The Effect Of Methanogens On The Flavor Compounds In The Pit Mud

The pit mud is a characteristic fermentation carrier of Luzhou-flavor liquor,and forms a variety of stable functional microorganisms including hexanoic acid bacteria,lactic acid bacteria,Clostridium,and methanogenic archaea.Methanogenic archaea is considered to be an important functional microorganism in the mud,but there is no systematic research on its metabolism in the mud and its interaction with other muddy functional microorganisms.In this paper,high-throughput sequencing was used to analyze the microbial diversity and metabolism in the mud,and the interaction between methanogenic archaea and other functional microorganisms was analyzed.The clotting separation of Clostridium and Methanogenic archaea in the mud was carried out by Hengate's anaerobic separation technique,and a phylogenetic analysis was performed on a strain having caproic acid-producing ability.The melon-forming archaea strain was used to treat the mud,and the fermentation of the strong-flavor liquor was carried out to analyze the effect of the methanogenic archaea on the flavor of the Luzhou-flavor liquor.The main research results are as follows:(1)The microbial community structure of pit mud at different pit ages was analyzed.The results showed that with the increase of pit age,thick-walled bacteria in pit mud were enriched and Proteus was eliminated.Methanobrevibacter,Lactobacillus and Curvibacter were the dominant microorganisms in pit mud of 5 years old.The dominant microorganisms of 25-year-old pit mud are Caproiciproducens,Petrimonas and Candidatus-Methanoplasma.The dominant microorganisms in 70-year-old pit mud are Caproiciproducens,Candidatus_Methanoplasma and Methanobacterium.Caproiciproducens and Lactobacillus are important sources of caproic acid,butyric acid and lactic acid in pit mud.Methanobacterium and Caproiciproducens,Methanobrevibacter and Lactobacillus have the same changing trend with the increase of pit age.Methanobacteria may play an important role in the composition of aroma components in pit mud.(2)The species distribution and metabolism of pit mud of 25 years old were analyzed.The results showed that ketone metabolism,methane metabolism,benzoate degradation and fatty acid metabolism were the most active in pit mud.Methanoculleus,Clostridium,Syntrophomonas and Lactobacillus are the most active species in pit mud.Methanogenic bacteria in pit mud are active.Macro-genome and macro-transcriptome analysis showed that there were key enzymes for caproic acid synthesis and they were highly expressed.Hydrotrophic methanogenic archaea can promote the synthesis of hexanoic acid by using the product H2 in the process of hexanoic acid synthesis as the metabolic substrate,so the methane metabolic pathway can promote the synthesis of hexanoic acid.Highly expressed mutants can promote the degradation of long-chain saturated fatty acids to acetic acid or propionic acid.Methanol nutritional methanogens can effectively reduce the methanol content in liquor and improve the quality of liquor.(3)116 strains of microorganisms were isolated from pit mud by directional isolation.Among them,53 strains were clearly classified and distributed in 3 families and 4 genera.The 16 S rRNA similarity of 63 strains of bacteria was less than 97%,and there were 7 potential new species.(4)The taxonomic status of JN-1 was analyzed.JN-1 was a Gram-positive bacterium with flagellum motility and sporulation.The optimum growth conditions of JN-1 were 0% NaCl,35 C and pH 6.5.The main metabolites of JN-1 are hexanoic acid,acetic acid and butyric acid.Based on the phylogenetic tree of 16 S rRNA,the similarity between JN-1 and its most similar strain was 94.2%.According to physiological,biochemical and molecular genetic characteristics,JN-1 is a new species of Clostridium,named Clostridium hexanoicum sp.nov.Removal of hydrogen and co-culture with methanogens can effectively promote the synthesis of Clostridium hexanoicum sp.Nov.hexanoic acid.(5)The pit mud was treated by adding methanogens,methanogens,bacterial cultures and inhibitors to complete liquor fermentation.After fermentation,Caproiciproducens and Methanobrevibacter were obviously enriched and their relative abundance increased.Mutual bacteria and methane oxidizing bacteria were inhibited in pit mud with inhibitors,and the growth of methanogenic bacteria and methanogenic bacteria with bacterial cultures could be effectively promoted.Adding methanogens,methanogens and bacterial cultures can significantly promote the content of hexanoic acid,butyric acid and acetic acid in yellow water and Luzhou-flavor liquor,effectively reduce the content of methanol and improve the quality of Luzhou-flavor liquor.