Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay

Abstract

A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50–60°C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56°C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6·7 deg C (D60°C=24·6 min) and 6·8 (D60°C=23·0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0·994) with those obtained by the colorimetric assay. A similar high correlation (r=0·998) was obtained when industrially thermized milks (62–67°C for 20–90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.