Yeast diversity during the fermentation of Andean chicha: a comparison between high-throughput sequencing and culture-dependent approaches.. ChichaEukarDiv26SrDNA

The diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Classical microbiological methods allowed isolation of five hundred eighty-two yeasts identified into 16 species by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing of D1/D2 26S rRNA gene amplicons from chicha samples allowed detection of more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical examination of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species, different from S. cerevisiae, dominated the pre-fermentative steps and abounded in fermentative samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with none or very few reads in the pyrosequencing analysis.

本研究针对阿根廷玉米基发酵饮料奇查（chicha）的发酵过程中关联酵母菌的多样性与动态变化展开了调查。研究人员对两批次奇查发酵生产过程中不同阶段采集的样本，分别采用依赖培养与非依赖培养的方法开展分析。通过经典微生物学方法共分离得到582株酵母菌，经限制性片段长度多态性（Restriction Fragment Length Polymorphism, RFLP）分析与D1/D2 26S核糖体RNA基因（D1/D2 26S rRNA gene）测序，将其鉴定为16个物种。对优势菌种酿酒酵母（Saccharomyces cerevisiae）的分离株进行δ元件PCR基因分型，共得到多达42种不同的分型模式。对奇查样本的D1/D2 26S核糖体RNA基因扩增子进行高通量测序，共检测到超过100种酵母菌与近50个丝状真菌类群。数据分析结果显示，酵母菌主导了整个发酵过程，尽管在发酵初始阶段存在占比可观的丝状真菌类群。对结果的统计学分析表明，在发酵过程的任一阶段，单样本中测序读段（reads）占比超过1%的类群极少。在发酵样本中，酿酒酵母（S. cerevisiae）的测序读段占比超过90%。与酿酒酵母不同的其他酵母菌类群，主导了发酵前阶段；且当酿酒酵母占比低于90%时，此类群在发酵样本中也会大量存在。通过焦磷酸测序（pyrosequencing）检测到的绝大多数酵母菌类群，均未通过培养法分离得到。与之相反，基于培养的方法仅检测到极少的酵母菌类群，且其中大多数类群在焦磷酸测序分析中对应的测序读段数极少甚至为零。