Microbial Community Structure And Microbial Source Tracking Of Chinese Light-flavor Liquor Fermentation In Different Environments

Chinese liquor brewing process is a complex spontaneous solid state fermentation process driven by diverse microorganisms.On the journey from grains to liquor,the raw materials encounter many microorganisms from not only Daqu,but also liquor-making environments.Thus,the microbial ecology of Chinese liquor fermentation involves the fermentation microbiota and the environmental microbiota.However,there is no systematic study on the community structure of environmental microbiota and the influences of environmental microbiota on liquor fermentation.In this study,we processed two batches of fermentation in new and old workshops with the same Daqu and fermentation parameters.Multiphasic metabolite target analysis,high-throughput sequencing and microbial source tracking were combined to study the community structure of liquor-fermentation environmental microbiota,the origins of liquor fermentation microbiota and the influences of environmental microbiota changes on liquor fermentation.Moreover,since the fermentation process of Daqu is also spontaneous solid state fermentation process,we further studied the influences of environmental microbiota on the microbial community structure of Daqu.The main contents are as follows:（1）Multiphasic metabolite target analysis was used to analyze the microbial metabolism of fermented grains in different environments.Results showed the changes of environments would result in the changes of flavor metabolic profile of fermented grains.Fifty-eight metabolites were identified from fermented grains samples,including 4 alcohols,10 acids,27esters,11 aromatics and 6 others.We found there were obvious changes between the metabolic profiles in the two batches of fermentation.At the end of fermentation,the content of ethanol in high quality fermented grains in old workshop(99.97±13.05 g?kg^-1 fermented grains)was significantly（p<0.01）higher than that in inferior fermented grains in new worshop(37.57±3.44 g?kg^-1 fermented grains).Meanwhile,the total content of volatile alcohols,aromatics and esters in fermented grains in fermented grains in old workshop were higher than those in new workshop,but the total content of acids in fermented grains in new workshop was higher than that in old workshop,mainly including acetic acid,lactic acid and malic acid.（2）High-throughput sequencing was used to analyze the microbial succession during fermentation in old workshop and new workshop.We found the microbial community structure in fermented grains at the beginning of fermentation was different between the two environments,which due to the discrepancy of microbial succession and metabolism during fermentation.Results showed that six bacterial genera and seven fungal genera were the main microorganisms in fermented grains,including Lactobacillus,Bacillu,Pseudomonas,Kroppenstedtia,Weissella,Acinetobacter,Pichia,Candida,Kazachstania,Saccharomycopsis,Saccharomyces,Wickerhamomyces and Aspergillus.At the beginning of fermentation,the relative abundances of Bacillu（20.78±1.08%）,Weissella（16.64±8.53%）,Pichia（55.10±12.37%）,Wickerhamomyces（3.35±0.35%）and Aspergillus（10.66±2.63%）in fermented grains in old workshop were higher than those in new workshop.However,the relative abundances of Pseudomonas（17.04±11.27%）,Acinetobacter（3.02±0.69%）,Candida（16.47±4.31%）and Kazachstania（1.31±0.76%）in new workshop were higher than those in old workshop.After fermenting for four days,the bacterial community in both fermented grains in new and old workshops were dominated by Lactobacillus（68.89⁹7.72%）.However,the fungal community in the two batches of fermentation were different.The dominant fungal community in new workshop was Candida（43.32⁷4.13%）,but in old workshop was Pichia（18.87⁶5.34%）.Canonical correspondence analysis and Pearson correlation analysis showed the relative abundances of Candida and Kazachstania were positive correlated with the acids metabolism,and were negative correlated with the esters metabolism.It was suggested that the differences of microbial community structure is the main reason of the differences of metabolic profile in fermented grains between the two environments.（3）We used high-throughput sequencing to illuminate the microbial community structure in liquor-making environments.Results showed there were more functional microbes for liquor making in the environments in old workshop than those in new workshop.Moreover,the functional microbes for liquor making were mainly found in the indoor ground and the tools in the workshop,mainly including Lactobacillus,Bacillu,Weissella,Pichia,Saccharomycopsis,Saccharomyces,Wickerhamomyces and Aspergillus.To test the influence of environmental microbiota on the liquor fermentation process,we processed two batches of in situ fermentation process without Daqu.Results showed that the distributions of Bacillus,Pseudomonas,Acinetobacter and Candida in the test fermentation system in the new and old workshop were similar with that in the normal fermentation system.This further indicated that the change of environmental microbiota would drive the microbial succession and metabolism in the liquor fermentation process.（4）Source Tracker was used to quantify the origin of microorganisms in fermented grains,and culture method was further used to identify the main reasons caused the differences of microbial community structure between the two environments.Results showed that the bacterial communities in fermented grains were mainly from environments（mainly tools and other unknown environments,72.61⁹0.90%）,mainly including Lactobacillus,Bacillu,Weissella,Pseudomonas,Acinetobacter and Kroppenstedtia.Meanwhile,the dominant bacterium Lactobacillus acetotolerans in fermented grains was only from environments.Moreover,the fungal communities in fermented grains were mainly from Daqu（61.06⁸0.00%）,mainly including Pichia,Saccharomycopsis and Aspergillus.Furthermore,we found the absence of Bacillus in environments（tools）led to the increase of Candida in environments of new workshop.（5）We used high-throughput sequencing and microbial source tracking to analyze the origins of the microbes in Daqu.We obtained 245 bacterial genera and 119 fungal genera in all samples.The bacterial community structure of new Daqu was most similar with that of raw materials,whereas the fungal community structure of new Daqu was most similar with that of indoor ground and tools.The dominant bacterial community’s succession during Daqu-making process was from Gammaproteobacteria and Alphaproteobacteria to Bacilli,and the dominant fungal community’s succession was from Saccharomycetes,Zygomycetes,Agaricostilbomycetes and Eurotiomycetes to Saccharomycetes and Zygomycetes.SourceTracker was used to analyze the efecct of raw materials and environments on the microbial community of Daqu.Results showed raw materials（82.67%）was the main source of bacterial communities,including Pantoea agglomerans,Enterobacter aerogenes,Pseudomonaskoreensis,Sphingomonasdesiccabilis,etc..However,Bacillus amyloliquefaciens and Pediococcus acidilactici,that were dominant in mature Daqu,were mainly from indoor ground and tools.Furthermore,indoor ground and tools were also the main sources of the fungal communities in Daqu,including Saccharomycopsis fibuligera,Rhizopus oryzae,Sterigmatomyces elviae,Aspergillus flavus/oryzae,Hyphopichia burtonii,Pichia kudriavzevii and Lichtheimia corymbifera.