Activité de la Phosphatase Alcaline (PAL) dans les laits de vache et les fromages au lait de vache

Activité de la Phosphatase Alcaline (PAL) dans les laits de vache et les fromages au lait de vache

Alkaline phosphatase is a naturally occurring enzyme in milk. Because thermal resistance of the enzyme is greater than that of non-spore forming pathogenic microorganisms commonly found in milk, the detection of alkaline phosphatase is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk (Aschaffenburg & Mullen, 1949, Murthy & Cox, 1988). However, the presence of alkaline phosphatase activity in properly pasteurized milk as a consequence of contamination with bacterial alkaline phosphatases has been reported (Hammer & Olson, 1941). To assist in identifying this microbially produced heat-resistant phosphatase activity, Knight & Fryer (1989) proposed to test again after laboratory re-pasteurization any sample failing the alkaline phosphatase activity test. However, Murthy & Kaylor (1990) demonstrated that it was not possible to differentiate between bovine milk and microbial alkaline phosphatase by the proposed re-pasteurization test, because both heat-labile and heat-stable alkaline phosphatase could be produced by microorganisms such as Bacillus anthracis, Bacillus cereus, Bacillus megaterium (Dobozy & Hammer, 1969), Micrococcus sadonesis (Glew & Heath, 1971), Saccharomyces cerevisiae (Gorman & Hu, 1969). Moreover, when testing alkaline phosphatase by phenol release, false positive results have been reported with pesticides, such as baygon (2-isopropoxy-phenyl-n-méthyl carbamate) and sevin (1-naphtyl-n-methyl carbamate) (Kumar et al. 1973), or antibiotics such as penicillin and oxytetracyclin (Manolkidis & Alichanidis, 1971). Conversely, false negative results can be due to the presence of pesticides such as phosphamidon and 0,0-dimethyl-2,2-dichlorovinyl phosphate (Kumar et al. 1973), or antibiotics such as streptomycin, erythromycin and neomycin (Manolkidis & Alichanidis, 1971).

To determine milk alkaline phosphatase without interference from microbial alkaline phosphatase, pesticides or antibiotics, an immunological approach based on polyclonal antibodies (PAbs) produced against bovine milk alkaline phosphatase has been proposed by Vega-Warner et al. (1999). The antibodies have been used to develop a competitive indirect ELISA specific for the bovine milk alkaline phosphatase (Vega-Warner et al. 2000). However, the PAbs cross-reacted with heat-denatured alkaline phosphatase and the ELISA was thus not specific enough to be used to verify adequate heat treatment of milk. Chen et al. (2006) used the same approach with PAbs developed in hens. The antibodies cross-reacted with Escherichia coli alkaline phosphatase and their reactivity against bovine alkaline phosphatase in raw milk was not established. Monoclonal antibodies specific of the bovine milk alkaline phosphatase have been recently obtained in mice from a raw bovine milk alkaline phosphatase preparation (Geneix et al, 2007). Coated in microtitre plates, these antibodies specifically capture the bovine milk alkaline phosphatase from dairy products. After washing (elimination of unbound components such as non-bovine milk PALs, antibiotics or pesticides) the enzymatic activity of the captured alkaline phosphatase is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with alkaline phosphatases of intestinal or bacterial origin and was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution Geneix et al, 2007). The test has been successfully applied to the study of the inactivation-denaturation kinetics of bovine milk phosphatase during mild heating (Levieux et al, 2007). The GTP Immuno Alkaline Phosphatase kit (PALTest) uses the monoclonal antibodies of Geneix et al (2007) with an improved two steps substrat which allows the detection of raw milk in boiled milk down to a 0.005% dilution (50 mU/L).


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