Enrichment of proteolytic activity relative to nitrogen in preparations from the rumen for in vitro studies

Three experiments compared treatment of rumen contents on recovery of proteolytic activity per ml rumen fluid (PA) and per mg nitrogen (PA/N). Proteolytic activity was determined by degradation of azocasein for 2 h, and N was determined by the Kjeldahl method. The first experiment compared effects of chilling, blending, rinsing, fractionating by centrifugation at 400g and 20 000g, and their interactions. The second experiment compared rumen fluid from a fistulated cow to the same rumen fluid that was incubated in continuous culture (dilution rate=0.07 h−1) in media with high sugar content. The third experiment compared effects of extracting centrifugation pellets of rumen fluid with buffered media, media with detergent, and acetone. For all experiments, most activity was found in the centrifugation pellets. Rinsing of rumen solids with cold buffer increased PA and PA/N. Chilling tended to increase PA slightly, but had no effect on PA/N. Blending rumen contents did not affect PA, but decreased PA/N. Incubation in continuous culture did not affect PA/N. Mixing rumen fluid precipitate with buffered media solubilized 18% of PA. Treatment with detergent or acetone solubilized 51% and 48% of PA respectively. Activity per mg N was 3.9 times greater in supernatant than in original sample after treatment with detergent or acetone. Proteolytic activity was enriched most by treatment with detergent or acetone, and additional recovery and enrichment were provided by rinsing of rumen solids.