The high protease activity strain is an important starter for promoting the maturation of Douchi. In addition, the ability to secrete flavor enzymes is also one of the main characteristics of a good starter. Screening strains with high protease activity and studying the functional genes related to flavor enzymes in the strains are of great significance for the development of high-quality Douchi starter. A high protease activity strain B3 was screened by the transparent circle and the protease activity assay method. Then, the strain was identified by 16S rDNA molecular, the enzymatic properties were studied, and the functional genes related to flavor enzymes and their encoded proteases were determined. The results showed that the dominant strain B3 was identified as Bacillus amyloliquefaciens (nos. MZ723401.1), and its protease activity reached 185.6 U/mL. Its enzymatic properties showed that the optimum temperature was 50 ℃, and the optimum pH was 7.0. By analyzing the whole genome sequence of strain B3, flavor protease genes AprX and ampS were retrieved. The amino acid sequences, physicochemical properties, and high-level structural properties of the encoded serine protease and aminopeptidase were predicted. At the same time, the establishment of phylogenetic tree and multiple sequence alignment were carried out. The full length of the AprX gene is 1 329 bp, encoding a total of 442 amino acids, and the full length of the ampS gene is 1 233 bp, encoding a total of 410 amino acids. Both of them encode hydrophilic proteins, and the amino acid sequences encoded by the genes have the highest similarity with the Bacillus amyloliquefaciens group. Therefore, the strain B3 has high protease activity and has characteristic functional genes encoding alkaline protease and depicroprotease, which is a potential good starter for fermented Douchi.
[1] 李德坤,李欣遥.关于豆豉生产工艺技术的探讨[J].中国调味品,2016,41(8):104-106.
LI D K, LI X Y.Discussion on production technology of fermented soybean[J].China Condiment, 2016, 41(8):104-106.
[2] LIU L B, CHEN X Q, HAO L L, et al.Traditional fermented soybean products:Processing, flavor formation, nutritional and biological activities[J].Critical Reviews in Food Science and Nutrition, 2020,62(7):1 971-1 989.
[3] CAO Z H, GREEN-JOHNSON J M, BUCKLEY N D, et al.Bioactivity of soy-based fermented foods:A review[J].Biotechnology Advances, 2019, 37(1):223-238.
[4] 贾璠,郭霞,何晨,等.传统发酵豆制品营养功能成分研究进展[J].中国酿造,2019,38(4):1-6.
JIA F, GUO X, HE C, et al.Research progress in nutritional and functional ingredients of the traditional fermented soybean products[J].China Brewing, 2019, 38(4):1-6.
[5] JAYACHANDRAN M, XU B J.An insight into the health benefits of fermented soy products[J].Food Chemistry, 2019, 271:362-371.
[6] 李小永,陈伟,程芳,等.一株细菌型豆豉发酵菌种的筛选及鉴定[J].食品工业科技,2011,32(11):212-215;219.
LI X Y, CHEN W, CHENG F, et al.Screening and identification of a strain for lobster sauce fermentation[J].Science and Technology of Food Industry, 2011, 32(11):212-215;219.
[7] 彭昀,杨富民,康红霞,等.庆阳豆豉优势菌株的分离鉴定[J].食品与发酵工业,2013,39(4):58-63.
PENG Y, YANG F M, KANG H X, et al.Isolation and identification of predominant strains in Qingyang douchi[J].Food and Fermentation Industries, 2013, 39(4):58-63.
[8] 叶慧兰,曲直,孙乔润,等.阳江豆豉菌种的筛选及前发酵工艺条件优化[J].食品科学,2012,33(19):264-268.
YE H L, QU Z, SUN Q R, et al.Screening of optimal fermentation starter and optimization of primary fermentation for Yangjiang lobster sauce production[J].Food Science, 2012, 33(19):264-268.
[9] TAN Y, ZHANG R F, CHEN G J, et al.Effect of different starter cultures on the control of biogenic amines and quality change of Douchi by rapid fermentation[J].LWT, 2019, 109:395-405.
[10] HE B, LI H R, HU Z H, et al.Difference in microbial community and taste compounds between Mucor-type and Aspergillus-type Douchi during koji-making[J].Food Research International, 2019, 121:136-143.
[11] 刘敏,张仁凤,陈光静,等.霉菌纯种制曲豆豉在传统后发酵中的成分变化及其与生物胺相关性的研究[J].食品与发酵工业,2019,45(5):51-60.
LIU M, ZHANG R F, CHEN G J, et al.Changes in relevant components of mold starter-making Douchi during traditional post-fermentation and their correlations with biogenic amines[J].Food and Fermentation Industries, 2019, 45(5):51-60.
[12] 魏丕伟,王凌云,罗惠波,等.大曲中高产酸性蛋白酶霉菌的分离鉴定[J].食品与发酵科技,2014,50(4):1-4;21.
WEI P W, WANG L Y, LUO H B, et al.Isolation and identification of acid-protease producing fungi from daqu[J].Food and Fermentation Technology, 2014, 50(4):1-4;21.
[13] 杨林,王筱兰,杨慧林,等.1株传统曲霉型豆豉中高活力蛋白酶产生菌的分离及其鉴定[J].江西师范大学学报(自然科学版),2015,39(5):497-501.
YANG L, WANG X L, YANG H L, et al.A strain of high activity of protease producing bacteria isolation and identification in the traditional type Aspergillus tempeh[J].Journal of Jiangxi Normal University (Natural Science Edition), 2015, 39(5):497-501.
[14] 周德庆. 微生物学教程[M].第3版.北京:高等教育出版社,2011:336-338.
ZHOU D Q.Microbiology Course[M].3rd Edition.Beijing:Higher Education Press, 2011:336-338.
[15] 布坎南, 吉本斯.伯杰细菌鉴定手册[M].8版.北京:科学出版社, 1984.
BUCHANAN, GIBBONS. Berger′s Handbook of Bacterial Identification[M].8th Edition.Beijing:Science Press, 1984.
[16] 权淑静,马焕,王一雯,等.青藏高原土壤低温产蛋白酶菌株的筛选及酶学性质研究[J].中国酿造,2018,37(5):66-70.
QUAN S J, MA H, WANG Y W, et al.Screening and enzymatic characterizations of a low temperature protease-producing strain from Qinghai-Tibet Plateau soil[J].China Brewing, 2018, 37(5):66-70.
[17] 张芹,王修俊,商景天,等.贵州三穗特色豆豉发酵优势菌种的筛选和鉴定[J].食品与机械,2018,34(7):43-47.
ZHANG Q, WANG X J, SHANG J T, et al.Screening and identification of dominant strains of fermented characteristic lobster sauce in Guizhou Sansui[J].Food and Machinery, 2018, 34(7):43-47.
[18] SOLANKI P, PUTATUNDA C, KUMAR A, et al.Microbial proteases:Ubiquitous enzymes with innumerable uses[J].3 Biotech, 2021, 11(10):428.
[19] YAN Z F, YUAN S, QIN Q, et al.Enhancement of rice protein hydrolysate quality using a novel dual enzyme system[J].LWT, 2022, 158:113110.
[20] SONG P, CHENG L, TIAN K M, et al.A novel aminopeptidase with potential debittering properties in casein and soybean protein hydrolysates[J].Food Science and Biotechnology, 2020, 29(11):1 491-1 499.
[21] 刘逸寒,王洪彬,刘夫锋,等.微生物碱性蛋白酶的研究与开发[J].天津科技大学学报,2017,32(2):1-6;12.
LIU Y H, WANG H B, LIU F F, et al.The research and development of microbial alkaline protease[J].Journal of Tianjin University of Science and Technology, 2017, 32(2):1-6;12.
[22] 林晓彤. 亮氨酸氨肽酶的基因挖掘和在黑曲霉中的高效重组表达及其酶学性质研究[D].广州:华南理工大学,2020.
LIN X T.Screening and expression analysis of leucine aminopeptidases genes in Aspergillus niger[D].Guangzhou:South China University of Technology, 2020.
