Studies on rumen metabolism VI. —in vitro hydrolysis of triglyceride and isolation of a lipolytic fraction

Hydrolysis of groundnut oil and synthetic glyceryl tri(oleate‐1‐14C) by bovine rumen contents was demonstrated under in vitro conditions. Fractionation by differential centrifugation of total rumen contents obtained from a cow grazing on fresh pasture showed that the organisms responsible for hydrolysis of triglycerides were closely associated with the particulate material in the rumen and homogenisation released much of the lipolytic activity. Cell‐free extracts of mixed rumen bacteria were prepared by subjecting mixed rumen bacteria to high frequency sonication or osmotic shock followed by isolation of the released lipolytic enzymes by high‐speed centrifugation. A method for assaying lipase activity is described which uses glyceryl tri(oleate‐1‐14C) as substrate, and thin‐layer chromatography to separate the free fatty acids, mono‐, di‐ and tri‐glycerides prior to radio‐chemical determination. The method was developed using pancreatic lipase and was applied to the isolation of lipases from rumen micro‐organisms.