Ecological Analysis Of The Amino Acid Metabolism In Pig Small Intestinal Bacteria

The small intestine is the major site for the digestion, metabolism and absorption of dietary nutrients. Increasing evidence indicate that the first-pass small intestinal metabolism of dietary amino acids (AA) play important role in the whole-body AA homeostasis. The low portal balance of essential AA (EAA) suggested that the small intestine is metabolic active with high metabolic requirements of AA. The lack of key enzymes in the catabolism of most of the EAA in pig enterocytes indicate that other factors might be responsible for the extraction of dietary AA during the first-pass metabolism. Meanwhile, recent findings revealed that the small intestinal microbiota might active in the metabolism of dietary AA thus regulate the compartmental AA metabolism of the host. However, the detail is largely unknown. Therefore, to unravel the protein and AA metabolism in small intestinal microbiota might help to develop new strategies to improve the nutrition and function of dietary proteins and AA in humans and animals. The present study hypotheses that the pig small intestinal bacteria is active in the metabolism of AA and the bacterial utilization and metabolism of AA is determined by both the composition and abundance of specific bacterial species and the luminal concentrations of AA in different compartment of the small intestine.Chapter1provides an overview of our current knowledge on the amino acid metabolism in the small intestine and in small intestinal microbiota. The aims of this study are listed and structure of the current study is outlined at the end of the chapter.Chapter2describes the research progress on the AA metabolism in intestinal bacteria at the context of the gut ecology and highlights on the diversity of intestinal bacteria and their species-specific AA metabolism with a special emphasis on interactions among dietary composition, gut environments, and host health. The functional aspects of the AA utilization in intestinal bacteria and the modulation strategies are discussed.The characteristics of the utilization of selected AA in pig small intestinal bacteria during in vitro incubation and subculture are described in Chapter3. Results of the in vitro cultivation experiment indicated that the24-h disappearance rates for lysine, arginine, threonine, glutamate, leucine, isoleucine, valine or histidine were50%to90%in the duodenum, jejunum or ileum groups. After the30times of subculture, the24-h disappearance rates for lysine, threonine, arginine or glutamate remained greater than50%. Meanwhile, results from the AA compositions in bacterial proteins in the pig small intestine bacteria and AA incorporation into bacterial proteins indicated that protein synthesis was one of the major pathways for AA metabolism in small intestinal bacteria. Furthermore, this study identified the possible AA that the utilization in pig small intestinal bacteria is fast, quantitatively significant and gut segment specific.Chapter4employs the16S rRNA gene based denaturing gradient gel electrophoresis (DGGE) and sequencing techniques to analysis the pig small intestinal bacteria community composition during in vitro incubation and subculture with individual AA. Results showed that Streptococcus sp., Mitsuokella sp., and Megasphaera elsdenii-like bacteria were predominant in subcultures for utilizing lysine, threonine, arginine and glutamate. In contrast, Klebsiella sp. was not predominant in the utilization of arginine or glutamate during subculture. Escherichia coli was not predominant in the utilization of threonine during subculture. The current work identified the possible predominant bacterial species responsible for the AA metabolism in the pig small intestine. The findings provide a new framework for future studies to characterize the metabolic fate of amino acids in the small intestinal bacteria especially streptococci and define their contribution to the AA metabolism in the small intestine and the nutritional significance for both animals and humans.Further, the metabolic fate of selected AA in pure bacterial strains and mixed bacterial cultures derived from the jejunum and ileum of pigs is compared and described in Chapter5. Results showed that the fast utilization of glutamine, lysine, arginine and threonine in pig small intestinal bacteria during the3-h incubation. The utilization of AA by pure cultures of E. coli and Klebsiella sp. during the3-h incubation were greater than small intestine derived mixed bacterial cultures or Streptococcus sp.. One average, the bacterial decarboxylation of lysine, threonine and arginine accounted for10%of their net utilization of corresponding AA. The decarboxylation of lysine was approximately15%to the net lysine utilization in both jejunal and ileal mixed bacteria while proportion of the decarboxylation of theonine to the net threonine utilization was greater in jejunal mixed bacteria than ileal mixed bacteria (5.5%vs.0.3%). The proportion of the decarboxylation of arginine to net arginine use was20%in ileal mixed bacteria, which was greater than that of jejunal mixed bacteria. Proportion of the AA used for bacterial protein synthesis to the net utilization of corresponding AA was the highest in leucine with50%-70%, followed by threonine, proline and methionine (25%), lysine and arginine (15%) and glutamine (10%). These results suggest that the AA metabolism in pig small intestinal bacteria was diverse and this diversity might contribute to host AA homeostasis in a species and gut compartment dependent manner.Moreover, Chapter6describes the regulatory role of glutamine on the short term (3h) in vitro utilization and metabolism of amino acids by pure bacterial strains and mixed bacterial cultures derived from the jejunum and ileum of pigs. Results showed that the utilization of glutamine in pig small intestinal bacteria increased (P<0.05) with increased dose of glutamine after the3-h incubation. The flux from glutamine to glutamate was observed in all bacterial cultures investigated, which was reflected by the reduced glutamate utilization and net glutamate production. Meanwhile, the production of ornithine was observed in pure bacterial strains and glutamine affected arginine metabolism in all the bacteria investigated. High requirements of asparagine, aspartate and serine were observed in pure bacterial strains with complete utilization of the corresponding AA after3h of incubation. Significant reduction (P<0.05) in the utilization of asparagine by both jejunal and ileal mixed bacteria were observed with the addition of glutamine to the media. Meanwhile, the addition of glutamine to the media reduced the net utilization of lysine, leucine, valine. ornithine and serine by both jejunal and ileal mixed bacteria. Overall, the presence and increased concentration of glutamine in the incubation system regulated the net AA utilization/production in pure bacterial strains and the reduction in the utilization of most AA in jejunal and ileal mixed bacterial cultures.In Chapter7, effect of arginine supplementation on the short term (3h) in vitro utilization and metabolism of amino acids by pure bacterial strains and mixed bacterial cultures derived from the jejunum and ileum of pigs is described. Results showed that the arginine utilization by bacteria was dose dependent and the rates of AA utilization by E. coli and Klebsiella sp. were higher than other bacteria. In pig small intestinal bacteria, the fluxes of arginine to ornithine and citrulline were affected by the increased dose of arginine, which was reflected by the decreased utilization and increased production of ornithine and citrulline. Net glutamine utilization increased in all the pure bacterial strains investigated with increased dose of arginine. High requirements of asparagine, aspartate and serine were observed in pure bacterial strains with complete utilization of the corresponding AA. Meanwhile, complete utilization of asparagine by jejunal mixed bacteria was observed with the addition of arginine. Net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P<0.05) in Streptococcus sp. and Klebsiella sp., however, decreased in E. coli with the addition of arginine. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by both jejunal and ileal mixed bacteria decreased (P<0.05) with the addition of arginine. However, dose effect was only observed for leucine and alanine in ileal mixed bacteria. Overall, the addition of arginine to the incubation system affected the metabolism of the arginine-family AA, serine-and aspartate-family AA in pig small intestinal bacteria and decreased the utilization of most AA in ileal mixed bacteria.Finally, Chapter8summarizes the knowledge collected in this study. The current work shows the bacteria is not only active in the utilization and metabolism of specific groups of amino acids but also in a intestinal compartment-and species-specific manner. Moreover, the core bacterial population (e.g., Streptococcus spp., Mitsuokella spp. and Megasphaera elsdenii, etc.) might play key role in the utilization and metabolism of most of the dietary amino acids in the pig small intestine, however, surrounded by certain groups of bacteria in the metabolism of specific AA. Furthermore, glutamine or arginine exerts regulatory effect on the fluxes of AA in the AA metabolic networks in pig small intestinal bacteria. The possible implications for the development of new strategies to improve amino acid nutrition and functionality in humans and animals are also discussed.In Conclusions1) The small intestinal microbiota is active in the compartmental metabolism of specific groups of dietary AA in the pig small intestine.2) The structures of the bacterial population associate with the metabolism of individual AA in the small intestine might be AA-dependent yet share the common core.3) Dietary supplementation of glutamine or arginine modulates the metabolic fluxes of AA and its metabolites in the AA metabolic networks in pig small intestinal microbiota and the regulatory effect is bacterial species and gut compartment specific.