Abstract:

【Objective】 Bacillus velezensis strain YH-18 is an excellent disease-resistant, growth-promoting microorganism. This microbial agent is harmless, efficient and environmentally friendly. This study aimed to increase the number of spores in the fermentation broth, prolong the storage time, reduce the fermentation cost, and improve its benefits within the context of industrial fermentation. The viable bacteria count of the fermentation broth was improved by optimizing the culture medium components and culture conditions of strain YH-18.【Method】 Using the spore number of the fermentation broth of B. velezensis strain as the quality index, single factor analysis was used to preliminarily screen the culture medium (i.e., in terms of carbon source, nitrogen source, inorganic salt and its concentration) and the culture conditions (i.e., temperature, rotational speed, initial pH, liquid loading, and inoculation amount). Based on the preliminary screening results and response surface design [plackett-burman (PB), steepest climbing test, and box-behnken design (BBD)], the most significant factors affecting spore production of strain YH-18 were screened for further verification, and the quadratic regression equation model of each significant factor was established. The culture medium components and culture conditions for strain YH-18 were systematically optimized. The inhibitory effects of the bacterial-free filtrate of the fermentation broth before and after optimization on the growth of Agrobacterium tumefaciens strain C58 were compared. The number of bacteria and spores in the optimized fermentation broth was determined every four months, and the shelf life of the optimized fermentation broth was investigated. 【Result】 After single factor screening, the medium components (mass fraction, same as bebw) were 1% dried corn steep liquor powder, 1% glucose, 0.4% CaCO₃, 0.4% K₂HPO₄ and 0.2% MgSO₄. The culture conditions were 40% liquid loading, 200 r/min rotation speed, 33 °C, 0.5% inoculation amount, and pH 7.3. The PB test was used to screen for the results of the single-factor test. The results showed that the concentration of dried corn steep liquor powder, liquid loading, and inoculation amount were the three most significant factors that affected the number of spores in the fermentation broth. The steepest climbing test determined that the center points of the response surface test factor level were 0.8% dried corn steep liquor powder, 1.1% inoculation amount, and 32% liquid loading. The box-behnken design (BBD) test determined that the medium was composed of 0.83% dried corn steep liquor powder, 1% glucose, 0.4% CaCO₃, 0.4% K₂HPO₄ and 0.2% MgSO₄. In addition, higher spore yields were obtained when the culture conditions were 31.53% liquid loading, 200 r/min rotation speed, pH 7.3, 33 °C culture temperature, and 1.12% inoculation amount. Under the optimized system, the spore concentration of the fermentation broth could reach 3.25×10⁹ cfu/mL, which was 9.48 times higher than that of LB basic medium. The bacteria-free filtrate of the optimized fermentation broth product significantly enhanced the inhibition of A. tumefaciens strain C58 compared to the non-optimized product. It could stop the growth of A. tumefaciens strain C58 in the first 16 h and still had a good inhibitory effect after 48 h. The number of viable bacteria and spores obtained by fermentation with the optimized formula in a 50 L fermenter was 87.5% and 89.74% of the initial value after one year of storage, while the number of viable bacteria in the original fermentation method was only 0.06% of the initial value after four months of storage. 【Conclusion】 The optimum medium and culture conditions for spore production by B. velezensis strain YH-18 in shaker fermentation were determined. After optimization, the number of spores in the fermentation broth was significantly increased; the antibacterial capacity of bacterial-free filtrate in the fermentation broth was enhanced; the product shelf life was prolonged; the production cost was reduced; and important technical support was provided for the industrial production, promotion and application of B. velezensis strain YH-18 in the future.

Key words: Bacillus velezensis, spore, culture medium, culture condition, response surface methodology, optimization of fermentation