Uncovering acid resistance genes in lactic acid bacteria and impact of non-viable bacteria on bacterial community during Chinese strong-flavor baijiu fermentation.

Chinese strong-flavor baijiu (CSFB) brewing is a spontaneously solid-state fermentation process for approximately 60 days. Numerous microorganisms grow, die, and spark a series of metabolic reactions during fermentation. In this study, the microbial community and structure between total and viable bacteria in zaopei from the 5- and 20-year pits of CSFB are revealed by amplicon sequencing. Metagenome sequencing was applied to investigate acid resistance genes in Lactobacillus and predict carbohydrate active enzyme in zaopei. Besides, SourceTracker was conducted to expose bacterial sources. Results revealed that there was no significant difference in the bacterial community and structure between the total and viable bacteria; Lactobacillus was the most dominant bacterium in zaopei of two types of pits. Meanwhile, acid resistance genes argR, aspA, ilvE, gshA, DnaK, and cfa were genes that sustained Lactobacillus survival in the late stages of fermentation with high contents of acid and ethanol, and glycosyltransferases were identified as the predominated enzymes during the CSFB fermentation which catalyzed the process of lactic acid generation via Embden-Meyerhof-Parnas pathway and Hexose Monophosphate Pathway. Moreover, the environment contributed most bacteria to zaopei of the 5- and 20-year pits. These findings will provide a deeper understanding of the microbial community structure of viable and total bacteria and the reason for the dominance of Lactobacillus in the later stages of CSFB fermentation.

Unraveling the composition and succession of microbial community and its relationship to flavor substances during Xin-flavor baijiu brewing.

A novel baijiu type, Xin-flavor, has been developed via combining Qu and craftsmanship from three famous baijiu types including light-flavor, sauce-flavor, and strong-flavor. However, the microbial community and its relationship to physicochemical factors and flavor substances were little known during the three-stage brewing processes including saccharification, stacking, and fermentation. In this study, 21 phyla, 407 genera, and 615 species distributed in bacteria and fungi were identified via high-throughput sequencing (HTS). For bacterial community, the dominant bacteria at saccharification stage were Weissella and Leuconostoc, which were superseded by Acetobacter and Gluconobacter at stacking stage, then quickly replaced by Lactobacillus at fermentation stage. For fungal community, Rhizopus, dominant at saccharification stage, was partially replaced by Thermomyces at stacking stage, then was completely superseded by Thermoascus and three yeasts (Saccharomyces, Kazachstania and Apiotrichum) at fermentation stage. The moisture, ethanol, and acidity, as key factors affecting the microbial community, explained this microbial succession. Interestingly, Apiotrichum was firstly detected in fermented grains. Furthermore, the rapid accumulation of ethanol mainly occurred in the first 10 days of fermentation stage, and highly correlated with Saccharomyces, while Kazachstania and Apiotrichum were related to the formation of flavor substances (acetic acid, lactic acid, caproic acid, ethyl lactate and ethyl caproate). Therefore, this study provides fundamental basis for the rational control of baijiu production and improving the craft and quality of Xin-flavor baijiu.

Detection of viable and total fungal community in zaopei of Chinese strong-flavor baijiu using PMA combined with qPCR and HTS based on ITS2 region.

BACKGROUND: Chinese strong-flavor baijiu (CSFB), one of the three major baijiu types, is the most popular baijiu type among consumers in China. A variety of microbes are involved in metabolizing raw materials to produce ethanol and flavor substances during fermentation, which fundamentally determined the quality of baijiu. It is of great importance to study microbial community of fermented grains (zaopei) during baijiu brewing process for improving its quality. In this study, we firstly used propidium monoazide (PMA) to treat zaopei samples from 5-year pit and 20-year pit for removing the interference of non-viable fungi, and analyzed the diversity of total fungi and viable fungi by quantitative PCR (qPCR) and high-throughput sequencing (HTS) based on ITS2 gene. RESULTS: The results showed that total fungi and viable fungi displayed no significant differences at OTU, phylum, or genus levels during fermentation within two kinds of pits. A total of 6 phyla, 19 classes, and 118 genera in fungi were found based on OTUs annotation in zaopei samples from 5-year pit and 20-year pit. Besides, non-viable fungi had little effect on the fungal community diversity during the fermentation cycle. It was found that the most dominant viable fungi belonged to Saccharomyces, Kazachstania, Naumovozyma, and Trichosporon, and Naumovozyma was firstly detected in zaopei samples of CSFB. Moreover, based on the variation of flavor substances in zaopei samples, the quality of CSFB produced from older pit was better than that produced from younger pit. CONCLUSION: The non-viable fungi had little effect on the fungal diversity, structure, and relative abundance in zaopei samples of CSFB, and Naumovozyma was firstly detected in zaopei samples of CSFB. Our findings can be applied as guidance for improving the quality and stability of CSFB.