| Cave-brewed sauce-flavor Baijiu(CBSB)was brewed by using natural caves formed in the karst landscape of Guizhou province as a natural brewing workshop.The CBSB is different of traditional sauce-flavor baijiu in that the brewing process is carried out entirely in the natural cave.To study the potential correlation between microbial communities and flavor substances in the fermentation process of CBSB,explore the main functional microorganisms that contribute to the formation of flavor substances,and provide theoretical basis for the production and quality control of CBSB.As the beginning of CBSB,the microorganisms and their metabolites accumulated during the production process,which have an important impact on subsequent baijiu production rounds.Therefore,it is particularly important to study the microbial diversity and flavor quality formation of Xiasha round of the CBSB.So,in this paper,fermented grains from different fermentation stages(stacking fermentation: 0 d,1 d,2 d and 3 d;cellar fermentation: 10 d,20 d and 30 d)in Xiasha round of CBSB were used as experimental materials.The community composition and succession of bacteria and fungi during fermentation were studied,and physicochemical properties,volatile flavor compounds,and taste compounds(organic acids)were analyzed.Then,multivariate statistical analysis was used to reveal the main functional microorganisms related to the formation of flavor substances.The research results were as follows:(1)The microbial diversity and community composition during the sand rotation fermentation process under CBSB were analyzed using Illumina High Throughput Sequencing(HTS)technology.The results showed that a total of 909,679 V3-V4 16 S r RNA original sequences and 890,292 ITS1 original sequences were obtained.Through quality filtration and splicing,a total of 644,027 bacterial effective sequences and561,344 fungal effective sequences were obtained.The average effective rates of bacteria and fungi were 74.51% and 65.34%,respectively.The bacterial abundance first decreased and then increased during the stacking fermentation stage,and significantly increased at the end of the fermentation stage in the cellar.Fungal richness tends to decrease during the stacking fermentation stage and increase during the fermentation process in the cellar.Overall,bacterial diversity was higher than that of fungi.At the phylum level,a total of 19 bacterial phyla and 6 fungal phyla were detected,of which the Firmicutes and Actinobacteria were dominant bacterial phyla,and the Ascomycota was dominant fungal phyla.A total of 185 bacterial and 102 fungal genera were detected at the genus level,with 10 dominant bacterial and 10 dominant fungal genera each,respectively.The dominant bacterial genera were Lactobacillus,Unspecified＿Leuconostocaceae,Weissella,Unspecified＿Bacillaceae,Unspecified＿Lactobacillaceae,Thermoactinomyces,Pediococcus,Bacillus,Oceanobacillus,Leuconostoc.Saccharomycopsis,Unspecified＿Phaffomycetaceae,Thermomyces,Debaryomyces,Wickerhamomyce,Byssochlamys,Rasamsonia,Unspecified＿Aspergillaceae,Thermoascus,Aspergillus were the dominant fungal genera.The correlation between microorganisms indicates that the positive correlation between dominant bacterial and dominant fungal genera was stronger than the negative correlation.We used Source Tracker,a Bayesian probability tool to trace the source of microorganisms in the fermented grains at the beginning of stacking fermentation.It was found that 73.37% of bacteria and 29.31% of fungi originated from hightemperature Daqu,and 20.50% and 9.43% of bacteria and fungi originated from raw material sorghum,respectively.Particularly,14.62% of fungi come from the ground of the fermentation environment,and the source of most fungi is unknown.(2)Volatile flavor compounds in different fermentation stages of CBSB Xiasha fermentation were analyzed.In total,72 VFCs were detected,including 23 esters,13 alcohols,11 acids,8 phenols,6 hydrocarbons,4 aldehydes,and 7 other compounds.With fermentation proceeds,the total content of VFCs increases.Using Linear discriminant analysis Effect Size(LEf Se)analysis,23 significantly different VFCs were obtained based on the difference level LDA=4,P < 0.05.The GC-IMS was used to analyze the VFC,and a total of 44 VFCs were identified.According to the twodimensional top view,with the extension of fermentation time,the VFCs in the fermented grains gradually increased.3-hydroxy-2-oxobutane,2-Methyl-1-butanol,2-Hexanone,Valeric acid,Valeraldehyde,1-Octen-3-ol,2-Pentanone,Ethyl isovalerate,2,5-dimethyl tetrahydrofuran,and 2-Butanone were the unique VFCs that mark the end of stacking fermentation.1-Hexanol,3-methyl-1-pentano,4-Methyl-1-pentanol,and 2-Heptanone were the unique VFCs that mark the end of cellar fermentation.(3)The physicochemical properties and organic acids were analyzed of different fermentation stages in the Xiasha round of CBSB.The results showed that during the stacking fermentation,temperature,acidity,and reducing sugar content were increased,while starch and moisture were decreased.During the fermentation stage of the cellar,the moisture and acidity increased significantly(P < 0.05),the temperature showed a downward trend,and the content of reducing sugar and starch decreased significantly(P < 0.05).A total of 7 types of organic acids were detected,including citric acid,malic acid,succinic acid,oxalic acid,acetic acid,tartaric acid,and lactic acid.Among them,citric acid,lactic acid,and succinic acid were important organic acids in the Xiasha round of CBSB.Principal component analysis found that organic acids mainly aggregate during the fermentation stage of the cellar.At the end of stacking fermentation,the organic acid content from high to low were: lactic acid,malic acid,citric acid,succinic acid,tartaric acid,acetic acid,and oxalic acid.At the end of fermentation in the cellar,the order of organic acid content from high to low were: citric acid,malic acid,lactic acid,succinic acid,tartaric acid,acetic acid,and oxalic acid.(4)The correlation between microbial community,physicochemical properties,and VFCs were analyzed in different fermentation process stages of Xiasha round of CBSB.The correlation analysis between dominant bacteria and physicochemical properties based on redundancy analysis(RDA)showed that starch was the main physicochemical properties affecting microbial succession during the stacking fermentation stage,while acidity,moisture,and reducing sugar were the key factors affecting microbial succession during the fermentation stage in the cellar.Based on Pearson correlation coefficient and P value,the correlation between dominant microorganisms and significantly different VFC was analyzed.The results showed that a total of 18 dominant bacterial genera were correlated with 23 significantly different VFCs,with a total of 62 positive correlations and 43 negative correlations.At the level of bacterial genera,the most positively and negatively correlated with significantly different VFCs were Lactobacillus and Weissella,which were positively and negatively correlated with 11 and 12 significantly different VFCs,respectively.At the level of fungi genera,Wickerhamomyces and Aspergillus were positively correlated with 10 significantly different volatile metabolites,respectively.The Byssochlamys was negatively correlated with 4 significantly different VFCs.The relative abundance of dominant bacteria positively correlated with VFCs in the cellar fermentation was higher than that in the stacking fermentation,indicating the cellar fermentation stage was the main stage for VFC accumulation.The names of all compounds were input into Metabo Analyst 5.0 for metabolic pathway enrichment,and a total of 13 metabolic pathways were obtained,indicating that these metabolic pathways were related to the fermentation process in the Xiasha round of CBSB.Based on relevant enzymes annotated by microorganisms,and combined with KEGG database and relevant literature,the metabolic profiles of dominant microorganisms during the Xiasha round of CBSB were predicted. |
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