| The brewing of Chinese baijiu is characterized by open inoculation and multi-bacterial mixed fermentation.As one of the twelve types of Chinese baijiu,the fermentation process involves more than 1 000 microbial species,and the metabolic activities of microorganisms during the production process produce a large number of flavor substances and form a unique baijiu style.The key microorganisms and community succession during the fermentation process are still unclear.Therefore,in this study,the dynamic changes of the fermentation microbial flora were analyzed using high-throughput sequencing coupled with flow cytometry,and a method for analyzing the dynamic community structure was proposed to facilitate the rapid study of the trends of active fermentation microorganisms,while the analysis of the change in microbial numbers was realized.Finally,the composition of the base wine of the rounds obtained after the completion of fermentation was analyzed to explore the relationship between important microorganisms and flavor components.The main findings of the study are as follows:(1)The results of high-throughput amplicon sequencing showed that 117 bacterial and 142fungal genera were detected in the stacked fermented grains,with 24 dominant bacterial genera and 14 dominant fungal genera.Among them,the contents of Lactobacillus,Acetobacter,Weissella,Pediococcus,Lactobacillales,Bacillaceae,Thermoactinomycetacea could account for 58.61%～89.28%of the total bacteria,which were considered to be the core dominant genera in the stacking The core dominant genera in the fermentation stage,Issatchenkia,Aspergillus,Wickerhamomyces,Pichia,and Monascus can account for 63.11%～99.16%of the total fungi,and are the core fungal genera in the stacking process.A total of 52 bacterial genera and 46 fungal genera were detected in the cellar fermentation stage,of which 7 were dominant bacterial genera and 18 were dominant fungal genera.lactobacillales was the absolute dominant genus in each round of cellar fermentation,with a relative abundance of 78.00%～97.71%in each stage,The relative abundance of Aspergillus,Issatchenkia,Pichia,Monascus,Penicillium,Zygosaccharomyces accounted for 66.86%～93.59%of the fermenting fungi in each stage,and were the core fungal genera in the cellar fermentation.The abundance of Lactobacillales decreased gradually(47.66%～0.08%)as the stacking rounds progressed,and the high abundance of Lactobacillus may have an important influence on the composition of microorganisms throughout the fermentation process due to the increase in acidity of the spirits at the pre-stacking stage.(2)Flow cytometry was used to detect the activity of the model microorganisms,and the positions of the three key categories of microorganisms bacteria,yeast and mold in the brewing process were determined in the flow analysis,further realizing real-time monitoring of the changes of various active microorganisms during the fermentation process,and finding that the percentage of active microorganisms in four rounds of in-cellar fermentation was up to 69.88%,and the lowest percentage of active bacteria in six rounds was only 21.99%.After sorting the active cells at the end of the third round of spirits stacking using flow cytometry,it was detected that the active dominant bacteria were Herbaspirillum,Chitinophaga,Ralstonia,etc.,and the more active dominant fungal genera were Aspergillus,Pichia,Exophiala,etc.,while the amplicon sequencing technique detected that the main dominant microorganisms at that time were Acetobacter,Weissella,Issatchenkia,Pichia,and Monascus,which indicates that although Acetobacter,Weissella,Issatchenkia,and Monascus were greatly enriched in the process of pile fermentation,the number of live cells at the end of pile The number of live cells was low.The absolute quantification of microorganisms showed that the stacking process enriched all brewing microorganisms to different degrees,and after one round of stacking,the number of bacteria in the stacked fermentation reached 2.54×10~6 cells/g from 5.24×10~5 cells/g at the beginning stage,yeast reached 1.6~5×10~5 cells/g from 2.75×10~4 cells/g,and mold from 2.12×10~4cells/g to 2.20×10~4 cells/g;after two rounds of sub-stacking,the number of bacteria enriched from 1.88×10~6 cells/g in the starting stage to 7.18×10~6 cells/g,yeast from 4.34×10~5 cells/g to1.10×10~6 cells/g,and mold from 2.53×10~4 cells/g enrichment to 2.77×10~5 cells/g;after three rounds of sub-stacking,the number of bacteria reached 2.7~5×10~6 cells/g from 1.21×10~6 cells/g enrichment in the starting stage,yeast reached 1.52×10~6 cells/g from 5.16×10~4 cells/g enrichment,and The above studies showed that flow cytometry,as a high-throughput,culture-free and sensitive technique,has good potential for microbial analysis in Chinese liquor brewing.(3)Qualitative and quantitative analysis of alcohols,esters aldehydes and acids in different rounds of base wines of sauce-flavor baijiu by gas chromatography-mass spectrometry(GC-MS)was carried out for a total of 33 substances,including 7 alcohols,6 acids,14 esters,4aldehydes and ethyl furfuryl ether and ethyl coupling.The alcohols were the most abundant in the base wines of the rounds,with an average content of 1498.78 mg/L,followed by 1189.70mg/L of esters,824.07 mg/L of acids,and 169.12 mg/L of aldehydes.This is consistent with the variation of Monascus content,which may indicate that Monascus plays an important role in the synthesis of ethyl lactate and ethyl caproate.Zygosaccharomyces started to become the dominant species in the third,fourth,fifth and sixth rounds of in-cellar fermentation,and the content of aldehydes also increased rapidly from the third round of base baijiu,with the yield of acetaldehyde,isovaleraldehyde,3-furaldehyde and 2-methylbutyraldehyde being enhanced,suggesting that Zygosaccharomyces may have a facilitating effect on the production of aldehydes. |
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