贝莱斯芽孢杆菌CC0955发酵培养基优化及其泡腾颗粒的研制

Optimization of fermentation medium and preparation of effervescent granules of Bacillus velezensis CC0955

摘要:

【背景】植物病害的生物防治及生防产品的开发一直是植物保护研究的重点方向，但现有的生防产品多为可湿性粉剂和水剂，存在剂型单一、货架期短及运输和使用不便等问题。【目的】优化贝莱斯芽孢杆菌CC0955菌株发酵培养基，并利用其开发出一种容易使用和保存的新生防产品——泡腾颗粒。【方法】采用Plackett-Burman设计、中心组合设计和响应面分析等方法，优化了贝莱斯芽孢杆菌CC0955的发酵培养基成分；采用L₉(3³)正交设计，以溶液pH和活菌数为指标，优化了贝莱斯芽孢杆菌CC0955泡腾颗粒配比，并评价了其物理性质和抑菌效果。【结果】贝莱斯芽孢杆菌CC0955最优发酵培养基成分为(g/L)：蛋白胨12.00，酵母粉1.00，葡萄糖15.00，MgSO₄×7H₂O 0.40，K₂HPO₄ 0.05。用此培养基发酵48 h，发酵液对立枯丝核菌的抑制率达到89.78%。泡腾颗粒最佳配比为：碱酸摩尔比为2.00，白炭黑1.50 g，黄腐酸钾0.03 g。泡腾颗粒的平均崩解时间为61.00 s，溶解后溶液pH值为5.44，对立枯丝核菌的EC₅₀为27.65 mg/ml，活菌数为6.05×10⁸ CFU/g。室温下放置6个月，细菌存活率为58.68%。盆栽实验表明，CC0955泡腾颗粒溶液对水稻纹枯病的防效为42.35%，对番茄灰霉病的防效为57.62%。【结论】贝莱斯芽孢杆菌CC0955泡腾颗粒是一种新型的抗植物病原真菌制剂，可用于农田病害的生物防治。

Abstract:

[Background] Biocontrol of plant diseases and development of biocontrol products have been the research focuses in plant protection. However, most of the existing biocontrol agents feature a few available dosage forms (wettable powder or aqueous solution), short shelf life, and inconvenient transport and use. [Objective] To optimize the fermentation medium of Bacillus velezensis CC0955 and develop a novel biocontrol agent “effervescent granules”, which is easy to use and preserve. [Methods] Plackett-Burman design, central composite design, and response surface methodology were used to optimize the composition of CC0955 fermentation medium. With the evaluation indicators of pH and bacterial viable count, the composition of CC0955 effervescent granules was optimized by L₉(3³) orthogonal design, and the physical properties and antifungal effect of the effervescent granules were assessed. [Results] The optimal fermentation medium for CC0955 was composed of peptone 12.00 g, yeast powder 1.00 g, glucose 15.00 g, MgSO₄·7H₂O 0.40 g, K₂HPO₄ 0.05 g, and distilled water 1 L. After 48 h fermentation with this medium, the inhibition of the culture broth on Rhizoctonia solani reached 89.78%. The optimal composition of effervescent granules is as follows: alkali-to-acid molar ratio of 2.00, silica of 1.50 g, and potassium fulvic acid of 0.03 g. The effervescent granules showed the average disintegration time of 61 s and the pH of the disintegrated solution was 5.44. In addition, the half maximal effective concentration (EC₅₀) against R. solani was 27.65 mg/mL, and the number of viable bacteria was 6.05×10⁸ CFU/g. The survival rate of bacteria was 58.68% after six months of storage at room temperature. Pot experiment showed that the control rate of CC0955 effervescent granule solution on rice sheath blight was 42.35%, and that on tomato gray mold was 57.62%. [Conclusion] The CC0955 effervescent granule is a novel formulation against phytopathogens, which can be used for the control of farmland diseases.