Analysis Of Black Glutinous Rice Wine Fermentation Flora And Flavor Metabolism Network Based On Metagenomic And Metabolomics

Guizhou black glutinous rice wine(BGRW),a traditional Chinese rice wine brewed from local specialty black glutinous rice as raw material,it was granted national GI product protection on August 23,2012.BGRW is produced by traditional Jiuqu fermentation,which has the problems from long production cycle,difficult fermentation conditions to control,poor quality stability,and low food safety.Thereby,it has great significance to provide theoretical for the industrial production and quality control of BGRW.In this study,the traditional Jiuqu was screened and inoculated with Rhizopus oryzae Q303 and Saccharomyces cerevisiae strains to prepare the fortified Jiuqu,and the same batch of uninoculated Jiuqu was the traditional Jiuqu.Then,BGRW of traditional and fortified fermentation were performed to study the microbial community succession pattern and the dynamic changes and differences between volatile and non-volatile flavor metabolites during fermentation process.Through with the data of correlation between microorganisms with physicochemical indexes and metabolites were combined to construct the microbiota and flavor metabolic network in BGRW fermentation,which revealed the core functional microorganisms related to the formation of flavor compounds in bio fortified fermentation.The main findings are as follows:(1)The non-volatile metabolites of Jiuqu were detected using GC-TOF-MS,whereas the classification and metabolic potential of the microbiota were investigated using metagenomic sequencing.Results showed that Firmicutes,Proteobacteria,and Actinobacteria represented the dominant bacterial phyla and Lactobacillus,Bacillus,Acinetobacter,Leuconostoc,and Weissella represented the dominant bacterial genera.Meanwhile,Ascomycota,Mucoromycota,and Basidiomycota were the dominant fungal phyla and Aspergillus,Saccharomyces,Pichia,Rhizopus,and Phycomyces were the predominant fungal genera.Functional annotation of the microbiota revealed a major association with metabolism of carbohydrates,cofactors and vitamins,as well as amino acids.A total of 39 ISDMs were identified to involve in 47 metabolic pathways,primarily that of starch and sucrose;glycine,serine,and threonine;glyoxylate and dicarboxylate;pyruvate;as well as biosynthesis of pantothenate and Co A.Further,based on Spearman's correlation analysis,Aspergillus,Saccharomyces,Lactobacillus,Acetobacter,Weissella,Pantoea,Desmospora,and Bacillus are closely correlated with production of physicochemical indexes and ISDMs.Moreover,the metabolic network of Jiuqu is mainly centered on carbohydrate metabolism and tricarboxylic acid cycle,which is used to decompose substrates to form ISDMs.(2)HTS was used to analyze the microbial community structure during traditional and fortified fermentation BGRW.A total of 1,635,863 and 1,623,855 pairs of raw reads(average length = 426 and 313 bp)were obtained from the bacteria and fungi of the 21 CTD group samples by Illumina Hi Seq sequencing,and after quality control,1,474,081 and 1,445,221 effective tags were generated and accounting for 90.11% and 89.00%,respectively.Meanwhile,1,671,092 and1,658,882 pairs of raw reads(average length = 425 and 332 bp)were obtained from the bacteria and fungi of the 21 QHD group samples,and after quality control,1,527,051 and 1,571,033 effective tags were generated and accounting for 98% and 97%,respectively.The microbial diversity changed greatly from 0 day to 4 day and from 11 day to 16 day.Judging from classification level of phylum,a total of four bacterial phyla and three fungal phyla(relative abundance > 0.01)were identified,namely Firmicutes,Proteobacteria,Cyanobacteria,and Bacteroidetes and Ascomycota,Mucoromycota,Basidiomycota,and Chytridiomycota,respectively.At the genus level,a total of 113 bacterial genera and 82 fungal genera were identified and classified in BGRW samples.Based on bacterial genera,the dominant bacterial communities were Pediococcus,Bacillus,uncultured?Enterobacteriaceae,Klebsiella,Acinetobacter,Lactobacillus,Weissella,Klebsiella,uncultured?Chloroplast,Acinetobacter,and uncultured?Mitochondria in CTD and QHD groups(relative abundance > 0.1).For the fungi,10 core fungal genera were detected(relative abundance exceeded 0.1),including: Rhizopus,Saccharomyces,Chaetomium,Penicillium,Aspergillus,Solicoccozyma,Mortierella,Stachybotrys,Trichocladium,and Schizothecium.The amplicon sequencing results showed that compared with traditional and fortified fermented BGRW,the dominant bacteria had no significant change,while the dominant fungi were disturbed greatly.During the fermentation process,PICRUSt function prediction showed that metabolism was the major important function of bacterial community,especially global and overview map,carbohydrate metabolism,and amino acid metabolism,and pathogenic genes showed a downward trend.(3)Physicochemical indexes and metabolites of BGRW during traditional and fortified fermentation were determined by physicochemical test with head space solid phase microextraction coupled with HS-SPME-GC-MS and UPLC-QTOF-MS,respectively.The results showed that the fermentation performance of CTD and QHD groups had a similar trend,but there were still differences in content between the two groups.The pH values of samples decreased rapidly from 2 nd to 4 th days of fermentation in CTD and QHD.Interestingly,the pH values of samples showed a slowly rising trend after 4 days of fermentation.The total sugar concentration decreased continuously from 2 day to 16 day of fermentation,and then increased to 22 day.However,alcohol,total acid,and amino acid nitrogen content gradually increased during the fermentation process.In addition,133 volatile metabolites and 789 non-volatile metabolites were detected.PCA and PLS-DA analysis showed that there were differences in the levels of metabolites between the samples.Combined with VIP > 1,p < 0.05,and compound matching degree,36 significantly different volatile metabolites and 48 significantly different non-volatile metabolites were examined.Pathway analysis showed that “Flavone and flavonol biosynthesis”,“Glycine,serine and threonine metabolism”,“Sulfur metabolism”,“Valine,leucine and isoleucine biosynthesis”,“Glyoxylate and dicarboxylate metabolism”,and “Cutin,suberine and wax biosynthesis” were the key metabolic pathways.(4)Based on Spearman analysis,the correlation between core microbiota with physicochemical indexes and significant difference metabolites were analyzed.Combined with core microbiota,physicochemical indexes,significant difference metabolites,network and literature data set,the network of BGRW fermentation microbiota and flavor metabolism was constructed.The results showed that Pediococcus,Bacillus,Acinetobacter,Lactobacillus,Weissella,Aspergillus,Rhizopus,Saccharomyces,Chaetomium,Penicillium,Aspergillus,Solidocozyma,Mortierella,Stachybotrys,Trichocladium,and Schizothecium had significant effects on the flavor quality of BGRW(| r | > 0.7,p < 0.05).Among them,Lactobacillus,Acinetobacter,Bacillus,Rhizopus,Saccharomyces,and Aspergillus are the main functional microorganisms.Based on the results of metaboanalyst 5.0 annotation of ISDMs and the literature data set,the network pathways of ISDMs in BGRW were predicted.The formation network of ISDMs was constructed by combining KEGG database and related enzymes annotated of microorganisms.