Screening Of Aspergillus Oryzae With High Protease Activity And Its Application In Doubanjiang

Aspergillus oryzae is the key strain in doubanjiang production since it has an important impact on fermentation.The ability of A.oryzae to secrete protease is closely related to the formation of flavor,which determines doubanjiang quality.Generally,the high protease activity of A.oryzae is beneficial to doubanjiang fermentation.However,its protease system is highly complex,and the protease plays a key role in the koji-making process has not been resolved.This thesis isolated a strain of A.oryzae named BL18 with strong protease production ability from doubanjiang mash and verified its safety to apply in doubanjiang fermentation through whole genome sequencing and modern analysis technique;Moreover,the influence of A.oryzae BL18 on doubanjiang quality during koji-making and fermentation was analyzed and compared with the commonly used starter A.oryzae 3.042.Finally,combined with genome analysis and transcriptomic analysis of A.oryzae BL18 during koji-making,the metabolic potential of A.oryzae BL18 was investigated.The protease system of A.oryzae BL18 was explored and its key protease genes during koji-making were predicted.The main findings are as follows:Through peptone media and simulated doubanjiang media,a strain of A.oryzae named BL18 was isolated and identified,which had significantly higher protease activity than A.oryzae 3.042.Moreover,the aflatoxin B1 gene cluster in A.oryzae BL18 genome was found incomplete and negative results were observed in different medium,koji-making and fermentation process of doubanjiang by A.oryzae BL18.The above results showed that A.oryzae BL18 with strong protease ability was safe to apply in doubanjiang fermentation.Compared with A.oryzae 3.042,A.oryzae BL18 had faster growth and sporulation production rate during koji-making;The total,acidic,neutral and alkaline protease activity of koji samples after koji-making for 64 h by A.oryzae BL18 were 4654.35 U/g,542.36 U/g,1828.41 U/g and 2283.58 U/g,which were 29.1%,52%,7.6% and 45.6% higher than those prepared by A.oryzae 3.042.During doubanjiang fermentation,the amino acid nitrogen concentration of pei samples prepared by A.oryzae BL18 was slightly higher than that by A.oryzae 3.042,among which the total concentrations of umami and sweet amino acids increased by 33.3% and 10.8%;Moreover,higher volatile flavor compounds concentration and sensory evaluation score was also observed in pei samples prepared by A.oryzae BL18.Based on whole genome sequencing and bioinformatics analysis,the majority of metabolic pathways enriched in A.oryzae BL18 were carbohydrate metabolism,amino acid metabolism and energy metabolism,indicating its strong metabolism potential in doubanjiang fermentation.Moreover,142 genes with protease function were identified in A.oryzae BL18 genome,among which 38 secreted protease genes were predicted.Through protease genes expression analysis by transcriptomics,the protease expression was mainly concentrated in the first 24 h during koji-making.After analysis of protease genes expressional levels and differentially expressed genes,16 protease genes were predicted to play key roles in koji-making,including 6 endopeptidases and 10 exopeptidases.