食品与发酵工业 >

2021 , Vol. 47 >Issue 23: 134 - 142

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.027476

生产与科研应用

鱼露中高产蛋白酶耐盐菌株的筛选、鉴定及产酶条件优化

  • 李文静 ,
  • 李春生 ,
  • 李来好 ,
  • 王悦齐 ,
  • 陈胜军 ,
  • 杨少玲
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  • 1(中国海洋大学 食品科学与工程学院,山东 青岛,266100)
    2(中国水产科学研究院南海水产研究所 农业农村部水产品加工重点实验室,国家水产品加工技术研发中心,广东 广州,510300)
    3(大连工业大学 海洋食品精深加工关键技术省部共建协同创新中心,辽宁 大连,116034)
硕士研究生(李来好研究员为通讯作者,E-mail:laihaoli@163.com)

收稿日期: 2021-03-23

  修回日期: 2021-04-25

  网络出版日期: 2021-12-31

基金资助

国家重点研发计划项目(2019YFD0901903)

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Isolation, identification and enzyme-producing condition of a salt-tolerant strain with high protease production

  • LI Wenjing ,
  • LI Chunsheng ,
  • LI Laihao ,
  • WANG Yueqi ,
  • CHEN Shengjun ,
  • YANG Shaoling
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  • 1(College of Food Science and Engineering,Ocean University of China,Qingdao 266100,China)
    2(Key Laboratory of Aquatic Product Processing,Ministry of Agriculture and Rural Affairs,National R&D Center for Aquatic Product Processing,South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510300,China)
    3(Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China)

Received date: 2021-03-23

  Revised date: 2021-04-25

  Online published: 2021-12-31

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摘要

利用微生物发酵剂和蛋白酶制剂对鱼露进行快速发酵具有广阔的应用前景。从鱼露发酵液中分离出的菌株B-2具有耐盐且高产蛋白酶的特性,经VITEK 2 COMPACT 生理生化鉴定和16S rRNA基因序列分析,确定该菌为Bacillus subtilis。利用扫描电子显微镜观察菌株在不同盐环境下的结构,并分析其生长情况,发现该菌株具有很强的耐盐性,在200~300 g/L的盐环境下仍能够保持生长和代谢,并维持较为完整的细胞形态。通过2次响应面试验,确定该菌最适培养基成分为0.57 g/L果糖、13.88 g/L豆粕、5.13 g/L NaCl,最适发酵条件为pH 9.4、温度28.4 ℃、接种量4.9%。菌株B-2的产酶条件经过优化后,蛋白酶活力为200.63 U/mL,比优化前提高了6.15倍。研究结果表明,菌株B-2具有高产蛋白酶活性和耐盐的特点,有望为鱼露的快速发酵提供一种高效的发酵剂和酶制剂。

关键词： 鱼露; 枯草芽胞杆菌; 耐盐; 产蛋白酶; 响应面优化

本文引用格式

李文静 , 李春生 , 李来好 , 王悦齐 , 陈胜军 , 杨少玲 . 鱼露中高产蛋白酶耐盐菌株的筛选、鉴定及产酶条件优化[J]. 食品与发酵工业, 2021 , 47(23) : 134 -142 . DOI: 10.13995/j.cnki.11-1802/ts.027476

Abstract

The salt-tolerant strain B-2 with high protease production ability was selected and identified as Bacillus subtilis. The cell structure of strain B-2 was still intact at 200-300 g/L of NaCl, indicating its high salt tolerance. The optimal conditions were as follows: 0.57 g/L of fructose, 13.88 g/L of soybean meal, 5.13 g/L of NaCl, pH 9.4, 28.4 ℃, and 4.9% of inoculum volume. Under the optimal conditions, the protease activity produced by strain B-2 was 200.63 U/mL, which was 6.15 times higher than that before optimization. These results suggested that strain B-2 has the characteristics of high salt tolerance and protease production ability, which is expected to provide an effective starter and enzyme for the rapid fermentation of fish sauce.

Key words： fish sauce; Bacillus subtilis; salt tolerance; protease production; response surface optimization

参考文献

[1] 李春生,王悦齐,李来好,等.传统鱼露发酵过程中细菌群落演替及对其挥发性风味形成的影响分析[J].食品科学,2018,39(24):85-92.
LI C S,WANG Y Q,LI L H,et al.Bacterial community succession during fermentation of Chinese fish sauce and its effect on formation of volatile flavor components[J].Food Science,2018,39(24):85-92.
[2] WANG Y Q,LI C S,LI L H,et al.Effect of bacterial community and free amino acids on the content of biogenic amines during fermentation of yu-lu,a Chinese fermented fish sauce[J].Journal of Aquatic Food Product Technology,2018,27(4):496-507.
[3] SUN J N,YU X H,FANG B H,et al.Effect of fermentation by Aspergillus oryzae on the biochemical and sensory properties of anchovy(Engraulis japonicus) fish sauce[J].International Journal of Food Science & Technology,2016,51(1):133-141.
[4] 黄紫燕,刘春花,罗婷婷,等.鱼露发酵液中产蛋白酶乳酸菌的筛选及其添加应用[J].食品与发酵工业,2010,36(11):88-92.
HUANG Z Y,LIU C H,LUO T T,et al.Screening of proteinase-producing lactic acid bacteria from fish sauce and the application[J].Food and Fermentation Industries,2010,36(11):88-92.
[5] KARPARVAR N,SAFAEI H J,DERAKHSHANDEH A,et al.Isolation and identification of lactic acid bacteria from a traditional fermented fish sauce(Mahyaveh) in Fars province,Iran[J].International Journal of Nutrition Sciences,2019,4(1):49-53.
[6] SINSUWAN S,RODTONG S,YONGSAWATDIGUL J.Production and characterization of NaCl-activated proteinases from Virgibacillus sp.SK33 isolated from fish sauce fermentation[J].Process Biochemistry,2008,43(2):185-192.
[7] MONTRIWONG A,RODTONG S,YONGSAWATDIGUL J.Detergent-stable salt-activated proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation[J].Applied Biochemistry and Biotechnology,2015,176(2):505-517.[PubMed]
[8] KUMAUNANG M,SANCHART C,SUYOTHA W,et al.Virgibacillus halodenitrificans MSK-10P,a potential protease-producing starter culture for fermented shrimp paste(Kapi) production[J].Journal of Aquatic Food Product Technology,2019,28(8):877-890.
[9] GAO R C,ZHOU J,LENG W J,et al.Screening of a Planococcus bacterium producing a cold-adapted protease and its application in low-salt fish sauce fermentation[J].Journal of Food Processing and Preservation,2020,44(8):e14625.
[10] AKOLKAR A V,DURAI D,DESAI A J.Halobacterium sp.SP1(1) as a starter culture for accelerating fish sauce fermentation[J].Journal of Applied Microbiology,2010,109(1):44-53.
[11] 伍蓉莉.嗜盐四联球菌的筛选及其发酵特性研究[D].广州:华南农业大学,2018.
WU R L.Isolation of salt-tolerant lactic acid bacteria in fish sauce and the research of its metabolic characteristics[D].Guangzhou:South China Agricultural University,2018.
[12] CHUEA-NONGTHON C,RODTONG S,YONGASAWATDIGU L J,et al.Draft genome sequences of Tetragenococcus muriaticus strains 3MR10-3 and PMC-11-5 isolated from Thai fish sauce during natural fermentation[J].Genome Announcements,2017,5(15):e00 198-17.
[13] FU X T,YOU S G,KIM S M,et al.Characterization of a salt-tolerant acid protease produced by Bacillus megaterium KLP-98 and its potential as a fermentation starter for the manufacture of fish sauce[J].Journal of Food Biochemistry,2008,32(3):279-298.
[14] DAROONPUNT R,ITOH T,KUDO T,et al.Bacillus piscicola sp.nov.,isolated from Thai fish sauce(Nam-pla)[J].International Journal of Systematic and Evolutionary Microbiology,2016,66(3):1 151-1 155.
[15] TOYOKAWA Y,TAKAHARA H,REUNGSANG A,et al.Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce[J].Applied Microbiology and Biotechnology,2010,86(6):1 867-1 875.
[16] KALWASINS K A,AGNIESZKAL,JANKIEWIC U,et al.Alkaline and halophilic protease production by Bacillus luteus H11 and its potential industrial applications[J].Food Technology and Biotechnology,2018,56(4):553-561.
[17] 冒鑫哲,彭政,周冠宇,等.枯草芽孢杆菌高产角蛋白酶发酵条件优化[J].食品与发酵工业,2020,46(17):138-144.
MAO X Z,PENG Z,ZHOU G Y,et al.Optimized fermentation for improving keratinase production by Bacillus subtilis WB600[J].Food and Fermentation Industries,2020,46(17):138-144.
[18] 陈茏,杨俊,马毛毛,等.产蛋白酶菌株的筛选及以菜籽粕为氮源的产酶条件优化[J].食品科学,2021,42(4):115-121.
CHEN L,YANG J,MA M M,et al.Strain screening and optimization of fermentation conditions for protease production using rapeseed meal as a nitrogen source[J].Food Science,2021,42(4):115-121.
[19] 王东,荣家萍,唐自钟,等.响应面法优化枯草芽孢杆菌产中性蛋白酶的发酵条件[J].基因组学与应用生物学,2016,35(1):143-151.
WANG D,RONG J P,TANG Z Z,et al.Response surface method optimization of fermentation conditions for Bacillus subtilis producing neutral protease[J].Genomics and Applied Biology,2016,35(1):143-151.
[20] VARELA E,MING T E.Effect of pH and oxalate on hydroquinone-derived hydroxyl radical formation during brown rot wood degradation[J].Applied and Environmental Microbiology,2003,69(10):6 025-6 031.
[21] 刘杨柳,魏东东,刘燕,等.枯草芽孢杆菌发酵蟹壳粉产蛋白酶的研究[J].食品研究与开发,2017,38(24):174-180;224.
LIU Y L,WEI D D,LIU Y,et al.Research on fermentation of crab powder for proteinase production by Bacillus subtilis[J].Food Research and Development,2017,38(24):174-180;224.
[22] PATEL R,DODIA M,SINGH S P.Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.:Production and optimization[J].Process Biochemistry,2005,40(11):3 569-3 575.
[23] 吴海波,江连洲,赵英,等.发酵低温豆粕生产碱性蛋白酶的工艺研究[J].食品工业科技,2011,32(3):195-200.
WU H B,JIANG L Z,ZHAO Y,et al.Alkaline protease produced by Bacillus Subtilis with defatted soy flour[J].Science and Technology of Food Industry,2011,32(3):195-200.
[24] 卢超,陈景鲜,王国霞,等.枯草芽孢杆菌L07产中性蛋白酶发酵条件优化[J].食品与发酵工业,2020,46(16):148-153.
LU C,CHEN J X,WANG G X,et al.Optimization of fermentation conditions for the production of neutral protease by Bacillus subtilis L07[J].Food and Fermentation Industries,2020,46(16):148-153.
[25] 刘晓艳,封健,韩傲,等.高产中/碱性蛋白酶的枯草芽孢杆菌W1菌株的筛选及条件优化[J].现代食品科技,2020,36(4):157-163.
LIU X Y,FENG J,HAN A,et al.Isolation and optimization of Bacillus subtilis W1 strain with high neutral/alkaline protease activity[J].Modern Food Science and Technology,2020,36(4):157-163.
[26] GAUVRY E,MATHOT A G,COUVERT O,et al.Effects of temperature,pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1[J].International Journal of Food Microbiology,2021,337:108915.
[27] PONGSETKUL J,BENJAKUL S,SUMPAVAPOL P,et al.Bacillus subtilis K-C3 isolated from Thai salted shrimp paste(Kapi):Its extracellular enzymes and use as a starter culture in Kapi production[J].Journal of Food Biochemistry,2018,42(6):1-12.
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