Determination of alkaline phosphatase inactivation in pasteurised milk under different heat treatment conditions

Raw milk is considered an excellent culture medium for a variety of pathogenic and non-pathogenic microorganisms. The effectiveness of raw milk pasteurisation is verified internationally by measuring residual alkaline phosphatase (ALP) activity. The aim of this study was to evaluate the performance of the official Brazilian qualitative method in comparison with Scharer's quantitative spectrophotometric method for assessing ALP inactivation. The evaluation was carried out on samples of raw milk subjected to different heat treatments (67, 70, 72, 75, and 77 °C for 20 s, and 65 °C for 30 min). In addition, the influence of fat content on the thermal protection of the enzyme was assessed, and detection limits were determined by adding phenol standard solutions to milk with inactivated ALP. The official qualitative method did not demonstrate sufficient sensitivity, with a visual detection limit of 4.13 μg phenol ml-1, which is four times higher than the internationally accepted limit for inadequate pasteurisation, thus generating false-negative results in under-pasteurised samples. A positive correlation was established between the fat content of the milk and the residual ALP activity, demonstrating that the membrane of milk fat globules provides significant thermal protection to the enzyme. Furthermore, Scharer's quantitative method produced false-positive results for properly pasteurised samples, suggesting that its established quantitative limit may be inadequate. In conclusion, the official Brazilian qualitative method proved inadequate for reliably verifying the safety of milk pasteurisation, and the limit established by the Scharer method requires optimisation to avoid false positives in milk testing.