Microbial Community Successionand Formation Of Higher Alcohols During The Fermentation Process Of Traditional Light-flavor Baijiu

Traditional liquor brewing mostly uses natural fermentation of microorganisms,lacks control over the activities of liquor microbial communities,and is easily affected by external environmental factors,and it is difficult to ensure homogenization and high-quality production.The demand for high-quality and standardized liquor production has driven the study of microbial metabolism regulation of liquor.In this study,the self-assembly mechanism of microbial communities during fermentation was studied by analyzing the changes of physicochemical indexes and the composition and dynamic changes of microbial communities in the fermentation process of traditional fragrant liquor.Furthermore,by designing short-chain zinc-dependent alcohol dehydrogenase(S.ADHs)degenerate primers,high-throughput sequencing was used to study the gene diversity,composition and abundance changes of S.ADHs during the fermentation of Qingxiang liquor,trace the microbial taxa related to higher alcohol synthesis,and explore the synthesis mechanism of higher alcohols during the fermentation process by combining the changes of microbial community structure during fermentation,in order to provide a theoretical basis for the synthesis mechanism of higher alcohols in liquor.The main results are as follows:(1)With the fermentation process,8 physical and chemical factors(moisture,temperature,acidity,p H,starch,amino acid nitrogen,protein and ethanol concentration,etc.)showed obvious dynamic changes.In the fermentation process,bacterial diversity first decreased and then increased with the fermentation process,while fungal diversity showed a change law of first increasing and then decreasing.Among them,Lactobacillus,Acetobacter,Pediococcus,Weiissella and Kazachstania are the dominant genera,Lactobacillus pontis and Lactobacillus paralimentarius and Lactobacillus acetotolerans and Kazachstania humilis are overwhelmingly dominant species.According to the differences between microbial communities at different fermentation times,the whole fermentation process was divided into three stages: I.(0d),II.(3-10d)and III.(12-28d).Among them,Acetobacter malorum,Lactobacillus paralimentarius,and Kazachstania humilis Lactobacillus pontis are different species in stages I,II and III,respectively.Starch,p H and temperature were positively correlated with most species,and other physicochemical factors were negatively correlated with microorganisms.Lactobacillus pontis,Lactobacillus acetotolerans,Kazachstania exigua and Kazachstania humilis.In addition,substitution contributes almost most of the microbial community succession,and ecological drift in the process of randomness is the most important process driving the self-assembly of microbial communities.At the same time,reducing sugars,temperature,and redox potential may be the main factors affecting the self-assembly of microbial communities during fermentation.(2)S.ADHs degenerate primers S.ADHs-F(GACGTCCTGGTCaayaaygc)and S.ADHs-R(GTCTACGGTTnacnckdat)were designed,and after high-throughput sequencing,it was found that the gene diversity of S.ADHs was highest at 0 d in fermentation and lowest at 28 d,and the number of S.ADHs gene species was the highest at 0 d and the least at 28 d.The distance matrix between samples showed significant differences in S.ADHs genes between fermentation 0 d and fermentation 3-28 days(p<0.05).The total relative abundance of OTUs in the top 30 relative abundances of S.ADHs gene fragments reached 75%,with OTU1 and OTU4 having higher relative abundances.With the fermentation process,OTU1 generally showed an increasing trend,and OTU4 showed a law of first decreasing and then increasing.Phylogenetic analysis divided the S.ADHs gene fragment into 8 clusters and found that a total of 19 OTUs were annotated to the species level,the vast majority of which were annotated to bacteria,and only 2 OTUs belonged to the genus Kazachstania.In addition,species information annotated by OTU35(Escherichia coli),OTU26(Komagataeibacter intermedius)and OTU61(Acetobacter pasteurianus)is also present during the fermentation of the mash.There were significant differences in community structure between the samples of fermentation 0d and fermentation 3-28 d,mainly because the S.ADHs gene fragments in Cluster III were different before and after fermentation,and the temperature affected the distribution of S.ADHs gene fragments in Cluster IV.(3)During fermentation,most bacterial species were positively correlated with propanol,the high-abundance species Lactobacillus pontis and Lactobacillus acetotolerans were negatively correlated with propanol,and both bacteria were positively correlated with other higher alcohols(n-butanol,n-hexanol,isobutanol,isoamyl alcohol,phenylethanol and 2-methylbutanol).Most bacteria are inversely associated with phenethyl ethanol.Most species in fungi were associated with higher alcohols,with Kazachstania humilis and Kazachstania exigua being significantly positively correlated with propanol(p<0.05).Most OTUs in Cluster I.-II were negatively correlated with higher alcohols,and OTU38 was significantly positively correlated with isobutanol,isoamyl alcohol,n-hexanol,n-amyl alcohol and n-butanol(p<0.05).Most OTUs in Cluster III were positively correlated with higher alcohols,and OTU1 and OTU4 were positively correlated with isoamyl alcohol(p<0.05).There were many microbial species related to higher alcohols found from many mixed fermentation microorganisms,among which OTU45(Kazachstania humilis)was significantly positively correlated with propanol,which was consistent with the correlation between dominant fungi and propanol during fermentation.