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Improving the Safety and Quality of Milk: Milk Production and Processing

Improving the Safety and Quality of Milk: Milk Production and Processing

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Improving the Safety and Quality of Milk: Milk Production and Processing

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Apr 8, 2010


Consumers demand quality milk with a reasonable shelf-life, a requirement that can be met more successfully by the milk industry through use of improved processes and technologies. Guaranteeing the production of safe milk also remains of paramount importance. Improving the safety and quality of milk provides a comprehensive and timely reference to best practice and research advances in these areas. Volume 1 focuses on milk production and processing. Volume 2 covers the sensory and nutritional quality of cow’s milk and addresses quality improvement of a range of other milk-based products.

The opening section of Volume 1: Milk production and processing introduces milk biochemistry and raw milk microbiology. Part two then reviews major milk contaminants, such as bacterial pathogens, pesticides and veterinary residues. The significance of milk production on the farm for product quality and safety is the focus of Part three. Chapters cover the effects of cows’ diet and mastitis, among other topics. Part four then reviews the state-of-the-art in milk processing. Improving the quality of pasteurised milk and UHT milk and novel non-thermal processing methods are among the subjects treated.

With its distinguished editor and international team of contributors, volume 1 of Improving the safety and quality of milk is an essential reference for researchers and those in industry responsible for milk safety and quality.
  • Addresses consumer demand for improved processes and technologies in the production, safety and quality of milk and milk products
  • Reviews the major milk contaminants including bacterial pathogens, pesticides and vetinary residues as well as the routes of contamination, analytical techniques and methods of control
  • Examines the latest advances in milk processing methods to improve the quality and safety of milk such as modelling heat processing, removal of bacteria and microfiltration techniques
Apr 8, 2010

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Improving the Safety and Quality of Milk - Elsevier Science



Since I first started my career in dairy research at the now defunct Hannah Research Institute in 1974, the dairy industry worldwide has faced many changes. With regard to food safety, it has witnessed the emergence of foodborne pathogens not previously associated with dairy products, such as Listeria monocytogenes and Escherichia coli O157:H7 along with the introduction of preventive food safety management systems (HACCP) to limit the impact of these pathogens. This year has seen the publication of the sequence of the entire cow genome, a feat that opens up innumerable possibilities. The use of this information will allow us, for example, to improve production costs through identification of traits related to feed conversion, to produce milk with specific characteristics and to impact animal welfare by selection of animals with increased disease resistance. These are just a few of the benefits the industry may reap. With more research it is also becoming apparent that milk and milk products are not the nutritional minefield that many would have us believe, in fact dairy products possess bioactive components that show substantial promise for health promotion.

It is the intent of this book to provide up-to-date coverage of several facets related to the production and processing of safe, wholesome and nutritious dairy products, not only from bovine milk but also from other domesticated ruminants. The first volume includes chapters related to milk safety and quality and focuses on the microbiological and chemical safety of raw milk and technologies for analyzing and processing milk. In the second volume, nutritional, sensory and sustainability issues are addressed as well as those associated with other milk- producing mammals and specific milk products.

I would like to thank all the contributing authors for their hard work and patience in waiting for edits. I would particularly like to acknowledge the contribution of Dr Richard Robinson, who sadly died during the production of this book. Dr Robinson was well known by all in the dairy industry for his research and the many books he edited.

On a personal note I would like to thank my wife, Susan, for her understanding and support, my two daughters, Megan and Bethan, and their respective husbands, Darren and Eric, and my four grandchildren, Rhys, Emma, Sophie and Evan, for keeping me young at heart.

I would also like to thank Dairy Farmers of Ontario for all the support they have given me over the last 20 years.

To all the readers of this book, I hope you learn from it and that it makes you realize that the proper production and processing of milk is complex and is carried out by professional and dedicated farmers and processors.

Part I

Key requirements for milk quality and safety


Milk biochemistry

A.L. Kelly,     University College Cork, Ireland

L. Bach Larsen,     University of Aarhus, Denmark


As milk is the key base raw material for all dairy products, the safety and quality of such products are heavily influenced by the characteristics of the milk. In this chapter, the key constituents of milk (fat, protein, salts, lactose and enzymes) and their properties are described, and the factors affecting the chemical composition and processing characteristics of milk, such as diet and lactation, are discussed in detail.

Key words:





1.1 Introduction

Milk is the fluid secreted by female mammals for the purpose of providing high-level nutrition to their offspring in the first days or weeks of life. Mankind has, for millennia, domesticated a small number of mammalian species, e.g., cows, goats, sheep and buffalo, for the purpose of producing their milk over an artificially lengthened season, and consuming it either directly or after conversion into a range of dairy products. Today, a very significant proportion of food consumed worldwide has its origins in mammalian milk, and a huge and diverse dairy industry is at the forefront of the global food industry in terms of scale, economic significance and technological sophistication.

It is well known that many people worldwide, e.g. in Asia, have problems tolerating milk due to lactose intolerance. It has recently been discovered that the ability of most European adults to tolerate milk is the result of a mutation in a single gene, which gave our ancestors bearing that mutation an advantage for survival, and furthermore that the tolerance for milk in the Saudi population is the result of a different mutation leading to the same adaptation to consumption of milk (Enattah et al., 2008). The mutation results in continuous production of the enzyme responsible for the cleavage of lactose, the lactase enzyme (β-galactosidase), which is produced by cells in the intestine. This mutation is thought to have originated in the Caucasus region before people migrated to Europe after the last ice age. Even though it is sometimes said by some people that ‘milk is not intended as food for adults’, the discovery of this mutation, which is thought to have its origin in a single person from whom it was spread, strongly indicated this to be very beneficial for the survival of our ancestors. During the last ice age, it gave them the possibility of exploiting the valuable nutrients in milk from domesticated animals, which was an obvious advantage for their survival at times with limited food alternatives.

The characteristics and quality of dairy products from market milk to cheese and yoghurt depend to a large extent on the primary stage of milk secretion within the mammal, and milk is a highly variable and complex raw material for processing. Hence, understanding of the mechanism of secretion of milk, the factors affecting the composition of milk, and ways in which milk composition and yield can be manipulated are of great interest to processors and farmers alike.

The objective of this chapter is to outline the major constituents of milk and their properties, to explore the manner in which they are secreted in the mammal, and to discuss factors affecting this production, and hence the quality of milk. The focus of the discussion will be on bovine milk, as the predominant milk-supplying species in most countries.

1.2 Milk composition and constituents

Milk is an enormously complex physicochemical system, with multiple constituents in different phases and states existing in a delicate balance of forces which exists on the brink of stability. It can readily be destabilised so as to collapse into separated or altered states; indeed, these phenomena had been exploited to produce dairy products long before their scientific mechanisms were understood.

In essence, milk is a solution of dilute salts, a simple sugar and vitamins, in which fat is emulsified as globules, and which contains a complex system of proteins, most of which exist in colloidal aggregates of thousands of molecules (casein micelles), an order of magnitude smaller than the fat globules. Studying milk under progressively higher microscopic magnification thus reveals a teeming multiphase system of complex biological molecules arranged in highly structured complex entities.

1.2.1 Lactose

In concentration terms, the dominant constituent of milk is generally lactose, a disaccharide consisting of one linked molecule each of glucose and galactose, which is present at 4.5–5.0% in bovine milk. The level of lactose in milk is relatively constant, and has an influential role on milk yield, as lactose is synthesised by the mammary gland, and determines how much water is drawn into the milk. The presence of lactose makes milk a highly fermentable medium, as a large number of bacterial species (collectively termed the lactic acid bacteria) can hydrolyse lactose to lactic acid, which reduces the pH of milk and, as we will see, results in coagulation if this drop is great enough (i.e., when the pH reaches 4.6, the isoelectric point of casein). While uncontrolled or unwanted fermentation clearly results in spoilage of the milk, controlling this fermentation is the basis of production of dairy products such as cheese and yoghurt. Lactose is also of interest due to its propensity, as a reducing sugar, to undergo Maillard reactions at high temperature, leading to colour changes in milk heated to very high temperatures (e.g., during sterilisation processes), and to its crystallisation behaviour, which is principally of significance in highly concentrated dairy systems, such as evaporated milk.

1.2.2 Milk fat

The next most abundant substance in milk is generally fat, although the level of fat can vary from below 3.0% to more than 5.0%, a much greater range than that of any milk constituent. The main constituent of milk fat is triglycerides (more than 95% of the milk fat), which consist of three fatty acid molecules esterified to a glycerol molecule. Milk contains several types of fatty acids, differing in the length of the chain of carbon atoms (and classified on this basis into short-, medium-and long-chain) and numbers of double bonds, i.e., whether saturated or unsaturated (Jensen, 2002; Huppertz et al., 2008). Compared to other types of food, milk fat is characterised by a great diversity of fatty acids, with chain lengths from four carbons up to more than 20 carbons, as well as branched fatty acids produced by microbes. The chemical properties of fatty acids have considerable consequences for both the nutritional quality of milk (in terms of the healthiness or otherwise of saturated fats) and its technological properties; chain length and degree of saturation both influence the melting point of the triglyceride, and hence the ultimate hardness of milk fat at, for example, refrigeration temperature. Compared to bovine milk fat, vegetable fats such as olive oil have a far higher proportion of unsaturated fatty acids, and hence provide both softness and perceived health benefits to consumers when added to products such as margarine and dairy spreads.

Milk fat also contains low levels of mono-and diglycerides, and minor constituents such as cholesterol, sphingolipids and phospholipids. Recently, attention has been drawn to some possible beneficial effects of some of the fatty acids in milk, including conjugated linoleic acid (CLA) and short-chain fatty acids (SCFAs) (Collomb et al., 2006; McIntosh et al., 2006; Bisig et al., 2007; Chilliard et al., 2007).

Milk fat is present in the milk as milk fat globules (MFG) with diameters ranging from 0.1 to more than 10 μm. The globules contain a nonpolar core of triglycerides and cholesterol esters. The milk fat in the core is protected and rendered (almost) stable in the aqueous environment of milk by the presence of a protective coating on the surface of the spherical globules, the milk fat globule membrane (MFGM), which stabilises the emulsion and protects the triglycerides from degradation (lipolysis). The structure and composition of milk fat were reviewed by Jensen (2002), and the physical stability of milk fat globules was reviewed by Huppertz and Kelly (2006). The MFGM consists of a double-layer phospholipid membrane into which different proteins are embedded, giving the MFGM specific characteristics. These proteins include some major proteins such as butyrophilin and the enzyme xanthine oxidase, and an increasing list of minor proteins are associated with the MFGM (Reinhardt and Lippolis, 2006; Fong et al., 2007). Studies on knock-out mice, in which functional genes coding for either xanthine oxidase or butyrophilin were lacking, have indicated some functions of the proteins associated with the MFGM. In both types of mice, large droplets of lipid without a proper outer membrane were secreted, and fused together into large aggregates of lipid; this strongly indicates that both of these proteins are essential for the production of intact milk fat droplets (Bauman et al., 2006; Huppertz and Kelly, 2006).

The size of most of the milk fat droplets (more than 80% in number) is below 1 μm, but in terms of volume most of the milk fat is made up of fat globules with average diameter of approximately 4 μm. The stability of the emulsion is challenged primarily by the density difference between milk fat and the surrounding aqueous serum; this leads to relatively rapid separation of unprocessed milk into a phase enriched in milk fat globules in much closer contact with each other (i.e., cream) and a phase largely depleted of such globules (i.e., skimmed milk). On processing of milk, this separation can be accelerated (by applying centrifugal force) or hugely retarded (by reducing the size of the fat globules, using homogenisation, which greatly slows their rate of separation, as described by Stokes’s Law). The globules do generally remain discrete, however, unless the integrity of the MFGM is severely compromised, either accidentally (through excessive agitation or pumping) or deliberately (in making butter, when the damaged globules are worked together into a homogeneous mass, from which most of the aqueous phase is drained).

The phospholipids mainly found at the outer side of the globules are more unsaturated than the triglycerides in the core of the fat globules (Jensen, 2002). Due to this, the level of unsaturated phospholipids is higher in milk with smaller fat globules. The levels of both phospholipids and triglycerides are affected by feeding (as will be discussed later).

Oxidation of lipids in milk is potentially a substantial problem for both milk and processed dairy products, as it gives rise to off-flavours and can reduce nutritional quality. The oxidative stability of milk is determined by a delicate balance between pro-and antioxidants in milk, where the concentration of polyunsaturated fatty acids, which are prone to oxidation, is believed to be an important factor for the stability (Barrefors et al., 1995). Oxidation is often measured during storage of milk but, in several cases, oxidation has been detected directly after milking, a phenomenon called spontaneous oxidation, where imbalance between pro-and antioxidants seems important for the development of off-flavour. Auto-oxidation is believed to be a major reason for the propagation of oxidation in milk during storage, and the concentrations of transition metal ions (Cu+, Fe²+) in milk play an important role as pro-oxidants (Ford et al., 1986).

Light-induced oxidation is another major reason for off-flavours in dairy products. Milk contains a relatively high concentration of riboflavin, which can act as a photo-sensitiser in two ways: it can either directly oxidise proteins and lipids, or it can react with oxygen, forming the reactive oxygen species, singlet oxygen, which can further oxidise lipids. Milk also contains a range of potential antioxidants, such as tocopherols, carotenoids and uric acid (Ostdal et al., 2000) formed by ruminal metabolism. However, there is an ongoing discussion of the significance of the antioxidants in relation to protection of milk from oxidation.

1.2.3 Proteins in milk

The proteins of milk are classed into two major groups, which differ fundamentally in their properties, in particular their solubility when the pH of milk is adjusted to 4.6. Under such conditions, the majority (typically around 75% by weight) of milk protein, called casein, is insoluble and either precipitates or forms a gel, depending on whether the rate of pH drop is rapid or slow, respectively. This casein fraction actually comprises four major caseins, called αs1-, αs2-, β-and κ-caseins, which are all relatively hydrophobic fibrous proteins, with little tertiary structure. The caseins associate in the aqueous environment of milk into exquisitely complex structures called micelles, containing thousands of molecules of each casein (for a review, see Fox and Kelly, 2004). The caseins are found in dairy cattle in different genetic variants (see Table 1.1 and Section 1.5.4). The caseins are phosphorylated to different extents and, in the case of κ-casein, may be glycosylated to different extents with carbohydrate groups including galactosamine, galactose and N-acetyl-nuraminic acid (Table 1.1). The variations in the amino acid sequences of the different genetic variants give the variant caseins slightly different molecular masses and, in some cases, also different isoelectric points (pI values). This is seen, for example, in κ-casein, where one of the differences between the variants is that the A variant has an aspartic acid residue at position 148, while variant B has an asparagine residue at that position. The presence of an extra aspartic acid provides an extra negative charge of the A variant compared with the B variant at the pH of milk, i.e. pH 6.7, and thereby some different processing characteristics of κ-casein type B milk compared with type A milk. This introduces, together with phosphorylation, further variations in the pI values of the different variants.

Table 1.1

Genetic variants and some molecular characteristics of the caseins

aMass of monomer.

Source: Data are according to Swaisgood (1992) and Holland et al. (2004). The pI values for A-2P and B-2P were calculated using the bioinformatics package at for the κ-casein sequence without the signal peptide.

Of the four caseins, only αs2-and κ-casein contain cysteine residues in their primary structure. Both molecules contain two cysteine residues per monomer. Due to the formation of disulphide bonds, the αs2-casein molecules in milk are found both as monomers and as dimers, linked together by two disulphide bridges. In contrast, κ-casein ranges in size from the monomer state to larger than a decamer, held together by an apparently randomised pattern of disulphide bonds between the cysteines (Rasmussen et al., 1999). In the monomers, the cysteines are occupied in intra-chain disulphide bridges.

Over the last decades, several different models for the inner structure of the casein micelle have been proposed, but it seems that the micelles are composed of a tangled mass of protein molecules, interacting by crosslinks either between hydrophobic regions of the casein molecules or through calcium bridges (De Kruif and Holt, 2003; Horne, 2003). A key feature shared by all models is that the κ-casein is mainly found at the surface of the micelles; this is where it exerts a protective force due to its glycosylated nature, which lends it an amphiphilic character and the ability to stabilise the hydrophobic micelle core in a manner analogous to that in which the MFGM stabilises the fat globules. κ-casein causes electrostatic and steric repulsion between micelles and, if this influence is negated, either by reducing the pH to the isoelectric point of casein (the afore mentioned value of 4.6) or by enzymatically removing the stabilising glycosylated part of the molecule (which is what the enzyme chymosin in rennet does during cheese-making), the unstable molecules aggregate into complex threedimensional structures, to yield the gels that give yoghurt and cheese their structure. Thus, the properties of the casein micelle, and the means by which it may be physicochemically destabilised, are key to the manufacture of a range of dairy products.

The proteins which remain stable at pH 4.6 are termed the whey proteins, and are a family of globular proteins. The principal whey proteins by weight are β-lactoglobulin and α-lactalbumin, while minor whey proteins include serum albumin, immunoglobulins, lactoperoxidase and lactoferrin, in addition to a growing list of further proteins identified in milk by new proteomic techniques (Fong et al., 2008). The main significance of the whey proteins, and β-lactoglobulin in particular, is that they unfold on heating and can subsequently interact and form complexes with themselves or other proteins. This is principally driven by the heat-induced exposure of a highly reactive free sulphydryl group in β-lactoglobulin, which can form disulphide bonds with any milk proteins containing disulphide bonds. Such reactions can lead to gel formation at sufficiently high whey protein concentrations (which is exploited during their use as food ingredients) and may be involved in phenomena such as the coagulation of milk on sterilisation, and the tendency of UHT-treated milk to gel during storage. In recent years there has been increasing focus on health-promoting proteins in milk, and on bioactive peptides derived by proteolytic cleavage, especially from the caseins.

1.2.4 Milk salts

Milk contains a wide range of mineral salts, some of which are associated with (and play a key role in maintaining the structure of) casein micelles, and some of which are in the serum phase of milk, in either ionised or non-ionised form; principal anionic salts in milk, in order of level, are potassium, calcium, sodium and magnesium, while principal cations include chloride, sulphate, carbonate, phosphate and citrate. The fraction of milk salts associated with the casein micelles is called colloidal calcium phosphate (CCP) and includes, as the name suggests, mainly calcium and phosphorus, but with lower levels of other species such as magnesium and citrate. The salt balance in milk between different forms and fractions is sensitive to processing conditions, in particular temperature and pH; reducing pH solubilises CCP progressively, while heating reduces the solubility of calcium and increases the CCP content of micelles.

Due to the content of lactose and salts in milk, the freezing point of milk is lower than that of water, milk with a freezing point between –0.516 and –0.545°C being considered normal. For example, a survey determined the mean value in Swedish dairy milk to be –0.529°C (Lindmark-Månsson et al., 2003). Changes in the levels of lactose in milk will be counteracted by changes in milk salts, and vice versa, in order to maintain constant osmotic pressure. Taken together, lactose, Cl–, K+ and Na+ contribute 80% of the freezing point depression of milk. The freezing point of milk is measured regularly by dairies and is used as a test for water addition, which would raise the freezing point.

1.3 Indigenous enzymes in milk

Milk of all species contains a heterogeneous population of enzymes of different types and activities; bovine milk probably contains almost 70 enzymes, of which around 20 have been investigated in some detail. Recent reviews on the enzymology of milk include those by Fox and Kelly (2006a,b) and Kelly and Fox (2006).

The four most important enzyme systems in milk, and their significance for product quality, will now be described.

1.3.1 Alkaline phosphatase

Alkaline phosphatase is arguably the best-known enzyme in milk, although its actual action in milk is probably of very little significance for milk quality. The reason for its importance lies rather in the serendipitous similarity of its thermal inactivation kinetics to those of the pathogenic bacterium Mycobacterium tuberculosis, and its ease of determination using assays based on colorimetric or fluorimetric substrates. As conventional pasteurisation is achieved using conditions designed to inactivate M. tuberculosis (heating at 72°C for 15 seconds), rapid determination of the presence or absence of activity of alkaline phosphatase is widely used as an indicator of the effectiveness of pasteurisation (Fox and Kelly, 2006b).

1.3.2 Plasmin

In blood, an enzyme called plasmin plays a key role in the process of control of blood clotting, and the activity must thus be tightly regulated, which is achieved by a system including the inactive precursor of the enzyme (plasminogen), a set of activators of plasminogen (grouped into tissue-and urokinase-type activators), and inhibitors of both plasmin and its activators. The entire blood system appears to also exist in milk, and levels of all components probably increase in circumstances where influx of blood constituents and somatic cells in milk increase (e.g., mastitis). Plasmin, a serine proteinase with a pH optimum of 7.5, is probably the principal proteolytic enzyme in milk from healthy cows. The total concentration of plasmin and plasminogen is in the range 1–3 μg/ml, of which approximately 10% is active plasmin (Richardson and Pearce, 1981; Benfeldt et al., 1994). Plasmin in milk can degrade the proteins in the milk through cleavage at bonds involving lysine or arginine residues in the polypeptide chains. Its significance arises from its hydrolysis of the caseins to yield the fractions called the γ-caseins and proteose peptones (fragments of β-casein) and λ-caseins (fragments of αs1-casein). Plasmin itself is relatively heat-stable, with low activity being detected even in UHT-treated milk (heated at 135–140°C for 3–4 seconds), partially due to activation of residual plasminogen by heat-stable plasminogen activators (PA) (Enright et al., 1999). In fact, the inhibitors of plasmin and plasminogen are probably more heat-labile than the enzyme or the activators, which can result in pasteurisation-like treatments actually increasing the net plasmin activity in milk, and consequent increases in proteolysis of casein (Richardson, 1983). The plasmin/plasminogen system in milk has been extensively studied, and has been shown to play a role in ripening of many cheese varieties (through initial or primary proteolysis of caseins to polypeptides which can be acted on by starter bacterial proteinases) and possibly also in gelation of UHT milk on storage (Kohlmann et al., 1991; Bastian and Brown, 1996; Kelly et al., 2006).

In raw milk, a high level of plasmin activity is normally not desirable, as it will result in a lower content of intact protein and, for example, can result in a lower cheese yield (Mara et al., 1998). In cheese, however, plasmin activity contributes positively to cheese ripening through initial proteolysis of the caseins, on which the microbial proteases can subsequently act, and furthermore may influence the taste and texture of many cheese varieties, although perhaps to a relatively minor extent (Farkye and Fox, 1992).

1.3.3 Somatic cell proteinases

Milk contains a variable number of somatic cells (white blood cells) and the number (somatic cell count, SCC) and types of cell present depend on a number of factors, principally the presence of infection such as mastitis (see Section 1.5.6). Several proteolytic enzymes and enzyme activities have been suggested as being associated with somatic cells in milk. The release of these enzymes from somatic cells can result from either active secretion or release from damaged cells. The final definitive proof that these enzymes are derived from somatic cells has not yet been provided, only that their presence and derived activities to some extent correlate with SCC. It is actually an alternative possibility that at least some of these proteases are secreted by mammary epithelial cells; this is an issue that awaits further research (Kelly et al., 2006).

Different cell types may have different enzyme profiles, and thus the enzyme profile of milk may be affected by both total SCC and also differential SCC (e.g., proportion of polymorphonuclear leucocytes, PMN, versus macrophages). For example, the proteases present during acute mastitis, where a large number of PMN are present in milk, may differ from the protease profile observed during chronic mastitis, where the majority of cells are macrophages.

The first so-called somatic cell protease suggested to be present in bovine milk was the lysosomal aspartic protease cathepsin D. It was later shown that the major part of the aspartic acid protease activity present in milk was derived from procathepsin D, and not from mature cathepsin D (Larsen and Petersen, 1995). At acid pH, at least at pH 3.5–5.0, milk procathepsin D can autoactivate into a proteolytically active intermediate form, called pseudocathepsin D (Larsen et al., 1993). As the pH of many cheeses is in the region of 5, it is interesting that cathepsin D and pseudocathepsin D can degrade the caseins into definite fragments, much like chymosin, and, furthermore, when added to milk in sufficient amounts, are actually able to coagulate milk (McSweeney et al., 1995; Larsen et al., 1996). Cathepsin D has been found to be more heat-stable in milk than in buffer, and approximately half of the activity derived from cathepsin D and procathepsin D in milk survived HTST pasteurisation at 72°C (Larsen et al., 2000; Hayes et al., 2001). As procathepsin D and cathepsin D in milk are mainly associated with the whey fraction, their main significance in relation to quality of dairy products is potentially in ultrafiltrated (UF) rennet-free cheeses, like UF-feta, quarg and cottage cheese. They may also be active in some Swiss-type cheeses, where the added rennet has been heat-inactivated, in addition to whey powders and potentially some fermented products. Indeed, activity derived from cathepsin D has been detected both in quarg and in extracts of UF-feta (Hurley et al., 2000; Larsen et al., 2000).

Cysteine protease activity has also been detected in bovine mik (Magboul et al., 2001) and different types of cysteine proteases have been fractionated. A partially purified fraction retained cysteine protease activity after heating at 72°C for 30 seconds, and immunoblotting revealed the presence of immuno-reactive cathepsin B. It is, however, likely that other types of cysteine proteases, apart from cathepsin B, are present in milk, due both to the heterogeneous nature of partly purified fractions, and to the fact that other types of cysteine proteases are present in the bovine lysosomes. The distribution between mature forms of cathepsin B and eventual pro-forms of the enzyme in milk also remains to be established. Like cathepsin D, cathepsin B is also able to hydrolyse the caseins, but the specificity is different (Considine et al., 2004).

It is very likely that other somatic cell proteinases remain to be identified in milk in the future. Some activities derived from unidentified milk proteases have been described (Larsen et al., 2006), potential candidates for which include the serine proteinases neutrophil elastase and cathepsin G, but other proteinases may also be present, e.g. metalloproteinases. Some new techniques, including mass spectrometry, have been employed recently for the detailed characterisation of the peptide profile in different types of high-cell-count milk and of healthy milk aiming at identifying responsible proteinases through the determination of cleavage sites (Wedholm et al., 2008), which are to a large extent enzyme-specific.

1.3.4 Lipoprotein lipase

Lipoprotein lipase (LPL) is the enzyme in milk responsible for enzymatic lipolysis, i.e., the hydrolysis of fatty acids from triglycerides and phospholipids in the milk; LPL is also involved in the biosynthesis of milk fat (Huppertz et al., 2008). Above a certain threshold, the released fatty acids can result in rancid off-flavours from short-chain fatty acids or from their oxidation to ketones. As long as the MFGM is intact, LPL cannot come into contact with its substrate, especially the triglycerides; as a result of this highly efficient partitioning, the extent of lipolysis of milk is a fraction of what it should theoretically be. However, when the MFGM is damaged, e.g. by excessive pumping of raw milk, especially uncooled milk, or homogenisation of raw milk, the triglycerides are rendered susceptible to lipolysis, resulting in an increase in the free fatty acid level (Wiking et al., 2003, 2005). In addition, some milk samples can undergo spontaneous lipolysis upon cooling of fresh, raw milk, which may depend on a balance of activating substances (e.g., apolipoproteins) and inhibiting substances (some proteins and peptides) present in milk. LPL in milk is reduced by pasteurisation, but more complete inactivation requires more severe heat treatments, as would be used for the processing of cream to be used in products where unwanted lipolysis could cause quality problems. The literature on lipoprotein lipases in milk was recently reviewed by Deeth (2006).

1.4 The secretion of milk

The production of milk involves a huge commitment of resources and energy by the mammal, and is mediated by the transport of raw materials from the blood into the udder, where the barrier between milk and blood is sufficiently porous to allow some constituents (lactose, minerals, enzymes, somatic cells) to transit in either direction. Other raw materials enter the mammary secretory cells for conversion and packaging into milk constituents and structures (e.g., fat globules, casein micelles), which then enter the milk.

The milk triglycerides are synthesised in the rough endoplasmic reticulum (ER) of the mammary cells. Small lipid droplets are released from the rough ER into the cytoplasm, where the lipid, coated with a bilayer membrane from the ER, becomes further coated with protein. Some of the droplets fuse with each other to form larger droplets on their way to the apical membrane of the cells (pathway A in Fig. 1.1), while others are secreted without fusion (pathway B in Fig. 1.1). When the lipid droplets arrive at the apical membrane, they are budded from the cell membrane (‘blebbing’), by which process they receive the second membrane bilayer. Interestingly, the enzyme xanthine oxidase plays a key role in the secretion of milk fat globules, but in this context does not rely on its enzymatic activity (Harrison, 2006).

Fig. 1.1 The pathways for secretion of major milk constituents in mammary epithelial cells. A: The cytoplasmic lipid droplet pathway. B: The microlipid droplet pathway. C: The secretory pathway for proteins and salts. MFGM: Milk fat globule membrane. ER: Endoplasmic reticulum. Adapted from Mather and Keenan (1998) and Bauman et al.

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