茯茶冠突散囊菌发酵生产吲哚生物碱的研究

摘要

冠突散囊菌是茯砖茶"发花"过程中的优势菌,其体内含有丰富的吲哚类生物碱类代谢产物.吲哚生物碱类化合物具有抗菌、抗过敏、抗辐射、抗紫外和抗肿瘤等重要的生物活性,开发前景广阔.为提高吲哚类生物碱类代谢产物的产量,本研究考察了冠突散囊菌繁殖、生长与发酵的影响因素,并利用薄层色谱(TLC)、高效液相色谱(HPLC)、液相色谱-质谱联用技术(LC-MS)对其发酵产物内含成分进行了分析.结果表明冠突散囊菌在察氏固体培养基上能快速繁殖,8 d内能产生大量的成熟孢子,液体培养的最佳条件与时长分别为pH 6.0、温度30℃、转速200 r·min-1和培养时长6 d;鲜茶干粉、鲜茶提取物、茯茶粉、茯茶提取物和色氨酸等能显著诱导吲哚生物碱的合成,其中色氨酸诱导效应最强;内含成分分析表明,TLC可分离出至少10种冠突散囊菌代谢物质,HPLC与LC-MS检测分析其代谢产物的主要成分为吲哚类生物碱,产量达到2.45 g·L-1.本研究为茯茶冠突散囊菌的高值化开发提供了技术支撑.%Abstact: Eurotium cristatum is the dominant fungi in Funzhuan tea, which produces indole alkaloids that have many important bioactivities, such as antibacterial, antiallergic, antiradiation, antiultraviolet, antitumor effects. In order to improve the production of indole alkaloids by E. cristatum, we investigated the factors affecting reproduction, growth and the fermentation of E. cristatum. The fermentation products of E. cristatum were analyzed using thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrum. The results showed that E. cristatum reproduced fast on Czapek-Dox solid medium with a large number of mature spores in 8 days, and the optimal growth conditions were obtained as following: pH 6.0, culture temperature 30℃, rotating speed 200 r·min-1 and culture time 6 days. Moreover, fresh tea leaf powder, fresh tea leaf extract, Funzhuan tea powder, Funzhuan tea extract and tryptophan obviously improved the biosynthesis of indole alkaloids, with the best enhancing effect of tryptophan. Furthermore, at least 10 compounds were observed in thin-layer chromatography,most of which were identified to be indole alkaloids by high-performance liquid chromatography and liquid chromatography-mass spectrum, with a yield up to 2.45 g·L-1. The present work provided technical support for utilization of E. cristatum in Fuzhuan tea.