Characterization of total proteolytic activity in alfalfa and red clover

Ensiled legumes will degrade 7-87% of their protein due to proteolysis during the ensiling process prior to feeding. The loss in forage protein needs to be corrected by supplementation and ration balancing which are additional expense to the dairy producer. It would be economically beneficial for producers to lower the extent of proteolysis in the silo.;Two forage legumes, different in their extent of proteolysis in the silo, were selected, ensiled into laboratory silos, and monitored for proteolytic activity. Red clover silages had 40% lower proteolysis than alfalfa after 7 days of ensiling. Characterization of proteolytic activity in red clover and alfalfa extracts showed that proteolytic activity in both legumes had similar rates, pH optima and stabilities and temperature optima and stabilities. It was concluded that the difference in extent of proteolysis of these two legumes during ensiling was not due to differences in their inherent proteolytic activity. It was concluded that a soluble protein was the factor.;Red clover is a forage that will brown upon tissue damage due to polyphenol oxidase activity. Activity of this polyphenol oxidase in a crude extract of red clover and red clover:alfalfa mixture will inhibit proteolysis. A method was developed to screen both different forage legumes and red clovers for browning and polyphenol oxidase activity. Red clover was the only legume to have both activities, and these activities were consistently present in all red clovers.;A soluble polyphenol oxidase was partially purified from red clover and characterized for pH effects (optima and stability) and temperature stability. Substrate staining of red clover tissue localized polyphenol oxidase activity to the epidermal region. It was concluded that the lower extent of proteolysis in red clover was due to the presence of this soluble polyphenol oxidase. Further research will be conducted to understand how polyphenol oxidase inhibits proteolysis and adapting the application of this method to other legume systems.