PCR-DGGE分析资中冬尖发酵过程中细菌多样性

摘要

Objective:To investigate dynamic changes in the microbial community structure during the fermentation of Zizhong Dongjian,a Chinese traditional fermented vegetable product,by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE).Methods:Four samples of Dongjian with different fermentation times were collected from Zizhong,Sichuan province.Total bacterial DNA was extracted from the samples,and nested PCR was applied to amplify the V3 region of 16S rDNA for identification based on DGGE fingerprints.Meanwhile,the dominant bands were cloned,selected and sequenced,and a phylogenetic tree was constructed.Results:During the fermentation process of Dongjian,rich bacterial diversity was observed and the microbial community structure was changed greatly.The similarity of bacterial community between Dongjian samples with different fermentation times ranged from 9％ to 67％.Among them,samples fermented for two and three years exhibited the highest similarity to each other,up to 67％.Furthermore,the bacteria involved in the fermentation process of Dongjian were mainly divided into 3 categories:Firmieutes,Proteobacteria,and unculturable bacteria.Among these,Firmieutes was the most dominant species followed by Proteobacteria,which accounted for 53％ and 37％ of the total population,respectively,while unculturable bacteria accounted for only 10％.Conclusions:DGGE fingerprint can provide a comprehensive and true reflection of dynamic changes in the microbial diversity of Dongjian during fermentation.Meanwhile,it also provides a theoretical basis for the production process for Dongjian and the formation of flavor substances.%目的:采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denatured gradient gel electrophoresis,PCR-DGGE)技术分析资中冬尖发酵过程中微生物群落结构及其动态变化.方法:从资中采集4份不同发酵年份冬尖样品,提取样品总DNA,采用巢式PCR法扩增细菌16S rDNA V3区,扩增产物采用DGGE分离,对细菌主要优势条带进行克隆、测序、构建系统发育树.结果:冬尖在发酵过程中细菌多样性较丰富,且群落结构发生了较大变化.不同发酵时间样品间,细菌群落结构相似性为9％～67％,其中第2年与第3年样品相似性最高,达67％.资中冬尖发酵过程中,所涉及的细菌主要分为3类,分别为厚壁菌门(Firmicutes)、变形杆菌门(Proteobacteria)和非培养菌,其中厚壁菌门为优势菌群,占53％;变形杆菌门占37％;非培养菌仅占10％.结论:采用PCR-DGGE技术能更全面、更真实地反映资中冬尖在发酵过程中微生物群落的多样性及其动态变化,为冬尖的生产工艺和风味物质的形成提供理论支撑.