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棉花学报 ›› 2016, Vol. 28 ›› Issue (3): 283-290.doi: 10.11963/issn.1002-7807.201603012

• 研究与进展 • 上一篇    下一篇

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

王全1,王占利2,高同国1,李术娜1*   

  1. 1. 河北农业大学生命科学学院,河北 保定 071001;2. 保定学院资源与环境系,河北 保定 071051
  • 收稿日期:2015-01-22 出版日期:2016-05-15 发布日期:2016-05-15
  • 通讯作者: bdlishuna@126.com
  • 作者简介: 王全(1980―),男,讲师,在读博士,wangquan210@163.com
  • 基金资助:
    河北省自然科学基金(398152)

Antagonistic Protein Producing Condition Optimization of Antagonistic Strain Bacillus amyloliquefaciens 12-7 against Verticillium dahliae Kleb. using Response Surface Methodology

Wang Quan1, Wang Zhanli2, Gao Tongguo1, Li Shuna1*   

  1. 1. College of Life Science, Agricultural University of Hebei, Baoding, Hebei 071001, China; 2. Department of Resources and Environment, Baoding University, Baoding, Hebei 071000, China
  • Received:2015-01-22 Online:2016-05-15 Published:2016-05-15

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