Paper No. 13 : FOOD ADDITIVES. Module 27 Enzyme application in dairy processing. Introduction

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1 Paper No. 13 : FOOD ADDITIVES Module 27 Enzyme application in dairy processing Introduction The increasing use of enzymes to produce specific products with characteristic attributes can be emphasized by the world-wide sale of industrial enzymes which was about US $ 3.0 billion in Principal among some enzymes that have important and growing applications are lipases and β-d-galactosidases. Enzymes with limited applications include glucose oxidase, superoxide dismutase, sulphydryl oxidase, etc. The usage of microbial enzymes is important for the development of milk and milk products with new physical and functional properties in the food industry. The native milk enzymes can be exploited in several ways during processing including an index of thermal treatment of milk and for consumer health and food safety, e.g., to combat bacterial invasion and growth. Enzymes native to milk There are over 70 enzymes in milk, encompassing a wide range of activities including lipases, proteinases, alkaline phosphatase, lactoperoxidase, lysozyme, cathepsin D, lysosomal enzymes, etc. The most highly characterized enzymes include lactoperoxidase, lysozyme, plasmin, lipoprotein lipase and xanthine oxido-reductase. Many processing technologies including heat treatment destroy many of these enzymes in milk. The enzymes are predominately associated with the milk fat globule membrane (MFGM) and vesicle membranes in milk. Use of enzymes as food additives Several enzymes are used to improve the quality of food products or to enhance yield or even to produce value-added ingredients for use in food industry. Some of the more frequently used enzyme in dairy industry is discussed herein. I. Rennet Technically rennet is the term for the lining of a calf's fourth stomach. The most common enzyme isolated from rennet is chymosin. Chymosin can also be obtained from several other animal, microbial or vegetable sources, but indigenous microbial chymosin (from fungi or bacteria) is ineffective for making Cheddar and other hard cheeses. The use of rennet in cheese manufacture was among the earliest applications of exogenous enzymes in food processing, dating back to approximately 6000 BC. Animal rennet (bovine chymosin) is conventionally used as a milk-clotting agent in dairy industry for the manufacture of cheeses with desired flavour and texture. Owing to an increase in demand for cheese production world wide ( i.e. 4% per annum over the past two decades), coupled with reduced supply of calf rennet, has led to search for rennet substitutes, such as microbial rennets. At present, microbial rennet is used for one-third of the cheeses produced world wide.

2 Rennet action in cheese making Chymosin has low general proteolytic activity, but high milk-clotting activity. The rennet coagulation of milk is a two-stage process. The first (primary) phase involves the enzymatic production of para-casein and glycomacropeptides, while the second (secondary) phase involves the calcium-induced gelation of para-casein at a temperature of o C. Proteolysis is essentially complete before the onset of coagulation. k-casein is cleaved by chymosin and for most of the other proteinases used as rennets, at the bond Phe105-Met106, which is many times more susceptible to hydrolysis by acid proteinases (which include all commercial rennets) than any other bond in milk protein system. Pepsins and most other acid proteinases used as rennets hydrolyze -casein at Phe105-Met106, but the acid proteinase of Cryphonectria parasitica hydrolyzes Ser104-Phe105. Rennet substitutes Many microorganisms are known to produce rennet-like proteinases which can substitute calf rennet. Microorganisms like Rhizomucor pusillus, Rhizomucor miehei, Endothia parasitica, Aspergillus oryzae and Irpex lactis are used extensively for rennet production in cheese manufacture. Only six rennet substitutes have been found to be more or less acceptable: bovine, porcine and chicken pepsins and the acid proteinases from Rhizomucor miehei, Rhizomucor pusillus and C. parasitica. Microbial rennets from various microorganisms (marketed under the trade names viz., Rennilase, Fromase, Marzyme, Hanilase, etc.) are being marketed since 1970s and have proved satisfactory for the production of different kinds of cheese. Recently Novo Nordisk has succeeded in expressing one proteolytic enzyme from the fungus Rhizomucor miehei in organism Aspergillus oryzae. This host organism is able to produce the single protease that cleaves -casein at phe105 met106 peptide bond (as for Chymosin). This mono component enzyme product is being sold under trade name Novoren. Limited supplies of calf rennet have prompted genetic engineering of microbial chymosin by cloning calf prochymosin genes into bacteria. Bioengineered chymosin is reported to be involved in production of up to 70% of cheese products. Properties of rennet substitutes vis-à-vis chymosin Among the microbial milk clotting enzymes, the ones produced by Rhizomucor miehei and Rhizomucor pusillus have gained wide industrial acceptance. However, the proteolytic specificities of these fungal coagulants are different from those of calf chymosin. Fungal rennet preparations have high milk clotting activity but they exhibit a degree of tertiary (or residual) proteolytic activity resulting in the production of bitter peptides during cheese ripening. Therefore, cleavage specificity as opposed to extended proteolysis (ratio of milk clotting activity/ proteolytic activity) defines a good milk coagulant.

3 Thermal stability of the coagulant is another important issue for being considered to be a good milk coagulant, depending on the cheese type. If the coagulant used is thermally labile, extensive unspecific proteolytic activities during cheese maturation could be prevented for the cheese varieties subjected to cooking process. Recombinant rennet One major drawback of using microbial rennet in cheese manufacture is the development of off flavour and bitter taste in the unripened as well as ripened cheeses. Hence, attempts have been made to clone the gene for calf chymosin, and to express it in selected bacteria, yeasts and molds. Due to shortage of calf stomachs and the economic value of cheese rennet, gene for calf chymosin was cloned and expressed in microorganisms. Several workers have cloned the gene for calf prochymosin in Escherichia coli. The enzymatic properties of recombinant E. coli chymosin are indistinguishable from those of native calf chymosin. The gene for calf chymosin has been cloned in selected bacteria, yeasts, and molds. Chymosin from genetically engineered Kluyveromyces marxianus var. lactis (Gist-brocades), Escherichia coli (Pfizer), and Aspergillus nidulans (Hansens) is commercially available and used extensively; however, these products are not yet permitted in all countries. The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and the filamentous fungi Aspergillus niger var awamori and Trichoderma reesei have been successfully used as hosts for the expression of recombinant calf chymosin which are now marketed commercially such as Maxiren (DSM Food Specialities, Netherlands) and Chymax (Chr. Hansen, Denmark). The gene for prochymosin has been cloned in Saccharomyces cerevisiae; the levels of expression are reported to be between 0.5 to 2.0% of total yeast protein. Microbial recombinant chymosin preparations do not contain any pepsin, whereas 5 50% of the milk-clotting activity of calf rennets may be due to pepsin. No major differences have been detected between cheeses made with recombinant chymosin or natural enzymes with regard to cheese yield, texture, smell, taste and ripening. Immobilized rennet Most (70 90%) of the rennet added to cheese milk is lost in the whey. About 6% of the added chymosin is retained in Cheddar cheese and up to 20 30% in high-moisture, low-cook, low-ph cheeses, such as Camembert. Therefore, the possibility of immobilizing rennet has been investigated as a means of extending its working life. Several rennets have been immobilized, but their efficiency as milk coagulants has been questioned. II. Proteinases/Proteases Proteinases have found additional applications in dairy technology, for example in acceleration of cheese ripening, modification of functional properties and preparation of dietetic products.

4 Proteolytic enzymes of Lactic Acid Bacteria in fermented milk products The proteolytic system of lactic acid bacteria (LAB) is essential for their growth in milk, and contributes significantly to flavour development in fermented milk products. The proteolytic system is composed of proteinases which initially cleave the milk protein to peptides; peptidases cleave the peptides to small peptides and amino acids; and transport system is responsible for cellular uptake of small peptides and amino acids. LAB has a complex proteolytic system capable of converting milk casein to the free amino acids and peptides necessary for their growth. These proteinases include extracellular proteinases, endopeptidases, aminopeptidases, tripeptidases and proline-specific peptidases, which are all serine proteases. Apart from lactic streptococcal proteinases, several other proteinases from non lactic streptococcal origin have been reported. There are also serine type of proteinases, i.e. from Lactobacillus acidophilus, L. plantarum, L. delbrueckii sp. bulgaricus, L. lactis, and L. helveticus. Amino-peptidases are important for the development of flavour in fermented milk products, since they are capable of releasing single amino acid residues from oligopeptides, formed by extracellular proteinase activity. Cheese ripening and flavor development Among the principal flavor compounds present in most cheese varieties are: peptides, amino acids, amines, acids, thiols and thioesters (derived from proteins); fatty acids, methyl ketones, lactones, esters, and thioesters (derived from lipids); organic acids (lactic, acetic and propionic); carbon dioxide, esters and alcohols (derived from lactose). At appropriate concentrations and combinations, these compounds are responsible for the characteristic flavor of the various cheese varieties. There is a good correlation between the intensity of Cheddar cheese flavor and the extent and depth of proteolysis. Four to five enzymes contribute to proteolysis in cheese during ripening: rennet or rennet substitute; indigenous milk enzymes, especially plasmin; starter bacteria and their enzymes, released on cell lysis; non-starter bacteria, which either survive pasteurization of the cheese milk or gain access to the pasteurized milk or curd during manufacture; and secondary inocula, e.g., propionic acid bacteria, Brevibacterium linens, yeasts, and molds (Penicillium roqueforti and Geotricum candidum), and their enzymes are of major importance in some varieties. Microbial enzymes in accelerated cheese ripening Cheese ripening is a complex process mediated by biochemical and biophysical changes during which a bland curd is transformed into a mature cheese with characteristic flavour, texture and aroma. The desirable attributes are produced by partial and gradual breakdown of carbohydrates, lipids and proteins during ripening, mediated by several agents, viz. (i) residual coagulants, (ii) starter bacteria and their enzymes, (iii) non-starter bacteria and their enzymes, (iv) indigenous milk enzymes, especially proteinases and (v) secondary inocula with their enzymes. Proteinases are utilized for accelerated cheese ripening for good flavour and textural development. Proteolysis is characteristic of most cheese varieties and is indispensable for good flavour and texture development. Combinations of individual neutral proteinases and microbial

5 peptidases intensify cheese flavour, and when used in combination with microbial rennet reduced the intensity of bitterness caused by the latter. Acid proteases in isolation cause intense bitterness. A balanced approach of accelerating cheese ripening involves using mixtures of proteinases and peptidases, attenuated starter cells or cell-free extracts (CFE). It is possible to develop an intense flavor in internal bacterially ripened cheeses, only if the moisture content is low and they are ripened for a long period, e.g., Parmesan, extra-mature Cheddar, or extra-mature Gouda, which are ripened for 2 3 years. Owing to the high cost of ripening facilities and stocks, the ripening of extra mature cheeses is expensive. Consequently, there is commercial interest in accelerating the ripening of these cheeses, provided quality can be maintained. Techniques for accelerating ripening may also be applicable to reduced-fat cheeses, which tend to ripen slowly. Methods for accelerating cheese ripening fall into six categories: (i) elevated ripening temperature, (ii) exogenous enzymes (mainly proteolytic enzymes), (iii) chemically or physically modified cells, (iv) genetically modified starters, (v) adjunct starters, and (vi) enzyme-modified cheeses. These methods either seek to make the conditions under which indigenous enzymes function more favorable (i.e., elevated temperature) or to increase the level of certain key enzymes which are considered to be particularly important in cheese ripening. A combination of exogenous proteinases and lactococcal cell-free extracts (rich in peptidases) accelerates ripening, but the results are equivocal. Uniform incorporation of the enzyme preparation into cheese curd poses problems. For Cheddar, the enzyme preparation, diluted with salt, may be added to milled curd. Addition of microencapsulated enzyme (to cheese milk) is technically feasible. The enzymes that have been used to accelerate cheese ripening include microbial serine proteinases, neutral proteinases, lactase and even lipases (see under Lipases). Enzyme modified cheese An extreme form of accelerated ripening is practiced in the production of enzyme-modified cheese (EMC). EMCs are produced by adding a cocktail of enzymes (proteinases, peptidases, lipases) and perhaps bacterial cultures to homogenized, pasteurized fresh curd or young cheese. The mixture is incubated for a requisite period and repasteurized to terminate the microbiological and enzymatic reactions. The preparation may be spray-dried or commercialized as a paste. Enzymes for debittering of cheese Flavorpro 937MDP (by M/s. Biocatalysts Ltd., Wales) is an exopeptidase preparation with low levels of endopeptidase activity. In EMC applications, the hydrolysis of cheese proteins by endopeptidases such as animal and bacterial proteases can give rise to unwanted bitter flavours. This is due to accumulation of small hydrophobic peptides. Flavorpro can be used to control bitterness by removing these bitter-tasting peptides. Due to its fungal origin, this enzyme is available as Kosher, Halal and Vegetarian versions.

6 Application of proteolytic enzymes for dietary purpose Proteases are used to produce hydrolyzed whey protein (i.e. formation of shorter polypeptide sequences). Hydrolyzed whey protein is less likely to cause allergic reactions and is used to prepare supplements for infant formulas and medical uses. Proteases are being used to reduce the allergic properties of cow milk products for infants. Enzymatic hydrolysis is a viable method to produce protein hydrolysates for use in soups, gravies, flavorings, and dietetic foods, but bitterness due to hydrophobic peptides is frequently encountered. There is an increasing interest in the production of casein-derived peptides with special nutritional or physiological properties. Apart from the interest in casein hydrolyzates for the nutrition of patients with digestive problems, interest has been focused also on phosphopeptides derived from casein which it is claimed stimulate the absorption of calcium and iron. Peptides with various physiological activities have been isolated from milk protein hydrolysates. The prominent ones are the β-caseinomorphins, a family of peptides containing 4 7 amino acids (representing β-cn f60 63/7) with opioid activity. These peptides are produced in the intestine in vivo. Peptides with opiate properties have been isolated from hydrolysates of s1- and - caseins, lactotransferrin, -lactalbumin and β-lactoglobulin. III. Lipases Lipases are extensively used in the dairy industry for hydrolysis of milk fat. The dairy industry uses lipases to modify the fatty acid chain lengths, to enhance the flavour of various cheeses. Current applications also include the acceleration of cheese ripening and the lipolysis of butterfat and cream. Lipases are utilized for the development of lipolytic flavours in speciality cheeses. A whole range of microbial lipase preparations have been developed for the cheese manufacturing industry from M. miehei, A. niger, A. oryzae and several others. In some cases microbial lipases have successfully replaced pre-gastric lipases. The free fatty acids (FFAs) take part in simple chemical reactions that initiate the synthesis of other flavour ingredients, such as acetoacetate, β- keto acids, methyl ketones, flavour esters and lactones. The cultures and secondary flora, such as P. roqueforti and P. camembertii in Blue-vein and Camembert cheeses respectively, are lipolytic and produce lipases which are responsible for lipolysis. In addition, lipases are usually added to Italian cheese viz. Parmesan, Provolone and Romano, to intensify their flavor. The characteristic piquant flavor of these cheeses is due primarily to short-chain fatty acids resulting from the action of lipase(s) in the rennet paste traditionally used in their manufacture. R. miehei secretes a lipase that gives satisfactory results in Italian cheese manufacture. The enzyme has been characterized and is commercially available as Piccantase. The lipases secreted by selected strains of Penicillium roqueforti, P. candidum, or A. niger are considered to be potentially useful for the manufacture of Romano, provolone and other cheese varieties. Extensive lipolysis also occurs in blue cheese varieties, and in addition to making a direct contribution to flavor, the FFAs serve as substrates for fungal enzyme systems in the biosynthesis of methyl ketones, which are the principal contributors to the typical flavor of blue cheese. P. roqueforti lipase

7 predominates the ripening of blue cheeses. However, blue cheese ripening may be accelerated and quality improved by the addition of exogenous lipases. Lipolysis makes an important contribution to Swiss cheese flavours, due mainly to the lipolytic enzymes of the starter cultures. The characteristic peppery flavour of Blue cheese is due to shortchain fatty acids and methyl ketones. Most of the lipolysis in Blue cheese is catalysed by Penicillium roqueforti lipase, with a lesser contribution from indigenous milk lipase. Exogenous lipase, traditionally pregastric esterase, is added to certain hard Italian cheeses, e.g., Romano and Provolone. It has been claimed that inclusion of selected lipases in the blend of exogenous enzymes accelerates the ripening of other cheeses, e.g. Cheddar and Ras. Animal lipases are obtained from kid, calf and lamb, while microbial lipase is derived by fermentation utilizing fungal species Mucor meihei. Microbial lipases are less specific based on the fats they hydrolyze, while animal enzymes are more specific to short and medium-length fats. Hydrolysis of the shorter chain fats is preferred because it results in desirable taste of many cheeses. Hydrolysis of the long chain fatty acids can result in either soapiness, or no flavour at all. The introduction of conjugated linoleic acid (CLA) in dairy foods has been made possible through the immobilization of lipases. IV. Lactase Lactase is a glycoside hydrolase enzyme that cuts lactose into its constituent sugars viz., galactose and glucose. Lactase can be obtained from various sources like plants, animal organs, bacteria, yeasts (intracellular enzyme), or molds. Some of these sources are used for commercial enzyme preparations. Lactase preparations from A. niger, A. oryzae and Kluyveromyces lactis are considered to be safe. Properties of lactase: Immobilization of enzyme, method of immobilization and type of carrier also influences the ph optima values. In general, fungal lactase have ph optima in the acidic range , and yeast and bacterial lactases in the neutral region of 6 7 and respectively. Hence, fungal lactases are used for acid whey hydrolysis, while yeast and bacterial lactases are suitable for milk (ph 6.6) and sweet whey (ph 6.1) hydrolysis. The enzyme from A. niger is more strongly inhibited by galactose than that from A. oryzae. Bacillus species derived lactases have a ph optima of 6.8 and a temperature optima of 65 C. Lactase from Bacillus species are superior with respect to thermo-stability, ph operation range, product inhibition and can tolerate high-substrate concentration. Thermo-stable enzymes (activity retained 60 C), have two advantages viz. they give shorter residence time for a given conversion rate and the process is less prone to microbial contamination. Without sufficient production of lactase enzyme in the small intestine, humans become lactose intolerant, resulting in discomfort (cramps, gas and diarrhea) in the digestive tract upon ingestion of milk products. Lactase is used commercially to prepare low-lactose or lactose-free products, particularly milk for lactose-intolerant individuals. It is also used in preparation of ice

8 cream to make a creamier and sweeter-tasting product. Another advantage of lactase-treated milk is the enhanced sweetness of the treated milk, thereby reducing the amount of externally added sugar in the manufacture of flavoured milk drinks. Manufacturers of ice cream, yoghurt and frozen desserts use lactase to improve scoop and creaminess, sweetness and digestibility, and to reduce sandiness in products (especially ice cream). Cheese manufactured from lactose hydrolyzed milk ripens more quickly than the cheese manufactured from normal milk. Galacto-oligosaccharides are synthesized commercially from lactose by microbial β- galactosidases. Lactose acts as galactosyl-donor as well as galactosyl-acceptor to convert lactose to indigestible oligosaccharides. High lactose concentrations (> 30%) favor oligosaccharides synthesis by β-galactosidases over hydrolysis. Galacto-oligosaccharides are indigestible (hence low caloric), stimulate colonic fermentation to short-chain fatty acids and increase the abundance of intestinal bifidobacteria (prebiotic activity). The enzymatic hydrolysis of lactose can be achieved either by free enzymes, usually in batch fermentation process, or by immobilized enzymes or even by immobilized whole cells producing intracellular enzyme. Large-scale systems which use free enzyme process have been developed for processing of UHT-milk and processing of whey using K. lactis lactase (Maxilact, Lactozyme). Snamprogretti process of industrial scale milk processing technology in Italy utilizes immobilized lactase system. They make use of fibre-entrapped yeast lactase in a batch process and the milk is previously sterilized by UHT. Processing of whey UF-permeate has been developed by Corning Glass, Connecticut, Lehigh, Valio and Amerace Corporation. The process by Corning Glass is being applied at commercial scale in the baker s yeast production using hydrolyzed whey. V. Phospholipase to modify phospholipid functionality Phospholipids (PL), despite constituting only ~0.5% of the total lipid in bovine milk, have a critical role in stabilizing milk fat globules against coalescence. Additional roles played in dairy products include, coating powder particles, provide higher foam volumes in aerated products and acting as co-emulsifiers. Phospholipases can be used to modify phospholipid functionality in dairy processing by improving fat stability or increasing product yield. Manufacture of part-skim Mozzarella cheese from milk hydrolyzed with fungal phospholipase A1 (PLA1), YieldMAX TM PL before renneting reduced the fat loss in whey and stretching water which led to increased cheese yield (> 1%, after moisture adjustment); improved fat and moisture retention was noted. The mechanism of yield improvement is ascribed to better o/w emulsification of lyso-pl, interaction of lyso-pl with protein and increased water binding of lyso-pl. There was improved retention of lysophospholipids in the cheese curd. lysophospholipids released from the fat globule membranes is supposed to act as surface-active agents in cheese curd, aiding emulsification of water and fat during processing and reducing syneresis. Phospholipase can also be used in the dairy industry to improve the foaming properties of whey protein. The reaction between lyso-pl (through use of phospholipase) and whey proteins (βlactoglobulin) increased the heat stability of whey proteins and show promise for preparing heat stable emulsions.

9 VI. Transglutaminase Transglutaminase (EC: ) catalyzes the acyl-transfer reaction between the γ-carboxamide group of peptide-bound glutamine residues and various primary amines, including the ε-amino group of protein lysine residues. The cross-links introduced by this enzyme changes the protein structure and improve its functional properties, like texture, viscosity and water-holding capacity without adversely affecting the nutritional quality of the lysine residue. The enzyme is found in almost all eukaryotic and prokaryotic organisms. It has been approved by food industry in some countries to improve quality of foods such as meat, fish, soy products, yogurt, ice cream and cheese. Use of transglutaminase enzyme to cause crosslinking of milk proteins led to higher gel firmness and incorporation of whey into the gel, resulting in increase in cottage cheese yield. The highest yield was obtained in the cottage cheese produced from acid gel pre-treated with 3.6 U transglutaminase/g of protein at 40 C for 1 h. In another report use of transglutaminase (@ 60 units/lit. of milk), in the manufacture of Soft cheese resulted in yield of 17.38% as against 16.02% for cheese obtained from untreated milk. VII. Catalase The enzyme catalase has found limited use in one particular area of cheese production. Hydrogen peroxide is a potent oxidizer and toxic to cells. It is used instead of pasteurization, when making cheeses like Swiss, in order to preserve natural milk enzymes that are beneficial to the end product, including flavour development. These enzymes are destroyed by high heat of pasteurization. However, residues of hydrogen peroxide in the milk inhibit bacterial cultures, so all traces of hydrogen peroxide must be removed. Catalase enzymes are typically obtained from bovine livers or microbial sources, and are added to milk to convert the hydrogen peroxide to water and molecular oxygen. Miscellaneous activity of enzymes The functional properties of milk proteins can be improved by limited proteolysis through the enzymatic modification of milk proteins. Acid-soluble casein, free of off flavour and suitable for incorporation into beverages and other acid foods has been prepared by limited proteolysis. The antigenicity of casein is destroyed by proteolysis, and the hydrolysate is suitable for use in milk protein based foods for infants, who are allergic to cow milk. The NOVO process for production of enzyme modified cheese (EMC) uses medium-aged cheese which is emulsified, homogenized and pasteurized, after which palatase (a lipase from R. miehei) is added, with or without a proteinase and the blend is ripened at high temperature for 1-4 days. The mixture is reheated which results in a paste which is suitable for inclusion in soups, dips, dressings or snack foods. EMC technology has been developed to produce a range of characteristic cheese flavours and flavour intensities e.g. Swiss, Blue, Cheddar, Provolo-nemor or Romano, suitable for inclusion at low levels in many food products.

10 The other minor enzymes having limited applications in dairy processing include glucose oxidase, superoxide dismutase, sulphydryl oxidase, lactoperoxidase and lysozymes. Glucose oxidase and catalase are often used together in selected foods for preservation. Superoxide dismutase is an antioxidant for foods and generates H 2 O 2, but is more effective when catalase is present. Thermally induced generation of volatile sulphydryl groups is thought to be responsible for the cooked off-flavour in UHT processed milk. Use of sulphydryl oxidase under aseptic conditions can eliminate this defect. The natural inhibitory mechanism in raw milk is due to the presence of low levels of lactoperoxidase (LP), which can be activated by the external addition of traces of H 2 O 2 and thiocyanate which enhances the keeping quality of milk. Cow milk can be added with lysozyme making it suitable for infant milk. Lysozyme acts as a preservative by reducing bacterial counts in milk, without affecting L. bifidus activity. Conclusions The global market for the production of microbial enzymes for use in dairy products manufacture is considerably large, but is being dominated only by a limited number of enzyme producers. In India, the microbial dairy enzymes requirement has been very limited till now. Presently, many microbial rennets and other enzymes are being imported. Hence, there is a scope for the production of enzymes such as microbial rennet, lactase, proteinases and lipases indigenously. In the near future, the requirement for these enzymes is bound to increase by leaps and bounds, basically due to requirement of value-added dairy products in the country.