Aspergillus oryzae is commonly used as starter for the fermentation of broad bean paste, and protease activity is an important indicator of finished koji. Protease degrades the protein in raw materials into oligopeptides and amino acids, providing important precursors for flavor formation of broad bean paste, so it is particularly crucial to improve the protease activity of A. oryzae. A. oryzae BL18 is a strain with high protease activity. It was applied in the fermentation of broad bean paste and compared with A. oryzae 3.042, and simultaneously unearthed the genetic basis behind the differences through genome sequencing. Compared with A. oryzae 3.042, the spore number of finished koji made by A. oryzae BL18 reached 4.19×108/g dry weight, and the activities of acidic, neutral and alkaline protease increased by 52%, 7.6% and 45.6%, respectively. The protease of A. oryzae BL18 had advantages in the early stage of fermentation, and increased umami amino acid and sweet amino acid content in broad bean paste by 33.3% and 10.8%, respectively. Gene annotation results showed that A. oryzae BL18 had strong ability of energy synthesis, protein synthesis and secondary metabolite synthesis, at the same time, compared with 3.042, 647 unique genes were found in A. oryzae BL18 genome. The dominant performance of A. oryzae BL18 in koji making and fermentation and the annotation of a large number of functional genes provide a strong theoretical basis for its application in broad bean paste fermentation.
[1] 冯宽, 孙群, 邓维琴, 等.一种同时测定豆瓣酱中的7种有机酸的HPLC方法[J].食品与发酵科技, 2019, 55(4):128-131;141.
FENG K, SUN Q, DENG W Q, et al.A HPLC method for determination of seven kinds of organic acids in broad bean paste with different fermentation year[J].Food and Fermentation Sciences ＆ Technology, 2019, 55(4):128-131;141.
[2] HE B, TU Y Y, JIANG C M, et al.Functional genomics of Aspergillus oryzae:Strategies and progress[J].Microorganisms, 2019, 7(4):103.
[3] YUE X Q, LI M H, LIU Y M, et al.Microbial diversity and function of soybean paste in East Asia:What we know and what we don’t[J].Current Opinion in Food Science, 2021, 37:145-152.
[4] ZHU Y Y, LIANG X L, ZHANG H, et al.A comparative secretome analysis of industrial Aspergillus oryzae and its spontaneous mutant ZJGS-LZ-21[J].International Journal of Food Microbiology, 2017, 248:1-9.
[5] ZHAO G Z, HOU L H, LU M F, et al.Construction of the mutant strain in Aspergillus oryzae 3.042 for abundant proteinase production by the N+ ion implantation mutagenesis[J].International Journal of Food Science & Technology, 2012, 47(3):504-510.
[6] XU D F, PAN L, ZHAO H F, et al.Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger[J].Journal of Industrial Microbiology and Biotechnology, 2011, 38(9):1 255-1 265.
[7] GAO X L, LIU E M, YIN Y Y, et al.Enhancing activities of salt-tolerant proteases secreted by Aspergillus oryzae using atmospheric and room-temperature plasma mutagenesis[J].Journal of Agricultural and Food Chemistry, 2020, 68(9):2 757-2 764.
[8] 钮成拓, 赵帅, 杜姝颖, 等.一株产高蛋白酶活的米曲霉及其在豆酱发酵中的应用:CN111808757B[P].2022-04-29.
[9] 高秀芝, 王小芬, 李献梅, 等.天源酱园豆酱发酵过程中营养及理化指标动态[J].食品科学, 2008, 29(9):352-354.
GAO X Z, WANG X F, LI X M, et al.Dynamics of nutrients and physical and chemical parameters during fermentation of Tianyuan Jiangyuan soybean paste[J].Food Science, 2008, 29(9):352-354.
[10] BIRNEY E, CLAMP M, DURBIN R.GeneWise and genomewise[J].Genome Research, 2004, 14(5):988-995.
[11] JOHNSON A D, HANDSAKER R E, PULIT S L, et al.SNAP:A web-based tool for identification and annotation of proxy SNPs using HapMap[J].Bioinformatics, 2008, 24(24):2 938-2 939.
[12] STANKE M, DIEKHANS M, BAERTSCH R, et al.Using native and syntenically mapped cDNA alignments to improve de novo gene finding[J].Bioinformatics, 2008, 24(5):637-644.
[13] TER-HOVHANNISYAN V, LOMSADZE A, CHERNOFF Y O, et al.Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training[J].Genome Research, 2008, 18(12):1 979-1 990.
[14] FU L M, NIU B F, ZHU Z W, et al.CD-HIT:Accelerated for clustering the next-generation sequencing data[J].Bioinformatics, 2012, 28(23):3 150-3 152.
[15] 林祖申. 米曲霉制曲过程中酶活性变化及其工艺优化[J].中国酿造, 2007, 26(5):56-59.
LIN Z S.Change of enzyme activity in Aspergillus oryzae cultivation and technical optimization[J].China Brewing, 2007, 26(5):56-59.
[16] BALTUSSEN T J H, ZOLL J, VERWEIJ P E, et al.Molecular mechanisms of conidial germination in Aspergillus spp[J].Microbiology and Molecular Biology Reviews:MMBR, 2019, 84(1):e00049-e00019.
[17] KUSUMOTO K I, YAMAGATA Y, TAZAWA R, et al.Japanese traditional miso and koji making[J].Journal of Fungi (Basel, Switzerland), 2021, 7(7):579.
[18] JIA Y, NIU C T, LU Z M, et al.A bottom-up approach to develop a synthetic microbial community model:Application for efficient reduced-salt broad bean paste fermentation[J].Applied and Environmental Microbiology, 2020, 86(12):e00306-e00320.
[19] HOANG N X, FERNG S, TING C H, et al.Optimizing the initial moromi fermentation conditions to improve the quality of soy sauce[J].LWT, 2016, 74:242-250.
[20] WU J R, TIAN T, LIU Y M, et al.The dynamic changes of chemical components and microbiota during the natural fermentation process in Da-Jiang, a Chinese popular traditional fermented condiment[J].Food Research International, 2018, 112:457-467.
[21] 王栋, 冯杰, 张丽敏, 等.耐盐米曲霉CICIM F0899在酱油酿造过程中的应用[J].食品与发酵工业, 2012, 38(5):1-6.
WANG D, FENG J, ZHANG L M, et al.Application of a salt-tolerant Aspergillus oryzae CICIM F0899 in soy sauce fermentation[J].Food and Fermentation Industries, 2012, 38(5):1-6.
[22] CHOU C C, LING M Y.Biochemical changes in soy sauce prepared with extruded and traditional raw materials[J].Food Research International, 1998, 31(6-7):487-492.
[23] HAN B Z, ROMBOUTS F M, NOUT M J R.Amino acid profiles of sufu, a Chinese fermented soybean food[J].Journal of Food Composition and Analysis, 2004, 17(6):689-698.
[24] ZHAO G Z, YAO Y P, QI W, et al.Draft genome sequence of Aspergillus oryzae strain 3.042[J].Eukaryotic Cell, 2012, 11(9):1178.
[25] KANG C, YU X W, XU Y.Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization[J].Journal of Industrial Microbiology and Biotechnology, 2015, 42(2):263-272.
[26] WANG B, GUO G W, WANG C, et al.Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing[J].Nucleic Acids Research, 2010, 38(15):5 075-5 087.
