白蚁巢拮抗放线菌BYC01代谢产物的分离和鉴定

Isolation and identification of termitarium antagonistic actinomycetes BYC 01 and its active metabolites

【目的】从白蚁巢中分离出具有抗菌活性的放线菌,并在其代谢产物中寻找具有抗菌活性的先导化合物。【方法】通过形态学观察和16S rRNA序列分析初步确定目标菌株BYC 01的分类地位。利用生长速率法和琼脂扩散法测定其代谢产物的抗菌活性。并通过大量发酵提取浸膏,运用多种色谱方法对发酵产物进行分离、纯化,利用质谱和核磁共振谱分析鉴定出化合物的结构。【结果】通过形态学观察和16S rRNA序列分析,菌株BYC 01被鉴定为紫红链霉菌(Streptomyces violaceoruber)。BYC 01发酵液不同极性溶剂萃取物的抗菌活性结果表明其有效抑菌物质主要存在于中等极性的乙酸乙酯部位。在供试浓度为100μg/mL时,BYC 01发酵液乙酸乙酯萃取物对苹果树腐烂病菌具有强烈的抑制作用,抑制率大于90%;对水稻纹枯病菌和杨树溃疡病菌具有较好抑制活性,抑制率均大于60%;在供试浓度为30μg/滤纸片时,与阳性对照相比,乙酸乙酯提取物对白色念珠菌、金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌和水稻白叶枯病菌有中等的抑制作用,其抑菌圈直径范围为11.3 mm-16.5 mm。经质谱和核磁共振谱分析,从BYC 01发酵产物中分离到的单体化合物被鉴定为fogacin;在供试浓度为30μg/滤纸片时,化合物fogacin对白色念珠菌生长的抑制作用与阳性对照两性霉素相当,其抑菌圈大小分别为19.3 mm和20.1 mm。【结论】菌株BYC 01具有开发为微生物杀菌剂的潜力。

[ Objective] We isolated actinomyces from the termitarium and studied its metabolites to find the antimicrobial compounds. [ Methods] We determined the taxonomic status of target strain BYC 01 by morphological observation and 16s rRNA sequence analysis. Growth rate method and agar disc diffusion assays were used to test the antimicrobial activities. Fermentation product was isolated and purified by various chromatographic methods, and the structure was determined by mass spectrum and nuclear magnetic resonance analyses. [ Results] BYC 01 was identified as Streptomyces violaceoruber. The main antimicrobial ingredients of BYC 01 fermentation broth consisted in the ethyl acetate fraction of moderate polar part. The ethyl acetate extract of BYC 01 had strong antifungal activities against Valsa mall with inhibition rate of more than 90% , and activities against Rhizoctonia solani and Dothiorella gregaria with inhibition rate of more than 60% under the concentration of 100 μg/mL. Furthermore, the extract showed the intermediate antimicrobial activities against Candida albicans, Staphyloccocus aureus, Escherichia coli, Bacillus subtilis and Xanthomonas oryzae with the mean halo diameters ranging from 11.3 to 16.5 mm under the concentration of 30 μg/filter paper. A monomer compound was purified from the fermentation products, and was identified as fogacin on the basis of mass spectrum and nuclear magnetic resonance analyses. The compound fogacin and the positive control had similar antimicrobial activities against C. albicans with inhibition zone of 19.3 mm and 20. 1 mm under the concentration of 30 μg/filter paper. [ Conclusion] Strain BYC 01 could be potentially developed as a new antimicrobial agent.