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