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