Fermentation, Isolation, Purification And Structural Identification Of Antimicrobial Substances Produced By Brevibacillus Brevis Xdh

A strain of antagonistic bacterium Brevibacillus brevis XDH,isolated from soil of Mountain Tai,has strong inhibition against several pathogenic bacteria. of animals and plants.My study is on fermentation,isolation and purification, structural identification.The effects of fermentation conditions and cultrue on production of antimicrobial substances produced by Brevibacillus brevis XDH were studied by statistical technology that includes PB(Plackett-Burman)design,Steepest ascent design and CCD(central composite design).The optimal fermentation conditions and concentrations of components were determined by response surface analysis.As follows:Glucose 15.6g/L,Starch 6g/L,Soybean powder 28.4g/L,CaCO₃ 2g/L,MgSO₄ 4g/L,Seed age 18h,Inoculation volume 2%, Fermentation time 48h,Volume of medium 50mL,Culture temperature 31.1℃, Rotation speed 212rpm,Initial pH 7.5.The titer of optimized medium and fermentation conditions got to 1419.30μg/mL,81.94%higher than that of basal medium and condition.Antimicrobial substances were scanned by UV-Vis within 190～400nm wavelength to determine the maximum absorption wavelength which is at 210nm finally.And then Ninhydrin reaction,Biuret reaction,Jack's Eight Solvent System Chromatography and pH Paper Chromatography were sequently used for identification of the antimicrobial substances.The results indicated that the main active components were not only water solubable but also weak acid. They probably belong to peptides.Antimicrobial substances from the fermentation of XDH were isolated through ammonium sulfate precipitation at first,secondly three fractions with high antibacterial activity were further purified by chromatography:cation exchange chromatography(CM Sepharose FF),gel filtration chromatography (Sephadex G25) and AKTA purifier HPLC system.At last the second fraction was showed a single peak in HPLC chromatography,and its purity got to 98.6%.By the analysis of ESI-MS and NMR spectra data,the structure of the second peak has been identified as peptide.The results showed that molecular weight was 1570.9Da.It is estimated that the amino acid sequence based on ESI-MS/MS technique maybe Ser-Ile/Leu-Tyr-Lys/Gln-Leu/Ile -Thr -Cys -Lys /Gln - Phe-469.54 that includes unknown components.Its ¹H-NMR spectrum had been got by NMR that is being unscrambled.