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Title:   Author  item
                                            EDWARDS, HOLLY - Texas A&M Universityitem
                                            Anderson, Robinitem
                                            TAYLOR, T - Texas A&M Universityitem
                                            MILLER, RHONDA - Texas A&M Universityitem
                                            HARDIN, MARGARET - Texas A&M Universityitem
                                            KRUEGER, NATHAN - Blinn Collegeitem
                                            Nisbet, David
  Submitted to: Agriculture, Food and Analytical Bacteriology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 7/26/2013
                                    Publication  Date: 12/1/2013
                                    Citation: Edwards, H.D., Anderson, R.C., Taylor, T.M., Miller, R.K., Hardin, M.D., Krueger, N.A., Nisbet, D.J. 2013. Development of non-forage based incubation system for culturing ruminal lipase-producing bacteria in vitro. Agriculture, Food and Analytical Bacteriology. 3:293-302.Interpretive Summary: Metabolism of dietary lipids within the gastrointestinal tract of cattle, sheep, and goats can differentially affect populations of gut microbes.  For instance, metabolism of dietary fats high in medium chain saturated fatty acids, such as the 12-carbon containing lauric acid and 14-carbon containing myristic acid, can cause significant inhibition of Gram-positive bacteria, such as undesirable Clostridium species.  Conversely, metabolism of fats containing large amounts of unsaturated long chain fatty acids, those containing 16 carbons or more, can cause broad spectrum inhibition of both Gram-positive and Gram-negative bacteria, but it is not known if this effect is specific or not specific to pathogens.  Studying the effects of lipids on the different types of gut bacteria is difficult, however, because oil substrates do not mix well in microbial incubation systems (i.e., oil and water do not mix).  The inclusion of gut digesta can markedly enhance rates of microbial lipid digestion in culture tubes by providing a solid support to catch and spread out the oil.  Reproducibility is compromised by adding the digesta, however, because digesta is not very uniform in size or structure.  Consequently, the objective of the present study was to develop a digesta-free method for culturing and assaying lipid metabolism of mixed and pure populations of known gut lipid-metabolizing bacteria.  Results showed a 95% increase in lipid metabolism by cultures incubated with 21 g of glass beads compared to cultures incubated in fluid without beads.  Addition of glass beads also increased lipid metabolism 73% over that of cultures incubated with digesta.  Results suggest that the use of glass beads is sufficient for examining lipid metabolism by mixed cultures.  Addition of glass beads to pure cultures of lipid-metabolizing bacteria did not show significant increases in free fatty acid release; however, results showed that there was substantial reduction in variation compared to pure cultures incubated without the glass beads.  Thus, inclusion of glass beads provides a clean and consistent incubation system for examining lipid metabolism by gut bacteria.  This new technology can help scientists better understand how different lipid substrates can affect microbes inhabiting the gastrointestinal tract of their animals.  Ultimately, this research may lead to new strategies to help farmers and ranchers continue to produce safe and wholesome foods for the American consumer.
                                Technical Abstract: Metabolism of dietary lipids within the ruminant gastrointestinal tract can differentially affect populations of gut microbes residing in the rumen or intestine.  For instance, hydrolysis of dietary fats in the rumen can liberate different mixtures of free fatty acids, depending on the dietary source.  If proportions of medium chain saturated fatty acids, such as lauric and myristic acid, are high, significant inhibition of Gram-positive bacteria can occur.  Conversely, if proportions of long-chain unsaturated fatty acids are high, then broad spectrum inhibition of both Gram-positive and -negative bacteria can occur.  Studying the effects of lipids on gut bacteria is difficult, however, because oil substrates do not mix into an aqueous in vitro system.  The inclusion of rumen digesta can markedly enhance rates of microbial lipolysis in vitro, but consistency is often compromised because digesta is not homogeneous in size or structure.  Consequently, the objective of the present study was to develop a digesta-free method for culturing and assaying lipolytic activity of mixed as well as pure populations of known ruminal lipase-producing bacteria.  The inclusion of 0, 11, and 21 g of glass beads was examined in place of digesta.  Results showed a 95% increase (P < 0.05) from the nonbead treatments to the 21 g treatments.  Activity was further increased (P < 0.05) in a separate study when tubes containing beads were incubated horizontally rather than vertically.  Addition of glass beads increased (P < 0.50) lipolytic activity over rumen digesta by 73%.  Results suggest that the use of glass beads is sufficient for examining lipolytic activity for mixed cultures in vitro.  Addition of glass beads to incubations of pure cultures of lipase-producing bacteria did not show significant increases in free fatty acid release; however, results showed that there was substantial reduction in variation in cultures incubated with the glass beads.  Thus, inclusion of glass beads provides a clean and consistent incubation system for examining lipase activity in vitro.


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