Abstract Cold raw milk with .1 ppm of added copper was pumped at a rate of 2 liters/h through a bed of 20 g of glass-bound trypsin for 5 h daily for 14 days. After each daily run the reactor was back washed with distilled water, flooded with 20% ethanol in phosphate buffer, and refrigerated. Samples were pasteurized, stored 6 days, and tested for oxidation. All copper-containing samples were protected from oxidation by the treatment. The bound enzyme showed no loss of activity over the 14 days as determined by N-benzoyl-L-arginine ethyl ester hydrolysis. Standard plate counts of ethanol solution after storage, aqueous washing, and the milk before and after the treatment showed that 20% ethanol was effective in limiting microbial growth, that there was no microbial build-up in the reactor during the 5 h of continuous milk passage, and that milk was not substantially contaminated by the treatment. Flow rate decreased with time whether the reactor was used as a packed column or a fluidized bed due to plugging of the column or the retaining screen. No increase in nonprotein nitrogen in the treated samples was detected. However, when casein was used as the substrate, increases in nonprotein nitrogen and free amino groups were appreciable.
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