[1]乔俊卿,刘邮洲,张荣胜,等.枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺优化[J].南京农业大学学报,2018,41(3):447-455.[doi:10.7685/jnau.201705017]
 QIAO Junqing,LIU Youzhou,ZHANG Rongsheng,et al.Optimization of fermentation process of Bacillus subtilis T-500 for production of lipopeptide antibiotics in flask[J].Journal of Nanjing Agricultural University,2018,41(3):447-455.[doi:10.7685/jnau.201705017]
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枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺优化()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
41卷
期数:
2018年3期
页码:
447-455
栏目:
出版日期:
2018-05-15

文章信息/Info

Title:
Optimization of fermentation process of Bacillus subtilis T-500 for production of lipopeptide antibiotics in flask
作者:
乔俊卿 刘邮洲 张荣胜 刘永锋
江苏省农业科学院植物保护研究所, 江苏 南京 210014
Author(s):
QIAO Junqing LIU Youzhou ZHANG Rongsheng LIU Yongfeng
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
关键词:
枯草芽胞杆菌脂肽类抗生素响应曲面法发酵培养基发酵条件
Keywords:
Bacillus subtilislipopeptide antibioticsresponse surface methodologyfermentation mediumfermentation condition
分类号:
S476+.9
DOI:
10.7685/jnau.201705017
摘要:
[目的]枯草芽胞杆菌(Bacillus subtilis)T-500是1株对水稻纹枯病和稻瘟病均有良好防治效果的生防菌,通过优化其摇瓶发酵工艺,从而提高发酵液中脂肽类抗生素的含量,为T-500菌株生防制剂的开发提供技术支撑。[方法]以水稻纹枯病菌(Rhizoctonia solani)为指示菌,利用酸沉淀法提取T-500菌株发酵液中的脂肽类抗生素,并进行脂肽抗生素粗提液的抑菌效果分析,筛选影响抑菌效果的发酵培养基主成分;随后通过Plackett-Burman试验设计、中心组合试验设计和响应曲面法,优化T-500菌株高产脂肽类抗生素的发酵培养基成分和发酵条件。[结果]T-500菌株高产脂肽类抗生素的最佳培养基为:黄豆饼粉7.00 g·L-1,蛋白胨4.92 g·L-1,酵母粉1.90 g·L-1,小麦粉5.00 g·L-1,玉米糊5.00 g·L-1,NaCl 1.00 g·L-1,MgSO4 0.20 g·L-1,MnSO4 5.0 mg·L-1,FeSO4 0.5 mg·L-1。最佳发酵培养条件为:装液量500 mL三角瓶装105 mL,接种量0.87%,发酵时间41.35 h,温度28℃,转速180 r·min-1。利用最佳摇瓶发酵工艺,T-500菌株所产生的脂肽类抗生素对纹枯病菌的抑菌带最宽,达(11.23±0.15)mm,菌含量达(7.41±1.18)×109CFU·mL-1。经摇瓶发酵试验和抑菌活性验证,理论预测值与实际值无显著差异。质谱和色谱检测表明:优化发酵工艺后产生的Surfactin含量较基础培养基提高了48.2%,Iturin含量较基础培养基提高了180.9%;优化发酵工艺后检测到了Fengycin的产生,但优化前未发现Fengycin的产生。[结论]利用响应曲面法成功优化了枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺;优化后,T-500产脂肽类抗生素产量增加,抑菌活性增强。
Abstract:
[Objectives] Bacillus subtilis T-500 showed strong biocontrol activities to rice sheath blight and rice blast bacteria. Here, the fermentation process was optimized to improve the yield of lipopeptide antibiotics, which will provide technical support for the development of biocontrol agent containing T-500. [Methods] According to the inhibition zoom of lipopeptide extraction against Rhizoctonia solani, the main nutritional components that are suitable for the production of lipopetides in Bacillus subtilis T-500 were screened by comparing the effects of 10 media that are commonly used for cycle lipopetide antibiotics production by Bacillus. After that, the Plackett-Burman method, the central composite design and response surface methodology were used to obtain optional medium and fermentation condition. [Results] The optimum medium of T-500 was soybean powder 7.00 g·L-1, peptone 4.92 g·L-1, yeast extract 1.90 g·L-1, wheat powder 5.00 g·L-1, corn powder 5.00 g·L-1, NaCl 1.00 g·L-1, MgSO4 0.20 g·L-1, MnSO4 5.0 mg·L-1, FeSO4 0.5 mg·L-1. The optimum culture condition was 28℃, inoculation volume 0.87% and the filling volume 105 mL medium in 500 mL flask for 41.35 h at 180 r·min-1. Under the optimum fermentation medium and culture conditions, the width of inhibition zoom was about(11.23±0.15)mm, and cell amount was up to(7.41±1.18)×109 CFU·mL-1. Fermentation experiments with shake flasks verified that there was no statistical difference between real and forecasted yields. The results of mass spectrometry and chromatography showed that Surfactin content increased by 48.2% and the content of Iturin increased by 180.9% compared with the basal medium after optimization of the fermentation process. Fengycin was detected after optimization of the fermentation process, but not in the basal medium. [Conclusions] Response surface methodology was used in the optimization of the fermentation of T-500 for production of lipopeptide. After optimization, the yield and the antifungal activity of lipopeptide was significantly enhanced respectively.

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备注/Memo

备注/Memo:
收稿日期:2017-05-11。
基金项目:国家重点研发计划项目(2016YFD0300706);江苏省科技计划重点项目(BE2015354)
作者简介:乔俊卿,博士,副研究员,主要从事植物病害及生物防治研究,E-mail:junqingqiao@hotmail.com。
通信作者:刘永锋,研究员,E-mail:liuyf@jaas.ac.cn。
更新日期/Last Update: 1900-01-01