DAIRY BIOTECHNOLOGY

Module 5. Application of biotechnology in dairying

Lesson 24
DESIGNER MILK

24.1 Introduction

Although, milk is often considered as nearly the most perfect and ideal food to meet our nutritional requirements, there is still considerable scope to improve its functional properties to suit to the needs of the consumers by introducing appropriate modifications in its composition. The attention is now focused on adding more value to milk and studying its health implications. With the recent developments in the Biotechnological techniques e.g. Genetic Engineering, rDNA technology, Protein Engineering and advances in animal cloning and transgenic techniques, it is now possible to alter milk composition at will for better manufacturing / technological properties to add variety to our traditional dairy products and also to produce variants of milk to cater to the needs of specific consumers from health and nutritional perspectives. With the advent of modern gene transfer and expression methodologies, new opportunities have been created for the modification of animal production traits including milk production with altered composition. Animal udder can virtually be used now as an efficient biological vat or Bioreactor for the production of homologous and heterologous proteins, sugars and fats. Transgenic animals which constitute a useful experimental tool for assessing the ability and effect of transgenic mammary gland specific expression are mainly concerned with either producing biologically important and active proteins such as pharmaceuticals in milk of transgenic animals or to alter the intrinsic properties and composition of milk itself by genetically adding a new or modified protein for better manufacturing properties for dairy industry. Our growing understanding of the lactation process in the ruminants at molecular level and continual innovations in dairy processing have presented exciting opportunities for genetic manipulations that are not possible through traditional, nutritional, and classical genetic approaches.

24.2 Biotechnology based strategies for altering the Properties of Milk

Advances in biotechnology and genetic engineering have led to exploring new initiatives that were hitherto not even thought possible in the field of dairying. It is now firmly established that a new generation of value-added products can be produced and harvested from milk and milk products. While until recently, emphasis has been on breeding large animals to produce more milk, the current interest of animal scientists is now on producing designer milk by expressing homologous/heterologous proteins and introducing appropriate alterations in the major milk constituents in the milch animals through animal cloning and transgenic technology. By a thorough understanding of the biochemistry, genetic traits and changes in the cows diet that affect milk synthesis and composition, ways and means to manipulate milk composition to suit specific needs can now be explored judicially. By combining the two approaches of nutritional and genetic interventions, researchers are now hoping to develop ‘designer milk’ tailored to consumer preferences or rich in specific milk components that have implications in health as well as milk processing. The current interest in the modification of milk composition include the healthful and therapeutic aspects of milk and milk products. To realize the full potential of these advantages, it would be desirable to have the opportunity to alter milk composition in several ways. For diet and human health measures, the actions that would be beneficial include: a) generate a greater proportion of unsaturated fatty acids (USFA) in milk fat b) reduce lactose content in milk in order to cater to persons suffering from lactose intolerance and c) remove β -lactoglobulin (β-lg) from milk. From a technological stand point, there exist vast opportunities in: a) alteration of primary structure of casein to improve technological properties of milk b) production of high-protein milk c) engineering milk meant for cheese manufacturing that leads to accelerated curd clotting time d) increased yield and/or more protein recovery e) milk containing nutraceuticals and f) replacement for infant formula.

Five basic areas that might be highly useful for introducing desired alterations in milk are listed in Table 24.1. Within these broad areas, a wide variety of modifications to milk can be exploited. These include; adding extra copies of an existing gene (αsl, κ - and β -casein), down regulating the expression of a gene (α-lactalbumin), adding new genes such as those encoding human lysozyme or lactoferrin, removal of a gene (β -casein, β -lactoglobulin or acetyl-CoA carboxylase), and adding a mutated gene (αsl, κ - and β -casein) etc. In this direction, preliminary research has already been carried out using transgenic mice as model systems in the first four of these categories.

Table 24.1 Potential areas for introducing desired manipulations in milk targeting its key components for value addition

24.2.1 Alteration/ Manipulation of milk proteins

24.2.1.1 Caseins

The major milk proteins comprise of four variants of caseins namely αsl, αs2-, β- and κ-caseins and two whey proteins viz. alpha lactalbumin (α-LA) and beta lactoglobulin (β-LG) along with serum proteins and immuno-globulins which are present in milk of most of the mammals. The relative abundance of the various protein constituents of cow’s milk varies between breeds and genotypes with the general proportions being approximately 31.5% αsl-casein, 29.5% β casein, 8.5% αs2-casein, 11% κ-casein, 10% β -lactoglobumin, 4% α-lactalbumin and 5.5% serum proteins and immuno-globulins. Caseins which include alpha, beta and kappa caseins are the major proteins of milk representing > 78% of the proteins. Normal Dairy Cattle have a single copy of alpha-CN, beta-CN and kappa-CN within each cell. Introduction of one extra copy of casein in the bovine genome can have a drastic impact on dairy industry. An increase of 20% in the content of αs1-CN of milk would result in an increase of over US$ 190 million/year for the dairy industry. Similarly increased β -CN content in milk reduces rennet clotting time and increases the extent of syneresis. Therefore, higher protein content obtained by over expressing additional copies of endogenous bovine casein genes would be a significant advantage to the dairy industry.

Further modifications at the level of milk proteins can be achieved through molecular cloning of milk protein genes using appropriate vector–host systems by introducing alterations in the nucleotide sequences before putting the construct in the milch animal for expression of the altered protein. This step allows for their genetic modification in order to improve the nutritional quality or functional properties of milk. By using this strategy, more economical cheese products could result from a more efficient cleavage of a κ -CN by chymosin. Storage time for cheese maturation may be considerably reduced by altering the chymosin cleavage site of αs1-CN to more efficiently hydrolysable peptide linkages. Other manipulations include alteration of physical properties particularly thermostability of casein. Thermostability can be achieved by increasing the expression of κ–casein genes and masking the expression of beta-lactoglobulin in the mammary gland. Since caseins are relatively low in sulphur containing amino-acids, their nutritional value could be improved by increasing their methionine content.

Desirable manipulations of the target milk proteins can also be introduced at protein sequence level as listed below with two specific examples

• Amino-acid sequence of αs1 casein can be manipulated by directed mutagenesis to make it more suitable for the reaction catalyzed by enzyme casein kinase which causes phosphorylation of caseins and this makes them highly thermostable.

• Amphiphilicity of the caseins can also be augmented by gene technology. This property is associated with the surface activities of the casein that govern their emulsification and foaming properties. This type of manipulation can be extremely useful in making ice-creams, softies, candies and whipped toppings.

24.2.1.2 Whey proteins

The two major whey proteins namely β -lactoglobulin and α-lactalbumin are separated during milk curdling. These proteins have been completely sequenced, crystallized and subjected to X ray diffraction studies. β -lactoglobulin is thought to function as a retinal binding protein that favors absorption of vitamin A in the gut of milch animals. β -lactoglobulin can specifically be targeted at the genetic level by site directed mutagenesis for extinction because its presence in milk confers some undesirable manufacturing properties. This would also aim towards utilization of the spared amino-acids pool in the cells made available to the protein synthesis apparatus to produce more desirable proteins.

The second most abundant whey protein in human milk, α-LA is a minor component in bovine milk. Because α-LA is a small protein of 123 amino-acids, it is amenable to genetic engineering by site directed mutagenesis to modify its amino-acid composition. This technique has been used to modify α-LA and make it consumable by patients with phenylketonuria who lack the enzyme that metaboilizes phenylalanine and thus require low phenylalanine diets. Since the three dimensional structure of α-LA is known, the sensitivity of four phenylalanine residues in α-LA can be engineered in-vitro without disrupting the structure of the molecule. The modified gene is then microinjected into the pronucleus of a fertilized egg and the protein is produced in milk and can be used as a supplement for an improved diet for phenyl-ketonuria patients.

The aforesaid manipulations can be explored by using the following approaches:

(i) Adding extra copies of an existing gene

The addition of more κ -casein to the milk protein system could affect the physical properties of the milk since κ -casein is directly involved with micelle formation, structure and size. An increase in κ -casein could increase the thermal stability of casein aggregates and act to decrease micelle size. A smaller micelle diameter would lead to a larger available surface area, which would result in a more consistent and firmer curd as well as an increase in cheese yield. These modified properties of milk could be of great benefit and interest to the dairy industry.

(ii) Down-regulating the expression of a gene

The expression of a gene can be down regulated in vivo in a developmentally and the tissue-specific manner using transgenes expressing antisense of ribozyme messages

(iii) Removal of a gene

In order to determine the consequences of milk protein system of deleting a major milk protein, a β -casein knockout line of mice was produced. It was concluded that β-casein was a non-essential component of the milk protein system, thus illustrating that profound changes can be made in the composition of milk without disrupting the general organization of the micellar systems.

24.2.2 Fat content

It is now possible to manipulate even the fat content in milk by disrupting the fat synthesis in the mammary gland. By using this strategy, milk with 2% fat content (40% reduction) might be achievable. It has been suggested that de novo fat synthesis in mammary glands might be reduced by blocking the expression of acetyl CoA-Carboxylase gene through stem cell (knock out), antisense or ribozyme technology. Natural milk with reduced fat content could be extremely beneficial for patients with heart ailments.

24.2.3 Removing components of milk by genetic manipulation

The essential availability of bovine ES cells should enable undesirable components of the milk to be removed by disrupting the target gene in the bovine genome using homologous recombination.

24.2.3.1 Lactose

Lactose intolerance is a serious problem in 70% of the world population due to deficiency in the intestinal lactase (β-galactosidase) needed to hydrolyze milk lactose into its constituent monosaccharides leading to gastrointestinal upset. Genetic manipulation can now be applied to reduce lactose content in milk by either removal of α-LA by ES cells and gene ‘knock out’ methodologies or by introducing a lactase enzyme (β-galactosidase) into milk via mammary gland specific expression. The important role of α-lactalbumin in lactose formation has been shown in experiments involving transgenic animals. Lactation is disrupted in α-lactalbumin knockout mice, but it can be restored by human α-lactalbumin gene replacement thereby making it an attractive target for genetic manipulation to produce low lactose milk. An alternative approach for suppressing the α-LA gene expression is the use of antisense and ribozyme sequences. It is, however, imperative that such studies are conducted in ruminant mammary tissues in order to bring about the actual reduction in lactose and the economic benefits.

24.2.3.2 β -Lactoglobulin (β-LG)

Whey proteins have nutritional benefit in fluid milk market but represent a less valuable milk component for cheese industry. β-LG is the most abundant (upto 50%) whey protein. Human milk is devoid of β-LG which is considered to be the main allergen in bovine milk. However, it is not required in the lactation process as β -LG has no known function in the process of milk secretion. Further upon heat treatment, β-LG forms gel aggregates with other β-LG molecules because of the exposure of a thiol group. This thiol group interacts with a disulphide group of k-CN and interferes with chymosin mediated hydrolysis which is important for curd formation and cheese production. It is possible now to manipulate the level of β-LG at genetic level. However, because β-LG is also the major source of cystine in milk, removal of β-LG may result in a milk of inferior nutritional value. Hence, it would be desirable to delete β-LG coding sequences and neutralize the endogenous regulatory elements to drive expression of other proteins i.e. casein and lactoferrin.

24.2.4 Humanization of bovine milk

Breast milk is nature’s perfect food for human infants providing them with all aspects of nutrition and protection against infections. However, a considerable number of infants are fed formulae based on bovine/buffalo milk. The composition of these infant formulations can be improved if the proteins contained therein resemble more closely to those of human milk. It is now possible to add human milk proteins including lactoferrin to bovine milk by genetic engineering to produce humanized milk and new functional foods that mimic human breast milk for providing better digestion and improved health and nutrition to the children. The shelf life of such products is also expected to be very high due to antimicrobial activity associated with some of these proteins.

24.2.4.1 Gene manipulation for enhanced shelf life

Milk and milk products have got a limited shelf life as they provide an ideal medium for the growth of a wide range of spoilage and pathogenic micro-organisms. The contamination of these perishable dairy foods with microbial contaminants would render them unfit for human consumption and also can cause huge economic losses to the dairy industry due to excessive food spoilage. However, the shelf life of these foods can be extended considerably by incorporating proteinaceous antimicrobial factors namely bacteriocins produced by food grade lactic acid bacteria. These bacteriocins can be expressed under the control of milk protein gene promoter in the mammary gland. Nisin is one such broad spectrum bacteriocin used extensively in dairy industry as a food-grade biopreservative. Nisin happens to be the first bacteriocin which was granted GRAS (Generally Regarded As Safe) status by FDA for application as food-gade biopreservative in canned processed cheese. It is produced by Lactococcus lactis subsp. lactis from where it can be isolated and purified. Nisin has been thoroughly characterized at molecular level. Hence, it can be explored as a potential target for its expression in milk through transgenic technology to provide inbuilt protection to milk and milk products against the common microbial contaminants without affecting the natural flora of milk, thereby, extending the shelf life and safety of raw milk considerably.

24.2.4.2 Adding new genes

In order to explore the possibility of introducing novel human genes encoding commercially important proteins such as lysozyme, attempts have been made to produce transgenic mice expressing human lysozyme in milk to determine the consequences of transgene on some basic rheological and antimicrobial properties of their milk. Lysozymes are ubiquitous enzymes found in avian egg whites and mammalian secretions such as tears, saliva and milk, that are positively charged at physiological pH and have an inherent antimicrobial activity. If human lysozyme is present in bovine milk at a significant level, two main effects could be expected. First, because of its antimicrobial activity, lysozyme may reduce the overall level of bacteria in milk thus decreasing disease in the udder and overall bacterial levels in the milk. As lysozyme is considered to be a part of the passive immunity and the natural defense against bacteria, viruses, parasites and fungi in human milk, it could also exert human health advantages as well. Second, due to the net positive charge, lysozyme may be able to interact with the negatively charged caseins to produce milk with altered functional and physical properties. In studies using transgenic mice expressing human lysozyme in their milk at an average concentration of 0.38 mg per ml, the rennet clotting time of milk was decreased by 35%, gel strength of rennet induced gels was significantly higher in milk from the transgenic mice than in milk from control mice, while the average size of the micelles tended to be smaller. Milk from these transgenic lines was found to be bacteriostatic against two cold spoilage organisms- Pseudomonas fragi and Lactobacillus viscous and a mastitis causing isolate of Staphylococcus aureus.

24.3 Animal Pharming

Animal pharming is defined as the use of transgenic animals as bioreactors for the production of pharmaceutical proteins and bioactive peptides for therapeutic applications in the treatment of different human diseases. Ever since this highly specialized technique was evolved, it was acclaimed as an highly efficient and cost effective method for the production of pharmaceuticals. The first therapeutic product produced in milk of transgenic live stock that got approval turned out to be recombinant human antithrombin-III which was produced by GTC Biothrapeutics under the brand name “ATryn”. This was indeed a new milestone with lot of impact on human health. Since then, quite a few other important therapeutic products have been developed through this technology by expressing these in the milk of cattle. However, there is some amount of skepticism whether animal pharming in the near future would be able to attract the new generation of investors to take up this technology at commercial scale and stimulate the society and the pharma industry to accept the transgenically derived live stock and products as an alternative to well established drug based production systems. Nevertheless, there is cause for optmism since pharmaceuticals represent a fast growing sector with lot of commercial stakes and has made considerable strides in this upcoming area of considerable health significance. Animal pharming seems to have immense potential and perhaps could be the future technology with bright prospects for managing chronic human diseases in accost effective manner.

24.4 Future Strategy

The ultimate goal of the dairy industry has been to create an efficient, healthy cow or buffalo that can serve all the needs of the industry in totality. Genetic engineering offers tremendous opportunity for a paradigm shift in reshaping of the industry from the producers to the processing plants. Dairy producers now find the prospects of applying advanced biotechnological tools a feasible strategy to add enormous value to milk by producing high protein milk, milk destined for cheese manufacture that has accelerated curd clotting time, milk containing neutraceuticals, orally administered biologicals that provide health benefits or a replacement for infant formulae. Such a scenario would be a radical change for the dairy industry. There is now little doubt that the products of genetic engineering will become a part of the dairy industry in the new millennium.

Hence, it can be concluded that it is now feasible to produce milk of altered chemical composition particularly in respect of homologous / heterologous proteins of considerable commercial value by appropriately manipulating genes encoding such commercially important proteins. Over production of such useful proteins in milk could not only improve the quality of dairy products made out of such milk but also be instrumental in increasing their shelf life and safety from public health point of view. Furthermore, it is possible to humanize bovine/buffalo milk by expressing human lysozyme, lactoferrin and other human milk protein in the target milch animal so that the milk produced from the transgenic animals mimic human milk in nutritional and therapeutic functionality and better digestibility.

Refrences

Internet resources