Study On The Isolation And Identification Of Methionine-producing Bovine Rumen Microorganism And Its Application

ObjectiveThe aim of this study was to screen out a rumen microorganism that could synthesize methionine and secrete methionine in the environment.The efficiency of synthesizing methionine was evaluated by in vitro fermentation simulation test,and its culture conditions were optimized,which laid the foundation for the development of microbial additives in ruminants.MethodsThe rumen contents of Chinese Holstein cows grazed by natural herbage from Yichun and the rumen fluid of Inner Mongolia steppe cattle raised by natural grazing were used as samples.Methionine deficiency medium was used to isolate and purify methionine.The methionine synthesis of the strains was tested,and the strains with higher content were identified by16SrRNA sequencing and biochemical reaction.The single factor experiment design and L₉?3³?orthogonal test were used to optimize the carbon source,nitrogen source and inorganic salt in the culture medium of the strain.The fermentation conditions such as fermentation time,fermentation temperature,sample injection and liquid loading were improved by L₉?3⁴?orthogonal design,and the linear regression equation between the number of live bacteria and OD_600nm value was established.The effects of the strain on rumen pH,total protein content and digestibility of rumen fermentation were tested by simulating rumen fermentation in vitro.The test data were analyzed by SPSS17.0 statistical software for LSD and Duncan.Results1.A microorganism was isolated from the rumen contents of cattle by a methionine deficient medium.The microorganism was identified as Candida tropicalis by Gram staining,colony morphology,16SrRNA sequencing and biochemical reaction.It was identified as Candida tropicalis and named Candida tropical TYM001.2.The standard growth curve of Candida tropicalis was established:y=12.15x-2.7124,R²=0.9495,the viable count of the culture broth can be determined by measuring the OD_600nm00nm value of the broth.3.Through the single factor experiments and the L₉?3³?orthogonal experiment,a more reasonable medium was obtained by orthogonal test.The optimum carbon source of the strain was glucose,and the addition amount was 20.0g/L.The optimum nitrogen source was amino acid free yeast with addition amount of 10g/L.The optimum inorganic salt and addition amount were NaCl 5.0g/L,MgSO₄ 7.0g/L and KH₂PO₄ 5.0g/L.4.The suitable fermentation conditions were obtained by L₉?3⁴?orthogonal test.The content of free amino acids in fermentation broth reached the maximum value of 27.815mg/L at 35?,3.5L,36h,180mL and 37?,2.5L,34h and 180mL respectively.The main and secondary relationships among the factors are as follows:Fermentation time>inoculation amount>fermentation temperature>liquid content.5.In vitro simulated rumen fermentation experiment showed that the pH value of the fermentation broth of the experimental group with Candida tropicalis decreased for a short time and began to rise from 6h to 18h and exceeded the initial pH value at 24h.The content of free methionine and the content of total protein also increased first,then decreased and then increased,and were superior to the control group.Conclusions1.A strain of Candida tropicalis was isolated from bovine rumen contents.It belongs to Candida tropicalis,and named Candida tropical TYM001.The synthesis of amino acids was4.386mg/L,which was significantly higher than that of the control group 1.262mg/L.2,The free methionine synthesis of the strain was 27.815 mg/L,23.429mg/L higher than before and 26.553mg/L higher than the control group.The optimized medium conditions were glucose 20.0g/L,non-amino acid yeast 10.0g/L,NaCl 5.0g/L,MgSO₄ 7.0g/L and KH₂PO₄5.0g/L.The culture conditions were as follows:fermentation temperature 35?,liquid loading3.5L,fermentation time 36h,inoculation amount 180mL or fermentation temperature 37?,liquid loading 2.5L,fermentation time 34h,inoculation amount 180mL.3.The strain of Candida tropicalis could increase rumen pH,and can improve the digestibility of diet and increase the total protein level of fermentation broth.