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 significanceENZYMOLOGY 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 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 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 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 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