舟山沿海有抑菌活性海洋微生物的筛选、鉴定及发酵条件等研究
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摘要
自从Flemming发现青霉素以来,相继己有350多种抗生素应用于临床,使多传染性疾病得到了控制,但随着抗生素的广泛使用和细菌耐药性的出现,寻找新的抗生素成为当务之急。海洋微生物由于其生存环境的特殊性(如高渗、高盐、低温、高压、低氧等),发展出独特的代谢方式,并使其成为海洋生物活性物质的重要来源,也使其成为新抗生素的重要来源,这类物质通常具备陆地微生物所没有的新的独特结构,在抗病菌、抗肿瘤、抗病毒等方面具有良好表现。
从舟山沿海海域采集海水、海泥、海绵、海带及几种鱼类肠道,分离纯化共得到196株海洋细菌、34株海洋放线菌、22株海洋真菌,采用双层琼脂扩散法和纸片法对目标菌株进行抑菌试验,筛选到具有较好抑菌作用的海洋细菌NF19、海洋放线菌a1、X6、X7、B2、B5。16S rDNA分子序列分析和生理生化鉴定结果显示,菌株NF19为乳杆菌属的唾液乳杆菌(L.salivarius),a1、X6为链霉菌属的浅玫瑰色链霉菌(Streptomyces roseus),B2、B5均为链霉菌属(Streptomyces sp.),X7为链霉菌属的玫瑰暗黃链霉菌(Streptomyces roseofulvus)。
对目标菌株进行发酵条件优化的研究,结果显示,将转速控制在120r/m时,摇瓶装量为50mL/250mL时候,菌株NF19的最适培养条件为:初始温度25℃、培养时间48h、初始pH为7.2、接种量为8%。菌株a1、X6、X7、B2、B5的最适培养条件为:初始温度25℃、培养时间96h、初始pH为7.0、接种量为2%,耐盐性分析结果表明X6菌株在NaCl浓度为5%具有最好的抑菌效果,其余菌株均在NaCl浓度为3%的抑菌效果最好。
Since penicillin was found by Flemming, more than 350 kinds of antibiotics were used in clinical application, which made many infectious diseases under control; however, with the wide application of antibiotics and the development of bacterial drug resistance, looking for new antibiotics becomes more and more important. Marine microorganism, for their special survival environment, high pressure, high salinity, low temperature and lack of oxygen, developed the unique metabolic ways, which made them become an important resource of marine bioactive substances and new antibiotics that have the new special structure, compared with the terrestrial microorganism, and be capable of antibacterial, antitumor and antivirus.
Antibacterial test were carried on the 196 strains of marine bacterium,34 strains of marine Actinomycetes and 22 strains of marine Fungi isolated from seawater , sea mud , sponge, kelp and several fish intestinal from Zhoushan coastal by double agar plate method and filter paper method. Strain NF19,a1,X6,X7,B2,B5 were screened for their good inhibition Identified by the 16S rRNA and physiology and biochemistry features, the strain NF19 belongs to L.salivarius of lactobacillus, the strain a1,X6 belong to Streptomyces roseus of Streptomyces, the strain B2,B5 belong to Streptomyces sp., the strain X7 belongs to Streptomyces roseofulvus of Streptomyces.
Fermentation condition research shows the best cultivation coditions of strain NF19 are 50ml medium in 250ml shaken flask,rotary rate 120rpm, 25℃,pH7.2,inoculation doses per cent 8, up to 48h,and the best cultivation coditions of strain a1、X6、X7、B2、B5 are 50ml medium in 250ml shaken flask, rotary rate 120rpm, 25℃,pH7.0, inoculation doses per cent 2,up to 96h. Analysis of salt tolerance shows the best inhibitory effect of strain X6 is at 5% of the sodium chloride concentration,another five strains is at 3% of the sodium chloride concentration.
从舟山沿海海域采集海水、海泥、海绵、海带及几种鱼类肠道,分离纯化共得到196株海洋细菌、34株海洋放线菌、22株海洋真菌,采用双层琼脂扩散法和纸片法对目标菌株进行抑菌试验,筛选到具有较好抑菌作用的海洋细菌NF19、海洋放线菌a1、X6、X7、B2、B5。16S rDNA分子序列分析和生理生化鉴定结果显示,菌株NF19为乳杆菌属的唾液乳杆菌(L.salivarius),a1、X6为链霉菌属的浅玫瑰色链霉菌(Streptomyces roseus),B2、B5均为链霉菌属(Streptomyces sp.),X7为链霉菌属的玫瑰暗黃链霉菌(Streptomyces roseofulvus)。
对目标菌株进行发酵条件优化的研究,结果显示,将转速控制在120r/m时,摇瓶装量为50mL/250mL时候,菌株NF19的最适培养条件为:初始温度25℃、培养时间48h、初始pH为7.2、接种量为8%。菌株a1、X6、X7、B2、B5的最适培养条件为:初始温度25℃、培养时间96h、初始pH为7.0、接种量为2%,耐盐性分析结果表明X6菌株在NaCl浓度为5%具有最好的抑菌效果,其余菌株均在NaCl浓度为3%的抑菌效果最好。
Since penicillin was found by Flemming, more than 350 kinds of antibiotics were used in clinical application, which made many infectious diseases under control; however, with the wide application of antibiotics and the development of bacterial drug resistance, looking for new antibiotics becomes more and more important. Marine microorganism, for their special survival environment, high pressure, high salinity, low temperature and lack of oxygen, developed the unique metabolic ways, which made them become an important resource of marine bioactive substances and new antibiotics that have the new special structure, compared with the terrestrial microorganism, and be capable of antibacterial, antitumor and antivirus.
Antibacterial test were carried on the 196 strains of marine bacterium,34 strains of marine Actinomycetes and 22 strains of marine Fungi isolated from seawater , sea mud , sponge, kelp and several fish intestinal from Zhoushan coastal by double agar plate method and filter paper method. Strain NF19,a1,X6,X7,B2,B5 were screened for their good inhibition Identified by the 16S rRNA and physiology and biochemistry features, the strain NF19 belongs to L.salivarius of lactobacillus, the strain a1,X6 belong to Streptomyces roseus of Streptomyces, the strain B2,B5 belong to Streptomyces sp., the strain X7 belongs to Streptomyces roseofulvus of Streptomyces.
Fermentation condition research shows the best cultivation coditions of strain NF19 are 50ml medium in 250ml shaken flask,rotary rate 120rpm, 25℃,pH7.2,inoculation doses per cent 8, up to 48h,and the best cultivation coditions of strain a1、X6、X7、B2、B5 are 50ml medium in 250ml shaken flask, rotary rate 120rpm, 25℃,pH7.0, inoculation doses per cent 2,up to 96h. Analysis of salt tolerance shows the best inhibitory effect of strain X6 is at 5% of the sodium chloride concentration,another five strains is at 3% of the sodium chloride concentration.
引文
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