Effects Of Bacillus Licheniformis Inoculation On The Microbial Community Structure And Metabolism During Strong Flavored Liquor Brewing Process

Chinese strong flavored liquor is characterized by multi-species solid-state fermentation.The core microbiota determines the efficiency and quality of fermented liquors.However,little is known about how to dig out the core microbiota in the multi-species fermentation system.It is still incomplete for the application of core microbes in the food fermentation.Therefore,it has become increasingly urgent to reveal out the core microbiota and apply it into liquor fermentation,which will be helpful for the directional control and quality improvement of Chinese liquor.This study firstly identified the core microbiota in Chinese liquor fermentation using amplicon sequencing and multivariate statistical analysis.By comparision of different groups of Daqu starters,we inoculated a core microbe into one Daqu fermentation,and systematically traced the whole process of liquor fermentation from the microbial and metabolic point of view.It is expected to be useful for constructing synthetic microbial communities and the further fortification of liquor production.The main results of this work are as follows:?1?The microbial community structure was characterized by Illumina Miseq sequencing.We revealed flavor-associated microbiota by the microbe-metabolite correlation approach,and identified co-occurring microbiota by co-occurrence network analysis.The flavor-associated microbiota consisted of Lactobacillus,Saccharomyces and Candida,which were associated with lactic acid,acetic acid,ethanol,a variety of volatile esters and alcohols.The core microbiota during the liquor fermentation process consisted of Lactobacillus,Saccharomyces,Candida,Rhizopus,Saccharomycopsis,Pichia,Dipodascus,Bacillus,Thermoascus and Lactococcus.In particular,Bacillus had 24 associations with other microbial genera,with a 4.49% incidence of co-occurrence in the network,second only to Lactobacillus?4.68%?,suggesting the importance of Bacillus in the microbial network.Comparison results suggested the amount of Bacillus in one Daqu?Daqu 3?was extremely lower?⁵0%?than the other two Daqu.Bacillus not only dominats the fermentation process,but also has good ability of enzyme and flavor production.While Bacillus licheniformis was the most abundant flavor-producing species among Bacillus strains.Consequently,we inoculated B.licheniformis into Daqu 3 for fortification.?2?Inoculated Daqu exihibited significantly higher amylase activity(1.7 g starch·g^-1·h^-1)than uninoculated Daqu(1.3 g starch·g^-1·h^-1).The glucoamylase activity decreased significantly from 627.6 mg glucose·g^-1·h^-1 to 445.6 mg glucose·g^-1·h^-1 after inoculation.However,no significant difference was observed for the acid protease activity.Inoculaition significantly decreased the esterase activity from 28.1 mg ethyl caproate·g^-1·100 h^-1 to 17.2 mg ethyl caproate·g^-1·100 h^-1.The aromatic compounds and pyrazines were enriched from 0.37 mg·kg^-1 and 0.35 mg·kg^-1 to 0.90 mg·kg^-1and 5.71 mg·kg^-1,respectively.The inoculation of B.licheniformis made the fungal community more sensitive than bacterial community.In the early stage,the inoculation affected only one genus?Weissella;decreased?,but influenced seven genera in the later stage.Among these genera,the growth of Bacillus was significantly enhanced after day 20.The other affected genera?Acetobacter,Acinetobacter,Brevibacterium,Corynebacterium,Lactobacillus and Staphylococcus?indicated their negative interaction with B.licheniformis.Nine of the fungal genera responded to inoculation in both stages,including the significant change in Aspergillus,Candida,Clavispora,Cyberlindnera,Dipodascus,Wickerhamomyces and Eurotium,indicating different interactions occurred when B.licheniformis was involved in Daqu fermentation.?3?The interaction between B.licheniformis and 12 strains?distributed in most of the responsive genera in Daqu?suggested that the inoculated strain inhibited the growth of Staphylococcus lentus,Brevibacterium flavum,Saccharomycopsis fibuligera and Pichia kudriavzeviii,while promoted the Candida parapsilosis,Clavispora lusitaniae and Aspergillus oryzae.This explained the variation of microbial community in Daqu.Interestingly,when B.licheniformis and Cl.lusitaniae were cocultured,the pyrazine content in cocultures was higher than the sum of pyrazines in each pure culture.Similar result was observed for aromatic compounds.Therefore,we concluded that the microbial members shifted their microbial activity and then metabolic status when interacting with B.licheniformis,explaining the variations in microbial communities and flavor components in Daqu fermentation.?4?The application of inoculated Daqu significantly reduced Chao1 richness,OTU number and taxa composition,including the decrease in Lactobacillus,Candida and Saccharomycopsis,and the increase in Bacillus,Lactococcus and Aspergillus.There were 10 significantly different compounds between the two pits,including the decrease in octanoate and isoamyl hexanoate,and the increase in nonanoic acid,acetic acid,pyrazines and some aromatic compounds.This indicates that inoculated Daqu exerted its effectiveness in pits.We identified 36 volatile compounds in final distilled liquor.There were 6 compounds with significant differences obtained by single-factor analysis of variance,including the increased isoamyl octanoate,ethyl octanoate,hexyl acetate,1-hexanol and tetramethylpyrazine,mostly with fruit fragrance,floral and baked fragrance.Whereas octanoic acid decreased.Sensory evaluation indicated that the inoculated liquor had a higher score of fragrant strength,acidity and coke paste,especially the empty cup fragrance score had a significant increase.