, for any gross contamination, for a rough estimation of cell numbers, and
for quantitative measurement of sugar content. After ascertaining these, the fermenter is
cooled. The cells are harvested either by centrifugation or by ultrafiltration. The cell
concentrate is obtained in the form of a thick liquid of the consistency of cream and is weighed
and rapidly cooled. Sterile preparations of cryoprotectants (glycerol, nonfat milk, monosodium
glutamate, sugars, etc.) are added, and uniformly mixed with the cell concentrate. The
concentrate may be filled as such into cans and frozen or frozen in droplet form in liquid
nitrogen (pellets), retrieved, and packaged. The concentrate as such or in pellet form may also
be lyophilized in industrial scale freeze dryers.
pH Control Systems
There are two main reasons for using pH control systems in propagating bulk starter cultures:
1. To minimize daily fluctuations in acid development and thereby prevent "over-ripening" of
the starter.
2. To prevent the cellular injury that may occur to some starters when the pH of the medium
drops below 5.0.
In the pH control systems, the acid produced by the starter culture is neutralized to maintain
the pH at around 6.0. The external pH control system, uses whey based medium fortified with
phosphates and yeast extract. The pH is maintained at around 6.0, by intermittent injection of
anhydrous or aqueous ammonia, or sodium hydroxide. This system has been used successfully
in the United States for production of most American-style cheeses. The internal pH control
system, developed uses a whey based medium containing encapsulated citrate-phosphate
buffers that maintain the pH at around 5.2. Unlike in the external pH control system, no
addition of ammonia or NaOH is necessary.
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Phage Inhibitory and Phage-Resistant Medium (PIM/PRM)
The PIM/PRM were developed following observations of Reiter64 that bacteriophage of lactic
streptococci were inhibited in a milk medium lacking in calcium. Hargrove364 reported on the
use of phosphates to sequester free calcium ions in milk or bulk-starter medium for inhibition
of bacteriophage. The effectiveness of phosphates in the formation of PIM/PRM for phage
control was confirmed by Christensen. The PIM/PRM consisting mainly of milk solids, sugar,
buffering agents such as phosphates and citrates and yeast extract have been widely used in
the United States, Canada, and Europe for about 20 years. However, the effectiveness of the
PIM/PRM in inhibiting bacteriophage and stimulating growth of the starter culture media is
somewhat limited. Despite the absence of calcium, some phages can infect the the starter
culture at its optimum growth temperature. Also, phosphates in the PIM/PRM can cause
metabolic injury to some starter cultures. The preparation of active bulk starter culture free of
phage contamination is essential for cheese manufacturing. If the pH is maintained in the
region 6.0 - 6.3 by neutralisation of the lactic acid produced by the starter bacteria then the cell
population can be increased about 10-100 fold depending on the neutraliser used. Both sodium
hydroxide and ammonium hydroxide have been used, use of the latter results in higher cell
densities. The cessation of growth of starters grown in fermentation media under pH control is
due to several factors including the accumulation of inhibitory concentrations of lactate,
hydrogen peroxide, nisin, D-leucine.
Higher cell densities (greater than 1010 CFU/g) can be obtained by harvesting the cells from the
fermenter medium by centrifugation, to give a starter population of 1011 - 1012 CFU/ml. Even
higher cell densities can be obtained by freeze drying the 'sludge' obtained by centrifugation.
Unfortunately, the increase in cell population for some strains does not necessarily parallel the
increase in the ability of the concentrated culture to produce acid. These strains are susceptible
to damage during the fermentation, centrifugation and freeze-drying/freezing and storage
stages.
Advantages of using cell concentrates
The following advantages have been claimed for the use of concentrates in cheese factories:
Centralised concentrate production enables a manufacturer to establish a team of technical
experts and to develop the necessary technology and protocols to produce a quality
product.
Concentrates can be produced at a central site, which is located at a place distant from
cheese manufacture thus avoiding the hazards of phage infection due to phage-leaden
whey aerosol particles in the environment.
Detailed quality control tests can be performed on each batch of concentrate and, in theory
at least, only batches meeting the manufacturer's specification are released for factory use.
338
No incubation or sub-culturing is required at the factory. This reduces the probability of
phage or other forms of contamination occurring since all the factory-staff have to do is to
thaw the concentrate or open a packet of freeze-dried concentrates and add it to the bulk
starter milk or to the vat milk.
Disadvantages of using starter concentrates
The following disadvantages have been claimed for the use of concentrates:
Not all starters respond well to the operations involved in concentrate production and/or
storage. This fact limits the number of strains suitable for concentrate production and can
create some difficulties for starter suppliers and factory laboratories in compiling rotations
of phage un-related strains. This reduction in the numbers of strains also results in a more
limited choice of starters that have the potential to produce good flavour in mature cheese.
To some extent this has prompted the development of adjunct cultures, some of which may
be used to enhance or even balance flavour in mature cheese.
Low temperature storage facilities are required for frozen concentrates at the production
point, during transit to the factory and at the factory. Power cuts and distribution problems
could obviously present difficulties. Some of these difficulties have been overcome by the
development of freeze-dried concentrates.
Although concentrate suppliers perform quality assurance on their products, starter
suppliers generally offer only limited guarantees of concentrate quality. In other words, if a
contaminated concentrate is used and an inferior quality cheese results, or worse, there
may be difficulties in getting the starter supplier to accept liability for all the resultant
economic loss. In fact, most starter suppliers recommend that concentrates should be pre-
tested at the factory before their use in cheese manufacture. Consequently, a decision to
replace the mother and the intermediate stages of bulk starter manufacture with
concentrates should be based on the knowledge that the responsibility for starter quality
has been taken from the factory laboratory and belongs to the starter supplier. However,
the accountability in the event of problems related to the starter may not have been fully
transferred to the supplier. For these reasons factories using concentrates should ideally
take representative samples from each batch of concentrates and pre-test them before
cheese and other ferment products manufacture. Factories lacking the facilities to do this
should take samples and in the event of problems and send concentrate samples unopened,
packaged properly and refrigerated to an independent laboratory for analysis.
Use of DVS cultures is expensive compared with bulk starter manufacture. This is
particularly so when the costs of modern, aseptically produced starter using pH control are
considered. The costs are well understood but this statement is only valid where companies
have well designed bulk starter facilities, qualified staff and good quality assurance
laboratories. In the absence of this combination, the economic losses resulting from poor-
339
quality cheese, means that use of DVI cultures is the logical choice for many small to
medium sized processing units.
Changes to the cheese making process may be required. Addition of traditional bulk starter
to cheese milk results in a drop of 0.1 to 0.2 pH units. This small drop in acidity has
significant even if subtle effects on subsequent proteolysis in the cheese, and has an effect
on coagulation. In addition, the culture starts producing acid virtually immediately. With
DVS cultures, there is no drop in acidity and there is a lag period before the cultures
commences growth and acid production. Consequently, small adjustments to the traditional
cheese making process are required to maintain cheese quality.
Table 3: Storage conditions and shelf lives of some concentrated cultures
Type of cultures Storage Shelf-life (Months)
1. Freeze dried (Direct Vat) -18°C 12
2. Deep frozen (Direct vat) -45°C 12
3. Freeze dried (Master culture) +5°C 12
Quality control of commercial cultures (DVS/DVI)
6) Viable cell numbers
7) Absence of contaminants, pathogens, and extraneous matter
8) Acid-producing and other functional activities
9) Package integrity, accuracy of label information on the package
10) Shelf life of the product according to specification
Starter organism 1010
-1012
cfu/g
Coliforms Absence in 1 g
Enterococci Less than 20 cfu/g
Yeasts and molds Absence in 1 g
Staphylococci (coagulase-positive) Absence in 10 g
Listeria Absence in 25 g
Salmonella Absence in 25 g
Conclusion
Commercial starter culture production is a highly demanding operation. It requires specialized
knowledge of microbiology, microbial physiology, process engineering, and cryobiology. In
addition to production knowledge, a full-fledged quality control program is necessary to test
incoming raw materials, design and maintain plant sanitation, test sterility of production
contact surfaces, monitor plant environment quality, and test every product lot for the pre-
scribed quality standards. The quality control section is also required to train and update plant
340
personnel on the importance of sanitation and strict adherence to process control protocols.
Suggested Readings
Cogan, T. M. and Hill, C. (1993). Cheese starter cultures, Ch.6 in: P.F. Fox, ed., Cheese: Chemistry, Physics and
Microbiology, Vol. 1, General Aspects, 2nd
ed., pp. 193-206. Chapman and Hall, London.
Lewis, J. E. (1987). The Lewis method, in: Cheese Starters, Development and Application of the Lewis System,
pp. 196-200.
Tamime, A. Y. and Robinson, R.K. (1999). Preservation and production of starter cultures, In: Yoghurt, Science
and Technology, pp. 486-514. CRC Press, New York and Woodhead Pub. Ltd., Cambridge, UK.
341
Microencapsulation – an Efficient Delivery System for Functional Food
Ingredients
Surajit Mandal, Sandip Basu, R. P. Singh, Chand Ram and Rameshwar Singh
Dairy Microbiology Division, National Dairy Research Institute, Karnal – 132001
Email: [email protected]
Introduction
Foods which promote health beyond providing basic nutrition are termed as ‘functional foods’.
The term ‘functional food’ refers to a food that has been modified or value-added. Significant
strategy in the development of functional foods evolves increasing the levels of specific
nutraceuticals that are known to have health benefits. This can be through enhancement of
levels of the desired component that is inherent in the food or by fortification of food products
with functional ingredients, such as dietary fibres, antioxidants, natural isoflavones, plant
sterols/stanols, other phytochemicals or phytonutrientvs, bioactive peptides, ώ-3, -6 PUFA,
probiotics, prebiotics, minerals and vitamins etc. (Table 1).
Table 1. Functional ingredients and the health benefits
Functional ingredients
addition/modifications of
foods
Functionality
Phytochemicals (as plant
ingredients or extracts)
Antioxidant, lower risk of CHD, cancer, and lower blood
pressure
Probiotics Improved gastrointestinal function, enhanced immune
system, lower risk of colon cancer and of food allergy
Prebiotics Improved gastrointestinal function, lower risk of colon
cancer, enhanced immune system
Bioactive proteins or peptides Enhanced immune function and bioavailability of
minerals, hypertensive function
Dietary fibers Prevention of constipation, lower risk of colon cancer
and lowering of blood cholesterol level
ώ-3 PUFA Lower risk of heart attack, lower risk of some cancers,
enhanced immune system
Removal of allergens Reduce or eliminate allergy to specific foods
Hydrolysis of lactose by adding
-galaoctosidase
Enable digestion of lactose by lactose-intolerant persons
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Functional food ingredients should be present in sufficient quantities/numbers at the time of
consumption and reach to the action site for functional activities. Thus, the ingredients should
withstand the food processing and preservation treatments and stable during storage and
gastrointestinal tract (GIT) transient. These should not be affected by food matrixes and
environmental factors prevailed in foods as well as should not react with food components.
However, most of the ingredients react with food components and also affected by food
matrixes and environments. These lead to the poor stability/survival of functional ingredients.
Different methods for stabilization or improvement of survival are including selection of
suitable and stable ingredients, food combinations, addition of protective agents, segregation
by physical barriers etc. The selection is highly probabilistic in nature. Alternatively, addition of
compatible protective/stabilizing agents and segregation by applying barriers are very suitable
and promising. Controlled release of food ingredients at the right place and the right time is the
key for the functionality of active ingredients. A timely and targeted release improves the
effectiveness of food additives, broadens the application range of ingredients and ensures
optimal dosage and cost-effectiveness. Among the different techniques, microencapsulation
offers advantages in improving the nutrient content of foods without affecting the sensory
qualities. Microencapsulation may is used for stabilizing a desirable component, reducing the
level of an undesirable component and enabling the targeted delivery of functional ingredints.
Hurdles affecting the functional food ingredients
Long chain poly-unsaturated fatty acids
Numerous challenges exist in the production, transportation and storage of poly-unsaturated
fatty acids (such as -3, -6 fatty acids) fortified foods as poy-unsaturated fatty acids are
extremely susceptible to oxidative deterioration. It has been a challenge for oil refiners to
inhibit oxidation -3 fatty acids during processing, shipping, and storage. Additional challenges
exist in preventing the oxidation of -3 fatty acids when these are incorporated into processed
foods.
Vitamin and minerals
Vitamin and mineral fortification has been used to improve nutrient content of foods. The level
of vitamins decreases during processing and storage. The interactions between the added
minerals and vitamins with other components in foods are important for fortification. pH, heat,
light, oxygen, oxidizing agents and enzymes decrease the stability and activity of many vitamins.
The addition of free mineral salts is having undesirable interactions between mineral salts and
components in milk and milk products can lead to precipitation, colour and flavour problems,
and the bio-availability. The fortification of milk with iron presents different challenges. The
343
most common iron salts (e.g. ferrous sulphate). The addition of these salts can affect the
sensory properties of the food due to the taste of the iron salt or the catalytic effect of iron the
oxidaiotn of fats. Some of the major problems encountered by direct addition of simple calcium
salts, such as loss of heat stability due to increase in calcium activity can be overcome by using
calcium complexing agents or insoluble calcium salts.
Probiotics
Probiotics are added as live cultures in a range of foods to improve the microbial balance of the
human gut. The survival of probiotics in foods during processing, preservation and storage as
well as during GIT transient is the determinant of probiotic functional foods. The consumption
of sufficient viable cells (108-109 cfu/ day) is required for functional activities. However,
probiotics survive poor in traditional fermented dairy products due to low pH, post-acidification
(during storage), hydrogen peroxide production, oxygen toxicity, storage temperatures, poor
growth in milk and lack of compatibility with traditional starter cultures, etc. and during GIT
transient. For increasing probiotic consumption, foods with probiotics need to be diversified to
non-fermented, where probiotics survival and compatibility is a big impediment.
Microencapsulation an efficient delivery system
In microencapsulation droplets/ particles of liquids, solids, or gases (core) are coated by thin
films (coatings), which protect the core from external environment. The core can be released at
different times as and when required by any desired mechanisms, such as disruption,
dissociation, dissolution or diffusion and with any desired rates. The coating on a core is semi-
permeable and protects the core from severe conditions and controls substances flowing into
the core and the release of metabolites from the core. Encapsulation in foods is also utilized to
mask odours or tastes. Various techniques are employed to form the capsules, including spray
drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome
entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational
suspension separation (Table 2). Number of food ingredients/substances have been
microencapsulated, such as acidulants, amino acids, antimicrobials, bases, colorants, edible oils,
flavour, enzymes, microorganisms, flavour enhancers, leavening agents, minerals, sugars, salts,
vitamins etc. The use of encapsulation for sweeteners such as aspartame and flavours in
chewing gum is well known. Fats, starches, dextrins, alginates, protein and lipid materials can
be employed as encapsulating materials. Various methods exist to release the ingredients from
the capsules such as site-specific, stage-specific or signalled by changes in pH, temperature,
irradiation or osmotic shock. In the food industry, the most common method is by solvent-
activated release. The addition of water to dry beverages or cake mixes is an example.
Liposomes have been applied in cheese-making, and its use in the preparation of food
emulsions such as spreads, margarine and mayonnaise is a developing area. Most recent
developments include the encapsulation of foods in the areas of controlled release, carrier
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materials, preparation methods and sweetener immobilization. New markets are being
developed and current research is underway to reduce the high production costs and lack of
food-grade materials.
Table 2: Various techniques for microencapsulation
Technique Major steps
1. Spray-drying a. Preparation of the dispersion
b. Homogenization of the dispersion
c. Atomization of the infeed dispersion
d. Dehydration of the atomized particles
2. Spray-cooling a. Preparation of the dispersion
b. Homogenization of the dispersion
c. Atomization of the infeed dispersion
3. Spray-chilling
a. Preparation of the dispersion
b. Homogenization of the dispersion
c. Atomization of the infeed dispersion
4. Fluidized-bed coating a. Preparation of coating solution
b. Fluidization of core particles
c. Coating of core particles
5. Extrusion a. Preparation of molten coating solution
b. Dispersion of core into molten polymer
c. Cooling or passing of core-coat mixture through dehydrating
liquid
6. Centrifugal extrusion a. Preparation of core solution
b. Preparation of coating material solution
c. Co-extrusion of core and coat solution through nozzles
7. Lyophilization a. Mixing of core in a coating solution
b. Freeze-drying of the mixture
8. Coacervation a. Formation of a three-immiscible chemical phases
b. Deposition of the coating
c. Solidification of the coating
9. Centrifugal suspension
separation
a. Mixing of core in a coating material
b. Pour the mixture over a rotating disc to obtain encapsulated tiny
particles
c. Drying
10. Co-crystallization a. Preparation of supersaturated sucrose solution
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b. Adding of core into supersaturated solution
c. Emission of substantial heat after solution reaches the sucrose
crystallization temperature
11. Liposome entrapment a. Micro-fluidization
b. Ultrasonication
c. Reverse-phase evaporation
12. Inclusion
complexation
Preparation of complexes by mixing or grinding or spray-drying
Consideration of materials for micro-encapsulation
The structure formed by microencapsulating agent around the core material is called the wall
material, which protects the core against deterioration, limits the evaporation of volatile core
materials. The encapsulating agents should have certain ideal characteristics, depending on the
objectives and requirements, process of encapsulation, chemical characteristics of the core
material, the intended use of the core material, the conditions under which the product will be
stored, and the processing conditions to which it will be exposed. Some general characteristics
of the encapsulating agent are that it is insoluble in and non-reactive with the core material,
have solubility in the end-product food system, and be able to withstand high temperature
processing. Some typical encapsulation agents are dextrans, gums, starches or proteins (Table
3). Many coating materials have been used for encapsulation of microorganisms. These include
a mixture of -carrageenan and locust bean gum, cellulose acetate phthalate, alginate, alginate-
starch mixture, -carrageenan etc.
Table 3: Coating materials used for encapsulation
Class of coating
materials Specific types of coatings
Gums Gum arabic, agar, sodium alginate, carrageenan
Carbohydrates Starch, dextran, sucrose, corn syrup
Celluloses CMC, methylcellulose, ethylcellulose, nitrocellulose, acetylcellulose,
cellulose acetate-phthalate, cellulose acetate-butylate-phthalate
Lipids Wax, paraffin, tristearin, stearic acid, monoglycerides, diglycerides,
beeswax, oils, fats, hardened oils
Inorganic materials Calcium sulfate, silicates, clays
Proteins Gluten, casein, gelatin, albumin
Additional treatments to microcapsules
Entrapment in hydrocolloid gels, such as alginate, -carrageenan etc have some limitations due
to less stability of microcapsules in the presence of chelating agents such as phosphate, lactate,
citrate etc., which share the affinity for ions such as Ca+2, K+, etc. and destabilize the gel. The
346
problems are encountered during lactic acid fermentation and cause cell release from the
beads. In other matrix material, such as chitosan, the entrapped cells can be released form the
beads during fermentation and cause low initial loading for the next fermentation. Therefore,
additional treatments, such as coating the beads, are applied to improve the properties of
beads. Coated beads not only prevent cell release but also increase mechanical and chemical
stability. Cross-linking with cationic polymers, coating with other polymers, mixing with starch
and incorporating additives improve stability of beads.
Applications
Protection of polyunsaturated fatty acids
Microencapsulation of long-chain polyunsaturated oils eliminates fishy odour and taste for
development of enriched fatty acids products. A supplement comprising a blend of omega-3
fatty acids, omega-6 fatty acids (gama-linoleinc acid C18: 3n-6 and arachidonic acid C20:4n-6)
and evening primrose oil encapsulated in gelatine may be provided for addition to infant
formulae to achieve a milk composition approximating to human milk. Infant formulae fortified
with microencapsulated spray dried marine oil powders have been successful in the market
place. Yoghurts, fermented milks and processed cheese with tuna oil encapsulated with
processed milk-protein-carbohydrate films (Driphorm 50) made using MicroMAX technology
have higher sensory scores than those fortified with an equivalent amount of non-encapsulated
oil. The development of spray-dried microencapsulated fish oil was under taken as part of EU
FAIR contact 9CT 95-0085 to establish a delivery system for fish oil in powdered form so that
there would be a degree of protection from oxidation during storage, containment of fishy
odour as far as possible, and finally to achieve the highest oil content possible in dry matter.
Deodorized sand-ell oil (fish oil) stabilized with natural antioxidants was emulsified with protein
and lactose (oil: protein: lactose: water in 10:10:10:70 ratio). Both the processing variables
(homogenization pressure and number of passes, and spray-drying effects) and packaging
(vacuum vs. nitrogen flushing) were studied. Based on physical indicator, it was concluded that
homogenization pressure and protein source (sodium caseinate, calcium caseinate, and skim
milk powder) influenced free fat and surface fat contents in the powder. Skim milk powder gave
better sensory scores. The resulting microencapsulated fish oil powder had very good sensory
properties and was stable for up to 6 months under refrigerated conditions.
Protection of vitamins and minerals
Many encapsulated preparations for addition to a range of beverage and foods have been
developed to overcome undesirable interaction of vitamins with the environment and food
components during processing and storage. Microencapsulated vitamins improve the of
vitamins’ stability during storage. Higher levels of the added vitamin D are entrapped into the
347
cheese curd when milk is fortified with liposome encapsulated vitamin as compared to
homogenizing in cream or milk (Table 4).
Table 4: Stability of various vitamin preparations in dairy products
Product Type of vitamin preparation % Loss
Instant skim milk
powder
Gelatin-encapsulated vitamin a (after
simulated instantising treatment)
60% in 40 weeks of storage
Gelatin-encapsualted vitamin A (Dry blends
with non-fat dry millk)
Approx. 10% in 40 weeks
storage
Cheddar cheese Water-soluble emulsion of vitamin D 16% in 7 months
Vitamin D homogenized in cream 11% in 7 months
Vitamin D entrapped in liposomes 40% in 7 months
Encapsulated mineral salts lessen the tendencies of undesirable interactions. The choice
between fortification with microencapsulated minerals or direct addition of mineral salts is
depend on their relative costs, the bioavailability and impact on sensory properties of foods.
Microencapsulated iron ingredients can prevent off-flavour development and maintain
bioavailability of the iron. Stearine-coated iron salts decrease fat oxidation in Harvati cheese
compared to unprotected iron salt. Liposome encapsulated iron may be used for fortification of
beverages for minimizing off-flavours and interactions with other food components. The use of
ferrous sulphate encapsulated in lecithin (SFE-171, Biofer) is claimed to allowed effective
fortification of fluid milk and dairy products, while preventing undesirable interactions with
milk components with higher bio-availability of iron. Iron absorption from milk with the use of
encapsulated ferrous sulphate SFE-171 is higher than that with the direct addition of ferrous
sulphate. A possible strategy for calcium fortification of fluid milk includes the use of micro-
crystalline cellulose-based ingredient co-processed with calcium carbonate and
carboxymethylcellulose, which results in good flavour and stability of milk. Alternatively,
liposomes may be used to protect calcium salts from interactions with proteins at higher
temperature, as these prevent precipitation of soy proteins in calcium-fortified soy milk.
Protection of probiotics
Viability of probiotics can be improved by appropriate selection of acid and bile resistant
strains, use of oxygen impermeable containers, two-step fermentation, stress adaptation,
incorporation of micronutrients such as peptides and amino acids, sonication of yogurt bacteria
and microencapsulation. Microencapsulation is the most suitable alternative technology to
offer the best protection to the probiotic cells resulting from the freeze-drying and milling and
such microencapsulated probiotics can be used in numerous food systems (Table 5 and 6).
Table 5: Encapsulation of cells for food and biotechnological applications
Cultures Encapsulating materials Product
B. bifidum, B. infantis Calcium alginate Mayonnaise
L. paracasei Milk fat Cheddar cheese
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Enterococcus faecium Milk fat Cheddar cheese
B. bifidum, B. adolescentis Cream White brined cheese
B. bifidum, B. infantis, and
B. longum
Calcium alginate gels Crescenza cheese
L. lactis subspp. Lactis k-Carrageenan and locust bean gum Fresh cheese
L. casei Liquid core alginate capsule Lactic acid
Lactobacilli Calcium alginate Frozen dessert
Lactococci Calcium alginate Cream
L. casei k-Carrageenan and locust bean gum Yoghurt
L. casei Calcium alginate Milk chocolate, kulfi
L. casei Skim milk-whey protein concentrate-
maltodextrin
Kulfi
Table 6: Survival of probiotics in milk and milk products
Products Storage
conditions
Form of bacteria added Counts (cfu/ml or g)
Initial After storage
Free B. bifidum 4.5x106 7.5x106
Yogurt 30 days at 4.4°C Free B. longum B6 1.51109 3.54x108
B. longum B6 enapsulated in k-
carrageenan
1.51x109 1.02x109
Free B. longum ATCC 15708 1.51x109 4.35x108
B. longum ATCC 15708
enapsulated in K-carragenna
1.51x109 1.48x109
Milk with 2% fat 12 days at 4°C Free B. longum Bb-46 1x107 1x104
B. longum Bb-46 encapsulated in
Ca alginate
4x107 3x105
Free B. lactis Bb-12 1x107 1x107
B. lactis Bb-12 encapsulated in Ca
alginate
2x108 2x108
Milk chocolate 60 days at 7°C Free L. casei NCDC 298 8.40 log
cuf/g
8.55 log
cuf/g
L. casei NCDC 298 encapsulated
in Ca alginate
8.38 log
cuf/g
8.48 log
cuf/g
30 days at room
temperature
Free L. casei NCDC 298 8.44 log
cuf/g
5.52 log
cuf/g
L. casei NCDC 298 encapsulated
in Ca alginate
8.46 log
cuf/g
6.88 log
cuf/g
Kulfi -10°C for 7 days L. casei NCDC 298 encapsulated
in skim milk – whey protein
concentrate-maltodextrin
107 cfu/g 107 cfu/g
349
-10°C for 35 days L. casei NCDC 298 encapsulated
in Ca alginate
108 cfu/g 108 cfu/g
Sustained and target release of functional ingredients
Immunoglobulins have potential in functional food development as they afford protection
against gastrointestinal infections. However, they are prone to inactivation in the gut.
Encapsulation of immunoglobulins may be used to preserve the activity in certain
environments. Milk immunoglobulin G (IgG) that was encapsulated in double emulsions, solid
agar or calcium alginate gels had improved stability in acid and alkali environments as well as
making them less susceptible towards the action of proteinases. Sustained release of amino
acids after ingestion of protein supplements is desirable in sports nutrition and for improved
exercise performance. Liposomal encapsulated ion-exchange whey protein as a protein
supplement maintains plasma amino acids at higher levels compared to when conventional
proteins supplements. Liposome encapsulated cholesterol-lowering plant sterols and stanols
are used in milk and dairy products and these preparations are alternative to the free stanols
and sterols having limited solubility in some foods.
Conclusion
Fine-tuned controlled release and stabilization of functional ingredients in foods and during GIT
transient is the key for development of functional foods. Among the different techniques,
microencapsulation is no longer just an added value technique, but the source of totally new
ingredients with matchless properties and can be applied in the development of new and novel
functional foods. It is only one of a suite of technologies that may be applied to enhance the
quality of healthy dairy foods and its suitability depends on the food product to be fortified, the
need for protection of food components and timed release of nutraceuticals.
References
Augustin, M. A. 2003. The role of microencapsulation in the development of functional dairy foods. The Australian Journal of Dairy Technology, 58(2): 156-160.
Desai, K.G.H and Park, H.J. 2005. Recent Developments in Microencapsulation of Food Ingredients. Drying Technology, 23(7): 1361-1394
Gibbs, B. F., Kermasha, S., Alli, I. and Mulligan, C. N. 1999. Encapsulation in the food industry: a review. International Journal of Food Sciences and Nutrition, 50 (3): 213-224.
Gouin, S. 2004. Microencapsulation industrial appraisal of existing technologies and trends. Trends in Food Science and Technology, 15(7-8): 330-347.
Hu, M., McClements, D.J., Decker, E.A. 2003. Impact of whey protein emulsifiers on the oxidative stability of salmon oil-in-water emulsions. Journal of Agricultural Food Chemistry, 51(5):1435–1439.
Makhal, S., Mandal, S., Kanawjia, S. K. and Singh, S. (2005). Value addition to foods for health. In: Indian Dairy Industry published by Chawla Dairy Information Centre Pvt. Ltd., New Delhi-110092, pp: 157-162.
Makhal, S., Mandal, S., Kanawjia, S. K. and Singh, S. (2005). Value addition to dairy products. In: Indian Dairy Industry: Annual dairy resource book. (Vol. I), 1
st edn. Eds. Chakraborty, G.C., Dr. Chawla Dairy Information
centre Pvt. Ltd., New Delhi – 110092, pp: 120-128.
350
Milk Bioactive Peptides and Their Immunomodulatory Role
Suman Kapila and Rajeev Kapila
Division of Animal Biochemistry, NDRI, Karnal
In recent years the role of protein in the diet has been acknowledged worldwide.
Dietary proteins become source of physiologically active components which have positive
impact on body’s function after gastrointestinal digestion. Milk remains one of the most
elaborately studied of human food. The benefit of milk in preventing infection has been
recognized since long time. Milk contains various components with physiological functionality.
Milk proteins are currently the main source of a range of biologically active peptides, even
though other animal or plant proteins contain potential bioactive substances. These peptides
have been obtained from casein as well as whey proteins. The bioactive peptides are inactive
within the sequence of parent proteins and can be released by enzymatic hydrolysis in vitro or
in vivo. Once these peptides released from parent proteins may act in the body as regulatory
compounds with a hormone-like activity [1]. Bioactive peptides usually contains 3-20 amino
acid residues per molecule [2].The sequence of amino acids of a particular peptide defines the
function of the peptide. Milk borne bioactive peptides have been found to exhibit various
physiological activities such as antihypertensive, immunomodulatory, antimicrobial,
antioxidative, antithrombotic as reviewed in many recent articles [3,4]. Several bioactive
peptides reveal multifunctional properties. Some regions of primary structures of caseins
contain overlapping sequences that exert different activities. These regions have been
considered as ‘strategic zones’ that are partially protected from further proteolytic breakdown
[5]. Due to various biological functions milk borne bioactive peptides are regarded as active
ingredient for preparation of various functional foods, nutraceuticals and pharmaceutical
drugs[6].
Bioactive peptides encrypted within precursor protein can be released in three ways: (a)
enzymatic hydrolysis by digestive enzymes like trypsin, pepsin etc. (b) food processing and (c)
proteolysis by enzymes derived from microorganisms or plants (fig.1). Starter lactic acid
bacteria generate bioactive peptides during milk fermentation and cheese maturation, thereby
enriching dairy products. Such dairy products under certain conditions carry specific health
effects when ingested as part of daily diet. Bioavailability of peptides most often requires that
they should not be digested in the gastrointestinal tract. The absorption of small peptides is
well known. Peptides can be absorbed through the gastrointestinal wall by different
mechanisms, such as by passive diffusion through the enterocytes, paracellularly through
cytosis or through carrier. Some peptides, such as caseinophosphopeptides, express their
activity in the gastrointestinal tract without being absorbed.
351
Fig. 1 Scheme of peptides release from precursor proteins by fermentation and/or
gastrointestinal digestion
Immunomodulatory peptides
The systems involved in the human body’s defense against invaders are rather complex;
diet is known to play an important role therein. The two main activities are the
immunomodulatory (stimulation of immune system) and antimicrobial (inhibition of bacterial
pathogens). Several casein and whey protein derived peptides display an immunomodulatory
role in which case a totally separate cascade of host defense responses is initiated (Table-1).
Immunomodulating peptides have been found to stimulate the proliferation of human
lymphocytes, the phagocytic activities of macrophages and antibody synthesis. The peptides
may stimulate the proliferation and maturation of T cells and natural killer cells for defense of
new born against a large number of bacteria, particularly enteric bacteria [7].
Milk
Fermentation
fermented milk
precursor protein
digestion GI-tract
Digestion
Encrypted bioactive peptides
352
Table 1: Immunomodulating peptides derived from milk proteins
Source Peptides Activity
Human β- Casein
Human α-
lactalbumin
Bovine β- Casein
Bovine αs1-casein
Bovine α-
lactalbumin
VEPIPY (54-59)
GLF (51-53)
PGPIPN (63-68)
LLY (191-193)
C-terminal
peptide
(192-209)
TTMPLW
(194-199)
YGG (18-20)
Activates phagocytosis of sheep red blood
cells by mice peritoneal macrophages, in vivo
protection against K.pneumonia.
Stimulates in vitro phagocytosis
Enhances proliferation of rat lymphocytes
Protection against infection.
Modulate proliferation of human peripheral
blood lymphocytes.
Inhibition of the proliferation of B
353
Bovine κ-casein
Bovine κ-casein
YG (38-39)
CMP (106-169)
FFSDK (17-21)
lymphocytes
Cytotoxic towards mouse spleen cells and
some mammalian cells including human
leukemic cell lines
Casein derived immunopeptides including fragments of αs1-casein and β casein
stimulate phagocytosis of sheep red blood cells by murine peritoneal macrophages and exert a
protective effect against Klebsiella pneumoniae infection in mice after intravenous
administration *8+. The C terminal β casein sequence 193-209 containing β casokinin-10 induced
significant proliferative response in rat lymphocytes *9+. Depending on peptide concentration β
casokinin-10 and β casomorphin-7 showed a suppression as well as stimulation of lymphocyte
proliferation. β casomorphin-7 inhibits the proliferation of human colonic lamina propria
lymphocytes where anti-proliferative effect was reversed by opiate receptor antagonist
naloxone [10]. Also, it has been suggested that immunomodulatory milk peptides may alleviate
allergic reactions in atopic humans and enhance mucosal immunity in the gastrointestinal tract
[2]. In this way immunomodulatory peptides may regulate the development of the immune
system in newborn infants. Furthermore, immunopeptides formed during milk fermentation
have been shown to contribute to the antitumor effects [11].
Recent studies have focused on immunoenhancing properties of caseinophosphopeptides. Hata
et al. (1998) reported on the immunostimulatory action of phosphopeptides αs1 –CN f(59-79)5P,
αs2 –CN f(1-32)4P and β CN f(1-25)4P which enhanced immunoglobulin IgG production in
mouse spleen cell cultures[12]. Moreover, the level of serum and intestinal antigen specific IgA
was higher in the mice fed the caseinophosphopeptides than those fed the control diet.
Another group of peptides which may be implicated in the stimulation of
immunosystem are ACE inhibitors. Inhibition of ACE favors bradykinin formation and thus acts
as immunomodulators. Bradykinin, known as mediator of the acute inflammation process, is
able to stimulate macrophages to enhance lymphocyte migration and increase secretion of
lymphokinines *13+. The peptide fragments αs1 –casein f(194-199) and β casein f(60-66) and
f(193-202) have shown to have both immunostimulatory and ACE inhibitory activities.
354
The structure-activity relationship and mechanisms by which milk-derived peptides
exert their immunomodulatory effects is not yet defined. It has been suggested that arginine in
the N-or C-terminal region of peptide is important structural entity recognized by specific
membrane bound receptors [13]. The immunostimulatory activity of caseinophosphopeptides
was attributable to phosphoseryl residues [14] and the phosphorylation site appears to be an
allergenic epitope in caseins [15]. The results obtained with human lymphocytes suggest that
opioid peptides may affect the immunoreactivity of lymphocytes via opiate receptor. Therefore
there is a remarkable relationship between the immune system and opioid peptides, because
opioid μ receptors for endorphins are present on lymphocytes [10]. It has been shown that
glutamine-containing peptides can substitute for the free amino acid glutamine which is
required for lymphocyte proliferation and utilized at a high rate by immunocompetent cells,
even in a resting state [16]. Therefore such peptides exert a non-specific immunostimulation as
a result of their trophic properties.
Conclusion
Immunopeptides have potential applications as supplements in the maintenance of
immune health. For example, they can potentially provide some protection against infections
involving bacteria, viruses and parasites. Alternatively, immunosuppressive peptides could be
considered in some medical applications such as the prevention of graft or transplants rejection
and in the regulation of inflammation process involved in various autoimmune disorders and
aging.
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Migliore-Samour, D.; Floc’h, F.; Jollés, P. J. Dairy Res. 1989, 56, 357–362.
Coste, M.; Rochet, V.; Leonil, J.; Molle, D.; Bouhallab, S.; Tome, D. Immunology lett.1992, 33,41-46.
Elitsur,Y.; Luke, G.D. Clin. Experiment. Immunol. 1991 85, 493-497.
Matar, C.; LeBlanc, J.G.; Martin, L.; Perdigón, G. (2003). Functional foods and nutraceuticals series, CRC Press, Florida, USA 177–201.
Hata, I.; Higashiyama, S. Otani, H. J. Dairy Res. 1998, 65, 569-578.
Pagelow, I.; Werner, H. Method. Findings Exp. Clin. Pharm. 1986 8, 91-95.
Hata, I.; Ueda, J.; Otani, H. Milchwissenschaft1999, 54, 3-7.
Bernard, H.; Meisel, H.; Creminon, C.; Wal, J.M. FEBS Lett. 2000, 467, 239-244.
Calder PC. Clin. Nutr.1994, 13, 2–8.
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Evaluation of Immunomodulatory Property of Milk Protein
Suman Kapila and Rajeev Kapila
Animal Biochemistry Division, NDRI, Karnal-132 001
1. Introduction
The systems involved in the human body’s defense against invaders are rather complex;
diet is known to play an important role therein. The two main activities are the
immunomodulatory (stimulation of immune system) and antimicrobial (inhibition of bacterial
pathogens). Several casein and whey protein derived peptides display an immunomodulatory
role in which case a totally separate cascade of host defense responses is initiated. Healthy
humans have two immune mechanisms: acquired (specific) immunity, which responds to
specific stimuli (antigens) and is enhanced by repeated exposure; and innate (nonspecific)
immunity, which does not require stimulation and is not enhanced by repeated exposure.
Innate immune mechanisms consist of physical barriers, such as mucous membranes, and the
phagocytic and cytotoxic function of neutrophils, monocytes, macrophages, and lymphatic cells
(NK cells). Immunomodulatory property of proteins/peptides can be studied in terms of their
affect on innate and humoral immunity. Innate immunity is analyzed by studying macrophage
function (phagocytic activity) whereas lymphocyte function is evaluated either by lymphocyte
proliferation index or by measuring immunoglobulin levels.
2. Phagocytosis
Phagocytosis is a process of binding and ingesting particles is a key part of the immune
response. In the 19th century Metchnikoff originally demonstrated the phenomenon, at the
macro level, by introducing a splinter into the body of a starfish larva. Phagocytosis is one type
of endocytosis the general term for the uptake by a cell of material from its environment. In
phagocytosis a cell’s plasma membrane expands around the particulate material, which may
include whole pathogenic microorganism to form large vesicles called phagosomes.
2.1 In vivo phagocytosis
Microorganisms, or their experimental equivalent of carbon, iron or latex particles, are
readily engulfed by circulating and tissue-fixed phagocytes. The cells of the reticuloendothelial
system are capable of ingesting and degrading foreign material by means of intracellular
enzymes in phagosomes, i.e. neutrophils (polymorphonuclear leucocytes), monocytes,
histiocytes or tissue macrophages (microglia-brain, kupffer cells-liver, glomerular mesangial
cells-kidney, synovial macrophages-joints, etc.) and vascular endothelial cells.
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Reticuloendothelial cell clearance can be monitored in vivo using colloidal carbon
particles or microorganisms. Following the intravenous injection of colloidal carbon the
clearance is determined by the light transmission through lysed blood samples. Similarly the
clearance of microorganisms can be estimated by culturing blood samples taken at time
intervals following intravenous injection.
2.2 In vitro phagocytosis
Phagocytosis is a two-stage process in which particles are first bound to the cell surface
and then ingested. In-vitro it is important to distinguish these two processes.
2.3 Phagocytosis of yeasts
Yeast bind to lectin-like receptors on the surface of phagocytic cells principally through
the mannose receptor, his binding being blockable with α-mannans. Moreover yeasts are also
potent activators of the alternative complement pathway and following exposure to fresh
serum, bind to CR1 and CR3 receptors for C3b and C3bi deposited on the yeast surface, this
binding not being blockable with α-mannans.
However, some of the problem with using yeasts to measure phagocytosis is
determining whether the organisms have been internalized or are simply binding to the
surface. With fresh yeast this is difficult but autoclaved yeasts exhibit staining properties which
allow the differentiation of ingested particles. Autoclaved yeasts stain light pink with May-
Grunwald/Giemsa unless pretreated with tannic acid, when they stain deep violet. Tannic acid
is unable to reach cell-ingested yeasts, therefore they stain light pink, whereas surface-bound
particles stain violet.
2.4 Preparation of phagocytic cells
Mouse macrophages can be obtained simply by washing out the peritoneal cavity.
Peritoneal–derived immune cells are essentially made up of macrophages and lymphocytes.
2.4.1 Isolation of normal peritoneal macrophages
Materials
Mice, DMEM Ham’s F-12 medium (without phenol red) supplement it with sodium bicarbonate
(1.2 g/L), bovine serum albumin (0.1%), penicillin (200 U/ml) and streptomycin (50 µg/ml) fluid.
Adjust the pH of the medium 7.2 using 1 N HCl or 1 N NaOH and then filter sterilize it through
0.22 µ Millex-GV disposable filter unit (Millipore), Needles (22 and 26 gauge), disposable
syringe.
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Method
1. Sacrifice the mouse by cervical dislocation and clean the abdominal skin with 70%
alcohol by swabbing.
2. Inject 6.0/8.0ml of tissue culture medium into the mouse’s peritoneal cavity.
3. Knead the abdomen gently to bring the cells into suspension.
4 Collect the peritoneal exudates.
5 Count the macrophages using neubauer chamber
A normal mouse will yield 5x106 peritoneal exudates cells, up to 50% of which will be
lymphocytes. This is not usually a problem as this method involves allowing the cells to
adhere to glass or plastic surfaces and this considerably enriches the preparation.
2.5 Phagocytic assay
Materials
Macrophages, Yeast-Saccharomyces cerevisiae, Potato dextrose broth, Autoclave,
Phosphate buffered saline (PBS), 35mm Petri plates, May-Grunwald and Giemsa stains,
Giemsa buffer, Microscope,1% tannic acid
Method
Preparation of yeast
1. Culture yeast in potato dextrose broth for 48h at 300C.
2. Autoclave at 1200C for 45min in culture medium.
3. Wash three times in PBS.
4. Aliquot and store at 40C.
5. Just before use, sonicate gently in a water bath to disrupt clumps and dilute to 108/ml in
DMEM- Ham F12 medium.
Assay
1. Add 1ml of macrophage suspension at105/ml to 35mm Petri plate.
2. Incubate at 370C for 2h.
3. Remove culture medium and wash with medium.
4. Add 1ml medium and incubate for 2h at 370C.
5. Add 100ul yeast suspension (108particles/ml).
6. Incubate for 1h at 370C in a 5%Co2 humidified incubator.
7. Wash twice gently with culture medium.
8. Add 1ml 1%w/v tannic acid solution and leave for 1min.
9. Wash with medium and dry in air.
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10. Stain with May-Grunwald freshly diluted 1:2 with buffer, for 5 min.
11. Rinse in buffer.
12. Stain in Giemsa solution, freshly diluted with buffer, for 15 min.
13. Rinse in buffer.
14. Observe at 1000X magnification.
15. Count 100 macrophages showing engulfed and attached yeast cell.
Percent Phagocytosis = No. of macrophages with yeast cell internalized/100 macrophages
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Concepts and Skills in Technical and Scientific Writing
Meena Malik
Assistant Professor (English), NDRI, Karnal
The function of language is to communicate, and any language that makes for clear and
accurate communication is a good language. Personality, gesture, and intonation all contribute
to the success of spoken communication. Written English, on the other hand, uses structure,
rather than the physical presence of the writer, to achieve clarity. Written English
communicates through the precision of its diction, the orderliness of its sentence and
paragraph structure, and the relative fullness of its detail.
Communication Skills
Communication is a process involving transferring of information and sharing of ideas from one
person to the other. “Communication" is a word with a rich history. It has been derived from
the Latin word communicare, meaning - to impart, share, or make common. This word entered
the English language in the fourteenth and fifteenth centuries. Besides the core competence
and knowledge in one’s specialized field, communication skills contribute a lot to the success of
an individual in any organization. These skills form an integral part of leadership and managerial
skills, one of the essential elements required for developing competence needed for career
success in the 21st Century. This is the Only Completely Portable Skill, used in every relationship
and required regardless of any career path. The history of civilization is the history of
information. Language and written documents facilitate the transfer of information and
knowledge through time and space.
Technical and Scientific Writing/Reporting
Technical and Scientific Writing/Reporting is a specialized branch of the field of communication.
This is the art of recording information on specialized fields accurately and effectively and
passing it on to those who have to use and process it.
Importance of Technical Reporting
Students: The typical undergraduate student regards the writing of reports as a dull and
superfluous chore. Consequently, he has little desire for instruction in technical writing. One of
the main reasons for this state of affairs is that the undergraduate-particularly in his/her earlier
years-seems to have very little to say. As he programs through college and on into graduate
school or industry, he develops a body of knowledge. At some time in his career, he acquires
some information or some idea that he wants to pass on to others. Only then does he wish for
instruction in technical reporting.
Big Organization: The complexity of an organization increases exponentially with its size. And
as the complexity goes up, soon too does the need for written records and communications.
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Only through a full exchange of information can the various divisions of large organization co-
ordinate their efforts effectively.
Small Organization: But even a small organization has a vital need for accurate technical
reporting. How was a special part fabricated last year? How was a test performed? What are
the precautions to be observed with seldom used instrument? Written records furnish
authoritative answers to many questions as these, and increase the efficiency of organization
that maintains vigorous reporting procedure.
Scientific Organization: And then some engineering and scientific organizations do nothing but
investigation, testing, experimentation, or research. Their only tangible product is the report. If
they are to have anything to show for their efforts, they must do thorough job of reporting.
Many industrial and research organizations nowadays place so much value on high quality
reports that they maintain separate editorial departments to write technical report or to edit
and polish them. Reports have achieved a recognized position of importance in our
technological world.
The Importance of Proficiency in Technical Reporting: In many engineering organizations,
particularly those doing experimental work or research, the young employee’s chief
communication with his superiors is through his written (or oral) reports. Often the superior
has no other criterion by which to judge an employee’s work.
Some students are admirably grounded in basic sciences, they are intelligent, and they are
capable of doing excellent work. But their education has left a serious gap; they are unable to
describe clearly and succinctly what they have done. This inability exists, I believe, not so much
because the engineering schools fail to offer instruction in this important subject, but because
the students lack sufficient motivation to apply themselves to it.
Functions of Technical Writing
Technical Reporting is different from creative writing because it deals with scientific facts and
does not present an imaginary view of reality. Scientific and Technical Writing is objective in
content and systematic in form. It is always precise, exact, and to the point so that it may have
the desired effect on the reader and lead to the required action.
Education and Research: Journals publish technical material on specialized fields and are
circulated amongst the scientists and scholars. All these writings must conform to the rules of
scientific and technical reporting so that they are properly understood and appreciated. All
types of articles such as Technical Articles; Semi-technical Articles; Popular Articles; Research
Papers; Dissertations and Theses, and Technical Bulletins are covered under the ambit of
Technical Writing.
Industry/ Service Sector: The written word is very important at every stage of Industrial
development. Industrial reports are must for spread of latest advances in the vast field of
Industry. They provide guidance to Industrial concerns and keep us abreast of the Information
about the products coming out of the Industrial unit. Service manuals and guidance manuals
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are efficient tools to provide specifications to the users. Technical Reports include all kinds of
reports such as Form Reports on a given proforma, Article Reports, Formal Reports such as
Annual Reports, Quarterly Reports; Manuals and Formal Correspondence such as letters,
memoranda etc
Form and Structure of Technical Reporting
The nature of the subject, the purpose of the report and the reader for whom the report is
written determine the form and structure of the report. Every written communication has a
specific purpose and a specific audience. It should be carefully planned and constructed to fit
both.
Special Purpose: Every technical communication has one certain clear purpose: to convey
information and ideas accurately and efficiently. This objective requires that the
communication be: (1) as clear as possible; (2) as brief as possible; and (3) as easy to be
understood as possible.
Specific Audience: Any communication, if it is to be effective and efficient, must be designed for
the needs and the understanding of a specific reader or group of readers. It must neither be
beyond their powers of comprehension, nor so far beneath their level of competence as to
bore them and thus, lose them. One must, therefore, have adequate knowledge of the
educational and professional background of the readers, their numbers, their interests and
involvement in the subject and their major interests in matters outside the subject of the
report.
Background Information: Are the people who will see or hear this report familiar with the
general field or do they need extensive general orientation? Are they familiar with the
circumstances of the present case, or do they need briefing? How much background
information must you supply them?
In other words the structure, form and layout of the report will be determined by the nature of
the work, the purpose of the report and the readers for whom it is intended.
Organization of Technical/Scientific Report
The Contents: The subject of the report primarily determines the nature of the contents. Report
writing is meaningless when the writer is not clear about the subject of his report. However, the
detailed aspects of the contents are determined by the purpose for which the report is written.
Basic questions (5 Ws i. e. What, Why, Who, Where, When, and How) need to be answered
satisfactorily before you set out to write the report. The answers depend on the usefulness of
the information to the reader and his interest in the subject, the details of the work carried out,
and the recommendations and suggestions you intend making and their implications.
There is no neat formula for the organization of technical reports. Each report must be
organized to fit its own subject, its own purpose, its own audience. But a few general principles
apply to most technical communications.
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Logical Progression toward conclusions: The material in any report should be presented in an
order that leads logically towards a conclusion or conclusions. This doesn’t mean, of course,
that everything in lengthy report will aim at one final climax; the various sections of the report
are organized so that each of them has its logical conclusions.
The three Parts: Almost every technical communication should have three functional elements.
This does not mean that it should be divided by boundaries into three distinct parts. But
functionally it should have a beginning, middle and an end.
The beginning orients the reader and supplies him with background material, so that he will see
how the subject of the paper fits into the general scheme of things. It prepares the reader for
the main presentation of information-the middle. The beginning is often called Introduction,
which states the purpose of the investigation and describes the basic scheme of the procedure
or methods used. It orients the reader by supplying as much historical background as necessary
and then describing the present problem. It may define the scope of the study, discussing
limitations or qualifications.
The middle is usually the longest part of the report. It can be organized in many different ways:
– It tells what you did. (Description)
– It tells what you found out. (Results)
– It analyzes, interprets and discusses these results. (Discussion)
The end is sometimes labeled conclusions. It brings together the various subjects that have
been discussed and shows their relationships with each other and with broader fields. This end
section makes the exposition come to a logical and an obvious termination, rather than simply
stop a note of detail. It ties a string around the bundle.
Skills in Technical Writing
Successful communication depends upon the correct use of language and a good style of
writing. One may learn the correct use of language, but has to cultivate a good style of writing.
The former concerns grammar, usage, spelling, capitalizations and punctuation, the latter
concerns the organization of ideas through proper choice of words, arrangement of words into
sentences, grouping of sentences into paragraphs, sections and chapters. The use of
abbreviations, your approach to the reader, your idiom, use of visual aids, the format and
layout of the report are all aspects of style.
Choice of words
The primary objective of Technical Writing is to transmit information briefly, clearly and
efficiently. This can be achieved only through simple, direct and unadorned style. The first step
towards a simple and clear style is to use simple language. One must choose a short word
rather than a long word, a plain and familiar word rather than a fancy or unusual word and a
concrete word rather than an abstract word.
The Short Word: The agreement was effected.
The agreement was made.
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The Plain Word or Familiar Word:
Everybody working near these tubes should be cognizant of the danger of explosion.
Everybody working near these tubes should be aware of the
danger of explosion.
The Concrete Word: Concrete nouns name objects or things that can be perceived by the
senses. Abstract nouns name qualities, ideas or conditions that are conceptions of mind.
Abstract nouns tend to be general and vague. As a result, expressions that contain abstract
nouns are less forceful, less direct, and less exact than their concrete counterparts.
Production engineers have found direct control of this operation to be a necessity.
Production engineers have found that this operation must be
directly controlled.
Verbosity (Wordiness)
For simple, clear style, eliminate from your writing every word that does not contribute to the
meaning or clarity of your message. Long-winded phrases should be avoided. Don’t use words
that add nothing. Don’t write “because of the fact that”, if simple “because” will suffice. On the
other hand, don’t eliminate so many words that the writing reads like a telegram. If a word
adds anything worthwhile to your sentence - meaning, grace rhythm, emphasis - let it remain.
Remove it if you don’t miss any of these.
• It is very correct that there are three unfilled vacancies in the directorate of the
company. (Omit)
• It should be noted that the factory will be closed on 31st May. (Omit)
Jargon
Jargon encompasses all technical terms. Such terminology is useful and often necessary in
technical communication restricted to people working on the same or similar subjects.
Technical terms become jargon only when carelessly used for wider audience. Jargon is a
special language of a particular field or profession. We can’t expect lawyers to say habeas
corpus in English just because the rest of us don’t understand. The Jargon of any given field is
often the most efficient means of communication within that field. It becomes offensive when
handy English equivalents are available or people outside the field are expected to understand,
what is said.
The Verb ‘Be’
The verb ‘be’ is often a cause of stylistic problems. Eight basic forms of verb ‘be’ are: am, are, is,
was, were, be, being, been. Avoid verb ‘be’ followed by adjectives or nouns that can be
turned into strong, economical verbs.
e.g. The new policy is violative of the Civil Right Act.
The new policy violates the Civil Right Act.
The Passive Voice
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In the passive voice, the subject is the receiver of an action rather than the doer of it. Passive
voice is employed by writers when they want to evade or conceal the responsibility for
someone’s behaviour. e.g.
I regret to inform you that your application has been rejected.
As the passive voice is sometimes vague and less economical than the active voice, good writers
tend to avoid it except when it is genuinely useful. The passive voice may be preferable, for
example, when the real doer of an action is either unknown or, in the context of a discussion,
relatively important.
Faulty Parallelism
In written English, word and phrases joined by ‘and’ are normally similar both in form and its
meaning. Violations of this convention are called “Faulty Parallelism”
My hobbies are hunting, fishing and to write.
My hobbies are hunting, fishing and writing.
Subordination
A common failing of technical writers is the expression of ideas of unequal importance in
constructions that seem to give equal weight. Meaning can be grasped more quickly and more
easily if subordinate ideas are indicated and put in subordinating constructions. A sentence
should express the main thought in a principal clause. Less important thoughts should be
expressed in subordinate clauses.
This machine has been imported from Japan and it is easy to operate.
This machine, which has been imported from Japan, is easy to operate.
Conclusions
Scientific and Technical Writing is objective in content and systematic in form. The primary
objective of Technical Writing is to transmit information briefly, clearly and efficiently. It is
always precise, exact, and to the point so that it may have the desired effect on the reader and
lead to the required action. This could only be achieved through simple, direct and plain style
using simple language. Every written communication has a specific purpose and a specific
audience. It should be carefully planned and constructed keeping the reader in mind.
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Novel Health Promoting Poly-functional Bioactive Peptide from Bovine Milk Fermented with Lactobacillus helveticus
Bhagat Singh*, Chand Ram**and Renu Singh*
Microbiology, Institute of Applied Medicines and Research, Duhai, Ghaziabad Utter Pradesh,
** Senior Scientist, Dairy Microbiology Division, National Dairy Research Institute, Karnal
Introduction
India has over one billion populations. Health care is essential for each age group as well for
rich and poor. Government’s National Health Policy, 1983 aimed at “Health for all by 2000” has
not helped the growth of the health and health care sector. The liberalization policy of
government of India in 1990’s started attracting private initiatives in the health care sectors.
The initiative taken by the private sector during this period has led to a steady growth of this
sector, because of large-scale investments and forays into the activities of health care. The
secondary and tertiary health care activities also need equal attention both in health and health
care system. The integrated efforts will help the health and health care system to become the
next revolution in India after telecommunica-tion, internet and biotechnology.
Milk is nearly a perfect food and is known to exhibit a range of biological activities those
influence digestive functions, development of specific organs and also resistance towards
diseases. Casein, one of the major constituents of milk. Not only provides amino acids and
nitrogen to the young mammals but also constitute an important part of dietary components
for adults. Thus intact milk protein has specific functions and physiological importance such as
its role in uptake of trace elements and vitamins. This protein in their native form is fragmented
into smaller peptides, which in turn exhibit biological activities in different physiological
systems. The peptides formed in the digestive system are limited only to 32mg/Kg of milk
intake; only a small fraction of these have some specific functional effects. With the help of
microorganisms especially lactic acid bacteria (LAB), the number of such bioactive peptides can
be increased many folds by the proteolytic action of enzymes produced by these organisms.
Milk fermented with LAB is enriched with a high level of peptides than original milk the actual
figure depends upon the proteolytic potential of the strain used. Such peptides have different
functions in vitro as well as in vivo for example anti-hypertensive, anti-microbial, anti-oxidative,
anti-thrombotic, mineral–carrying and opioid etc. If we look at the work done during the last
decade, main stress has been laid on the identification and characterization of bioactive
peptides exhibiting these unique properties individually. It has been reported that there are
certain regions in the primary structure of casein those contain overlapping peptide sequence
possessing bi- or tri- functional physiological effects. These regions are known as ‘strategic
zones’ and are partially protected from proteolytic enzymes, owing to their higher proline
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content. However reports on detailed molecular characterization of such peptides exhibiting
multifunctional nature are very scanty. Recently, there has been an increasing commercial
interest in the production of poly-functional bioactive peptides for the purpose of using them
as active ingredients of food for health promotion. This concept has resulted in to the
emergence of potential functional food market with lots of commercial stake
Milk being a source of well-balanced nutrients, shows a range of biological activities;
influencing digestive functions metabolic responses to absorbed nutrients, growth and
development of specific organs and disease resistance. Much of the biological activity in milk is
due to peptides and proteins secreted into milk in active form by the mammary gland,
examples being epidermal growth factor, transforming growth factor, nerve growth factor,
insulin and insulin-like growth factors I & II. However, some of the biological activity is latent in
that it is only related by proteolytic action, whether this occur during digestion in the gut or
during the fermentation and processing of milk. It is due to these latent biological activities and
their effects on metabolism that this research is concerned. A large number of potential
biological activities are encoded in the primary structures of milk protons, the picture is farther
complicated by the time being that many milk-derived peptides show multifunctional features
in that a specific peptide sequence can exert different biological effects.
Hypertension is major risk factor for cardiovascular disease, such as coronary heart disease,
congestive heart failure and stroke, by lowering high blood pressure with antihypertensive
treatment; the incidence and severity of these complications can be decreased. In addition to
pharmacological treatment, changes in life style factor have beneficial effects in the treatment
of high blood pressure and its complications. These factors may also have a favourable role in
prevention of hypertension. Non-pharmacological treatment of hypertension includes
diminished use of salt (NaCl) and alcohol, and decreased over weight. Increased in take of
potassium, magnesium and calcium may also be advantageous. Recent recommendations for
prevention and treatment of hypertension, by the Sixth Joint National Committee, report on
detection, evaluation and diagnosis of high blood pressure, recommendations by World Health
Organisation and the International Society of Hypertension as well as the Finnish Hypertension
Society, emphasize the role of non- pharmacological therapy, which should also be considered
the foundation for treating hypertension patients receiving anti hypertension medication.
There is now a common understanding that apart from nutritional value the proteins possess
also biological and physico-chemical properties. For example, milk is known to contain a wide
range of proteins, which either provides the protection against enteropathogens or is essential
for the manufacture and characteristics of certain dairy products. Research carried out during
last two decade show that both major milk protein groups, kappa casein and whey protein can
also be an important source of biologically active peptides. These peptides are in an inactive
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state inside the protein molecule and can be released during (in vitro or in vivo) enzymatic
digestion.
Protein and Blood Pressure
Milk proteins are divided in to casein and whey protein. Casein, which comprises approximately
80% of total protein content in bovine milk, is in turn divided in to alpha, beta, and kappa
casein. Major whey proteins -lactalbumin and -lactoglobulin, account for 2-5% and 7-12% of
the total protein in bovine milk respectively.
A few investigations trials evaluating the effect of dietary proteins on blood pressure have been
performed. Recently an eight week randomized controlled trial was performed in mildly
hypertensive men and women (n =36) aged at least 20 years, who received antihypertensive
medication. When compared to low- protein diet (12.5% of energy), a diet supplemented with
soy protein (protein intake 255 of energy) lowered Systolic Blood Pressure (SBP) by 5.9mm Hg
and DBP by 2.6mm Hg. The Multiple Risk Factor Intervention Trials (MRFIT) conducted in the
USA with over 11,000 middle- aged men at high risk of coronary heart disease found that at
high intake of total protein was inversely associated with DBP.
Formation of Bioactive Peptides by Microbial Fermentation:
Peptides with biological activity can be produced from milk proteins in three wings
(a) Enzymatic hydrolysis with digestive enzymes,
(b) Fermentation of milk with proteolysis starter cultures and
(c) Through the action of enzymes derived from proteolysis microorganism
During controlled fermentation of milk with certain dairy starters, peptides with various
bioactivities can be formed and are detected as an active form even in the final products, such
as fermented milks and cheese.
Milk Protein Derived Peptides and Blood Pressure
The first peptide having opioid-like activity from milk protein was discovered in 1979. Other
properties of milk-derived peptide include Angiotensin Converting Enzyme (ACE) inhibitory
activity as well as mineral binding, anti-thrombotic activity, anti-microbial activity and
immunomodulatory activity. The cardio-vascular effects of milk protein derived peptides have
not been extensively studied to date, but along with other component of milk, they appear to
have beneficial effects on blood pressure.
The release by microbial fermentation of various bioactive peptides from both caseins and
whey proteins has been reported in many studies. Nakamura in1995 identified two angiotensin
converting enzyme (ACE) inhibitory peptides (Val-Pro-Pro, Ilu-Pro-Pro) in milk, which was
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fermented with a starter culture of Lactobacillus helveticus and Saccharomyces cerevisiae. This
enzyme plays a crucial role in the regulation of blood pressure in mammals Master et al., in
1996 detected immunstimulatory properties in milk fermented with a Lactobacillus helveticus
strain. Yamamoto et al, in 1990 identified an ACE inhibitory di-peptide (Try-Pro) from a yoghurt-
like product fermented by Lactobacillus helveticus CPN4 strain. This peptide sequence is
present in all major casein fractions, the concentration of Try-Pro peptide increased during
fermentation and reached about 8.1 /g of whey in the yoghurt-like product. In humans, the
antihypertensive effect of milk protein- derived peptide has yet to be demonstrated. However,
some studies have investigated the effect of fermented milk products containing IPP and VPP
on blood pressure. In a small controlled randomized clinical trial, daily intake of a fermented
milk product (Calpis) for eight weeks (95ml/d) lowered blood pressure in mildly hypertensive
patients (n=30).
Ability of Milk-Peptides to Modulate Immune Function
The systems involved in the body's defence are very varied and complex. Diet plays an
important part in this, particularly fermented dairy products as a result of their protein content
and the activity of live ferments. The investigation of the role of functional peptides in this field
is a fairly recent and very promising line of research.
Biopeptides exhibiting an i S1- - - caseins are
S1-casokinins, -casokinins and glycomacropeptide, respectively. Peptides of a similar nature
are also obtained from the whey protein, -lactalbumin. These peptides have been shown to
stimulate the phagocytic activities of murine and human macrophages, and enhance resistance
against certain bacteria. They also stimulate the proliferation and maturation of immune
system cells, such as T-cells and B-cells.
Low temperature processed whey protein containing a high concentration of specific di-
peptides (glutamyl cysteine) has been found to promote the synthesis of glutathione, an
important anti-oxidant involved with cellular protection and repair. Consumption of Yoghurt
has been associated with a reduced incidence of colon cancer.
The structure-activity relationship and the mechanism by which milk-protein derived peptides
exert their immunomodulatory effects is not yet defined. However, it is suggested that opioid
peptides may affect the immunoreactivity of lymphocytes via the opiate receptor. There is
indeed a remarkable relationship between the immune system and opioid peptides, because
-lymphocytes
and human phagocytic leukocytes. Furthermore, it is known that lymphocytes and
macrophages express receptors for many biologically active mediators. It has been suggested
that an arginine residue at the N- or C- terminal region may be the dominating entity
recognized by specific surface membrane receptors.
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Investigating the role of biologically active peptides is a very promising line of research. The
milk fermented with LAB release components that possess immunomodulatory activity was
investigated in several culture supernatants arising from LAB cultured in a medium
composed primarily of UF permeate etc. of bovine milk, only Lab. helveticus supernatant
allowed the modulation of lymphocytes proliferation in vitro on human peripheral blood
lymphocytes.
Over the last decade the effects of LAB on the immune system have been the topic of
extensive research. The immunomodulatory effect of many peptides has been demonstrated
in vitro as well as in vivo. The Try-Gly and Tyr Gly-Gly peptides, potentially derived from K-
casein and LsI ,
- La have shown to modulate the lymphokine production in vitro. Peptide
derived from sI and -casein and from L- lactalbumin enhance phagocytosis and modulate
proliferation and differentia-tion of lymphocytes. Opioid peptides also may affect the
immunoreactivity of lymphocytes via the opiate receptor. Opioid -receptors for endorphin
is known to be present on lymphocytes and human phagocytic leucocytes (Meisel, 1999).
The hydrolysate derived from sodium casinate through 30 min trypsinization yielded highest
quantity of immunoprotective proteins. Casein derived immunopeptides including fragments
of sI - casein (residues 194-199; Thr-Thr-Met-Pro- Leu- Trp ) and -casein (Residues 63-68;
Pro–Gly-Pro-Ile-Pro-Asn and 191-193; Leu-leu-Tyr) have been shown to stimulate
phagocytesis of sheep red blood cells by murine peritoneal macrophages, and to exert a
protective effect against Klebsiella pneumonia infection in mice after intravenous treatment.
The immunohexapeptide derived from -casein represents the C-terminal part of -
casomorphin-II. The immunopeptide sequence in human -casein corresponds to residues
54-59.
The C-terminal sequence 193-209 of -casein (containing -casokinin– 10) obtained from a
pepsin-chymosin digest of bovine casein induced a significant proliferate response in rat
lymphocytes. Kayser and Mesel in 1996 reported that the immunoreactivity of human
peripheral blood lymphocytes (PBL) was either stimulated or suppressed by various bioactive
peptides derived from milk proteins. The peptides Tyr-Gly and Tyr-Gly-Gly corresponding to
fragments of bovine -lactalbumin (eg. the N-terminal end of -lactorphin) and k-casein,
respectively, significantly enhanced the proliferation of PBL at concentrations ranging from
10-11 to 10-4 mol L-1. The peptide Tyr-Gly exhibited 93% of maximal stimulation at 10-12 mol L-
1. Depending in peptide concentration, -caskokinin–10 and -casomorphin–7 showed a
suppression as well as stimulation of lymphocytes proliferation of human colonic lamina
propria lymphocytes (LPL) where the anti-proliferative effect was reversed by the opiate
receptor antagonist naloxone.
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The activity of releasing components that possess immunomodulatory activity was
investigated in several culture supernatants arising from LAB cultured in a medium
composed primarily of UF permeate of bovine milk with -CN was added as the sole source.
Only a Lactobacillus helveticus supernatant allowed the modulations of lymphocyte
proliferation in vitro on human peripheral blood lymphocytes. With possible explanation for
mechanism of action, it is presumed that lymphocytes and macrophages express receptors
for many biologically active mediators. It bas been further suggested that an argenine
residue at the N-terminal region of peptides may be the dominating entity recognized by
specific surface membrane receptors.
Antioxidant Activity of Peptides
Lipid oxidation deteriorates the colour, flavour and qualities of food. Lipid oxidation turns into
biologically active compounds such as active radicals or low molecular carbonyl compounds,
which has relation with ageing. It is therefore essential to prevent the lipid oxidation for food
stability (Kim et al., 2002). It is well established that elevated levels of low density lipoprotein
(LDL) cholesterol are associated with increased risk of coronary heart disease (CHD). The
mechanism of the atherogenic effect of LDL has become 3-10 times more atherogenic
compared to native LDL. In LDL particle the unsaturated fatty acid in the cholesterol esters and
phospholipids are an important substrate for oxidation. Fat-soluble antioxidants, which are
transported in the plasma through LDL, protect acids from oxidation. There is considerable
experimental and clinical evidence for this theory and it is hypothesised that low antioxidant
levels may increase CHD risk through oxidation.
Zommara et al. 1994 proved that milk whey as well as fermented milk wheys is effective for
suppressing the elevation of lipid hydroperoxide induced by bile duct ligation. Rats fed on milk
whey and its fermentation product exhibited lower levels of mitochondria hydroperoxide as
compared with bile duct ligated rats fed on the control diets. An elevated serum hydroperoxide
was also suppressed in the rats fed on milk whey and its fermentation products.
The culture supernatant of L. acidophilus and Bifidobacterium adolesceins exhibited
antioxidative property that prevented lipid per oxidation. Likewise, Lin and Yen (1999) studied
reactive oxygen species and lipid per oxidation product-scavenging ability of yoghurt organism.
Terahara et al. 2000 confirmed that radical scavengers were produced in the culture of L.
delbrueckii subsp bulgaricus 2038 and administration of freeze-dried powder of this organism
prevented the oxidation of lipoprotein in rats. It was observed that the ability of peptides
released during milk fermentation to sequencing the lipid oxidation products and thus
protecting the lipid oxidation.
Anti-microbial Peptides
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With the recent restriction of antibiotic use in food producing animals, the greater concerns
about antibiotic resistant bacteria in both the animal and human population and the desire to
reduce food borne pathogen levels, an increasing need to develop effective but human and
animal compatible, antibiotic alternatives for the medical industry has arisen. Recently, natural
proteins have been identified that possess these attributes. In vitro, these proteins upon
degradation by digestive enzymes or microbial enzymes have been shown to release
antimicrobial peptides (AMPs), which stimulate endogenous synthesis of AMP by the animal
and induce immune responses favorable to bacterial removal. Milk proteins (e.g. Lactoferrin,
lactoferricin, and -lactalbumin) functions as a natural bacterial barrier for the pathogen-
susceptible neonate, embryo and animal cells or organs, respectively.
Some AMPs exhibit unique mechanism for killing bacteria compared with current antibiotics.
These AMPs selectively bind to the outer lipid membrane of the bacterium and form blisters
and pores, which eventually result in lyses of the cell and cellular death. AMPs also have the
ability to stimulate the production of II-I. The stimulation of IL-I would create an increase in
chemotaxis of the neutrophils to that area. These neutrophils contain AMPs produced from the
animal, which would serve as a secondary source of AMPs for the host.
Based on data it is hypothesized that the feeding of these natural proteins results in the
production of AMPs, which function as effecting antibiotics via the direct antimicrobial activity
of the peptides, and the peptides indirect enhancement of the immune response of the
animals. Because of their unique mechanism for killing bacteria, it is also believed that the
AMPs or their precursor may be effective in killing antibiotic-resistant, as well as antibiotic-
sensitive, bacteria.
Antimicrobial Peptides from Milk
Common feature of antimicrobial peptides is their net positive charge & properties for forming
highly ordered amphipathic conformation, such as helices or -sheets upon interaction with
the negatively charged phospho-lipids of the bacterial cell membrane.
The first antimicrobial peptides have been derived from the whey protein lactoferrin. The
peptide derived from casein also have antimicrobial activity Casocidin released by chymosin
digestion of casein at neutral pH, was the first defensive peptide actually purified and exhibited
activity in vitro against Staphylococcus aurius, Serracia marcecens, Bacillus subtilis Diplococcus
pneumonniae & Streptococcus pyogens.
Casocidin I, a cationic s2 casein derived peptide inhibited growth of E. coli and Staphylococcus,
Isracidin, a N- terminal segment of -s1 – casein has been reported to protect mice against
Staphylococcus aureus and Candida albicans. This peptide also safe guards sheep and cow
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against mastitis Dionysisus and Milne 1997 have identified two peptides from the N- terminal
of lactoferrin which displayed antimicrobial activity towards a number of pathogenic and food
spoilage micro-organisms. A potent bactericidal peptide specifically generated by pepsin
degradation of lactoferrin, so named lactoferricin-B, also displayed antimicrobial activity
towards Gram-positive and Gram- negative microorganisms. These properties appear to be
correlated with the net positive charge of the peptide, which may kill susceptible
microorganism by increasing cell membrane permeability.
Opioid Effects of Bioactive Peptides
Exorphins or formons (Food hormone) have pharmacological properties similar to opium
(morphine) and exert naloxone – inhibitory activities. The absorption and degradation of
natural -casomorphine and their analogues has been intensively studied. -casomorphines are
resistant to enzymes of the gastrointestinal tract and have been detected in vivo in and human
small intestines. Opioid receptors (, and K. type) are widely distributed, being found in the
nervous, endocrine and immune systems as well as in the gut. These receptors interact with
endogenous legends as well as with exogenous opioid agonists and antagonists.
Three peptides derived from –casein, corresponding to bovine s1–casein fragments 90-96
(Arg-Tyr-Leu-Gly-Tyr-Teu-Glu), 90-95 and 91-96 have been identified as -opioid receptor
legends (Lou Kas et al., 1990). Other opioid derived from milk proteins induce -Lactorphin
(Tyr-Gly-Leu-Phe-NH2) and - lactorphin (Tyr-Leu-Phe-NH2) from - lactalbumin and -
lactoglobulin respectively (Chiba, et al., 1986). In addition to these opioid agonists, opioid
antagonists have also been identified in peptide sequences in bovine and human -casein
(casoxius) and in s1-cascin. The casoxins are opioid receptor legate of the -type but they are
of relatively low potency compared with the opioid antagonist, naloxone. Most of the peptides
have common structural feature containing N-terminal tyrosin residue. This is absolutely
essential for activity.
Lactobacillus helveticus Amino Peptidase
Cell surface bound amino peptidase from Lactobacillus helveticus LHE-511 was purified and
characterized by Hiroshi in1990. The enzyme was found to have a monomeric structure and a
molecular mass of 92 KD. The optimal pH and temperature for activity were 7.0 and 370C
respectively. The enzyme was strongly activated by CO2+, completely inhibited by EDTA and
1,10-phenantroline, and weakly inhibited by p-chloromercuribenzoate, suggesting that it is a
metalloenzyme possessing a thiol group at its active site. The enzyme showed its high activity
with p-nitro aniline derivatives or di-peptides and tri-peptides that have a hydrophobic amino
acid (leu, Ala or Phe) or di-amino mono carboxylic acid (Lys or Ary) at N Terminus.
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Fermented milk prepared by Lactobacillus helveticus showed significant antihypertensive effect
in spontaneously hypertensive rats (SHR) while other species of lactic acid bacteria did not
show so significant antihypertensive effects. Most of the why fractions of the milk fermented
by L. helveticus or Lacto delbrueckii subsp bulgaricus showed higher angiotensin 1-converting
enzyme inhibitory activity than the activity of milk fermented by other species. Proteolytic
activity in cell wall and peptide content of the milk fermented by L. helveticus strains were
higher than other species. The cell density of milk formed by L. helveticus was also higher than
the milk fermented by other strains and the pH was lower than the other.
References:
Kim, S. M., Shin, I. S. and Kim, W. J. 2002. Faculty of Marine Bioscience and Technology, Kangnung
National University, 123 Jibyundong, Kangnung, Kangwondo, 210-702
Meisel, H. 1986. Chemical characterization and opioid activity of an exorphin isolated from in vivo
digestion to casein. FEBS Lett. 196: 223-227.
Meisel, H. 1997. Biochemical properties of bioactive peptides derived from milk proteins: Potential
nutraceutical for food and pharmacological applications. Livestock Production Science. 50:125-138.
Meisel, H. and Frister, H. 1989. Chemical characterization of bioactive peptides from in-vivo digestion
of casein. J.Dairy Res. 56: 343-349.
Meisel, H. and Bockemann, W. (1999), Bioactive Peptide Encrypted in Milk Proteins: Proteolytic
Activation and Thropho-functional Properties. Antonie von Leeuwenhoek. 76: 207-215.
Reed D, McGee D, Yano K, Hankin J. Diet, blood pressure, and multicollinearity.
Rodgers A, Ni Mhurchu C, Clark T. 1999 World Health Organization-ternational
Vijayalakshmi, A., Tandon, H. K. L. and S. M. Dutta 2000. Immunoenhancing effect of bioactive
peptides from milk. Indian J. Dairy Sci. 54 (1): 14-19.
Wong D. W. S, Camirand W. M, Pavlath A. E. Structures and functionalities of milk proteins. Crit Rev Food
Sci 2000; 36: 807-844.
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Gene Expression Microarrays in Livestock Genomics
M Mukesh and Monika Sodhi
National Bureau of Animal Genetic Resources, Karnal-132001
In last decade or so DNA microarrays has revolutionized the study of gene expression and has
given rise to an unprecedented increase in the rate of data acquisition in identifying gene
transcript regulation at whole genome level. Microarrays have now been evolved as a very
powerful technology that enables large numbers of genes, up to the order of tens of thousands,
to be evaluated simultaneously. The objective of a microarray experiment might be to
investigate genes which are differentially up or down regulated in cells between, say, a control
group and cells which have undergone some treatment, or between cells of animals of different
genetic background {e.g., control mice compared to knockout mice) or between cells in healthy
tissue and diseased tissues, or between cells at different time points {e.g., developmental
biology).
Numerous studies have been published addressing the critical issues of microarray
experimental design, data analyses, and application of microarray technology to investigate
normal physiology and disease pathogenesis. The method is based on the phenomenon of
preferential complementary base pairing, known as hybridization, and produces its signal by
parallel hybridization of labeled targets to specific probes that have been immobilized on a solid
surface in an ordered array. Thus, DNA microarrays are an orderly array of ‘‘target’’ DNA
material immobilized onto a substrate, normally a coated glass microscope slide in a precise,
well-known pattern. Each probe corresponds to either a complete transcript or to part of a
transcribed sequence which is tethered onto the array and the target is a labelled pool of DNA
that is complementary to mRNA. There are two principle DNA microarray methods based
upon the nature of the ‘‘target’’ arrayed DNA material (cDNA or oligonucleotide microarrays)
and method of spotting DNA (mechanical microspotting or photolithography). The number of
‘‘target’’ genes that make up an array can range from a small number of specific well-
characterized genes to a pool of thousands of genes that may comprise entire genomes. For
certain model organisms including Arabidopsis, yeast, mouse, and human, both cDNA and
oligonucleotide arrays are commercially available and are suited to medical diagnostics and
drug discovery applications. Whole genome arrays allow researchers to monitor expression of
all genes simultaneously taking advantage of the full power of microarray experimentation. For
many non-model organisms used in physiological studies, custom arrays can be constructed
from a number of different ‘‘target’’ DNA sources including: cDNAs clones obtained from
normalized libraries, ESTs, oligonucleotides, genomic clones or genomic DNA. Obtaining this
‘‘target’’ DNA material remains a costly barrier to employing microarray technology for a large
number of non-model physiologically interesting organisms. These days, oligo arrays and whole
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genome arrays have superseded the cDNA arrays in terms of quality, reliability and spot
uniformity and avoid some of the technical pitfalls of cDNA arrays. There are various types of
microarray platforms that are commercially available for different species. Arrays can be tissue
specific (mammary, macrophage specific) or whole genome (representing all genes expressed
in an organism).Two of the major requirements of any microarray platform are system
reproducibility, which provides the means for high confidence experiments and accurate
comparison across multiple samples; and high sensitivity to detect even small fold changes
across multiple gene sets. Agilent whole genome bovine 44K chip harboring 60 mer oligos is
one such very popular platform for detecting accurate differential expression. The oligos
representing transcripts/genes are physically spotted or printed onto a solid surface. Bovine
whole genome platforms from Affymatrix are coming with shorter oligos (25-35 mer) built by
photolithographic masks. Microarray platforms from Illumina are also available for bovine and
other species. The bead chip from Illumina consist of 50 mer oligos attached to beads
randomly. Generally the cost of spotted arrays is lower than that of Affy- or Illumine arrays.
Usually, microarrays allow for the direct comparison of expression patterns of all the
‘‘target’’ genes spotted on an array between samples taken under two conditions or
treatments. Different fluorophores are used to label cDNA prepared from either total RNA or
messenger RNA, typically representing control and experimental conditions. The most common
dyes for microarray studies, Cy3 and Cy5. The fluorescently labeled cDNAs are mixed, and this
‘‘probe’’ is hybridized to ‘‘target’’ DNA samples on the array, where labeled messenger
sequences will quantitatively anneal to ‘‘target’’ DNA sequences. However, the two dyes have
non-linear sample labeling and hybridization kinetics, which means that they do not provide
equal sensitivity across the whole range of transcripts in a sample. More specifically, they have
differential labeling and scanning efficiencies and also exhibit gene-specific bias. To combat
this, the roles of the dyes are often exchanged and the procedures of hybridization and
scanning repeated, known as a dye-swap. Taking a suitable average of both dye-swap pair
ratios removes dye-bias, giving more reliable results. If a dye-swap has not been performed,
gene-specific dye-bias cannot easily be removed. The contribution and cause of gene-specific
dye-bias to the underlying variation has not been properly characterized however there has
been recent research in this area aimed at modeling this effect.
In general, between and within slide replication, as well as the use of well-characterized
control genes are used to ensure accuracy. Automated processes calculate a relative measure
of gene expression within the two samples for each of the ‘‘target’’ DNA samples present on
the array. The overall expression pattern of all genes collectively is known as an ‘‘expression
profile’’. Genes that are upregulated or downregulated can easily be identified.
Earlier microarray technology in mammals has been limited primarily to mice and
humans. In contrast, there had been a substantial delay in the application of microarray
technologies in the area of functional genomics to the investigate the biological questions in
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species of veterinary importance like cattle, sheep, goat swine and poultry. Only recently, a
variety of commercial microarrays are now available for different livestock species to
characterize specific cell signaling pathways or biological functions as routine tools to address
hypotheses in basic research and clinical trials. Before the development of microarrays in
livestock species, some groups used heterologous human microarrays as cross-species
hybridization studies. With recent developments in sequencing of genome for different
livestock species, the availability of species specific microarray platform has enabled the
researcher to utilize this powerful technology to discover genes and address a variety of
questions relating to normal physiological processes, such as cell differentiation, pregnancy,
lactation, and parturition in different livestock species. Very recently, results from global
transcriptomics studies have started unfolding critical aspects in bovine health, normal
physiology or pathology.
Several microarray based attempts were made to understand the host-pathogen
interaction in animal species to better understand the immune functions and regulation of
genes controlling immunity trait (Wilson et al., 2005; Jiang et al., 2008). Also substantial
progress has been made in understanding the physiology and tissue (mammary gland, liver)
genomic responses of high producing Holstein Frisian cattle during the stressful periparturient
stage of animal (3 week before and 3 week after calving), infectious disease like mastitis and
metabolic disorder like ketosis (Loor et al 2007; Moyes et al., 2010). Using bovine microarray
chip, Loor et al (2007) highlighted the changes in key metabolic and signaling network
signatures during nutrition induced ketosis and liver lipidosis in peripaturient dairy cows. In
their study, several genes playing key roles in hepatic metabolism adaptations to negative
energy balance and changing physiological state near time of parturition were identified.
Some insights into bovine muscle biology (beef biology) have been obtained by cattle muscle profiling utilizing microarray studies. Byrne et al., (2005) undertook gene expression profiling of muscle tissue in Brahmen steers to understand the processes associated with remodeling of muscle tissue in response to nutritional stress. Gene expression profiling was also conducted in different muscle types to better understand the muscle characteristics which determine meat quality traits across muscles, and is a major factor of variability of meat tenderness. Australian and Japneese scientists undertook a microarray-based comparision of the longissimus muscle (LM) from Japanese black and Holstein cattle over an extended intensive feeding period to identify genes that may be involved in determining the unique ability of Japanese black cattle to deposit intramuscular fat with lower melting temperature (Wang et al., 2005). Other transcriptomic studies of bovine muscle were reported to identify some markers of meat tenderness and insight into muscle growth in cattle (Sudre et al., 2005; Reecy et al., 2006). Gene expression profiles were compared in Charolias bulls between high and low meat quality scores of tenderness, flavour and juices. Out of several differentially expressed genes, 14 of them were highly correlated with flavour and juices and one of them (DNAJA1) had a strong negative correlation with tenderness (Bernard et al., 2007).
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Microarray technology has also been extensively used to unravel key insights of reproductive biology in different livestock species. Caetano et al. (2004) identified differentially expressed genes in ovaries and ovarian follicles of pigs selected for increased ovulation rate to seek new insights into ovarian physiology and the quantitative genetic control of reproduction in swine. Ushizawa et al. (2004) undertook cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period to identify genes involved in embryonic development. Recently, Hayashi et al. (2010) carried out differential genome-wide gene expression profiling of bovine largest and second-largest follicles to identify genes associated with growth of dominant follicles. With a goal to better understand bovine mammary gland biology, Suchyta et al., (2004) compared the gene expression profiles of lactating bovine mammary gland against non-lactating tissue on a bovine microarray chip that yielded many novel and interesting genes expressed specifically in lactating mammary tissue. One of the long-term objectives in area of mammary gland biology of lactating dairy animal is to identify all genes responsible for lactation and to understand the underlying genomic and physiological adaptations occurring in the mammary tissue of dairy animal. To understand the complexity that underlies mammary gland development and function, microarray expression data may provide insight into the mechanisms that ultimately allow mammary gland to function in a coordinated fashion throughout puberty, pregnancy, lactation, and involution. Initiatives like elucidating the signaling mechanisms underlying the functional development of mammary gland and regulation of milk fat/protein synthesis through out the lactation cycle by generating whole genome expression pattern coupled with metabolic/hormonal pathways has become high priority area of research in animal genomics that can yield a wealth of information on as yet unknown molecular adaptations in response to physiological stage of the animal. Such inputs can relate functional development of mammary gland of dairy animals with coordinated changes in the global expression pattern to understand the basic biology of mammary gland development that is far from complete. Microarrays have also become a standard tool of any modern microbiology laboratory to generate genowide data set. The longer term goals of functional genomics and microarray technology in infectious diseases include describing the host-pathogen interaction and identifying critical target molecules and pathways for diagnosis and intervention. Microarrays promise to accelerate our understanding of the host side of the host-pathogen interaction. The few published studies represent what is certain to be the beginning of a deluge of genome-scale pathogen data. At Stanford University alone, microarray-based studies of Bordetella pertussis, Salmonella, H. pylori, Campylobacter jejuni, V. cholerae, M. tuberculosis, and E. coli, as well as the nonpathogenic microbes Streptomyces coelicolor and C. crescentus, are under way. Microarray based whole genome transcriptome analyses is also contributing to our understanding of bacterial behaviour in the environment and pathogenesis in the host. While transcriptomics have not been used frequently to investigate the response of bacteria in milk, this approach has been used to examine the growth and stress responses of bacteria under conditions relevant to the production, treatment, and storage of milk The continuous development of bioinformatics approaches for improved array annotation combined with new data analysis tools that enable cross-species comparisons will
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greatly enhance the extraction of biological information from species specific microarrays and advance our understanding of livestock biology. From the economic point of view, the importance and impact of genome wide tools in modern dairy sector is likely to increase in coming years. Over the longer term, this high-throughput technology would reshape the livestock biology in terms of functional annotation and discovery of new gene regulating trait of economic importance, complete description and understanding of cellular pathways (e.g., metabolism, proliferation, cell-cell interaction), understanding genomic-environment interaction (e.g., developmental pathways, abiotic stress, nutritional genomics and infectious diseases).
References
Bernard C et al. (2007). New indicators of beef sensory quality revealed by expression of specific genes. J Agric Food Chem. 55:5229–5237
Byrne KA, Wang Y H, Lehnert S A, Harper G S, McWilliam S M, Bruce H L and Reverter A (2005). Gene expression profiling of muscle tissue in Brahman steers during nutritional restriction. J. Anim. Sci. 83:1-12
Caetano AR, Johnson RK, Ford JJ, Pomp D. (2004). Microarray profiling for differential gene expression in ovaries and ovarian follicles of pigs selected for increased ovulation rate. Genetics, 168: 1529-1537
Hayashi KG, Ushizawa K, Hosoe M and Takahashi T. (2010). Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles. Reproductive Biology and Endocrinology, 8:11
Jiang L, Sorensen P, Rontved C, Vels L and Ingvartsen KL. (2008). Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide, BMC Genomics 9, p. 443
Loor JJ, Everts RE, Bionaz M, Dann HM, Morin DE, Oliveira R, Rodriguez-Zas SL, Drackley JK, and Lewin HA (2007). Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows. Physiol. Genomics 32: 105-116
Moyes K M, Drackley J K, Morin DE, Rodriguez-Zas SL, Everts R E, Lewin HA, and Loor JJ. (2010). Mammary gene expression profiles during an intramammary challenge reveal potential mechanisms linking negative energy balance with impaired immune response. Physiol Genomics, 41(2): 161 – 170
Reecy J M, Moody SD and CH Stah (2006). Gene expression profiling: Insights into skeletal muscle growth and development. Journal of Animal Sciences, 84:E150-E154
Suchyta SP, Sipkovsky S, Halgren RG, Kruska R, Elftman M, Weber-Nielsen M, Vandehaar MJ, Xiao L, Tempelman RJ, Coussens PM (2003) Bovine mammary gene expression profiling using a cDNA microarray enhanced for mammary-specific transcripts. Physiol Genomics, 16:8–18
Sudre K, Cassar-Malek I, Listrat A, Ueda Y, Loroux C, Jurie C, Auffrag C, Renand G, Martin P, and Hocquette JF. (2005). Biochemical and transcriptomic analyses of two bovine skeletal muscles in Charolais bulls divergently selected for muscle growth. Meat Sci. 70:267–277
Ushizawa K, Herath CB, Kaneyama K, Shiojima S, Hirasawa A, Takahashi T, Imai K, Ochiai K, Tokunaga T, Tsunoda Y, Tsujimoto G, Hashizume K. (2004). cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period. Reprod Biol Endocrinol. 2, 77
Wang, YH, Byrne KA, Reverter A, Harper GS, Taniguchi M, McWilliam S M, Mannen H, Oyama K, and Lehnert S A. (2005). Transcriptional profiling of skeletal muscle tissue from two breeds of cattle. Mamm. Genome, 16:201–210
04/04/2011
1
EVALUATION METHODS FOR QUALITY
OF MILK AND DAIRY PRODUCTS
Prof. Purshotam Kaushik Department of Botany & Microbiology
Gurukul Kangri University Hardwar, U.K.
INDIA Webpage: purshotam.kaushik.googlepages.com
Production of Quality Milk and Its Products
� Fresh and normal milk from healthy, properly fed and milked
animals, is a white, opaque liquid with a slightly sweet taste which has practically no odor (Le Jaouen, 1987).
� Production of quality milk should start at every farm level, because flavor and quality of the milk cannot be improved later in the processing stage (Park and Guo, 2006).
� The basic principle is that the better the milk, the better the processed products (Peters, 2000; Park and Guo, 2006).
� Good management of the entire farm system leads to good quality milk. The recommended milking procedure has to be practiced in a daily routine, maintain functioning and sanitary equipment, have healthy animals, and use recommended detergent, acid and sanitizers for cleaning and milking equipment.
Production of Quality Milk and Its Products
� Milk quality is negatively affected by improper handling from many factors such as feeding, handling of animals prior and during milking, handling of the milk during and after milking, cooling and transportation, pasteurization, processing, packaging, and processing utensils (Peters, 1990; Haenlein, 1992).
� Off-flavor in goat milk can be attributed to the feeds, weeds, forages, chemicals, building materials, colostrum, estrus, mastitic milk, filthy utensils and strainer, unclean milking equipment, slow cooling, odors from bucks, barn and/or milk room.
Five major parameters are routinely checked by regulatory agencies for
quality raw milk production
1. Nutritional constituents in milk.
2. Somatic cell counts as related to mastitis.
3. Bacteria counts as related to sanitary practices.
4. Adulteration and pesticide residue contents.
5. Flavor, taste, appearance and temperature.
Quality of Raw Milk tested by Individual Dairy Processing Plants
1. Standard plate count (SPC)
2. Direct microscopic count (DMC)
3. Freezing point determination (Cryoscope)
4. Presence of inhibitory substances (antibiotic screening test)
5. Sensory evaluation
6. Preliminary incubation (PI) – SPC
7. Direct microscopic somatic cell count (DMSCC)
8. Acid degree value (ADV)
9. Laboratory pasteurization count (LPC)
10. Thermoduric spore count
11. Fat content
12. Total solids content (can also include protein content)
13. Sediment test
3M Petrifilm Plate Techniques
04/04/2011
2
Total Aerobic Plate Count E. coli and Coliform counts
Yeast and Mold counts Staphylococcus aureus count
Grade A raw milk for pasteurization
� Temperature: Cooled to 45oF (7oC) or less within two hours after milking, provided that the blend temperature after the first and subsequent milkings does not exceed 50oF (10oC).
� Bacterial limits: Individual producer milk not to exceed 100,000 per ml. prior to commingling with other producer milk. Not to exceed 300,000 per ml. as commingled milk prior to pasteurization.
� Antibiotics: Individual producer milk: No detectable zone with the Bacillus subtilies method or equivalent. Commingled milk: No detectable zone by the Sarcina lutea Cylinder Plate Method or equivalent.
� Somatic cell count: Individual producer milk. Not to exceed 1,500,000 per ml.
Grade A pasteurized milk and
milk products
� Temperature: Cooled to 45oF (7oC) or less and maintained thereat.
� Bacterial limits: 20,000 per ml.*
� Coliform: Not to exceed 10 per ml.: Provided that , in the case of bulk milk transport tank shipments, shall not exceed 100 per ml.
� Phosphatase: Less than 1 microgram per ml. by the Scharer Rapid Method or equivalent.
� Antibiotics: No detectable zone by the Sarcina lutea Cylinder Plate Method or equivalent.
04/04/2011
3
Terms for Milk Quality – Cont’d B. Measurement of acidity of milk: 1. Titratable Acidity:
a. It is determined by adding NaOH (0.1 N) solution to raise the pH of the milk to about 8.3.
b. One ml of the base equals 0.1% lactic acid.
c. %TA = ml 0.1 N NaOH x .009 x 100/gram of sample
2. SH (Soxhlet-Henkel) value:
a. It indicates how many ml of NaOH (25 mol/ml) are required to neutralize 100 ml of milk. One ml of 2% alcoholic phenolphthalein solution is added as indicator.
b. SH value of fresh milk ranges 6.4 – 7.0 c. SH value of raw milk <5.0 indicates mastitis. d. SH values of 8.0-9.0 gives sour taste, and
coagulate.
Minimum Pasteurization Temperature and Times __________________________________________________________________________________________________________________ Product Temperature Time _______________________________________________________________ 1. Milk 145oF (62.8oC) 30 minutes LTLT 161oF (71.7oC) 15 seconds STHT 191oF (88oC) 1 second UHT 194oF (89oC) 0.5 second 201oF (94oC) 0.1 second 204oF (96oC) 0.05 second 212oF (100oC) 0.01 second 2. Milk products of 150oF 30 minutes 10% fat or more 166oF 15 seconds or added sugar 191oF 1 second (half/half, cream, 194oF 0.5 second chocolate milk) 201oF 0.1 second 204oF 0.05 second 212oF 0.01 second 3. Eggnog and 155oF 30 minutes Frozen dessert 175oF 25 seconds Mixes 180oF 15 seconds
Quality Evaluation of Dairy Products/Cheeses
� Quality of dairy products are changed during manufacturing, refrigeration, distribution and storage.
� Qualities of all dairy products including cheeses are influenced by several parameters, such as chemical, microbiological, rheological and sensory scores of the products.
� Proteolysis and lipolysis are two primary processes in cheese ripening with a variety of chemical, physical, microbiological, textural, and rheological changes which occur under controlled environmental conditions.
� Studies showed that cheese quality is greatly influenced by levels of peptides, amino acids, and free fatty acids resulting from proteolysis and lipolysis.
3.5
4
4.5
5
5.5
6
0 2 4 8 16 24
pH
Aging Period (wk)
Plain soft
Pepper soft
Caciotta
Monterey Jack
Goat Cheddar
Cow Cheddar
Ripened CowCheddar
04/04/2011
4
CONCLUSIONS
1. The basic principle for production of
quality dairy products is the better the
original milk, the better the processed
products.
2. Milk is highly perishable, and its quality
is easily deteriorated by improper
handling of feeding, animals prior and
during milking, handling of the milk
during and after milking, cooling and
transportation, pasteurization, processing,
packaging, and processing utensils, etc.
CONCLUSIONS – Cont’d
3. Each processing plant should establish appropriate quality control systems for each point of manufacturing facilities.
4. All personnel involved (farm level, transport, dairy plants) in production, processing, distribution, and marketing of dairy products must follow the required regulations (PMO) enforced by appropriate regulatory agencies (e.g. FDA, APHA).
5. Four important requirements for Grade A
dairy products are: i) safe to drink, ii) good flavor, iii) relatively free from spoilage bacteria and somatic cells, and iv) composition.
THANK YOU!!
LIST OF PARTICIPANTS
ADVANCED COURSE IN FACULTY TRAINING IN DAIRY PROCESSING on
Advances in Processing and Quality Assurance of Dairy Foods (22nd March - 11th April, 2011)
1. Dr. Srinivasan, R Assistant Professor Dept. of Dairy and Food Microbiology, College of Dairy and Food Science Technology, Maharana Pratap University of Agriculture & Technology, Udaipur- 313 001 (Rajasthan) [email protected], 09950547446
5. Mr. K. Ramesh Department of Biotechnology Manonmaniam Sundaranar University, SPKCES Campus, Alwarkurichi, District- Tirunelveli PIN- 627 412 (TN) [email protected], 09943112125
2. Dr. P. Selvan Assistant Professor Livestock Research Station (TANUVAS) Kattupakkam- 603 203 (TN) [email protected], 09790813709
6. Dr. Surajit Mandal Scientist, Dairy Microbiology Division, National Dairy Research Institute, Karnal- 132 001 (Haryana) [email protected], 09991423316
3. Dr. (Mrs) Reeta Mishra SMS (Home Sci Food & Nutrition) KVK, RVS Krishi Viswavidyalaya, Near Commissioner Office, AB Road, Morena- 476 001 (MP) [email protected], 09425795028
7. Mr. Raghu H V Scientist, DM Division, National Dairy Research Institute, Karnal- 132001 ( Haryana) [email protected] , 09729488649
4. Dr. Bhagat Singh Assistant Professor & Head, Department of Microbiology Institute of Applied Medicines & Research, Delhi Meerut Road, Duhai, Ghaziabad -201 206 (UP) [email protected], 09457671259
8. Dr. Pranav Kumar Singh Assistant Professor (DT) College of Dairy Science & Technology, Near Verka Milk Plant, Ferozpur Road, GADVASU, Ludhiana- 141 004 (Punjab) [email protected], 09417300374
9. Dr. Pralhad
Assistant Professor (Animal Science), Krishi Vigyan Kendra,Raichur University of Agricultural Sciences, Raichur- 584 102 (Karnataka) [email protected], 09448777357
12. Prof. Digamber Govindrao More Assistant Professor Deptt. Animal Husbandry & Dairy Science, College of Agriculture, Latur, Marathawada Krishi Vidyapeeth Parbhani- 431 402 (MS) [email protected], 08087011336
10. Dr. Vishal Kumar Deshwal HOD Microbiology, Doon (PG) Paramedical College, 28 Chakarata Road , Dehradun-248 001 (UKD) [email protected], 09897538555
13. Mr. Surinder Kumar T-9, KVK/DTC National Dairy Research Institute, Karnal- 132001 ( Haryana) [email protected] 09812077005
11. Dr. (Mrs.) Renu Singh Sr. Lecturer, Department of Microbiology, Institute of Applied Medicines & Research, Delhi Meerut Road, Duhai, Ghaziabad -201206(UP) [email protected], 09897736479
14. Dr. Aarti Bhardwaj Lecturer, Department of Microbiology/Biotechnology MIET, Meerut (UP) [email protected], 09045044802
