黄瓜内生放线菌Hhs.015发酵液抑菌活性成分的研究

Studies on the anti-microbial active substances from the fermentation broth of cucumber endophytic actinomyce Hhs.015

为了明确放线菌Hhs.015发酵液中的抑菌活性成分,试验采用硅胶柱层析和凝胶柱层析等分离手段,以苹果树腐烂病菌为靶标菌进行活性跟踪,对发酵液中抑菌活性成分进行提取、分离和纯化,得到2种活性物质。采用ESI-MS、NMR等对活性物质进行结构解析,确定化合物为苯乙酸和染料木素。对苯乙酸和染料木素分别进行抑菌活性测定,结果表明,苯乙酸对苹果树腐烂病菌、小麦全蚀病菌和小麦纹枯病菌具有明显抑制作用,其EC_(50)分别为57.21,166.92和238.23μg/mL,。就对苹果树腐烂病菌菌丝生长的抑制作用而言,苯乙酸(EC_(50)=57.21μg/mL)优于市售药剂70%甲基硫菌灵可湿性粉剂(EC_(50)=154.39μg/mL),因此与阳性对照相比,苯乙酸尚具有一定的开发应用前景。染料木素对玉米大斑病菌和番茄早疫病菌具有抑制作用,其EC_(50)分别为522.40和311.53μg/mL,。利用HPLC方法测定了苯乙酸在发酵过程中的含量变化,发现发酵5 d时发酵液中苯乙酸含量较高。以上结果明确了放线菌Hhs.015发酵液中的部分抑菌活性物质及其抑菌效果,为后续研究提供了参考。

In order to identify the antimicrobial constituent in the fermentation of Hhs. 015, normal phase silica gel, Sephadex LH-20 column chromatography and other separation methods were used to separate the anti-microbial components against the target plant pathogen Valsa mall. Two substances were isolated from the fermentation broth. The exact structures were phenylacetic acid, and genistein, identified through ESI-MS, NMR. Anti-microbial results showed that the EC50 of compound phenylacetic acid against Valsa mali ,Gaeumannomy- ces graminis var tritici and Rhizoctonia cerealis were 57.21, 166.92, 238.23μg/mL. Pheny- lacetic acid showed better antimicrobial activity than 70% Thiophanate-methyl on mycelial growth of Valsa mali, thus phenylacetic acid may have some development and application prospects. The EC50 of genistein against Setosphaeria turcica and Alternaria solani were 522.40,311.53 μg/mL. The exact quantity of phenylacetic acid in fermentation broth was analyzed by HPLC： the content of phenylacetic acid reached the highest at the fifth day. This report clarified parts of the anti-microbial substances and effects from fermentation broth of Hhs. 015, supplied some basis for further study.