【作者】 何亮；

【导师】 宋新华； 刘训理；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2007， 硕士

【摘要】 从泰山土壤中分离获得一株拮抗细菌(菌株编号:C332),前期研究结果表明:该菌株对多种动植物病原真菌具有较强拮抗作用,菌株发酵性状优异,发酵液性质稳定,具有一定的应用前景。本研究对该菌株进行了鉴定,研究了摇瓶发酵产生抗细菌活性物质的最佳发酵配方和条件,对抗细菌活性物质进行了分离纯化,并研究了其部分理化性质。主要研究结果如下。通过菌体形态特征观察、生理生化指标测定、16S rDNA序列及其系统进化树分析,鉴定C332菌株为蜡状芽孢杆菌(Bacillus cereus)。该菌株的16S rDNA序列已在GenBank注册,登录号为AY756511。体外拮抗试验表明C332菌株对烟赤星病菌(Alternaria alternata)、茶轮斑病菌(Phyllosticta thesefolia)、棉花立枯病(Rhizoctonia solani)、黄瓜枯萎病菌(Fusarium oxysporum)、棉花枯萎病菌(Fusarium oxysporum f.sp. vasinfectum )、大肠杆菌(Escherichia coli)、黑胸败血(Bacillus sp.)等多种植物和动物病原菌具有拮抗作用。采用单次单因子法(one factor at a time)对C332菌株发酵产生抗细菌活性物质的培养基配方及发酵条件进行了研究,进一步采用响应面法对发酵培养基进行了优化,首先用一次回归试验设计对培养基组分进行了评价,筛选出影响产量的三个主要因素:牛肉膏、葡萄糖和土豆。再根据试验结果进行最陡爬坡试验逼近最大产量区域,最后用中心组合设计和响应面分析确定主要影响因子的最佳浓度。优化后最佳发酵条件:初始pH5.0,装液量50mL,接种量2%,发酵温度30℃,种龄20h。最佳培养基配方:葡萄糖0.99%,牛肉膏1.4%,土豆4.0%,MgSO4 0.3%,KH2PO4 0.2%,CaCO3 0.15%。运用该配方和条件进行摇瓶发酵,发酵液效价比初始培养基提高2.08倍。通过20%~40%硫酸铵分级盐析、CM-Sepharose离子交换柱层析、Sephadex G-50柱层析、高效液相色谱以及薄层层析对C332菌株产生的抗细菌活性物质进行了分离纯化,通过硅胶G薄板层析(TLC)最终将抗菌物质分离为Rf值分别为0.39,0.53,0.72的3种有效成分。3个有效组分在单组分状态下表现不稳定。抗细菌活性物质粗物在pH2.5~10.0稳定,耐高温,对蛋白酶敏感,对紫外线不敏感,对家蚕安全无毒。更多还原

【Abstract】 C332, a strain of antagonistic bacterium, was isolated from Mountain Tai soil. C332 strain shows strong inhibition against many pathogens, excellent fermentation characterization, and stability culture characterization.The strain was identified, The component of medium and ferment condition was optimized, The antibacterial was purified step by step and it`s physical chemical characterizations were studied. The results were as follows.Strain C332 was identified as bacillus cereus by study of phenotypic characteristics, physiological and biochemical properities and phylogenetic analysis of 16Sr DNA sequences. The sequence of 16S rDNA obtained in this study has been registered at GenBank database and its accession number was AY756511.In vitro, We testing the antagonistic spectrum of strain C332. Strain C332 shows inhibition against many pathogens such as Alternaria alternate, Phyllosticta thesefolia, Rhizoctonia solani, Fusarium oxysporum, Escherichia coli, Bacillus sp.et al.After one factor at a time experiment, response surface methodology was applied to optimize the culture medium of antibacterial production by strain C332.In the first optimization step, the influence of each factor was evaluated by first-order design. The significant factors at the analysis range were potato, glucose and beef-extract. Then a steepest ascent design was used to approach the optimal region of the medium composition. Finally, the optimal concentration of the medium was determined by central composite design and response surface analysis, and the study showed that glucose 0.99%, beef-extract 1.4%, potato 4.0%, MgSO4 0.3%, KH2PO4 0.2%, CaCO3 0.15% were the optimal medium composition.The production of antibacterial was improved by 208% compared with original medium, with the optimum ferment condition: Initial pH 5.0, Liquid volume 50 mL, Pre-inculation time 20 h, Temperature 30℃, Inculation volume 2%.Antibacterial produced by strain C332 was separated and purified with 20%~40% ammonium sulphate precipitation and column chromatography on CM-Sepharose, Sephadex G-50 and HPLC.Finally, we isolated the antibacterial for three single component by TLC with the Rf of 0.39, 0.53 and 0.72.We supposed the pure antibacterial should be a kind of substancewhich instable and often lose its activity in a single component. The crude antibacterial substance kept active under high temperature and pH 2.5~10.0, sensitive to protease, unsensitive under UV, harmless to silkworm.更多还原