Dairy At Glance
Paratuberculosis

Usage Of Improved Enzymes In The Manufacture

Of Value Added Functional Dairy Products

Arvind Kumar, M.S. Bhadwal and Sakshi Sharma

Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Jammu

 

Introduction

Usage of improved enzyme in dairy food products is an integral part of dairy industry. The dairy foods industry traditionally uses enzymes in the manufacture of various dairy products. The best known dairy enzyme preparation is, of course, rennet, which is a collective name for commercial preparations containing acid proteases extracted from calf abomasum tissues [1]. These products clot milk by removing a highly charged peptide fragment from kappa-casein on the surface of micellar casein, the major form of milk protein. Destabilized casein micelles aggregate and form the structure of milk clot, which is then acidified by lactobacillus cultures to make cheese curd [2]. Although this use of enzymes is the single most important in the dairy sector, modern production methods have made possible other applications to meet changing needs of societies and individual priorities. The shortage of calves from which to source traditional rennet has led to the development of enzyme production technology, with yeasts, moulds and fungi as the primary source. Over half of all milk coagulating enzymes used is microbial in origin, mostly from genetically-modified food yeast and mould containing the calf gene for the production of chymosin, the main acid protease involved in milk clotting. The other main type of microbial rennet (coagulant) is made from Rhizomucor miehei, a non-GM mould.

In addition to the use of milk clotting enzymes to make cheese, the dairy industry also makes use of enzymes such as lipases, non-coagulant proteases, amino-peptidases, lactase, lysozyme and lacto-peroxidase. Some of these applications are traditional (lipase for flavour enhancement) while others are relatively new (lactose hydrolysis, accelerated cheese ripening, control of microbiological spoilage, modification of protein functionality)

Enzymes used in dairy technology

Acid proteinases: Milk coagulation    

 Neutral proteinases: Accelerated cheese ripening: de-bittering; enzyme modified cheese:  and peptidases production of hypoallergenic milk-based foods           

Lipases: Quick cheese ripening: enzyme-modified cheese production:  flavour- modified cheese production: structurally-modified milk fat products

beta-Galactosidase: Lactose-reduced whey products

Lactoperoxidase :  Cold sterilization of milk: milk replacers for calves

Lysozyme :  Nitrate replacer for washed-curd cheeses and cheeses  with eyes

The nature and identity of rennets and coagulants

The first commercial standardized rennet preparation was made and sold by Chr. Hansen A/S, Denmark in 1874, and was the first commercial enzyme of any type. It was then and still is by definition an extract of ruminant abomasum, ideally containing mainly chymosin, the enzyme which is specific for kappa-casein hydrolysis and casein destabilization. However, dependent on the age of the calves from which it is extracted, rennet can contain more or less pepsin, another acid protease with a wider casein substrate range. Both chymosin and pepsin, and indeed all of the milk coagulating enzymes used in cheese technology, are classified as aspartic proteinases. There are several types as well as sources of milk clotting enzymes in the market, the name 'rennet' be reserved for enzyme preparations from ruminant stomach, and other milk clotting enzymes [1].

Main characteristics of rennets and coagulants from different sources

The calf rennet is widely regarded as the ideal milk-clotting enzyme for cheese making. This preference arises partly through traditional familiarity with the product through long use, but also has a sound scientific basis in that calf rennet is typically 80-90% chymosin. This means that most of the casein breakdown in the cheese vat is directed very specifically at kappa-casein to clot the milk, and not at the other caseins. Non-specific proteolysis of alpha- and beta-casein during curd formation can result in the loss of casein nitrogen in the whey, reducing the yield of the cheese process. Indeed, the cheese makers now make excellent quality long-hold cheeses (especially Cheddar) using pure chymosin from genetically modified yeast and fungi expressing cloned copies of the calf (pro) chymosin gene. Sheep, goats and pigs can provide rennet preparations that are enzymatically similar to calf rennet, but not ideally suited to clotting cows' milk [3]. Rennet 'paste' is a crude form of rennet made from the macerated stomachs of suckling calf, lamb or kid, and containing pre-gastric lipase to add piquancy to the flavour of the cheese. It is mainly used in traditional Italian cheeses. Many plants produce proteinases that clot milk. However, plant coagulants are not produced on a commercial scale, but made locally (mainly in Portugal) for artisanal cheese making [1].

The best-known and most widely used microbial coagulant is that produced from Rhizomucor miehei. The commercial preparation is a mixture of aspartyl proteinases and is commercially available in three types. The native, unmodified enzyme ('type L') is very heat stable and hydrolyses all of the caseins, not just kappa-casein. Although this has been used successfully to make soft, short-hold cheese varieties, its non-specific proteolytic action reduces yields of cheeses whose curd spends a long time in the whey (hard and semi hard cheese), and caused bitterness in long-hold cheeses. The heat resistance of the enzyme is also a potential drawback in cheese plant from which the whey is processed as a food ingredient. The heat treatment and processing does not eliminate the activity of the coagulant and it can cause protein breakdown in other food products in which whey protein is a supplementary ingredient. To substantiate these problems, dairy enzyme producers have developed heat labile versions of R. miehei coagulant ('TL' and 'XL') using chemical oxidation to modify methionine side chains. These enzymes can be denatured by pasteurizing, and they are also less generally proteolytic than the native proteinase. These coagulants can be an alternative to fermentation-produced chymosin in the manufacture of 'vegetarian' cheeses, but the texture of hard cheese made with them became crumbly more quick than that of cheese made with chymosin. The flavour profile of hard cheese made with fungal rennet is not the same as that cheese made of chymosin. The most widely used alternative to calf rennet in the cheese industry worldwide is fermentation-produced chymosin. It is produced by large-scale fermentation of genetically modified Kluyveromyces lactis or Aspergillus niger. In both cases the microorganism has been modified using gene technology by incorporation of pro-chymosin gene from the calf into the host organism with a suitable promoter to ensure its efficient secretion into the growth medium. The enzyme is relatively easy to harvest and purify from the culture, unlike the earlier production system using E. coli to produce chymosin in inclusion bodies [1, 4].

Production of rennets and coagulants

Animal rennets are secreted from the stomach mucosa as inactive pro-enzymes that can easily be extracted by maceration with water, weak brine or a buffer solution. A preservative (usually sodium benzoate) is normally added at this stage to prevent microbial growth during the next stages of production, involving filtration and acidification to activate the proenzymes. After neutralization to pH 5.5 and a second filtration to clarify the extract, the preparation is standardized to the 'advertised' milk clotting activity, sterile-filtered and packaged as a liquid enzyme product to be transported and stored refrigerated. Animal rennets are not purified, but contain whatever enzymes that is secreted by the mucosal tissue. However, the enzymes in good quality calf abomasal tissue are mainly chymosin and pepsin and only standardization is necessary. Microbial coagulants are produced by fermentation of production organisms viz. Rhizomucor or Cryphonectria. The fermentation is usually for several days, after which the enzyme is recovered as a crude filtrate, concentrated by ultra-filtration and finally standardized. No attempt is being made by the manufacturer to purify the product by removing other co-produced enzymes such as lipases and starch hydrolases, though the production strains of the mould are selected to minimize these contaminants. Nevertheless, it is important to note that Rhizomucor coagulants contain significant amounts of starch-degrading enzymes which pass through to the cheese whey and are not removed by whey processing. Whey protein concentrates from cheese plants using microbial coagulants should not therefore be used in the formulation of sauces and other food products containing starch as a thickener. Fermentation-produced chymosin is mainly sourced from either genetically modified yeast (Kluyveromyces lactis) or the genetically modified filamentous fungus (Aspergillus niger). Both organisms have a long history of safe usage in fermentations, enzyme production and the genetic modifications in various foods [6]. Special emphasis is put on an acidification production process to ensure that the source organism is killed and that the DNA is broken down before the enzyme is isolated from the fermentates. The acidification produced from Aspergillus is purified by chromatography to minimize the activity of non-coagulant enzymes and to provide added product purity assurance for cheese makers and consumers [1].

Formulation and standardization of rennets and coagulants

The most common type of rennet/coagulant product is the liquid form, inexpensive to produce, easy to measure out for addition to the cheese milk, and easy to mix. All products are formulated in a similar way irrespective of their source, and the same types of stabilizers (sodium chloride, buffer, sorbitol, glycerol) are used. The only permitted preservative is sodium benzoate, but some manufacturers also sterilize their products by filtration to prevent microbial growth in the stored liquid products. Some rennets and coagulants are sold as powders or tablets, especially for shipment to hot countries. Whether dry or liquid, the products are all standardized so that a particular volume or weight always has the same milk-clotting activity. Rennet/coagulant strength is determined by an international standard method, IDF standard 157A [7], developed jointly by IDF, International Standards Organisation (ISO) and the Association of Official Analytical Chemists (AOAC). This standard uses International Milk Clotting Units (IMCU) and measures clotting at the pH of most milk (6.5). Standard 157A uses a calf rennet preparation as the reference standard and is used to standardize rennets and FPC. A similar standard method is also available for microbial coagulants (IDF Standard 176; [8]). The standard method for determining rennet composition (% chymosin and pepsin) is IDF 110B [9, 5].

Lactoperoxidase

Lactoperoxidase occurs naturally in raw milk, colostrums and saliva; it is thought to be part of the protective system for suckling animals against enteric infections. It is bactericidal to Gram-negative bacteria, and bacteriostatic to Gram positives. It is a peroxidase that used hydrogen peroxide to oxidise thiocyanate ion to hypothiocyanate [10].  LP 'system' (LP + thiocyanate + hydrogen peroxide) irreversibly inhibits the membrane energizing D-lactate dehydrogenase in Gram negative bacteria, leading to cell death. In Gram-positive bacteria, the membrane ATP-ase is reversibly inhibited and may be the basis of bacteriostasis, rather than death. Although all raw milk contains LP and thiocyanate, there is not sufficient natural hydrogen peroxide to activate the enzyme system (LPS), and several methods have been devised to increase hydrogen peroxide levels in commercial raw milk supplies to provide a 'cold sterilization' system for countries with insufficient energy resources for heat treatment to preserve raw milk before consumption. Its efficacy in eliminating psychrotrophic Gram-negative spoilage bacteria from raw milk stored at 4°C [11]. Although hydrogen peroxide alone can be used as a preservative in these countries, it must be used at a dosage of 300-500 mg/1 to be effective, and at this concentration it destroys some vitamins and impairs the functionality of the milk proteins. With the LPS, hydrogen peroxide can be generated in situ using glucose oxidase, and free hydrogen peroxide levels are too low to damage the milk. Even if chemical peroxide dosage is used instead of glucose oxidase, it need to be added at only 10 ppm to activate the LPS.

 

Cheese Ripening Enzymes

 

Types Of Enzyme Available Commercially

The enzymes and enzyme 'packages' used to modify, enhance or accelerate the maturation of cheese are generally composed of more than one class of enzyme, and for the sake of clarity they are discussed here as a technological group, rather than as individual classes. The enzymes used in commercial ripening technology include many hydrolases represented by proteinases, peptidases and lipases, and extend to metabolic enzymes such as acetyl-CoA synthases and amino acid-catabolising enzymes to generate volatile esters and sulfur compounds. Considering the very extensive worldwide research effort and literature on the enzymology of cheese ripening [12], few enzyme companies have successfully developed commercial enzyme packages for cheese technology, other than the ageing Enzyme-Modified Cheese production methods used to make flavour ingredients for processed cheese and cheese-like foods [13,14].

This product was developed and applied research into the role of starter enzymes and cell lysis in flavour development [15, 16]. The efficacy of the commercial system made up of a food grade microbial endopeptidase (proteinase) active against all of the casein components in cheese, together with general and specific LAB amino-peptidases, and undefined esterases and flavour enzymes present in LAB cell homogenates [17]. Extensive trial data from commercial cheese manufacture suggest that, when the product is added to cheese curd, the cheese reaches the equivalent of nine months' maturity in only five months. In addition, this enzyme treatment is claimed to reduce bitterness due to certain cultures, and to enhance flavour notes such as 'sulfur', acid and Cheddar. The precise mechanism of flavour enhancement is not defined but, like its research prototype, this product increases the amino acid pool in cheese, providing taste enhancement directly, and increasing the supply of flavour and aroma precursors, in the presence of added LAB biomass. The emerging research on amino acid catabolising enzymes in LAB also suggest that Accelase® may not only increase the cheese amino acid pool but also increase its enzymatic turnover to flavour and aroma compounds. 'Rulactine' (Rhone-Poulenc) and 'Flavorage'  enzymes have been marketed as cheese ripening enzymes. 'Rulacine' is a proteinase from Micrococcus sp. and 'Flavorage' contains a lipase from Aspergillus sp. together with proteolytic enzymes. The research literature suggests that one day cheese makers will knowingly benefit from enzymes that convert amino acids into sulfur volatiles, esters, aldehydes, amines, ammonia and fatty acids. However, many obstacles stand in the way of commercialization, not least the instability and low production levels of these enzymes in their natural host microorganisms. Also, some of these conversions require enzyme/cofactor complexes that are only sustainable in whole cell environments, and variants of the wild-type cheese bacteria with selectively-enhanced activities may be the only route to the technology. The increased plasmin activity accelerates proteolysis during maturation, and would presumably accelerate texture development in hard and semi-hard cheese as a consequence. If this enzyme technology could be economically viable, it could link up well with different forms of peptidase enrichment ('Accelase' and GM peptidase mutants). The activation of plasmin as an indigenous milk proteinase component of a ripening system, after whey separation and curd formation, would also overcome one of the practical difficulties of incorporating enzymes intimately into the cheese matrix. [18].

Enzyme Addition Technology

The stage in manufacture of hard and semi-hard cheese that could be the addition points for ripening enzymes. Proteinases added to break down casein in cheese are needed only in very small amounts because, like all enzymes, they are catalysts, and a small quantity will convert a large amount of substrate. This is fine from the point of view of cost and conversion efficiency, but it means mixing grams of the active enzyme with tonnes of cheese. Putting enzymes evenly into the complex cheese matrix is difficult enough in itself, but the problem of distributing such small amounts is far from trivial. These peptides are lost into the whey when the cheese curd is separated, causing unacceptable losses to cheese yield. Also, the early breakdown of caseins disrupts their orderly structure, prevents proper gel formation, and renders the curds to soft and unworkable in the later stages of curd acidification, prior to salting and pressing into cheese. Add to these problems the loss of added enzymes into the whey (at a rate of about 95%) and it is clear that addition of proteinases directly to the milk is not an option. If peptidase preparations were                                    

very inexpensive, they could be added by this route, but most large cheese plants sell their whey as concentrates to be added to foods for their functionality. Any carry-over of ripening enzymes would have to be removed or destroyed before the whey was processed and sold. Enzyme microencapsulation is the obvious solution to the above problem, to protect caseins in the milk and ensure their physical entrapment in the curd gel matrix. Options include fat, starch or gelatine capsules, but none of these has a satisfactory 'release' mechanism in cheese. The author's research group has developed a special type of phospholipid liposomes [19,20] as an effective technology for overcoming this problem. Proteinases and peptidases were entrapped in the liposomes and added to cheese milk. Most of the ripening enzymes were entrapped in the water spaces between the curd particles and very little was lost in the whey. The liposomes were degraded naturally in the cheese matrix and allowed full contact between the protein matrix and the ripening enzymes. However, the technique has since been adopted in numerous experimental cheese trials [21], the high cost of the pure phospholipids necessary to make stable, high-capacity liposomes rules this out as a large-scale commercial technology [22],

In dry-salted cheese varieties, such as Cheddar, the addition of enzymes to milled curd with optimum salt level was originally proposed for laboratory-scale cheese making [23], and this was successfully adapted to a 1801 vat scale [24]. However, this technique is difficult to adapt and scale up to automated salting equipment in large throughput cheese plant, and although enzymes can be granulated with dry salt, this is an expensive process for cheap ingredients and is not widely used. An alternative method of enzyme addition was patented recently, involving mixing curds and enzyme in a vessel to which 'negative pressure' is applied, so that the enzyme is 'sucked in' to the curd matrix [25]. However, it is not clear how the problems of even distribution and alteration of curd moisture and structure are solved by this invention. Whatever physical method is employed to place enzymes into cheese curd, some kind of vehicle is needed to disperse them and this is either water, or some other natural constituent of cheese such as salt or fat.

Thus, this whole area of enzyme addition technology is in urgent need of radical new ideas from the research base, but researchers also need feedback from cheese technologists and business economists. For example, there is sufficient expertise in molecular and applied enzymology to devise matrices and support materials to create micro-particulate enzyme complexes which could both liberate and metabolise amino acids, fatty acids and sugars to known flavour and aroma compounds. Such expertise has been generated in the fields of low-water enzymology, immobilized enzyme science, cellular enzymology and membranology, but as yet there has been no incentive to apply this to cheese ripening research. This is understandable in current circumstances, because the logical route to the use of complex enzyme systems that are easy to put into cheese is via whole cell technology. Nature designed microbial cells for efficient life processes, not for efficient cheese ripening technology, and the natural microbial cell chemistry and architecture needs modification to put the (technologically) right combinations together. Gene technology achieves this within the whole cell technology concept but however safe this technology is made, the tide of media and consumer opinion is firmly against developments along this route. [12]

Enzyme-modified cheese (EMC) technology

Enzyme-modified cheese is not really cheese from the consumer food point of view. It is a highly-flavoured ingredient for processed cheese, cheese flavoured snack foods and sauces, made by incubating emulsified cheese homogenates with animal or GRAS (generally recognised as safe) microbial lipases and proteinases. All of the major dairy ingredient suppliers now have extensive and relatively sophisticated EMC based flavour product lines; their production methods are based on patents and a large body of proprietary knowledge [14] but the general flow of the process is universal.The raw material for EMC manufacture is young bland hard or semi-hard cheese, cheese off-cuts and/or fresh salted Cheddar cheese curd. This is blended to homogeneity with emulsifying salts as a semi-liquid slurry (40–45% solids), pasteurised for 10 min at 72°C, then cooled ready for enzyme treatment. For example, a temperature of 25-27°C is suitable for making a blue cheese flavour product using a Penicillium roqueforti mould culture to grow and metabolise milk fatty acids to the characteristic methyl ketones. However, the slurry would need a pre-treatment at 40–45°C with a lipase to generate sufficient short and medium chain free fatty acids for rapid development of blue cheese flavour. The most common emulsifiers and stabilizers used in EMC production include monoglycerides and diglycerides, phosphates, citric acid and Xanthan gum. Antioxidants are usually added in the form of plant oils and fat-soluble vitamins (e.g. tocopherols). The basic flavours generated enzymatically can be 'topped up' using food-grade natural aroma compounds or refined by fermentation with dairy cultures of lactic acid bacteria and moulds. Cheddar, Parmesan, Romano, Swiss-type and Gouda flavoured EMCs require a composite enzyme treatment, using lipases, proteinases and peptidases to develop the characteristic savoury, pungent and lipolysed notes in balance. The choice of incubation temperature (or indeed temperatures) is critical for the balance of these flavour notes, but is generally in the range of 40–55°C. This range is also governed by a compromise between the need for a short incubation time at high temperature for process efficiency and reduction of microbiological spoilage on the one hand, and the need to avoid temperature denaturation of the

enzymes. EMC producers would like to have more robust microbial enzymes that could produce the required flavour biochemicals from the raw material in a few hours at temperatures up to 70° C. However, this might create new shelf life and users' problems from residual enzyme activity in the product. Currently, EMC products are pasteurized at 72°C for 30 min after enzyme incubation to destroy residual activity and eliminate spoilage microorganisms. EMCs are spray-dried or packaged as pastes of different water content, dependent on customer preference and intended food use.

Lysozyme

Lysozyme is a hydrolase widely distributed in Nature; it is bactericidal to many Gram-positive species because it breaks down their cell walls. The enzyme is a mucopeptide W-acetyl murarnoyihydrolase, available commercially from hen egg white or Micrococcus lysodiekticus. The food-grade preparations are from egg albumin. Lysozyme is sold by the major dairy enzyme suppliers as an alternative control agent for 'late blowing', the textural defect of slits and irregular holes caused by the butyric fermenation in Gouda, Danbo, Grana Padano, Emmental and other important hard and semi-hard cheese varieties. Traditionally the defect, caused by Clostridium tyrobutyricum in raw milk, has been controlled by the addition of potassium nitrate to the cheese   milk. However, this practice will be phased out because it is associated with the production of carcinogens, and lysozyme has become the preferred control agent [26,6]. Clostridium tyrobutyricum is a spore former and as such, cannot be killed by pasteurisation, hence the need to

treat the milk by alternative methods. Lysozyme kills vegetative cells and also inhibits outgrowth of spores in cheese; it is stable for long periods in the cheese matrix and because it binds to the cheese curd, little of the enzyme is lost on whey separation. Although lysozyme also inhibits the lactic acid bacteria used as starters in cheese making, they are less sensitive than the Clostridia, and a typical enzyme dose rate of 500 units/ml is sufficiently selective (commercial lysozyme preparations contain about 20,000 units/mg). Nevertheless, some thermophilic lactobacilli used in Grana cheese making are very sensitive, but can be 'conditioned' by unknown mechanisms by repeated growth on lysozyme containing media [26].

Lysozyme also inhibits the growth of Listeria monocytogenes in yoghurt and fresh cheese with high acidity (< pH 5.0), but the effect is not consistent enough to rely on in commercial fermented milk products, and in any case high acidity is usually sufficient in itself to inhibit these pathogens.

Lactoferrin

Lactoferrin, an iron-binding glycoprotein, was first isolated from cow's milk and subsequently from human milk. It is present in large quantities in mammalian secretions such as milk, tears, saliva, seminal fluid, and in some white blood cells. It is one of the minor proteins naturally occurring in cow milk at an average concentration of about 0.2 grams/liter. In colostrum, the lactoferrin content can be as high as 0.5 to 1 grams/liter. During the dry period, its concentration in mammary secretions from dry cows increases until about 30 days after drying off. The highest lactoferrin concentration found in cow mammary secretions is about 50 to 100 grams/liter. In human milk and colostrum, the reported concentrations of lactoferrin are 2 to 4 grams/liter and 6 to 8 grams/liter, respectively. It has many proposed biological functions, including antibacterial, anti-inflammatory activities, defense against gastro-intestinal infections, participation in local secretory immune systems in synergism with some immunoglobulins and other protective proteins, provision of an iron-binding antioxidant protein in tissues, and possibly promotion of growth of animal cells such as lymphocytes and intestinal cells. A role for milk lactoferrin in iron absorption by the intestine has long been postulated, but remains unproven. Most micro-organisms need iron for growth and lactoferrin has the potential to inhibit the growth of bacteria, and even kill them by depriving them of iron. The effectiveness of its antibacterial activity depends on the iron requirement of the organism, the availability of exogenous iron, and the concentration and degree of iron-saturation of lactoferrin. It has been shown that 'natural' lactoferrin is bacteriostatic against a wide range of micro-organisms, including gram-negative bacteria with high iron requirements (coliforms, which are major mastitis pathogens), and also against some gram-positive organisms such as Staphylcoccus aureus (also a major mastitis pathogen), bacillus species, and Listeria monocytogenes. Lactic acid bacteria in the stomach and intestine have low iron requirements and are generally not affected.

N-Acetyl-ß-D-Glucosaminidase

N-Acetyl-ß-D-glucosamindase (NAGase) is an enzyme whose activity has been implicated as an indicator of tissue damage during mastitis. It is a lysosomal enzyme that is secreted in large quantities in the mammary gland during involution and inflammation. The NAGase enzyme has also been found in other bovine secretions, such as uterine fluids. The specific function of its in in the mammary gland is not known, however, recent research has suggested that it may exhibit some antimicrobial activity. During lactation, cow milk normally has low its activity. Similarly, it is also low in mammary secretions in the early dry period, coinciding with the period of highest incidence of new intra-mammary infection. By the mid-dry period, however, its activity is at its highest in mammary secretions, concurrent with the lowest incidence of new intra-mammary infection. Therefore, the high levels of its activity, along with elevated lactoferrin concentrations, in the mammary gland during the mid-dry period may contribute to increased antibacterial activity found in mammary secretions. There is a relationship between the presence of pathogens in the udder and its levels in milk. Marked increases in its activity resulting from the presence of major mastitis pathogens have been observed. Since it has been found in uterine fluids, it has been suggested that it may have a role in the bactericidal function of the uterus as well. Researchers have studied the bactericidal effect of its on several bacterial pathogens commonly found to infect the cow uterus. Of these pathogens, Actionmyces pyogenes, Staphylococcus aureus, Strepto-coccus agalactiae, and Pseudomonas aeroginosa were inhibited by NAGase, while the Escherichia coli and Enterobacter aerogenes were not inhibited. Although these results cannot be directly extrapolated to bacterial strains that cause mastitis, they do lend support for such an antimicrobial function of it’s in the mammary gland.

Transglutaminase

With the recent availability of commercial microbially derived transglutaminase preparations, there has been considerable interest in their application to the gelation of caseins and whey proteins. However, transglutaminase is effective in reducing syneresis in acid milk gels and has been investigated as a method of improving the texture and shelf life of yoghurt. It is an enzyme catalyzing the formation of covalent bonds between the amino groups of similar or different proteins. It helps for the polymerization and their cross-linking. The higher the content of proteins is, produced in greater the effect of TG. The bonds formed by TG exhibit visco-resistance to proteolytic reduction, which improves the properties of the proteins in food. In addition to that, the cross-linking process of the molecules of the proteins leads to increase of the water binding in the end product, and thus to a higher yield. The better consistency of the product is accomplished in the short run, and the ready product is made significantly faster due to polymerization and cross linkings.TG is a white to light grey powder without peculiar smell, used in the food industry - meat, fish or seafood, bread and milk processing. The modern production of various types of cheese consists of series of different processes. There are no same types of cheese which are produced in the same way. In the raw materials the cheese has an abundance of casein and lacto-albumen. TG stimulates the binding of casein and lacto-albumen, reducing the loss of lacto-albumen in the wheyleads to increase in production of cheese up to 12-15 %. [27]

Lipase

Although lipases are used in cheese flavour technology as components of the ripening systems also used to produce modified milk fat products for other food applications [28].

Lipolysed milk fat (LMF)

It has a creamy, buttery and cheesy aroma derived from short to medium chain fatty acids and fatty acid chemical derivatives released from milk fat by lipases. The raw material substrate for manufacturing LMF is either condensed milk or butter oil. Lipases are added and left in contact with the substrate at the optimum temperature for the enzymes used, until the required flavour/aroma is achieved, or until a predetermined acid degree value is reached, corresponding to a measurable release of fatty acids by the lipase. The product is pasteurized, spray dried or otherwise adjusted to standard solids content, and packaged. It also include chocolate coatings and syrups, butter flavours for margarine, artificial creams and sauces, flavourings for coffee whiteners, and cheese flavour additives [29]. The type of lipase used to make LMF products depends on the intended food application. Generally, good LMF products for use in baked products can be made using pancreatic lipase preparations, lamb and kid pregastric esterase and fungal/mould lipases from Aspergillus niger, Geotrichum candidum and Penicillium roqueforti. Some bacterial lipases are also suitable (Achromobacter lipolyticum and Pseudomonas lipase) but the LMFs for bread baking should not be prepared with Achromobacter, Penicillium or Geotrichum lipase to avoid soapy and musty flavours, and the pregastric esterases also produce too high a proportion of butyric acid for bread making, in which they tend to produce rancid, sweaty flavour notes [28].This technology and its food applications has been comprehensively reviewed recently, and the interested reader can find detailed information therein [30].

Lipase-catalysed intra - and intermolecular modification of milk fat

Chemical inter-esterification, acidolysis, alcoholysis and transesterification have been used for many years to modify the physical/functional properties of milk fat, but more recently lipase technology has replaced this chemical technology to give more precise and 'cleaner' processing [30–32]. In particular, milk fat substitutes have been prepared as a partial replacement for the milk fat in baby foods [33]. However, fat fractionation by physical methods is the commercially preferred option for milk fat modification in dairy product applications, and for coverage of lipase-catalysed molecular modification reference should be made to the reviews cited above.

Lactase

Lactase (beta-galactosidase) hydrolyses lactose to its constituent monosaccharide sugars, galactose and glucose. The enzyme is widespread in animals, but it has only become important technologically since microbial sources have become readily available [34]. The principal commercial preparations are sourced from Aspergillus niger, Aspergillus oryzae. Candida pseudotropicalis and Kluveromyces lactis. Lactase applications are in batch and immobilised enzyme technology, favouring the Aspergillus and Kluyveromyces sources, respectively.

Commercial dairy products of lactase technology

Lactase in tablet form is sold as an in vitro remedy for lactose intolerance, a widespread condition caused by a deficiency of lactase in the digestive tract. Suffers experience stomach cramps, bloating and diarrhoea due to the accumulation of lactose in the gut lumen. Lactaid® tablets deliver active lactase to the gut to break down the ingested lactose and alleviate the intolerance symptoms. Hydrolysed whey syrup is produced from whey, the by-product of cheese making and casein production. The hydrolysis step can be on the whey itself, or on the permeate from UF plant used to make whey protein concentrate. The UF permeate still contains some whey protein but is enriched in lactose. It is concentrated to 15-20% total solids (TS), de-mineralised, usually by ion exchange, though electro-dialysis or nano-filtration can be used for this, then heated according to the type of lactase treatment to be used. The hydrolysis step can be

by batch treatment with yeast lactase, though the use of immobilized enzyme reactor columns is more efficient, using Aspergillus lactase. Immobilised lactase reactors can achieve up to 90% lactose hydrolysis in whey permeate, though the batch process converts only about 70%. However, both processes produce sufficient free glucose and galactose to make the product sweet, and this property is enhanced by evaporation of the hydrolysate to 60% TS to make the final syrup. The whey syrup is made sticky by the high concentration of glucose and galactose, so it is not dried, but sold and used in that form. It is used in food manufacture to replace sweetened condensed milk, sugar and skim milk in many products such as ice cream, milk desserts and sauces. The syrup is also an excellent caramel ingredient and as a sweetener/binder in cereal bars.

Conclusion

The tremendous knowledge gathered on enzyme structures and function in recent years has opened up a new era for milk processing applications with enzymes. A new phase of enzyme technology may revolutionize the ways foods are prepared and processed in the near future. It is becoming easier to obtain relatively large numbers of naturally rare proteins, crystallography requires smaller amounts of purified proteins and smaller crystals are easier to work with than ever before. The database of known protein structures is expanding rapidly leading these to be more meaningful questions that need to be answered at the atomic level of resolution.

References

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2. Lomholt, S.B., Qvist, K.B. The formation of cheese curd. In: Law, B.A. (ed). Technology of Cheese Making. Sheffield: Sheffield Academic Press; 1999: 66-98.

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