Keywords

Bacillus Subtilis, Lipopeptide Antibiotics, Response Surface Methodology, Fermentation Medium, Fermentation Condition

Abstract

[Purpose] Bacillus subtilis T-500 is a biocontrol strain that has good control effects on rice sheath blight and rice blast. By optimizing its shake flask fermentation process, the lipopeptides in the fermentation broth can be increased. The content of antibiotics provides technical support for the development of T-500 strain biocontrol preparations. [Method] Using Rhizoctonia solani as the indicator bacterium, acid precipitation method was used to extract lipopeptide antibiotics in the fermentation broth of T-500 strain, and the antibacterial effect of the lipopeptide antibiotic crude extract was analyzed and screened. The main components of the fermentation medium that affect the antibacterial effect; then, through Plackett-Burman experimental design, central combination experimental design and response surface method, the fermentation medium components and fermentation conditions of the T-500 strain with high production of lipopeptide antibiotics were optimized. [Result] The optimal culture medium for T-500 strain to produce lipopeptide antibiotics is: 7.00 g of soybean cake powder? L-1, peptone 4.92 g? L-1, yeast powder 1.90 g? L-1, wheat flour 5.00 g? L-1, polenta 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 optimal fermentation culture conditions are: 500 mL triangular flask with 105 mL liquid volume, 0.87% inoculum, 41.35 h fermentation time, 28¡ãC temperature, and 180 rpm rotation speed? min-1. Using the optimal shake flask fermentation process, the lipopeptide antibiotics produced by the T-500 strain have the widest inhibitory band against sheath blight, reaching (11.23¡À0.15) mm, and the bacterial content reaches (7.41¡À1.18)¡Á109CFU? mL-1. After shake flask fermentation test and antibacterial activity verification, there was no significant difference between the theoretical prediction value and the actual value. Mass spectrometry and chromatographic detection showed that: the Surfactin content produced after optimizing the fermentation process increased by 48.2% compared with the basic medium, and the Iturin content increased by 180.9% compared with the basic medium; the production of Fengycin was detected after optimizing the fermentation process, but Fengycin was not found before optimization. of production. [Conclusion] The response surface method was used to successfully optimize the shake flask fermentation process of lipopeptide antibiotics produced by Bacillus subtilis T-500. After optimization, the yield of lipopeptide antibiotics produced by T-500 increased and the antibacterial activity was enhanced. [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¡æ, 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¡À0.15)mm, and cell amount was up to (7.41¡À1.18)¡Á109 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

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Original research done by Qiao Junqing, Liu Youzhou, Zhang Rongsheng, Liu Yongfeng

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