Fermentation Succession And Metabolic Characteristics Of Clostridia Community In Pit Mud Of Luzhou-Flavor Baijiu

Luzhou-flavor baijiu(Strong-flavour baijiu)was famous for its unique style of flavor,which produced by distinctive fermentation pit.Microorganisms inhabiting in pit mud had a key impact on the quality of strong-flavour baijiu.Studies had shown that as an important functional microflora in pit mud,the role of obligately anaerobic Clostridia had the functions of synthesizing butyric acid,caproic acid et al.However,their species diversity,fermentation succession and metabolic function in fermentation process were not fully clear.In this study,the high-quality 100-year pit mud of Luzhou-type liquor was taken as the research object,and the species diversity and fermentation succession of Clostridia community in pit mud during brewing process were analyzed by microbial molecular ecology technology;Meanwhile,Clostridial community in pit mud were screened by pure culture of microorganisms,and their metabolic functions were analyzed;The metabolic characteristics of a new Clostridium species were studied in detail.The main results obtained were as follows:(1)Using high-throughput sequencing platform and other molecular ecology techniques to analyze the community changes and its fermentation succession of Clostridia in pit mud during the fermentation process of strong-flavour baijiu liquor.It could be concluded that the relative abundance and biomass of Clostridial increased firstly,then decreased and remained relatively stable.According to the composition of Clostridial community structure,the whole fermentation cycle could be roughly divided into two stages,the middle stage of fermentation(7-22 days)was one stage,and the initial and final stage of fermentation was another stage.The results of 16 S rRNA gene amplifier sequencing showed that the dominant groups of Clostridia in pit mud mainly included 10 families,including Heliobacteriaceae,Ruminococcaceae,Clostridiales incertae sedis,Syntrophomonadaceae,Clostridiaceae,Clostries vadinBB60,Christensenellaceae Lachnospiraceae,Peptococcaceae and Caldicoprobacteraceae.Clostridial specific primer sequencing results showed that the dominant families in pit mud were Clostries Incertae Sedis,Heliobacteriaceae,Clostridiaceae,Lachnospiraceae,Syntdiaceae.Phomonadaceae,Christensenellaceae,Ruminococcaceae,Peptococcaceae,Clostridiales vadinBB61 group,Caldicoprobacteraceae,Eubacteriaceae and Thermoanaerobacteraceae.Especially Clostridiales Incertae Sedis,Heliobacteriaceae,Clostridiaceae.At genus level,Hydrogenispora,Sedimentiter,Clostridium,Unclas.Clostridiaceae 1,Syntrophomonas,Tissierella and other twenty-five genera were dominant community,especially Hydrogenispora(18.3%),Sedimentibacter(17.1%)and Clostridium(5.0%).The above dominant flora had different trends during fermentation.According to the PICRUSt prediction of Clostridia,it was preliminarily considered that metabolic activity of Clostridia in pit mud was concentrated in the stage of 11-26 day during fermentation cycle.(2)The composition of Clostridiaceaes in pit mud and jiupei were significantly different.The top three families with the highest average relative abundance in jiupei were Christensenellaceae(49.8%),Peptostreptococceae(25.9%)and Peptococceae(15.3%).The top three families with the highest average relative abundance in pit mud were Heliobacteriaceae(52.6%),Ruminococcaceae(12.6%)and Clostridiales incertae sedis(11.9%).(3)113 Clostridial strains were screened by pure culture of microorganisms from pit mud,which were divided into 2 orders,4 families,6 genera and 29 species.The main volatile components of fermentation broth of 29 representative strains were detected.Acids(acetic acid,propionic acid,butyric acid,hexanoic acid,heptanoic acid,octanoic acid,2/4-methylvaleric acid,n-decanoic acid,n-nonyl acid,etc.),alcohols(ethanol,phenylethanol,propanol,n/iso butanol,2-pentanol,hexanol,n-octanol,n-decanol,geraniol,undecanol,etc.),esters(ethyl acetate,ethyl hexanoate,ethyl hexanoate,S-methyl butanethioate,S-methyl thioacetate,isobutyl phenylacetate),indole(2-methylindole,methylindole),phenols(4-methylphenol,3-methylphenol,2,4-ditert-butylphenol,etc.)and aldehydes(benzaldehyde,etc.)showed that Clostridia were closely related to the quality of Luzhou-flavor baijiu.(4)The strain JN500901 was identified as a new species and named Clostridium fermenticellae.It had the ability to produce butyric acid,hexanoic acid and p-methylphenol.The results of gene annotation showed that the strain had the potential to utilize hemicellulose and pectin which were relatively difficult to be used in Jiupei.