【作者】 严和送；

【导师】 唐欣昀；

【作者基本信息】 安徽农业大学 ， 微生物学， 2004， 硕士

【摘要】 用双层琼脂扩散法检测海洋放线菌M023、M056、M058、M066、M220、M230的抗菌谱，部分菌株具有一定抑菌活性，其中M058对金黄色葡萄球菌（革兰氏阳性球菌）、假单胞菌有较强的抑菌作用，对大肠杆菌（革兰氏阴性细菌）、白色念珠菌（酵母状真菌）也有抑制作用，而对铜绿假单胞菌抑制作用相对较弱，以对金黄色葡萄球菌的抑菌活性尤为明显，是一株具有广谱抗菌活性的菌株，因此对M058进行了进一步的研究。通过观察菌丝形态、孢子形状、在不同培养基上的培养特征及明胶液化、淀粉水解、纤维素上生长情况、H2S的产生、碳源利用一系列生理生化特征，按照《链霉菌鉴定手册》将M058菌株初步归类为链霉菌属金色类群，类似菌：金色链霉菌Streptomyces aureus。为提高抗生素产量，对M058的液体培养基成分进行了优化。通过杯碟法检测发酵液中抗生素的含量, 筛选出最佳碳源为可溶性淀粉, 最佳氮源为硝酸钾, 通过改变碳、氮比及进行碳、氮源及磷酸盐的正交实验，获得碳源、氮源及磷酸盐配比为：可溶性淀粉1%，KNO30.15%，K2HPO40.075%。发酵工艺条件的研究结果表明最适初始pH为7.0~7.5，最适温度为25℃，最适接种量为10%，250ml的三角瓶装入50 ml为最佳装液量。在优化后的培养基和最佳发酵条件下，36~48小时抗生素的分泌量达到最高峰，为获得大量抗生素，采取工艺放大，进行罐发酵，在发酵液中抗生素活性达到高峰时结束发酵，放罐。将发酵液除菌体后进行浓缩、采用乙酸乙酯分级萃取、活性炭吸附除盐，丙酮溶液洗脱的方法获得酯溶性抗生素粗品A和水溶性抗生素粗品B，经检测粗品A和B均对金黄色葡萄球菌均有抑制作用，同时通过GF254高效硅胶板检测出A、B所含组分，选用200-300目硅胶柱，通过柱层析的方法使粗品A和B各个组分得到了初步分离，探索了抗生素粗品A、B的初步纯化方法，获得抗生素粗品A、B的部分理化性质，为进一步纯化奠定基础。更多还原

【Abstract】 Antimicrobial activity of marine antinomycetes M023、M056、M058、M066、M220、M230 strains were studied by method of double layers agar and the results showed some strains had an antimicrobial activities. Strain M058 had strong activity against Staphylococcus aureus(Gram-positive bacteria) and Pseudomonas, also had activity against Escherichia coli(Gram-negative bacteria) and Candida albicans(fugal), and had low activity against Pseudomonas aeruginosa , Marine antinomycete M058 was a strain of yielding broad-spectrum antibiotic, so strain M058 was studied further.The morphology of mycelium and spores in Gause’s syntetic No1 media, growth situation and physiol-biochemical characteristics in different media were observed. According to Identification Manual of Streptomyces, strain M058 was identified to be Aureus group of genus Streptomyces, and similar to Streptomyces aureus.For increasing production of antibiotic, the liquid media composition were studied. Antibiotic content of the fermented liquid was determined by cylinde-plate method, the screening result indicated that the optimal carbon source was soluble starch, and the optimal nitrogen source was KNO3. By changing the ratio of carbon source and nitrogen source , and orthogonal tests of carbon, nitrogen and phosphate , the optimal proportion of these component was obtained and percentage of these component was soluble starch 1%, KNO3 0.15%， K2HPO4 0.075%.The suitable technical parameters in fermentation were: initial pH 7.0~7.5, 25℃, 10% inoculum, loading liquid volume 50 ml(in 250ml flask). On the conditions of the optimal media and the suitable fermentation condition, the antibiotic yield of strain M058 reached maximum in 36~48 hours. Fermentation process of strain M058 in 20L fermenter was designed to yield more antibiotic for further study, and fermented liquid was determined by cylinde-plate method ,and stopped fermenting when the antibiotic yield of strain M058 reached maximum.The fermented liquid was filtrated, the filtrate was concentrated. The concentrated product was extracted by ethyl acetate, then the concentrated product was adsorbed by active carbon, and the active carbon was eluted by acetone solution. Oil-soluble antibiotic A and water-soluble antibiotic B were gained. Both oil-soluble antibiotic A and water-soluble antibiotic B had activity against Staphylococcus aureus. Components of A and B were determined by silica GF254( HPTLC). Components of A and B were purified <WP=7>primarily by 200~300 sizes silica. Method of purifying oil-soluble antibiotic A and water-soluble antibiotic B were studied primarily. Part physico-chemical properties of A and B were found, which were bases for purifying of A and B further.更多还原