COURSE COMPENDIUM

TECHNOLOGICAL ADVANCES IN THE UTILIZATION OF DAIRY BY-PRODUCTS

February 27 – March 18, 2008

CENTRE OF ADVANCED STUDIESDairy Technology Division

NATIONAL DAIRY RESEARCH INSTITUTE(Deemed University)

KARNAL – 132 001 (Haryana), India

2008

COURSE COMPENDIUM

TECHNOLOGICAL ADVANCES IN THE UTILIZATION OF DAIRY BY-PRODUCTS

22nd Short Course

Organised under the aegis of

Centre of Advanced Studies in Dairy Technology

February 27 – March 18, 2008

Course Director Dr. Vijay Kumar Gupta

CENTRE OF ADVANCED STUDIES

Dairy Technology Division NATIONAL DAIRY RESEARCH INSTITUTE

(Deemed University)

KARNAL – 132 001 (Haryana), India 2008

Published by

Dr. A. A. Patel Head, Dairy Technology Division &

Director, CAS

Course Director

Dr. Vijay Kumar Gupta

Editing and Compilation

Dr. Vijay Kumar Gupta

All Rights Reserved ©

No part of this lecture compendium may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photography, recording, or any information storage and retrieval system without the written permission from the Director, NDRI, Karnal.

Cover Design & Page Layout

Dr. Vijay Kumar Gupta

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An important criterion in the economic choice of any process is the efficient utililization of by-product that is produced along with the main product. Main by-products of dairy industry are buttermilk, whey, ghee residue and sometimes skim milk. The techno-economic problems associated with the utilization of dairy by-products, especially whey, have been receiving considerable attention and remarkable advancements have been made in processing equipments and techniques. In the past 25 years, whey has transformed from a waste by-product to a valued functional co-product with the perfection of membranes and ion-exchange as well as an increased understanding of whey itself. Further the market for dairy by-products as ingredient in food products has been growing. A number of factors account for this. On the demand side, there is a growing demand of prepared consumer foods, including ready meals, snacks and microwaveable foods. On the supply side, more efficient, cost effective and sophisticated technologies in processing of dairy by-products has opened up further markets, hitherto closed.

The production of other derived by-products like casein, caseinates, co-precipitates, protein hydrolysates, whey protein concentrates, lactose, whey beverages, low lactose powder and many others have prominence in advanced dairy countries and Indian dairy industry is trying to make advancement in this direction. A number of by-products based dairies with large automatic and continuous manufacturing plants have been set up in India and quite a few more are in line.

Scientists of Dairy Technology Division have themselves received advance training abroad on their own or through UNDP, DAAD, NARP, DANIDA etc. programmes. They have done extensive work in the area of dairy by-products and developed a number of new technologies suited to Indian dairy situations that merit sharing with the teaching faculty of other Agricultural Universities and ICAR Institutes. I am happy that they are doing so in a commendable manner by organizing this twenty second CAS Short Course now on "Technological Advances in the Utilization of Dairy-By-Products". The Dairy Technology Division received good response from the participants for this short course, as had been the case in earlier ones, which shows that the Division and its programmes are held in high esteem by the State Agricultural Universities and other institutions.

Scientists have deftly compiled the lecture material of their own faculty and of guest speakers. The topic of the seminar is very important since nearly one third of the total milk produced in the country ends up as by-products. Their utilisation is of utmost importance. Each and every component of milk must be judiciously processed into edible form for the obvious reason of its unique nutritional value. In this endeavour, I wish to commend the Dairy Technology faculty for their splendid effort first on doing the research on processing and utilisation of the by-products, publishing the work and now compiling into lecture material, of which I am sure, all the participants will be proud of. It is hoped that the compendium so ably brought out by the course organisers will serve as a reference work of immense importance to the participants of the course in their research and teaching endeavours. 27th February, 2008 (Dr. Sushil Kumar) DIRECTOR

FOREWORD FOREWORD

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Dr. A.A. Patel, CAS Director

Dr. R. S. Mann

Dr. S. K. Kanawjia

Dr. D. K. Thompkinson

Dr. V.K. Gupta, Course Director

Dr. S. K. Kanawjia, Chairman Dr. V. K. Gupta, Chairman

Dr. R.R.B. Singh Dr. B. B. Verma

Dr. Latha Sabikhi Dr. A.K. Singh

Dr. R. S. Mann, Chairman Dr. D. K. Thompkinson, Chairman

Dr. G. K. Goyal Dr. Dharam Pal

Dr. A.K. Singh Mr. F. C. Garg

COMMITTEES FOR THE COURSE ORGANIZATION

ORGANIZING COMMITTEE

RECEPTION COMMITTEE TECHNICAL COMMITTEE

COMMITTEES FOR THE COURSE ORGANIZATION

HOSPITALITY COMMITTEE PURCHASE COMMITTEE

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Short Course on

TECHNOLOGICAL ADVANCES IN THE UTILIZATION OF DAIRY BY-RODUCTS FEBRUARY 27 - MARCH 18, 2008

FEBRUARY 27, 2008 09.30 AM - 10.00 AM Registration 10.30 AM - 11.30 AM Inaugural function 11.30 AM - 11.45 AM Inaugural tea 11.45 AM - 12.45 PM Orientation and visit of the institute Dr. A. K. Singh 01.00 PM - 02.30 PM Lunch break 02.30 PM - 03.30 PM Overview of production, processing

and utilization of dairy by-products Dr. V.K. Gupta

03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Nutritional qualities of dairy by-

products Dr. V.K. Kansal

FEBRUARY 28, 2008 10.00 AM - 11.00 AM Developments in the manufacturing

technology of caseins Dr. V.K. Gupta

11.00 AM - 11.15 AM Discussion 11.15 AM - 01.00 PM Mechanisation/automation in

manufacture of edible casein Mr. S.P.S Sawhney

01.00 PM - 02.30 PM Lunch Break 02.30 PM - 03.30 PM Non-food uses of caseins Dr. V.K. Gupta 03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Developments in the manufacturing

technology of co-precipitates Dr. R.S. Mann

FEBRUARY 29, 2008 10.00 AM - 1.00 PM Manufacture of edible acid casein

(Practical) Dr. R.S. Mann

02.30 PM - 03.30 PM Developments in the manufacturing technology of caseinates

Dr. V.K. Gupta

03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Preparation of fermented beverages

from whey Dr. D.N. Gandhi

MARCH 1, 2008 10.00 AM - 11.00 AM Developments in the use of casein

products in food products Dr. V.K. Gupta

11.00 AM - 11.15 AM Discussion

COURSE PROGRAMME COURSE PROGRAMME

iv

11.15 AM - 12.15 PM Bioprocessing of whey for preparation of products of industrial importance

Dr. D.N. Gandhi

01.00 PM - 02.30 PM Lunch break 02.30 PM - 03.30 PM Developments in the manufacture of

whey protein products Dr. V.K. Gupta

03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Functional properties of milk

proteins Dr. R.B. Sangwan

MARCH 2, 2008 SUNDAY

MARCH 3, 2008 10.00 AM - 01.00 PM Evaluation of functional proper-ties

of milk proteins (Practical) Dr. R.B. Sangwan

01.00 PM - 02.00 PM Lunch Break 02.30 PM - 03.30 PM Developments in the manufacture of

condensed whey and whey powder Dr. V.K. Gupta

03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Technological developments in whey

based non-fermented beverages and soups

Dr. A.K. Singh

MARCH 4, 2008 10.00 AM - 01.00 PM Preparation of fruit-whey beverage

(Practical) Dr. A.K. Singh

01.00 PM - 02.00 PM Lunch Break 02.00 PM - 05.00 PM Preparation of tomato soup from whey

(Practical) Dr. A.K. Singh

MARCH 5, 2008 10.00 AM - 11.00 AM Separation and application of bioactive

whey proteins Dr. Rajesh Bajaj

11.00 AM - 11.15 AM Discussion 11.15 AM - 12.15 PM Separation of bioactive whey proteins

(Practical) Dr. Rajesh Bajaj

01.00 PM - 02.00 PM Lunch Break 02.00 PM - 03.45 PM Separation of bioactive whey proteins

(Practical) Dr. Rajesh Bajaj

03.45 PM - 05.00 PM Utilization of whey for the production of microbial biomass proteins

Dr.(Mrs.) Shilpa Vij

MARCH 6, 2008 HOLIDAY

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MARCH 7, 2008 10.00 AM - 01.00 AM Production of microbial biomass

proteins from whey (Practical) Dr.(Mrs.) Shilpa Vij

01.00 PM - 02.30 PM Lunch Break 02.30 PM - 05.00 PM Production of ethanol from whey

(Practical) Dr.(Mrs.) Shilpa Vij

MARCH 8, 2008 HOLIDAY

MARCH 9, 2008 SUNDAY

MARCH 10, 2008 10.00 AM - 11.00 AM Demineralization of whey for use in

value added products Dr. V.K. Gupta

11.00 AM - 11.15 AM Discussion 11.15 AM - 12.15 PM Technology of manufacture of milk

protein hydrolysates Dr. S.K. Kanawjia

01.00 PM - 02.30 PM Lunch break 02.30 PM - 05.00 PM Preparation of eggless cake using

Whey protein concentrates (Practical) Dr. A.K. Singh

MARCH 11, 2008 10.00 AM - 11.00 AM Developments in utilization of WPC

in dairy products Dr. V.K. Gupta

11.00 AM - 11.15 AM Discussion 11.15 AM - 12.15 PM Technological advances in the

preparation of whey cheeses Dr. S.K. Kanawjia

01.00 PM - 02.30 PM Lunch break 02.00 PM - 05.00 PM Manufacture of Ricotta cheese

(Practical) Dr. S.K. Kanawjia

MARCH 12, 2008 10.00 AM - 01.00 AM Preparation of soft serve ice cream

using whey protein concentrates (Practical)

Dr. G.K. Goyal

01.00 PM - 02.00 PM Lunch break 02.30 PM - 03.30 PM Advances in manufacture of lactose

from whey Dr. A.K. Dodeja

03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Hydrolysis of lactose for application

in food industry Dr. R.K. Sharma

MARCH 13, 2008 10.00 AM - 01.00 PM HPLC analysis of lactose hydrolysed

products (Practical) Dr. R.K. Sharma

01.00 PM - 02.30 PM Lunch break

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02.30 PM - 05.00 PM Immobilization of enzyme/ cells by entrapment method (Practical)

Dr. R.K. Sharma

MARCH 14, 2008 10.00 AM - 11.00 AM Ghee residue: processing, properties

and utilization Dr. B.B. Verma

11.00 AM - 11.15 AM Discussion 11.30 AM - 01.30 PM Preparation of chocolate burfi using

ghee residue (Practical) Dr. B.B. Verma

01.30 PM - 02.30 PM Lunch break 02.30 PM - 03.30 PM LIBRARY 03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Utilization of whey products in dairy

analogues Dr. A.A. Patel

MARCH 15, 2008 10.00 AM - 11.00 AM Application of buttermilk in the

manufacture of value added dairy products

Dr. Dharam Pal

11.00 AM - 11.15 AM Discussion 11.15 AM - 12.15 PM LIBRARY 01.00 PM - 02.30 PM Lunch break 02.30 PM - 03.30 PM Application of dairy by-products in

bakery and confectionery products Dr. A.K. Singh

03.30 PM - 03.45 PM Discussion 03.45 PM - 04.45 PM Application of dairy by-products in

the formulation of infant foods Dr. D.K. Thompkinson

MARCH 16, 2008 SUNDAY

MARCH 17, 2008 10.00 AM - 11.00 AM Application of dairy by-products in

meat industry Dr. R.R.B.Singh

11.00 AM - 11.15 AM Discussion 11.15 AM - 12.15 PM Export requirements of dairy by-

product plants Mr. H.K. Mondal

01.00 PM - 02.30 PM Lunch 02.30 PM - 03.30 PM Role of whey components in de-

signer dairy foods Dr. (Mrs.) Lata Sahikhi

03.45 PM - 04.45 PM Advances in packaging of dairy by-products

Dr. G.K. Goyal

MARCH 18, 2008 10.30 AM - 11.30 PM Valedictory function

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Foreword Dr. Sushil Kumar i Committees for course organization ii Course programme iii

I INTRODUCTION

1 Overview of production, processing and utilization of dairy by-products

Dr. V.K. Gupta 1

2 Nutritional qualities of dairy by-products Dr. V.K. Kansal 83 Export requirements of dairy by-product plants Mr.H.K. Mondal 14

II CASEIN PRODUCTS

4 Developments in the manufacturing technology

of caseins Dr. V.K. Gupta 29

5 Non-food uses of caseins Dr. V.K. Gupta 356 Developments in the manufacturing technology

of caseinates Dr. V.K. Gupta 40

7 Developments in the manufacturing technology of co-precipitates

Dr. R.S. Mann & Dr. V.K. Gupta

46

8 Technology of manufacture of milk protein hydrolysates

Dr. S.K. Kanawjia Mr. Vikash Gupta Mr. Hitesh Gahane

51

9 Development in the use of casein products in food products

Dr. Vijay Kumar Gupta

60

III FUNCTIONALITY OF MILK PROTEINS

10 Functional properties of milk proteins Dr. R.B. Sangwan Rajesh Kumar and Bimlesh Mann

67

11 Role of whey components in designer dairy foods Dr.(Mrs.) Lata Sahikhi 75

IV BUTTER MILK

12 Application of buttermilk in the manufacture of value added dairy products

Dr. Dharam Pal Mr. P. Narender Raju

81

V WHEY BASED BY-PRODUCTS

13 Technological developments in whey based non-fermented beverages and soups

Dr. A.K. Singh 91

CONTENTSCONTENTS

viii

14 Preparation of fermented beverages from whey Dr. D.N. Gandhi Mr. Kalpana Dixit

101

15 Bioprocessing of whey for preparation of products of industrial importance

Dr. D.N. Gandhi Mr. Krishan Kumar

105

16 Developments in the manufacture of condensed whey and whey powder

Dr. V.K. Gupta 109

17 Developments in demineralization of whey Dr. V.K. Gupta 11618 Developments in the manufacture of whey

protein products Dr. V.K. Gupta 124

19 Developments in utilization of whey protein concentrates in dairy products

Dr. V.K. Gupta 131

20 Separation and application of bioactive whey proteins

Dr. Rajesh Bajaj Dr. R.B. Sangwan Dr. B. Mann

138

21 Advances in manufacture of lactose from whey Dr .A.K. Dodeja

143

22 Hydrolysis of lactose for application in food industry

Dr. R.K. Sharma

149

23 Technological advances in the preparation of whey cheeses

Dr. S.K. Kanawjia Mr. Hitesh Gahane

156

24 Utilization of whey for the production of microbial biomass proteins

Dr.(Mrs.) Shilpa Vij

162

25 Utilization of whey products in dairy analogues Dr. A. A. Patel 169

VI GHEE RESIDUE

26 Ghee residue: processing, properties and utilization

Dr. B.B. Verma Mr. P. Narender Raju

176

VII UTILIZATION OF DAIRY BY-PRODUCTS

27 Application of dairy by-products in bakery and

confectionery products Dr. A.K. Singh

184

28 Application of dairy by-products in the formulation of infant foods

Dr. D. K. Thompkinson 192

VIII PACKAGING OF DAIRY BY-PRODUCTS

29 Advances in packaging of dairy by-products Dr. G.K. Goyal 198

List of Course participants

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SECTION - I

INTRODUCTIONINTRODUCTION

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

Skim milk, buttermilk, whey and ghee residue are the various basic dairy by-products. Each and every component of milk must be judiciously processed into edible form for the obvious reason of its unique nutritional value. However, dairy plants, particularly in India, are usually confronted with the problem of by-products utilization, especially that of whey and ghee residue in an economical manner. In the past 25 years, whey has transformed from a waste by-product to a valued functional co-product. Whey technologies have grown exponentially during the past 25 years with the perfection of membranes and ion-exchange as well as an increased understanding of whey itself.

The western dairy industry especially American and European and also of Australia and New Zealand have been the harbingers in the utilization of by-products. They have developed technologies for the utilization of by-products, developed large automated plants and also developed users for by-products in dairy and food industry.

The production of other derived by-products like casein, caseinates, co-precipitates, protein hydrolysates, whey protein concentrates, lactose, whey beverages, low lactose powder and many others have prominence in advanced dairy countries and Indian dairy industry is trying to make advancement in this direction. It is only after economic liberalization and de-licensing of the dairy industry in 1993 that dairy by-products, which were mostly neglected by the dairy plants earlier started receiving due attention. A number of by-products based dairies with large automatic and continuous manufacturing plants have been set up and quite a few more are in line. India is still to emerge as a global player in the international scenario. A low per capita availability of milk, higher proportion of buffalo milk, poor quality of raw milk, lack of organized manufacture of products, lack of adequate technology, high cost of new technologies, lack of in-house R & D, lack of proper infrastructure, lack of equipment and plants indigenously etc. are the host of problems associated with the production and utilization of by-products in India.

2. SKIM MILK AND ITS BY-PRODUCTS

Skim milk is a by-product obtained during the manufacture of cream. It is rich in solids-not-fat content and has high nutritional value. In dairy plants, it is mostly utilized either in standardization for the manufacture of main dairy products or preserved by removing moisture in spray dried form. The skim milk when utilized in either of these two forms or consumed as liquid is not considered a by-product. It is regarded as a by-product only when it is either not economically utilized or utilized for derived by-products like casein and related products, co-precipitates, protein hydrolysates etc.

OVERVIEW OF PRODUCTION, PROCESSING AND UTILIZATION OF DAIRY BY-PRODUCTS

OVERVIEW OF PRODUCTION, PROCESSING AND UTILIZATION OF DAIRY BY-PRODUCTS

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2.1 Casein and caseinates

Casein has a long history of technical use in industries producing paper, textile, paint, leather, rubber etc. Edible casein and caseinates are also long established dairy by-products finding use in many dairy and food products. The world production of caseins/caseinates is hard to define due to lack of a significant data. However estimation could be about 3.5 lakh tonnes. The large producers are New Zealand (1.4 lakh tonnes), Netherlands (80,000 tonnes), and Germany (24,000 tonnes). The world market of casein/caseinates used in the food industry fluctuates between 2 to 2.5 lakh tonnes. The biggest importer of casein is United States of America, where food casein demand can be estimated at 20,000 tonnes per year and caseinates demand at 27,000 tonnes per year. About 20% of this demand is for nutraceutical applications. A lot of casein is utilized for the manufacture of imitation cheese. The second biggest importer is Japan.

Production of edible casein is an economically feasible proposition, only when the whey thereby produced is efficiently and economically utilized. This has been one of the main reasons why edible casein was not produced seriously in India before 1995. Most of the requirement of this by-product, even for industrial uses, was met through import. During the last 12 years, there has been an entry of a few large automatic and continuous manufacturing plants in India for the manufacture of edible casein, lactose and whey protein concentrates. Presently, most of the casein produced is being exported, but eventually with the assured Indian market, the product would be diverted for internal consumption also.

The production of soluble form of casein, i.e., caseinates has not picked up in India. The economic constraints for the production of spray dried caseinates are their high drying cost, low bulk density and high packaging, storage and transportation costs.

2.2 Co-precipitates

The manufacture of co-precipitates has several advantages like increased yield and flexible functional properties and higher nutritional value over that of casein. However, even though its production was standardised in sixties and seventies,it has never been commercially exploited to any great extent. A poor solubility, especially of the higher calcium co-precipitates, is a particular limitation in its functionality. 2.3 Milk protein hydrolysates

Today protein hydrolysis has assumed a new dimension in the food industry. Protein hydrolysates find extensive use in nutritional, dietetic and formulated foods, where a pre-digested form of protein is needed. Protein hydrolysates are boon to the people who are suffering from protein allergy or stomach disorders and to those who require easily digestible foods. In India, there lies a great scope for making improvement in the quality of the protein hydrolysates being prepared. 3. BUTTER MILK

Buttermilk is the by-product obtained during the manufacture of butter. Sweet cream buttermilk resembles skim milk in gross chemical composition and is usually admixed with bulk of skim milk for further spray drying or even product manufacture in dairy plants. Desi

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buttermilk, on the other hand, has long been an important domestic beverage in India. It has high nutritive and therapeutic value. It also finds its way in the preparation of a host of items such as kadhi, dhokla and idli. Also a number of federations and private plants sell salted and spiced buttermilk in 200 ml pouches (Goel and Choudhary, 1996). Surat-based Sumul does business out of selling buttermilk (chhach) in and around the city. "Sumul chhach" in 500ml packs reaches practically very nook and corner of Surat, covering over 850 retail outlets. In the summer, sales average around 45,000 litres a day (Gupta, 1997).

4. WHEY AND ITS BY-PRODUCTS

Whey is a dilute, highly perishable greenish yellow fluid and the largest by-product of the dairy world produced during the manufacture of cheese, casein, chhana, paneer, chakka and co-precipitates Its composition and acidity varies widely (Table 1). It contains about half of the total solids of milk, and is a source of precious nutrients like lactose, whey proteins, minerals and vitamins. Whey proteins, though present in small quantity, have high protein efficiency ratio (3.6), biological value (104) and net protein utilisation (95) and are next only to egg protein in terms of nutritive value (Renner, 1990). Further, being a rich source of lactose, whey is a good fermentation media for a number of fermented products. In many applications, lactose in whole or deproteinised whey is hydrolysed to glucose and galactose, thereby increasing its sweetness. Such lactose hydrolysed syrups, generally after condensing, are mostly utilised in sweet confectionery products and in ice cream. The market for whey products is estimated at about $ 6.5 billion in sales globally. Future growth is expected to be led by the industry’s increasing focus on nutritional products, particularly in the dietary, sports and clinical segments of the market. Table 1. Composition of different whey systems

Constituents Cheddar cheese Acid casein Rennet casein Chhana and paneer

Co-precipitates

Total solids (%) 7.0 7.0 6.8 6.5 6.2 Fat (%) 0.3 0.1 0.1 0.5 0.1 Protein (%) 0.9 1.0 1.0 0.4 0.3 Lactose (%) 4.9 5.1 5.1 5.0 5.1 Ash (%) 0.6 0.7 0.5 0.5 0.6 T.A. (%) 0.2 0.4 0.2 0.4 0.3

The continuing annual growth in the production and consumption of the cheese and coagulated milk products represents the generation of extremely large additional quantities of whey because of the huge base. The current world production of whey is estimated at about 165 million tonnes. Cheese whey accounts for nearly 95% of total whey. In India, the major source of whey is from the production of chhana and paneer. In the absence of systematic surveys/statistics, the predicted value for whey production is estimated at 5 million tonnes per annum. In view of the low solids content of whey, there has been a gross lack of interest in its utilisation compared to other fluid by-products of dairy industry.

Utilization of whey has been of a great concern in the dairy industries engaged in manufacturing of cheese and coagulated milk products. The techno-economic problems associated with the utilisation of whey have been receiving considerable attention and remarkable advancements have been made. For more than 25 years, a virtual explosion of

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papers and reports has appeared about whey and its by-products, by and for scientists in industry and academia. Whey proteins, together with lactose, have provided an excellent area for research. Today, modern industrial processing techniques such as ultrafiltration (UF), reverse osmosis (RO), new drying methods, hydrolysis, electrodialysis, ion-exchange, fermentation and protein fractionation, among others, have converted whey into a major source of ingredients with differing functional and nutritional properties, that could be used in various branches of food and dairy industry. The global market for whey derivatives is growing at an average of around 10% annually. The predominant driving force behind the development of whey utilisation has been stringent regulations imposed by the environmental pollution agencies all over the world. Other aspect relates to economic return from whey, which contains almost half the solids of original milk. Food manufacturers are increasingly viewing whey products as an ideal means of achieving added value.

Despite significant gains in the amount of whey being processed, a large amount of current whey production still is disposed of as raw whey. Much of this represents production of small plants, where the cost of purchasing, processing as well as the subsequent transportation and handling clearly exceeds the value of any whey product that might be produced. In small plants, the choice remains some form of disposal, be it municipal treatment, spreading raw whey on local farm lands for its nutrient value or feeding to local livestock. Further, acid whey, because of high mineral content and low pH pose considerable difficulties in utilization and, therefore, mostly remain unutilized. 4.1 Condensing and drying

By far the single largest use of whey solids on global basis is in the form of whole dry whey and it continues to grow. This is whole whey that has been condensed and spray dried as such or after blending with certain other liquid ingredients. These powdered whey products are marketed as commodity ingredients for a variety of food and animal applications. The feed industry may be the largest consumer of dried whey and whey products.

Considerable advancements have been made in condensing and drying equipments for energy conservation and for the production of better quality product. A significant trend in the last two decades has been the increasing interest in reverse osmosis for removing water from whey. Small plants concentrate whey by RO for shipment to the larger plants. Medium sized plants concentrate whey by evaporation for large drying plants. Large plants concentrate their own whey plus outside sources whey to high solids for lactose crystallization and drying. 4.2 Demineralization

At the other end of the spectrum, a small percentage of utilized whey (less than 5%) is demineralized by ion exchange or electrodialysis prior to spray drying to produce dry demineralized whey for specialized uses. These include whey protein based infant formulas and other medical and nutritional products that require lactose, special nutritional quality of whey proteins and low mineral content.

4.3 Whey drinks A variety of beverages consisting of plain, carbonated, alcoholic and fruit flavoured have been successfully developed and marketed all over the world, because they hold great

5

potential for utilizing whey solids. In India also, a number of refreshing and low cost whey drink 'Whevit' or 'Acidowhey' is in the market. These drinks are prepared from paneer/chhana whey which is acidic and has low protein content (0.4%).

Whevit, an orange, pineapple, lime or mango flavoured alcoholic drink from whey, was developed at National Dairy Research Institute, Karnal by Bambha et al. (1975). For its manufacture, fresh whey is efficiently separated in cream separator, deproteinised by steaming for half an hour and cooled to room temperature. To the deproteinised and clarified whey, 22-23% of 50% sugar solution is added followed by 2-2.1% of 10% citric acid, colour and flavour. It is then fermented by incubation at 22oC for 14-16 hr with a 1% culture of Saccharomyces cerevisae. The product is bottled, pasteurised (75oC/30 min), cooled and stored at low temperature (5-10oC). For the manufacture of Acidowhey that is a non-alcoholic whey drink, deproteinised whey is fermented with a culture of Lactobacillus acidophilus and Lactobacillus bulgaricus (1:1) (Paul, 1990). 4.4 Lactose production

The production of lactose from whey continues to be one of the most important form of whey utilisation. The global market for lactose based goods is valued at over $3 billion. The market for lactose ingredients is likely to be strengthened as the dairy industry focuses more on healthy and nutritional products. The renewed research interest in application of lactose is expected to drive interest in lactulose, lactitol and galactooligosaccharides. A significant quantity of food and pharmaceutical grade lactose is produced by conventional process. During the process, a protein-mineral precipitate is segregated, which is dried and sold as a by-product for animal feed. It has limited application as a food ingredient because of the high mineral content and the less functional (insoluble) property of the denatured whey proteins. 4.5 Ultrafiltration processing

By 1981, UF had become the most widely used process for recovery of soluble whey protein concentrates (WPC). By this process, a highly functional WPC is produced as the primary end product for a wide variety of applications as a substitute for non-fat dry milk and other protein ingredients. The largest potential use of WPC is as a replacement for non-fat dry milk (NFDM) in the food industry. 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.

However, WPC constitutes a very small proportion (10%) of protein utilisation in food industry. More product formulation work, especially in the food industry, is needed to move WPC into the general market place. Whey proteins are also being used for reactive extrusion to supplement polyethylene - a common non-biodegradable plastic.

4.6 UF permeate stream Although UF of whey has been in commercial use since 1972, the industry has been slow to adopt it. This is because the process generates a UF permeate as a by-product rich in lactose and minerals that creates a waste disposal problem, equal in magnitude to the disposal of whole

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raw whey. Of concern too is the fact that a great deal of milk permeate is now coming on stream (the by-product from UF processing), adding to the problem. The fuel crisis of the seventies and perhaps the possibility of another looming on the horizon in the 1990's provided stimulants to look at an array of fermentation possibilities of using whey or milk permeates to produce alcohols, methane, organic acids, microbial biomass protein and other useful products. To some extent, lactose and alcohol is produced from the UF permeate but the identification of the most cost effective means for its utilization is still awaited by most dairy companies world wide. In India, all the lactose, the total production capacity being about 15,000 tonnes per annum, is produced from UF permeate. 4.7 Alcohol

The lactose in whey can be converted by fermentation by a variety of organisms to products ranging from lactic acid to flavouring materials. Three plants in New Zealand.

4.8 Milk Mineral products

Milk mineral products rich in natural calcium and phosphate are valuable nutritional supplements in today’s osteoporosis-sensitive world. These products are prepared by precipitation of calcium phosphate in whey UF permeate under suitable conditions of concentration, pH, time and temperature. The crystals that first precipitate quickly undergo solid state transitions depending on the conditions to which they are subjected. It is necessary to grow calcium phosphate particles to sufficient size to recover them in a good yield by centrifugation and filtration. Milk mineral is used as a natural calcium supplement in a growing range of food products including milks, yoghurts, canned milk powders and confectionary and health foods.

5. GHEE RESIDUE

Ghee residue is a by-product of ghee industry and is produced in large quantity in India. This nutritious by-product has been studied for its physico-chemical characteristics and for its utilization in a number of food products like chocolate burfi, samosa filling, chapatis etc. However, most dairy plants in India have not been utilizing ghee residue profitably except for fat extraction. Most of the ghee residue goes to waste. A sincere R & D work and a strong willingness on the part of manufacturer is required to develop food uses of ghee residue and put it in the market place. 6.0 CONCLUSION The utilisation of skim milk and buttermilk has attracted sufficient successful attention in India and abroad. During the last two decades, lot of technological advances have been made for effective utilisation of whey. However, just technical feasibility of whey processing into interesting by-products does not ensure its utilisation. Processing technologies need to be economically attractive before any attempt of commercial utilisation of this dilute liquor can be made. Therefore, further emphasis needs to be put on identifying and developing cost effective processing technologies. Commercial utilisation of whole ghee residue is also yet to pick up.

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7. REFERENCES Bambha, P.P., Nambudripad, V.K.N. and Srinivasan, M.R. 1975. NDRI Bull. No. 136, NDRI, Karnal. Goel, B.K. and Chouhdary, V.K. 1996. Techno-economic aspects of production of salted spiced buttermilk: A

promising profit making by-product utilization. Indian Dairyman, 48(5): 29-31. Gupta, P.R. 1997. Dairy India. P.R. Gupta Publisher, N. Delhi. Gupta, V.K. and Mathur, B.N. 1989. Current trends in whey utilization. Indian Dairyman, 41: 165. Horton, B.S. 1995. Whey processing and utilization. Bull. of the IDF No. 308 : 2-6. Huffman, L.M. 1996. Processing whey protein for use as a food ingredient. Food Technol. 50(2): 49-52. Khanna, R.S. and Gupta, V.K. 1996. Process optimization for the production of buffalo milk casein hydrolysate.

Indian J. Dairy Sci., 49: 386-397. Mann, E.J. 1993. Whey utilization. Part 2. Dairy Ind. Int., 58 (6): 19-20. Mann, E.J. 1996. Dairy ingredients in foods. Part 2. Dairy Ind. Int. 61(2): 10-11. Mann, E.J. 1996. Casein, caseinates and hydrolysates. Dairy Ind. Int. 61(9): 13-14. Paul, S.C. 1990. Nutritive Beverages for product diversification in dairy industry. Indian Dairyman, 42:

282. Pittis, E. 1995. The European market for dairy ingredients. J. Soc. Dairy Technol. 48(3): 79-86. Southward, C.R. 1994. Utilization of milk components: Casein. In: Modern Dairy Technology, Vol. 1. Advances

in Milk Processing (ed. R.K. Robinson), Chapman & Hall, London, U.K. P.. 375-432. Zadow, J.G. 1994. Utilization of milk components. Whey. P. 313-374. Zall, R.R. 1992. Sources and composition of whey and permeate. In: Whey and Lactose Processing (Ed. Z.G.

Zadow), Elsevier Science Publishers Ltd., New York, USA. P. 1-72.

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Vinod K. Kansal

Principal Scientist & Head Division of Animal Biochemistry, N.D.R.I., Karnal – 132 001

1. INTRODUCTION

Milk occupies a special position among foods in being an animal food that has a vegetarian connotation. For children, adolescent, elderly people pregnant and nursing mothers, milk plays an important role in meeting the requirements of many essential nutrients, and hence milk is considered as a protective food. Milk helps to balance human diet by supplementing good quality protein, calcium and vitamins particularly, vitamin A, riboflavin, niacin and folic acid. In addition, milk contains several bio-protective molecules that ensure health security to humans. Casein prepared from skim milk, being high quality protein with good supplementary value, can be used to fight protein deficiency. During processing, there is partition of nutrients. A considerable portion of nutrients pass into whey during manufacture of cheese and paneer. Whey protein concentrate (WPC) is indeed very rich in high quality proteins, minerals and vitamins. Besides having functional properties, WPC improves the nutritional attributes of the product. Milk sugar, lactose, has therapeutic value in improving intestinal flora and absorption of calcium. 2. NUTRITIONAL QUALITY OF CASEIN AND WHEY PROTEINS

Milk proteins are rich in essential amino acids. Whey proteins that constitute 20% of milk proteins especially are of high nutritional quality, containing 51% essential amino acids, compared to 45% in casein. The sulphur amino acids are higher in whey proteins then in casein. The Protein Efficiency Ratio (3.1), Biological Value (91) and Net Protein Utilization (82) of milk protein are very close to that of egg proteins (3.8, 100 and 94, respectively). Lactalbumin is superior to casein having Biological Value (BV), Net Protein Utilization (NPU) and Protein Efficiency Ratio (PER) 100, 92 and 3.6, respectively. The corresponding values for casein are 77, 76 and 2.5, respectively. Only 14.5g of lactalbumin or 28.5g milk proteins is sufficient to meet the daily requirement of essential amino acids for adult humans. 2.1 Supplementary value and digestibility of whey proteins

Milk proteins contain a surplus of certain essential amino acids (lysine and threonine), so they can raise the BV of vegetable proteins. Milk proteins can be added to cereal based products to increase their lysine and threonine content. A breakfast consisting of milk, egg and bread has an excellent NPU value, and provides balanced amounts of minerals and vitamins. Whey proteins have even better supplementary value. A mixture of whey protein, wheat, rice or maize has PER value even greater than that of whey proteins. Whey proteins can raise the BV of soy proteins because of their high concentration of sulphur amino acids. Whey obtained as a by-product in cheese manufacture has thus a great potential for incorporation in cereal based products.

The digestibility of milk proteins is rated higher (96%) then that of plant proteins (74-78%). Because of their high BV, the milk proteins are useful in the diet of patients suffering from liver and gall bladder diseases, hyperlipidaemia and diabetes. Patients with impaired kidney

NUTRITIONAL QUALITIES OF DAIRY BY-PRODUCTS NUTRITIONAL QUALITIES OF DAIRY BY-PRODUCTS

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functions rely on protein with high BV for relieving strain on the excretory function of the kidney. The milk proteins are also used in slimming diets.

2.2 Immunoprotective properties of milk proteins

Immunoglobulins, lactoferrin, lysozyme, lactoperoxidase and vitamin B12-binding protein have antimicrobial effect. The immunoglobulins mainly 1gA are not broken down by the digestive enzymes. Thus, they not only act against the microorganisms in the intestine, but also prevent the absorption of foreign proteins.

Lactoferrin is an iron binding glycoprotein that occurs in cow milk at a level of 0.2 mg/ml. Lactoferrin plays an important role in the resistance against intestinal infection, particularly E. coli. The bacteriostatic effect of lactoferrin is due to its iron binding ability making iron unavailable for iron requiring bacteria. Lactoferrin has also been shown to have bactericidal effect. Unsaturated vitamin B12-binding protein competes with bacteria that have a vitamin B12.

A number of enzymes are also involved in the milk immune system. These are lactoperoxidase, xanthin oxidase and lysozyme. Lysozyme has a direct effect by breaking down the cell wall of gram-positive bacteria. The lactoperoxidase-thiocyanate-H2O2 system is an antibacterial system. Lactoperoxidase and thiocyanate are found in milk and other tissue secretions, and H2O2 is produced by lactic acid bacteria or by the action of xanthin oxidase. Thiocyanate is oxidized by H2O2 and lactoperoxidase to an intermediate product that destroys the microorganisms. 2.3 Functional Peptides

Many milk-derived peptides possess functional properties. Several peptides with opium like (sleep inducing) activity have been extracted from the degradation products of milk proteins. These include ß-casomorphins (from ß-casein), exorphin (from ∝S1 casin), ß-lactostensin (from lactoglobulin) and serorphin (from serum albumin). These opium-like peptides have been shown to prolong gastrointestinal transit time exerting anti-diarrhoeal effect. These also stimulate secretion of insulin and somatostatin.

Glycomacro peptide (GMP) derived from K-casein induces production of cholecystokinin, a hormone associated with satiety. GMP and other fractions of K-casein digest inhibit the adhesion of oral actinomycetes and streptococci to erythrocytes, and binding of cholera toxins to its receptor. GMP is free from aromatic amino acids, and therefore a suitable protein substitute for those suffering from hereditary disorder of aromatic amino acid metabolism, such as phenyl ketonuria.

Angiotensin-converting enzyme (ACE) located in different tissue, splits two amino acids from C-terminal end of angiotensinogen I converting it into angiotensinogen II, which is a highly hypertensive octa-peptide. Peptides with anti-hypertensive activity that act through inhibition of ACE have been identified in the sequence of bovine and human ß-and ∝S1 casein. Recently ∝-lactalbumin and ß-lactglobulin fragments that inhibit ACE have also been characterized.

Several immune-stimulatory peptides have been identified from both bovine and human casein and whey proteins. These peptides have been shown to stimulate the phagocytic activities of murine and human macrophages and enhance resistance against certain bacteria. Certain

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peptides from casein stimulate the production of immunoglobulins.

Casecidin, a chymosin digest of casein in vitro, inhibits Sarcina, Bacillus subtitis, Diplococcus pneumoniae and Streptococcus pyrogenes. Similarly, fragments of human ß-casein have a protective effect against Klebsiella pneumoniae. Iracidin, an ∝S1 -casein fragment has both therapeutic and prophylatic effect. Lactoferricin, an acid-pepsin digestion product of lactoferrin, has stronger bactericidal activity compared to the native molecule.

3. CHOLESTEROL LOWERING FACTORS

Several studies have shown that milk reduces serum cholesterol levels of the consumers. Experiments with volunteers have shown that cholesterol levels do not rise when as much as 2 litres of milk is consumed daily. On the contrary, the cholesterol level is reduced. Experiments with animals have shown that even buffalo milk, containing 7% fat, lowers plasma cholesterol levels. Both decreased formation and increased breakdown of cholesterol are responsible for the cholesterol lowering effect of milk. Orotic acid and another nucleotide associated with proteose-peptone fraction of milk proteins and calcium are suggested to have cholesterol reducing properties. Milk slows down the biochemical processes leading to atherogenesis in rabbits fed on atherogenic diet. It has been suggested that the regular intake of milk keeps blood vessels healthy.

4. NUTRACEUTICAL ATTRIBUTES OF MILK CONSTITUENTS

Milk proteins have high buffering power; therefore it is useful in the treatment of inflammation of mucous lining of stomach and of stomach ulcers, preventing hyperacidity. Milk and milk products are used as a source of proteins in hyperuricaemia and goat disease. In contrast to other foods, they do not contain purines, which are precursors in the synthesis of uric acid that causes gout when deposited in the joints or may lead to formation of urinary calculi.

Short and medium-chain fatty acids with 4-12 carbon atoms, which occur in a relatively high concentration in milk fat, are reported to have antibacterial and fungicidal activity against gram negative bacteria and certain moulds. Milk fat has a protective effect against human tooth decay. This effect has been ascribed in part to adsorption of milk fat onto the enamel surface and in part to antimicrobial effect of milk fatty acids.

Recently much attention has been directed towards an unusual fatty acid, conjugated linoleic acid (CLA), which is naturally present in milk and dairy products. Several bio-protective properties of CLA have been demonstrated such as anti-carcinogenic activity in mouse fore-stomach, mammary cancer prevention in rats, anti-carcinogenic activity in rat colon, anti-carcinogenic activity in skin, anti-diabetic activity in rats and reduction in body fat and anti-atherogenic activity in rabbits and mice and immuno-modulation in rats. 5. NUTRITIONAL BENEFITS OF MILK FAT

Compared to other fats and oils, milk fat is easily digestible. The digestibility of milk fat is 99%, while that of natural palm oil is 91%. The excellent digestibility of milk fat is due to dispersion of fat globules in the aqueous phase of milk forming an emulsion. They are absorbed directly unlike other dietary fats that have to be emulsified by bile, pancreatic enzymes and intestinal lipases before they can pass through intestinal well. Also, milk fat is rich in short and

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medium-chain fatty acids, which are more easily absorbed than long chain fatty acids. The ester bonds involving short-chain fatty acids are more easily cleaved by lipases. The easy digestibility of milk fat makes it a valuable dietary constituent in diseases of stomach, intestine, liver, gall bladder, kidney and disorders of fat digestion. In infant and child nutrition, milk fat is of immense benefit. It helps them in meeting their energy requirements by increasing energy density of the diet. A sufficient fat supply is essential for thriving babies, a rosy and smooth skin and also resistance to bacterial infections. 5.1 No Justification for replacing milk fat with vegetable oils

Milk fat has a low content of essential fatty acids (EFA), linoleic and linolenic acid. The EFA requirement is only 3% of total calories, two-third of which is met from invisible fat present in dietary cereals, pulses and vegetables. Therefore there is no justification to replace milk fat with another fat having higher linoleic acid content.

5.2 Milk fat and misconceptions about its role in coronary heart diseases (CHD)

Milk fat has often been implicated in CHD because of its cholesterol content and composition of its fatty acids. It is however, not correct to judge the implication of milk fat in development of CHD solely on the basis of its fatty acid composition and cholesterol content. The average cholesterol content in cow and buffalo milk is only 2.8 and 1.9 mg/g fat respectively. Moreover, humans absorb 10-14% of dietary cholesterol, thus only 20-40 mg cholesterol will be absorbed from 50g of dietary milk fat. On the other hand, the body itself synthesizes cholesterol (1-4g daily) in much higher amounts than what is absorbed from the diet.

The so called "Lipid hypothesis" states that there is a connection between fatty acid composition of the diet and cholesterol content of serum in that the saturated fatty acids (SFA) increase cholesterol and polyunsaturated fatty acids (PUFA) decrease it. Since increased cholesterol levels are believed to play an important role in the development of CHD, the demand is often made that the dietary fat having low proportion of PUFA be replaced with oil that are rich in PUFA. The question that naturally arises is should one avoid milk or milk fat because it contains high content of saturated fat. The major saturated fatty acids in milk fat are palmitic acid (24-28%), myristic acid (13-14%) and stearic acid (11-12%), and the major unsaturated fatty acid is oleic acid (23-28%). Milk fat has high proportion of short and medium chain saturated fatty acids, which do not raise serum cholesterol levels, nor does stearic acid. Only palmitic acid has some effect.

The idea that excess of SFA and/or cholesterol were associated with the development of CHD arose from epidemiological association between high total and animal fat intake, high serum cholesterol levels and incidences of CHD, in some countries. The conclusion made from such empirical studies have been criticized by many researchers in that the population that consumed fat with higher PUFA/ SFA ratio also consumed less calories from sugars and total fat.

An excessive intake of energy, resulting in excess weight is one of the major reasons of altered cholesterol metabolism and atherosclerosis. Indeed, a diet containing optimum amounts of calorie and essential nutrients, wherein the type of fat has no significance, is a real safeguard against high mortality from atherosclerosis.

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6. MILK AND SKIM MILK ARE RICHEST NATURAL SOURCE OF CALCIUM

Recent research has shown that poor nutritional status with respect to calcium is related to diseases like osteoporosis, hypertension and colon cancer. The hypertensive patients have shown significant reduction in blood pressure in response to increased calcium intake. The effect of calcium on blood pressure is mediated by (1) increase in urinary excretion of sodium, (2) preventing the rise in vitamin D hormone which increases blood vessel resistance (3) relaxing smooth muscle cells which lines the blood vessels (4) suppressing the renin-angiotensin system and (5) increasing production of endothelial relaxing factors.

Introduction of increased dietary calcium through dairy products has been shown to reduce incidences in colon cancer and hyper-proliferation in the colonic mucosa in rodents. Calcium produces these effects by neutralizing deconjugated bile acids and free acids, thereby removing their mitogenic/ toxic influence.

Milk and dairy products are the most important source of calcium in readily available form. In western countries milk and dairy products provide up to 75% of total calcium intake. A 250 ml serving of cow milk contains calcium equivalent to 60% of ICMR’s Recommended Dietary Allowance (RDA) for adults. Equal amount of buffalo milk contains 95% of calcium RDA for adults. Calcium in all forms of milk is equally well utilized. Incorporation of milk in the diet also improves the bioavailability of calcium from vegetable foods. The factors that contribute to better availability of calcium from milk include lactose, protein and phosphorus.

The sulphur amino acids, methionine and cystine produce acidic urine, causing calcium loss. Milk has lower amounts of sulphur amino acids, and also has higher amounts of lysine that enhances diffusion of calcium across the intestine. A part of calcium in milk is associated with phosphorylated casein. The casein phosphopeptides, released in the gastrointestinal tract during digestion, form soluble complex with calcium phosphate salts and improves the diffusion of calcium across the intestine. Milk is rich in phosphorus that reduces urinary calcium excretion, and counter balances, at least in part, the calciuric effect of dietary proteins. Milk and most dairy products, except some processed cheese, have a near 1:1 calcium to phosphorus ratio considered to be ideal for retention of calcium in the body.

7. MILK AND WHEY ARE RICH SOURCE OF VITAMINS

Milk is a rich source of vitamins not only in terms of their contents but also their better bioavailability. Milk is one of the richest natural sources of riboflavin (vitamin B2). A 250 ml serving of cow milk contains riboflavin equivalent to 50% of the daily requirement of a pre-school child. Although milk contains only small amounts of preformed niacin (Vitamin B3), nevertheless, it is a very good source of this vitamin. Niacin can be synthesized in the body from tryptophan, which is present in milk proteins in good amount (480 mg/L). Sixty mg tryptophan is metabolised in the body to give rise to one mg niacin. Indeed, milk is used as dietary ingredient for patient suffering from pellagra, a niacin deficiency disease. For vegetarian, milk is sole natural source of vitamin B12, as this vitamin is present only in animal foods. Milk is also a good source of folic acid. Vitamin A deficiency is a major cause of widespread blindness among children in India. A 250 ml serving of cow milk contain vitamin A sufficient to meet 75% daily vitamin A requirement of pre-school child.

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8. THERAPEUTIC ROLE OF LACTOSE

Lactose, the principal milk sugar, is slowly metabolised and therefore, a considerable portion of it passes into the large intestine where it promotes the growth of lactic acid producing bacteria. Lactic acid creates a desirable condition that inhibits the growth of proteolytic and putrefying bacteria in the intestine and replaces them gradually with acidophilic bacteria. Lactulose, a disaccharide, originally not present in milk, is produced from lactose during heating or long storage. It is not hydrolysed by intestinal lactase, and hence passes almost completely into large intestine where it is fermented by Bifidobacterium bifidus and L. acidophilus to produce lactic acid. Thus lactose very effectively favours the growth of acidophilus bacteria. Microbial flora in the intestine of infant fed on mother's milk consists almost entirely of anaerobic lactobacilli, e.g., B. bifidus. Glycoproteins and oligosaccharides, present in mother's milk in high concentration, promote the growth of bifidus bacteria and thus provide baby resistance against intestinal infection.

Several investigations have shown that lactose promotes the utilization of calcium, magnesium and phosphorus. The acidic condition created by fermentation of lactose by intestinal bacteria may increase the solubilization and absorption of calcium. Part of the effect may also be due to the ability of lactose to form soluble complex with calcium. The lactose effect on calcium absorption has been observed in human infant and rats, but not in human adults. Lactose promotes the diffusion of calcium across the intestinal wall, which is the only route of calcium transport in human infant, since the active transport system is not developed at the time of birth. In adults, the calcium transport is predominantly via calcium carrier protein, induced by Vitamin D-hormone, which is not influenced by lactose.

Since lactose is slowly absorbed, it has slight laxative effect. It is due to lowering of the pH that increases the peristalsis of the intestine. The blood glucose does not rise rapidly on lactose diet. Milk consumption, therefore, enables the diabetic person to obtain the biologically highly valuable milk proteins without running the risk of rise in blood glucose levels.

9. SUGGESTED READING Kansal, V.K. 1990. Milk keeps blood vessels healthy. Indian Dairyman, 42: 353-355. Kansal, V.K. 1992. Essentialities of milk proteins in human diet. Indian Dairyman, 44: 328-331. Kansal, V.K. 1992. Lactose in human health. Indian Dairyman, 44: 497-500 Kansal, V.K. 1994. Milk fat and human health. Indian Dairyman, 46: 345-350. Kansal, V.K. 1995. Advances in milk fat and its role in human health. Indian Dairyman, 47: 20-27. Kansal, V.K. 1998. Milk offers dietary calcium in best available form. Indian Dairyman 50: 23-26. Kansal, V.K. 1999. Nutritional quality of milk in relation to milk processing. In: Advances in processing and

preservation of milk. Centre of Advance Studies in Dairy Technology, National Dairy Research Institute, Karnal pp 57-62.

Runner, E. 1991. Cultured dairy products in human nutrition. International Dairy Federation Bulletin No. 255. Kansal, V.K. (2004) Milk: A nutritious and protective food, good for every thing-a rejoinder to anti-milk campaign.

Indian Dairyman 56: 39-47.

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Mr. H. K. Mondal Deputy Director, Export Inspection Agency, New Delhi

1. INTRODUCTION

The Export Inspection Council (EIC) is the official certifying body for Exports. The organization is a statutory body set up by Govt. of India, Ministry of Commerce & Industry under the export (Quality Control & Inspection) act 1963. In order to ensure sound development of Export trade of India through quality control and Inspection. EIC provides mandatory certification for various food items namely, Fish & fishery products, Dairy Products, Egg products, Meat & Meat products, poultry and poultry meat products and honey and while other food and non food items are being certified on voluntary basis. The certification is given against the standards of importing countries or in absence of this, international standards / Indian National Standards. Export Certification is carried out through its field organization, Export Inspection Agency (EIA) located at Mumbai, Kolkata, Kochi, Delhi and Chennai and 38 sub offices and is based on a system approach to include GMP/GHP/HACCP and also tailored to meet the requirements of the importing country. EIC certification is recognized by several of Indian Trading partners while with others the dialogue is on for seeking recognition.

Though, India is the largest milk producer in the word, its exports are relatively insignificant. Major factors for low export of milk products are the quality and safety aspects. Consumer of all over the world is showing their preference for high quality of products. Beside with the establishment of WTO and further increase in global trade, due to removal of quantitative restrictions, the Governments have realized their role in protecting the health and safety of their populations by imposing stringent restrictions relating to pesticides residue, heavy metals, contaminants, microbiological parameters as well as various aspects of hygiene control.

Many importing countries such as U.S.A., E.U., insist on implementation of Food Safety Management System (FSMS) such as H.A.C.C.P. / G.H.P. rather then depending on final product inspection. On the similar approach E.U. has issued directive on general hygienic condition for processing storage, packaging and transportation of dairy products for approval of dairy processing unit to produce while some and safe dairy products. 2. NOTIFICATION OF DAIRY PRODUCTS

In light of the development in the International Market, Ministry of Commerce and Industry, Govt. of India has issue order / notification wherein dairy products have been brought under compulsory quality control inspection and certification vide S.O. No. 2719 and 2720 dated 28/11/2000 under the export of milk products (Quality Control, Inspection and Monitoring) Rules 2000. The dairy product processing units are required to meet the sanitation and hygiene and other food safety requirement as laid down in the said notification for getting approval from E.I.C. / E.I.A.s for export.

EXPORT REQUIREMENTS OF DAIRY PRODUCTS PLANT EXPORT REQUIREMENTS OF DAIRY PRODUCTS PLANT

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3. SPECIFICATION FOR DAIRY PRODUCTS FOR EXPORT

Specification for dairy products recognized under section 6 of the Export (Quality Control and Inspection) Act 1963 shall be:-

a. National Standard of Importing Countries or Codex Standards of Codex Alementerious Commissions. Or

b. Contractual Specification agreed to between the foreign buyer and the exporter provided the same is not below the National Standard of the Importing Countries.

c. In absence of a or b above, the National Standard Specification as notified shall apply. d. In case any dairy products for which no standard is available at a. b. & c. above, the

standard formulates for the said product by the standing committee. 4. BASIS OF COMPLIANCE

a.) Responsibility of the processor to ensure that the dairy products intended for export are handled, Processed at all stages of production, storage and transported under hygienic condition so as to meet the health requirement laid down the rules and that product confirms to the Specification recognized.

b.) CA shall ensure that the processor comply with the requirement by regular monitoring the plant as per prescribed control measures.

4.1 Conditions to be fulfilled in order to export Dairy Products

a) Any statutory restriction imposed by any State/Central Govt. with respect to commercial / environment / conservation measures from time to time.

b) They must have been obtained milk from dairy animals, which are apparently healthy and do not show any visible symptoms to infectious disease likely to be transferred to human through milk. Further, animals, which have been treated with antibiotics, or other veterinary drugs, which can be transferred to milk, shall not be brought to the collection center unless the retention period of drug is over. Raw milk shall meet the standard of heavy metals, pesticide residues, aflatoxin, antibiotic residues, contaminants and adulterants.

c) They must contain only the permissible food, additives / processing aids other than milk, which are fit for human consumption within the specified limit.

d) They must have been treated and prepared in an approved plant. e) They must have been processed and or manufactured under hygienic conditions. f) Undergone processing treatment which enable meet the analytical specification laid down

such as:

i. Pathogenic micro organism – should be absent. • Listeria monocytogen. • Salmonella spp • Shigella.

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ii. Organism indicating poor hygiene-within prescribed limit • Staphylococcus aureus • Escherchia coli

iii. Indicator organism-within prescribed limit • Coliform • Plate count

g. Undergone a health marking and labeling on the packaging as required by importing country.

h. They must have been packed by observing:

• Wrapping and packaging must take place under satisfactory hygiene conditions in room provided for that purpose.

• Bottling, filling of containers with liquid milk products and sealing of containers and packaging must be carried out automatically

• Wrapping on packaging may not be reused for the products with the exception of certain types of containers, which may be reused after through cleaning and disinfecting.

i. They must have been stored at the temperature established by the manufactures to ensure their durability. In particular, the maximum temperature of 6oC, at which pasteurized milk may be kept until it leaves the establishment and during transportation. When stored under cooled conditions the storage temperature must be registered and the cooling rate are must be such that the product reaches the required temperature as quickly as possible.

j. The processor shall indicate the period during which the dairy products are fit for human consumption and storage.

k. The results of the various checks and tests are recorded and kept for presentation to the competent authority for a period of two years.

l. To detect any residues of substances having a pharmacological or hormonal action and of antibiotic, pesticide, detergents and other substances should not be present in the milk, which might alter the organic characteristic of dairy products or make their consumption harmful or dangerous to human health.

m. If the dairy products examined show traces residues in excess of the permitted levels fixed, they must not be allowed either for the manufacture of food stuff or for directs human consumption.

n. Tests for residues must be carried out in accordance with National / International, recognized methods.

o. If the plant meets the requirements with regard to the nature of activities carried out, CA shall accord approval of such plant.

p. CA may take the assistance of a representative from EIC and other departments in the matter of approval of processing plant.

q. CA shall draw of a list of approval plant each of which has an official number.

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r. Inspection and monitoring of plant and packing centers shall be carried out regularly by competent authority. The competent authorities have free access at all times to all part of the plant to check that the rules are being strictly complied with. The health checks and supervision of the production shall be carried out by CA as follows:

o The cleanliness of the premises, equipment and hygiene.

o The efficacy of the checks carried out by the establishment.

o The microbiological and hygiene condition of the milk based products.

o The efficacy of treatment of the milk based products.

o The hermetically sealed containers by means of random sampling. o The appropriate health making of the milk based products whenever applicable

o Storage and transport condition

o Take sample required for laboratory tests.

o Make any other checks it considers necessary to ensure compliance with this order.

5. CERTIFICATION

a. CA shall issue health certification in prescribed Performa after satisfying that the dairy products are processed in approved processing plant having valid approval number and after satisfying the relevant requirements

b. The CA shall also issue any other certificate on request from processor / exporter after satisfying itself that the requirements of the relevant standards are met.

6. GENERAL CONDITIONS OF DAIRY PROCESSING ESTABLISHMENT FOR

APPROVAL FOR EXPORT

6.1 Premises

• In open, clean and healthy surrounding, away from the roadside, garbage dumps, cattle sheds, open sewage drains.

• Free from sources of obnoxious fumes, smokes, odor or excessive dust

• Surrounding roads to be cemented, tarred or turned 6.2 Building

• Paramount in nature, suitable in size, construction, designs to facilitate maintenance and hygienic condition and unidirectional flow.

• Sufficient space for housing equipments & storage materials.

• Shall not be used for domestic purpose

• Rodents, flies & birds proof.

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6.3 Ventilation & lighting • Adequate lighting in accordance with factory act.

• Light to be covered with protective cover

• Adequate ventilated in according to the number of worker with fly proofing arrangement.

6.4 Floor, Walls & Ceiling

• Hard smooth. Washable, slopped to the drain, not affected by weak acid, alkali, stream.

• Cement concrete floor for store room and go down also

• Walls and ceiling shall be smooth, non-absorbent light colored,

• Free from crevices and sharp angle

• Wall washable up to 1.5 meter

• Junction of floor with walls and two walls to be rounded off 6.5 Processing Room

• Shall be made fly proofing & rodent proof

• Floor shall have sufficient slop to the drain

• Drains to be cemented and covered with detachable covers

• For effective drainage 15 to 30 cm half circular drains with glazed tiles on the bottom to be provided.

• Screen to be provided at the opening of the drain to prevent solid matter from logging the drain, end of the drain to be made rodent proof by providing screen.

• Doors to be provided with self closing system

• Exhaust fan to be provided whenever necessary

• Windows, ventilators and Exhaust fan’s openings to be provided fly proofing arrangements

• Facilities for washing & cleaning

• Adequate numbers of wash basis to be provided with non hand operated taps

• Hose holders with hoses to be provided near all water outlets

• Hose shall be kept always rolled condition

• Self closing containers to be provided for waste collection

• Fly catchers

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6.6 Change Room • Adequate numbers separately for males & females

• Smooth water proof washable walls & floor

• Wash basins with non-hand operated taps, soap solution

• Flash lavatories, door must not open directly to the work room

• Single used towels for hand drying, foot operated bins nail brush

• Arrangements for changing shoes

• Separate cupboard for company cloths and personal belongings 6.7 Entrance

• Hand washing and sanitation facilities

• Arrangement for hand drying

• Foot dip in sufficient size

• Air curtains / fly catchers 6.8 Plant & Equipments

• Smooth, free form pits & crevice • Non absorbent, corrosion resistance materials (St. Steel) and easily cleanable • Gasket to be food grade, non-porous & non-absorbent • Place at least 45 cm from walls and ceiling • Drains & catch pans to be provided where ever necessary to collect spill & drip,

easily cleanable. • All electrical connection switch boxes, control boxes, cables to be installed 45cm

away from the equipment walls to facilitate cleaning • All cable wires to be covered • All switch boards to be covered with washable covers • Equipment coming in contact with milk to be kept always clean • All equipment, machines to be serially numbered • Heat treatment equipment to be fitted with

o An automatic temperature control and recording thermometer o An automatic safety device preventing insufficient heating or cooling medium o An automatic safety system preventing the mixture of pasteurized or sterilized

milk with incompletely heated milk.

• All the temperature / pressure measuring apparatus, balance shall be calibrated from authorized authority.

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6.9 Water suppler • Adequate safe & potable water to be supplied • Hot, cold water to be supplied for cleaning the plant equipments machineries • Storage tank to be kept always lockable condition • Manhole of storage tank to be sufficient for easy cleaning • Cleaning of storage tank to be carried out at least once in six months. • Date of cleaning, next date of cleaning, capacity, procedure of cleaning to be

displayed on the storage tank • Water to be periodically tested (once in a year) for physically, chemically and

microbiologically as per IS 4251 to check portability • Plumbing arrangements to be such type so that potable water should not be

contaminated

• All taps and water outlets to be serially numbered

6.10 Dairy hygiene • Arrangements to be made to protect the entry of Birds & domestic animals in the

plant.

• Collect waste and refuse in covered receptacles and not allow to scatter on the floor

• Prevent mould growth on equipments and internal structure of the plant

• Steps to be taken to prevent infestation of cockroaches and other pests.

• Care to be taken to prevent contamination of equipments, raw materials etc if pesticides used.

• Pesticides not to be used during processing in side the processing hall.

• Processing room not to be used as store & eating room

• Premises to be well lit and ventilated, sufficient number of exhaust fans to be provided

• Ventilators to be covered with fly proofing nets

• Safety type bulbs & fixture to be used.

• Glass windows & light fitting to be cleaned at regular intervals

• All pumps and waste disposal lines to be large enough to carry peak loads

• Mechanism to be provided to treat dairy effluent

• Effluent not to be disposed on the road or open area outside the plant

• Store rooms, brushes, buckets and other cleaning gears in separate place.

• Dry & wet chemicals to be stored in separate room to avoid contamination.

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• Tankers / cans washing facilities to be provided in separate place away from the receiving area.

• Top and side of raw milk unloading area shall be covered.

• Dry & wet production area to be divided to avoid cross contamination.

• Equipments, containers, installations which come in contact with milk & milk products to be cleaned & disinfected at the end of each work phase.

• Treatment area to be cleaned at least once in each working day. 6.11 Employee Hygiene

• Medically examined once in a year or more frequently

• Test of blood, urine, stools and X-ray to be carried out once in a year to ensure that the employee is medically fit and free from communicable diseases

• Inoculated and vaccinated against the enteric groups of diseases once in a year and small pox once in two years.

• Free from boils, cuts, sores etc.

• Keep nail short and clean

• Wash and sanitize hands before commencing work and after each absence

• Wash feet through foot dip before entering the unit

• Provided clean uniform (Preferably white) or apron or both and clean washable cap.

• Uniform not to be worn outside the units and to be put before starting the work and changed when leaving.

• Provided clean footwear

• Eating, spitting, nose cleaning, use of tobacco in any form including smoking, chewing betel leaves shall be prohibited within the plant.

• Wrist watch, ring, chain, bangles, earring shall not be allowed in the plant. While working.

• Personal hygiene to be checked before start of work and after each absence

• Sufficient numbers of wash basin with non-hand operated taps, nail bushes soap solution, towels, latrines and urinals in prescribed manner to be provided conveniently situated and accessible to workers at all the time while they are in the dairy.

• High degree of personal hygiene and cleanliness to be maintained. 6.12 Laboratory

• To be installed for testing chemicals and microbiological parameters separately as per requirements.

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6.13 Storage

• Store the materials away from the walls & ceiling and on the pellets

• Packaging materials to be transported to the processing unit with protective coverers to avoid dust and damaged.

• Automatic temperature recording device to be provided for cold room / cold storage

• Light to be provided with safety covers

• Rodent control system to be provided

6.14 Transportation

• Tanks, cans, tankers, and other containers which are used for transport or pasteurized milk must comply the following: o Inside part made by SS304, smooth easy to clean and disinfect o Designed such a way to drain completely o If fitted with tap, which must be easy to remove, dismantle, wash clean and

disinfect. o Washed, cleaned and disinfected immediately after each use and before reuse. o Must be sealed before and during transport by means of watertight sealing

device. o Vehicles & containers must be designed and equipped in such a way to

maintain temperature through out the period. o Vehicles transporting milk in small containers or in churns must be in good

condition and must not be used to transport any objectionable products to cause the milk to deteriorate or contaminate.

7. REQUIREMENT FOR APPROVAL OF PROCESSING PLAN FOR DAIRY

PRODUCT • HACCP / GMP/GHP

• Minimum test facilities

• Waste disposal/Effluent treatment mechanism

• Record keeping mechanism

• Competence of technical man power

• Conformance of products to standard

• Facilities as per GOI notification

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8. PROCEDURE FOR APPROVAL OF A PROCESSING PLANT • The processor seeking approval of their plant submits an application in prescribed

format along with relevant documents and HACCP manual including SSOP to concerned Export Inspection Agency (EIA) of their region. Any discrepancies/shortcomings observed in the application are immediately communicated to the applicant for rectification. Desk audit of HACCP manual including SSOPs are also carried out and any deficiencies observed are communicated to the applicant for rectification.

• Applications complete in all respects will be forward to convener of Inter Departmental Panel (IDP), the convener will be from the concerned EIA. The members of IDP are from APEDA, NDRI, Ministry of Agriculture, Ministry of food Processing, NDDB-New Delhi, Indian Dairy Association.

• The IDP will visit the plant to adjudge the facilities available in the plant and give their specific recommendations for approval or otherwise. The minimum corium of the IDP will be three members including the convener.

• The recommendation of the IDP will be placed to In charge of EIA for issuance of approval letter to the processing plant or otherwise.

• Certificate of approval will be issue by the Director (Q/C & I), EIC New Delhi.

• The validity of certificate of approval will be for a period of 2 years from the date of issue of the letter of approval.

8.1 Documents required

• HACCP Manual with SSOP and organizational Chart

• Water test report as per IS 4251

• Plan lay out

• Details Plumbing Diagram

• Flow Diagram of the product

• Legal identity of the unit

• Lease agreement if necessary

• Bio-data of technologist/chemist/Supervisor

• Valid consent order for air and water issued by pollution control board

• Certified copy of IEC number 8.2 Marking on The Export Packaging

It is mandatory for the approved plant to put approval number & Q-Mark on all export packages by printing/stenciling, besides the requirements as stipulated in the export contact or the requirements of the importing country.

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8.3 Addition of New Products

The exporters will request for expansion of scope of approval along with required documents to the Export Inspection Agency. If the application and documents are in order, the Incharge of agency will arrange assessment of the unit. Assessment will be carried out by the officer of EIA, nominated by Incharge of Agency. On satisfactory report, Incharge of agency will approve the inclusion of additional product in scope of approval.

8.4 Monitoring and Control by Processor to Produce the Safe Dairy Products

It is the primary responsibility of the processor to ensure compliance with the requirements of the notification and to ensure safety and wholesomeness of the product. Processing plants shall exercise all controls required as per notification and maintain records thereof in respect of following broad areas.

• Hygienic requirements relating to the premises. • Structure & layout • Pest control (Prevention, Extermination, Use of Chemicals) • Maintenance • Cleaning and Sanitation • Personal Hygiene • Rest Room • Water Management • Chemicals • Lighting and ventilation • Waste disposal including effluent treatment. • Good Manufacturing Practices (GMP)

8.5 Processor needs to implement HACCP system to control

• Raw Materials including Raw milk by testing in own/approved laboratory for required parameters

• Raw Milk to be tested for residues as per RMP at prescribed frequency. • Online process control to be conducted by competent personnel of the plant. • Finish product control; test of samples are carried out by the unit in their own

laboratory or E.I.C. Approved Laboratory as per buyer’s requirement / GOI notification / National specification.

• Sanitary and Hygiene Control by testing sanitary samples in their own laboratory. • Temperature control • The storage control • Transportation control • Documentation

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8.6 Surveillance by E.I.C. / E.I.A.s

Three tier Surveillance System is being followed By EIC/EIAs to check the compliance to laid down requirements by the approved Dariy Processing plant.

8.6.1 Monitoring by EIA official

• Verify the raw material, process and product control

• Verify sanitary and hygiene practice.

• Verify parameters tested as specified in the notification are within the tolerance limit and observe testing by laboratories.

• Verify the records

• Verify implementation of HACCP plan.

• Draw sample of raw milk, swabs from workers and equipments, in process and finish products for ensuring safety and wholesomeness of the product.

8.6.2 Supervisory visit to verify

• Compliance to norms by the processors

• Quality and correctness of monitoring by EIA officers

8.6.3 Corporate Audit by EIC

• Examine the operations of scheme by EIAs as per documented system. • Visit by audit team at least 10% of the approved units.

9. SOME IMPORTANT REQUIREMENTS FOR DAIRY PRODUCTS.

9.1 Microbiological criteria for Dairy Products

A) Pathogenic micro organisms – should be absent

• Listeria monocytogenes

• Salmonella spp

• Shigella

B) Organisms indicating poor hygiene-within prescribed limits

• Staphylococcus aureus (Milk powder- absent, others dairy products – 100/g)

• Eschericia coli (Dairy product - absent)

C) Indicator Organisms – within prescribed limits

• Coliforms (Milk powder – absent, other dairy product <200/g)

• Plate Count (Pasteurized milk - <20000/g, Dairy products - <40000/g, Ice-cream - <250000/g)

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9.2 Residue Monitoring Plan (RMP)- Testing of Raw Milk by Processor

Dairy processing plants shall exercise suitable control on quality of incoming raw milk. They shall test or arrange to get tested the raw milk in outside EIC recognised laboratories for the following parameters as prescribed by EIC.

1. Pesticide residues

2. Drugs ;Total residues antibiotic (as Beta Lactum)

3. Heavy Metals a. Lead b. Arsenic c. Cadmium d. Tin e. Zinc f. Mercury

4. Aflatoxin

a. Aflatoxin M1

10. ISSUUANCE OF CERTIFICATE OF INSPECTION

The printed blank certificates of inspection are issued to the approved plant. The approved plant will issue the certificate of inspection for every export consignment & submit two copies of the same to the concern EIA. The certificate of inspection can be issued only by the authorized signatories of the plants. Validity of the certificate of inspection will be 45 days from the date of issue.

11. ISSUANCE OF HEALTH CERTIFICATE

The health certificate can be obtained by an approved plant for the products for which they are approved for, by making a request on a prescribed format to concerned EIA along with the following documents

i. Copy of certificate of inspection for the concern consignment issued by the processor.

ii. Testing data of residues of pesticides, drugs and heavy metals for the period of production of the consignment.

iii. Laboratory test report for the additional parameters to be indicated in health certificate of clearly indicating about compliance of the consignment as per the requirement of importing country.

E.I.A.s also draw the sample of raw milk from approved processing unit for testing the following parameters under residue monitoring plan (RMP).

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PARAMETERS MRL

Veterinary Drugs • ChloramPhenical ND • Nitrofurans (including Metabolites) ND • Ronidazole ND • Metronidazole ND • Albendazole 100ppb • Fenbendazole 100ppb • Phenyl butazone ND

Heavy Metals

• Lead 0.02 ppm • Arsenic 0.1 ppm • Mercury 1.0 ppm • Tin 250 ppm • Cadmium 1.5 ppm • Zinc 50 ppm

Aflatoxin M1 0.5 ppb

Total Antibiotic as (Beta lactum) 10.0 ppb

Pesticide Residue • Organochlorine Group 0.01 ppm

• Organiophosphorus Group 0.01 ppm

The above parameters shall be tested as per methods given in the latest AOAC/Codex/Internationally recognized methods. 12. NEW EU DIRECTIVES ON FOOD-STUFFS

• Regulation (EC) No. 852/2004 on the hygiene of food stuffs requires food business operators to put in place, implement and maintain a permanent procedure based on Hazard Analysis and Critical Control Point (HACCP) principles.

• HACCP system is generally considered to be a useful tool for food business operators in order to control hazards through out the food chain from raw material to distribution that may occur in food.

• Regulation (EC) No. 852/2004 allows HACCP based procedures to be implemented with flexibility so as to ensure that they can be applied in all situations and in particularly in small businesses operator.

• Primary objective of Regulation (EC) No. 852/2004 is to ensure a high level of consumer protection with regard to food safety.

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• General hygiene requirements are specified for food business operator in Annexure of regulation No. 852/2004. o General requirements for food premises. o Specific requirements for processing rooms o Transport o Equipment Requirements o Food Waste o Water Supply o Personal Hygiene o Provisions applicable to foodstuffs o Provisions applicable to wrapping and packaging o Heat Treatment o Training

• Regulation (EC) No. 853/2004 is on specific hygiene rules for products of animal origin. It contains detailed hygiene rules for products of all animal origin including dairy products recognizing the specific microbiological and chemical hazards associated with such foods.

• Products of animal origin are also subject to the general hygiene rules in Regulation (EC) No. 852/2004 and thus the requirements for procedures are based on the HACCP principles.

• Regulation (EC) No. 854/2004 is on specific rules for the organization of official controls for products of animal origin intended for human consumption.

• Regulation (EC) No. 882/2004 is on official control performs to ensure the verification of compliance with feed and food law, animal health and animal welfare rules.

• Regulation (EC) No. 2073/2005 is on microbiological criteria for food stuffs.

• Regulation (EC) No. 183/2005 is laid down requirements for feed hygiene. It ensures the primary responsibility for feed rests with feed business operator.

• Regulation (EC) No. 178/2002 is on general principles and requirements of food law. It contains food safety requirements, responsibility, traceability and liability.

2

SECTION - II

CASEIN PRODUCTSCASEIN PRODUCTS

29

Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

Edible casein is a long established dairy by-product finding its use as an ingredient in many dairy and food products. The general development in technologies and the new uses in foods have ever increased the production and demand of this by-product. Its manufacture differs from that of non-edible casein (also called industrial casein) in that it is produced under sanitary conditions. Further, during its manufacture, food grade chemicals are to be used and sufficiently heat treated to make it safe for human consumption. Appropriate national and international standards for this by-product (Table 1) call for rigorous control during its manufacture. The intensive investigation in manufacturing technologies over the years and the introduction of efficient plant designs, have immensely improved the technology of edible casein. 2. MANUFACTURING PROCESSES

Processes for the manufacture of edible casein from cow milk are well known to dairy processes all over the world. Efficient separation of fat from milk is essential. For this, filtered and warmed milk (40-45°C) should be separated in a hermetic cream separator so that fat in skim milk is reduced to less than 0.05%. Achievement of the microbiological standards for edible casein requires pasteurization of either or both the milk and the curd. Heat treatment tends to give higher yield of casein. Some authorities hold that heat treatment of milk for casein manufacture causes slight insolubility and other defects. 2.1 Precipitation

Casein exists in milk as a calcium caseinate-calcium phosphate complex. When an acid is added to milk, this complex is dissociated. As the pH of milk is lowered, the calcium is displaced from the casein molecules by hydronium ions, H3O+ and the calcium phosphate associated with the complex is converted into soluble Ca2+ ions and H2PO4

- ions. At about 5.3 pH the casein begins to precipitate out of solution and at the isoelectric point of casein (about pH 4.6), maximum precipitation occurs. At this pH all the calcium is solubilized. Not only is the calcium from the caseinate molecule removed but also the calcium phosphate is liberated to the soluble form. This makes it possible to wash these soluble salts from the curd and achieve a low ash content in the final product.

It might be expected that all the casein in a sample of milk would be precipitated simply by adding sufficient acid to bring the pH value to approximately 4.6. However, the reaction of acid with caseinate complex is not instantaneous and the pH will tends to rise slowly with time. Therefore, ample time should be allowed for achieving equilibrium conditions. When casein is precipitated from skim milk by the direct addition of acid, the temperature and pH of precipitation

DEVELOPMENTS IN THE MANUFACTURING TECHNOLOGY OF CASEIN DEVELOPMENTS IN THE MANUFACTURING TECHNOLOGY OF CASEIN

30

and the mechanical handling of the curd during its formation are very important in determining the subsequent properties of the curd.

Table 1. National and international standards for edible casein Requirement for edible casein International standards

(FIL-IDF 45:1969) ISI standards (IS:1167-1965)

Extra grade Standard grade Moisture, % by weight, max. 12 12 10 Total ash, % by weight, (on dry basis), max.

- - 2.5

Copper, max. 5 ppm 5 ppm - Lead, max, 5 ppm 5 ppm - Iron, max. 20 ppm 20 ppm - Acid insolutble ash, % by Weight (on dry basis) max.

- - 0.1

Fat % by weight (on dry basis) max.

1.7 2.25 1.5

Nitrogen, % by weight (on dry basis), min.

-

-

14.5

Protein, % by weight (on dry basis), min.

95 90 -

Total acidity in terms of ml. of 0.1 N NaOH/g.

- - 6-14

Free acidity in terms of ml of 0.1 N NaOH, max.

0.20/g 0.27/g 5.6/10 g

Lactose, % by weight, max. 0.2 1 - Bacterial counts, per g, max. 30,000 100,000 50,000 Coliform count, max. Negative in 0.1g 10 per g Mould count, per g, max. - - 50 Moulds and Yeasts, per g, max. 50 100 - Thermophillic organisms, per g, max.

5,000 5,000 -

Optional Requirements Staphylococci (beta haemolytic coagulase positive), per g.

Negative Negative -

Salmonella, per 100 g. Negative Negative - Casein precipitated by acid usually includes the name of the acid in its description e.g. hydrochloric acid casein, lactic acid casein etc. but may simply be called acid casein. Any of the acid precipitation processes (Hydrochloric acid casein, sulphuric acid casein or lactic casein process) can be used to produce edible quality casein. The choice of method for reducing the pH of skim milk to precipitate casein is largely governed by economics. In terms of cost of acid, a lactic fermentation process is attractive especially when, with large-scale processing by modern methods, the tendency for higher capital and operational costs are minimized. For lactic acid casein, the pasteurized milk is cooled to 22-26°C and inoculated with about 0.5% starter of mixed strain (S. cremoris) being the

31

major culture) and incubated for 14-16 hr. during which the pH reduces to 4.6 giving a coagulum. The slow coagulating cultures exhibit less proteolysis and increased protein yield (Heap and Richardson, 1985). The precise rate of acid production by the starter is not important as coagulation usually takes place several hours before processing begins and at about 4.5 pH, the culture is in stationary phase of growth. The coagulum is cooked to 50-55°C to create a curd firm enough for subsequent processing. The acid and heat help in synersis of whey.

The use of mineral acids, on the other hand, has the advantage of completely continuous operation with no holding time for coagulation. Hydrochloric acid has been found, with experience, to be a superior coagulating agent. When sulphuric acid or hydrochloric acid is used to precipitate curd, it should be diluted before being added to the skim milk; otherwise local action of the acid may injure the curd, even though the agitation is rapid. Within reasonable limits, the more dilute the acid, the better will be quality of casein produced. In practice, hydrochloric acid is used in dilutions ranging from 1 part in 3 to 1 part in 9 and sulphuric acid is diluted 1 part in 20. 2.1.1 Temperature of Precipitation

The kind of curd formed is quite sensitive to heat. Curd precipitated at temperature below 35°C is very soft and fine, and consequently, is slow to settle and difficult to wash without loss. Precipitated at temperatures between 35 and 38°C, the curd is coarse provided stirring is not too fast. Stirring is necessary to distribute the acid uniformly, but rapid string at temperatures below 38°C produces a curd so fine that it settles very slowly during drainage and washing and may be lost to some extent in the whey and washings. Much more rapid equilibrium, more complete precipitation and, therefore, better yields are obtained by rapid and complete mixing before precipitation. The curd can be made firm in either of two ways; by heating to a temperature above 38°C; or the pH lowered to 4.1. Curd precipitated at about 43°C has a texture resembling chewing gum, being stringy, lumpy and coarse, containing practically no fine particles, and separating cleanly from the whey.

A high-grade casein, low in ash and readily soluble, is made by the grain-curd process, provided pH value and temperature are closely controlled. The best product is made by the use of hydrochloric acid, but lactic and sulphuric acids may be used successfully. The temperature of the skim milk should be held close to 35°C for hydrochloric acid curd. The pH value of 4.1 is attained by adding dilute acid slowly with slow stirring of the milk. this pH produces a granular curd of casein which is easy to drain and wash. 2.2 Drainage of Whey

After the precipitation has been completed and the curd has settled, whey should be removed from contact with the curd as soon as possible. The longer the curd stands in contact with the whey, the more difficult it is to wash out acids, salts, whey protein and lactose, as the freshly broken curd tends to anneal itself, thereby enclosing these constituents within a protein film. 2.3 Washing

The most positive quality improvement in casein is achieved through efficient washing. Relatively large amounts of lactose, minerals and acids are trapped within the curd, which prevents their ready removal during washing of the curd. It is necessary to allow sufficient

32

holding time during each washing stage to permit diffusion of these whey components from the curd into wash water. The diffusion rate depends on the size and permeability of the curd particles, and the purity, amount and rate of movement of the wash water. Smaller size and better permeability of the curd particles are important for efficient washing. Three separate washes of casein curd are required with contact times of 15-20 min. each. As soon as the whey is removed from the curd, wash water should be added equal in quantity to whey that has drained off. The curd should be well stirred in the wash water, either by rakes or by mechanical agitators, but care should be taken not to break the curd into fine particles. Firm and friable curd particles are required to avoid creation of excessive fines. Rubbery and plastic curds cannot be washed effectively. A marked increase in the efficiency of washing can be achieved by removal of as much whey as possible at the whey off stage. Even small amounts of whey contamination in wash water can cause a sharp decrease in washing efficiency. 2.3.1 pH of Wash Water

The pH of wash water should be about 4.6 for first two washings to avoid the formation of a gelatinous layer over the curd particles in excessively acid water and softening and redispersion of the curd in alkaline waters. Gelatinous layer if formed over the curd particles, inhibits drainage of salts and lactose from the particles. The adjustment of same pH of wash water as that of casein facilitates in maintaining the equilibrium. For pH adjustment, sulphuric acid is preferred, as casein is much less soluble in this acid, than in hydrochloric acid. The third wash should be given with neutral water. 2.3.2 Temperature of Wash Water

Casein curd has the usual property of acting somewhat like a sponge in water, contracting to expel water when heat is applied (Synersis) and relaxing when the water temperature is lowered. On the application of heat, the curd also becomes hard and rubbery, while cold water softens it and causes the curd to be quite fragile and readily broken. The temperature of the first wash should be about the same as the precipitation temperature to give good curd shrinkage. With lactic casein, higher temperature (70°C or more) is necessary at some stage of washing to reduce the bacteria which multiply during incubation of milk with starter. In practice, it is usual to adjust the temperature of last wash water to 32-40°C for better expulsion of water during subsequent pressing. 2.4 Pressing

Efficient pressing for dewatering of washed casein curd is important in minimising the energy required for removal of remaining water by drying. If the pressing has not been adequate, the subsequent grinding will give lumps of curd that will dry on the outside to give hard, impervious surface that prevents the escape of moisture from the inside, a condition known as case hardening. Further, mechanically removal of water is cheaper than thermal vaporization. The batch pressing operation is usually an overnight operation. The pressing of the curd should not be for less than 12 to 15 hr. with 34 kg/cm2 pressure.

The proportion of water in washed curd and its ease of removal depend upon the type of curd made. Precipitation of the curd at pH of 4.1-4.3, and the curd well-washed in waters, also of

33

the proper pH and at temperatures of 41°C would give a firm, friable curd which would drain well and press well. The final moisture content is usually 55-60%. 2.5 Milling and Drying

Pressed curd is liable to deterioration by action of moulds and bacteria and therefore, should be shredded and dried as promptly as practicable. The pressed curd is milled to produce particles of uniform size and surface for drying. Otherwise, uneven drying occurs. Large particles or lumps may dry on the outside forming a hard, impervious outer surface that prevents the diffusion of the remaining moisture from the interior of the particle. The kind of grinder used is dictated by the kind of drier used.

The ground curd may be spread on trays by hand or mechanically, should be spread evenly and not more than 0.9 to 1.1 kg of curd should be placed on each standard tray of 75 by 75 cm. The bottom tray on each truck should be of finer mesh than the others, or should be covered with a cloth to catch fine particles that may sift through the other trays.

Proper control of temperature and humidity of air coming in contact with the curd are the essentials of efficient drying of casein. A temperature of 52-57°C for the air entering a tunnel drier is suitable for any type of curd. Higher temperatures may be used for well-washed curd, but they are not recommended because of the risk of discolouring the casein and impairing its solubility. Especially during the early stages of drying, it is desirable to circulate a portion of the air so that the surface of the particle will not become fully dried while the interior is still moist. Care should be taken that the temperature in the drier does not rise above 57°C towards the end of the drying when comparatively small amounts of water are being evaporated. Drying once started should not be interrupted, but should continue until the percentage of moisture is approximately 8%. Properly dried casein has mush the same fine, granular characteristics as the properly ground curd from which it is made. 2.6 Tempering, Grinding, Sieving and Bagging

Tempering means holding of the casein for a period (24 h) to allow efficient cooling and hardening of the casein and evenness of moisture throughout the batch. Casein shows variation in moisture content during a day's run as it comes from the drier. Agitation is necessary for efficient tempering. The most efficient tempering consists of recalculating the dried casein by pneumatic conveyance. It has the advantage that air used for transport of the casein assists in cooling the curd.

The cool, tempered casein is ground. The casein must be cooled before grinding because warm casein is plastic and causes "burn on" of the rollers. An object of the grinding and sieving operation is to produce the highest proportion of the product in the size range desired by the buyer. The grinding may be done by roller mills, pin mills or hammer mills. For production of 60 and 80-mesh casein, pin mills are much more efficient than hammer mills for grinding casein. The grinding operation is followed by sieving into various mesh sizes and bagging. Common mesh sizes are 30-40 mesh casein, 60-mesh casein and 90 mesh casein. The casein is packed in sacks or bags of 100 or 200 pounds capacity and as prescribed by grade classification of the casein. Burlap sacks lined with closely woven cloth or with heavy papers or by three ply paper bags may also be used.

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3. CONTINUOUS MANUFACTURE OF EDIBLE CASEIN

Due to the advance in technology and automation, continuous casein manufacturing plants have taken over the batch processes for large production. These plants, not only, reduced manufacturing costs improving the value of milk proteins relating to that of dried milk, but also elevated the status of casein for both industrial and food uses.

A large casein plant with continuous hydrochloric acid precipitation having a capacity of 14000 l per hr. requires only one person to operate it. There are provisions of accurate measuring pumps to ensure a constant flow of milk and acid, mixing acid with skim milk at controlled temperature below, even 25°C to ensure attaining of equilibrium conditions before coagulation begins, automatic regulation of steam injection to achieve the coagulation temperature and a holding tube to obtain complete coagulation and a well structured curd. Such system ensures less than 1% losses of casein in whey. After precipitation, casein curd is concentrated by passage over stationary, inclined and fine mesh screens, which remove between 70 to 90% of the whey. Several dairy companies have installed and are successfully operating, roller presses and lately decanters for dewheying. Hydrocyclones may be employed to recover fine particles from whey and wash water. For continuous washing of casein curd, the most common procedure now adopted is a counter flow which reduces both the volume of water needed and the loss of casein fines - technique involving as many as five washing stages (mostly 3 stages). These tanks are of sufficient capacity to permit an average holding time of 20 to 30 min. Continuous curd pressing is done in mechanically driven roller presses, belt or by passing through decanters, where water is sufficiently expelled for subsequent economical drying.

There have been a number of types of equipment developed for drying casein. Probably the most widely used in recent years is a vibratory type of drier developed in New Zealand. The curd passes through a mill to reduce it to even sized particles, which then travel by means of a vibratory action over trays of perforated stainless steel, transferring to successively lower trays. The heated air flows through the beds of curd from the bottom to the top, thus encountering layers of curd of increasing water content and giving improved efficiency of heat utilization. Of particular interest is a new drying system - attrition drying. The drier features a fast-revolving (1800-2100 rpm) multi-chambered rotor and a stator with a serrated surface. The action involves grinding the curd to very small particles thus exposing a large surface area to the hot air which conveys the curd through the drier. Drying is very fast (1-2 sec) and gives a product similar to ground spray dried casein. 4. REFERENCES Caric, M. 1994. Casein, in concentrated and dried dairy products, P. 199-225. VCH Publishers, Inc., New York. Gupta, V.K. (1989) Technology of edible casein. Indian Dairyman, 41, 643. Mulvihill, D.M. 1989. Caseins and caseinates: Manufacture, in Developments in Dairy Chemistry 4, P.F. Fox (Ed.), P.

97-130, Elsevier Science Publishing Co., Inc., New York. Southward, C.R. 1985. Manufacture and applications of edible casein products. 1. Manufacture and properties. N.Z. J.

Dairy Sci. Technol., 20: 79-101. Southward, C.R. 1994. Utilization of Milk Components: casein, in Modern Dairy Technology. Vol.1. Advances in

milk processing, R.K.Robinson (Ed.) 2nd Edn.,Champman Hall, U.K. P. 375-432. Vijay Kumar (1982) Studies on the utilization of sour buffalo milk for the manufacture of edible casein. Ph.D. thesis

submitted to Kurukshetra University, Kurukshetra.

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Casein is unique amongst dairy products in that it has had a long history of use in foods and in non-food industrial or technical applications. The major use of casein until the 1960s was in technical, non-food applications. Considerable efforts are being made to prepare more and more food and pharmaceutical grade casein during the past 35 years, not only because it gives better return, but also because it is an excellent protein nutritionally and functionally. Still, a sizable amount of casein for non-food uses is being prepared for industrial and technical applications. 2. CASEIN IN GLUES

Casein glues came to be used extensively in Europe probably in the 1890s, but they did not become widely known in the United States until 1917. When that country became involved in the First World War, a need arose for a water-resistant glue for the construction of military aircraft which were made mostly of wood. Interest was aroused in casein glue, especially for plywood, and this led to a thriving industry in its manufacture. Although animal glues had been commercially important for wood gluing long before casein was used, it was difficult to make them water-resistant and their durability was consequently often poorer than that of casein. By the time aircraft construction had shifted largely to metal, casein glue was firmly established in other woodworking industries.

For marketing purposes, casein glues have been classified as (a) prepared glues, and (b) wet-mix glues. Prepared glues were sold in the form of dry powders, which contained all the necessary ingredients except water. The proportion of powder to water was usually about 1:2 by weight and even large batches could be prepared with a mechanical mixer in less than 30 min. Once mixed, the glues generally had to be used within a working day. Wet-mix glues were prepared by mixing together ground casein, water and additional chemicals according to the formula.

Besides casein and water, an alkali must be used to dissolve the casein. This is often sodium hydroxide and provides the third ingredient in a simple-glue. Lime may be added if the glue is to be water resistant. The lime promotes cross-linking of the casein and, over a period of several hours, will cause a casein glue to form an irreversible jelly, which is insoluble in water. Various additives may be employed to change the properties of casein glues, e.g., sodium silicate prolongs the working life of the glue while addition of a soluble copper salt such as copper chloride increases the water resistance of the glue after it has dried. Preservation of solutions of casein glue against putrefaction and mould growth may be accomplished by using chlorophenol derivatives, for instance, and the viscosity of solutions of casein glues may be reduced by addition of a viscosity-modifying agent such as urea or ammonium thiocyanate.

Casein glue is used for gluing timber in internal woodwork, such as laminated beams and arches and in interior doors, plywood, wood particle board and in bonding of Formica laminate to

NON-FOOD USES OF CASEINS NON-FOOD USES OF CASEINS

36

timber. Casein adhesives may also be used for bonding paper, in packaging and in foil laminating, in holding the seam of a cigarette together, in the seaming of paper bags, in the assembly of milk cartons and in securing the abrasive strip on the covers of match boxes.

3. CASEIN AS AN ADHESIVE IN COATING PAPER AND CARDBOARD

Coated paper was developed to satisfy the needs of printers for a paper upon which illustrations, especially fine halftones, could be reproduced satisfactorily. The coating is prepared by mixing mineral material with a solution of an adhesive and applying this mixture in a thin, even layer to the surface of a sheet of paper. the function of the adhesive is to bind the coating material so firmly that it will not be removed or 'picked' off during printing. The coating material, which covers the individual fibres on the paper surface and also fills any hollows between them, forms a surface, which is receptive to printing ink. After calendering (polishing), the surface of the coated paper is smooth, even and continuous which is needed for high quality reproduction of illustrations, in particular. Casein is still a preferred binder for cast-coating and in enamel grades of coating. It is also used in coating stock, label stock and bleached Kraft board for food cartons.

4. CASEIN IN SIZING

Casein glue has long been used as a sizing material, sometimes for the sealing of absorbent surfaces prior to subsequent treatment. The film-forming ability of casein is retained even when deposited from a very dilute solution, and casein sizings have been used on such diverse products as shot-gun shells, heels of ladies' shoes, in varnishes, in paper making, leather finishing and textile manufacture. Casein has also been applied to wool to reduce its felting properties and to artificial textile materials such as Nylon in order to ensure successful wearing or knitting.

The casein film may be given a high degree of water resistance by the inclusion of 'hardeners', either in the solution or by post-application to the film. Casein then becomes a permanent finish which can be applied to paper to enhance its snap, lustre or stiffness. 5. CASEIN IN PAINTS

Casein has a long history of use in paints. As paint technology evolved, synthetic resin emulsions were produced in which the ratio of casein to drying oil was much lower than in the oil-phase reinforced casein paints. The casein became the thickener and emulsion stabilizer and only a minor portion of the binder. After World War II, styrene-butadiene latex paints were developed in which casein was used mainly as a thickener and stabilizer, generally at a level of 1-2% by weight of the finished paint.

In general, casein is used in paints for its ability to disperse both white and coloured pigments and its power to thicken the binder. It may also be used as a protective colloid, as a film former and to improve flow and levelling properties of the paint. The paints are marketed in both powder and paste form.

6. CASEIN IN THE LEATHER INDUSTRY

The use of casein in the leather industry is confined almost entirely to the last of the finishing operations, which consist in coating leather with certain preparations and then subjecting it to mechanical operations such as glazing, plating, brushing and ironing. After finishing in this

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way, leather is said to have been seasoned. For application to leather, casein is first dissolved in alkalis such as ammonia, borax, sodium hydroxide or trisodium phosphate or acids. An acid solution of casein (concentration, 1-6% by weight) produces a clear, bright finish for naturally-finished vegetable tanned leather, commonly known as russet leather, to which up to five coats may be applied. The particular properties of casein which make it desirable for this application are its film-forming properties, adhesive strength and viscosity-enhancing characteristics, which prevent the film from running before it has set. Casein is hard, but tends to be brittle, and it is for this reason that oils and glycerol may be added to the casein solution to increase its plasticity and reduce the brittleness of the film. A casein product, treated with a chlorocarbonate plasticizer, has been proposed to overcome the natural brittleness of casein on its own. Casein may also be incorporated with other binders such as shellac, carnauba wax, blood albumin, gelatin and sulphonated castor oil. More recently, various acrylates have been used with casein (as graft co-polymers) in leather finishing.

7. CASEIN IN RUBBER PRODUCTS

One of the less well-known applications of casein is its use as a reinforcing agent and stabilizer for rubber used in motor vehicle tyres. Casein which had been hardened by formaldehyde was used to replace part of the carbon black used in the vulcanizing of rubber. Measurement of such properties as resistance to breaking, extensibility, resistance to tearing, hardness and abrasion of rubber which contained, for instance, 18% carbon black and 10% casein were either similar to or better than those for rubber containing 28% carbon black and no casein.

The Dunlop Rubber Company Ltd. used casein with paraformaldehyde as a protective colloid to improve the stability of a dispersion of a resin-latex composition which was used to treat textile fibres intended for reinforcing rubber products. Casein gave significantly greater adhesion of textile fibres to rubber compared with fibres without any added casein.

8. USE OF CASEIN IN TEXTILE FIBRES

For producing casein fibres for textiles, acid casein is dissolved in an alkali, such as sodium hydroxide, at a concentration of about 200 g/ltire, and the solution is then forced through a spinneret into a coagulating bath. The bath usually contains acid, inorganic salts and often heavy metal salts. The fibres thus formed resemble wool, except that they have a lower tensile strength and do not 'felt' (i.e. shrink on washing) like wool. The dissolved salt produces a large osmotic pressure and causes a considerable shrinkage in the diameter of the freshly extruded filaments. It also reduces the tendency of the filaments to stick together. Aluminium sulphate is used in such coagulating baths and can also be employed with formaldehyde for stretching and hardening the fibres. The hardening of the fibres is very important since the strength of the wet casein fibres is generally less than half that of the dry fibres. Amongst the more successful hardening process developed is acetylation.

The principal proprietary casein fibres which were developed throughout the world in the decade from 1936 to 1945 included: Aralac (National Dairy Products Corp., USA), Casolana (Co-op Condensfabriek Friesland, Netherlands), Fibrolane (Courtaulds Ltd., UK), Lanital and Merinova (Snia Viscosa, Italy). Of these, only Fibrolane and Merinova were still in production by 1971, though two other casein fibre products were being produced elsewhere (Wipolan, in Poland and Chinon, in Japan).

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Casein fibres were used during and after the war years, usually in combination with wool and other fibres, such as cotton, viscose, rayon, etc., in a variety of products, such as flannel, woolen spun cloth (overcoats, blankets), felt hats (up to 25% casein fibre with wool), filling materials such as artificial horsehair, and in carpets and rugs. Bristles were also produced from casein fibres for use in brushes of various types. The importance of casein fibre for textiles has now declined in the face of competition from other fibres. However, co-polymer fibres containing casein have been prepared in Japan as a substitute for silk, which was still being undertaken during the preparation of this article.

9. RENNET CASEIN PLASTICS

Rennet casein produces a plastic, which is far superior to that of acid casein. In the production of casein platic, the casein (if unground) is milled, sieved through a screen with apertures of about 600 µm and mixed thoroughly with water to a final moisture content between 20 and 35%. At this stage, a filler such as titanium dioxide or zinc oxide may be added to produce either a white or opaque plastic, and dyes may also be included to produce coloured plastic. The wet casein is then stored for several hours or overnight to allow the water and casein to come to equilibrium and ensure a uniform moisture content throughout the whole mixture. The extrusion mixture is then placed in a hopper feeding the extruder, which consists of a screw rotating in a water-cooled barrel. The casein mixture is delivered by the screw into a heated nozzle section, where it undergoes several compression and expansion stages and is consequently formed into casein plastic. Extrusion of the plastic mass generally occurs in the temperature range of 60-100°C to produce a smooth rod or strip sheet from the nozzle section. Extruders can be equipped with up to three screws, which may feed casein with different dyes into a single nozzle section. By manipulation of the feed rates of the casein in each barrel and alteration of the design of the mixing head where the various streams of casein platic merge, it is possible to produce many beautiful and intricate designs in the plastic.

The warm plastic, which emerges from the nozzle of the extruder is initially soft and pliable. This is immediately immersed in cold water, which has the effect of hardening the plastic and preventing or reducing the development of internal stresses. Rods of casein plastic are subjected to 'dowelling' after they have cooled. This process trims the surface irregularities to produce smooth rods of uniform diameter. These are sliced into discs (or 'button blanks'), which are subsequently placed into a dilute solution of formaldehyde for several weeks in order to 'cure' or harden the plastic. The cured blanks are later dried, machined into butons and finally polished, usually by mechanical tumbling in the presence of wood chips and oil seeds.

If sheet plastic from casein is required, rods of casein plastic are placed side by side in a heated hydraulic press and then subjected to high pressure. The casein plastic sheet so produced must still be cured in formaldehyde for periods varing from 1 week to 6 months, depending on the thickness of the plastic. Once cured, the plastic sheets may be dried carefully to avoid rupturing the material or setting up stresses and strains within it during the expulsion of excess moisture and formaldehyde. Sheets of casein plastic still tend to warp during this process and must be straightened in low pressure hydraulic presses. Even when hardened, casein plastic can absorb moisture and its moisture content can vary according to changes in humidity. This limits its use in large panels or long rods which can warp badly. Even though many different treatments of casein and casein plastic have been made in an effort to overcome the problems outlined above, they have not caused a significant increase in the commercial production of casein plastic.

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Although articles fashioned from casein plastic have included knife handles ('imitation ivory'), combs, imitation tortoise shell, pens, shoehorns and dominoes, the present range of casein plastic articles is somewhat more limited and includes buttons, buckles, novelties and knitting needles. In spite of this limitation, casein plastic articles do take up a great range of dyes to produce very attractive patterns which are somewhat more difficult to reproduce in the more common plastics made from petrochemicals. 10. MISCELLANEOUS TECHNICAL APPLICATIONS FOR CASEIN

Amongst the large number of other technical (non-food) applications where casein has been used (or at least claimed to be used) are in cleaners and dish washing liquids, hair setting products and cosmetics and cheese marks. In building and civil enginering, uses for casein are claimed in the preparation of bitumen emulsions, in light weight concrete, in gypsum wallboards, in the preservation and restoration of old stone buildings and as a foaming agent for de-icing equipment, roads and runways. In printing, casein is claimed to be used as a film-forming transfer regulator in a thermal printing adhesive ink and as a binder for a printable coating on a foamed polystyrene sheet. Casein has been used in photo etching for the production of shadowmasks for colour television sets, computer circuitry and electronic ignition components for motor vehicles. Cross-linked casein has been used for water purification and for recover of chromium from waste electroplating liquors.

In agriculture and horticulture, casein has also been used in insecticide sprays (as a spreader), in fungicides (as an adhesive), as a fertilizer and in coated seeds (as an adhesive).

11. REFERENCES Anon. 1987. Food Technol. N.Z., 22 (6) : 5. Fox, K.K. 1970. In : By-products from milk, B.H. Webb and E.O. Whittier (eds), 2nd edn, AVI Publishing Co., Inc.,

Westport, Connecticut, pp. 331-355. Munro, P.A., Southward, C.R. and Elston, P.D. 1976. N.Z. J. Dairy Sci. Technol. 11 : 40. Salzberg, H.K. 1997. In : Handbook of Adhesives, I. Skeist (ed.), 2nd edn, VAn Nostrand Reinhold Company, New

York, pp. 158-171. Southwood, C.R. 1989. Uses of Casein and Caseinates, in Developments in Dairy Chemistry-4, P.F. Fox (Ed. ),

Elsevier Science Publishers Ltd., England. P.173-244.

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Dr. Vijay Kumar Gupta Principal scientist

Dairy Technology Division, N.D.R.I., Karnal -132001

1. INTRODUCTION

The soluble form of casein, caseinates may be prepared from freshly precipitated acid casein curd or from dry acid casein by reaction with dilute solution of alkali (such as sodium, potassium, calcium or ammonium hydroxide). Sodium caseinate is the most commonly used water soluble form of casein and is used in wide range of processed food products as a source of protein, and for their physico-chemical, nutritional and functional properties. Next to sodium caseinate, calcium caseinate is common and finds use in both pharmaceutical preparations and as a food ingredient. It functions as supplier of both calcium and protein. The specifications for this product vary with its end use, but they frequently include a limitation of the calcium content to within the range of 1.0-1.5%. 2. MANUFACTURING PROCESS

For the manufacture of caseinates, fresh acid casein curd is preferred over dried casein as raw material, since the former yields caseinates with blander flavour than does the latter. Caseinates prepared from dry casein will also incur the additional manufacturing costs associated with drying, dry processing, bagging and storage of the casein prior to its conversion to sodium caseinate. However, in countries that import casein, buyers may still prefer to purchase casein and produce their own sodium caseinate. Casein should have a low calcium content (< 0.15% dry basis) in order to produce a caseinate solution with a low viscosity, and a low lactose content (< 0.2% dry basis) to produce sodium caseinate with the best colour, flavour and nutritional value. Control of the curd characteristics is also important to ensure rapid dissolution. 2.1 Sodium caseinate

Irrespective of the starting material used, the manufacture of sodium caseinate consists of the formation of a casein suspension, solubilization of casein using sodium hydroxide, and drying the sodium caseinate produced. 2.1.1 Casein suspension and solubilization

The main difficulties experienced in the conversion of acid casein to sodium caseinate are: (a) the very high viscosity of sodium caseinate solution of moderate concentration, which limits the solids content for spray drying to 20%, (b) the formation of a relatively impervious, jelly-like, viscous coating on the surface of casein particles, which impedes their dissolution on addition of alkali. To overcome the former difficulty, it is essential that the pH and temperature are controlled during conversion as these influence viscosity, while the latter can be overcome by reducing the particle size by passing the casein and water mixture through a colloid mill prior to addition of alkali.

DEVELOPMENTS IN THE MANUFACTURING TECHNOLOGY OF CASEINATES

DEVELOPMENTS IN THE MANUFACTURING TECHNOLOGY OF CASEINATES

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After the final casein wash, the curd may be dewatered to about 45% solids and then mixed with water (to 25-30% solids) prior to entering the colloid mill. The temperature of the emerging slurry, which may have the consistency of 'toothpaste', should be below 45o C, since it has been observed that milled curd can re-agglomerate at higher temperatures. 2.1.1.1 Addition of Alkali and pH control

The commonest alkali used in the production of sodium caseinate is sodium hydroxide in the form of 2.5 M solution. The quantity of sodium hydroxide required is generally 1.7-2.2% by weight of the casein solids. Other alkalis such as sodium bicarbonate or sodium phosphates may be used, but the amounts required and their cost are both greater than those of sodium hydroxide. Hence, they would generally be used only for specific purposes such as in the manufacture of citrated caseinate. The addition of the dilute alkali (preferably by dosing into the recirculating line just prior to the pump (Fig. 2D.) must be carefully controlled with the aim of reaching a final caseinate pH of 6.6-7.0 (generally about 6.7). The recommended technique for achieving the correct caseinate pH is to add sufficient alkali to bring the pH close to, but below the specified value and then add the additional alkali needed towards the end of the dissolving operation. This technique is used for two main reasons firstly, because reduction of the pH of a sodium caseinate solution by addition of acid is likely to cause localised precipitation of casein and, secondly, the development of any off-flavours associated with localised conditions of high alkalinity is minimized. A third reason is the potential for formation of lysinolamine when the pH is excessively high (e.g.>10). 2.1.1.2 Dissolving The viscosity of sodium caseinate solutions is a logarithmic function of the total solid concentration. Each dissolving vat, therefore, must be equipped with a powerful agitator and a high- speed recirculating pump (Fig. 2.F1, F2). In addition to concentration, other factors, which affect viscosity of sodium caseinate solutions are temperature (semi-logarithmic), pH, calcium content of the curd, type of alkali used and seasonal and genetic factors. Once the alkali has been added to the casein, it is important to raise the temperature as quickly as possible to 60-75oC to reduce the viscosity. However, care should be taken to avoid holding the hot (> 70oC) concentrated sodium caseinate solution for extended periods prior to drying, since brown colour may develop in the solution due to reaction between the protein and residual lactose. During the dissolving operation, the incorporation of air should be kept to a minimum, since caseinate solutions form very stable foams. Therefore, all joints on pipes, especially on the suction side of pumps, must be airtight and the recirculation line must discharge below the surface of the liquid in the dissolving vat. In view of the many variables, which can affect the viscosity of sodium caseinate solutions, it is considered desirable to standardize them to a constant viscosity, rather than to a constant concentration, prior to drying. 2.1.2 Drying of sodium caseinate solution

The homogeneous sodium caseinate solution is usually spray dried in a stream of hot air. In order to ensure efficient atomization of the sodium caseinate solution, it must have a constant viscosity as it is fed to the drier. It is common practice to minimise the viscosity by preheating the

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solution to a temperature of 90-95oC just prior to spray drying (Fig. 2H). However, care should be taken to minimise the time for which the caseinate solution is at high temperature.

Fig.1. Suggested plant layout for dissolving casein in sodium caseinate manufacture

The total solid content of the solution destined for spray drying ranges between 20 and 22% and only occasionally may be as high as 25%. The highest possible caseinate concentration is determined experimentally for every spray drier. With 20% sodium caseinate solution, about 4 kg of water needs to be evaporated to produce 1 kg of powder. Hence, the solids output from a drier used for caseinate is about one quarter of that when used for drying of skim milk (feed concentration usually 45-50% solids). However, it is possible to increase the inlet air temperature in order to increase the water evaporation rate. Thus, while steam radiators may produce inlet air temperatures of up to 170oC, it is possible (with indirect oil heating or direct gas firing) to produce inlet air temperatures of up to 260oC. The warm spray dried sodium caseinate powder is cooled in a fluid bed drier. The low solids content of the feed solution produces a spray-dried powder with a low bulk density. Bulk density may vary from 0.25 g/ml to 0.40 g/ml. Generally, pressure nozzle dryers, operating at 100-250 bar, produce caseinate with a higher bulk density than that from disc atomizing dryers. The powder particles produced on disc dryers tend to be in the shape of hollow spheres. Since the powder is so light, the losses from the product recovery cyclones may be rather high, and it is, therefore, considered prudent to install bag filters for improved recovery.

The moisture content of spray-dried sodium caseinate should be less than 5% for satisfactory storage and this appears to be consistent with many product specifications. Other

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methods used to reduce cost, increase processing rate during caseinate manufacture and control the properties of the resulting powders include:

• Production of roller dried sodium caseinate by feeding a mixture of curd (50-65% moisture) and an alkaline sodium salt (Na2CO3 or NaHCO3) onto the drying drum of a roller-drier. Sodium caseinate with good flavour and a high bulk density could be produced by using the roller drier at relatively low steam pressure (i.e. low drying temperature).

• Production of granular sodium caseinate by lowering the moisture content of acid casein curd to < 40%, reacting the curd with Na2CO3 with agitation for up to 60 min and drying the resultant caseinate in a pneumatic ring drier or a fluidized bed drier. The resulting caseinate has a higher bulk density and improved dispersibility compared to spray and roller dried product.

• Drying a mixture of acid casein curd (45% dry matter) and Na2CO3 in an attrition drier to produce a product that looks like spray dried sodium caseinate but which has a much higher bulk density.

• Spray drying sodium caseinate solutions of higher solids content (up to 30%) in a drier fitted with a modified atomizer disc or preparation of concentrated caseinate solutions (33-47% solids) by a modified procedure and drying these solutions in spray or roller dryers or by an extrusion drying method.

• Conversion of casein to caseinate in the presence of a limited amount of water using extrusion techniques. Caseinate in non-soluble powder form is prepared continuously from casein. In one procedure, casein in powder form is introduced continuously into an extruder machine, where in the first step, it is transported with water. An alkaline reagent is introduced in second step and the whole mixture is subjected to intense kneading under pressure with a rise in temperature to initiate the chemical reaction between casein and the alkaline reagent. The mixture is subjected to second intense kneading under pressure and intense shear in order to finish off the chemical reaction with the temperature of the mixture rising so as to cause the mixture to melt and so as to obtain a viscous caseinate paste. It is then cooled with a degassing operation to reduce and adjust its temperature and the viscosity. Finally caseinate paste is extruded at 70-90oC with a moisture content lying in the range of 30-40% to form a continuous strand of caseinate paste at the outlet from the extruder machine. A continuous thin sheet of caseinate paste is formed from the strand and said sheet of caseinate is simultaneously cooled to a temperature below 20oC. The sheet is cut longitudinally into a plurality of parallel strips and the strips are cut into small sized pieces of caseinate. Acid casein conversion to sodium caseinate using extrusion is used commercially.

2.2 Ammonium/Potassium/Citrated Caseinates

Ammonium and potassium caseinates may be prepared by a method similar to that used for the production of sodium caseinate by substituting Ammonium hydroxide or potassium hydroxide for sodium hydroxide. Granular ammonium caseinate may be prepared by exposing dry acid casein to ammonia gas and removing excess ammonia with a stream of air in a fluidized bed degassing system. Citrated caseinate has been prepared by a method similar to that used for the

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preparation of spray dried sodium caseinate by using a mixture of trisodium citrate and tripotassium citrate in place of sodium hydroxide. 2.3 Calcium caseinate

In contrast to the translucent, viscous, straw-coloured sodium, potassium and ammonium caseinate solutions, calcium caseinate forms micelles in water, producing an intensely white, opaque, 'milky' solution of relatively low viscosity. Calcium caseinates are much less soluble and have poorer functional attributes than sodium caseinate. Its preparation follows the same general process as that used for sodium caseinate with one or two important exceptions. Calcium caseinate solutions may be destabilized on heating especially at pH values below 6. This sensitivity decreases with an increase in pH or a decrease in concentration and is manifested as a reversible heat-gelation. During the dissolving process, it has been found that the reaction between acid casein curd and calcium hydroxide (the alkali most commonly used in the production of calcium caseinate) proceeds at a much slower rate than that between curd and sodium hydroxide. The temperature of conversion is a particularly important factor in determining the completeness of solubility (assessed from the amount of sedimentable matter) of the calcium caseinate. Therefore, the dissolving process must be closely monitored to ensure production of calcium caseinate with a good solubility. The optimum process as recommended by Roper (1977) is: pass 'soft' casein curd through a mixer to give evenly-sized particles, mix with water to about 25% total solids, pass the mixture through a colloid mill and adjust the temperature to give a milled slurry at 35-40oC; mix the slurry with a metered volume of 10% aqueous calcium hydroxide to give the desired final pH; agitate and recirculate in a low-temperature conversion tank until conversion is complete (> 10 min); heat the dispersion in a tubular heat exchanger to 70oC and pump directly to a spray drier. The common associated problems in the manufacture of calcium caseinate have led to an examination of alternative methods of producing calcium caseinate. Calcium hydroxide is soluble in sugar solutions and may, therefore, be used in this form for reaction with acid casein. In order to increase the rate of reaction between casein and calcium (hydroxide), the casein may first be dissolved completely (and rapidly) in ammonia. Calcium hydroxide solution of sugars (sucrose, glucose, galactose, lactose or fructose) is then added and the solution of calcium caseinate dried by means of a roller drier. Most of the ammonia evaporates during processing, leaving a relatively pure calcium caseinate (moisture 4.5%, protein 84%, sucrose 5.8%, calcium 1.0%) with nutritional properties similar to those of the original casein. 2.4 Other caseinates

Magnesium caseinate is prepared from casein and a magnesium base or basic salt such as magnesium oxide, magnesium hydroxide, carbonate or phosphate or by ion exchange. Compounds of casein with aluminium may be prepared for medicinal use or for use as an emulsifier in meat products. Heavy metal derivatives of casein, which have been used principally for therapeutic purposes include those containing silver, mercury, iron and bismuth. Iron and copper caseinates have also been prepared by ion exchange for use in infant and dietetic products.

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3. COMPOSITION OF CASEINATES

The quality standards for sodium caseinate vary from country to country. The typical composition of sodium and calcium caseinate, produced from well-washed acid casein, is shown in Table 1. The ash content of the caseinates includes approximately 1.8% derived from the organic phosphorus, which forms an integral part of the casein molecule. With a pH generally in the range 6.5-7.0, sodium caseinate will usually contain 1.2-1.4% sodium, while the calcium content of calcium caseinate is generally in the range, 1.3-1.6%. Table 1. Typical composition of caseinates

Sodium caseinate Calcium caseinate

Moisture (%) 3.8 3.8 Protein (N x 6.38) (%) 91.4 91.2 Ash (%) 3.6 3.8 Lactose (%) 0.1 0.1 Fat (%) 1.1 1.1 Sodium (%) 1.2 -1.4 < 0.1 Calcium (%) 0.1 1.3-1.6 Iron (mg/kg) 3-20 10-40 Copper (mg/kg) 1-2 1-2 Lead (mg/kg) <1 <1 pH 6.5-6.9 6.8-7.0 4. REFERENCES Caric, M. 1994. Casein, in concentrated and dried dairy products, P. 199-225. VCH Publishers, Inc., New York. Mulvihill, D.M. 1989. Caseins and caseinates: Manufacture, in Developments in Dairy Chemistry 4, P.F. Fox

(Ed.), P. 97-130, Elsevier Science Publishing Co., Inc., New York. Roeper, J. 1977. N.Z.J. Dairy Sci. Tekchnol., 12: 182 Southward, C.R. 1985. Manufacture and applications of edible casein products. 1. Manufacture and properties.

N.Z. J. Dairy Sci. Technol., 20: 79-101. Southward, C.R. 1994. Utilization of Milk Components casein, in Modern Dairy Technology. Vol.1. Advances in

milk processing, R.K.Robinson (Ed.) 2nd Edn.,Champman Hall, U.K. P. 375-432. Vijay Kumar (1982). Studies on the utilization of sour buffalo milk for the manufacture of edible casein. Ph.D.

Thesis submitted to Kurukshetra University, Kurukshetra.

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Dr. R.S. Mann and Dr. V.K. Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Following precipitation of caseins from milk by acidification or renneting, the whey proteins remain soluble (in the whey). However, these can be precipitated in combination with the casein by first heating milk to temperatures that denature and induce complexation of the whey proteins with casein, followed by precipitation of the milk protein complex by acidification to pH 4.6 or by a combination of added CaCl2 and acidification. Precipitation of casein and whey proteins together from heated skim milk by acidification was termed as "co-precipitate' first by Scott (1952). Previously this name was confined to milk proteins only, but recent work has widen the term "Co-precipitates" in the sense that it covers the combination of milk proteins as well as the proteins derived from other biological systems. A co-precipitate of milk proteins may be defined as the product which separates as the solid phase after the heat treatment and precipitation of dairy fluid, or mixtures of dairy fluids, which contain both casein and heat coagulable whey proteins. The resultant product contains a significant proportion of the whey proteins and almost all the casein present in the raw material.

Co-precipitates may be used as an ingredient in the preparation of various food products such as in dairy, meat, baked, confectionary, snack and animal and pet foods, either to contribute the desired functional properties or to improve the nutritional qualities of the products. 2. MANUFACTURING PROCESS

Early developments in the manufacture of co-precipitates occurred mainly in the USA and USSR. In the USA, milk was heated to 90°C and the proteins precipitated by direct acidification while in the USSR, milk at 80-90°C was treated with CaCl2 to precipitate the protein.

Buchanan et al. (1965) published the first report on an Australian method for co-precipitate manufacture. In this method, skim milk was heated to about 90°C and CaCl2 added to a level of 0.25%. After holding for 1 min, the curd was separated from the supernatant and washed once at 29-36°C. The wet curd was redispersed in 2% sodium tripolyphosphate and spray-dried.

A method for the recovery of co-precipitates from milk using acid and/or CaCl2 was described by Muller et al. (1966). Skim milk was heated at 92°C for 15 or 5 min prior to protein precipitation by acid or CaCl2, respectively. The resulting precipitates were washed once, pressed and dried. Approximately 90% of total proteins was recovered by these processes. The manufacture of a range of co-precipitates with diffeent calcium contents was described by Muller et al. (1967). The terms high-, medium- and low-calcium were used to define co-precipitates with calcium contents in the range 2.5-3.0, 1.0-2.0 and 0.5-0.8%, respectively. The level of calcium in the product was controlled by varying the pH of precipitation, the amount of CaCl2 added and the length of time for which the milk was held at 90°C. For a low-calcium co-precipitate, milk was

DEVELOPMENTS IN THE MANUFACTURING TECHNOLOGY OF MILK PROTEIN CO-PRECIPITATES

DEVELOPMENTS IN THE MANUFACTURING TECHNOLOGY OF MILK PROTEIN CO-PRECIPITATES

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heated at 90°C for 15-20 min in the presence of 0.03% CaCl2 and precipitated at pH 4.6; for a medium-calcium co-precipitate, milk was heated at 90°C for 10-12 min in the presence of 0.06% CaCl2 and precipitated at about pH 5.4; for a high-calcium co-precipitae, milk was heated at 90°C for 1-2 min and 0.2% CaCl2 added to precipitate the protein. Yields of protein were reported to be 95-97% of total milk protein.

Laboratory and pilot scale studies to determine the effects of skim milk pH (6.2-6.8), heat treatment conditions (85°C, 2-16 min), quantity of added acid or CaCl2 (giving whey pH varying from 4.0 to 6.0) and coagulation temperature (48-78°C) on curd characteristics and the yield of protein were performed. The firmest curd and the highest protein yield were obtained when skim milk at pH 6.4 was heated at 85°C for 6 min prior to protein precipitation. Acid (low-calcium) and medium-calcium co-precipitates were precipitated from the cooled milk at a coagulation temperature of 58-60°C and 65°C, respectively, and at whey pH's of 4.9-5.1 and 5.1-5.5, respectively, while high-calcium co-precipitate was precipitated from the cooled milk at a coagulation temperature of 77°C by adding 0.2% CaCl2. Yields of co-precipitates using the conditions described above were 92.7% for the acid, 94.1% for the medium-calcium and 98.5% for the high-calcium co-precipitates. These findings were confirmed by Vattula et al. (1979). The Austrialian plant design for the continuous manufacture of coprecipitates is illustrated in Fig. 1.

Co-precipitates produced by the methods described have poor solubility properties and processes for the manufacture of similar products but with good solubility and giving protein yields higher than those of conventional co-precipitates have been described. Connolly (1982) prepared a product referred to as 'total milk protein' by heating skim milk to between 40 and 70°C, alkalizing it to pH 9.5-10.5 and holding for 3 min. The milk was then acidified to pH 3.5 and held for 5 min before the protein was precipitated by adjustment to pH 4.7. The whey protein in the product was claimed to be undenatured and protein yield to be as high as about 99% of true protein.

Lankveldt (1984) described a process for the production of 'soluble lactoprotein'. In this process, skim milk is adjusted to pH 7-7.5, heated to temperatures in the range 80-145°C for a period ranging from several seconds to about 20 min and then cooled to a temperature between 4 and 45°C. The pH of the milk is then adjusted to 4.4-4.7 to precipitate the proteins. The curd is separated, washed, dispersed in water, dissolved at pH 6.7 and spray-dried.

More recently, Grufferty and Mulvihill (1987) showed the maximum protein yield from skim milk was obtained by adjusting milk to pH 7.5, heating at 90°C for 15 min, cooling to 30°C and precipitating the protein at pH 4.6. Heating at a pH above or below pH 7.5 for a period shorter than 15 min at 90°C at pH 7.5, reduced the protein yield. Heating at 140°C for 4.8-9.6 s also gave a reduced protein yield. Protein yields from milk heated in the range 40-70°C at pH 9-10.5 (a process similar to that of Connolly) were much lower than when milk was heated at pH 7.5. Furthermore, the solubilities of the protein isolates prepared by acid precipitation of heated milks were about 100% when skim milk was adjusted to pH > 7.5, before heating. 3. COMPOSITION OF CO-PRECIPITATES

The proximate compositional analysis of granular (insoluble) co-precipitates, prepared from curd, which is washed twice in water, is shown in Table 1. The data show that the calcium content of co-precipitates decreases steadily as the ash content decreases. Both of these effects are caused by a decrease in the pH of precipitation. The fat content of the co-precipitates increases

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from 0.6% to 0.9% as the pH of precipitation is reduced from 5.9 to 4.9. The high ash content of high calcium co-precipitate leads to a consequential reduction of 3-5% in the protein content of the product relative to lactic and acid caseins and acid co-precipitate.

Fig. 1. Co-precipitate manufacture - Australian plant design. ____, Product flow line: --

---, water flow line: - - -, acid or CaCl2 flow line. Table 1. Proximate compositional analysis of granular co-precipitates

Co-precipitates High calcium Medium calcium Acid Moisture (%) 9.5 9.5 9.5 Fat (%) 0.6 0.7 0.9 Ash (%) 7.7 3.7 2.4 Protein (N x 6.38) (%) 81.7 85.6 86.7 Protein (dry basis) (%) 90.3 94.5 95.8 Lactose (%) 0.5 0.5 0.5 Calcium (%) 2.81 1.13 0.54 pH 6.5-7.2 5.6-6.2 5.4-5.8 pH of whey after separation of curd 5.8-5.9 5.1-5.3 4.9-5.1

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Proximate compositional analysis and physical properties of soluble and dispersible co-precipitates are presented in Table 2. Soluble high calcium co-precipitate has very high ash content (13.5%) due in part to the presence of sodium tripolyphosphate and contains almost 2% sodium. Soluble acid co-precipitate has a composition similar to that of sodium caseinate (Table 2). In order to render medium calcium co-precipitate substantially (> 90%) soluble at pH 7.5, it is necessary to use both, sodium hydroxide and sodium tripolyphosphate (or some other complex phosphate). Table 2. Proximate compositional analyses and physical properties of spray-dried soluble co-precipitates.

Analysis Product A B C D E Moisture (%) 4 4 4 4 4 Fat (%) 0.6 0.6 0.7 0.7 0.9 Ash (%) 13.5 10 5 6.7 4.1 Protein (N x 6.38) (dry basis) (%) 84.5 88.0 93.0 91.5 94.0 Lactose (%) 0.5 0.5 0.5 0.5 0.5 Calcium (%) 2.9 2.9 1.2 1.2 0.5 Sodium (%) 1.9 0.6 0.6 - 1.1 pH 7.1-7.2 6.5-6.9 6.6-7.2 6.6-7.2 6.6-7.2 Solubility (%) 92 72 70-90 95-98 97-98 Farinograph water absorption (%) 278 129 193 282 292

Product: A High calcium co-precipitate rendered soluble by the addition of sodium tripolyphosphate

(6% w/w). B High calcium co-precipitate rendered dispersible by the addition of sodium

tripolyphosphate (2% w/w). C Medium calcium co-precipitate, neutralized with sodium hydroxide. D Product C containing additional sodium tripolyphosphate (2% w/w). E Acid co-precipitate, neutralized with sodium hydroxide.

The successful and potential uses of co-precipitates in food products will depend upon the satisfactory functional properties of the proteins that are added in the food system so that in body, texture, flavour and nutritive characteristics etc. of the finished product are not adversely affected and remain acceptable to the consumers. the investigations conducted so far indicated that the co-precipitates are used mostly in a variety of food products in small proportions to improve their functional and nutritive properties. 4. REFERENCES

Buchanan, R.A., Snow, N.S. and Hayes, J.F. 1965. The manufacture of calcium Co-precipitate Aust. J. Dairy Techno., 20 : 139.

Lankveldt, J.M.G. In : Milu Proteins' 84. Proc. Int. Congr. on milk proteins. Grufferty, M.B. and Mulvihill, D.M. 1987. J. Soc. Dairy Technol. 40 : 82. Mulvihill, D.M. 1989. Caseins and caseinates: Manufacture, in Developments in Dairy Chemistry 4, P.F: Fox

(Ed.), P. 97-130, Elsevier Science Publishing Co., Inc., New York. Muller, L.L. 1982. Manufacture of casein, caseinates and coprecipitates, in Development in Dairy Chemistry, P.

F. Fox (ed.), Vol I, pp. 328-330.

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Muller, L.L., Hays, J.F. and Snow, N. 1967. Studies on coprecipitates of milk proteins. Part I. Manufacture with varying calcium contents. Aust. J. Dairy Technol. 22, 12.

Muller, L.L.; Snow, N., Hayes, J.F. and Buchanan, R.A. 1966. The manufacture of specialized casein products. Scott, E.C. 1952. Casein-Lactalbumin coprecipitate and method of its preparation. US Palent 2, 623,038. Cited

in Dairy Sci. Abstr. 15:962 (1953). Sen, D.C. 1985. Usage of Co-precipitates in Foods. Indian Dairyman 37 : 579-585. Southward, C.R. and Goldman, A. 1975. Co-precipitates - A review. N.Z. J. Dairy Sci. Technol. 10, 101-112. Southward, C.R. 1985. Manufacture and applications of edible casein products. 1. Manufacture and properties.

N.Z. J. Dairy Sci. Technol., 20: 79-101.

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S.K. Kanawjia* and Vikash Gupta** Hitesh Gahane*** *Principal Scientist, **Research Associate, ***Research Scholar

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION The manufacture of protein hydrolysates is a well-established process for the utilization of protein rich food by-products, wastes and non-conventional food proteins. Historically, soy sauce was the first protein hydrolysate. The fundamental flavour characteristic of a protein hydrolysate is suggestive of meat flavour. Therefore, hydrolysates are used by packer, canners and other manufacturers, who wish either to accentuate or to suggest the flavour of meat. Fish protein concentrates and egg albumin are two popularly used animal products as substrate for hydrolysates, while soy protein is the extensively used vegetable source. Casein is an important substrate for the preparation of milk protein hydrolysates. The hydrolysate of each protein has distinct flavour characteristic. Different proteins may be blended to give a specific flavour characteristic. Lighter and more delicately flavoured hydrolysates are derived from casein, corn and rice proteins and are preferred for fish and pork as well as poultry. Darker and heavier hydrolysates, on the other hand, are derived from soy, yeast and gluten and are preferred for beef and mutton. 2. PROCESSES OF CASEIN HYDROLYSATES MANUFACTURE Usually, functional properties of a milk protein product are specific parameters which determine its applicability in a diverse array of food commodities. Casein hydrolysates can be manufactured by acid, alkaline and enzymatic hydrolysis of casein. 2.1 Acid Hydrolysis In this process, casein or caseinates are allowed to react with arsenic-free hydrochloric acid (35-45%) or sulphuric acid for a period of 4-18h. The reaction is carried out at higher temperature (80-100°C). Thereafter, the content is neutralised to pH 6.0-7.0 by an alkali. The product is then either concentrated or dried. Although acid hydrolysis has been used for preparing casein hydrolysates, the process has some limitations. The acid hydrolysis ordinarily entails the complete or partial destruction of some of the amino acids like tryptophan. This process also poses problem of removing residual acid from the products of hydrolysis. Neutralisation of acid employed for hydrolysis result in the formation of salts which in turn becomes another limiting factor in food/dietic applications. 2.2 Alkaline Hydrolysis This is another process of manufacturing protein hydrolysates. However, it is not effective method as it leads to the destruction of some and the recemisation of most amino acids. It also

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TECHNOLOGY OF MANUFACTURE 0F MILK PROTEIN HYDROLYSATES

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results in partial chemical hydrolysis, oligomerisation and destruction of functional groups of amino acid residues. 2.3 Enzymatic Hydrolysis Proteolytic enzymes have ability to hydrolyse proteins to peptides and amino acids. The chain length of peptides formed is dependent upon the extent of hydrolysis, condition of hydrolysis, type, concentration and activity of enzyme, and type of protein to be hydrolysed. This process of hydrolysis of casein results in product with high content of water soluble nitrogen and low salt. Another advantage of this process is that enzymatic hydrolysis causes no destruction or recemisation of the amino acids and nutritional quality of the original protein is retained. Proteolytic enzymes could be obtained from plant (Papain, ficin, bromolain), animal (Pepsin, trypsin, Rennin) or microbial origin (Neutrase, Alcalase, Esperase, Pronase, Naturage, etc.). Hydrolysis of casein has been performed both as a single stage process and a two-stage process. The extent of casein hydrolysis, which represents the extent of protein breakdown to peptides and amino acids is expressed either as percent amino nitrogen or as degree of hydrolysis (DH). Degree of hydrolysis is the ratio of the number of peptide bonds cleaved and the total number of peptide bonds in the intact protein. In the manufacture of casein hydrolyates, per cent DH is one of the important controlling factors which reflects on the product quality. Proteolytic enzymes have the ability to hydrolyse proteins into peptides and amino acids and could be obtained from plant (Papain, ficin, bromolain), animal (Pepsin - from stomach secretion, trypsin - from pancreatic secretion, rennin - from calf stomach) or microbe (Neutrase, Alcalase, Esperase, Pronase, etc.). As per their specificity of action they can be classified into exopeptidases and endopeptidases.

• Exopeptidases refer to enzymes, which split terminal amino acids from one end of the chain by hydrolysis of peptide bond - mostly microbial enzymes. One can distinguish the Carboxy (exo) peptidases which act on the terminus of chain carrying free carboxyl group and the Amino (exo) peptidases which start from the other end i.e. the terminus of chain carrying free amino group.

• Endopeptidases have preference for certain side chains on amino acids adjoining peptide bond and are usually divided into three groups: (i) The pepsin type of protease is characterized by a preference for amino acids with free carboxyl groups, (ii) Trypsin types are characterized by a preference for amino acids with basic group, and (iii) Chymotrypsin types are characterized by a preference for amino acids with aromatic or bulky chains. Papainase type enzymes (Papain, chymopapain, ficin, bromelain) are endopeptidases, but are difficult to classify under this scheme.

Choice of the protease for protein hydrolysis depends mainly on its specificity and also on its pH optimum, heat stability and the presence of activators or inhibitors. The production of protein hydrolysates for flavour purposes requires enzymes of broad specificity, which lead to extensive hydrolysis into low molecular weight peptides and amino acids. Individual endopeptidases do not split all or even a majority of the peptide bonds in a protein system leading

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to the formation of bulky, hydrophobic acid chains, which give bitter taste. Exopeptidases are reported to hydrolyse carboxyl and amino terminal amino acids of such peptides, thus eliminating the bitter taste. Hydrolysis of protein has been carried out both as a single stage process (where enzyme is added once during the hydrolysis period) and a two-stage process (addition of two or more enzymes are added at subsequent intervals of hydrolysis). 2.3.1 Single-stage hydrolysis

In single-stage process, Kodjev et al. (1974) used 0.4% pancreatin as the proleolytic enzyme at pH 8.2-8.7, temperature 65°C, yeast preparation 0.2% and sodium triphosphate 6% as the optimal conditions for the manufacture of casein hydrolysate with improved flavour qualities. In other study three enzymes, pancreatin, proteinase of Aspergillus oryzae and Lactobacillus helveticus were added to 10% casein solution and incubated at 50°C for 24 h. Inactivation of proteolytic enzymes was done by heating at 85°C for 15 min (Takase, et al., 1979).

Vegarud and Langsrud (1989) manufactured hydrolysates from commercial sodium caseinate using various commercial proteolytic enzymes: Corolase PS, Corolase L10, Maxatase LS 400,000 and Novozym 257. Hydrolysis was carried out in a pH-stat at constant pH using IM NaOH for 5 h at 40°C at pH 6.7-7.0 in a vessel, with substrate ratio in the range of 0.1 to 5%. The hydrolysis process was terminated by inactivating the enzyme by heating at 100°C for 5 min. 2.3.2 Two stage hydrolysis

A process was described for the production of casein hydrolysate on commercial scale with 12 Kg casein suspended in 220 l. water at pH 6.2-6.3 and digested with papain at 40°C for 18h and then with a pig kidney homogenate at pH 7.8-8.0 for 24 h. The hydrolysate produced was passed through a separator to remove insoluble material followed by heating at 83-85°C for 3-5 min and spray drying (Clegg et al., 1974). Clegg and McMillan (1974) used first endopeptidase papain (4%) and then with a exopeptidase leucine aminopeptidase (0.015%) obtained casein hydrolysates with reduced bitterness and high (46%) free amino acids.

Cogan et al. (1981) suggested use of papain and pepsin. Treatment with Rhozyme enzymes was performed with specified enzyme concentrations under the following conditions of pH and temperature: Rhozyme P-11 and Rhozyme 41 at pH 8.5, 50°C. Rhozyme P-33 at pH 7.5, 60°C and Rhozyme 62 at pH 8.3, 60°C.

Problems of bitter taste in casein hydrolysates prepared by enzyme hydrolysis of casein substrate were overcome by using enzymes with high peptidase activity especially proteinase 3545 from Rhizopus sp to hydrolyse the bitter peptides and removal of residual bitter peptides and free amino acids. Electrophoresis improved the taste of the casein hydrolysates, which had antigenicity of about 1/10,.000 of intact milk casein. The antigenicity of whey protein concentrate hydrolysates was reduced significantly by treatment with papain and proleather and by heat treatment before and during hydrolysis (Nakamura, 1994).

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3. MANUFACTURE OF WHEY PROTEIN HYDROLYSATES The pure whey protein is dissolved in distilled water to a concentration of 6.7g/l. For hydrolysis pepsin is added at pH 2.5, keeping the pH constant by the controlled addition of HCl 0.050 mol/l with a pH-stat, the mass ratio of enzyme to substrate (E/S) is 1/500. Incubations with papain is carried out at pH 8.0 at ratio of 1/200, keeping the pH constant with NaOH 0.050 mol/l. All incubations are at 40°C. The degree of hydrolysis is calculated. After optimum hydrolysis, the product is further concentrated and dried.

Whey protein isolate was modified by proteolysis using a broad spectrum proteinase in combination with heat treatment of the hydrolysate. The degree of hydrolysis and the adjustment of pH during heat treatment governed the emulsifying and foaming properties of the modified protein. The emulsion resistance to coalescence increased when the hydrolysate was heated at pH 4.0 and 8.0 while the reverse trend was observed when heating pH was 6.0. Emulsions showed evidence of age thickening during a 4 week storage period. However change in viscosity during storage decreased with increasing degree of hydrolysis.

Whey protein concentrate was hydrolysed using the technical food grade enzyme Corolase 7092 in order to eliminate the allergenicity of whey proteins. Ultrafiltration of the hydrolysates appeared to be necessary to obtain a hypoallergenic product. The minimum molecular weight capable of eliciting immunogenicity and allergenicity of whey protein hydrolysates appeared to be between 3000 and 5000 Da, so the molecular weight cut off value of the filters required must be in this range. Although there was no evidence that extensively-hydrolysed whey protein is nutritionally inferior to casein, the slightly bitter taste might reduce food intake (Beresteijn et al., 1994).

A process has been developed for hypoallergenic and low-bitterness hydrolysates from whey proteins that are suitable for use in infant formula. Of the proteinases tested, Alealase 2.4 L, papain W-40 and proleather were found to be effective in reducing the antigenicity of WPC. The hydrolysate prepared from papain W-40, had no bitterness and was thus selected as the most suitable treatment for reducing antigenicity (Nakamura et al., 1993).

Ziajka et al. (1994) prepared whey protein concentrate by adding trypsin or pepsin to dried whey protein and distilled water at enzyme: substrate ratios of 0.1, 0.2, 0.3 and 0.4 and incubating for 6h at 37°C followed by heating at 85°C for 20 min and spray drying. The bitterness in all hydrolysates was not strong, probably because of the masking of salts formed during hydrolysis. The hydrolysates could be used for the production of specific dietetic formulae or for protein fortification of acidic beverages.

Whey protein isolate (WPI) with or without preheating (90�C for 5 min) was hydrolyzed for 0.5 to 6 h using four pure enzymes (pepsin, papain, trypsin, and chymotrypsin) and three commercial crude proteases.After determining the degree of hydrolysis, the hydrolysates were incubated (37�C, 1 h) with a liposome oxidizing system (50 mM FeCl3/0.1 mM ascorbate, pH 7.0). Lipid oxidation was measured by determining the concentrations of TBA-reactive substances (TBARS). The degree of hydrolysis of WPI ranged from 4 to 37% depending on the enzymes used and whether the substrate was heated or not. WPI hydrolysates prepared by pure enzyme treatments did not prevent TBARS formation in the oxidative model system, but WPI hydrolyzed by the commercial crude enzymes, especially protease F, exhibited antioxidant activity. The antioxidative potential of hydrolyzed WPI was not affected by the degree of hydrolysis, and it was

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improved by preheat treatment in only some samples (Pena-Ramos and Xlong, 2001). 4. DEBITTERING OF PROTEIN HYDROLYSATES

Bitterness is the main problem for practical use in food preparations. Bitterness is generally considered to be due to high hydrophobicity especially if the C-terminal residue is hydrophobic. Bitterness can be eliminated or at least reduced to an acceptable level by treatment with activated carbon, carboxy-peptidase A, leucine aminopeptidase, Ultrafiltration or via the plastein reaction.

Most of the work done on the preparation of soluble protein hydrolysates for food use has naturally been concentrated on either preventing the formation of bitter peptides or on removing them if already present. Hydrophobic chromatography on the phenolic resin, Duolite S-761, removes bitter hydrophobic peptides and amino acid including phenylalanine, which may be useful in the preparation of diets for management of phenylketonuria. Debittering by hydrophabic chromatography on hexyl Sepharose 6B was described by Lalasidis and Sjoberg (1978) who also showed that solvent extraction of hydrolysates with azeotropic secondary butanol is very effective in reducing bitterness. The bitter peptides and amino acids were soluble in the organic phase which extracted 5-10% of total nitrogen, the bitterness of the extract, which was rich in essential amino acids, may be reduced by the plastein reaction.

The debittering of skim milk and casein hydrolysates (Produced by various proteinases) by a variety of hydrophobic and absorption chromatographic media was assessed by Helbig et al. (1978). While most of the systems investigated were effective, activated carbon and glass fibre were considered the most practically feasible. The saltiness of the dibittered hydrolysates was decreased by ion-exchange chromatography on Amberlite IR-45. Hydrophobic chromatography based on delay of the emergence of bitter compounds was successfully employed by Roland et al. (1978). However, Ma et al. (1983) reported this method to be impractical and costly for application to casein hydrolysates.

The enzymatic process of debittering includes further hydrolysis of bitter peptides by exopeptidases and peptidase from Ps. fluorescens. An approach involved the sequential use of papain and of pig kidney homogenate, the latter serving as a source of exopeptidases However, this is not an efficient process as it is costly, time consuming and results in production of significant amount of free amino acids (Clegg et al. 1974, Fujimaki et al. 1970, Helbig et al., 1980).

Aminopeptidase T hydrolyse hydrophobic amino acid residues at N-terminal of peptides and proteins and hence remove bitter components from bitter peptides (Minagawa et al.,1989). Suzuki et al. (1981) and Szejthl (1982) suggested addition of 10% ß-Cyclodextrin for removal of bitterness. Attempts are being made to establish its safety as a food additive. Among different adsorption methods employed, activated carbon talc and ß-Cyclodextrin are found to be more effective. This method has been found safe, promising and most effective in the elimination of the bitter taste which develops during the course of hydrolysis.

Cogan et al. (1981) successfully eliminated the bitterness in the hydrolysates obtained from Rhozyme 62 by employing 0.5 g of activated carbon per g protein and storing it for 60 min at 25°C. However, such treatment was accompanied by a selective loss of tryptophan (63%), phenylalanine (36%), arginine (30%) and 26±2% of protein nitrogen due to adsorption of peptides

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and amino acids on to the activated carbon. As remedial measure, supplementation of the treated hydrolysates with the proper amounts of tryptophan and phenylalanine was suggested for the production of casein hydrolysates of acceptable taste and high nutritive quality.

Helbig et al. (1980) obtained soluble casein hydrolysates for use in acid beverages by stirring 5% casein hydrolysates solutions with 10% carbon of various kinds and mesh sizes at 22°C of 2 h or at 90°C for 10 min. From the results, it has been established that activated carbon treatment is an effective, promising and practically most feasible technique for debittering casein hydrolysates.

Khanna (1991) optimised process for enzymatic production of casein hydrolysates free from any bitterness from buffalo milk. The yield and per cent recovery of liquid casein hydrolysate prepared with optimised process was 47.98% and 46.23%, respectively. The liquid product had 10.25% TS, 1.93% nitrogen, 1.19% ash and a low viscosity of 1.99 cP at 20°C. 5. BENEFITS OF PROTEIN HYDROLYSATES

There are many benefits of ingesting milk proteins hydrolysates.

5.1 Already Digested

The benefit is obvious for any person with impaired digestives functions: amino acids are more easily available than from whole proteins. Pre-digested proteins may allow faster intestinal absorption. This is of great interest for athletes who are at risk of negative nitrogen balance, and need to get fast absorbed amino acids

The digestion of dietary proteins results in a mixture of amino acids and small peptides. Some peptides escape digestion and are absorbed intact, through specific transport systems. In 1982, the characteristics of di-and tripeptide absorption were investigated in man. A consistent finding was that amino acid residues were absorbed more rapidly from di-and tripeptides than from free amino acids. Given the greater palatability of oligopeptide-based nitrogen (N) sources versus free amino acid mixtures, the authors considered protein hydrolysates potentially useful for clinical nutrition, when digestive and absorptive capacities are diminished.

Silk et al. also showed that alpha-amino N absorption was greater from two casein hydrolysates and a lactalbumin hydrolysate than from the respective free amino acid mixtures. Shorter the peptides are, faster the AA are absorbed. In healthy adult individuals, di- and tripeptides were shown to induce a faster N absorption than tri- to pentapeptides. These data confirm the existence of di- and tripeptide transport systems, while longer peptides need to be digested before being absorbed. 5.2 Reduced allergenicity

Dietary amino acids are mainly provided as whole proteins. However, for many reasons, like dietary allergies, protein hydrolysates containing various peptides and free amino acids are manufactured. Dietary allergens are often represented by proteins mainly glycoproteins from 14,000 to 40,000 Da. The antigenicity of a protein, i.e. its ability to induce an allergic reaction, depends on its amino acid sequence and spatial conformation. This second factor determines the epitope accessibility. Technological treatments, when modifying the spatial structure, may decrease antigenicity or, contrary, increase it, by revealing new epitopes. Proteins, from milk for

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example, are hydrolyzed in order to reduce their antigenicity and allergenicity. Protein hydrolysates globally induce good clinical results. The hydrolysis process, sometimes associated with a thermic treatment must be advanced enough to be efficient. It destroys not only major allergenic determinants, but whole protein. The hydrolysate composition depends on the protein and on the enzymes used for the hydrolysis.

The weight of the peptides obtained after hydrolysis is very important. An antigenic sequence included in a long peptide presents a stronger allergenic power than the same sequence included in a shorter peptide. A 3,500 Da molecular weight seems to be determinant.

The hydrolysis process depends on food applications. For example, there are infant formulas for allergy prevention, dedicated for non-allergic children (mainly trypsic hydrolysates from lactic proteins). Others are dedicated for allergic children (advanced hydrolysis to obtain shortest peptides and long peptide suppression by ultrafiltration. 5.3 Improve growth and nitrogen balance

The comparison of diets containing either whole protein, peptide or free AA, showed that total body weight gain was better with the peptide diet, followed by the whole protein and, last, the free AA diets. In an animal model of realimentation following 72 hours of starvation, a whey protein hydrolysate diet induced better weight gain, higher nitrogen retention than did whole protein and free AA mixture diets. 5.4 BCAA and Fatigue hypothesis : the importance of the composition of an hydrolysate

During sustained exercise, muscle BCAA is used for energy and NH3 production. The subsequent increase of free tryptophan to BCAA ration is thought to increase the tryptophan availability for serotonin synthesis, which can cause sleep and could increase the mental effort necessary to maintain athletic activity. Research suggests that regular supplementation with branched chain amino acids can prevent central fatigue by preventing tryptophan from entering the brain. 5.5 Milk protein Hydrolysate for addresing a bone or dental disorder

A composition for prevention or treatment of a bone or dental disorder comprises of a milk protein hydrolysate. In preferred embodiments, the milk protein hydrolysate is a hydrolysate of casein, in particular a caseinoglycomacropeptide (CGMP), a mimetic, homologue or fragment thereof in a bioavailable form which retains the ability of CGMP to inhibit bone resorption or bone loss; or favour calcium absorption, retention or calcification; or a combination thereof.

6. CONCLUSION

Protein hydrolysates, especially from casein and whey proteins, are increasingly finding commercial application in a number of formulated foods. Diets that are suitable for geriatrics, high energy supplements, weight control diets, hypoallergenic infant formulas and therapeutic or enteric diets are some of the areas in which protein hydrolysates are most useful. The DH can be used as the controlling index for the hydrolysis of the milk proteins. Selection of appropriate

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enzyme is also important for obtaining desirable functionalities. Among the technological advantage of proteins hydrolysates are improved solubility, gelation, emulsification, foaming, heat stability and relatively greater resistance to precipitation by many agents as pH or metal ions. Nutritionally protein hydrolysates are particularly useful for the management of patients with various digestive disorder, pre- and post—operative abdominal surgical patients, diabetes and geriatrics. 7. REFERENCES Boopathy, R. 1994 Enzyme technology in food and health industries. Indian Food Industry, 34:22-37 Beresteijn, E.V., Peeters, R.A., Kaper, J. Meijer, R.J.G.M., Robben, A.J.P.M., Schmidt, D.G., and Van, B.E.C.H.

1994 J. Food Prot. 57 : 619-615. Clare, D. A. andSwaisgood, H. E.2000. Bioactive milk peptides: A prospectus. J. Dairy Sci. 83:1187-1195 Clegg, K.M. and McMillan, A.D. 1974. Dietary enzymic hydrolysates of protein with reduced bitterness. J Food

Technol, 9:21-25 Clegg, K.M., Smith, G. and Walker, A.L. 1974. Production of an enzymic hydrolysate of casein on a kilogram scale.

J. Food Technol., 9:425-430. Cogan, U., Moshe, M. and Mokady, S. 1981. Debittering and nutritional upgrading of enzymic casein hydrolysates. J.

Sci. Food Agric., 32:459-564. Fujimaki, M., Yamashita, M., Arai, S. and Kato, H. 1970. Plastein reaction Agric. Biol. Chem., 34:483-487. Helbig, N.B., HO, L., Christy, G.E. and Nakai, S. 1980. Setittering of skim milk hydrolysates by adsorption for

incorporation into acid beverages. J. Food Sci. 45:331-337. Gupta, V. K. 2000. Enzymatic production of protein hydrolysates for food application. New Concepts in Dairy

Technology-Lecture Compendium, CAS in Dairy technology, NDRI, Karnal. Kanawjia,S. K. 1997. Technology of protein hydrolysates, Technological Advances in Dairy Byproducts-Lecture

Compendium, CAS in Dairy Technology, NDRI, Karnal. Khanna, R. 1991. Process optimisation for the enzymic production of casein hydrolysates. M.Sc. Thesis, National

Dairy Research Institute, Karnal. Kodjev, A., Ratchev, R. and Panova, V. 1974. Methods for obtaining the protein hydrolysates with improved flavour

qualities. IV Int. Congr. Food Sci. Technol., Id : 3, Cited : Dairy Sci. Abstr., 37 : 5411. Lalasidis, G. and Sjoberg, L. 1989. Two new methods of debittering protein hydrolysates and a fraction of

hydrolysates with exceptionally high content of essential amino acids. H, Agric Food Chem. 26:742-756. Ma, C.Y., Amantea, G.F. and Nakai, S. 1983. Production of non-bitter, desalted milk hydrolysate from milk casein by

amino peptidase T. J. Food Sci., 54:1225-1231. Minagawa, E, Kaminogawa, S., Tsukasaki, F, and Yamauchi, K. 1989. Debittering mechanism in bitter peptides of

enzymic hydrolysates from milk casein by aminopeptidase T. J. Food Sci, 54 : 1225-1231. Nakamura, T. 1994. Studies on enzymatic production of hypoallergenic food from milk protein. Technical Research

Report, Snow Brand Milk Products Co. Ltd. Japan. Nakamura, T., Sado, H, Syukunobe, Y and Hirota, T. 1993. Antigenicity of whey protein hydrolysates prepared by

combination of two proteases. Milchwissenchaft, 48 : 667-670. Panyam, D., and Kilara, A. 1996. Enhancing the functionality of food proteins by enzymic modification. J. Trends

Food Sci. Technol. 7:120-125. Pena-Ramos, E. A. and Xlong, Y. L. (2001) Antioxidative activity of whey protein hydrolysates in a liposomal

system. J. Dairy Sci.84: 2577-2883. Roland, J.F., Matlis, D.L., Kiang, S. and Alm, W.L. 1978. Hydrophobic Chromatography. Debittering protein

hydrolysates J. Food Sci. 43:1491-1496. Schimidt, D.G. and Van Markwijk, B.W. 1993. Enzymatic hydrolysis of whey proteins. Influence of heat treatment of

a-Lactalbumin and ß lactoglobulin on the proteolysis by pepsin and papain. Neth. Milk Dairy J. 47:15-22. Suzuki, Y., Fukuza, T. and Shindai, H. 1981. Method for removing bitter taste of protein hydrolysate. Japanese Patent

5, 612084 cited Dairy Sci. Abstr., 44:5937. Szejthl, J. 1982. Cyclodextrins in foods, cosmetic and toiletries. In Proc. I. Int. symp. on 'Cyclodextrins'. Budapest,

Hungary, Cited Dairy Sci. Abstr., 46:3927. Takase, M., Fukuwatari, Y, Karwase, K and Kiyosawa, I. 1979. Antigenicity of casein enzymatic hydrolysate. J.

Dairy Sci., 62 : 1570-1576.

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Vegarud, G.E. and Langsrud, T. 1989. The level of bitterness and solubility of hydrolysates produced by controlled proteolysis of caseins. J. Dairy Res., 56:375-379.

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I. Karnal-132 001 1. INTRODUCTION

With a content of 0.7 - 0.9% phosphorus, covalently bound to the casein by a serine ester linkage, casein as a phospho-protein is a member of a relatively rare class of proteins. Moreover, due to high proportions of essential amino acids, casein is nutritionally excellent protein. Its protein efficiency ratio reported is 2.5, which is mostly unaffected by the processing conditions usually employed during the dairy operations. Casein has some rather unique properties and cannot be replaced by other proteins in certain food application. Edible casein and caseinates are long established dairy by-products finding use in many dairy and food products.

Sodium caseinate is the most commonly used water-soluble form of casein and is used in the food industry. Sodium caseinate is announced by FAO and WHO as unrestricted food additive. The two main reasons for using sodium caseinate as an ingredient in foods are its functional properties and nutritive value. Sodium caseinate is valued for its ability to emulsify fat in the production of modified dairy products such as coffee whiteners, whipped cream and ice cream. It also possesses very good water binding and whipping properties. Industries of meat processing, baking and modified dairy products are the largest consumer of sodium caseinate. The various food products in which sodium caseinate is used consist of various kinds of sausages, meat-based and milk-based instant breakfasts, modified milk, whipped cream, coffee whiteners, non-dairy creams, desserts, sauces, soups, bread, doughs, crackers (biscuits), dietetic products and various protein-enriched products. Other casein products, used in a descending order in the food industry are calcium caseinate, potassium caseinate, other caseinates, and, finally, pure casein.

Other soluble forms of casein are produced using phosphates, carbonates, and other salts as the solubilizers. Magnesium caseinate is prepared from casein and a magnesium base or basic salt such as magnesium oxide, magnesium hydroxide, carbonate or phosphate or by ion exchange. Compounds of casein with aluminium may be prepared for medicinal use or for use as an emulsifier in meat products. Heavy metal derivatives of casein, which have been used principally for therapeutic purposes, include those containing silver, mercury, iron and bismuth. Iron and copper caseinates have also been prepared by ion exchange for use in infant and dietetic products.

One another form of casein that is commonly used in the food industry is the hydrolyzate (obtained by acid hydrolysis), which has a meat-like flavor. Casein hydrolyszates are, therefore, used to accentuate the meat flavor in heat-treated canned and dried meat products such as soups. The hydrolyzates are also used as ingredients in crackers, snack foods, and other food products. Today, casein hydrolysates have assumed a new dimension in food industry. They find use in wide ranges of soups, gravies, sauces, drinks, vegetable and fruit juices, flavourings and nutritional, dietetic and formulated foods.

The highest nutritional efficiency in the dairy protein field can be obtained by balanced blending of casein and whey proteins. The latter being richer in some of the important amino

DEVELOPMENT IN THE USE OF CASEIN PRODUCTS IN FOOD PRODUCTS

DEVELOPMENT IN THE USE OF CASEIN PRODUCTS IN FOOD PRODUCTS

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acids, such blending preparations result in an excellent amino acid profile and render highly nutritious protein supplements as coprecipitates contain 97% protein as against 80% in casein (Jones, 1973). The functional properties such as water binding capability, viscosity and solubility of these coprecipitates can be altered to a greater extent for the best technological performance by making changes in the manufacturing method. This enhanced water binding property may prove superior to caseinate in chopped meats. 2. BAKERY PRODUCTS

Casein and casein derivatives are mainly used in bakery products to enhance flavour and other sensory properties and also for nutritional fortification of the wheat flour. The limiting amino acid (lysine) in most cereal proteins can be very well be complemented with dairy proteins. Casein/caseinates can be added to breakfast cereals, milk biscuits and protein-enriched bread. The PER of wheat flour is only 1.1, compared with 2.5 for casein. By supplementing the wheat flour with casein, it is possible to increase considerably the PER of the mixture. For instance, for a 50:50 mixture of casein and wheat protein, the PER can be raised to 2.2 - 2.3. One of the most important functional characteristics of casein products in bakery products is its water binding capacity. Table 1. Use of casein products in bakery products

Food product Casein derivative % in food product

Purpose/effect

Pastry glaze

Soluble high-calcium co-precipitate

63.4 Colour, shine (with lactose)

Breakfast cereal Casein 1.5-10 Nutrition Milk biscuits

Calcium caseinate or high-ca. co-ppt.

16-20 Nutrition

Protein-enriched bread Calcium caseinate 5-10 Nutrition Cake mix for diabetics High Ca. co-ppt. 6.5 Nutrition and cake vol Frozen baked cake Sodium caseinate 0.5-5 Texture, emulsifier Protrein-enriched milk biscuits

Sodium caseinate 12.5 Nutrition

Biscuits Calcium caseinate 20 Nutrition High-protein bread Casein 2-6 Nutrition Fortified bread Co-precipitate 3-10 Dough consistency and sensory

prop. Cookies Casein and co-ppt. 6.5 Nutrition and texture Acid and rennet casein, sodium caseinate, calcium caseinate can be used in bread making and are added at a level of 15-20% of the wheat flour. A satisfactory loaf volume can be obtained by the use of casein products. The physical structure of bread reflects the unique properties of the major proteins of wheat flour. Upon hydration, gluten forms a stretchable viscoelastic network that can entrap gas produced by yeasts. The structure stabilizes during baking. Bread, with milk proteins added in one form or another, shows a good crumb structure, bread yield, flavour and keeping quality. In the manufacturing of high protein biscuits, milk proteins play an important role as they increase the nutritive value and also the texture.

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Milk proteins are often incorporated into the base flour for pasta manufacture for the purpose of enhancing nutritional quality and to improve texture. Products fortified by addition of sodium or calcium caseinate prior to extrusion include macaroni and pasta. 3. MODIFIED DAIRY PRODUCTS

The use of sodium caseinate in the dairy industry and in the manufacture of modified milk products has increased all around the world. The addition of 1% sodium caseinate into UHT low-fat milk fortifies this dietetic drink with protein. The addition of sodium caseinate to cultured milk products simultaneously increases the nutritive value and improves the technological quality by products.

Milk Protein products are widely used to supplement the protein content and, therefore, enhance sensory characteristics of conventionally processed dairy products and are also used in the production of a range of imitation dairy products. Imitation cheeses are made from vegetable fat, caseins, salts and water and are used in pizza, lasgne and sauces and on burgers, grilled sandwiches, macaroni etc., at a significant cost-saving compared to the use of natural cheese. The functional properties of caseins that favour their use in imitation cheese include fat and water binding, texture enhancing, melting properties, stringiness and shredding ability.

Sodium caseinate is used in powdered coffee creamers, which also contain vegetable fat, a carbohydrate source and added emulsifier and stabilizers. These creamers are cheaper, have a longer shelf life and are more convenient to use (e.g. they require no refrigeration) than fresh coffee creams. In these products, sodium caseinate acts as an emulsifier/fat encapsulator and whitener, imparts body and flavour and promotes resistance to feathering.

Sodium caseinate is used to increase gel firmness and reduce synersis in yoghurts, and is added to milk shakes for its emulsifying and foaming properties. Sodium caseinate is also used as an emulsifying and fat encapsulating agent in the manufacture of high-fat powders for use as shortenings in baking or cooking. Dry whipping fats or whipping creams contain casein products.

A procedure for the manufacture of a soluble casein concentrate suitable as an ingredient in baby foods and dietetic foods was developed in the former USSR.31 Precipitated casein is dissolved by adding 2.8% sodium citrate, 3.2% calcium citrate, and 5% sodium bicarbonate to it and the product is dried and ground.

4. BEVERAGES

Casein products are used as stabilizers or for their whipping and foaming properties in drinking chocolate, fizzy drinks and fruit beverages. There is also alarge market for sodium caseinate as an emulsifier in cream liqueurs and to a lesser extent in wine aperitifs. Cream liqueurs typically contain 16% (w/w) milk fat, 3.3% sodium caseinate, 19 % added sugar and 14 % ethanol. Trisodium citrate is also added to prevent calcium-induced age gelation. Casein products have also been used in the wine and beer industries as fining agent, to decrease colour and astringency and to aid in clarification.

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5. CONFECTIONERY

Caseins are used in toffee, caramel, fudge and other confections as they form a firm, resilient, chewy matrix on heating and they contribute water binding and aid emulsification.Casein hydrolysates are used as foaming agents in place of egg albumen in marshmallow and nougat as they confer stability to high cooking temperatures and good flavour and browning properties. 6. MEAT PRODUCTS

The possibility of the production of simulated meat by using artificial protein fibers based on casein has been investigated for several years. This field is protected by a variety of patents. The basic principle described in one of them consists of the extrusion of a sodium caseinate solution through a die with small openings into an acidic medium. Care must be taken that the continuity of casein fibers is maintained at all times. The patent covers the production facility scheme and 26 products based on sodium caseinate. Using characteristic flavors, coloring agents, and other ingredients, it is possible to produce simulated beef, chicken meat, pork, bacon, ham, and even fish, all of which have the taste and texture of the products being simulated.

The use of caseinates in meat products is traditional and has been used for 25 years as a potential functional protein ingredient in meat industry and it has gone far since then. Today the best-documented non-meat ingredient in meat products is caseinates round the globe. Besides nutritional value, they are mainly used due to their excellent water binding and emulsifying properties and, therefore, the major application field is that of the rather comminuted meat products. Amongst many other functional properties, the bland flavour and neutral colour of caseinates deserve a special mention. The caseinates get their functionality from their unique molecular characteristics. They have a random coil structure with a low percentage of helix. They show no heat gelation or denaturation and have a high viscosity in solution. The ability of caseinates to bind moisture through H-bonding and entrapment thereby enhancing the yield of end products, has been used beneficially in various meat and sausage preparations. In addition, their salt tolerance and high protein content attract many meat traders.

The functional behaviour of milk proteins in comminuted meat products have been studied by Hung and Zayas (1992). Milk proteins have been utilised as fillers, binders and extenders in cooked comminuted meat products to reduce cook shrink and formulation cost, as well as to improve emulsifying capacity, emulsion stability, water binding, potential nutritive value and slicing characteristics. The dairy proteins can also improve or alter the consumer acceptance (flavour, mouth-feel, colour, appearance etc.) of the finished product. These proteins significantly increase the gel strength of meat proteins and it has been shown that there is a synergistic effect between milk proteins and salt soluble meat proteins, through covalent cross-linkages. The functionally designed dairy ingredients, especially milk protein products, have held their position in this competitive sector because they exhibit good functionality features and affecting the final product in a desirable way. Choice of dairy by-products in meat industry is often more guided by economic criteria. Surplus caseins have been used for substituting more expensive meat and egg proteins in Western countries. Even in this area, competition from vegetable proteins is increasing. Milk proteins, however, are preferred ingredients for their functional supremacy, their good flavour, colour and nutritional profile, because of which they are able to keep their foothold with vegetable protein products.

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In comminuted meat products, a considerable amount of free fat is released during the manufacture of these products, which must be stabilised. Caseinates, when used in the desired way, not only stabilize the fat, but also impart water binding and consistency. They do retain a part of the soluble fraction of meat proteins in their native form, which otherwise prone to denaturation at the interface, enabling it available for gel formation upon heating (Schut and Brouwer, 1971) possibly through protein-protein interactions of meat and milk analogues. Consequently caseinates, a milk protein derivative, is a widely accepted functional protein giving the meat product a better stability. This kind of protein also has many hydrophobic groups. This could be one of the several reasons why they are perfect emulsifiers, and are active at the fat-water interface to prevent fat separation. Besides they are perfectly water soluble, a truly desirable characteristic in several restructured meat products. It further supplements and complements the native meat proteins and does not doubt the naturality of products when utilised in functional proportions, i.e. less than 3 percent (Visser, 1984).

Though caseinates may be employed in dry or pre-solubilised form at the beginning of the comminuting process, optimum stability results are obtained when they are processed in the form of previously prepared caseinate/fat/water emulsion (Schut and Brouwer, 1975; Visser, 1984). Sodium caseinate is the most versatile of all milk proteins and disperses well in water or melted fats. Upon addition of warm water during processing, sodium caseinate dispersion hydrate and the resulting colloidal solution forms a base for subsequent emulsions. The technologically more difficult and cheaper type of fat, such as of beef and sheep, may be perfectly used by pre-emulsifying with caseinates. The rationale of using sodium caseinate in emulsion technology is based on its manifold chemical reactivity. The reactivity of caseinates lies in the unique distribution of the electrical charges on the polymeric molecule, its hydrogen bonding ability and its richness of hydrophilic as well as lipophilic bonding sites. The more complex colloidal chemical performance of caseinates can be accentuated by its reactivity with lecithin and carrageenan.

Addition of caseinate stabilizes the meat emulsion as required in the sausage mix. It thickens the gravy during frying and prevents it running out, but excess incorporation of caseinate may result in drying up of the sausages. Further addition of water-absorbent materials becomes essential when sodium caseinate concentration in sausages exceeds 5% (Salavatulina et al., 1983). The greater water holding capacity, lower viscosity and lower cooking losses of sausage batters containing 2% sodium caseinate in comparison to all meat control were observed by Hung and Zayas (1992).

The nutritional value of sausage products, in which part of the meat protein replaced with other proteins was studied by Safronova (1983). The nutritional value of proteins in sausages depended directly on the levels of replacement; for sodium caseinate 50% of the meat protein could be replaced without any adverse effect on it. Usage of a very high viscosity sodium caseinate as an effective meat binder has been found to be desirable in some meat products, because of its greater water binding and gelling properties.

The effect of sodium and calcium caseinates on the functional quality of nuggets made from spent hen meat has been studied by Rao et al. (1994a,b). They observed that with 2% incorporation of sodium caseinate or 1% calcium caseinate, the yields were improved, protein content was higher and brightness scores were higher for nuggets containing caseinates as compared to control nuggets. The frying losses were also lesser for caseinate added nuggets. The texture profile analysis revealed higher firmness and springiness for sodium caseinate containing

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chicken nuggets and higher cohesiveness and gumminess for nuggets having calcium caseinate (Rao et al., 1996). 7. DIETARY, PHARMACEUTICAL AND MEDICAL APPLICATION

Since milk protein products are of high nutritional quality, they are used extensively in dietary preparations for people who are ill or convalescing, for malnourished children in developing countries on a therapeutic diet and for people on weight-reducing diets. Caseins are used in special preparations to enhance athletic performance and have been incorporated into formula diets for space feeding.

While casein products are not generally used in infant formulae, they are used extensively in specialized preparations for infants with specific nutritional problems. Caseinates are used in low-lactose formulae for lactose-intolerant infants while various types of caseinates have been used in infant foods with a specific mineral balance, e.g. low-sodium infant formulae for children with specific renal problems. Casein hydrolysates are used in specialized foods for premature infants, in formulae for infant suffering from diarrhea, gastroenteritis, galactosaemia and malabsorption. A special casein hydrolysate, low in phenylalanine, has been prepared for use in formulae for feeding infants with phenylketonuria. Casein products are also added to various foods for children and infants and to drinks as a nutritional supplement.

Diets that are suitable for geriatrics, high-energy supplements, weight-control diets, hypoallergenic infant formulas and therapeutic or enteric diets are some of the areas in which casein hydrolysates are most useful. casein hydrolysates are boon to people who are suffering from protein allergy or stomach disorders and to those who require easily digestible foods. The production of hydrolysed protein provides an opportunity for the dietary management of patients with various digestive disorders as a result of pancreatic malfunction, pre- and post-operative abdominal surgical patients, patients on geriatric and convalescent feeding and others who for various reasons are not able to ingest a normal diet. Casein hydrolysates also have pharmaceutical applications in intensive care foods, anemia treatment, prevention of blood cholesterol, treatment of dental diseases and in administration of amino acid mixture intravenously.

Specific drugs have been produced from casein ; β-casein is used as raw material for production of β-casomorphins, tetra- to heptapeptides which can regulate sleep, hunger or insulin secretion. Sulphonated glycopeptides prepared from casein have been used for the treatment of gastric ulcers. It is claimed that the use of casein in toothpaste prevents dental caries, in cosmetics it conceals facial wrinkles and in special therapeutic creams it heals wounds. 8. REFERENCES Hung, S.C. and Zayas, J.F. (1992) Functionality of milk proteins and corngerm protein flour in comminuted meat

products. J. Food Qual., 15: 139-152. Mann, E.J. (1989) Dairy ingredients in meat products. Dairy Indus. Inter., 54(2): 9-10. Rao, K.V.S.S., Anjaneyulu, A.S.R., Singh, R.R.B., Rao, K.H. and Yadav, P.L. (1994a) Effect of sodium caseinate

on the fuctional quality of nuggets from spent hen meat. Ind. J. Poultry Sci., 29(1): 115-116. Rao, K.V.S.S., Anjaneyulu, A.S.R., Singh, R.R.B., Rao, K.H. and Yadav, P.L. (1994b) Effect of calcium

caseinate on the quality of nuggets from spent hen meat. Paper presented at the Silver Jub. Nat. Symp. on meat and milk industry: Trends & Developmental Strategies, CCS Har. Agri. Univ., Hisar, Aug. 25-27.

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Rao, K.H., Singh, R.R.B., Anjaneyulu, A.S.R., Rao, K.V.S.S. and Yadav, P.L. (1996) Evaluation of caseinates and refined wheat flour as emulsion stabilizers in chicken nuggets. Proc. XX World's poultry Cong., Vol-IV, PP 435.

Visser, F.M.W. (1984) Milk proteins in meat products and soups. In Milk Proteins-84, Proc. Inter. Cong. Milk Proteins, Luxumberg, 7-11 May, Galesloot, T.E. and Tinbergen, B.J. (Eds.) Pudoc, Wageningen, The Netherlands.

Schmidt, D.G. (1986). "Association of Caseins and Casein Micelle Structure." In Developments in Dairy Chemistry. 1. Proteins, P.F. Fox, ed. Elsevier, London, pp. 61-86.

Mulvihill, D.M. (1992) "Production, functional properties and utilization of milk protein products." In Advanced Dairy Chemistry. Vol. 1. Proteins, P.F. Fox, ed. Elsevier Applied Science, London, pp. 369-404.

Caric, M. (1990). Technology of Concentrated and Dried Dairy Products, 3rd ed. Naucna Knjiga, Beograd, Yugosla via, 293 pp. (in Serbian).

Mann, E.J. (1991). Dairy Ind. Int. 56, 13-14. Fichtali, J., Voort van de, F.R., and Khuri, A.I. (1990). J. Food Process Eng. 12, 247-258.

3

SECTION - III

FUNCTIONALITY OF MILK PROTEINS FUNCTIONALITY

OF MILK PROTEINS

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R B Sangwan, Rajesh Kumar and Bimlesh Mann Dairy Chemistry Division, N.D.R.I., Karnal-132 001

1. INTODUTION

The choice of milk proteins in food system is guided by their potential functional

properties - physicochemical properties that govern the performance and behaviour of a protein in food systems during preparation, processing, storage and consumption, which in turn are monitored by their constituents, composition and processing conditions employed for their preparation. Milk proteins are available in various forms such as skim milk powder, total milk protein, milk protein concentrate, caseinates, co-precipitates, sweet whey, acid whey, modified whey, reduced lactose whey, demineralized whey, whey protein concentrates (34-80% protein), and whey protein isolates (>90% protein), which are among the most commonly used milk protein products for incorporation in food product formulations. Milk protein offer excellent functional properties such as swelling, dispersibility, solubility, opacity, acid stability, heat stability, water holding, fat binding, viscosity, gelation, emulsification activity, emulsion capacity, emulsion stability, foaming capacity, foam stability, adsorption at air-water surface and oil-water interfaces. The functionality of milk proteins for an application can be further tailored by altering the physicochemical properties of proteins by physical (heat treatment, acidification, addition of mineral salts, homogenization, and shear), chemical (use of chemical agents and the Maillard reaction), and enzymatic modification (renneting, hydrolysis, and transglutamination), and high pressure processing. 2. MILK PROTEINS AS INGREDIENTS 2.1 Casein

Casein is a well-defined group of phosphoproteins found in milk, constituting about 80% of the proteins in cow's milk. Addition of acid or rennet to milk will cause casein to join together and separate from other milk components. This separation happens during cheese/chhanna/paneer manufacture where the casein portion is separated and the remaining milk components are known as whey. The four casein families; αs1- caseins(molecular weight 23,000; 199 residues, 17 proline residues, two hydrophobic regions, containing all the proline residues, separated by a polar region, which contains all but one of eight phosphate groups and can be precipitated at very low levels of calcium), αs2-caseins( molecular weight 25,000; 207 residues, 10 prolines, concentrated negative charges near N-terminus and positive charges near C-terminus and can also be precipitated at very low levels of calcium), ß -casein(molecular weight 24,000; 209 residues, 35 prolines, highly charged N-terminal region and a hydrophobic C-terminal region, very amphiphilic protein, acts like a detergent molecule, self association is temperature dependant; will form a large polymer at 20° C but not at 4° C, less sensitive to calcium precipitation), and k-casein(molecular weight 19,000; 169 residues, 20 prolines, very resistant to calcium precipitation, stabilizes other caseins, rennet cleavage at the 105-106 bond eliminates the stabilizing ability, leaving a hydrophobic portion- para- k-casein, and a hydrophilic portion called glycomacropeptide (GMP) or

FUNCTIONAL PROPERTIES OF MILK PROTEINS FUNCTIONAL PROPERTIES OF MILK PROTEINS

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caseinomacropeptide). The conformation of caseins is much like that of denatured globular proteins. The lack of ordered secondary and tertiary structure accounts for the stability of caseins against heat denaturation because there is very little structure to unfold. There is considerable exposure of hydrophobic residues which results in strong association of caseins and renders them insoluble in water. It can be manufactured into a range of extremely versatile protein products with excellent functional properties.

2.2 Caseinates

Caseinates are the soluble salts of casein and are produced by neutralizing acid or rennet casein with alkali and then drying the resulting product. There is a wide range of caseinates e.g. sodium caseinate, calcium caseinate, potassium caseinate, ammonium caseinate, etc. Specifically, casein is most commonly bound to calcium (Ca2+) or sodium (Na+) since both of these metals are found naturally in milk. Sodium caseinate can also be polymerised with small amount of formaldehyde to obtain high viscosity, having greater water binding and gelling properties to make efficient stabilizer. Potassium caseinates are used in food systems where the functional properties of sodium caseinate are desired, but without the sodium content. 2.3 Milk protein concentrates

Milk protein concentrates (MPC > 50% protein) are typically prepared by ultrafiltration/diafiltration of skim milk prior to drying. The drying of these high protein concentrates is known to cause a loss of functionality. This is typically exemplified by poor hydration properties, loss in solubility, and poor reconstitutability. MPC powders with improved solubility are made by the addition of monovalent salts to the ultrafiltered retentate or by the removal of calcium ions prior to drying. 2.4 Coprecipitates

Coprecipitates are manufactured by heating skim milk to denature most of the whey proteins, treating with acid to isoelectrically precipitate the caseins and whey proteins and further processing as in casein manufacture. Recent development in the manufacture of milk co-precipitate offers excellent potential in the process for coprecipitating casein and whey proteins in an undenatured complex that contains all of the protein components in a highly functional form. High and low calcium coprecipitates show synergistic effect with other food proteins. At low pH, coprecipitates improve emulsification performance, while water binding capacity is improved at both high and low pH. Sausages containing milk coprecipitates at 40% level of substitution are of equal organoleptic quality to all meat sausages Addition of sodium tripolyphosphate improves the solubility of coprecipitate for increasing its viscosity, thereby improving water holding/binding properties and making it more suitable for sausage industry. The coprecipitates are also reported to have good potential in frankfurters, luncheon meats as meat replacers or extenders. 2.6 Whey Proteins

20 per cent of milk proteins, are globular, more ordered and undergo heat denaturation, exposing hydrophobic amino acids to the aqueous phase, which increase their hydrophobic interactions. The major whey proteins are α-la (123 aa, 8 half-cystine residues that are linked as 4-

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intra molecular disulphide bonds, binds calcium, which may stabilize against the irreversible denaturation.) and β-lg (162 aa, conformation is pH dependent, structure is 15 percent of α-helix, 50 percent β-sheet and 15-20 percent reverse turn, consists of 8 strands of anti parallel β-sheets wrapped around to form a β-barrel. 2.7 Whey protein concentrates

Whey protein concentrates are whey protein products containing more than 35 per cent whey proteins and are manufactured from the whey, obtained from cheese/paneer/chhanna or casein preparation, by ultrafiltration/diafiltration to remove major portion of water, lactose and ash after which these are spray dried. By introducing ion-exchange as pretreatment to ultrafiltration, whey protein isolate with 90% protein can be manufactured. WPCs prepared by different processes vary in their functionality. Whey protein concentrates are highly nutritious, with a protein efficiency ratio (PER) of 3.0-3.2 against 2.5 of casein and contain significant amount of essential amino acids. 3. FUNCTIONAL PROPERTIES 3.1 Solubility

Solubility is one of the important prerequisite for other functionalities. Casein is almost completely insoluble over the PH range 4.0-5.0 but is dispersible at pH >5.5 and pH < 3.5 but at this pH it is much more viscous than at neutral pH. Lactic, mineral acid and casein powders are, therefore, insoluble as manufactured, though they do take up water and swell. For virtually all-commercial applications, they are dissolved in alkali before use. Na, K, and ammonium caseinate are completely soluble at neutral pH. Calcium caseinate forms a white, milky colloidal dispersion, provide low viscosity, exhibit minimum water absorption and are ideal for use in infant formulas, nutritional powders and bars, calcium fortifications and cream soups. Rennet casein is insoluble in water at pH 7, but can be dissolved by raising the pH above 9.5 with NaOH or by using polyphosphates or citrates. Whey proteins are soluble over a wide range of pH. Unique solubility of WPC at low pH allows it to function in acid foods and beverages. So WPC is a source of protein in such products as fruit jams, jellies, fruit juices, carbonated beverages, cream liqueurs, wine aperitifs, and other soft drinks. 3.2 Heat stability

Heat stability is an important property of caseinate in soups and gravies, in coffee creamers and in reported nutritional products. Na, K, and ammonium caseinates are highly heat stable, for example, a 3% solution of Na caseinate at pH 7 will coagulate after 60 min at 140degree C. However, lowered pH and the presence of multivalent cations, whey proteins and other food components generally reduce its heat stability. Calcium caseinates have much lower heat stability than sodium caseinate.

Heating WPCs leads to the unfolding and association to form aggregates. α-la heated alone does not form aggregates while β-lg forms large aggregates, while their mixture interact to form soluble aggregates, as well as larger particles, by means of both disulfide bonds, and hydrophobic interaction. This step leads to the formation of small aggregate precipitates or gel lattice structures. Small aggregates remain soluble; they bind increased amounts of water, increase

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viscosity and add body or improve the product texture. However, gelation and some flocculating can occur. Lowering the pH of WPCs has been shown to increase their heat stability. Some precautions to follow include keeping the temperature below 75ºC while the system is dilute. Denaturation of WPCs can be decreased by controlled heating and adjusting calcium levels with chelators. 3.3 Water binding/sorption

Water sorption properties of casein products are important in bakery and cereals product, pasta products, meat products, or imitation cheese. For baked products, a suitable method for assessing water sorption measures water uptake by flour dough’s to which a milk protein product has been added, this method give values of 0.97 – 1.15 g water/g casein, and 1.59 and 2.95 g water/g caseinate for Ca caseinate and Na caseinate, respectively. Efficient water binding is an important function provided by whey protein products in reduced and fat free salad dressings. The retained water improves texture and reduces cost by replacing oil with water. This provides improved texture and mouthful in low fat products.

3.4 Viscosity

Viscosity is important in soups, sauces and gravies where thickening is a required functionality and texture building is important in imitation cheeses, extruded snacks and confectionery. The flexible nature of casein molecules gives their solution much higher viscosity then those of whey proteins. Sodium caseinate solutions are highly viscous even at 15% and are pseudo-plastic with viscosity increasing exponentionally with concentration and with reciprocal of absolute temperature. Calcium caseinate dispersions are much less viscous because of the aggregation of much of the protein into compact micelles. Rennet casein solutions solubilized by polyphosphate or citrates are even more viscous than sodium caseinates. 3.5 Gelation

Caseinates at neutral pH and under suitable ionic conditions coagulate or gel on adding acid to reduce the pH to about 4.6 or by rennet addition. Therefore, caseinates generally lack acid stability, though good quality sodium caseinate can be used as a major component of coffee creamers, which demand stability in a moderately acidic environment. Sodium caseinate solutions at high salt concentration show different reversible gelation behavior, forming gels in cold that liquefy on reheating. Whey proteins unfold on heating and build intermolecular disulphide bonds resulting into extended 3-dimensional net work to form heat-induced irreversible gels. Gelation entraps water within capillaries of the gel matrix, thus providing additional water holding capacity. Heating time, temperature, pH, and salt, all influence the texture of WPC gels. High gelling WPCs function particularly well in reduced fat meats. Whey proteins have to be pre-treated by heating to at least 70oC to achieve cold gelling ability for use in salad dressings, meat and mayonnaise type products. 3.6 Film formation

The film forming ability of casein is important in many food applications, particularly bakery and applications that involve emulsification or foaming. Caseins are highly surface active. They are readily adsorbed at both air \ water surfaces and oil\water interfaces and act to reduce

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surface or interfacial tension. This surface activity of caseins is attributed to their molecular properties, in particular their ampiphillic nature with a mixture of polar and non-polar side chain. These molecular properties make it easy for the casein to unfold at an interface. 3.7 Oil binding

The capacity of proteins to bind oil is an important property in formulated foods, in which the absorption of oil by proteins enhances the flavour retention and improves mouthfeel. The oil absorption capacity of buffalo milk proteins are in the order: coprecipitate, whey protein concentrate followed by casein. Phosphorylation increases the net negative charge of proteins thereby decreasing their oil absorption capacity. 3.8 Emulsifying properties

Emulsification of fat or oil is an important functional property in almost all the food applications of caseins or caseinates, e.g. coffee creamers, whipped toppings, imitation cheeses, soups, gravies and meat products. At a molecular level, the performance of protein in providing stability against emulsion coalescence is more strongly related to the protein rheology at interface, e.g. interfacial shear viscosity, than it is to the surface activity of the protein. Whey proteins contain both hydrophobic and hydrophillic regions to provide emulsifying properties that are more irreversible than with corresponding caseinate emulsions. In comminuted meat products, WPC contributes to fat emulsification, water binding and improved consistency; as it releases meat proteins for gel formation and water binding. WPC and WPI can be used effectively to improve the texture properties of surimi-based seafood, fish balls, fish and shellfish and other similar products. Fat replacers are fat substitutes and fat mimics. Fat substitutes have fat like physico-chemical properties, while, fat mimics are usually protein and / or carbohydrate based ingredients for producing emulsion based reduced fat products. Micro particulate WPC provides fat like mouth feel and provides consumers a lower dietary fat without sacrificing sensing qualities WPC is a fat replacer in ice cream and meat products. 3.9 Foaming properties

Formation of foam depends on partial unfolding of protein chains at the air-liquid interface and incorporates air to form a stable structure This functionality is assessed by foam volume and foam stability. Foaming properties depends on factors such as protein concentration, ionic environment, and the presence of other food components. Foaming and whipping is an important functional property of caseinates for application in ice cream, whipped toppings, whipped desserts such as mousses, and foamed confections such as marshmallows. Denatured whey proteins have poor whipping properties and to have a better whipping property, severe heat treatment of whey proteins should be avoided. But mild heat treatment tends to improve the whipping ability of the whey proteins. The highest foaming capacity is obtained when the whey protein is at its highest solubility in which there will be mild but not zero denaturation. The whey protein concentrate is a better substitute for egg white, the use of which in food is objected to by strict vegetarians. Residual fat in whey protein concentrate is detrimental to foaming properties

Dairy proteins have been used as valued ingredients by the confectionery industry for many years as they help achieve the required flavour, colour and texture in many products including chocolate coating, caramels, aerated confections, and toffee. WPC and blend provides a

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brilliant surface and a moisture barrier coating to high quality confectionaries. Milk proteins play an important role in the manufacture of high protein biscuits, as they increase the nutritive value and also the texture. Bread, with milk proteins added, shows a good crumb structure, bread yield, flavour and keeping quality. Whey protein concentrates may find better applications as ingredients in minced and ground meat products such as Wiener, Frankfurter, Hotdogs, Bologna (large sausages) and meat loaves or luncheon meat.. A textured whey protein meatless patty containing WPC-80 results in mushroom and vegetable flavored textured patties which re as acceptable as commercial soy patty. Traditional ragi products like ragi-malt and ragi dosa with good sensory attributes can be prepared by incorporating up to 30% WPC. 4. MODIFICATION OF MILK PROTEINS FOR IMPROVED FUNCTIONALITY

Proteins have unique functional properties. However, most proteins still have scope for further improvement in their functional properties. The application of several simple physicochemical and hydrolytic treatments to caseins and whey proteins can change significantly their functional properties. Protein modification usually refers to alteration in the structure by means of physical, chemical and enzymatic treatments. 4.1 Enzymatic Modification

Enzymatic processes such as renneting, hydrolysis, and cross-linking with transglutaminase change the integrity of the proteins, resulting in their physicochemical and functional changes. Enzymatic modification of proteins results in increase in the ionizable groups (NH4

+, - COO-), hydrophilicity and the net charge of the product, decrease in molecular weight of the peptide chain and possible alteration in the molecular configuration leading to exposure of hydrophobic interior to the aqueous environment.. Different enzymes have different rate of hydrolysis and the changes are also varying in terms of functional properties. Depending on the sites cleaved by enzymes, a range of peptides with altered ratios of hydrophobic to hydrophilic groups are obtained. Functional properties that are affected by hydrolysis include heat stability, gelling properties, foaming, and emulsification. Hydrolysis is now commonly used to make physiologically functional bioactive peptides. In comparison to intact proteins, the smaller peptides in hydrolysates form a less cohesive film at the interface and this can affect the stability of the emulsions and foams.

Hydrolysis of globular proteins results in the exposure of buried hydrophobic groups. This enhances surface hydrophobicity that improves surface properties. The degree of hydrolysis needs to be optimized for good surface properties. This is governed by the type of protein used, the extent of hydrolysis, and the enzymes used. Limited hydrolysis of whey protein concentrate results in improved foaming and emulsification properties. Sodium caseinate hydrolysed to a limited degree (1% DH) with the commercial enzyme at optimum temperature and pH for the enzyme reduces water loss during baking and increases bread yield with a significantly finer crumb structure. Hydrolysates give higher emulsion activity at alkaline pH than at acid pH, lower emulsion stability (ES) at acidic pH values but higher in the alkaline pH range, higher whipping and foaming capacity but a lower foam stability, lower water-binding capacity and extensive hydrolysis results in very poor emulsification and foaming/whipping properties. Hydrolysis of proteins can be used to manipulate gel properties of whey proteins. The hydrolysis of whey proteins (>18%) can lead to gel formation. The treatment of whey proteins with a protease from B.

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licheniformis has been shown to induce gelation of both unheated and heated whey proteins. Increasing the degree of hydrolysis results in earlier gelation and increase in gel firmness.

Transglutaminase catalyzes an acyl-transfer reaction and cross-links are formed between glutamine and lysine residues. The introduction of new cross-links has important consequences for the functionality of proteins. The use of transglutaminase increases the strength of acidified whey gels. Dephosphorylated caseins are more soluble in NaCl at pH 3 and in the presence of Ca at pH 7. Emulsions made with modified caseins are similar to native casein emulsions in initial turbidity and emulsion activity index values but have lower oil capacities and tend to be less stable. Dephosphorylated caseins form less foam volume and the foams are very unstable.

4.2 Chemical Modification

Chemical modification of proteins, such as acylation, succinylation, esterification, chemical hydrolysis, and phosphorylation cause changes in the physical properties by changing the ionization of polar amino acid side-chains or by blocking α-amino groups and hence their functional attributes. The manipulation of disulfide bridges of α-lactalbumin and β-lactoglobulin create intermolecular arrays of disulfide connections. By varying applied pressure, temperature, pH, concentrations and initiating reducer sulfide, different viscosities or gel types of the protein solutions can be obtained.

Acylation (e.g., acetylation and succinylation) modifies the charge of proteins. When succinylation is carried out, positive amino groups are replaced by negative succinyl groups, inducing a greater increase in negative charge compared to acetylation where the amino groups are replaced by neutral acetyl groups. The main effects are increased dissolution of the calcium and phosphate and increased solubilization of proteins as a consequence of acylation, affecting their functionality.

Esterification of milk proteins (β-lactoglobulin, α-lactalbumin, β-casein) with methanol, ethanol, or propanol improves their solubility in the pH range 3–6 and emulsifying activity and stability at low pH (3–5). Esterified dairy proteins can become almost unlimited substrates for the production of diversified and amplified functional peptides.

Phosphorylation of caseins and whey proteins creates novel functionality in these proteins. Phosphorylated whole casein using POCl3 alters the net charge of substituted proteins and also grafts significant amounts of arginine and lysine through phosphoamide bonds. Solutions (0.2–0.7% protein) containing the superphosphorylated caseins are more resistant to thickening in high Ca2+ solutions at low protein concentrations. Phosphorylation improves the stability of the whey protein to heat at pH 7. Gels made with phosphorylated proteins are firmer, more resilient, and had better water-holding capacity compared to untreated whey protein isolate gels.

Maillard reaction is employed for improving the emulsifying properties of proteins. The introduction of a sugar or polysaccharide group changes the charge on the protein. This has an impact on its emulsifying capacity and solubility. Conjugation of caseins with maltodextrins improves the emulsifying capacity and stability of caseins at low pH. Emulsifying capacity and stability is improved in glycated β-casein and β-lg in aqueous system. Whey protein isolate that is conjugated to low methoxy pectins under dry heat conditions have superior emulsion stabilization properties. . Glycoconjugates of casein with inulin and reducing sugars have higher viscosity compared to unmodified casein.

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4.3 Physical Modification

Whey proteins are denatured on heating and the extent of denaturation depends on the temperature and pH at the time of heating. In contrast to the whey proteins, caseins are more stable to heat. The heat-induced changes to proteins and their states of association have significant consequences for the functionality of proteins. The foaming, emulsifying and gelling properties of whey protein are dictated by pH, time and temperature of heating, presence of salts, concentration of protein, and order of processing, Fine-stranded gels have good water-binding activity. The ability to manipulate acid gelation properties of heated whey polymers enables their incorporation into yoghurt formulations. A combination of heat and shear is used for microparticulation of thermally denatured whey protein for the production of a whey-based fat replacer. 5. SUGGESTED READINGS

Augustin, M A and Udabage, P (2007). Influence of Processing on Functionality of Milk and Dairy Proteins. Advances Food Nutr Res, 53 ;1-30

Chobert, J M (2003). Milk Protein Modification to Improve Functional and Biological Properties. Adv.s Food Nutr. Res. 47: 1-71

Damodaran,S and Paraf,A (1997) Food Proteins and their Applications. Marcel Dekkar, New York Fox, P F ( 1982). Development in Dairy Chemistry, Applied Science Publishers, U. K. Fox, P F and McSweeney, P L H (1998). Dairy Chemistry and Biochemistry, Blackie, London. Fox, P F and McSweeney, PLH(2003). Advanced Dairy Chemistry: Proteins. Kluwn Acad, New York Mangino, ME (1992). Properties of Whey Protein Concentrate. In: Whey and Lactose Processing pp 230-270, Elsevier

Applied Science, London. Mann, EJ (1996). Dairy Ingredients in Foods. Dairy Ind. Inter. 61: 14-15.

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Latha Sabikhi Senior Scientist

Dairy Technology Division, N.D.R.I., Karnal 132 001

1. INTRODUCTION

The healthfulness of components from whey has been discovered in the past three decades and is being used in pharmaceuticals as well as food industries now. These components have now found a novel avenue as ingredients in designer foods. Designer foods are those foods tailored to consumer preferences or rich in specific milk components that have implications in health as well as processing. This has found support from the research community with clinically established linkages between diet and chronic diseases that encourage search for new links between food and disease. Diets ‘designed’ to suit patients of specific diseases or made to consumer order are available in global markets today. Milk and milk products have also been brought into this array of research owing to reported extra-nutritional therapeutic attributes of milk. Producing ‘designer milk’ by altering its composition by nutritional management or through the manipulation of naturally occurring genetic variation among cattle may have health benefits or have a positive influence on processing.

2. HOW CAN WHEY COMPONENTS HELP?

The global appeal of milk as a healthy beverage that is good for adults as well as infants has prompted much investigation on the commodity. Altering the composition of milk in a manner that suits health and processing needs forms the basis of the current research interests in the area. For example, a reduced lactose content in milk for lactose-intolerant people and/or milk free from β-lactoglobulin (β-Lg) would benefit human diet and health. Milk that contains nutraceuticals and replacement ingredients for infant formula are other interesting avenues. Medicines may be naturally produced in the milk of cows. For example, GM cows could produce milk with a clotting factor for haemophiliacs, milk containing human serum albumin for blood transfusions or milk with a hepatitis vaccine. Several of these medicines could be produced much more efficiently than with the technologies currently used. Some of the potential changes that can be brought about in milk with particular reference to whey ingredients are listed in Table 1.

3. ROLE OF WHEY COMPONENTS IN LACTOSE MANIPULATION

Lactose is synthesized in the secretory vesicles of the mammary glands by the lactose synthase complex. It is unable to diffuse out of the vesicles and therefore, draws water into the vesicles by osmosis. So, the volume of milk produced is directly dependent upon the amount of lactose synthesized.

Lactose can be absorbed only after its enzymatic hydrolysis to the monosaccharides glucose and galactose by intestinal lactase (β-galactosidase). More than 75% of the human adult population suffers from deficiency of β-galactosidase and are therefore lactose-intolerant. When such individuals ingest milk or milk products, the lactose remains undigested and mal-absorbed in the gut, where it causes retention of water by its osmotic action. This along with the large volumes of carbon dioxide produced by intestinal microflora leads to intestinal upsets and dehydration.

ROLE OF WHEY COMPONENTS IN DESIGNER DAIRY FOODS ROLE OF WHEY COMPONENTS IN DESIGNER DAIRY FOODS

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Table 1. Selected applications of altering whey components for ‘designing’ milk

Modification Benefits* Removal of α-lactalbumin, production of lactase by transgenic technology

Reduced synthesis of lactose

Over-expression of β-galactosidase enzyme Better lactose digestibility, caters to the lactose-intolerant customers

Removal of β-lactoglobulin

Less milk allergies, better processing properties

Modification of bovine milk to simulate human milk

Better infant health, less mortality, less problems due to milk allergy

Introduction of human therapeutic proteins Used in the therapy of various ailments Production of antibodies and antimicrobials against pathogens in milk

Safer food, prevention of mastitis and other diseases

* - Compiled from different sources

The ill-effects of lactose intolerance can be reduced by two methods: a) pre-harvest - use of β-galactosidase-replacement or b) post-harvest - hydrolysed low-lactose products. The preharvest methodologies of reducing lactose involve either the introduction of β-galactosidase enzyme into milk via mammary gland specific expression or the removal of α-lactalbumin (ALA) and gene removal methods. ALA is one of the major whey proteins present in almost all mammalian milks. It interacts with β-1, 4-UDP-galactosyl transferase (UDP-gal) to modify substrate specificity of this enzyme. It creates a unique binding site for glucose and leading to the synthesis of lactose. The disadvantage of these methods is that they reduce the overall sugar content of the milk, resulting in highly viscous milk. Studies on mice have revealed that reduction of lactose via ALA deletion was inappropriate because it impaired milk volume regulation. The milk of such mice was highly viscous with very high protein (88%) and fat (60%), no ALA and no lactose (Karatzas and Turner, 1997). Knocking out the UDP-gal gene in mice also produced milk with no lactose but very high viscosity (Vilotte, 2002). In an in vivo technique for low-lactose milk production, Jost et al. (1999) claimed that it is possible to achieve at least two-fold greater levels of lactose-reduction. They generated transgenic mice that selectively produced a biologically active β-galactosidase in their milk. This caused a significant decrease in milk lactose (50-85%) without obvious changes in fat and protein concentrations, thus maintaining a balanced nutrient supply. It may be technically feasible to produce transgenic livestock carrying this transgene and probably, similar or better expression levels could be achieved.

4. MANIPULATING WHEY PROTEINS FOR INFANT HEALTH

There is no doubt that breast milk is the best mode of nourishment for babies because human milk provides optimal nourishment and growth during infancy and also for supplies certain bio-protective factors that afford protection against commonly occurring infections. However, in certain situations it becomes imperative to have infant formulas which closely imitate human milk. The composition of these formulas could be greatly improved to suit the needs of the infant by incorporation ingredients present in human milk.

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4.1. Lactoferrin

Lactoferrin (LF) is a component of mammal milk and is responsible for the natural host defence mechanism. It has excellent antimicrobial activities and is an iron-binding protein and also has a role in regulating various components of the immune system. Human milk has about one g/l and in human colostrums, about 7 g/l of LF. The level of LF in cow milk is only about one tenth of that in human milk. Therefore, it has found a place in designing infant formulas. Several infant formulas containing bovine LF are marketed in Japan under brand names such as Hagukumi, Chilmil Ayumi, Non-Lact, E-Akachan, GP-P and New-NA-20- Morinaga. The consumption of such formulas may result in anti-infection, improvement of oro-gastro-intestinal microflora, immunomodulation, anti-inflammation and antioxidation (Wakabayashi et al., 2006). Pharming, a company in Leiden (Netherlands) developed the first transgenic bull in the late 1980s and a line of transgenic cows to produce several proteins including hLF (Subramanian, 2004). The company claims that that as human gut receptors have better affinity to a human protein than a bovine one, the ingredient would be more effective in boosting gut health.

4.2. Lysozyme

Lysozyme is an antibacterial enzyme that destroys bacterial cell walls by hydrolyzing the polysaccharide component of the cell wall. Human milk contains 0.4 g/l of lysozyme (LZ), an enzyme that contributes to antibacterial activity in human milk. A group of researchers in China also developed two lines of transgenic mice that expressed fully active recombinant human LZ (hLZ) in the mammary gland (Yu et al., 2006). Maga et al. (2006) designed a line of transgenic goats that expressed hLZ in the mammary gland. The authors aim to produce cows that will produce lysozyme in their milk. Such lysozyme-fortified milk has the potential to reduce udder infections in dairy cows and intestinal ailments in humans who drink milk (Bailey, 2001). Feeding young goats and pigs with this lysozyme-enriched milk produced by transgenic goats altered their intestinal bacterial profile (www.eurekalert.org, 2006). The intestinal microbial ecology of both species was altered positively. Both animal groups were healthy and exhibited normal growth patterns. The researchers anticipate that these results will pave the way for protection of infants and children against diarrhoeal illnesses through milk-feeding programmes.

Table 2. Selected applications of transgenesis to produce human therapeutic proteins in milk

Sr. No.

Details of research Benefit Reference

1 Human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system

Blood transfusions Shani et al. (1992)

2 Increasing the amount of α-LA in milk at the expense of β-LG

Alleviating cow milk allergies, a better nutritional source

Coleman (1996)

3 Replacing the four phenylalanine residues in α-LA with other amino acids

Treatment of phenylketonuria (PKU), a congenital disease occurring in those without the enzyme that metabolizes phenyalanine

Coleman (1996)

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4 Expression of recombinant human fibrinogen (rHF) to the mammary gland of transgenic mice

Blood clotting Prunkard et al. (1996)

5 Transgenic cattle containing human α-glucosidase

Treating Pompe's disease, a hereditary, lethal muscle disorder

Biotech Patent News (2000)

6 Insertion of a copy of the human myelin basic protein gene (MBP) in cattle

Treatment of multiple sclerosis www.agresearch. co.nz (2001)

7 Two mouse strains genetically engineered to produce large quantities of malaria vaccine in their milk

Controlled the disease in monkeys vaccinated with the same. May be extrapolated to livestock

www.sciam.com (2001)

8 Recombinant human α-antitrypsin (hAAT), a recombinant human albumin and a CD137 antibody in goat milk

Stimulation of immune system as a potential treatment for solid tumors

www.sciam.com (2001)

9 Insulin and growth hormone from milk of transgenic cows, sheep, or goats

Pharmaceutical interest Margawati (2003)

10 Recombinant human anti-thrombin III in goat milk

Anti-coagulant protein meant for those suffering from a deficiency in anti-thrombin in their blood

Subramanian (2004)

11 Produce bovine milk with enhanced quantities of immunoglobulin (IgA) antibodies

Protection against pathogens in infants

AgResearch Now (2005)

12 Transgenic cows that secreted lysostaphin (a peptidoglycan hydrolase) in milk

Potent antistaphylococcal activity and its secretion into milk offers considerable resistance to mastitis

Wall et al. (2005)

13 HSA produced by calves created from foetal cells

Blood transfusions Johnston (2006)

14 Sheep to produce in milk a therapeutic protein

Reduces lung damage – treatment of cystic fibrosis

Morgan (2006)

15 Rabbits and sheep to produce lipase Pancreatic insufficiency in digesting dietary lipids

Morgan (2006)

16 Genetically altered sheep created to produce milk that contained a therapeutic protein

Treatment of human haemophilia

Pettus (2006)

17 Transgenic goats and cows produce > 60 therapeutic proteins, including plasma proteins, monoclonal antibodies and vaccines

Health management www.transgenics. com /news.html

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4.3 Cow milk allergy

Casein as well as whey proteins are reported to be responsible for these allergic responses to cow milk. This ailment is rare in adults (0.1% - 0.5%). Usually, infants and young children (about 2%) suffer from this ailment and outgrow it by the age of 5. Cow’s milk protein allergy is characterised by one or more of cutaneous (e.g., eczema, rashes), gastrointestinal (e.g., nausea, vomiting, diarrhoea), or respiratory (e.g., asthma, rhinitis, wheezing) symptoms. The only effective management strategy for cow’s milk protein allergy is avoidance of cow’s milk and its products, which in turn negatively influences dietary nutrition. Cow milk allergenicity in children is caused by the presence of β-Lg, which is absent in human milk. Besides β-Lg the caseins, α-LA, serum albumin, and immunoglobulins and digests of these proteins are also allergenic in infants and children. Elimination of β-Lg by knocking out its gene from cow’s milk is might help to overcome many of the major allergy problems associated with cow’s milk. 5. MILK WITH HUMAN THERAPEUTIC PROTEINS

The preparation of therapeutic proteins which are high in value though low in volume in the milk of domestic animals through transgenic technology is now a possibility. The major benefit of transgenic technology is that it produces proteins 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/g of htPA to be 20000, 10000 and 10 US dollars respectively (Karatzas and Turner, 1997). Table 2 illustrates some applications of transgenic technology in producing human therapeutic proteins in milk.

6. CONCLUSION

Novel research in genetic engineering may target only individual animals and not generations together or it may modify the genetic line wherein the altered traits can be inherited by the progeny. Whey components have a great future in are useful in the enhancement of animal productivity, faster growth, improved feed conversion, better quality of animal products and improved resistance to diseases through transgenesis. 77.. RFERENCES AgResearch Now. (2005). Your pasture, our pasture. 30% more milk. Interested?

www.agresearch.cri.nz/publications/now. June. Issue 3. pp. 1-20. Bailey, P. (2001). Why milk? UC Davis Magazine Online. Spring. Vol.18. No.3.

www.ucdmag.ucdavis.edu/sp01/feature_1.html Biotech Patent News. (2000). September Issue. www.allbusiness.com. Coleman, A. (1996). Production of proteins in the milk of transgenic livestock: Problems, solutions and successes.

Am. J. Clin. Nutr. 63, 639S-645S. Johnston, I. (2006). What did Dolly do for us? The Scotsman. Science & Technology. July 5.

news.scotsman.com. Jost, B., Vilotte, J-L., Duluc, I., Rodeau, J-L. and Freund, J-N. (1999). Production of low-lactose milk by ectopic

expression of intestinal lactase in the mouse mammary gland. Nature Biotechnol. 17, 160-164. Karatzas, C. N. and Turner, J. D. (1997). Toward altering milk composition by genetic manipulation: Current

status and challenges. J. Dairy Sci. 80, 2225-2232. Maga, E. A., Shoemaker, C. F., Rowe, J. D., BonDurant, R. H., Anderson, G. B. and Murray, J. D. (2006).

Production and processing of milk from transgenic goats expressing human lysozyme in the mammary

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gland. J. Dairy Sci. 89, 518-524. Margawati, E. T. (2003). Trangenic animals: Their benefits to human welfare. www.actionscience.org. Morgan, J. (2006). The clone arrangers. The Herald. Science & Technology. June 27.

www.theherald.co.uk/features/64780.html. Pettus, P. (2006). Straight from the sheep's mouth. The New York Sun. June 19. www.nysun.com/article/34616. Prunkard, D., Cottingham, I., Garner, I., Bruce, S., Dalrymple, M., Lasser, G., Bishop, P. and Foster, D. (1996).

High-level expression of recombinant human fibrinogen in the milk of transgenic mice. Nature Biotechnol. 14, 867-871.

Shani, M., Barash, I., Nathan, M., Ricca, G., Searfoss, G. H., Dekel, I., Faerman, A., Givol, D. and Hurwitz, D. R. (1992). Expression of human serum albumin in the milk of transgenic mice. Transgenic Res. 1, 195-208.

Subramanian, S. (2004). Transgenic therapeutics - Medicines in milk. Biotechnology and Society - Part 22. www.chennaionline.com.

Vilotte, J-L. (2002). Lowering the milk lactose content in vivo: potential interests, strategies and physiological consequences. Reproduction Nutrition Development. 42, 127–132.

Wakabayashi, H., Yamauchi, K. and Takase, M. (2006). Lactoferrin research, technology and applications. International Dairy Journal. 16, 1241-1251.

Wall, R. J., Powell, A. M., Paape, M. J., Kerr, D. E., Bannerman, D. D., Pursel, V. G., Wells, K. D., Talbot, N. and Hawk, H. W. (2005). Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nature Biotechnol. 23, 445 – 451.

Yu, Z., Meng, Q., Yu, H., Fan, B., Yu, S., Fei, J., Wang, L., Dai, Y. and Li, N. (2006). Expression and bioactivity of recombinant human lysozyme in the milk of transgenic mice. J. Dairy Sci. 89, 2911-2918.

4

SECTION - IV

BUTTER MILK BUTTERMILK

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Dr. Dharam Pal and Mr. P. Narender Raju Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

Buttermilk is an important byproduct obtained during the manufacture of butter. The exact amount of buttermilk production in India is not estimated. However, based on conversion of 6.5% of total milk production into creamery butter, it can be estimated that about 3.2 million tonnes of buttermilk is produced annually as a by-product. In addition, a substantial amount of lassi (sour buttermilk) is also produced during the manufacture of makkhan directly from fermented milk (curd). Sweet cream buttermilk is almost similar in composition to skimmed milk except for their high amount of phospholipids and milk fat globular membrane proteins. Buttermilk solids are untapped till recently and with the worldwide research focused on their utilization their role in health is now recognized. On the basis of the estimated availability of buttermilk and the scope for the development of health-based value added foods, it can be concluded that buttermilk, as a byproduct, needs proper attention for its judicious utilization. 2. TYPES OF BUTTER MILK

Normally three types of buttermilk are produced in our country, viz. (a) sweet cream buttermilk (SCBM) – obtained by churning of fresh/pasteurized cream with little or no developed acidity, (b) sour buttermilk –obtained by churning naturally sour milk or cream, and (c) desi buttermilk (chhachh or lassi) –obtained by churning of curd (dahi) during the manufacture of makkhan. The sweet and sour buttermilks are produced in the organized sector and lassi at the household levels in small quantities. 3. CHEMICAL COMPOSITION OF BUTTERMILK

The chemical composition of buttermilk varies to a great extent, depending on the amount of water added to cream. Some of the butter manufacturers standardize cream with water, thereby decreasing the total solids level of buttermilk. The chemical composition of buttermilk produced under ideal conditions is almost similar to that of skim milk (Table 1).

Sour buttermilk differs from SCBM in respect of titratable acidity. The acidity in SCBM varies from 0.10 to 0.14 per cent, whereas in sour buttermilk it is more than 0.15% and even as high as 1%. However, there is not much difference in the chemical composition of two types of buttermilk. Desi buttermilk has wide range of composition depending on the quality of milk used for making curd and levels of addition of water during churning. Desi buttermilk on an average contains 4% total solids comprising of 0.8% fat, 1.29% protein and 1.2% lactic acid. The colour of desi buttermilk is brownish due to prolonged heating of milk before culturing and the body not as homogeneous as that of factory produced buttermilk. When kept undisturbed for sometime, curdy material deposits at the bottom of cultured buttermilk.

APPLICATION OF BUTTERMILK IN THE MANUFACTURE OF VALUE ADDED DAIRY PRODUCTS

APPLICATION OF BUTTERMILK IN THE MANUFACTURE OF VALUE ADDED DAIRY PRODUCTS

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Table 1. Average gross composition and physico-chemical properties of buffalo sweet cream buttermilk and buffalo skim milk

Characteristic Buttermilk Skim milk T.S. (%) 9.88 10.38 Fat (%) 0.59 0.09 Total proteins (%) 3.73 4.27 Lactose (%) 4.81 5.2 Ash (%) 0.75 0.82 Total phospholipids (mg %) 78.56 8.65 Titratable acidity (% LA) 0.12 0.16 pH 6.86 6.69 Curd tension (g) 18.84 66.85 Relative viscosity (cP at 30°C) 1.80 1.64

4. DIFFERENCE BETWEEN SWEET CREAM BUTTERMILK AND SKIM MILK

Buttermilk contains higher fat content than skim milk, which can be reduced to some extent by subjecting it to centrifugal separation. Buttermilk also contains a larger proportion of protein mixture sloughed from the fat globule-milk-serum interface by churning process. The amount of milk fat globule membrane (MFGM) protein is, however, not large in comparison with total buttermilk proteins. The MFGM proteins are hydrophilic and hydrophobic in nature and their physical properties, nitrogen content and amino acid composition do not correspond with any other milk proteins. These proteins exert emulsion in milk and milk products during manufacture and storage (King, 1955). Recently, Roesch and Corredig (2002) investigated and demonstrated the potential of MFGM proteins, rich in buttermilk, to release biologically active peptides. The MFGM protein also contributes a complex mixture of glycerophospholipids to buttermilk. SCBM contains about nine times higher phospholipids than skim milk (Table 1). It has been noticed that phospholipids in buttermilk do not have short chain fatty acids. The principal fatty acids are C16 (palmitic) and higher acids. Of the total phospholipid fatty acids, about 40% by wt. are saturated acids and the rest are non-conjugated di- to penta-unsaturated acids (Garton, 1963). Phospholipids of buttermilk include more or less equal proportions of lecithin, sphingomyelin and cephalin together with a small proportion of cerebrosides.

Various physico-chemical properties of buttermilk also differ from that of skim milk (Table 1). SCBM has lower acidity and curd tension but higher viscosity as compared with skim milk. These differences in physico-chemical properties of buttermilk and skim milk provide many choices for their selective applications in dairy products manufacture.

5. PROCESSING AND DRYING OF BUTTERMILK

Since the composition of sweet cream buttermilk is like that of skim milk, no problems are encountered during its processing, i.e., separation, clarification, pasteurization, concentration and drying. Rather the heat stability of SCBM is considered to be better than skim milk thereby making it more suitable for processing to very high heat treatments (Bratland, 1972). Concentration and spray drying of SCBM can also be achieved by adopting the same standard conditions as those used for skim milk. The gross composition and physico-chemical properties

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of spray dried SCBM and skim milk are given in Table 2.

The striking differences between two types of powders are the high total lipids including phospholipids and low bulk density in SCBM powder in comparison with skim milk. The spray dried buttermilk powder is less free flowing and dusty because of high fat content in comparison with skim milk powder. Though the high fat content reduces the shelf-life of the powder during storage, the high phospholipids will provide better oxidative stability to dried buttermilk.

Table 2. Physico-chemical characteristics of spray powders

Characteristis Sweet cream buttermilk powder

Skim milk powder

Moisture (%) 2.59 2.75 Fat (%) 6.38 1.05 Total protein (%) 37.09 40.29 Lactose (%) 47.00 48.15 Ash (%) 6.94 7.76 Total phospholipids (mg %) 625.25 97.1 Titralable acidity (% L.A.) 1.17 1.39 Solubility index (ml) 0.15 0.30 Bulk density (g/ml) 0.345 0.544

6. UTILIZATION OF BUTTERMILK IN DAIRY AND FOOD INDUSTRY

6.1 Sweet Cream Buttermilk 6.1.1 Market Milk

The undiluted SCBM produced in the organized dairies is admixed either with the whole milk for fluid milk supply or with skim milk for drying. It has been observed that use of SCBM in the market milk for toning of buffalo milk improves the palatability, viscosity and heat-stability and reduce the curd tension without adversely affecting the keeping quality (Pal and Mulay, 1983). In addition to plain fluid milk, it can also be used for the preparation of flavoured milks and milk beverages. The powder made from the mixture of skim milk and SCBM is treated as a skim milk powder and used for reconstitution purposes. 6.1.2 Fermented Dairy Products

6.1.2.1 Dahi: Curd prepared by incorporating SCBM into whole milk has soft-body which is probably due to the change in the electric charge on the casein during churning, the presence of phospholipids and other MFGM materials, and the free fat in the buttermilk. Shreshtha and Gupta (1979) have recommended 1-2% skim milk powder for improving the body of dahi made from buttermilk. As an alternative to curd making, SCBM can be successfully utilized in the manufacture of cultured buttermilk and lassi in which the firmness is not of much consideration.

6.1.2.2 Yoghurt: Increasing the total solids in yoghurt milk to around 14-16g 100g-1 is one of the essential steps in the process of yoghurt making. Traditionally, the fortification of the total solids in the yoghurt mix is achieved by boiling to reduce the volume of the milk to two-thirds of its original or by the addition of skimmed milk powder (SMP). Replacement of SMP with buttermilk

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powder up to 50% in the manufacture of low-fat yoghurt was found to be acceptable and similar to the control product (Vijayalakshmi et al., 1994). Buttermilk powder when added to low-fat yoghurts up to 4.8% yielded a soft and smooth product (Trachoo and Mistry, 1998). Among the various dairy ingredients used in the manufacture of yoghurt, dried buttermilk was found to reduce the susceptibility of syneresis (Guinee et al., 1995).

6.1.2.3 Cheeses: The preparation of hard varieties of cheese such as, Cheddar and Gouda involve the adjustment of casein and fat ratio with the help of skim milk. The replacement of skim milk with SCBM resulted into soft body which was ascribed to the presence of higher amount of fat globule membrane materials in buttermilk. Joshi and Thakar (1996a) however, were successful in improving the firmness and producing acceptable quality of Cheddar cheese from blends of buffalo milk and SCBM. They maintained the setting temperature at 33°C, added starter @ 2% and rennet @ 4.5 g/100 kg milk. The cheddar cheese so prepared was also used for making processed cheese of satisfactory quality (Joshi and Thakar, 1996b).

Gokhale et al, (1999) attempted to substitute water with SCBM for standardization of moisture content in processed cheese and reported that no significant effects were observed in the composition up to 75% level of substitution. However, higher levels of substitutions were reported to result in reduction of total volatile fatty acids. Attempts were also made to replace whole milk in cottage cheese production by Shodjaodini et al, (2000) who reported that 30-40% replacement improved flavour and yielded a softer cheese. Recently, Govindasamy-Lucey et al, (2006) studied the effect of using condensed SCBM on the quality of pizza cheese and reported that at low levels (2%) of addition SCBM improved cheese yield without affecting the quality of cheese. But at higher levels (4% and 6%) the product showed less melt and stretch than control.

With the growing awareness of dietary fat on human health, worldwide research was focused on the development of low-fat or reduced fat cheeses as most cheeses contain high fat. However, defects related to flavour, flavour development, body and texture were reported in low-fat cheeses. Along with milk proteins milk fat also contributes to the texture of cheese. Lowering fat in reduced fat cheese lowers fat globule membrane, which in turn affects cheese texture and flavour. With a view to improve the quality of reduced fat Cheddar cheese, Mistry et al. (1996) added ultrafiltered (UF) buttermilk to cheese milk and reported that UF-buttermilk @ 5% produced softer cheese with improved body and texture and increased yield than the control. Similar results observations were made when 5% UF-buttermilk was added to reduced fat Mozzarella cheese (Poduval and Mistry, 1999). Reduced-fat processed cheese manufactured using reduced-fat Cheddar cheese made with pasteurized milk supplemented with 5% UF-SCBM was reported to contain less fat (14.48%) than the control (15.10%) but also resulted in hard body owing to compositional differences (Raval and Mistry, 1999).

6.1.3 Indian Traditional Dairy Products 6.1.3.1 Chhana: 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). Recently, Kumar (2006) reported that admixture of SCBM and buffalo milk in the proportion of 60:100, on total solids basis, adjusting fat and SNF ratio to 1:2.1 and coagulating at a pH of 5.2 and 75°C produced highly

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acceptable chhana.

6.1.3.2 Paneer: Buffalo milk has to be standardized to a fat and SNF ratio of about 1:1.65 to meet the PFA requirements for the manufacture of paneer. The replacement of skim milk with SCBM for the standardization of buffalo milk has been found to increase the yield of paneer by about one per cent without altering the organoleptic and textural properties (Pal and Garg, 1989). It is also possible to prepare good quality paneer from low fat milk by incorporating buttermilk solids to buffalo milk. Attempts were made by Sharma et al, (1998) to substitute buffalo milk with butter milk up to 10% in the preparation of fried paneer and reported that oil uptake was found to be higher and the product had soft texture than the control.

6.1.3.3 Rasogolla: Rasogolla 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. Recently, Kumar (2006) attempted to develop rasogolla from buffalo milk by admixing SCBM. The worker reported that good quality rasogolla can be produced by kneading refrigerated chhana (7±2°C for 2-3 hr) obtained by admixing SCBM and buffalo milk in the proportion of 60:100, along with 1% arrowroot, 1% maida and 1% semolina and forming into smooth balls followed by cooking in 50% sugar syrup for 20 min and soaking into 40% sugar syrup for 3-4 hr.

6.1.3.4 Sandesh: 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. Recently, Kumar (2006) attempted to use SCBM in sandesh making and reported that kneading of chhana obtained by admixture of SCBM and buffalo milk in the proportion of 60:100, by passing through wire mesh to obtain uniform smooth dough, adding 30% sugar by weight to chhana, heating with continuous stirring and scraping at 75°C for 10 min for first pat formation and 60°C for 10 min for final pat formation produced product close to cow milk sandesh.

6.1.3.5 Basundi: Basundi, a partially desiccated sweetened milk product, is prepared traditionally from buffalo milk. Attempts were made by Patel and Upadhyay (2004) to replace buffalo milk solids by SCBM solids in basundi making and reported that 100% replacement of buffalo milk solids resulted in a significant decrease in the lactose and ash contents and adversely affected the physico-chemical properties such as free fatty acids (FFA) and hydroxyl methyl furfural (HMF) contents. However, it was recommended by the workers that replacement up to 25% would check such adverse effects on the product. 6.1.3.6 Chakka and Shrikhand: Chakka, a semi-solid mass obtained after draining whey from dahi, is an intermediate product for shrikhand manufacture. Karthikeyan et al, (2000) studied the effect of replacement of buffalo skim milk by SCBM on various technological parameters and reported that chakka made from 50% replacement of skim milk by SCBM had improved flavour and with smoother body and texture, and no adverse effect on chemical composition. Karthikeyan et al, (1999) attempted to prepare shrikhand from SCBM with varying total solids content and reported that shrikhand prepared from SCBM with 15% total solids was similar to that of control product prepared from buffalo skim milk.

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6.1.4 Frozen Dairy Products

The buttermilk powder has been extensively used in preparation of ice cream and other frozen desserts. Tirumalesha and Jayaprakasha (1998) reported that 100% replacement of skim milk solids in the form of a blend of spray dried whey protein concentrate (WPC) and dried SCBM (50:50) in ice cream resulted in better quality product than the control.

6.1.5 Beverages

Addition of fruit juices or fruit pulps is an attractive avenue for the utilization of buttermilk. With the availability of a variety of region-specific and season/climate-specific fruits country wide, a large variety of beverages can be formulated and marketed as ready-to-serve drinks. Shukla et al, (2004) studied the suitability of blending apple, banana, guava, litchi and mango juice/pulp with buttermilk at different levels and reported that apple juice, litchi juice, banana pulp, guava pulp and mango pulp could be added up to 30%, 30%, 20%, 10% and 20% levels respectively in buttermilk to make refreshing drinks. Recently, Kankhare et al, (2005) attempted to develop fruit flavoured buttermilk and reported that cashew (Anacardium occidentale) syrup and kokum (Garcinia india choisy) syrup at a level of 15% and 10% produced best quality flavoured buttermilk.

Rao and Kumar (2005) attempted to develop a spray dried mango-buttermilk powder and reported that the blend with 20:80 ratio of mango juice and buttermilk possessed desired sensory characteristics. It was further reported that there was a steep increase in the yield of such powder when the mango solids were added up to 35% level. 6.1.6 Probiotic Drinks

The growing interest worldwide in probiotic foods led the researchers to find all possible ways of developing health foods and buttermilk is no exception. Various workers have developed buttermilk-based probiotic drinks. El-Fattah and Ibrahim (1998) investigated the health benefit of fresh buttermilk which was fermented with Lactobacillus acidophilus for 100 days and mixed with regular feed and fed to 50-day old Japanese quail hens and found that serum and liver cholesterol were reduced while the high density lipoproteins were increased. Rodas et al, (2002) developed probiotic buttermilk by adding the probiotic strain of Lactobacillus reuteri at a rate of 1% and reported that there were no significant differences in composition and sensory quality compared with control and the viability remained greater than 106 cfu/ml for 10 days of storage. Several other workers have also reported the development of probiotic buttermilk with different probiotic bacteria (El-Shafei, 2003; Antunes, et al, 2007). 6.2 Utilization of desi buttermilk

Desi buttermilk (chhach / lassi) is an important domestic beverage in India. 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

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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. It has high nutritive and therapeutic value. In addition to normal milk constituents, lassi is also a rich source of vitamins. It is considered to be an excellent thirst quenching and nourishing beverage, particularly during summer months. It is also used for making some popular traditional preparations e.g. karhi, rabri (fermented), raita etc. The industrial utilization of lassi cannot be exploited due to lack of proper collection system and day-to-day variations in the composition and quality. 6.3 Utilization of sour buttermilk

During the manufacture of butter, a considerable amount of sour buttermilk is obtained either from natural souring of milk/cream or by controlled fermentation. Sour buttermilk obtained from first category is not preferred for human consumption. This may, however, be converted into casein by adopting modified processing conditions for other uses. A process has been standardized for the manufacture of paneer from the mixture of buffalo milk and sour buttermilk (Fig.1) (Pal and Garg, 1989). The paneer prepared by this method gave higher yield by retaining more moisture in comparison with control paneer. The organoleptic quality and shelf life of the buttermilk extended paneer were comparable to control paneer. 7. FUTURE SCOPE

The role of buttermilk solids in health foods is being investigated by many workers. Recently, El-Sayed et al, (2006) reported that processed cheese spread containing 30% buttermilk concentrate when fed to rats significantly decreased the total and LDL-cholesterol contents. With the recent studies suggesting the role of buttermilk phospholipids in many health-related functions, successful attempts were made to isolate the MFGM material from skim milk retentates rich in phospholipids (Corredig et al, 2003). Such isolates can be used as functional ingredients in the development of nutraceuticals. Buttermilk solids were also demonstrated to possess antioxidant activity (Wong and Kitts, 2003) and have been suggested for use in stabilizing food matrixes against lipid peroxidation reactions. Further, great scope lies in the effective use of buttermilk or buttermilk solids in the manufacture of some of our popular indigenous Indian dairy products, e.g., khoa, kheer and rabri. The dried buttermilk can replace the SMP in the manufacture of gulabjamun mix powder. Because of high lecithin content in buttermilk, it may improve the textural properties of rasogolla.

8. CONCLUSIONS

The high nutritional value of buttermilk and increasing public awareness concerning the environmental pollution warrants for the economic utilization of this important byproduct. SCBM can be used in the fluid milk industry as a milk extender with specific benefits over skim milk. The other potential uses of buttermilk solids are in manufacture of soft varieties of cheese, paneer, fermented milks and traditional milk products. Judicious use of buttermilk solids in the development of functional foods is another promising area.

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9. REFERENCES Antunes, A.E.C., Marasca, E.T.G., Moreno, I., Dourado, F.M., Rodrigues, L.G. and Lerayer, A.L.S. 2007.

Development of a probiotic buttermilk. Ciencia-e-Tecnologia-de-Alimentos (in Portuguese). 27(1): 83-90. Bratland, A. 1972. Production of Milk. Cited in Dairy Sci. Abstr., 40: 1294. Corredig, M., Roesch, R.R. and Dalgleish, D.G. 2003. Production of a novel ingredient from buttermilk. J. Dairy Sci.

86(9): 2744-2750. El-Fattah, A.M.A. and Ibrahim, F.A.A. 1998. Effect of feeding fresh and fermented buttermilk on serum and egg yolk

cholesterol of Japanese quail hens. Bulletin of Faculty of Agriculture, University of Cairo. 49(3): 355-368. El-Sayed, M.S., Hamed, I.M., Asker, A.A., Hamzawi, L.F. and El-Sayed, M.M. 2006. Plasma lipid profile of rats fed

on processed cheese spread enriched with buttermilk concentrate. Egyp. J. Dairy Sci. 34(2): 169-174. El-Shafei, K. 2003. Production of probiotic cultured buttermilk using mixed cultures containing polysaccharide

producing Leuconostoc mesenteroides. Egyp J. Dairy Sci. 31(1): 43-60. Garton, G.A. 1963. The composition and biosynthesis of milk lipids. J. Lipid Res. 6: 237. Gokhale, A.J., Pandya, A.J. and Upadhyay, K.G. 1999. Effect of substitution of water with sweet cream buttermilk on

quality of processed cheese spread. Ind. J. Dairy Sci. 52(4): 256-261.

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Govindasamy-Lucey, S., Lin, T., Jaeggi, J.J., Johnson, M.E. and Lucey, J.A. 2006. Influence of condensed sweet cream buttermilk on the manufacture, yield, and functionality of pizza cheese. J.Dairy Sci. 89(2): 454-467.

Guinee, T.P., Mullins, C.G., Reville, W.J. and Cotter, M.P. 1995. Physical properties of stirred-curd unsweetened yoghurts stabilized with different dairy ingredients. Milchwissenschaft. 50(4): 196-200.

Joshi, N.S. and Thakar, P.N. 1996a. Utilization of buttermilk in manufacture of buffalo Cheddar cheese – Standardizing the conditions for curd setting. Ind. J. Dairy Sci. 49(5): 350-352.

Joshi, N.S. and Thakar, P.N. 1996b. Utilization of buttermilk Cheddar cheese made using butter milk solids in processed cheese manufacture. Ind. J. Dairy Sci. 49(5): 353-355.

Kankhare, D.H., Joshi, S.V., Toro, V.A. and Dandekar, V.S. 2005. Utilization of fruit syrups in the manufacture of flavoured buttermilk. Ind. J. Dairy Sci. 58(6): 430-432.

Karthikeyan, S., Desai, H.K. and Upadhyay, K.G. 1999. Effect of varying levels of total solids in sweet cream buttermilk on the quality of fresh shrikhand. Ind. J. Dairy Sci. 52(2): 95-99.

Karthikeyan, S., Desai, H.K. and Upadhyay, K.G. 2000. Study on the quality of chakka made from the partial and full replacement of buffalo skim milk by sweet cream buttermilk. Ind. J. Dairy Biosci. 11: 62-66.

King, N. 1955. The milk fat globule membrane. Commonwealth Agri. Bureau, Farnhan, Royal Bulk, England. Kumar, J. 2006. Admixture of buttermilk to buffalo milk for production of chhana and chhana based sweets. Ph.D.

Thesis submitted to National Dairy Research Institute (Deemed University), Karnal. Mistry, V.V., Metzger, L.E. and Maubois, J.L. 1996. Use of ultrafiltered sweet buttermilk in the manufacture of

reduced fat cheddar cheese. J. Dairy Sci. 79(7): 1137-1145. Pal, D. and Garg, F.C. 1989. Studies on utilization of sweet cream buttermilk in the manufacture of paneer. J. Fd Sci.

Technol. 26: 259-264. Pal, D. and Garg, F.C. 1989. Utilization of sour buttermilk in the manufacture of paneer. Ind. J. Dairy Sci. 42: 589-

594. Pal, D. and Mulay C.A. 1983. Influence of buttermilk solids on the physico-chemical and sensory properties of market

milks. Asian J. Dairy Res. 2: 129-135. Patel, H.G. and Upadhyay, K.G. 2004. Substitution of milk solids by sweet cream buttermilk solids in the manufacture

of basundi. Ind. J. Dairy Sci. 57(4): 272-275. Poduval, V.S. and Mistry, V.V. 1999. Manufacture of reduced fat Mozzarella cheese using ultrafiltered sweet

buttermilk and homogenized cream. J. Dairy Sci. 82(1): 1-9. Rajorhia, G. S. and Sen, D. C. 1988. Technology of chhana – a review. Ind. J. Dairy Sci. 41(2): 141 – 148. Rao, H.G.R. and Kumar, A.H. 2005. Spray drying of mango juice-butter milk blends. Lait. 85(4/5): 395-404. Raval, D.M. and Mistry, V.V. 1999. Application of ultrafiltered sweet buttermilk in the manufacture of reduced fat

process cheese. J. Dairy Sci. 82(11): 2334-2343. Rodas, B.A., Angulo, J.O., Cruz, J.De-La. And Garcia, H.S. 2002. Preparation of probiotic buttermilk with

Lactobacillus reuteri. Milchwissenschaft. 57(1): 26-28. Roesch, R.R. and Corredig, M. 2002. Production of buttermilk hydrolyzates and their characterization.

Milchwissenschaft. 57(7): 376-380. Sabikhi, L. 2006. Developments in the manufacture of lassi. In: Lecture compendium of the short course on

“Developments in Traditional Dairy Products”, Centre of Advanced Studies, NDRI, Karnal. Pp: 64 – 67. Sen, D. C. and Rajorhia, G. S. 1990. Production of soft grade sandesh from cow milk. Ind. J. Dairy Sci. 43(3): 419 –

427. Sharma, H.K., Singhal, R.S. and Kulkarni, P.R. 1998. Characteristics of fried paneer prepared from mixtures of

buffalo milk, skimmed milk, soy milk and buttermilk. Int. J. Dairy Technol. 51(4): 105-107. Shodjaodini, E.S., Mortazavi, A. and Shahidi, F. 2000. Study of cottage cheese production from sweet buttermilk.

Agri. Sci. Technol. (Iran). 14(2): 61-70. Shreshtha, R.G. and Gupta, S.K. 1979. Dahi from sweet cream buttermilk. Indian Dairyman, 31: 657-60. Shukla, F.C., Sharma, A. and Singh, B. 2004. Studies on the preparation of fruit beverages using whey and buttermilk.

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Tirumalesh, A. and Jayaprakasha, H.M. 1998. Effect of admixture of spray dried whey protein concentrate and dried butter milk powder on physico-chemical and sensory characteristics of ice cream. Ind. J. Dairy Sci. 51(1): 13-19.

Trachoo, N. and Mistry, V.V. 1998. Application of ultrafiltered sweet buttermilk and sweet buttermilk powder in the manufacture of nonfat and low-fat yoghurts. J. Dairy Sci. 81(12): 3163-3171.

Vijayalakshmi, R., Khan, M. M.H., Narasimhan, R. and Kumar, C.N. 1994. Utilization of butter milk powder in preparation of low-fat yoghurt. Cheiron. 23(6): 248 – 254.

Wong, P.Y.Y. and Kitts, D.D. 2003. Chemistry of buttermilk solid antioxidant activity. J. Dairy Sci. 86(5): 1541-1547.

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SECTION - V

WHEY BASED BY PRODUCTS WHEY BASED

BY-PRODUCTS

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Dr. Ashish Kumar Singh Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

Use of cheese whey as a beverage in human nutrition, especially for therapeutic purpose can be traced back to 460 BC. Hippocrates, the legendary Greek physician, is reported to have prescribed whey for an assortment of human ailments. Liquid whey has been utilized for the manufacture of wide range of beverages and soups over the years as result of which a number of such products are available to consumers. However, in India, despite demand for natural nutritious drinks or beverages the commercial production of whey based drinks is still in infancy. Some organized manufacturers have initiated production of whey based beverages and soups and these products are fast becoming popular. In this article an attempt has been made to provide an insight of whey utilization for beverage and soup production.

2. WHEY AS BASE FOR FRUIT BEVERAGES

2.1 Technological Developments in Whey Based Fruit Beverages

The ready-to-serve (RTS) type beverage may be prepared by mixing an appropriate fruit juice or concentrate and minimally processed whey along with other minor additives to improve the sensory characteristics and thermally processed to make it shelf-stable (Figure 1). Processing, physico- chemical and nutritional aspects of whey beverages have been well documented in the literature (Kosikowski, 1979; Krarvchenko, 1988; Gandhi, 1989; Driersen and Vandenberg, 1990, Jelen, 1992; Mann, 1994). Singh et al (1994) standardized the formulation of whey-fruit beverages using cheese or paneer whey with three different fruits viz. banana, lemon and pineapple and found 5 and 20% juice content optimum for lemon, pineapple or banana, respectively. Among the beverages mango beverage scored maximum for all sensory attributes and it contained 15% mango pulp, 7% sugar, 78% whey and the pH of the beverage was kept at below 4.5. Jayaprakasha et al (1986) found 10 percent sugar, citric acid and orange flavour as optimum level for whey fruit flavoured beverage. Reddy et al. (1987) reported 8% lemon juice and 14% sugar level for developing acceptable quality beverage. In general the total solid level above 15%, sugar level between 7-10% and pH below 4.5 is required for developing acceptable quality beverage. In-package thermization above 800C and sterilization above 1150C often results in shelf-life enhancement at ambient storage. UHT processing of such beverages increases the shelf-life up to a year at ambient storage conditions. The pH adjustment should always be compatible with typical flavour profile of the used fruit pulp/juice. For example pH level in the range of 3.6-3.8 maximizes the flavour perception of citrus fruit beverages. UHT processing of the finished product, especially while using direct-steam injected systems often results in loss of flavour.

TECHNOLOGICAL DEVELOPMENTS IN WHEY BASED NON-FERMENTED BEVERAGES AND SOUPS

TECHNOLOGICAL DEVELOPMENTS IN WHEY BASED NON-FERMENTED BEVERAGES AND SOUPS

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Fig. 1 Process Flow Chart for Production of Whey-Fruit Beverage

The tetra-pack paper (Laminates) based containers are mostly used for packaging of whey fruit based beverages in India. Plastic cups with aluminum linings are frequently used in Western countries (Singh and Kumar, 1997). Metal cans, glass or rigid containers are rarely used for these products, because of the acidic pH and presence of minerals. 2.2 Quality Characteristics of Whey Based Fruit Beverages

Despite its relative simplicity, finding a successful flavour combination to mask the unpleasant whey taste is difficult. Several workers have reported cheesiness or saltiness as two most dominant flavours in processed whey beverages. The presence of residual lipids also sometime pose flavour problem in whey beverages especially if the product is thermally processed at elevated temperature. The whey flavour was observed to be most compatible with citrus flavour

Clarification in cream

Addition of sugar and Stabilizer

Addition of Fruit Juice/Pulp

Filtration

pH adjustment with Acidulant

Pasteurization/Sterilization/UHT processing

Cooling and Storage

Whey – Fruit Beverage

Paneer or cheese whey

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(Holsinger et al. 1978). Branger et al. (1999) evaluated the sensory characteristics (grapefruit, sweetness, saltiness, sourness, cheesiness and astringency) of cottage cheese whey and grapefruit juice blends. They observed that increasing the proportion of whey increased the cheesiness and saltiness in blends. Deproteination had no effect on sensory attributed however vacuum stripping reduced cheesiness with subsequent improvement in grapefruit flavour and perception of sweetness. Lactose hydrolysis also enhanced the sweetness if whey is added above 50% level. Consumer prefer whey beverages that are sparkling clear, hence clarification of whey is essential to remove residual lipids, casein fines and partially denatured whey proteins. Acid whey possesses fewer proteins as compared to sweet whey and that make them more suitable base for beverages base. Major problems reported in whey beverages are turbidity and sedimentation after heat treatment and during subsequent storage. During thermal treatment whey proteins coagulate near their isoelectric point (at acidic pH) which causes development of turbidity and subsequent sedimentation in whey-fruit juice beverage (Gagrani et al., 1987). Shekilango et al. (1997) noted that the other major problems often encountered in whey-fruit beverage are cloudiness and high viscosity. These are caused by the interaction between whey components (protein and calcium) and fruit components (pectin, tannins and starch). The Hunter colour parameter i.e a* (redness), b* (yellowness) and C* (saturation) of whey-fortified banana beverage increased with time at elevated storage temperature while lightness (L*) remained same (Koffi et al; 2005). Sensory testing of beverage using consumer panel indicated that sourness and acidity are critical quality parameters. The storage temperature had significant effect on sedimentation and serum separation of beverage and higher storage temperature maximize these defects.

According to Jelen (1992), such quality problems often lead to failure of these products in market. Several approaches have been tried to minimise these problems. Passing whey through a centrifugal classifies removed casein fines and residual fats and improved the clarity (Jelen et al., 1987). Another approach adopted was to homogenize the product after pasteurization (Barabas and Albrecht, 1988). According to them whey drinks processed in this way were rated as homogenous with excellent flavour quality. De-proteination of whey (Reddy et al., 1987; Mathur et al., 1988; Jayaprakasha et al., 1986; Krishnaiah et al., 1989) and protein coagulation by addition of 0.5% CMC and 0.08% pectin in UHT-sterilized product (D'yachenko and Suarez, 1984) were the other methods adopted to obtain sparkling clear beverages. 2.3 Concentrated Fruit Based Whey Beverages

The purpose of developing concentrated fruit based whey beverages is to deliver the product in more convenient form, minimize the transportation and packaging requirement and improve the protein content. For preparing such products whey is concentrated in vacuum pan or evaporators, mixed with fruit juice concentrate, sugar and other additives, heat treated and packaged. A British patent (1975) described a method for preparation of a concentrate with 40-50% whey proteins in dry matter and the product contained citric acid, natural orange flavour, sorbitol, sodium saccharin and colouring matter as other ingredients. Singh et al (2003) found that the whey-mango concentrate obtained by mixing 15% mango pulp (25o Brix), 77% paneer why concentrate (37% TS), 8% sugar and a pH of 4.2 was most acceptable for developing whey-mango concentrate. Whey-apple concentrate was developed at this Institute using 15% apple juice concentrate, 7% sugar, 78% concentrated paneer whey and the pH of the beverage was maintained at 3.7 using glucono-delta-lactone (GDL). The product could keep well for 45 days at 250C. Khamrui (2000) utilized reverse osmosis concentrated Kinnow juice (23%), concentrated cheddar

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cheese whey (45% TS), sugar, acidulant, CMC, pectin and other minor additives to manufacture kinnow-whey concentrate. The most acceptable formulation consisted of 40% kinnow juice, 53% whey, 7% sugar, 0.05% pectin, 0.15% CMC and had a pH of 4.5. Whey based Kinnow juice concentrate (WKJP) packed in metallized polyester pouches at 250C storage temperature had a shelf-life of more than 6 months (Khamrui and Pal, 2003). The major problems in whey-fruit juice concentrates are as follows:

• Lactose crystallization during storage specially under refrigeration storage

• High viscosity of concentrates hinder their effective thermal processing

• Whey protein coagulation during thermal processing

• Low storage stability at elevated temperatures These points should be investigated for developing acceptable Shelf-stable nutritious

whey-fruit juice concentrates. 3. WHEY PROTEIN ENRICHED WHEY BEVERAGES

Development of “Functional whey beverages” is an attractive possibility to utilize whey nutrients for human beings. Whey proteins of late have been discovered with many nutritional and therapeutic attributes. Whey proteins are one of the highest quality food proteins with a high PER (3.6), biological value (104), NPU (95) and highest Protein Digestibility Corrected Amino Acid Score (PDCAAS) score that make then an ideal protein source for fortification of fruit juices, beverages and specialty foods. Dietary whey proteins have a number of putative, biological effects when ingested (Horton, 1995). The ability of whey proteins to increase the level of natural anti-oxidants within the body and possibly in stabilizing DNA during cell division is emerging as premier contribution to population health (Bonous et al., 1989) Steinys. Whey protein may find applications in both dairy type as well as fruit based acidic drinks. Some of the technological advantages associated with utilization of whey proteins in beverages include:

• Their solubility over a broad range of pH (3-8), even in their iso-electric pH.

• They have bland flavour so their inclusions in formulation do not cause cheesiness, saltiness, in developed products. Instead they are carrier of aroma compounds and help in developing flavour to their full potential.

• They possess excellent buffering capacity that is advantageous particularly in probiotic drinks where it help in survival of “live” bacteria in stomach

• Their addition improve the viscosity of beverages, hence they can substitute stabilizers in beverages and therefore enhance the “mouthfeel”

Several attempts have been made to increase protein content of whey-based beverages. Holsinger et al. (1973) added dried cottage cheese whey to concentrate soft drinks to improve their nutritional value without any detectable changes in their flavour or appearance. In another experiment, orange juice was combined with dried cheese whey, which contained 74 % protein by weight and had a bland flavour with high PER (3.1-3.2). The resultant beverage was found to have appearance and flavour similar to orange juice with protein content approximately equal to that of milk. Nazare et al., (1979) added dried cheese whey (12.06% protein) at 4.2-20.8% levels to passion fruit juice to obtain a final protein concentration of 0.5-2.5% in finished product. The

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product was processed at 85-90°C for 30 min, bottled and stored at room temperature up to 60 days. A refreshing beverage could be prepared by adding the concentrate with 2-3 volumes of water. Swedish Dairy Cooperative developed and marketed a protein rich fruit drink called “Nature’s Wonder”. It was produced by mixing a high-grade whey protein, hydrolyzed lactose and pineapple, orange and passion fruit juices (Hakansson, 1983).

Visco-de-Velez (1986) filed a patent for a process for preparing a nutritional drink from whey. This process consisted of concentration of whey 5-fold by ultrafilteration, inoculating of the retentate with 1-5% lactic acid bacteria and incubating at 35-50°C for 8-18 hours. Stabilizers and pasteurized cream were added to give 0.5-5% fat content in finished product. The product with or without fruit juice concentrates preservatives, and 7-12% sugar was heat treated to partially denature the soluble proteins and homogenized at 50-300 kg/cm2 and at 60-80°C. The drink had a shelf life of up to 45 days at refrigerated temperature. Bangert (1976) developed a nutritious orange drink concentrate of about 10% protein content by using whey protein concentrate. Benea and Contarelli (1984) worked on the development of a beverage based on frozen mandarin concentrate and whey proteins. They found whey proteins to be the most stable at low pH. The formulations contained 2.5 or 5% whey protein, 15% sucrose, 33% orange concentrate, and 0.2% guar meal. The spray-dried formulations showed better solubility, dispersibility and organoleptic quality than vacuum dried formulations. But the dried product lack typical flavour as observed in liquid formulation. Most consumers preferred pasteurized beverage to sterilized one. Whey protein fortified beverage was developed by Sharma et al. (1998) using 4% whey protein isolate, 3% hydrolyzed guar gum, 11% orange juice concentrate, 10% sucrose, 1.2% calcium gluconate and 0.2% citric acid. The pH of beverage was adjusted to 3.75 and other minor ingredients were also added. Beverage was processed in a pulsed electric field (PEF). Singh (2002) optimized the formulation of whey-protein-enriched orange beverage by utilizing ultrafiltered whey retentate, range juice, sugar and a stabilizer mix (guar gum and propylene glycol alginate (PGA)). Addition of stabilizer mix improved the mouthfeel and prevented the coagulation of why proteins during in-bottle sterilization. Storage investigations for determining the quality parameters affecting the acceptability of beverage revealed that discoloration and sedimentation were the two critical defects have major impact on beverage organoleptic quality. Thermally processed beverage was stable up to 6 months storage in glass bottles. 3.1 Problems in processing of whey protein-enriched fruit based beverages

Tuohy et al. (1988) reported that pH less than 4 were necessary to prevent protein coagulation during pasteurization. Sharma et al. (1998) evaluated the feasibility of pulsed electric field (PEP) treatment, as an alternative to conventional heat treatment for to check/retard tendency of sedimentation. PEF treated protein fortified fruit beverages had less protein denaturation and no problem of sedimentation during storage but microbiologically less stable as compared to the heat-treated product and had shorter shelf- life. Whey proteins complexed with acidic polysaccharides can be used to fortify acidic fruit juices and this approach was used to develop whey protein-enriched Bael (Aegle marmeols) beverage. The CMC-WPC complex addition increased the protein level to 1.75% and this complex was observed better in comparison to pectin-WPC complex (Singh and Nath, 2004). Further Singh at al. (2005) reported that CMC and pectin can be used to stabilize whey proteins under acidic conditions.

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4. WHEY BASED SPORTS AND THIRST QUENCHING BEVERAGES

Wagner et al. (1975) reported that whey drinks could stabilize the osmolar system in the body and had a thirst quenching effect. Whey being rich in lactose and minerals can serve as base for development of sports as well as electrolyte drinks of varying composition. Whey permeate which is obtained during whey protein preparation using ultrafiteration process, can be an ideal starting material for the development of such products. Further more addition of other additives and process of carbonation may provide thirst quenching properties and consumer appeal. Milk permeate was utilize for the production of electrolyte beverage after hydrolyzing the lactose, adding sugar, lowering pH (3.5-3.8) by citric acid and UHT processing to make it shelf-stable. The beverage contained minerals (in ppm) calcium 150, phosphorus 157, magnesium 43, potassium 1166, sodium 286, iron 17, copper 8, zinc 3.4 (Geilman et al, 1992). However dilution was suggested to overcome salty flavour due to high mineral content in permeate. Beucler et al. (2005) utilized whey permeate (WP) with or without hydrolyzed lactose for development of thirst quenching beverages and compared with commercially available beverages for various sensory attributes using descriptive sensory analysis. They observed that beverages made with 25-50% substitution of hydrolyzed or un-hydrolyzed whey permeate were similar to commercial beverages in visual and flavour characteristics than those made with higher level of substitutions. All beverages made with WP were higher in electrolytes (Na, K, Zn, Mg, P) compared with commercially available sports beverage.

An attempt has been made at our Institute to develop sports beverage using whey. Optimum conditions for the effective hydrolysis of paneer and cheese whey by using Maxilact-2000 L were 0.4% enzyme concentration (v/v), temperature of 400C, pH of 6.75 and incubation period of 180 min. Under optimized conditions 85-90 percent hydrolysis of lactose occurred. Hydrolysis of lactose in whey resulted in increased sweetness of whey and was comparable to 2.5% sucrose solution. The hydrolyzed paneer whey was utilized for the preparation of lemon and pineapple based sports beverages (Figure 3). Acceptable lemon based sports drink can be made with 7.43% sugar, 3.1% lemon juice, 0.07% lemon flavour and 0.07% CMC. Addition of 0.35% salt enhanced the overall acceptability, but protein enrichment of the beverage using hydrolyzed whey protein resulted in lowering of product acceptability. The best formulation for pineapple sport beverage were 20% pineapple juice, 7.49% sugar, 0.15% stabilizer mix and 0.12% salt mix. The stored sports drink was acceptable up to 6 months.

The formulation for the manufacture of “Panna” like beverage utilizing hydrolyzed whey permeates consisted of 15%, un-ripened mango pulp, 7% sugar, 1% salt mix, 0.2% commercial spice mix, 0.1% stabilizer and hydrolyzed whey permeate. Similar product in concentrated for was also developed and its formulation included 25% un-ripened mango pulp, 1% citric acid, 40% sugar, 0.4% commercial spice mix and 33% hydrolyzed whey (Gupta, 2005).However, these beverage suffered with heavy sedimentation during storage and homogenization was found to be effective in controlling this problem.

Whey proteins contain higher amounts of branched chain amino acids that serves as rapid source of energy in muscle during fatigue hence can be used in formulation of sports drink meant for athletes. Many dairy-like beverage formulations flavoured with either vanilla or strawberry or pineapple have been developed using whey protein concentrate @ 8-10% level.

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Paneer Whey

Clarification/Filtration

Pasteurization

Hydrolysis of Lactose in Whey

at 80O C for 1 min

Hydrolyzed Whey

Addition of Sugar & Stabilizer Mix

Heating to 50OC

Salt mix Fruit JuiceFruit Juice

Mixing, Filtration & Heating to 80OC

Filling in Sterilized Bottles & Thermal Processing

Sports Beverage

Cooling

Fig: 2 Flow Diagram for Manufacture of Sports BeverageFig: 2 Flow Diagram for Manufacture of Sports Beverage

5. WHEY BASED SOUPS

Soups are served as appetizers before meals as they stimulate the secretion of gastric enzymes that leads to feeling of hunger. In market a large number of ready-to-make soup mixes are available to suit the palate of consumers. But certain additives in such soups mixes are considered harmful particularly to children. Moreover apparently they do not seem to provide quality nutrients and utilization of whey for soup preparation is attractive possibility.

The process for the manufacture of whey based soup involves blending of vegetables in whey and cooking of corn flour followed by heating. The time-temperature combination of cooking of vegetables, corn flour and seasoning is important for dispersion of vegetables, gelatinization of starch and flavour perception of soup respectively (Singh and Kumar, 1997). The process for manufacturing whey tomato soup is outlined in figure 3. The developed product could be stored for a week under refrigeration and UHT treatment can be adopted to improve the shelf-stability.

Paneer and cheese whey were utilized for the potato-carrot-tomato and spinach soups. Cheese whey was preferred for the manufacture of vegetable soups than paneer whey (Singh et al.

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1994). The reason could be the low pH of paneer whey that resulted in acidic product not usually compatible with most vegetables. Whey based soups have been reported to be more viscous as compared to water based most probably gelation of whey proteins on heating. Whey based soups require less amount of salt, thickener and fat and technology for manufacture of retort processed low fat tomato-whey soup has been developed recently at out Institute. Alam et al (2002) reported the technological aspects for the manufacture of tomato whey soup using paneer whey. Few years back AMUL has introduced UHT processed tomato-whey soup in Tetra-Pak and last year VITA has launched tomato-whey soup in polystyrene cup in Haryana.

Paneer whey / Tomatoes Frying of seasoning cheese whey (onion , ginger and garlic in oil)

Cooking under pressure

Grinding Addition of corn flour

Straining Gelatinization of starch (80-85° C/ 2 min)

Tomato Pulp

Tomato-whey-corn flour suspension

Addition of salt and permitted colour

Boiling for 2 min

Tomato-whey soup

Fig. 3 Process Flow diagramme for the Manufacture of Whey-Tomato Soup

Mushroom-whey soup powder was prepared by cooking mushroom with concentrated cheese whey and blending in it. The seasonings (onion, garlic and ginger) were fried separately in hydrogenated fat, and cornflour was added if thickening was desirable. After the addition of salt and additional quantity of concentrated whey, the soup mix was again blended. It was then spray-dried to produce a mushroom-whey soup powder. The moisture content, loose and packed bulk density, wettability, insolubility index, thiobarbituric acid and hydroxy methyl furfural content, increased during storage. However, dispersibility and reflectance value decreased during storage. The soup powder reconstituted well when boiled in water for 2 min. The reconstituted soup was considered acceptable, with an overall acceptability score of 7.1 on a nine-point hedonic scale, even after 8 months of storage of the soup powder at 30 °C when packed in metalized polyester (Singh et al. 2003).

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6. CONCLUSION

Beverages and soups based on whey continue to receive a considerable amount of attention nowadays. These indicate the growing awareness among consumers and manufacturers alike for the enormous potential these offered for diversifying product profile. Technological packages are available for wide range of whey based such products and with better understanding regarding the functionality of different ingredients it would be possible to develop “functional” whey products for specific target groups.

7.0 REFERENCES Alam, M. T., Singh, Sudhir and Broadway, A. A. 2002. Utilization of paneer whey for the preparation of tomato whey

soup. Egyptian J. Dairy Sci. 30: 355 – 361. Bangert, J.C. 1976. Orange drink concentrate, US Patent. 3949098. Cited in Food Sci and Tech Abstr. 1977. 9 (9): 74. Barabas, J. and Albrecht, P. 1988. Production technology for whey drink. Technologia Vyroby Srvatkovych napojov.

10: 283- 289 pp. Benea, F. and Contarelli,C. 1984. New beverages based on fruit juice and milk protein. Industria-delle-Bevande, 13

(71) 202-210. cited from Food Sci. Technol. Abstr. (1985) 05-H0104. Beucler, J., Drake, M.; and Foegeding, E.A. 2005. Design of beverage from whey permeate. Journal of Food science

70 (4), 5277-5285 Bounous, G., Batist, G. and Gold, P. (1989). Immunoenhancing property of dietary whey proteins in mice: role of glutathione. Clin. Invest. Med. Vol.12: 154 – 161. Branger, E.B.; Sims, C. A., Schmidt, R. H.; O’Keefe, S. F. And Cornell, J. A. 1999. Sensory characteristics of cottage

cheese whey and grapefruit juice blends and changes during processing. Journal of Food Science, 64 (1), 180-184 D’yanchenko, L. F. and Saurez, V. 1984. Technology of fruit/whey beverage. Molochanya Promy Shlennost 29 (7):

27-29. Driersen, F.M. and Van Den Berg, M.G. 1990. New developments in whey drinks. Bull. IDF 250, pp11-19. Gagrani, R.L.; Rathi, S.D. and Ingle, U.M. 1987. Preparation of fruit flavour beverage from whey. J. Food Sci. and

Tech., 24: 93. Gandhi, D.N. 1989. Whey utilization for beverage production. Indian Dairyman. 41 (1): 35-37. Geilman, W.G., Schmidt, D., Herfurth-Kennedy, C., Path, J nad Cullor, J. 1992. Production of electrolyte beverage

from milk permeate. Journal of Dairy Science, 75: 2364-2369. Gupta, R. 2005. Development of raw mango whey beverage. M. Sc. Thesis. Bundelkhand University, Jhnasi. Hakansson, H. 1983. Nature’s Wonder: The secrete of mixing. Milk and juice to taste good. Nordik – Mejeriindustry,

10 (6): 401-403. Holsinger, V.H. Postali, L.P.; Devilbliss, D. and Pollansch, M.J. 1973. Fortifying soft drinks with cheese whey

protein. Food Technology 27 (2):59-60. Hoogstratan, J.J. 1987. Trends in whey utilization. IDF Bulletin No. 17: 212-215pp. Horton, B. S. (1995). Commercial utilization of minor milk components in the health and food industries. J.Dairy Sci.

Vol.78: 2584 – 2589. Horton, B.S. 1995. Commercial utilization of minor milk components in the health and food industries. J. Dairy Sci .,

78: 2584-2589. Jayaprakasha, H.M. and Anathakrishnan, C.P.; Atmaram, K. and Natarajan, A.M. 1986. Preparation of soft drink

from clarified and deproteinized whey. Cherion, 15 (1): 16-19. Jelen, P. 1992. Whey cheeses and beverages. In: Whey and Lactose processing. J. Zadow, (ed.) Elsevier Applied

Science, London and New York, pp 157-194. Jelen, P., Currie, R. and Kadie, V.W. 1987. Compositional analysis of commercial whey drink. J. Dairy Sci., 70 (4):

892-895. Khamrui K and Pal, D. 2003. Effect of storage temperature on microbiological and sensory characteristics of whey

based Kinnow juice powder. Indian Journal of Dairy Science, 56 (2) 77-80. Khamrui, K. 2000. Development of technology for concentrated and dried whey based fruit juice mixes. Ph. D.

Thesis, National Dairy Research Institute (Deemed University), Karnal, India. Koffi, E.; Shesfelt, R. and Wicker, L. 2005. Storage stability and sensory analysis of UHT-processed whey-banana

beverage. Journal of Food Quality, 28 (4), 386-401

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Kosikowski, F.V. 1979. Whey utilization and whey products. J. Dairy Sci., 62(7): 1149-1160. Krishanaiah, M; Reddy, C. R; Sastry, P. M. and Rao M. R. 1989. Studies on keeping quality of whey beverages. Asian

Journal of Dairy Research, 8 (1): 8-14. Krorvchenko, E.F. 1998. Whey beverages. IDF Ball No. 238 pp 61-67. Mann, E.J. 1994. Dairy beverages. Dairy Ind. Intl., 59 (11): 16-17. Mathur, B.N.; Kumar, A. and Ladkani, B.G. 1988. UHT- Processed beverage paved way for economic utilization of

whey. Indian Dairyman, 40 (10): 533- 535. Nazare, R.F.R., Jeixera, M. A.; Code, A.R. and Colletro, D.T. 1979. Addition of dried cheese whey for enrichment of

passion fruit juices. Revista CFTRI, 76(143); 13-15, 26-52 Cited in Food Sci. &Tech., Abstr. 1980. 12(5); 89. Parekh, J.V. 2007. Small-scale utilization of whey. In Dairy India (2007). Sixth Edition. Ed. Gupta, S. Dairy India

Year Book, New Delhi, 245-246 Reddy, G.L., Rao, B.V.R., Reddy, K.R.S. and Venkayya, D. 1987. Development of a whey beverage. Indian J. Dairy

Sci., 40 (4): 445-450. Sharma, S.K., Zhang, Q.H., and Chism, G.W. 1998. Development of a protein fortified fruit beverage and its quality

when processed with pulsed electric field treatment. Journal of Food Quality, 21: 459-473. Shekilango, A; Jelen, P, and Bagdan, G. C. 1997. Production of whey banana beverage from acid whey and overripe

bananas. Milcharissns Chalt 52 (4): 209-212. Singh, Ashish Kumar and Nath, Nirankar. 2004. Development and evaluation of whey protein-enriched ‘BAEL’

beverage. Journal of Food Science and Technology, 41 (4), 432-436 Singh, Ashish Kumar. 2002. Development and evaluation of protein-rich fruit based beverages. Ph. D. Thesis

submitted to G. B. Pant University of Agriculture and Technology, Pantnagar Singh, S. and Kumar, A. 1997. Whey in soups and fruit beverages. In compendium of Short Course on “

Technological advances in dairy by-products” organized under the aegis of CAS in Dairy Technology. 64-68 Singh, S. Ladkhani, B. G., Kumar A and Mathur, B. N. 1994. Development of whey based beverages. Indian Journal

of Dairy Science 47 (7) : 586-590. Singh, S.; Singh, Ashish Kumar and Gandhi, D. N. 2000. Formulation of whey-mango concentrate. Paper presented in

Conference on Biotechnological Strategies in Agro Processing” on 9-11th Feb. 2000. Organized by Punjab State Council for Science and Technology, Chandigarh.

Singh, S.; Singh, Ashish Kumar and Kumar, Abhay. 2005. Effect of pH and hydrocolloids on the thermal stability of whey proteins in model beverage system. Journal of Food Science and Technology, 42 (5), 407-410

Singh, Sudhir, Ghosh, Subhajit and Patil, G.R 2003. Development of mushroom – whey soup powder. International Journal Food Science & Technology. 38: 217 – 224.

Singh, Sudhir, Singh, A. K. and Patil, G. R. 2002. Whey utilization for health beverages. Indian Food Industry 21 (4), 38 – 41.

Tuohy, H.Y., Fitzgerald, A. and Nash, P. 1988. Utilization of whey for beverage. Farm and Food Research, 19(4): 8-10.

Visco-de-Velez, S. M. 1986. Process for preparing a nutritional drink from whey. French- Patent-Application. FR (2567366 A1)

Wagner, K.H., Albig, K., Ali, A. and Maicold, K.G. 1975. The effects of whey based drinks on osmolarity. Milchwissenschatt. 30(12): 724-729.

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D.N. Gandhi* and Kalpana Dixit *Principal Scientist

Dairy Microbiology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Whey is a good tasting, versatile and highly functional ingredient and possesses benefits to food and beverage manufacturer as they create nutritious and delicious products as per the demand of the consumers. Historically, whey has some interesting beginning. In fact whey has been an important food and medium for thousands of years. The father of modern medicine, Hippocrates is said to have insisted on a daily dose of two liters of whey. By the year 1600, whey was used to treat jaundice infected lesions of skin, gonorrhea and epilepsy. Indeed, most of the dairy industries today consider whey as mere by product of cheese making. Well, with development in Research and Development on whey utilization on beverages, the evolution of whey from waste to commercial product is a real story of turning lemons in to lemonade. In view of increasing demand of beverage in the form of cold drinks and fruit juices, whey has a promising future in the beverage formulating world and formulator’s particularly cheese and coagulated milk products industries are looking at whey right now.

Manufacturing of fermented whey beverages such as soft drinks, whey wine, beer like products and low alcoholic beverages appear to be most economical and viable process for returning the wasted milk nutrient into value added products among various innovative microbiological process of whey utilization. Several authors have reviewed processing of whey in the preparation of wide varieties of palatable beverages through microbial fermentation since whey has been found to be a suitable growth medium for the proliferation of selected strains of lactic acid bacteria and yeast. These products have been categorized as functional group, pleasure, health, nutrition etc. Recently commercial interest in fermented whey beverages has increased in several European countries notable Germany, Holland, Australia and Switzerland, possibly as a result of health consciousness of the modern consumers. In India there has been a tremendous increase in the production of cheese and coagulated milk products resulting in a proportionate increase in whey. Keeping in view an increased trend in consumption of soft drinks, various type of fermented whey beverages have been developed and some of them have been marketed successfully because of their distinctive characteristics as compared to other beverage. 2. BIPROCESSING OF WHEY INTO BEVERAGES

Whey has been found to be useful as a base for growth of some of the lactic acid bacteria (LAB) in the preparation of beverages. These beverages are obtained due to lactic acid bacteria fermenting lactose in whey to form mainly lactic acid. The lactic acid imparts fresh flavor and is known suppress the growth of pathogenic spoilage organisms. In addition some LAB particularly L. acidophilus enhances nutritional properties and adds certain therapeutic attributes to the beverages. Antibacterial substances produced during fermentation are reported to have curative properties in controlling several gastro-intestinal disorders (Prasher and Gandhi 1990).

PREPARATION OF FERMENTED BEVERAGES FROM WHEY

PREPARATION OF FERMENTED BEVERAGES FROM WHEY

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An attempt has been made at National Dairy Research Institute at Karnal (India) to convert whey into palatable refreshing probiotic in nature and low cost beverage named as ‘Acidowhey’ and its concentrate (Gandhi, 1989 and Gandhi and Patel 1992). This product is free from any preservative and synthetic color. The process has been found to be commercial feasible since the process has been adopted by the Experimental Dairy for the sale of the product at Milk Parlor of the Institute and has also been licensed to the dairy industries engaged in paneer making.

2.1 Significant Features of Fermentation of Whey

Fermentation is one of the oldest forms of food preservation and biological upgrading of dairy by-product into value added food has been well established. Process of manufacturing of fermented whey beverages have developed following transfer of technology from related fermentation of milk in to yoghurt, acidophilus milk, kefir and kumis, which possess nutritional and probiotic properties in addition to their palatability. Presence of valuable nutrients in whey is considered to be the suitable to convert whey into lactic and alcoholic beverages through selective lactic acid bacteria and yeast respectively. Drissen and Berg (1998) emphasized the significant feature of adopting the process of manufacturing of fermented whey beverages by dairy industries for the following advantages.

• The composition of whey make it a suitable base for fermentation • The dairy industry possesses the know how regarding fermentation such as cheese

and yoghurt making • The dairy industry has the equipment for fermentation process • The dairy industries being trusted by the consumers • As a result of these features the dairy industry can manufacture a nutritious and

palatable health whey drinks by their own identity rather than being associated with just another soft drink manufacture.

2.2 Selection of Lactic Acid Bacteria

Selection of Lactic acid bacteria suitable as starter culture for the production of fermented whey beverages was according to two main criteria.

• The strain should be able to grow and survive in whey giving a product with suitable organoleptic and probiotic properties, and

• The strain should possess antibacterial properties against organisms responsible to cause gastro intestinal disorders.

Nutritional, non-carbonated beverages may be manufactured using whey as a functional ingredient. Utilization of cheese whey for beverage manufacture appear to be the most obvious and logical avenue for returning the wasted milk nutrient in to food chain. .However, the basic approach for the acceptability of whey based beverages may become quite complex in light of consumers preferences in the competitive beverage market. Commercial interest in whey beverage technology in Europe increased considerably in the early 1980, partly in response to consumer’s demand which stimulated by several medically oriented programs in German television on medical use of whey for various ailments includes tuberculosis and arthritis.

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2.3 Development of Acidic, Alcoholic and Non-alcoholic Whey Beverages

Work on non-alcoholic whey beverages is quite limited. A patent was granted for the production of a kumis-like product form a mixture of cow’s milk, whey, and lactose (Jagielski, 1871). A combined lactic acid and alcohol fermentation was employed to produce a sparkling product containing both lactic acid and alcohol. Nearly 50 year later a United States patent was issued to Laessing (1961) in which he described a procedure for producing nonalcoholic beverages from whey. The Second World War stimulates German work on the production of beverages consisting, in part, of whey. More recently a whey beverage designated as “Rivella” was developed in Switzerland. This product has met with considerable acceptance as a soft drink and its manufacture, during the early 1960s spread to other European countries. Rivella is a clear, amber colored liquid with a refreshing acidic flavor.

In last three decades procedures for the manufacture of acidic and alcoholic beverages have been reported. Khrulkevich (1959) returned to the idea of preparing a kumis-like beverage combined equal volume of whey and butter milk and inoculated the mixture with of a culture comprised of kumis yeasts, Lactibacillus bulgaricus and Lactobacillus acidophilus. The resulting beverage is claimed to closely resemble kumis and to have a refreshing sour alcoholic taste. Its stability and consistency were improved by adding gelatin. Another product designated as “Milone” and characterized as mildly alcoholic and sweet sour in flavor.

Use of whey as a substrate for the production of alcoholic beverages has also been exploited. A variety of beer like products has been produced from whey. Interest in these beverages was greatest in Germany during and after the Second World War when sources of fermentable carbohydrates were in shortage .Description of four beers like beverages which have been made from whey are: a) An alcoholic whey beer b) A malted whey beer c) A whey malt beer and, d) A whey nutrient beer.

Several research workers have shown keen interest in the production of wine from cheese whey after deprotienization of whey and heating at 82 C for 5 minutes approximately 22% dextrose is added depending upon the amount of alcohol desired in wine Fermentation is completed in 7 days at 22-25 C using the yeast Saccharomyces cerevisiae. As the whey itself contains sufficient nutrients for the growth of yeast, no additional nutrients are added. 2.4 Preparation of Fermented Whey drinks ‘Acidowhey’

The process of Acidowhey making has been developed at NDRI, Karnal. In this process, whey from paneer, cheese, chhana or casein manufacturing is first separated to remove fat and traces curd particles. Clarified whey is heat treated to 85-900C for 10 min. or its equivalent combination. Clarified whey after cooling to 400C is inoculated with an active culture of L. acidophilus and incubated at 39±100C for 20-22 hr. After incubation, fermented whey is clarified to remove precipitated cellular mass, whey protein and minerals formed during the process. Sugar is then added to the product @10-12% in the form of 50% sugar syrup which had been earlier pasteurized. At this stage desirable amount of pine apple flavor or any compatible citrus flavor is added. The beverage is now chilled to 40C and packaged (Fig.1). Beverage should be pasteurized before packing or alternatively pasteurized in the container. Additional studies demonstrated antibacterial properties of ‘Acidowhey’ against certain gram positive and gram negative organisms such as Micrococcus flavus, Staphylococcus aureus, Escherichia coli and Bacillus subtilis.

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3. CONCLUSION

The largest by product whey obtained from different sources such as cheese and coagulated milk products can be processed into value added fermented whey beverages .These beverages have been found to be nutritious, palatable, economical , therapeutic in nature and well accepted by the producers and consumers.

4. FURTHER READING Gandhi, D.N. (1989), Utilization of cheese and paneer whey for the preparation of acidophilus whey drink. Indian

Dairyman, 37:511 Gandhi, D. N. and R. S. Patel (1992), Technological and keeping quality of fermented whey concentrate Cult. Dairy

Products. J. 29:25 Gandhi, D.N. (2002) Potential application of LAB in development of fermented milk products and for biprocessing of

whey; Indian Dairyman; 54 pp 64-67 Holsinger, V. H., L. P. Posti and E. D. Velbiss (1974), Whey utilization- a Review: J. Dairy Sci. 97:849 N. Kosaric and Y. J. Asher.(2006) The utilization of cheese whey and its components N. Kosaric1 and Y. J. Asher.

Advances in Biochemical Engineering /Biotechnology. Springer Berlin / Heidelberg.pp 25-60. Suresh, K. B. and H.N.Jayaprakasha (2004); Process optimization for preparation of beverages from lactose

hydrolyzed whey permeate; J.Fd.Sci.Technol; 41; 27-32.

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D.N.Gandhi* and Krishan Kumar *Principal Scientist

Dairy Microbiology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

Annually, approximately 1.2 million tons of lactose and 200,000 tons of milk protein are transferred into whey worldwide, of which less than 60% are utilized for human food and animal feed. Large quantities of whey are unsuitable to be processed to lactose powder due to the presence of lactic acid and sulphate ions which interfere with the crystallization process. Some progress has been made in utilizing whey, whey solids, and whey protein concentrates in the manufacture of products of industrial importance in dairy, bakery, and specialized products such as organic acids (lactic and citric acid) and ethyl alcohol, single cell protein (SCP) and for the synthesis of gums. The new concept of handling and increasing volumes of liquid whey is strongly influencing the processing sector in developed countries. Surplus whey problem has increased mainly due to lack of capital, poor infrastructure, and inadequate marketing and utilization channel.

2. INDUSTRIAL UTILIZATION OF WHEY

One approach to transform whey and lactose into value-added products is to use the whey as a fermentation feedstock. Fermentation should be used as a means of modifying the functional properties of the whey to add value. Uses of whey or whey permeate as a fermentation medium reduces the lactose content, resulting in a decrease in the BOD and associated disposal costs. The substances that can be produced by the fermentation of the lactose of whey can be produced by fermentation of cane, beet or corn sugar. Whether it is practical to utilize whey in making fermentation product depends in general on whether a suitable organism is available to convert lactose in to desired product and whether whey is a less costly source of fermentable sugar than molasses or corn sugar. Methods of whey utilization through microbial fermentation are

• Ethyl alcohol • Organic acid 1)Lactic acid and 11) Citric acid • Vitamins Biosynthesis- (Vit B Group) • Fertilizer- (Ammonium Lactate) • Fermented beverages • Oral Rehydration Solution • Baker’s Yeast • Xanthan Gum(Polysaccharides Products) • Flavor concentrate • Single Cell Protein

BIOPROCESSING OF WHEY FOR PREPARATION OF PRODUCTS OF INDUSTRIAL IMPORTANCE

BIOPROCESSING OF WHEY FOR PREPARATION OF PRODUCTS OF INDUSTRIAL IMPORTANCE

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2.1 Ethanol Production

Production of ethyl alcohol from whey has been studied extensively in the past. Among most widely used raw materials for ethanol fermentation are cellulose materials (straw, baggase, and waste paper), starch containing materials (corn, wheat, and rice), sugar cane, and sugar beet molasses. Utilization of whey material for ethanol formation offers special advantages by providing cheap raw material and simultaneous waste treatment with ethanol production.

Waste biomass has been the most widely used raw material but ethanol production by using waste biomass is expensive since the process requires separation from lignin from cellulose, hydrolysis of cellulose to sugars, fermentation of sugar solution to ethanol and separation of ethanol from water. Production of ethanol form starch containing materials such as corn technically more feasible but high cost of corn and starch containing grains makes process economically less attractive. Among the inexpensive and highly available raw material for ethanol production are molasses and cheese whey, which are the waste byproducts of sugar and dairy industries. Cheese whey has been used by many investigators for production of ethanol because of its high carbohydrate content and availability. Typical cheese whey contains 5-6% lactose, 0.8-1% protein, and 0.06% fat. Not very but many yeast strains are capable of fermenting lactose to ethanol. Most of the Saccharomyces species cannot ferment lactose to ethanol because of lack of galactose fermenting enzymes. Most Kluyveromyces species are capable of fermenting lactose present in cheese whey to ethanol. Cheese whey control 5-6% lactose which yields nearly 2.5% ethanol upon direct fermentation, which is not economically feasible due to low lactose and resulting ethanol concentrations. Ultrafiltration is usually used to remove the protein content of cheese whey and to concentrate the lactose for feasible fermentation. The use of cheese whey powder (CWP) instead of cheese whey eliminates costly ultrafiltration steps and improves process economics for ethanol fermentation. Besides, CWP is concentrated form of cheese whey with considerable advantage as substrate such as reduced volume, concentrated lactose content, long term stability, easy storage and transportation.

For the production of ethanol, whey is heated to boiling, acidified to pH 5.0 with sour whey or acid, and the precipitated protein removed by filtering. After the filtrate has cooled to 28-300C, I-2% of the yeast is added for each 100 lts of whey and the fermentation continued at constant temperature until it is complete, usually for about 50 hours. The yeast is removed and the alcohol recovered by distillation as depicted in Fig. 1b. The protein, spent yeast and residues from the still are suitable for feed.

2.2 Lactic acid production

It is produced commercially from whey by means of mixed culture of Lactobacillus spp. The efficiency of conversion is greater than 90%, the acid is the inactive mixture of the dextro and levo forms, and no objectionable by-products are formed. A starter culture is built up by successive inoculations and incubations of batches of whey of increasing size. Five hundred gallons of starter are added to 5000 gallons of raw whey maintained at 430C (110o F). Every 6 hour or whenever the reaction approaches pH 5.0, a slurry of slaked lime is added in quantity sufficient to bring the reaction to pH 6.0 but not higher. When chemical tests show that practically all the sugar has been fermented, or when the quantity of lime consumed indicates the conversion is complete, the whey is neutralized to pH 6.5 to 7.5 with lime slurry and heated to the boiling

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point. After 10 minutes at the boiling point the coagulum is allowed to settle; the cleared liquid is run to the filter press and is followed by the sludge. The hot filtrate is treated with the small percentage of decolorization carbon, stirred, and brought to a pit value of 10.0 by addition of lime slurry. As soon as the sample shows that sedimentation will be rapid, the precipitate is allowed to settle and the batch again is filtered. The filtrate is neutralized with lactic acid and concentrated in vacuum pan at 150 Baume. The concentrate is run to jacketed crystallizers, and by circulating cold water in jacket, it is cooled to 10-15oC. After 12 hours, the crystallized mass is spun in a basket centrifuge until no more filtrate is obtained and the crystals are washed lightly with cold water. The mother liquor and washings are concentrated to 13.5o B to obtain second crop of crystals. The calcium lactate obtained may be recrystallized to produce calcium lactate or it may be treated with sulfuric acid to convert it to lactic acid either before or after crystallization as shown in Fig 1a. Lactic acid is available in the market principally as 22 to 44% crude, 50% edible, and 65% purified acid.

Fermented whey broth Whey Broth

↓ ↓ pH adjustment with 1% (CaOH)2 Concentration by Heating and Cooling ↓ ↓ Neutralization with 10% lactic acid Addition of yeast ↓ ↓ Concentration and 20% H2SO4 treatment Fermentation for 50hrs ↓ ↓ Filtration and 10% H2SO4 treatment Distillation of Fermented Whey

↓ ↓ Distillation and HPLC quantification of LA Ethanol

Fig. 1a: Production of lactic acid Fig. 1b: Production of ethanol 3.3 Lactase production

A strain of Fusarium moniliforme, previously used for microbial protein production, excreted lactase when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced by both media has pH and temperature optima 4-5 and 50-600C respectively and was particularly suitable for processing acid whey. In the whey culture, maximum lactase yield was observed after 95 hours of growth at 300C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3nanomoles o-nitrophenyl-D galactopyranoside sec-1 ml-1 of growth medium, at pH 5 and 600C, was obtained.

3.4 Flavor Concentrates and Vitamin B12

Use of flavor concentrate has been suggested by many workers for improving flavor in dairy products. Techniques for the production of such flavor concentrate milk and citrate both fermented by aroma producing Streptococci have been successfully used. Since whey is capable of supporting growth of microorganisms and is rich in citric acid it has also has also been considered in the development of flavor in fermented dairy products (Madan Lal et al. 1974). The pattern of

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growth and vitamin B12 production by Propionibacterium shermonii has been established in several types of substrates including cheese whey. The organisms grow anaerobically and produce propionic acid which accumulates in medium. Most industrial process for the production of Vit. B12 by P. shermonii require neutralization of accumulated propionic acid to keep the culture growing logarithmically. The formation of vitamins take place during the later part of the fermentation after the organism approached maximum growth. Whey has also been reported to act as a base for the production of antibacterial substances (Gandhi and Darshan Lal, 1987). Efforts to utilize whey and whey permeate for the production of no-conventional baker’s yeast having maximum dough raising capacity have been reported by Champagne et al (1990) and Vij and Gandhi (1993).

3.5 Xanthan Gum and Microbial Oil

Xanthan gum is microbial polysaccharides of great commercial interest in the food industry. Its production from a number of whey based media has been evaluated using lactose utilizing yellow pigment, gram negative bacterium Xanthomonas campestris C7L isolate. The effect of xanthan gum addition on physicochemical properties of 2 wt % whey protein isolate (WPI) stabilized oil in water (O/W) emulsions containing 20% v/v menhaden oil was studied by measuring droplet size, viscosity, microstructure, creaming profile and oxidative stability. For a number of years, various workers have carried out the fermentation of cheese whey and whey permeates to produce microbial oil. It has been studied that the most efficient lipid producer, C. curvata D, may accumulate up to 60% of its dry weight as lipid. It has been studied whey permeate is better substrate than whey for oil production. Also the whey permeate obtained from various types of cheese is a best substrate when the studies were conducted using batch culture fermentation and continuous culture systems to study lipid accumulations in a number of yeasts. Continuous culture system of whey permeate to produce lipid has been suggested to be the more efficient and potentially more economical than batch fermentation.

. 4. SUGGESTED READING Gandhi,D.N., R.S.Patel, B.K.Wadhwa, Neena Bansal, Manjit Kour and C.Ganesh Kumar (2000) Effect of agro-based

by-products on production of lactic acid in whey permeate medium; J .Fd.Sci.Technol .37 :292-295 Proceedings of the Second International Whey Conference. Held in Chicago, USA, 27-29 October 1997. Brussels,

Belgium: International Dairy Federation, 1998. Naidu, A.S. (Ed). Lactoferrin: Natural, Multifunctional, Antimicrobial. Boca Raton, FL: CRC Press, 2000. Salman Zafar, Mohammad Owais, Mohammed Saleemuddin and Sattar Husain (2005) Batch kinetics and

modeling of ethanolic fermentation of whey. International Journal of Food Science & Technology.40 (6).597-604

Shilpa Vij and D.N.Gandhi (1993) Whey –an alternate substrate for production of bakers yeast. Indian Food Industry 12; 41-43.

Panesar, P.S., J.F.Kennedy, D.N.Gandhi and K.Bunko (2007); Bioutilisation of whey to lactic acid production; Food Chemistry; 105; 1-14

Manual for U.S. Whey and Lactose Products. Arlington, VA: U.S. Dairy Export Council, 2003. www.usdec.org/publications/ PubDetail.cfm?ItemNumber=587

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

By far the single largest use of whey solids on global basis is in the form of whole dry whey and it continues to grow. This is whole whey that has been condensed and spray dried as such or after blending with certain other liquid ingredients. The production of whey powder including demineralized whey powder have prominence in advanced dairy countries, but Indian dairy industry is still a novice in this field. These powdered whey products are marketed as commodity ingredients for a variety of foods for human and animals. The feed industry may be the largest consumer of dried whey and whey products.

Since whey is a highly perishable raw material and its use in liquid form is limited to only small quantities, the concentration and drying of whey assumes a special significance. Whey can be preserved as plain or sweetened condensed whey. With the development of membrane technology (reverse osmosis or, RO), it became economically feasible to manufacture RO whey concentrates with 28% TS retaining all functional properties of whey proteins. The potential use of RO can be as a pre-concentration step prior to transportation of whey concentrate to a central processing facility for further processing for the manufacture of whey powder, whey protein powder, demineralised whey powder and lactose. The trend in Western Europe is to preserve whey solids in the form of demineralised and delactosed whey powders. 2. CONCENTRATION OF WHEY

Whey concentration is carried out with the following objectives: a) Volume reduction for transport to other processing facilities. b) Pre-concentration for drying into whey powder. c) Manufacture of condensed whey: plain or sweetened.

Various steps involved in the production of concentrated whey are as follows: 2.1 Clarification

The whey always has curd fines, which confer serious risk of blocking heat exchangers' channels or fouling ultrafiltration or RO membranes. These curd fines also adversely affect the solubility properties and flavour of the end product. These curd fines are usually removed by a combination of treatments such as settling, screening and clarification. For large-scale operation, it is usual to employ a self-discharging clarifier with an enlarged solid-holding capacity. Also used commonly are the hydrocyclones, where whey is made to flow tangentially into the upper

DEVELOPMENTS IN THE MANUFACTURE OF CONDENSED WHEY AND WHEY POWDER

DEVELOPMENTS IN THE MANUFACTURE OF CONDENSED WHEY AND WHEY POWDER

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cylindrical part. The centrifugal force causes the particles to move toward the cyclone wall and they get deposited on the bottom.

2.2 Separation and pasteurization

In order to obtain flavour stability in concentrated whey, the whey is separated to remove fat. Then the whey is pasteurized for optimum microbiological quality, to inactivate rennet and to ensure storage stability and stored at 5°C till concentration and drying.

2.3 Concentration

The clarified and defatted whey may be concentrated economically with the integration of RO for pre-concentration, and multi-effect evaporator. From economic point of view, a 2.0 fold concentration (50% volume reduction) of paneer whey and 2.5 fold concentration (60% volume reduction) of cow or buffalo cheese whey using RO have been suggested by Jayaprakasha (1992). An energy saving of 50.79, 63.09 and 63.99 per cent by using RO along with conventional evaporator to concentrate 1000 l of paneer whey, cow milk cheese whey and buffalo milk cheese whey, respectively, to 50 per cent TS, has been reported. The costs of concentrating 200,000 kg whey per day are 20 and 25 per cent lower at concentration factor of 2 and 3, respectively by RO than by evaporation. RO is reported to be 3 times more energy efficient than either a 7-effect evaporator or a new evaporator fitted with mechanical vapour recompression. The long tube falling film evaporators used for this purpose are characterized by short residence time (5-30 sec), high heat transfer coefficients and efficient energy use. The most modern concentration installation consists of a 5-7 stages evaporation system. By adding more stages to the evaporator, the specific steam consumption is reduced. A seven-stage plant consumes 50 % less steam than a three-stage plant. Acid whey foams less during evaporation in evaporators than does sweet whey. When sweet whey is difficult to manage in the evaporator a small quantity of acid or a little fat may be added to break the foam.

There is spontaneous lactose crystallization in vapour separator while concentrating whey to levels above 55% TS. This may be avoided by immediate removal of concentrated whey from the evaporator apart from controlling final total solids contents of whey and increasing the evaporation temperature in the last stage, where lactose is in most concentrated form. For this, the whey evaporators are sequenced in such a way that the higher solids are reached at higher temperature than that prevails in the last stage. For instance, a 7-Stage falling film evaporator can be sequenced 1-2-3-4-7-6-5, where the temperature (°C) in the respective stages is 68, 65, 61, 57, 50, 45, and 39. 2.4 Plain condensed whey

The desirable degree of concentration of plain whey is 35- 50% total solids. Whey condensed to higher solids content (more than 55% TS) forms a gel after cooling and is not recommended to be used in any food or feed products due to its coarser texture and low solubility.

Plain condensed whey is also prepared as an intermediate product for whey powder making. The whey is concentrated to about 40-60% solids and pre-crystallized before drying.

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2.5 Sweetened condensed whey

For the preparation of sweetened condensed whey, sweet cheese whey is mixed with sugar equal to the weight of solids in whey (about 6.7 kg sugar for 100 kg whey). The mixture is concentrated using multistage evaporators to at least 76% TS. The specific gravity of sweetened condensed whey at 50°C is 1.360 (38.4° Bé). The concentrate is always cooled to 30°C using flash coolers and seeded with lactose crystals. The seeded mixture is stirred for 1 to 3 hr to crystallize the lactose. It may then be packed in barrels or cans. The product does not require any refrigeration for storage.

Sweetened condensed whey darkens and thickens in storage, but these changes do not develop to an objectionable level during the first year. The product must be utilised within one year of production to get full advantage of its functional and nutritional properties.

Due to high salt content, condensed whey products are somewhat salty and have a characteristic whey flavour. The saltiness may be reduced by partly desalting the original whey by nanofiltration process. A typical composition of different types of condensed whey is given in Table 2. 2.6 Uses of condensed whey

Whey solids in form of condensed whey are used in dairy products, bakery goods, baby food, meat products, beverages, soups, sauces, dressings and creams. Concentrated whey has worldwide industrial application in bread and other bakery products. The addition of whey solids in macaroni and spaghetti improves their viscosity and dough properties. Condensed whey is also used with fruits and jams in various toppings and spreads in confectionery. In addition, whey solids are used in animal feed mixes, as it is a cheap source of high-quality proteins and carbohydrate. Table 2. Proximate composition of condensed whey

Product Constituent (%) Sugar Water Ash Fat Protein Lactose Sucrose Plain condensed whey 51.3 - 32.0 6.0 0.6 10.1 Sweetened condensed whey 28.7 38.0 24.0 3.4 0.3 5.6 Condensed acid whey 49.0 - 33.5 8.2 0.6 7.7

Plain condensed whey and sweetened condensed whey may be used to make various whey candies such as 'wheyfers', whipped whey fudge, whey caramel and 'whey' taffy. The whipping properties of sweetened condensed whey is of value in many food preparations, say for example, ice cream, shakes, sherbets and bakery and confectionery products. Uses of whipped sweetened condensed whey include making of fruit whips, certain candies and frozen dessert preparations. A sweet spread of good keeping quality, may be made by mixing equal weights of sweetened condensed whey and peanut. Condensed sweetened whey is also used in caramel production, which is 38% whey solids, 38% sucrose and 24% water.

In Norway, "Mysost" and "Primost" cheeses are produced from concentrated whey with 84% total solids. Dulce de leche, yoghurt and whipped cream substitute may also contain condensed whey.

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2.7 Lactose crystallization

To avoid the very undesirable caking properties of ordinary whey powder, it is of great industrial importance to get the major part of the lactose content in a crystalline form. The advantages of this lie both in energy saving and in improved powder properties. In the spray drier, it is possible to dry whey concentrate containing up to around 60% TS, when the lactose content has been subjected to a crystallization degree of 85-90%. On the other hand, for non-crystallized concentrate, it is not possible to attain more than 42-45% TS for drying. Obviously, this low degree of concentration has negative effect on the process economics. Controlled crystallization can be initiated by immediate flash cooling of condensed whey after evaporation to about 30°C. As far as possible slow agitation should start immediately and fine-grained α-lactose monohydrate at a level of about one kg per tonne of concentrate should be added. The holding time under these conditions should be 3-4 hours. Cooling of the concentrate should then start, the rate being about 3°C/h until 10°C is reached.

Important factors which whey powder manufacturers should keep in mind for efficient and rich crystallization are.

• As the crystallization rate is proportional to the existing surface area of the seeding crystals, it is important that the seeding material is very fine grained. The crystal size aimed at in the concentrate is 20-30 µm, and the biggest crystals should not exceed 50 µm.

• Sufficient agitation is imperative. This means that fresh, supersaturated solution is available continuously for interaction with crystal surface.

• A high viscosity of the concentrate affected by the proteins and their time/temperature history-has a negative influence on crystallization. Therefore, every heat treatment from the very start of the manufacturing process should be taken into consideration.

• It is also necessary to control crystallization in order to create a maximum number of small crystals to give the largest total crystal surface and consequently, most rapid and efficient crystallization.

3. SPRAY DRYING

Recent trend in drying of milk and product is extensive use of spray drier that may be single stage, two-stage and more recently three-stage drying. Processes pertaining to the spray drying of whey for the manufacture of various types of whey powder are depicted in Fig. 2. 3.1 Single-stage process

In single-stage drying, the product is dried to its final moisture content in the spray-drying chamber alone; some time pneumatic conveying system is adopted with one-stage drying system. Ambient air provides both the conveying and cooling of powder. If climatic conditions prevent powder temperature/residual moisture contents to be achieved with ambient air, the conveying air must be dehumidified and cooled to usually 8°C.

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3.2 Two-stage process

The principle of two-stage drying is a combination of spray drying at the first- stage drying and fluid bed drying at the second stage. By two stage drying, it has been possible to obtain good quality powders and also with advantage regarding drying economy in the manufacture of non-agglomerated products. In this process normally, powders leave the chamber and enter the attached vibrating fluid bed drier with a moisture content of 5-6%. The excess moisture is removed in the fluid bed, where the moist powder is first met by a cold air stream thus avoiding lumping of the thermoplastic powder, and then by a hot air stream for final drying. By introducing cold air in the last stage of the fluid bed the powder is cooled to the desired temperature. Fines are recovered in the spray drier and fluid bed cyclones, collected and returned to the atomizing zone for agglomeration. This agglomeration gives a free flowing powder. In this process typical drying conditions are: feed preheating to 80°C, feed concentration of 50-60%, crystallization, and then drying at an inlet temperature of approximately 185°C.

Whey Powder

Ordinary Precrystallized Non-caking (One stage) (Two stage) (Belt process)

↓ ↓ ↓ ↓ Pretreatment

↓ ↓ ↓ ↓ Evaporation

42-45 % TS About 40 % TS 50 % TS ⏐ ↓ ↓ ⏐ ⏐ High-concentration ⏐ ⏐ 50-60 % TS ⏐ ⏐ ↓ ↓ ↓ ⏐ Pre-crystallization ⏐ 4-16 hours 10-24 hours ↓ ↓ ↓ ↓

Spray drying Ti= 180 °C Ti=200 °C Ti=185 °C Ti=150 °C

⏐ ⏐ ⏐ ↓ ⏐ ⏐ ⏐ After crystallisation⏐ ⏐ ↓ ↓ ⏐ ⏐ Fluid bed drying ↓ ↓ ↓ ↓

Pneumatic transport/cooling Fluid bed cooling

Fig. 2. Different processes for the manufacture of whey powder

3.3 Belt process

Lactic acid in dry form is very thermoplastic even at low temperature and difficult to dry by conventional spray drier. This means that this type of powder is extremely sticky during the

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spray drying process. The belt process is especially advantageous in drying lactic acid whey. In the process, typical drying conditions involve preheating to 80°C, feed concentration to 50%, spray drying at inlet temperature of 150°C and outlet temperature of 55°C to a moisture content of 12-15%. In order to avoid condensation in the cyclones, hot air is introduced into the exhaust system of the spray dryer to increase the relative humidity.

The high moisture content of powder leaving the spray drying chamber ensures that crystallization will continue in the powder to an even greater extent than in the two-stage process using fluid bed drier. This crystallization is improved if the wet powder is kept at high moisture content for 10-15 minutes. This is conveniently done on a belt conveyor mounted between the chamber outlet and a fluid bed, where the final drying takes place. The resulting powder consists of large agglomerates, which has a low bulk density, but is extremely instant and have excellent non-caking properties. 3.3 Three stage drying system with integrated fluid bed using rotary atomizer

The difficulty of the moist, sticky powder is to transfer it from the drying chamber to the secondary stage of drying. The principle of these newly developed types of spray dryers is to spray dry the powder to high moisture content, but at the same time to avoid any contact with hot metal surfaces by handling the powder, directly on a fluidized powder layer in a fluid bed, at the base of the spray drying chamber. This problem has to certain extent, been overcome by vibrating the fluid beds. In the new drying concept, a fluidization technique without vibration is developed introducing a type of perforated plate - so called grill plate - and a secondary air intake. The stationary fluid bed operates with high fluidizing velocities and high bed depths, about 400-500 meters, which is also possible to adjust according to the product to be processed. Two types of spray dryer are made according to this principle, namely the compact spray dryer (Fig-4) and the multi stage dryer. 4. CHEMICAL COMPOSITION OF WHEY POWDER

Composition of whey powder varies depending on the type of whey from which it has come, pretreatment given to the whey, and the various processing steps followed in the production, Analysis of commercially produced sweet and acid type dry whey revealed the values for lactose, total protein, NPN, total ash and fat for sweet and acid type whey powders were 69.4 and 63.40 per cent, 13.0 and 11.7 per cent 0.5 and 0.58 per cent, 8.3 and 10.6 % and 1.03 and 0.48 per cent respectively. The moisture content varied from 3.7 to 6.0 %. Other properties are shown in Table below: Table - 1. Properties of Whey Powder.

Properties Ordinary Whey

Powder

Precrystallized whey powder

Non caking whey powder

Non caking whey powder

Total moisture (%) 3-4 3-5 5-6 4-5.5 Free moisture (%) 3-4 1-2 1.5-2.5 1-2 Bulk density (g/cm3) 0.6-0.7 0.6-0.7 0.55-0.65 0.4-0.5 Degree of cakeness (%) 100 40-100 0-5 0-5 Degree of Crystallization %) 0.1 50-75 75-85 85-90

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5. STORAGE OF WHEY POWDER

Non enzymatic browning via Maillard reaction is one of the important modes of deterioration in whey powder, which limit shelf life. Whey powders contain relatively high concentration of lactose and protein, in the presence of moisture these components readily participate in the maillard reaction. This interaction may result in a decrease in protein quality which is accompanied or followed by undesirable colour changes.

Rennet and acid whey powder can be kept for a maximum of 50-80 days at 20°C if intended for use in the food stuff industry. If whey powder is to be kept in an acceptable condition over a 3 month period or longer, the storage should be at 15 to 20°C with 10-15 % relative humidity and under air tight conditions. 6. REFERENCES Boessen, A.C. (1990) Spray drying Technology. J. Soec. Dairy Technol. 43(1) : 5-8. Jensen, G.K. (1987) New Dairying Technologies IDF Bulletin No. 212 : 27-37. Kjaeegared Jensen, G. and Oxlund, J.K. (1988) concentreation and drying of whey and permeate IDF Bulletin 238 : 4-

20. Jayaprakash, H.M. (1992) Membrane processing application for production of whey powder and whey protein

concentrates. Ph.D. Thesis submitted to NDRI deemed university, Karnal. Patel R.S., Jayaprakasha H.M. and Singh. S. (1991). Recent Advances in concentration and drying of whey Indian

Dairyman 43:417. Sienkiewicz, T. and Riedel, C.L. (1990). In whey and whey utilization. Published by verlag-Th. Mann,

Gelsenkirschen-Buer. Germany. Second revision and extended edition. Westergaard, (1983) Milk Powder Technology. Evaporation and spray drying cited in IDF Bulletin 233, 4.

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

The high level of minerals (0.7-0.8%) present in whey restricts their commercial utilization in many applications. A major problem with many whey based products is their salty flavour owing to their high mineral content. Demineralized sweet whey (25-65% demineralization) can be used in foods such as coffee whitener, soft serve ice cream, milk shakes, whey drinks and caramel, citrus drinks, salad dressing, animal feeds, bakery goods, is demineralised to produce dry demineralised whey for specialised uses. These include whey protein based infant formulas and other medical and nutritional products that require lactose, special nutritional quality of whey proteins and low mineral content. A range of demineralized spray-dried, whey-based products for use in infant feeds and dietetic applications have been developed based on products manufactured by demineralization and ultrafiltration of whey. Processes that have been utilized for the demineralization of whey include electrodialysis, ion exchange, loose reverse osmosis and counter diffusion (Hoppe and Higgins, 1994).

Demineralised sweet whey (25-65% demineralization) can be used in foods such as coffee whitener, soft serve ice cream, milk shakes, whey drinks and caramel, citrus drinks, salad dressing, animal feeds, bakery goods, confectionery coatings and dry mixes. 2. ELECTRODIALYSIS

Electrodialysis is a separation process in which membranes are used to remove ionic (electrically charged) species from non-ionic species. The key to the electrodialysis process is the use of ion-selective membranes. These membranes are essentially ion exchange resins cast in sheet form. Ion selective membranes that allow passage of positively charged cations (Na+, K+) are called cation membranes. Membranes that allow passage of negatively charged ion (Cl-, PO4

3-

) are called anion membranes. The ion removal medium is generally a brine solution prepared from hydrochloric acid. This solution is continuously replaced to prevent high concentrations of ions being accumulated, leading to high osmotic pressure.

To achieve separation by electrodialysis, cation and anion membranes are altered with plastic spaces in a stack configuration with positive electrode (anode) at one end and cathode at other end. The spacers, whilst creating the flow channel, also include turbulence promoters. When a DC voltage is applied across the electrodes, electrical potential created causes anions to move in the direction of anode and cations towards cathode. The ion-selective membranes form barrier to ions of opposite charge. The result is: anions attempting to migrate to anode will pass through anion membranes and are stopped by cation membranes: cations trying to migrate to cathode pass through cation membranes but are stopped by anion membranes. Hence, memberanes form alternate compartments of ion-diluting cells and ion-concentrating cells. By circulating whey through diluting cells and brine solution through concentrating cells, free mineral ions leave the whey and collect in brine stream. The flow is generally co-current to prevent the development of

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large pressure differences. The electrodes are rinsed with their own rinse solution to avoid the possibility of scale formation. The preferred solution for rinsing of electrodes is sulphuric acid, rather than hydrochloric acid, which can lead to chlorine production.

The process is ion-type selective, generally resulting in a higher loss of monovalent ions. Thus the mineral profile of the product is significantly different from the feed stock. The level of demineralisation is determined by initial ash content, whey viscosity, current density and residence time. Electrodialysis is generally used for applications where low levels of demineralization are sufficient since power consumption at high level of demineralization is excessive. In practice, levels of demineralisation of about 50% are viable. 3. ELECTRODIALYSIS MEMBRANES

Electrodialysis membranes are thin sheets (0.15-1.00 mm thick) of cation or anion exchange resins, usually reinforced with synthetic fibres necessary to give mechanical strength. An example of a cation membrane is styrene-divinyl benzene copolymer with sulfonic acid groups as active sites. The corresponding anion-selective membrane has the same copolymer with quaternary ammonium groups as anion exchanging active sites. Most commercially available membranes are having effective pore sizes of 0.7-2 nm, which are slightly greater than atomic dimensions and therefore, impermeable to flow of liquids and solids and to diffusion of large molecules. 3.1 Electrodialysis Process

There are three possible modes for electrodialysis of whey: batch, continuous and feed and bleed processing. In the batch method, pre-treated whey is fed batch-wise to the electrodialysis unit, where it is recirculated until the required degree of demineralization is achieved. This method is quite convenient when a high and uniform level of demineralization is required (i.e. 90% whey demineralization for infant formulations). The typical batch-wise layout consists of several stacks, each consisting of 200 pairs of cells in parallel connection. The line is completed with pumps, tanks, valves and instrumentation for controlling and recording flow and pressure in the stack, as well as pH, temperature, and conductivity of whey and brine. The constant electrode rinse solution and DC power supply are controlled, too. To lower the cost, a high whey pre-concentration is necessary (20-28% TS) when the temperature of 30-45oC provides maximal electric conductivity. However, lower temperatures (20oC) and feed concentrations (even 12% TS) may be used, but a higher voltage is then required to provide the corresponding current density. The operation time is 4-6 hours.

In continuous operation, feed is passed once through a series of modules to achieve the required demineralisation. The continuous electrodialysis method is advantageous when products with lower levels of demineralization are desired. The brine cells as well as the line for electrode rinsing are connected in parallel. Operation temperature is relatively low (as low as 20°C) and the pH remains constant (about 4.7) during processing. Whey at 6% TS can also be processed, with no pre-concentration. Continuous electrodialysis has a longer period of uninterrupted operation (10-12 hours) than batch-wise processing, which is convenient for large-scale production.

In feed and bleed method, feed is recycled continuously around the module with whey being fed in and demineralised whey being bled off.

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The main operating costs for electrodialysis include membrane and spacer replacement, recoating of the electrodes with platinum, effluent treatment, electric power, labour for operation and maintenance, water for rinsing, chemicals (brine, HCl, H2SO4 , CIP chemicals), and steam, depending on operating temperatures. 2.3 Some Developments

Classical electrodialysis processes with anion/cation membranes have the following problems, as a consequence of membrane fouling, associated with them:

• Deposition of calcium phosphate on the cathode side of the cation-selective membrane. This deposition can be removed by a normal cleaning process using acid.

• Deposition of denatured, negatively charged protein molecules which pass through the membranes and are deposited as a thin film in the membrane pores. At a current density of 20-25 mA/cm2, and with continuous operation, there is a danger of irreversible protein deposition.

Effective cleaning of the anionic membranes can only be achieved by washing with alkaline solutions. As a consequence, however, of the alkaline effect, the lifespan of the anionic membranes is shorter than of the cationic. The polarization reversal technique, which is also used for electrodialysis membrane cleaning, is somewhat less effective.

In order to avoid membrane fouling a new commercial electrodialysis process, called transport depletion, has been developed. This method involves the substitution of selective anion membranes by non-ionic membranes of regenerated cellulose. In this way, no concentration polarization of the whey constituents occurs on the membrane surface. Higher current densities can be applied, the lifespan of the membranes is longer and the cleaning process is simpler. This electrodialysis modification is, however, only half as effective as the classical methods.

Another modification of electrodialysis is ion substitution. When applying this method, a third liquid stream, containing an ion selected for substitution, passes through the apparatus in addition to the usual whey and brine stream. A particular feature of this method is the structure of the ion membranes as compared to those used for conventional electrodialysis. The whey runs between two cation-selective membranes and the sodium is separated, into the brine, on the cathode side of the apparatus. At the same time, another cation, e.g., potassium, travels through the membrane into the whey stream. The anions remain in the whey and form new salts.

A further modification of electrodialysis is electro-osmosis, which has been industrially applied in the USA, Japan and Norway since 1967. The size of an electrodilaysis apparatus is a function of the applied membrane surface. A typical structure with 100 pairs of cells represents a space of 50 mµ and can remove 60% of salts from a 25% whey concentrate at a rate of flow of 500 l/h. The ions which are removed by electrodialysis are Na- K- and C-. Calcium, magnesium phosphate and citrates are not transported as they are probably present, at the pH value of the electrodialysis, in the form of soluble complexes.

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3. ION EXCHANGE

Ion exchange is now the most mature of the demineralization technologies. It has the ability to remove nearly all of the minerals present in whey and whey permeate. 3.1 Ion Exchange Resin

Modern ion exchange resins are macro-molecular porous plastic materials formed into beads with diameter in the range of 0.4-0.8 mm having a large number of attached bonds on their surface which can absorb (reversibly) one specific type of ions. Chemically they act as insoluble acids or bases, which when converted into salts, remain insoluble. A wide range of base polymers may be used in ion exchange resins (Table 1) depending on the specific physical and chemical requirements of the system. The average pore size within the polymer is normally less than 40Ao. Macroporous resins are prepared by re-dissolving of part of the matrix of the polymer to controlled pore sizes up to 1000Ao. The matrix of the resin contains the charged sites involved in the ion exchange process. These sites may be occupied by anionic or cationic groups (Table 2). Exchange capacities of resins typically range between 1 and 4 equivalents per litre of resin.

Table 1. Resin Matrix Materials

Silicates - inorganic cellulose base Cross-linking agent, e.g. Phenol formaldehyde divinyl benzene (DVB)

Polystyrene Polyacrylate

3.2 Ion Exchange Process

Ion exchange processing of whey is based on the ability of macromolecular resins to exchange their surface-bound ions for mobile ions of the same charge from the treated whey. As the result of experience over many years, it has been found that whey processing is best carried out using strong acid cation and medium to weak base anion resins.

There are only a few options available for the application of ion-exchange resins for the practical demineralization of whey. Column systems are the most widely employed. In such systems, either each resin is packed into its own column, or alternatively, sometimes, the resins are used in a 'mixed bed' column. The simplest system is a continuously-stirred reactor tank.

Table 2. Ion Exchange Resin Classification

Charge type Affinity level Functional group Cation exchange resin Strong acid

Weak acid SO3

- sulphonate Coo- carboxyl

Anion exchange resin Strong base Weak base

NR3+ quatery ammonium

NH2.HNR, NR2 substituted amino groups

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Before demineralization, whey has to be pasteurized, clarified and separated, because contamination of the resin with fat, and/or fine cheese curd, results in a shortening of the production life of the resins. Whey pasteurization should be as mild as possible (65°C, 15 sec.), since any contact with fresh ion-exchange resins will shift the pH and may cause protein destabilization. Whey is first introduced into a cation exchanger, where all the positively charged ions (Na-, Ca2

-, Mg-, etc.) are replaced with H+. The whey is then pumped in an anion exchanger where all the negatively charged ions (C1-, PO4

3-, SO42-, etc.) are replaced with OH-. The ion

exchange of whey can be carried out at higher temperatures (> 50°C), if it has previously been deproteinized. However, if the whey is not deproteinized, protein destabilization and loss during ion exchange can be avoided by using lower temperatures such as 5-12°C.

Ion exchange is actually a batch process. The transfer of whey through the resin beds continues, until the resins are saturated with cations and anions. This point is controlled by means of a conductivity meter, after which the resin beds are purged of whey, washed with water and regenerated by means of acid and alkali solutions. These solutions should be sufficiently concentrated to remove the absorbed cations and anions and replace them by H+ and OH- bringing them back to their previous state. The treatment with acid and alkaly results at the same time in a sufficient cleaning. After the regeneration , the resins are washed with clean water, preferably condensate from an evaporator. After this, the process can start again. The production line consists of ion-exchange vessels, storage tanks for chemicals, feed and product storage, pumps, valves and dosing systems, a water supply, and a refrigeration system. If continuous production is required it is necessary to erect 2 demineralization plants. Putting one plant into operation allows the regeneration of the other. Each regeneration can process 10-15 bed volumes of whey; a figure which is based on the volume of the cation exchanger (Herrmann et al., 1988).

With a one-line configuration consisting of typical 2 hours of production and 4 hours of regeneration, four cycles can be performed per day, i.e., 8 hours of productive work. Two parallel lines provide 16 hours of production, and three parallel lines provide 24 hours of production. Usually, one or two lines are installed.

After its passage through both exchanger columns the whey is demineralized, depending on its type, from 90 to 98%. The processing/holding time is 20 min. The processing temperature is <10oC and therefore no bacteriological growth is experienced. Whey that has been demineralized up to 90% can be directly concentrated and dried. The obtained powder is non-hygroscopic. This advantageous phenomenon has as its probable cause the partial hydrolysis of the lactose at the cation exchanger. For some applications, though, a degree of demineralization of only 50-60% is desirable. In such a case the demineralized whey is automatically mixed, in appropriate proportions with the untreated whey (pH control).

After completion of the operation or on exhaustion of the resin, the residual whey is flushed out. The flushing normally uses 2 bed volumes. Back washing way be employed, which results in expansion of the bed as a result of reverse flow and allows easier removal of physically entrapped material. After washing, the resins are regenerated through introduction of the regenerate solutions (generally 3 to 10 bed volumes for cation exchangers and 3 to 5 bed volumes for anion exchangers). The regenerant solution is then finally resumed. This takes place automatically and, for the cation exchanger, uses HCl. The deionized water used for the final rinse is derived from the condensation water supply of the evaporator (up to 10 bed volume).

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Na2CO3 and NH4OH are used as regeneration solution for anion exchangers. Sanitization is possible after the regeneration stage using sodium isocyanurate solution. Counter current regeneration minimises the amount of the regenerating agent. 3.3 Process variations (SMR Process)

A variant of the traditional ion exchange process is the Swedish SMR (Svenska Mejeriernas Riksferening or Swedish Dairy Association) process. It is designed primary to reduce the consumption of regeneration chemicals, which apart from saving money also leads to a better waste (reduced salt load) situation from demineralization plant. Instead of using cation resin of H+-form and anion resin on OH- from, This process uses a cation resin on NH4

+-form and an anion resin on HCO3

--form.

In SMR process, whey first enters anion column where whey anions are exchanged for HCO3

- ions. After this, the whey enters cation column, where cations of whey are exchanged for NH4

+ ions. After the process, whey salts are thus exchanged for ammonium bicarbonate. Ammonium bicarbonate is thermolytic salt which decomposes to recoverable NH3CO2 and water when heated during subsequent evaporation of whey. A plant using the SMR system at Arjang, Sweden, has achieved 70% recovery of NH3 and 90% recovery of CO2. The details of the process have been described by Batchelder (1987) and Hoppe and Higgins (1992). Table 3. Composition of 2 different demineralized whey powders

Whey Powder 50% Demineralized 90% Demineralized

Fat Protein Lactose Mineral Salts Sodium Potassium Chloride Calcium Phosphorus pH in 5% Sol. Solubility Index Total counts Yeasts of Moulds B. cereus Faecal streptococci Coagulose +ve staphylococci E. Coli Coli aerogenes Salmonella, shigella

0.6 - 0.9% 12.0 - 14.5% 75 - 80% 4.0 - 5.0% 0.4 - 0.5% 0.6 - 1.0% 0.1% 0.6% 0.4% 6.4 - 6.9 < 0.1 ml < 3000/g < 10/g < 200/g < 100/g Nil in 1 g Nil in 10 g Nil in 0.1 g Nil in 50 g

0.6 - 0.9% 13.0 - 15.5% 80 - 85% 0.7 - 1.0% 0.06 - 0.12% 0.25 - 0.3% 0.05% 0.15% 0.14% 6.4 - 6.9 < 0.1 ml < 3000/g < 10/g < 200/g < 100/g Nil in 1 g Nil in 10g Nil 0.1g Nil in 50 g

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4. PREPARATION OF DEMINERALIZED WHEY POWDER

Demineralized whey is evaporated to about 58% TS and then rapidly cooled to force as much of the lactose to crystallize as possible into fine crystal form. The cooled slurry is spray dried. 5. ELECTRODIALYSIS V/S ION EXCHANGE PROCESS

Electrodialysis is very capital intensive whereas ion exchange has high operating cost. Based on economic studies, the recent trend is towards installation of combined electrodialysis/ion exchange plants for high level demineralisation of whey. The cost for electrodialysis demineralization is greatly dependant on conductivity and, therefore, increases with reduced ash content. Realistic cost effective demineralization figures are 50% with electrodialysis. A consumption of 10 to 28 KWH of electrical energy must be considered for each kg of dimineralized whey powder.

The costs for ion exchange deminralisation are essentially linear with ash removal. Lower the ash content, lower is the cost of ion exchange demineralization. In Industrial practice, electrodialysis demineralization of 50% is followed by further 50-95% demineralization by ion exchange process. The typical composition of demineralised whey powder is given in Table. 3. 5. LOOSE REVERSE OSMOSIS

Electrodialysis and ion-exchange processes are effective but are limited by high capital cost, high running cost and high level of effluents. Nanofiltration (also known as loose reverse osmosis or ultra-osmosis) is a membrane separation process which allows selective passage of water, salts and very low molecular weight organic molecules. The membrane pore size used is 10-50A° and the operating pressure used is about 300 PSIG. The adoption of this technology was earlier inhibited because of the poor retention characteristics of many membranes for lactose. However, recent developments have resulted in the availability of membranes which combine low rejection of salt with very high rejection of lactose.

The first installation of this process in the United States was in 1986, for the reduction of the salt content of whey. Today, there are many plants utilizing this technology for the partial demineralization of sweet whey, hydrochloric acid casein whey and ultrafiltration permeates.

About 50% demineralization is economically feasible with this technology. An added advantage of the process is that water is also removed from the product during operation, and thus a significant degree of concentration (> 20% solids) is also achieved. Diafiltration in conjunction with loose RO further in creases the effectiveness and degree of demineralization. Nanofiltration represents a unique opportunity for the dairy industry in relation to whey processing and a large number of plants have been installed since the process was introduced in the mid-1980s. 6. REFERENCES Batchelder, B.T. (1987). Electrodilaysis in whey processing. IDF Bulletin No. 212. Delaney, R.A.M. (1976). Demineralization of whey. Aust. J. Dairy Technol., 31 : 12. Hoppe, G.K. and Higgins, J.J. (1992). Demineralization, In Whey and Lactose processing, J.G. Zadow (ed.)

Elsevier Applied Science; London.

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Jenson, G.K. and Orelund, J.K. (1988). Concentration and drying of whey and permeates. IDF Bulletin No. 233, P.4.

Sienkiewicz, T. and Rieder, C.L. (1990). Whey and Whey utilization. Verlag Th. Mann, Germany.

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Procedures for the manufacture of whey protein products are based on known behaviour of whey components under defined conditions.Properties that have been exploited commercially include: molecular size differences (Ultrafiltration, gel filtration), insolubility of protein at high temperature, charge characteristics (demineralization, protein removal by ion exchange), aggregation (by polyphosphates) and crystallization of lactose. By 1981, Ultrafiltration (UF) had become the most widely used process for recovery of soluble whey protein concentrate (WPC). WPC is commonly characterised by its protein content on dry basis (e.g. WPC-80 has 80% protein on dry basis). The development of robust, synthetic and cleanable membranes and the refinement of continuous operation using multi-stage recycle loops, and diafiltration have been significant factors contributing to the success of this process. 2. ULTRAFILTRATION PROCESS

Membrane configurations designed to date have included rubular, plate and frame, spiral wound, hollow fiber, and flat leaf. Examples of each type are in commercial operation. The permeation behaviour and the characteristic of whey protein concentrate obtained by ultrafiltration, widely vary with the type of feed stream, pH, ionic strength and various other constitutional make up of the system The economy of the process and the characteristics of the resultant WPC is dependent on the permeation behaviour of the constituents during ultrafiltration. The upper limit to fractionation will be set by the design of the plant, but 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. Diafiltration means that water is added to the retentate, thereby the viscosity is reduced, and the concentration of lactose, ash, and NPN is decreased by further UF. Both sweet and acid whey may be used in the production of WPC. 2.1 Pre-treatment of whey to increase the permeate flux rate

Fouling of membranes during UF normally is observed. This can be caused by concentration polarization and by progressive accumulation of materials, such as calcium phosphate, on UF membranes during processing. The design of modern UF plants is directed toward better control of concentration polarization, primarily through maximizing shear at the membrane surface. To minimize progressive fouling in continuous plants (e.g., at constant concentration factor), pre-treatment techniques can be sequestration, demineralization, heating plus calcium addition or pH adjustment replacement of calcium with sodium, clarification and filtration. Other pre-treatments include protein interaction, which aids in larger aggregate or apatite formation, which are non-fouling. Heating whey to a temperature of 80ºC/15 sec resulted

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DEVELOPMENTS IN THE MANUFACTURE OF WHEY PROTEIN PRODUCTS

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in better flux than at 60ºC/30 min. Muller and Harper (1979) also observed an increase in flux by 50% when cheese whey was heated to 80ºC/15 sec instead of pasteurization temperature.

Gupta and Reuter (1987) investigated the manufacture of WPC from sweet cheese whey. Clarification of Tilsit cheese whey, prior to Ultrafiltration (UF), was found essential for the effective operation of an UF plant. UF permeation rates were greater with higher UF temperature and with preholding of the whey for 30-40 min at temperatures (68-72°C), higher than the UF temperature (50°C). The preheating probabely holding of whey at a temperature higher than the UF temperature causes the main precipitation of calcium phosphate to take place in the balance tank itself. Therefore, there is much less tendency for the precipitation of calcium phosphate in the membrane system. New (unused) UF cartridge also showed much improved permeation rates compared to the old (used) one. The increase in total solids concentration in the retentate was slow during early UF stages, but improved considerable in advanced stages. 15, 20 and 25% TS whey protein concentrates were obtained at about 92.5, 95.35 and 96.8% Whey volume reduction (WVR), respectively, irrespective of the preheating and UF temperatures used. In diafiltered concentrates, 15, 20, and 23% TS were achieved at greater WVR, i.e. 95.2, 96.7 and 97.35%, respectively. Preheating and UF temperatures treatments affected the consituents of concentrates. The effect was most distinguished on calcium content. The preheating treatment of 68-72°C for 30-40 min, before Ultrafiltration of whey at 50°C, increased calcium/TS of concentrates considerably higher with increased UF concentration compared to lower preheating temperatures (equivalent to UF temperatures). In addition, concentrates produced at higher UF temperatures had higher calcium/TS. Ash/TS of concentrates decreased during Ultrafiltration and further with diafiltration. The decrease was more rapid in cases where lesser calcium was retained. During both UF and diafiltration, lactose/TS of concentrates reduced drastically. Protein purity of whey protein concentrates improved greatly with increased UF concentration, but at a diminishing rate during later UF stages. 50, 60, 70 and 75% protein/TS in concentrates were obtained at about 12, 15.5, 21 and 25.5% TS concentration respectively. With diafiltration, the protein purity of the product improved significantly. The results can be used to produce WPC with different protein: Lactose: calcium ratios. The acidity/TS of concentrates showed a slight decrease during UF. The diafiltration, however, reduced acidity/TS of concentrates significantly. 2.2 Manufacture of spray dried WPC

After UF, the concentrate is heat-treated, after which it is cooled and then preheated again for drying. To achieve acceptable powder densities (0.35 to 0.5 g/cm3), it is normally necessary to concentrate in evaporators the WPC 35-50% prior to spray drying. WPC 50-80% often has 27-30% TS and can be pumped directly to spray drying. UF plant concentrate is evaporated to 25 to 40 per cent solids, depending on the concentrate composition. For example, concentrates for 35 per cent protein powders are typically evaporated to 40 per cent solids; concentrates for 75 per cent protein powders are evaporated (if necessary) to 25 per cent or less total solids. The evaporated concentrate is then spray dried. The spray drying process for this product is conventional. Drying of WPC is preferably carried out in a all form dryer with nozzle atomisation. The advantages of this type of dryer are a well defined residence time and gentle heat treatment of the heat sensitive product. The typical inlet drying air temperature is 175-190oC. Between 90 and 95% of the protein in the whey is recovered. The resulting powder (typically 4% moisture or less) may be blended to ensure good product uniformity, and then bagged. Low-temperature

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processing is necessary because of the heat sensitivity of the product, but suitable equipment is readily available. 2.3 Chemical composition of dried WPC

The UF concentrate of a 35 per cent protein product would have about 10 per cent total solids. The concentrates for 50-and 75 per cent products would have about 15.5 percent total solids and 25% total solids, respectively. The protein content of products achievable with a given UF plant without diafiltration is tied to the hydrodynamics of the specific system design. In essence, the higher the total solids level attainable in the concentrate, the higher the protein purity achievable without diafiltration. The range for various UF equipment suppliers is 50-65%.

Fig 1. Process scheme for the manufacture of Whey Protein Concentrate by UF process 2.4 Process optimization for the production of WPC from buffalo milk whey

Jayaprakash (1987) reported the process optimization for the production of WPC from buffalo milk whey (BW). When the calrified BW was subjected to 80ºC/15 sec heating at various pH levels, the flux rate appeared to be better at extreme pH values. At pH 3.0 and 7.2, flux was significantly higher than at any other pH levels. The change in pH and heat treatments affected the status of calcium salts and the configuration of protein and hence the flux. At pH 4.5, the net charge on the proteins is low and hence dispersion of protein is poor. They get adsorbed on the surface of the membrane, forming gel layer which results in the lowest flux rate. As the pH is lowered from 4.5, the dispersion of proteins improves calcium gets solubilized and pass through the membrane without much fouling, thus increasing the flux rate significantly.

The ultrafiltered whey was also spray dried. The major components such as moisture, protein, fat, lactose and ash contents of the dried WPC were determined. It was observed that

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various components varied with the protein content of the samples. The samples WPC 26, WPC 45, WPC 70 and WPC 80 had protein content of 25.11, 43.46, 71.16 and 80.60 per cent. The lactose content of these samples was 53.64, 42.76, 14.37 and 4.45%, respectively. The respective fat content of WPC 26, WPC 45, WPC 70 and WPC 80 was, 1.42, 4.45, 6.70 and 7.17%. The respective ash content of the samples was found to be 15.03, 5.58, 3.36 and 4.01%. 3. GEL FILTRATION PROCESS

This useful laboratory technique for separation of solution components also has been used commercially for recovery of WPC. The hydrated gel acts as a molecular sieve; small molecular weight components are able to enter the solvent phase within the gel beads. Protein molecules remain in the solvent phase surrounding the beads. High and low molecular weight fractions then can be recovered. Products of 30 to 80% protein can be manufactured. The process is expensive to install and operate, and the yield, at 65% of the protein in whey, is low. It also is subject to fouling and microbial contamination. It appears that it is no longer in commercial operation. 4. HEAT PRECIPITATION PROCESS

Whey proteins may be precipitated (and thereby rendered insoluble in water) by heating whey at acid or near-neutral pH. The critical variables that affect these process have been described. Acid whey must be heated to at least 90oC and maintained at such temperatures for at least 10 min to achieve maximum yields. For sweet wheys, good yields can be obtained by heating at pH between 6.0 and 6.5, although products so derived have higher mineral concentrations than those of acid whey unless pH adjustment to 4.6 is effected prior to protein removal. The precipitate so formed is firmer and more readily separated than that formed in unacidified whey.

Processes for recovery of lactalbumin have been described. Whey is heated and held, precipitated protein is removed by settling (static or accelerated), and the precipitate then is washed, reseparated, and dried. In modern plants, high speed centrifuges such as clarifiers and decanters are used to effect both primary and secondary (after washing) separations. Ring, fluid bed, roller, and spray driers have been used to obtain the finished product. Typical yields are 4.2 to 5.2 kg/m3.

Process refinements investigations have included demineralization prior to heating, pre-concentration by reverse osmosis and Ultrafiltration, and continuous, high temperature reaction (120°C for 8 min at pH 6). Most processes result in an insoluble product, but through heating whey to 95oC at pH 2.5 to 3.5, then adjusting to pH 4.5 prior to separation, it has been claimed that a product soluble at pH 5 can be produced. The use of additives such as salts (NaCl, CaCl2) and cysteine to improve product yields has been investigated. 5. PRECIPITATION BY COMPLEXING AGENTS

Numerous complexing agents have been used experimentally to recover protein from whey; of these, polyphosphates appear to be the only group to be used commercially for this purpose. Long-chain polyphosphates will precipitate protein from whey at low pH e.g., 2.5. Typically, potassium polymetaphosphate and sodium hextametaphosphate are used. The precipitate so formed is removed by centrifugation, washed, and then subjected to pH alteration

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Table 1. Protein yieldsa and concentrations of principal classes of dried whey protein products Basis of recovery Heat

Precipation Molecular sizeb ----------------- UF GF

Adsorptionc -------------- Cell. Sil.

Phosphate Complex

Removal of d -------------------------

Lac Min. Lac+ MinYield % 70-80 90 50-90 50-90 70-85 90 90 90 90 Concentration % 65 30-80 30-80 30-80 30-85 40 20 45 Product namee WPC WPC WPC WPC WPC WPC DLW DMW DLDMW

a Expressed as precentage of (total-nonprotein N) X 6.38 in original whey. b Ultrafiltration, UF; gel filtration, GF c Regenerated cellulose, cell; silica, sil. Pilot-scale data only. d Lactose, Lac; Minerals, Min. e Whey protein concentrate, WPC; delactosed whey, DLW; demineralized whey, DMW; delactosed, demineralized whey, DLDMW. Table 4. Percentage composition of whey protein concentrates Composition/Source of Whey Method of Isolation

Protein Carbohydrate Fat Ash Moisture

CW AW PW CW AW PW CW AW PW CW AW PW CW AW PWGel filtration 83.1 86.2 65.6 1.4 1.3 4.1 2.4 1.6 6.2 0.04 0.04 2.48 8.2 7.1 11.4Ion Exchange 77.4 85.4 46.6 0.4 0.4 1.9 0.3 0.1 4.1 13.2 13.0 28.8 7.5 5.3 14.3Hexmetaphosphate complex

62.3 59.4 33.9 9.7 12.8 14.5 6.8 1.5 35.0 17.8 19.6 19.1 3.1 4.5 6.1

Carboxy methyl Cellulose complex

60.99 69.63 28.0 17.8 18.0 32.8 15.6 4.5 19.0 1.9 1.2 3.6 2.2 5.1 2.1

CW = Cheese whey; AW = Acid whey; PW = Paneer whey and calcium addition to remove the phosphate. Cation (particularly calcium) removal prior to phosphate addition reduces the amount of phosphate required and results in recovery of up to 90% of the original whey protein. Further refinements also have been described.

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6. ADSORPTION METHOD

Adsorption techniques, based on the ion exchange properties of whey protein, are currently under serious investigation. Because whey proteins are amphoteric, solid phase charged adsorption media can be used to remove them from solution under appropriate conditions. Media suitable for this purpose have included regenerated cellulose, titania plus alumina, and silica. Of these, cellulose and silica-based systems have progressed to semi-commercial operation..

Regenerated cellulose is used in the "Vistec" (Biolsolats) process. The development of sulfopropyl cellulose resins of high charge density (1.1 meq/g) has enhanced its commercial propects. Whey is first decationized, then mixed with resin in a stirred tank reactor. After separation of the protein-resin complex from the deproteinated whey, the protein is disrobed at pH 9. Ultrafiltration is used to concentrate (and demineralize) the protein solution, which then is spray dried. Protein yield is 85%, and the dried WPC may contain as much as 95% protein. Because lipid molecules are not adsorbed by the media, such products have low fat concentrations.

Silica-based adsorbents are used in the "Spherosil" process. For acid whey, an adsorbent with strong cation exchange properties is used. For sweet whey, an anion exchange properties is used. For sweet whey, an anion exchanger is necessary, and as immunoglobulins are not adsorbed at this higher pH, a second (weak cation) exchanger must be used. Eluants used are ammonium hydroxide (from cation exchangers) and hydrochloric acid (from anion exchangers). As with the Vistec process, Ultrafiltration and spray drying are then necessary. Protein yield is 90%. 7. REMOVAL OF LACTOSE AND MINERALS

The product, known as delactosed whey powder, contains about 25% protein. It has been used as a stock food because of the extensive protein denaturation in the process. In more modern plants, with lower temperature-shorter residence time evaporators, the product is more functional and, therefore, of greater value. Delactose whey powder has a high mineral concentration (up to 25%). Processes have been described whereby preconcentrated whey (up to 30% TS) is subjected to Electrodialysis, the whey is concentrated to 60% TS, lactose is crystallized and removed, and the remaining liquid is concentrated and spray dried. The resultant product may contain up to 35% protein. 8. REFERENCES Gupta, V.K. and Reuter, H. 1987. Studies on the Ultrafiltration of cheese whey for the manufacture of whey protein

concentrates. Keiler Milchw forsch. Btr., 39, 39. Jayaprakasha, H.M. 1992. Membrane processing applications for production of whey powder and whey protein

concentrates. Ph.D. thesis submitted to N.D.R.I. Deemed Univ., Karnal. Kuo, K.P. and Cheryan, M. 1983. Ultrafiltration of acid whey in a spiral wound unit. Effect of operating parameters

on membrane fouling. J. Food Sci., 48, 113. Marshall, K.R. and Harper, W.J. 1988. Whey protein concentrates IDF, bulletin No.233, 21. Maubois, J.L. 1980 Ultrafiltration of whey. J. Soc. Dairy Technol., 33(2), 55.

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Matthews, M.E. 1984. Whey protein recovery processes and products. J. Dairy Sci. 67 : 2680-2692. Renner, E. and Abd-El-Salam 1991. Application of Ultrafiltration in the dairy industry. Elsevier. Applied Sci.,

London.

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Dr. Vijay Kumar Gupta Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

The most common forms of whey protein used in high protein bars, beverages and supplements are the whey protein concentrates (WPC) or the whey protein isolates (WPI). There has been a continuous increase in the production of whey protein concentrates (WPC) since the introduction of the latest ultrafiltration process about three decades ago. It is now a major means of WPC production throughout most of the dairy countries of the world. Increased production of WPC warrants its greater application in food products. Though soluble WPC have been found to be technically suited to a wide range of products, its use is not cost effective in all cases. Presently, WPC constitutes a very small proportion (10%) of protein utilisation in food industry. More product formulation work, especially in the food industry, is needed to move WPC into the general market place. The largest potential use of WPC is as a replacement for non-fat dry milk (NFDM) in the food industry. WPC with 35% protein is perceived to be a universal substitute for NFDM, because of the similarity in gross composition and its dairy character. Superiority of WPC over NFDM is also due to cost advantage. WPC can also be seen competing with casein, egg albumin and soya proteins within the existing markets. The PER value of whey proteins (3.2) is very high compared to standard casein (2.5). Whey proteins are also finding use in reactive extrusion to supplement polyethylene - a common non-biodegradable plastic. 2. HEATH AND DIETETIC FOODS

Whey proteins have been promoted as having a number of health benefits. Today whey proteins are supplemented to provide antimicrobial activity, immune modulation, improved muscle strength and body composition, and to prevent cardiovascular disease and osteoporosis. Numerous studies have shown that whey proteins can help enhance the body’s overall immune system and protect tissue from the effect of ageing. Whey proteins have become a staple supplement for many bodybuilders and other athletes.

There are numerous commercial sports nutrition products available in the US today that use whey proteins as a protein source. Whey protein is a complete, high quality protein with a rich amino acid (AA) profile. It contains the full spectrum of AA including essential AAs (EAAs) and branched chain AAs (BCAAs), which are important in tissue growth and repair. The EAAs and BCAAs in whey protein are not only present in higher concentrations than in other protein sources, but also they are efficiently absorbed and utilized. Many atheletes consume whey proteins because the demand for BCCAs increases with endurance exercise. Whey proteins are ideal for enhancing muscle protein synthesis because of identical AA profile and muscle growth during the recovery period. In addition, the relatively high levels of EAA in whey proteins are effective at stimulating protein synthesis in adult muscle.

DEVELOPMENTS IN UTILISATION OF WHEY PROTEIN CONCENTRATES DEVELOPMENTS IN UTILISATION OF WHEY PROTEIN CONCENTRATES

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Whey proteins have been touted as potentially being helpful in a number of medical conditions. Whey proteins are unique in their ability to optimize a number of aspects of the immune system, primarily by boosting glutathione (GSH) levels in various tissues. GSH, the centre piece of the body’s antioxidant defence system, protects cell against free radical damage, pollution, toxins, infection and UV exposure. GSH levels are typically depressed in individuals with cancer, HIV, chronic fatigue syndrome and other immune-compromising conditions. GSH also decreases with age and may be partially responsible for diseases such as Alzheimer’s disease, cataracts, Parkinson’s disease and arteriosclerosis. Thus, incorporating whey proteins into the diet may protect the health of not just those with a compromised immune system but those of all ages. Whey proteins may also be helpful in blood sugar control.

Due to various reasons, buffalo and cow milks are being humanised and used partly or exclusively for feeding human infants throughout the world. For humanisation, apart from making other modifications, whey proteins proportion needs to be increased in these milks. For this, a great potential lies in the application of WPC. In two of the three patents for humanisation of cow milk, Morinaga Milk Industry Co. Ltd. of Japan indicated the addition of whey proteins and lysozyme. Ivanov et al. (1971) also described the supplementation of high quality whey proteins for formulation of two humanised milk formulae "Bebe O" and "Babe 1" from cow milk.

A UK patent covers whey protein preparations, which are heat stable at neutral pH and suitable for increasing the protein contents of infant formulae and beverages. Whey which might have been demineralised or ultrafiltered, is processed to adjust its salt balance before or after concentration by evaporation. Adjustments in minerals concentration are achieved by additions of citrate, phosphate, a food acid or by diluting the whey proteins with water.

A high whey protein intake can help many people restrict food intake since protein has satiating effect 3. VEGETARIAN FOOD SYSTEM

For both nutritional and organoleptic reasons, there is continuous interest in fortifying or substitution of egg with whey. Whey proteins provide functionality as well as enhance sensory attributes and improve the nutritional profile of bakery products such as cakes, cookies and muffins. WPC replaces some or all the eggs in cakes. Totally 100% vegetarian cake can be prepared with exceptional nutritional value. 4. CHEESES

The cheese manufacturing industry has considerable interest in developing applications to use WPC. Ultrafiltered and denatured liquid WPC have been added to milk for cheese making. A patent from Netherlands described the production of fresh cheese or products containing fresh cheese with high protein content by adding a WPC obtained through ultrafiltration. Kuipers and Schroder (1980) reported the dispersion of WPC (pH 2.5-3.7, heated to 80-95°C) in unripened cheese curd. The yield of cheese is improved but the quality of cheese made has been generally unsatisfactory. Addition of WPC (10-12% protein, 6-6.5% lactose and heated to 85-87oC) to milk resulted in poor structure of Danbo cheese (Birkkjaer et al., 1974). Abrahamsen (1979) manufactured Saint Paulin cheese with added liquid WPC containing 14% total solids and 8.75% protein. All experimental cheeses exhibited a loose and doughy body and some cheese had a sour

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off flavour. Yields were 1-17% greater. Brown and Ernstrom (1982) reported an increase in the average yield of cheddar cheese by 4% with the addition of WPC (9.8 and 20.3% total solids and denatured) to milk. Experimental and control cheese did not differ significantly in any flavour or body-texture defects except acid. Banks and Muir (1985) manufactured Cheddar cheese with added acidified WPC and observed a maximum increase in yield of 7%.

Baldwin et al. (1986) reported the reconstitution of two WPC powders containing 35 and 55% protein to a 15% (w/v) suspension, heat treatment at 70°C for 15 min. and addition of 5-10% denatured WPC suspension to milk for Cheddar cheese manufacture. Cheese yield increase was 1.4-6.2% above those of the control on a 63% total solids basis. With increased WPC, there were more flavour defects in cheese. The most common criticism was atypical (unclean) cheese flavour.

Important functional properties of whey proteins in cream cheeses include the binding of water and fat without affecting the taste of the product. To achieve this, heat denatured whey proteins are pulverized and subsequently homogenized with fat to give non-syneresing emulsions. These emulsions are used as a protein base for the preparation of all dairy formulated cream cheese and cream cheese spreads. New types of cream cheese spreads have been developed from 59% fat cultured cream blended with up to 60% whey proteins (on a protein basis) with no differences in firmness or smoothness from a commercial cheese spread (Modler et al., 1985).

Georgakis (1975) reported the preparation of good quality processed cheese from Feta cheese with the addition of 5-20% WPC. At Federal Dairy Research Centre in Federal Republic of Germany, Gupta and Reuter (1986) standardised the manufacturing process of processed cheese foods with 20% of their cheese solids replaced by WPC. Among the different emulsifiers tested (a combination of trisodium citrate and disodium phosphate and disodium phosphate and trisodium citrate alone) only trisodium citrate was able to produce a smooth texture. An increased amount of WPC and trisodium citrate improved the firmness in a highly significant manner (p <0.01), but had a highly significant deleterious effect (p <0.01) on the melting quality of processed cheese foods (Gupta and Reuter, 1993). With the increase of moisture content over a wide range, the firmness of processed cheese foods decreased in a highly significant manner (p <0.01), while melting quality increased in a highly significant manner. It was also observed that increased amount of WPC in processed cheese foods imparted milder flavour in the final product (Thapa and Gupta, 1996). Trisodium citrate at 2.5% and with a moisture content of 45.2% resulted in processed cheese foods with the best sensory characteristics (out of a total of 7, the scores were as follows: flavour, 5.5; consistency, 6; appearance, 5.8; overall acceptability, 5.6. With less than 43.4% moisture, processed cheese foods were judged to be short, dry, hard, brittle and crumbly in body, while with more than 46% moisture, the product was judged to be soft, pasty, sticky and weak in body. WPC with a high UF concentration (26.1% TS) and low calcium content (0.7% on dry basis) were found to be the most suitable for incorporation in processed cheese foods. Diafiltration of WPC had a negative effect as regards suitability for the product. The standardized technique for processed cheese manufacture is: take a mixture of 25% 6.5-7.5 month-old and 55% 2-3 month old grated cheddar cheese), WPC equivalent to 20% of cheese solids, dry salt and water; heat the contents with thorough stirring by indirect steam heating. At a temperature of about 490C sprinkle 2.5% dry trisodium citrate and continue heating until the temperature reaches 820C, and maintain this temperature for 3-4 min. In processed cheese foods, on dry basis, there was observed a decrease in fat (12.4-15.65%), total ash (7.46-11.55%), calcium (30.54-36.46%)

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and free fat (10.32-36.0%), but increase in protein (18.23-20.15%) with 20% cheese solids replacement by WPC solids. The shelf life of processed cheese foods was observed to be only 42 days that was highly significantly lower (P<0.01) compared to about 78 days of processed cheese (Thapa and Gupta, 1992b) at 37+10C in cheese tins.

Thapa and Gupta (1992a) reported the preparation of processed cheese foods with 15 and 20% cheese solids replacement by solids of WPC (27.41% TS), obtained through ultrafiltration of cheddar cheese whey. Tri-sodium citrate emulsifier was tried at two levels (2.0 and 2.5%). Instron measurements revealed that increased levels of WPC and emulsifier and decreased moisture level imparted greater hardness, springiness, adhesiveness, gumminess and chewiness in processed cheese foods. Cohesiveness of the product, however, did not show any definite correlation with these variables.

Canadian Scientists at the University of Guelph reported the development of WPC, which has been utilised successfully in cheese spreads and other cheese foods. 5. FERMENTED PRODUCTS

Fermented milk products play a significant role in human nutrition all over the world. To increase the utility of fermented products like yoghurt, kefir and frolkren, the milk supplemented with whey proteins is used. Chojnowski et al. (1978) reported the production of yoghurt, kefir and frolkren by adding WPC Products had better structure and organoleptic property than traditionally made products.

The formulation of yoghurt products with optimum consistency and stability to synersis (whey separation) is of primary concern to the dairy industry. The viscosity and stability of yoghurt is almost wholly dependent on the protein content of the milk. A combination of whey protein addition and heat (pre-) treatment of the milk improves the viscosity and water binding properties of yoghurt. With heat treatment, whey proteins get denatured leading to interactions between B-Lg and KCn, which is presumed to be the reason for the formation of branched casein network during fermentation, showing a firm structure and little susceptibility to synersis. Up to 60% denaturation of whey proteins, the firmness of the gel increases considerably (Dannenberg and Kessler, 1988).

Jelen et al. (1987) reported decreased viscosity of yoghurt with increasing amount of WPC. Greig and Harris (1983) also observed that the replacement of 40% of liquid milk with WPC gave Yoghurt of lower viscosity. Odour, taste, appearance and consistency were not affected by substitution of 10% WPC. Later, workers at Massey University, New Zealand concluded that the addition of up to 15% WPC (20%, if the preheating step is modified) would enhance the desirable property of natural set yoghurt. In general, the addition of WPC resulted in firmer yoghurt with less synersis, but yoghurt made with more than 20% WPC exhibited slight graininess. Abd-Rabo et al. (1987) reported increase in sensory score of yoghurts manufactured with 15-40% WPC (3.9% protein) addition.

In Germany, a natural whey protein concentrate Lactalbumin 70 SHG has been developed to improve the gelling structure of both set and stirred yoghurts (Anon, 1992). In order to achieve more stable gelling structure, a part of the skim milk powder added to yoghurt was replaced by whey protein concentrates. The lactalbumin 70 SHG is put through a heating stage before use in

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order to develop the gelling properties of the whey protein. Lactalbumin increases protein levels and also reduces synersis in the final product.

Gupta and Renner (1993) experimented with preparation of yoghurt samples using 3.5% fat UHT milk with different quantities (0, 2.5 and 4.0%) of two types of WPC, namely Biolan P-35 (35% protein) and lactalbumin 80 (80% protein) and with different temperature pretreatments (50°C, 72°C/2 min, 75°C/5 min, 80°C/5 min and 90°C/10 min). All yoghurt samples were subjected to chemical, sensory (aroma, taste, consistency and appearance) and flow property evaluation. During incubation, a slightly slower development of acidity was observed in yoghurt with added WPC than in control yoghurt. Addition of about 33% more yoghurt culture was required in all WPC added yoghurt samples for achieving the similar final pH as in control after 4 - 4.5 h of incubation. There was an increase in protein as well as total solids content in yoghurt with the addition of WPC's. Further, it was observed that the higher the amount of WPC and the higher the temperature pretreatment of WPC added milk, the firmer was the consistency of the yoghurt. Higher protein WPC (80% protein) gave firmer yoghurt than did the 35% protein WPC. In general, it was observed that firmness on higher side to some extent was well accepted in yoghurt samples. It was concluded that the deleterious effect of higher protein in yoghurt in terms of too firm body could be countered by resorting to lower heat pretreatment of WPC added UHT milk. With added 4% of Biolan P-35 (representing about 30% on dry matter basis) and 2.5% of lactalbumin 80 (representing about 19% on dry matter basis), yoghurt of good quality, comparable to the control, could be prepared by a temperature pretreatment of UHT milk to 75°C/5 min and adding 10% yoghurt as culture. Yoghurt with 4% Lactalbumin 80 could be prepared of comparable consistency and pH by heat treatment of UHT milk to 72°C/2 min and with the addition of 10% yoghurt as culture. But the final flavour of this yoghurt was observed to be musty and was, therefore, unacceptable. 6. ICE CREAM AND FROZEN DESSERTS

WPC have been used to replace milk solids-not-fat in a variety of ice cream formulations. With the addition of WPC, there is an increase of albumin and globulins in the mix which improve the quality of ice cream and frozen desserts by eliminating shrinkage problems. Lando and Dahle (1949) attributed the shrinkage of ice cream to lower level of abumins and globulins in ice cream mix. Hugunin (1987) reported that ice cream in USA can include up to 25% of milk solids-not-fat as WPC. Jayaprakasha et al. (1999) successful prepared kulfi by replacing skim milk solids with whey protein concentrates to an extent of 80% without affecting any of the quality attributes.

Ice cream prepared with 50% skim milk powder and 50% WPC and lactose hydrolysed skim milk powder scored best in creaminess, smoothness and full of flavour (Huse et al., 1984). Addesso and Kleyn (1986) observed low sodium content (30.5 mg/100 g compared to 81.5 mg/100 g) in high quality vanilla ice cream, prepared with the partial replacement of dried skim milk with WPC. In a European patent, production of desserts having good flavour and texture characteristics have been claimed by partially replacing dried skim milk and stabilisers with modified WPC (denatured). Zall (1992) pointed out that encapsulating WPC proteins gives a fat-like mouth-feel which makes this form of WPC useful for making non-fat or very low-fat frozen desserts.

Ultrafiltered WPC has been tested in ice-cream in liquid and powder form. Although the use of ultrafiltered whey proteins in liquid form is not recommended, an addition of native whey

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protein powder, up to a replacement level of 30% (protein content of the powder 50%), is possible. Higher replacement levels produce a sensory devaluation, an increase in mix viscosity and a grey colouration of the samples. The danger that a "cooked" taste will develop still remains, as does that of a change in the meltdown behaviour (Sienkiewicz and Riedel, 1990).

WPC can be used in milk chocolate flavoured coatings. Compound coatings are used on frozen desserts to give proper coating, hardening and texture (Edwards, 1984). Hugunin (1987) advocated the incorporation of WPC in juice bar mixes. The author pointed out that protein provides lubrication to water ice mixes and reduces wear on continuous freezers. Further, WPC facilitates low over run into juice bar mix and thus reduces the hardness of the bar. In addition, level of protein can be adjusted from giving no discernible flavour change to that which produces a different dairy like flavour. 7. OTHER PRODUCTS

McDonough et al. (1976) used WPC as milk extender. Up to 40% of WPC from sweet whey or 20% from acid whey could be blended with skimmed milk without adversely affecting the organoleptic quality.

Gupta et al. (1994) investigated the manufacture of khoa from buffalo milk with added 0, 5, 10 and 18% WPC (27.40% TS), prepared by ultrafiltration of sweet cheese whey. A higher than 18% WPC was not possible without the addition of cream for maintaining the desired fat/TS ratio of 0.38 in the product. The addition of WPC to buffalo milk prior to heat processing gave more uniform product. Slow heating during manufacture of khoa gave better quality product in terms of white colour and softness of grains. The flavour score of 18% WPC added khoa was marginally higher (6.5) than 6.25 of the control khoa. The increased amount of WPC gave khoa with increased grain size, mainly due to which the overall acceptability score of 18% WPC added khoa was lower (6.1) to 6.9 of control khoa. However, such khoa is desirable for the preparation of kalakand. Good quality kalakand could be prepared by adding 0.3% k-carrageenan, which was necessary to improve the cohesiveness of the product. The total solid (TS) and WPC contents of khoa had signigicant effect on its rheological characteristics. The addition of WPC increased hardness and adhesiveness, but decreased cohesiveness, springiness, and chewiness in khoa. Further, The WPC added khoa bound more moisture, as a result of which, it was difficult to concentrate it to the same TS level as the controlled khoa. In other words, the desired body of the WPC added khoa could be obtained at a lower TS level than the controlled khoa, which resulted in substantial increase in the yield of the former product.

Cow milk khoa is generally not favoured due to its excessive smooth and pasty body, slight sandy texture, yellowish colour and salty taste. Patel et al. (1992) observed that addition of 5% WPC solids to cow milk improved the flavour, body and texture and colour of khoa prepared. The WPC incorporated cow milk khoa compared well with the traditional buffalo milk khoa. 8. REFERENCES Abd Rabo, F.H.R., Partridge, J.A. and Futedo, M.M. (1987) J. Dairy Sci., 70: 93. Abrahamsen, R.K. (1979) Milchwissenschaft, 34, 65. Addesso, K.M. and Kleyn, D.H. (1986) J. Food. Sci., 51, 1467. Baldwin, K.A., Baer, R.J., Parsons, J.G., Seas, S.W., Spurgeon, K.R. and Torrey, G.S. (1986) J. Dairy Sci., 69, 2543. Banks, J.M. and Muir, D.D. (1985) J. Soc. Dairy Technol., 38, 27.

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Brikkjaer, H.E., Forsingdal, K. and Thomson, D. (1974) XIX Int. Dairy Congr., IE, 304. Chojnowski, W., Poznanski, S., Smietona, Z. and Bednarski, W. (1978) XX Int. Dairy Congr., E, 946. Edwards, W.P. (1984) J. Soc. Dairy Technol., 37, 122. Georgakis, S.A. (1975) Milchwissenschaft, 30, 680. Greig, R.I.W. and Harris, A.J. (1983) Dairy Ind. Int. , 48 (10), 17. Gupta, V.K. (1996) The Proceedings of the Short Course on "Recent advances in membrane processing" under the

aegis of Centre of Advanced Studies, DT Division, NDRI, Karnal, March 20 - April 4. P. 57-61. Gupta, V.K. (1996) The Proceedings of the Short Course on "Recent advances in membrane processing" under the

aegis of Centre of Advanced Studies, DT Division, NDRI, Karnal, March 20 - April 4. P. 177-84. Gupta, V.K., Renner, E. and Renz-Schauen A. (1994) Brief Communication, 24th Int. Dairy Congr., Melbourne,

Australia, p. 404. Gupta, V.K. and Reuter, H. (1992) Lait, 72, 201. Gupta, V.K. and Reuter, H. (1993) Lait., 73, 381 Gupta, V.K. and Thapa,. (1991) Indian J. Dairy Sci., 44, 104. Patel, R.S., Gupta, V.K. and Singh, S. (1991) NDRI Newsletter. Gupta, V.K., Patel, R.S., Singh, S. and Reuter, H. (1994) Abstracts, Poster Session, Silver Jubilee National

Symposium on "Meat and milk industry: Trends and developmental strategies", Hisar. Gupta V.K. and Reuter, H (1992) Lait, 72 : 201. Hugunin, A.G. (1987) IDF Bulletin No.212, P.17. Huse, P.A., Towler, C. and Herper, W.J. (1984) N.Z.J. Dairy Sci. Technol., 19, 225. Ivanov, I.G., Velev, S. and Trifonova, S. (1971) Izvestiya, Nauchuoizsledovatchski Inst. PO Mlechna Promishlenast

Vidin. Cited in Dairy Sci. Abstr. (1973) 35, 336. Jayaprakasha, H.M., Sathyanarayana, H and Shobha, B. (1999) Effect of whey protein concentrates on the quality

attributes of kulfi. India j. Dairy Biosci., 10, 64-70. Jelen, P., Buchheim, W. and Peters, K.H. (1987) Milchwissenschaft, 42, 418. Kuipers, A. and Schroder, K. (1980) US Patent 4, 188 411. Cited in Dairy Sci. Abstr. (1980) 42, 7059. Kravchenko, E.F. (1988) IDF Bulletin No.233, p.61. Lando, J.C., Dehle, C.D. (1949) Ice cream Trade J. 49, 96. McDonough, F.E., Alford, J.A. and Momack, M. (1976) J. Dairy Sci., 59, 34. Patel, R.S., Gupta, V.K., Singh, S. and Reuter, H. (1993) J. Food Sci. Technol., 30(1), 64. Sienkiewicz, T. and Riedel, C.L. (1990) Whey and whey utilization. Verlag Th. Mann, Germany. Thapa, T.B. and Gupta, V.K. (1992a) Indian J. Dairy Sci., 45, 86. Thapa, T.B. and Gupta, V.K. (1992b) Indian J. Dairy Sci., 45, 140. Thapa, T.B. and Gupta, V.K. (1996) Indian J. Dairy Sci., 49, 129. Zall, R.R.(1992) In: Whey and lactose processing (editor, J.G. Zadow). Elsevier Applied Science, London, P.1.

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Rajesh Kumar, R.B. Sangwan and B. Mann Dairy Chemistry Division. N.D.R.I., Karnal -132 001

1. INTRODUCTION

Milk is the source of a wide range of proteins that deliver nutrition to the most promising new food products today. Isolated milk proteins are natural, trusted food ingredients with excellent functionality. Separation technologies provide the basis for adding value to milk through the production of proteins that provide the food industry with ingredients to meet specific needs, not possible with milk itself or with other ingredients. The global functional food and nutraceutical market is currently worth about US$50 billion and is growing at some 8% annually. This huge and rapidly growing market, driven by consumer demands for health-promoting foods, is creating an almost insatiable desire on the part of food manufacturers for new and novel ingredients with which to formulate these foods. Dairy constituents, notably the proteins and peptides, provide the food technologist with a rich selection of potential ingredients for functional foods.

2. BIOLOGICAL ACTIVITY OF WHEY PROTEINS

Milk contains two major protein groups - caseins and whey proteins, which differ greatly with regard to their physico-chemical and biological properties. The whey proteins accounts for 20% of total proteins, represents an excellent source of functional & nutritious proteins and exhibit a wide range of biological activity such as antimicrobial, antioxidative, immunomodulation, iron absorption, anticarcinogenic, retinol carrier and precursor of bioactive peptides.

3. SEPARATION TECHNIQUES OF BIOACTIVE WHEY PROTEINS

Separation technologies used to produce protein ingredients derived from whey include screening based on size differences: centrifugation based on density differences; membrane processes based on size differences, such as ultrafiltration, diafiltration, nanofiltration, and reverse osmosis; ion exchange based primarily on charge differences; and affinity chromatography based on specific binding to a matrix. Owing to unique functional and biological properties of many of the whey proteins, a number of pilot and industrial scale technological methods have been developed for their isolation in a purified form. Improved separation technologies and emerging markets have resulted in fractionation of whey proteins into ingredients that are enriched in specific proteins, or peptides, or both to fill those new opportunities. This is especially true for whey protein fractions that are in very low concentrations in the native state that require further concentration. These ingredients may be especially useful in the developing market of physiologically functional foods, or nutraceuticals. Separation technologies are available to prepare fractions that are enriched in the following milk components: β-Lg, α-La, lactoferrin, lactoperoxidase, immunoglobulins and other minor proteins with special functional properties. Many of these products are commercially available in limited quantities.

SEPARATION AND APPLICATION OF BIOACTIVE WHEY PROTEINS

SEPARATION AND APPLICATION OF BIOACTIVE WHEY PROTEINS

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3.1 Chromatographic techniques

Chromatographic techniques have been widely used for the isolation of milk proteins and high performance methodology now forms the basis for several accurate methods of analysis. Different types of separation chemistries are used for chromatographic separation of milk proteins. In its simplest form, a chromatographic separation system consists of a column filled with separation adsorbent beads. Chromatography has been known since the turn of the century, but its primary use has been in the analytical sector, where the excellent separation capability has been a valuable investigative tool. The industrial use of this technology has however, been fairly limited and mainly used for high value added products in the pharmaceutical industry. Low processing rates and difficulties in scaling up chromatographic separation from laboratory to production scales has hampered the broader use and acceptance of the technology. However, now with the advancement in chromatography based separation technology, it has greatly improved the profitability of dairy industry through the best possible utilization of raw material especially whey in a cost effective manner.

3.1.1 Ion Exchange Chromatography

Proteins bind to ion exchangers by electrostatic forces between the adsorbent charged beads and the charged groups of the protein. The charges need to be balanced by counterions such as metal ions, chloride ions and sometimes, buffer ions. A protein has to move the counterion to be attached; the net charge of the protein will usually be the same as the counterions displaced. That is why this type of chromatography is called “ion exchange”. There are two kinds of ion exchangers: anion exchangers, which have positively charged matrix, and will adsorb the proteins with negative charge; cation exchanger, which have negative charged matrix, and will adsorb the proteins with positive charge. The most common anion exchangers are DEAE- ,TEAE- and QAE-, and the cation exchangers often being used are CM- , S- and SP-. The variety in adsorbent types and the range of applications are however far beyond what is known for ion exchange, and this has created a need for more sophisticated systems such as membrane adsorber based chromatography and expanded bed chromatography.

4. FRACTIONATION OF WHEY PROTEINS

Chromatography offers a large variety of options for production of whey protein fractions in dairy industry. In each of the processes, a tailored adsorbent with optimal protein binding characteristics for the specific protein is used.

4.1 Production of WPI

The whey is adjusted to pH 3.5 and loaded on the column, i.e. whey is passed through the column. The bulk of the whey proteins are adsorbed. Following the adsorption step, the column is washed with water. Then the column is eluted by means of a pH gradient solution. These conditions result in a salt free protein solution, which can be concentrated to approx. 25 % solids by ultrafiltration and spray dried to a protein powder with more than 95% protein in total solids.

4.2 Production of α-la

The whey is loaded on the column after adjustment of pH to 3.0. At this pH the tailored adsorbent mainly binds α-la together with some minor fractions of other proteins. More than 80%

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of α-la is adsorbed and after the column has been washed with water the elution takes place using a pH gradient system resulting in a salt free protein solution, which can be concentrated and dried as described above. The purity of the a-la is approx. 70%. The whey protein that flows through the column and is not adsorbed onto the resin can be further processed to WPC or WPI.

4.3 Production of lactoferrin and lactoperoxidase

The whey is loaded on the column at pH 6.5, which is close to the normal pH of cheese whey. Due to the relatively high isoelectric point of lactoferrin and lactoperoxidase they will be captured by the adsorbent, while the other components including proteins will pass through the column. Lactoperoxidase is eluted with a NaCI buffer and recovered as a 70-90% pure solution, while lactoferrin is eluted using a pH gradient and recovered as a 90-95% pure solution. Both products are concentrated by ultrafiltration and freeze dried. The flow through is whey depleted from lactoferrin and lactoperoxidase and may be further processed to WPC or by any of the other process options shown in the diagram.

4.4 Production of β-Ig, immunoglobulins and α-la

The whey is loaded on the column after adjustment of pH to 4.7. At this pH β-Ig and immunoglobulins are captured by the tailored adsorbent, while the rest of the components pass through. The flow through is then adjusted to pH 3.0 and loaded on a second column, which captures α-la. Following a wash both columns are eluted with a pH gradient and the recovered solutions are concentrated by ultrafiltration and dried. Different purity levels of β-Ig and immunoglobulin fractions can be obtained according to specific applications. In general the purity of the immunoglobulins is in the area of 50- 70%.

5. APPLICATIONS OF BIOACTIVE WHEY PROTEINS

5.1 β-lg:

β-lg is approximately 50% of the total whey protein content in bovine milk. It is capable of binding hydrophobic molecules and may function in vivo as a transport mechanism for retinol into small intestine. It also binds fatty acids and may stimulate the activity of pancreatic lipases. β- lactoglobulin has numerous binding sites for minerals, fat soluble vitamins and lipids and can be used to incorporate desirable lipophilic compounds such as tocopherol and vitamin A into low fat products.

5.2 α-la

It represents about 25% of the total whey protein in bovine milk. Seventy percent of protein in human milk is like whey protein and 41 % of that protein is α-la. α-la accounts for 28% of the total proteins in human milk. It is a calcium modulating protein and may function as a metal carrier. It also plays an important role in the newborn. Recently it has been associated with apoptosis of transformed cancer cell lines in vitro and is under consideration in design of new anti tumor agents. Addition of bovine α-la is strongly advocated to humanize infant formulae and create other products for people with limited or restricted protein intake.

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5.3 Lactoferrin

The major use of lactoferrin is in Infants formula with the expectation of contributing to the babies’ defense system in the gut against harmful microorganisms, i.e., inhibition of the growth of Enterobacteriaceae and promotion of the growth of bifidobacteria. Several kinds of lactoferrin tablets are now available to promote or maintain human health. Lactoferrin saturated with excess iron is expected to be useful supplement to prevent anemia. Many kinds of commercial products containing lactoferrin are now being developed and some have appeared in the market such as makeup and cosmetics for facial skin, chewing gum and Infant formula, yoghurt, sports foods, nutritional supplements and special therapeutic diets for fish and animals.

The worldwide production of bovine lactoferrin has increased tremendously in the last decade with current estimates ranging from 50 to 100 metric tonnes per year for product purities over 90%. The production from cow’s milk, or the whey from cheese factories, is mainly due to the economy of scale in the dairy industry together with industrial developments in chromatographic separation technology. Lactoferrin is currently manufactured on an industrial scale by various companies in Europe, USA, Japan and Germany. 5.4 Lactoperoxidase

The most widely recommended industrial application of the LP system in food production is in the dairy industry for the preservation of raw milk during storage and/or transportation to processing plants. Antimicrobial agents of the LP system in milk cause inhibition of various spoilage and pathogenic organisms, thus enhancing the microbiological quality of milk. The antibacterial activity of the LP system in milk against psychrotrophic and mesophilic spoilage organisms has been widely investigated. Experiments conducted in India showed that activation of the lactoperoxidase system extended the keeping quality of raw buffalo milk at 300 C under both farm and field conditions. Apart from its importance in the preservation of raw milk, the LP system can also be used to extend shelf-life of pasteurized milk. However, other novel applications of the LP system are being explored. If the LP system is activated immediately prior to application of approved thermal processes, the shelf-life of dairy products may be extended significantly and high-temperature processes may be replaced with more economical lower temperature treatments. In addition to energy savings, LP low temperature thermal processes may provide better nutrient and/or quality retention for highly heat-sensitive foods such as salad dressings, spreads, beverages, dips and desserts. The shelf life of paneer is extended on treatment with LP-system. The LP system can provide a broad-spectrum antimicrobial activity against bacteria, yeasts and moulds and have the potential for use in cosmetics, oral hygiene, toothpastes and mouth rinses, fish farming, meat, milk replacers for calves and functional foods.

5.5 Immunoglobulins

Recent commercial and industrial applications have involved the targeted immunization of cows, such that bovine milk can be used to recover high levels of specific antibodies raised against predetermined species of bacteria and viruses. Such hyperimmunized milk products are currently being developed for use within both the pharmaceutical and feed industries. Also, preparations containing specific colostral Ig’s (antibodies) may in the future find applications in the prevention and treatment of human microbial diseases. Further, Ig-enriched preparations have been produced in many countries as calf milk replacers.

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6. CONCLUSION

Dairy manufacturing technology has expanded tremendously in recent years and the emphasis on identifying, recovering, and/ or supplementing bioactive proteins and peptides as functional ingredients will remain at forefront of future. Ultimately these approaches will improve the quality of food products containing such constituents. 7. REFERENCES Bajaj, R.K. and Sangwan, R.B. (2002) Health enhancing potential of whey proteins – a review. Indian J. Dairy Sci.,

55(5): 253-260. Clare, D.A. , Catignani, G.L. and Swaisgood, H.E. (2003) Biodefense properties of milk: The role of antimicrobial

proteins and peptides. Current Pharmaceutical Design, 9: 1239-1255. Expanded bed adsorption; principles and methods, Amersham Pharmacia Biotech, (1997). Hauffman, L.M. and Harper, W.J (1999). Maximizing the value of milk through separation technologies. J. Dairy

Sci., 82(10): 2238-2244 Korhonen, H. (1998) Colostrum immunoglobulins and the complement system- potential ingredients of functional

foods. IDF bulletin, 336: 36-40. Nielsen, W.K., Morten, A.O. and Lihme, A. Expanding the fronteiers in separation technology in Scandinavian dairy

Information 2 / 02. Olander, M.A., Jakobsen, U.L. , Hansen M.B. and Lihme, A. Fractionation of high value whey proteins. in

Scandinavian dairy Information 2 / 01. Roe, S. (ed) Protein purification techniques, 2nd edition, Oxford university, Oxford (2001) Roper, D.K. andLightfoot, E.N. (1995) Separation of biomolecules using adsorptive membranes. J. Chromatography

A 702: 3

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A.K. Dodeja Principal Scientist

Dairy Engineering Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Manufacture of lactose from whey solves both the problems of improving economics of whey utilization and of pollution. Lactose has various applications in food and pharmaceutical industries due to its multiple functional properties. Lactose does not lead to the formation of tooth plague, stimulates calcium assimilation and intestinal peristalsis and reduces the formation of liver fat in human body. Therefore, it is particularly suitable for diabetics.

While considering the economics of whey utilization for lactose manufacture, the concentration of whey to high total solids is the cost limiting factor. The running costs of the process are dominated by the energy required to remove the water from the whey. The whey price has barely managed to keep pace with the dramatic rise in the price of heavy fuel oil over past ten years. This had the effect of making people look closely at the costs of evaporation. This has resulted in the design of six stage evaporators and the development of mechanical vapor recompression evaporators. These are manifested with fouling problems and the limits to which the total solids can be reached. Further, developments in limiting the costs of whey evaporation are necessary to make the process of lactose manufacture more economically feasible (Kapil 1989).

2. MANUFACTURE OF LACTOSE

The conventional process for lactose manufacture consists of four basic steps a) deproteinization, b) concentration, c) crystallization and d) separation and drying. The main drawbacks of the traditional process of lactose manufacture are with respect to low yield and purity of recovered lactose and the high cost of manufacture and energy consumption. The purity of recovered lactose depends on the extent to which the proteins and minerals are removed from whey. Some processing steps have to be included so as to increase the purity but these lead to increase the purity but these lead to increased loss of lactose and add to the cost. Subsequently it yields mother liquor, which is less useful for food or feed. Thus a compromise has to be made between the yield and purity aspects in the process to be adopted for lactose manufacture.

Lactose can be manufactured from either sweet whey resulting from the manufacture of products where rennet type enzymes are used (such as cheddar, swiss, blue and similar cheeses) having a minimum pH of 5.6 usual range being 5.9 to 6.3 or from acid whey where the coagulum is formed by acidification (such as cottage, cream, Italian cheese and casein whey manufactured with mineral acids) with a maximum pH of 5.1, in the usual range of 4.4 to 4.6. In Indian context, channa and paneer whey, whose pH lies between 5.1 to 5.6 can also be used as a good raw material for lactose manufacture. The different methods of lactose manufacture can be classified in two groups a) Crystallization of the lactose in the original whey and, b) Crystallization of the lactose in deproteinized whey.

ADVANCES IN MANUFACTURE OF LACTOSE FROM WHEY ADVANCES IN MANUFACTURE OF LACTOSE FROM WHEY

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The deproteinized whey at the same concentration contains more lactose and is thus more supersaturated than concentrated whey. Moreover, due to the decreased viscosity, a higher concentration can be obtained in evaporator equipped with a suitable finisher. If the whey were not to be deproteinized then due to the greatly increased viscosity of concentrated whey, the separation of the crystallized lactose would be exceedingly difficult or in extreme cases the lactose may not crystallize out at all. On the other hand, adding processing steps of material to clarify the whey has limitations. It may result in loss of lactose through occlusion with the extraneous matter being removed, add to the cost of materials used and yield less useful mother liquor because of the materials added. Thus, purity may be improved at the expense of reduced yield of lactose and less desirable mother liquor for food or feed use. However, both methods, after the recovery of the crystallized lactose, result in a by-product, the mother liquor. This may be either dried as a fodder product or can be improved either by demineralization or by protein enrichment with a cheap vegetable protein.

Protein precipitation may be affected in several ways. The most commonly used methods in industrial processes are:

2.1 Heat Deneturation

The protein is heat denatured and then precipitated by acidifying to the isoelectric point with protein harvesting by means of separator. A simple and inexpensive method of removing protein from the cheese whey is to heat it to 93C and adjust the PH to 4.7 with acetic acid, and remove the denatured protein by centrifugation.

The methods for deproteinizing whey by heat denaturation are:

a) Heating to 90-95oC.

b) As a) but with acidification to 30-35oC with HCl or acetic acid.

c) As a) but with acidification to 30-35oC holding for 20 minutes and neutralization with NaHCO3 to 10-15oC

d). As a), but with the addition of 20% CaCl2 solution at 1%.

The precipitation is recovered with centrifugation. The overall protein recovery for different techniques is 85.1% for d), 79.1% for c) followed by 74.6% for b) and 52.3% for a). The disadvantage in d) is the high ash content in the mother liquor.

2.2 Ultrafilteration

Today UF is an interesting alternative. The advantage of the same over previously mentioned methods is an almost complete recovery of more denatured proteins. On the other hand, the disadvantages are:

a). Increased ash content of the permeate.

b). Delays in production due to membrane failure or unexpected clogging may occur.

c). The pretreatment of the UF whey requires expensive additional equipment.

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2.3 Concentration of whey

According to Pallansch (1972), the concentration of whey to a particular total solids, is very critical because, a high total solids concentrate will be too viscous to pump, while a lower total solids concentrate will result in insufficient lactose crystallization.

Different workers have recommended different processes for concentration up to 65 percent total solids. Thermal evaporators have been used for concentration in the ranger of 45 to 70 percent total solids. Concentration of whey in dairy industry is presently using triple effect evaporator equipped with mechanical vapor compression (MVR) systems. The basic limitations of these systems are: high cost of operation, problem of foaming, fouling, viscosity and browning due to high residence time. Although the energy cost for preconcentration by RO or MVR is about the same, evaporation cost more than RO system. The key comparison point is the RO systems sanitation and membrane replacement cost.

In lactose manufacture the total solids content of the concentrate is critical, since higher solids content of the concentrate is critical, since higher solids become too viscous to pump after holding and lower solids result in insufficient lactose crystallization. The limits to which this can be done are defined by changing physical properties of the whey concentrates on water removal. The viscosity of the whey concentrates increases rapidly as their total solids content raises about 40%. This increases further on holding. However, since whey concentrates can be considered pseudothixotropic and their viscosity changes with stirring rate, vigorous stirring can be done to prevent excessive viscosity buildup in highly concentrated whey when holding is necessary during processing.

To confront all the above-mentioned problems associated in handling whey in conventional evaporators and to increase steam economy thin film scraped surface heat exchanger (SSHE) seems to be a better proposition because of its unique performance characteristics.

In SSHE the working fluid is spread in the form of a thin film over the heat transfer surface by rotating blades. Each blade scoops a certain amount of fluid from the pool and accelerates it along the heat exchanger surface. At any given instant the fluid picked up by the blade is partly in the form of a film behind the blade and partly in the form of a fillet in front of blade. The blade action, which is similar to that of a plough, causes part of the fluid in the film to mix that in the fillet, simultaneously restoring the film thickness by allowing an equal amount of fluid to squeeze past the tip of the blade. (Abichandani et. al 1989)

2.4 Lactose Crystallization

If the aim is to manufacture alpha lactose then crystallization should be allowed to take place in a supersaturated lactose solution at a temperature below 93.5oC. However if β-lactose is to be manufactured the crystallization should be allowed to take place above 93o5 C.

In case of lactose manufacture for harvesting of crystals by centrifugation, the average crystal size must be sufficiently large to ensure the quick settling of the crystals. Regular size distribution is also important for adequate drainage of the mother liquor during separation process. Easy recovery is obtained with an average size of 0.2 mm. In order to create the optimum conditions for lactose crystallization, the concentrate is subjected to a fast cooling by passing through a flesh cooler to a temperature of 30 C. After this it is transferred to a crystallization tank.

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The crystal must be added in the form of finely grained lactose monohydrate at a level of about 1 kg per ton of concentrate. The holding time under these conditions should be 3-4 hours. Cooling of the concentrate should then start, the rate being about 3 C per hour until 10 C is reached. The entire crystallization lasts between 15-24 hours and takes place under constant agitation.

2.5 Harvesting lactose crystals

Crystal recovery can be carried out batch wise in basket centrifuge, which have the advantage of permitting complicated wash crystals. Washing of the crystals is generally done by ordinary tap water at the rate of 1.5 kg water/ kg. However continuous decanters equipped with a screw conveyor for crystal discharge are used more frequently. The washed crystals recovered have a moisture content of approximately 10% and about 99% lactose in dry matter. They can consequently be dried directly as edible milk sugar. Edible lactose is normally dried to 0.5% moisture content. The drying process is limited to a product temperature of 93oC, since at temperature above 100oC, the water of crystallization of the alpha-hydrate will be driven out. The drying is usually carried out in tunnel driers or tray driers. The lactose is finally ground to 80 to 200-mesh size before bagging(Kapil et. al. 1991b).

2.6 Lactose Refining

Especially in the case of the manufacture of pharmaceutical lactose, which requires a high degree of purity the recovered crystals must be refined. The lactose (dried or directly from the separator) is dissolved in hot water to a 50%-60% concentration, depending on the purity of the raw sugar. Quick dissolving requires heating to boiling point i.e. 105 C. In the dissolving tank active carbon and a small amount of chemicals, such as super phosphate, are added, plus a suitable amount of filter aid.

The slurry after being pumped through self cleaning filters at a high temperature is introduced into crystallization tanks and seeded as before. The crystals are cooled to approximately 20 C in about 6 hours. After completion of cooling the crystals are reseparated, either in a basket centrifuge or in a single decanter, where they are slightly washed. Finally the lactose crystals of pharmaceutical grade are dried to 0.1% moisture content.

3. MEMBRANE TECHNOLOGY FOR LACTOSE MANUFACTURE

The membrane technology has an edge over the conventional technology because of improved product yield, improved product consistency, continuous processing operation, minimum man power and energy requirement, greater efficiency due to decreased processing time and over and above, the whey proteins can be recovered in their natural form (Sachdeva et al. 1992).

An APV Pasilic process involves the ultrafilteration of milk or whey for the separation of proteins. The utilization of membranes with a high degree of protein retention ensures that the resulting permeate is very pure and, thus, provides the best possible raw material for production of crystalline lactose. The UF permeate is preconcentrated in the reverse osmosis plant and further concentrated in a falling film evaporator. The concentrated mass is then crystallized, separated, refined and dried in the fluid bed drier.

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Guu and Zall (1992) used nanofilteration membranes to process sweet whey and skim milk ultra filtration permeate and reported an increased lactose yield by about 10 and 8 %, respectively at a concentration factor of 3.0. These increases are attributed to depletion of minerals, especially monovalent cations such as sodium and potassium, by the partial demineralization effect of the nanofilteration.

A cost effective technology for the production of lactose using ultrafilteration for deproteinisation of whey, and reverse osmosis and scraped surface heat exchanger for concentration of the UF permeate, has been developed by Bhattacharjee and Sachdeva (1994). Crystallisation followed by washing with demineralised water and tray drying with 99% pure lactose with a yield of nearly 75 %.

4. USES OF LACTOSE

The major use for lactose, and one of the oldest, is the production of simulated human milk or infant formula. Human milk contains some 7% lactose. Lactose while processing a pleasantly sweet mild taste of its own has been found to enhance the natural flavors of food in general. The fact that lactose is much less sweet than the other commercial sugar expands its potential use as a flavor enhancer, since it will bring out other flavors without causing excessive sweetness. For these reasons it is used such diverse products as dusting powders to flavor potato chips; barbecue sauces etc. Because of its nonhygroscopic property, crystalline lactose powder is used as an anticaking agent and dispensing aid in powdered products of many types. In other cases, flow and dispersibility characteristics are improved by instantizing certain foods. Preparations containing 15% to 50% lactose are spray dried to form a lactose glass, which causes the food particle to agglomerate when they are moistened and redried. Such foods are well suited as items to be dispended from vending machines, or for easy preparations of convenience foods. The hygroscopic properties of the lactose glass are used to advantage in coating tablets or food pieces, as in pan coating.

Lactose is used to achieve desirable characteristics in certain types of candies, where it changes the crystallization habit of the other sugars. It is particularly useful in caramels or fudges to improve the body, texture, chewiness or shelf life.

There are number of applications of lactose to the improvement of bakery products. It can contribute to the flavor, appearance, texture, shelf life, and toasting qualities of baked foods. Another property of lactose of interest to the baker is that it improves the emulsifying efficiency of shortenings. This results in a uniform cell structure and a desirable texture, promoting good distribution of shortening with minimum mixing of pie crusts.

There are incidental industrial uses for lactose, such as in silvering mirrors, controlling the rate of burning in fireworks, or as polishing agent in toothpaste. It has been used extensively for many years by pharmaceuticals industry as a filler and inert carrier for capsules and tablets formed by direct compression.

5. CONCLUSION

The Indian cheese industry is rapidly growing and there is a growing concern for possible outlets of economic utilization of whey. The economy of lactose production depends upon the supply of raw materials, the operating costs and the marketing opportunities. The process designed

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and adopted for lactose production should aim at maximum yield, increased purity and should be energy efficient. Membrane technology offers distinct advantages in this regard. 6. REFERENCES Abichandani, H. and Sarma, S.C. 1987. Evaporation in horizontal thin film scraped surface heat exchanger. J. Fd.

Proc. Engg. 14(3): 173. Abichandani, H., Dodeja, A.K. and Sarma, S.C. 1989. Applications of thin film scraped surface heat exchangers.

Indian Dairyman. 41(1): 21. Kapil, V., Dodeja, A.K.and Sarma, S.C.1991a. Lactose manufacture. A review. Indian Food Packer. 2:52. Kapil V., Dodeja, A.K. and Sarma, S.C. 1991b. Manufacture of lactose: Effect of processing parameters on yield and

purity. J. Food Sci. Techno. 28:167. Kapil, V.1989. Application of thin film scraped surface heat exchanger for lactose manufacture from paneer whey.

MSc. Thesis, submitted to Kurukshetra university., Kurukshetra. Bhttacharjee, P.P.and Sachdeva, S. 1994. Membrane processing for lactose manufacture. 24th Intern. Dairy Congr. Kb

5, 486. Guu, Y.K.and Zall, R.R. 1992. Nanofilterationconcentration : Effect on thfe efficacy of lactose crystallization. J. Food

Sci. 57:735. Pallansch, M.J. 1972. New methods for drying acid whey. In Proc. Whey products Conference, Chicago, p. 100. Sachdeva, S., Reuter, H., Prokopek, D. and Klobes, H.1992. Ultrafilteration of heated, acidic and coagulated skim

milk with different modules.

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Dr. R.K. Sharma Principal Scientist

Animal Biochemistry Division, NDRI, Karnal-132 001

1. INTRODUCTION

Lactose, a disaccharide, is the predominant sugar of milk. It is not as sweet as sucrose, glucose, or fructose. It has various applications in food and pharmaceutical industries due to its multiple functional properties. The usual commercial source of lactose is whey, a by-product of cheese industry. The hydrolytic conversion of lactose to glucose and galactose represents one way of adding value to whey and whey-derived products. By enzymatic hydrolysis of lactose, whey can be processed to produce sweeteners, whey drinks etc. β-Galactosidases, used in enzymatic hydrolysis of lactose, are also able to catalyze a series of transferase reactions using lactose and its hydrolysis products resulting in the formation of galactose rich oligosaccharides, functional food ingredients. Ingestion of these galactooligosaccharides has been suggested to have a number of beneficial effects on human health (Mahoney, 1998; Sako et al., 1999). The wide range of applications of lactose has been reviewed by various authors (Thelwall, 1985; Pritzwald-Stegmann, 1986; Harper, 1992, Mahoney, 1998). 2. LACTOSE HYDROLYSIS

The hydrolysis of lactose provides various nutritional and technological advantages. In the process of lactose hydrolysis, the disaccharide lactose is converted either chemically or enzymatically, to its component monosaccharide units, glucose and galactose. This technique can be applied to all lactose-containing fluids, including milk, whey and permeates. However, some hydrolysis systems cannot be used in practice to process each one of these streams. Several excellent reviews exist in which the application of lactose hydrolysis technology to dairy products has been described (Coton et al, 1982; Hobman, 1984; Mahoney, 1985; Gekas and Lopez-Leiva, 1985; Harju, 1987; Zadow, 1992: Mahoney, 1997).

2.1 Acid Hydrolysis

Hydrolysis of lactose can be carried out under conditions of low pH and high temperatures. This procedure is of value only for protein-free streams such as permeates or protein-free wheys. The adjustment of pH can be made either by direct addition of acid to the system, or by treatment with a cation exchange resin. Homogenous or single-phase hydrolysis uses hydrogen ions in solution, with a heat treatment (ranging from about 60°C for 24 h to 140°C for 11 min at a pH of about 1.2). The hydrogen ions are provided either by adding mineral acid or by treatment with a cation exchange resin. In heterogenous or two-phase hydrolysis the demineralized product is passed at 90-98°C through a bed of cation exchange resin in the hydrogen form. There is continuous-flow processing. The products typically are brown and may require neutralization, demineralization and decolourization before use. There has been no commercial adoption of both types of processes.

HYDROLYSIS OF LACTOSE FOR APPLICATION IN FOOD INDUSTRY HYDROLYSIS OF LACTOSE FOR APPLICATION IN FOOD INDUSTRY

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2.2 Enzymatic Hydrolysis

β-Galactosidase, commonly known as lactase, catalyzes the hydrolysis of lactose to its component monosaccharides, glucose and galactose. The major products of hydrolysis are, in combination, sweeter, more soluble, more easily fermented and directly absorbed from the mammalian intestine. These changes are the basis for the production of new foodstuffs such as lactose-hydrolysed milk and whey, and the products derived therefrom. The use of β-galactosidase enzyme has been suggested as a solution to problem of lactose–intolerance among certain human populations and lactose crystallization during preparation of frozen, concentrated milk products. By using β-galactosidase technology, whey can be processed to produce sweeteners, whey drinks etc. In addition to hydrolytic activity, β-galactosidase also shows transferase activity by which a family of galactose rich oligosaccharides (galactooligosaccharides) are formed (Mahoney, 1998; Kumar, 2005). More recently, there is an increased interest in these galactooligosaccharides which have been recognized as prebiotics. A prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thus improves host health (Gibson and Roberfroid, 1995). Beneficial physiological properties are attributed to different prebiotics including galactooligosaccharides. Galactooligosaccharides have been suggested to have a number of beneficial effects on human health which include bifidogenic activity, stimulation of mineral absorption, hypolipidemic effect, prevention of colon cancer (Sako et al., 1999).

2.2.1 Sources and characteristics of enzymes

Microorganisms are considered the important sources for industrial applications. Enzymes from microbial cells are generally more useful than enzymes derived from plant or animals because of the great variety of catalytic activities available, the high yields possible, ease of cultivation and manipulation of cultural conditions optimum for the production of the enzyme in minimum space and time. For use in processing dairy foods, the enzyme must be derived from a source that is 'recognized as safe' and acceptable to regulatory bodies in each of the countries of interest. Various enzyme preparations from microbial sources have been described in the literature. The most widely used microbial sources of β-galactosidase are Kluyveromyces lactis, K. fragilis, Aspergillus oryzae. The application of many known β-galactosidases is however partly hampered due to the moderate thermal stability and narrow pH profile of enzyme activity as well as due to the inhibition by galactose. Many companies are producing yeast β-galactosidases. The yeast enzymes have neutral pH optima and are, therefore, suitable for the hydrolysis of sweet wheys. They are less temperature stable than the fungal enzymes and are, therefore, used at lower temperatures. They are, however, generally less affected by reaction products. The potassium ion is an activator and calcium and sodium ions inhibitors to the yeast β-galactosidases. Fungal enzymes generally have acidic pH optima in the range of 2.5–5.4 and with high optimum operating temperatures are especially suitable for the hydrolysis of acid wheys. They are, however, more strongly inhibited by galactose, thus slowing the rate of conversion and limiting their effectiveness in the manufacture of products with a high degree of hydrolysis.

2.2.2 Soluble/single use enzyme systems

In general, the manufacturer has two choices. The least costly is to utilize low levels of soluble enzyme, and allow an extended contact time (24-48 h). It is necessary for the substrate

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to be refrigerated if it is to be held for such a long period, which effectively reduces the activity of the enzyme. Such system needs extensive holding tanks and may result in microbiological problems with some substrates. Alternatively, the time required for hydrolysis can be reduced significantly by the use of much higher levels of added enzyme and of temperatures closer to that of the enzyme optimum (say about 40-50°C). In s uc h circumstances, holding times are generally of the order of 3-5 h. Again, care must be taken to monitor microbial growth in the substrate during the holding period. In practice, the enzyme is added directly to the reactor, in such an amount as to obtain the required degree of hydrolysis within the given holding period. An alternative approach is to use a continuous stirred reactor, in which feed and enzyme are continuously added to the reactor and the hydrolysed product (containing some of the enzyme) is continuously removed. This system has the advantage of allowing reduction in reactor size, but is less cost effective in enzyme use than a batch reactor.

The commercial attraction of the single-use process lies in its simplicity and flexibility, both in enzyme selection and in operating conditions. The major drawbacks are the cost, and the fact that the enzyme is lost in the product. Although the activity of enzyme is normally destroyed by pasteurization of product, its presence even in a denatured form may create functional, legal and marketing problems.

Jelen (1993) proposed the use of sonicated dairy cultures to produce a relatively impure source of β-galactosidase for potentially more economical process of lactose hydrolysis. Bury and Jelen (2000) evaluated the technical and economical feasibility of lactose hydrolysis by the use of a crude source of β-galactosidase from a disrupted dairy culture (L. delbrueckii subsp. bulgaricus). The production of partially lactose-hydrolysed milk was feasible given an appropriate process optimisation. However, the production of whey or permeate syrup did not appear feasible.

2.2.3 Sterile enzyme injection system

The process permits the use of a much smaller quantity of enzyme which is then allowed to work for a longer period of time. The enzyme is ultrafiltered to sterilize it and then mixed with UHT-sterilized milk immediately before the milk is aseptically packaged into containers. Only minute amounts of enzyme are needed (about 10 parts per million) to get almost complete hydrolysis in 7-10 days at room temperature (Anon., 1981). However, the enzyme must be very pure, i.e., protease-free or else the milk will deteriorate during storage. Whilst the system has considerable cost advantages, it has the disadvantage that the level of hydrolysis is uncontrolled, the enzyme is still undenatured and presumably active when the product is ingested.

2.2.4 Enzyme recovery systems

These systems utilize ultrafiltration membranes to allow the low molecular weight lactose and its hydrolysis products to pass through the membrane, whereas the comparatively high molecular weight enzyme is retained. The complexity and cost of such operations appear to make the process commercially uneconomic.

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2.2.5 Permeabilized whole cells

The use of whole cells as biocatalyst has an important advantage in terms of reducing the cost of process. Permeabilized whole microbial cells can also be used as a crude enzyme preparation for hydrolysis of lactose (Mahoney, 1997; Somkuti et al., 1998). The microbial cells are pretreated with chemicals to stop growth without influencing the activity of lactose enzyme inside the cells. Treated cells allow free diffusion of lactose into the dormant cells where the enzyme catalyses the reaction followed by release of products from the cells. The microbial cells used in the process must be safe, food grade.

2.2.6 Immobilized biocatalyst systems

An important factor in use of biocatalysts is their cost. When used in free (soluble) form, the enzymes are lost with the product. Enzymes can be immobilized on water-insoluble materials (matrices). Water-insoluble derivatives of enzymes are of practical value because they can be readily added to or removed from the reaction mixture, permitting close control of reaction and reuse of the biocatalyst. Using an immobilized enzyme the process can be operated continuously.

β-Galactosidase enzyme and the cells containing β-galactosidase can also be immobilized on a wide variety of supports using different immobilization techniques such as adsorption, covalent attachment and entrapment (Greenberg and Mahoney, 1981; Gekas and Lopez-Leiva, 1985; Sharma and Dutta, 1990; Roy and Gupta, 2003). However, there are advantages and disadvantages associated with different types of techniques. In general, supports which are likely to be useful in commercial operations should have a very even particle size, and should be resistant to degradation at operating temperatures, pH and cleaning regimes. They should not support microbial growth and should be safe for use in food processing operations.

In general, two types of reactor systems may be used for immobilized enzyme systems: either fixed bed or fluidized bed. Fixed bed systems have the advantage that they are simple to operate, and in general obtain a higher rate of hydrolysis per litre of enzyme in comparison to fluidized bed systems. Their major disadvantage lies in the problem of microbial growth. Cleaning a fixed bed reactor may also pose difficulties. There is generally a build-up of material on the surface of the bed. Fluidized bed systems attempt to overcome the difficulties by ensuring the continuous movement of enzyme particles. The commercial adoption of any system depends on a number of factors, including activity per litre, operating temperature, operating pH values, effective time of operation before cleaning is required and the ability of the system to withstand sanitation and cleaning operations without loss of activity or change in physical characteristics. Li et al. (2007) immobilized β-galactosidase from Kluyveromyces lactis on cotton fabric using glutaraldehyde as the crosslinking reagent and a pilot-scale module with a 10-l packed-bed reactor applied to hydrolyse lactose in whole milk. About 95% of lactose conversion was achieved after 2 h of batch operation.

Permeabilized cells with β-galactosidase activity have been immobilized in different gel forms: a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen (Genari et al., 2003); cells of S. thermophilus containing β-galactosidase entrapped in calcium aginate, κ-carrageenan and gellan-xanthan gel beads (Goel et al., 2006); Kluveromyces marxianus cells entrapped in alginate gel (Panesar et al., 2007).

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2.2 Commercial Systems

2.2.1 Soluble enzyme systems

Soluble enzymes are used in many countries for the manufacture of lactose hydrolysed whey and permeates. In the United States and Canada, products known as 'Lacteeze' and 'Lactaid' are available, which are hydrolysed milks prepared using soluble lactase. Lacteeze is 90% hydrolysed and after a type of UHT treatment, has a shelf-life of 30 days. Recently, Valio has developed a lactose free milk drink by membrane ultrafiltration and enzymatic removal of lactose from milk.

2.2.2 Immobilized Systems

A. Valio process: This process for hydrolysing whey uses enzyme immobilized on a phenol-formaldehyde resin. The enzyme is simply adsorbed onto the washed resin and then fixed in place by cross-linking with glutaraldehyde. This support has good mechanical/physical properties and is capable of binding high levels of enzyme activity with high binding efficiency. For hydrolysis, the resin is packed into a fixed-bed column reactor. Highest productivity is obtained by working at temperatures well below the optimum for the enzyme, e.g. 20-30°C, pre-concentrating the feed, operating at pH 3.5.

B. Sumitomo system: This immobilized system can hydrolyse the lactose in milk, sweet or acid wheys, and permeates. The system utilizes an enzyme from Aspergillus oryzae immobilized onto an ion-exchange resin. The optimum pH of the system is about 5, but it retains about 50% of its activity at pH 6.8 and may, therefore, be used for hydrolysis of milk. The operating temperature of system is about 35-40°C. The system was designed to result in levels of hydrolysis of the order of 70%. To achieve this level, contact times of 3-6 min are required for milk, 1.5-2 min for whey and about 1 min for permeate. A commercial plant utilizing this process was installed in an Australian factory in 1986.

C. Other system: Hydrolysis of lactose in milk has also been done with immobilized lactase using SNAMprogetti technology in which enzyme is entrapped in cellulose triacetate fibers. This process was used in Italy. A similar process was used in Japan by Snow Brand Milk to produce lactose hydrolysed milk. 3. APPLICATIONS

For commercial viability, the lactose-hydrolysed products must offer the food manufacturer commercial advantages over other competitive products. The major benefits of hydrolysed whey and permeate relate to their ability to replace sucrose, whereas hydrolysed milk offers a wider range of valuable attributes as a raw material for manufacturing purposes. In flavoured milk products, the amount of sucrose can be reduced by 20 to 40% without loss of sweetness by using lactose hydrolysed milk in the formulation of the product. In sweetened condensed milk, partial hydrolysis of lactose (25-30%) reduces the tendency for lactose crystallization. However, high reactivity of lactose-hydrolysed milk to the Maillard reaction and a wide variability in the quality of commercial

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samples of lactose-hydrolysed milk has been reported (Messia et al., 2007). The low solubility of lactose often leads to its crystallization during storage of frozen milk products. This phenomenon observed as 'Sandiness' in ice-cream can be prevented by the use of lactose hydrolysed milk. Use of lactose hydrolysed milk for yoghurt manufacture results in accelerated acid development by the starters. This leads to a reduction in the set time of 15-20%. Adding β-galactosidase to milk causes faster acidification by some of the starter organisms used in cheese manufacture. Accelerated ripening has been reported for a number of cheese varieties. Lactose-hydrolysed wheys and permeates can also be used in manufacture of ice-cream, confectionary, bakery products, fermented beverages, etc. Attempts are also being made towards the production of milk, infant formulas etc enriched with galactooligosaccharides. Chen (2002) reported optimisation of the enzymic process for manufacturing low-lactose milk containing oligosaccharides. This involved ultrafiltration, beta-galactosidase catalysed transglycosylation treatment of permeate and subsequent reconstitution with the retentate to obtain the final milk product.

4. REFERENCES Anon. 1981. Process Biochem., 16(2): 36. Cited: Developments in Dairy Chemistry -3 : Lactose and Minor

Constituents 1985. P.F. Fox (ed.), P. 85. Elsevier Applied Science Publishers, London. Bury, D. and Jelen, P. 2000. Lactose hydrolysis using a disrupted dairy culture: Evaluation of technical and

economic feasibility. Can. Agri. Engg. 42 (2): 75-80. Chen, C.S., Hsu, C.K. and Chiang, B.H. 2002 Optimization of the enzymic process for manufacturing low-lactose

milk containing oligosaccharides. Process Biochem., 38(5):801-808. Coton, S.G., Poynton, T.R. and Ryder, D. 1982. Utilization of lactose in the food industry. Bull. Int. Dairy Fed.

147: 23-30. Gekas, V. and Lopcz-Lciva, M. 1985. Hydrolysis of lactose: A literature review. Process Biochem. 20: 2-12. Genari, A.N., Passos, F.V. and Passos, F.M.L. 2003 Configuration of a bioreactor for milk lactose hydrolysis. J. Dairy

Sci. 86: 2783-2789. Gibson, G.R. and Roberfroid, M.B. 1995. Dietary modulation of the human colonic microbiota: Introducing the

concept of prebiotics. J. Nutr. 125: 1401-1413. Goel, Anita, Sharma, R.K. and Tandon, H.K.L. 2006. A comparison of different polymeric gels for entrapment of

cells of Streptococcus thermophilus containing β-galactosidase. J Food Sci. Technol. 43 (5): 526-531. Greenberg. N.A. and Mahoney, R.R. 1981. Immobilization of lactase (β-galactosidase) for use in dairy

processing: A review. Process Biochem. 16(2): 2, 4, 6-8, 49. Harju, M. 1987. Lactose hydrolysis. Bull. Int. Dairy Fed. 212: 50-55. Harper, W.J. 1992. Lactose and lactose derivatives, in Whey and Lactose Processing, J.G. Zadow (ed.), P.317-

360. Elsevier Applied Science, London. Hobman, P.G. 1984. Review of processes and products for utilization of lactose in deproteinized milk serum.

J. Dairy Sci. 67: 2630-53. Jelen, P.J. 1993. Lactose hydrolysis using sonicated dairy cultures. Bull. Int. Dairy Fed. 289:54-56. Kumar, P. 2005. Study on oligosaccharide formation by β-galactosidase of lactic acid bacteria. M.Sc. Thesis,

N.D.R.I., Karnal, India. Li, X., Zhou, Q. Z..K., and Chen, X.D. 2007. Pilot-scale lactose hydrolysis using β-galactosidase immobilized on

cotton fabric. Chem. Engg. Process. 46 (5): 497-500. Mahoney. R.R. 1985. Modification of lactose and lactose-containing dairy products with β-galactosidase, in

Developments in Dairy Chemistry-3: Lactose and Minor Constituents, P.F. Fox (ed.), P. 69-109, Elsevier Applied Science Publishers, London.

Mahoney, R.R. 1997. Lactose: Enzymatic modification. in Advanced Dairy Chemistry. Vol.3. Lactose, Water, Salts and Vitamins, P.F. Fox(ed.), P 77-125, Chapman Hall, UK.

Mahoney, R.R. 1998. Galactosyl-oligosaccharide formation during lactose hydrolysis: a review. Food Chem. 63(2):147-154.

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Messia, M.C., Candigliota, T. and Marconi, E. 2007. Assessment of quality and technological characterization of lactose-hydrolyzed milk. Food Chemistry. 104(3): 910-917.

Panesar, R., Panesar, P.S., Singh, R.S., and Bera, M.B. 2007. Applicability of alginate entrapped yeast cells for the production of lactose-hydrolyzed milk. J. Food Process Engg. 30 (4): 472-484.

Pritzwald-Stegmann. B.F. 1986. Lactose and some of its derivatives. J. Soc. Dairy Technol. 39: 91-96. Roy, I. and Gupta, M.N. 2003. Lactose hydrolysis by LactozymeTM immobilized on cellulose beads in batch and

fluidized bed models. Process Biochem. 39:325-332. Sako, T., Matsumoto, K. and Tanaka, R. 1999. Recent progress on research and applications of

non-digestible galacto-oligosaccharides. Int. Dairy J. 9: 69-80 Sharma, R.K. and Dutta, S.M. 1990. Immobilization of β-galactosidase of Streptococcus thermophilus on DEAE-

cellulose. Indian J. Dairy Sci. 43(2): 213. Somkuti, G.A., Dominiecki, M.E. and Steinberg, D.H. 1998. Permeabilization of S .thermophilus and

Lactobacillus delbrueckii subsp. bulgaricus with ethanol. Curr. Microb. 36: 202-206. Thelwall. L.A.W. 1985. Developments in the chemistry and chemical modification of lactose, in

Developments in Dairy Chemistrv-3: Lactose and Minor Constituents, P.F. Fox (ed.), P. 35-67. Elsevier Applied Science Publishers, London.

Zadow, J.G. 1992. Lactose hydrolysis, in Whey and Lactose Processing, J.G. Zadow (ed.), P. 361-408. Elsevier Applied Science, London.

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S. K. Kanawjia* and Hitesh Gahane** *Principal Scientist ** Research Scholar

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Whey Cheeses are solid, semi-solid, or soft products which are principally obtained through either of the following processes:(i) the concentration of whey and the moulding of the concentrated product;(ii) the coagulation of whey by heat with or without the addition of acid. In each case, the whey may be pre-concentrated prior to the further concentration of whey or coagulation of the whey proteins. The process may also include the addition of milk, cream, or other raw materials of milk origin before or after concentration or coagulation. The ratio of whey protein to casein in the product obtained through the coagulation of whey shall be distinctly higher than that of milk. The product obtained through the coagulation of whey may either be ripened or unripened. Whey Cheese obtained through the concentration of whey is produced by heat evaporation of whey, or a mixture of whey and milk, cream, or other raw materials of milk origin, to a concentration enabling the final cheese to obtain a stable shape. Due to their relatively high lactose content, these cheeses are typically yellowish to brown in color and possess a sweet, cooked, or caramelized flavor. Whey Cheese obtained through the coagulation of whey is produced by heat precipitation of whey, or a mixture of whey and milk or cream, with or without the addition of acid. These whey cheeses have relatively low lactose content and a white to yellowis color.

2. RATIONALE OF USING WHEY IN CHEESE MAKING

Whey is being touted as a functional food with a number of health benefits. The biological components of whey, including lactoferrin, β-lactoglobulin, α-lactalbumin, glycomacropeptide, and immunoglobulins, demonstrate a range of immune-enhancing properties. In addition, whey has the ability to act as an antioxidant, antihypertensive, antitumor, hypolipidemic, antiviral, antibacterial, and chelating agent. Whey proteins have also exhibited benefit in the arena of exercise performance and enhancement. Today, whey proteins are the popular dietary protein supplement purported to provide antimicrobial activity, immune modulation, improved muscle strength and body composition, and to prevent cardiovascular disease and osteoporosis. Whey is commercially used for manufacture of various soft cheeses having great nutritional significance.

3. WHEY CHEESES

Whey cheeses are very popular in Norway, Greece and Italy. Whey cheeses like Gjetost, Mysost and Gudbrandsdulsost are produced in Norway, while Manouri, Anthotryos, Cryzittroa and Giza in Greece. The names of whey cheeses in Greece indicate their quality. For example, Manouri contains 30% fat and 65% TS while the fat content of Antotryos is only 19.25%. Ricotta cheese is another cheese which is popular in Italy and in many other countries. Whey cheeses are

TECHNOLOGICAL ADVANCES IN THE PREPARATION OF WHEY CHEESES

TECHNOLOGICAL ADVANCES IN THE PREPARATION OF WHEY CHEESES

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also known by different names in different countries. Some of the popular names are, Schoftenzieger, Schabzieger (Germany); Mascarpone (Switzerland); Klila (Tunisia); Nicotta (North Africa); Zieger (Romania); Kaukaz (USSR); Anan (Cyprus); Lour (Iraq); Ricotta Fresca (Brazil); Urda (Israel and Czechoslovakia); Zinicica (Czechoslovakia) and Karicha (Lebanon). 4. PRINCIPLE OF CHEESE MAKING FROM WHEY

The basic principle involved in making most of the whey cheeses involves coagulation and subsequent separation of whey proteins by heating of whey which may also be supplemented with milk or milk fat. Whey cheeses may be classified as Brown whey cheese and re-cooked whey cheese. The classification of Brown whey cheese as cheese is somewhat misleading as they contain all the whey constituents including lactose which otherwise is eliminated in cheese. From the standpoint of alleviating disposal problem of cheese whey Brown cheeses offer a much better alternative as they utilize all the whey solids in comparison to re-cooked cheeses which result in a significant amount of partially deproteinised whey. In a cheese plant, two types of whey, i.e., sweet and acid whey are produced as a by-product. Sweet whey results from the production of ripened cheeses like Cheddar, Swiss, Blue etc. and is recommended for most of the whey cheeses. Acid whey obtained from soft cheeses such as cottage, cream, paneer etc. can be used for cheese making after neutralization but some of the cheese quality is lost. Acid whey below a pH 6.0 results in a coarse texture, reddish colour and sour flavour of cheese. Different methods by which whey can be used in cheese making are as follows:

(a) Heating of whey with or without acidifying and separation of proteins and fat as curd which after drainage gives a cheese.

(b) Heating of whey, separation of coagulated whey proteins and its addition to cheese milk to increase yield. Whey proteins must be denatured before addition to cheese milk. Addition of acid whey, or acid whey powder and/or lactic, citric or acetic acid improves precipitation of whey proteins. Whey protein concentrates should be added to cheese milk in liquid form and the powdered preparations dissolved and heat treated prior to use to minimise bitterness in cheddar cheese.

(c) Heating of whey for obtaining curd with denatured proteins which are added in different proportions to processed cheese blend or are converted into soft cheese like products.

(d) Condensation of whey by heating or by reverse osmosis for making Brown whey cheese. Modifications in the method of heating include heating of whey with a large number of fine tubes inside the vat or by making special arrangements in the cheese vat that permits the use of hot serum for pre-heating the whey. Considerable saving in labour could be achieved by using a screen to drain the mixture of whey and curd or by providing a mechanical system for collection and transfer of curd into moulds for subsequent drainage. 5. MANUFACTURE OF WHEY CHEESES

Norwagian Brown whey cheeses are made from goat's milk, cow's milk or a combination of both. Gjetost is made only from goat's milk while cow's milk is used for Mysost. Addition of cream to Gjetost yields a quality cheese known as Primost which has a light tan colour and smooth and creamy body texture. Gjetost is darker Brown in colour with coarser texture. The flavour is

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similar to cream caramel. A mixture of 88% goat's milk and 12% cows milk is used for another Brown cheese known as Gudbrandsdulsost. The unit operations involved in the manufacture of Brown cheeses include evaporation, rapid cooling with vigorous stirring, packaging and solidification. The whey (sweet or acid) after filtration is centrifuged and pasteurised. Milk and cream to the extent of 35 to 40% are added to increase the protein and fat content in the cheese. Mixture is then concentrated to 50 to 55% TS in conventional vacuum evaporator. Second stage concentration to 80-84% TS is carried out in a kettle under reduced vacuum pressure. The vacuum is thereafter released and temperature raised to allow the concentrate to develop the desired brown colour and flavour. The concentrate is then filled into containers where it is cooled with stirring. In a modified method, the concentrate from the evaporator is further concentrated to 75 to 82% TS in a specially designed kettle (Gryta) at a temperature of 95-96°C and then transferred to scraped heat exchangers for kneading and rapid cooling to 75°C or lower. Vigorous stirring in both the methods is important to prevent formation of lactose crystals aggegates and the sandiness defect. Finally the viscous mass is packed in Al-foils, plastic bags or cups and cooled overnight so that it solidifies. It is now a standard practice to add 10 mg ferrus sulphate per 100 g cheese to increase the iron content.

Greek whey cheeses are manufactured in a similar way from sheep's milk but other types of whey can also be used. The manufacturing steps include filtration of sweet whey (pH above 6.0) followed by heating to 90°C for 40 to 45 min. Cream and milk (optional) are added at 40°C and salt @ 1.0-1.5% at 75°C. The curd after coagulation is cooked at 88-92°C for 15-30 min and then transferred to moulds and drained. Manouri and Myzithra that are to be used fresh are heated at lower temperatures. When the products are to be dehydrated afterwards, heating is done to a higher temperature.

Reverse osmosis and addition of dry butter milk to liquid unprocessed whey have been used to develop hard as well as spreadable Mysost like products. Lactose content of buttermilk containing spreads can be reduced by using lactose reduced buttermilk powder produced by ultrafiltration process (UF). Hydrolysis of lactose in condensed rennet whey using Maxilact enzyme helps in controlling sandiness and also in the production of optimally caramelized whey cheeses. A new technology involves admixing of highly ultrafiltered whey with cream followed by packaging of the mix and heating at 90°C for 50 to 90 min. Denaturation of proteins occurs in the packages and the cheese is formed. UF retentate from cottage cheese whey mixed with butterfat without any additional source of milk solids has also been used.

The best known whey cheese on universal scale is Ricotta cheese which is also known as albumin quarg. It is known as Ricotone in certain parts of U.S.A. if made only from whey. The traditional Italian style Ricotta cheese is primarily a heat coagulated whey protein. However, in some countries, modern Ricotta (Re-cooked cheese) is made from milk-whey or buttermilk-whey mixture or even from whole milk by heating the mix to 80° to 90°C followed by acidification to a pH value below 5.9. The coagulated curd is separated and filled in perforated metal cans for draining. Salt and lactic starter may be added after the curd is cooled to 30°C. The drained curd is ready for consumption. In one of the mechanised methods separation of coagulum from the surface of the heating vat is accomplished by a series of peddles which transfers the curd into perforated conveyor. However, this device is suitable for Ricotta cheese produced from whey-milk mixture in which the high casein content provides cohesiveness to the otherwise fragile whey protein coagulum. In a separate development, heating vat has been replaced by a tubular holding

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section which uses whey after concentration of whey protein to about 2% by UF. A fully automated line which combines the heating, drainage and filling of moulds has also been developed for Ricotta cheese.

A method for the manufacture of whey cheese from buffalo milk whey known as Requeson has been developed by using calcium chloride or citric acid and subsequent heat precipitation of whey proteins. Another whey cheese from a mixture of whey and cream is manufactured by concentrating the whey to 10-11% TS by reverse osmosis process followed by addition of cream. The mix is further concentrated in two steps to the desired level under vacuum. 6. COMPOSITION OF WHEY CHEESES

Whey cheeses differ in composition mainly according to fat and moisture content. The differences in technology and composition of whey, addition of whole milk and type of milk, influence the composition of cheese. Other factors which affect the quality of whey cheese include quality of whey, method and degree of heating the whey, whey acidity and addition of calcium chloride and sodium chloride. The compositional data is presented in the Table 1. The amount of fat in Ricotta cheese is determined by the type of cheese from which the raw material i.e. whey was obtained, and by the amount of milk fat contributed through any added milk. Table1. Chemical Composition of Whey Cheeses

Whey Cheese % Chemical Composition Fat Protein Lactose Ash Salt Moisture Mysost 28-30 11.5-12.0 36 4.2-4.5 -- 16.0-18.5 Manouri 36.5 11.0 2.5 1.7 0.8 48.0 Myzithra 16.0 13.0 3.3 1.7 0.8 66.5 Dry Myzithra 20.8 25.4 4.0 9.9 8.7 38.6 Ricotta 30.5 8.7 3.2 0.6 -- 56.9 Ricotta from can's 19.6 23.1 4 2.1 -- 50.9 Ricotta from sheep's whey

22.4 6.4 4.3 0.5 -- 66.9

Ricotta 2.5 16.0 3.5 1 -- 77.8 Dry Ricotta 5 19 4 -- -- 60.0 7. NUTRITIVE VALUE OF WHEY CHEESES

Whey cheeses are distinguished in that they have different kind of proteins than the other cheeses. Whey proteins are rich in the amino acids, methionine and cystine and their essential amino acids content is more than sufficient to cover the needs of man as recommended by FAO. The biological value of whey proteins is 1.0 as against 0.8 of casein and 0.9 of coprecipitates of casein and whey proteins. When cheese is made by heating whey there is no appreciable reduction in biological value of proteins. The biological value of cheese proteins is 0.91 as against 0.92 of fresh whey, 0.91 of protein powder of UF whey and 0.94 of lactalbumin. Also, the cheese was not inferior in available lysine against other products and it had true digestibility 0.97. The diet of children could be supplemented with whey cheese.

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Norwagian whey cheeses are quick source of energy (18,000 kJ/kg) as they contain high amount of lactose. The spreadable product, however, has lower energy content. The high moisture content of Ricotta cheese reduces its energy value and, therefore, it is known as slimming food. It is a healthful food because of high protein content of good quality.

8. SHELF-LIFE OF WHEY CHEESES

The high lactose content of Mysost types of cheeses is the primary reason for their excellent shelf-life and microbiological stability. The whey cheeses produced in Greece in salted form (2.5%) and with the addition of starter (1%) have a shelf life of 6 months. Ricotta cheese is very susceptible to rapid spoilage by moulds, yeast and bacteria because of its high moisture content, high pH and the contamination to which the surface is exposed. However, pH adjustment and filling under heat application improve the keeping quality of the product. Fresh Ricotta packaged in plastic bags has a shelf life of 2-3 weeks. Packaging in an atmosphere of nitrogen or carbon dioxide increases its storagebility to 6 weeks. High moisture (50-80%) whey cheeses are, therefore, usually consumed fresh. The cheeses are marketed one day after their manufacture and disposed within a week. 9. USES OF WHEY CHEESES

Whey cheeses are traditional in several countries. Norwagians commonly consume brown whey cheeses in Sandwiches or Scandinavian-type crispbread. Brown whey chees has a sweet taste. Manouri and Anthotyros are consumed as table cheeses in Greece. Unsalted cheeses are eaten with honey. Myzithra is sometimes used as a table cheese but more often in preparations of certain foods and cheese pies. Fresh Ricotta cheese is white, moist and grainy. Its appearance resembles cottage cheese curd but the consistency is very dry and crumbly. It has a bland to semi-sweet flavour. Fresh Ricotta is ideal for cooking purposes as it does not develop lumps, and forms a smooth emulsion with fat. The potential uses of fresh Ricotta cheese are in combination with fruits or honey for breakfast dishes, in creamy desserts, cake fillings, baked products, Italian pasta products and Indian sweets. Dried Ricotta is suitable for grating and as a compliment for other cheeses for more pronounced flavour. Processed cheese is also produced (pH 5.9, 23% FDM) from Ricotta and Cheddar cheese.

10. CONCLUSION

Whey, a protein complex derived from milk, is being touted as a functional food with a number of health benefits. The biological components of whey, including lactoferrin, beta-lactoglobulin, alpha-lactalbumin, glycomacropeptide, and immunoglobulins, demonstrate a range of immune-enhancing properties. In addition, whey has the ability to act as an antioxidant, antihypertensive, antitumor, hypolipidemic, antiviral, antibacterial, and chelating agent. Several types of whey cheeses are being manufactured commercially and consumed throughout the world. These cheeses are nutritionally sound and enjoyed for its functional attributes. 11. REFERENCES Abrahamsen, R.K. 1986. Production of Brown cheese. Int. Dairy Fed. Bull. 202: 125-130. Baldwin, K.A., Baer, R.J., Parsons, J.G., Seas, S.W., Spurgeon, K.R. and Torrey, G.S. 1986. Evaluation of yield

and quality of Cheddar cheese manufactured from milk with added WPC. J. Dairy Sci., 69: 2543-2550.

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Banks, J.M. 1988. Elimination of the development of bitter flavour in Cheddar cheese made from milk containing heat denatured whey proteins. J. Soc. Dairy Technol. 41: 37-41.

Cabrera, Mc-del, Menendez, T., Ootega, O. and Real, E. 1995. Manufacture of Requeson from buffalo milk whey. Alimentaria. 32: 260, 275-278. C.F. Dairy Sci. Abstr. 57(6): 3378.

Jelen, P. 1992. Whey cheeses and beverages. CF Whey and Lactose Processing. Ed. by Zadow, T.G. Pub. by Barking, U.K. Elsevier Science Publishers Ltd. P.157-171.

Jelen, P. and Buchheim, W. 1976. Norwagian whey cheese. Food Technol., 30(11): 62-74. Kanawjia, S. K., Makhal, S. and Harpreet, K. K (2004). Say cheese- for health and nutrition. Science Reporter, 41

(10): 46-49. Kanawjia, S.K., Makhal, S. Harpreet, K.K. (2003). Novel bioactive cheese: fads and fantasy to facts and figures. In:

Lecture Compendium, 16th Short Course on “Application of Biotechnology in Dairy and Food Processing” Nov.4-24, 2003, Org. by Dairy Technology Division, NDRI, Karnal-132001.pp: 168-178.

Kandarakis, T.G. 1986. Traditional whey cheeses. Int. Dairy Fed. Bull. 202: 118-124. Knoop, T.K. 1988. Whey utilization in cheese. Int. Dairy Fed. Bull. 202: 118-124. Knoop, T.K. 1988. Whey utilization in cheese. Cult. dairy Products J. 23 (2): 14-18. Kosikowski, F.V. 1977. Cheese and Fermented Milk Products. Edward Brothers Inc. Ann/Arbor/Michigan. 2nd

Edn. P.367-374. Makhal, S. and Kanawjia, S. K. (2005). Developments in cheese technology: A mini assessment. Food and Pack, 5:

28-31. Makhal, S., Mandal, S. and Kanawjia, S.K. (2004). Development of bioactive fermented dairy products with special

reference to cheese: scope and challenges. Indian Food Industry, 23 (6): 25-35. Marshall D. Current Concepts on Whey Protein Usage. http://www.cfids-cab-inform/ Optimist/ marshall 97.html Scott, R. 1986. Cheese Making Practice. Applied Science Publishers, London & New York. 2nd Edn. P.24, 31-

34, 302-319, 382, 481-484. Sienkiewieze, T. and Riedel, C.L. 1990. Whey and whey utilization. Verlag, T.H. Mann Gelsenkirchen-Buer,

Germany. 2nd edn. P.193-203. Streift, P.J., Nilson, K.M.; Duthie, A.H.; Atherton, H.V. 1979. Whey Ricotta cheese manufactured from fluid and

condensed whey. J. Food Protection. 42: 552-554. Tiwari, B. D.1997. Technology of protein hydrolysates, Technological Advances in Dairy Byproducts-Lecture

Compendium, CAS in Dairy Technology, NDRI, Karnal. Walzem RL, Dillard CJ, German JB.2002. Whey components: millennia of evolution create functionalities for

mammalian nutrition: what we know and what we may be overlooking. Crit Rev Food Sci Nutr 2002;42:353-375.

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Shilpa Vij, Senior Scientist

Dairy Microbiology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

The main solute in cheese whey is lactose, present at a concentration of about 3-8% (Speer, 1998). Other components are protein, salts and vitamins that are present in minor amounts. Whey is a considerable organic carbon and energy source but, at the same time, it represents an important environmental problem, so that its utilization, opposed to its disposal, has been the subject of several studies (Ben-Hassan and Ghaly, 1994; González Siso, 1996). There has been a greater emphasis all over the World on utilization of whey solids in different manners. Since the amounts of purified lactose produced world-wide would require the only 5% of the available whey, alternative uses have been explored (Mawson, 1994; Mahoney, 1998; Povolo and Casella, 2003). The process based on microbial cultivation on cheese whey permeate may offer a suitable alternative for the transformation of cheese whey surplus. In this respect, the number of microorganisms of commercial interest that are able to metabolize glucose and galactose is notably higher than the ones able to directly utilize lactose as a carbon source. Therefore, the preliminary hydrolysis of disaccharide into the two monosaccharides could significantly increase the number of biological systems to be utilized to obtain biomass. It is known that acid hydrolysis shows some important drawbacks, thus suggesting investigating enzymatic hydrolysis as a feasible solution. The soluble enzyme (β-galactosidase) is normally used for batch processes, while the immobilized system may be used in continuous and offers the possibility to recycle the enzyme. The immobilized enzyme reactor has been successfully used for easy and efficient utilization of whey for yeasts biomass production as well as to reduce the environmental pollution from this by-product of cheese making. The use of whey for the production of yeast biomass has the advantages that it is a simple treatment process and the final discharge of the whey is facilitated since the pollutant load is significantly reduced and the whey lactose is converted into yeast biomass. It has been established that whey can serve as a substrate for production of food grade and feed grade yeast and yeast whey products. The microbial biomass could be produced from whey using yeasts such as Kluyveromyces, Candida and Trichosporon, as they are naturally able to metabolize lactose (Galvez et al. 1990; Mansour et al. 1993). Lactose fermenting yeast utilize lactose without hydrolysis to glucose and galactose though it is capable of using these sugars, whereas,, Sachharomyces cerevisiae can not utilize lactose as a carbon source, therefore, prior hydrolysis of whey is required in this case. 2. MICROBIAL BIOMASS

Many processes have been developed to produce microorganisms able to use organic material as a source of carbon and energy and to convert inorganic nitrogen into high-food value proteins. These can be used in human foodstuffs or animal feed to replace traditional plant or animal sources. Culture of microorganisms for their nutritional value started at the end of the First

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World War. The Germans developed yeast culture for use in animal and human diets. The term “fodder yeast” was coined After the Second World War, Yeast biomass has been produced commercially by whey fermentation from 1940s until present strains of K.marxianus var lactis or var. marxianus are those most commonly used, although other strains may also be produced successfully. (Mansour et al. 1993; Moresi et al. 1989; Grba et al. 2002) Thus the biomass produced by unicellular or multicellular organisms is processed and used as human food or animal feed supplement is called ‘microbial biomass protein’ which is dried dead cells. The microbial biomass used as food is Candida utilis, K. fragilis and S. cerevisiae. 3.0 MICROORGANISMS

Microbial cells are produced for two main applications, (a) as a source of protein for animal or human food, as source of microbial Protein) or (b) for use as commercial inoculums in food fermentations and for agriculture and waste treatment. As a commodity, microbial biomass must be competitive with commercial animal and plant proteins, in terms of process and nutritional value and must conform to human and animal food safety requirements. Productivity, yield and selling price are the major factors affecting the economics of biomass protein production. Microbial inoculants, which are used as a process aid, generally have a higher value. Four types of microorganisms are generally used to produce biomass: bacteria, yeasts, fungi and algae, but the choice of a microorganism for whey utilization depends on the ability of the organism to ferment the lactose as carbon source and other criteria such as:

• Nutritional: energy value, protein content, amino acid balance;

• Technological: type of culture, nutritional requirements, type of separation;

• Toxicological.

The ideal microorganism for microbial biomass production should possess the following characteristics:

• High specific growth rate (m) and biomass yield (Yx/s) to ensure high productivity

• High affinity for the substrate, the strain must not be affected by whey proteins

• The strain must be suited to continuous culture

• The strain must be good tolerance to temperature and pH.

• Large cell size and uniform morphology to aid cell separation and concentration.

• Low nutritional requirements, i.e., few indispensable growth factors;

• Ability to develop high cell density;

• Stability during multiplication;

• Capacity for genetic modification;

In addition, it should have a balanced protein and lipid composition. It must have a low nucleic acid content, good digestibility and be non-toxic.

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3.1 Yeasts

Yeasts were the first microorganisms known, the best studied and generally best accepted by consumers. Yeasts are rarely toxic or pathogenic and can be used in human diets. Although their protein content rarely exceeds 60%, their concentration in essential amino acids such as lysine (6 to 9%), tryptophan and threonine is satisfactory. In contrast, they contain small amounts of the sulfur- containing amino acids methionine and cysteine. They are also rich in vitamins (B group), and their nucleic acid content ranges from 4 to 10%. They are larger than bacteria, facilitating separation. They can be used in a raw state. However, their specific growth rate is relatively slow (generation time 2 to 5 hours).Example: Candida melivii, C. oleophila, C. parapsilosis, C. tropicalis, Hansenula sp., Pichia polymorpha, Saccharomyces uvarum, Schizosaccharomyces pombe.

3.2 Bacteria

The specific growth rate and biomass yield of bacteria are greater than those of the other categories of microorganisms. Total protein content may reach 80%. Their amino acid profile is balanced and their sulfur-containing amino acid and lysine concentrations are high. In contrast, their nucleic acid content (10 to 16 %) is greater than that of yeasts. A limited number of bacterial species can be used in foodstuffs as many are pathogenic. In addition, separation is difficult because of their small size. Bacillus amyloiquefaciens, B. cereus, B. firmus, B. lentus, B. macerans, B. megaterium, B. subtilis have been found suitable organisms for microbial biomass production.

3.4 Fungi

The use of fungi as biomass is relatively new. They are more conventionally used for producing enzymes, organic acids and antibiotics. Their generation times (5 to 12 hours) are distinctly longer than those of yeasts and bacteria. This is generally an apparent generation time as they grow through elongation of mycelium; growth is not really exponential. Their protein content (50 %) is often smaller than that of yeasts and bacteria, and they are deficient in sulfur-containing amino acids. There are also problems of wall digestibility. However, the nucleic acid content is low (3 to5%).The principal merits of fungi are their ability to use a large number of complex growth substances such as cellulose and starch and easy recovery by simple filtration, reducing production costs. The fungi Aspergillus carbonarius, A. flavus, A. glaucus, A. ostianus, A. parasiticum, A. terreus, Neurospora (-), Neurospora (+) are able to grow in whey medium for biomass production (Omar and Sabry, 1991).

4.0 TYPES OF BIOMASS PRODUCTION ON WHEY

The whey as carbon substrate is used by bacteria and yeast for microbial biomass production. The lactose fermenting organisms such as Kluyveromyces fragilis, Candida intermedia and Lactobacillus are able to grow in whey. Saccaromyces cerevisiae is non lactose fermenting yeast hence; enzymatically hydrolyzed whey is preferred for S. cerevisiae. Two types of yeast biomass has been produced from whey are baker’s yeast and Single cell protein (Vij and Gandhi 1993).

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4.1 Baker’s yeast

Two processes have been developed for the production of baker’s yeast to overcome the limitation of S. cerevisiae not being able to utilize lactose. In the first process, the lactose is hydrolyzed using β-galactosidase, and the glucose and galactose are consumed simultaneously by the yeast in fed batch or continuous culture. The second process utilizes a two stage fermentation system. In the initial stage, lactic acid bacteria convert lactose to lactate and this is consumed in the subsequent stage by the yeast. Although the baker’s yeast so produced appear comparable in quality to that from conventional processes, only very limited quantities are produced by these methods.

4.2 Microbial biomass protein

Whole milk whey or deproteinised whey is a carbohydrate source, for biomass production are usually insufficient substrate, seasonal supply variations and its high water content (>90%) which makes transport prohibitively expensive. While most organisms do not grow on lactose as a carbon source, strains of the yeast Kluyveromyces marxianus readily grow on lactose. The key criteria used in selecting suitable strains for biomass production should consider these parameters - High specific growth rates, productivity and yields on a given substrate, pH and temperature tolerance, aeration requirements and foaming characteristics, growth morphology in the reactor, safety and acceptability – non pathogenic, absence of toxins, ease of recovery, Protein, RNA and nutritional composition of the product, structural properties of the final product. Bacteria, in general, have faster growth rates than fungi and grow at higher temperatures, thereby reducing fermenter cooling requirements. Bacterial and yeast fermentations are easier to aerate. Recovery of bacteria and yeast require the use of sedimentation techniques and centrifugation. Bacteria, in general produce a more favorable protein composition than yeast or fungi. Protein content in bacterial can range from 60-65% whereas fungi selected for biomass production and yeast have protein contents in the range of 33-45%. However, associated with the higher bacterial protein levels is a much higher level of nutritionally undesirable RNA content of 15-25% Microorganisms involved in biomass production must be safe and acceptable for use in food. Organisms should be stable genetically so that the strain with optimal biochemical and physiological characteristics may be maintained in the process through many hundreds of generations. 5.0 PRODUCTION OF MICROBIAL BIOMASS ON WHEY

Large scale fermenters are required for high biomass productivity requires high oxygen transfer rates which promote high respiration rates which in turn increase metabolic heat production and the need for an efficient cooling system (Moeini et al. 2004) In order to maximize fermentation productivity it is essential to operate continuous fermentation processes. Different processes have adopted different fermenter designs with respect to process requirements.

The biomass production processes essentially contain the same basic stages irrespective of the carbon substrate or microorganism used.

5.1. Medium preparation

The main carbon source may require physical or chemical pretreatment prior to use. The substrates are often deproteinized / hydrolyzed before being incorporated with sources of nitrogen, phosphorus and other essential nutrients.

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5.2. Fermentation

The fermentation may be aseptic or run as a ‘clean’ operation depending upon the particular objectives. Continuous fermentations are generally used, which are operated at dose to the organism’s maximum growth rate to fully exploit the superior productivity of continuous culture.

5.3 Separation and downstream processing.

The cells are separated from the spent medium by filtration or centrifugation and may be processed in order to reduce the level of nucleic acids. This often involves a thermal shock to inactivate cellular proteases. RNase activity is retained and degrades RNA to nucleotides that diffuse out of the cells. Depending upon the growth medium used, further purification may be required, such as a solvent wash, prior to pasteurization, dehydration and packaging.

Bel Fromageries process is most commonly used for biomass production by Kluyveromyces marxianus from whey. The worldwide dairy industry generates over 80 million tones of whey each year. This byproduct of cheese manufacturing has a high pollution load with a chemical oxygen demand (COD) of 60 g oxygen per liter. Consequently, it usually has to be disposed of at a high capital cost to the dairy industry. Whey contains approximately 45g/L lactose and 10g/Lprotein. It is particularly suitable for the production of biomass using lactose utilizing yeast. The Bel process was developed with the aim of reducing the pollution load of dairy industry waste, while simultaneously producing a marketable protein product. A number of plants are operated using Kluyveomyces lactis or K. marxianus (formerly K. fragilis) to produce a protein, Protibel, which is used for both human and animal consumption. These processes initially involve whey pasteurization, during which 75% of whey proteins are precipitated. The lactose concentration is adjusted to 34g/L. Whey is limiting in nitrogen source for yeast growth, so ammonium salts are added to maintain a high nitrogen contents and growth rate and trace metals (Fe, Cu, Mn, and Zn) may also be added. This supplemented whey is introduced into a 22 m3 continuous fermenter, maintained at 38°C and pH 3.5, with an aeration rate of 1700 m3/h. The yeasts utilize the lactose and attain biomass concentrations of 25 g/L, with a biomass yield of 0.45-0.55g/g lactose. Yeast cells are recovered by centrifugation, then resuspended in water, recentrifuged finally roller-dried to 95% solids. Levels of residual sugar remaining in the spent medium are less than 1g/l. 6. CHARACTERISTICS OF MICROBIAL BIOMASS

The food value of microorganisms is directly related to their protein and amino acid composition and their lipid, vitamin and nucleic acid contents. Various analyses must also be performed.

Overall analysis: water, lipid, protein, fiber and mineral contents; • Lipid analysis: proportions of fatty acids, sterols and phospholipids; • Analysis of nitrogen compounds: total nitrogen, amino acid profile, nucleic acid

nitrogen, purine and pyrimidine base, quantification of RNA and DNA; • Analysis of minerals: major elements (Na, K, Mg, Ca, Cl) and trace elements (Mn, Zn, Cu,

Fe, Co, Mo, As, Pb, Hg);

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• Analysis of carbohydrates • Analysis of vitamins

7. COMPOSITION

The compositions of microorganisms used for biomass production for bacteria is 50-85% protein and 10-16% nucleic acid and for yeast is 45-55% protein and 5-12% nucleic acid. Important aspects of the quality of the biomass produced are as follows:

• Nutritional value of the product, • Safety of the product, • Production of protein concentrate free of nucleic acid and toxic substances.

Three parameters are used to establish the nutrient value of biomass: digestibility, biological value and protein efficiency ratio. Digestibility (D) is the percentage of total nitrogen consumed in relation to the nitrogen in the food ration: Dp[(IPF)/I ] 100. The total quantity of microbial protein ingested by animals is measured and the nitrogen content (I ) is analyzed. Nitrogen contents in feces (F) and urine (U) are collected and measured. Digestibility of bacteria is 83 to 88% and that of yeasts ranges from 88 to 96%). Biological value (BV) is the percentage of total nitrogen assimilated that is retained by the body, taking into account the simultaneous loss of endogenous nitrogen through urinary excretion: BVp[(IP(FcU))/ (IPF)] 100.The protein efficiency ratio (PER) is the proportion of nitrogen retained by the animal in comparison with reference proteins, e.g., egg albumin. Numerous nutritional and toxicological tests on animals are obligatory before any use in human or animal foodstuffs.

8. BIOMASS SAFETY AND QUALITY

Microbial biomass has applications in animal feed, human food and as functional protein concentrates. Some bacterial biomass protein has amino acid profiles similar to animal/plant protein. Yeast, fungal and Soya bean proteins tend to be deficient in methionine. Ingestion of RNA from non-conventional sources should be limited to 50g per day. Ingestion of purine compounds arising from RNA breakdown, leads to increased plasma levels of uric acid, which can cause gout and kidney stones. High content of nucleic acids causes no problems to animals since uric acid is converted to allantoin which is readily excreted in urine. Nucleic acid removal is not necessary from animal feeds but is from human foods. A temperature hold at 64C inactivates fungal proteases and allows RNA-ases to hydrolyse RNA with release of nucleotides from cell to culture broth. A 30 min stand at 64C reduces intracellular RNA levels in Fusarium graminearum from 80mg/g to 2mg/g.

9. ECONOMICS OF BIOMASS PRODUCTION

The initially, reason for biomass production was to produce, at low cost, high value SCP from abundantly available cheap substrate for addition to animal feed. The intention was to replace imported protein additives such as soybean meal. Agricultural crops, the major competitor to single cell protein for animal feed, manifest a remarkable ability to respond to market forces and maintain price stability. By producing human protein supplements, the end value of the product can be appreciated; however the production process has to utilize more conventional and costly substrates for production (glucose). These products take advantage of the high fibre content

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in certain fungi to produce a product that is a good meat analogue. This product is also low in sodium and fat. , the economics of production can be improved by either increasing the value of the product or reducing the production costs through:

• Use of cheaper substrates such as whey • Improvements in the efficiency of the organism; • enhanced nutritional value/composition of the microbial protein; • Marketing the protein as a premium product for human rather than animal

consumption; • Production of other valuable byproducts, i.e. development of a multi product process • Lowering downstream processing costs, e.g. by reducing endogenous RNA levels

10 REFERENCES Ben-Hassan, R.M. and Ghaly, A.E. Continuous propagation of Kluyveromyces fragilis in cheese whey for pollution

potential reduction. Applied Biochemistry and Biotechnology, 1994, vol. 47, p. 89-105. Caballero, R.; Olguin, P.; Cruz-Guerrero, A.; Gallardo, F.; Garcia-Garibay, M. And Gómez-Ruiz, C. Evaluation of

Kluyveromyces marxianus as baker’s yeast. Food Research International, 1995, vol. 28, no. 1, p. 37-41. Carlotti, A.; Jacob, F.; Perrier, J. And Poncet, S. Yeast production from crude sweet whey by a mixed culture of

Candida kefyr LY496 and Candida valida Casella, 2003, Lactose hydrolysis from whey permeate by free and immobilised lactase, Enzyme engineering XVII,

Santa Fe, New Mexico,USA González Siso, M.I., 1996, The biotechnological utilization of cheese whey: a review, Bioresour. Technol. 57, 1-11. Mahoney, R. R., 1998. Galactosyl-oligosaccharide formation during lactose hydrolysis: a review. Food Chem. 63, 2,

147-154. Mansour, M.H.; Ghaly, A.E; Ben-Hassan, R.M. and Nassar, M.A1993, Modelling batch production of single cell

protein from cheese whey. I: Kluyveromyces fragilis growth. Applied Biochemistry and Biotechnology,. 43, ( 1), 1-14.

Mawson, A.J. Bioconversions for whey utilization and waste abatement. Bioresource Technology, 1994, vol. 47, no. 3, p. 195-203.

Moeini H Nahvi I Tavassoli M 2004, Improvement of SCP production and BOD removal of whey with mixed yeast culture. Process Biotechnology 7 ( 3) ,15

Moresi, M.; Patete, M. And Trunfio, A. Scaling-up of a batch whey fermentation by Kluyveromyces fragilis. Applied Microbiology and Biotechnology, 1989, vol. 31, p. 495-501.

Omar S. and Sabry,S. 1991, microbial biomass and protein production from whey. Journal of Islamic Academy of Sciences 4:2, 170-172.

Povolo S. and S. Casella, 2003, Bacterial production of PHA from lactose cheese whey permeate, Macromolecular Symposium 197, 1-9.

Speer, E. Milk and dairy product technology. New York: Marcel Dekker, 1998. Vij .S and Gandhi D.N.1993, Whey- an alternative substrater for the production of baker’s yeast. Indian Food

industry !2 (5) 41-43. Yang, S.T. and E.M. Silva, 1995, Novel Products and new technologies for use of a familiar carbohydrate, milk

lactose, J. Dairy Sci. 78, 2541-2562.

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Dr. A.A. Patel Head

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

The nutritional value of by-products from milk is one of very strong reasons for their utilization in food applications. One such application is in dairy-product analogues or substitutes. The constituents of by-products responsible for their nutritional virtues also provide some of desirable sensory attributes to these products, where non-dairy ingredients tend impart atypical sensory characteristics, particularly flavour. For instance, Childs et al. (2007) noted that meal replacement bars (a.k.a. ‘energy bars’ or ‘food bars’) and beverages made with whey protein displayed sweet aromatic and vanillin flavour notes whereas those with soy protein had cereal/grainy flavours, the former scoring higher in consumer acceptance studies. Further, certain health attributes of whey proteins (e.g. their greater effect on immune response as reported by Parker & Goodrum,1990) make whey and whey-protein products particularly favourable for use in various non-dairy foods including dairy analogues. Incidentally, whey proteins have found application in imitation products as fat re-placer, or the so-called 'fat mimetics' besides their role as a supplementary protein, which makes them particularly relevant to vegetable protein-based dairy analogues.

Dairy analogues, also called ‘Imitation dairy products’ are products which, in appearance, flavour and texture, are similar to their regular dairy counterparts but comprise one or more non-dairy ingredients. Often serving as low-cost substitutes for genuine milk products, dairy analogues are also intended to provide alternatives which are not allergenic, or are free from cholesterol and low in saturated fat. According to IDF (Anon., 1989), an ‘imitation product’ is a substitute for milk or for a milk product which in general composition, appearance, characteristics and intended use is similar to milk or the milk product, and in which the milk solids constituents are wholly or partly replaced with non-milk ingredient(s). Even the products prepared from casein or caseinates are termed imitation products. Similarly, products containing whey protein products, e.g. whey protein concentrate (WPC) as the only dairy ingredients can be considered as imitation products.

The ingredients of dairy analogues may be wide ranging, but vegetable fat and/or protein products are commonly employed to replace the corresponding milk constituents. Among protein products, soy protein concentrates or isolates, and soy flour are frequently used in dairy analogues primarily on account of heir nutritional superiority to other vege-table protein products. Like whey proteins, the healthfulness of soy proteins realized in the last two decades has made them choice ingredients in various food applications.

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2. WHY WHEY PROTEINS IN COMBINATION WITH VEGETABLE PROTEINS ?

In terms of nutritive value, whey protein is fairly close to egg protein, ahead of most other first-class proteins, its biological value being 104 (vs. 100 for egg protein), protein efficiency ratio (PER) 3.6 (vs. 3.8) and net protein utilisation (NPU) 92 (vs. 94) (Renner, 1990). Their richness in the essential amino acids such as lysine, methionine and cystine (Swaisgood, 1995) render whey proteins especially suitable for combining with vegetable proteins in order to enhance the nutritive value of the latter in which the said amino acids are the 'limiting' ones. Thus, the use of whey protein products in combination with soy and cereal ingredients in dairy analogues can synergistically upgrade the protein quality of these products (Puranik and Rao, 1996). The healthfulness of the two proteins also favour combining the two in various foods; studies have shown that diets containing soy protein isolate could protect against chemically induced tumours in rats, and whey protein was at least twice as effective as soy protein in reducing both tumour incidence and multiplicity (Hakkak et al., 2000). As a further example, soy proteins have been demonstrated to have an antioxidant effect in humans (Bazzoli et al., 2002). It is, therefore, not surprising that mixtures of whey solids and soy protein isolates/concentrates are extensively used to replace non-fat dry milk in many food applications, and account for an appreciable quantum of whey solids utilization. 3. WHEY-BASED MILK-LIKE DRINKS/BEVERAGES

Most frequently, the milk-like beverages are flavoured and sweetened ones, i.e., flavoured milk analogues, although occasional reports refer to plain, milk-like beverages as, for example, 'La Colina R', a Venezuelan dried milk substitute obtained from whey, soy protein isolate and vegetable oil (Cioccia et al., 1995).

Whey solids have been used in various forms to produce milk-like beverages. Sweet whey or rennet whey is often preferred to acid whey because of its bland flavour. Rennet whey, however, must be so heat processed as to inactivate the residual enzyme in order to eliminate the problem of bitterness (Klostermeyer, 1988). The use of acid whey necessitates neutralization.

Recently, Nakamura et al. (2004) studied the interactions between milk proteins and certain non-protein soy components in oil-in-water (O/W) emulsions and observed for the first time interactions occurring between soy soluble polysaccharides (SSPS) and caseinate or whey protein isolate, the latter showing an electrostatic interaction. The competitive adsorption of SSPS in O/W emulsions could be significant in certain soy beverages containing milk proteins. 3.1 Vegetable Protein-Based Imitation Milks

Vegetable protein products, primarily soy protein products, have been blended with whey/whey solids to produce flavoured milk-like beverages. Ingredients and processing aspects of whey-soy beverages have been reviewed by Patil et al. (1984). Workers at the Philadelphia Centre of the USDA found that sweet cheese whey was most suitable for combining with full-fat soy flour for the production of nutritious and palatable drinks, although cottage cheese whey could be successfully used in citrus-flavoured beverages. Lactose hydrolysed whey was found to improve the beverage flavour. Spray drying has been frequently used to obtain a ready-to-reconstitute powder, but vacuum-shelf drying and foam drying have also been gainfully employed.

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A high-protein beverage process developed at this Institute (Patil and Gupta, 1981) utilized whole soybeans and cheese whey: blanched beans were ground in lactose-reduced condensed whey prior to two-stage homogenisation, pasteurization and spray drying. The methionine- and vitamin-fortified flavoured beverage had a high PER (3.1) could be stored well in polyethylene bags under ambient conditions. Pandya (1988) employed acid whey from paneer-making as a medium for grinding blanched soybeans for production of pineapple flavoured beverage, which was sterilized in-bottle for extended shelf-life at room temperature.

Occasionally, other protein products, such as peanut and sesame cake have also been used in sweet or acid whey-based milk-like beverages (Sienkiewicz and Riedel, 1990). 3.2 Beverages Without Vegetable Proteins

Application of sweet or neutralized whey as a diluent in flavoured milk drinks, in some cases at commercial levels (Odendahl et al., 1983) has been reported. Aseptically canned concentrated chocolate milk-like product (35% TS) was produced by Edmondson et al. (1968). Presence of casein in such whey-milk systems makes it possible to use stabilizers such as k-carrageenan to improve the colloidal stability (Driessen and van den Berg, 1990).

While cloudiness may be undesirable in many whey beverages, it is desired in milk-like ones. Rajesh Kumar (1982) dispersed whey protein precipitate from (acidified rennet whey) in whey neutralized to pH 7.0 followed by homogenization to produce a beverage base suitable for the preparation of a flavoured milk analogue. Lactose-reduced whey powder could also be used for producing a similar drink (Rajesh Kumar et al., 1987).

Sienkiewicz and Riedel (1990) reported that a commercial vitaminized milk-like drink, 'Waymil', was obtained by incorporating vegetable oils, hydrocolloids and emulsifiers into neutralized whey. In a variation of this process liquid, condensed or dried whey was used together with cream. 4. YOGHURT-TYPE PRODUCTS

As for whey-extended milk drinks, yoghurt and similar fermented products, e.g., stirred yoghurt (Otero et al., 1995) have also been prepared by part replacement of milk solids with whey solids. Particularly relevant in this regard are the attempts to substitute the skim milk solids normally added to the yoghurt milk with delactosed whey powder, mixtures of whey powder and caseinate or WPC. The 'all-whey' beverage base developed by Rajesh Kumar (1982) could also be cultured into a ‘lassi-like’ product containing 3.0% protein.

A more important role is played by whey solids in vegetable protein-based imitation yoghurt owing to the presence of fermentable carbohydrate, lactose in whey (Patel et al., 1980). Since soymilk is particularly deficient in simple sugars, addition of lactose, glucose etc., or liquid or dried whey has been found to greatly improve its suitability by lactic fermented products. Soymilk fortified with cheese whey solids (2%) and sucrose (4-5%) could be fermented into a yoghurt-like product using Lactobacillus acidophilus (Kanda et al., 1976). The improved flavour of cultured whey-soy milk (Patel and Gupta, 1982) can be particularly valuable in enhancing the palatability of soy-based yoghurt analogues. Rasmy et al. (2000) observed that ‘soyoghurt’ fortified with Swiss cheese whey and sodium caseinate showed higher viscosity than that of the unfortified product; the whey-fortified product also exhibited a lower syneresis.

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Probiotic beverages without undesirable flavours associated with soybeans could be obtained by fermenting mixtures of cheese whey and soy preparation with bifidobacteria, lactobacilli and Streptococcus thermophilus, followed by fruit supplementation (Dalev et al., 2006). Karleskind et al. (1991), however, earlier reported that partial replacement of isolated soy protein with fresh cheese whey or whey protein isolate in soy-based yoghurt imparted physical instability to the product. A whey-soy yoghurt could be fortified with calcium in the form of slats such as calcium carbonate, citrate, phosphate, gluconate or lactate (600 mg/lit) without adversely affecting sensory properties of the product (Umbelino et al., 2001).

Maity and Misra (2001) prepared ‘acido soy milk’, a soy-based fermented beverage obtained using Lactobacillus acidophilus, S. thermophilus, and L. delbrueckii subsp. bulgaricus as starters, and reported that the antibacterial activity of the product against Escherichia coli, Staphylococcus aureus and Shigella dysentariae was greater when whey was used than without whey. It was also noted that use of a mixed-strain starter comprising the three starter organisms resulted in a greater antibacterial activity than any single strain. 5. IMITATION CHEESE

In recent times, a considerable interest has been evinced in the use of particulated whey proteins (e.g. Simplesse) as a fat substitute in imitation cheese, as discussed elsewhere in this volume. However, equally important is application of whey solids as a protein substitute in various imitation cheeses.

Based on the meltability and stringiness of imitation Mozzarella cheese, Nishiya (1991) concluded that 25% of the casein could be replaced by WPC. Hsieh et al. (1993) observed that like caseinate, egg white and soy protein, whey protein also altered the viscoelastic properties of Mozzarella cheese. Soy protein isolate induced the strongest gel network structure in the cheese.

Pereira et al. (1992) standardised the process for the manufacture of a fresh-type soft cheese analogue from soy extract (89%) mixed with whey (6%) and cow milk (5%), coagulating the mixture by lactic fermentation and calcium sulphate. Several Russian cheese varieties including cream-type cheese of acceptable quality have also been obtained from vegetable oil emulsified in reconstituted whey (Snegireva and Sokolov, 1993). The moisture retaining capacity of these cheeses was higher than those made by the conventional methods.

Tofu, a product obtained by coagulation of soymilk, is a typtical paneer analogue. Soy flour (4%) dispersed, in whey, or soymilk mixed with whey or WPC (1:1) could be coagulated to produce tofu (Wu and Peng, 1983). An increasing level of WPC (3-6%) added to soymilk increased the product's stiffness. 6. MARGARINE, FAT SPREADS AND OTHER IMITATION PRODUCTS

Margarine, an analogue of table butter, is a water-in-oil emulsion. Though the aqueous phase in normal margarine is relatively small (20%), it is important for the product's physical properties. The solubility characteristic of un-denatured whey proteins at a low pH makes them valuable as a functional ingredient in margarine. Thus, the aqueous phase of margarine is often derived from delactosed whey or whey protein products. According to Morr (1990), nearly 1% of total whey powder was used by the US margarine industry in 1987.

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Camejo et al. (2006) prepared a butter-like spread enriched with protein by using whey powder, whole milk powder, soy protein isolate or soy meal, but the formulations containing the last two protein products were found sensorily acceptable. In a 'non-spread' application of margarine, with lipoprotein concentrate obtained from an oilseed such as rapeseed has been claimed to be useful as a margarine substitute in bread and other bakery products (Paschenko, 1991).

In low-fat spreads as also in reduced-fat margarine, the non-fat component is even more critical with respect to the emulsion stability and product texture as well as flavour. Spurgeon et al. (1973) observed that dried whey used in a 40% fat dairy spread resulted in a soft body, but lactose crystallization adversely affected the texture. Obviously, lactose-reduced or delactosed whey products should be more suitable in such products. Also, liquid whey would not provide the required level of non-fat solids in low-fat spreads as observed by Prajapati (1988), a weak and leaky body being one of the defects in such products.

Razavi et al. (2001) found that partial replacement of SNF with sweet cheese whey, buttermilk or skim milk in soft-serve soy ice cream resulted in decreased protein content, relative viscosity as well as melting resistance; overrun in the product containing whey or buttermilk was lower than that of the product with skim milk or control. Whey solids as whey powder, WPC or lactose hydrolysed syrup have been frequently used for partial substitution of milk SNF in ice cream and frozen desserts including water ices. Recently, Del-C-Cabrera et al. (1995) noted that 20% of milk solids in the Cuban dessert, 'Chocoleche' could be replaced with neutralized acid whey solids. Coffee whiteners especially of the non-dairy type offer yet another avenue for utilization of whey solids, WPC in particular. 7. CONCLUSION

While the high nutritional status of whey proteins makes a strong case for their use in dairy analogues, their increasing importance on health grounds is even a greater reason for utilization of whey and whey-protein products in a variety of dairy-products substitutes. Since the health attributes of soy are also being realized more and more in recent times, the combination of the two food proteins in dairy analogues is quite natural as seen from the trends during the last two decades. The most noteworthy application of whey solids is in vegetable protein-based products because of the remarkable improvement in the protein quality of these otherwise low-quality products. Also, the problem of poor culture growth-promoting properties of vegetable protein extracts, e.g., soymilk, employed as a base for imitation yoghurt can largely be overcome by incorporating whey solids which provide the fermentable carbohydrate, lactose. Whey solids can serve as a valuable ingredient for many other dairy analogues such as milk-like beverages, margarine, cheese etc. and in some cases their application has been commercially successful, although much of the potential needs to be exploited by the food industry.

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8. REFERENCES Anon 1989. The Present and Future Importance of Imitation Dairy Products. IDF Bull. No. 239, p. 19. Intern. Dairy

Fed., Brussels. Bazzoli, D.L., Hill, S. and DiSilvestro, R.A. 2002. Soy protein antioxidant action in active, young adult women. Nutr.

Res. 22(7) : 807-815. Camejo, J., Rodriguez, T., Garcia, A., Bencomo, E., M’Boumba, A. Seivanes, A. and Fernandez, A. 2006. Spreadable product enriched with protein. Ciencia y Tecnologia de Alimentos, 16(3):49-54. Childs, J.L., Yates, M.D. and Drake, M.A. 2007. Sensory properties of meal replacement bars and beverages made from whey and soy proteins. J. Food Sci., 72(6) : S425-S434. Cioccia, A.M., Pinero, D., Carias, D., Brito, O., Waggle, D.H. and Hevia, P. 1995. Nutritional evaluation of the soy-

whey milk analog, La Colina. Plant Foods Human Nutr. 47(2): 139. Dalev, D., Bielecka, M., Ziajka, S. and Lamparski, G. 2006. Sensory quality of new probiotic beverages based on

cheese whey and soy preparation. Polish J. Food Nutr. Sci., 15(Suppl.1): 71-77. Del-C-Cabrera, M., Martinez, G., Espinosa, B., Ortega, O., Real, E. and Cabrera, M. del, C. 1995. Alimentaria

32(260): 107. Drissen, F.M. and van den Berg, M.G. 1990. New developments in whey drinks. IDF Bull., 250: 11. Edmondson, L.F., Dvants, J.K. and Douglas, F.W. Jr. 1968. Utilization of whey in sterilized milk products. J. Dairy

Sci. 151: 931. Hakkak, R., Korourian, S., Shelnutt, S.R., Lensing, S.,Ronis, M.J.J. and Badger, T.M. 2000. Diets containing whey

proteins or soy protein isolate protect against induced mammary tumours in female rats. Cancer Epidemiol Biomarkers and Prevention, 9(1) : 113-117.

Hsieh, Y.L., Yun, J.J. and Rao, M.A. 1993. Rheological properties of Mozzarella cheese filled with dairy, egg, soy proteins and gelatin. J. Food Sci. 58: 1001.

Kanda, H., Wang, H.L., Hesseltine, C.W. and Warner, K. 1976. Yoghurt production by Lactobacillus fermentation of soy milk. Process Biochem. 11(4): 23.

Karleskind, D., Laye, I., Halpin, E. and Morr, C.V. 1991. Improving acid production in soy-based yoghurt by adding cheese whey proteins and mineral salts. J. Food Sci., 56(4): 999-1001.

Klostermeyer, H. 1988. Proteolysis in milk and whey. In: Inst. f. milchwist. Qualilactsfragen modene Labortechnik. Verlag Th. Mann, Gelsenkirehin-Buer, Germany. P. 119.

Maity, T.K. and Misra, A.K. 2001. Antibacterial activity of soy-based fermented beverage (acido soy milk) using L.acidophilus, S. thermophilus and L. delbrueckii Subsp. bulgaricus as starters. J. Interacadicia, 5(2): 232-235.

Morr, C.V. 1990. Whey Utilization. In: Whey and Lactose Processing, by J.G.Zadow (Ed.) P.133, Elsevier Applied Science, London.

Nakamura, A., Maeda, H. and Corredig, M. 2004. Competitive adsorption of soy soluble polysaccharides in oil-in-water emulsions. Food Res. Intern. 37(8): 823-831.

Nishiya, T. 1991. Study on the functional properties of imitation cheese. Reports of Research Laboratory, Snow Brand Milk Products Co., No. 95: 1.

Odendahl, W.A. 1983. The nutritional value and utilization of whey milk. South African J. Dairy Technol. 15: 3. Otero, M., Rodriquez, T., Camejo, J. and Eardoso, F. 1995. Cultured milk drink. Alimentaria 32(260): 93. Pandya, A.J. 1988. Technological studies on the manufacture of soymilk. Ph.D. Thesis, Kurukshetra Univ., NDRI,

Karnal. Parker, N.T. and Goodrum, K.J. 1990. A comparison of casein, lactalbumin and soy protein effect on the immune

response to a T-cell antigen. Nutr. Res., 10(7): 781-792. Paschenko, I.P. 1991. Biotechnological aspects of the use of a lipoprotein concentrate and albumin milk. Pishchevaya-

Promyshlennost No. 10: 60. Patel, A.A. and Gupta, S.K. 1982. Fermentation of blanched been soymilk with lactic cultures. J. Food Protect. 45:

620. Patel, A.A., Waghmare, W.M. and Gupta, S.K. 1980. Lactic fermentation of soymilk: A review. Process Biochem.,

15(5): 9. Patil, G.R. 1981. Manufacture of high-protein beverage from andcheese whey soybean and soybean. Ph.D. Thesis,

Kurukshetra Univ., NDRI, Karnal Patil, G.R. 1982. High protein beverage from cheese whey and soybean. II. Storage stability. Indian J. Dairy Sci. 35:

418.

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Patil, G.R., Patel, A.A., Gupta, S.K. and Rajor, R.B. 1984. Manufacture of whey-soy beaverages: A review. J. Food Sci. Technol., 21: 340.

Prajapati, P.S. 1988. Technology of low-fat spread manufacture. Ph.D. Thesis, P. 193, Kurukshetra Univ., NDRI, Karnal.

Pereira, G.V., Antunes, I.A.F. and Ferreira-da-Silva, R.S. dos-S. 1992. Development and characterization of a cheese analogue containing aqueous soya extract (soya milk), whey and cow milk. Arquivos-de-Biologia-e-Tecnologia, 35(1): 99.

Puranik, D.B. and Rao, H.G.R. 1996. Potentiality of whey protein as a nutritional ingredient. Indian Dairyman, 48(11): 17.

Rajesh Kumar (1982). Studies on the fermentation of protein-rich beverages from cheese whey. M.Sc. diss., Kurukshetra Univ., NDRI, Karnal.

Rajesh Kumar, Patil, G.R. and Rajor, R.B. 1987. An economical approach to utilize lactose-reduced whey powder for the manufacture of soft drink type beverage. Indian Dairyland, 1:14.

Rasmy, N.M.H., Ibrahim, M.T., Basyony, A.E. and Saad, G.M.K. 2000. Annals Agril. Sci. – Moshtohor (Egypt) 38(3): 1539-56.

Razavi, S.M.A., Habibi, M.B. and Nsyebzadeh, K. 2001. Effect of dairy substituents and stabilizers on chemical and physical properties of soy ice cream (Paravin). Iranian J. Agri. Sci., 32(3):615-624.

Renner, E. 1990. Nutritional aspects. In: Whey and Lactose Processing. J.G. Zadow (ed.), Elsevier Appl. Sci., London, P. 449.

Sienkiewicz, T. and Riedel, C.L. 1990. Whey and whey utilization, P. 164. Verlag Th. Mann, Gehenbirohen-Buer, Germany.

Snegireva, T.A. and Sokolov, A.M. 193. Use of food emulsions in the manufacture of processed cheeses. Molochnaya Promyshlennost, No. 5-6: 17.

Spurgeon, K.R., Seas, S.W. and Dalaly, B.K. 1973. Effect of non-fat milk solids and stabilizers on body, texture and water retention in low fat dairy spreads. Food Prod. Development 7(3): 34.

Swaisgood, H.E. 1995. Protein and amino acid composition of bovine milk. In: Handbook of Milk Composition, R.G. Jonsen (ed.), Academic Press, San Diego (USA), P. 464.

Umbelino, D.C., Rossi, E.A.,cardello, HMAB, and Lepera, J.S. 2001. Sensorial and technological aspects of calcium enrichment of soy-whey-yoghurt. Ciencia e Tecnologia de Alimentos, 21(3): 267-280.

Wu, H.M. and Peng, A.C. 1983. Production and textural properties of soy-cheese whey curd. J. Food Sci., 48: 1562.

6

SECTION - VI

GHEE RESIDUE GHEE RESIDUE

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Dr. B. B. Verma and Mr. P. Narender Raju Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

Ghee residue is a brownish solid mass obtained as a byproduct during ghee manufacture. It contains considerable amounts of milk fat, protein and minerals. Depending on the intensity of the heat treatment used during the ghee manufacture, colour of ghee-residue (GR) may vary from light to dark brown. It has smooth to granular texture with glossy exterior due to the presence of excessive free fat. According to one estimate about 33 percent of total milk produced in the country is diverted for ghee making (Dairy India, 2007). Taking an average yield of GR as one-tenth the quantity of ghee produced, at present level of ghee production (30.4 million tones), the bulk of GR produced works out to more than 3 million tonnes per annum.

2. CHEMICAL COMPOSITION

A look at the chemical composition of GR obtained from various sources (Table 1) will give an idea of the huge quantity of nutrients in terms of fat, protein and mineral that goes in GR. The yield of GR varies with the variation in the serum solid content of the raw materials used for ghee manufacture.

Table 1. Chemical composition and yield of ghee-residue (Hand pressed)

Chemical Composition (%) Source of Ghee-residue

Moisture Fat Protein Lactose Ash Yield

(Kg/100 Kg) Makkhan (indigenous butter) 13.4 33.4 32.8 15.4 5.2 1.6

Creamery butter (unsalted) 5.7 65.0 25.5 Trace 3.8 1.2 Sweet cream 4.1 63.2 18.0 12.3 2.4 7.7 Sour cream 8.0 38.8 41.6 7.3 4.3 5.1 Washed sweet cream 1.7 80.8 16.2 Trace 1.3 3.5

3. TREATMENT AND PROCESSING OF GHEE-RESIDUE

Ghee-residue has soft and smooth body but gets progressively hardened during storage. The change in the textural characteristics is much faster particularly during the first 15 days and by the end of a month it becomes hard and gritty. In order to eliminate the undesirable characteristics it is necessary to process it so as to yield a soft and smooth texture essential for edible preparations. Before subjecting the residue to any-treatment, its lumps are broken and then pulverized by passing through 40 mesh sieve. A number of processing treatments for GR as suggested by Prahlad (1954) are given in Table-2. All the treatments make the processed

GHEE-RESIDUE: PROCESSING, PROPERTIES AND UTILIZATION GHEE-RESIDUE: PROCESSING,

PROPERTIES AND UTILIZATION

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Table 2. Comparison of chemical composition of ghee-residue subjected to various processing treatments.

Particulars Treatment I

Treatment II

Treatment III

TreatmentIV

Treatment V

TreatmentVI

Before After Before After Before After After Before After After Acidity (ml N/10 NaOH/g) 18.8 9.2 20.6 --- 18.1 10.3 --- 20.6 5.0 ---

Moisture 13.3 49.7 13.8 65.0 15.3 61.5 70.7 13.8 49.0 68.0 Fat 52.2 26.7 49.8 18.5 46.8 18.2 15.0 49.8 22.0 15.4 Protein 19.7 17.6 19.9 10.8 19.9 16 10.1 19.9 23.6 12.0 Lactose 11.5 3.8 12.5 2.5 13.1 1.1 1.6 12.5 1.2 1.4 Ash 3.3 2.2 4.0 3.2 4.3 2.3 2.6 4.0 3.2 3.2

Treatment I: Loosely tying the residue in the form of bundle and cooking in boiling water for 30 min.

Treatment II: Cooking the residue in boiling 1.0% sodium bicarbonate for 30 min. Treatment III: Washing the residue with 50% alcohol and then cooking in boiling water

for 30 min. Treatment IV: Washing the residue with 50% alcohol followed by boiling in 1% sodium bicarbonate. Treatment V: Autoclaving the residue (15 PSI/10 min) obtained from III after incorporating 2% vinegar Treatment VI: Autoclaving the residue obtained from IV after incorporating 2% vinegar.

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residue soft and smooth. It was reported that the trend of changes brought about in the constituents of the residue remained same. Residues absorb considerable amount of moisture, its acidity reduces; in case of treatments II, IV and VI acidity reduced to nil. Fat and lactose contents of the residue also reduced considerably. Washing of residue with 50% alcohol followed by cooking in baking soda, i.e., treatment IV was reported to be the best as far as removal of excess fat from the residue was concerned. Autoclaving of this residue after incorporating 2% vinegar lowered the moisture content and improved the texture of the product. Keeping quality of all types of GR clarified at 120°C is 3 months (Prahlad, 1954). Its shelf life can further be extended to more than 4 months by pressing it in cake form (Viswanathan et al, 1973). Sripad et al, (1996) reported that addition of ethanol extracts of browning compounds (1%) from defatted GR to cow and buffalo ghee clarified at 100°C and stored at 37°C extended the shelf life by about 1.5 and 2 months respectively.

3.1 Recovery of ghee from ghee residue

In dairy plants, attempt is made to recover as much ghee as possible from GR. Two methods of recovery of ghee from GR viz. centrifugal process and pressure technique have been developed (Viswanathan et al., 1973). The centrifugal process consists of heating GR in water (65°C) so as to transfer the occluded ghee of the residue to water. Ghee is subsequently recovered by centrifugation. This method is reported to yields 25% ghee (46% efficiency). In the pressure technique, the heated GR (65-70°C) is subjected to a limited pressure in hand screw or hydraulic press. This method gives an yield of about 45% (extraction efficiency of about 67%). The pressure technique method has been recommended for adoption as it is simple, efficient, economical and practical and requires no electricity or sophisticated equipment.

4. PHYSICO-CHEMICAL PROPERTIES

4.1 Particle size and density

Ghee-residue is generally removed by bag filters or muslin cloth, but for the continuous ghee clarification process, centrifugal clarifiers have been designed. The average particle diameter of GR is 104.79 microns and average density is 1.14 g/cm3 (Wadhwa, 1997). 4.2 Lipids in ghee-residue

The lipids of GR have lower Reichert and Polenske values (24.4., 1.3) but have higher iodine value (43.4) in comparison to those of the corresponding ghee. The lipids of GR contain 53% lower chain fatty acids (C4.0 to C12.0), 58.7% total saturated fatty acids and 41.3% unsaturated fatty acids. Irrespective of the method of preparation, polyunsaturated fatty acids (PUFA) content of GR lipids (4.4%) is higher than those of corresponding ghee (2.8%).

Ghee-residue is rich in phospholipids (1-9%). Phospholipid acts synergistically with reducing substances in GR and protects it from oxidative defect. Higher phospholipid (a good emulsifier) content of GR is beneficial in developing certain products where emulsification of fat and aqueous phase is desired. The phospholipid content of GR is dependent upon the method of preparation. It is highest in creamery butter GR lipids (17.39%) followed by desi butter GR lipids (4.95%) and the least in direct creamery GR lipids (1.57%) (Santha and Narayanan, 1978a). These levels are much higher than those in ghee (0.004-0.08%). The fatty acid composition of

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phospholipids shows that it has no fatty acids lower than 12 carbon atoms (Santha and Narayanan, 1978b). The phopholipid content of GR decreases as the period of heating increases due to the transfer of phospholipids from GR to ghee. While heating cream/butter, only a small fraction of the phospholipids gets transferred to ghee, most of the phospholipids remain with the residue because of their polar character. The differences observed in the physico-chemical constant, fatty acids and PUFA contents between lipids of GR and ghee are due to the high phospholipid content of GR (Wadhwa, 1997).

4.3 Proteins in ghee-residue

Soluble nitrogen content of GR prepared from cream or creamery butter decreases with heating time. This decrease is due to the denaturation of the proteins. By paper chromatographic technique, 11 free amino acids, 2 amines and 8 unidentified spots were detected in GR (Wadhwa, 1997). The total reducing capacity expressed as mg of cysteine hydrochloride/g of creamery butter GR (26.0) and free sulphydryl content (µm/g) of GR (2.90) are much higher than those in ghee (0.075 and 0.02, respectively). These substances are liberated from protein during heat treatment and because of their polar nature the are mostly retained in the GR. Whey proteins, especially ß-lactoglobulin are the main source for these sulphydryl compounds (Santha and Narayanan, 1979a).

4.4 Milk sugars in ghee residue

Ghee-residue prepared at 120oC contains sugars (wt %) such as lactose (76.6), galactose (14.1), glucose (5.3), and two unidentified sugars. As the period of heating is increased, the lactose content of GR decreases with a corresponding increase in galactose and glucose content. The unidentified sugars are also found to increase as the period of heating is increased. The unidentified sugars might be due to some of the breakdown products or sugar fragments formed during browning or caramelization reactions. (Santha and Narayanan, 1979b).

5. ANTIOXIDANT PROPERTIES

The development of peroxides in ghee samples containing 2% of GR at 80°C is slower than in the control showing thereby that GR contain antioxygenic substances. The overall antioxygenic properties are due to both lipid and non-lipid constituents (Santha and Narayanan, 1979c). In lipid constituent of GR, phospholipids show the maximum antioxidant activity followed by �-tocopherol and vitamin A. Among the various phospholipid fractions, cephalin shows the greatest antioxidant activity. The oxidative stability of ghee can be increased by increasing its phospholipid content (0.1%) either through heat treatment process or through solvent extraction process. It has been observed that heating GR with ghee in the ratio of 1:4 at 130°C have maximum transfer of phospholipids from GR to ghee. These antioxidant concentrates can be added to ghee to give about 0.1% phospholipids so as to increase the keeping quality of ghee. Among the non-lipid constituents, the amino acid proline, lysine, cysteine hydrochloride and tryptophane show the antioxidant properties. The contribution of proline as antioxidant is maximum though less than BHA at 0.02% level. Further, the addition of lactose, glucose, galactose and their interaction products with protein and phospholipids to ghee also increase the oxidative stability of ghee. As ghee-residues contain large amount of reducing substances including free sulphydryls, such compounds may also contribute to the antioxidant properties of

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GR (Santha and Narayanan, 1979 b). Santha and Narayanan (1978 c) showed that the antioxidant efficiency of GR decreases with increase in the temperature of classification of ghee. The addition of GR obtained from ghee prepared at lower temperature (110°C) results in lesser development of peroxides than the addition of GR prepared at higher addition of GR prepared at higher temperature (150°C). Santha and Narayanan (1978 c) observed that creamery butter GR has the maximum antioxidant properties followed by desi butter and direct creamery GRs.

Hence, GR is a rich source of natural antioxidants and its antioxidant properties are due to the mixture of its constituents affected by various technological parameters. Wadhwa et al. (1991 a & b) have observed that oxidative stability imparted by GR to flavoured butter oil was parallel to that imparted by BHA and BHT. Thus, GR can be used as a source of natural antioxidants for improving the shelf-life of food products including dairy products where use of synthetic antioxidants is generally not preferred because of their toxic effects. 6. FLAVOURING PROPERTIES

Ghee-residue is a rich source of natural flavouring compounds viz. free fatty acids (FFA), carbonyls and lactones (Galhotra and Wadhwa, 1993). The level of FFA, carbonyls and lactones in GR are higher than those in ghee Table-3. Wadhwa and Bindal (1995) simulated ghee flavour in vegetable fat. Vegetable fat is mixed thoroughly with water (20%) at 20-25°C to obtain butter-like product. To this, GR (10%) is mixed well and clarified at 120°C/flash, filtered through 4-fold muslin cloth and centrifuged at 3000 rpm for 10 min. The flavour score of vegetable fat was reported to be 7.0 as against 5.0 for untreated vegetable fat and 8.0 for pure ghee. Butter oil can also be simulated with ghee flavour by addition of GR (10%) essentially following the above procedure. This treatment is also said to enhance the keeping quality of the product. The oxidative stability of flavoured vegetable fat (FVF) in comparison to vegetable fat and milk fat (Ghee) at 80°C has been investigation. Induction period (IP) of FVF is exceptionally high (288hr) i.e. 11 times that of VF (26 hr) and 2.4 times that of ghee (120 hr). The studies have thus established the flavouring as well as antioxidant properties of GR.

Table 3. Flavour potential of ghee and ghee residue

FFA (µm/g) Carbonyls (µm/g) Lactones (µg/g) GR Ghee GR Ghee GR Ghee

627.5 53.6 43.7 4.3 3992.9 30.3

7. NUTRITIVE VALUE OF GHEE RESIDUE

Ghee residue has poor quality of protein because of its lower lysine content. Relwani (1978) reported that supplementation of GR with some good quality protein such as skim milk powder (SMP) sharply increased its protein efficiency ratio (PER) from 0.66 to 2.4. Supplementation with 8% lysine, 2.5% methionine and 1.4% tryptophane strikingly improved its nutritive value which was even slightly higher than SMP (Table 4). Supplementation of GR with other proteins and/or amino acids can be taken up for developing value added edible products from it.

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Table 4. Nutritive value of ghee-residue

Diet Protein Efficiency Ratio (PER)

Digestibility Coefficient

Biological Value (%) NPU (%)

Skim Milk Powder 3.92 93.61 91.99 86.12 Ghee-residue 0.75 92.65 65.07 40.77 Ghee-residue + 8%lysine 3.24 82.48 77.87 64.23 Ghee-residue + 8% lysine + 2.5% Methionine + 1.4% Tryptophane

4.11 86.24 93.84 80.64

8. APPLICATIONS OF GHEE-RESIDUE

8.1 Preparation of confections

The physico-chemical properties of processed GR make it suitable for preparation of confections. It contains the major constituents in suitable proportion and possesses fine texture that impart requisite body to such products. Further the treatment during processing of these confections involves heating to such an extent that it completely arrests enzymic activity and flavour deterioration in the final product. The higher fat content in the residue quite often obviate the need for addition of oils and fats in its preparation (Prahlad, 1954). 8.2 Preparation of candy

The ingredients required for candy preparation include processed GR (1 kg), sugar (500-625g), and coconut powder (125-250g). The candy making process involves thorough mixing of processed GR with 50% sugar syrup with the help of suitable laddle followed by evaporation to remove moisture by heating on low fire with continuous stirring. When the mass becomes sufficiently sticky, coconut powder is added. The candy is evenly spread on a plate and cooled (5-10°C) for about an hour and cut into small cubes and wrapped in parchment paper.

8.3 Preparation of chocolate

Recipe for preparation of chocolate consists of: processed GR 1 kg, sugar 500 to 625 g, cocoa powder 60 to 90 g and skim milk powder 250 g. Processed GR is added to 50% sugar syrup, and thoroughly mixed with a laddle. The contents are desiccated on a low flame till dough is formed. At this stage cocoa and skim milk powder are added and stirred vigorously till pat is formed. Finished product is spread on a plate and cooled overnight and cut into slabs or cubes and wrapped in parchment paper. The product has a shelf life of more than 3 months. 8.4 Preparation of edible pastes

For preparation of edible paste for sandwich, processed GR is first mixed with salt @ 2.5-3% and then with marmite (a yeast product) @ 0.1-0.5%. The whole mass is heated on a low fire for about 5 min till a paste is formed. An edible paste for dosa and samosa can be prepared if chutney powder @ 2-4% is used instead of marmite. Both these preparations, if properly packaged, can remain marketable for 2 months (Prahlad, 1954).

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8.5 Preparation of burfi-type confection

Verma and De (1978) prepared burfi-type sweet from GR processed in 0.5 per cent sodium bicarbonate for 30 min. Processed GR was mixed with khoa in the proportion of 1:1 (total solids basis). Sugar was added @75% of the total solids (khoa + ghee residue). The whole mass was heated and worked vigorously for 10-15 minutes so as to dissolve the added sugar completely. At that stage about two-third of the mixture was taken out and spread as a thick layer on a well-greased tray. In the remaining one-third of the sweetened mass, chocolate powder @ 8% of the total solids (khoa + ghee residue) was thoroughly worked. This portion containing the chocolate was applied as a thin layer over the two-third portion of the mixture in the tray taken out earlier. The mass was cooled and when set cut into pieces of uniform size and shape. The product got a sensory score of 7.5 (like moderately to very much) on 9-point Hedonic scale.

8.6 Preparation of bakery products

Borawake and Bhosale (1996) prepared nankatai type cookies and sponge cake from processed GR obtained from ripened cream. In this study a part of vanaspati fat used in preparation of these products was replaced by ghee-residue fat. Replacement of vegetable fat upto a level of 30 and 20% for cookies and sponge cake respectively resulted in acceptable quality of products. The product is reported to have a sensory score of 7.15 and 7.45 respectively. Use of GR enriched protein content of both the bakery products as compared to control. Bajwa and Kaur (1995) reported that incorporation of GR up to 30% into cookie dough increased the spread factor and also improved the sensory attributes of cookies.

8.7 Enhancing flavour of ghee

Ghee, especially one prepared from fresh creamery-butter (without ripening) has mild flavour. GR can be used to enhance flavour in such ghee. Unripened creamery butter mixed thoroughly with ghee-residue (10%) and clarified at 120°C, filtered through 4-fold of muslin cloth and subsequently centrifuged to get residue free ghee enhances flavour.

9. CONCLUSION

Ghee-residue being rich source of fat and proteins is valuable for its nutritive value as human dietary supplement. The flavour potential of ghee-residue is much greater than that of ghee. Hence it can be utilised for flavouring bland fats and also enhance their keeping quality. Ghee residue, thus, by virtue of its chemical composition, bulk of production, physical characteristics, long shelf life permitting its collection and centralized handling has great potential and is more amenable to exploit its utilization in food industry. 10. EFERENCES Bajwa, U. and Kaur, A. 1995. Effect of ghee (butter oil) residue and additives on physical and sensory characteristics

of cookies. Chemie,-Mikrobiologie,-Technologie-der-Lebensmittel. 17(5/6): 151-155. Borawake, K.N. and Bhosale, D.N. 1996. Utilization of ghee residue in preparation of nankatai type cookies and

sponge cakes. Indian J. Dairy Sci. 49(2): 114-119. Dairy India. 2007. Milk utilization pattern. Dairy India 2007. Dairy India Yearbook. New Delhi. Pp: 33. Galhotra, K.K. and Wadhwa, B.K. 1993. Chemistry of ghee-residue, its significance and utilisation - a review. Indian

J. Dairy Sci. 46: 142-146. Prahlad, S.N. 1954. By-Products of Indian Dairy Industry-Ghee residue. M.Sc. thesis, Bombay University, Bombay.

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Relwani, I. 1978. Assessment of Nutritive value of ghee-residue as human dietary supplement. M.Sc. Thesis, Kurukshetra University, Kurukshetra.

Santha, I.M. and Narayanan, K.M. 1978a. Composition of ghee-residue J. Fd. Sci. Technol. 15(1): 24-27. Santha, I.M. and Narayanan, K.M. 1978b. Composition of ghee-residue lipids. Indian J. Dairy Sci. 31(4): 365-369. Santha, I.M. and Narayanan, K.M. 1978c. Antioxidant properties of ghee-residue as affected by temperature of

clasification and method of preparation of ghee. Indian J. Anim. Sci. 48(4): 266-271. Santha, I.M. and Narayanan, K.M. 1979a. Changes taking place in proteins during the conversion of butter/cream to

ghee. Indian J. Dairy Sci. 32(1): 68-74. Santha, I.M. and Narayanan, K.M. 1979b. Free sugars of ghee-residue. Indian J. Dairy Sci. 32(2): 189-191. Santha, I.M. and Narayanan, K.M. 1979c. Studies on the constituents responsible for the antioxidant properties of

ghee-residue. Indian J. Anim. Sci. 49(1): 37-41. Sripad, S., Kempanna, C. and Bhat, G.S. 1996. Effect of alcohol extract of deffated ghee-residue on the shelf life of

ghee. Indian J. Dairy Biosci. 7: 82-84. Verma, B.B. and De, S. 1978. Preparation of chocsidu Burfi from ghee residue. Indian J. Dairy Sci. 31(4): 370-374. Viswanathan, K., Rao, S.D.T. and Reddy, B.R. 1973. Recovery of ghee from ghee residue. Indian J. Dairy Sci. 26:

245. Wadhwa, B.K. 1997. Functional properties of ghee residue. In lecture compendium of short course on “Technological

advances in dairy by-products”. Centre of Advanced Studies in Dairy Technology, NDRI, Karnal. Pp: 141-146. Wadhwa, B.K. and Bindal, M.P. 1995. Ghee-residue : A promise for simulating flavours in vanaspati (Hydrogenated

edible vegetable oils) and butter oil. Indian J. Dairy Sci. 48: 469-472. Wadhwa, B.K. Surinder Kaur and Jain, M.K. 1991a. Enhancement in the shelf-life of flavoured butter oil by natural

antioxidants. Indian J. Dairy Sci. 44(1): 119. Wadhwa, B.K., Surinder Kaur and Jain, M.K. 1991b. Enhancement in the shelf-life of flavoured butter oil by synthetic

antioxidants. J. Food Quality. 14(2): 175-182.

7

SECTION - VII

UTILIZATION OF DAIRY BY-PRODUCTS

UTILIZATION OF DAIRY BY-PRODUCTS

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Dr. Ashish Kumar Singh Scientist

Dairy Technology Division, N.D.R.I., Karnal – 132 001

1. INTRODUCTION

Milk solids are an essential ingredient in formulations of wide range of bakery and confectionery products. Their inclusion in recipe enhances the quality and acceptability of processed foods because of the nutritional and functionality they impart to products. Skim milk, casein and its derivatives and whey solids are important dairy by-products commonly used as ingredient in bakery and confectionery products. During early periods the main purpose of including milk solids in bakery or confectionary products was to enhance the nutritional profile of resultant product. However, later on with increase in milk production, processing and technological developments it became possible to make available wide range of milk components that can be used in these products at economical level. Now with the better understanding regarding the functionality of milk molecules in bakery and confectionery products, it has become possible to produce tailor-made ingredients utilizing dairy by-products for specific application. 2. FUNCTIONAL PROPERTIES OF MILK COMPONENTS IN BAKERY AND

CONFECTIONERY PRODUCTS

Milk molecules are endowed with many functional properties in bakery and confectionery products (Table 1). These functionalities in specific products are required to develop an optimum formulation and better acceptability by consumers. The majority of these reactions are imparted by milk proteins i.e. casein and whey proteins and lactose. The functional properties of milk proteins have been extensively reviewed by Kinsella (1984) and Morr & Ha (1993). The Milk fat also contributes towards the quality enhancement of bakery and confectionary products, but due to its higher cost, susceptibility to adverse chemical interactions and slightly negative health perceptions in majority of consumers, its use in these products has been discouraged. Moreover, milk lipids are usually present in minor amounts in dairy by-products. The functionality of milk proteins depends upon the intrinsic properties of native milk proteins such as their amino acid composition and sequence, molecular weight, charge hydrophobicity and their configuration. It is greatly influenced by extrinsic factors including temperature, pH, salts and the concentration (Henning et al., 2006). The processing techniques, parameters like homogenization, fore-warming, freeze concentration and drying also affect the functional properties of milk proteins. The best known applications of dairy by-products include high heat skim milk powder in biscuits & fermented bakery products and whey proteins in foamed bakery products.

APPLICATION OF DAIRY BY-PRODUCTS IN BAKERY AND CONFECTIONARY PRODUCTS

APPLICATION OF DAIRY BY-PRODUCTS IN BAKERY AND CONFECTIONARY PRODUCTS

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3. DAIRY BY-PRODUCTS IN BAKERY PRODUCTS

3.1 Casein and casein derivatives

Casein and casein derivatives are mainly used in bakery products to enhance flavour and other sensory properties and also for nutritional fortification of the wheat flour. By supplementing the wheat flour with casein, it is possible to increase considerably the PER of the mixture. The casein addition not only improves the amino acid profile of resulting products specially by providing the essential amino-acids. One of the most important functional characteristics of casein products in bakery products is its water binding capacity and all casein products have high water binding capacities if compared with wheat protein. Acid and rennet casein, sodium caseinate, calcium caseinate can be used in bread making and are added at a level of 15-20% of the wheat flour. A satisfactory loaf volume can be obtained by the use of casein products. Caseins are less soluble and they improve the dough making properties and texture of baker products, pasta, snack foods and breakfast cereals (Swaiswood, 1985). However before using Caseinates appropriate water requirements must be assessed, otherwise it may lead to the problem of sogginess.

Table 1: Functional Properties of milk Components in Bakery and Confectionary Products

S. No.

Functional Properties

Mechanisms Products

1 Water absorption & Binding

Hydrogen, covalent bonding Cakes, Caramel, Toffee

2 Gelation Heat induced denaturation and formation of protein matrix, setting of structure

Bakery products, Caramel, Toffee, Taffy, Muffins

3 Cohesion-Adhesion Chemical interactions between milk protein and other ingredients

Bakery & pasta products

4 Elasticity Hydrophobic binding in gluten disulfide link, in gels

Bakery products (Fermented baker products)

5 Emulsification Formation and stabilization of fat emulsion

Confectionary products, cakes, muffins

6 Whipping Formation of boundary film to entrap air bubbles

Cakes, Fudge

7 Flavour formation Interaction between milk proteins and lactose (Maillard reaction)

Caramels, toffee, toffee, cakes, biscuits, cookies

8. Colour Maillard reactions, caramelization of lactose at higher temperature

Cakes, caramel. Toffee, fudge, biscuits, cookies

9. Nutrition Supply of quality proteins, amino acids, milk minerals, water soluble vitamins and lactose

High protein biscuits, cakes, confectionary products etc.

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3.2 Whey Ingredients and Their Application in Bakery Products

Whey ingredients like liquid whey concentrates, whey powder, lactose, whey protein concentrate and isolated whey proteins have been attempted in wide range of bakery products. Whey powders because of high lactose content and higher amount of minerals are rarely used in bakery products because of saltiness and formation of lactose glass in products. Addition of whey powders often produces more brown products. Now a day’s whey protein preparations either as whey protein concentrate (WPC) or Whey Protein Isolates (WPI) are available for various bakery products. Commercial WPCs are available in 34 or 70% protein content and depending upon protein content, levels of lactose, milk minerals and residual lipids varies. deWit (1998) reviewed the nutritional and functional characteristics of whey proteins in food products and concluded that the functional properties of whey proteins are governed by the composition and structure of the protein and influenced by the prevailing environmental conditions, prior treatments, processing conditions and methods of their characterization which is difficult to evaluate. Bakery and confectionary products require specific functionality of whey proteins including solubility, foaming, emulsification, water binding and gelling characteristics to develop desirable characteristics (Dewani, 2004).

In the bakery industry egg protein is primarily used on account of its unique functional properties; it influences the texture of many bakery products as a result of its emulsifying, leavening, tenderizing and binding qualities. Whey proteins have a number of functional properties similar to egg proteins. Many attempts have been made to substitute WPCs for egg white or whole egg in bakery products. In particular the capability of whey proteins of producing foam when whipped, and of inducing structural properties due to heat setting, indicates that these proteins are very similar to egg white. Also nutritional and organoleptic reasons contribute to the continuing interest in fortifying or substituting wheat flour in bakery products (de Wit. 1984). However, before the utilization of whey protein preparations in food products, its functional properties must be evaluated carefully specially lot of variations exists in composition that influences functionality. 4. DAIRY BY-PROUDCTS IN BAKERY PROUDCTS

4.1 Fermented Bakery Products.

The major role of milk by-product in fermented bakery products viz bread, pizza base etc. include improvement in dough handling properties and quality such as crust colour, flavour and crumb texture (Kenny et al., 2000).The structure of bread stabilizes during baking due to coagulation of proteins and gelatinization of starch and inclusion of milk proteins added in one form or another, shows a good crumb structure, bread yield, flavour and keeping quality. Skim milk powder or condensed skim milk is used in many bread formulations and reported to have beneficial effects on bread quality.

Whey proteins interfere with the normal structure of gluten and its incorporation usually decreased elasticity of dough (Lupano, 2000). In bread making, some denaturation of the whey proteins is necessary to avoid adverse reactions between whey proteins and other components of the system. Addition of undenatured WPC resulted in weaker, less elastic doughs that yield loaves of reduced volume. When the proportion of undenatured protein was reduced to about 30% by heat treatment of UF retentate, the loaf volume increased from 22 to 28 and the loaf score from 66

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to about 80 (Zadow and Marston, 1984). Breads receiving the highest scores were those made from WPC that had received a heat treatment of either 85oC for 15 s or 75oC for 60 s (Harper and Zadow, 1984).

Srivastava and Rao (1993) evaluated the effect of different sources of milk solids including the SMP on the quality of bread and found that all of these milk products, reduced the Farinograph water absorption, increased the dough stability and made the dough stiffer. Incorporation of any type of milk solids at 6.0 per cent level (on dry basis) lowered the loaf volume by 4.8 to 12.4 per cent, hardened the texture and made the grains coarser. However to improve the functionality of added milk by-products in bread some additives like reducing agents, sugar, emulsifiers are required. Hydrolyzed whey permeate concentrated to suitable total solid may be a cheap substitute of sugars in bread formulations and it can also be effectively utilized by yeasts. 4.2 Biscuits, Cookies and Crackers

In the manufacturing of high protein biscuits meant for community nutrition or for the patients suffering with protein-energy malnutrition, milk proteins are considered an essential ingredient. The nutritional profile of milk proteins makes them an ideal component of such specialized formulations. Awasthi and Yadav (2000) reported that incorporation of addition of 70% liquid whey or 50% skim milk along with 14% defatted soy flour (DSF) improved the protein, mineral, fiber content in biscuits. Whey powders because of their lower cost extensively used in biscuit in place of SMP and it exhibits similar functionality as SMP. Lactose is mainly used in less sweet and savory type biscuits. However high mineral content of whey powder may contribute a salty flavour and it can be solved by using deminerlaized whey powder. In such biscuits WPC can replace 10-20% of the wheat flour. The unbalanced amino acid composition of wheat proteins, especially the deficit of lysine, can be improved by supplementation. Raju et al (2007) substituted refined wheat flour (maida) with WPC (70% protein) in biscuit manufacture and observed significant increase in protein and mineral levels. WPC substitution up to 30% level had no adverse effect on sensory quality of biscuits. WPC incorporation in biscuit dough resulted in decreased consistency and firmness and increase in cohesiveness of dough. The fracture stress of biscuit also decreased and all these observation indicate positive impact of WPC addition on biscuit quality. Casein and Caseinates may also be used in development of cookies with improved textural properties; however particle size and method of drying affect their application in cookies. In crackers based on fermented dough addition of hydrolyzed lactose syrup can be added to improve the flavour and reduction in fermentation period. 4.3 Cakes

Some researchers have investigated the possibility of replacing egg white with whey proteins in the manufacture of cakes with varying degree of success. A situation quite different from that in bread exists in some cakes, where the main functions of egg proteins are encapsulation during the mixing process, stabilization of acqueoes foam in the intermediated baking stage and coagulation of the egg proteins in the heat-setting stage of the cake batter. In this case, whole eggs cannot simply be replaced by WPC, but good results have been obtained when the butter and eggs are completely replaced in cake-making by a fat-WPC-emulsion containing the same amounts of fat and protein (de Wit, 1984).

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Richert (1973) studied the complete egg replacement with whey protein isolates for making white layer cakes. The resulting product exhibited similar cake volumes and profiles as the cakes made with egg. However, cakes prepared with whey protein isolates appeared to be more tender, moist and crumbly.

Mann (1974) studied the formulation of the “Egg Substitutes” from milk protein complexes for use in cake making. He observed that the carbodymethyl cellulose protein complexes (as 5% solution in water) had superior form stability when compared to that of whipped egg. Replacement of egg solids was attempted by using WPC, skim milk retentate powder (SRP) and buttermilk retentate powder (BRP) in cakes and cake-mixes. Maximum porosity and specific volume was obtained by using WPC-60, whereas SRP & BRP addition also produced cakes similar to control (with egg) (Puranik, 2003). Substitution of eggs up to 50% level using WPC-790 resulted no significant lowering in volume and weight of cakes (Singh et al, 2003). Further they observed that WPC at 6% level along with emulsifiers i.e. lecithin and GMS can be used to develop egg-less cake of excellent quality. Puranik (1997) developed ready-to-bake cake mix by dry blending of 12% WPC, 20.30% fat, 32% maida, 2.90% baking powder and 0.80% flavour. For the preparation of SRP or BRP cake mix, 17% SRP or BRP, 22.10% fat, 30% maida, 3.00% baking powder and 0.90% flavour. 4.4 Pasta Products

Milk proteins from by-products are often incorporated into the base flour for pasta manufacture for the purpose of enhancing nutritional quality- and to improve texture Products fortified by addition of sodium or calcium caseinate, low calcium co-precipitate or WPC prior to extrusion include macaroni and pasta. (Towler, 1982)

Undenatured whey protein products a strong final cooked noodle which is also more freeze-thaw stable and is suitable for microwave cooking ‘Imitation or synthetic’ pasta-type products containing a substantial proportion of milk protein have also been manufactured. When WPC is used for manufacturing macaroni, two important processing aspects should be taken into account: dough stickiness which is observed when WPC with low and medium denaturation degree is added, and dough softening when the percentage of supplemented WPC is increased and the denaturation degree is decreased; similarly, mechanical resistance of the dry noodles is affected by both factors. The cooking loss increases as the amount of WPC and its protein content are increased, while taste is lonely affected by the amount of WPC added; however, this effect may be neutralized by the usual flavouring used for pasta in cooked meals.

5.0 MILK BY-PRODUCTS IN CONFECTIONERY PRODUCTS

Term Confectionery is quite ambiguous and applied to wide rage of processed foods where sugar is principle ingredient. Confectionery products can be classified as sugar confectionery, chocolate confectionery and flour confectionery. Dairy by-products such as skim milk solids, whey solids and milk proteins are added in various forms in confectionery products.

Milk solids impart three important characteristics to caramels: they produce the background milk flavour; the protein and fat aid in producing the texture and chew; the proteins and lactose interact during heating to produce the characteristic caramel flavour. Generally, the more milk solids in sugar confectionary such as caramel the better the flavour colour and standup

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qualities will be. Whey powders and sweetened whey have gained popularity as economical substitute for nonfat milk solids (Bauzas, 1999). Greater concentration of lactose present in whey enhances the golden brown colour of caramel, and up to 50% of the nonfat milk solids can be replaced by whey. The resulting caramel is slightly softer. Lactose treated whey has been suggested to produce the desired caramel colour and flavour more rapidly while permitting reduction in sucrose and eliminating lactose crystallization problems (Hugunin, and Nishikawa., 1978)

5.1 Lactose Powder

Lactose can have an important effect on the flavour, colour, and texture of confection. Flavours depend on cooking temperature and time, acidity of mixture, and presence of free amino group. Lactose, the least sweet of the principal sugars used in confections, can be added to establish flavour balance. A lesser known but important attributes is its ability to hold flavour, odors, and pigments. These substances, absorbed on the surface as lactose crystals, tend to be grained until the lactose is dissolved. Because lactose is far less soluble than sucrose of dextrose, it can greatly affect texture. Depending on temperature of processing, one of two types of crystals forms. Above 200oF, β-anhydride crystals form, but as the candy mass cool and during storage, α anhydride crystals develop. α – crystals are hard and less soluble than the β form, and if they reach palpable size, they become gritty of sandy in texture. In addition to enhancing, absorbing and holding flavour, lactose decreases vapour pressure, retard moisture loss, and prolong freshness. When added to a solution of sucrose and corn syrup, lactose can increase viscosity. (Webb, 1966). 5.2 Casein, Caseinates and Whey Proteins

Milk proteins can have pronounced effects on the texture and rheological properties of a food system, by forming complexes among themselves and with the other constituents in the system. The physical characteristics of food products attributed to milk protein functionality have been reported by numerous researches (Kinsella, 1984a; Kinsella, 1984b). Depending on the individual protein or protein mixtures and on various environmental and processing factors, proteins can impart a wide range of physical properties to foods. These physical properties are in most cases related to the functional properties of proteins. The functional properties of milk proteins result from their interactions at three levels: 1) protein-water interactions; 2) protein-protein interactions; 3) protein surface interactions.

Caramel is an example of a complex food product in which milk proteins are expected to play a major role in controlling the textural properties. However, in caramel, the role that milk proteins play in establishing the textural characteristics has not been clearly elucidated. The milk protein affects the firmness of caramel (Lees and Jackson, 1975). The functional properties of milk proteins in food systems can be categorized into solubility, viscosity, water binding, gelation, cohesion/adhesion, elasticity, emulsification, foaming, fat and flavour binding (Kinseilla, 1985). Except for foaming, all of the above functionalities are exhibited in caramel.

Caseins and whey proteins, the two major classes of proteins in proteins in milk, aid in blending by promoting emulsification and improving emulsion stability. Caseins have the ability to bind more than twice their weight of water, thus producing a drying effect. Undenatured whey proteins, on the other hand, bind very little water, but their water absorption capability can be increased by heat denaturing.(Campbell and Pavlasek 1987) The foaming properties of whey

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protein exceed those of caseins. Milk proteins yield many of their beneficial attributes upon denaturation during cooking. The resulting network of fibrous structure imparts much of final texture of confection. Caseins can produce a firm chewy body, neither sticky nor tough, while whey proteins form a soft coagulum with less resilience.

The final colour and flavour of confection also reflect contribution from milk proteins. Caseinates, largely responsible for milk’s white colour, transfer their whitening effect to many food products. Caramel is a multi-component system, in which interactions between casein and whey proteins and the interactions of these proteins with the other ingredients are inevitable. The textural properties of caramel are related to these interactions in some fashions. The association of proteins with other ingredients takes place, in varying pattern in the process of the structural stabilization of protein in a food system. Therefore, predicting the textural changes of caramel from the physico-chemical properties of milk proteins alone is not possible.

The firmness of caramel was significantly affected by both whey and casein proteins. The maximum firmness can obtained when casein and whey protein were present in the highest amounts. The increased firmness is not only attributed to higher amount of protein but this is due to physical and/or chemical interaction between these two proteins. Furthermore, there appeared to be a synergistic effect between whey and casein proteins on the increase in caramel firmness.

Mixing skim milk powder and whey protein concentrate at 10% concentration and in 50:50 ratio produced a synergistic effect on gel firmness. Sodium caseinate was also reported to improve the firmness of WPC gels. These results suggest that WPC interact with other milk proteins and improve the structure of the end product. Similar interactive effects of whey and casein proteins could be involved in increasing the firmness of caramel. This increase in firmness is dependent on both proteins and heating temperature. However, Condensed whey, lacking the casein protein, can be used o produce a cheaper confection provided no more than 10% substitution is used (Lees, 1973). High albumin milk products produce a soft textured confection which is highly suited to cup depositing.

6.0 CONCLUSION

Very high value is placed on ingredients and if the food label does not offer the right mix of ingredients that offer flavour and nutritional value, the manufacturer might be the ultimate looser. The ingredient suppliers are also keeping a close eye on trends impacting consumer purchase. Marketing ingredients require equal emphasis and functionality. In last few years there has been a paradigm shift in dairy and food processing sector interface. The interdependence of these tow processing sectors is generally confined to only few areas like in application of milk proteins in nutritional products however there are many promising areas where both can work together in an integrated approach with the common objectives of improving the functionality, nutritional quality and health promoting effects of the food supply. 7.0 REFERENCES Awasthi P. and Yadav, M.C. (2000) Effect of incorporation of liquid dairy by-products on chemical characteristics of soy-fortified biscuits. J. Food. Sci. tEchnol., 37:158-161. Bouzas, J. 1999. Whey products and lactose in confectionery applications. Application Monogrpah, US Dairy Export Council, 2-8 Campbell, L. B. and Labuza, S. J. 1987. Dairy products as ingredients in Chocolate and confections. Food

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Technology, October: 78-85 Confonti, P.A. and Lupano, c. E. 2004. Functional properties of Biscuits with whey protein concentrate and Honey. International Journal of Food Science & Technology 39 (743-753). De Wit. J.N. (1984) Functional properties of whey proteins in food systems. Neth. Milk Dairy J., 38: 71-76 Dewani, P. P. 2004. Whey protein concentrate and its application in bakery industry. Beverage and Food World, 31 (9) 56-58. Evans, E. (1980) Whey research J. Soc. Dairy Technol., 33: 95-98. Harper, W.J. and Zadow, J. G. (1984). Heat Induced changes in whey protein concentrates as related to bead manufacture. New Zealand J. Dairy Sci. Technol. 19: 229-227. Henning, D. R. Baer, r. J. , Hassan A. N. And Dave, R. 2006. Major Advvances in concentrated and dry milk products, cheese and milk fat based spreads. J. Dairy Science, 89: 1179-1188. Howarth, S. J. (1991) Tailored dairy ingredients in bakery and confectionary applications Bakers Digest cited in Dairy Sci. Abstr., 55 (10): 6280. Hugunin, A. G. and Nishikawa, R. K., 1978. Milk derived ingredients lend flavour,texture, nutrition to confection. Food product development, 73:46-50. de Wit , J. N. (1998). Nutritional and functional characteristics of whey proteins in food products. J. Dairy Sci 81: 597-608. Kenny S., Wehrke, K. and Stanton; C. 2000. Incorporation of dairy ingredients into wheat bread: effects on dough rheology and bread quality. European Food Research and Technology, 210: 391-396. Kilara, A. (1984) Standardization of methodology for evaluating whey proteins. J. Dairy Sci. 67:2734-2738 Kinsella, J. E. 1984b. Functional properties of food proteins: Thermal modifications involving denaturation and gelation. Food Sci Technol., 5: 226-246. Kinsella, J. E. l984a. Milk protein: physicochemical and fùnctional properties. CRC Crit. Rev. Food Sci. and Nutr. 21(3): 197-262. Lees, R. 1973. Manufacture of caramel. Food manufacture, 48(3):45-46. Lees, R. and Jackson, E.B. 1975. Milk and milk product. In: Sugar confectionery and chocolate manufacture. Chemical Publishing Co., New York, 66-72. Lupano, C. E. (2000). Gelation of mixed systems whey protein concentrates gluten in acidic conditions. Food Research International 33:691-696. Mann, R. S. (1974) Studies on the formulation of “Egg Substitutes” from milk protein complexes for use in cake making, M.Sc. Thesis submitted to Punjab University, Chandigarh. Morr CV, Ha EYW 1993. Whey protein concentrates and isolates: processing and functional properties, CRC Crit Rev Food Sci Nutr 33: 431-176. Purnaik DB 2003. Milk byproducts as function ingredients in bakery products. Bev Food World 30: 21-22. Puranik, D. B. 1997. Development of cake mixes using milk by-products as egg substitutes. Ph. D. Thesis. National Dairy Research Institute, Kanral. Raju P. N., Rao, K.H. and Devi N. L. 2007. Development and evaluation of high protein biscuits containing whey protein concentrate, J. Food Sci Technol 21: 236-219. Richert S. H. (1973) Dissertation Abstr. Int. B, 33, 3128. Cited in Dairy Sci. Abstr., 1974, 36: 5992. Singh S., Chauhan G. S., Raghvanshi R., Sharma P, Chauhan O. P. and Bajpai A. (2003) Replacement of Egg Solids with whey protein concentrate and optimization of its levels in cake making. J. Food Sci. Technology 40(386-388). Srivastava, A. K. and Rao, A. P. (1993) Effect of using different sources of milk products on the quality of bread, J. Food Sci. Technol., 30 (2): 109-113. Swaisgood, H.F., 1985. Characteristics of edible fluids of animal orgin: Milk in “Food Chemistry” 2nd Edn. Fenemma O.Ed. Marcel Dekker Inc., New York. Towler, C. (1982) Utilization of whey protein products in pasta. N. Z. J. Dairy Sci., Technol., 17: 229-233. Webb, B. H. 1966. Whey- A low cost dairy product for use in candy. J. dairy sci., 49:1310-1313. Zadow, J. G. and Marston, P.E. (1984) Food Technol. Australia, 36 (6): 278. Cited in IDF Bulletin No. 212, p. 135-137.

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Dr. D.K. Thompkinson Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001 1. INTRODUCTION

Mother milk is the ultimate designer food for the new born infant. Nature has designed human milk for optimal nourishment and growth during infancy and also for supplying certain bio-protective factors that afford protection against commenly occurring infections. However, in the event of lactation failure, insufficient milk secretion and where mothers are infected / suffer with transmittable diseases, infant food as a substitute of breast milk serves as saver of precious life during vulnerable stages of infancy. Most of the breast milk substitutes utilize bovine milk due to its easy availability. Nearly 40-50% of the total calories in infant formula are provided by carbohydrates. Whey, a rich source of lactose, is widely used to enhance carbohydrate content of infant formulations in order to adjust the fat: protein:carbohydrate ratio and in specific, the ratio of casein:whey protein to the level that exist in human milk. Lactose helps in establishing intestinal environment suited to the development of a favourable lactic acid producing flora, which may inhibit growth of pathogenic organism. Apart from this, the minor constituents present in whey play an important role in bestowing nutritional and therapeutic benefits. 2. MODIFICATION OF WHEY

Although lactose is present in both human and bovine milks, the variations in its concentration as well as higher incidence of mono- and oligo- saccharides, imparts a superior status to human milk in infant nutrition. The higher carbohydrate level in human milk plays a significant role in infant nutrition. Human breast-milk contains both digestible and indigestible carbohydrates. For infant formulas and follow-on formulas, on the contrary, only digestible carbohydrates are required and regulated in the infant formulas. It is well documented that lactose is assimilated from the digestive tract more slowly than other carbohydrates. The change of lactose into glucose and galactose when feeding human and bovine milk proceeds so slowly that some lactose remains intact almost up to the last section of the digestive tract (Engfer and others 2000). This is of considerable importance in breast-feeding as it leads to the production of lactic acid in the large intestine under the influence of bifidus bacteria, making the intestinal environment acidic with pH values between 5.50 and 6.00. The acid medium is also conducive to the growth of lactose fermenting rather than putrefactive bacteria, thus decreasing the likelihood of infection. This effect is more remarkable with breast-fed infants. Moreover, the extra lactose present in breast milk is helpful in the synthesis of certain vitamins. Lactose also increases the absorption of calcium and iron. Bovine milk differs from human milk in several aspects. Casein and whey protein ratio and mineral content in bovine milk is higher whereas lactose content is lower as compared to human milk. Therefore, for industrially prepared infant formula the lactose content is raised through addition of rennet whey, however, the resultant product turns out to be higher in mineral content. It is desirable to have an appropriate level of minerals to avoid mineral load on infantile kidney during early years of life. Therefore, it is necessary to modify whey

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APPLICATION OF DAIRY BY-PRODUCTS IN THE FORMULATION OF INFANT FOODS

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before it is ideal for use in infant formula. The proceeding text describes possible technological modifications needed for the processing of whey before its utilisation for manufacture of infant formula. 2.1 Demineralisation

Formulation for physiologically suitable infant foods necessitates reduction of protein and mineral levels from bovine milk. Rich in lactose and whey proteins and containing appropriately low levels of essential minerals, demineralised whey is an ideal ingredient for infant formula. The major contribution, that the reduced mineral whey fraction brings to infant formula are:

• It provides 70% of total carbohydrate requirement. • Helps in achieving adjustment of whey protein to casein ratio. • Provides mineral composition with enhanced Ca and Mg concentration in relation to

sodium and chloride. • Low phosphorous content is achieved with desired Ca:P ratio without excessive calcium

supplimentation. • Provides 40% calories.

Demineralisation of whey can be achieved by ion-exchange or electrodialysis. In ion-exchange, a loss of about 5-30% whey proteins and even their denaturation occurs. Electrodialysis is the preferred technique and depending upon type and freshness of whey and on seasonal variation in ash content of milk, the level of demineralisation varies typically between 88-92%.

In the manufacturing process directed at achieving whey protein :casein ratio in infant formula similar to human milk, Singh and Mathur (1989) performed electrodialysis on the mixture of skim milk and whey (15:85). By mixing appropriate amount of skim milk (to provide casein) and demineralised whey (to provide whey proteins and lactose) a casein:whey protein ratio similar to that of human milk can be achieved. The demineralisation process removes substantial amount of all minerals and helps in adjustment of ratios of several important minerals in the system. Evidences exist that feeding of formula having casein:whey protein ratio similar to human milk provide better growth and nitrogen absorption amongst low birth weight babies during first three months (Berger et al., 1979). Demineralised whey formulae have been available at advanced countries for many years. However, in India, the process could not be adopted due to very high cost of infrastructure involved and availability of sufficient amount of good quality whey. 2.2 Lactose hydrolysis

Lactase non-persistent individuals do not have the ability to synthesize lactase and therefore experience flatulation and diarrhoea on consumion or milk rich in lactose. Infants with such physiological disorders require special formulations having lactose in easily digestible and absorbable form. To achieve this goal it is necessary to break the milk disaccharide into digestible mono-saccharides through hydrolysis using acid or the enzyme ß-galactosidase. The deproteinised whey concentrated to 25-30% total solids is subjected to hydrolysis to attain 50-60% hydrolysis, and then mixed with rest of the mix for preparation of infant formulae. A pilot scale process for enzymic hydrolysis of lactose in skim milk was standardised by Paul and Mathur (1989). They suggested that 20% TS feed was most suitable for hydrolysis as it requires moderate time and

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provides viscosity suitable for spray drying. Paul and Mathur (1991) attempted preparation of low lactose infant formula with lactose hydrolysis level ranging from 20-50% and reported that 35% hydrolysis product was suitable for infant formula. They further studied the effect of processing parameters on protein profile of low lactose infant formula in relation to degree of hydrolysis and reported that β-lactoglobulin was denatured most readily than other components of whey protein irrespective of degree of hydrolysis. Components belonging to casein and whey protein fractions displayed varying but lesser degree of modification in their electrophoretic behaviour. Both casein and whey protein components showed definite evidence of protein aggregation during processing. 2.3 Whey Proteins

Human milk contains higher proportion of whey proteins than casein which markedly affects the curd tension of milk that aids to easy digestibility by infants. Whey proteins have been found to alter iron availability. Human studies show superior bioavailability of iron from human milk as compared to infant formula. This is presumably due to the presence of minor whey proteins and amino acids viz. cystein, taurine etc. which are present in higher concentration in human milk. Zhan and Mohanty (1989) reported that maximal iron bioavailability of 73% was found when diet rich in ferripolyphosphate-whey protein complex was fed to anemic weanling rats, as compared to iron casein complex. A complex of whey protein with ferric iron was 92-100% effective in restoring haemoglobin level in iron depleted rats by making ferrous sulphate readily available through the diet containing this complex. Recently importance of the characteristic amino acid profile of whey protein (α−La) in enhancing the immune system has been shown. It has been demonstrated that addition of whey protein concentrate from bovine source in a 50% ratio to soy protein induces marked increament in immune reactivity in comparison to pure soy protein diet. 2.4 Nutritional and Therapeutic Considerations

It is tempting to postulate that predominance of whey proteins in human milk constitute a characteristic of evolutionary significance by providing enhanced resistance. With the advancement of knowledge in human milk components and technological feasibility of fractionating whey constituents in undenatured form, it is now possible to use various fractions in a most profitable manner so as to provide maximum nutritional and immunological qualities to industrially produced infant formula as a near perfect human milk substitute. 2.4.1 Lactoferrin

The role of lactoferrin in infant feeding is concentrated due to its ability to bind iron and inhibit the growth of enteropathogens. Lactoferrin demonstrate consistent bactericidal activity against Gram negative bacteria (Yamauchi et al., 1993). 2.4.2 Immunoglobulins

At birth neonates are immunologically immature and do not have sufficient self defence mechanism. A number of attempts have been made in the past for the enhancement of immunoglobulins content of infant formulae, but without much technological success. It is imperative that bovine milk antibodies remain intact during technological treatments involved

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during manufacture of infant formula so that it can effectively participate with the pathomechanisms of entropathogens. Further such preparations should be resistant to proteolytic enzymes of the digestive tract. It is for these limitations that such approaches could not be effectively utilized for enhancement of bio-immune characteristics of infant formulae. 2.4.3 Lysozyme

In view of the antibacterial effect and possibly the general immune system of neonates, there has been a considerable interest in developing food applications of lysozyme. It is presumed that hydrolysate of cell walls of other bacteria stimulate the growth of bifidobacteria in the intestinal tract. The concentration of lysozyme in human milk is 3000 times higher which has a marked influence on the bacterial flora of infantile intestine. It has recently attracted renewed interest as a component of antibacterial system of milk. Reports are that rats fed on diet containing lysozyme increases the bifidobacterial population in the intestine thereby bestowing a clinical benefit. Lysozyme seems to be useful in the treatment of anaemia and pneumonia. Thus, application of lysozyme in infant formula could be quite useful in view of its influence under the ecological system of the intestinal tract. 2.4.4 Lactulose

During the early infancy the physiological activity are under-developed and due to no enzymic activity in small intestine, lactulose is transported, without hydrolysis, to large intestine where it promotes growth of selected intestinal microbes, providing clinical benefits against enteropathogens. Addition of 1.2-1.5% lactulose in diet has been found to cause increase of bifidobacteria in large intestine. It is now revealed to be very important humanising factor and is applied for commercial infant food products in Japan. 3. EFFECT OF PROCESSING ON NUTRITIONAL QUALITY OF INFANT

FORMULA

Different minerals display varying degree of diffusion rates during electrodialysis (ED) process. This would have significant effect on the pattern of mineral profile of both skim milk and whey. The levels of Fe, Ca, Na and K decreased progressively with increase in ED membrane surface area in a stream of skim milk having 28% TS. With 51% demineralisation of skim milk, K, Na, Ca, Fe and P, it were observed to be 92.8, 73.2, 36.8, 29 and 27.3% with respect to respectively. Stepwise increase in the membrane area was found to increase extent of depletion significantly. The pattern of diffusion of minerals from whey showed varying rate of diffusion of these minerals. It was also observed that Cu, Mn, and Zn resisted ED process and did not exhibit any depletion.

The diffusion pattern of minerals from skim milk and whey systems had a close resemblance. Higher potassium and lower sodium levels in human milk appears to be physiologically beneficial to the infant. Maternalised infant formula to be prepared from ED processed milk or whey would necessitate exogenous incorporation of potassium to maintain the ratio of K:Na to 3.8:1 as is present in human milk. Similarly, it becomes critical to adjust the ratio of Ca:P in the infant formula to provide physiologically optimum levels. The depletion of Ca is more during ED process and hence, the same need to be added exogeneouly to maintain the optimum ratio of Ca:P in the resultant product.

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Nutritional evaluation of low lactose infant formula based on assessment of PER, NPU and lysine content signify that there is a measurable loss of the nutritional value of the product with progressively higher level of lactose hydrolysis. However, the extent of loss is within the nutritionally acceptable limits for infant formulation. Keeping in view the above parameters for nutritional evaluation, 35% hydrolysis of lactose may be recommended for manufacturing nutritionally adequate formula for the dietary management of lactose intolerant infants. 4. EFFECT OF STORAGE ON PRODUCT QUALITY

The shelf life behaviour of processed food system, such as spray dried maternalised infant formula, is mainly determined by the nature and extent of physico-chemical changes, as governed by temperature and length of storage period. 4.1 Colour

Various colour changes observed during storage as characterised by reflectance spectrophotometric technique revealed that with the progress of storage period violet, blue, greenish blue and bluish green colours deminished whereas, yellowish green, yellow, orange and red hues were found to intensify progressively. These changes in colour are due to certain pigments like melanoidins, malto-5-hydroxymethyl 1,2-furfuraldehyde and furfural alcohol as a sequel to the interaction between proteins and reducing sugars during storage. Paul and Mathur (1992) observed lower reflectance values with the progress of storage period, specially with higher degree of hydrolysis.

4.2 Wettability

The wettability characteristics of infant formula having casein: whey protein ratio similar to human milk has shown a decrease from 43 sec. to 48 sec. during storage of 12 months at 37°C. It was observed that irrespective of lactose hydrolysis the wetting time of low lactose infant formula increased with the progress of storage. However, whey containing infant formula showed better wettability compared to commercial infant formula even after 12 months storage period. 4.3 Sinkability

The sinking characteristics of maternalised infant formula was studied by continuous plotting the percent transmission against the time spectrophotomertrically during storage There was observed a steady increase of transmission values from 4.5% to 10, 30, 36 and 38% during 6, 8, 10 and 12 months respectively. In case of low lactose infant formula it was noticed that irrespective of level of hydrolysis the sinking time of samples increased with the progress of storage. 4.4 Dispersibiliy

The changes in dispersibility behaviour of maternalised infant formula was observed and found to decrease from 92.2% to 87.9% during 12 months of storage. The overall decrease was only 4.3% during the period. This loss was, however, within the limits of BIS standards. The variation in the dispersibility characteristics of infant formula was found to follow a linear trend

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irrespective of degree of hydrolysis of lactose and did not affect the reconstitution property of such infant formulae. 4.5 Solubility index

The solubility index of infant formula with added whey was found to increase steadily with the progress of storage time, but only marginally. Further, when lactose was hydrolysed to different levels highly significant changes in solubility of such infant formula was observed indicating an interdependent phenomenon towards increase of solubility index during storage. 4.6 Free fat

The rate of increase in free fat levels of low lactose infant formula was progressive with the storage period however, the magnitude of changes were not critical from practical point of view. The free fat content was observed to be higher in the later half of the storage period. 5. REFERENCES Kawakami, H.; Hiratsuka, M. and Dosako, S. (1988) Effect of iron saturated lactoferrin on iron absorption. Agric. Biol. Chem., 52: 903. Paul, S.C. and Mathur, B.N. (1989) Development of low lactose infant formula. Ind. J. Dairy Sci.,42: 437. Paul, S.C. and Mathur, B.N. (1991) Effect of processing on the protein profile of low lactose infant formula in relation

to degree of hydrolysis. Ind. J. Dairy Sci., 44: 547. Paul, S.C. and Mathur, B.N. (1992) Development of low lactose infant formula- Physico-chemical charecteristics

during storage. Ind. J. Dairy Sci., 45: 540. Reiter, B.(1985) Protective proteins in milk. Biological significance and expoitation- Lysozyme, lactoferrin,

lactoperoxidase, xanthineoxidase. IDF Bull. No.191 pp. Singh, M.N. and Mathur, B.N. (1989) Mineral modification of buffalo milk for infant formula employing

electrodialysis Part-1 Optimisation of processing variables. Ind. J. Dairy Sci., 42: 1989. Singh, M.N. and Mathur, B.N. (1991) Development of maternalised infant formula Part-V Storage related physico-

chemical changes. Ind. J. Dairy Sci., 44: 614. Thompkinson, D.K.; Sabikhi,L. and Mathur,B.N. (2005) Annual Dairy Resource Book, Vol.1:150. Zhan, D. and Mohanty, A.W. (1989) Bioavailability of iron milk protein complex. J. Dairy Sci., 72: 2845.

8

SECTION - VIII

PACKAGING OF DAIRY BY-PRODUCTS

PACKAGING OF DAIRY BY-PRODUCTS

198

Dr. G.K. Goyal Principal Scientist

Dairy Technology Division, N.D.R.I., Karnal-132 001

1. INTRODUCTION

In modern times packaging has been identified as an integral part of processing in the food industry. Packaging is the science, art & technology of protecting products from the adverse effects of the environment. Packaging is a technique of using the most appropriate packaging media for the safe delivery of the contents from the centers of production to the points of consumption. Packaging of products materially contributes to trade promotion. World wide packaging industry estimated to be $300 billion. In India, around 15,000 units are engaged in packaging industry and the projected growth rate of demand and consumption for packaging is 10%.

2. IMPORTANCE OF PACKAGING OF DAIRY BY- PRODUCTS

The proper packaging of dairy by-products is important not only to preserve edible or industrial quality, protecting nutritive value and savings from wastages, but also for competitive marketing and getting better returns, It acts as the vital link between the manufacturer and consumer for the safe delivery of the by-product through the various stages of storage, transportation and marketing. All the precautions and care taken to produce high-grade dairy by - products will go in vain, unless they are delivered in a fresh, sound and convenient form to the consumer. Here lies the importance of packaging because this loss can be minimized to a great extent by adequate protective packaging to withstand the hazards of storage, transportation, handling and environmental influences. Factors to be taken into consideration for selection of packaging material:

• It must protect and preserve the commodity from the time it is packed to the point of consumption.

• It must be compatible for the chosen selling and distribution system.

• It must be attractive to the consumer, easy to open, store and dispose.

• It must cost not more than the market can bear.

The packages mainly perform three functions viz. to contain, to protect and to sell the product. It is essential to know the nature and composition of the product, its desired shelf life under specified conditions of storage in terms of light, temperature, humidity, presence of oxygen and the types and causes of deterioration including mechanical stress, which the product may undergo during handling and storage. The packaging material must not impart its own odour to the product. It should be inert to food and non-toxic. The consumer expects that package should not only protect the product but also give information about the contents, storage conditions, methods of use, date of manufacture and expiry, price and nutritional considerations.

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3. CHOICE OF AN APPROPRIATE PACKAGING MATERIAL

Choice of an appropriate packaging material is governed by several factors such as:

• The specific sensitivities of the contents, e.g. moisture, oxygen, etc.

• Factors affecting the contents viz. temperature, % RH, pH, and the reaction mechanism involved.

• Weight and shape of the container.

• Effect on filling and sealing speeds.

• Contamination of food by constituents of the packaging material.

• Storage conditions -- How long the product needs to be protected?

• Bio-degradability and recycling potential.

• Many more peculiarities, which could be identified under the following for determining the packaging of beverages:

I. Product range II. Market

III. Consumer needs IV. Operating margins

4. PACKAGING OF DAIRY BY-PRODUCTS 4.1 Whey Products

Whey by-products are generally manufactured at the plant, where whey is mostly produced and processed. Hence, as such it is stored in refrigerated tanks and seldom packaged for sale. Products obtained from whey include whey powder, Whey Protein Concentrates (WPC), lactose, ricotta cheese, whey beverages etc.

4.1.1 Dried Whey

Whey powder can be packed in same form of bulk container used as for packaging of WMP or SMP which are usually of two types: 25 kg multi-wall paper bag with PE liner and 500-l000 kg bulk tin. Greater quantity of powder is packed in multi-wall bags but there is increasing interest in the development of larger packs due to the potential savings in freight and handling costs. The key feature of multi-wall bags is that it is robust, relatively cheap and capable of being kept at high production rates e.g. 9-10 tones/hr being achievable with modern filling-sealing equipments it is very convenient for major end use such as recombining plants and repacking stations. Specifications for multi-bags vary from country to country, but a 4-5 ply bag with a minimum 420 g/m2 basis weight with an inserted PE liner of 15 µm is considered to be adequate. Any of the following constructions have been shown to be adequate for export purposes:

4-ply construction: Outer layer of crepe laminate of 168 g/m2 basis weight; 3 inner plies of 84 g/m2 natural Kraft paper1 inserted liner of 80-90 µm LDPE.

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5-ply construction: One outer layer of 70 g/m2; 1 inner layer of 70 g/m2 plus 15 g/m2 polythene; 3 inner layers of Kraft of 70 g/m2.

Organoleptic properties of whey powder are affected more than color or consistency during storage. Jayaprakash (1992) observed that these changes were more in market Samples stored in PE paper laminate as compared to the samples stored in metalized PET packages or PE pouches. PE/paper laminate is more permeable to oxygen and vapour and this might be the reason of deterioration. He concluded that whey powder should be packed in materials having low OTR and WVTR.

4.1.2 Whey Protein Concentrates (WPC)

Dried WPC can be packed similar to SMP, i.e., the packaging material should be moisture-proof because WPC contains some amount of lactose, which may lump, if it comes in contact with moisture vapors, and some protein-lactose interaction may also take place. 4.1.3 Lactose Powder

Dried lactose powder is non-hygroscopic if all the lactose is in crystalline form. It has no special requirement as such, and so can be packed in bags used for SMP. As Lactose is mostly used for industrial purposes, it is generally packed in bulk packages i.e. Kraft paper bags with polyethylene lining.

4.1.4 Whey Beverages and Drinks

The beverage market is a diverse business involving brands and private label, refillable and recyclable one-way packaging, licensers and licensees, turnkey or industry standard packaging and custom designs for gift-wrapped and commodity favourites. The impact of Efficient Consumer Response (ECR) is increasing and will affect packaging sizes and formats, particularly in respect of secondary and tertiary packaging. Today’s consumers want more than just refreshment from their drinks. They must be functional as well as satisfying. Packaging serves a number of important functions in the beverage industry. It must protect the beverage throughout its lifetime, from the filling of the container to consumer use. Beverage packaging consists of beverage cans, bottles of glass and plastic, pouches and cartons.

Some of these (whey beverage) types of products are manufactured by mixing appropriate fruit juice concentrates with processed whey. Other types include the fermented whey beverages, both alcoholic and non-alcoholic (acidic), carbonated thirst-quenching beverages and whey-based flavoured milk drinks. Whey drinks are very popular in western countries and most of the non-carbonated beverages are sold in tetra-brick or gable top paperboard containers. Various types of packages for whey beverages are:

i. Cans ii. Glass Bottles

iii. PET Bottles iv. Plastics Bottles v. Drink Cartons

vi. Foil Pouches

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4.1.5 Whey Cheeses

The cheeses prepared from cheese or casein whey include, Ricotta, Mysost, Gjetost, Primost etc. These cheeses contain whey proteins, some fat and very high moisture content. The packaging material should have low WVTR and low oxygen transmission rate (OTR) to prevent moisture loss and fat oxidation. The highly soft nature of the product makes it unsuitable for vacuum packaging. Modified Atmosphere Packaging (MAP) may be very successful because of tire ‘cushioning’ effect of the gas. Gas flushing with CO2/N2 mixture has been reported to be successful in prolonging the shelf life of Ricotta cheese. Abrahmsen (1986) recommended hot filling of Norwegian brown goat whey cheeses in aluminum foil or Plastic bags at 75-90ºC. Cheese had to be temporarily supported by rectangular aluminum boxes because of the soft nature of the cheese. Shukla and Brar (1986) reported that plastic, parchment paper; polyethylene and wax-coated papers may be used for packaging of Ricotta cheese. However, wax coated paper checks the hardening of outer surfaces not satisfactorily on storage as compared to polyethylene papers.

4.2 By-products from Skim Milk

The various by-products made from skim milk are casein and co-precipitates. Casein can absorb moisture and becomes soft and gummy, which leads to the deterioration of the quality of the casein. So the packaging material should have a low WVTR, should be tamper-proof dried casein, either ground or ungrounded, is packed in sacks or bags. Burlap sacks lined with closely woven cloth or with heavy paper or heavy three-ply paper bags may be used and the filled containers should be tightly closed and transferred to a clean dry storage room maintained at uniform temperatures. If casein has to be stored for a considerable time, it is best to store it ungrounded or else the rodents prove to be a great nuisance. Mann (1982) reported the use of 300 gauge polyethylene bags for packing and storage of co-precipitates.

4.3 By-Products from Butter Milk

Though there are lot of products that can he made from buttermilk, only few of them are economically viable and produced in substantial quantities in dairy advanced countries important among them are dried buttermilk, casein and beverages. For practical and economic reasons the packaging material used for skim milk may be used for packaging of buttermilk.

4.3.1 Buttermilk Powder

Buttermilk powder is hygroscopic and hence the packaging material must be air tight and

totally moisture proof. The composition of this product is almost similar to skim milk powder (SMP) so the packaging material used is generally the same as that for SMP. The beverages manufactured from buttermilk are almost similar to whey drinks except the fact that they have high acidity. Hence, the packaging used for buttermilk based beverages are almost same as that for whey beverages.

4.4 Ghee Residue

Ghee residue contains fat, denatured protein, burnt lactose and minerals, and considered as highly nutritious. The main defect during the storage of ghee residue is related to the development of tallowiness. If prolonged storage is required, removal of fat by boiling in hot

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water followed by boiling in 10% sodium bicarbonate solution has been recommended (De, 1980). As ghee residue is hardly marketed in a packed form, literature concerning this is scanty. In a study on shelf life evaluation of ghee residue, Subbulakshmi et al. (1990) observed that ghee residue samples packed in plastic, glass and tin container showed no significant changes in free fatty acid content, Peroxide value and Tintometer reading after 90 days of storage. The glass and tin containers are superior packages than plastic containers.

5. REFERENCES Abrahamsen.R.K.1986. Production of brown whey cheese. Bulletin of IDF, No. 202: 125-130. De. S. 1980. Outlines of dairy technology. Oxford university press. New Delhi. Jayaprakash. H.M. 1992. Membrane processing applications for producing whey powder and WPC. Ph.D.Thesis

submitted to NDRI Deemed Univ., Karnal. Mann. R.S. 1982. Technological aspects of high medium and low calcium co-precipitates from buffalo milk. Ph.D

thesis submitted to Kurukshetra Univ., Kurukshetra. India. Subbulaksmi. G. Periwal.S. And Ravi. P.J.1990. Studies on shelf life and utilization of ghee residue ,Indian

J.Food sci. & Technol., 27(3): 165-166. Shukla. F.C. and Brai. M.K. 1986. Manufacture and significance of ricotta cheese. Indian j. Dairy Sci. 39(4): 347-

355.

Dr. P.T. Dhole Prof., Department of Animal Sci & Dairy Sci. Mahatma Phule Krishi Vidyapeeth Rahuri-413 722 (Maharashtra)

Mr. K.B. Suresha Asst. Prof. of DT PHT Scheme, UAS, Gandhi Krishi Vignana Kendra Bangalore-560 065 (Karnataka)

Bhanudas M. Patil Sr. Research Assistant Department of Food Engineering College of Food Technology M.A.U. Parbhani-431 402 (Maharashtra)

Mrs. Geeta Chauhan Scientist (Senior Scale) Division of Livestock Products Technology Indian Veterinary Research Institute, Izatnagar, Bareilly - 243 122 (UP)

Devaraja Naika.H, Asst.Professor Dept. of Livestock Products Technology Veterinary College, KVAFSU, BIDAR -585 401 (KARNATAKA)

Dr. Sunil Kumar Asstt. Professor Department of Livestock Products Technology C.V.Sc. & A.H., NDUAT, Kumarganj Faizabad-224 229 (U.P.)

Dr. K.D. Chavan C/o Prof. of Animal Sci & Dairy Sci. Agriculture College Dairy Farm Behind Mariai Gate, Pune-Mumbai Road, Khadki, Pune-441003 (Maharashtra)

Mr. Hiral Kumar M. Modha Asstt. Professor, Dairy Technology Dept. S. M. C. College of Dairy Science, AAU, Anand -388 110

Dr. Bhimsen Department of Animal Husbandry & Dairying R.B.S. College, Bichpuri Agra (U.P.)

Mr. F.L Pathan Asstt. Prof. Maeer’s MIT College of Food Technology Loni-Pune-412 201 (Maharashtra)

Dr. Ram Binay Singh Reader, A.H & Dairying Udai Pratap Autonomous College, Varanasi-221 002 (U.P.)

Dr. Mojpal Singh Reader Chhotu Ram (P.G.) College Muzaffar-Nagar-251 001 (U.P.)

Dr. Surajit Mandal Scientist Dairy Microbiology Division NDRI, Karnal-132 001

Mr. R.J. Desale E-35 (750), MPKV Rahuri-413 722 Distt. Ahmednagar (Maharashtra)

LIST OF COURSE PARTICIPANTS LIST OF COURSE PARTICIPANTS

Mr. Maske Damodhar Narayanrao Dy. Director Research (Ani. Science.) Office of Directorate of Research M.A.U. Parbhani-431 402 (Maharashtra)

Dr. B. Dhanalakshmi Associate Professor Department of Dairy Science Madras Veterinary College Chennai-600 007

Dr. S.G. Narwade Asstt. Professor Aditya Agricultural College Telgaon Road Beed-431 122 (Maharashtra)

Mr. Saraff Sripad Lecturer in Dairy Technology College of Dairy Technology Govt. College Campus SVVU, Kamareddy-503 111 Distt Nizamabad (Andhra Pradesh)

Mr. Lehri Singh Technical Officer Quality Control Lab. Dairy Technology Division NDRI, Karnal-132 002

Prof. Kailash S. Gadhe Dept. of Food Chemistry and Nutrition College of Food Technology, M.A.U. Parbhani-431 402 (Maharashtra)