Fluorimetric determination of alkaline phosphatase in solid and fluid dairy products

A new assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products. The method proposed is simple, rapid and directly applicable to solid and liquid dairy samples. ALP in the test sample hydrolyzes a non fluorescent substrate, trifluoromethyl-β-umbelliferone phosphate, to its highly fluorescent phenolate product. The assay is performed in a reverse micellar medium composed of mixed buffer (2-amino-2-methyl-1-propanol buffer pH 9.0 and borate buffer pH 9.0) in AOT/isooctane, at a temperature of 38 °C. Total test time is 450 s. Reaction rates are linear (except for butter) up to 8.5 and 11% (v/v) raw milk, for whole milk and chocolate milk, respectively. The detection limits are 0.04, 0.4 and 0.22% (v/v) raw milk, for whole milk, chocolate milk and butter, respectively. The precision of the fluorimetric method was assessed by repeated analysis of a pasteurized milk sample spiked with mixed herd raw milk. The accuracy of the method was evaluated by comparison with an official colorimetric assay using p-nitrophenylphosphate as ALP substrate.