CHANGES IN THE LEVELS OF PROTEOLYTIC ENZYMES IN ENSILED ALFALFA FORAGE

Abstract

The activities of several proteolytic enzymes were quantified in ensiled alfalfa (Medicago sativa) forage and their activities were related to proteolysis in the ensiled crop. The enzymes studied were carboxypeptidase at pH 5.2, aminopeptidase at pH 7.0, and acid proteinase against hemoglobin at pH 4.5 and azocasein at pH 5.5. Proteolysis, which was measured as the increase in trichloroacetic acid-soluble, non-protein nitrogen, occurred during the first 1–2 days after ensiling. Silage pH was above 5.5 during the time period of proteolysis. The rate of proteolysis in the ensiled herbage was approximately 25 μmoles amino acid released per gram dry weight per hour, and can be accounted for by the activity of proteolytic enzymes in alfalfa herbage without consideration of microbial enzymes. The activity of the alfalfa proteolytic enzymes declined with time after ensiling, but each enzyme declined independently. For example, the proteinase at pH 4.5 was inactive after 2 days whereas carboxypeptidase retained 45% of its activity even at 21 days after ensiling. The cessation of proteolysis after 2 days ensiling was not due to the lack of proteolytic enzyme activity nor was pH completely inhibitory. The results are further discussed in regard to the regulation of proteolysis during ensiling.