Study On The Analysis And Separation Of The Core Microbial Flora Of Maotai-flavor Liquor Mas

The brewing process of Maotai-flavor liquor is complicated,which is completed by a variety of microorganisms under the open environment,and the open environment is easy to cause the liquor quality instability.Analyzing the microbial community structure of Maotai-flavor liquor,revealing the core microbial community in the brewing process,and artificially constructing the fermentation system of the synthetic core microbial community are important methods to study the microbial community performance and improve the unstable quality of Maotai-flavor liquor.Moreover,the fifth round is one of the best rounds of Maotai-flavor liquor fermentation,and pit fermentation is the key link of Maotai-flavor liquor solid-state fermentation.It is of great significance to analyze the bacterial community structure in the fifth round of cellar-pit fermentation and study the relationship between key bacteria and flavor substances for the targeted regulation of Maotai-flavor liquor production.In this study,High-throughput sequencing and culture methods were combined to analyze the bacterial community structure and core bacteria composition and function in the fermentation process based on Isolation Chip(Ichip)in-situ culture and traditional culture,which provided a theoretical basis for in-depth understanding of the microbial community and fermentation mechanism of Maotai-flavor liquor brewing.Firstly,the physical and chemical parameters in the fermentation process were detected,and the bacterial community structure of fermented grains in the fifth round of pit fermentation of Maotai-flavor liquor was revealed by high-throughput sequencing technology,and the formation of volatile flavor substances in the pit fermentation was analyzed by headspace solid-phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GCMS).Comprehensive abundance,breadth,degree of interaction and the ability to synthesize volatile flavor substances determine the key bacterial communities.Secondly,the culturable bacteria in liquor were separated by traditional culture and Ichip in-situ culture,and the key bacterial species of core bacterial community were determined.Finally,the fermentation characteristics of the selected key bacterial species were discussed.Main conclusions are as follows:(1)In the fermentation process,the contents of moisture,ethanol and acidity showed an increasing trend,the content of lactic acid increased in the early stage and slightly decreased in the late stage,and the p H,reducing sugar and saccharification power showed a decreasing trend.A total of 11 phyla and 230 genera were detected by high-throughput sequencing.At the phylum level,Firmicutes accounted for 95.85%,which was the dominant phylum.At the genus level,Achromobacter,Bacillus,Kroppenstedtia,Scopulibacillus,Anaerosalibacter,Oceanobacillus,Virgibacillus,unclassified＿f＿＿Bacillaceae,Lactobacillus,Clostridium＿sensu＿stricto＿1,Thermoactinomyces were more than 1% and distributed in all samples,which were high abundance and widely distributed bacterial communities.In the fermentation process of cellar,bacterial diversity increased first and then decreased,with the highest diversity on the 10 th day.A total of 77 volatile flavor compounds were detected by HS-SPME-GC-MS,including23 esters,7 acids,7 alcohols,23 aromatic families,9 heterocyclic and 8other volatile compounds.The overall content showed an increasing trend.The contents of aromatic family and ester were the highest and increased with the increase of time.Correlation analysis showed that the Lactobacillus and Bacillus were in the key node and had strong ability to synthesize flavor substances.By comprehensively analyzing bacterial abundance,width degree,correlation degree and production capacity of volatile flavor substance,it was determined that Lactobacillus and Bacillus were the key bacteria in the fifth round fermentation of Maotai-flavor liquor.(2)Ichip in-situ culture method and traditional culture method were used to isolate and screen the culturable bacteria in fermented grains of pit,and analyze the bacterial structure and diversity.Fourteen species of 5bacterial genera were obtained by traditional culture,belonging to Firmicutes.The results showed that 22 species from 12 bacterial genera were obtained by Ichip in-situ culture,belonging to Firmicutes,Proteobacteria and Actinomyces,among which Firmicutes accounted for the largest proportion.Therefore,Ichip in-situ culture isolated and screened more bacterial species than traditional culture.Bacillus is the dominant bacterium genus screened by the two methods,and Bacillus amyloliquefaciens is the most abundant strain of Bacillus.The Lactobacillus plantarum obtained by traditional culture and in situ culture screening was high in content.B.amyloliquefaciens and L.plantarum were determined to be the key bacterial species,which may play an important role in the fermentation of liquor.(3)B.amyloliquefaciens and L.plantarum were used as the core bacteria to explore the fermentation characteristics through proportional and sequential fermentation.The p H value of Mbl fermentation broth of the sequential fermentation samples was low and the lactic acid content was the highest,indicating that Mbl had better acid production.L.plantarum inhibited the growth of B.amyloliquefaciens,which may have promoted the production of lactic acid.A total of 56 volatile flavor compounds were detected in the fermentation liquid samples,including 13 kinds of aromatic family,10 acids,12 alcohols,8 esters,8 heterocyclic cyclics and 5 other volatile compounds.The content of total volatile substances in Mbl was higher than that in other treatments,mainly reflected in acids,alcohols,aromatic family and aldehydes and ketones.The results showed that B.amyloliquefaciens and L.plantarum inoculated successively may be more beneficial to flavor substance transformation.A further comparative analysis of flavor substances of Mbl and fermented grains showed that there were 22 common flavor substances of Mbl and fermented grains,and the highest content of phenyl ethanol was found in common flavor substances,and there were many volatile flavor precursors in Mbl.The results showed that L.plantarum and B.amyloliquefaciens contributed significantly to the formation of liquor flavor substances.