Metaproteomic Reveals The Difference Of Microbial Community Structure And Metabolic Function In Different Fermented Soybean Paste

Soybean paste is a traditional fermented soybean food product that has been consumed for centuries as flavoring ingredient in Asian countries for thousands of years.It as a flavoring ingredient and source of protein contains rich in nutrients and with unique flavor.During the fermentation process,the dynamic changes of the microbial community structure cause certain effects on the fermentation and function of soybean paste,which then directly affects the flavor and quality.Bacteria and fungi included in the soybean paste are responsible for producing various primary metabolites.As such,it is of great research value to research microbial assortments and biological functions of dajiang.To our best knowledge,this study is the first united meta-omics analysis to investigate protein sources and functional properties of the microbial communities in soybean paste meju,and investigate differences in microbial assortments,functional properties and metabolic modes between traditional and commercial dajiang through the metaproteomic approach.Our results also identified microbial proteins with critical functions involved in several main fermentation-related pathways.We put forward active metabolic pathways based on the proteins being identified,which is a suitable method to disentangle the role of each member in dajiang microbial community:1.In this work we created such a database from the metagenomic sequences we obtained from the DNA isolated from the soybean paste-meju samples.We sequenced the metagenome of 12 soybean paste-meju samples and analyzed the microbial functional diversity by metaproteomics.The metagenome analysis showed that the bacterial communities of fermented dajiang-meju have a high diversity level,however,most of the identified proteins were assigned to fungi during the entire fermentation period,this may be related to the selective expression of microbial genes.Total of 7785 proteins from 12 meju samples were identified with false discovery rates.The results demonstrated that the proteins among the 12 meju samples were mainly derived from Ascomycota,Eukaryota?norank,Proteobacteria,Firmicutes and Streptophyta at the phylum level.At the genus rank,fungal proteins were mainly derived from Rhizopus,Penicillium,Geotrichum,while bacterial proteins mainly came from Enterobacter,Enterococcus,Leuconostoc,Lactobacillus,Citrobacter and Leclercia.According to the COG annotation,the DEPs were mainly associated with“global and overview maps”?“carbohydrate transport and metabolism”?“energy production and conversion”?“protein turnover and Translation”and“translation,signal transduction”.2.This part of work sequenced the metagenome of 9 naturally fermented dajiang samples,were collected on the 14^th?H2?,28^th?H4?and 42^nd?H6?day of fermentation from three households located in Liaoning province.A total of 3493 proteins have been identified consulting with bacteria and fungi databases.The results demonstrated that the proteins were mainly derived from Firmicutes and Ascomycota at the phylum level.At the genus rank,proteins were mainly derived from Bacillus,Penicillium,Rhizoctonia,Geotrichum,Tetragenococcus,Duganella,Pneumocyslis,Lactobacillus,Mucor,Aspergillus and Fusarium.GO analysis indicated that 1368 kinds of identified differential proteins were mainly related to catalytic activity and binding force.According to the COG annotation,the DEPs were mainly associated with genetic information processing and metabolic pathways,such as“carbohydrate metabolism”,“amino acid biosynthesis”,“energy metabolism”and“nucleic acid biosynthesis”.These core functions are mainly concerned with the interaction between microorganisms and the possible access to nutrients by microorganisms.The core proteins of related pathways mainly come from Bacillus,Penicillium and Tetralococcus in fungi.3.Representative samples?n=9?of commercial dajiang were collected on the 14^th?G2?,28^th?G4?and 42^nd?G6?day from three fermentation batches between March and June 2017 in Qinghua food brewing co.LTD?Shenyang,China?.The label-free quantification identified1987 proteins.The detected proteins were classified into 19 phyla and 125 genera.Proteins that were assigned to fungi were predominant to a similar extent as the fermentation,which were mainly derived from Basidiomycota and Ascomycota at the phylum level.At the genus rank,the microbial protein source was mainly composed of the genus Hypholoma,Aspergillus followed,and a small amount of Bacillus,Mitosporidium,and Eubacterium were also present.According to the COG annotation,the DEPs were mainly associated with“posttranslational modification”,“protein conversion and translation”,“ribosome structure and biogenesis”,“carbohydrate transport and metabolism”,and related pathway enzyme systems are mainly from Aspergillus,Penicillium and Mucor in fungi,with little effect by bacteria.4.This part of work contrast differences in metaproteomic between traditional and commercial dajiang.The results indicated that the traditional and commercial dajiang communities demonstrated overlaps but also considerable heterogeneity at the proteome level and regarding functional categories of identified proteins.By contrast,there are 48 genera that belong exclusively to the commercial dajiang.According to the KEGG annotation,these DEPs were enriched in the“biological regulation”,“cellular component tissue”,“biogenesis,metabolic processes,response stimuli”,and“protein-containing”?p<0.5?.There were also some pathways related to human diseases in traditional dajiang,so the monitoring of harmful microbes during the fermentation process should be strengthened.5.The whole spectrum metabolic scan of soybean paste was performed by UPLC-QTOF/MS.Metabolites were the result of the action of functional proteins.Combined with the metaproteomic results,the core microorganisms of the main metabolites were annotated from three aspects:bean paste quality,food safety and functionality.The results showed that the core microorganisms involved in carbohydrate metabolism in H group soybean paste samlpes were Penicillium,Tetracocci,Aspergillus,Lactobacillus,which are core saccharification microbial populations.Especially Penicillium,the most abundant glucoamylase.Tetracocci and Leuconostoc spp.have a great influence on the flavor of bean paste.The core microorganism of lipid metabolism is Agaricus.The core microorganisms that affect the safety of bean paste are mainly cryptococcus.In addition,we confirmed from the protein level.Ornithine carbamoyltransferase produced by Tetragenococcus halophilus?Tetragenococcus muriaticus?is a key enzyme for inhibiting harmful substances.In the commercial soybean paste,the core microorganisms are single,mainly Aspergillus oryzae,Rasamsonia emersonii.The study of microbes in traditionally fermented soybean paste not only elucidates the biological functions of these microbes,but it could also elevate the commercial product by bringing the knowledge of how the unique flavor is formed into the industrially efficient production and improves the understanding of microbial metabolic patterns with respect to the metaproteomes of soybean paste and provides a powerful tool for studying the fermentation process of soybean products.