“发花”过程中黑曲霉对茯茶微生物群落及主要成分的影响及作用机制

Fu brick tea is a kind of fermented tea that is mainly formed by the fungi of the Eurotium cristatum. Previous studies have shown that there is a certain amount of Aspergillus niger existing in the fermentation process of Fu brick tea. The mechanism that A. niger affects the microflora and the main components of the tea is unknown. This study would inoculate the fungi of A. niger JW23 which isolated from Fu brick tea to carry out the fermentation experiments of Fu brick tea. Firstly, the high-throughput sequencing technology was used to reveal the effect of A. niger on the microbial community and the richness of E. cristatum in the fermentation process of Fu brick tea. Followed, HPLC method would be employ to explore the effect of A. niger on the main component of the tea. And then, the studies concerned about the mechanism of A. niger affect the quality of Fu brick tea would carried out. Based on the Metatranscriptomic method, to explore the molecular mechanism of A. niger affecting the microorganism community, the transcriptional level of functional genes related to microorganis in fermentation process of Fu brick tea is analyzed. Then the relationship of active microorganisms population and main component of the tea would also been revealed. The active microbial groups that affected by the A. niger would also been analyzed. Through the culture based methods, pathway of A. niger affect the E. cristatum would be revealed. Finally, the contribution of A. niger to the main components of Fu brick tea would be studied with the inoculation of pure A. niger to the raw material of Fu brick tea. Based on the analysis above, the role mechanism of A. niger on the formation of quality of Fu brick tea would be clarified. This project will provide an important theoretical basis for the researches of the factors affecting the quality of Fu brick tea.

茯茶是一种主要由冠突散囊菌（俗称“金花菌”）催化形成的发酵茶。本课题组前期研究表明，茯茶发酵过程中存在一定数量的黑曲霉。而黑曲霉影响茯茶微生物及主要成分的机制尚不明确。本课题接种分离自茯茶的黑曲霉Aspergillus niger JW23进行茯茶发酵实验。首先采用高通量测序揭示黑曲霉对茯茶“发花”过程中微生物群落及“金花菌”丰富度的影响。其次运用HPLC探究黑曲霉对茯茶主要成分含量变化的作用。之后展开黑曲霉作用机制的研究：基于宏转录组学方法，研究茯茶微生物相关功能基因转录水平差异，揭示黑曲霉影响茯茶微生物群落的分子机制；分析茯茶主要成分含量与代谢活性微生物之间的相关性，并揭示黑曲霉对活性微生物类群的影响；通过纯菌株培养，探讨黑曲霉对冠突散囊菌的作用方式；最后通过黑曲霉单菌种发酵实验，阐明黑曲霉催化产生茯茶主要成分的组成及含量。本项目为完善茯茶品质影响因素及作用机制研究提供重要的理论依据。

本研究主要开展了茯茶加工过程中关键微生物、主要代谢产物组成和变化规律的研究。探索了影响茯茶品质的关键因素及其作用机制。首先对茯茶“发花”过程中微生物群落及主要成分含量的变化规律开展研究。茯茶加工过程中主要的真菌类群为Aspergillus、Cyberlindnera、Pichia、Candida等，主要细菌类群为Klebsiella、Lactobacillus、Bacillus等。茯茶的渥堆和“发花”两个主要加工过程中的真菌和细菌类群有着明显的区别。茯茶加工过程中，茶多酚含量整体呈现下降趋势，茶多糖和游离氨基酸呈现波动性变化趋势，茶红素和茶褐素整体呈现增加趋势。茯茶“发花”中代谢物种类发生明显变化，尤其在“发花”后期，茯茶中代谢产物种类和含量上调明显。茯茶中关键的真菌和细菌类群与茯茶中代谢成分的产生有显著相关性。第二，开展黑曲霉对茯茶“发花”过程中微生物群落及主要成分的影响的研究。黑曲霉对茯茶中关键的冠突散囊菌具有较明显的影响作用，并且作用时间越长，影响越大。“发花”不同阶段接种黑曲霉促进茯茶中茶多糖含量大幅度的上升，而对茶多酚和茶氨酸和咖啡碱的含量变化影响不够明显。最后，对影响茯茶品质形成的关键因素及其作用机制展开研究。结果发现黑曲霉主要是通过菌落相互作用对冠突散囊菌产生影响，进而影响茯茶品质的。研究过程中发现提高培养基蔗糖或NaCl浓度，可以显著促进“金花菌”闭囊壳的形成。培养基中添加3.0%浓度NaCl能明显促进“金花菌”闭囊壳的数量和直径增加。通过转录组分析和定量PCR方法揭示NaCl通过调控关键基因的表达量促进“金花菌”有性生殖进行，产生子囊孢子，形成子囊果，即茯茶中的“金花”结构。本研究对于提升茯茶品质、进一步优化茯茶加工工艺，促进茶叶主产区夏秋茶资源综合利用，推动茯茶行业快速发展有着重要的意义。

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