Study Of The Diversity And Metabolic Characteristics Of Microbial Community Harbored In Fermented Grains For The Chinese Strong-flavor Baijiu Production Based On Metatranscriptome

Chinese strong-flavor Baijiu(CSFB)is a typical representative of traditional Chinese distilled Baijiu,it is produced by a unique multi-strain spontaneous and solid-state fermentation process.The fermented grains(FG)mainly refers to a mixture of various materials in the entire fermentation process,which is used for distillation to prepare raw wine after fermentation.FG serves as the growth substrate of microorganisms and the direct source of aroma substances in the fermentation process,the structure of microbial community,physiological activity and metabolic processes in FG directly affect the quality of Baijiu.At present,most studies use DNA-level technology to analyze the structure and function of the microorganisms in the fermentation process of CSFB.However,the results cannot accurately reflect the true physiological activity and metabolic characteristics of the microbial community in the FG environment.While the RNA level-based metatranscriptome technology studies the active members of the microbial community in FG,which can more truly reflect the contribution of microorganisms.In order to further reveal the community diversity,functional characteristics and metabolic pathways of the active microorganisms in strong-flavor FG,this study used the metatranscriptome technology for the first time to analyze the microorganisms in the FG samples at different times during the fermentation process of CSFB.The following are the main findings:(1)The established SDS-phenol method for total RNA extraction from FG,compared with the kit method,Trizol method,CTAB method and sodium laurate method,the average concentration(647.6 ng/?L))and purity(OD₂₆₀/OD₂₈₀=1.88,OD₂₆₀/OD₂₃₀=2.05)of total RNA were high,the electrophoresis band was complete,and the RNA can be effectively used for reverse transcription and high-throughput sequencing analysis.(2)The results of the microbial community composition and function in FG(7 days)obtained by using the two methods of metatranscriptome(RNA level)and 16S/ITS gene sequencing(DNA level)had certain similarities and differences.The most common bacterial and fungal phyla was Firmicutes and Ascomycota at the two levels respectively,the fungi with the highest relative abundance was Saccharomyces(RNA:83.15%,DNA:89.74%)at the two levels.The most abundant bacterium was Kroppenstedtia(37.09%)detected only at the DNA level,while it was Streptococcus(93.75%)at the RNA level.Moreover,many genera with transcriptional activity(Aphanizomenon,Prevotella,Amanita,naumovozyma,etc.)can only be detected by metatranscriptome technique.Most genes in the KEGG pathway were related to“Metabolism”,and especially enriched in“Carbohydrate metabolism”at the two levels.The pathways with highest proportions under“Environmental information processing”,“Organismal systems”and“Genetic information processing”functions,were“Membrane transport”,“Environmental adaptation”and“Replication and repair”at the DNA level,respectively,while they were“Signal transduction”,“Endocrine system”and“Translation”at the RNA level,respectively.Finally,on the 7th day of fermentation,the synthesis of ethanol,acetic acid and lactic acid started from starch or dextrin,and go through the conversion of a series of substances,such as glucose,pyruvic acid,(lactic acid),acetyl-Co A,acetic acid,acetaldehyde and ethanol.Saccharomyces cerevisiae was the main contributor in the whole process and interacted with other microorganisms(Torulaspora dellbrueckii?Pichia kudriavzevii,etc.)to promote the synthesis of substances.(3)By using the metatranscriptome technology to annotate the biological community in FG during the whole fermentation process of CSFB,it was found that there were 6kingdoms,135 phyla,239 classes,690 orders,1621 families,3942 genera,and 14800species in all FG samples,including 4 dominant phyla(Firmicutes,Ascomycota,Basidiomycota,Proteobacteria),4 dominant classes(Bacilli,Saccharomycetes,Clostridia,Agaricomycetes)and 5 dominant genera(Lactobacillus,Saccharomyces,Bacillus,Staphyoclococcus,Streptocloccus).The diversity of the microbial community in the bacteria kingdom showed an upward and then downward trend during the fermentation processs,while the diversity of the microbial community in the fungus kingdom showed an upward trend.The fermentation period can be divided into early(3d),middle(7-15d)and later(25-60d)stage,the fungus kingdom was the predominance in the early stage,and the bacteria kingdom was the predominance in the middle and later stages.The high content of Firmicutes and Ascomycota played an important role in driving the changes in the active microbial community of FG.Bacilli and Saccharomycetes were the main dominant classes in the whole fermentation process.In the later stage of fermentation,Lactobacillus also had an absolute advantage at the RNA level.The metatranscriptomics technology can confirm whether the microorganisms detected by culture are really viable.The active microorganisms in FG were mainly affected by total acid and p H,followed by ammonia nitrogen and water content.(4)In terms of gene expression,the fermentation period can also be divided into three stages:early(3d),middle(7-15d)and later(25-60d)stage.Both egg NOG and COG annotation results showed that there are more genes involved in“Translation,ribosomal structure and biogenesis”,“Energy production and conversion”,“Carbohydrate transport and metabolism”and“Amino acid transport and metabolism”.The metabolic pathways with a large number of enriched genes in the KEGG annotation results were“Carbohydrate metabolism(9439 genes)”,“Translation(6110 genes)”and“Signal transduction(3674genes)”.These results showed that the cell growth,productivity and metabolism of the microbial community in FG were relatively active.In the GO annotation results,the functions with a larger number of genes under the category of“Biological process(85603genes)”were“metabolic processes(24803 genes)”and“cellular process(24053 genes)”,it showed that there are a large number of genes involved in regulating cell metabolism and physiological processes in the fermentation process.The changes in gene expression in the early and middle stages were the most significant,there were 157 and 5435 up-regulated and down-regulated genes between B3 and B7,and 4029 and 95 genes between B7 and B15,respectively.The 7th day of fermentation was the most active period of gene expression,and the expression level of genes related to gene information processing and productivity activities increased significantly at 7th day of fermentation,then the expression level of microbial metabolic activities decreased after 7th day,indicating that the 7th day of fermentation was a key time node for FG.(5)There were 76 enzymes,965 genes,20 classes and 152 genera in butyric acid metabolism network,and Lactobacillus,Clostridium,Rhodococcus,Herbaspirillum,Saccharomyces and Bacteroides were the main contributors.And the butyric acid metabolism network were divided into 7 different pathways:glutamate,butanol,lysine,3-butenoyl-Co A,oxaloacetate,acetoacetic acid and acetyl-Co A(main pathway).During the synthesis process,crotonyl-Co A was the intermediate hub,buk and ato D were the final enzymes.The presumed synthetic pathway of caproic acid was that butyryl-Co A and acetyl-Co A first form caproyl-Co A,then acetic acid and caproyl-Co A form caproic acid.The synthesis and metabolism pathway of 3-methylbutyric acid involved 4 enzymes and 155genes,mainly based on the synthetic route of leucine,?-ketoisocaproic acid,3-methylbutyraldehyde and 3-methylbutyric acid.Saccharomyces was the main contributor in the early stage of fermentation,then it became Lactobacillus in the middle and later stages.This study not only revealed the difference between the results of the microbial flora in FG based on the two methods at the level of DNA and RNA,but also clarified the changes in the diversity and functional characteristics of the active microbial community during the fermentation process.It has important theoretical and practical significance for evaluating the quality and safety of FG,controlling and optimizing the community structure.Moreover,it could provide a theoretical basis for further revealing the fermentation mechanism and fermentation process of CSFB.In addition,systematicly studied the metabolic pathways of the important fragrant fatty acids synthesized by the microbial flora of FG can further reveal its synthesis mechanism,with a view to controlling the content of important fragrant fatty acids and their corresponding esters in CSFB from the source through microbial targeted enhancement technology.