The reactivation of milk alkaline phosphatase after heat treatment

Abstract

A simple system was developed, consisting of a solution of jS-glycerophosphate, jS-lactoglobulin and magnesium ions, in which alkaline phosphatase isolated from milk became strongly reactivated following heat treatment for 45 sec in boiling water. From 10 to 30% of the original enzyme activity reappeared after incubation at 37 °C. Variations in the components of the system, and factors affecting it, were studied. Salts of Hg, Zn and Cd inhibited reactivation at low concentrations.Milk which, after the same heat treatment, became reactivated to a much smaller extent (about 1 %), was found to contain a dialysable, heat labile inhibitor whose presence is thought to be largely responsible for the low level of reactivation, though other factors, e.g. the suboptimal concentration of phosphate esters must be considered as contributory causes.Milk contains also a non-dialysable, heat stable activator of the reactivation process, capable of replacing /Mactoglobulin in the simple system and active at very low concentration, e.g. 0-2% (v/v).When milk or cream is exposed briefly to high temperatures, cooled and stored, the alkaline phosphatase activity, which is absent immediately after heating, tends to reappear. The conditions under which reactivation occurs have been studied by Wright & Tramer (1953a, b, 1954, 1956), Fram (1957a and McFarren, Thomas, Black & Campbell (1960). Reactivation has been observed after heating milk to temperatures ranging from 82 °C, and cream from 74 °C to 180 °C for times varying from a few seconds to a few minutes and after subsequent storage at between 5 and 40 °C. The effect is not due to bacterial growth. Reactivation does not occur in milk that has been pasteurized by the accepted H. T. S. T. process [161 °F (71-7 °C) for 15 sec], so that the efficacy of that treatment can be controlled by the phosphatase test. With cream, however, the application of this test presents difficulties since more severe heat treatment of this product is often favoured and since cream has been observed to become reactivated after being heated to as low a temperature as 74 °C.Milk is a complex medium, and in the hope of clarifying the nature of the processes involved in reactivation we have prepared alkaline phosphatase in a highly purified state and studied its reactivation both in systems simpler than milk and in milk itself.

Indigenous enzymes in milk: Overview and historical aspects—Part 2

“…A number of attempts have been made to explain the mechanism of reactivation of AlP (see Andrews et al, 1992;Copius Peereboom, 1970;Fox, 2003;Fox & Morrissey, 1981;Fox et al, 2003;Kresheck & Harper, 1967;Linden, 1979;Lyster & Aschaffenburg, 1962;Murthy, Cox, & Kaylor, 1976;Shakeel-ur-Rehman et al, 2003). There is evidence that the form of the enzyme that becomes reactivated is membrane-bound and several factors which influence reactivation have been established.…”

Ultra-High-Temperature Processing of Milk and Milk Products

“…showed that reactivation did not take place on storage at lOoe or less, but that at temperatures above 20 0 e reactivation was considerable, and was complete after about 14 days. Lyster & Aschaffenburg (1962) examined the phenomenon, and postulated that the activity of alkaline phosphatase was determined by the combined effects of an inhibitor and an activator, both being heat labile but with different kinetics.…”

Use of a fluorimetric test for bovine alkaline phosphatase to demonstrate under-pasteurisation of skimmed milk and cream

“…All samples with an alkaline phosphatase activity of greater than 100 mU L À1 were retested to confirm the result and, in addition, were tested for bacterial phosphatase (Knight & Fryer, 1989;BS ISO 11816-1, 1997) and reactivation of phosphatase enzyme (Lyster & Aschaffenburg, 1962;AOAC, 1995, Chapter 33). If either bacterial phosphatase or reactivation were detected in samples the sample results were excluded.…”