Study Of Acid Production Of Lactic Acid Bacteria And Labeled With Enhanced Green Fluorescent Protein

Lactic acid bacteria possessed antimicrobial activity by producing organic acids,beneficial to intestinal tract of host.A strain of Lactobacillus casei was screened and preserved by our laboratory,total acid yield of this strain was determined.With the purpose of a more particular knowledge of kinds and concentration of organic acid metabolized by this strain,chromatographic conditions used as determining organic acid were optimized in this study.The effect on growth and acid production of L.bulgaricus CICC 6047 by abscisic acid was also studied in this paper.Vector which could express enhanced green fluorescent protein was constructed to give a marker to lactic acid bacteria,this might provide reference for studying the beneficial effect of lactic acid bacteria on intestinal tract.Principal results are as follows.Aminex HPX-87H column was used as analytical column,the conditions of column temperature and flow rate of mobile phase as well as the composition of mobile phase were changed to make a thorough inquiry of the influence of separation of formic,acetic,propionic,lactic,n-butyric,succinic,citric,pyruvic and isobutyric acids.Analytical condition of these nine organic acids was established in this study.Results demonstrated that the column temperature was 50?,0.6 mL/min was the flow rate of mobile phase.The mobile phase was composed by 3.5 m mol/L sulfuric acid and acetonitrile(V/V)99:1.The correlation coefficient of these nine kinds of organic acids was from 0.99982 to 0.99999.Coefficient of recovery and degree of precision of this analytical method were ranged from 97.28%to 107.33%and from 0.47%to 2.07%respectively.These eight strains owned different ability of production of organic acids.Lactic and pyruvic acids were the common product.These eight strains could not product citric,succinic,isobutyric acids.Along with the growth of L.casei,lactic and propionic acids were constantly produced.Different concentration of abscisic acid was added to investigate the effect of abscisic acid on growth and acid production of L.bulgaricus CICC 6047.Result demonstrated that lower concentration of abscisic acid not obvious affected the growth and acid production of L.bulgaricus CICC 6047.The concentration of abscisic acid reached at 132?g/mL,the growth and acid production of L.bulgaricus CICC 6047 was reduced.pH value was higher compared with control group.Ability of producing lactic,formic,propionic acids was decreased,the accumulation of pyruvic acid was increased in the meantime.Plasmid pEGFP-N1 used as the template,primer was designed to amplify the gene egfp by PCR.Plasmid pMG36e and PCR product were digested by restriction endonucleases Pst ? HF and Hind ? HF.Fragments was connected by T4 DNA ligase.Connected product was transferred to E.coli DH5a strain by heat shock.Plate with erythrocin was used to screen the positive converter.Recombined plasmid pMG36e-egfp used electroporation was transferred to L.bulgaricus CICC 6047 and L.lactis JW 18.Plate with erythrocin was used to screen positive colony.Result demonstrated that gene egfp was linked to plasmid pMG36e,recombined plasmid pMG36e-egfp was obtained.Converter E.coli DH5?/PMG36e-egfp could express the EGFP.Plasmid pMG36e-egfp was transferred to L.bulgaricus CICC 6047 and L.lactis JW 18,green florescence was also detected.