【目的】 贝莱斯芽孢杆菌(Bacillus velezensis)菌株YH-18是优良的抗病促生微生物,其制成的微生物菌剂具有无害、高效、环保等特点。通过优化菌株YH-18的培养基组分和培养条件,提高发酵液中的有效活菌数,以期增加发酵液中的芽孢数,延长储存时间,降低发酵成本,提高其在工业化生产发酵中的质量效益。【方法】 以贝莱斯芽孢杆菌(B. velezensis)菌株YH-18发酵液芽孢数为质量指标,采用单因素分析法对发酵液培养基(碳源、氮源、无机盐及其浓度)和培养条件(温度、转速、初始pH、装液量、接菌量)进行初步筛选。将初筛结果结合响应面试验设计[plackett-burman(PB)、最陡爬坡试验、box-behnken design(BBD)],筛选出对贝莱斯芽孢杆菌菌株YH-18产芽孢影响最为显著的因素进一步验证,建立各显著性因素的二次回归方程模型,完成对菌株YH-18培养基组分和培养条件的系统优化。对比优化前后发酵液产品的去菌体滤液对根癌农杆病菌(Agrobacterium tumefaciens)菌株C58生长的抑制效果。每隔4个月测定优化后发酵液中的活菌数和芽孢数,考察优化发酵液的货架期。【结果】 单因素试验初步筛选后得到培养基组分为1.0%(质量分数,后同)玉米浆干粉、1.0%葡萄糖、0.4%碳酸钙、0.4%磷酸氢二钾、0.2%的硫酸镁,培养条件为装液量50 mL三角瓶的40%、摇床转速200 r/min、培养温度33 ℃、接菌量0.5%、初始pH 7.3。将单因素试验后的结果进行PB试验筛选得到玉米浆干粉质量分数、装液量和接菌量是影响发酵液芽孢数最显著的3个因素。最陡爬坡试验确定了响应面试验因素水平的中心点为0.8%玉米浆干粉、接菌量1.1%、装液量32%。BBD试验最终确定,培养基组分(质量分数)为0.83%玉米浆干粉、1.0%葡萄糖、0.4%碳酸钙、0.4%磷酸氢二钾、0.2%的硫酸镁,培养条件为装液量31.53%、摇床转速200 r/min、培养温度33 ℃、接菌量1.12%、初始pH 7.3时,可获得较高发酵液芽孢产量。在本优化体系下,发酵液产品的芽孢数可达3.25×10⁹ cfu/mL,较LB基础培养基芽孢数提高了9.48倍。菌株优化后发酵液产品的去菌体滤液对根癌农杆病菌菌株菌株C58的抑制能力较未优化的明显增强,能在前16 h使根癌农杆菌菌株C58基本不生长,且在48 h后仍有较好的抑制效果。在50 L发酵罐中使用优化配方发酵得到的发酵液产品储存1 a后有效活菌数和芽孢数仍然分别为初始值的87.50%和89.74%,原始发酵方法的发酵液产品在储存4个月后有效活菌数仅为初始值的0.06%。【结论】 确定了贝莱斯芽孢杆菌菌株YH-18摇瓶发酵产芽孢的最适培养基和培养条件。优化后显著提高了发酵液芽孢数,增强了发酵液去菌体滤液的抑菌能力,延长了产品储存时间,降低了生产成本,为贝莱斯芽孢杆菌菌株YH-18未来的工业化生产和推广应用提供了重要的技术支持。

【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.