Isolation Of Clostrium From Pit Mud Of Luzhou-flavor Liquor And Their Metabolic Profiles

Microorganisms play an important role in the formation of flavor compounds in Luzhou-flavor liquor.There were many studies have showed that Clostridia bacteria were the main functional bacteria involved in the formation of liquor flavor compounds in pit mud.However,there is still a lack of intensive research on the diversity of community composition and metabolic function.In this study,we used high-throughput sequencing to detect Clostridium communities in pit mud.And then we analyzed the correlation between flavor compound abundance and Clostridium community structure in pit mud.Besides that we also used pure culture to isolated and identified Clostridium in pit mud.Finally,we evaluated metabolic profiles of Clostridium.The main results of this study were as follows:The microbial community structure in pit mud was analyzed by high-throughput sequencing.The α-diversity of microbial community showed that the diversity index of microbial community in B pit mud was significantly higher than that in A pit mud.β-diversity showed that Clostridia occupied the dominant position of microbial community in A and B pit mud at class level,the relative abundance were 53.67% and 73.95% respectively.At genus level,the dominant microbial were Desulfurispora(15.90%),Caproiciproducens(14.24%),Clostridium sensu stricto 12(4.96%)and Hydrogenispora(4.96%)in A pit mud,and were Caproiciproducens(51.71%)and Clostridium sensu stricto 12(9.05%)in B pit mud.According to the correlation analysis,the microorganisms of Caproiciproducen and Clostridium sensu stricto 12 were significantly positively correlated with physical and chemical factors,such as butyric acid and caproic acid abundance.So we could speculated that the microorganisms of these two genera were probably the main Clostridium bacteria producing butyric acid and caproic acid in pit mud.We screened 63 strains from pit mud,and they belonged to five different classes.32 strains belonging to Clostridia were devided into 14 clusters,which were C.tepidum,C.botulinum,C.sporogenes,C.tyrobutyricum,C.scatologenes,C.carboxidivorans,C.senegalense,C.homopropionicum,C.butyricum,C.beijerinckii,C.aminovalericum,Clostridium sp.,C.sphenoides and C.celerecrescens,respectively.The blast results showed that C.sporogenes and C.scatologenes were classified as Clostridium sensu stricto 12 in pit mud,and the two strains had high relative abundance in Clostridium of pit mud.Finally,we used headspace solid-phase microextraction(HS-SPME)and gas chromatographymass spectrometry(GC-MS)to detect the volatile profiles of representative Clostridium strains.The results showed that 239 volatile flavor compounds were detected,including 43 esters,17 acids,56 alcohols,44 aldehydes and ketones,and 79 others.According to the metabolic capability,these strains could be divided into two main categories,the first group included C.carboxidivorans,C.sporogenes,C.tyrobutyricum,C.aminovalericum and C.homopropionicum,which were good at producing acid,another group contained C.beijerinckii,C.butyricum,C.senegalense,C.celerecrescens and C.sphenoides,which were good at producing alcohol.Interestingly,the content of hexanoic acid produced by C.sporogenes accounted for more than 80% of the total metabolites of the strain,and that of butyric acid produced by C.tyrobutyricum accounts for more than 83% of the total metabolites of the strain.