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

by IDBiotech, 13 juillet 2015

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 IDBiotech 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).

 


References

Aschaffenburg R & Mullen JEC 1949 A rapid and simple phosphatase test for milk. Journal of Dairy Research 16 58-67

Black R, Kuzyk M & Duggan J 1992 Evaluation of a fluorometric assay for alkaline phophatase in fluid dairy products. Australian Journal of Dairy Technology 47 64-67

Blel M, Guingamp MF, Gaillard JL & Humbert G 2002. Studies on the thermal sensitivity of γ-glutamyl transpeptidase measured with a modified test procedure and compared with that of alkaline phosphatase and lactoperoxidase in milk. Lait 82 555-566

Chen CC, Tai YC, Shen SC, Tu YY, Wu MC & Chang HM 2006 Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase. Food Chemistry 95 213-220

Dobozy K & Hammer BW 1969 Some properties of alkaline phosphatase in Bacillus species. Acta Microbiologica of the Academy of Sciences Hungarian 16 181-184

Geneix N, Dufour E, Vénien A & Levieux D 2007 Development of a monoclonal antibody-based immunoassay for specific quantitation of bovine milk alkaline phosphatase. Journal of Dairy Research 74 290-295

Glew RH & Heath EC 1971 Studies on the extracellular alkaline phosphatase of Micrococcus sadonesis. II : Factors affecting secretion. Journal of Biological Chemistry 246 156-132

Gorman J & Hu W 1969 The separation and partial characterization of L-histidinol phosphatase and an alkaline phosphatase of Saccharomyces cerevisiae. Journal of Biological Chemistry 244 1645-1654 Hammer BW & Olson HC 1941 Phosphatase production in dairy products by microorganisms. Journal of Milk and Food Technology 4 83-101

Knight AH Fryer SM 1989 The development of heat resistant phosphatase activity in raw milk. Journal of the Society of Dairy Technolology 42 8186-8193

Kumar P, Sud RK & Gupta KG 1973 Interference of some pesticides in the milk phosphatase pasteurization test. Journal of Dairy Science 56 553-557

Levieux D, Geneix N, Levieux A 2007 Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay. Journal of Dairy Research 74 296-301

Manolkidis KS & Alichanidis ES 1971 Effects of some antibiotics on the milk phosphatase pasteurization test. Journal of Dairy Science 54 335-338

Murthy GK & Cox S 1988. Evaluation of APHA and AOAC methods for phosphatase in cheese. Journal of the Association of Official Analytical Chemists 71 1195-1199

Vega-Warner AV, Wang CH, Smith DM & Ustunol Z 1999 Milk alkaline phosphatase purification and production of polyclonal antibodies. Journal of Food Science 64 601-605

Vega-Warner AV, Gandhi H, Smith D & Ustunol Z 2000 Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase. Journal of Agriculture and Food Chemistry 48 2087-2091

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