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 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 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-nitrophenyl￾phosphate 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 phenol￾phthalein 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