Evaluation of APHA and AOAC II methods for phosphatase in butter and differentiation of milk and microbial phosphatases by agarose-gel electrophoresis.

Salted and unsalted butters with 3 levels of phosphatase were prepared with both raw and pasteurized cream containing 36% fat. Test samples were analyzed for phosphatase by the modified method of the American Public Health Association (APHA) and the official AOAC method, 16.256 (1984, 14th Ed., 1990 15th Ed., 946.02). In the APHA method, weighing of solid frozen butter for testing yielded repeatable results. Addition of 0.0-1.0 mg magnesium to the butter had little effect on phosphatase activity in the APHA modified rapid colorimetric method (MRCM), but caused the phosphatase activity to decrease in the AOAC method. Phosphatase in salted and unsalted butters was quite stable at -17 +/- 1 degrees C and at 3.0 +/- 0.5 degrees C; however, within 2 to 4 days, freshly prepared butters stored at 22 +/- 1 degrees C developed reactivated and/or microbial phosphatases that were both heat-labile and heat-stable. At 22 +/- 1 degrees C, frozen butters showed decreased milk phosphatase activity before producing microbial phosphatase. Heat-labile phosphatases in salted and unsalted butters were inactivated at 62.8 degrees C for 10 min, and the phosphatase lability was partially due to the heat-denaturing effect of NaCl in salted butter. Some heat-stable phosphatases in unsalted butter survived at 66 degrees C for 30 min. Differentiation of milk phosphatase from microbial phosphatases was difficult by both methods; however, they were successfully differentiated by the agarose-gel electrophoretic technique.

Melanogenesis in murine B16 cells exposed to Aeromonas hydrophila cytotoxic enterotoxin.

Specific markers (growth, melanogenesis) of B16 murine melanoma cells in culture were used as indicators of toxin production by Aeromonas hydrophila. Cytotonic enterotoxinlike activity (inhibited growth, raised tyrosinase activity, and melanin accumulation) occurred at cytotoxic end points of purified beta-hemolysin and several culture filtrates. Antihemolysin rabbit serum inhibited this activity. A hemolysin-neutralized culture filtrate concentrate (10X) failed to elevate tyrosinase relative to untreated and cholera toxin treated controls. Similar dilution profiles using Chinese hamster ovary cells showed limited cell extension only at cytotoxic end points with antihemolysin inhibiting this activity. Cytotoxicity of Chinese hamster ovary cells and B16 cells was proportional to hemolytic activity, with B16 cells showing about 100-fold greater sensitivity on a per cell basis. Cell culture cytotoxicity attributed to beta-hemolysin correlated with reactivity in rabbit ileal loop assays. The ADP-ribosyl transferase activity of concentrated (10X) A. hydrophila culture filtrates and fractions thereof was negative. Apparently sublethal doses of A. hydrophila beta-hemolysin can nonspecifically stimulate cyclic adenosine monophosphate mediated events in melanoma and Chinese hamster ovary cell assays, producing lower activities than cholera toxin with shorter lag times.