Edwards, Holly Danielle (2012-07). Development of Methodology and Characterization of Ruminal Lipase-Producing Bacteria In Vitro. Master's Thesis.
Thesis
Hydrolysis of dietary lipids to free fatty acids (FFA) is a prerequisite for ruminal biohydrogenation, a bacterially mediated process that extensively saturates unsaturated FFAs thus limiting the absorption and ultimate assimilation of these healthy nutrients into ruminant produced foods. Three experiments were conducted to learn how to better enrich, isolate and study lipolytic bacteria from the rumen while providing further characterization of four prominent lipase-producing bacteria that are known to be major contributors of lipolysis in the rumen. In experiment one the effects of various physical treatments on ruminal lipase activity were investigated by comparing incubation positions, glass bead levels, transfer techniques and combinations of headspace gasses. Based on results from this experiment an incubation system was established as a standard for subsequent studies for culturing and transferring mixed and pure cultures of ruminal bacteria. In experiment two the effect of glycerol on lipolysis by Anaerovibrio lipolyticus 5S, Butyrivibrio fibrisolvens 49, Propionibacterium avidum, and Propionibacterium acnes was examined. Two levels of glycerol were examined on lipase activity and results showed that glycerol inhibited rates of FFA accumulation at both levels. In addition the mechanism behind glycerol inhibition was also examined by culturing and assaying activity of the four bacteria to determine if glycerol inhibition is a result of equilibrium displacement or lipase gene expression inhibition. Results indicated that higher and constitutively expressed lipase activity of A. lipolyticus 5S and P. avidum probably contribute more to lipolysis in ruminants than P. acnes and B. fibrisolvens 49. In the case of P. acnes and B. fibrisolvens 49 cells, results suggest that lipase gene expression is down-regulated in these bacteria. Experiment three was conducted to further characterize the lipase activity of the four different bacteria by growing them with four different energy substrates and measuring enzyme activity at early logarithmic and stationary phase. Results from this study showed that diets containing a high content of oleic acid and linolenic acid promoted higher rates of lipolysis in the rumen. In accordance with findings in experiment two these results support that P. avidum may contribute to a higher amount of lipolysis than previously considered.
