8 Enzymology of milk and milk products 8. 1 Introduction indigenous enzymes which are constituents of the milk as secreted. The principal constituents of milk(lactose, lipids and proteins) can be modified by exogenous enzymes, added to induce specific changes. Exogenous en zymes may also be used to analyse for certain constituents in milk. In addition, milk and most dairy products contain viable micro-organisms which secrete extracellular enzymes or release intracellular enzymes after the cells have died sed. Some of these enzymes may cause undesirable changes, e.g. hydrolytic rancidity of milk and dairy products, bitterness and or age gelation of UHT milks, bittiness in cream, malty flavours or bitterness in fluid milk, or they may cause desirable flavours, e. g. in ripened cheese r is devoted mainly to the significance of indigenous enzymes in milk. The principal applications of exogenous enzymes have been deal chapters, e.g. rennets and lipases in cheese production Chapter 10), B-galactosidase to modify lactose(Chapter 2). Some minor or potential applications of exogenous enzymes are presented here. Enzymes derived from contaminating bacteria, which may be significant in milk and some dairy products, will not be discussed. The interested reader is referred to McKellar(1989) for a comprehensive review of enzymes produced by psychrotrophs which are the principal spoilage microorganisms in refrig erated milk and milk products. The significance of enzymes from microbial cultures in cheese ripening is discussed in Chapter 10 8.2 Indigenous enzymes of bovine milk 8.2.1 Introduction As many as 60 indigenous enzymes have been reported in normal bovine milk. With the exception of a-lactalbumin, which is an enzyme modifier in lactose synthesis(Chapter 2)most, if not all, of the indigenous enzymes in milk have no obvious physiological role. They arise from three principal sources the blood via defective mammary cell membranes
8 Enzymology of milk and milk products 8.1 Introduction Like all other foods of plant or animal origin, milk contains several indigenous enzymes which are constituents of the milk as secreted. The principal constituents of milk (lactose, lipids and proteins) can be modified by exogenous enzymes, added to induce specific changes. Exogenous enzymes may also be used to analyse for certain constituents in milk. In addition, milk and most dairy products contain viable micro-organisms which secrete extracellular enzymes or release intracellular enzymes after the cells have died and lysed. Some of these enzymes may cause undesirable changes, e.g. hydrolytic rancidity of milk and dairy products, bitterness and/or age gelation of UHT milks, bittiness in cream, malty flavours or bitterness in fluid milk, or they may cause desirable flavours, e.g. in ripened cheese. This chapter is devoted mainly to the significance of indigenous enzymes in milk. The principal applications of exogenous enzymes have been dealt with in other chapters, e.g. rennets and lipases in cheese production (Chapter lo), P-galactosidase to modify lactose (Chapter 2). Some minor or potential applications of exogenous enzymes are presented here. Enzymes derived from contaminating bacteria, which may be significant in milk and some dairy products, will not be discussed. The interested reader is referred to McKellar (1989) for a comprehensive review of enzymes produced by psychrotrophs which are the principal spoilage microorganisms in refrigerated milk and milk products. The significance of enzymes from microbial cultures in cheese ripening is discussed in Chapter 10. 8.2 Indigenous enzymes of bovine milk 8.2.1 Introduction As many as 60 indigenous enzymes have been reported in normal bovine milk. With the exception of cr-lactalbumin, which is an enzyme modifier in lactose synthesis (Chapter 2) most, if not all, of the indigenous enzymes in milk have no obvious physiological role. They arise from three principal sources: 0 the blood via defective mammary cell membranes;
318 DAIRY CHEMISTRY AND BIOCHEMISTRY secretory cell cytoplasm, some of which is occasionally entrapped within fat globules by the encircling fat globule membrane(MfGm)(Chapter 3) the mfgm itself, the outer layers of which are derived from the apica membrane of the secretory cell, which, in turn, originates from the golgi membranes( Chapter 3); this is probably the principal source of indigen Thus, most enzymes enter milk due to peculiarities of the mechanism by which milk constituents, especially the fat globules, are excreted from the secretory cells. Milk does not contain substrates for many of the enzymes present, while others are inactive in milk owing to unsuitable environment conditions, e.g. pH t: Many indigenous milk enzymes are technologically significant from five 1. Deterioration (lipase(commercially, probably the most significant en- zyme in milk), proteinase, acid phosphatase and xanthine oxidase)or preservation(sulphydryl oxidase, superoxide dismutase)of milk quality 2. As indices of the thermal history of milk: alkaline phosphatase, r-glutamyl transpeptidase, lactoperoxidase 3. As indices of mastitic infection: catalase, N-acetyl-B-D-glucosaminidase acid phosphatase; the concentration of several other enzymes increases on mastitic infection 4. Antimicrobial activity: lysozyme, lactoperoxidase(which is exploited as a component of the lactoperoxidase-H2O2-thiocyanate system for the cold pasteurization of milk 5. As commercial source of enzymes: ribonuclease, lactoperoxidase with a few exceptions(e.g. lysozyme and lactoperoxidase), the indigenous milk enzymes do not have a beneficial effect on the nutritional or organo leptic attributes of milk, and hence their destruction by heat is one of the objectives of many dairy processes The distribution of the principal indigenous enzymes in milk and their atalytic activity are listed in Table 8.1. In this chapter, the occurrence, distribution, isolation and characterization of the principal indigenou enzymes will be discussed, with an emphasis on their commercial signifi ance in milk 8. 2. 2 Proteinases(EC3.4.--) The presence of an indigenous proteinase in milk was suggested by Babcock and Russel in 1897 but because it occurs at a low concentration or has low activity in milk, it was felt until the 1960s that the proteinase in milk may be of microbial origin. Recent changes in the dairy industry, e.g. improved hygiene in milk production, extended storage of milk at a low temperature
318 DAIRY CHEMISTRY AND BIOCHEMISTRY 0 secretory cell cytoplasm, some of which is occasionally entrapped within fat globules by the encircling fat globule membrane (MFGM) (Chapter 3); 0 the MFGM itself, the outer layers of which are derived from the apical membrane of the secretory cell, which, in turn, originates from the Golgi membranes (Chapter 3); this is probably the principal source of indigenous enzymes. Thus, most enzymes enter milk due to peculiarities of the mechanism by which milk constituents, especially the fat globules, are excreted from the secretory cells. Milk does not contain substrates for many of the enzymes present, while others are inactive in milk owing to unsuitable environmental conditions, e.g. pH. Many indigenous milk enzymes are technologically significant from five viewpoints: 1. Deterioration (lipase (commercially, probably the most significant enzyme in milk), proteinase, acid phosphatase and xanthine oxidase) or preservation (sulphydryl oxidase, superoxide dismutase) of milk quality. 2. As indices of the thermal history of milk: alkaline phosphatase, y-glutamyl transpeptidase, lactoperoxidase. 3. As indices of mastitic infection: catalase, N-acetyl-P-D-glucosaminidase, acid phosphatase; the concentration of several other enzymes increases on mastitic infection. 4. Antimicrobial activity: lysozyme, lactoperoxidase (which is exploited as a component of the lactoperoxidase - H,O, - thiocyanate system for the cold pasteurization of milk). 5. As commercial source of enzymes: ribonuclease, lactoperoxidase. With a few exceptions (e.g. lysozyme and lactoperoxidase), the indigenous milk enzymes do not have a beneficial effect on the nutritional or organoleptic attributes of milk, and hence their destruction by heat is one of the objectives of many dairy processes. The distribution of the principal indigenous enzymes in milk and their catalytic activity are listed in Table 8.1. In this chapter, the occurrence, distribution, isolation and characterization of the principal indigenous enzymes will be discussed, with an emphasis on their commercial significance in milk. 8.2.2 Proteinases (EC 3.4.-.-) The presence of an indigenous proteinase in milk was suggested by Babcock and Russel in 1897 but because it occurs at a low concentration or has low activity in milk, it was felt until the 1960s that the proteinase in milk may be of microbial origin. Recent changes in the dairy industry, e.g. improved hygiene in milk production, extended storage of milk at a low temperature
Table 8.1 Indigenous enzymes of significance to milk Enzyme Off flavours in milk glycerides +glycerol Proteinase(plasmin Hydrolysis of peptide bonds, particularly Reduced storage stability of UhT products: Catalase 2H2O2→O2+2H2O sis of mucopolysaccharides Aldchyde+H2O+O2- Acid+H2O2 ro-oxidant; cheese ripening Sulphydryl oxidase 2RSH+O2→RSSR+H2O2 Amelioration of cooked favour utase 2O2+2H+→H2O2+O2 Lactoperoxidase H2O2+AH2→2H2O+A non bacteriocidal agent; cid ieosphsphonmoeterase olysis of phosphoric acid esters nization rolysis of phosphoric acid esters educe heat stability of milk
Table 8.1 Indigenous enzymes of significance to milk Enzyme Reaction Importance Lipase Proteinase (plasmin) Catalase Lysozyme Xanthine oxidase Sulphydryl oxidase Superoxide dismutase Lactoperoxidase Alkaline phosphomonoesterase Acid phosphomonoesterase Triglycerides + H,O 4 fatty acids +partial Hydrolysis of peptide bonds, particularly 2H,O, + 0, + 2H,O Hydrolysis of mucopolysaccharides Aldehyde+H,O+O, + Acid+H,O, 2RSH + 0, + RSSR + H,O, 20;+2H+ 4 H,O,+O, H,O,+AH, +2H,O+A glycerides +glycerol in bcasein Hydrolysis of phosphoric acid esters Hydrolysis of phosphoric acid esters Off flavours in milk; Reduced storage stability of UHT products; Index of mastitis; pro-oxidant Bacteriocidal agent Pro-oxidant; cheese ripening Amelioration of cooked flavour Antioxidant Index of pasteurization; flavour development in Blue cheese chccse ripening bacteriocidal agent; index of mastitis; pro-oxidant Index of pasteurization Reduce heat stability of milk; cheese ripening
DAIRY CHEMISTRY AND BIOCHEMISTRY at the farm and or factory and altered product profile, e.g. UHT processing of milk, have increased the significance of indigenous milk proteinase which has, consequently, been the focus of considerable research Milk contains at least two proteinases, plasmin (alkaline milk proteinase) and cathepsin d(acid milk proteinase)and possibly several others, i.e. two thiol proteinases, thrombin and an aminopeptidase. In terms of activity and technological significance, plasmin is the most important of the indigenous proteinases and has been the subject of most attention. The relevant literature has been reviewed by Grufferty and Fox(1988)and Bastian and Brown(1996) Plasmin(EC 3.4.21.7) The physiological function of plasmin(fibrinolysin) is to dissolve blood clots. It is part of a complex system consisting of plasmin, its zymogen (plasminogen), plasminogen activators, plasmin inhibitors and inhibitors of plasminogen activators( Figure 8.1). In milk, there is about four times as much plasminogen as plasmin and both, as well as plasminogen activators, are associated with the casein micelles, from which they dissociate when the pH is reduced to 46. The inhibitors of plasmin and of plasminogen ctivators are in the milk serum. The concentration of plasmin and plas- minogen in milk increase with advancing lactation, mastitic infection and number of lactations Plasmin is usually extracted from casein at pH3.5 and purified by precipitation with(NH4)2SO4 and various forms of chromatography, in- cluding affinity chromatography. Plasmin is optimally active at about H7. 5 and 35 C; it exhibits c. 20% of maximum activity at 5 C and is stable over the ph range 4 to 9. Plasmin is quite heat stable: it is partially nactivated by heating at 72C x 15s but its activity in milk increases following HTST pasteurization, probably through inactivation of the indig- enous inhibitors of plasmin or, more likely, inhibitors of plasminogen activators. It partly survives UHT sterilization and is inactivated by heating at 80C x 10 min at pH 6.8: its stability decreases with increasing pH in the range 3. 5-9.2 Inhibitors of plasminogen (casein micelles) Figure 8.1 Schematic representation of the plasmin system in milk
320 DAIRY CHEMISTRY AND BIOCHEMISTRY at the farm and/or factory and altered product profile, e.g. UHT processing of milk, have increased the significance of indigenous milk proteinase which has, consequently, been the focus of considerable research. Milk contains at least two proteinases, plasmin (alkaline milk proteinase) and cathepsin D (acid milk proteinase) and possibly several others, i.e. two thiol proteinases, thrombin and an aminopeptidase. In terms of activity and technological significance, plasmin is the most important of the indigenous proteinases and has been the subject of most attention. The relevant literature has been reviewed by Grufferty and Fox (1988) and Bastian and Brown (1996). Plasmin (EC 3.4.21.7) The physiological function of plasmin (fibrinolysin) is to dissolve blood clots. It is part of a complex system consisting of plasmin, its zymogen (plasminogen), plasminogen activators, plasmin inhibitors and inhibitors of plasminogen activators (Figure 8.1). In milk, there is about four times as much plasminogen as plasmin and both, as well as plasminogen activators, are associated with the casein micelles, from which they dissociate when the pH is reduced to 4.6. The inhibitors of plasmin and of plasminogen activators are in the milk serum. The concentration of plasmin and plasminogen in milk increase with advancing lactation, mastitic infection and number of lactations. Plasmin is usually extracted from casein at pH 3.5 and purified by precipitation with (NH,),SO, and various forms of chromatography, including affinity chromatography. Plasmin is optimally active at about pH 7.5 and 35°C; it exhibits c. 20% of maximum activity at 5°C and is stable over the pH range 4 to 9. Plasmin is quite heat stable: it is partially inactivated by heating at 72°C x 15s but its activity in milk increases following HTST pasteurization, probably through inactivation of the indigenous inhibitors of plasmin or, more likely, inhibitors of plasminogen activators. It partly survives UHT sterilization and is inactivated by heating at 80°C x 10 min at pH 6.8; its stability decreases with increasing pH in the range 3.5-9.2. Plasminogen activator(s) - Inhibitors of plasminogen (milk serum) (casein micelles) activators I Plasminogen - Plasmin Plasmin inhibitors (casein micellesl (casein micelles) (milk serum) Figure 8.1 Schematic representation of the plasmin system in milk
ENZYMOLOGY OF MILK AND Plasmin is a serine proteinase(inhibited by diisopropylfluorophosphate, phenylmethyl sulphonyl fluoride and trypsin inhibitor) with a high specific ity for peptide bonds to which lysine or arginine supplies the carboxyl group. Its molecular weight is about 81 Da and its structure contains five intramolecular disulphide-linked loops(kringles) which are essential for its activity Activity of plasmin on milk proteins. B-Casein is the most susceptible milk protein to plasmin action; it is hydrolysed rapidly at Lys28-Lys29 Lys,05-His1os and Lys107-Glu,os, to yield 7 (B-CN f29-209),7 2(B-CN f106-209)and ?(B-CN f108-209)caseins and proteose-peptone(PP)5 B-CN f1-105/7), PP8 slow (B-CN f29-105/7)and PP8 fast(B-CN f1-29) (Chapter 4). In solution, B-casein is also hydrolysed at Lys13-Tyru14 and Lys183-Asp1s4, but it is not known if these bonds are hydrolysed in milk -Caseins normally represent about 3% of total n in milk but can be as high as 10% in late lactation milk; the concentration of proteose peptones is about half that of the r-caseins as2-Casein in solution is also hydrolysed very rapidly by pla bonds Lys21-GIn22, Lys24-Asn2s, Argu14-Asnu15, Lys,49-Lys15o, Lys,5( Thr151, Lys181-Thr182, Lys187-Thr188 and Lys188-Ala1s9(see Bastian and Brown, 1996)but it is not known if it is hydrolysed in milk. Although less Isceptible than %s2- or B-caseins, a,r -casein in solution is also readily hydrolysed by plasmin(see Bastian and Brown, 1996)but it does not appear to be hydrolysed to a significant extent in milk although it has beer suggested that A-casein is produced from a,r -casein by plasmin. Although K-casein contains several Lys and Arg residues, it appears to be quite resistant to plasmin, presumably due to a relatively high level of secondary and tertiary structure. B-Lactoglobulin, especially when denatured, inhibit plasmin, presumably via sulphydryl-disulphide interactions which rupture the structurally important kringl Significance of plasmin activity in milk. Plasmin and plasminogen accor pany the casein micelles on the rennet coagulation of milk and are concentrated in cheese in which plasmin contributes to primary proteolysis of the caseins, especially in cheeses with a high-cook temperature, e. g. Swiss and some Italian varieties, in which the coagulant is totally or largely inactivated( Chapter 10). Plasmin activity may contribute to age gelatic UHT milk produced from high-quality raw milk (which contains a low level of Pseudomonas proteinase). It has been suggested that plasmin activity contributes to the poor cheesemaking properties of late-lactation milk but proof is lacking. The acid precipitability of casein from late lactation milk is also poor but evidence for the involvement of plasmin is lacking. Reduced yields of cheese and casein can be expected to result fro plasmin action since the proteose peptones are, by definition, soluble at
ENZYMOLOGY OF MILK AND MILK PRODUCTS 321 Plasmin is a serine proteinase (inhibited by diisopropylfluorophosphate, phenylmethyl sulphonyl fluoride and trypsin inhibitor) with a high specificity for peptide bonds to which lysine or arginine supplies the carboxyl group. Its molecular weight is about 81 Da and its structure contains five intramolecular disulphide-linked loops (kringles) which are essential for its activity. Activity of plasmin on milk proteins. 8-Casein is the most susceptible milk protein to plasmin action; it is hydrolysed rapidly at LyS28-Lys,g, Lys,,,-His,,, and Lys,,7-Glulo8, to yield y1 (8-CN f29-209), yz (P-CN f106-209) and y3 (P-CN f 108-209) caseins and proteose-peptone (PP)5 (P-CN fl-105/7), PP8 slow (P-CN f29-105/7) and PP8 fast (8-CN fl-29) (Chapter 4). In solution, p-casein is also hydrolysed at Lys, 13-Tyr1 14 and Lys,,3-Asp,84, but it is not known if these bonds are hydrolysed in milk. ?-Caseins normally represent about 3% of total N in milk but can be as high as 10% in late lactation milk; the concentration of proteose peptones is about half that of the y-caseins. a,,-Casein in solution is also hydrolysed very rapidly by plasmin at bonds Lys,,-Gln,,, Lys,,-Asn,,, Arg, 14-ASn1159 LY~~~,-LY~~,,, LY~~- Thr,,,, LyS18,-Thr182, Lys187-Thr188 and Lys188-Ala1,g (See Bastian and Brown, 1996) but it is not known if it is hydrolysed in milk. Although less susceptible than z,- or ,&caseins, a,,-casein in solution is also readily hydrolysed by plasmin (see Bastian and Brown, 1996) but it does not appear to be hydrolysed to a significant extent in milk although it has been suggested that /.-casein is produced from us,-casein by plasmin. Although K-casein contains several Lys and Arg residues, it appears to be quite resistant to plasmin, presumably due to a relatively high level of secondary and tertiary structure. P-Lactoglobulin, especially when denatured, inhibits plasmin, presumably via sulphydryl-disulphide interactions which rupture the structurally important kringles. Signijicance of plasmin activity in milk. Plasmin and plasminogen accompany the casein micelles on the rennet coagulation of milk and are concentrated in cheese in which plasmin contributes to primary proteolysis of the caseins, especially in cheeses with a high-cook temperature, e.g. Swiss and some Italian varieties, in which the coagulant is totally or largely inactivated (Chapter 10). Plasmin activity may contribute to age gelation in UHT milk produced from high-quality raw milk (which contains a low level of Pseudomonas proteinase). It has been suggested that plasmin activity contributes to the poor cheesemaking properties of late-lactation milk but proof is lacking. The acid precipitability of casein from late lactation milk is also poor but evidence for the involvement of plasmin is lacking. Reduced yields of cheese and casein can be expected to result from plasmin action since the proteose peptones are, by definition, soluble at pH4.6
322 DAIRY CHEMISTRY AND BIOCHEMISTRY Cathepsin D(EC 3. 423. 5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile ted by 70C x 10 min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd; its specificity on z,,and B-caseins is quite similar to that of chymosin but it has very poor milk-clotting activity(McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level Other proteinases. The presence of low levels of other proteolytic enzymes in milk has been reported(see Fox and McSweeney, 1996). Most of these originate from somatic cells, and their level increases during mastitic infection. The presence of cathepsin D, a lysozomal enzyme, in milk suggests that all the lysozomal proteinases are present in milk although they may not be active. These minor proteinases are considered to be much less significan than plasmin, but more work on the subject is necessary 8.2. 3 Lipases and esterases(EC 3. 1.1.-) es catalyse the development of hydrolytic rancidity in milk, and, uently, lipases and lipolysis in milk have been studied extensively Ik contains three types of esterase 1. A-type carboxylic ester hydrolases(arylesterase; EC 3. 1.1.2),which hydrolyse aromatic esters, e.g. phenylacetate; they show little activity on tributyrin, and are not inhibited by organophosphates 2. B-type esterases(glycerol tricarboxyl esterases, aliphatic esterases, lipases; EC 3.1.1.3): they are most active on aliphatic esters although they show some activity on aromatic esters; they are inhibited by organophosphates 3. C-type esterases(cholinesterase; EC 3.1. 1.7: EC 3. 1. 1.8): they are most active on choline esters but hydrolyse some aromatic and aliphatic esters slowly; they are inhibited by organophosphates In normal milk, the ratio of A: B: C esterase activity is about 3: 10: 1 but the level of A-esterase activity increases considerably on mastitic infection.a and C esterases are considered to be of little technological significance in Classically, lipases hydrolyse ester bonds in emulsified esters, i.e. at a water/oil interface, although some may have limited activity on soluble esters;they are usually activated by blood serum albumin and Ca2+ which bind free fatty acids, which are inhibitory. Little lipolysis normally occurs in
322 DAIRY CHEMISTRY AND BIOCHEMISTRY Cathepsin D (EC3.4.23.5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH x 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile (inactivated by 70°C x 10min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd; its specificity on zsl- and p-caseins is quite similar to that of chymosin but it has very poor milk-clotting activity (McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level. Other proteinases. The presence of low levels of other proteolytic enzymes in milk has been reported (see Fox and McSweeney, 1996). Most of these originate from somatic cells, and their level increases during mastitic infection. The presence of cathepsin D, a lysozomal enzyme, in milk suggests that all the lysozomal proteinases are present in milk although they may not be active. These minor proteinases are considered to be much less significant than plasmin, but more work on the subject is necessary. 8.2.3 Lipases catalyse the development of hydrolytic rancidity in milk, and, consequently, lipases and lipolysis in milk have been studied extensively. Lipases and esterases (EC 3.1.1.-) Milk contains three types of esterase: 1. A-type carboxylic ester hydrolases (arylesterases; EC 3.1.1.2), which hydrolyse aromatic esters, e.g. phenylacetate; they show little activity on tributyrin, and are not inhibited by organophosphates. 2. B-type esterases (glycerol tricarboxyl esterases, aliphatic esterases, lipases; EC 3.1.1.3): they are most active on aliphatic esters although they show some activity on aromatic esters; they are inhibited by organophosphates. 3. C-type esterases (cholinesterase; EC 3.1.1.7; EC 3.1.1.8): they are most active on choline esters but hydrolyse some aromatic and aliphatic esters slowly; they are inhibited by organophosphates. In normal milk, the ratio of A : B : C esterase activity is about 3 : 10: 1 but the level of A-esterase activity increases considerably on mastitic infection. A and C esterases are considered to be of little technological significance in milk. Classically, lipases hydrolyse ester bonds in emulsified esters, i.e. at a water/oil interface, although some may have limited activity on soluble esters; they are usually activated by blood serum albumin and Ca2+ which bind free fatty acids, which are inhibitory. Little lipolysis normally occurs in
ENZYMOLOGY OF MILK AND MILK PRODUCTS milk because more than 90% of the lipase is associated with the casein micelles while the triglyceride substrates are in fat globules surrounded, and protected, by the fat globule membrane(MFGM). When the MFGM is damaged, lipolysis occurs rapidly, giving rise to hydrolytic rancidity Lipase was first isolated from skim milk and characterized by Fox and Tarassuk in 1967. The enzyme was optimally active at pH9.2 and 37"C and found to be a serine enzyme(inactivated by organophosphates). A lipo- protein lipase(LPL; activated by lipoprotein co-factors)was demonstrated in milk by Korn in 1962 and was isolated by Egelrud and Olivecrona in 72. LPL is, in fact, the principal indigenous lipase in milk and most recent work has been focused accordingly The molecule has been characterized at he molecular, genetic, enzymatic and physiological levels(see Olivecrona et a,1992) In addition to LPL, human milk contains a bile salts-activated lipase which probably contributes to the metabolism of lipids by breast-fed babies who have limited pancreatic lipase activity. Bovine milk and milks from other dairy animals do not contain this enzyme The lipolytic system in most milks becomes active only when the milk MFGM is damaged by agitation, homogenization or temperature fluctu ations. However, some individual cows produce milk which becomes rancid spontaneously, i.e. without apparent activation. Spontaneous rancidity was considered to be due to a second lipase, termed membrane lipase, which was believed to be associated with the mfgm, but recent evidence suggests that LPL is responsible for spontaneous rancidity following activation by a lipoprotein(co-lipase)from blood serum; normal milk will become sponta neously rancid if blood serum is added, suggesting that'spontaneous milks contain a higher than normal level of blood serum. Dilution of'spontaneous milk with normal milk prevents spontaneous rancidity, which consequently not normally a problem with bulk herd milks; presumably, dilution with normal milk reduces the lipoprotein content of the mixture to below the threshold necessary for lipase adsorption atural variations in the levels of free fatty acids in normal milk and the susceptibility of normal milks to lipolysis may be due to variations in the level of blood serum in milk Significance of lipase. Technologically, lipase is arguably the most signi ficant indigenous enzyme in milk. Although indigenous milk lipase may play a positive role in cheese ripening, undoubtedly the most industrially impor tant aspect of milk lipase is its role in hydrolytic rancidity which renders liquid milk and dair ducts unpalatable and eventually unsaleable Lipolysis in milk has been reviewed extensively(Deeth and Fitz-Gerald, 1995). As discussed in Chapter 3, all milks contain an adequate level of lipase for rapid lipolysis, but become rancid only after the fat globule membrane has been damaged
ENZYMOLOGY OF MILK AND MILK PRODUCTS 323 milk because more than 90% of the lipase is associated with the casein micelles while the triglyceride substrates are in fat globules surrounded, and protected, by the fat globule membrane (MFGM). When the MFGM is damaged, lipolysis occurs rapidly, giving rise to hydrolytic rancidity. Lipase was first isolated from skim milk and characterized by Fox and Tarassuk in 1967. The enzyme was optimally active at pH 9.2 and 37°C and found to be a serine enzyme (inactivated by organophosphates). A lipoprotein lipase (LPL; activated by lipoprotein co-factors) was demonstrated in milk by Korn in 1962 and was isolated by Egelrud and Olivecrona in 1972. LPL is, in fact, the principal indigenous lipase in milk and most recent work has been focused accordingly. The molecule has been characterized at the molecular, genetic, enzymatic and physiological levels (see Olivecrona et al., 1992). In addition to LPL, human milk contains a bile salts-activated lipase, which probably contributes to the metabolism of lipids by breast-fed babies who have limited pancreatic lipase activity. Bovine milk and milks from other dairy animals do not contain this enzyme. The lipolytic system in most milks becomes active only when the milk MFGM is damaged by agitation, homogenization or temperature fluctuations. However, some individual cows produce milk which becomes rancid spontaneously, i.e. without apparent activation. Spontaneous rancidity was considered to be due to a second lipase, termed membrane lipase, which was believed to be associated with the MFGM, but recent evidence suggests that LPL is responsible for spontaneous rancidity following activation by a lipoprotein (co-lipase) from blood serum; normal milk will become spontaneously rancid if blood serum is added, suggesting that ‘spontaneous milks’ contain a higher than normal level of blood serum. Dilution of ‘spontaneous milk’ with normal milk prevents spontaneous rancidity, which consequently is not normally a problem with bulk herd milks; presumably, dilution with normal milk reduces the lipoprotein content of the mixture to below the threshold necessary for lipase adsorption. Natural variations in the levels of free fatty acids in normal milk and the susceptibility of normal milks to lipolysis may be due to variations in the level of blood serum in milk. Sign8cance of lipase. Technologically, lipase is arguably the most significant indigenous enzyme in milk. Although indigenous milk lipase may play a positive role in cheese ripening, undoubtedly the most industrially important aspect of milk lipase is its role in hydrolytic rancidity which renders liquid milk and dairy products unpalatable and eventually unsaleable. Lipolysis in milk has been reviewed extensively (Deeth and Fitz-Gerald, 1995). As discussed in Chapter 3, all milks contain an adequate level of lipase for rapid lipolysis, but become rancid only after the fat globule membrane has been damaged
324 DAIRY CHEMISTRY AND BIOCHEMISTRY 8.2.4 Phosphatases Milk contains several pho he principal ones being alkaline and acid phosphomonoesterase are of technological significance, and ribonuclease which has no function or significance in milk. The alkaline and acid phosphomonoesterases have been studied extensively( see Andrews(1993)for references) alkaline phosphomonoesterase (EC 3. 1.3.1). The existence of a phospha- tase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed and a test procedure based on alkaline phosphatase inactivation was developed control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl- phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively X-O-P-OH-------NN PO 3.+ XOh where XOH = phenol, p-nitrophenol or phenolphthalein The release of inorganic phosphate may be assayed but the other product sually determined. Phenol is colourless but forms a coloured complex on reaction with one of several reagents, e.g. 2, 6-dichloroquinonechloroimide, with which it forms a blue complex. p-Nitrophenol is yellow while phenol- hthalein is red at the alkaline ph of the assay (10)and hence the concentration of either of these may be determined easily Isolation and characterization. Alkaline phosphatase is concentrated in the fat globule membrane and hence in cream. It is released into the buttermilk on phase inversion; consequently, buttermilk is the starting material for most published methods for the purification of alkaline phos phatase. Later methods have used chromatography on various media to give a homogeneous preparation with 7440-fold purification and 28% yield The characteristics of milk alkaline phosphatase are summarized in Table 8.2. The enzyme appears to be similar to the alkaline phosphatase of mammary tissue
3 24 DAIRY CHEMISTRY AND BIOCHEMISTRY 8.2.4 Phosphatases Milk contains several phosphatases, the principal ones being alkaline and acid phosphomonoesterases, which are of technological significance, and ribonuclease, which has no known function or significance in milk. The alkaline and acid phosphomonoesterases have been studied extensively (see Andrews (1993) for references). Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacteriurn tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenylphosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively: where XOH = phenol, p-nitrophenol or phenolphthalein. The release of inorganic phosphate may be assayed but the other product is usually determined. Phenol is colourless but forms a coloured complex on reaction with one of several reagents, e.g. 2,6-dichloroquinonechloroimide, with which it forms a blue complex. p-Nitrophenol is yellow while phenolphthalein is red at the alkaline pH of the assay (10) and hence the concentration of either of these may be determined easily. Isolation and characterization. Alkaline phosphatase is concentrated in the fat globule membrane and hence in cream. It is released into the buttermilk on phase inversion; consequently, buttermilk is the starting material for most published methods for the purification of alkaline phosphatase. Later methods have used chromatography on various media to give a homogeneous preparation with 7440-fold purification and 28% yield. The characteristics of milk alkaline phosphatase are summarized in Table 8.2. The enzyme appears to be similar to the alkaline phosphatase of mammary tissue
MILK AND MILK PRODUCTS Table 8.2 Characteristics of milk alkaline phosphatase Casein: 6.8 p-nitrophenylphosphate: 9.65 p-nitrophenylphosphate: 10.5 0.69 mM on p-nitrophe hate Activators 170-190kDa 2 subunits of molecular weight 85 k Da formed on heating (100C for 2 min or acidification to pH 2.1) Polymorphic forms Reactivation of phosphatase. Much work has been focused on a phe- nomenon known as 'phosphatase reactivation, first recognized by wright and Tramer in 1953, who observed that UHT-treated milk was phos phatase-negative immediately after processing but became positive on tanding microbial phosphatase was shown not to be responsible. Bulk HTST milk never showed reactivation, although occasional individual-cow samples did; HTST pasteurization after UHT treatment usually prevented reactivation and reactivation was never observed in very severely heated milk. Reactivation can occur following heating at temperatures as low as 84C for milk and 74 C for cream; the optimum storage temperature for eactivation is 30 C, at which reactivation is detectable after 6 h and may continue for up to 7 days. the greater reactivation in cream than in milk may be due to protection by fat but this has not been substantiated. Mg. and Zn2+ strongly promote reactivation; Sn*, Cu2, Co2+ and EDTA are inhibitory, while Fet has no effect ulphydryl-(SH) groups appear to be essential for reactivation; perhaps this is why phosphatase becomes reactivated in UHT milk but not in HTST milk. The role of-SH groups, supplied by denatured whey proteins, is considered to be chelation of heavy metals, which would otherwise bind to renaturation. The role of Mg2+ or Zn'* is seen as causing a conformational change in the denatured enzyme, necessary for renaturation Reactivation of alkaline phosphatase is of considerable practical signifi- cance since regulatory tests for pasteurization assume the absence of phosphatase activity. An official AOAC method used to distinguish between renatured and residual native alkaline phosphata based on the increase in phosphatase activity resulting from addition of Mg2: the ac renatured alkaline phosphatase is increased about 14-fold but that of the native enzyme is increased only two-fold Although it can dephosphorylate casein under suitable conditions, as far as is known, alkaline phosphatase has no direct technological significance
ENZYMOLOGY OF MILK AND MILK PRODUCTS 325 Table 8.2 Characteristics of milk alkaline phosphatase Characteristic Conditions pH optimum Casein: 6.8 p-nitrophenylphosphate: 9.65 p-nitrophenylphosphate: 10.5 0.69 mM on p-nitrophenylphosphate Ca2+, Mn2', Zn2+, Co2+ 3g M 2+ 2 subunits of molecular weight 85 kDa formed on heating Temperature optimum 37°C Km Activators Molecular weight 170- 190 kDa Association/dissociation Polymorphic forms 4 (100°C for 2min or acidification to pH2.1) Reactivation of phosphatase. Much work has been focused on a phenomenon known as 'phosphatase reactivation', first recognized by Wright and Tramer in 1953, who observed that UHT-treated milk was phosphatase-negative immediately after processing but became positive on standing; microbial phosphatase was shown not to be responsible. Bulk HTST milk never showed reactivation, although occasional individual-cow samples did; HTST pasteurization after UHT treatment usually prevented reactivation and reactivation was never observed in very severely heated milk. Reactivation can occur following heating at temperatures as low as 84°C for milk and 74°C for cream; the optimum storage temperature for reactivation is 30°C, at which reactivation is detectable after 6 h and may continue for up to 7 days. The greater reactivation in cream than in milk may be due to protection by fat but this has not been substantiated. Mg2+ and Zn2+ strongly promote reactivation; Sn2+, CuZ+, Coz+ and EDTA are inhibitory, while Fe2+ has no effect. Sulphydryl -(SH) groups appear to be essential for reactivation; perhaps this is why phosphatase becomes reactivated in UHT milk but not in HTST milk. The role of -SH groups, supplied by denatured whey proteins, is considered to be chelation of heavy metals, which would otherwise bind to -SH groups of the enzyme (also activated on denaturation), thus preventing renaturation. The role of Mg2+ or Zn2+ is seen as causing a conformational change in the denatured enzyme, necessary for renaturation. Reactivation of alkaline phosphatase is of considerable practical significance since regulatory tests for pasteurization assume the absence of phosphatase activity. An official AOAC method used to distinguish between renatured and residual native alkaline phosphatase is based on the increase in phosphatase activity resulting from addition of Mg2+: the activity of renatured alkaline phosphatase is increased about 14-fold but that of the native enzyme is increased only two-fold. Although it can dephosphorylate casein under suitable conditions, as far as is known, alkaline phosphatase has no direct technological significance
326 DAIRY CHEMISTRY AND BIOCHEMISTRY in milk and milk products; perhaps its pH optimum is too far removed from that of milk; it is also inhibited by inorganic phosphate Acid phosphomonoesterase(EC 3. 1.3.2). Milk contains an acid phospha tase which has a pH optimum at 4.0 and is very heat stable(LTLT pasteurization causes only 10-20% inactivation and 30 min at 88C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7 he enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+(as is alkaline phosphatase), but it is slightly activated by Mn and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography; purification up to 40000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 k Da and an isoelectric point of 7. 9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it a num ber of cha Although casein is a substrate for milk acid phosphatase, the major caseins, in the order as(as1 +as2)>B>K, also act as competitive inhibitors of the enzyme when assayed on p-nitrophenylphosphate, probably due to binding of the enzyme to the casein phosphate groups(the effectiveness of the caseins as inhibitors is related to their phosphate content) Significance. Although acid phosphatase is present in milk at a much wer level than alkaline phosphatase, its greater heat stability and lower pH optimum may make it technologically significant. Dephosporylation of casein reduces its ability to bind Cazt, to react with K-casein, to form micelles and its heat stability. Several small partially dephosphorylated peptides have been isolated from Cheddar and Parmesan cheese. However is not known whether indigenous or bacterial acid phosphatases are mainly responsible for dephosphorylation in cheese. Dephosphorylation may be rate-limiting for proteolysis in cheese ripening since most pro teinase and peptidases are inactive on phosphoproteins or peptides. It has been suggested that phosphatase activity should be included in the criteria for starter selection
326 DAIRY CHEMISTRY AND BIOCHEMISTRY in milk and milk products; perhaps its pH optimum is too far removed from that of milk; it is also inhibited by inorganic phosphate. Acid phosphomonoesterase (EC 3.1.3.2). Milk contains an acid phosphatase which has a pH optimum at 4.0 and is very heat stable (LTLT pasteurization causes only 10-20% inactivation and 30min at 88°C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7, the enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+ (as is alkaline phosphatase), but it is slightly activated by Mn2+ and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase; activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation. Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography; purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. Although casein is a substrate for milk acid phosphatase, the major caseins, in the order cts(ctsl + ~1,~) > p > K, also act as competitive inhibitors of the enzyme when assayed on p-nitrophenylphosphate, probably due to binding of the enzyme to the casein phosphate groups (the effectiveness of the caseins as inhibitors is related to their phosphate content). Signijicance. Although acid phosphatase is present in milk at a much lower level than alkaline phosphatase, its greater heat stability and lower pH optimum may make it technologically significant. Dephosporylation of casein reduces its ability to bind Caz+, to react with K-casein, to form micelles and its heat stability. Several small partially dephosphorylated peptides have been isolated from Cheddar and Parmesan cheese. However, it is not known whether indigenous or bacterial acid phosphatases are mainly responsible for dephosphorylation in cheese. Dephosphorylation may be rate-limiting for proteolysis in cheese ripening since most proteinases and peptidases are inactive on phosphoproteins or peptides. It has been suggested that phosphatase activity should be included in the criteria for starter selection