Revealing The Core Brewing Microbes And Their Metabolic Mechanisms In Chinese Maotai-flavor Baijiu

The brewing process of Chinese Maotai-flavor liquor(Baijiu)is a spontaneous solid-state fermentation process with artificial regulation.The complex microbiota is usually affected by some e exogenous fermentation conditions including environmental microbiota,raw materials,environmental temperature,and environmental moisture.Moreover,it is also influenced by some fermentation conditions including the microbial succession,metabolites accumulation,bio-heat accumulation,and humidity change.However,it is unclear that how to determine the core functional microbes and how the fermentation conditions influenced the microbial structures and functions in the fermentation microbiota.Therefore,in this study,we processed two batches of Chinese Maotai-flavor Baijiu production in A and B factories with the same manual operation.This study systematically analyzed the change of fermentation parameters,microbes,metabolic pathways,and flavor-related metabolites in microbiota.The main contents are as follows:(1)The modern method of flavor chemistry was used to analyze the changes of different fermentation parameters.We found that the trend of the change ratio about fermentation temperature and ethanol content were basically the same.In this study,the change ratio of fermentation temperature showed a trend of first decreasing and then increasing in the samples of fermented grains from A and B factories during pit fermentation process.The change of production ratio in ethanol content showed a trend of first increasing and then decreasing in the samples of fermented grains from A and B factories during pit fermentation process.(2)We used amplicon and metatranscriptomic sequencing technologies to analyze the structure and function of microbiota.In this study,we reveled the major function of microbiota and established a way to identify the core functional microbes in the process of microbial succession and functional shifts during production.We also found that the core microbes from microbiota were Pichia kudriavzevii,Saccharomyces cerevisiae,Schizosaccharomyces pombe,Zygosaccharomyces bailii,Lactobacillus spp.,Desmospora spp.,Byssochlamys spectabilis,Bacillus spp.,and Aspergillus spp.in the samples of fermented grains from two factories.In the Maotai-flavor Baijiu production,P.kudriavzevii,S.cerevisiae,Schi.pombe,Z.bailii and Lactobacillus spp.expressed a large number of genes related to the production of ethanol,acetic acid and lactic acid in pyruvate metabolism.It indicated P.kudriavzevii,S.cerevisiae,Schi.pombe and Z.bailii are core alcohol-producing yeasts in the early stage,and Lactobacillus spp.are core acid-producing bacterium in the late stage during pit fermentation.(3)We combined metatranscriptomic sequencing and simulated fermentation experiments to illustrate that Schi.pombe improved alcohol dehydrogenase adh P and yia Y to maintain the ethanol yield in the fermentation microbiota during Baijiu production.In the study,we analyzed the expression of AKR1A1,ADH5,adh P and yia Y related to the core microbes P.kudriavzevii,S.cerevisiae,Schi.pombe,Z.bailii in the samples of fermented grains from factory A and B.Schi.pombe adh P and yia Y genes in the samples of fermented grains from factory B were higher expressed than those from factory A at day 15.Moreover,the expression level of adh P(average5.93,log₂ fold change)and yia Y(average 4.21,log₂ fold change)about Schi.pombe C-11 in the samples from co-culture fermentation was higher than those in the samples from mono-culture(average 1.69 and 3.02,log₂ fold change,respectively).(4)We also combined metatranscriptomic sequencing and simulated fermentation experiments to illustrate that Schi.pombe degraded acetic acid by activating the mevalonic acid pathway in fermentation microbiota.In the study,we analyzed the enzyme expression of ADH5,ACH1,ACS2,HXT1,VMA7,ATF1,ACCAT1,HMGCS1 and HMGCR1 related to the core yeasts P.kudriavzevii,S.cerevisiae,Schi.pombe and Z.bailii(fragments per kilobase per million,FPKM)in the samples of fermented grains from factory B.The P.kudriavzevii,S.cerevisiae and Z.bailii expressed HMGCS1(average 0.30,0.12,and 0.75 FPKM,respectively)and HMGCR1(average 0.03,0.08,and 0.09 FPKM,respectively).Compared with the expression level of them,Schi.pombe has higher expression levels of HMGCS1(average 6.99FPKM)and HMGCR1(average 1.04 FPKM).Moreover,in the laboratory simulated fermentation experiments,we found that the microbial biomass of Schi.pombe C-11 showed a significant difference between the test(+acetic acid 10 g·L^-1)and control(no addition)group.The acetic acid content from the test group showed a downward trend with a minimum of 6.40±0.18 g·L^-1 during fermentation.Besides,the content of mevalonic acid increased significantly from 36 h to 60 h in the simulated fermentation(Mann-Whitney U test,p<0.001).The expression levels of ACS2,ACCAT1,HMGCS1 and HMGCR1 related to the acetic acid conversion were significantly upregulated(Mann-Whitney U test,p<0.01).(5)We revealed Schi.pombe could increase the content of propanoic and butyric acid by improving the key genes that influenced by bio-heat and acetic acid accumulation in microbiota,thereby promoting the production of other volatile flavor-related metabolites.In this study,there were 16 volatile components with large differences,including 1-butanol,propionic acid,butyric acid and other"framework"components between the samples of fermented grains from A and B factories.In the samples of fermented grains from B factory,P.kudriavzevii,S.cerevisiae,Schi.pombe and Z.bailii could increase the content of propanoic and butyric acid by improving the key genes related to the propionate and butanoate metabolism,thereby promoting the production of flavor-related metabolites.In the laboratory fermentation experiments,the ethanol content of the test group(without Schi.pombe C-11)was up to 26.49±0.32 g·L^-1,whereas the ethanol content of the control group(with Schi.pombe C-11)was up to 29.36±0.31 g·L^-1.In addition,there were five volatile alcohols and acids components with large differences,including 1-butanol,valeric acid,nonanoic acid,hexanoic acid,2-methylpropionic acid and heptanoic acid.This study increased our understanding of the population mechanism in the fermentation process of Maotai-flavor Baijiu.It made a solid foundation to improve the stability of process scientifically,effectively and systematically.It also enhanced the ability of producer to predict and regulate the fermentation process during Chinese Maotai-flavor Baijiu production.