【作者】 夏尚远；

【导师】 刘训理；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2008， 硕士

【摘要】 本试验室从泰山土壤中分离获得一株对多种病原菌具有较强拮抗作用的细菌—短短芽孢杆菌（Brevibacillus brevis）XDH。本研究对XDH菌株抗细菌物质的发酵、分离纯化及其结构进行了研究,主要研究结果如下:通过Plackett-Burman试验、最陡爬坡试验、中心组合试验并结合响应面法确定了最佳发酵培养基配方为:葡萄糖15.6g/L、黄豆饼粉28.4g/L、淀粉6g/L、CaCO₃ 2g/L、MgSO₄ 4g/L;最佳发酵条件为种龄18h、接种量2%、发酵时间48h、装液量50mL、发酵温度31.1℃、转速212rpm、pH7.5。优化后发酵液效价达到了1419.30μg/mL,较初始发酵工艺（780.08μg/mL）提高了81.94%。抗菌物质粗物的最大吸收波长为210nm,茚三酮反应、双缩脲反应呈阳性,结合捷克八溶剂纸层析、pH纸层析结果,推测抗菌物质属于弱酸性物质,极有可能是肽类物质。通过硫酸铵盐析、CM Sepharose FF离子交换层析、Sephadex G25凝胶层析、AKTA Explore10高效液相层析系统对抗菌物质进行了分离纯化。结果为:CM Sepharose FF水洗样品得到至少两种物质,生测表明不具抗菌活性,而通过0～1mol/L Nacl梯度洗脱时得到一个峰,具有抗菌活性。收集该峰进行Sephadex G25凝胶层析,层析图谱中有两个色谱峰,表明至少含有两个组分,其中第一峰为有效峰,收集该峰,冷冻干燥后利用AKTAExplore10高效液相层析系统对其进行纯化,得到三种具有抗菌活性的物质,分别记为A、B、C组分;通过反相高相液相色谱对其中的B组分进行了检测,结果为单一峰,纯度为98.6%。利用质谱及核磁共振对B组分进行结构解析。一级质谱表明该组分的分子量为1570.9Da,结合二级质谱图推测其为肽类抗菌物质,其一级结构为:Ser-Ile/Leu-Tyr-Lys/Gln-Leu/Ile-Thr-Cys-Lys/Gln-Phe-469.54,其C末端含有一分子量为469.54的未知组分。已经获得了其核磁共振氢谱图,解谱工作正在进行中。更多还原

【Abstract】 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.更多还原