Deciphering Succession Patterns Of Functional Microbial Communities In Chinese Xiaoqu Light-flavor Baijiu Brewing Process

Fermented foods have a long history of production and are consumed by people all over the world every year.Traditional fermented foods are produced by spontaneous fermentation,which lacks the regulation of microbial community during the fermentation process,resulting in unstable and low-safety fermented foods.The demand for high-quality and standardized fermented food production has promoted attempts to regulate the fermented functional microbial metabolism,which requires our understanding of the microbial community succession mechanism during fermentation process.In this study,the brewing technology of traditional solid-state Xiaoqu light-flavor baijiu was selected as the research model in which sorghum grains were firstly aerobic saccharified,and then anaerobic fermented after being stirred and acidified to explore the multi-stage community succession patterns during the brewing process.In the same process and production condition,two batches inoculated with two different starters namely pure starter(PS)and traditional starter(TS),were carried out for brewing experiments.Amplicon sequencing,physicochemical factor analysis,and targeted metabolites analysis were performed in combination with microbial source tracking,?diversity partition,and community assembly quantitative analysis to systematically analyze the succession patterns of functional microbial community during the brewing process of Xiaoqu light-flavor baijiu and its impact on the flavor compounds metabolism.The main results are following:(1)The environmental factors of fermentation system directly related to the microbial community during brewing process were analyzed.The trends of the two batches are consistent.During saccharification,moisture,temperature,and reducing sugar increased while p H decreased.In the first 3 days of fermentation,moisture,temperature and ethanol increased rapidly while reducing sugar decreased quickly.After that,temperature dropped slowly while moisture and ethanol remained relatively stable.Acidity increased throughout the whole fermentation stage.(2)Bacterial and fungal biomass during brewing process was measured by q PCR.The change trend was consistent between two batches.Both bacterial and fungal biomass was increased during saccharification.During fermentation,the bacterial biomass in two batches remained relatively stable while the fungal biomass changed significantly.The fungal biomass of PS and TS batches rapidly increased from 10^9.39±0.25 copies/g grain and 10^9.17±0.25copies/g grain to 10^10.14±0.07 copies/g grain and 10^10.37±0.09 copies/g grain during the first three days of fermentation,respectively.In the post-fermentation stage,it decreased slowly to10^8.84±0.31 copies/g grain and 10^9.14±0.23 copies/g grain,respectively.(3)The microbial compositions of starters,saccharified grains,and fermented grains were analyzed by amplicon sequencing.Weissella(44.0%),Pseudomonas(11.4%),Rhizopus(64.9%),and Saccharomyces(28.0%)were the dominant microorganisms in PS.Lactiplantibacillus(43.2%),Acetobacter(12.8%),Levilactobacillus(11.9%),Monascus(64.9%),Saccharomycopsis(11.4%),Saccharomyces(11.0%),and Rhizopus(9.6%)dominated the microbial community of TS.The microbial community composition in the saccharification stage was consistent with that of starter in each batch.The microbial communities during fermentation in both batches were consisted of Lactobacillaceae(75.6%for PS batch,and 86.8%for TS batch)and Saccharomyces(97.6%for the PS batch,and 96.3%for TS batch).Microbial source tracking analysis showed that the microorganisms during saccharification were mainly derived from starters while environmental microorganisms dominated the fermentation community.The proportion of environmental microorganisms was gradually increased over brewing process.(4)?diversity partition was used to analyze the microbial community succession pattern during brewing process.Similar succession pattern was observed in both batches.Turnover(species replacement)dominated bacterial community succession,and 54%turnover in PS batch and 52%turnover in TS batch were related to the temporal bacterial succession.Succession pattern of fungal communities was attributed to both turnover and nestedness(species loss).Species loss mainly occurred at the beginning of the fermentation stage.More than 50%of turnover and nestedness were related to the temporal fungal succession.(5)The community assembly quantitative analysis framework was used to analyze the ecological process and determinants of microbial community construction during the brewing process.Both batches shared same patterns.In the saccharification stage,homogenization selection dominated bacterial community assembly.Temperature,p H,and reducing sugar were the determinants leading to homogenization selection.Ecological drift dominated the assembly of fungal communities in the saccharification stage.Grain acidification,mechanical stirring,and lack of oxygen in the fermentation stage promoted the succession of microbial communities.For the bacterial community,the acidification of the grain resulted in homogenization selection.For fungi,the reduction of oxygen led to the reduction of Rhizopus,Monascus,Saccharomycopsis.Saccharomyces dominated the fungal community by homogenizing dispersal during the fermentation stage.(6)HS-SPME-GC-MS was used to analyze the dynamics of main flavor compounds during brewing process.A total of 32 volatile metabolites were identified,including 10alcohols,8 acids,9 esters,1 aromatic compound and 4 others.Most of the flavor compounds were produced in the end of the saccharification stage and the fermentation stage,of which esters were mainly produced at the end of the saccharification stage.The metabolic of two batches was similar in composition but different in content.PS batch produced more alcohols while TS batch produced more acids and esters.