响应面法对解淀粉芽孢杆菌(Bacillus amyloliquefaciens)12-7产抗菌蛋白条件的优化

doi:10.11963/issn.1002-7807.201603012

摘要/Abstract

摘要： 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)12-7是从发病棉田土壤中分离得到的1株生防细菌，其对棉花黄萎病致病菌大丽轮枝菌Verticillium dahliae Kleb. V190具有明显的拮抗作用，已证明其抗菌物质为蛋白质。采用响应面法对该生防菌株的产抗菌蛋白条件进行优化，以期为棉花黄萎病生物农药的研制开发以及抗菌蛋白的后续研究奠定基础。以发酵液上清液对大丽轮枝菌的抑菌圈面积为考察指标，首先采用单因子试验考察培养时间、温度、初始pH、碳源、氮源、无机盐等因素对菌株12-7发酵产抗菌蛋白的影响；再根据响应面分析法的基本原理和方法，对发酵条件进行优化。得出该菌株产抗菌蛋白的最优发酵培养基和培养条件为：1.5%（质量分数）蔗糖、1.5%（质量分数）大豆蛋白胨、0.3%（质量分数）NaCl、0.1%（质量分数）K₂HPO₄、初始pH 7.3的培养基，31 ℃发酵培养60 h。优化后抑菌圈面积从试验设计初期的171.95 mm²提高至320.3 mm²，且经过验证试验证实预测值与实际值之间具有较好的拟合度。

关键词: 响应面法; 大丽轮枝菌; 拮抗细菌; 抗菌蛋白; 发酵条件; 优化

Abstract: In a previous study, the novel strain Bacillus amyloliquefaciens 12-7, which has a high antifungal activity against Verticillium dahliae Kleb., was isolated and screened from the soil of a diseased cotton field. It was demonstrated that antifungal materials produced by 12-7 were proteins. In this study, response surface methodology (RSM) was used to optimize antagonistic protein fermentation parameters. This work lays the foundation for the development of antifungal protein biologicals as pesticides, and for follow-up studies. Taking the inhibition zone area induced by the fermentation supernatant as standard, single factorial experiments were designed to determine effects of incubation time, temperature, incipient pH value, different carbon and nitrogen sources, and inorganic salt on the antifungal activity of the 12-7 strain. Then, RSM was used to optimize the fermentation parameters. This revealed the optimum fermentation of strain 12-7 to produce antagonistic protein was as follows: medium with 1.5% (mass fraction) sucrose, 1.5% soy peptone, 0.3% NaCl, 0.1% K₂HPO₄, and incipient pH value 7.3; with cultivation at 31 ℃ for 60 h. Antifungal activity value increased from 171.95 mm² in the initial experimental design to 320.3 mm² under the optimum cultivation condition. The experimental data under various conditions thus validated the theoretical values.

Key words: response surface methodology; Verticillium dahliae Kleb.; antagonistic bacterium; antagonistic protein; fermentation condition; optimization

中图分类号:

S482.7

王全, 王占利, 高同国, 李术娜. 响应面法对解淀粉芽孢杆菌(Bacillus amyloliquefaciens)12-7产抗菌蛋白条件的优化[J]. 棉花学报, 2016, 28(3): 283-290.

Wang Quan, Wang Zhanli, Gao Tongguo, Li Shuna. Antagonistic Protein Producing Condition Optimization of Antagonistic Strain Bacillus amyloliquefaciens 12-7 against Verticillium dahliae Kleb. using Response Surface Methodology[J]. Cotton Science, 2016, 28(3): 283-290.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/issn.1002-7807.201603012

http://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2016/V28/I3/283

参考文献

[1] 陈旭升, 陈永萱, 黄骏麒. 棉花黄萎病菌致病性生理生化研究进展[J]. 棉花学报, 2001, 13(3): 183-187. Chen Xusheng, Chen Yongxuan, Huang Junqi. Advances of studies on physiological and biochemical properties of pathogenesis caused by Verticillium dahliae in cotton[J]. Cotton Science, 2001, 13(3): 183-187. [2] 陈旭生, 陈永萱, 黄骏麒. 棉花黄萎病菌鉴定技术进展[J]. 棉花学报, 1997, 9(2): 64-67. Chen Xusheng, Chen Yongxuan, Huang Junqi. The developmental trend of Verticillium identification technique[J]. Cotton Science, 1997, 9(2): 64-67. [3] 袁洪水, 马平, 李术娜, 等. 棉花黄萎病拮抗细菌的筛选与抗菌物分析[J]. 棉花学报, 2007, 19(6): 436-439. Yuan Hongshui, Ma Ping, Li Shuna, et al. Screening of antagonistic bacteria against Verticillium dahliae and characteristic analyses of antagonistic substance[J]. Cotton Science, 2007, 19(6): 436-439. [4] 郭秀君, 郑平, 王蔚, 等. 蜡质芽孢杆菌(Bacillus cereus)芽孢的形成与PHB的关系[J]. 山东大学学报(自然科学版), 1994, 29(1): 101-108. Guo Xiujun, Zheng Ping, Wang Wei, et al. Relationship between spore formation and PHB synthesis by Bacillus cereus[J]. Journal of Shandong University (Natural Science Edition), 1994, 29(1): 101-108. [5] Shih L, Kuo C Y, Hsieh F C, et al. Use of surface response methodology to optimize culture conditions for iturin A production by Bacillus subtilis in solid-state fermentation[J]. Journal of the Chinese Institute of Chemical Engineers, 2008, 39(6): 635- 643. [6] El-Sersy N A, Ebrahim H A H, Abou-Elela G M. Response surface methodology as a tool for optimizing the production of antimicrobial agents from Bacillus licheniformis SN 2[J]. Current Research in Bacteriology, 2010, 3(1): 1-14. [7] Reddy P R M, Ramesh B, Mrudula S, et al. Production of thermostable β-amylase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrient levels using response surface methodology[J]. Process Biochemistry, 2003, 39: 267-277. [8] Guneet K, Sanjeev K, Satyanarayana T. Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis[J]. Bioresource Technology, 2004, 94: 239-243. [9] 郝学财, 余晓斌, 刘志钰, 等. 响应面方法在优化微生物培养基中的应用[J]. 食品研究与开发, 2006, 27(1): 38-41. Hao Xuecai, Yu Xiaobin, Liu Zhiyu, et al. The application of response surface methodology in optimization of microbial media[J]. Research and Development of Food, 2006, 27(1): 38-41. [10] Singh P, Shera S S, Banik J, et al. Optimization of cultural conditions using response surface methodology versus artificial neural network and modeling of L-glutaminase production by Bacillus cereus MTCC 1305[J]. Bioresource Technology, 2013, 137: 261-269. [11] 李术娜, 姜军坡, 朱莹, 等. 大丽轮枝菌拮抗细菌枯草芽孢杆菌12-34菌株发酵产抗菌蛋白的条件优化[J]. 植物保护, 2009, 35(3): 51-56. Li Shuna, Jiang Junpo, Zhu Ying, et al. Optimization of the fermentation conditions of the antagonistic strain 12-34 of Bacillus subtilis against Verticillium dahlia[J]. Plant Protection, 2009, 35(3): 51-56. [12] 赵斌, 何绍江. 微生物学实验[M]. 北京: 科学出版社, 2002: 23. Zhao Bin, He Shaojiang. Experiment of microbiology[M]. Beijing: Science Press, 2002: 23. [13] 李术娜, 杜红方, 袁洪水, 等. 棉花黄萎病拮抗细菌LC-04菌株的抗菌蛋白产生条件研究[J]. 棉花学报, 2006, 18(4): 233- 237. Li Shuna, Du Hongfang, Yuan Hongshui, et al. Fermentation conditions of antagonistic strain LC-04 against Verticillium dahliae[J]. Cotton Science, 2006, 18(4): 233-237. [14] Douglas C M. 实验设计与分析[M]. 汪仁官, 陈荣昭, 译. 北京: 中国统计出版社, 1998: 123. Douglas C M. Design and analysis of experiments[M]. Translated by Wang Renguan, Chen Rongzhao. Beijing: China Statistics Press, 1998: 123. [15] 齐东梅, 梁启美, 惠明, 等. 棉花枯萎、黄萎病拮抗芽孢杆菌的抗菌蛋白特性[J]. 微生物学通报, 2005, 32(4): 42-46. Qi Dongmei, Liang Qimei, Hui Ming, et al. Characteristics of antagonistic proteins from Bacillus against cotton Fusarium wilt and Verticillium wilt[J]. Microbiology, 2005, 32(4): 42-46. [16] Lin H Y, Rao Y K, Wu Wenshi, et al. Ferrous ion enhanced lipopeptide antibiotic iturin A production from Bacillus amyloliquefaciens B128[J]. International Journal of Applied Science and Engineering, 2008, 5(2): 123-132. [17] 郭成栓, 谢和. 枯草芽孢杆菌B1⑥菌株产蛋白酶液体发酵条件优化[J]. 山地农业生物学报, 2007, 25(6): 554-557. Guo Chengshuan, Xie He. Optimizing liquid fermentation conditions of protease by Bacillus subtilis B1⑥[J]. Journal of Mountain Agriculture and Biology, 2007, 25(6): 554-557. [18] 姜云, 黄丽丽, 陈长卿, 等. 一株拮抗番茄叶霉病菌的放线菌筛选、鉴定及发酵条件研究[J]. 微生物学报, 2007, 47(7): 622- 627. Jiang Yun, Huang Lili, Chen Changqing, et al. Screen, identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva[J]. Acta Microbiologica Sinica, 2007, 47(7): 622-627.