Study On Microbial Diversity And Flavor Quality Of Black Glutinous Rice Wine During Fermentation

Black glutinous rice wine,a traditional Chinese rice wine,is made from black glutinous rice with starters and yeast.Fermented black glutinous rice wine is a bilateral fermentation with the participation of multiple species in an open environment.Study the potential relationship between flavor and microorganisms during the fermentation of black glutinous rice wine,and reveal the main functional microorganisms that contribute to the formation of flavor substances,which is of great significance for guiding the production of black glutinous rice wine and controlling the quality of products.At present,there are many studies on the changes of flavor substances in black glutinous rice wine,but there are few studies on the composition and changes of microbial community during the fermentation of black glutinous rice wine and the relationship between microorganisms and the formation of flavor substances is not clear.This study aims to research the dynamic changes of flavor substances and microbial community during the fermentation of black glutinous rice wine and their potential correlation,and reveale the functional microorganisms related to the formation of flavor substances.The results are as follows:(1)High throughput sequencing(HTS)technology was used to analyze the physicochemical characteristics and microbial flora of three unique starters in Guizhou province.The analysis of physicochemical characteristics showed that the water content in starter A was the highest,the acidity and liquifying enzyme activity were the highest in starter B,and the saccharifying enzyme activity and fermentability were the highest in starter C.The diversity analysis of bacteria and fungi in three starters showed that the species richness was the highest in starter A and the community structure was the most complex in starter C.At the level of phylum,Proteobacteria was the dominant bacteria phylum and Zygomycetes was the dominant fungi phylum in the starter A.Firmicutes was the dominant bacteria phylum and Zygomycetes was the dominant fungi phylum in the starter B.Proteobacteria was the dominant bacteria phylum and Ascomycota was the dominant fungi phylum in the starter C.Atthe genus level,the dominant bacteria of starter A was Rhodococcus,and the dominant bacteria of starters B and C were Bacillus.The dominantfungi of starters A and B were Rhizopus,while the dominant fungi of starter C was Saccharomyce.There was a certain correlation between the physicochemical indicators and the bacterial population structure of starters.Based on the analysis of physical and chemical indexes and microbial diversity,the starter C was selected to ferment black glutinous rice wine for further study.(2)The microbial community succession during the fermentation of black glutinous rice wine was analyzed by HTS.A total of 1,671,743 pairs of original sequences were obtained by bacteria,with an average length of 396 bp.A total of 1,679,460 pairs of original sequences were obtained by fungi,with an average length of 292 bp.The α-diversity analysis showed that the microbial community diversity and richness indices of bacteria and fungi were unstable.The analysis ofβ-diversity showed that the succession of bacterial community was divided into 5 stages,while the succession of fungal community was divided into 3 stages.Taxonomic analysis revealed that 5phyla and 23 genera were identified in the bacterial community structure,in which,Firmicutes and Proteobacteria were dominant bacteria phylum,while Leuconostoc,Pediococcus,Bacillus and Lactobacillus were dominant bacteria genera.A total of 3 phyla and 10 genera were identified in the fungal community structure,among which Mucoromycota,Ascomycota,Rhizopus,Saccharomyce and Saccharomycopsis were dominant fungal genera.(3)The physical and chemical indices in the fermentation process of black glutinous rice wine were detected.The results showed that the contents of amino acid nitrogen,alcohol and total acid showed an overall increasing trend,and the p H decreased first and then increased,while the total sugar content remained relatively stable after a sharp decline after 4d.HS-SPME-GC-MS and HPLC were used to dynamically monitor volatile flavor substances and organic acids in the fermentation process.The results showed that 44 volatile flavor substances were detected,including 17 esters,7 alcohols,7 alkanes,4 acids,3 aldehydes and 6 other compounds.Through principal component analysis(PCA),it was found that there were 23 volatile substances in the wine which had a great influence on the overall flavor and taste.A total of 9 organic acids were detected,including oxalic acid,tartaric acid,pyruvate,malic acid,ketoglutaric acid,lactic acid,citric acid,succinic acid and acetic acid.Among them,lactic acid and succinic acid were the main organic acids in the wine.(4)Gas chromatography-time-of-flight mass spectrometry(GC-TOF-MS)metabolomics and multivariate statistical analysis were used to explore the relationship between metabolites and fermentation time.The result showed that 159 metabolites were identified during the whole fermentation process.The PCA analysis displayedthat there was a clear separation between the samples after 4 days and 2 days,while the samples after 4-24 were clustered together,indicated that the wine fermentation progresses rapidly in the first 48 hours of fermentation.A total of 40 metabolites were identified as differential metabolites during fermentation(VIP > 1 and p < 0.05),including 12 organic acids,4 amino acids,1 fatty acid,17 sugars and sugar alcohols,1 alcohol and5 other metabolites.Pathway analysis showed that differential metabolites involved 28 metabolic pathways,showing the most influenced pathways(impact value > 0.1 and p < 0.05)were galactose metabolism,pyruvate metabolism,starch and sucrose metabolism,alanine,aspartic acid and glutamate metabolism,the tricarboxylic acid cycle,glyoxylic acid and dicarboxylic acid metabolism,and amino sugar and nucleotide sugar metabolism.Moreove,the integrated metabolic pathway was generated to understand the transformation and accumulation of differential metabolites.(5)The analysis of the generic relationship among microbial communities by correlation analysis.The bacterial correlation analysis showed that there were 51 positive correlations and 4negative correlations between the bacterial genera.Fungal correlation analysis showed that there were 23 positive correlations and 22 negative correlations in the fungal genera.The correlation analysis between microorganisms and physicochemical indicators showed that Lactococcus and Pediococcus were affected by total acid,p H,alcohol and amino acid nitrogen.In fungal community structure,Aspergillus was affected by total acid,alcohol content,p H and amino acid nitrogen,while Rhizopus was correlated with total sugar content.The correlation analysis of microorganisms belonging to volatile compounds showed that a total of 13 microbial genera had important influence on volatile flavor(VIP > 1.0 and p < 0.05),namely Leuconostoc,Bacillus,Lactobacillus,Cronobacter,Pantoea,Kosakonia,Weissella,Enterococcus,Rhizopus,Saccharomycopsis,Myceliophthora,Bacillus,Lactobacillus,Cronobacter,Pantoea,Kosakonia,Weissella,Enterococcus,Rhizopus,Saccharomycopsis,Myceliophthora,Cystofilobasidium and Aspergillus.Among them,Cronobacter,Pantoea and Weissella were the main core functionalbacteria.A total of 71 positive correlations and 37 negative correlations were found between microorganisms and non-volatile metabolites.A total of five bacterial genera have a greater impact on non-volatile metabolites,namely Cronobacter,Pantoea,Weissella,Klebsiella,Acinetobacter.