Analysis Of Microbiota And Screening Of Functional Microbes In Douchi Fermentation Process

Aspergillus-type douchi is a kind of Chinese traditional fermented food,there were a lot of advantages including nutrient-rich and special flour in aspergillus-type douchi.At present,the production model of traditional aspergillus-type douchi is still natural fermentation,which would lead to unstable quality,pathogen contaminated easily and so on.This paper focused on the microbiota diversity in the whole douchi fermentation process;Screening and identifying the functional dominant bacteria in douchi koji making and fermentation stage;Optimization of technological conditions for douchi fibrinolytic enzyme;Purification and characterization of a novel fibrinolytic enzyme from douchi fermentation process.The results were as follows:(1)We used an Illumina MiSeq approach to investigate bacterial and fungal community diversity during both douchi-koji making and fermentation.A total of 181,443 high quality bacterial 16 S rRNA sequences and 221,059 high quality fungal internal transcribed spacer(ITS)reads were used for taxonomic classification,revealing eight bacterial and three fungal phyla.Firmicutes,Actinobacteria and Proteobacteria were the dominant bacterial phyla,while Ascomycota and Zygomycota were the dominant fungal phyla.At the genus level,Staphylococcus and Weissella were the dominant bacteria,while Aspergillus and Lichtheimia were the dominant fungi.Principal coordinate analysis showed structural separation between the composition of bacteria in koji production and fermentation.However,multivariate analysis of variance based on unweighted UniFrac distances did identify distinct differences(p <0.05),and redundancy analysis identified two key genera that are largely responsible for the differences in bacterial composition between the two steps.Staphylococcus was enriched in koji making,while Corynebacterium was enriched in fermentation.(2)Using plate separation method to screen and identify functional dominant bacteria,and successfully screened a protease producing strain Bacillus amyloliquefaciens,fibrinolytic enzyme producing strain Bacillus amyloliquefaciens,alchohol producing strain Saccharomyces cerevisiae,lactic acid producing strain Pediococcus lolii.Then we focused on the fibrinolytic enzyme producing strain Bacillus amyloliquefaciens Jxnuwx-1.In this research,single factor tests and response surface analysis was applied to optimize the fermentation conditions for Douchi fibrinolytic enzyme production by Bacillus amyloliquefaciens Jxnuwx-1.Based on single factor tests,the three significant factors: corn starch,beef powder and pH were applied to the response surface methodology design by the Design-Expert software.As the result,the optimum fermentation conditions were: corn starch concentration was 2.91%;the concentration of the beef powder was 1.76%;the optimum pH was 7.47.Under this condition,the enzyme activity was 346.62 IU/mL,increased 8.9 times before optimized.(3)Using AKTA explore 100 system to purify the fibrinolytic enzyme from submerge fermentation,after a series of chromatographic steps including(NH)4SO4 precipitation,anion exchange and gel filtration chromatographic.The molecular mass of the enzyme was 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The optimal pH and temperature of the enzyme were 7.6 and 41 ?,respectively.The enzyme was inhibited by phenylmethylsulfonyl fluoride(PMSF),SBTI,EDTA,Fe3+,and Fe2+.The highest affinity of the enzyme was towards N-Succinyl-Ala-Ala-Pro-Phe-pNA.These results indicated that it is a subtilisin-like serine metalloprotease.The enzyme had a highest degrading activity for A?-chains of fibrin(ogen),followed by B? chains and C? chains,and also activated plasminogen,which indicated it was able to degrade the fibrin in two ways:(a)by active plasminogen into plasmin and(b)by directly hydrolyzed.It degraded thrombin,suggested that it can act as an anticoagulant and prevent thrombosis.This paper investigated the microbiota diversity in traditional aspergillus-type douchi fermentation process,Screening and identifying the functional dominant bacteria in douchi koji making and fermentation stage;Optimization of technological conditions for douchi fibrinolytic enzyme;Purification and characterization of a novel fibrinolytic enzyme from douchi fermentation process,which laid a foundation for industrialization of traditional aspergillus-type douchi.