普洱熟茶后发酵过程细菌多样性的16S+rRNA基因文库的研究.pdfVIP

普洱熟茶后发酵过程细菌多样性的16S rRNA 基因文库研究 赵 明，马 燕，张冬莲，苏小琴 （云南农业大学普洱茶学院，云南昆明 650201 ） 摘要：普洱熟茶是云南地理标志产品，微生物在其品质形成中起着关键作用，但已有基于纯培养的研究未能完全 揭示普洱熟茶发酵过程微生物群落结构与变化，尤其是对发酵过程细菌的种类研究较少。本文应用16S RNA基因 文库技术，研究了1次普洱熟茶发酵过程细菌群落的结构与动态变化。建立了发酵原料（LD-0 ）、5天 LD-5 、10 天 LD-10 、15天 LD-15 和20天 LD-20 发酵样品的5个克隆文库，每个文库挑选克隆子测序分别获得115-174序列， 共747个序列。经分析按97% 的相似性，每个文库分别归于4-20单一的可操作的分类学单位 OTUs 。经序RDP Naive Bayesian rRNA Classifier Version 2.5 检索Ribosomal Database Project 鉴定发现这些OTUs属于 Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria 4 个门，进一步分为Alcaligenaceae, Bacillaceae, Enterobacteriaceae等10个纲。Enterobacteriaceae是原料、5天发酵样和10天发酵样的优势菌；随着发酵进行，温度 升高为40–60oC，Bacillaceae成为优势菌。有趣的是我们发现15天发酵样和20天发酵样的优势菌为凝结芽孢杆菌 （Bacillus coagulans ），该菌是一种经FDA认证的公认安全的耐热益生菌。研究提示今后可以人工接种B. coagulans 发酵普洱茶，生产含有益生菌的普洱熟茶。 关键词：茶学；凝结芽孢杆菌；细菌多样性；益生菌；普洱熟茶 Bacterial diversity in Fermentation of the Traditional Chinese Post-fermented Pu-erh Tea Revealed by 16S rRNA Gene Clone Library? ZHAO Ming, MA Yan, ZHANG Donglian, SU Xiaoqin College of Pu-erh Tea, Yunnan Agricultural University, Kunming, Yunnan 650201, China Abstract: Microbes are thought to have key roles in the development of the special properties of post-fermented pu-erh tea pu-erh shucha , a well-known traditional Chinese tea; however, little is known about the bacteria during the fermentation. In this work, the structure and dynamics of the bacterial community involved in the production of pu-erh shucha were investigated using 16S rRNA gene clone libraries constructed from samples collected on days zero LD-0 , 5 LD-5 , 10 LD-10 , 15 LD-15 and 20 LD-20 of the fermentation. A total of 747 sequences with individual clone library containing 115-174 sequences and 4-20 unique operational taxonomic units OTUs were obtained. These OTUs were grouped into four phyla Actinobacteria, Bacteroidetes, Fi