Microbial Diversity In Traditional Pit Mud For Strong-flavor Baijiu,and Relationship Between Succession Of Microbial Community Structure In Artificial Pit Mud And Quality Variation Of Baijiu During The Production Process

Chinese strong-flavor Baijiu（CSFB）dominates the Chinese Baijiu-making industry and plays a central role in the national economic development.CSFB is produced via fermentation of grains in cellars lined with pit mud.This pit mud contains significant numbers of functional microbes,which are important for the production of aromatic compounds in CSFB.Thus,the composition of microbial communities in pit mud largely determines the CSFB quality.According to microbial enrichment and the time required to reach maturity,pit mud can be divided into traditional（TPM）and artificial pit mud（APM）.Although previous studies that focused on the microbial communities in pit mud have obtained a number of achievements,several problems remain:the actual diversity of microbial communities within pit mud has not been explored in detail.Moreover,the microbial community succession and function change during CSFB brewing in APM are also not well understood.These issues limit the further exploration and utilization of beneficial microbial resources,and restrict the improvement of the APM culture technique.The contents and achievements of this study can be summarized as follows:（1）The microbial community diversities in TPM samples with ages of 5 and 100years were analyzed using a high-throughput sequencing technique,and a total of 65bacterial and 111 fungal genera were detected.Despite the non-significance of the differences in microbial species diversity between the 5-and the 100-year TPM samples,their community structures differed significantly.Among the dominant genera,for example,Hydrogenispora,Caproiciproducen,Saccharomyces,Candida,and Wickerhamomyces greatly increased in the 100 year-TPM samples.The results of high performance gas chromatography（HPGC）,high performance liquid chromatography（HPLC）,and conventional physicochemical analysis indicate that the levels of organic matter,NH₄⁺,caproic acid,and pH were significantly increased in the 100 year-TPM samples（p<0.05）.Moreover,the organic matter content is the main factor that influences the microbial community distribution in TPM samples,and correlates positively with the abundances of Caproiciproducens,Aspergillus,Candida,and Saccharomyces,as well as with the caproic acid content,butyric acid content,and fungal Shannon index（p<0.05）.The result of BLAST in Gen Bank indicates that the TPM samples contain both abundant and new taxa that differ from any other pure culture previously characterized.21.7%of the bacterial operational taxonomic units（OTUs）and 20.75%of the fungal OTUs are distant to cultured counterparts,respectively.At the high rank classification level,the classes Clostridia and Eurotiomycetes presented the highest proportion of novel OTUs.Most novel OTUs were initially isolated from diverse environments,the most abundant of which originated from Chinese Baijiu brewing ecosystems,thus highlighting the importance of these OTUs for CSFB production.However,their taxonomic status and physiological function still require further identification.（2）The bacterial community structure in APM samples derived from new cellars during the 1st to the 4th CSFB brewing batch was investigated using high-throughput sequencing and real-time quantitative PCR.A total of 34 dominant bacterial genera were detected in the APM samples in each batch.Among these,Clostridium,Hydrogenispora,Lactobacillus,and Bacillus remained dominant during different phases of the brewing.With extended brewing,the bacterial community structure in the APM showed a dramatic succession,which was driven by Clostridia and Bacilli.Moreover,the result of PICRUSt indicates that the bacterial succession is closely related to the metabolic function of Baijiu brewing.For example,the gene copy numbers of fatty acid degradation,propanoate metabolism,and butanoate metabolism were significantly increased during brewing（p<0.05）.The results of redundancy and Pearson correlation analysis indicate that the contents of total N,available K,and available P are the main factors that influence the bacterial community distribution of APM samples.These three physicochemical properties have significant correlations with the abundance of 18 dominant genera.Of these genera,Clostridium,Desulfitobacterium,Fonticella,Haloimpatiens,and Desulfosporosinus were significantly positively correlated with these three physicochemical properties and are most closely related with the aromatic compound content in CSFB.Their abundances all significantly correlated with the contents of more than eight main aromatic compounds.Mainly positive correlations were found with contents of ethyl caproate,ethyl butyrate,caproic acid,acetic acid,isovaleric acid,and n-hexanol（p<0.05）.Negative correlations were found with contents of ethyl acetate,n-propanol,3-methyl butanol,and 2-butanol（p<0.05）.This result suggests that an appropriate increase in the contents of total N,available K,and available P in APM can promote the growth of beneficial microbes,improve the CSFB quality,and the comfort degree after drinking.Finally,analysis of the distribution of dominant bacteria from APM samples in the APM-starter culture and APM-raw materials indicates that the dominant genera in the APM samples that are closely related to aromatic compounds are consistent with those in the APM-starter culture.Thus,improving the quality of the APM-starter culture is essential to enhance the APM efficacy.（3）The APM samples from the 1st and 4th CSFB brewing batch were used as study material,and changes of proteome and metabolome between both groups were compared using combined tandem mass tags（TMT）,gas chromatography-mass spectrometer,（GC-MS）,and liquid chromatograph-mass spectrometer（LC-MS）.A total of 213 proteins and 7478 metabolites with quantitative information were identified in APM samples.The proteins originated from 69 bacterial groups and mainly belong to Lactobacillus and Clostridium.A total of 47 differentially expressed proteins and 1014 differentially expressed metabolites were screened,among which 16 proteins and 483 metabolites were significantly up-regulated,while 31 proteins and 531 metabolites were significantly down-regulated in the 4th batch-APM samples.We integrated 11 KEGG pathways directly linked with the synthesis of caproic acid,butyric acid,acetic acid,and lactic acid.The integration pathway includes 13 differentially expressed proteins and 32 differentially expressed metabolites.The differentially expressed proteins were mainly derived from Lactobacillus,while acetyl-CoA acetyltransferase from Syntrophomonas was involved in the largest number of KEGG pathways.Based on the result of the integration pathway,it could be deduced that with extended brewing,the main function of the bacterial community in the APM can be transformed from sugar metabolism to organic acid metabolism.Furthermore,the decrease in lactic acid synthesis capability and the enhancement of the butanoate metabolism are directly correlated with the improvement of the CSFB quality.Moreover,the obtained results indicate that succinate semialdehyde and acetyl-CoA are important links in multiple metabolic pathways that drain into the butanoate metabolism.Acetyl-CoA acetyltransferase is a critical protein that catalyzes acetyl-CoA,thus promoting the transformation from pyruvate metabolism,propanoate metabolism,and fatty acid degradation to the butanoate metabolism.Overall,the results of this study significantly increase the number of microbes known to be associated with CSFB production,and provide a basis for further understanding the CSFB fermentation mechanism and exploring microbial resources for biotechnological applications.Moreover,the integrated metabolic pathway related to the synthesis of aromatic compounds in CSFB obtained will be helpful for further optimization of APM culture technique.