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Microbiologicalandbiochemicalcharacterizationofseera:atraditionalfermentedfoodofHimachalPradesh
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INTERNATIONAL JOURNAL OF FOOD ANDFERMENTATION TECHNOLOGY
VOL. 2, NO. 1, JUNE 2012
PRINT ISSN NO. : 2249-1570ONLINE ISSN NO.: 2277-9396
Editor-in-Chief
Prof. VK JoshiProfessor and Head,
Fermentation Technology Lab, Department of Food Science and TechnologyDr YS Parmar University of Horticulture and Forestry
Nauni, Solan, Himachal Pradesh, IndiaE Mail:[email protected]
NEW DELHI PUBLISHERS90, Sainik Vihar, Near Lakshmi Narayan Mandir, Mohan Garden, New Delhi – 110059 (India)
Email: [email protected] Website: www.ndpublisher.inPhone: 91-11-25372232 Mobile: 9971676330, 9582248909
INTERNATIONAL JOURNAL OF FOOD ANDFERMENTATION TECHNOLOGY
ABOUT THE JOURNAL
The Journal would publish research papers on all the subjects related to the Food and Fermentation Technology. All aspects offood and fermentation technology would be considered. Papers on bio-technology, bio-chemical, toxicological aspects havingdirect bearing or are related with food would be welcomed. R & D work related to the fermentation covering microbiology, bio-chemical, genetics, indigenous fermented foods, toxicology or nutritive value would also be included. Articles highlighting thefood standards and safety issues will be given special emphasis. Preparation and evaluation of alcoholic beverages would be animportant aspect of the articles published. The review on any aspect of food processing, composition, nutrition, and fermentationwould be considered. Management of food processing industrial waste would be an integral component of the papers published.The other aspects of food processing like low temperature preservation, by dehydration, thermal processing, irradiation, emergingtechnologies viz., ohmic preservative, pulse electric field, high pressure preservation, enzymology, microbiological quality, foodsafety and standards and food engineering, will also be considered.
International Journal of Food and Fermentation Technology, a half yearly journal, publishes original research papers, short
communications and review papers on topics which include in brief :
• Food microbiology • Bio-chemical aspects of food • Genetic and genetically modified foods
• Enology • Indigenous fermented foods • Toxicology, safety and quality • Food processing
• Fermentation technology • Food engineering • Quality assurance • Food preservation • Food additives
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NEW DELHI PUBLISHERS90, Sainik Vihar, Near Lakshmi Narayan Mandir, Mohan Garden, New Delhi – 110059 (India)
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EDITORIAL BOARD
Editor-In-chief
Prof. V.K. JoshiProfessor and Head,
Fermentation Technology Lab,Department of Food Science and Technology,Dr YS Parmar University of Horticulture and
Forestry, Nauni, Solan, Himachal Pradesh, [email protected]
Dr (Mrs) Sunita Garg,Editor, Natural Product Radiance,NISCAIR, New Delhi, India [email protected]
Dr ( Mrs) Devina Vaidya Department of Food Science and TechnologyDr. Y.S. Parmar University of Horticulture andForestry, Nauni, Solan, Himachal Pradesh, [email protected]
Dr NS ThakurDepartment of Food Science and TechnologyDr. Y.S. Parmar University of Horticulture andForestry, Nauni, Solan, Himachal Pradesh, [email protected]
Dr JK GuptaDepartment of Entomology and ApicultureDr YS Parmar University of Horticulture and ForestryNauni-Solan , Himachal Pradesh, [email protected]
Dr B.L.AttriCentral Institute of temperate Horticulture,Regional Station ,Mukteshwar,-Kunam,Nainital, Uttarakhand [email protected],
Dr Wamik AzmiDepartment of Biotechnology,Himachal Pradesh University,Summer Hill, Shimla, Himachal Pradesh, [email protected]
Dr Neerja S. RanaDepartment of Basic SciencesDr. Y.S. Parmar University of Horticulture andForestry, Nauni-Solan, Himachal Pradesh, [email protected]
Dr Shashi BhushanDivision of Biotechnology,Institute of Himalayan Bio-resource Technology (CSIR),Palampur,DisttKangra, Himachal Pradesh, [email protected],
Dr Satish Kumar SharmaG B Pant University of Agriculture & Technology, Hill campus,Ranichauri, Distt Tehri-Garhwal,Uttarakhand, [email protected]
Dr Om Prakash ChauhanScientistFruits and Vegetables Technology DivisionDefence Food Research LaboratorySiddarthanagar, Mysore, [email protected]
Dr PS PanesarAssociate ProfessorBiotechnology Research LaboratoryDepartment of Food Engineering and TechnologySL Institute of Engineering and Technology,Longowal, Punjab,[email protected]
Executive Editorial Board
Dr Sumit Arora ( Dairy Science)Dairy Chemistry Division, National Dairy Research InstituteKarnal,[email protected]
Prof Tek Chand BhallaDepartment of Biotechnology,Himachal Pradesh University,Shimla, Himachal Pradesh, [email protected]
Dr M C Pandey, ( Meat and Meat products)Deptt of Freeze Drying and Animal Product Technology,Defense Food Research lab, Mysore, [email protected]
Dr V M Pratape, (Grain Science and Technology),Deptt of Grain Science and Technology,CFTRI Mysore, India [email protected]
Dr Pura Naik J (Plantation crops)Division of Plantation, Spices and Flavour Tech.,CFTRI Mysore, India [email protected]
Professor Pradeep KhannaCoordinatorCollege of Basic Science, PAU Ludhiana, Punjab, [email protected]
Dr Y S Dhaliwal ( Food and Nutrition)Head, Department of Food Science and Nutrition, College of HomeScience, CSK HPKV, Palampur , Himachal Pradesh, [email protected]
Dr R.S. Singh ( Food fermentation and enzyme technology)Department of Biotechnology,Punjabi University, Patiala, Punjab, [email protected]
Dr SS KanwarDepartment of Microbiology, CSK HPKV Palampur, Distt KangraHimachal Pradesh, India [email protected]
Dr. RC RayPrincipal Scientist (Microbiology)Regional Centre of Central Tuber Crops Research InstituteDumuduma Housing Board, Bhubaneswar, Orissa, India
Dr. Rintu Banerjee ( Microbial Technology) Microbial Biotechnology and Downstream ProcessingLaboratoryAgricultural & Food Engineering DepartmentIndian Institute of Technology, Kharagpur ,West Bengal, India rin_ [email protected]
Dr Eveline Bartowsky (Wine Microbiology)The Australian Wine Research InstituteP.O. Box 197, Glen Osmond, SA, 5064, [email protected]
Dr L. Rebordinos ( Food Microbiology)Laboratorio de Microbiología y Genética.Facultad de Ciencias del Mar y Ambientales.Universidad de Cádiz. Polígono del río SanPedro. 11510 Puerto Real, Cádiz, [email protected]
Dr Aline Lonvaud (Wine and Brandy)Faculty of Enology, University Victor SegalenBordeaux 2, [email protected]
Dr Creina S. Stockley ( Wine and Health)The Australian Wine Research Institute [email protected]
Dr M. Remedios MarínUniversidad Publica de Navarra NafarroakoUnibertsitste Publikoa Area de Tecnologia deAlimentos Universidad Publica de NavarraCampus Arrosadia S/N 31006 Pamplona, (Navarra), [email protected]
Dr Philippe JeandetLaboratory of Enology and Applied Chemistry,Unité de Recherche sur la Vigne et le Vin deChampagne, Research Unit N°2069 University ofReims, Faculty of Science, [email protected]
Dr Luca CocolinDipartimento di Scienze degli Alimenti,Università degli studi di Udine,Facoltà di Agraria, via MarangoniUdine, [email protected]
Dr Ginés NAVARRODepartamento de Química Agrícola,Geología y Edafología, Facultad de Química.Universidad de Murcia. Campus Universitario deEspinardo, Murcia. Spain. [email protected]
EDITORIAL ADVISORY BOARD
Contents
International Journal of Food and Fermentation TechnologyVol. 2 No. 1, June 2012
Review paperA Panorama of lactic acid bacterial fermentation of vegetables 1-12V.K. Joshi and Somesh Sharma
Research PaperApplication of thermostable ααααα-amylase in Streptomyces erumpens liquefaction of cassava bagasse for production of lactic acid 13-18Ramesh C. Ray and Shaktimay Kar
Changes in phytochemicals during fermentation of wine grapes 19-25A.K. Sharma, S.V. Navale, S.N. Aute, G. S. Karibasappa, D. P. Oulkar and P. G. Adsule
Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach 27-36Balvir Kumar, P. P. Balgir and B. Kaur
Effect of growth conditions on extracellular ααααα-amylase production by Bacillus thuringiensis 37-41V.R. Tembhurkar, M.K. Bannatwala, S.S. Udare and M.G. Kalyankar
Design of cascade membrane filtration process for clarification of whey proteins and membrane fouling 43-48Pranav Kaushik Pidatala and Senthil R. Kumar
Microbiological and biochemical characterization of Seera: A traditional fermented food of Himachal Pradesh 49-56Savitri, N. Thakur, D. Kumar and T.C. Bhalla
Preparation and evaluation of Mahua (Bassia latifolia) Vermouth 57-61Preeti Yadav, Neelima Garg and Deepa Dwivedi
ααααα-amylase production from Endomyces fibuliger – an indigenous yeast isolate of Western Himalayas 63-69Keshani Bhushan, Anamika Jain, O.P. Sharma, B. Singh and S.S. Kanwar
Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7 71-79Baljinder Kaur and Rajinder Kaur
Effect of nitrogen source on the fermentation behaviour of musts and quality of wine from two Cvs. of pineapple 81-86B.L. Attri
Research NoteEffect of Solid state fermentation and yeast species on composition of apple pomace: Application of PCA 87-91V.K. Joshi and D.K. Sandhu
Processing potential of newly introduced Amla cultivars grown in lower Himalayan Region of Himachal Pradesh 93-97Manisha Kaushal and Shashi K. Sharma
Evaluation of the stability of plum anthocyanin powder in RTS based model solution 99-101M. Preema Devi, V.K. Joshi and Y. Indrani Devi
Book Review 103
From the Desk of Editor- in-Chief
It is with great pleasure and a sense of fulfilment to state that we have successfully brought out two issues ofinaugural volume of our journal “International journal of Food and Fermentation Technology” during 2011 tocater to the needs of food scientists and R and D workers, and the academicians in the fields. The inauguralissue of the journal was released by the honourable Vice-chancellor of Dr YS Parmar university of Horticultureand Forestry, Nauni, Solan (India) Dr K.R.Dhiman on 26th June 2012 in a function held in the university to markthe beginning of the ICAR sponsored training on “Advance in Fruit and Vegetable Processing and Preservation”with me as a Course Director.
I am equalty happly to inform you that the journal has received an overwhelming response from the scientistsand academician from the field of food and fermentation technology.The journal during the past one year haspublished papers on various aspects of food technology including food fermentation technology. Form thisissues, we could be achieved only with the help of all the members of editorial board, the contributors and thepublisher. I hope you would have relished the style, general get-up, cover page, coverage of the journal; scientificand technical contents, with an attractive of look A4 Size format. At the same time, we would welcome suggestionsfrom the readers, contributors and peers of the field. Now, I am placing before you the first issue of Vol. 2Number 1of the journal. I would like to express my deep gratitude to all those who have extended their help,guidance and co-operation in bringing out this issue.
I siege this opportunity to request all the members of editorial board to exert more so that the journal getshigh recognition among the peers and we are able to get the NAAS ratings to the journal. I am sure through yourefforts we would get more number of papers of good quality. They should themselves contribute papers of highquality to encourage others to do so. From this issue, we are introducing conceptual editorial for which I invitethe members of editorial board to contribute liberally. Certainly, there would not be any page charges from themfor this contribution. The scientists abroad can certainly contribute to improve the quality of this journal being ininfancy. On behalf of the editorial board and the publisher, I assure the readers and the contributors that wewould strive hard to ensure high level of scientific integrity, transparency, impartiality and accuracy in selectionof articles and finally, production of the journal. I earnestly call upon all the scientists of food and fermentationtechnology to contribute their valuable research articles to the journal as a vehicle of transmission of theirscientific findings. The contributors of review or mini-review can consult the editor- in- chief, member of theeditorial board for the topic of review to contribute. At the same time, I must express very clearly that justbecause of some page charges we will not accept and publish substandard articles, but only peer reviewed andaccepted papers would be published. I understand very well that the final scientific quality of any journal wouldbe determined by the type of papers submitted to the journal so, the cooperation of all would be a pre-requisite inthis regard and would be welcomed from the core of my heart.
V.K. Joshi
Modern Post-Harvest TechnologyA Panacea for Developing Countries
The Green Revolution and subsequent efforts made through the application of science and technology for increasing foodproduction in India and world have brought self-reliance in food. With advent of advanced agriculture practices, the production ofvarious crops have increased linearly. This phenomenon has been recorded not only from the developed countries but also in thedeveloping nations of the world. At present, the world production of wheat is 653654525 MT, maize 840308214 MT, rice andpaddy 696324394 MT, Buffalo milk, whole, fresh milk 92473371 MT, Cow milk, whole, fresh 600838992 MT, Indigenous cattlemeat 63782689 MT, Indigenous pig meat 109100198 MT, Indigenous chicken meat 85860953 MT, Indigenous sheep meat8689557 MT, fruits 609213509 MT, vegetables 965650533 MT. Out of these, milk, meat, fruits and vegetables highly perishablecrops. Food being a living commodity respire and therefore, is liable to be spoiled. The major causes of spoilage of food includespoilage by microorganisms, enzymes of microorganisms or the food. The non-enzymatic or purely chemical reactions, environmentalconditions. After the crop is harvested, these factors lead to a considerable postharvest spoilage. In the developed countries, thequantam of spoilages is bare minimum but in the developing countries, like India. The postharvest losses are staggering high.These have been estimated to be as high as 25-30% leading to a loss of Rs. 52,000 crores annually. It may be astonishing but isa fact that the United Kingdom produces the amount of the fruit crop equal to what India wastes. The major difference lies in lackof proper infrastructure in the developing countries in contrast to the developed nations.
Postharvest handling is the stage of crop production immediately following harvest. It largely determines the final quality andit includes cooling, cleaning, sorting and packing. After harvest, foods (e.g. fruits, vegetables, milk, meat, fish) are liable toaccelerated physiological, chemical, and microbial processes that invariably lead to deterioration and loss of wholesomeness. It isthen, necessary to institute some measure of processing such as reduction in moisture content, denaturation of endogenous
Conceptual Editorial
Figure 1: World production of various agricultural commoditiesSource: FAO, Stat: 2010
enzymes and microorganisms, or packaging in order to curtail the loss of perishables. Utilizing improved postharvest practicesthus often results in reduced food losses, improved overall quality and food safety, and higher profits for growers and marketers.The most important goals of post-harvesting handling are to keep the product cool, to avoid moisture loss and slow downundesirable chemical changes, and avoiding physical damage such as bruising, to delay spoilage. The way the grains are storedafter harvest becomes the major cause of contamination with fungi some of which are producer of toxin like aflatoxin. The fruitlike apple after contamination has a toxin called patulin, if not stored properly. Sanitation is also an important factor, to reduce thepossibility of pathogens that could be carried by fresh produce, for example, as residue from contaminated washing water.Consumption of such food results in the diseases of gastro-intestinal track which sometimes prove to be fatal.
Postharvest loss reduction technology encompasses the usage of optimum harvest factors, reduction of losses in handling,packaging, transportation and storage with modern infrastructure machinery, processing into a wide variety of products, homescale preservation with low cost technology. Use of thermal processing, low temperature storage drying chemical and biologicalreactions coupled with other preservation techniques are applied to enhance the storability of the perishables.
There are different constraints for postharvest management like: large number of small and marginal farmers with primitivesystem of cultivation, poor infrastructure in terms of handling, transport, storage, processing and marketing, lack of adequate qualitysystems and procedure like grading and sorting, hot and humid climates, cost of installation of post-harvest treatment facilities isexpensive, lack of awareness training for the rural farmers and inconsistency in supply due to seasonality of the produce.
Adoption of latest techniques could make available a large quantity of food by avoiding losses and provide quality food andnutrition, more raw materials for processing. Postharvest technology has the capability to meet food requirement of growingpopulation. By adopting improper and inefficient methods of storage we are losing a substantial portion of production. If bettermethods of processing and storage are adopted, the losses could be reduced to a large extent. The developing countries needproper infrastructure for this and a strong will to adopt modern postharvest technology to save the wastage. Thus, it couldcertainty prove to be a panacea for the problems faced by the developing countries.
V K Joshi
Figure 2: Production of various agricultural commodities in IndiaSource: FAO, Stat: 2010
Intl. J. of Food. Ferment. Technol. 2(1): 1-12, June, 2012
A Panorama of lactic acid bacterialfermentation of vegetables
V.K. Joshi1* and Somesh Sharma2
1Department of Food Science and Technology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, HimachalPradesh,India2Department of Biotechnology, School of Bioengineering and Food Technology, Shoolini University, Bajhol, Solan, HimachalPradesh,India
*Email: [email protected]
Paper no: 31 Received: 14 February,2011 Received in revised form: 19 April, 2012 Accepted: 17 May, 2012
Abstract
Vegetables occupy a prominent position in our diet and form the most nourishing diet.Preservation ofvegetables is carried out to prevent their postharvest losses of functional quality whilst providing safeand stable products. In general, preservation process consists of a combination of mild heat stress andlow concentration of chemical preservatives to control the food spoilage and the growth of pathogenicspore- forming bacteria. But, consumer demand today is for natural and minimally processed foods, witha fresh like appearance and taste, easy-to-eat and with high level of safety.It is also dependent on a widerange of technologies to ensure that food is maintained at an acceptable level of quality from the time ofmanufacture till the time of consumption. As a result, research and development of new products isleading to the reduction or even replacement of heat treatments and encouraging the use of traditionalpreservatives as these are capable of assuring the sensory and nutritional properties of the productwithout reducing food safety. Non-thermal preservation methods are thus gaining interest as alternativetreatments in the food industry due to their capability of assuring the quality and safety of foods.Among them, use of natural antimicrobials from plants (essential oil) and microorganisms (LAB andBacteriocins) have gained wide range of popularity as bio-preservatives. Traditionally, microorganismshave been used in fermentation from ages but now modern large scale production of foods exploits useof defined strains to ensure consistency and quality in the final product. The biopreservation offerpotential application in food preservation and helps in reducing the addition of chemical preservatives aswell as the intensity of heat treatment, resulting in foods which are more naturally preserved and richer insensory and nutritional qualities. Beside this, biopreservation also provides health benefits such asreduction in cardiovascular diseases, improving gastro intestinal microorganisms and immune system,helps in curing diarrhea and lowering cholesterol in the consumers of lactic acid fermented foods. Thepreservation is accomplished through the production of antimicrobial substances such as lactic acid,diacetyl, hydrogen peroxide, ethanol reutrin and bacteriocin. The bacteria from the genera Lactobacillus,Leuconostoc, Pediococcus and Streptococcus are the main species involved in the lactic acid fermentation.Thus,the lactic acid fermentation of vegetables is an important method of bio-preservation and providinghealthful foods to the consumers.
©2012 New Delhi Publishers. All rights reserved
Keywords: Vegetables,Lactic acid bacteria, LAB fermentation, Biopreservation, Bacteriocin
Review paper
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Vegetables form the most nourishing diet, especially to toneup our digestive system and for providing vigour. Regular useof vegetables, supplies many of the essential health buildingand protective substances, such as vitamins and minerals.India ranks second in vegetable production. Therefore,theabundant production of vegetables during the season, resultsin a glut in the market. Consequently, a large quantity ofvegetables gets spoiled resulting in a considerable loss ofnatural resources. Therefore, preservation can be the only toolto save the wastage of vegetables and make them available inoff-season.
Fermentation is one of the oldest forms of food preservationin the world. Earlier, the term ‘Fermentation’ was used for theproduction of alcoholic beverages like wine but it encompassesall the foods made by the application of microorganismsincluding lactic acid bacteria (LAB) or their enzymes. Lacticacid fermentation can be one of the methods to preserve thesevegetables and to provide variety to the diet in the form ofdiversified products and is employed throughout the world, inconjunction with chemical preservation, using salt and acid.Until the development of curing and freezing during the past180 years, lactic fermentation (LA) was one of the mostimportant method for preserving foods (Joshi et al., 2011) Inconsistence with the changing life style of consumers attitudetowards food products with fresh and convenience food, lacticacid fermentation provides food with diversified taste andflavour characteristics.
The Chinese were the first to ferment the vegetables and thereis evidence to show that they had prepared such vegetablesat the time of building the great wall of China (Pederson, 1971).Today, because of the development of efficient heat sterilizingand refrigeration systems, lactic acid fermentation, to someextent has lost its significance as a food preservation methodin industrialized countries, but is being used as a tastediversification tool. It is extensively used in developingcountries, but is gaining importance more in developedcountries as a food with therapeutic value. It is true especiallyfor vegetables, where either natural fermentation orfermentation by pure culture of lactic acid bacteria is practicedeven in the developed countries (Pederson, 1971; Fleming andMcfeeters, 1981; Anderson et al., 1990).
Role and advantages of Vegetables Fermentation
Fermented foods including fruits and Vegetables play animportant role in providing food security, enhancing livelihoodsand improving the nutrition and social well being of millions ofpeople around the world, particularly the marginalized andvulnerable groups (Table 1). This is achieved through improvedfood preservation that combines salting to selectively controlthe microorganisms and fermentation to stabilize the treated
materials. Lactic acid fermentation is used for commercial bulkstorage, to increase the availability of seasonal vegetablesand to obtain desired sensory product quality (Plate 1.). Lacticacid fermentation removes fermentable sugars, prevents growthof pathogenic microorganisms, stabilizes the product andenhances the flavour (Fleming et al., 1983). Although mostvegetables can be lactic acid fermented, cabbage, olives garlic,red beet, gourd, mixture of cabbage, carrot, onion, red beetand cucumber are the vegetables that are fermentedcommercially in large volumes for human consumption(Fleming, 1982; Karovicova and Kohajdova, 2005; Sharma,et al., 2011). These vegetable contain sugars and nutritionallyadequate substances as substrate for the growth of lactic acidbacteria and other organisms. Recently, lactic acid fermentationhas been used in vegetables such as lupins, peas, lentils,cassava, carrot and radish to achieve technological ornutritional advantages (Kim et al., 1987 and Buckenhuskes,2001). Besides lactic acid fermentation of vegetable juices,applied as a preservation method for the production of finishedand half-finished products, is again being ranked as animportant technology and it is being further investigatedbecause of the growing amount of raw materials processed inthis way in the food industry. In Korea, fermented vegetablesknown as kimchi is an almost ubiquitous accompaniment tomeals (Adams and Moss, 1996; Ha Jae-Ho et al., 1989). Chinesecabbage or radish is generally used as a source vegetable forkimchi, adding various ingredients such as red pepper, garlic,
Plate 1: Fermented vegetables
A Panorama of lactic acid bacterial fermentation of vegetables
3
onion, ginger, jeotkal (salted and fermented sea foods) (Haweret al., 1988). Lactic acid fermentation reduces the pungency ofradish kimchi (Kim and Rhee, 1993). It was once the onlymethod for successful preservation of cucumbers pickling inbrine. It increases the range of raw materials that can be usedto produce edible food products and removing anti- nutritionalfactors to make food safe- to- eat. It is definitely a cheap andenergy efficient means of preserving perishable raw materials
Table 1: Role of vegetable fermentation
• Improving food security• Increasing income and employment• Improving nutrition• Providing Medicinal benefits• Creates new products for new markets• Develop characteristic sensory properties
There are several options for preserving fresh vegetablesincluding drying, freezing, canning and pickling. However,many of these are inappropriate for use on the small-scale indeveloping countries. Freezing of vegetables is noteconomically viable at the small-scale. Fermentation requiresvery little sophisticated equipment, either to carry out thefermentation or for subsequent storage of the fermentedproduct. Many fermented foods have played a very significantrole in preserving food to enhance food security. About 60%of the fermented foods of Sudan are famine or survival foods.Gundruk is a very important food product in Nepal ensuringfood security for many Nepali communities especially in remoteareas. It is served as a side dish with the main meal and is alsoused as an appetiser in the bland, starchy diet.
Many vegetables contain naturally occurring toxins and anti-nutritional compounds. These can be removed or detoxifiedby the action of micro-organisms during fermentation. Forinstance the fermentation process that produces the Sudaneseproduct Kawal removes the toxins from the leaves of Cassiaobtusifolia and fermentation is an important step in ensuringthat cassava is safe to eat. The production of fermentedvegetable products provides income and employment tomillions of people around the world.
The optimum health and nutrition of individuals is dependentupon a regular supply of food and a balanced diet. When dietsare sub-optimal, the individual’s capacity for work andachievements are greatly reduced. The most vulnerable groupsare women, children and weaning infants. Availability of food,dietary restrictions and taboos, misconceptions, limited timeavailable for feeding or eating contribute to create a group ofindividuals who are nutritionally disadvantaged.
Approximately, 30% of women consume less than their dailyrequirements of energy and at least 40% of women world-widesuffer from iron-deficiency anaemia. Fermentation enhancesthe nutritional value of a food product though increasedvitamin levels and improves digestibility, especially of somelegumes, enriches products with desirable microbial metabolitese.g. lactic acid or amino acids.
There are many traditional beliefs about the medicinalproperties of fermented food products. The Fur ethnic groupin Sudan strongly believe that the consumption of fermentedfoods protects them from diseases (Dirar, 1992). Koumiss (afermented milk product in Russia) has been used to treattuberculosis. Pulque (a fermented fruit sap) is felt to havemedicinal properties in Mexico. The beneficial health effectsof lactic acid bacteria on the intestinal flora are well documented(Ottogalli and Galli, 1997). Substances in fermented foods havebeen found to have a protective effect against the developmentof cancer (Frohlich et al, 1997). A study in Tanzania has shownthat children fed with fermented gruels had a 33% lowerincidence of diarrhoea than those fed unfermented gruels,owing to the inhibition of pathogenic bacteria by lactic acidforming bacteria (Svanberg, 1992).
Technology of Vegetable Fermentation
In vegetables, lactic acid fermentation is commonly employedfor the effective utilization of vegetables and providingdiversified taste and flavoured vegetable products. Thefermentation in vegetables is usually carried out by a class ofbacteria called Lactic acid bacteria.
Lactic Acid Bacteria
The lactic acid bacteria are a group of Gram positive bacteria,non-respiring, non-spore forming, cocci or rods, which producelactic acid as the major end product of the fermentation ofcarbohydrates. They are the most important bacteria indesirable food fermentations, being responsible for thefermentation of sour dough bread, sorghum beer, all fermentedmilks, cassava (to produce gari and fufu) and the most “pickled”(fermented) vegetables. Historically, bacteria from the generaLactobacillus, Leuconostoc, Pediococcus and Streptococcusare the main species involved. Several more have beenidentified (Table 2), but they play a minor role in lacticfermentation.
Lactic acid bacteria carry out their reactions - the conversionof carbohydrate to lactic acid plus carbon dioxide and otherorganic acids - without the need for oxygen. They are describedas micro-aerophilic as they do not utilise oxygen. Because ofthis, they do not cause drastic changes in the composition ofthe food. Some of the menber of lactic acid family are homo-
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Joshi and Sharma
fermentative, i.e. they only produce lactic acid, while othersare hetero-fermentative and produce lactic acid plus othervolatile compounds and small amounts of alcohol.Lactobacillus acidophilus, L. bulgaricus, L. plantarum, L.caret, L. pentoaceticus, L brevis and L. thermophilus areexamples of lactic acid-producing bacteria involved in foodfermentations. All species of lactic acid bacteria have theirown particular reactions and niches, but overall, L. plantarum–a homo-fermenter produces high acidity in all vegetablefermentations and play the major role. All lactic acid producersare non-motile gram positive rods that need complexcarbohydrate substrates as a source of energy. The lactic acidthey produce is effective in inhibiting the growth of otherbacteria that may decompose or spoil the food. Because thewhole group are referred to as ‘lactic acid bacteria’ it mightappear that the reactions they carry out are very simple, withthe production of one substrate. However,the lactic acidbacteria are a diverse group of organisms with a diversemetabolic capacity. This diversity makes them very adaptableto a range of conditions and is largely responsible for theirsuccess in acid food fermentations. Despite their complexity,the whole basis of lactic acid fermentation centres on the abilityof lactic acid bacteria to produce acid, which then inhibits thegrowth of other non-desirable organisms. Species of the generaStreptococcus and Leuconostoc produce the least acid. Nextare the heterofermentative species of Lactobacillus whichproduce intermediate amounts of acid, followed by thePediococcus and lastly, the homofermenters of theLactobacillus species, which produce most of the acid.Homofermenters, convert sugars primarily to lactic acid, whileheterofermenters produce about 50% lactic acid plus 25% aceticacid and ethyl alcohol and 25% carbon dioxide. The othercompounds are important as they impart particular tastes andaromas to the final product. Leuconostoc mesenteroides is a
bacterium associated with the sauerkraut and picklefermentations. This organism initiates the desirable lactic acidfermentation in these products. It differs from other lactic acidspecies in that it can tolerate fairly high concentrations of saltand sugar (up to 50% sugar). L. mesenteroides initiates growthin vegetables more rapidly over a range of temperatures andsalt concentrations than any other lactic acid bacteria. Itproduces carbon dioxide and acids which rapidly lower the pHand inhibit the development of undesirable micro-organisms.The carbon dioxide produced replaces the oxygen, making theenvironment anaerobic and suitable for the growth ofsubsequent species of lactobacillus. Removal of oxygen alsohelps to preserve the colour of vegetables and stabilises anyascorbic acid that maybe present. Organisms from the Grampositive Propionibacteriaceae family are responsible for theflavour and texture of some fermented foods, especially Swisscheese, where they are responsible for the formation of ‘eyes’or holes in the cheese. These bacteria break down lactic acidinto acetic, propionic acids and carbon dioxide.
Lactic Acid Fermentation
The pathways of lactic acid production differ for the twotypes of fermentation i.e., the homofermenters and theheterofermenters. Homofermenters produce mainly lactic acid,via the glycolytic (Embden–Meyerhof) pathway).Heterofermenters produce lactic acid plus appreciable amountsof ethanol, acetate and carbon dioxide, via the 6-phosphoglucanate/phosphoketolase pathway. The glycolyticpathway is used by all lactic acid bacteria except Leuconostoc,group III Lactobacilli, oenococci and weissellas. Normalconditions required for this pathway are excess sugar andlimited oxygen. Axelsson et al (1998) gives an in-depth accountof the biochemical pathways for both homo- and hetero-fermenters.
Table 2: Major lactic acid bacteria in fermented plant or vegetable products
Homofermenter Facultative homofermenter Obligate heterofermenter
Enterococcus faecium Lactobacillus bavaricus Lactobacillus brevisEnterococcus faecalis Lactobacillus casei Lactobacillus buchneriLactobacillus acidophilus Lactobacillus coryniformis Lactobacillus cellobiosusLactobacillus lactis Lactobacillus curvatus Lactobacillus confusesLactobacillus delbrueckii Lactobacillus plantarum Lactobacillus coprophilusLactobacillus salivarius Lactobacillus sake Lactobacillus fermentatumPediococcus pentocacus Lactobacillus sanfranciscoStreptococcus thermophilus Leuconostoc dextranicumPediococcus acidilactici Leuconostoc mesenteroidesPedicoccus damnosus Leuconostoc paramesenteroides
(Source: Beuchat 1995; Sharma et.al., 2011).
A Panorama of lactic acid bacterial fermentation of vegetables
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Factors efffecting vegetables Fermentation
Microorganisms
As described earlier ,lactic acid bacteria (LAB) responsible forvegetable fermentation. Micro-organisms vary in their optimalpH requirements for growth. Most bacteria favour conditionswith a near neutral pH . The varied pH requirements of differentgroups of micro-organisms is used to effect fermented foodswhere successions of micro-organisms take over from eachother as the pH of the environment changes. Certain bacteriaare acid tolerant and will survive at reduced pH levels. Notableacid-tolerant bacteria include the Lactobacillus andStreptococcus species, which play a role in the fermentationof vegetable products. Oxygen requirements vary from speciesto species. Many researchers have studied the changingpattern of microflora involved in the fermentation of kimchialong with the taste and odour produced during fermentation.Major microflora isolated from kimchi were; Lactobacillusplantarum, Lactobacillus brevis, Streptococcus faecalis,Leuconostoc mesentroides, Pediococcus cerevisae andAchromobacter. Recently, most of the fermented vegetablejuices are manufactured by “lactoferment Process” throughthe use of pure starter cultures (Buckenhuskes, 1993)
Temperature
Different bacteria can tolerate different temperatures, whichprovides enormous scope for a range of fermentations. Whilemost bacteria have a temperature optimum of between 20 to30ºC, there are some (the thermophiles) which prefer highertemperatures (50 to 55ºC) and those with colder temperatureoptima (15 to 20ºC). Most lactic acid bacteria work best attemperatures of 18 to 22ºC. The Leuconostoc species whichinitiate fermentation have an optimum of 18 to 22ºC.Temperatures above 22ºC, favour the lactobacillus species.
Additives
Salt concentration
Lactic acid bacteria tolerate high salt concentrations. The salttolerance gives them an advantage over other less tolerantspecies and allows the lactic acid fermenters to begin metabolism,which produces acid, that further inhibits the growth of non-desirable organisms. Leuconostoc is noted for its high salttolerance and for this reason, initiates the majority of lactic acidfermentations. Majority of vegetables are fermented in the saltconcentration range of 2-5 per cent (Table 3).
Nutrients
Some of ingredients, added more or less empirically to lacticacid acid fermented vegetables, seem to enhance the
development of lactic flora, which generally requires veryspecial condition to proliferate. It was reported that lactic acidbacteria depends essentially on the plant sugars for growth(Montet, et.al., 1999). For some vegetables with low nutrientcontent, such as turnip and cucumber, the addition of sugarpromotes bacterial growth, thereby accelerating fermentationas jaggery is added during lactic acid fermentation of sweetturnip (Anand and Das, 1971). Adding 1-2 percent sucrose orjiggery increased lactic acid production during fermentation.Spices are also added to most of the lactic acid fermentedvegetables to improve the flavour of finished product, besidesthese spices have an antimicrobial effect, which means thatthey have a selective role in the development of bacteria duringfermentation (Laencina et.al., 1985). Mustard seeds are also ofinterest as they contain volatile aromatic compounds withantibacterial and antifungal properties. Adding 6-10 percentof mustard seed powder to turnip and 6 percent salt mixtureincreased lactic acid levels during fermentation (Anand andDas, 1971). Sethi, (1990) recommended addition of 3 % salt, 1.0% mustard, 0.015% sodium benzoate and 0.01% potassiummetabisulphite to 1:1 carrot water mixture for making blackcarrot beverage called ‘Kanji’.
Water activity
In general, bacteria require a fairly high water activity (0.9 orhigher) to survive. There are a few species which can toleratewater activities lower than this, but usually the yeasts andfungi will predominate on foods with a lower water activity.
Hydrogen ion concentration (pH)
The optimum pH for most bacteria is near the neutral point (pH7.0). Certain bacteria are acid tolerant and will survive at reducedpH levels. Notable acid-tolerant bacteria include theLactobacillus and Streptococcus species, which play a vitalrole in the fermentation of dairy and vegetable products.
Oxygen availability
Some of the fermentative bacteria are anaerobes, while othersrequire oxygen for their metabolic activities. Some, lactobacilliin particular, are microaerophilic i.e they grow in the presenceof reduced amounts of atmospheric oxygen. In aerobicfermentations, the amount of oxygen present is one of thelimiting factors. It determines the type and amount of biologicalproduct obtained, the amount of substrate consumed and theenergy released from the reaction.
Production Technology and Preservation of FermentedVegetables
Lactic acid fermentations are carried out under three basic
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types of condition dry salted, brined and non-salted. Saltingprovides a suitable environment for lactic acid bacteria to growwhich impart the acid flavour to the vegetable. Sauerkraut isone example of an acid fermentation of vegetables. The namesauerkraut literally translates as acid cabbage. The ‘sauerkrautprocess’ can be applied to any other suitable type of vegetableproduct. Because of the importance of this product in theGerman diet, the process has received substantial research inorder to commercialize and standardize production. Table 4summarises the fermented vegetables developed along withthe microorganisms involved.
The fermentation process
Vegetables are washed and shredded and placed in a jar andsalt is added (Figure 2). Mechanical pressure is applied to the
vegetable to expell the juice, which contains fermentable sugarsand other nutrients suitable for microbial activity. The firstmicro-organisms to start acting are the gas-producing cocci(L. mesenteroides). These microbes produce acids. When theacidity reaches 0.25 to 0.3% (calculated as lactic acid), thesebacteria slow down and begin to die off, although their enzymescontinue to function. The activity initiated by the L.mesenteroides is continued by the lactobacilli (L. plantarumand L. Cucumeris) until an acidity level of 1.5 to 2% is attained.The high salt concentration and low temperature inhibit thesebacteria to some extent. Finally, L. pentoaceticus continuesthe fermentation, bringing the acidity to 2 to 2.5% thus,completing the fermentation. The end products of a normalfermentation are lactic acid along with smaller amounts of aceticand propionic acids, a mixture of gases of which carbon dioxide
Table 3: Salt concentration used during lactic acid fermentation of vegetables
Vegetable Salt concentration (% wt) Type of salting References
CabbagesSauerkraut 2-3 Dry salt Fleming, 1982Korean Kimchi 3 Dry salt Steinkraus et al., 1983Cucumbers 5-8 Brine Fleming, 1982Green olives 4-7 Brine Fleming, 1982Carrots Raddish 1.5-32.52.5 BrineDry saltDry salt Niketic-Aleksic et al., 1973
Joshi, et. al., 2008Sharma and Joshi, 2007
Black olives 2-5 Brine Battcock and Azam-Ali, 1998Over-night Dill pickles 5.3 Brine Frazier and Westhoff, 1998Genuine Dill pickles 7.5-8.5 Brine Frazier and Westhoff, 1998Sauerrruben (turnip) 2.2 Dry salt Frazier and Westhoff, 1998Pickled leafy vegetables 2.5 Dry salt Frazier and Westhoff, 1998
Table 4: Fermented vegetables developed along with the microorganisms involved.
Fermented Vegetables Microorganisms involved Reference
Fermented fruit juices Lactobacillus casei Karovicova and Kohajdova et al., 1986Fermented vegetable juices Lactobacillus strains Sethi, 1990Pickled leafy vegetablesPak-Gard-Dong Lactobacillus brevis,Pediococcus Boonlong, 1986;Joshi et al., 2003
cerevisiaeLactobacillus plantarumGundruk Lactobacillus plantarum,Pediococcus Karki, 1986
pentosaceusBossam kimchi (Pickled cabbage kimchi) Lactobacillus plantarum,Lactobacillus Joshi et al., 2003
brevis,Streptococcus faecalis,Leuconostocmesenteroides,Pediococcus pentosaceus
Sunki (Otaki turnip) Lactobacillus plantarum,Lactobacillus Makayama, 1957brevis,Bacillus coagulansPediococcus pentosaceus
Kawal (Sickle pod plant) Bacillus subtilis,Lactobacillus plantarum Battcock and Azam-Ali, 1998
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is the principal gas, small amounts of alcohol and a mixture ofaromatic esters. The acids, in combination with alcohol formesters, which contribute to the characteristic flavour offermented vegetable. The acidity helps to control the growthof spoilage and putrefactive organisms and contributes to theextended shelf life of the product. Besides acidity othermetabolites are also formed which have inhibitory effect onother competing bacteria and pathogenic microorganisms(Table 5). Changes in the sequence of desirable bacteria, orindeed the presence of undesirable bacteria, alter the tasteand quality of the product.
Use of starter cultures
To produce vegetables of consistent quality, starter cultures(similar to those used in the dairy industry) have beenrecommended. Not only do the starter cultures ensureconsistency between batches, they speed up the fermentationprocess as there is no time lag while the relevant microfloracolonise the sample. Because the starter cultures used areacidic, they also inhibit the undesirable micro-organisms. It ispossible to add starters traditionally used for milk fermentation,such as Streptococcus lactis, without any adverse effect onfinal quality. Because these organisms only survive for a shorttime (long enough to initiate the acidification process) in thefermenting medium, they do not disturb the natural sequenceof micro-organisms. On the other hand, if Leuconostocmesenteroides is added in the early stages, it gives a goodflavour to the final product, but alters the sequence ofsubsequent bacterial growth and results in a product that isincompletely fermented. Joshi et al. (2007) reported sequentialculture fermentation for production of fermented radish andcarrot by using cultures of Streptococcus lactis, Pediococcuscerevisiae, Lactobacillus plantarum at the rate of 2 % each.The cultures were added in sequence to the shredded carrotsafter 24, 48 and 72 h of fermentation, respectively (Sharma,2005; Joshi, et al., 2007)
It is possible to use the juice from a previous fermentation asa starter culture for subsequent fermentations. The efficacy ofusing old juice depends largely on the types of organismspresent in the juice and its acidity.
Figure 2: Natural carrot fermentation
Table 5: Metabolites of lactic bacteria which may be inhibitory to other pathogenic and food spoilage organisms
Product Main target organisms
Organic acidsLactic acid Putrefactive and Gram-negative bacteria, some fungiAcetic acid Putrefactive bacteria, clostridia, some yeasts and some fungiHydrogen peroxide Pathogens and spoilage organisms, especially in protein rich foodsEnzymesLactoperoxidase system Pathogens and spoilage causing bacteria (milk and diary products) with hydrogen peroxideLysozyme(by recombinant DNA) Undesired Gram-positive bacteriaLow molecular weight metabolitesReuterin Wide spectrum of bacteria, yeasts and moldsDiacetyl Gram-negative bacteriaFatty acids Different bacteriaNisin Some LAB and Gram-positive bacteria, notably endospore-formersOther Gram-positive bacteria, inhibitory spectrum according to producer strain and bacteriocin type
Source: Breidt & Fleming, 1997, Joshi et al., 2006
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Non- salted, lactic acid fermented vegetables
Some vegetables are fermented by lactic acid bacteria, withoutthe prior addition of salt or brine. Examples of non-saltedproducts include gundruk (consumed in Nepal), sinki and otherwilted fermented leaves. The detoxification of cassava throughfermentation includes an acid fermentation, during which timethe cyanogenic glycosides are hydrolysed to liberate the toxiccyanide gas. The fermentation process relies on the rapidcolonisation of the food by lactic acid producing bacteria,which lowers the pH and makes the environment unsuitablefor the growth of spoilage organisms. Oxygen is also excludedas the Lactobacilli favour an anaerobic atmosphere. Restrictionof oxygen ensures that yeasts do not grow. For the productionof sinki, fresh radish roots are harvested, washed and wiltedby sun-drying for one to two days. They are then shredded,re-washed and packed tightly into an earthenware or glass jar,which is sealed and left to ferment. The optimum fermentationtime is twelve days at 30ºC. Sinki fermentation is initiated byL. fermentum and L. brevis, followed by L. plantarum. Duringfermentation the pH drops from 6.7 to 3.3. After fermentation,the radish substrate is sun-dried to a moisture level of about21%. For consumption, sinki is rinsed in water for two minutes,squeezed to remove the excess water and fried with salt, tomato,onion and green chilli. The fried mixture is then boiled in ricewater and served hot as soup along with the main meal (Joshiand Thakur, 2000).
Lactic fermented Vegetable products
Preparation of fermented foods using LAB has been suggestedto improve their acceptability. A special fermented vegetablebeverage traditionally known as Kanji has been prepared fromcrimson coloured carrots supplemented with salt and mustardpowder (Sethi, 1990). Lactic acid fermentation is employed toprepare various products such as fermented cucumber extract,fermented carrot, radish and cucumber based appetizer, sauce,RTS chutney, sauce and other products (Sharma, 2011, Joshiand Sharma, 2009; Joshi and Sharma, 2010; Joshi et al.,2011a&b)) .
Lactic acid fermentation is used to stabilize beverages byacidification. The process renders the lactose more digestiblefor people with lactose intolerance. Traditional East-Africanbeverages such as “Muratina” (Sauce tree) or“mnazi”(fermented coconut palm) have also been studied. OnlyLactobacillii were isolated from “Muratina” and onlyStreptococcii from “Mnazi”.
A method for extension of postharvest life of mushroom byinducing lactic acid fermentation was developed and sauce
from fermented mushroom was prepared (Joshi et al., 1996).The sauce has a typical mushroom flavour though lactic, aceticacid and other ingredients used in sauce preparation alsoimparted their respective tastes and aromas to the product.The original texture of unfermented mushroom was spongyand the use of lactic acid fermentation brought the consistencyof mushroom sauce to the desirable level besides improvedflavour. Different varieties of radish have been evaluated forlactic acid fermentaton (Sharma et al., 2011). While responsesurface methodology has been employed to optimize thenutrients and stimulators needed for this fermentation (Joshiet al., 2012).
Preservative Action in Fermented Vegetables
Lactic acid bacteria are inhibitory to many othermicroorganisms when they are cultured together (Adams, 1990)and this the basis of the extended shelf-life and improvedmicrobiological safety of lactic-fermented foods. Lactobacillusspecies can produce a variety of metabolites that are inhibitoryto competing bacteria, including psychrotrophic pathogens.
Antagonistic behavior of lactic acid bacteria may be eitherdue to their major metabolic end products or via production oflow molecular weight compounds. The low molecular weightsubstances elaborated by LAB, capable of exhibitingantagonism are termed as bacteriocins. These are proteins innature, which exert a bactericidal mode of action on susceptiblebacteria (Tagg et al., 1976). Lactic acid bacteria (LAB) arecapable of producing inhibitory substances in smaller amountsas discussed earlier. Bacteriocin or other substances elaboratedby LAB are listed in Table 6 while the bacteriocin producedand their antimicrobial spectrum is shown in Table 7.
Consumer awareness against the hazardous and ill effect natureof synthetic chemicals used for preservation of food productsand development of the process for the production ofconvenient food or semi-processed products forced theresearch workers to focus on the development of natural,stable, antibiotic and safe substances, such as bacteriocin asan antimicrobial agent or biopreservative. It can be useddirectly as a pure compound in unfermented processedproducts or by the use of lactic acid bacteria that producedbacteriocin as a starter culture in fermented fruits andvegetables like cabbage, cauliflower, olives, cucumber andpeppers (Lucke and Earnshaw, 1991). If food is going to befermented by LAB, the use of bacteriocin-producing starterculture provide added value to the product. The presence of anisin producer among the strains used to make cheedar cheeseprovides enough nisin to increase the shelf-life of pasteurized
A Panorama of lactic acid bacterial fermentation of vegetables
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processed cheese made from it from 14-87 days at 220C (Robertsand Zottola, 1993).
Table 6: Bacteriocins or bacteriocin-like substances elaborated bylactic acid bacteria
Producing microorganism Bacteriocin or bacteriocin –like substances
Lactobacillus plantarum Plantacin B, Plantaricin C,Plantacin N
Lactococcus lactis sub sp. lactis Nisin, Lacticin, LactococcinG
Lactococcus lactis sub sp. cremoris Diplococcin, LactococcinPediococcus acidilactici Pediocin AcH, Pediocin
P-A-1Leuconostoc mesenteroides UL5 Mesenterocin 5Lactobacillus acidophilus Lactocidin, AcidophilinLactobacillus caseii Caseicin
Source: Sarkar and Misra, 2001
Some bacteria possess the genetic capability to synthesizebacteriocin. Nisin (a polypeptide antibacterial substanceproduced by lactic acid bacterium, Lactococcus lactis formallyStreptococcus lactis, during fermentation) is one of the earliestknown bacteriocin, showing antimicrobial activity against arange of Gram-positive bacteria, particularly spore forming(Broughton, 1990). A bacteriocin producing strain Lactococcuslactis sub sp. cremoris R was isolated from radish anddesignated bacteriocin as Lactococcin R (Yildirim and Johnson,1998). It was active against many food borne pathogenic andfood spoilage bacteria such as Clostridium, Staphylococcus,Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus,Leuconostoc, Sterptococcus and Pediococcus spp. but wasinactive against Gram-negative bacteria. Reuterin a broadspectrum antimicrobial agent active against certain bacteria,yeasts and fungi (Axellson et al., 1998), was produced byheterofermentative organism Lactobacillus ruterii. Reuterinmay have application in the preservation of food stuffs byreducing pathogenic and spoilage causing organisms(Daeschel, 1989). The pediococci used as a starter culture inthe vegetable and meat fermentation have been of much recentinvestigations with regard to their bacteriocin producing abilityalthough, pediococci was known to produce a range ofbacteriocins and other metabolites with broad spectrumantibacterial activity (Skytta et al., 1993). Pediocins producedby Pediococcus spp. can inhibit a wide range of pathogensincluding Listeria monocytogenes and the Gram-negativePsedomonas fluorescens (Singhal and Kulkarni, 1999). Butpedicin AcH produced by Pediococcus acidilactici has beenshown to have antibacterial activity against Staphylococcusaureus, Listeria monocytogenes and Clostridium perfringes
(Bhunia et al., 1998). The use of pure starter culture (LAB) hasbeen reported for the inhibition of spoilage organismPseudomonas fluorescens and Staphylococcus typhimurium(Raccach et al., 1979). The growth of Staphylococcus aureusis also inhibited when grown with Pediococcus cerevisiae andLactobacillus plantarum. A wide variety of raw foods arepreserved by LAB fermentation including milk, meat, fruit andvegetables (Daeschel, 1989). Reduction of pH and removal ofa large amount of carbohydrates by fermentation are the primarypreserving actions that these bacteria provide, though, LABare capable of producing inhibitory substances that areantagonistic towards other microorganisms as described earlier(Joshi and Thakur, 2000; Battcock and Azam-Ali, 1998).
Table 7: Bacteriocin producing microorganisms and their inhibitoryspectrum
Microorganism BacteriocinInhibitory Spectrum
Bacillus subtilis Subtilin Gram +vebacteria
Lactococcus lactis sub sp. lactis Nisin Many gram+ve bacteria
Lactococcus lactis sub sp. lactis CNRZ Lacticin-481 Lacticacid bacteria(LAB)andClostridium
Pediococcus acidilactici Pediocin PA1Some LAB, Listeria
Lactobacillus sake Sakacin A SomeLAB, Listeria
Leuconostoc mesenteroides UL5 Mesenterocin 5Some LAB, Listeria
Propionibacterium shermanni Microgard Gram–vebacteria, some yeastsand moulds
Lactobacillus sakei Sakacin P E. coli,Lactic acid bacteria
Source: Barnby-Smith, 1992; Vaughan et al., 2001
Healthful effects of lactic bacterial fermented Products
Probiotic effect
One of the reasons for the increasing interest in fermentedfoods is their ability to promote the functions of the humandigestive system in a number of positive ways. As early as1900, Metchnikoff pointed out the use of fermented milks inthe diet for prevention of certain diseases of thegastrointestinal tract and promotion of healthy day-to-day life.Since then, a number of studies have shown that the fermentedfood products do have a positive effect on health status inmany ways. A fermented food product or live microbial food
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Joshi and Sharma
supplement, which has beneficial effects on the host byimproving intestinal microbial balance is generally understoodto have probiotic effect (Fuller, 1989).
Flatulence reducing activity
The lactic acid fermented products do have flatulence reducingeffect. During fermentation of the beans for preparation oftemphe, the trypsin inhibitor is inactivated and the amount ofseveral oligosaccharides which usually cause flatulence aresignificantly reduced (Hesseltine, 1983). Bean flour inoculatedwith Lactobacillus and fermented with 20% moisture content,showed a reduction of the stachyose content (Duszkiewicz-Reinhard et al., 1994)
Hypercholesteremic effect
Hepner et al. (1979) reported hypercholesteremic effect ofyoghurt in human subjects receiving a one-week dietarysupplement. Studies on supplementation of infant formulationwith Lactobacillus acidophilus showed that the serumcholesterol in infants was reduced from 147 mg/ml to 119 mg/100 ml (Harrison and Peat, 1975). The ability of yoghurt tolower the cholesterol in serum by controlled human trials hadalso been reported (Poppel and Schafsma 1996). Possible roleof LAB in lowering cholesterol concentration and variousmechanisms by which it may be possible has also beendiscussed (Haberer et al., 1997).
Anticarcinogenic effect
Apart from this, there are interesting data on anticarcinogeniceffect of fermented foods showing a potential role of lactobacilliin reducing or eliminating procarcinogens and carcinogensfrom the alimentary canal (Shahani, 1983; Mital and Garg, 1995).
Mutagenicity
The fermentation of foods is also reported to reduce themutagenicity of foods by degrading the mutagenic substancesduring the process. Lactic acid bacteria isolated from dadih, atraditional Indonesian fermented milk, were found to be ableto bind mutagens and inhibit mutagenic nitrosamines. Milkfermented with Lactobacillus acidophilus LA-2 wasdemonstrated to suppress faecal mutagenicity in the humanintestine.
Immune system
Some LAB present in fermented milk products, are found toplay an important role in the immune system of the host aftercolonisation in the gut (De Simone, 1986). The mechanism ofthis effect is not clearly known, but it is speculated that the
lactobacilli, their enzymes or the metabolic products presentin the fermented food product may act as antigens, activatingproduction of antibodies.
Future Trends
Vegetable fermentation employing Lactic acid bacteria is oneof the oldest forms of food preservation technology in theworld is strongly linked to culture and tradition, especially inrural households and village communities. It is a relativelyefficient, low energy preservation process, which increasesthe shelflife and decreases the need for refrigeration or otherforms of food preservation technology. Thus, a highlyappropriate technique for use in developing countries andremote areas where access to sophisticated equipment islimited. It is therefore, essential to increase the knowledge andunderstanding of the methods of preparation, in order toimprove the efficiency of fermentation, especially the traditionalprocesses. The potential areas for improvement of lactic acidfermented products are: better understanding of fermentedproducts; refining the process; creating a supportiveenvironment for production of fermented food products;development of new starter cultures. With many of thefermented products knowledge of the processes involved ispoor. It is likely that the basic principles apply across the world,but production conditions vary enormously from region-to-region, giving rise to numerous variations of the basicfermented product. It is not the intention or the desire tostandardise the process and thereby lose this huge diversity,rather it is to harness the tremendous potential these methodshave to contribute to increasing not only the quantity, butquality of food available to the world’s population. If theprocesses are to be refined, with a view to production on alarger scale, it is essential to have a scientific understandingof the fermentation processes. This can be developed by theisolation and characterization of the essential micro-organismsinvolved; determination of the role of external factors infermentations and the effects of these on the metabolism ofmicro-organisms; investigation on the effects of pre-treatmentsof raw materials on the fermentation process; identification ofthe options for further processing and how these affect thetaste and texture of the product; biotechnologies need to bedeveloped which are affordable by the poor, since it is theywho are likely to benefit most by improvements in the traditionalprocesses and product incorporate objective methods ofprocess control and to standardize quality of the final products.Fermented foods should be recognised as part of eachcountries heritage and culture and efforts are made to preservethe methods of production. More research has to be done toascertain the factor or combination of factors responsible forthe positive consequence of fermented diets. Consumers need
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to be made aware of the numerous benefits of fermented foodsand their prejudices against fermented foods, especially thosetraditionally produced at the home scale are dispelled.
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Intl. J. of Food. Ferment. Technol. 2(1): 13-18, June, 2012
Application of thermostable ααααα-amylase in Streptomyceserumpens liquefaction of cassava bagasse
for production of lactic acid
Ramesh C. Ray* and Shaktimay Kar
Microbiology Laboratory, Regional Center of Central Tuber Crops Research Institute, Bhubaneswar, India
*Email: [email protected]
Paper no: 32 Received: 14 Jan 2012 Received in revised form: 19 April 2012 Accepted: 19 May 2012
Abstract
Application of thermostable α- amylase (crude and purified) produced by Streptomyces erumpens onliquefaction of cassava bagasse into sugars for further conversion to lactic acid (LA), was comparedwith that of a commercial enzyme Termamyl (Novozyme, Denmark). By applying crude S. erumpensamylase (10%, v/v), 21.89 g LA was achieved per 100g bagasse at 96h of incubation, which was 44.01%less than LA yield obtained with Termamyl (2%, v/v). However, when partially purified S. erumpensamylase was applied at 5% (v/v) concentration or Termamyl at 2% (v/v) concentration to liquefycassava bagasse, the LA yield was similar, i.e., 38.45 LA/100g bagasse. The purified form of S. erumpensamylase was found as effective as commercial Termamyl enzyme in liquefying cassava bagasse tosugars for further conversion to LA.
©2012 New Delhi Publishers. All rights reserved
Keywords: α- amylase, cassava bagasse, Lactic acid, Streptomyces erumpens
Lactic acid (LA) is a versatile organic acid having variousapplications in food industry such as acidulant,in pickling asa, flavour and preservative (Oh et al., 2005). LA can be obtainedon an industrial scale either by microbial fermentation orchemical synthesis. In recent years, the fermentation approachhas become more successful because of the increasing marketdemand for naturally produced LA. Of the 80,000 tonnes of LAproduced world wide every year, about 90% are made bymicrobial fermentation employing LA bacteria (LAB) (Kadamet al., 2006). LAB (Lactobacillus spp., those include L.acidophilus, L. plantarum, L. casei, L. gasseri, etc) aretraditionally fastidious microorganisms and have complexnutritional requirements due to their limited ability tobiosynthesize B-vitamins and amino acids (Fitzpatrick and O’Keeffe, 2001). Generally, LA is produced using costly substratesuch as cane or beet sugar as the carbon source. Thus, a low
cost of feed stock is very much desirable to reduce theproduction cost by replacing sugar with starchy agriculturaland forest residues (John et al., 2005, 2006, 2007). Starch is themost abundant carbon compound in the world, which can behydrolyzed to fermentable sugars by acid and enzymehydrolysis. Among the agro-industrial residues, cassavabagasse, one of the major solid waste released during extractionof starch from cassava (Manihot esculenta Crantz) proved tobe an alternative ideal bio-resource for production of variousend products such as enzymes (Kar and Ray, 2008; Kar et al.,2011), organic acids (Jyothi et al., 2005) and ethanol (Ray etal., 2008), because of its high starch content (55-65 % on dryweight basis) (Ray et al., 2008).
Amylases constitutes one of the important group of enzymesthat are widely used in starch processing industries. Amongthe starch hydrolyzing enzymes, thermostable α-amylase is of
Research Paper
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Ray and Kar
utmost significance (Haki and Rakshit, 2003) as it breaks onlyα-1, 4 glucosidic bonding in starch (Tonkova, 2006). Further,the enzyme possesses high temperature stability that canovercome high viscosity and mass transfer problems commonlyassociated with starch slurry and facilitate higher conversionof starch into fermentable sugars.
But many lactobacilli are unable to degrade starch as theyeither do not produce or produce in less quantity of starchdegrading enzymes such as α-amylase, pullulanase andglucoamylase (Gupta et al., 2003). Thus, in order to utilize thestarchy residues such as cassava bagasse, it is necessary toadd starch-degrading enzymes to the fermentation medium tocatalyze conversion of starch in to sugars. However stepinvariably escalates the production cost. To overcome thisproblem, an indigenously produced thermostable α-amylasefrom an actinomycete, Streptomyces erumpens MTCC 7317was effectively demonstrated to degrade both soluble andcassava starch into sugar in our previous studies (Kar et al.,2009; Kar et al., 2010).
The present investigation was carried out to study further theapplication of (1) crude and (2) partially purified α-amylasefrom S. erumpens on LA production using cassava bagasse asthe substrate. The LA yield and conversion efficiency of S.erumpens α-amylase was also compared with that of acommercial amylase (Termamyl®).
Materials and methods
Chemicals
All the chemicals used were of analytical reagent grade andwere procured from M/s Merck Specialties,Mumbai, India.
Microbial strains and inoculum preparation
Lactobacillus plantarum MTCC 1407 was used for theproduction of LA. The culture was obtained from.The Instituteof Microbial Technology, Chandigarh, India and wasmaintained on Mann Rogassa Sharpe (MRS) agar slants at4ºC. The bacterial inoculum was prepared in 250 ml Erlenmeyerflask containing 100 ml of MRS liquid medium by transferringone loop full of organism (L. plantarum) from stock cultureand incubated at 35°C for 48h at 120 rpm in an orbital incubatorshaker (Remi Pvt. Ltd., Mumbai, India).
Streptomyces erumpens MTCC 7317 was previously isolatedfrom a brick kiln soil and it possessed thermostable α-amylaseactivity. The optimum enzyme activity was obtained at pH,6.0-7.0 and temperature, 50ºC (Kar and Ray, 2008). This organismalso produces thermostable pectinase (Kar and Ray, 2011) andamylopullulanase (Kar et al., 2011). S. erumpens inoculum wasprepared in soluble starch-beef extract (SB) broth (pH 7.0)
[soluble starch, 1.0%; beef extract, 1.0%; yeast extract, 0.2%;MgSO
4, 0.1%, glycerol, 0.02% and 100 ml double distilled H
2O],
by transferring a loop full of organism from a fresh culture andincubated at 50°C for 24h at 120 rpm in an orbital incubator.
Enzyme sources
Commercial enzymes
Termamyl® is a thermostable (>90°C) α- amylase enzyme andDextrozyme (>45°C) is a fungal amyloglucosidase(glucoamylase) enzyme. The enzymes were procured from Arunand Co. Mumbai, India.
S. erumpens enzyme
Crude α-amylase was prepared from S. erumpens as follows;.Sterile SB medium (50 ml) taken in 250 ml Erlenmeyer flasks (intriplicate) were inoculated with 2% inoculum and agitated at120 rpm in an incubator shaker (Remi Pvt Ltd, Mumbai, India)at 50°C. After 36h, the culture broths from the flasks werepooled and centrifuged at 8000 rpm in a refrigerated centrifuge(Model C-24, Remi Pvt. Ltd, Mumbai, India) for 20 min at 4°C.The clear cell free supernatant was used as the crude enzyme.
Partially purified α-amylase was prepared as follows; A totalof 100 ml of culture filtrate (crude enzyme) was brought to 60%ammonium sulphate saturation at 4°C in an ice bath forovernight was carried out. Precipitated protein was collectedby centrifugation at 8000 rpm at 4°C for 20 min and dissolvedin minimum volume of phosphate buffer (0.1M, pH 6.0). Enzymesolution was dialyzed against the same buffer for 24h at 4°Cwith continuous stirring and three changes of the same buffer.The dialyzed enzyme solution was used as partially purifiedenzyme.
Cassava bagasse
Cassava bagasse [(g/100g dry residue); moisture, 11.2; starch,63.0; crude fibre, 10.8; crude protein, 0.9 and total ash, 1.2](Ray et al., 2008) was used as the substrate for LA production.Cassava bagasse was collected during starch extraction fromcassava using the mobile starch extraction plant, developedby our institute (Edison et al., 2006). Because of its high waterand starch content, the residue was de-watered, sun dried for6-8 days and then was oven–dried at 80 °C for 24h to preventmicrobial deterioration and stored in air-tight container, untilrequired.
LA production
The starch slurry was prepared in 500 ml Erlenmeyer flask bymixing cassava bagasse (25g) with 250 ml of distilled water. It
Application of thermostable α-amylase in Streptomyces erumpens liquefaction of cassava bagasse for production of lactic acid
15
was liquefied either with Termamylâ (2%, v/v) or with differentlevels of S. erumpens crude α-amylase (2, 5 and 10%, v/v) at50°C for 1h. The liquefied broth was subsequently cooleddown to 45oC and then, saccharified with amyloglucosidase(AMG, 2%, v/w) by incubating for 24h at 45°C in an incubator.After saccharification process was over, the volume of thebroth was adjusted to 500 ml with distilled water and to it,constituents of MRS liquid medium except glucose (carbonsource) [(gl-1): peptone, 10.0; beef extract,10.0; yeast extract,5.0; Na
2HPO
4, 2.0; sodium acetate, 5.0; tri-ammonium citrate,
2.0; MgSO4, 0.2; MnSO
4,0.2; CaCO
3, 4.0; Tween 80, 0.1ml and
pH adjusted to 6.8] were added in appropriate proportions,and autoclaved at 15 lb pressure for 20 min. Then, the flaskswere cooled to room temperature (30 ±20C) and were inoculatedwith 2% L. plantarum inoculum, and incubated at 35°C for 96h
in an incubator shaker at 120 rpm. Triplicate flasks weremaintained for the experiment. At interval of 24h, culture brothfrom individual flask was taken out and centrifuged at 8000rpm in a refrigerated centrifuge for 20 min at 4°C. The clearsupernatant was used for LA estimation. The flow- chart forLA production from cassava bagasse is shown in Figure 1.
In another set of experiment, in lieu of crude enzyme, partiallypurified S. erumpens α-amylase (2, 5 and 10%) was used forliquefaction of cassava bagasse, and the remaining steps werethe same as described in Figure 1 for production of LA.Triplicate flasks were maintained for this experiment. LA yieldfrom cassava bagasse was calculated as follows:
LA yield (%) = LA produced x 100 Starch present in cassava bagasse
Figure 1: Flow chart for lactic acid production from cassava bagasse
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Ray and Kar
Starch conversion efficiency (%) was calculated as follows;
Starch conversion efficiency (%): Amount of starch converted to LA
x 100Initial starch present in cassava bagasse
Biochemical analysis
LA content was estimated by the method described by Amerineand Ough (1984) using UV-Vis spectrophotometer (Model noCE 7250, Cecil Instrument, UK) and expressed as g/100g. Thereducing sugar content in fermented broth (before and afterfermentation) was estimated using Nelson’s method(Mahadevan and Sridhar, 1998). Enzyme activities [S. erumpens(crude and partially purified) amylase and Termamyl] weremeasured by incubating 0.2 ml of either S. erumpens amylaseor Termamyl with 0.5 ml of 0.2% soluble starch in 0.1Mphosphate buffer (pH 6.0) at 50°C for 30 min. Reducing sugarsformed were measured by Nelson’s method (Mahadevan andSridhar, 1998). One unit of enzyme activity was defined as theamount of enzyme which produced 1µm/min of reducing sugarwith glucose as the standard under the conditions described.
Statistical analysis
The data of enzyme production were analyzed using one wayANOVA. Where significant difference in ANOVA (p< 0.05) wasdetected the Fisher’s Least Significant Difference (LSD)multiple comparison test was applied to compare the factorlevel difference. The analyses were performed using MSTAT-C (Version 2.0, Michigan State University, Michigan, USA).
Results and discussion
Cassava bagasse was hydrolyzed by three sets of thermostableα – amylases: crude, partially purified enzyme (from S.erumpens) and Termamyl and the hydrolysates were used asthe sole carbon source for LA production. In the firstexperiment, by applying 2, 5 and 10% of crude amylase oncassava bagasse, 10, 15 and 17 g reducing sugars /100gbagasse were produced (showing 15.0, 22.7 and 25.7%conversion efficiency respectively), which were furtherconverted to LA by L. plantarum (Figure 2). LA production
was proportional to the applied enzyme concentration as wellas duration of incubation (fermentation). Maximum LAproduction (21.89g LA/100g bagasse) was achieved byapplication of 10% crude amylase at the end of 96 h incubation,showing 36.5% yield (assuming 63% starch present in 100gbagasse) and 31.5% starch conversion efficiency. When theperformance of crude enzyme was compared with Termamyl,the production of LA and conversion efficiency were 44.01%and 43.2% less in crude enzyme(10%) than that of Termamyl.This was due to the formation of less fermentable sugar (17 gsugar) from the hydrolysis of 100g cassava bagasse by crudeenzyme in comparison to that of Termamyl (30 g sugar/100 gbagasse). To obtain highest LA production, a key factor is torelease more available sugar into the medium for fermentation.In this context, purified S. erumpens α-amylase was applied inthe next experiment in order to increase the starch hydrolysisefficiency.
The crude extract of a-amylase was purified with 60%ammonium sulphate precipitation, which contained 2.5 mg/mlprotein and showed a specific activity of 136.0 Units/ml protein.After dialysis, specific activity increased to 408.0 Units/mgprotein with yield of 24.24% and 3.0 fold purification (Table 1).
When partially purified S. erumpens amylase (PSEA) at 2, 5and 10% concentration were applied to cassava bagasse withAMG at a constant rate (2%, v/w), it gave 66-94% morefermentable sugars (20, 29 and 33 g sugar/100g bagasse,respectively) in comparison to crude enzyme (Figure. 3).Highest LA production (42.5 g/100 g bagasse) was observedwith 70.8% yield after 96h of incubation by the application of10% Partially α-amylase.John et al. (2007) reported LAproduction (54.0 g/100 g cassava bagasse) obtained at 60hwith the application of 8.3% commercial enzyme (α-amylase).Likewise, the optimum LA production for Lactobacillusdelbrueckii and L. plantarum were found to be 32.0 and 46.0g/100g, respectively using alfalfa fibre as the carbohydratesource, whereas with that of soya fibre, it was 44.0 g/100 g(Hassan et al., 2001). Further, in our study, 5% PSEA and 2%Termamyl showed no significant difference (Fisher’s LSD testp< 0.05%) on fermentable sugar (29 g/100 g) after enzymatichydrolysis and subsequently, on LA production (38.45 g LA).Likewise, by the application of 5% partially enzyme, the starch
Table 1: Partial purification of amylase for liquefaction of cassava bagasse
Purification steps Total Total enzyme Total Yield Specific activity Fold ofvolume (ml) activity (Units) protein (mg) (%) (Units/mg protein) purification
Culture filtrate (crude) 100 34000 250 100 136 0Ammonium sulphate precipitation 15 6747 24.8 19.8 272 2.0After dialysis 16 8241.6 20.2 24.24 408 3.0
Application of thermostable α-amylase in Streptomyces erumpens liquefaction of cassava bagasse for production of lactic acid
17
to LA conversion efficiency (54.0%) was similar with that of2% Termamylâ (55.0%). However, a higher starch to LAconversion ratio of 61.1% was obtained in case of 10% PSEA.
Generally, LA bacteria are deficient in cellulolytic and amylolyticcharacters necessitating the prior hydrolysis of cellulosic andstarchy wastes for their better utilization. Enzymatic hydrolysisis preferred to acid hydrolysis for this purpose (John et al.,2007). Woiciechowski et al. (2002) reported 97.3% conversionof starch into sugar in cassava bagasse by treatment withTermamyl and AMG. Simultaneous saccharification and lacticacid production is done with the addition of amylolytic enzymes(Termamyl and AMG) into starch and inoculated with L.delbrueckii (Anuradha et al., 1999). The yield of LA was 45%.Co-immobilization of starch-degrading organisms likeAspergillus awamori and lactic acid-producing bacteriaStreptococcus lactis was also done for the simultaneoussaccharification and lactic acid production (Kurusava etal.,1988); however, the yield of LA was poor. Further, almostall commercial amylases available in market are in purified form;the purification results in removal of impurities from the enzymethereby enhancing its catalyzing activity. In our findings, thePSEA (5%) was found equally efficient as commercialα-amylase (Termamyl, 2%) in converting starch in cassavabagasse into sugar.
Conclusion
It was concluded that application of partially purified S.erumpens α-amylase was more effective than crude amylase
Figure 2: Lactic acid production and residual sugars from cassavabagasse with application of S. erumpens crude amylase (2, 5 and10%) and Termamylâ (2%)
Figure 3: Lactic acid production and residual sugars from cassavabagasse with application of S. erumpens purified amylase (2, 5 and10%) and Termamylâ (2%)
as it produces 94% more LA from cassava bagasse. Likewise,the conversion efficiency and LA yield using 5% purifiedamylase was found to be similar to that of 2% Termamyl. Hence,the indigenous application of PSEA for conversion of starchin cassava bagasse have potential to save considerable amountof LA production cost.
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Woiciechowski, A.L., Nitsche, S., Pandey, A. and Socool, C.R. 2002.Acid and enzymatic hydrolysis to recover reducing sugarsfrom cassava bagasse: an economic study. Braz. Archi. Biol.Technol., 45:393-400.
Intl. J. of Food. Ferment. Technol. 2(1): 19-25-, June, 2012
Changes in phytochemicals during fermentationof wine grapes
A. K. Sharma*, S.V. Navale, S.N. Aute, G.S. Karibasappa, D.P. Oulkar and P. G. Adsule
National Research Centre for Grapes, Pune, Maharashtra, India
*Email: [email protected]
Paper no: 33 Received: 12 Jan,2012 Received in revised form: 14 April, 2012 Accepted: 19 May, 2012
Abstract
Changes in various phytoemicals during fermentation of grape cultivars for red wine (CabernetSauvignon and Zinfandel) and white wine (Sauvignon Blanc and Charark-4; a cross of Chardonnayand Arkavati) were determined. The fermentation was performed in food grade plastic vessels, usinga commercial wine yeast strain Premier Cuveeat 20±1°C. The data were recorded on total phenols,anthocyanin,Ferric ion reducing antioxidant power (FRAP), DPPH assay and individual phenoliccontent and amino acids. At the initial stage of fermentation, total phenols were higher in Zinfandelthan in Cabernet Sauvignon, but increased to 4-folds at the completion of fermentation. Higher levelsof total phenols and antioxidant activities were noted in red wines than in white ones. Free radicalscavenging activity (DPPH) was higher in Cabernet Sauvignon than in Zinfandel. However,resveratrolcontent was more than twice in Zinfandel wine than in Cabernet Sauvignon wine. White winesshowed low levels of phenolic compounds and some phenolics viz.; epicatechingallate, resveratroland kaempferol were found absent. During the process of fermentation quantities of various aminoacids were changed.Alanine, serine, lysine and vaniline amino acids were found absent at beginningbut on the completion of fermentation presence of these amino acids was confirmed and quantified.
©2012 New Delhi Publishers. All rights reserved
Keywords: Wine, phytochemicals, phenols, resveratrol, amino acids
The quality of wine is decided by consumer preference.Themost important factor that contributes to wine character is thekind of grapes used, while the fermentation has been recognizedto be essential for the quality and stability of wine. Red wine isthe result of must fermentation and extraction of variouscompounds from the pulp, seeds and skin. Grape skinfermentation helps in the extraction of colour compoundsrelevant to the wine structure, its body, bouquet and aromaperception, besides the extraction of various substances frompolyphenolic to nitrogen compounds, polysaccharides,pectins, mineral substances, pyrazine, terpenes, etc. (Klenar,et al., 2004). Usually, large amounts of phenolic compounds,mostly the flavonoids as well as their byproducts are presentin grapes at high concentrations (Kanneret, et al., 1994), but
their presence is affected by number of factors including grapevariety, skin colour, sun exposure, vinification technique andaging (Price et al., 1995, McDonald et al., 1998; Burns et al.,2001 and Pakhale et al., 2007). The phenolics profile of wine isnot the same as those of fresh grapes because of significantchanges occurring during winemaking process from the berrycrushing stage, to subsequent fermentation and wine aging(Meyer 1997). The phenolic compounds, anthocyanins ofgrapes and their antioxidant activities have been extensivelystudied and a strong correlation exits between antioxidantcapicity and total phenols (Rrice-tvans 1995; Kalt et al., 1999;Netzel et al., 2003) and represents about 20–50% of the totalnitrogen in the must. The amino acid profile of wine can beused to differentiate various wines according to vine variety,
Research Paper
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Sharma et al.
geographical origin and year of production (Huang and Ough.1991; Hollendet et al., 1995). Proline makes the majority of thefree amino acids in wine and its largest amount is found infresh grapes. The present investigation was conducted tostudy the changes in phytochemicals and antioxidant activitiesof wine grapes during process of winemaking.
Materials and methods
Raw material
The study was carried out during cropping season of 2009 atthe National Research Center for Grapes, Pune. The bunchesof grape cultivars for red wine (Cabernet Sauvignon andZinfandel) and white wine (Sauvignon Blanc and Charark-4; across of Chardonnay and Arkavati) were harvested when TotalSoluble Solids (TSS) content reached around 20 °B. The berrieswere destemmed manually and crushed to extract juice. Tosuppress the development of natural microflora 100 ppmpotassium metabisulpite (KMS) was added to the must/juicewhich was kept at 0 °C for overnight.
Fermentation
A commercial wine yeast strain Premier Cuvee was used forfermentation. The must/juice was fermented in food gradeplastic vessels which were kept at 20±1°C. After recording ofinitial observations, samples were taken during fermentationon 2nd, 3rd, 6th and on 11th day when fermentation was completedand skin separation was observed. During the fermentationprocess, the must of red grapes was punched and fermentingwhite juice was mixed two times every day. The samples werecollected and each sample was replicated three times foranalysis of various parameters. The generated data wereanalyzed as per CRD using SPSS program.
Physico-chemical analysis
The collected samples were analyzed following standardoperating procedures. Total phenols were quantified byfollowing the procedure of Singleton and Rossi (1965).Content of anthocyanins in samples was estimated by usingmethod of Fulekiand Francis (1968). Antioxidant activities weremeasured by Ferric ion reducing antioxidant power (FRAP),and Free radical scavenging activity (DPPH assay). Inmeasuring of FRAP method of Benzie and Strain (1996) wasadopted and in case of DPPH assay method of Arnouset al.(2001) was followed. LC-MS/MS (Agilent Technologies withseries hyphenated to API 4000 Qtrap (ABS Sciex) massspectrometer equipped with electrospray ionization (ESI+)probe 1200) was used for quantification of individual phenoliccontent in samples. For amino acids, the HPLC (Perkin Elmer200 series) of column G8 (50 x 4.6 mm ID x 1.8 µm) was used,with mobile phase A: 0.1 % pentadecaflurooctonoic acid and0.1 % formic acid in Water: Methanol (90:10), B: 0.1% formicacid in water: methanol (10:90). Calibration curves wereobtained by plotting the peak areas against differentconcentration of amino acids and poly phenolic compounds.
Results and discussion
Significant differences were noted in total phenols content ofdifferent samples at various stages of fermentation of winegrape varieties except Sauvignon Blanc (Table 1) where samevalue i.e. 230 mg/ was recorded on 3rd and 6th day. Total phenolswere recorded higher in red varieties than white ones. Valuesof 3760 and 1670 mg/L were recorded in Cabernet Sauvignonand Zinfandel, respectively while in Sauvignon Blanc andCharak-4, these values were 220 and 370 mg/L, respectively on11th day. Zinfandel recorded initially higher concentration oftotal phenols (1050 mg/L) than Cabernet Sauvignon (950 mg/L) but experienced only slight increase during fermentation.
Table 1: Changes in total phenols and anthocyanin content at different intervals during fermentation process of wine grapes.
Days Total Phenols (mg/L) Anthocyanin (mg/L)
Red White Red White
CabernetSauvignon Zinfandel Sauvignon Blanc Charark-4 CabernetSauvignon Zinfandel Sauvignon Blanc Charark-4
Initial 950 1050 330 652 1070 3400 - -2 1770 1330 260 680 8330 7940 - -3 1990 1740 230 545 14330 10210 - -6 3880 1850 230 393 19080 12350 - -11 3760 1670 220 370 15030 8340 - -SEM± 5.362 3.162 2.828 6.251 2.236 3.162 - -CD at1% 16.16 9.53 8.52 18.84 6.74 9.53 - -
Changes in phytochemicals during fermentation of wine grapes
21
On the other hand, in both the white varieties, total phenolswere significantly declined as the fermentation progressed. Ingeneral, concentration of phenolics rises during grapefermentation. But results of present study were different incase of white grapes. During the fermentation of white winegrapes decrease of gallic and protocatechuic acids was noticedby Budic-Leto and Lovric (2002).In wine, there are two groupsof phenolic acids; hydroxybenzoic acids and hydroxycinnamicacids (Cabrita et al. 2008). Tian et al. (2009) found that theevolution of hydrobenzoic acids during the fermentation ofdry wine, semi-sweet wine and ice wine. Higher concentrationsof total hydroxybenzoic acids were noted in all the wines. Theconcentrations of total phenols in all varieties were different.In case of Cabernet Sauvignon about 4 times and in Zinfandelonly 0.6 time total phenols were increased during fermentation.These results are confirmatory to those of Sulc and Lachman(2005) who also found significant differences in total phenolsamong the varieties. Red wines contained on an average 2times more total phenols in comparison with grape must at thebeginning of winemaking. White and red wines differedsignificantly in total phenols content during vinificationprocess. The red and white wines differ not only in finalcontents of phenolics, but also in their extreme increase in redwines during fermentation.The phenolic compounds in winerange from relatively simple compounds to complex tannin-type substances with antioxidant activity. Wine phenoliccomposition is conditioned by the grape used and by thewinemaking processes that determine their extraction into themust and their further stability in wine. Grape phenolics dependon the variety and other factors that affect berry developmentsuch as soil, geographical location and weatherconditions.Wine samples of Cabernet Sauvignon showedhigher levels of Catechin (117mg/L) and Epicatechin (59.8 mg/L) and similar trend was observed in Zinfandel. Lower levelsof Kaempferol (0.316 mg/L), Resveratrol (0.503 mg/L) and p –Coumaric acid (0.521 mg/L) were recorded in wine of CabernetSauvignon. On the other hand, Kaempferol in Zinfandel hasminimum quantity i.e. 0.139 mg/L and Epicatechin Gallate (0.386mg/L). More than double the quantity of Resveratrol wasfound in Zinfandel wine than Cabernet Sauvignon wine (Table2). White wines showed very low concentration of differentphenolic. Epicatechin Gallate, Resveratrol and Kaempferol wereabsent in white wines from Sauvignon Blanc and Charark-4.p–Coumaric acid was absent from Sauvignon Blanc winehowever, only 0.807 mg/l was noted in wine of Charark-4. Thewines of Sauvignon Blanc contained lower concentration ofthe phenolic compounds as well as total phenolics than winesfrom Charark-4. Content of total and individual phenols werefound to be characteristic of the grape variety. Red winescontained higher total as well as individual phenols than white.Phenolic compounds have long been considered to be the
basic components of wines and over 200 compounds havebeen identified. Resveratrol is mainly contained in the skins ofgrapes (Schmandke, 2002) due to this reason resveratrol wasabsent from white wines.Flavonols, such as quercetin,kaempferol, and myricetin, are localized in grape skin and occurin glycosidic forms. They exist in trace amount in white wines(Singleton, 1992). Our findings are also matched with theseresults.
The anthocyanin contents were higher in Cabernet Sauvignonthan in Zinfandel at all levels of fermentation and remainedabsent in white varieties (Table 1). Maximum concentration of
Figure 1: Total ion chromatograph of 100 ng/mL calibration stan-dard (A), red wine (B), white wine (C)
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Sharma et al.
anthocyanins in both the varieties was recorded on 6th daythis finding is same as recorded by Sims and Bates (1994). whoobserved the maximum rate of anthocyanins extraction betweendays 4 and 6 of fermentation of crushed grapes.The increasein anthocyanin concentrations, recorded in given time intervals,was closely correlated with the production of alcohol, whichwas the most important factor of maceration of colouringmatters. Balik (2006) recorded maximum concentrations ofanthocyanins one day earlier than those of alcohol. Theanthocyanins are localized in the skin tissues of most grapecultivars; fermentation and maceration have a profound effecton the amount of anthocyanins present in the final wine. Anextreme example of this would be the separation of the solidparts of the grape berry from the juice with little or no macerationresulting in a wine with little or no red colour (Kennedy, 2008).
Ferric ion reducing antioxidant power (FRAP) values (Table 3)were registered more than double on the last day (11th day)
sample in comparison to the initial value in CabernetSauvignon while more than four times increase was noted inZinfandel. However, higher FRAP values were noted inCabernet Sauvignon at initial and final fermentation thanZinfandel. Very low FRAP values were noted from both thewhite varieties and declining trend was noted. The red winesamples showed higher free radical scavenging activities thanwhite varieties as estimated by DPPH assay. The antioxidantvalues were increased with advancement of fermentation inboth the red varieties. Wines made from Cabernet Sauvignonrecorded with higher antioxidant as compared to Zinfandel.The FRAP and DPPH values were higher in red wine varietiesthan white. Katalini et al. (2008) also noted that the red wineshave greater reducting power capacity than white wines.Significant differences in reducing power capacity of somered wines can be related to differences in flavonoid content,especially anthocyanins which represent a numerous flavonoidsub-group of very efficient free radical scavengers with
Table 3: Changes in antioxidant property during fermentation process at the interval of days
FRAP (mg/l) DPPH (mM)
Red White Red White
CabernetSauvignon Zinfandel Sauvignon Blanc Charark-4 CabernetSauvignon Zinfandel Sauvignon Blanc Charark-4
Initial 290 130 0.33 1.7 1.24 1.19 0.39 0.602 280 380 0.32 2.3 1.51 1.23 0.42 0.743 340 510 0.19 1.5 1.46 1.60 0.35 0.616 320 600 0.28 1.1 2.10 1.76 1.13 0.5211 660 570 0.26 1.3 2.29 1.90 0.36 0.63SEM± 5.477 10.624 0.010 0.028 0.054 0.053 0.019 0.079CD at1% 16.51 32.02 0.03 0.09 0.16 0.16 0.06 NS
Table 4: Instrument parameters for the individual phenolic compounds.
Sr.no. Name of compound RT (min) ESI+ Quantifier Qualifier
Q1 Q3 DP CE CXP Q3 CE CXP
1. Epicatechingallate 7.87 [M+H]+ 443 123.0 51 19 5 273 12 52. Caffeicacid 7.42 [M+H]+ 181 89.1 26 43 6 135 27 83. Epicatechin 7.14 [M+H]+ 291 123.0 50 23 5 139,165 20 ,18 64. p-Coumaricacid 7.86 [M+H]+ 165 147.0 56 16 6 119 , 91 25, 35 5,35. Quicetrin 8.24 [M+H]+ 449 303.2 45 15 9 129, 85 22, 35 66. Syringicacid 7.29 [M+H]+ 199 155.0 56 14 7 123, 77 18,38 5,27. Vanillicacid 7.21 [M+H]+ 169 125 55 14 5 93 21 38. Quercetin 9.63 [M+H]+ 303.0 153.0 55 45 5 137, 69 45,80 5,19. Resvaretrol 9.24 [M+H]+ 229.0 135 29 22 6 107, 119 30,55 4,5
10. Catechin 6.84 [M+H]+ 291.0 165.0 38 17 7 139,123 22 7, 211. Kaempferol 7.46 [M+H]+ 287.0 165.0 53 40 2 121 50 5
* Q1- Precursor ion, Q3- product ion, DP- declusturing potential, CE- collision energy, CXP- collision exit potential
Changes in phytochemicals during fermentation of wine grapes
23
confirmed excellent antioxidant properties. The value of FRAPand DPPH increased during course of fermentation. However,DPPH and FRAP values in Charark-4 and Sauvignon Blancwere low and declined non-significantly during winemakingprocess.
The concentrations of amino acids in both red and white wineswere measured at initial and final fermentations. Theconcentrations of different amino acids were changeddramatically during the fermentation process (Table 5). Argininewas decreased in both the red wine grapes (CabernetSauvignon and Zinfandel) but was increased in white wine
Table 2: Concentration (mg/L) of different phenolic compounds in wines
Phenolic compounds Concentration (mg/L)
Red White
Cabernet Sauvignon Zinfandel Sauvignon Blanc Charark-4
Epicatechingallate 1.10 0.39 0.00 0.00Caffeic acid 2.14 2.47 0.35 0.33Epicatechin 59.80 59.30 0.11 1.54p- Coumaric acid 0.52 2.65 0.00 0.81Quicetrin hydrate 9.10 1.65 0.60 4.07Rutin hydrate 7.98 1.97 0.24 0.22Syringic acid 5.27 5.29 0.16 0.29Quercetin 7.08 2.71 0.11 0.18Resveratrol 0.50 1.08 0.00 0.00Catechin 117.00 71.40 0.07 3.51Kaempfero 0.316 0.14 0.00 0.00Total Phenolics 4160.00 1050.00 20.00 50.00
Table 5: Changes in quantity of amino acids (mg/l) during fermentation of wine grapes
Amino acid Red wine grapes White wine grapes
Cabernet Sauvignon Zinfandel Sauvignon Blanc Charak-4
1st day 12th day 1st day 12th day 1st day 12th day 1st day 12th day
Arginine 113 79.9 702 80.1 34.4 80.4 37.3 126Aspartic Acid 0 5.7 21.8 26.1 0.274 29 2.49 69.4Glutamic Acid 0 23 70.9 41.6 0.105 30.1 0.203 67Alanine 0 94.5 242 54.5 0.94 58.3 15.3 189Histidine 5.08 36.9 35.4 12.9 2.97 8.74 3.39 16.5Hydroxyproline 15.5 32.4 5.33 8.22 10.3 20.2 9.93 18.2Leucine 1.36 32.7 15.1 31.9 0.367 25.8 0 107Lysine 9.53 30.2 0 33.4 0 29.2 0 64.3Methionine 1.53 0 1.58 12.5 0 11.4 1.55 29.3Ornithine 4.17 31.3 9.37 8.19 0.831 10.9 2.61 52.7Phenylalanine 0 9.06 26.9 13.1 0 9.22 0.156 26.8Proline 448 1150 421 462 940 928 200 733Serine 0 13.5 81.3 19.2 4.39 21.1 0 39.2Tryptophan 0 0.285 6.19 0 0 0.946 0.063 0Tyrosine 1.37 11.4 25 24.2 1.05 20.1 3.37 55.4Vaniline 0 10.5 30 21.8 0.646 18.3 0 55.3
grapes (Sauvignon Blanc and Charak-4). The concentration ofglutamic acid and aspartic acid was decreased at final level offermentation of Cabernet Sauvignon but the concentration ofglutamic acid, Histidine, Ornithine, Phenylalanine, Tryptophan,Tyrosine, and Vaniline was declined in Zinfandel. Thequantities of Leucine and Lysine amino acids were increasedat final level of fermentation of Cabernet Sauvignon and Charak-4. Amino acids in wine originate from various sources such asindigenous compounds in grapes, metabolites by yeast duringgrowth phase exerted by living yeast or released by proteolysisduring the autolysis of dead yeast or by enzymatic degradation
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Sharma et al.
of grape protein (Cataldiet al. 2003). Yeast and bacteria usethese amino acids to grow and ferment the must and can releasesome of these amino acids back into the wine after fermentation.In general, in white wines average levels of free amino acidswas higher and more variable than those of the red musts.Proline makes the majority of the free amino acids in wine andis the largest amount found in fresh grapes. Arginine is animportant nutrient for yeast growth (Ough. 2009). But duringfermentation of white grapes, quantity of arginine wasincreased. During fermentation, yeasts assimilate between 1and 2 g/L of amino acids but towards the end of fermentation,yeasts excrete significant but variable amounts of differentamino acids.The pH values lower than 3.5 have been found toinhibit arginine consumption (Terrade and Mira, 2006). ThepH of white wines was less than 3.5 that might resulted haveincreases of arginine content. Finally, at the end of alcoholicfermentation, a few hundred mg/L of amino acids remain;proline generally represents half. Our results are similar to thefindings of other authors. Proline is major amino acid in allwine grape varieties studied. Tryptophan disappeared in thewinemaking process of Zinfandel and Charak-4 and very lowquantity of this amino acid was generated in CabernetSauvignon and Sauvignon Blanc. During the fermentationprocess, some amino acids were excreted in variableamount.The yeast strains release some amino acids includingL-threonine, L-tryptophan, L-cysteine, and L-methionine duringgrape fermentation (Mauricio, et al. 2001). However, the contentof amino acid in grape must or wine were affected by variousfactors like variety, yeast strain, stage of fermentation processetc.
From this study it may be concluded that the total phenolsand anthocyanin content increased in red wine grape varietiesalong with advancement of fermentation. Maximumanthocyanin content was recorded on sixth day offermentation. Red wines contained higher total as well asindividual phenols than white. The presence of importantphenolic compounds viz.; resveratrol and kaempferol wasfound in red wines only where the grape skin was fermented.The total phonels have significance as an antioxidantcompound
Acknowledgements
Authors are thankful to Dr. K. Banerjee (I/C National ReferralLaboratory) for extending use of LCMS/MS and HPLC foranalysis of wine samples.
References
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Holland, M.V., Bernreuther, A., and Reniero, F. 1995. The use ofamino acid as a fingerprint for the monitoring of Europeanwines. Magnetic Resonance in Food Sciencepp 136 – 145.Belton, P. S., Delgadillo, I., Gil, A. M., and Webb, G. A.(Eds.), The Royal Society of Chemistry, Cambridge, UK.
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Intl. J. of Food. Ferment. Technol. 2(3): 27-36-, June, 2012
Cloning and expression of rec-pediocin CP2 inEscherichia coli using OmpA and TAP gene
fusion approach
Balvir Kumar*, P. P. Balgir and B. Kaur
Department of Biotechnology, Punjabi University, Patiala, Punjab, India
*Email: [email protected], [email protected]
Paper no: 34 Received: 26 Jan, 2012 Received in revised form: 17 April,2012 Accepted: 19 May,2012
Abstract
Study was aimed at cloning, expression and characterization of rec-pediocin CP2 in Escherichia coliBL21(DE3) using ompA and tap gene fusion expression approach. pET32(b)-pedA vector containingan inframe fusion of sequences encoding E. coli ompA secretion signal, two tandem affinity purificationtags and rec-pediocin synthetic gene was introduced into a pediocin CP2 resistant strain of E. coliBL21(DE3). Rec-protein was accumulated in periplasmic fraction as well as inclusion bodies oftransformed E. coli cultures and was extracted by cell lysis. Crude fusion protein did not show anybiological activity. Purification of rec-pediocin involved two tandem affinity chromatographic stepsbased on 6XHis-tag and Strep-tag. In between these, enterokinase cleavage was carried out. Afterdigestion, rec-pediocin displayed a very strong bactericidal activity against Listeria monocytogenes.The process based on T7-driven pET expression system and tandem affinity chromatography resultedin approximately 10 times higher yield of rec-pediocin (in terms of specific activity) as compared to thenative P. acidilactici MTCC5101.
©2012 New Delhi Publishers. All rights reserved
Keywords: Rec-pediocin, Pediococcus acidilactici, Pediocin CP2, Heterologous expression,Cloning, Bacteriocin
Bacteriocins of lactic acid bacteria have long been used forfermentation and preservation of meat and milk (Gillor et al.,2008). Pediocins are prone to proteolytic digestion in thegastrointestinal tract and seem to be non-toxic and lack anypotential antigenicity/toxicity in animals (Bhunia et al., 1990;Guerra et al., 2001; Kheadr et al., 2010). Native hosts sufferfrom low fermentation yields and poor recovery due to in vitrointeractions of bacteriocins with other proteins (Daba et al.,1994; Elegado et al., 1997; Stein et al., 2003; Beaulieu et al.,2007). Keeping this in view, a large number of heterologoussystems have been developed for controlled expression ofpediocins including Bifidobacterium longum (Moon et al.,2005), Enterococcus faecalis (Somkuti and Steinberg, 2003),Escherichia coli (Halami and Chandrashekar, 2007; Liu et al.,2011), Lactococcus lactis (Halami and Prakash, 2008), L. sakei(Johnsen et al., 2005), Streptococcus thermophilus (Somkuti
and Steinberg, 2003), Saccharomyces cerevisiae (Schoemanet al., 1999) and Pichia pastoris (Beaulieu et al., 1999). Rec-pediocins were secreted out in the culture medium by attachingspecialized secretary signals such as Bifidobacterial α-amylasesignal peptide (Moon et al., 2005), lactococcin A secretoryapparatus (Arques et al., 2008) and yeast mating factor α-1Ssignal peptide (Schoeman et al., 1999). Attachment of highlyspecific affinity tags facilitated their detection by affinitypurification, immuno-fluorescence, immuno-precipitation andWestern blotting. Many reports indicated decrease in viabilityof recombinant cells (Miller et al., 1998), low expression levels(Horn et al., 1998; 1999), loss of recombinant plasmids fromexpression host (Coderre and Somkuti, 1999) and aggregationof rec-pediocin with other host proteins (Beaulieu et al., 2007).Expression of inactive rec-pediocin in inclusion bodiesfollowing over expression in the recombinant host (Halami
Research paper
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Kumar et al.
and Chandrashekar, 2007; Liu et al., 2011) further complicatedthe purification as additional steps of isolation, purificationand refolding are needed.
Pediocin CP2 is a class IIa bacteriocin produced byPediococcus acidilactici MTCC 5101 (Kaur and Balgir, 2004;Balgir et al., 2010). It has a great commercial importance owingto its remarkable heat stability (121oC for 15min), activity overa wide pH range (2.5 to 9.5), higher specificity andeffectiveness at very low concentrations. Pediocin productionin P. acidilactici MTCC 5101 has been linked to a 8.9 kb plasmidpCP289 (Kaur and Balgir, 2007). It inhibits growth of Aspergillusflavus, Clostridium sporogenes, Enterococcus faecalis,Escherichia coli, Lactobacillus brevis, L. bulgaricus,Leuconostoc mesenteroides, Listeria monocytogenes,Micrococcus flavus, Neisseria mucosa, Pediococcusacidilactici, P. pento-saceus, Pseudomonas putida, P.aeruginosa, Salmonella typhimurium, Staphylococcus albus,S. aureus, Streptococcus mutans and S. pyogenes. It killssensitive bacterial strains in a bactericidal manner andsusceptible fungal cultures by inhibiting their sporulation (Kaurand Balgir, 2008; Kumar et al., 2011). Here, we report high levelexpression of rec-pediocin in E. coli BL21(DE3) followed bytandem affinity purification and glycine/β-mercaptoethanol(β-ME) mediated in vitro refolding of rec-pediocin CP2. Thismethod provides approximately 10 times higher specific activityof rec-pediocin than native pediocin CP2, produced in a shorttime to be used for biophysical and in vitro therapeutic studies.
Materials and methods
Procurement and maintenance of cultures
P. acidilactici MTCC5101 was grown in de Man, Ragosa andSharpe medium (MRs) (Lactobacillus Heteroferm Screen Broth,Himedia) containing 0.1% Tween-80 and pH6.5 at 37ºC. E. coli
DH5α MTCC1652, E. coli BL21(DE3) MTCC1679 and Listeriamonocytogenes MTCC657 were procured from MTCC,Chandigarh, India. E. coli cultures were revived and maintainedin LB medium (containing tryptone 10g/l, yeast extract 5g/l,NaCl 10g/l) at 37°C for 24h. L. monocytogenes MTCC657 wasmaintained in Brain Heart Infusion broth (Himedia) at 37ºC. P.acidilactici LB42 procured from Prof. R. K. Malik (NDRI, Karnal,India) was grown in MRS medium at 37ºC and maintained asglycerol stocks. E. coli expression vector pET32(b) was kindlyprovided by Prof. R.K. Jethi, Ambala College of Engineeringand Applied Research, Ambala Cantt., India.
Designing synthetic rec-pediocin fusion gene construct
Pediocin CP2 gene cluster was designed according to codonusage of E. coli using various bioinformatics tools of SequenceManipulation Suite version 2 available online (Stothard, 2000).Novagen’s pET32(b) vector was selected for expression ofrec-pediocin in E. coli BL21(DE3). To facilitate periplasmicexpression, a 22 amino acid long secretion signal of outermembrane protease (OmpA) of E. coli BL21(DE3) wasincorporated at N-terminus of rec-protein. To enhance recoveryof rec-protein from cell lysates, two affinity tags were added intandem (TAP-tandem affinity purification technique). Twoproteolytic cleavage sites were added downstream of eachaffinity tag to facilitate their selective removal. Pediocinsequence was manipulated to enhance antimicrobial range,production and secretion of rec-pediocin in heterologous host(Tominaga and Hatakeyama, 2007). Figure 1 comparativelyillustrates sequence features of the designed rec-pediocinfusion protein and native pediocin CP2. Parental pediocin CP2sequence (KYYGNGVTCGKHSC) was changed toTKYYGNGVSCTKSGC. The designed 94 amino acid longsequence of rec-pediocin was then reverse translated usingSMS reverse translate tool. Finally, KpnI site and SalI restrictionsites were added 5’ and 3’ and it was further polished using
Figure 1: Comparison of sequence features of rec-pediocin fusion protein and native pediocin CP2
Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach
29
oligo analysis tool of Generunner. Sequence features ofdesigned fusion gene construct are shown in Figure 2. Pediocingene construct has been synthesized by a German basedcompany (GENART). It was supplied in the lyophilized powderform which was reconstituted and multiplied in recombinantE. coli DH5α after its transformation.
Isolation and analysis of plasmid DNA
Expression vector pET32(b) and GENART carrier plasmid wereisolated using mdi plasmid DNA miniprep kit. Isolated plasmidswere analyzed on 0.8% agarose gels containing 5µg/ml ethidiumbromide. Electrophoresis was carried out in 1X TBE buffer(containing 0.089M Tris, 0.089M Boric acid and 0.002M EDTA,pH8.3) at 50V for 1h. Gels were finally visualized on UV-transilluminator.
Construction of recombinant pET32(b)-pedA
Sequential double digestion with KpnI and SalI was carriedout for linearization of vector and isolation of syntheticpediocin gene construct. Digestions were performed using100µg plasmid DNA, 50U KpnI and SalI, 1X restriction enzymebuffers and 1X BSA at 37°C for 3 to 4h. Reactions were stoppedby keeping reaction vials at 65°C for 20 min. Separatedrestriction fragments, bearing rec-pediocin fusion geneconstruct (300bp) and linearized pET vector (5843bp), wereextracted from agarose gels using Bangalore Genei gelextraction kit. Ligation reaction was performed using 1 unit T4DNA ligase per µg of restricted DNA and IX cohesive endligation buffer. 5µg recombinant pET32(b)-pedA vector wasused to transform E. coli DH5α (primary storage host) and E.coli BL21(DE3) (expression host). Cultures were multiplied inLB medium containing ampicillin (100µg/ml).
Screening and selection of recombinant E. coli BL21(DE3)-pedA
Transformed E. coli BL21(DE3)-pedA was spread on Luria Agarplates containing 100mg/ml ampicillin and incubated at 37°Cfor 24h. Around 100 isolates were picked up, multiplied byrepeated subculturing and maintained as glycerol stocks.Isolates were then screened for recombinant pET32(b)-pedAby comparative plasmid profiling and PCR assay. A sequencespecific pair of primers constituting a forward primer5’TGGCGCTGGCGGGCTTTG3’ and a reverse primer5’CTGATGGCCGCCGGTCG3’ was used to amplify a 239bpfragment of rec-pediocin gene construct. PCR reactions wereset up using 1ng template DNA, 250pM each of forward andreverse primer, 2U iTaq DNA polymerase (Invitrogen), 1X iTaqBuffer, 10µM MgCl
2, and 50µM dNTP’s. GENART carrier
plasmid and native pET32(b) were incorporated as positiveand negative controls respectively. Their reaction mixturescontained 1U Vent DNA polymerase (Invitrogen) and 1X VentDNA polymerase buffer in addition to standard components.PCR was carried out in Techne thermal cycler in four segments.Segment I consisted of initial denaturation at 95ºC for 3 minfollowed by segment II having 30 cycles of denaturation at95ºC for 1 min, annealing at 51ºC for 1 min and synthesis at72ºC for 1 min. Segment III included a final extension of 5 minat 72ºC and PCR products were kept at 4ºC for 5 min duringsegment IV. PCR products were analyzed on 1.2% agarose gelsin the presence of Novagen’s molecular weight marker. Finalconfirmation of insert DNA was carried out by sequencing239bp amplicon outsourced at Bangalore Genei.
Expression and purification of rec-pediocin fusion protein
Expression of rec-pediocin was induced in early log phase
Figure 2: Sequence and features of rec-pediocin fusion gene construct
30
Kumar et al.
cultures (3-4h) of E. coli BL21(DE3) by adding 1mM IPTG.Cultures were allowed to grow for additional 4h after inductionat 37ºC. E. coli BL21(DE3) transformed with native pET32(b)was incorporated in the study as a negative control. Cellswere harvested by centrifugation at 6000g, washed with 0.9%NaCl and resuspended in 50mM Tris (pH7.5) containing 2mMEDTA and 0.1% Triton X100. Resuspended cells were freezethawed twice and disrupted by sonication for 1min (Halamiand Chandrashekar, 2007). Inclusion bodies (IBs) wereprecipitated by centrifugation, washed with 2M urea andresuspended in 6M urea, 1mM EDTA and 50mM Tris-HCl,pH8.0. The urea solublized IBs were resuspended slowly inrefolding buffer consisting of 50mM Tris (pH7.5), 50mM NaCl,1mM EDTA, 5mM β-mercaptoethanol, 5mM immidazole and1M glycine and stirred for 18-20h at room temperature thatwould assist in protein refolding and solublization (Eisenmesseret al., 2000). His-tag protein purification column (BangaloreGenei) was charged with refolding buffer containing 5mMimmidazole and solublized IBs were loaded onto it. Unboundproteins were washed in 50mM Tris (pH7.5), 50mM NaCl, 1mMEDTA, 5mM β-mercaptoethanol, 20mM immidazole and 1Mglycine. Nickel bound poly-his tagged rec-pediocin fusionprotein was eluted in 50mM Tris (pH7.5), 50mM NaCl, 1mMEDTA, 5mM β-mercaptoethanol, 0.5M immidazole and 1Mglycine.
Fusion protein was digested with recombinant humanenterokinase in a reaction mixture containing 1mg His-taggedfusion protein, 5 units enterokinase, 1X enterokinase cleavagebuffer, and 1X enterokinase dilution buffer at 20ºC for 16h.Enzyme was inactivated by keeping the reaction mixture at65oC for 20min. Digested fusion protein was further separatedon 500µl Strep-tactin affinity resin. Resin was packed undergravity flow in a polypropylene syringe plugged with glasswool. It was equilibrated using 2 volumes of 1X wash buffer(containing 1M Tris-HCl, pH8.0, 1.5M NaCl, 10mM EDTA).Rec-protein was loaded and washed five times each with 1volume 1X wash buffer. Flow throughs and washings werecollected in separate vials to check for presence of rec-protein.N-terminal TAP fragment was eluted from the resin 6 timeseach with 0.5 volume 1X elution buffer (containing 1M Tris-HCl, pH8.0, 1.5M NaCl, 10mM EDTA, 25mM Desthiobiotin).Elution samples were collected and OD was checked at 280nm.Strep-tactin resin was regenerated using regeneration buffer(comprising of 1M Tris-HCl, pH8.0, 1.5M NaCl, 10mM EDTA,10mM HABA).
Periplasmic expression was checked in induced cell cultureswhich were harvested by centrifugation at 6500Xg for 15minat 4oC and resuspended thoroughly in 0.8ml sucrose lysis buffer(30mM Tris-HCl, 20% sucrose, pH8.0) and 1.6µl of 0.5M EDTA,pH8.0. Suspension was stirred slowly at room temperature for
10 min and centrifuged at 10,000Xg at 4oC for 10min. Supernatantwas separated very carefully to remove all the liquid, becauseresidual EDTA might interfere with binding to the metal affinityresin. Pellet was suspended very gently in 0.8 ml ice cold 5mMMgSO
4 and stirred slowly for 10min on ice. Shocked cells of
recombinant E. coli appeared round instead of rod shapedunder the light microscope. Finally, cells were separated bycentrifugation at 10,000Xg at 4oC for 10min and supernatantwas collected in a new vial and stored at 4oC.
Biophysical characterization of recombinant pediocin CP2
Analysis of the urea lysates, periplasmic extracts, poly-Histagged rec-pediocin fusion protein and digested rec-pediocinwas carried out on 20% SDS-polyacrylamide gels in presenceof medium range protein molecular weight marker (BangaloreGenei) run at 100V for 90 min. Biophysical analysis of unfoldedand refolded rec-pediocin CP2 was carried out by semi-preparative C-18 reverse phase HPLC using the protocoldescribed by Elegado and others (1997). Gradient elution wasperformed using solvent B (99.9% acetonitrile with 0.1% TFA)against solvent A (water with 0.1% TFA) at a flow rate of 1.5ml/min (Halami and Chandrashekar, 2007). All the protein peakswere collected manually, concentrated by vacuum evaporationand subjected to bioassay to identify the rec-pediocin peak.UV absorption spectrum (from 190 to 350nm) of the purifiedrec-pediocin was also compared with that of rec-pediocinfusion protein.
Preparation of native pediocin CP2 of P. acidilacticiMTCC5101
Native pediocin CP2 preparation was prepared by growing theP. acidilactici MTCC5101 in MRS broth at 37°C for 24h. It wasfurther purified by Adsorption-Desorption method describedpreviously by Yang and others (1992).
Pediocin CP2 activity assay and protein estimation
Bacteriocin activity assay was performed against indicators L.monocytogenes MTCC657 and P. acidilactici LB42 as perstandard method (Bhunia et al., 1988). Bacteriocin activity wascalculated as arbitrary unit (AU) and expressed as AU/ml.Protein estimation was carried out by measuring OD at 280nmusing BSA as a standard.
Results
Designing synthetic rec-pediocin gene construct
Rec-pediocin CP2 fusion peptide carries 5 point mutationsincluding Thr
1, Ser
9, Thr
11, Ser
13 and Gly
14 in N-terminal region
as compared to native pediocin CP2 produced by P. acidilactici
Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach
31
MTCC5101, pediocin PA-1 produced by P. acidilactici PAC1.0and pediocin AcH produced by P. acidilactici AcH videGenBank accession numbers ACS70931.1, P29430.2 andAAA98337 respectively. Periplasmic expression andpurification of rec-protein was facilitated by ompA secretionsignal of E. coli, and tandem affinity tags. E. coli codon choiceand Novagen’s pET32(b) further were maximized expressionof rec-pediocin CP2 in heterologous system.
The theoretical molecular weight of rec-pediocin CP2 waspredicted as 9.87 kDa with a pI of 8.67 (using Compute pI/Mwand TagIdent tools), which was approximately three timeshigher than the native pediocin CP2. Sequence manipulationsuite tool predicted its “Test Code” value as 1.063 which ismore than 0.95 for a protein coding sequence (Fickett, 1982).ProtParam tool (Expasy proteomics server) estimated half-lifeof recombinant pediocin CP2 as 30h (in mammalianreticulocytes, in vitro), >20h (in yeast, in vivo), >10h (inE. coli, in vivo). The instability index (II) of 10.12 classified itas stable. Simple Modular Architecture Research Tool(SMART) identified a bacteriocin domain in the designedconstruct with a significant e-value.
Construction of recombinant pET32(b)-pedA vector
pET32(b)-pedA of 6143bp was constructed using sequencesof native pET32(b) and pediocin synthetic fusion geneconstruct. A 300bp fragment of synthetic pediocin geneconstruct was ligated to 5843bp fragment of double digestedpET32(b) (Figure 3). E. coli DH5α and E. coli BL21(DE3) weretransformed with 6.1 kb recombinant vector and multiplied inLB medium containing ampicillin.
Screening and selection of recombinant E. coli BL21(DE3)
Plasmids were extracted from transformed cultures andanalyzed on 1.2% agarose gels. Most of the isolates indicatedpresence of 6.1kb plasmids which were subjected to PCR. APCR product of 239 base pairs further confirmed the presenceof rec-pediocin fusion gene construct in them (Figure 4).Sequence analysis of the amplified products also confirmedpresence of original insert DNA i.e. rec-pediocin synthetic genein correct orientation.
Expression and purification of rec-pediocin CP2
Rec-protein was purified by TAP technology where a sequential
Figure 3: Analysis of double digested plasmids in 2% agarose gel.Lane 1, native pET32(b), lane 2, double digested pET32(b), lane 3,medium range DNA ruler (Bangalore Genei), lane 4, double digestedGENART plasmid with pediocin fusion gene construct and lane 5,undigested gene carrier plasmid”
Figure 4: Analysis of PCR products on 1.2% agarose gel. Lane 1,0.05 to 10kb DNA ruler (Novagen) and lane 2, 239bp PCR productfrom recombinant pET32(b)-pedA
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Kumar et al.
metal affinity and strep-tactin affinity chromatographies werecarried out. A 9.87 kDa rec-protein was eluted from His-tagaffinity matrix with 0.5 M immidazole as indicated in Figure 5.Over expression of rec-protein was observed in periplasmicfraction as well as IBs (Figure 6). However, induced cells wereunable to synthesize rec-protein as evidenced by lack ofprotein band of the expected size (Figure 5). Upon over-expression in heterologous systems, rec-protein accumulatedin IBs of E. coli as a result of reducing conditions of thecytosol. Cells were disrupted, and IBs were harvested to extractrec-protein. IBs were subsequently washed and resolublizedfor proper folding of rec-proteins (Kane and Hartley, 1991;Mukhopadhyay, 1997).
Subsequenly, native pediocin CP2 obtained using adsorption-desorption approach from Pediococcal culture broth and rec-pediocin extracted from recombinant E. coli using tandemaffinity purification approaches are compared in Table 1. NativePediococcal host produced pediocin CP2 with a specificactivity of 30,612 AU/mg that increased to 95000 AU/mg afterpurification by adsorption-desorption method. There was 3.1fold increase in specific activity of pediocin CP2 with only31.66% recovery. On the other hand recombinant system
offered approximately 10 times higher yields of rec-pediocin(9,00,000 AU/mg specific activity) as a result of over-expressionin T7-driven system and a very high purification level achievedthrough tandem affinity purification with an intermediateproteolytic digestion. Recovery of rec-protein form urea celllysates was 675% and 29.4 fold purity was displayed by TAPpurification. Fractions containing rec-pediocin fusion proteinobtained from the 1st metal affinity matrix were collected andactivity assay was performed against L. monocytogenes andP. acidilactici LB42. No activity was observed in any of theelution fraction against tested indicators. They were digestedwith enterokinase and N-terminal fusion tags were separatedfrom rec-pediocin using strep-tactin affinity purification (Figure7) that improved purity level without interfering with final yieldof the product. Rec-pediocin recovered after digestion of fusionprotein with enterokinase successfully inhibited growth ofindicator strain in a disc diffusion bioassay (Figure 8). Anti-microbial activity of rec-pediocin was also comparatively higher(128AU for 103 cfu/ml indicator) than native pediocin CP2(400AU for 103 cfu/ml indicator) when tested against L.monocytogenes. Thus, it indicated that engineered proteindisplayed lower MIC value against tested indicator strain as
Figure 5: Analysis of rec-protein on silver stained polyacrylamidegel. Lane 1, His-tag purified rec-pediocin fusion protein, lane 2, urealysate of E. coli BL21(DE3) transformed with native pET32 (b),lanes 3 and 5, urea lysates from recombinant isolates and lane 4,protein molecular weight marker (Bangalore Genei)
Figure 6: SDS-PAGE analysis of periplasmic extract from hyperexpressing E. coli. Lane 1, periplasmic fraction from induced cells,lane 2, protein molecular weight marker (Bangalore Genei) and lanes 3and 4, Ni-NTA purified rec-pediocin fusion protein
Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach
33
reported previously (Tominaga and Hatakeyama, 2007).
Biophysical characterization of rec-pediocin CP2
In a semi-perperative RP-HPLC, refolded rec-pediocin waseluted as a single major peak at 3.036 min with a shoulder at3.202 min as compared to unfolded rec-pediocin that showedlate elution at 3.25 min (Figure 9). Minor peaks at 2.559 and2.702 min indicated contamination of rec-pediocin with TAPfragment. Another cycle of Strep-tactin affinity purificationwas therefore incorporated to remove protein contaminants
from pure rec-pediocin preparation. Refolding of rec-pediocinto its active confirmation in refolding buffer was confirmed intwo separate experiments including growth inhibition of L.monocytogenes and P. acidilactici LB42 in a disc diffusionbioassay and a complete loss of activity in undigested sample.The MIC values of engineered pediocin bearing five pointmutations were lower than native pediocin CP2. UV absorptionspectrum of the purified rec-pediocin was also compared withthat of rec-pediocin fusion protein. Absorbance maximum ofthe recombinant pediocin was observed at 195 nm with ashoulder at around 220 nm as compared to 205 nm for rec-
Figure 7: Gel analysis (20% SDS-PAGE) of rec-pediocin purified byTAP technology. Lane 1, strep-tactin purified rec-pediocin, lane 2,protein molecular weight marker (Bangalore Genei), lane 3, His-tagpurified rec-pediocin fusion protein and lane 4, fusion product di-gested with enterokinase
Table 1: Purification of native and rec-pediocin using different approaches
Bacteriocin Sample Total Volume Total Protein Total Bacteriocin Specific Folds of % Recovery(ml) (mg) Activity Activity Purity
(AU) (AU/mg)
Cell free supernatant 400 392 1,20,00,000 30,612 a - 100Native pediocin CP2 10 40 38,00,000 95,000 a 3.103 31.66(Adsorption-Desorption method)Culture broth 400 ND Nil ND - -Rec-pediocin 10 90 8,10,00,000 9,00,000 a 29.4 675(Tandem affinity purification)
aData is an average of two experimental sets
Figure 8: Bacteriocin disc assay plate showing inhibition ofindicator bacteria around bacteriocin discs numbered 1 to 3and 6 to 10
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Kumar et al.
pediocin fusion protein (Figure 10). The difference may beattributed to the N-terminal TAP fragment.
Discussion
expression is controlled by lacI repressor protein. It is deficientin lon protease and ompT an outer membrane protease thatcan degrade expressed proteins during purification (Grodbergand Dunn, 1988). By adjusting concentration of IPTG,expression can be regulated from very low up to robust, fullyinduced levels. Additionally, the heterologous expression ofclass IIa pediocins in E. coli as fusion proteins will improveprotein solublization and avoid possible toxic effects on thehost cell (Quadri et al., 1997; Moon et al., 2006; Beaulieu et al.,2007).
This bioprocess offered approximately 10 times higher yieldsachieved collectively through PT7 based pET32(b)-pedA andtandem affinity purification. Fusion protein itself did not showany biological activity, but upon cleavage by an enterokinase,biologically active pediocin PA-1 was obtained. As it wasexpected, rec-pediocin displayed 3.125 times more antimicrobialactivity than its native counterpart. This confirms the findingsof Tominaga and Hatakeyama (2007) that pediocins withmultiamino acid substitutions prove more useful in terms oftheir final yields and bacteriocin activity against food spoilageorganisms. A PT5 based pQE32 has been previously used forover-expression of pediocin F of P. acidilactici F in E. coli(Osmanagaoglu et al., 2000). Similarly, Beaulieu et al. (2007)cloned and expressed thioredoxin-pediocin PA-1 fusion proteinin E. coli. Moon et al. (2006) coexpressed pedA with His-taggedDHFR using vector pQE40PED in E. coli M15. Recombinantsdisplayed very high pediocin activity upon overexpressionwith IPTG and subsequently fusion protein was purified byNi-NTA metal affinity chromatography. Native pediocins wererecovered from their fusion partners by enzymatic digestion.
Many reports suggested that the refolding buffer containing5-15 mM β-mercaptoethanol, and 1M glycine facilitatesrenaturation of proteins, allows correct formation of disulfidebonds and reduces protein aggregation during refoldingprocess (Rogl et al., 1998; de Bernardez Clark, 2001; Halamiand Chandrashekar, 2007; Tominaga and Hatakeyama, 2007).In accordance with earlier reports, present strategy usingrecombinant pET32(b)-pedA and E. coli BL21(DE3) allowedcopious accumulation of recombinant protein in IBs oftransformed cells and slow dilution of urea solubilized IBs in arefolding buffer consisting of â-ME along with glycine assistedin proper folding of the rec-pediocin in its proper secondarystructure.
Spectroscopic analysis is a widely exploited method to studyrefolding of rec-proteins. Previously, Patra et al. (2000) hadused this technique to study refolding of recombinant humangrowth hormone. Similarly, Halami and Chandrashekar (2007)compared UV absorption spectra of native and recombinantpediocin PA-1 to study refolding of rec-protein. Their UVabsorption spectra were almost identical except for a shoulder
Figure 9: Reverse phase chromatograms of A) rec-pediocin fusionprotein and B) pure rec-pediocin
Figure 10: UV absorption spectra of rec-pediocin and its fusionproteinE. coli BL21(DE3) was selected for achieving high levelproduction of rec-proteins as extensive workout has alreadybeen done to study regulation of gene expression and toachieve biological activity of rec-proteins (Makrides, 1996). Inaddition, a variety of highly specific fusion partners areavailable that facilitate detection of recombinant proteins byaffinity purification, immuno-fluorescence, imm-unoprecipitation, Western blotting (Kumar et al., 2011). Inpresence of selection pressure, E. coli cells usually maintain avery high copy number (approximately 40 /cell) of pET vectorand its derivatives that carry a strong T7 phage promoter whose
Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach
35
at 220 nm in case of recombinant pediocin which was attributedto the N-terminal extention of pre-peptide sequence and tagfragment. Our results support these observations asbiophysical changes in rec-pediocin and its refolding can beeasily studied by a simple spectroscopic analysis.
Conclusion
Pediocins have attracted health conscious society because oftheir remarkable stability, broader antimicrobial spectrum,higher specificity and effectiveness in very low concentrations.Pediocin CP2 has a great potential to contribute in food, healthand pharmaceutical industry. Large scale production ofpediocin CP2 using a fastidious strain of P. acidilaciticiMTCC5101 is highly uneconomical and its purification usingconventional methods are cumbersome and time consumingtoo. In recent years, a great deal of diverse expression systemswere exploited for cloning, expression and purification ofpediocins at laboratory scale but data are lacking for industrialpro-cesses. Hence, it necessitated for the development of lowcost, industrially viable and con-tinuous system in order toexploit this natural bioactive compound in food andpharmaceutical industry. A bioprocess is now in place whererec-pediocin fusion protein can be expressed in large quantityusing a cheaper culture medium, recovered from IBs, solubilizedand refolded to its biologically active conformation and purifiedeasily by TAP purification described in the study. Thepreliminary characterization that has been done with rec-pediocin has revealed its several desirable properties as invivo antimicrobial agent. What remains unexplored is to usethis knowledge for formulating novel therapeutics and studytheir suitability as in vivo antimicrobial agents.
Acknowledgement
Authors acknowledge the UGC, New Delhi, India, for providingfinancial assistance in the form of Rajiv Gandhi NationalFellowship to Mr. Balvir Kumar.
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purification of a fusion-typed pediocin PA-1 in Escherichiacoli and recovery of biologically active pediocin PA-1. Int. J.Food Microbiol., 108: 136-140.
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Intl. J. of Food. Ferment. Technol. 2(1): 37-41, June, 2012
Effect of growth conditions on extracellular ααααα-amylaseproduction by Bacillus thuringiensis
V.R. Tembhurkar1*, M.K. Bannatwala2, S.S. Udare3 and M.G. Kalyankar4
1* Department of Microbiology, ASC College, Jalna, India2Department of Medical Microbiology, Greater Kailash Hospital, Indore, India3Department of Biotechnology, MGM College, Aurangabad, India4Deptepartment of Biotechnology, University of Houston, Clear Lake, USA
*Email: [email protected].
Paper no: 35 Received: 19 December 2011 Received in revised form: 14 April 2012 Accepted: 17 May 2012
Abstract
Influence of few growth conditions and media components on alpha amylase production by Bacillusthuringiensis in lab scale batch process were determined. B. thuringiensis produced maximum amylaseafter 72hrs of incubation. The optimum temperature and pH for amylase production were found to be31oC and 7, respectively. The optimized media composed of [g/l] 10.0g Rice flour; 5.0 Peptone; CaSO
4
4.0; MgSO4 2.0; NaCl 1.0; FeSO
4 0.5 and pH 7. The alpha amylase produced was working optimally at
60oC, pH 5, [S] 20 mg/mL, [E] 2.0 mL. Many of these parameters are in compliance with the insecticidalcrystal protein production by B. thuringiensis. Therefore, the process can be used for simultaneousproduction of alpha amylase and bioinsecticide in a single fermentation cycle.
©2012 New Delhi Publishers. All rights reserved
Keywords: Bacillus thuringiensis, Alpha amylase, Insecticide Crystal proteins,growth
Bacillus thuringiensis (Bt) is a popular microbial insecticideextensively applied in agriculture and forestry for controllinginsect pests (Ninfa and Rosas-García, 2009). These insecticidalproteins are products of cry genes whose expression peaks instationary phase of growth. These insecticidal proteins generallyaccumulate in the mother cell compartment (intracellular) to forma crystal inclusion (ICP) that can account for 20 to 30% of thedry weight of the sporulated cells (Schnep et al., 1998). The ICPof Bt has been reported to be active against certain insectspecies among the orders Lepidoptera, Diptera, Coleoptera.Hymenoptera, Homoptera, Orthoptera, Mallophaga and againstnematodes, mites, and protozoa (Feitelson et al., 1992; Beegleand Yamamoto, 1992; Feitelson, 1993). Bt is already a usefulalternative or supplement to synthetic chemical pesticideapplication in commercial agriculture, forest management, andmosquito control (Schnep et al., 1998).
Although Bt is well known for bioinsecticide production it isalso a potential producer of alpha amylase. A few strains of Btdo produce and secrete several isozymes of alpha amylases(Maity et al., 2011). But alpha amylase production parametersof Bt are largely unexplored thus, its use for large scaleproduction of alpha amylase is still obscure. Microbial amylaseshave completely replaced chemical hydrolysis of starch in starchprocessing industries (Vidyalakshmi et al., 2009). Both ICPand alpha amylase are of immense commercial importance. Theprevious one in intracellular and later is secreted out of thecell, additionally their synthesis is independent. The objectiveof this work is to optimize growth conditions and mediadesigning for maximum production of alpha amylase by Bt. Ifthe results match with ICP production parameters, this willallow simultaneous production of alpha amylase and ICP insingle fermentation cycle. Such “Simultaneous fermentation”
Research paper
38
Tembhurkar et al.
of two products will be a cost effective option for their largescale production. The results obtained are discussed in thiscommunication.
Materials and methods
Test Culture: Bacillus thuringiensis ATCC 10752 was used inpresent studies and its amylase production ability wasconfirmed by plating on starch agar composed of [g/l] 5.0gBeef extract; 0.5 g NaCl; 1.0 g Starch, 2.0g Agar agar, pH 7.After 48hr of incubation, starch hydrolysis was detected byreacting with Lugal’s iodine (Harley and Prescott, 2002).
Enzyme assay: Alpha amylase activity was measured in termsof hydrolysis product, the reducing sugars released by enzymeaction. The reducing sugars released were measured byBernfeld method that uses 3,5-di-nitrosalicylic acid as couplingagent (Bernfeld, 1955). One enzyme unit (U/ml) of alphaamylase is the amount of enzyme required to liberate 1µmoleof product (Dey et al., 2003).
Optimization of growth conditions: Alpha amylase productionwith respect to incubation time, incubation temperature andmedia pH was estimated in independent batch experiments.The composition of fermentation medium used was [g/l] 5.0 gBeef extract; 0.5 g NaCl; 1.0 g Starch, pH 7. The fementationmedium was inoculated with 1% inoculum. Inoculum wasprepared in one step of 48hr incubation in media of compositionsimilar to the method used for fermentation medium. In timecourse experiment, amylase activity was assayed after every24hrs till activity was dropped after constant rise. Fortemperature optimization the fermentation medium flasks wereincubated at different temperatures viz. 28ºC, 31ºC, 34ºC and37ºC, 41ºC. Similarly, for pH optimization, media pH wasadjusted in the range of 4 to 9 using 0.1N NaOH/HCl.
Media formulation: Effect of carbon source, nitrogen sourceand micronutrient was determined. For carbon sourceoptimization media composition was [g/L] 10.0g Carbon source;5.0 Beef extract; pH 7. Carbon sources used were rice flour,wheat flour, corn flour, potato and pure starch. Following this,nitrogen source was optimized in media composed of [g/L]10.0g Optimum carbon source; 5.0 Nitrogen source; pH 7. Yeastextract, tryptone, beef extract, peptone, urea, ammonium nitrateand ammonium sulphate were tested as nitrogen sources.Lastly effect of micronutrients (Na+, Mg2+, Ca2+, Fe2+) at variousconcentrations (0.05, 0.1, 0.2, 0.4%) on amylase productionwas analyzed. Production media used contained [g/L] 10.0gOptimum carbon source; 5.0 Optimum nitrogen source; Salt tobe tested (NaCl/MgSO
4/CaSO
4/FeSO
4) and pH 7.
Enzyme characterization: The optimum temperature, pH,substrate concentration, enzyme concentration, reaction timeand kinetic constants (K
m and V
max) were determined. To
determine effect of temperature, the reacting mixture wasincubated at different temperatures range 30 to 100 oC for 15minfollowed by sugar liberated estimated by DNS method (Bernfeld,1955). For pH optimization, buffers of different pH were used;acetate buffer for pH 4 & 5; phosphate buffer for pH 6, 7 & 8;glycine/NaOH buffer of pH 9. Effect of substrate concentrationwas analyzed using 1 to 10% starch. 0.1 to 2.0 ml of enzymewas used to study the effect of enzyme concentration. Optimumreaction time was determined by measuring reducing sugarproduced after every 5min for 60min. Values of kineticconstants were estimated by plotting Lineweaver-Burk doublereciprocal plot of 1/[S] Vs. 1/[V].
Result and discussion
Optimization of growth conditions: Alpha amylase productionby Bt reached to maximum level on third day of incubation afterwhich amylase concentration progressively decreased till fifthday of incubation (Table 1). The amylase production was 10fold higher at 31oC than at other temperatures tested (Table 2).Deviation of initial media pH from neutral pH (7) negativelyaffected alpha amylase production (Table 3). According tofindings of Yezza et al. (2005) entomotoxicity of Bt reached peakat 72hrs of incubation . This observation is in agreement withfindings of Vidyalakshmi et al (Vidyalakshmi et al., 2009). Ozkanet al. (2003). concluded in his study of toxin production by B.thuringiensis HD500 that the highest concentrations of Cry4Bawas produced at 25°C, while the optimum temperature forproduction of Cry11Aa was 30°C (Ozkan et al., 2003). KhanhDang Vu et al.(2009) also reported a pH value of 7 was optimumfor bioinsecticide production. Abdel-Hameed et al., (1991) statedthat if the pH of a culture medium is not between the range 6.5-7.5, sporulation and δ-endotoxin formation could be adverselyaffected (Khanh Dang Vu et al., 2009; Abdel-Hameed et al.,1991).
Table 1: Time course of amylase production.
Incubation Time (hr) Amylase activity (U/mL)
24 22.7748 15.9272 112.1496 88.96
120 16.66
Table 2: Effect of incubation temperature on amylase production.
Incubation Temperature (oC) Amylase activity (U/mL)
28 7.4031 122.234 11.1137 7.4041 7.40
Effect of growth conditions on extracellular α-amylase production by Bacillus thuringiensis
39
Table 3: Effect of initial media pH on amylase production.
pH Amylase activity (U/mL)
4 7.405 11.116 14.817 122.28 14.819 14.81
Table 4: Optimization of carbon source.
Carbon Source Amylase activity (U/mL)
Starch (Pure) 90.72Rice flour 104.79Wheat Flour 102.67Corn Flour 103.12Potato 101.61
Table 5: Optimization of nitrogen source.
Nitrogen Source Amylase activity (U/mL)
Yeast Extract 66.66Tryptone 96.29Beef extract 77.77Peptone 107.40Urea 155.55Ammonium Nitrate 66.66Ammonium Sulfate 48.14
Table 6: Effect of metal ions on amylase production.
Metal Ion Concentration (%) Amylase activity (%)*
Na+ 0.05 920.1 1160.2 720.4 48
Mg++ 0.05 800.1 760.2 1000.4 48
Ca++ 0.05 720.1 1400.2 1240.4 176
Fe++ 0.05 1160.1 68
* Note: In table 6 percent amylase activity in presence of metal saltswith respect to amylase activity in absence of metal salts is presented.The activity higher than 100% indicates stimulation of amylaseactivity. And values less than 100% indicate suppression of amylaseactivity.
Media formulation: Four carbon sources were tested assubstitute for pure starch. The values presented in table 4show that all the four carban sources enhanced amylaseproduction than pure starch. It has been reported that cornstarch was better than pure starch for stimulation of amylaseproduction (Ajayi et al., 2006). Amylase production was betterwhen peptone was used as nitrogen source compared to otherorganic nitrogen sources (Table 5). In case of inorganicnitrogen sources, nitrate was better than ammonium sulphatefor stimulation of amylase production. The support for thisobservation is the report of Dharani Aiyer (Dharani Aiyer, 2004).But we found that in all the nitrogen sources tested urea wasfound to be the best. In the presence of urea, amylase yieldwas almost 2.34 times when nitrate was used as a source ofnitrogen and 1.45 when peptone was used as nitrogen source.Earlier starch medium had been used successfully forproduction ICP by Bt and their finding was that starch basedmedium give higher yield of δ endotoxin compared to yieldobtained in glucose based defined medium (Ming Chang etal., 2008). Effect of metal ions on amylase production by Btwas studied. Four concentrations of metal salts were used.The results (Table 6) show that Na+ stimulated amylaseproduction when 0.1% NaCl was added in fermentation medium.MgSO
4 was required at 0.2% concentration. Increasing
concentration of CaSO4 linearly increased amylase production.
FeSO4 at 0.05% concentration was acting as stimulant but
higher concentration was toxic to amylase production. It hasbeen reported that Ca++ and Mg++ stimulate amylase activitywhereas Fe++ was inhibitor of amylase production (Elif Sarikayaet al., 2000). Sikdar et al. (1991) identified Fe++ one of therequirements for the production of ICP. It was proved thatMg++ was essential for the synthesis of the ICP as the level ofcrystal protein synthesis was almost zero when Mg wasomitted from the medium (Ozkan et al., 2003). But Ca++ had noeffect on toxin production. Whereas their studies with B.thuringiensis var. israelensis HD500 showed that Fe++
negatively influenced the synthesis of the ICP and Mg and Cafavoured the toxin production. It has also been reported thatMg, Ca, Fe and Na were required for toxin production by Bt(Fernando Hercos Valicente et al., 2010).
Enzyme characterization: Effect of various parameters on alphaamylase activity was studied. The enzyme activity wasmaximum at 60oC (13.17 U/mL) after which activity of alphaamylase was reduced (Figure 1). At 90oC the amylase activitywas very poor (1.03 U/mL). The optimum pH was 5 (Figure 2),at this pH amylase activity was 17.97 U/mL. Enzyme activitywas the highest when 20 mg/ml substrate concentration wasused (Figure 3) and with increase in enzyme concentrationamylase activity also increased (Figure 4). Effect of reactiontime (Figure 5) showed that amylase activity reached a peak at
40
Tembhurkar et al.
Figure 1: Effect of temperature on amylase activity Figure 2: Effect of pH on amylase activity
Figure 3: Effect of substrate concentration on amylase activity Figure 4: Effect of enzyme concentration on amylase activity
Figure 5: Effect of reaction time on amylase activity. Figure 6: Lineweaver Burk plot for determination of Vmax and Km
Effect of growth conditions on extracellular α-amylase production by Bacillus thuringiensis
41
25th min. (48.14 U/mL) after that activity reduced slightly andremained stable up to 60min. Lee et al., characterized alphaamylase from 19 serotypes of B. thuringiensis (Lee et al., 1988).They found amylase worked optimally in temperature range 55to 60 oC and optimum pH range was 6.7 to 7.2. Kinetic constantsfor Bt alpha amylase were determined by plotting LineweaverBurk double reciprocal plot (Figure 6). The values obtained forVmax and Km were 666.67 U/ml and 3.49 mg/mL respectively.These values are fairly high than reported by Elif Demirkan(2011). It was found Km and Vmax of B. subtilis amylase were1.08 mg/ml and 100 U/ml, respectively. After randommutagenesis he could isolate a strain that produced amylasewith higher Km (1.43 mg/ml) and Vmax (151 U/ml).
Conclusion
Bacillus thuringiensis ATCC 10752 is potential producer of alphaamylase. Studies of lab scale batch process indicate optimumfermentation time was 72hrs and optimum temperature was 31oC.The optimized media composed of [g/l] 10.0g Rice flour; 5.0 Peptone;CaSO
4 4.0; MgSO
4 2.0; NaCl 1.0; FeSO
4 0.5; pH 7. The alpha amylase
produced was partially characterized. The enzyme worked efficientlyat 60 oC, pH 5. The alpha amylase activity was maximum whensubstrate concentration was kept 20 mg/mL. With increase in enzymeconcentration upto 2.0mL the enzyme activity linearly increased.The optimum reaction time was 25 min. The values of Vmax and Kmwere 666.67 U/ml and 3.49 mg/mL respectively. Most of the optimumproduction parameters matched with the production parameters ofICP by B. thuringiensis previously reported by other workers. ThusB. thuringiensis can be used for production of alpha amylase andICP simultaneously in single fermentation cycle.
Acknowledgement
We express our gratitude to MGM’s Institute of Biosciences andTechnology, Aurangabad for providing all the necessary financial andtechnical support.
References
Abdul-Hameed, A., Carlberg, G. and El-Tayeb, O.M. 1991. Studieson the Bacillus thuringiensis H-14 strains isolated in Egypt-IV Characterization of fermentation conditions for ä endotoxinprodtucion. World. J. Microbiol. Biotechnology. 7: 231-236.
Ajayi, A.O. and Fagade, O.E. 2006. Growth pattern and structuralnature of amylases produced by some Bacillus species instarchy substrates. Afr. J. Biotechnol. 5(5): 440-444.
Beegle, C.C. and Yamamoto, T. 1992. History of Bacillusthuringiensis Berliner research and development. Can.Entomol. 124:587–616.
Bernfeld, P. 1955. Amylases, alpha and beta in: Methods inEnzymology. Academic Press, USA, pp 49-158.
Dey, G.; Singh, B. and Banerjee, R. 2003. Immobilization of á-amylaseproduced by Bacillus circulans GRS 313. Braz. Arch. Biol.Technol. 46(2): 167-176.
Dharani Aiyer, P.V. 2004. Effect of C:N ratio on alpha amylaseproduction by Bacillus licheniformis SPT 27. Afr. J.
Biotechnol. 3(10): 519-522.Elif Demirkan. 2011. Production, purification and characterization
of á-amylase by Bacillus subtilis and its mutant derivates.Turk. J. Biol. 35: 705-712.
Elif Sarkaya. and Velittin Gurgun. 2000. Increase of the a-amylaseyield by some Bacillus strains. Turk. J. Biol. 24: 299–308.
Feitelson, J.S. 1993. The Bacillus thuringiensis family tree inAdvanced engineered pesticides. New York, USA, pp 63-71.
Feitelson, J.S, Payne, J. and Kim, L. 1992. Bacillus thuringiensis:insects and beyond. Biotechnology. 10: 271–275.
Harley, J.P.; Prescott, L.M. 2002. Carbohydrates III: StarchHydrolysis in Laboratory exercises in microbiology. McGrawHill, pp 139-140.
Khanh Dang Vu; Tyagi, R.D.,Vale´ro, J.R. and Surampalli, R.Y. 2009.Impact of different pH control agents on biopesticidal activityof Bacillus thuringiensis during the fermentation of starchindustry wastewater. Bioprocess. Biosyst. Eng. 32: 511–519.
Kuppusamy, M. and Balaraman, K. 1990. Extra cellular hydrolyticenzyme secretion in Bacillus thuringiensis H14 & B.sphaericus & their significance in media design. Indian. J.Med. Res. 91: 149-50
Lee, K.J.; Park, D.W.; Lee, H.H. and Lee, Y.J. 1988. Examination ofproduction of alpha amylase by Bacillus thuringiensis, 19serotypes. Kor. J. Appl. Microbiol. Bioeng. 16(5): 348-351.
Maity, C.; Samanta, S.; Halder, S.K.; Das, P.K.; Pati, B.R.; Jana, M.and Mondal, K.C. 2011. Isozymes of á-amylases fromNewly Isolated Bacillus thuringiensis CKB19: Productionfrom Immobilized cells. Biotechnol. Bioproc. E. 16: 312-319.
Ming Chang; Shun-Gui Zhou; Na Lu and Jin-Ren Ni 2008. Starchprocessing wastewater as a new medium for production ofBacillus thuringiensis. World. J. Microbiol. Biotechnol. 24:441–447.
Ninfa, M. and Rosas-García 2009. Biopesticide Production fromBacillus thuringiensis: An Environmentally FriendlyAlternative. Recent. Pat. Biotechnol. 3: 28-36.
Ozkan, M.; Dilek, F.B.; Yetis, U. and Ozcengiz, G. 2003. Nutritionaland cultural parameters influencing antidipteran delta-endotoxin production. Res. Microbiol. 154(1), 49-53.
Schnep, E.; Crickmore, N., Van Rie J., Lereclus, D., Baum, J.,Feitelson, J., Zeigler, D.R. and Dean, D.H. 1998. Bacillusthuringiensis and its pesticidal crystal proteins. Microbiol.Mol. Biol. Rev. 62(3): 775–806.
Sikdar, D.P., Majumdar, M.K. and Majumdar, S.K. 1991. Effect ofMinerals on the production of the delta-endotoxin by Bacillusthuringiensis subps. israelensis. Biotechnol. Lett. 13:511-514.
Vidyalakshmi, R., Paranthaman, R. and Indhumathi, J. 2009. AmylaseProduction on Submerged Fermentation by Bacillus spp.World. J. Chem. 4(1): 89-91.
Valicente, F.H., Edmar de Souza Tuelher; Santos Leite, M.I.; Freire,F.L. and Viera, C.M. 2010. Production of Bacillus thuringiensisbiopesticide using commercial lab medium and agriculturalby- products as nutrient sources. Braz. J. Maize. Sorg. 9(1):1-11.
Yezza, A., Tyagi, R.D., Vale, J.R. and Surampalli, R.Y. 2005.Production of Bacillus thuringiensis based biopesticides inbatch and fed batch cultures using wastewater sludge as araw material. J. Chem. Technol. Biotec. 80: 502–510.
Intl. J. of Food. Ferment. Technol. 2(1): 43-48, June, 2012
Design of cascade membrane filtration process forclarification of whey proteins and membrane fouling
Pranav Kaushik Pidatala* and Senthil R. Kumar
Downstream Processing Lab, Department of Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India
*Email: [email protected]
Paper no: 36 Received: 29 April ,2012 Received in revised form: 17 April, 2012 Accepted: 14 May, 2012
Abstract
Whey protein isolates are processed by membrane separation techniques - Micro-filtration, Nano-filtration and Ultra-filtration. Proteins, Glyco-macropeptide, Lacto-albumin, Lacto-globulin and BovineSerum Albumin are collected by employment of 10, 30, 50 and 100 kilo-Daltons (kDa) ultra-filtrationmembranes, respectively based mainly on protein molecular weights. Pressure drop, trans-membranepressure, flux and feed volume were optimized for lab scale. Volume and concentration values for allstreams were calculated for micro-filtration, nano-filtration and 4 ultra-filtration membranes andsatisfactory yield values were obtained. Through scale-up criteria, a cascade design has been proposedfor pilot scale whose pressure values fall within the range of standard pressure values. Foulingstudies were carried out in lab scale by calculating fouling and membrane resistances for all membranesand effect of anti-fouling chemical agents – SDS and NaOH on the membrane cleaning has beenanalyzed. The study has given on overall idea about the design corresponding to 6 membranesarranged in series to clarify 4 proteins from milk.
©2012 New Delhi Publishers. All rights reserved
Keywords: Whey Proteins, Membrane Filtration, Flux, Scale-up, Membrane Fouling
Whey is liquid portion of the milk left out in cheese industryafter milk is treated with rennet. In cheese manufacturingindustry, it is a by-product, released as dairy waste water intothe surrounding environment. The dischangecreatesecological imbalance by altering the biochemical oxygendemand (BOD) value of nearby water bodies which in turnaffecting the aquatic life. By processing the whey at industrylevel, pure proteins are obtained and served as foodsupplements and biological oxygen demand value can also becontrolled. At industrial level, the problem of proteinclarification is addressed with the purification process takingplace under the action of certain buffering systems thatmaintain the particular pH conditions that correspond to theiso-electric points of the desired proteins. This does ensuresseparation of the protein but the recovery of the proteins atindustrial scale from the buffer systems becomes difficult
because separation of proteins is done by precipitationmethods does buffers. The proteins get precipitated, butrequire chemicals to bring back to their native states. Atindustrial level, it is very expensive to use the chemicals forpurifying the proteins. Beside, a few proteins are sensitive toprecipitation methods. So, a need arises to design a muchsimpler method of purification.
Microfiltration →1kDa Nano-filtration →10kDa Ultra-filtration
→ 30kDa Ultra-filtration → 50kDa Ultra-filtration →100kDaUltra-filtration
The above mentioned series is a cascade mode filtrationprocess where, at each junction, each of the 4 desired proteinsare isolated. Particles are separated on the basis of theirmolecular size with the use of pressure and specially designedsemi-permeable membranes. This process at the industry level
Research paper
44
Pidatala and Kumar
involves handling of bulk volumes of fluid and requires a largenumber of filtration procedures. The current process attemptsto come up with an innovative cascade mode by implementingonly mechanical methods.
From Figure 1, Composition of Milk Solids: 1 ml = 1.03 gm. So,in 1 liter, solids = (13% of 1litre * 1.03) = 133.9gm. Proteins: 27*133.9 = 36.15gm. Whey proteins: 0.198*36.15 = 7.158 gm.
Cascade Mode: MF → → → → →NF 1kDa → → → → →UF 10kDa → → → → → UF 30kDa→→→→→ UF 50kDa → → → → → UF 100kDa
The proteins were confirmed qualitatively and quantitatively;and yield values are calculated.
Scale up Criteria: Scale up is linear as per the real time industrylevel pilot plants.
Ratio of Feed Volume to Membrane Area and Permeate Fluxvalues for pilot and lab scales are constant and Pilot scale
Figure 1: Composition of Milk
Materials and Methods
The whole methodology involved the clarification of wheyproteins into separates fractions and then, subsequent designstudies of the membranes involved, in cascade mode. Buffalomilk was boiled. Then fat, casein calcium and phosphorouswere removed. The left-out product was passed through 6membrane filters arranged in series to clarify proteins fromother impurities.
A. Pretreatment of Whey
One liter of buffalo milk was boiled for 15 minutes at 50 0C andcentrifuged for 6000rpm for 12 minutes to remove fat from thetop layer. RPM Centrifuge was be maintained below 8000rpmby the shearing force to ovoid alternation of the native proteinconformation centrifuge will not alter the native proteinconfirmations. Then HCl was added to precipitate casein.
Minerals – Calcium and Phosphorous were removed byprecipitation in the form of salt i.e., Calcium Phosphate. Thewhey was sent into the membrane filters for clarification.
B. Membrane Filtration Process in Cascade Mode (Figure 2)
Each of the samples, after the respective filtration, was subjectedto subsequent filtration process. This sequential process istermed as cascade membrane filtration as shown in Figure 3.
The methodology applied in the Figure 3, is depicted assequential representation shown below:
Figure 2: Pump connected to membrane cartridge ends and pressuretransducers for protein separation
Figure 3: Cascade Membrane Filtration Process
Design of cascade membrane filtration process for clarification of whey proteins and membrane fouling
45
Pressure Drop = 3 * (Lab scale Pressure Drop). These werescaled up from lab scale results to pilot plant scale.
C. Fouling of Membranes
Due to continuous filtration and prolonged usage ofmembranes, the pores are blocked and thick layer of feedparticles get deposited on the inner surface of membrane. Thusdecreasing the efficiency of the membrane decreases andevaluationaly the recovery of permeates. As the proteinconcentration in the feed solution increases, the maximumachievable flux decreases. Fouling effect should be controlledas it decreases the efficiency of protein purification on industrialscale. The flux equation for diffusion of solvent through themembrane is
Flux = P * (TMP) where, Permeability, P is reciprocal of productof viscosity and membrane resistance. For proteins withconcentration less than 1 % (grams of whey proteins per 100ml of milk), above equation is valid. The fouling resistance canbe calculated by:
Flux= Tans-membrane Pressure/ (Permeate Viscosity*(Foulingresistance + Membrane Resistance)
Hence TMP, permeate viscosity and flux values are to be knownfor calculating the resistance.
Results and discussion
A. Pre-treatment of whey
The results obtained and summarized in Table 6.
B. Optimization
Optimization of parameters gives an insight on deciding theoptimum conditions for effective and efficient purification ofthe proteins. Rotational speed of the pump was varied anddifferent time values were noted. Lower speed values reducedprotein yield and higher speed values denatured the proteins.So for each membrane separation process, the average speedvalue was taken and flux values were calculated. These fluxvalues were found to be within the standard flux range values.Pressure transducers were fixed to inlet and outlet ends ofmembrane cartridge and pressures at these points are noteddown. As permeate is open to atmosphere, permeate pressureis taken as atmospheric pressure. For each membrane, pressureand flux values were optimized.
1. TMP = 0.5 * (Pin + Pout) – P permeate
2. Pressure Drop = (Pin – Pout)
3. Flux = Volume of permeate collected / (Membrane area *Collection Time)
Permeate Yield = Permeate Recovery * (Concentration ofpermeate/Concentration of feed)
Only for NF 1kDa, instead of permeate; retentate values areused as the proteins exist in retentate. Permeate Yield is thedesign parameter which measures the extent of protein purified.
Table 1: Volume statistics for Pre-treatment of whey
Sl. no Volumes used Amount (ml) Comments
1. Initial Raw milk 1,000 From Murrah breed buffalo2. Defatted milk 738 Separated 262ml – fat3. 2N HCl for precipitation of casein 22 Total 760 ml product4. After casein removal from whey 563 Separated 197 ml – casein5. Calcium chloride of 1.2 g/lt 32 Total 595 ml product6. NaOH of 6N 25 Total 620 ml product7. Final Volume of the Purified Whey 608 Ca3 (PO4)2 – 12ml removed
Table 2: Flux, TMP and Pressure Drop values for whey
Sl no Type Whey (Cascade) Pressure (Whey - cascademode) in bar
Time(sec) Flux(lt/sq.mt-hr) Pin Pout TMP Pressure Drop
1 M F 166 67.77 2.347 1.297 0.822 1.052 NF 1 kDa 1328 8.47 2.443 1.313 0.878 1.133 UF 10 kDa 744 18.25 2.312 1.122 0.782 1.064 UF 30 kDa 667 18.78 2.320 1.130 0.725 1.195 UF 50 kDa 621 18.12 2.250 1.020 0.635 1.236 UF 100 kDa 133 52.05 2.250 0.980 0.615 1.27C. Design of Cascade Membrane Separation Process
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Pidatala and Kumar
D. Proposing values for pilot plant using Scale-up criteria:MF Membrane
1. (Volume of feed/ Area) for pilot scale => (Volume of feed/ Area) for lab scale = 608/16 sq.cm
= 380lt/sq.mt. As volume of feed for pilot scale is 500 lt(Basis); Area => (500/380) = 1.315sq.mt
2. (Volumetric Flow Rate/ Area) of permeate for pilot scale= 67.78 ml/sq.mt-sec
So volumetric flow rate, Q per => 67.78*1.315 = 89.18lt/hr
3. Pilot scale Pressure Drop =3*(Lab scale Pressure Drop).
Table 3: Volume & Concentration values for feed, retentate & permeate of all 6 membranes
Type MF-F MF-R MF-P / NF-F NF-P NF-R /UF 10-F UF 10-P UF 10-R /UF 30-F
Volume (ml) 608 103.9 504.1 101.3 402.8 204 198.8Concentration(g/l) 0.75 0.50 0.75 0.75 0.75 1.25 1.50MF- Microfiltration, NF- Nanofiltration, UF- Ultrafiltration, F- Feed, P- Permeate, R- Retentate
Type UF 30-P UF 30–R/UF 50-F UF 50-P UF 50–R/UF 100-F UF 100-P UF 100-R
Volume (ml) 121 77.8 47.2 30.6 26.7 3.9Concentration (g/l) 1.625 2.75 3.00 0.75 0.50 0.25
Basis for pilot plant scale was taken as 500 liters as for linear scale up, there should be an initial basis value as per the real time industry requirement.
Table 4: Permeate Recovery and Yield values
Membrane Flu TMP Pressure Permeate Yield Lab Scale Pilot Scalex(lt/sq.mt-hr) (bar) Drop(bar) Recovery Permeate Volume-ml Volume-lt
MF(0.1um) 67.77 0.822 1.05 79.76 79.76 608 500NF 1kDa 8.47 0.878 1.13 79.91 79.91 504.1 414.550UF 10kDa 18.25 0.782 1.06 50.746 84.57 402.8 331.250UF 30kDa 18.78 0.725 1.19 60.865 65.73 198.8 163.486UF 50kDa 18.12 0.635 1.23 60.668 73.054 77.8 63.980UF 100kDa 52.05 0.615 1.27 87.25 58.166 30.6 25.164
Permeate Recovery = (Volume of permeate / Volume of feed) * 100
Table 5: Proposed Pilot Scale Values
Sl no Parameters MF NF UF 10 UF 30 UF 50 UF100
1. Pilot plant area (mt^2) 1.32 1.32 1.32 1.32 1.32 2.142. Volumetric flow rate(lt/hr) 89.18 11.14 24.01 24.7 23.84 68.483. Pressure drop, (bar) 3.15 3.39 3.18 3.57 3.69 3.8
Based on the equations, the pilot plant values were calculated.E. Fouling of membranes
lab= 1.05 bar; so pilot = 3.15 bar lab should be (1 to
3.5 bars) & pilot should be (1 to 7 bars). So pressuredrop is satisfied.
Inference from Tabular Values
Initially, pure water was sent into the membranes to note theflux values. Then whey was sent into the membranes to checkflux values. Again, water was sent and flux values were noted.Water flux values were decreased when both experiments werecompared. Due to the continuous purification process, foulingphenomena occurred. Hence, the flux value decreased. Thefouling phenomena were measured in terms of fouling andmembrane resistance values. Membrane cleaning wasperformed to observe alteration in flux values and reduction of
Design of cascade membrane filtration process for clarification of whey proteins and membrane fouling
47
Table 6: Membrane Fouling studies – Flux and resistance values for whey and watera) Before Fouling with i).Pure water and ii).Whey
Sl. no Pure water M F NF UF 10 UF 30 UF 50 UF 100
1. Time for 5 ml permeate rise(sec) 40 1121 618 596 468 1012. Flux (lt/sq.mt-hr) 281.25 10.04 18.2 18.88 24.04 68.54
After cleaning with water for 20 minutes, whey is sent into the filtration cartridges.1. Time for 5 ml permeate rise(sec) 158 1305 679 668 623 1412. Flux (lt/sq.mt-hr) 71.2 8.62 16.57 16.84 18.06 49.09
After whey filtration is done for about 3 hours, the membranes were tested for fouling phenomena.b) After Fouling with i).Pure water and ii).Whey
1. Time for 5 ml permeate rise(sec) 51 1206 635 743 529 1382. Flux (lt/sq.mt-hr) 220.59 9.33 17.72 15.14 21.27 50.16
Again membranes were treated with whey to calculate the resistances.1. Time for 5 ml permeate rise(sec) 172 1425 685 692 648 1892. Flux (lt/sq.mt-hr) 65.41 7.89 16.43 16.26 17.36 36.63
fouling. Due to continuous filtration of NaOH and SDS reagents,the membrane pores were unblocked and fouling was reducedto minimum.
By finding out flux and TMP values, the membrane resistanceand fouling resistance values for all 6 membranes werecalculated. In order to prevent fouling, chemical agents likeNaOH, SDS are circulated through membranes to clear the pores.
As per the cleaning process, NaOH is circulated for 2 hoursand then pure water flux is calculated.
Fouling of all membranes was studied in lab scale by estimatingpure water flux, whey flux in cascade mode, fouling water flux(after protein separation). The decrement in the water flux hadshown that fouling took place. Cleaning of membranes restores
c) Membrane resistance and fouling resistance values in sq.mt/lt units
Sl no Whey M F NF UF 10 UF 30 UF 50 UF 100
1. Permeate viscosity(milli. Pa-sec) 1.01095 1.01095 1.01825 1.02373 1.0438 1.00732. Membrane Resistance(* 10^6) 11.44768 10.109716 4.64071 4.20543 3.38443 1.247763. Fouling Resistance 10,09,416 8,72,687.9 2,75,888 1,31,987 1,14,394 4,29,859
the flux values near to the initial values indicating that themembranes were washed properly and pores were cleared.These flux values are inversely proportional to total resistanceof the membranes. So, membrane resistance and foulingresistance for each membrane were found out as otherparameters were known on lab scale. Using the correlations,the pilot plant fouling resistance for each membrane was foundout. Usually pilot plant scale operations either go for Water-Back Washing or Air Flushing for the reduction of fouling asuse of reagents is very expensive and these reagents cannotbe reusable on pilot scale.
Conclusion
Whey proteins have a high nutritional value. They are
Table 7: Cleaning of membranes
Sl no Water M F NF UF 10 UF 30 UF 50 UF 100
1. Time for 5 ml permeate rise (sec) 46 1262 628 672 502 1242. Flux (It/sq.mt-hr) 244.56 8.91 17.1 16.74 22.41 55.83
Then SDS was circulated for 2 hours and again pure water flux was calculated.1. Time for 5 ml permeate rise (sec) 42 1191 621 601 483 1122. Flux (lt/sq.mt-hr) 267.86 9.45 18.12 18.72 23.29 61.81
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Pidatala and Kumar
biologically essential molecules which provide instant energywhen consumed and hence, a major commercial interest at theindustrial level. Preparation of low fat milk, low sodium milkand low potassium milk using ultra filter and nano filtermembranes, recovery of whey proteins in cheese productionby membrane separation are few applications of the presentwork. The milk protein constituents of whey are clarified intodifferent fractions and their respective percentage yields havebeen estimated by quantitative studies - concentration valuesand permeate yields. The purification process is cheaper,economical and produces efficient results. Design of asimplified cascade membrane filtration process has beenstudied and the results have been proposed for a pilot scaleplant. Subsequent fouling studies were conducted to calculatethe membrane and fouling resistances. Cleaning procedureswere carried out to find out the flux changes and efficiency ofmembranes. Over all, the work gives a clear idea about labscale cascade membrane filtration process of hollow fibermodule in clarifying of milk whey proteins.
References
Alomirah, H.F., Alli. I. 2004. Separation and characterization oflactoglobulin and lactalbumin from whey and whey proteinpreparations, Int. Dairy J. 14 411.
Bottomley, R.C., 1989. Process for obtaining concentrates havinghigh á-lactalbumin content from whey Eur. Patent Appl. EP311 283 A2.
Carol Ann Patterson., 2005. Membrane Processing: State of The ArtTechnology, The Pathfinders Research Limited, NationalResearch Council, Canada.
Cheryan, M 1986. ‘Ultrafiltration Handbook’, TechnomicPublication, Lancaster.
Cowan, S. and S. Ritchie 2007. PES membrane for whey proteinseparation. Separation Science and Technology, 42:2405-2418.
Esther W.Y.Li, Yoshinori Mine, Comparision of chromatographicProfile of Glyco-macropeptide from cheese whey isolatedusing different methods, J. Food Science.
Field, R.W., Wu, D., Howell, J.A.. Gupta, B.B. 1995. Critical fluxconcept for microfiltration fouling. Journal of MembraneScience 100 259–272.
Helicon. 2003. Protein Concentration and Dia-filtration by TangentialFlow Filtration. In issue special of Millipore Corporation,Billerica.
Hinrichs, J. 2004. Fractionation of whey proteins by ultra-highpressure, Bull. Int, Dairy Federal. 389.24.
Jonsson, A.S. Tragardh, (1990). Ultrafiltration applications -Desalination, 77-78:135–179.
Kirk, D.E., Montgomery, M.W. and Kortekaas, M.G., 1983.Clarification of pear juice by hollow fiber ultrafiltration. J.Food Sci., 48:1663-1666.
M. Carmen Alm ecija, Rub en Ib a nez, Antonio Guadix, Emilia M.Guadix. Effect of pH on fractionation of whey proteins witha ceramic ultrafiltration membrane.
Marshal, K.R. 1982. Industrial Isolation of Milk Proteins, WheyProtein Development in Dairy Chemistry. Applied SciencePublisher, London.
Marshall, A.D., Munro, P.A. and Tragardh, G. 1993. The effect ofprotein fouling in microfiltration and ultrafiltration onpermeate flux, protein retention and selectivity: a literaturereview, Desalination, 91: 65–108.
Maubois, J. L. and Ollivier, G. 1997. Extraction of Milk Proteins.Food Proteins and Their Applications. New York Publishers,p 579.
Mehra, R. and Kelly, P.M. 2004.Whey protein fractionation usingcascade membrane filtration. International Dairy FederationBulletin: ‘Advances in Fractionation and Separation:Processes for Novel Dairy Applications’, 389:40-44.
O. Wallberg, 2003. Fractionation and concentration of whey proteinsby ultrafiltration. J. Chem Engg.
Pongsathon Limsawat, Suwattana Pruksasri. 2010. Separation oflactose from milk by ultra-filtration. J.Food Ag-Ind.
Quinones H.J., and Phillips, 2007. Influence of ProteinStandardization by Ultra-filtration on the viscosity, colorand sensory properties of skim milk. J. Dairy Sci., 80:3142-3151.
Scopes R.K., 1993. Protein Purification, Principles & Practice.Springer–Verlag, NY.
Van R. Reis, and A. Zydney, 2007. Bio-process membranetechnology, Journal of Membrane Science 297:16–50.
Van Reis, R., Gadam, S., Frautschy, L.N., Orlando, S., Goodrich,E.M., Saksena, S., Zydney, A.L. 1997. High PerformanceTangential Flow Filtration, J. Bioengg. 56:71-82.
Zeman, L.J. and Zydney, A.L. 1996. Microfiltration andUltrafiltration: Principles and Application, Marcel Dekker,New York Publications.
Zydney, A.L. 1998. Protein separations using membrane filtration:new opportunities for whey fractionation, Int. Dairy J. 8243.
Intl. J. of Food. Ferment. Technol. 2(1): 49-56, June, 2012
Microbiological and biochemical characterization ofSeera: A traditional fermented
food of Himachal Pradesh
Savitri*1, N. Thakur1, D. Kumar2 and T.C. Bhalla1
1Department of Biotechnology, Himachal Pradesh University, Summer hill, Shimla, Himachal Pradesh, India2Shoolini University of Biotechnology and Management Sciences, Bhajol, Solan, Himachal Pradesh, India
*Email: [email protected]
Paper no: 37 Received: 26 April, 2012 Received in revised form: 17 May, 2012 Accepted: 19 June, 2012
Abstract
Seera is a nutritious, easily digestible traditional fermented food made from wheat grains in Bilaspur,Kangra, Hamirpur, Mandi and Kullu districts of Himachal Pradesh, India. Samples during seerafermentation were analysed for various microbiological and biochemical parameters. The microfloraisolated from seera mainly comprised of Saccharomyces cerevisiae, Cryptococcus laurentii andTorulospora delbrueckii among yeasts and Lactobacillus amylovorus, Cellulomonas sp.,Staphylococcus sciuri, Weisella cibaria, Bacillus sp., Leuconostoc sp. and Enterobacter sakazakiiamong bacteria. Protein content decreased from 14.9% on day 1 to 8.2% on 5th day of fermentation.However, final product obtained had higher protein content i.e. 10.4%. Total sugars decreased withthe time of fermentation.Seera had 11.9 mg reducing sugars/g dry matter. Starch content decreasedinitially from 72.1 to 57.0 % (w/w) on dry weight basis from first to fourth day. After steeping anddrying of seera, the starch content increased to 87.4% (w/w) on dry weight basis. Amylase andprotease activity increased with the fermentation up to 4th day, then it started decreasing and very lowamylase activity was recorded in the final product. A significant increase in thiamin, riboflavin, nicotinicacid and cyanocobalamin was observed during fermentation of seera. The level of essential aminoacids especially methionine, phenylalanine, threonine, lysine and leucine also increased during seerafermentation.
©2012 New Delhi Publishers. All rights reserved
Keywords: Fermented foods, Seera, Himachal Pradesh, Traditional.
Indigenous fermented foods such as bread, cheese and winehave been prepared and consumed since antiquity and someof these products have become an integral part of culture andtradition (Battcock and Azam-Ali, 1998). The high nutritivevalue, sensory characteristics and easy digestibility made thefermented foods an integral part of diet of people almost everypart of the world (Sankaran, 1998). Fermented foods generallypreserve pleasant flavor, aroma, texture, enhanced nutritivevalues and good keeping quality under ambient conditions
(Law et al., 2011). Fermented cereals and pulses along withmillets have been the staple food of people of Asian and Africansub-continent since the time immemorial (Narashimahan andRajalakshmi, 1999) and the cereal foods are the most dominantgroup of fermented foods consumed in India. Diversity offermented foods in Asia is directly related to food culture ofeach and every community, and also availability of raw materials(Tamang, 2011).
Research paper
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Savitri et al.
In Himachal Pradesh, a number of traditional fermented foodsand beverages are popular which are unique in comparison toother parts of the country. Bhatooru, chilra, seera, siddu,gulgule, marchu, sepubari, pickles (of local fruits andvegetables) and fermented beverages (kinnauri, chhang, sura,behmii, etc.) are some of the indigenous fermented productsof Himachal Pradesh (Savitri and Bhalla, 2007). Seera also calledNishasta is a traditional fermented food prepared in Bilaspur,Kangra, Hamirpur, Mandi, Shimla and Kullu districts ofHimachal Pradesh. It is a starch based food made by soaking,crushing and fermenting wheat grains used to prepare sweetdish/snack generally served in breakfast or for people duringreligious fast. It holds special significance to the village peopleof Himachal Pradesh where during drought seera is offered tothe God of water for rain. Seera is also recommended for peoplesuffering from jaundice/ hepatitis and to the post-natalwomen.Similar starchy food tapai made form cassava is verypopular and is used to prepare sweet delicacies in Malaysia(Law et al., 2011).Village based cooperatives in HimachalPradesh market seera in small packets (Plate I) and thus it alsoserves as a source of revenue for many rural people.
In the traditional method of preparation of seera, wheat grainsare soaked in water for 2-3 days to allow natural fermentation.After fermentation, grains are ground and steeping is done toallow the starch grains and some proteins to settle down, branis removed. The settled solids are then,sun dried and the driedmaterial is called seeraAlthough seera is a popular traditionalfermented food of Himachal Pradesh, there is no informationabout its microbiological and biochemical aspects. So, thepresent investigation was carried out to evaluate themicrobiological and biochemical properties of seera and theresults are discussed in this communication.
Materials and methods
Chemicals
All the chemicals used were of analytical grade and wereprocured from Hi-media, sd-fine, Loba Chemie andMerck.Wheat grains for preparation of seera were purchasedfrom local market of Shimla, Himachal Pradesh.For HPLC,Lichrosorb RP-18 (5 µM) and for gas chromatography,Chromosorb WHP 15% SE-30, 1/8’’x 2 m column were used.
Preparation of seera
The details about seera and the traditional method of seerapreparation were studied by visiting various places in HimachalPradesh and by active interaction with local people.. To makeseera under laboratory conditions, 200g of wheat grains werecleaned thoroughly and soaked in 300ml tap water without the
addition of any inoculum. This was kept at 250C for naturalfermentation to take place. On fourth day water was decantedand the grains were washed with tap water. The softened wheatgrains were gently mashed and fermented overnight. Wheatbran was removed by filtration through muslin cloth and starchwas allowed to settle down. Water was decanted off and solidssettled at the bottom of the vessel were dried to form seera.During fermentation of seera, the samples were drawn everyday during six days and were analysed for microbiological andbiochemical aspects.
Microbial profile during the fermentation
One gram of soaked wheat sample was withdrawn at an intervalof 24 h and was plated on Nutrient agar, Czapek Malt agar,Lactobacillus Selection Agar Base and Czapek Dox Agar platesfor isolation of bacteria, yeasts, lactic acid bacteria and fungi.The number of colonies of bacteria, yeasts and moulds thatappeared on plates were counted and expressed as log cfu(colony forming units) g-1 of the sample. The microorganismsisolated were identified at and submitted to Microbial TypeCulture Collection and Gene Bank (MTCC), Chandigarh.
Biochemical analysis
Samples during seera fermentation were analysed for variousbiochemical parameters viz., total acidity by Amerine et al.(1980) while pH was measured by using digital pH meter (MAC).Total proteins were estimated by using the methods of Lowryet al. (1951) and total carbohydrates were estimated by phenolsulphuric acid method (Dubois et al., 1956). Reducing sugarswere estimated by DNSA method given by Miller (1959). Starchwas estimated according to Hedge and Hofreiter (1962). Theamount of different enzymes during the course of seerafermentation was also studied. Protease activity was assayedby the method given by Manachini et al. (1988) and amylaseactivity of the samples during seera fermentation was assayedaccording to Bernfield (1955).
Vitamin and amino acid analysis
Vitamins in the samples during seera preparation were analysedaccording to Šnajdrova et al. (2004). For assay of water solubleB-vitamins the samples were filtered through 0.45µ pore sizefilters. The mobile phase comprised of acetonitrile: HPLC water(75:25) and 0.1% orthophosphoric acid. Samples (5 µl) of thesolution of water-soluble vitamins were injected in to the HPLCcolumn. Identification of vitamins was made by comparingtheir retention times and UV spectra with those of standards.
For amino acid analysis sample was first hydrolysed (Schillinget al., 1996)and then derivatization (Hûsek, 1991)was done.The derivatised amino acids were then analysed using GC.
Microbiological and biochemical characterization of Seera
51
Gas chromatographic analysis was carried out on aNetelChromatograph GC (MICHRO-9100)equipped withChromosorb WHP 15% SE-30 column coupled with FlameIonization Detector. The GC was operated at the oventemperature 170-295°C, injector temperature 170-280°C, ramprate 5oC and carrier flow (nitrogen) 5 ml/min.
Results and discussion
Microbiological analysis of seera
In order to study microflora during seera fermentation, thesamples were periodically drawn at interval of 24 h for 6 daysand were analysed for viable counts.
During fermentation
The microbial counts increased substantially (Figure 1).Themicroflora isolated from seera comprised of yeasts mainlySaccharomyces cerevisiae, Cryptococcus laurentii andTorulospora delbrueckii and bacteria including Lactobacillusamylovorus, Cellulomonas sp., Staphylococcus sciuri,Weisella cibaria, Bacillus sp., Leuconostocsp. andEnterobacter sakazakii (Table 1). Out of these, Saccharomycescerevisiae and Lactobacillus sp. persisted in the finalproduct.Lactic acid bacteria have been reported as the mostimportant group of microorganisms involved in spontaneousor natural fermentation of foods (Kalui et al. 2010). Bacillussp., Enterobacter sakazaki and Staphylococcus sciuri werealso present in early stages of fermentation probably originatedfrom air or water used for steeping of wheat.
Increase in total bacterial count was found to be higher ascompared to that of yeast count. Bacterial fermentation duringsoaking is found to be important for the acidification.Lactobacillus and Leuconostoc sp. predominate the microfloraas the fermentation progressed. Similar profile of lactic acidbacteria, aerobic mesophiles and enterobacteriaceae, whichconstituted the primary microflora of pozol (prepared bysoaking of maize) have been reported by Wacher et al. (1993).The lactic acid bacteria present in fermented foods have been
found to produce antimicrobial products that lead to safe andlong storang of foods (Corganet al., 2007; Kalui et al., 2009;Parvezet al., 2006; Steinkraus, 2002). The presence ofEnterobactersakazakiiin case of seera fermentation is not amatter of concern as its population starts declining with thefall in pH and finally, it disappears (Savitri, 2007).
Chemical and biochemical analysis of seera
The pH and titrable acidity of seera prepared by soaking wheatgrains in water was estimated and the results are presented inFigure 1. There was a sharp decrease in initial pH from first tosecond day. This decline in pH was observed up to 4th day andafter that pH again increased because of washing given to thesamples. The low pH supports the growth of various yeastand lactic acid bacteria important in food fermentation. Withthe decrease in pH, total acidity increased from 0.009% initiallyto 0.45% in the final product (Table 2). The main factorsregulating acidification and changes in pH are the amount of
Table 1: Microbial population during seera fermentation
Time Predominant microorganisms(days)
1. Bacillus sp.,Enterobactersakazakii, Cellulomonas sp., Saccharomyces cerevisiae, Staphylococcus sciuri2. Leuconostoc sp., Lactobacillus amylovorus, S. cerevisiae, Cryptococcus laurentii, Torulspora delbrueckii, Staphylococcus sciuri3. Leuconostoc sp., L. amylovorus, S. cerevisiae, C. laurentii, T. delbrueckii, Weisella cibaria4. Leuconostoc sp., L. amylovorus, S. cerevisiae5. S. cerevisiae, Leuconostoc sp., Lactobacillus sp.6. (Seera) Lactobacillus sp., S. cerevisiae
Figure 1: Changes in pH, titrable acidity and total count during seerafermentation
52
Savitri et al.
fermentable carbohydrates.Acidification of wheat basedsubstrate during food fermentation is an essential requirementfor the prevention of harmful microorganisms and theassociated risks of poisoning and spoilage (Katina, 2005).
The change in total titrable acidity from 0.072% at 0 h to 0.25%after 96 h was reported in fermentation of fufu i.e. a cassavabased fermented food of Nigeria (Oyewole, 1990). Similar risein acidity, decrease in pH, increased solubility and digestibilityhave also been reported during the preparation of Indian warri,oncom, tempeh, gari, pozol, miso, mahewas, kenkey and idli(Soni and Sandhu, 1990b; Beuchat, 1983; Soni et al., 2001).
The level of protein decreased from 14.9-8.2 mg/g of dry matterfrom first to fifth day of fermentation (Figure 2). This may bedue to the increase in the activity of proteolytic enzymes withthe progress of fermentation. However, the higher proteincontent in seera as compared to the 5th day of fermentationwas due to the concentration of various components duringits drying (Table 2).Niba (2003) reported that controlledfermentation can be applied for the production ofphysiologically beneficial components i.e. for increasingamount of proteins.
With the progress of fermentation, the level of total sugarsdecreased from 74.8% to 67.5% (Figure 2). The decrease maybe due to the utilization of sugars by the fermentingmicroorganisms. The sugars are rapidly metabolized to acids,ethanol, biomass, carbon dioxide and other metabolites requiredfor the growth of microorganisms with concomitant decreasein total sugars during fermentation (Mensah, 1997). However,higher total sugar content in the final product may be due toits concentration during drying (Table 2) of Seera.
The level of reducing sugars doubled initially from 8.3 mg/g
dry matter to 16.3 mg/g dry matter in 24 h. After 2nd day offermentation, the level of reducing sugars started decreasingtill day 4 and again increased on 5th day (Figure 2). Increase insugar content in the final product was either due toconcentration of reducing sugars by drying of seera or due tothe activity of amylolytic enzymes (Table 2).Mosha andSvanberg (1983) have reported the hydrolysis of starch andoligosaccharides present in the substrates of fermentationresulting in an increase in reducing sugars due to the activityof amylases present in cereal grains or secreted bymicroorganisms. The decrease in reducing sugars withprolonged fermentation was attributed to their utilization byfermenting microflora as observed by Daeschel et al. (1987).
Table 2: Biochemical analysis of seera
Parameters Values*
pH 3.45±0.055Titrable acidity (% of dry matter) 0.44±0.006Total count (log cfu/g dry matter) 5.39Protein (mg/g of dry matter) 10.4±0.20Total sugars (% of dry matter) 89.0±0.43Reducing sugars (mg/g of dry matter) 11.9±0.53Starch (% of dry matter) 87.4±1.51Amylase** (U/g of dry matter) 3.6±0.36Protease***(U/g of dry matter) 1.02±0.05
* Values are mean ± SD of three observations** One unit of amylase activity was defined as the amount of enzyme required to release one µg of maltose/mg of substrate/min under theassay conditions***One unit of protease activity was defined as the amount of enzyme required to release one µg of tyrosine/mg of substrate /min under theassay conditions.
Figure 2: Change in total protein, total sugars, reducing sugars andstarch during seera fermentation
Microbiological and biochemical characterization of Seera
53
The starch content during fermentation of seera was decreasedinitially from 72.1-57.0 % (w/w) from 1-4th day (Figure 2). Aftersteeping and drying of seera, the starch content increased to87.4% (w/w) on dry weight basis (Table 2). The decrease in thestarch content initially may be due to the degradation of starchduring fermentation by the action of amylases.Giraud et al.(1994) reported the degradation of cassava starch by theamylolytic activities of Lactobacillus plantarum duringfermentation of raw cassava.
Amylase and protease activity
As shown in Figure 3, the amylase activity increased with theincrease in fermentation up to 4th day and then, it startsdecreasing. A very low amylase activity was observed in thefinal product (Table 2). Similar trend in amylase activity wasreported in fermentation of dosa, and Punjabi warri (Soni andSandhu, 1999). Increase in amylase activity might have beencontributed by fermentative microorganisms and then due todecline in pH, the activity decreased. Low pH has also beenshown to slow down the amylase activity of malt (Abegaz etal. 2002)
During initial 24 h, there was a very small increase in proteaseactivity (0.71-1.25U/gon dry weight basis); however, theactivity increased drastically i.e. 7.37 U/g (on dry weight basis)on 3rd day (Figure 3). This increase in activity may be due tothe decrease in pH and activation of protease at low pH.Cerealproteases have been shown to be generally active at lowpH.Katina (2005) reported that protease in sourdough are
active at low pH i.e. below 5.5. After 3rd day, protease activitystarted decreasing and finally the activity was 1.02 U/g on dryweight basis (Table 2). Decrease in activity may be due to thedisappearance of microorganisms responsible for theproduction of protease. The degree of proteolysis and theamount of acids and volatile compounds formed depend bothon the kinds of microorganisms present in the starter and onthe process parameters. Lactic acid bacteria possessingproteolytic and amylolytic properties have been consideredto be most effective in delaying staling in bread (Corsetti etal., 1998).
Vitamin and amino acid content of seera
The vitamin content of seera and wheat grains were analyzedby HPLC (Figure 4) and results are presented in Table 3. Asignificant increase in thiamine, riboflavin, nicotinic acid andcyanocobalamin was observed during fermentation of seera.Pyridoxine (0.079 mg/g of dry matter) was observed in seerawhich was absent in wheat grains taken at the start of thefermentation. The rise in the level of various vitamins especiallythiamine and riboflavin appears to be due to increase inmicroflora and yeasts, most of which have the ability to producevitamins from simple precursors.Fermentations, which involveyeasts and particularly those in which the yeasts are consumedalong with the substrate, tend to be enriched in the water-soluble B vitamins (Steinkraus, 1998). The changes in thiamin,riboflavin and niacin content in case of idli fermented withpure cultures than with natural microflora has also been
Figure 3: Amylase and protease profile during seera fermentation
Figure 4: HPLC chromatogram of vitamins content of 1) wheat grainsand 2) Seera (Thiamine- RT=2.08 min, pyridoxine- RT=2.23 min,nicotinic acid- RT=2.24 min, cyanocobalamin- RT=2.34 min, ribofla-vin- RT=3.02 min)
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Savitri et al.
observed earlier (Steinkraus, 1998). Rajalakshmi and Vanaja(1967) had also reported that thiamin and riboflavin contentincreased during idli fermentation. Akolkar (1977) has alsoobserved an increase in thiamine, riboflavin and niacin contentduring the idlifermentation.
Thefermentation brought about an increase in essential aminoacid content (Table 3). A significant increase in methionine(2.7-8.4 mg/g) and phenylalanine (2.8 to 12.2 mg/g) wasobserved. However, threonine (1.7 to 2.4 mg/g), lysine (1.1 to1.4 mg/g) and leucine (4.1 to 4.2 mg/g) exhibited marginalincrease in seera as compared to the substrate (wheat grains).The increase in these amino acids in seera fermentation is anindication of proteins hydrolysis caused by the activities ofproteolytic enzymes as well as the addition of such aminoacids by the fermentative microbes due to their metabolic
activities (Figure 5).
El-Tinay et al. (1979) have observed a significant increase inthreonine and methionine level during kisra (sorghum product)fermentation. During tempeh fermentation (soaking ofsoybeans in water), a fivefold increase in free amino acidcontent has been observed by Hering et al. (1991).
Conclusion
The present study has demonstrated for the first time thatduring soaking of wheat grains, a mixed microbial fermentationof complex ecology develops. This fermentation adds qualityto seera by way of enhancing its protein content, vitamin andessential amino acids as compared to the control.While theWestern world can afford to enrich its foods with syntheticvitamins, the developing world must rely upon biological
Table 3: Vitamin and amino acid content in wheat grains and seera
*Vitamins and amino acids (mg/g dry matter) *Wheat *Seera
Thiamine (per 100 g) 0.62±0.03 3.7±0.17Riboflavin 0.003±0.0002 0.129±0.005Nicotinic acid 0.049±0.003 0.56±0.04Pyridoxine ND 0.079±0.004Cyanocobalamin 0.006±0.0008 0.33±0.04Methionine 2.7±0.26 8.4±0.3Phenylalanine 2.8±0.43 12.2±1.2Threonine 1.7±0.062 2.4±0.26Lysine 1.1±0.08 1.4±0.026Leucine 4.1±0.09 4.2±0.36
*Values are mean ± SD of three observationsND= Not Detected
Figure 5: GC chromatogram of amino acids content of 1) wheat grains and 2) Seera (Peaks 1- chloroform, 2,3- leucine, lysine and threonine,4- methionine and 5-phenylalanine)
Microbiological and biochemical characterization of Seera
55
enrichment for its vitamins and essential amino acids. As seerais biologically enriched with vitamins and amino acids duringfermentation so it forms a good source of nutrition to thepeople who consume it.
Acknowledgement
One the author (Savitri) gratefully acknowledges Council ofScientific and Industrial Research (CSIR), New Delhi, India forfinancial support in the form of Junior Research Fellowshipand Senior Research Fellowship.
References
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Akolkar, P.N.., 1977. Studies on soyidli fermentation.Ph.D. Thesis,M.S. University of Baroda, Baroda, India.
Amerine, M.A., Berg, H.W.,Kunkee, R.E.,Ough, E.S., Singleton, V.L.and Webb AD. 1980. The technology of wine making. AVIPublishing Co Inc, Westport.
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Corgan, T.M., Bresford, T.P., Steele, J., Broadbent, J., Shah, N.P.and Ustunol, Z. 2007.Advances in starter cultures.J Dairy
Sci., 90: 4005-4021.Corsetti, A., Gobbetti, M., Balestrieri, F., Paoletti, F., Russi, L. and
Rassi J. 1998. Sourdough lactic acid bacteria effects on breadfirmness and staling.J Food Sci., 63: 347-351.
Daeschel, M.A., Andersson, R.E. and Fleming, H.P. 1987.Microbialecology of fermenting plant materials.FEMS Microbiol.,46:357-367.
Dubois, M., Gilles, K.A., Hamilton, J.K.,Rebers, P.A. and Smith, F.1956.Colorimetric methods for determination of sugar andrelated substances.Anal Chem.,28: 350-356.
El-Tinay, A.H., Abdel Gadir, I.A.M. and El-Hidal, M. 1979. Sorghumfermented kisra bread: nutritive value of kisra. J Sci FoodAgric.,30: 859.
Giraud, E. L., Champailler, A. and Raimbault M. 1994. Degradationof raw starch by a wild amylolytic strain of Lactobacillusplantarum. ApplEnvMicrobiol., 60:4319-4323.
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Hering, L., Bisping, B. and Rehm, H.J. 1991. Pattrens and formationof fatty acids at tempe fermentation by several strains ofRhizopus sp. Fat Sci Technol.,93: 303-308.
Hûsek, P. 1991.Amino acid derivatization and analysis in fiveminutes.FEBS Lett., 280: 354-356.
Kalui, C.M., Mathara, J.M., Kutima, P.M., Kiiyukia, C. and Wongo,L.E. 2009. Functional characteristics of Lactobacillusplantarum and Lactobacillus rhamnosus from ikii, a Kenyantraditional fermented maize porridge. Afr J Biotechnol.,8(17):4363-4373.
Kalui, C.M., Julius, M.M. and Kutima, P.M. 2010. Probioticpotential of spontaneously fermented cereal based foods- Areview. Afr J Biotechnol., 9(17): 2490-2498.
Katina, K. 2005. Sourdough: a tool for the improved flavour, textureand shelf –life of wheat bread. VTT Publications, 569: 1-92.
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Law, S.V., Bakar, A.F., Hashim, M.D. and Hamid , A.A. 2011. Popularfermented foods and beverages in Southeast Asia. Int FoodRes J., 18: 475-484.
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Manachini, P.L.,Fortina, M.G. and Parinic, C. 1988.Thermostablealkaline protease produced by Bacillus thermoruber-a newspecies of Bacillus. ApplMicrobiolBiotechnol.,28: 409-413.
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Rajalakshmi, R. and Vanaja, K. 1967. Chemical and biologicalevaluation of the effects of fermentation on the nutritive valueof food prepared from rice and gram. Brit J Nutr., 21:467-473.
Sankaran, R. 1998. Fermented foods of Indian subcontinent. In:Microbiology of Fermented Foods(Ed. BJB Wood),vol 2,Blackie Academic and Professional, New York, pp. 753-789.
Savitri, 2007. Microbiological and biochemical characterization ofsome fermented foods (bhatooru, seera and siddu) of HimachalPradesh. Ph.D. thesis submitted to Faculty of Life Sciences,Himachal Pradesh University, Shimla.
Savitri and Bhalla, T.C. 2007. Traditional foods and beverages ofHimachal Pradesh. Ind J TradKnowl.,6:17-24.
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Soni, S.K. and Sandhu, D.K. 1990b. Biochemical and nutritionalchanges associated with Indian Punjabi warrifermentation. JFood Sci Technol., 27: 82-85.
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Intl. J. of Food. Ferment. Technol. 2(1): 57-61, June, 2012
Preparation and evaluation of Mahua(Bassia latifolia) Vermouth
Preeti Yadav1*, Neelima Garg2 and Deepa Dwivedi3
1Microbiology Lab, Division of Postharvest Management, Lucknow, India2Division of Postharvest Management Central Institute for Subtropical Horticulture, Lucknow, India3Department of Applied Plant Sciences, Babasaheb Bhimrao Ambekar, University, Lucknow, India
*Email: [email protected]
Paper no: 38 Received: 19 Jan, 2012 Received in revised form: 14 April, 2012 Accepted: 17 May, 2012
Abstract
Mahua is used for preparation of distilled liquors since time immemorial. Vermouth was prepared fromyoung mahua wine after its fortification and flavouring with traditional Indian herbs, viz. black pepper,cinnamon, clove, cumin, fenugreek, large cardamom, nutmeg, fennel and Indian Cassia. Initially, mahuawine had 6.00 B TSS, 0.63 % acidity, 2.11 mg/L 100 ml ascorbic acid, 0.09 % tannins, 0.19 % reducingsugars and 10.5 % alcohol. The fortified mahua vermouth after one year of ageing had 6.40 B TSS, 0.57% acidity, 1.19 mg/L 100 ml ascorbic acid, 0.11 % tannins, 0.27 % reducing sugars and 18.4% alcohol.The vermouth was organoleptically more acceptable than the mahua wine. Phenolics, viz. gallic acid,caffeic acid, p–coumaric acid and kaempferol as well as the flavanols, (+)-catechins and (-)-epi-catechinwere detected in both the mahua wine and vermouth.
©2012 New Delhi Publishers. All rights reserved
Keywords: Mahua, Vermouth, Bassia latifolia, Saccharomyces cerevisiae, Aromatic herbs,spices
Mahua (Bassia latifolia) the Kalpavriksha, is the most valuedtree among tribal communities; whose every part is of serviceto the people of Central India. Its flower has been used forethanol fermentation. Fruit pulp is a good source of sugar,whereas dry husk makes a good source of absolute alcohol.Earlier wine had been prepared from mahua flowers (Yadav etal., 2009). However, one limiting factor with mahua wine is itsburnt starchy flavour. Vermouth is basically, a fortified wineflavored with aromatic herbs and spices including, cardamom,cinnamon, chamomile, cloves and marjoram (Pilon, 1954). Evenginger, citrus peel, cloves, corriander may be used for thispurpose (Jarczyk and Wzorek, 1977). Onkarayya (1985)reported mango vermouth having high acceptability. Joshi etal. (1991) prepared plum vermouth while Attri et al. (1993)developed sand pear vermouth and reported that addition ofspices/herbal extract increased the total phenols, aldehyde
and ester contents. Sweet apple vermouth with 15 % ethanolwas reported by Joshi and Sandhu, (2000). Flavour of wine isof utmost significance and is one of the quality parameter ofits evaluation. To mask the burnt starchy flavor of mahua wineand improve its acceptability, mahua vermouth was prepared.It was matured and evaluated at different intervals for variousphysico-chemical, biochemical and sensory qualitycharacteristic and the results have been discussed in this paper.
Materials and methods
Mahua flower collection
In the month of April, mahua flowers from Rehmankhera areawere collected in morning hours on polythene sheets (20m X20m) laid down under the trees, filled in clean polythene bagand brought to lab under hygienic conditions.
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Juice extraction
Mahua flowers were washed thoroughly with tap water.Diseased, rotten and damaged flowers were sorted out, theclean and undamaged flowers were crushed in a fruit mill,pressed with hydraulic press and the juice (from all the partsof the flower) was collected in clean stainless steel utensils.
Yeast culture
The yeast culture Saccharomyces cerevisiae (var.ellipsoideus), used in the present investigation, was obtainedfrom the culture collection of microbiology laboratory of CISH,Lucknow. The culture was maintained on Yeast Extract PeptoneDextrose (YEPD) Agar slants which was re-cultured everymonth.
Mahua base wine
Mahua base wine was prepared as per the method describedby Yadav et al. (2009).
Mahua vermouth
1000 ml of mahua base wine was distilled and different spiceand herbs (Table 1) were added to distillate which were keptfor extraction for two days at 20oC. The content was filteredand added @ 10% to the base wine, mixed well and kept foraging for two months. The vermouth was transferred to glassbottles; crown corked, pasteurized and kept under ambientconditions for further aging. The entire process of mahua wineand vermouth preparation is shown in Figure 1.
Physicochemical Analysis
Vermouth was analyzed for various biochemical and sensoryparameters at 3 months interval. Total soluble solids (TSS)content of vermouth were recorded using hand refractometer(Erma, Japan). The titratable acidity, tannins, ascorbic acid,non – enzymatic browning was determined using the methodsdescribed by Ranganna (1986). The reducing sugar wasdetermined using the methods of AOAC (1985). Ethanol
concentration in the vermouth was determinedspectrophotometrically by the method of Caputi et al. (1968).Phenolics in mahua vermouth were analyzed by highperformance liquid chromatography as described by Basha etal. (2004) with slight modification. Microbial count was carriedout as per method of Speck (1985).
Statistical Analysis
The data recorded during the course of investigation weresubjected to statistical analysis as per the method of Analysisof Variance (ANOVA) with two factors in CompletelyRandomised Design (CRD). All determinations were run intriplicate and values were averaged. The standard deviation(SD) was also calculated. The significant differences on themean value were compared by the least significant difference(LSD) test at a significance level of 0.01. The data were subjectedto statistical analysis using Statistical Package for AgriculturalWorkers developed by O.P. Sheoran of CCSHAU, Hisar.
Sensory Analysis
The sensory analysis of mahua vermouth was conducted by apanel of semi-skilled judges (Amerine, 1980). The attributesconsidered on a 10 point scale for colour (20%), clarity (20%),aroma (10%), taste (10%), astringency (10%), freedom fromacetic acid (10%), sweetness (10%) and body (10%). The overallrating was obtained by calculating the average of the scores.
Results and discussion
Biochemical analysis of mahua flower juice is shown in Table2. It shows that the juice is rich in sugar and may be utilized forproduction of wine. No microbial growth could be observed inmahua vermouth after one year of aging. The vermouthprepared by mahua had 18.4 % alcohol, 1.26 mg/100g tannincontent compared to the base wine where it was 10.50 %; 0.09%, (Table 3). Non-significant changes were observed inbiochemical parameters of mahua vermouth (Table 4) duringmaturation. It had TSS of 6.40 B, acidity 0.57 %, ascorbic acid
Table 1: Spices and herbs used in mahua vermouth preparation
Common name Gram/ litre mahua distillate Botanical name Part used
Black pepper 15.5 Piper nigrum L. FruitCinnamon 9.2 Cinnamonum zeylanicum Beryn. BarkClove 7.0 Syzygium aromaticum L. FruitCumin 12.8 Cumis cyminum L. SeedsFenugreek 6.0 Zingiber officinale Rosc. Dried rootsLarge cardamom 6.5 Amomum subulatum Roxb. SeedsNutmeg 8.2 Myristica fragrans Houth. SeedsFennel 50 Foeniculum vulgare SeedIndian Cassia 8 Cinnamomum tamala Leafs
Preparation and evaluation of Mahua (Bassia latifolia) Vermouth
59
Figure. 1: Flow sheet for the preparation of mahua flower wine andvermouth
mg/100g) as well as the flavanols (+)-catechins (35.31 mg/100g)and (-)-epi-catechin (39.51 mg/100g) (Table 5). Attri et al. (1993)reported that addition of herbs/spices improves the phenoliccontent of vermouth. Goldberg et al. (1998) have reported highconcentrations of (+)-catechin and (-)-epicatechin in redBurgundy and Canadian wines. Arts et al. (2000) reported thepresence of (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate in 8 types of black tea, 18 types of redand white wines, apple juice, grape juice, iced tea, beer,chocolate milk, and coffee using HPLC. Sirohi et al. (2005)developed nutritionally rich and therapeutically value addedwhey based mango herbal beverage. Heinonen et al. (1998)reported that berry and fruit wines contain phenoliccompounds, some of which are capable of protecting lipidsagainst oxidation also in a hydrophobic lipid system.
Table 2: Biochemical composition of mahua flower juice
Parameters Mahua juice
T.S.S (0B) 13.0Acidity (%) 0.11Ascorbic Acid (mg/100ml) 3.15Tannins (%) 0.11Reducing sugar (g %) 1.04
Table 3: Biochemical and nutritional characteristics of mahua wine
Parameters Mahua wine
T.S.S (0B) 6.0Acidity (%) 0.63Volatile acidity (%) 0.07Ascorbic acid (mg/100ml) 2.11Tannins (%) 0.09Reducing Sugar (%) 0.19Alcohol (%) 10.50NEB 0.021
Table 4: Biochemical changes in mahua vermouth during one year of maturation
Storage SO2
aTSSb Acidityc Ascorbic Tanninse Reducing Total Alcoholh Non-enzymatici
periods (ppm) (0B) (%) acidd (mg/100g) sugarg (g %) sugarg (%) sugarf(g %) (%) browning
0 107 6.2 0.59 1.26 0.32 0.16 0.39 18.40 0.0203 105 6.2 0.58 1.24 0.29 0.17 0.37 18.40 0.0296 103 6.4 0.58 1.23 0.28 0.19 0.35 18.40 0.0359 100 6.4 0.57 1.20 0.20 0.23 0.33 18.40 0.03812 97 6.4 0.57 1.19 0.11 0.27 0.33 18.40 0.040
Data followed by different letters for each column are significantly different by statistical (p > 0.01).Statistical analysis due to storage periods: a 0.001; b 0.001; c non-significant; d 0.001; e 0.001; f non-significant; g 0.001; h non-significant; i
0.002
1.19 mg 100-1 ml at the time of bottling which decreasedmarginally during storage while the reducing and total sugarsincreased from a level of 0.27 % to 0.33 %, respectively. Thereare reports of loss of ascorbic acid and antioxidants duringstorage of fruit wines (Patras et al., 2009). The phenoliccompounds reflected through HPLC in mahua vermouthincluded gallic acid (122.59 mg/100g), caffeic acid (10.76 mg/100g), p–coumaric acid (4.18 mg/100g) and kaempferol (282.16
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Vermouth is an aromatized wine having added sugar, roots,herbs, spices and flowers (Fessler, 1971). It contains ethylalcohol, sugars, acid, tannins, aldehyde, esters, amino acids,vitamins, anthocyanins, fatty acids and minor constituentslike flavouring compounds (Joshi, 1997; Joshi et al., 1999).The additives don’t boost the alcohol content, but they dosculpt the flavor of the wine. Fessler (1971) has also
recommended use of herbs, roots and spices for developmentof flavoured wines having distinctive character. Vermouth canhave alcohol content between 15 % and 19 % by volume (Joshiand Sandhu, 2000). Since mahua flower have burnt starchyflavours, herbs were used to mask the same and replace withsweet spicy flavor.
Table 5: Changes in phenolics compoundsb during storage of mahua vermouth
Storage Source of variationa
periods Total phenols Gallic acid (+)-Catechin (-)-Epi-catechin Caffeic acid p-coumaric acid kaempferol
0 494.54±10.28a 122.59±25.76b 35.31± 1.45b 39.51±6.98a 10.76±3.06a 4.18±0.95a 282.16±49.4a3 475.31±19.23a 112.45±13.76ab 32.56±1.65b 35.26±6.34ab 11.98±2.95b 5.35±1.45b 281.19±46.4b6 436.20±39.11a 107.38±18.67ab 30.85±1.78b 32.83±4.96ab 13.11±2.25b 5.87±1.34b 280.97±53.1b9 417.17±19.03a 99.19±14.98ab 30.19±2.98b 29.74±4.77ab 16.87±1.98b 5.99±1.75b 280.91±51.9b
12 409.45±7.72a 92.87±24.74ab 28.75±2.57a 29.18±4.29b 18.35±1.59b 6.18±1.11b 280.57±49.6b
a Data followed by different letters for each column are significantly different by LSD test (p<00.1). b Values are expressed as mg/100gram ± standard error (n=33).
Figure 2: Sensory evaluation of mahua wine and vermouth during storage
Preparation and evaluation of Mahua (Bassia latifolia) Vermouth
61
The sensory results clearly reflected improvement in its aromaand acceptability during storage (Figure 2).
Conclusions
Mahua vermouth, after one year of aging, was found to besuperior to the base wine based on physio-chemical andsensory qualities. Aging further improved its acceptability. Avariety of phenolic compounds found in the Mahua wine hasadded its value as an antioxidant rich beverage.
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preparation and storage of whey-based mango herbal pudina(Mentha arvensis) beverage, Journal of Food Science andTechnology, 42(2):157-161
Yadav, P., Garg, N. and Diwedi, D.H. 2009. Standardization of pre-treatment conditions for mahua wine preparation, J.Ecofriendly Agric., 4(1):88-92.
Yadav, P., Garg, N. and Dwivedi, D.H. 2009. Effect of location ofcultivar, fermentation temperature and additives on thephysico-chemical and sensory qualities on mahua (Madhucaindica J.F. Gmel.) wine preparation, Natural ProductRadiance, 8:406-418.
α-amylase production from Endomyces fibuliger – an indigenous yeast isolate of Western Himalayas
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Intl. J. of Food. Ferment. Technol. 2(1): 63-69, June, 2012
ααααα-amylase production from Endomyces fibuliger – anindigenous yeast isolate of Western Himalayas
Keshani Bhushan¹, Anamika Jain¹, O.P. Sharma², B. Singh² and S.S. Kanwar¹*
1Department of Microbiology, Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India2Regional Station, Indian Veterinary Research Institute, Palampur, Himachal Pradesh, India¹* Department of Microbiology, Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India
*Email: [email protected]
Paper no: 39 Received: 26 April, 2012 Received in revised form: 17 May, 2012 Accepted: 19 June, 2012
Abstract
Amylase production, its purification, characterization and optimization have been studied in Endomycesfibuliger. The enzyme was purified to about 13-fold by using gel permeation chromatography (SephadexG-25, Sephadex G-100). The purity of enzyme was checked by the use of Native-PAGE and SDS-PAGE.The molecular weight of enzyme was 55KD with the presence of two polypeptide units. Enzyme wasmost active at pH 7.0, temperature 30 °C with optimum incubation time of 30 minutes. The maximumactivity of the enzyme was at 2 % (w/v) soluble starch concentration. The enzyme was para-constitutivein nature. The parameters for amylase production were optimized by Response Surface Methodology(RSM) . The best carbon source was soluble starch, the optimum pH values were 5.0 and 8.0, and theoptimum temperatures were 30 °C and 37 °C. Ca+² and Mg+² had enhancing effect on its production.
©2012 New Delhi Publishers. All rights reserved
Keywords: Endomyces fibuliger, Amylase, Sephadex, Starch, Response surface methodology
Starch degrading enzymes (amylases) are the most importantmicrobial enzymes and are of great significance in food industry.Now-a-days, the microbial amylases are consideredindispensable in meeting the industrial demand of enzymes(Pandey et al., 2000). Amylases constitute a class of industrialenzymes having approximately 25 per cent of the enzyme market(Rao et al., 1998). The amylase family of enzymes has beenwell characterized through the study of variousmicroorganisms. Two major classes of amylases identifiedamong the microorganisms, are alpha-amylase (endo-1,4-alpha-D-glucan glucohydrolase, EC-3.2.1.1), and glucoamylase(exo-1,4-alpha-D-glucan glucanohydrolase, EC-3.2.1.3). Inaddition, beta-amylase (α-1,4-glucan maltohydrolase, EC-3.2.1.2), which is generally of plant origin, has also beenreported from a few microbial sources.
A considerable amount of information pertaining to amylases
from bacteria and filamentous fungi is available but little isknown on the amylases of yeasts despite the fact that a numberof them can grow on starch. Yeasts have received considerableattention on account of their importance in brewing and bakingindustries. One of the major applications is their ability toproduce a variety of industrially important enzymes which haveapplications in food, fermentation, textile and paper industry(Walsh, 2002).
In food industry (brewing and baking), starch as a raw materialneeds pretreatment for liquefaction and saccharification forits conversion. Common alcohol producing yeast,Saccharomyces cerevisiae lacks the enzyme amylase, whichis needed to hydrolyze starch into simple monomeric units(Banerjee et al., 1988). Thus, more attention has been paidtowards amylase producing yeasts especially of food origin.Amylolytic yeasts are potentially valuable in utilization of
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Bhushan et al.
starchy substances for direct conversion into single cell protein(SCP) or ethanol (Soni et al. 1991). Now-a-days, recombinantyeast strains are also produced for better yield of the amylases(Galdino et al., 2011). Yeasts are known to produce at least twotype of amylases i.e. alpha-amylase (E.C. 3.2.1.1) andglucoamylase (E.C. 3.2.1.3) which differ from each other withrespect to their mechanism of action causing starch liquefactionand saccharification, respectively (DeMot and Verachtert,1985). The assay methods to determine these amylases alsodiffer which help to differentiate between different type ofamylases.
It is well documented that extracellular amylase production bymicrobes is greatly influenced by media components, especiallycarbon and nitrogen sources, minerals and physical factorssuch as pH, temperature, agitation, dissolved oxygen andinoculum density (Gigras et al., 2002). Response surfacemethodology (RSM) is an experimental strategy for seekingthe optimum conditions for a multivariate system (He et al.,2004). Application of RSM has gained attention of researchersfor optimizing media components and process parameters(Vohra and Satyanarayana, 2002).
Forty-three yeast isolates derived from various fermented foods,alcoholic beverages and traditional inocula of WesternHimalayas, and characterized by using traditional and molecu-lar techniques (Pathania et al., 2010), were investigated in thepresent study for the production of amylases. The best strainwas employed to produce amylases and important parametersfor production were optimized by Response surface methodol-
ogy (RSM).
Materials and methods
Organism, growth conditions and enzyme production
Forty-three yeast isolates derived from various fermented foodswere screened for amylolytic activity. Starch hydrolysis test(Shaw et al., 1995) was performed in order to identify theamylolytic strains among the available isolates. Enzyme activitywas checked on starch agar medium. The surface of starchagar plate was point inoculated and incubated at 28°C for 72hours. The growth obtained was flooded with 5-10 ml of iodinesolution for twenty minutes. The presence of clear zone showsamylase production. The most active strain, Endomycesfibuliger was then, used for further study. This strain wasisolated from dhaeli used as traditional inoculum by the localpeople of Lahaul and Spiti (Himachal Pradesh) and wasmolecularly characterized as reported earlier (Pathania et al.,2010). The organism was grown at 28°C on PDA. Enzymeproduction was carried out in a 250 ml Erlenmeyer flaskcontaining standard production medium (Kwan et al., 1993).
The medium was inoculated with 1% inoculum grown in potatodextrose broth (PDB). The medium was then, incubated at 27°Cfor five days. After incubation, culture broth was filtered andcentrifuged at 8000g for 15 minutes at 4°C. The supernatantobtained was then, used as the crude enzyme for extracellularamylolytic activity. The enzyme was assayed by measuringthe increase in reducing sugars by complex formation withdinitrosalicylic acid (Miller, 1950). However, this assay methoddid not work here due to interference by the reducing sugarsin the enzyme extract leading to high blank values. Therefore,amylase assay was done by the starch-iodine method (Xiao etal., 2006) which is specific to α-amylase. One unit of amylaseactivity is defined as the disappearance of an average of 1mgof iodine binding starch material per minute in the assay reaction(Vandyk and Caldwell, 1956). Protein content was determinedat 280nm by using spectrophotometric method.
Enzyme purification and characterization
The purification was carried out first by freezing the crudeenzyme extract and lyophilizing it at -60 °C in a freeze dryer(Edwards EF4 Modulyo). The lyophilized protein was dissolvedin acetate buffer (0.2M, pH 5.2) and applied to a sephadex G-25column. The protein was eluted by applying the same buffer.Twenty fractions of 5ml each were collected and pooled on thebasis of results of enzyme activity. The fractions with maximumactivity were lyophilized and again purified by sephadex G-100 superfine column.
The protein on electrophoretic separation showed two bands.Sodium dodecyl sulphate- polyacrylamide gel electrophoresis(SDS-PAGE) was used for the determination of molecular weightof the desired amylolytic enzyme with glyceraldehydes 3-phosphate dehydrogenase (36KD), ovalbumin (45KD),glutamic dehydrogenase (55KD), fructose-6-phosphate kinase(84KD), phosphorylase b (97KD) and galactosidase (116KD)as molecular weight markers and native polyacrylamide gelelectrophoresis (Native PAGE) was used to determine thenumber of proteins present in the sample. PAGE was performedon 1.5mm and 0.75mm thick plate with a current of 30-40 volts.The gel, buffer and samples were prepared according to theprocedure of Laemmli (1970). Staining of SDS-PAGE was doneby coomassie blue stain and Native-PAGE was stained by silverstain.
The purified enzyme was further characterized for activity atvarying temperature (10°C-80°C), pH (4-9), substrateconcentration (0.5-4g/100ml) and incubation time.
Optimization by applying response surface methodology(RSM)
The chemically defined standard medium (Kwan et al., 1993)
α-amylase production from Endomyces fibuliger – an indigenous yeast isolate of Western Himalayas
65
was first optimized by one variable at a time (OVAT) approachfor alpha-amylase production. This medium was used forfurther optimization by applying RSM of central compositedesign (CCD). The levels of four independent variables (starchconcentration (A), temperature (B), pH (C) and divalent ionconcentration (D) chosen for this study were optimized byRSM. Each factor in the design was studied at five differentlevels (Table 1). A set of 30 experiments was performed. Eachexperiment was carried out in triplicates. The full experimentalplan with respect to their values in actual and coded form islisted in Table 2. Upon completion of experiments, the averageof alpha-amylase production was taken as the dependentvariable or response (Y).
Table 1: Range of values for the RSM
Independent variables -2 -1 0 +1 +2Starch (%) 2 4 5 6 8Temperature(ÚC) 25 30 31 37 43pH 4 5 6 8 10
CaCl2 (mg) 4 6 8 12 16
Statistical analysis and modeling
The data obtained from RSM on alpha-amylase productionwas subjected to the analysis of variance (ANOVA). Theresults of RSM were used to fit a polynomial equation (1), as itrepresents the behavior of such a system more appropriately,
Y=β0+β1A+β2B+β3C+β4D+β11A²+β22B²+β33C²+β44D²
+β12AB +β13AC+β14AD+β23 BC+β24BD+β34CD (1)
Where y is response variable, β0 is intercept, β1, β2, β3 and β4are linear coefficients, β11, β22, β33 and β44 are squaredcoefficients, β12, β13, β14, β23, β24 and β34 are interactioncoefficients and A,B,C,D,A², B², C², D², AB, AC, AD, BC, BDand CD are level of independent variables.
Statistical significance of the model equation was determinedby Fischer’s test value, and the proportion of variance explainedby the model was given by the multiple coefficient of
Table 2: Experimental design and results of CCD of RSM
Experiment No. Starch Temperature pH CaCl2 Activity(U/mg)concentration(%) (A) (°C) (B) (C) (mg) (D) Predicted Experimental
1 4 25 8.0 12 0.1 0.0392 2 37 3.0 4.0 0.56 0.3663 2 25 4.0 12 0.006 0.04 8 25 4.0 12 -0.067 0.05 5 30 5.0 12 0.53 1.1336 8 37 8.0 12 0.91 0.92287 8 37 5.0 16 1.13 1.12638 8 37 8.0 4.0 1.01 0.96849 5 43 6.0 8.0 -0.033 0.0
10 6 30 6.0 8.0 0.67 0.804311 2 25 4.0 12 0.006 0.012 2 37 4.0 4.0 -0.014 0.013 8 37 4.0 12 0.098 0.014 2 37 4.0 12 -0.007 0.015 5 25 6.0 8.0 0.33 0.016 6 31 6.0 8.0 0.7 0.804317 5 30 6.0 6.0 0.7 0.80318 5 31 10 8.0 0.027 0.019 5 31 6.0 12 0.74 0.79620 8 25 8.0 12 0.47 0.49721 8 25 8.0 4.0 0.56 0.54422 5 37 8.0 4.0 0.76 0.9223 5 30 6.0 12 0.71 0.78824 5 37 4.0 4.0 -0.037 0.025 5 31 6.0 8.0 0.67 0.026 5 31 6.0 16 0.99 0.827 5 31 4.0 12 0.21 0.028 2 37 8.0 12 0.36 0.43729 2 25 8.0 4.0 0.29 0.48930 5 30 6.0 8.0 6.64 0.8
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determination, R squared (R²) value. Design Expert (Ver 7.0,STATEASE. Inc., Minneapolis, USA) was used in thisinvestigation.
Results and discussion
Enzyme production and assay methods
The production of extracellular alpha-amylase from Endomycesfibuliger was first checked on starch agar medium and 1cmzone of clearance was observed. In order to obtain sufficientamount of enzyme, standard medium designed by Kwan et al.(1993) was used. The enzyme assays were done in the nativestate, heat inactivated state, after dialysis and after autoclavingusing DNS method. The enzyme showed activity both ininactivated state as well as after stopping the reaction. Theseresults indicated that the enzyme was probably heat stable. Tocheck the accuracy of the DNS method in the present study,salivary amylase was used as a test case. Salivary amylase didnot show measureable activity after stopping the reaction.These results suggest that the enzyme assay by the DNSmethod was not correct probably due to the interference byreducing sugars existing in the enzyme extract. Thus, it wasnecessary to look for an alternate method where such type ofinterference could be avoided. So, the starch-iodine methodwas used which is based on assay for the substrate and notthe product of enzyme reaction. This study was also carriedout with salivary amylase and the results were found to be inaccordance with that of yeast amylase. Therefore, starch iodinemethod was found to be more appropriate in detecting theamylolytic activity in the present study. The specific activity
obtained was 0.8545U/mg. The total protein in the crude enzymeextract was found to be 1.59mg/ml.
Purification of enzyme
During purification process, the culture supernatant wasconcentrated (34 fold) and low molecular weight compoundswere removed from the enzyme extract by chromatography onsephadex G-25 gel filtration (Figure.1). The amylolytic enzymewas further purified by sephadex G-100 gel filtration. A 13-foldpurification of amylolytic enzyme was achieved with a yield of8.2% of the original activity (Figure. 2). The enzyme preparationobtained after sephadex G-100 column had a specific activityof 7.57.
The pooled fractions from gel permeation chromatographydeveloped using coomassie brilliant blue (CBB) stainingindicated the presence of two bands. These two bands indicatepolypeptide subunits in the enzyme fraction. The molecularmass established by SDS-PAGE developed using CBB wasfound to be 55KD. To detect the low concentration of proteinsthe pooled fractions from GPC were developed using silverstaining. Native-PAGE indicated the presence of two bandsshowing two polypeptide units. One of them was the majorband while other one was minor.
Characterization of enzyme
The purified enzyme of Endomyces fibuliger was tested for itsactivity on soluble starch at its different concentrations andthe maximum activity of amylolytic enzyme was noticed at 2 %(w/v) soluble starch concentration. The optimum incubationtime was found to be 30 min. The enzyme was most stable at
Table 3: Summary of purification process
Purification step Volume of Protein Total activity Specific activity Recovery Purification fold extract (ml) (mg) (units) (U/mg) (%)
Crude extract 100 159 95 .8545 100 1Sephadex G-25 40 2.8 18.47 6.59 19.2 11.04Sephadex G-100 25 1.03 7.8 7.57 8.2 13
Table 4: Anova for alpha-amylase production
Source Sum of square Degree of freedom Mean square F-value P-value
Model 3.9 14 0.28 3.48 0.0112Error 0 1 0Total 5.1 29
R²=0.7645Adjusted R²=0.5447Predicted R²=0.3713Adequate precision=5.996
α-amylase production from Endomyces fibuliger – an indigenous yeast isolate of Western Himalayas
67
pH 7.0 with 3.084U/mg of activity. The optimum temperaturefor enzyme activity was 30°C. It was active over a wide rangeof temperature i.e. from 20°C to 50°C after which a suddendecline in activity was observed.
Response surface methodology for optimization ofparameters
By ‘one variable at a time ‘approach, the medium was optimizedfor enzyme production. Carbon sources used for amylaseproduction were amylum, sorbose, arabinose, maltodextrin,dextrose, sucrose, xylose and soluble starch. The organismshowed enzyme production with all carbon sources but themaximum enzyme production was obtained with soluble starch.Since, enzyme production was seen in glucose containingmedium, although less than starch, it appeared that the enzymehad para-constitutive nature. Enzyme production was checkedwith different divalent ions: Ni, Mn, Cu, Ca, Zn, Ba, Fe, Sr andMg. All ions showed inhibition to enzyme production exceptCa and Mg ions. Ca was found to be more effective than Mgbut the presence of both ions was required for better enzymeproduction. Ca produces stabilizing effect during enzymeproduction and hence, favours the process (Kar and Ray, 2008).Similarly, temperature (25oC-43oC) and pH (4-10) ranges werestudied for enzyme activity. The results of CCD experimentsfor studying the effect of four independent variables are
Figure 1: Elution profile of Endomyces fibuliger amylase on SephadexG-25 column (2.5 × 12 cm bed size). The concentrated crude extractcontaining 159 mg protein was loaded on the column and elutedisocratically with 0.2 M acetate buffer pH 5.2. Fractions (5.0 ml each)were collected and assayed for amylolytic activity. Elution for pro-teins was monitored as absorbance at 280 nm. Amylase activity isexpressed as Units/mg.
Figure 2: Elution profile of Endomyces fibuliger amylase on SephadexG-100 column. Sample: Pooled fraction (2.8 mg protein) from SephadexG-25 column chromatography (Figure 1) positive for amylase activ-ity. Elution was with 0.2M acetate buffer pH 5.2. Fractions (5.0 mleach) were collected and assayed for amylolytic activity.
Figure 3: 3-D surface diagram of amylase production shows on z-axis against the effect of temperature and pH plottted on x-axis and y-axis, respectively.
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Bhushan et al.
presented along with the mean predicted and observedresponses in Table 4. The regression equations obtained afterANOVA gave the kind of alpha-amylase production as afunction of the initial values of starch concentration,temperature, pH and divalent ion concentration.
The final response equation that represented a suitable modelfor alpha-amylase production is given below:
Y=0.67+0.093×A+0.011×B+0.28×C-0.021×D+ 0.024×A²-0.23 ×B²0.30×C²+0.091×D²+0.045×AB+0.11×AC+0.026×AD + 0.069 ×BC-0.001×BD-0.052×CD (2)
Where Y is enzyme production, A is starch concentration (%),B is temperature (oC), C is pH and D is divalent ionconcentration (mg).
The coefficient of determination (R²) was calculated as 0.7645for alpha-amylase production, indicating that the statisticalmodel can explain 76.45% of variability in the response. The R²value is always between 0 and 1. The closer the R² is to 1.0, thestronger the model and the better it predicts the response(Haaland, 1989). The purpose of statistical analysis is todetermine the experimental factors, which generate signals thatare large in comparison to the noise. Adequate precisionmeasures signal to noise ratio. An adequate precision of 7.404for alpha-amylase production was recorded. The predicted R²of 0.3713 is in reasonable agreement with the adjusted R² of0.5447. This indicated the good agreement between the
experimental and predicted values for alpha-amylaseproduction. The adjusted R² corrects the R² value for the samplesize and for the number of terms in the model. If there are manyterms in the model and the sample size is not very large, theadjusted R² may be noticeably smaller than the R².The modelF-value of 3.48 and values of probability > F (<0.05) indicatedthat the model terms are significant. For alpha-amylaseproduction C, B² and C² are significant model terms.
Response surface was generated by plotting the response(alpha-amylase production) on the z-axis against any twoindependent variables while keeping the other independentvariables at their ‘O’ levels. Figure 3 depicts three-dimensionaldiagram and a contour plot of calculated response surfacefrom the interaction between temperature and pH while keepingall other variables at their ‘O’ level. At specific temperatureand pH combinations i.e. 30 °C and 5.0; 37 °C and 8.0; 37 °Cand 5.0, better enzyme production was seen. Starchconcentration and temperature had linear relationship (Figure5). Enzyme production was found to be independent of thedivalent ion concentration effect but at least 4 mg of divalention concentration is required for better enzyme production(Figure 4). Thus, fourth parameter shows very little interactionto other parameters.
The mathematical model generated during RSMimplementation was validated by conducting check pointstudies. The experimentally obtained data were compared with
Figure 4: Graph showing the effect of starch concentration (x-axis)and CaCl
2 concentration (y-axis) on amylase production
Figure 5: Interaction graph showing the effect of interactions ofstarch concentration and temperature on amylase production.
α-amylase production from Endomyces fibuliger – an indigenous yeast isolate of Western Himalayas
69
the predicted one and the prediction error was calculated. Theobserved and the predicted values obtained were quite close(Table 2) and therefore, showed good predictability of themodel.
Conclusion
Endomyces fibuliger is an excellent producer of alpha-amylaseenzyme, which is para-constitutive in nature. Organism showedenzyme production in both alkaline and acidic conditions.Enzyme is active at a wide range of temperature. So, it can playan important role in various industrial processes viz. brewing,baking, distillery etc. The indigenous yeast isolate can also bedirectly supplied to the local people by co-immobilizing it withalcohol producing strains for cereal based beverages.
References
Banerjee, M., Debnath, S. and Majumdar, S.K. 1988. Production ofalcohol from starch by direct fermentation. BiotechnologyBioengineering, 32:831-834.
De Mot, R. and Verachtert, H. 1985. Purification and characterizationof the extracellular amylolytic enzymes from the yeastFilobasidium capsuligenum. Appl Environmental Microbiol,50: 1474-1482.
Galdino, A.S., Silva, R.N, Lottermann, M.T., Alvares, A.C.M.,Moraes LMP, Torres FAG, de Freitas SM, and Ulhoa CJ.2011. Biochemical and Structural Characterization of Amy1:An Alpha-Amylase from Cryptococcus flavus Expressed inSaccharomyces cerevisiae. Enzyme Research, doi:10.4061/2011/157294.
Gigras, P., Sahai, V. and Gupta, R. 2002. Statistical media optimizationand production of its alpha-amylase from Aspergillus oryzaein a bioreactor. Curr Microbiol, 45: 203-208.
Haaland, P.D. 1989. Statistical problem solving. In ExperimentalDesign in Biotechnology ed. Haaland, PD pp. 1-18. NewYork and Basel: Marcel Dekker, Inc.
He, G.Q., Kong, Q. and Ding, L.X. 2004. Response surfacemethodology for optimizing the fermentation medium ofClostridium butyricum. Lett Applied Microbiol, 39: 363-368.
Kar, S. and Ray, R. 2008. Statistical optimization of α-amylaseproduction by Streptomyces erumpens MTCC 7317 cells incalcium alginate beads using response surface methodology.Polish Jour Microbiol, 57: 49-57.
Kwan, S.H., So, K.H., Chan, K.Y. and Cheng, S.C. 1993. Productionof thermotolerant alpha amylases by Bacillus circulans. WorldJour Microbiol Biotechnol, 9: 50-52.
Laemmli, U.K. 1970. Cleavage of structural protein during theassembly of the head of bacteriophage T4. Nature, 227: 680-685.
Miller, G.L. 1950. Use of Dinitrosalicylic acid reagent fordetermination of reducing sugar. Anal Chem, 32: 426-428.
Pandey A, Nigam P, Soccal RC, Soccal TV, Singh D and Mohan R.2000. Review: Advances in microbial amylases. BiotechnolAppl Biochemistry, 31: 135-152.
Pathania, N., Kanwar, S.S., Jhang, T., Koundal, K.R. and Sharma,T.R. 2010. Application of different molecular techniques fordeciphering genetic diversity among yeast isolates oftraditional fermented food products of Western Himalayas.World Journ Microbiol Biotechnol 26:1539-1547.
Rao, M.B., Tanksale, A.M., Gathe, M.S. and Despande, V.V. 1998.Molecular and Biotechnological aspects of microbialproteases. Microbiol Molecul Biolog Reviews, 62: 597-635.
Shaw, F.J., Lin, P.F., Chen, C.S. and Chen, C.H. 1995. Purificationand characterization properties of an extracellular alphaamylase from Thermus sp. Botanical Bulletin of AcademiaSinica, 36: 195-200.
Soni, S.K., Boparai, P.K. and Sandhu, I.K. 1991. Amylase productionby Lipomyces starkeyi. Indian Journ Microbiol, 31: 285-289.
Vandyk, J.W. and Caldwell, M.L. 1956. A study of the kinetics of waxymaize starch by crystalline pancreatic amylase from swine. JournAmeric Chemic Socie, 78: 3345-3350.
Vohra, A. and Satyanarayana, T. 2002. Statistical optimization ofthe medium components by response surface methodologyto enhance phytase production by Pichia anomala. ProcessBiochemistry, 37: 999-1004.
Walsh. 2002. Protein Biochemistry and Biotechnology. 547p. JohnWiley and Sons, England.
Xiao, Z., Storms, R. and Tsang, A. 2006. A quantitative starch-iodine method for measuring alpha amylase and glucoamylaseactivities. Analytic Biochemis, 351:146-148.
Intl. J. of Food. Ferment. Technol. 2(1): 71-79, June, 2012
Statistical screening of media components for theproduction of arginine deiminase by
Weissella confusa GR7
Baljinder Kaur* and Rajinder Kaur
Department of Biotechnology, Punjabi University, Patiala, Punjab, India
*Email: [email protected]
Paper no: 40 Received: 17 Jan, 2012 Received in revised form: 14 April,2012 Accepted: 17 May, 2012
Abstract
A lactic acid bacterial strain capable of metabolizing arginine via arginine deiminase pathway wasisolated from lassi, an Indian fermented food. On the basis of biochemical analysis it was identified asWeissella confusa. Response surface methodology (RSM) was used to optimize media componentssuch as carbon and nitrogen sources, minerals, inducers and metal ions for enhancing its specificarginine deiminase activity. A Placket-Burman design was applied to identify significant factors affectingenzyme production in Weissella confusa GR7. Central composite rotatable design was applied furtherto optimize the concentrations of sucrose, copper, magnesium, manganese and arginine for maximizingarginine deiminase activity. Statistical design has suggested a Two Factor Interaction model whichwas validated experimentally where it showed 10 fold increases in specific arginine deiminase activityover basal medium. By solving the regression equation and analyzing the response surface cartons,optimal concentration of various components was determined as 2.5g/l sucrose, 5g/l fructose, 10g/ltryptone, 5mM MgSO
4, 5mM MnSO
4, 25mM arginine, 5mM Copper, 50mg/ml CTAB and 50mM Tween
80. The model was found satisfactory as the coefficient of determination was 0.83. Results indicate animproved arginine deiminase production in statistically optimized medium using response surfacemethodology.
©2012 New Delhi Publishers. All rights reserved
Keywords: Arginine deiminase, Weissella confusa, Central composite rotatable design, Responsesurface methodology, Lactic acid, Bacteria
L-Arginine is a semi-essential amino acid that plays a key rolein cellular metabolism. It not only serves as a building block ofproteins and as a precursor of polyamines, but also as a sourceof carbon, nitrogen, and energy through a variety of metabolicpathways in bacteria. Microorganisms catabolize argininemainly through arginase pathway, ADI pathway, argininesuccinyltransferase (AST) pathway and arginine transaminase/oxidase/dehydrogenase pathway. The ADI pathway wasestablished in lactic acid bacteria which were described in
strains belonging to genera of Enterococcus, Lactobacillus,Leuconostoc, Oenococcus, Streptococcus and Weissella (Crowand Thomas, 1982; Zuniga et al., 2002; Liu et al., 2003; Ammorand Mayo 2007; Spano et al., 2007; Yu et al., 2010).
Weissella species are Gram-positive, non-spore-forming,heterofermentative, non-motile, and irregular or coccoid rodshaped organisms. Members of the genus Weissella have beenisolated from a variety of sources, such as fresh vegetables,fermented cabbage, fermented foods, and fermented silage,
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meat or meat products and soil (Niven et al., 1957; Milbourne,1983; Dellaglio et al., 1984; Dellaglio and Torriani, 1986; Collinset al., 1993; Magnusson et al., 2002; Katina et al., 2009).Weisella confusa has been detected in sugar cane, carrot juiceand occasionally, in raw milk and sewage (Hammes & Vogel,1995). Several strains of Weissella have been exploited forproduction of exopolysaccharides and dextran from wheatsourdough baking (Katina et al., 2009). Weissella kimchiiPL9023 is a well established probiotic that can be utilized as afood source (probiotic) (Lee, 2005). The RSM, formerly knownas Box-Wilson methodology, is the most widely used statisticaltechnique to evaluate relationship between a set of controllableexperimental factors and observed results (Kennedy andKrouse, 1999; Lee and Chen, 1997; Liu et al., 2003). RSM iscapable of finding optimum set of experimental factors thatproduce maximum or minimum value of response and representthe direct and interactive effects of process variables throughtwo dimensional and three dimensional graphs.
The present study was aimed to statistically screen mediacomponents that increase ADI activity of Weissella species.There are no reports on the optimization of media componentsfor ADI enzyme production from Weissella species using RSM.Media optimization was carried out in two steps: (i) Plackett-Burman design used for selection of most influential mediacomponents, (ii) CCRD design was used for furtheroptimization to enhance ADI activity using influential processvariables.
Materials and methods
Bacterial strain and culture medium used
A lactic acid bacterial (LAB) isolate Weissella confusa GR7(phenotypic identification by MTTC, Chandigarh) isolatedfrom dairy sample collected from local market was used in thepresent study. MAM medium consisting of tryptone-10g;yeast extract-5g; arginine-3g; KH
2PO
4-0.5g; MgSO
4-0.2g;
MnSO4-0.05g; Tween80-1ml; glucose-5g; Agar-2g; H
2O-
1000ml, (pH 6.0) was used for enzyme production. Opticaldensity of the inoculums was adjusted to 1.0 and 1% (v/v)inoculum was used. Cultures were incubated at 37oC for 24 hand sub-cultured thrice in MAM broth, before subjecting toADI assay.
Enzyme assay
To determine enzyme activity, 24h old cultures were centrifugedat 8,000 rpm for 10min. Cell free supernatant (CFS) was assessedfor extracellular protein and enzyme activity. For assayingintracellular ADI activity, cell pellet was resuspended in lysisbuffer. Total protein was estimated by measuring absorptionat 280nm using standard curve of BSA. The assay of ADI
activity was based on standard method of Angelis et al. (2002).Assay mixture consisted of 150µl of 50 mM arginine, 2.3ml of50 mM acetate buffer (pH 5.5), 50 µl of cell wall or cytoplasmpreparation, and 3.6 µl of sodium azide (0.05%). Controlswithout substrate and without enzyme were included. Afterincubation at 37oC for 1h, the reaction was stopped by adding0.5ml solution of 2N HCl and precipitated protein was removedby centrifugation. Citrulline content in CFS was determinedby Archibald’s method (1944). One enzyme unit was definedas the amount of enzyme required to catalyze formation of1µmol citrulline per min. One milliliter of supernatant was addedto 1.5ml of an acid mixture of H
3PO
4-H
2SO
4 (3/1 v/v) and 250 µl
of diacetyl monoxime (1.5% 2, 3 butanadiona monoxime) in10% (v/v) methanol, mixed and boiled in dark for 30 min. Aftercooling for 10 min, the absorbance at 460 nm was measured.Finally, specific activity was calculated as international enzymeunits present per mg (IU/mg) of protein.
Experimental designs
In present study, MAM broth was used as basal medium andeffect of various carbons, nitrogen sources, minerals, inducersand metal ions, was studied for enhancing ADI activity of W.confusa GR7. RSM was adopted for improving enzyme activityusing Design Expert Software version 8.0.2 trial statisticalsoftware (State-Ease Inc., Minneaopolis, MN, USA). Plackett-Burman design is an efficient tool, especially when a largenumber of variables are involved, for screening variables thatare able to determine the influence of various factors withminimal number of experiments, which is time saving andmaintain convincing information on each component (Krishnanet al., 1998; Abdel-Fattah et al., 2005). It is a saturatedorthogonal design work at two-levels, and can be constructedon the basis of fractional replication of a full factorial design.Total number of experiments to be carried out in Plackett-Burman design are n+1, where n is the number of variables.Each variable was represented at two levels, upper and lowerdenoted by “high (+)” and “low (-)” respectively (Plackett andBurman, 1946). Here, a total of eleven factors or variables werescreened in twelve trials. 11 variables namely fructose, sucrose,tryptone, meat extract, MnSO
4, MgSO
4, copper,
arginine, CTAB,
tween 80, CO2
were screened as shown in Table1 whereas,phosphate buffer, pH-6, temperature at 37oC and incubationtime of 24 h were kept constant. Effect of each variable isquantified as difference between the average of measurementsmade at the high and the average of measurements made at thelow level of that factor. It is possible to calculate effect of eachvariable on ADI activity following equation no. 1.
E (Xi) = (2 (∑ Pi+ - ∑ Pi-) / N.....................................................(1)
Where E (Xi) is the concentration effect of tested variables;P
i+ and P
i- represent ADI activities obtained in various trials,
Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7
73
where the variable (Xi) measured was present at the high orlow level, respectively, and N is the number of trials(experiments) performed.
Standard error (SE) of the concentration effect was the squareroot of the variance of an effect, and the significance level(P-value) of the effect of each constituent was determinedusing student’s t-test as given by the equation no. 2;
t (xi)=E(xi)/SE........................................................................(2)
Where E (xi) is the effect of variable Xi
This model does not describe the interaction among factorsand it is used for preliminary screening and evaluates importantfactors that influence the response. From the regressionanalysis of the variables, the factors having a significant effecton ADI activity were further optimized by central compositerotatable design (CCRD). Total 50 experiments were employedin CCRD to estimate curvature and interaction effects ofselected five variables i.e. sucrose (A), copper (B), MgSO
4 (C),
MnSO4 (D) and arginine (E). All experiments were carried out
at 37oC and pH-6 for 24 h. Fructose (5g/l), CTAB (50mM), tween80 (50mM) and phosphate buffer (50mM) were kept as constantfactors as they are known to influence ADI activity.
Results and discussion
When grown in unoptimized modified MAM broth, Weissellaconfusa GR7 showed 0.27 IU/mg of specific ADI activity.Specific ADI activity was further optimized using two statisticaltools (Plackett-Burman design and CCRD). The Plackett-Burman design screens important variables that may affectproduction of this therapeutic protein i.e ADI, but does notconsider the interactive effects among the variables as in CCRD.In CCRD, each selected variable is studied at five differentlevels along with other variables, and therefore, interactionsamong these variables at different levels could be studied. Inorder to enhance enzyme activity, experiments were designedto optimize media constituents using Plackett- Burmanexperimental design. Table 1 presents Plackett–Burman designfor 11 culture variables and their corresponding response interms of specific enzyme activity. When the concentration effectvalue (E (Xi)) of the tested variable was positive, the influenceof the variable was greater at the high concentration tested,and when negative, the influence of the variable was greater atlow concentration. The variation in ADI specific activity indifferent sets ranged from -0.0138 to 2.0455 IU/mg, reiteratingthe importance of selection and identification of importantfactors. On the basis of their percent contribution, fructose,sucrose, MgSO
4, CTAB, tween 80 and copper are proved to be
very effective for maximizing enzyme activity from this lacticacid bacterial isolate. Other variables had only a minor
contribution towards enzyme activity. The experimental resultsof Plackett-Burman design were fitted in central compositerotatable design (CCRD) of experiments to be employed tooptimize concentration of most contributing factors for ADIactivity (Table2). Fructose (5g/l), CTAB (50mM), tween 80(50mM), phosphate buffer (50mM), pH-6, temperature 37ºCand incubation time of 24 h were constant factors as wereinfluencing ADI activity, therefore kept at specific value duringCCRD design. Sucrose (5g/l), copper (10mM), MgSO
4 (10mM),
MnSO4 (10mM) and arginine (50mM) concentrations
significantly enhanced ADI activity in W. confusa GR7.
Results so obtained were fed into Design-Expert software andanalyzed using analysis of variance (ANOVA) as appropriateto the experimental design used. Based on the CCRD, theexperimental levels of specific ADI activity under each set ofcondition were determined and compared with thecorresponding predicted levels (Table 2). The maximumexperimental value for ADI specific activity was 2.8081 IU/mg,while RSM predicted response as 1.76 IU/mg. The closecorrelation between the experimental and predicted dataindicates the appropriateness of the experimental design. Thequality of the model can also be checked using various criteria.The calculated regression equation for the optimization ofmedia constituents assessed the specific activity (Y) as afunction of these variables. Multiple regression analysis ofthe experimental data was carried out and statistical equationwas generated that gives ADI enzyme activity according toequation no. 3 as shown below:
Y = +0.68+0.12*A-0.15*B-0.011*C-0.11*D+0.11*E-0.17*A*B+0.046*A*C-0.18*A*D+0.12*A*E+0.051*B*C+0.19*B*D-0.11*B*E-0.010*C*D+0.064*C*E-0.18*D*E........................(3)
Where, Y is the specific enzyme activity and A, B, C, D, E iscoded values of sucrose, copper, magnesium, manganese, andarginine respectively.
Table 3 shows ANOVA results for the RSM Two FactorInteraction (2FI) for response Y. According to the present modelA, B, D, E, AB, AD, AE, BD, BE and DE are significant modelterms.2FI model was found to be the ‘best fit model’ for thespecific enzyme activity with the highest F-value whencompared to other models (Table 4). ANOVA for ADI specificactivity (Y, IU/mg) indicated the ‘F-value’ to be 11.10, whichimplies the model to be significant. Model terms having valuesof ‘Prob > F’ less than 0.0500 indicate model terms aresignificant, whereas those greater than 0.10 are insignificant.The “Lack of Fit F-value” of 138.35 implies the Lack of Fit issignificant. There is only a 0.01% chance that a “Lack of Fit F-value” this large could occur due to noise. ANOVA indicatedthe R2 value of 0.8305 for response Y. This ensured a good
74
Kaur and Kaur
Tabl
e 1:
Exp
erim
enta
l run
and
res
ults
obt
aine
d in
Pla
cket
t-B
urm
an d
esig
n
Run
Fru
ctos
e)Su
cros
eM
eat
Ext
ract
Try
pton
e C
uSO
MgS
OM
nSO
CTA
BT
wee
n-80
Arg
inin
e%
Spec
ific
AD
I(g
/l(g
/l)(g
/l)(g
/l)4(
mM
)4
(mM
)4
(mM
)(m
g/m
l)(m
M)
(mM
)C
O2
acti
vity
(IU
/mg)
Exp
erim
enta
lP
redi
cted
valu
eva
lue
10
50
1010
010
5050
00
0.43
0.57
25
00
010
010
500
505
1.62
0.57
30
510
100
00
500
505
0.4
0.57
45
010
1010
00
050
05
0.27
0.57
55
50
00
100
5050
05
2.04
0.57
60
00
00
00
00
00
-0.0
10.
577
00
010
010
100
5050
50.
580.
578
55
010
1010
00
050
00.
380.
579
00
100
1010
050
5050
00.
440.
5710
50
1010
010
1050
00
00.
150.
5711
55
100
00
100
5050
00.
20.
5712
05
100
1010
100
00
50.
250.
57
Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7
75
Table 2: Experimental run and results obtained in CCRD design
Run Sucrose CuSO4
MgSO4
MnSO4
Arginine Specific ADI activity (IU/mg)(g/l) (mM) (mM) (mM) (mM) Experimental Value Predicted Value
1 5 10 0 0 0 0.34 0.682 5 0 0 0 50 2.33 0.283 0 10 10 10 50 0.7 0.974 2.5 5 5 5 25 0.79 0.565 5 0 10 0 50 2.81 1.766 8.4 5 5 5 25 0.76 1.037 2.5 -6.9 5 5 25 1.05 1.048 2.5 5 -6.9 5 25 0.88 0.689 5 10 10 0 50 0.93 0.71
10 0 10 0 0 50 0.47 2.1711 2.5 5 5 5 84.5 0.47 2.0512 2.5 5 5 -6.9 25 0.77 0.4413 2.5 5 5 5 25 0.77 0.1714 0 0 10 0 50 0.71 0.4815 5 10 10 10 0 0.52 0.3116 5 10 0 10 0 0.52 1.0517 0 10 0 0 0 0.32 0.4518 0 10 0 10 0 0.72 1.1619 0 0 0 10 50 0.51 0.5720 -3.4 5 5 5 25 0.51 0.9521 0 10 10 0 50 0.52 0.3922 5 0 0 10 0 0.51 0.2423 2.5 5 5 5 25 0.78 0.924 5 0 10 0 0 0.78 06925 5 0 0 10 50 0.59 0.3126 0 0 10 0 0 0.31 0.3927 5 0 10 10 0 0.42 0.4128 0 0 10 10 0 0.33 0.429 5 0 0 0 0 0.98 0.6830 2.5 5 16.9 5 25 0.51 0.5831 5 0 10 10 50 0.66 0.3332 0 0 0 0 50 0.73 0.6833 5 10 0 10 50 0.21 0.7134 2.5 5 5 5 25 0.73 1.0235 0 10 10 0 0 0.29 0.2136 0 10 10 10 0 0.83 0.6837 0 0 0 10 0 0.67 0.3238 2.5 5 5 5 25 0.74 0.8339 5 10 0 0 50 0.57 0.4240 0 10 0 10 50 0.53 0.4341 2.5 5 5 16.9 25 0.56 0.6842 2.5 5 5 5 25 0.74 1.2143 2.5 5 5 5 25 0.77 0.4444 5 10 10 0 0 0.32 0.2545 2.5 5 5 5 -34.5 0.51 0.9646 2.5 16.9 5 5 25 0.31 0.6847 2.5 5 5 5 25 0.78 0.3248 0 0 0 0 0 0.57 0.6549 5 10 10 10 50 0.55 0.9350 0 0 10 10 50 0.28 0.68
76
Kaur and Kaur
Table 3: One way ANOVA analysis of Response Surface designs
Source Sum of squares Degree of freedom Mean squares F- value p-value prob > F
Model 7.87 15 0.52 11.1 < 0.0001 significant A-Sucrose 0.61 1 0.61 12.87 0.001 B-Copper 1.02 1 1.02 21.51 < 0.0001 C-Magnesium 5.42E-03 1 5.42E-03 0.11 0.737 D-Manganese 0.56 1 0.56 11.9 0.0015 E-Arginine 0.48 1 0.48 10.18 0.003 AB 0.91 1 0.91 19.25 0.0001 AC 0.067 1 0.067 1.43 0.2407 AD 1.03 1 1.03 21.71 < 0.0001 AE 0.47 1 0.47 9.88 0.0035 BC 0.082 1 0.082 1.74 0.1964 BD 1.14 1 1.14 24.2 < 0.0001 BE 0.37 1 0.37 7.89 0.0082 CD 3.39E-03 1 3.39E-03 0.072 0.7903 CE 0.13 1 0.13 2.8 0.1033 DE 0.99 1 0.99 21 < 0.0001Residual 1.61 34 0.047Lack of Fit 1.6 27 0.059 138.35 < 0.0001 significantPure Error 3.01E-03 7 4.29E-04Cor Total 9.48 49
Coefficient of determination (R2) = 0.8305
Table 4: Statistical summary of model
Source Sum of Squares Degree of freedom Mean Square F-value p-value Prob > F
Mean vs Total 23.07 1 23.07Linear vs Mean 2.67 5 0.53 3.46 0.0101 Linear vs Mean2FI vs Linear 5.2 10 0.52 11 < 0.0001 SuggestedQuadratic vs 2FI 0.13 5 0.026 0.5 0.7734Cubic vs Quadratic 1.34 15 0.089 9 < 0.0001 AliasedResidual 0.14 14 9.93E-03Total 32.54 50 0.65
correlation between observed and expected values of the 2FImodel indicated that this model could explain 83% responsevariability. The adequate precision which measures the signalto noise ratio of 16.229 indicates an adequate signal. A ratiogreater than 4 is desirable.
The three-dimensional response surface plots based oninteractions between the variables showed an increase inspecific ADI activity as the concentration of each variablereached an optimum level, beyond which a decline wasobserved (Figure 1a to 1e). 3D response surface plots wereobtained by plotting response i.e specific ADI activity (IU/mg) on the Z-axis against any two variables on X and Y-axiswhile keeping other three variables at their central values, were2.50g/l sucrose, 25 mM arginine, 5.0 mM CuSO
4, 5.0 mM
MgSO4, 5.0 mM MnSO
4. Results indicated that concentration
of sucrose in the culture medium had profound effect on specificactivity of arginine deiminase (Figure 1a to 1c ). CCRD employedhas signified that sucrose at concentration of 2.5 g/l wasoptimal for maximum enzyme activity. Similar effect wasobserved in case of arginine which was used as an inducer ofADI activity. As its concentration is raised there is concomitantincrease in specific ADI activity (Figure 1a, 1d and 1e). Resultsindicated a central value of arginine as 25 mM for optimalenzyme production. Presence of CuSO
4 and MnSO
4 caused
marginal increase in ADI activity as compared to other twocomponents (Figure 1b to 1e).
Weissella confusa GR7 showed 0.27 IU/mg specific ADI activityin unoptimized MAM broth, which was raised to 2.8081 IU/mg
Statistical screening of media components for the production of arginine deiminase by Weissella confusa GR7
77
(a)
Figure1: Three-dimensional response cartons showing effects and interaction of (a) arginine (mM) and sucrose (g/l), (b) copper sulphate (mM)and sucrose (g/l), (c) manganese sulphate (mM) and sucrose (g/l), (d) arginine (mM) and manganese sulphate (mM) and (e) arginine (mM) andcopper sulphate (mM) on specific ADI activity of W. confusa GR7.
78
Kaur and Kaur
in RSM optimized media (run 5). It was improved by optimizingconcentration of arginine, sucrose, copper, manganesesulphate and magnesium sulphate concentration. Arginineinfluenced ADI activity as it acts as an inducer for ADI activityas previously reported in Streptococcus lactis, Lactobacillussanfrancisciscensis CB1, Weissella koreensisMSI-3, Weissellakoreensis MSI-14, Lactobacillus buchneri CUC-3 (Crow andThomas, 1982; Mira de Orduña et al., 2000; Angelis et al.,2002; Yu et al., 2010). The nature and concentration of sugarsalso affect ADI activity as reported earlier in Lactobacillussanfrancisciscensis CB1 and did not grow in MAM mediumwith arginine as sole carbon source (Angelis et al., 2002). InStreptococcus lactis ADI activity was increased 5- to- 10 foldin galactose grown cultures as compared to glucose or lactosegrown cultures (Crow and Thomas, 1982). Higher concentrationof sugars may repress ADI activity as reported in Lactobacillussake (Montel et al., 1987). In our case the effect of sugars,inorganic nutrients and surfactants on the activity of ADI wasinvestigated and results showed maximum specific activity atconcentration of 2.5g/l sucrose, 5mM MgSO
4, 5mM Copper,
and 50mg/ml CTAB. Similar, findings are also reported in aEnterococcus faecalis NJ402 isolate (Li et al., 2005). Based onthe results obtained, mediums consisting of sucrose (2.5g/l),fructose (5g/l), tryptone (10g/l), MgSO
4 (5mM), MnSO
4 (5mM),
arginine (25mM), CuSO4 (5mM), CTAB (50mg/ml) and tween
80 (50mM), pH 6, at 37 oC for 24 h was optimized.
Conclusion
Sequential statistical strategies, Placket- Burman designfollowed by CCRD were used successfully to find out optimumvalues of the significant factors to achieve maximum ADIactivity in W.confusa GR7. The experimental result (2.8081IU/mg) in a medium optimized for ADI production was in closeagreement with the predicted value of 2FI model (1.76 IU/mg)and hence, the model was successfully validated. The specificenzyme activity showed about 10 fold increases over the basalmedium. Further, it is important to discover newer lactic acidbacterial strain via ADI pathway that produces therapeuticenzyme of importance in healthcare industry.
Acknowledgement
Authors acknowledge UGC, New Delhi for funding MaulanaAzad National Fellowship for Minority Students to Mrs.Rajinder Kaur (vide letter no.F.40-116(M/S)/2009(SA-III/MANF).
References
Abdel-Fattah, Y.R., Saeed, H.M., Gohar, Y.M. and El-Baz, M.A.2005. Improved production of Pseudomonas aeruginosauricase by optimization of process parameters through
statistical experimental designs. Process Biochem., 40:1707-1714.
Ammor, M.S. and Mayo, B. 2007. Selection criteria for lactic acidbacteria to be used as functional starter cultures in dry sausageproduction: an update. Meat Sci., 76:138-146.
Angelis, M.De., Mariotti, L., Rossi, J., Servili, M., Fox, P.F., Rolla,G., and Gobbetti, M. 2002. Arginine catabolism by sourdoughlactic acid bacteria: purification and characterization of thearginine deiminase pathway enzymes from Lactobacillussanfranciscensis CB1. Appl. Environ. Microbiol., 68: 6193-6201.
Archibald, R.M. 1944. Determination of citrulline and allantoin anddemonstration of citrulline in blood plasma. J. Biol. Chem.,156: 121-142.
Collins, M.D., Samelis, J., Metaxopoulos, J. and Wallbanks, S. 1993.Taxonomic studies on some leuconostoc-like organisms fromfermented sausages: description of a new genus Weissella forthe Leuconostoc paramesenteroides group of species. J. Appl.Bacteriol., 75: 595-603.
Crow, V.L. and Thomas, T.D. 1982. Arginine metabolism in lacticStreptococci. J. Bacteriol.,150:1024-1032.
Dellaglio, F. and Torriani, S. 1986. DNA-DNA homology,physiological characteristics and distribution of lactic acidbacteria from maize silage. J. Appl. Bacteriol., 60: 83-93.
Dellaglio, F., Vescovo, M., Morelli, L. and Torriani, S. 1984. Lacticacid bacteria in ensiled high-moisture corn grain: physiologicaland genetic characterization. Syst. Appl. Microbiol., 5: 534-544.
Hammes, W.P. and Vogel, R.F. 1995. The genus Lactobacillus. In:The Genera of Lactic Acid Bacteria, Ed. B. J. B. Wood and W.Holzapfel. Glasgow: Blackie Academic Professional. pp. 19-54.
Katina, K., Maina, N.H. and Juvonen, R. 2009. In situ productionand analysis of Weissella confusa dextran in wheat sourdough.Food Microbiol., 26:734-743.
Kennedy, M. and Krouse, D. 1999. Strategies for improvingfermentation medium performance: a review. IndustrialMicrobiol. Biotech., 23:456-475.
Krishnan, S., Prapulla, S.G., Rajalakshmi, D., Misra, M.C. andKaranth, N.G. (1998). Screening and selection of mediacomponents for lactic acid production using plackett-burmandesign. Bioprocess Engg., 19:61-65.
Lee, S.L. and Chen, W.C. 1997. Optimization of medium compositionfor the production of glycosyltransferase by Aspergillus nigerwith response surface methodology. Enzyme Microbiol. Tech.,21:436-440.
Lee, Y. 2005. Characterization of Weissella kimchii PL9023 as apotential probiotic for women. FEMS Microbiol. Lett., 250:157-162.
Li, J., Cao, Y., Liu, Y., Qian, S., Jiao, Q. 2005. Activity and stabilityof arginine deiminase for producing L-Citrulline. Chinese J.Chem. Engg. 13(6):841-844.
Liu, S.Q. Holland, R. and Crow V.L. 2003. The potential of dairylactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination. Int.J. Food Microbiol., 86:257-269.
Magnusson, J. Jonsson, H. Schnurer, J. and Roos, S. 2002. Weissella
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Milbourne, K. 1983. Thermal tolerance of Lactobacillus viridescensin ham. Meat Sci. 9: 113-119.
Mira de Orduña, R., Liu, S.Q., Patchett, M.L. and Pilone, G.J. 2000.Ethyl carbamate precursor citrulline formation from argininedegradation by malolactic wine lactic acid bacteria. FEMSMicrobiol. Lett., 183:31-35.
Montel, M.C. and Champomier, M.C. 1987. Arginine catabolism inLactobacillus sake isolated from meat. Appl. Environ.Microbiol., 53:2683-2685.
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Intl. J. of Food. Ferment. Technol. 2(1): 81-86, June, 2012
Effect of nitrogen source on the fermentation behaviourof musts and quality of wine from two Cvs. of pineapple
B.L. Attri
Central Institute of Temperate Horticulture, Regional Station, Mukteshwar - 263 138 (Uttarakhand), India
*Email: [email protected]
Paper no: 41 Received: 19 Jan, 2012 Received in revised form: 25 April, 2012 Accepted: 19 May, 2012
Abstract
The composition of the musts prepared from the pineapple fruits cvs. Kew and Queen by ameliorationof TSS by addition of KMS (100ppm), with and without DAHP (0.1%) as nitrogen source was foundto vary significantly. The fermentation rate of the pineapple musts having nitrogen source, carried outby the yeast Saccharomyces cerevisiae var. ellipsoideus was comparatively faster than those withoutDAHP and over a period of 10 days. After six months of storage the physico-chemical characteristicsof the pineapple wines revealed significantly higher alcohol (10.56 and 11.44 %) in the treatmentshaving nitrogen source with better fermentation efficiency of yeast (48 and 52%). The acidity andvolatile acidity of these treatments was significantly higher. The pineapple wines having more alcoholwere found to have lower aldehydes (112.78 and 117.47 mg/litre) whereas total esters (94.65 and 104.77mg/litre) and phenolic contents (148.70 and 156.54 mg/litre) were found to be higher. From the sensoryquality point of view, the wine prepared from cv. Queen was preferred to cv Kew. The costs of the finalproducts were Rs. 35.38 and Rs. Rs. 31.82 per 650 ml bottle in cv. Kew and Queen, respectively.
©2012 New Delhi Publishers. All rights reserved
Keywords: Keywords: Pineapple nitrogen source, must, fermentation, wine, phenolic content,Saccharomyces cerevisiae, sensory
Introduction
The pineapple (Ananas comosus (L.) Merr. Syn. Ananas sativusSchult.f) originated from Paraguay belongs to familyBromeliaceae (Bertoni 1919). It is one of the most importantand wanted tropical fruits because of good source of vitaminA, B, C and calcium. Apart from its various products viz., slices,juice, squash, jam, and mixed fruit jam, other by-products arealcohol, calcium citrate, citric acid and vinegar. The dried wasteafter juice extraction is valuable cattle feed. It is being cultivatedin different countries like Hawaiian Islands, Philippines,Malaysia, Thailand, Brazil, Ghanna, Kenya, Mexico, Taiwan,S. Africa, Australia and India. It was introduced in India in
1548 and is coming up well with a total area of about 25,000 hain North- Eastern states viz., Assam, Tripura, and Meghalaya,W. Bengal, Kerala, Tamilnadu, Goa, Karnataka and Orissa (Sen,1985). The warm and humid tropical climatic conditions ofAndaman and Nicobar Islands, situated between 100 31’ and130 42’ North latitude and 920 14’ and 940 16’ East longitude inthe Bay of Bengal, are congenial for its cultivation. Although,the fruits is used as fresh as well as processed into variousforms but glut production is high, producers do not getremunerative returns and are forced to sell their produce atvery low prices. The composition as well as the nutritionalstatus of pineapple fruits grown in these islands is at par withthe fruits being produced elsewhere in other parts of the
Research paper
82
Attri
country. Apart from other products that can be processed intoalcoholic beverages from the extracted juice which is a goodalternative that in turn will increase the socio-economic statusof local farmers. The Kew and Queen cultivars of pineappleare performing well in these islands. The area and productionof pineapple fruits is expected to increase substantially becauseof adoption of year round production technology (Singh, 1996).Different products prepared from pineapple have been welldocumented (Anon. 1985) but information on pineapple winepreparation is meager. Therefore, the present investigationswere carried out to utilize the pineapple fruits of these cultivarsgrown in Andaman and Nicobar Islands for wine production.
Materials and methods
Collection of pineapple fruits
The physiologically matured fruits of pineapple cvs. Kew andQueen were harvested from the research farm of CentralAgricultural Research Institute, Port Blair . The harvested fruitswere allowed to ripen in the laboratory at a room temperatureof 25-300 C and after getting the required colour of the skin ofthe fruits, these were analysed for various physical characters.
Extraction of juice
The fully ripened fruits of both the pineapple cvs. were peeledmanually with steel knife and after cutting into small piecesand removing the central core, the juice was extracted bypassing the pineapple pieces through a juicer. The extractedjuice was filtered through a muslin cloth followed by heatingupto 85-90o C for 5 minutes in a steel container.
Preparation of must
To prepare pineapple must, the total soluble solids (TSS) ofthe juice were ameliorated to 220 B with 70 per cent sugar syrup.The prepared must was filled in glass containers having 100ppm potassium metabisulphite (KMS). Further, di-ammoniumhydrogen phosphate (DAHP) @ 0.1% was added as a nitrogensource in two treatments (T
1 and T
3) whereas other two (T
2
and T4) musts were devoid of any nitrogen source.
Fermentation and maturation
An active culture of yeast Saccharomyces cerevisiae var.ellipsoideus was added @ 5% (v/v) containing approximately104-105 cells/ ml in the glass containers having must, equippedwith air locks at a temperature of 22±1oC. Samples of thefermenting musts were taken periodically to determine thechanges in total soluble solids (oB). The fermentation rate (fallof TSS/24hour) was recorded till the TSS became stable. Thepineapple wine thus prepared was siphoned, filtered and stored
at room temperature for ageing/maturation upto six months.The flow diagram for the preparation of pineapple wines hasbeen illustrated in Figure 1.
Analysis of juice and wine
Figure 1: Flow diagram for the preparation of pineapple wine
The different physico-chemical characteristics i.e., TotalSoluble Solids (0Brix), titratable acidity (% malic acid), pH ofextracted juice and moisture (%) of the fruit were measured bystandard methods described by Ranganna (1986) whereasascorbic acid (mg/100g) and carotenoid contents (µg/100g)were estimated as per the methods of AOAC (1975).
After storage, different treatments of pineapple wine were
Effect of nitrogen source on the fermentation behaviour of musts and quality of wine
83
analyzed for various physico-chemical characteristics andoverall sensory quality. The TSS, acidity, pH and ascorbicacid were recorded as described earlier. The alcohol (%) wasestimated by a standard colorimetric methods given by Caputiet al (1968), volatile acidity (% Acetic acid) by the methods ofPilone et al (1972) whereas aldehydes and total esters weredetermined according to the methods described by Amerineand Ough (1979) and Libraty (1961) respectively. The totalphenols were estimated by the method of Singleton and Rossi(1965).
Sensory analysis
For overall sensory quality, a panel of 10 judges was asked togive score out of 20,keeping in view the colour, appearance,aroma and taste of the wines.
Statistical analysis
The available data for physico-chemical characteristics ofdifferent pineapple musts and wines were analysed in aCompletely Randomized Block Design (CRBD) as describedby Panse and Sukhatme (1989).
Cost of production of pineapple wine
The cost of production of pineapple wines from both the cvs.Kew and Queen was also estimated keeping in view the presentrates of various items used in the fermentation and thereafter.The overhead charges as well as additional charges onfermentation were also added along with sales tax and profit ofthe producer.
Results and discussion
The composition of both the cvs. of pineapple (Table 1) wascomparable to those which are being cultivated elsewhere inthe country. The length, breadth, weight, volume, juice (%),TSS, brix acid ratio and moisture (%) of the fruits recorded wascomparatively more in Kew than Queen cultivar whereas theacidity (%), ascorbic acid, carotenoid contents and dry matterwere higher in the latter than the former. The difference in thephysico-chemical characteristics of the pineapple cultivarsmay be due to their inherent genetic characteristics. Thecomposition of the musts of two cvs. with and without nitrogensource (DAHP) was found to be different appreciably (Table2). The variation may be attributed to the initial composition ofthe pineapple fruit juice. The fermentation of the musts wasvery fast and the TSS became stable just after 10 days (Figure2). It is apperent that the fermentation was comparatively fasterin the treatments having DAHP than those without DAHP.The higher fermentation rate in the musts having nitrogensource may be due to fast growth and multiplication of the
yeast cells which has also been reported earlier by Joshi et al.(1991). The fermentation rate however declined towards thecompletion of fermentation which may be due to more alcoholproduction inhibiting the fermentation efficiency of the yeast(Attri et al 1994). The fermentation efficiency of the yeast wasfound 48 and 52 per cent in T
1 and T-
3 (nitrogen source) as
compared to 45 and 50 per cent in T2 and T-
4 (without nitrogen
source) which has been depicted in Fig. 3. The higher efficiencymay be attributed to the addition of nitrogen source for theyeast during fermentation.
Table 1: Composition of different cultivars of pineapple
Characters \CultivarsKew Queen
Length (cm) 15.70±0.040 10.50±0.092Breadth (cm) 12.50±0.040 9.80±0.092Weight (g) 1060.00±8.16 680.00±4.08Volume (ml) 1099.00±7.55 707.00±1.53Specific gravity (w/v) 0.964±0.0008 0.962±0.0002Juice (%) 56.00±0.489 52.00±0.408Waste (%) 44.00±0.489 48.00±0.408T.S.S. (0B) 15.50±0.081 12.00±0.081Acidity (% MA) 0.938±0.0016 1.045±0.0008Brix acid ratio 16.52±0.057 11.48±0.069Ascorbic acid (mg/100g) 39.13±0.257 45.00±0.326Carotenoids (µg/100g) 38.99±0.608 58.00±0.347Moisture (%) 78.83±0.048 76.980±0.122Dry matter (%) 21.17±0.048 23.10±0.122
The physico-chemical characteristics of the wines revealedsignificant differences in all the treatments. The TSS of thewines prepared by adding DAHP was lower as compared tothose without it. It might be due to higher utilization of thesugars by yeast during fermentation. The acidity (%MA) ofall the pineapple wines del to increased during fermentation.Attri (2009) reported that initial sugar concentration increasedthe acidity in the cashew apple wine. The pH of the wines werefound in corrobation with acidity. Similarly, the ascorbic acidwhich was 39.13 and 45.00 mg/100g in extracted juice of boththe pineapple cvs. was reduced to 15.40 and 19.50 mg/100g,respectively in T
2 and T-
4 whereas 18.70 and 21.76 mg/100g,
respectively in T1 and T-
3. The reduction may be attributed
due to the loss during boiling of the extracted juice. The alcoholproduction was significantly better in the treatments having Nsource than others which may be due to the more utilization ofthe sugars during fermentation. Likewise acidity, the volatileacidity (%AA) was also recorded significantly higher in thewines with more alcohol content.
The other constituents viz., aldehydes, total esters andphenols varied significantly among the wines of both cultivars
84
Attri
Table 2: Physico-chemical characteristics of pineapple musts
Cultivars Treatments Characteristics of musts
TSS (oB) Acidity (%MA) pH Ascorbic acid (mg/100g) Carotenoids (µg/100g)
Kew T1
22.0 0.850 4.12 34.35 27.65T
222.0 0.875 4.08 35.50 29.57
Queen T3
22.0 1.010 3.98 41.20 43.87T
422.0 1.014 3.92 42.45 45.28
CD=0.05 - NS 0.004 0.046 0.465 0.254
T1=Must with 0.1% DAHP, T
2= Must without DAHP, T
3= Must with 0.1% DAHP, T
4=Must without DAHP
Table 3 : Physico-chemical characteristics of pineapple wines
Cultivars Treatments Characteristics of wines
TSS Acidity pH Ascorbic acid Alcohol Volatile Aldehydes Total esters Total(oB) (%MA) (mg/100g) (% v/v) acidity (mg/litre) (mg/ litre) (mg/litre) phenols
(%AA)
Kew T 1 4.8 1.012 4.00 18.70 10.56 0.009 112.78 94.65 148.70T 2 5.0 0.975 4.06 15.40 9.90 0.007 119.45 86.97 141.63
Queen T 3 4.6 1.045 3.89 21.76 11.44 0.010 117.47 104.77 156.54T 4 5.0 1.020 3.94 19.50 11.00 0.008 123.86 101.20 151.87
CD=0.05 0.399 0.0023 0.113 0.118 0.143 0.012 0.330 0.173 0.131
Figure 2: Fermentation rate of different treatments of pineapplemust
Figure 3: Comparison of fermentation efficiency of two musts byyeasts
(Table 3). The aldehydes contents were recorded significantlylower 112.78 and 117.47 mg/litre in T
1 and T-
3 having more
alcohol as compared to 119.45 and 123.86 mg/litre in T2 and T-
4
respectively in Kew and Queen. The total esters weresignificantly better (94.65 and 104.77 mg/litre) in T
1 and T-
3
than T2 and T-
4 (86.97 and 101.20 mg/litre). Likewise total esters,
total phenolic contents were also recorded higher in T1 and
T-3 (148.70 and 156.54 mg/litre) than T
2 and T-
4 (141.63 and
151.87 mg/litre). The results are in confirmation with those ofAttri (2009) in cashew apple wine where higher alcoholproduction have reduced aldehydes and increased total estersand phenolic constituents.
In the overall sensory quality, the pineapple wine from cv.Queen had an edge over than that of Kew (Figure 4) whichmay be due to better colour of the final product because ofinitial colour of juice.
Effect of nitrogen source on the fermentation behaviour of musts and quality of wine
85
Further, for developing any product, the most important factoris its cost of production. It has been calculated on lab scale(Table 4) which indicated that the product can be sold at areasonable cost with good profit margin. The cost of productioncalculated for the product (Rs. 35.38 and 31.82 for Kew andQueen pineapple wine) is quite reasonable for any low alcoholicbeverage available in the market. The product prepared fromthe pineapple fruits is comparable to other fruit wines describedby Joshi et al (1990) and Attri et al (1994).
Table 4: Cost of production of pineapple wine
S. No. Material required Kew Queen
Quantity Rate (Rs.) Amount (Rs.) Quantity Rate (Rs.) Amount (Rs.)
1. Pineapple fruit 100kg 10.00/kg 1000.00 100 kg 8.00/kg 800.002. Transportation - - 50.00 - - 50.003. Juice extraction 2 mandays 60.00/day 120.00 2 mandays 60.00/day 120.004. Sugar 4.0 kg 18.00/kg 72.00 5.0 kg 18.00/kg 90.005. Potassium metabisulphite @ 100 ppm 6.5g - 15.00 6.5g - 15.006. DAHP @ 0.1% 65.0g - 15.00 65.0g - 15.007. Glass bottles (650 ml) 100 3.00/bottle 300.00 100 3.00/bottle 300.008. Crown corks 100 0.25/cork 25.00 100 0.25/cork 25.009. Total cost - - 1597.00 - - 1415.0010. Overhead charges @20% - - 320.00 - - 283.0011. Additional charges @ 15% on fermentation - - 240.00 - - 212.0012. Excise duty - 3.00/bottle 300.00 - 3.00/bottle 300.0013. Grand total - - 2457.00 - - 2210.0014. Sales tax @20% - - 491.00 - - 442.00
Profit @ 20% - - 590.00 - - 530.0015. 3538.00 3182.0016. Sale price/bottle - - 35.38 - - 31.82
Figure 4: Overall sensory quality of different pineapple wines(maximum score = 20, NS = Nitrogen source)
Conclusion
The wine made by the addition of nitrogen sources had betterfermentability than those without it. The physico-chemical andsensory qualities of both the wines were acceptable but theproduct from cv. Queen was preferred. The cost of productionof pineapple wine was also preferable. It can be concludedthat there is an ample scope for the utilization of pineapplefruits for production of wine which are good in nutrients andavailable throughout the year in the islands by the methoddescribed here.
References
Amerine MA and Ough CS. 1979. Wine and Must Analysis, 2nd Edn.John Wiley and Sons, New York.
Anonymous. 1985. Home Scale Processing and Preservation of Fruitsand Vegetables. 7th Edn. CFTRI, Mysore. Pp: 28-29.
AOAC. 1975. Official Methods of Analysis. Association of OfficialAnalytical Chemists. 12th Edn. Washington, D.C.
Attri BL, Lal BB and Joshi VK. 1994. Technology for the preparationof sand pear vermouth. Indian Food Packer, 48(1):39-45.
Attri BL. 2009. Effect of initial sugar concentration on the physico-chemical characteristics and sensory qualities of cashew applewine. Natural Product Radiance, 8(4):374-379.
Bertoni MS. 1919. Ancient Paraguay, Series II, 4: 250-332.Caputi A, Ucda Jr M and Brown T. 1968. Spectrophotometric
determination of ethanol in wine. Am. J. Enol. Vitic., 19:160-165.
Joshi VK, Attri BL, Gupta JK and Chopra SK. 1990. Comparativefermentation behaviour, physico-chemical characteristics and
86
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sensory qualities of fruit honey wines. Indian J. Hort., 47(1): 49-54.
Joshi VK, Sharma PC and Attri BL. 1991. A note on deacidificationactivity of Schizosaccharomyces pombe in plum must ofvariable compositions. J. Applied Bacteriol, 70: 385-390.
Libraty V. 1961. Ester determination and their application in wine.M.Sc. Thesis. University of California, Davis.
Panse VG and Sukhatme PV. 1989. Statistical Methods forAgricultural Workers. ICAR Publication, N. Delhi.
Pilone GJ, Rankine BC and Hatcher CJ. 1972. Evaluation of animproved method of measuring volatile acid in wine. BrewingSpirit Rev., 91:62-66.
Ranganna S. 1986. Handbook of Analysis and Quality Control forFruit and Vegetable Products. 2nd Edn. Tata McGraw – HillPub. Co. Ltd., N. Delhi.
Sen SK. 1985. Pineapple, In Fruits of India: Tropical and Sub Tropical,edited by T.K. Bose and S.K. Mitra. Nayaprakash Publishers,Calcutta. pp: 296-319.
Singh DB. 1996. Year round pineapple production. The Hindu, Dec.,26, 1996. pp. 28.
Singleton VL and Rossi JA Jr. 1965. Colorimetetry of total phenolicswith phosphomolybdic-phosphotungustic acid reagents.Am.J. Enol.Vitic., 16:144-158.
Intl. J. of Food. Ferment. Technol. 2(1): 87-91, June, 2012
Effect of Solid state fermentation and yeast species oncomposition of apple pomace: Application of PCA
V.K. Joshi1* and D.K. Sandhu2
Department of Food Science and Technology Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, HimachalPradesh,India2Department of Microbiology,Guru Nanak Dev University,Amritsar,Punjab,India
*Email: [email protected]
Paper no: 42 Received: 19 Jan, 2012 Received in revised form: 14 April, 2012 Accepted: 21 May, 2012
Abstract
Apple pomace (AP) fermented powders were made after solid state fermentation with different yeasts(Saccharomyces cerevisiae,Candida utilis and Torula utilis) and after removal of ethanol was madeinto powders. Application of Principal Component Analysis (PCA) to the compositional data clearlyseparated the unfermented apple powders from the fermented apple fomace powders. The PCA separatedthe fermented apple pomace from unfermented based upon the parameters selected viz. total sugars,TSS, vitamin C, crude proteins, moisture, crude fat and soluble protein. The summary of Eigenanalysisclearly shows that the first two PCs’ accounted for most of the variations found in the composition ofsamples. Out of the two PCs, the PC 1 was defined by TSS, vitamin-C, crude fat and titratable aciditywhile PC 2 contrasted the treatment based upon the total sugar and crude proteins. However, yellowcolour and moisture content were less related as shown by their intensities. The unfermented AP wasdistinct for total sugar and TSS while fermented apple pomace samples were specific in vitamin-C,crude protein, crude fat and soluble protein. Based on the mineral analysis, the PCA failed to differentiatebetween unfermented and fermented APP when treatment and attributes (vector loadings) were plottedtogether. Ferrous defined the PC 1 strongly while as shown by their intensities K, Cu, Na and Ca, wereweekly correlated. The PC-2 was defined by Zn and Mn contents.
©2012 New Delhi Publishers. All rights reserved
Keywords: Solid state fermentation, PCA, Apple pomace, Protein, Minerals, Yeast
Apple pomace (AP) - a byproduct of apple juice processingindustry, being highly biodegradable, poses a problem for itsdisposal. It is a good source of sugar, acid and mineral besidesthe fibre content (Smock and Neubert, 1950; Downing, 1989;Joshi et al. 2009) and its disposal into the environment is aloss of precious resource. Utilization of the pomace usingfermentation technology for concomitant production of animalfeed and ethanol could be one of the promising alternativesavailable especially due to its low protein contents making it apoor animal feed supplement (Hang, 1988). Microbial
fermentations had been used previously for the production ofprotein enriched feed and food products from highcarbohydrate raw materials by direct solid state fermentation(SSF) of cassava meal with a food fungus, Aspergillus niger(Raimbault and Alazard, 1980). Recently, efforts have also beenmade to use AP as a substrate for the production of ethanol,biogas, citric acid and edible fibres (Hang, 1987 Joshi et al.2009). Hang (1988) also reported that using SSF with foodyeast Candida utilis, nutritive value of apple pomace couldbe increased considerably.
Research note
88
Joshi and Sandhu
Solid substrate or state fermentation (SSF) refers to any processin which the substrate is a solid and there is no freely availablewater. It has been used for centuries, in the preparation of avariety of food products such as meso, soysauce, tofu andtempeh (Hessltine, 1972). Solid state fermentation has alsobeen applied for ethanol production. (Hang et al., 1982). Guptaet al. (1990) conducted SSF of apple pomace for ethanolproduction. Ghanum (1992) cultivated mixed cultures ofTrichoderma and yeasts, and a combination of T.reesei andKluyveromyces maxiams offered a maximum yield of 51 percent and efficiently converted leaf pulp into proteins (39.4%).But there is a limited information available on the effect ofsolid state fermentation on the physico-chemical characteristicsof apple pomace fermented with different yeasts and driedinto powder after removal of ethanol and analysis the datausing maltivariete analysis tool. The results of studies carriedout on the aspects of the application of PCA for analysis ofdata obtained from the solid state fermentation of apple pomacehave been discussed in this communication.
Materials and methods
Solid State fermentationUsing the optimized conditions, solid state fermentations ofapple pomace with three yeasts were carried out as reportedearlier (Joshi and Sandhu, 1996). After the completion ofrespective fermentations, samples for ethanol estimation wereremoved and the remaining fermented pomace was extractedwith water and the extract was distilled. The left-over materialwas dried at 60±l°C in a dryer followed by grinding into powder.The powder was packed in polyethylene bags of 200 gaugeand sealed by a pouch sealing machine.Analysis of driedfermented apple pomace for various physico-chemicalcharacteristics viz., TSS, total sugar, pH, crude proteins, solubleproteins, crude fat, vitamin C, mineral, total acid, rehydrationratio etc. were carried out as reported earlier (Joshi and Sandhu,1996). Parameters like total ash, calorific values, crude fibres ofapple pomace powders were got analysed from the CentralFood Technological Research Institute, Mysore (India) whilethe remaining parameters were analysed in the departmentallaboratories of the then Postharvest Technology Department,UHF, Nauni, Solan, (H.P.) India.
Principal Component analysis
The means of Physico – chemical Characteristics were usedfor Principal Components Analysis (PCA) as per theinstructions given for this computer package, PCA. BAS(Ludwig and Reynolds, 1988). Various treatments and thecompositional values constituted the data. The data ofchemical characteristics were analysed for significance ofvariation by CRD. The output was obtained in the form of
Principal Components (first three), correlation coefficients,matrix and eigen -vectors. The analysis was performed withoutrotation as per the soft wase. The interpretation of data fromPCA was made by plotting Principal Components I vs 2 or 1 vs3 and attributes loading as vectors along with treatments(species), simultaneously.
Results and discussion
Physico- chemical characteristics
The results show that there was a considerable reduction inthe total sugar content in the FAPPs (fermented apple pomacepowders), though these still contained residual sugar insufficient amount. Compared to the control, the FAPP containedsignificantly less TSS and no difference due to the yeaststype could be recorded. The fermented and dried apple pomacecontained significantly more acid content than the dried andunfermented apple pomace. The fermented pomace made byfermentation with different yeasts contained similar amount oftotal sugar. Compared to the CP content of AP, the FAPPscontained almost 3 times more crude proteins CP. Among thethree yeasts tried, Candida gave the highest crude protein.Increase in the protein content by fermentation is highlydesirable as apple pomace has been described as low proteinand high carbohydrates food having low palatability anddigestibility in rumen (Sharma et al., 1986; Hang, 1988). But,there was only a slight increase in the soluble proteins of theFAPPs compared to the APP without fermentation. A significantincrease (1.5 to 2.0 fold) in crude fat in the fermented applepomace powder (FAPPs) as compared to the unfermented(APP) also took place. Similar to these observations, productionof fat by oleaginous yeasts including Candida curvata hasbeen described earlier (Ratledge, 1989; Holdsworth andRatledge, 1991).
Nutritionally important vitamin-C showed 1.5 to 2.0 foldincrease in the fermented apple pomace compared to theunfermented one. According to Lee and Mattick (1989) thoughaverage ascorbic acid content of apple is about 5 mg/100 g,the peel contained more ascorbic acid than pulp and it couldhave been the reason for higher vitamin-C content of dried APas it contains a considerable amount of peel. There was asignificant increase in the crude fibres of AP after fermentationapparently due to the increase by drying due to loss of water.The increase is desirable as the crude fibres, constitute animportant part of the nutritionally balanced diet. There weresignificant differences in the total ash content of theunfermented and fermented apple pomace. The increase intotal ash is clearly the contribution made by drying where lossof water resulted in comparative increase in variouscomponents in the dried matter.
Effect of Solid state fermentation and yeast species on composition of apple pomace: Application of PCA
89
Mineral composition of the fermented and unfermented APshows that K content increased significantly in all the fermentedsamples compared to the unfermented. There was nodifference in the K contents of the dried apple pomace fermentedby different yeasts as was the case of Na. Magnesium contentdecreased in the fermented APP while Ca levels werecomparable. Copper concentration registered an increase in tothe fermentated powder. The increase in Zn and Mn was 1.5and 3 to 5 fold respectively. The enhancement of Fe content inthe fermented powder was more than 2 fold. The increase inthe mineral content could be attributed to the effect of drying.
Principal component analysis
The basic chemical data of fermented and unfermented applepomace were analysed by PCA. The projection of the yeastsand the vector loading (chemical parameters) is shown inFigure 1. The PCA separated the fermented apple pomace fromunfermented clearly based upon the parameters selected. Thesummary of Eigenanalysis (Table 1) clearly shows that thefirst two PCs accounted for most of the variations found in thesamples. Out of the two PCs, the PC 1 was defined by TSS,vitamin-C, crude fat and titratable acidity while PC 2 contrastedthe treatment based upon the total sugar and crude proteins.However, yellow colour and moisture were less related asshown by their intensities. The unfermented AP was distinctfor total sugar, TSS while fermented apple pomace samples
were specific in vitamin-C, crude protein, crude fat, solubleprotein. Out of the selected parameters, total sugar, crudeproteins, vitamin-C and TSS were related and separated thefermented apple pomace from non-fermented. Out of theseparameters, total sugars and TSS were highly correlated withthe unfermented apple pomace while crude proteins andvitamin-C were related with fermented apple pomace powders.
Based on mineral analysis, the PCA could not differentiatebetween unfermented and fermented APP (Figure 2) whentreatment and attributes (vector loadings) were plotted. Asper the Kaiser criterion, the PC accounting for the highestvariation was PC-1 with Eigenvector value of more than 1.Ferrous defined the PC 1 strongly while as shown by theirintensity K, Cu, Na and Ca, were weekly correlated. Along thefirst PC, Fe contrasted the fermented poamce by Candida utilisand Torula utilis from unfermented and that fermented bySaccharomyces. The PC-2 was defined by Zn and Mn contents.
To sum up, in the SSF of apple pomace, compared to theunfermented, the apple pomace powders after fermentationwith different yeasts were found to be enriched with severalnutritionally important components. The application of PCAtechnique have proved to be effective in interpreting the datasuch as that obtained from solid state fermentation of applepomace and have clearly charaterized and separated fermentedapple pomace from fermented.
Figure 1: Projection of physico-chemical analysis data of apple pomace powders obtained with or without fermentation by different yeastsin planes defined by principle component 1 and 2 (1) = unfermented apple pomace, Apple pomace fermented by (2)= Sacchamyces,(3)=Candida,(4)= Torula
90
Joshi and Sandhu
Table 1: Principal Components Analysis output of data of physico-chemical parameters of apple pomace fermented by different yeasts
Eigen values Per cent of trace Accumulated & trace
(a) All parameters except minerals (12 attributes)1 = 3.486 87.2 87.22 = 0.502 12.5 99.73 = 0.011 0.3 100.04 = 0.001 0.0 100.0
(b) All minerals (8 attributes)1 = 3.998 100.00 100.02 = 0.001 0.00 100.03 = 0.00 0.00 100.04 = 0.00 0.00 100.0
Figure 2: Projection of mineral analysis data of apple pomace powders obtained with or withot fermentation by different yeasts in planesdefined by principal component 1 and 2 (1) Unfermeted apple pomace, Apple pomace fermented by (2) = Saccharomyces, (3) = Candida,(4) = Torula
Effect of Solid state fermentation and yeast species on composition of apple pomace: Application of PCA
91
References
AOAC 1980. Association of Official Analytical Chemists. OfficialMethods of Analysis. ed. Hortwitz. W. 13th edn, Washington.D.C.
Downing, D.L. 1989. Processed Apple Products. AVI Van NostrandReinhold, Newyork. p. 433.
Fontenot, J.P., Bovard K.P., Oltzen, R.R., Rumsey T.S. and Driode,B.M. 1977. Supplementation of apple pomace with non-protein nitrogen for gestating cows. J. Anim . Sci .,46:513-522.
Ghildyal, N.P., Lonsane, B.L., Sreekantiah, K.R. andSreenivasamurthy, V. 1985. Economics of submerged andsolids state fermentation for the production ofamyloglucosidase. J. Food Sci. Technol. 22:171-176.
Gupta, L.K., Pathak, G. and Tiwari, R.P. 1990. Effect of nutritionvariables on solid state alcoholic fermentation of apple pomaceby yeasts. J. Sci. Food. Agric. 50:55-62.
Hang, Y.D. and Walter, R.H. 1989. Treatment and utilization ofapple processing waste.In; Processed Apple Products, ed.Downing D.L. AVI Van Nostrand Reinhold, New York,365-376.
Hang, Y.D., Lee, C.Y. and Woodams, E.F. 1982. A solid statefermentation system for production of ethanol from applepomace. J. Food Sci. 47:1851-1852.
Hang, Y.D. and Woodams, E.F. 1984. Apple pomace : A potentialsubstrate for citric acid production by Aspergillus niger.Biotechnol Lett. 6:763-764.
Hang, Y.D. 1988. Improvement of the nutritional value of applepomace by fermentation. Nutrition Reports International,38(1): 207-209.
Holdsworth, J.E. and Ratledge, C. 1991. Triglyceride synthesis inthe oleaginous yeast Candida curvata D. Lipids, 26(2):111-118.
Joshi, C. and Joshi, V.K. 1990. Food Processing Waste ManagementTechnology, Need for an integrated approach. Indian FoodPacker, 44: 56-67.
Joshi, V.K. and Sandhu, D.K. 1994. Solid state fermentation of applepomace for production of ethanol and animal feed. In SolidState Fermentation. Ashok Pandey, Eastern Wiley Ltd., NewDelhi, pp. 93-98.
Joshi, V.K.and Sandhu, D.K. 1996. Composition of the distillates,from the solid state fermentation of apple pomace by differentyeasts. Nat. Acad. Sci. letter,49(1),1-13
Joshi, V.K. and Sandhu, D.K. 1996. Preparation and evaluationof animal feed using solid state fermentation of applepomace. Bioresource Technol., 56:251-255.
Joshi, V.K. and Rana S. Neerja. 2009. Microbial Technology for theProduction of Value Added Products from Apple Pomace,Agriculturally Impotant Microorganisms (Vol.II),(Eds.George, G.K., D.K. Arora, T.P.R., A.K.SrivatavaAcademic World (13) pp.271-286.
Kaur, K. 1989. Microbial transformation of apple pomace to recoverindustrial Products. M.Sc. (Hons.) Thesis, Punjab University,Chandigarh.
Keunedy, M.J. 1994. Apple pomace and Kiwifruit : Processingoptions. Australasian Biotechnol. 4:43-49.
Lee, C.Y. and Mattick, L.R. 1989. Composition and Nutritive valueof apple; products. In: Processed Products. (C.D.L.Downing, ed). AVI Novanstrand Reinhold, New York, pp.303-322.
Lammel, S.A., Heimsch, R.C. and Edwards, L.L., 1979. Optimizingthe continous production of Candidta utilis andSaccharomyces fibuliger on potato processing waste water.Appl. Environ. Microbiol, 37:227-232.
Ngadi, M.O. and Corrota, L.R. 1992. Solid state fermentation ofapple pomace as affected by moisture and bioreactor mixingspeed. J. Fd. Sci., 57(3):667-670.
Ranganna, S. 1986. Hand book of Analysis and Quality Control forFruit and Vegetable Products. 2nd Edn. Tata McGraw HillPublishing Co., Ltd. New Delhi.
Ratledge, C. 1989. Microbiol Technology of Lipids : Lipid Technol.1:34-39.
Intl. J. of Food. Ferment. Technol. 2(1): 93-97, June, 2012
Processing potential of newly introduced Amla cultivarsgrown in lower Himalayan Region of Himachal Pradesh
Manisha Kaushal1* and Shashi K. Sharma2
1*Department of Food Science and Technology, Nauni, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan,Himachal Pradesh,India2 Institute of Biotechnology and Environmental Science, Neri, Hamirpur, Dr YS Parmar University of Horticulture and Forestry,Nauni, Solan, Himachal Pradesh,India
*Email: [email protected]
Paper no: 43 Received: 20 March ,2012 Received in revised form: 19 April ,2012 Accepted: 16 June, 2012
Abstract
Varietal performance studies of wild and introduced variety of Amla were conducted at RegionalHorticultural and Forestry Research Station, Bhota, Himachal Pradesh. The selections of amla (NA-6,NA-7 and NA-10) were made from Faizabad (UP) and introduced at Research station Bhota, Hamirpurand at farmer’s field during the year 2002 to evaluate their performance. It was inferred that cultivarNA-7 and NA-10 have outperformed the traditional local strain for almost all the characteristics studied.These cultivars were having higher survival rate, shorter juvenile phase, better tree canopy and yield.The fruit of Local (wild) variety was found smaller than the introduced variety like NA-7 and NA-10. Interms of chemical characteristics, the berries of local cultivars had comparatively higher soluble solids(11.36oB) and ascorbic acid (275.7 mg/100g) than NA-7 and NA-10 varieties. However, on the basis ofantioxidant potential NA-7 had higher phenol content (21.02 g/100g) as compared to the local and NA-10 cultivars. The fruits of local variety were used to prepare pickle while NA-7 and NA-10 wereemployed to prepare preserve.
©2012 New Delhi Publishers. All rights reserved
Keywords: Amla, Introduced, Desi, Performance studies, Pickle, Preserve
The Amla/Aonla or Indian Gooseberry (Emblica officinalisGaertn., family Euphorbiaceae) is a minor sub-tropical treeindigenous to Indian sub-continent. Owing to its hardy nature,it has been successfully grown in dry and marginal regionsextending from the base of the Himalayas to Sri Lanka andfrom Malacca to South China. Added to it, the plants andfruits of amla are regarded as sacred in Hindu mythology.Probably, it is the only fruit recognized by the ‘AyurvedicSystem of Medicines’ for complete sound health. Being one ofthe richest sources of vitamin C and several active tannoidprinciples (Emblicannin A, Emblicannin B, Punigluconin andPedunculagin), it possess expectorant, purgative, spasmolytic,anti-bacterial, hypoglycemic properties (Rastogi 1993, Rao et
al. 1985, Jamwal et al 1959, Jayshri and Jolly 1993).
Lower Himalayan region of Himachal Pradesh hascharacteristic features of skewed rainfall distribution andsloppy terrains which render the region vulnerable to droughtlike situations during most part of the year. Under such anagro-ecological situation, the status of commercial fruit cropslike mango, litchi etc is quite weak. Amla performs well in theregion but its plantations of local strains are sporadic. Duringthe recent past a number of varieties have been introduced inthe region which is also giving outstanding performance withrespect to yield and quality of the fruit. These introducedcultivars are producing an average yield of more than 100 Kg/
Research note
94
Kaushal and Sharma
tree/year. But, because of high acidic and astringent nature ofamla fruits, very little quantity of the harvest is utilized forfresh consumption and most of the crop is wasted. Thus, amlahas not been able to make significant contribution to the socialeconomic upliftment of the region despite of its high yield andnutritional potential. The present studies were therefore,designed for evaluating the high yielding varieties of amla forvarious physico-chemical properties and thereby, working outtheir potential for value addition and income generation forthe rural livelihood.
Materials and methods
The varietal performance studies were conducted at RegionalHorticultural and Forestry Research Station, Bhota, Hamirpur(Himachal Pradesh) on different introduced and locally grownstrains of amla. Data were recorded for 10 year old plantationof NA-7, NA-10 cultivars and Local strains of aonla on theirsurvival, tree size, juvenile period and average yield duringtheir 9th and 10th year of growth. The study on physico- chemicalevaluation and value addition potential of these cultivars wasconducted at the Department of Food Science and Technology,Dr Y S Parmar University of Horticulture and Forestry, Nauni,Solan during 2010-11 and 2011-12.
For physical parameters, the fruit were evaluated for berrycolour, weight, pressure, size and pulp:stone ratio. The chemicalcharacteristics such as moisture, total soluble solids (TSS),titratable acidity, sugars, ascorbic acid, carotenoids, phenols,crude fibre and ash were estimated as per the standardprocedure described by Ranganna (1986) and AOAC (1995).For value addition studies, the products like preserve andpickle were prepared as per the recipe of Lal et al. (1959).Smallsize amla fruit (Local/Desi) which are not suitable forpreparation of preserves and other confectionary items, wereutilized for pickle making. The procedure for amla preservepreparation (Figure 1) involves washing and selecting firmand sound amla fruit followed by dipping in 2% common saltsolution until the green fruit changes to a creamish green colour,with replacement of the brine solution on alternate days. Thefruit were thoroughly washed, pricked with a stainless steelpricker and then, blanched in boiling water for 4 to 5 minutes.Sugar equal to the weight of fruits was sprinkled over the fruitand kept overnight. The next day one boiling was given to thewhole mass and the syrup was then drained out. The syrupwas thoroughly boiled and concentrated by adding more sugarto 54-55°Brix strength and mixed with fruit. The following daythe fruit were taken out and syrup is concentrated to 75°Brixby adding sugar and boiling. Amla fruit were added back andallowed to stand in syrup for couple of days. When the oBrixof the syrup stabilized at around 70°, the preserve was packedin clean, sterilized, dry glass jars and stored at ambient room
temperature away from direct sunlight. Preparation of pickleinvolves the steps outlined in Figure 2.
Selection of fruit
↓Washing and blanching
↓Sun drying (3-4hrs) to remove surface water
↓Add spices, oil & mix well
↓Keep in sun for few days
↓Store amla pickle at room temperature
↓Amla pickle
Figure 2: Flow diagram of amla/aonla pickle preparation
For the preparation of aonla candy, mature fruit were washed,pricked and dipped in 2 percent salt solution for 24 hours. Thefruit were thoroughly washed and blanched in boiling waterfor 5 minutes and steeped in 50° Brix syrup solution for 24hours. The next day steeping was done in 60° Brix for 24 hours.Again steeping was done in 70° Brix for 72 hours. Excess syrupwas drained. The fruit were dried to 15% moisture contentand coated with powdered sugar/pectin. Packaging was donein polythene pouches (400 gauge).
Amla preserve: Various steps involved in the preparation of amlapreserve are depicted in Figure 2.
Selection of fruit
↓Washing and dipping in salt (2%) for 2-3 days
↓Pricking, blanching in boiling water (4-5 minutes)
↓Sugar equal to weight of amla sprinkled on fruit dipping and
boiling in sugar solution by raising the TSS on alternate days tillfinal TSS reaches 70oB
↓Dipping of amla segments in sugar syrup
↓Storage in jars at cool and dry place
Figure 1: Flow diagram of alma preserve preparation
Processing potential of newly introduced Amla cultivars grown in lower Himalayan Region of Himachal Pradesh
95
Results and discussion
Performance studies
The performance data (pooled averages) of different amlacultivars presented in Table 1 revealed that survival per centwas recorded highest for local strains though statistically itwas at par with cultivar NA-10 (71%). The length of juvenileperiod (years) was observed almost similar in all the cultivars.As far as tree growth was concerned the tree height wasrecorded highest for NA-7 which was significantly greater thanthe local strains. The height of NA-10 was statistically at parwith NA-7. Tree spread was almost similar among the differentcultivars. Per tree yield data indicates that highest yield wasobtained for cultivar NA -7 which was significantly higherthan that of local. From the results presented it can easily beinferred that cultivar NA-7 and NA-10 have out-performed the
traditional Local strain for almost all the characteristics studied.These cultivars were having higher survival rate, shorterjuvenile phase, better tree anchorage and yield as well. Thefindings are contrary to those of Rao and Subramanyam (2009)who evaluated ten year old plantations of different aonlacultivars and reported better performance in NA-10 under redsandy loam conditions of Andhra Pradesh. But, the presentfindings are in close proximity with those of Patil et al. (2010)who evaluated eight varieties of Regional Fruit ResearchStation, Katol, Maharastra and reported that variety NA-7 gavethe highest fruit yield, followed by Kanchan and Chakaiya.Regional variability in performance of different cultivars ofaonla has also been described by Pathak (2003).
On the basis of the results the two cultivars (NA-7, NA-10)were taken further for evaluation and product development.
Plate 2a: Amla pickle (Local variety) Plate 2b: Amla preserve (Introduced variety)
Plate 1: Amla cultivars (Local, NA-7 and NA-10)
96
Kaushal and Sharma
Physical characteristics
The comparative study of three cultivars of amla viz., NA-7,NA 10 and Local showed that the fruits were round, ribbedand pale green. The average fruit weight and seed weight variesfrom 6.58 to 33.8g and 0.94 to 2.70g respectively (Plate 1). Thedifferent components of amla fruit accounted for 6.07-14.26per cent peel, 69.77-85.49 per cent pulp and 7.10- 15.12 per centseed. The maximum and minimum fruit weight was attained byNA-10 (33.8g) and Local (6.58g), while the maximum seed weightby NA-7 (2.70g) with a minimum of 0.94 g by Local variety. Outof the three varieties, the skin of NA-7 cultivar was glossy andshiny with colour parameters of Yellow Green 144C as readfrom Horticulture Colour Charts, while the other two cultivarswere having Yellow Green 145A colour. The size parameters ofthree cultivars showed that the Local, NA-7 and NA-10 had alength (mm) of 21.8, 36.44 and 33.47 with a diameter measuring22.19, 38.08 and 38.47 mm respectively. Thus, the average sizeof NA-7 aonla cultivar was found larger than the other two (Table 2). The data were in conformity with those obtained byGoyal et al (2008), Mishra (2009) and Kalra (1988) for differentamla cultivars.
Table 2: Physical analysis of different varieties of Amla (Emblicaofficinalis)
Parameters Local NA-7 NA-10Weight (g) 6.58 32.8 33.8
Size (mm)Length (V) 21.8 36.44 33.47Breadth (H) 22.19 38.08 38.47
Berry Quotient (V/H) 0.96 0.95 0.86Peel wt (%) 14.26 6.07 10.89Pulp wt (%) 69.77 85.49 77.32Seed wt (%) 15.12 7.10 11.77Stone:pulp ratio 1:5.2 1:12.5 1:9.2Colour Yellow Green Yellow Yellow
145A Green 144C Green 145CGreen & Glossy
Pressure (lbs/in2) - 19.5 27.0
Table 3: Chemical analysis of different varieties of Amla
(Emblica officinalis)
Parameters Local NA-7 NA-10
Moisture (%) 84.6 82.2 86.18Total solids (%) 15.4 17.8 13.82TSS (oB) 11.36 9.8 9.15Titratable acidity 2.73 2.97 2.17 (% citric acid)Brix/acid ratio 4.16 3.29 4.21Sugars (%) Reducing 4.76 3.17 3.21 Total 5.54 4.54 5.12Ascorbic acid (mg/100g) 275.7 205.0 200.3Total phenols (g/100g) 20.63 21.02 20.99Fibre (%) 2.70 3.83 3.41Juice recovery (%) 40.0 42.0 44.0
Chemical characteristics
Data pertaining to the chemical composition of amla cultivarsare presented in Table 3 indicate that moisture content in amlaberries (Local, NA-7 and NA-10) ranged between 82.2 to 86.18per cent with a maximum in NA-10 and minimum in NA-7cultivars. In all the three cultivars of amla, the total solublesolids (oB) and titratable acidity (% citric acid) lied between9.15 to 11.36 and 2.17 to 2.97 respectively, where Local andNA-7 cultivar had highest TSS and acidity respectively. Thebrix/acid ratio was maximum in NA-10 (4.21) and minimum inNA-7 (3.29) cultivars. As reported earlier, the amla fruit isregarded as a rich source of ascorbic acid besides other vitalnutrients. The level of vitamin C in amla berries varied from200.3 to 275.7mg/100g with the maximum in Local cultivar.Besides vitamins, the amla fruit also contained good proportionof total phenols, in different cultivars; their respective valueswere recorded as 20.63, 21.09 and 20.99 g/100g for Local, NA-7 and NA-10, respectively. The fibre content in the amlacultivars was found maximum in NA-7 (3.83%) with a minimumin Local (2.70%). Thus, the berries of amla were regarded as afruit containing good proportion of vitamin C and phenols
Table 1: Performance of introduced amla cultivars under low hill region of Himachal Pradesh (Pooled data for tree size and yield for twoyears)
Cultivar Survival Juvenile Tree Size (m) Yield (%) Period (years) Height Spread (kg)
NA-7 5 3 4.75 4.92 92NA-10 71 3 4.40 4.63 83Local 72 5 3.3 3.90 52CD
(0.05)7.2 NS 1.33 NS 18.7
Processing potential of newly introduced Amla cultivars grown in lower Himalayan Region of Himachal Pradesh
97
besides other components which can be used for value addition(Kumar et al. 2006).
Processed Products
Preserve: On the basis of sensory evaluation, it was observedthat amla preserve scored 7.0, 7.5 and 7.5 for colour, flavourand body characteristics respectively (Plate 2a). On the basisof taste, the sample scored 8.0 thus emphasizing its overallacceptability (Table 4).
Amla pickle: To improve the texture of the fruit and also toremove astringency, brining is important in pickling. Sensoryevaluation of the amla pickle showed that the pickle scored 7.0for colour and flavour on the basis of 9 point Hedonic Rating(Table 4). However, the taste perception of the product scored7.85 out of 9 (Plate 2b). Thus, on the basis of overallacceptability, amla pickle prepared by using local cultivarshighly acceptable (7.93) apart from meeting the specificationslaid down in FPO (1955).
Table 4: Sensory evaluation of processed amla products
S.No Attributes* Pickle Preserve
1. Colour 7.0±0.2 7.0±0.102. Flavour 7.0±0.15 7.5±0.183. Body 7.45±0.08 7.5±0.114. Taste 7.85±0.10 8.0±0.125. Overall Acceptability 7.93±0.10 8.0±0.15
*on 9 point Hedonic Scale
Conclusion
It has been concluded from the studies that newly introducedcultivars (NA-7 and NA-10) aonla has great yield potential forquality fruit production (in terms of fruit colour, appearance,weight, length, and diameter the fruits, and other physico-chemical features) under the lower Himalayan region.
References
AOAC. 1995. Official Methods of Analysis 16th edn, Association ofOfficial Analytical Chemists, Washington, DC.
Goyal, R.K., Patil, R.T., Kingsly, A.R.P., Walia, H., and Kumar, P.2008. Status of postharvest technology of aonla in India- areview. American Journal of Food Technology. 3 (1):13-23
Jamwal, K.S., Sharma, I.P.,and Chopra, L. 1959. Pharmacologicalinvestigations on the fruits of Emblica officinalis. J. Sci.Ind.Res. 18: 180-181.
Jayshri, S, and Jolly, C.I. 1993. Phytochemical , antibacterial andpharmacological investigations on Monordica chiranlia andEmblica officinalis. Ind. J. Pharm. Sci. 1: 6-13.
Kalra, C.L. 1988.The chemistry and technology of Amla (Phyllanthusemblica) - a resume. Indian Food Packer. 42(4): 67-82.
Kumar, G. S., Nayaka, H., Dharmesh, S. M. and Salimath, P. V. 2006.Free and bound phenolic antioxidants in amla (Emblicaofficinalis) and turmeric (Curcuma longa). Journal of FoodComposition and Analysis. 19: 446-452.
Lal, G, Siddappa, G.S. and Tandon, G.L. 1959. Preservation of Fruitsand Vegetables, Indian Council of Agriculture Research, NewDelhi, p 483.
Mishra, P., Srivastava, V., Verma, D., Chauhan, O. P. and Rai, G. K.2009. Physico-chemical properties of Chakiya variety ofAmla (Emblica officinalis) and effect of different dehydrationmethods on quality of powder. African Journal of FoodScience. 3(10): 303-306.
Pathak, R. K. 2003. Status Report on Genetic Resources of IndianGooseberry - Aonla (Emblica officinalis Gaertn.) in Southand Southeast Asia. IPGRI Office for South Asia NationalAgriculture Science Centre (NASC). 99p.
Patil, S. R., Suryawanshi, A. B., and Phad, G. N. 2010. Performanceof some aonla (Emblica officinalis Gaertn) cultivars underVidarbha condition of Maharashtra. International Journal ofPlant Sciences, 5 (1): 36-37.
Rao, K. D. and Subramanyam, K. 2009. Growth and yield performanceof aonla varieties under scarce rainfall zone. Agric. Sci. Digest,29 (2): 45-47.
Ranganna, S. 1986. Handbook of Analysis and Quality Control forFruit and Vegetable Products, 2nd edn, Tata McGraw HillPub. Co. Ltd. New Delhi. p 1110.
Rao, T.S., Kumari, K.K., Netaji, B., and Subhokta, P.K. 1985.Ayurveda Siddha. J Res. 6:213-224.
Rastogi, R.P. 1993. Compendium of Indian Medicinal plants, CDRI,Lucknow and ID, New Delhi 1: 530.
Intl. J. of Food. Ferment. Technol. 2(1): 99-101, June, 2012
Evaluation of the stability of plum anthocyaninpowder in RTS based model solution
M. Preema Devi2, V.K. Joshi2 and Y. Indrani Devi1
1Department of Postharvest technology of Hort. Crops, Bidhan Chandra Krishi Viswavidyalaya,West Bengal,India2Department of Food Science and Technology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, HimachalPradesh,India
*Email: [email protected]
Paper no: 44 Received: 15 Jan, 2012 Received in revised form: 19 April, 2012 Accepted: 14 May, 2012
Abstract
Colour of a foodstuff is one of the most important elements determining its acceptance besidesenhancing its delicacy. Storage stability of the dried plum anthocyanin powder with 300B maltodextrinas a carrier in RTS beverage model solution showed more effect of temperature at 350C than at 00C.There were less changes in dark light condition than in day and UV light. The change in colour wasrapid in the first 1 month than in the later period of storage. By the use of plum pomace and with theabove optimized conditions, crude anthocyanin pigments can be produced and used in processedfood.
©2012 New Delhi Publishers. All rights reserved
Keywords: Plum, anthocyanin, RTS, Model solution
Colour of a food is not a label flavour type, but it also providesinformation on its quality and condition. In many food systems,colour acts as an indicator of condition of food such as fitnessto eat. Because of consumer awareness and concern for healthfuland fully balanced food, now-a-days people have developedincreasing interest in food colourants of natural origin.Anthocyanins are water-soluble pigments which exist in thecell sap. The total amount of anthocyanin in plum ranges from44.1 to 231.29 mg as cyanidin 3-glucoside per 100g fresh tissue(Casal et al 2002) The waste of the plum after processing isgenerally thrown out by the processing industries which containsufficient quantity of anthocyanin pigment. It can be used forextraction of biocolour. The anthocyanins in general are verysensitive to different factors including storage conditions andtherefore, how the plum anthocyanins would behave is notknown. Before commercializing these biocolours the evaluationof their stability in model solution is very important keeping
these in view,anthrocyanins were extracted and their stabilityin RTS based model solution was determined and reportedhere.
Materials and methods
Anthocyanins from plum pomace were extracted using 50%ethanol and 0.2% citric acid, which was further concentratedupto 10 fold. Spray drying of the concentrate was done using30 0B maltodextrin as a carrier. Concentration of anthocyaninpowder was optimized separately. RTS beverage (modelsolution) were prepared as per the FPO specifications(Devi2008). Each treatment was analysed for TSS, total sugar,titratable acid, colour measurement, tintometer and pH afterevery one month for 3 months of storage and changesoccurring were recorded. Analysis was carried as per thestandard method reported elsewhere (Ranganna , 1986) Thestorage conditions were 0 0C Day Light, 35 0C Day Light, 35 oC
Research note
100
Devi et al.
Tabl
e1:
Per
cent
chan
gein
vari
ous
phy
sico
-ch
emic
alan
dco
lour
char
acte
rist
ics
ofth
eR
TS
base
dm
odel
solu
tio
naf
ter
thre
em
onth
sof
stor
age
Tre
atm
ents
TS
S%
chan
geT
otal
suga
r%
chan
geA
cidi
ty%
chan
gepH
%ch
ang
e
L%
chan
gea
%ch
ange
b%
chan
geR
edu
nit
%ch
ange
Yel
low
unit
%ch
ange
03
03
03
03
03
03
03
03
03
T1:0
0 CD
ay14
.014
.21.
4012
.70
12.9 0
0.50
0.30
0.28
6.60
3.1
53.
222.
20
78.5
073
.4 0
2.90
7.70
7.30
7.80
19.0
016
.0 0
15.8
03.
601.
70
52.7
03.
203.
95
23.4
0
T2:3
50 CD
ay14
.013
.9
0.70
12.7
0
12.8 0
0.30
0.30
0.27
10.0
0
3.15
3.24
2.8
078
.50
73.7 0
2.50
7.70
6.60
14.3
019
.00
16.5 0
13.2
03.
601.
10
69.4
03.
204.
10
28.0
0
T3:3
50 CU
V14
.013
.90.
7012
.70
12.8 0
0.30
0.30
0.27
10.0
03.
153.
222.
20
78.5
073
.9 0
2.50
7.70
5.70
26.0
019
.00
16.6 0
12.6
03.
601.
00
72.2
03.
204.
10
28.0
0
T4:0
0 CD
ark
14.0
14.2
0.40
12.7
0
12.9 0
0.50
0.30
0.29
3.30
3.15
3.21
1.90
78.5
073
.3 0
3.00
7.7
07.
403.
9019
.00
15.8 0
16.8
03.
601.
80
50.0
03.
203.
92
22.5
0
T5:2
50 CD
ark
14.0
14.1
0.70
12.7
0
12.8 0
0.30
0.30
0.29
3.30
3.15
3.20
1.6
078
.50
73.6 0
2.80
7.70
7.00
9.00
19.0
016
.3 0
14.2
03.
601.
80
50.0
03.
203.
99
24.7
0
T6
:350 C
Dar
k14
.014
.10.
7012
.70
12.8 0
0.30
0.30
0.29
3.30
3.15
3.20
1.6
078
.50
73.6 0
2.80
7.70
6.90
10.4
019
.00
16.4 0
13.7
03.
601.
30
63.9
03.
204.
03
26.0
0
Tim
es (
Mon
ths)
Evaluation of the stability of plum anthocyanin powder in RTS based model solution
101
U.V. Light, 0 0C Dark Light, 25 0C Dark Light, 35 0C Dark Light.The RTS was packed in a 30 ml bottle.The concentration of theplum anthocyanin powder used was 4% which wasstandardized in earlier experiment. Three replications weremade for each treatment. The data were analysed statisticallyby CRD as per the precribed method (Cochran and Cox, 1963)
Results and discussion
Physico-chemical characteristics
There was a slight increase in the TSS, total sugar and pHduring the different storage conditions and storage interval inRTS based model solution. However, the titratable acidityshowed a slight decrease during different storage conditionsand storage intervals. Although, statistically no significantdifferences were observed in this parameters as the variationwere too narrow that almost a constant TSS, total sugar,titratable acidity and pH were observed along the experiment.These findings are in accordance with those of Viguera et.al.(1998). Attri (2004) also conducted stroage study of microbialPigment and reported that there were no significant changesin these parameters in RTS beverage prepared with microbialpegments during storage at 20, 30 and 370C.
There was a significant change in the colour measurement ofthe RTS based model solutions in different light andtemperature conditions as well as during storage for 3 monthsinterval, which is also in line with the findings of Attri (2004).The ‘a’ value which gives the depth of redness was found tobe decreasing as the storage interval increases. These findingsare in agreement with those of Pilando et. al. (1985) whoreported that anthocyanins, the red pigments in raspberry andother fruits, degrade and polymerized easily with passage oftime with time. These findings are further in conformity withTimberlake and Bridle (1976) who studied in model system,found that mixtures of anthocyanins and catechin duringstorage gradually lost colour in the red region of the spectrumwhile increasing in the brown region.According to Skrede(1985) day light storage of syrups lowered half-life of Hunter‘a’ values by 10-30% as compared with dark storage which isin line with the observations recorded in our study.
It is also clear from the values given in table 1 that changes inTSS and total sugar during storage was not even 1%.It isdesirable from food processing point of view. However, it isvery much evident that the changes in acidity, pH, ‘L’, ‘a’, ‘b’values of colour measurement and the red and yellow units oftintometer colour unit were very drastic. The fading of colourwas very much apperent during 3 month of storage. Similarly,effect of storage in light was greater than in lower temperatureand in dark conditions.The contrasting effect of storagerevealed the occurrence of faster changes during the first one
month than the later periods.Thus,the studies need to bedirected so that the anthrocyanin after extraction andconcentration remain stable after their addition to the foodproducts. The pigment have to withstand the drasticconditions of food processing,if they are to be used inprocessing.
Conclusion
The storage studies have clearly shown that high temperatureof 35 oC, storage in Day and UV light adversely affected thebiocolour. The visual light proved to be more destructive thanDark .In brief, storage studies reflected that the products werestable and the pigment did not induce any biochemical change.As anthocyanin is water soluble, the plum anthocyanin canbe used commercially in food products where water is themain solvent through more studies on stability would beneeded.
References
Attri, D.,2004. Production and evaluation of microbial colours usingapple pomace. Dr Y S Parmar University of Horticulture andForestry, Nauni, Solan, HP, India.
Casal, B.A.C., Byrne D.H., Zevallos L.C. and Okie W. R., 2002.Total phenolic and anthocyanin content in red fleshed peachesand plums Acta Horticulturae. 5:592-589.
Cochran,W.G. and Cox,G.M., 1963. Experiment Designs.14th ed.P613. Asia Publishing House,Bombay
Pilando, L. S., Wrolstad, R. E. and Heatherbell, D. A.,1985. Influenceof fruit composition, maturity and mold contamination onthe colour and appearance of strawberry wine. Jour FoodScience. 50: 1121-1125.
Skrede, S., 1985 Color quality of blackcurrant syrups during storageevaluated by hunter L’, a’, b’ values. Journ Food Science. 50:514-525.
Timberlake, C. F. and Bridle, P.,1976 Interactions betweenanthocyanins, phenolic compounds and acetaldehyde andtheir significance in red wines. Amer Jour Enol Viticult. 27:97-99.
Viguera, C. G., Zafrilla P., Artes F., Romero F., Abellan P. and BarberanF. A. T.,1998. Colour and anthocyanin stability of redraspberry jam. 78: 565-573.
Joshi, V.K.,Mutum Preema Devi and Attri,Devender,2011. Bicolour:Chemistry,Production,Safety and Market Potential.In:FoodBiotechnology:Priciples and Practices.Joshi,V.K. and Singh,R.S.(Eds),I.K Publishers,New Delhi. Pp 641-689
Joshi,V.K.,Devender,Attri, B,Anju and Bhushan,Shashi. 2003.Microbial Pigment. Indian Journ Biotechnol 2: 363-369
Ranganna S. 1986.Handbook of Analysis of Quality Control forfruit and Vegetable Products,2nd Edn. Tata Mcgraw Hill Publ.Co, New Delhi
Devi Mutum Preema,2008.Refinement of extraction method andevauation of anthrocyanins from plum.M.Sc Thesis,Dr.Y.S.Parmar University of Horticulture andForestry,Nauni,Solan, H.P 1997
Book Review
Bio-processing of Foods
ASIA TECH PUBLISHERS INC, NEW DELHI
This book is based on the proceedings of the conference on ‘New Horizons in Bo-Processing of Foods organized by Departmentof Food Engineering and Technology held at Sant Longowal Institute of Engineering and Technology from 25th -26th February2011. The selected papers that were presented in th conference have been compiled and edited in the form of book. It providesan in-depth understanding on different topics that are of great relevance to food and fermentation technology. This indispensibleoutcome is the result of combined efforts of different academic credentials in the area of food technology and biotechnology. Tomake the concept more clear, the contents of this treatise have been divided into following sections:
• Section I: Bio-processes for Value added Products
• Section II: Bio-Safety and Quality system in Food Processes
• Section III: Bio-Preservation and Bio -Packaging of food products
• Section IV: Bio-processing and Bio- management of Agro industrial waste
The each sections comprises of many chapters. The chapters clearly demonstrate the recent developments and advances madein the field of food Industry and applications of biotechnology especially its latest innovations in food processing. Theprominent topics covered in the book are
• Potential application of lactic acid bacteria in functional food
• Genetically modified food
• Importance and Nutitive value,Sole present status and future strategies in Fruit wines in India
• The production of organic acids and enzymes using solid-state fermentation
• Bio preservation: A novel approach to food preservation.
• PlumRTS : Screening cultivars
• Biosensors in food processing
• Nutritive value and importance of fruit wines
• Storage quality of Olive oil
• Enzymatic preparation of high Fructose syrup from Inulin.
• Biotechnological tools :potential in food quality and safety
Nowadays bio technology plays an important role in the food industry as integral part of processing. This field for foodapplications has expanded rapidly, particularly in the last two decades, aiming to optimize processing parameters, design novelfunctional foods, alternative applications for several agricultural products and/or improve the safety, health impact and thequality of foods. During the last years, much research has focused on the improvement of enzyme behaviour in the conditionsin which they were to be used, and especially on the increase of their thermal and operational stability. This book attempts topresent and update account of the most recent efforts and technologies to manipulate and improve the versatility and effectivenessof enzyme and biotechnological interventions in food technology. In this regard, the use of food enzymes have been coveredin the three chapters of this volume.
The text is supported by a number of clear, informative diagrams for better understanding. The book is highly useful for post-graduate students and researchers of food technology, biotechnology, applied biology, microbiology and biochemical engineering.It presents the basic and applied aspects from all the possible facets to serve as a text-cum-reference book. The book includesnumerous informative diagrams supporting the text. The book provides information about the latest research and advances onthe topics biotechnology in food processing which would be an asset to all the readers.
Dr ( Mrs) Neerja RanaAssistant Professor
Department of Basic SciencesDr Y.S. Parmar University of Horticulture and Forestry Nauni Solan(HP)
