《乳品生物化学》（英文版） 4 Milk proteins

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 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 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 disulphide￾linked 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 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, 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 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

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) (Chap￾ter 5). 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 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 frac￾tion and the non-protein nitrogen (NPN) fraction. These fractions were recognized as early as 1938 by Rowland but until recently very little was

Skimmilk(Kjeldahl I) Acidify to pH 4.6, teat at 10°Cx20min fake to 12% ool and acidify to pH 4.6, atural with Precipitate (all proteins) (globulins) Total nil K Proteose peptone N- KjeldahL Noseiprotkin dthogernkjekieldah m n= kjeldahl Il-Kjeldahl Iv Figure 4.4 Scheme for quantifying the principal protein fractions in milk

Skimmilk (Kjeldahl I) II Heat at loO°C x 20 min. Make to 12% cool and acidify to pH 4.6, filter trichloroacetic acid, filter I Acidify to pH 4.6, filter Precipitate Filtrate (casein) (non-casein N: serum proteins non-protein N; Kjeldahl 11) Neutralize and saturate with filter MgS04 Precipitate (globulins) Filtrate (albumin and non-protein N; Kjeldahl V) Total nitrogen = Kjeldahl I Casein = Kjeldahl I - Kjeldahl II Non-protein nitrogen = Kjeldahl 111 Precipitate Filtrate (casein and (proteose peptone heat-labile and non-protein N; semm proteins) Kjeldahl IV) I Precipitate (all proteins) 1 Filtrate (non-protein nitmgen; Kjeldahl 111) Proteose peptone N = Kjeldahl IV-Kjeldahl III Serum protein = Kjeldahl 11- Kjeldalil IV Figure 4.4 Scheme for quantifying the principal protein fractions in milk

DAIRY CHEMISTRY AND BIOCHEMISTRY known about them. rowland observed that when milk was heated to 95.c for 10 min, 80% of the nitrogenous compounds in whey were denatured and co-precipitated with the casein when the pH of the heated milk was adjusted ubsequently to 4.6. He considered that the heat-denaturable whey proteins represented the lactoglobulin and lactalbumin fractions and designated the remaining 20%proteose-peptone. The proteose-peptone fraction was precipitated by 12% trichloracetic acid(TCA) but some nitrogenous com- pounds remained soluble in 12% TCA and were designated as nonprotei based on that of Rowland, is shown in Figure 4y groups of milk proteins, A scheme for the fractionation of the principal 4.3 Preparation of casein and whey proteins Skim milk prepared by mechanical separation(see Chapter 3)is used as the starting material for the preparation of casein and whey proteins 4.3.1 Acid ( isoelectric) precipitation Acidification of milk to about pH 4.6 induces coagulation of the casein Aggregation occurs at all temperatures, but below about 6.C the aggregates are very fine and remain in suspension, although they can be sedimented by low-speed centrifugation. At higher temperatures(30-40oC), the aggregates are quite coarse and precipitate readily from solution at above about 50 C, the precipitate tends to be stringy and difficult to handle. For laboratory-scale production of casein, HCI is usually used for cidification; acetic or lactic acids are used less frequently. Industrially, HCI is also usually used; H, SO4 is used occasionally but the resulting whey is not suitable for animal feeding(MgSO4 is a laxative). Lactic acid produced in situ by a culture of lactic acid bacteria is also widely used, especially in New Zealand, the principal producer of casein time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2C, holding for about 30 min and then warming to 30-35.C. The fine precipitate formed at 2 C allows time for the colloidal calcium phosphate to dissolve( Chapter 5). A moderately dilute acid(1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of

152 DAIRY CHEMISTRY AND BIOCHEMISTRY known about them. Rowland observed that when milk was heated to 95°C for 10 min, 80% of the nitrogenous compounds in whey were denatured and co-precipitated with the casein when the pH of the heated milk was adjusted subsequently to 4.6. He considered that the heat-denaturable whey proteins represented the lactoglobulin and lactalbumin fractions and designated the remaining 20% 'proteose-peptone'. The proteose-peptone fraction was precipitated by 12% trichloracetic acid (TCA) but some nitrogenous com￾pounds remained soluble in 12% TCA and were designated as nonprotein nitrogen. A scheme for the fractionation of the principal groups of milk proteins, based on that of Rowland, is shown in Figure 4.4. 4.3 Preparation of casein and whey proteins Skim milk prepared by mechanical separation (see Chapter 3) is used as the starting material for the preparation of casein and whey proteins. 4.3.1 Acid (isoelectric) precipitation Acidification of milk to about pH 4.6 induces coagulation of the casein. Aggregation occurs at all temperatures, but below about 6°C the aggregates are very fine and remain in suspension, although they can be sedimented by low-speed centrifugation. At higher temperatures (3O-4OcC), the aggregates are quite coarse and precipitate readily from solution. At temperatures above about 5OCC, the precipitate tends to be stringy and difficult to handle. For laboratory-scale production of casein, HCl is usually used for acidification; acetic or lactic acids are used less frequently. Industrially, HC1 is also usually used; H,SO, is used occasionally but the resulting whey is not suitable for animal feeding (MgSO, is a laxative). Lactic acid produced in situ by a culture of lactic acid bacteria is also widely used, especially in New Zealand, the principal producer of casein. The inorganic colloidal calcium phosphate associated with casein in normal milk dissolves on acidification of milk to pH 4.6 so that if sufficient time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2"C, holding for about 30min and then warming to 30-35°C. The fine precipitate formed at 2°C allows time for the colloidal calcium phosphate to dissolve (Chapter 5). A moderately dilute acid (1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of

MILK PROTEINS 153 lactose will interact with casein on heating via the maillard browning reaction, with undesirable consequences The procedure used for the industrial production of acid (isoelecti asein is essentially the same as that used on a laboratory scale, except for many technological differences(section 4. 15.1).The whey proteins may be recovered from the whey by salting out, dialysis or ultrafiltration 4.3.2 Centrifugation Because they occur as large aggregates, micelles, most (90-95%)of the casein in milk is sedimented by centrifugation at 100 000 g for 1 h. Sedimen tation is more complete at higher (30-37'C)than at low(2C)temperature, at which some of the casein components dissociate from the micelles and are non-sedimentable. Casein prepared by centrifugation contains its original level of colloidal calcium phosphate and can be redispersed as micelles with properties essentially similar to the original micelles demented milk Addition of CaCl 2 to about 0. 2 M causes aggregation of the casein such that it can be readily removed by low-speed centrifugation. If calcium is added at 90C, the casein forms coarse aggregates which precipitate readily. this principle is used in the commercial production of some 'casein co-precipi- tates'in which the whey proteins, denatured on heating milk at 90 C for 10 min, co-precipitate with the casein. Such products have a very high ash 4.3.4 Salting-out methods Casein can be precipitated from solution by any of several salts. Addition of NH4)2SO4 to milk to a concentration of 260 g l- causes complete precipi tation of the casein together with some whey proteins(immunoglobulins, Ig). MgSO4 may also be used. Saturation of milk with Nacl at 37C precipitates the casein and Igs while the major whey proteins re emain soluble, provided they are undenatured. This characteristic is the basis of a commercial test used for the heat classification of milk powders which contain variable levels of denatured whey proteins 4.3.5 Ultrafiltration The casein micelles are retained by fine-pore filters. Filtration through large-pore ceramic membranes is used to purify and concentrate casein on a laboratory scale. Ultrafiltration(UF)membranes retain both the caseins

MILK PROTEINS 153 lactose will interact with casein on heating via the Maillard browning reaction, with undesirable consequences. The procedure used for the industrial production of acid (isoelectric) casein is essentially the same as that used on a laboratory scale, except for many technological differences (section 4.15.1).The whey proteins may be recovered from the whey by salting out, dialysis or ultrafiltration. 4.3.2 Because they occur as large aggregates, micelles, most (90-95%) of the casein in milk is sedimented by centrifugation at 100 000 g for 1 h. Sedimen￾tation is more complete at higher (30-37°C) than at low (2°C) temperature, at which some of the casein components dissociate from the micelles and are non-sedimentable. Casein prepared by centrifugation contains its original level of colloidal calcium phosphate and can be redispersed as micelles with properties essentially similar to the original micelles. Cen tr ifuga t ion 4.3.3 Centrifugation of calcium-supplemented milk Addition of CaCI, to about 0.2 M causes aggregation of the casein such that it can be readily removed by low-speed centrifugation. If calcium is added at 90"C, the casein forms coarse aggregates which precipitate readily. This principle is used in the commercial production of some 'casein co-precipi￾tates' in which the whey proteins, denatured on heating milk at 90°C for lOmin, co-precipitate with the casein. Such products have a very high ash content. 4.3.4 Salting-out methods Casein can be precipitated from solution by any of several salts. Addition of (NH,),SO, to milk to a concentration of 260 g 1- causes complete precipi￾tation of the casein together with some whey proteins (immunoglobulins, Ig). MgSO, may also be used. Saturation of milk with NaCl at 37°C precipitates the casein and Igs while the major whey proteins remain soluble, provided they are undenatured. This characteristic is the basis of a commercial test used for the heat classification of milk powders which contain variable levels of denatured whey proteins. 4.3.5 Ultrajiltration The casein micelles are retained by fine-pore filters. Filtration through large-pore ceramic membranes is used to purify and concentrate casein on a laboratory scale. Ultrafiltration (UF) membranes retain both the caseins

154 DAIRY CHEMISTRY AND BIOCHEMISTRY and whey proteins while lactose and soluble salts are permeable; total milk protein may be produced by this method. The casein micelles permeate the membranes used in microfiltration(pore size 0.05-10 um)but bacteria are retained by membranes with pores of less than 0.5 um, thus providing a method for removing more than 99.9% of the bacteria in milk without heat treatment; microfiltration is being used increasingly in several sectors of the dairy industry. Industrially, whey proteins are prepared by ultrafiltration or diafiltration of whey(to remove lactose and salts), followed by spray drying; these products, referred to as whey protein concentrates, contain 30-80% protein 4.3.6 Gel filtration ( gel permeation chromatography Filtration through cross- linked dextrans(e.g. Sephadex, Pharmacia, Upp sala, Sweden)makes it possible to fractionate molecules, including proteins on a commercial scale. It is possible to separate the casein and whey proteins by gel filtration but the process is uneconomical on an industrial 4.3.7 Precipitation with ethanol The caseins may be precipitated from milk by c. 40% ethanol while the whey proteins remain soluble; lower concentrations of ethanol may be used at lower pH values 4.3.8 Cryoprecipitation Casein, in a mainly micellar form, is destabilized and precipitated by freezing milk or, preferably, concentrated milk, at about-10'C; casein prepared by this method has some interesting properties but is not produced commer cially at present. 4.3.9 Rennet coagulation Casein may be coagulated and recovered as rennet casein by treatment of milk with selected proteinases(rennets ). However, one of the caseins, K-casein,is hydrolysed during renneting and therefore the properties of rennet casein differ fundamentally from those of acid casein. Rennet casein which contains the colloidal calcium phosphate of milk, is insoluble in water at pH 7 but can be dissolved by adding calcium sequestering agents, usually citrates or polyphosphates. It has desirable functional properties for certain food applications, e. g. in the production of cheese analogues

154 DAIRY CHEMISTRY AND BIOCHEMISTRY and whey proteins while lactose and soluble salts are permeable; total milk protein may be produced by this method. The casein micelles permeate the membranes used in microfiltration (pore size - 0.05-10 pm) but bacteria are retained by membranes with pores of less than OSpm, thus providing a method for removing more than 99.9% of the bacteria in milk without heat treatment; microfiltration is being used increasingly in several sectors of the dairy industry. Industrially, whey proteins are prepared by ultrafiltration or diafiltration of whey (to remove lactose and salts), followed by spray drying; these products, referred to as whey protein concentrates, contain 30-80% protein. 4.3.6 Geljltration (gel permeation chromatography) Filtration through cross-linked dextrans (e.g. Sephadex, Pharmacia, Upp￾sala, Sweden) makes it possible to fractionate molecules, including proteins, on a commercial scale. It is possible to separate the casein and whey proteins by gel filtration but the process is uneconomical on an industrial scale. 4.3.7 Precipitation with ethanol The caseins may be precipitated from milk by c. 40% ethanol while the whey proteins remain soluble; lower concentrations of ethanol may be used at lower pH values. 4.3.8 Cryoprecipitation Casein, in a mainly micellar form, is destabilized and precipitated by freezing milk or, preferably, concentrated milk, at about - 10°C; casein prepared by this method has some interesting properties but is not produced commer￾cially at present. 4.3.9 Rennet coagulation Casein may be coagulated and recovered as rennet casein by treatment of milk with selected proteinases (rennets). However, one of the caseins, K-casein, is hydrolysed during renneting and therefore the properties of rennet casein differ fundamentally from those of acid casein. Rennet casein, which contains the colloidal calcium phosphate of milk, is insoluble in water at pH 7 but can be dissolved by adding calcium sequestering agents, usually citrates or polyphosphates. It has desirable functional properties for certain food applications, e.g. in the production of cheese analogues

MILK PROTEINS 4.3. 10 Other methods for the preparation of whey proteins Highly purified whey protein preparations, referred to as whey protein isolates(containing 90-95% protein), are prepared industrially from whey by ion exchange chromatography. Denatured (insoluble) whey proteins, referred to as lactalbumin, may be prepared by heating whey to 95C for 10-20 min at about pH 6.0 the coagulated whey proteins are recovered by centrifugation. The whey proteins may also be precipitated using FeCl3or polyphosphates( section 4. 15.6) 4.4 Heterogeneity and fractionation of casein Initially, casein was considered to be a homogeneous protein Heterogeneity was first demonstrated in the 1920s by Linderstrem-Lang and co-workers, using fractionation with ethanol-HCl, and confirmed in 1936 by Pedersen using analytical ultracentrifugation, and in 1939 by Mellander, using free boundary electrophoresis. Three components were demonstrated and named a-, B- and 2-casein in order of decreasing electrophoretic mobility and represented 75, 22 and 3%, respectively, of whole casein. These caseins were successfully fractionated in 1952 by Hipp and collaborators based on differential solubilities in urea at c. pH 4.6 or in ethanol /water mixtures; the former is widely used although the possibility of forming artefacts through interaction of casein with cyanate produced from urea is of concern n 1956, Waugh and von Hippel showed that the a-casein fraction of Hipp et al. contained two proteins, one of which was precipitated by low ncentrations of Ca*and was called a- casein(s= sensitive) while the other, which was insensitive to Ca2+, was called K-casein. a-Casein was later shown to contain two proteins which are now called x,,.and as2-caseins. Thus, bovine casein contains four distinct gene products, desig nated &, 1", %s2"B- and K-caseins which represent approximately 37, 10, 35 and 12% of whole casein, respectively Various chemical methods were developed to fractionate the caseins but es homogeneous preparations. Fractionation is now usually achieved by ion-exchange chromatography on, for example, DEAE-cellu lose, using urea-containing buffers; quite large (e.g. 10 g)amounts of caseinate can be fractionated by this method, with excellent results(Figure 4.5a, b). Good results are also obtained by ion-exchange chromatography using urea-free buffers at 2-4C. High performance ion-exchange chromatography(e.g Pharmacia FPLC M on Mono Q or Mono S)gives excellent results for small amounts of sample(Figure 4.5c, d ). Reversed phase HPLC or hydrophobic interaction chromatography may also be used but are less effective than ion-exchange chromatography

MILK PROTEINS 155 4.3.10 Highly purified whey protein preparations, referred to as whey protein isolates (containing 90-95% protein), are prepared industrially from whey by ion exchange chromatography. Denatured (insoluble) whey proteins, referred to as lactalbumin, may be prepared by heating whey to 95°C for 10-20 min at about pH 6.0; the coagulated whey proteins are recovered by centrifugation. The whey proteins may also be precipitated using FeCl, or polyphosphates (section 4.15.6). Other methods for the preparation of whey proteins 4.4 Heterogeneity and fractionation of casein Initially, casein was considered to be a homogeneous protein. Heterogeneity was first demonstrated in the 1920s by Linderstrsm-Lang and co-workers, using fractionation with ethanol-HC1, and confirmed in 1936 by Pedersen, using analytical ultracentrifugation, and in 1939 by Mellander, using free boundary electrophoresis. Three components were demonstrated and named a-, ,!?- and y-casein in order of decreasing electrophoretic mobility and represented 75, 22 and 3%, respectively, of whole casein. These caseins were successfully fractionated in 1952 by Hipp and collaborators based on differential solubilities in urea at c. pH 4.6 or in ethanol/water mixtures; the former is widely used although the possibility of forming artefacts through interaction of casein with cyanate produced from urea is of concern. In 1956, Waugh and von Hippel showed that the a-casein fraction of Hipp et al. contained two proteins, one of which was precipitated by low concentrations of Ca2+ and was called a,-casein (s = sensitive) while the other, which was insensitive to Ca2+, was called k--casein. a,-Casein was later shown to contain two proteins which are now called uSl- and a,,-caseins. Thus, bovine casein contains four distinct gene products, desig￾nated ctsl-, rs2-, /I- and K-caseins which represent approximately 37, 10, 35 and 12% of whole casein, respectively. Various chemical methods were developed to fractionate the caseins but none gives homogeneous preparations. Fractionation is now usually achieved by ion-exchange chromatography on, for example, DEAE-cellu￾lose, using urea-containing buffers; quite large (e.g. 10 g) amounts of caseinate can be fractionated by this method, with excellent results (Figure 4Sa, b). Good results are also obtained by ion-exchange chromatography using urea-free buffers at 2-4°C. High performance ion-exchange chromatography (e.g. Pharmacia FPLCTM on Mono Q or Mono S) gives excellent results for small amounts of sample (Figure 4.5c, d). Reversed￾phase HPLC or hydrophobic interaction chromatography may also be used but are less effective than ion-exchange chromatography