| Chinese liquor has a long history and is one of the six famous distilled spirits in the world.In the process of liquor brewing,many microorganisms play significant role and are the decisive factors of liquor quality.Advances in gene sequencing and other technologies have promoted the in-depth study of microorganisms in liquor production.With a brewing history of more than 200 years,Zhijiang Daqu(founded in 1817)is a typical representative of Chinese strong flavor liquor in the middle reaches of the Yangtze River.Those liquor-brewing microorganisms,under the geographical and climatic characteristics,have an important research value.This study focuses on Zhijiang liquor brewing related microorganism.The viable microorganism composition and dynamic change regularity of microorganisms related to liquor fermentation were systematically studied,as well as their relationship with liquor quality.At the same time,the influence of the presence of dead microorganisms in the wine-making system on the microbial community in daqu,pit mud and fermented grains was analyzed,and the question that the interference of dead microorganisms in the wine-making system might lead to the research results and cognition distorted was answered.The main results are as follows:1.There were obvious differences in the flavor components of the strong flavor liquor respectively brewed from the old and new cellars.In this study,flavor liquor produced in cellars at age of 5 and 20 years was used as material.The differential flavor substances in liquor were characterized by gas chromatography and ion chromatography coupled with multivariate statistics.and the sensory quality was evaluated by radar chart.A total of 30 different aroma compounds were screened out.Compared with those produced from the new cellars,the content of ethyl caproate in the liquor brewed from the old cellars was higher,the content of acetaldehyde and acetal was lower,the pungency and irritation were weaker,the alcohol thickness and the sense of freshness were better,therefore the sensory quality was better.2.There were dead cells found in Daqu,pit mud,and fermented grains of strong flavor liquor.Propidium monoazide(PMA)can be used to remove dead microorganisms.Using PMA combined with q PCR,we found that:(1)there were few dead bacteria and many dead fungi in Daqu,accounting for 71.3% of total fungi.(2)There were a lot of dead bacterium cells in pit mud at the age of 5 years and 20 years,accounting for 50.8% and 71.8% of total bacteria,respectively.The total bacteria and viable bacteria in 20-year pit mud were higher than those in 5-year pit mud.(3)On day 7 during fermentation,a large number of dead bacteria were observed in the two kinds of fermented grains respectively from 5-year-old cellar and 20-year-old cellar,each accounting for about 50% of the total bacteria,but few dead bacteria were observed on day15,day 30 and day 60;and few dead fungi were observed in the whole fermentation process;both viable bacteria and fungi reached their peak on day30 during fermentation.3.The diversity and structure of microbial community in Daqu,pit mud and fermented grains for strong flavor liquor production were not affected by dead microorganism cells.However,the diversity and structure of viable microbial community in fermented grains changed with fermentation time.The microbial community diversity and structure of Daqu,5-year and 20-year pit mud,and fermented grains from the cellars at age of 5-year and 20-year were studied through PMA and amplification sequence analysis.The results indicated that the diversity and structure of bacteria and fungi in Daqu were not affected by dead microorganisms,that the diversity and structure of bacterial communities in two kinds of pit muds were not affected by dead bacteria,but affected by cellar age,and that the diversity and structure of bacteria and fungi in the two kinds of fermented grains were not affected by dead microorganism cells.However,during the fermentation process,the diversity of viable bacteria in the two kinds of fermented grains were decreased during the whole fermentation.There were significant differences in the structure of viable bacteria in fermented grains between day 7 and day 15.However,no significant difference in the structure of viable bacteria in the fermented grains was observed between day 30 and day 60.The diversity of viable fungi in fermented grains in cellars at age of 5 years was stable during the whole fermentation,but that in cellars at age of 20 years fluctuated greatly.The fungal structure of fermented grains from the cellar at age of 5 year was relatively stable during the whole fermentation process,whereas the viable fungal structure of fermented grains from the cellar at age of 20years tended to be stable 30 days after fermentation.4.The viable microorganism community compositions in Daqu,pit mud,and fermented grains were different,and the bacteria from environment had the greatest influence on fermented grains.PMA combined with amplified fragment sequencing was used to analyze the composition of viable microorganisms in Daqu,pit mud,and fermented grains.The main findings are as follows:(1)The dominant bacterium genera in Daqu were bacillus(16.6%),Kroppenstedtia(15.1%),and 14 other bacterium genera.High abundance fungus genera were Aspergillus(40.7%),Thermoascus(24.6%),Thermomyces(11.0%),and 5 other genera.(2)There were 18 dominant bacterial genera in pit mud.Significant difference in bacterial composition was observed between old and new pit mud.In the 5-year pit mud,Lactobacillus and new genera with unknown taxonomic status exhibited the greatest abundance,accounting for 81.6% of the total abundance.In the 20-year pit mud,except Lactobacillus,Clostridium and Capriciproducens displayed the highest abundance,which were 11.9% and 12.8%,respectively.(3)In the fermented grains,35 bacteria and 9 fungi were dominant.On day 0-15 post fermentation,the dominant viable bacteria included Lactobacillus(8.0%-70.9%),Pseudomonas(2.3%-7.7%),Bacillus(1.2%-17.1%),and Acetobacter(0.002%-12.52%),etc.On day 30 post fermentation,Lactobacillus(83%-92%)was found to be dominant bacteria in fermented grains.For fungi,Saccharomyces(40,7%-80.7%)was the main component.Source tracker software was used to trace the microorganism source in fermented grains.The results showed that the bacteria from the environment had the greatest impact on the fermented grains,followed by Daqu and pit mud.The influence of bacteria in pit mud from old cellars on fermented grains was greater than that from new cellars.Bacillus in fermented grains mainly came from Daqu and environment,while Lactic Acid Bacteria mainly came from pit mud.5.Lactic acid bacterium genome was dominant bacterium genome in the fermented grains from new and old cellars,and the key genes exhibited change with the fermentation time.The 161 single-bacterium genomes were obtained from fermented grains in new and old cellars by macrosequencing technology and Binning technology,of which 19 genomes with average relative abundance > 1% were dominant,and they exhibited the highest Lactic acid abundance.The functional analysis of microbiomics indicated that the relative abundance of key genes producing ethanol,butyric acid,and caproic acid was high in the early stage of fermentation,but low in the late stage of fermentation.In fermented grains from the cellars at age of 5years,the key genes producing acetic acid and lactic acid accounted for a high proportion in the whole fermentation process.However,in fermented grains from the cellars at age of 20 years,,the relative abundance of the key genes producing acetic acid and lactic acid was high in the early stage and low in the later stage.In addition,we also found that acid resistant genes arg R,asp A,ilv E,cfa of Lactic acid bacterium played important role in achieving acid resistance in the early stage of fermentation,and Dna K gene in the late stage of fermentation.In this study,the influence of dead microorganisms on microbial community structure related to strong flavor liquor fermentation was evaluated,and the characteristics of viable bacterial community were described in detail.Based on it,the source of viable bacteria in fermented grains was analyzed,and the relationship between viable bacteria in fermented grains,in Daqu,and in pit mud was quantitatively analyzed,which is conducive to more accurate description and understanding of liquor-brewing-related microbial community.Furthermore,the aroma production-related key genes and acid tolerance mechanism of lactic acid bacteria in the fermentation process of strong flavor liquor were revealed on the gene level.This study will provide insight into the role of microorganisms in the flavor production and acid resistance of lactic acid bacteria during the process of liquor fermentation.Our results will provide strong guidance for improving liquor quality. |
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