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Abstract：
The fermentation process of Chinese Baijiu\'s fermented grains involves the intricate succession and metabolism of microbial communities, collectively shaping the Baijiu\'s quality. Understanding the composition and succession of these living microbial communities within fermented grains is crucial for comprehending fermentation and flavor formation mechanisms. However, conducting high-throughput analysis of living microbial communities within the complex microbial system of fermented grains poses significant challenges. Thus, this study addressed this challenge by devising a high-throughput analysis framework using light-flavor Baijiu as a model. This framework combined propidium monoazide (PMA) pretreatment technology with amplicon sequencing techniques. Optimal PMA treatment parameters, including a concentration of 50 μM and incubation in darkness for 5 min followed by an exposure incubation period of 5 min, were identified. Utilizing this protocol, viable microorganism biomass ranging from 8.71 × 106 to 1.47 × 108 copies/μL was successfully detected in fermented grain samples. Subsequent amplicon sequencing analysis revealed distinct microbial community structures between untreated and PMA-treated groups, with notable differences in relative abundance compositions, particularly in dominant species such as Lactobacillus, Bacillus, Pediococcus, Saccharomycopsis, Issatchenkia and Pichia, as identified by LEfSe analysis. The results of this study confirmed the efficacy of PMA-amplicon sequencing technology for analyzing living microbial communities in fermented grains and furnished a methodological framework for investigating living microbial communities in diverse traditional fermented foods. This technical framework holds considerable significance for advancing our understanding of the fermentation mechanisms intrinsic to traditional fermented foods.
摘要：
白酒酒糟的发酵过程涉及微生物群落的复杂演替和代谢,集体塑造白酒的品质。了解发酵谷物中这些活微生物群落的组成和演替对于理解发酵和风味形成机制至关重要。然而,在发酵谷物的复杂微生物系统中对活微生物群落进行高通量分析提出了重大挑战。因此，本研究通过设计一个以淡味白酒为模型的高通量分析框架来应对这一挑战。该框架结合了单叠氮化物丙啶(PMA)预处理技术与扩增子测序技术。最佳PMA处理参数，包括浓度为50μM，在黑暗中孵育5分钟，然后暴露孵育5分钟，已确定。利用这个协议，在发酵谷物样品中成功检测到的活微生物生物量范围为8.71×106至1.47×108拷贝/μL。随后的扩增子测序分析揭示了未处理组和PMA处理组之间不同的微生物群落结构。相对丰度组成存在显著差异，特别是在优势物种中，如乳杆菌，芽孢杆菌,片球菌,酵母菌,Issatchenkia和Pichia,通过LEfSe分析确定。这项研究的结果证实了PMA-扩增子测序技术用于分析发酵谷物中的活微生物群落的功效，并为研究各种传统发酵食品中的活微生物群落提供了方法学框架。该技术框架对于促进我们对传统发酵食品固有的发酵机制的理解具有重要意义。
