Unrevaling The Community Structure Of Butyrate-Producing Bacteria In The Fermentation Pit Of Luzhou-Flavour Liquor

As the typical model of Chinese liquor, Luzhou-flavour liquor, as known as Chinese strong flavor liquor, is famous for its unique manufacturing process and aroma style. Butyrate and its derivatives are key aroma compounds to the integral flavour of Luzhou-flavour liquor, thus, analyzing the generating mechanism of butyrate during the links in the production chain of Luzhou-flavour liquor, especially the in-pit fermentation phase is important to understanding the generating mechanism of the integral flavour of Luzhou-flavour liquor. In this thesis, molecular tenichques including the construction of clone libraries, high-throughput sequencing, denaturing gradient gel electrophoresis and real-time quantitive PCR combined with microbial culture technique are applied to study the structure and dynamics of butyrate-producing bacteria community in the fermenting pit of Luzhou-flavour liquor. This study possesses an important significance for both understanding the generating mechanism of the integral flavour of Luzhou-flavour liquor and locating, isolating the core functional microorganisms in the fermenting pit of Luzhou-flavour liquor. Main results showed as follows:1. In fermented grains, butyrate kinase pathway is found more widely distributed than butyryl-CoA:acetate CoA-transferase pathway. Butyrate-producing bacteria are mainly distributed in three classes namely Bacilli, Clostridia and Bacteroidia; among them, Clostridia butyrate-producing bacteria is dominant. The decreasement of Bacilli butyrate-producing bacteria is found closely related to the increasement of lactate content during the fermentation process(r =-0.812,P < 0.05). The mean relative abundance and the copy number of Clostridia 16 S rRNA sequences is 2.21% and 6.53×106 copies?g-1-3.46×107 copies?g-1, respectively, and showed a trend of rise first, then fall during the whole fermentation process. Compared with other samples, a significant difference is observed among Clostridia community in sample day 7; 17 OTUs are regarded as biomarkers, most of these OTUs remains unclassified. The decreasement of Clostridium and increasement of Sedimentibacter are also observed in in sample day 7. Clostridiaceae and [Tissierellaceae] are not only the two dominant Clostridia families but also the two Clostridia families that butyrate-producing bacteria centralized in; relative abundance of the two families are 38.12%and 12.30%, respectively.2. In pit mud, butyrate kinase pathway is also found more widely distributed than butyryl-CoA:acetate CoA-transferase pathway. Butyrate-producing bacteria are distributed in Firmicutes classes including Bacilli, Clostridia and Bacteroidia and Fusobacteriia belonging to Fusobacteria. Clostridia butyrate-producing bacteria are still considered as the dominant group. The relative abundance of Bacilli butyrate-producing bacteria is higher than that in fermented grains. 9 genera including Clostridium, Tissierella, Sedimentibacter, Dethiobacter, Natronincola, Lutispora, Sporosalibacterium, Gracilibacter and Cellulosibacter from 5 families namely [Tissierellaceae], Clostridiaceae, Syntrophomonadaceae, Gracilibacteraceae and Ruminococcaceae are observed. The copy number of Clostridia 16 S rRNA sequences ranges from 3.48×107 copies?g-1 to 1.85×109 copies?g-1, which is 10 to 100 times higher than that in fermented grains. A bimodal pattern of the Clostridia-biomass is detected.3. Two butyrate-producing strains are isolated from pit mud and furtherly identified as Clostridium butyricum and Clostridium kluyveri. Several kinds of volatile compounds namely 1-butanol; 1-butanol, 3-methyl-; 1-pentanol; 1-pentanol, 4-methyl-; benzyl alcohol; benzeneethanol; benzenemethanol, 3,5-dimethyl-; ethanethioic acid, S-methyl ester; octyl formate; acetic acid; butanoic acid; butanoic acid, 3-methyl-; pentanoic acid; pentanoic acid, 4-methyl-; disulfide, dimethyl; pyrazine, 2,5-dimethyl-; nonanal and Benzaldehyde are detected in the broth of C. butyricum in RCM. Only ethanol, butanoic acid and hexanoic acid are detected in the broth of C. kluyveri in ES medium in which hexanoic acid occupies 80%of total amount. The copy number of C. kluyveri 16 S rRNA sequences account for 0.1~1.0%of Clostridia 16 S rRNA sequences in both fermented grains and pit mud, and the mean biomass of C. kluyveri in pit mud is 0.86 times larger than that in fermented grains. Increasing trends of C. kluyveri 16 S rRNA sequences are observed in both fermented grains and pit mud during the whole fermentation process.