Screening and Optimization of Bacillus subtilis HMB19198 fermentation medium
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摘要: 枯草芽孢杆菌Bacillus subtilis HMB19198菌株能有效防治番茄灰霉病，脂肽类抗生素泛革素(fengycin)是该菌株产生的主要抑菌活性物质。为提高HMB19198菌株的发酵水平，本研究以活芽孢浓度为检测指标，通过对8种常用工业培养基进行筛选，获得了利于HMB19198菌株芽孢形成的基础培养基。采用单因素试验筛选HMB19198菌株芽孢产量较高的碳源和氮源组合，结果表明，以可溶性玉米淀粉和糖蜜作为碳源、以花生饼浸粉和蛋白胨作为氮源时，有利于HMB19198菌株芽孢的形成。进一步运用Plackett-Burman及响应曲面分析相结合的方法，对碳源物质玉米淀粉和糖蜜，氮源物质花生饼浸粉和蛋白胨，以及无机盐K2HPO4•3H2O和MgSO4•7H2O的适宜浓度进行了优化。优化后发酵培养基配方中各成分的质量浓度分别为：可溶性玉米淀粉67.0 g/L、糖蜜14.1 g/L、花生饼浸粉41.6 g/L、蛋白胨10 g/L、K2HPO4•3H2O 9.2 g/L以及MgSO4•7H2O 1.5 g/L。在优化培养基中，HMB19198菌株发酵液的芽孢浓度可达到6.92 × 109 个/mL；同基础培养基相比，采用优化后的培养基，菌株发酵周期缩短了40%，发酵液中芽孢浓度提高了107%，泛革素浓度提高了39.4%。Abstract: Bacillus subtilis strain HMB19198 is a promising biocontrol agent for tomato gray mold. Fengycin, a kind of lipopeptide, plays an important role in strain HMB19198 suppressing tomato gray mold. To improve the fermentation level of strain HMB19198, the basic medium benefiting for the sporulation of strain HMB19198 was selected from eight kinds of industrial mediums. The carbon and nitrogen sources suitable for sporulation of strain HMB19198 were screened by a single factor test. Results showed that soluble corn starch and molasses as carbon sources, peanut cake powder, and peptone as nitrogen sources were conducive to the sporulation of strain HMB19198. The Plackett-Burman analysis and response surface analysis were used to optimize the concentrations of soluble corn starch, molasses, peanut cake powder, peptone, K2HPO4•3H2O, and MgSO4•7H2O in the medium. The optimized medium included 67.0 g/L soluble corn starch, 14.1 g/L molasses, 41.6 g/L peanut cake powder, 10 g/L peptone, 9.2 g/L K2HPO4•3H2O and 1.5 g/L MgSO4•7H2O. In the optimized medium, the spore concentration of strain HMB19198 reached 6.92 × 109 spore/mL. Compared with the basic medium, using the optimized medium, the fermentation period of the strain was shortened by 40%, the spore concentration in the fermentation broth was increased by 107%, and the fengycin concentration was increased by 39.4%.
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Key words:
- Bacillus subtilis /
- fermentation solution /
- sporulation /
- medium optimization /
- response surface analysis /
- fengycin
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图 1 不同基础培养基对枯草芽孢杆菌HMB19198菌株芽孢浓度的影响
注：图中培养基序号所对应培养基同表1。不同小写字母表示在0.05水平差异显著。
Figure 1. Effect of different basic mediums on the spore concentration of B. subtilis strain HMB19198
Note：The medium numbers in the figure correspond to the medium in Table 1 and different letters indicate the significant differences at P = 0.05 level.
图 2 不同碳源 (A) 和氮源 (B) 对枯草芽孢杆菌 HMB19198 菌株芽孢形成的影响
注：图中不同小写字母表示在0.05水平差异显著。
Figure 2. Spore concentrations of B. subtilis strain HMB19198 in basic medium supplemented with different carbon (A) and nitrogen (B) sources
Note：The different letters indicate the significant differences at P = 0.05 level.
图 4 影响芽孢形成主要因子的预测模型
Figure 4. Predictive models of the main factors affecting spore formation
图 5 可溶性玉米淀粉与花生饼浸粉双因素最适浓度及配比模拟结果
Figure 5. Simulation results of the optimum concentrations and ratios of soluble corn starch and peanut cake powder
图 6 糖蜜与花生饼浸粉双因素最适浓度及配比模拟结果
Figure 6. Simulation results of the optimum concentrations and ratios of molasses and peanut cake powder
表 1 芽孢杆菌基础培养基
Table 1. Basic mediums for Bacillus
培养基
Medium配方
Formula1 玉米粉 30.0 g/L，葡萄糖 2.0 g/L，黄豆粉 20.0 g/L，麸皮 5.0 g/L，NaCl 4.0 g/LCorn powder 30.0 g/L, Glucose 2.0 g/L, Soybean powder 20.0 g/L, Wheat bran 5.0 g/L, NaCl 4.0 g/L 2 玉米粉 30.0 g/L，黄豆粉 30.0 g/L，K2HPO4•3H2O 4.0 g/L，MgSO4•7H2O 1.5 g/LCorn powder 30.0 g/L, Soybean powder 30.0 g/L, K2HPO4•3H2O 4.0 g/L, MgSO4•7H2O 1.5 g/L 3 葡萄糖 10.0 g/L，黄豆粉 10.0 g/L，NaCl 5.0 g/L，MnSO4•7H2O 0.6 g/LGlucose 10.0 g/L, Soybean powder 10.0 g/L, NaCl 5.0 g/L, MnSO4•7H2O 0.6 g/L 4 蛋白胨 10.0 g/L，酵母浸膏 5.0 g/L，葡萄糖 1.0 g/L，K2HPO4•3H2O 0.2 g/L，MgSO4•7H2O 0.2 g/LPepton 10.0 g/L, Yeast extract 5.0 g/L, Glucose 1.0 g/L, K2HPO4•3H2O 0.2 g/L, MgSO4•7H2O 0.2 g/L 5 玉米粉 50.0 g/L，葡萄糖 8.0 g/L，豆粕粉 20.0 g/L，麸皮 20.0 g/L，NaCl 4.0 g/LCorn powder 50.0 g/L, Glucose 8.0 g/L, Soybean meal powder 20.0 g/L, Wheat bran 20.0 g/L, NaCl 4.0 g/L 6 玉米粉 17.0 g/L，葡萄糖 10.0 g/L，黄豆粉 23.0 g/L，麦芽糖 10.0 g/L，酵母浸膏 5.0 g/LCorn powder 17.0 g/L, Glucose 10.0 g/L, Soybean powder 23.0 g/L, Maltose 10.0 g/L, Yeast extract 5.0 g/L 7 玉米粉 5.0 g/L，葡萄糖 3.0 g/L，豆饼粉 5.0 g/L，鱼粉 3.0 g/LCorn powder 5.0 g/L, Glucose 3.0 g/L, Bean cake powder 5.0 g/L, Fish meal 3.0 g/L 8 玉米粉 40.0 g/L，豆饼粉 30.0 g/L，酵母粉 5.0 g/L，K2HPO4•3H2O 5.0 g/L，MgSO4•7H2O 1.5 g/LCorn powder 40.0 g/L, Bean cake powder 30.0 g/L, Yeast powder 5.0 g/L, K2HPO4•3H2O 5.0 g/L, MgSO4•7H2O 1.5 g/L 
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表 2 Plackett-Burman试验因子与水平设计
Table 2. Experimental design of the factors and levels of Plackett-Burman
因子
Factor水平
Level/(g/L)−1 1 可溶性玉米淀粉 Soluble corn starch 30.0 45.0 糖蜜 Molasses 10.0 15.0 花生饼浸粉 Peanut cake powder 30.0 45.0 蛋白胨 Peptone 10.0 15.0 磷酸氢二钾 K2HPO4•3H2O 5.0 7.5 硫酸镁 MgSO4•7H2O 1.5 3.0
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表 3 响应曲面试验因子与水平设计
Table 3. Experimental design of the factors and levels of RSA
因子
Factor水平
Level/(g/L)−1 0 1 可溶性玉米淀粉 Soluble corn starch 45.0 56.0 67.0 糖蜜 Molasses 10.0 12.5 15.0 花生饼浸粉 Peanut cake powder 30.0 37.5 45.0 磷酸氢二钾 K2HPO4•3H2O 7.5 9.4 11.3
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表 4 Plackett-Burman试验设计与芽孢浓度响应
Table 4. Experimental designs of Plackett-Burman and corresponding spore concentrations
运行序
Run可溶性玉米淀粉Soluble corn starch/
(g/L)糖蜜Molasses/
(g/L)花生饼浸粉Peanut cake powder/
(g/L)蛋白胨Peptone/
(g/L)磷酸氢二钾K2HPO4•3H2O/(g/L) 硫酸镁MgSO4•7H2O/
(g/L)芽孢浓度Spore conc. (×109)/
(spore/mL)1 45.0 10.0 45.0 10.0 5.0 1.5 3.76 2 30.0 15.0 45.0 10.0 7.5 1.5 3.58 3 30.0 15.0 30.0 10.0 5.0 3.0 2.86 4 45.0 15.0 45.0 10.0 7.5 3.0 3.80 5 30.0 15.0 45.0 15.0 5.0 3.0 3.18 6 45.0 10.0 30.0 10.0 7.5 3.0 4.75 7 45.0 10.0 45.0 15.0 5.0 3.0 4.12 8 45.0 15.0 30.0 15.0 7.5 1.5 4.40 9 30.0 10.0 30.0 15.0 7.5 3.0 4.63 10 30.0 10.0 30.0 10.0 5.0 1.5 4.10 11 45.0 15.0 30.0 15.0 5.0 1.5 4.29 12 30.0 10.0 45.0 15.0 7.5 1.5 3.90
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表 5 Plackett-Burman试验统计分析
Table 5. The regression analysises of Plackett-Burman experiments
因子
FactorF 值
F valueP 值
P value重要性排序Importance
ranking模型 Model 5.78 0.037 — 可溶性玉米淀粉 Soluble corn starch 7.83 0.038 2 糖蜜 Molasses 9.49 0.027 1 花生饼浸粉 Peanut cake powder 6.94 0.046 4 蛋白胨 Peptone 2.71 0.161 6 磷酸氢二钾 K2HPO4•3H2O 7.27 0.043 3 硫酸镁 MgSO4•7H2O 0.45 0.534 5
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表 6 响应曲面分析试验设计及结果
Table 6. Experimental designs and results of RSA
运行序
Run可溶性玉米淀粉Soluble corn
starch/ (g/L)糖蜜Molasses/
(g/L)花生饼浸粉Peanut cake
powder/(g/L)磷酸氢二钾K2HPO4•3H2O/(g/L) 芽孢浓度Spore conc.
(×109)/(spore/mL)1 0 0 0 0 6.50 2 −1 0 0 −1 6.00 3 0 −1 0 1 4.89 4 0 0 0 0 7.23 5 0 0 0 0 7.07 6 0 0 0 0 7.02 7 −1 0 0 1 4.42 8 −1 0 1 0 3.24 9 0 0 1 1 4.93 10 0 −1 1 0 3.13 11 0 1 1 0 5.23 12 0 0 −1 1 6.35 13 0 0 1 −1 4.79 14 −1 1 0 0 4.08 15 1 0 −1 0 4.48 16 1 0 0 −1 7.85 17 0 1 0 −1 5.06 18 1 0 0 1 6.32 19 −1 0 −1 0 5.47 20 0 1 0 1 5.93 21 0 −1 0 −1 4.17 22 1 1 0 0 7.93 23 −1 −1 0 0 4.02 24 0 −1 −1 0 5.81 25 1 −1 0 0 4.75 26 1 0 1 0 6.21 27 0 0 −1 −1 6.04 28 0 1 −1 0 3.26 29 0 0 0 0 7.04
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表 7 响应曲面试验回归分析结果
Table 7. The regression analysis of RSA
来源
Source平方和
SS自由度
df均方
MSF 值
F valueP 值
P value显著性
Significance模型
Model4378.30 14 312.74 5.870 0.0011 ** A 885.80 1 885.80 16.626 0.0011 ** B 185.65 1 185.65 3.485 0.0830 C 125.45 1 125.45 2.355 0.1472 D 9.54 1 9.54 0.179 0.6786 A × B 243.36 1 243.36 4.568 0.0507 A × C 392.04 1 392.04 7.359 0.0168 * A × D 0.06 1 0.06 0.001 0.9732 B × C 540.56 1 540.56 10.146 0.0066 ** B × D 0.56 1 0.56 0.011 0.9196 C × D 0.72 1 0.72 0.014 0.9089 A2 211.79 1 211.79 3.975 0.0660 B2 1244.55 1 1244.55 23.360 0.0003 ** C2 1096.50 1 1096.50 20.581 0.0005 ** D2 68.06 1 68.06 1.277 0.2774 残差 Residual 745.87 14 53.28 失拟项 Lack of fit 715.28 10 71.53 9.354 0.0226 纯误差 Pure error 30.59 4 7.65 总误差 Total error 5124.17 28 R2=0.8544；adj R2=0.7089；精密度 Adeq precision=8.840 注：A. 可溶性玉米淀粉；B. 糖蜜；C. 花生饼浸粉；D. K2HPO4•3H2O。*表示在0.05水平显著相关；** 表示在0.01水平极显著相关。Note: A. Soluble corn starch；B. Molasses; C. Peanut cake powder; D. K2HPO4•3H2O. *Means significant correlation at 0. 05 level; **means extremely significant correlation at 0. 01 level.
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表 8 培养基优化前后发酵液中泛革素浓度
Table 8. Relative contents of fengycin in fermentation mediums before and after optimization
培养基类型
Media type泛革素峰面积
Fengycin peak area/mAU芽孢浓度
Spore conc. (×109)/(spore/mL)泛革素浓度
Fengycin conc. (×109)/(mAU/cfu)优化培养基 Optimized media 551.5 ± 22.4 a 6.10 ± 0.18 a 90.9 a 2 号基础培养基 No.2 basic medium 215.3 ± 17.2 b 3.30 ± 0.17 b 65.2 b 注：同列数据后不同小写字母表示在 0.05 水平差异显著。Note: Different letters within the same column represent significant differences at 5% level.
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