The Metabolic Pattern Of Pig Hindgut Bacteria On Aromatic Amino Acids And FOS On Its Metabolism In Vitro

Some of the proteins in the diet and endogenous metabolites of mucus were metabolized in colon,produced harmful substances that seriously affect the health of the intestines.Amino acids Aromatic is a kind of substances containing aromatic ring,including phenylalanine,tyrosine and tryptophan.A large number of studies have indicated there were a large number of metabolites of aromatic amino acids in the colon,such as phenolic and indolic compounds.This thesis aimed to explore the fermentation characteristics of aromatic amino acid in pig hindgut,isolate special amino acid utilizing bacteria,research the effects of additional FOS on aromatic amino acid metabolism and provide the data reference and theoretical support for further research on the microbial metabolism and transport mechanism of amino acid.It was described in the following three sections.Expt.l.The metabolic pattern of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation methodThis experiment was conducted to study the different metabolic pattern of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method.Ileum,cecum and colon chyme in Duroc,Landrace and Yorkshire goods hybridization pigs were taken as inoculum.The single aromatic amino acid concentration was kept 10 mmol/L in fermentation flask.Then the fermentation flask was incubated at 37? of 24 h.Gas production was measured at 4,8,12,16 and 24 h,while the samples of fermentation collected at 0 and 24 h were used to measure NH3-N and MCP.Denaturing gradient gel electrophoresis(DGGE)and real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid.The concentrations of ammonia nitrogen(NH3-N)and microbial protein(MCP)were significantly affected by aromatic amino acids and intestinal segments(P<0.01).Intestinal segments also affected gas production(GP)significantly(P<0.01).And NH3-N,MCP and GP were affected by interaction of aromatic amino acids and intestinal segments.DGGE analysis showed bacteria of aromatic amino acids shared amount of bands together,especially similarity analysis of DGGE profile of Phe and Tyr in ileum,Tyr and Trp in colon were 87.9%and 80.5%separately.Shannon diversity indices analysis revealed that aromatic amino acids in cecum and colon varied significantly(P<0.05).Real-time PCR results showed that the quantity of total bacteria were affected by aromatic amino acids and intestinal segments significantly(P<0.05).The results implied that the potential as proportion of different aromatic amino acids are different.Compared with Trp and Phe,the diversity of bacteria utilizing Tyr in cecum or colon is low;compared with Tyr and Trp,a large number of Phe participated in synthesizing bacteria.The fermentation pattern of Specific aromatic amino acids in different intestinal segment was unique.Compared with ileum and cecum,much more aromatic amino acids participated in the synthesis of bacteria in colon.Expt.2.Effects of additional FOS on metabolism of phenylalanine or tryptophan in pig hindgut bacteria fermentation brothsIn order to study the effect of FOS on metabolic pattern of pig hindgut bacteria utilizing phenylalanine or tryptophan by in vitro fermentation method.Ileum,cecum and colon chyme in Duroc,Landrace and Yorkshire goods hybridization pigs were taken as inoculum.The Phe or Trp concentration was kept 10 mmol/L in fermentation flask.The additional dose of FOS were 0,0.5 and 0.75 g respectively.Then the fermentation flask was incubated at 37? of 24 h.The samples of fermentation collected at 24 h were used to measure the concentrations of NH3-N,MCP,indole and skatole.Real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid.The results showed as follows:1)there existed Compartmentalization effects of enteric fermentation of Phe and Trp.2)The concentrations of NH3-N and MCP were significantly affected by FOS(P<0.05).3)Trp was the main influence factor on indole and skatole in fermentation broths,while the concentration of skatole was significantly affected by FOS(P<0.05),but no indole.4)Real-time PCR results showed that the quantity of total bacteria were affected by FOS significantly(P<0.05).The results implied that FOS could alter the metabolic pattern of pig hindgut bacteria utilizing Phenylalanine or Tryptophan,while decreased the conversion of tryptophan to skatole.Expt.3.Isolation and identification of phenylalanine or tryptophan utilizing bacteria from fattening pig colonIn order to research and analysis species of amino acid utilizing bacteria and utilization efficiency,phenylalanine and tryptophan utilizing strains were isolated by modified Hungate technique from fattening pig colon.Amino acids utilizing bacteria were identified by 16S rRNA sequence analysis and blast analysis.These amino acid utilizing strains were taken as inoculum into single amino acid fermentation flask.Then the fermentation flask was incubated at 37? for 30 h.OD600 was measured at 0,4,9,12,16,20,24 and 30 h for drawing growth curve,while the samples of fermentation collected at 0 and 24 h were used to measure the concentration of free amino acid in culture media by amino acid analyzer,which calculated and expression as disappearance rates.The results showed as follows:three strains were isolated,of which two Phe utilizing strains were named JP1 and JP2,which Phe disappearance rate were 23.48%and 33.33%respectively;one Trp utilizing strains was named JT,which Trp disappearance rate was 21.59%.By 16S rRNA sequence analysis and blast analysis,JP1 and JT were identified as Enterococcus and JP2 was identified as Klebsiella.