Study On Amino Acid Metabolism By Rumen Microorganism And Methionine Degradable Bacterium

Methionine and lysine are the limited amino acids in corn and soybean-meal diets of ruminant,and improving their utilization rate is very important to improve the performance of ruminant.But owing to the parts of feed protein and amino acids what had been in rumen were fermented and degraded by rumen microorganism,most of protein and amino acids were intercepted in rumen.A lot of predecessors had been investigated many modus operandies for improving amino acids to get into small intestine,but these methods didn't begin with metabolic pathway of microorganism,and they were only the palliative methods and the cost price were much higher.So it was expected to control the activities of the main enzymes what participated the amino acid metabolism in rumen and the quantities of the methionine degradable bacterium in rumen microorganism by studying of the metabolism regularity of amino acid by rumen microorganism and isolating of the methionine degradable bacterium,accordingly lowed the degradation rate of the methionine in rumen and increased the utilization rate of limit amino acid for animals.1,Effect of the sampling and culturing time in vitro on amino acid concentration of ruminal fluid.The aim of this study was to investigate the influence of the different sampling and culturing time on amino acid concentration of ruminal fluid by mixed rumen microorganisms in vitro.Three fistulated Salon goats used as the experimental animals in this experiment,and two different sampling times(before feeding and after feeding)were designed.This aim was to determine the effect of the different culturing time on metabolism of the amino acid in vitro.Rumen fluids from three perpetually fistulated Salon goats were collected on different time points(before feeding and after feeding),then divided into 18 bottles and allocated into two treatments,sampled after culturing 0,8,16h in vitro.The result indicated:there was significant influence between the different sampling time on the change regulation of some amino acid (P＜0.05);but there were great effect on the glutaminic acid(Glu),valine(Val),phenylalanine(Phe),lysine(Lys)and the total amino acid(P＜0.05)between different culturing time in vitro.2,Studies on the Utilization of Methionine and Lysine by Mixed Rumen Microorganisms in vitro.The aim of this study was to investigate the utilization of supplementary methionine and lysine by mixed rumen microorganisms in vitro,and further effects of adding methionine and lysine on urea nitrogen(UN)and enzyme activities(γ-glutamyltransferaseγ-GT or GGT,glutamic pyruvic transaminase GPT, glutamic oxalacetic transaminase GOT and glutamic acid dehydrogenase GLDH)were studied.Mixed rumen fluids from three perpetually fistulated Salon goats were collected, then divided into 18 bottles and allocated into three treatments arranged in a randomized complete block design with 6 which were added methionine(2mM),or lysine(2mM) and then added double distilled water as control groups,at last all treatments incubated for 16 hours at 39℃in vitro.Results indicated that the content of the UN in mixed rumen fluid increased when added with methionine or lysine compared with that in control group(P＜0.05).The activities of the GLDH increased rapidly with culture time getting longer.GLDH activity in methionine group was highly significant higher than that in other two groups(P＜0.01),however,GLDH activity in lysine group was highly significant lower than that in other two groups(P＜0.01).Culture time and added amino acid had no effect on GPT activity.GOT activities in both methionine and lysine groups were significantly lower than that in control.(P＜0.05),and culture time had no significant influence on them.Lysine was degraded more quickly than the methionine by rumen microorganisms during culturing in vitro.The UN was increased after adding amino acids,however activities of GOT and GPT were inhibited,and activities of GLDH was increased after adding methionine.3,Isolation and identification of Methionine utilization bacterium from rumen fluid. In order to research and analyze species resource of the methionine utilization bacterium from goats rumen fluid,after strict anaerobe isolation and purity for rumen microorganism using methionine as the sole sources of nitrogen and carbon,the bacterium which can degrade the methionine were screened,and the 16S rDNA encoding gene(16S rDNA)from the three strains MB6-1,MB6-2,MB7-1 of methionine utilization bacterium were amplified by PCR,and their nucleotide sequences were determined,and identified the characters by partial physiology and biochemistry experiments.The 16S rDNA gene sequences of the strains MB6-1,MB6-2,MB7-1 had been accepted by GenBank,and the GenBank accession numbers were DQ436917,EF581856,DQ453797 respectively;On the basis of the homology and phylogenetic analysis of 16S rDNA,the homology of the strains MB6-1 and providencia rettgeri was up to 96.7%,so it was considered that strain MB6-1 may be a novel species of the providencia family;The homology of the strains MB6-2 and the Paenibacillus cookii was up to 99.1%,so it was considered that strain MB6-2 just may be a subspecies of the Paenibacillus cookii;The homology of the strains MB7-1 and other strains was low,so it can not be confirmed the genus categorization.The influences of sampling and culturing time on the partial amino acid concentration and the change tendency were very significant,but the concentrations of methionine and lysine presented to descend tendency all the time,rumen microorganism were priority to use lysine,and the speed of the rumen microorganism degraded the two amino acids was quicker than the speed of the rumen microorganism synthesized the two amino acids;The effect of adding amino acids and extending the culture time in vitro on the content of UN and the activities of GGT,GLDH were significant,but there were no significant influences on GPT,GOT,it means that oxydasic activities had an extensive changing scope during the course of rumen microorganism fermentation,so the genetic probability of the oxidative deamination reaction was fluctuated,but transaminase activities were stable corresponding to oxydasic activities,so the genetic probability of the transamination was low.We got the strains MB6-1,MB6-2,MB7-1 by strict screening and adding the methionine as sole sources of nitrogen and carbon;It was profit to research and analyze more species resource of the methionine utilization bacterium from rumen fluid of goat in the future,and it was expected to increase the utilization rate of the Methionine in rumen by regulating the quantities of the Methionine degradable bacterium in rumen microorganism.