4 Milk proteins 4. 1 Introduction Normal bovine milk contains about 3. 5% protein. The conce changes significantly during lactation, especially during the first ft post-partum(Figure 4. 1); the greatest change occurs in the whey fraction(Figure 4.2). The natural function of milk proteins is to supply young mammals with the essential amino acids required for the develop- ment of muscular and other protein-containing tissues, and with a number of biologically active proteins, e.g. immunoglobulins, vitamin- binding, metal-binding proteins and various protein hormones. The young of differ ent species are born at very different states of maturity, and, consequently have different nutritional and physiological requirements. These differences are reflected in the protein content of the milk of the species, which ranges Weeks of lactation Figure 4.1 Changes in the concentrations of lactose(O), fat(O)and protein(O)in bovine milk during lactation
4 Milk proteins 4.1 Introduction Normal bovine milk contains about 3.5% protein. The concentration changes significantly during lactation, especially during the first few days post-partum (Figure 4.1); the greatest change occurs in the whey protein fraction (Figure 4.2). The natural function of milk proteins is to supply young mammals with the essential amino acids required for the development of muscular and other protein-containing tissues, and with a number of biologically active proteins, e.g. immunoglobulins, vitamin-binding, metal-binding proteins and various protein hormones. The young of different species are born at very different states of maturity, and, consequently, have different nutritional and physiological requirements. These differences are reflected in the protein content of the milk of the species, which ranges 6 d5 2 8 4 3 0 10 20 30 40 50 Weeks of lactation Figure 4.1 Changes in the concentrations of lactose (O), fat (0) and protein (0) in bovine milk during lactation
MILK PROTEINS 147 Days postpartum igure 42 Changes in the concentration of total protein(A)and of casein(O)and whey proteins ()in bovine milk during the early stage of lactation. from c. I to c. 24%(Table 4.1). The protein content of milk is directly related to the growth rate of the young of that species(Figure 4.3), reflecting the requirements of protein for growth The properties of many dairy products, in fact their very existence, depend on the properties of milk proteins, although the fat, lactose and especially the salts, exert very significant modifying influences. Casein products are almost exclusively milk protein while the production of most cheese varieties is initiated through the specific modification of proteins by proteolytic enzymes or isoelectric precipitation. The high heat treatments to which many milk products are subjected are possible only because of the exceptionally high heat stability of the principal milk proteins, the caseins Traditionally, milk was paid for mainly on the basis of its fat content but nilk payments are now usually based on the content of fat plus protein Specifications for many dairy products include a value for protein content. Changes in protein characteristics, e.g. insolubility as a result of heat denaturation in milk powders or the increasing solubility of cheese proteins during ripening, are industrially important features of these products. It is assumed that the reader is familiar with the structure of proteins; for convenience, the structures of the amino acids found in milk are given in
MILK PROTEINS 147 10 - 0: I I I 0 10 20 30 Days postpartum Figure 4.2 Changes in the concentration of total protein (A) and of casein (0) and whey proteins (W) in bovine milk during the early stage of lactation. from c. 1 to c. 24% (Table 4.1). The protein content of milk is directly related to the growth rate of the young of that species (Figure 4.3), reflecting the requirements of protein for growth. The properties of many dairy products, in fact their very existence, depend on the properties of milk proteins, although the fat, lactose and especially the salts, exert very significant modifying influences. Casein products are almost exclusively milk protein while the production of most cheese varieties is initiated through the specific modification of proteins by proteolytic enzymes or isoelectric precipitation. The high heat treatments to which many milk products are subjected are possible only because of the exceptionally high heat stability of the principal milk proteins, the caseins. Traditionally, milk was paid for mainly on the basis of its fat content but milk payments are now usually based on the content of fat plus protein. Specifications for many dairy products include a value for protein content. Changes in protein characteristics, e.g. insolubility as a result of heat denaturation in milk powders or the increasing solubility of cheese proteins during ripening, are industrially important features of these products. It is assumed that the reader is familiar with the structure of proteins; for convenience, the structures of the amino acids found in milk are given in
148 AIRY CHEMISTRY AND BIOCHEMISTRY Table 4.1 Protein content (%)in the milks of some species Casein Whey protein Total on Camel(bactrian) Domestic rabbit 39 11.2 House mouse Human Indian elephant Polar bear 109 Sheep white-tailed jack rabbit 197 40 23.7 8EEE Horse Days to double birth weight Figure 4.3 Relationship betw he growth rate(days to double birth weight) of the yo,f from rotein content(expressed as of total calories derive protein) of the milk of that species(from Bernhart, 1961)
148 DAIRY CHEMISTRY AND BIOCHEMISTRY 30 - h 20- E .- e, K - E bz .z 10- - 8 s 0 Table 4.1 Protein content (YO) in the milks of some species Cat Kat ** Rabbit Dog PIS cow Sheep Goat . Horse Reindeer Buffalo 0 Man 4 Species Casein Whey protein Total ~ ~~ Bison 3.7 0.8 4.5 Black bear 8.8 5.7 14.5 Camel (bactrian) 2.9 1 .o 3.9 Cat 11.1 cow 2.8 0.6 3.4 Domestic rabbit 9.3 4.6 13.9 Donkey 1 .o 1.0 2.0 Echidna 7.3 5.2 12.5 Goat 2.5 0.4 2.9 Grey seal 11.2 Guinea-pig 6.6 1.5 8.1 Hare 19.5 Horse 1.3 1.2 2.5 House mouse 7.0 2.0 9.0 Human 0.4 0.6 1 .o Indian elephant 1.9 3.0 4.9 Pig 2.8 2.0 4.8 Polar bear 7.1 3.8 10.9 Red kangaroo 2.3 2.3 4.6 Reindeer 8.6 1.5 10.1 Rhesus monkey 1.1 0.5 1.6 White-tailed jack rabbit 19.7 4.0 23.7 - - - - - - Sheep 4.6 0.9 5.5 Days to double birth weight Figure 4.3 Relationship between the growth rate (days to double birth weight) of the young of some species of mammal and the protein content (expressed as YO of total calories derived from protein) of the milk of that species (from Bernhart, 1961)
MILK PROTEINS 149 Appendix 4A. We have retained the term cystine to indicate two disulphide linked cysteines 4.2 Heterogeneity of milk proteins Initially, it was believed that milk contained only one type of protein bi about 100 years ago it was shown that the proteins in milk could be fractionated into two well-defined groups On acidification to pH 4.6(the oelectric pH)at around 30C, about 80% of the total protein in bovine milk precipitates out of solution; this fraction is now called casein. The protein which remains soluble under these conditions is referred to as whey or serum protein or non-casein nitrogen. The pioneering work in this area was done by the German scientist, Hammarsten, and consequently isoelec- tric(acid) casein is sometimes referred to as casein nach Hammarsten The ratio of casein; whey proteins shows large interspecies differences; in human milk, the ratio is c. 40: 60, in equine(mare's)milk it is 50: 50 while in the milks of the cow, goat, sheep and buffalo it is c. 80: 20. Presumably, hese differences reflect the nutritional and physiological requirements of the young of these species There are several major differences between the caseins and whey proteins, of which the following are probably the most significant, especially from an industrial or technological viewpoint 1. In contrast to the caseins, the whey proteins do not precipitate from solution when the pH of milk is adjusted to 4.6. This characteristic is used as the usual operational definition of casein. This difference in the properties of the two milk protein groups is exploited in the preparation of industrial casein and certain varieties of cheese(e. g. cottage, quarg and 2. Chymosin and some other proteinases(known as rennets) produce a very ight, specific change in casein, resulting in its coagulation in the presence of Ca-t. Whey proteins undergo no such alteration. The coagulability of casein through the action of rennets is exploited in the manufacture of most cheese varieties and rennet casein; the whey proteins are lost in the whey. The rennet coagulation of milk is discussed in Chapter 10 3. Casein is very stable to high temperatures; milk may be heated at its natural pH (c. 6.7)at 100C for 24 h without coagulation and it withstands heating at 140C for up to 20 min. Such severe heat treat ments cause many changes in milk, e.g. production of acids from lactose resulting in a decrease in pH and changes in the salt balance, which eventually cause the precipitation of casein. The whey proteins, on the
MILK PROTEINS 149 Appendix 4A. We have retained the term cystine to indicate two disulphidelinked cysteines. 4.2 Heterogeneity of milk proteins Initially, it was believed that milk contained only one type of protein but about 100 years ago it was shown that the proteins in milk could be fractionated into two well-defined groups. On acidification to pH 4.6 (the isoelectric pH) at around 30°C, about 80% of the total protein in bovine milk precipitates out of solution; this fraction is now called casein. The protein which remains soluble under these conditions is referred to as whey or serum protein or non-casein nitrogen. The pioneering work in this area was done by the German scientist, Hammarsten, and consequently isoelectric (acid) casein is sometimes referred to as casein nach Hammarsten. The ratio of casein : whey proteins shows large interspecies differences; in human milk, the ratio is c. 40 : 60, in equine (mare's) milk it is 50: 50 while in the milks of the cow, goat, sheep and buffalo it is c. 80 : 20. Presumably, these differences reflect the nutritional and physiological requirements of the young of these species. There are several major differences between the caseins and whey proteins, of which the following are probably the most significant, especially from an industrial or technological viewpoint: 1. In contrast to the caseins, the whey proteins do not precipitate from solution when the pH of milk is adjusted to 4.6. This characteristic is used as the usual operational definition of casein. This difference in the properties of the two milk protein groups is exploited in the preparation of industrial casein and certain varieties of cheese (e.g. cottage, quarg and cream cheese). Only the casein fraction of milk protein is normally incorporated into these products, the whey proteins being lost in the whey. 2. Chymosin and some other proteinases (known as rennets) produce a very slight, specific change in casein, resulting in its coagulation in the presence of Ca2+. Whey proteins undergo no such alteration. The coagulability of casein through the action of rennets is exploited in the manufacture of most cheese varieties and rennet casein; the whey proteins are lost in the whey. The rennet coagulation of milk is discussed in Chapter 10. 3. Casein is very stable to high temperatures; milk may be heated at its natural pH (c. 6.7) at 100°C for 24h without coagulation and it withstands heating at 140°C for up to 20min. Such severe heat treatments cause many changes in milk, e.g. production of acids from lactose resulting in a decrease in pH and changes in the salt balance, which eventually cause the precipitation of casein. The whey proteins, on the
DAIRY CHEMISTRY AND BIOCHEMISTRY other hand. are relative ely heat labile, being completely denatured by heating at 90C for 10 min Heat-induced changes in milk are discussed in Chapter 9. 4. Caseins are phosphoproteins, containing, on average, 0.85% phosphorus, while the whey proteins contain no phosphorus. The phosphate groups are responsible for many of the important characteristics of casein, especially its ability to bind relatively large amounts of calcium, making it a very nutritionally valuable protein, especially for young animals. The phosphate, which is esterified to the protein via the hydroxyl group of serine, is generally referred to as organic phosphate. Part of the inorganic phosphorus in milk is also associated with the casein in the form of colloidal calcium phosphate(c. 57% of the inorganic phosphorus)( Chap The phosphate of casein is an important contributor to its remarkably high heat stability and to the calcium-induced coagulation of rennet altered casein(although many other factors are involved in both cases) 5. Casein is low in sulphur(0.8%)while the whey proteins are relatively rich 1.7%). Differences in sulphur content become more apparent if one considers the levels of individual sulphur-containing amino acids. The sulphur of casein is present mainly in methionine, with low concentra- tions of cysteine and cystine; in fact the principal caseins contain only methionine. The whey proteins contain significant amounts of both cysteine and cystine in addition to methionine and these amino acids are responsible, in part, for many of the changes which occur in milk on heating, e. g. cooked flavour, increased rennet coagulation time (due to interaction between B-lactoglobulin and K-casein) and improved heat stability of milk pre-heated prior to sterilization 6. Casein is synthesized in the mammary gland and is found nowhere else in nature. Some of the whey proteins(B-lactoglobulin and a-lactalbumin) are also synthesized in the mammary gland, while others(e.g. bovine serum albumin and the immunoglobulins) are derived from the blood 7. The whey proteins are molecularly dispersed in solution or have simple quaternary structures, whereas the caseins have a complicated quaternary structure and exist in milk as large colloidal aggregates, referred to as micelles, with particle masses of 10% D: 8. Both the casein and whey protein groups are heterogeneous, each containing several different proteins 4.2.1 Other protein fractions In addition to the caseins and whey proteins, milk contains two other groups of proteins or protein-like material, i.e. the proteose-peptone frac and the non-protein nitrogen(NPN) fraction. These fractions were gnized as early as 1938 by rowland but until recently very little was
150 DAIRY CHEMISTRY AND BIOCHEMISTRY other hand, are relatively heat labile, being completely denatured by heating at 90°C for 10min. Heat-induced changes in milk are discussed in Chapter 9. 4. Caseins are phosphoproteins, containing, on average, 0.85% phosphorus, while the whey proteins contain no phosphorus. The phosphate groups are responsible for many of the important characteristics of casein, especially its ability to bind relatively large amounts of calcium, making it a very nutritionally valuable protein, especially for young animals. The phosphate, which is esterified to the protein via the hydroxyl group of serine, is generally referred to as organic phosphate. Part of the inorganic phosphorus in milk is also associated with the casein in the form of colloidal calcium phosphate (c. 57% of the inorganic phosphorus) (Chapter 5). The phosphate of casein is an important contributor to its remarkably high heat stability and to the calcium-induced coagulation of rennetaltered casein (although many other factors are involved in both cases). 5. Casein is low in sulphur (0.8%) while the whey proteins are relatively rich (1.7%). Differences in sulphur content become more apparent if one considers the levels of individual sulphur-containing amino acids. The sulphur of casein is present mainly in methionine, with low concentrations of cysteine and cystine; in fact the principal caseins contain only methionine. The whey proteins contain significant amounts of both cysteine and cystine in addition to methionine and these amino acids are responsible, in part, for many of the changes which occur in milk on heating, e.g. cooked flavour, increased rennet coagulation time (due to interaction between P-lactoglobulin and K-casein) and improved heat stability of milk pre-heated prior to sterilization. 6. Casein is synthesized in the mammary gland and is found nowhere else in nature. Some of the whey proteins (P-lactoglobulin and cr-lactalbumin) are also synthesized in the mammary gland, while others (e.g. bovine serum albumin and the immunoglobulins) are derived from the blood. 7. The whey proteins are molecularly dispersed in solution or have simple quaternary structures, whereas the caseins have a complicated quaternary structure and exist in milk as large colloidal aggregates, referred to as micelles, with particle masses of 106-109 Da. 8. Both the casein and whey protein groups are heterogeneous, each containing several different proteins. 4.2.1 Other protein fractions In addition to the caseins and whey proteins, milk contains two other groups of proteins or protein-like material, i.e. the proteose-peptone fraction and the non-protein nitrogen (NPN) fraction. These fractions were recognized as early as 1938 by Rowland but until recently very little was