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生防枯草芽孢杆菌HMB19198发酵培养基的筛选及优化

曲远航 郭庆港 李社增 宣立锋 张晓云 马平

曲远航, 郭庆港, 李社增, 宣立锋, 张晓云, 马平. 生防枯草芽孢杆菌HMB19198发酵培养基的筛选及优化[J]. 农药学学报, 2022, 24(3): 509-519. doi: 10.16801/j.issn.1008-7303.2022.0009
引用本文: 曲远航, 郭庆港, 李社增, 宣立锋, 张晓云, 马平. 生防枯草芽孢杆菌HMB19198发酵培养基的筛选及优化[J]. 农药学学报, 2022, 24(3): 509-519. doi: 10.16801/j.issn.1008-7303.2022.0009
QU Yuanhang, GUO Qinggang, LI Shezeng, XUAN Lifeng, ZHANG Xiaoyun, MA Ping. Screening and Optimization of Bacillus subtilis HMB19198 fermentation medium[J]. Chinese Journal of Pesticide Science, 2022, 24(3): 509-519. doi: 10.16801/j.issn.1008-7303.2022.0009
Citation: QU Yuanhang, GUO Qinggang, LI Shezeng, XUAN Lifeng, ZHANG Xiaoyun, MA Ping. Screening and Optimization of Bacillus subtilis HMB19198 fermentation medium[J]. Chinese Journal of Pesticide Science, 2022, 24(3): 509-519. doi: 10.16801/j.issn.1008-7303.2022.0009

生防枯草芽孢杆菌HMB19198发酵培养基的筛选及优化

doi: 10.16801/j.issn.1008-7303.2022.0009
基金项目: 河北省重点研发计划(19226510D);河北省农林科学院现代农业科技创新工程(2019-1-1-7).
详细信息
    作者简介:

    曲远航,quyuanhang1990@163.com

    通讯作者:

    郭庆港,gqg77@163.com

  • 中图分类号: S482.2;S432.4

Screening and Optimization of Bacillus subtilis HMB19198 fermentation medium

Funds: the Key Research and Development Program of Hebei Province (19226510D), the HAAFS Agriculture Science and Technology Innovation Project (2019-1-1-7).
  • 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%。
  • 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

    图  7  枯草芽孢杆菌HMB19198菌株的生长曲线

    Figure  7.  Growth curve of B. subtilis strain HMB19198

    表  1  芽孢杆菌基础培养基

    Table  1.   Basic mediums for Bacillus

    培养基
    Medium
    配方
    Formula
    1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
    下载: 导出CSV

    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)
    −11
    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
    下载: 导出CSV

    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)
    −101
    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
    下载: 导出CSV

    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)
    145.010.045.010.05.01.53.76
    230.015.045.010.07.51.53.58
    330.015.030.010.05.03.02.86
    445.015.045.010.07.53.03.80
    530.015.045.015.05.03.03.18
    645.010.030.010.07.53.04.75
    745.010.045.015.05.03.04.12
    845.015.030.015.07.51.54.40
    930.010.030.015.07.53.04.63
    1030.010.030.010.05.01.54.10
    1145.015.030.015.05.01.54.29
    1230.010.045.015.07.51.53.90
    下载: 导出CSV

    表  5  Plackett-Burman试验统计分析

    Table  5.   The regression analysises of Plackett-Burman experiments

    因子
    Factor
    F
    F value
    P
    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
    下载: 导出CSV

    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)
    100006.50
    2−100−16.00
    30−1014.89
    400007.23
    500007.07
    600007.02
    7−10014.42
    8−10103.24
    900114.93
    100−1103.13
    1101105.23
    1200−116.35
    13001−14.79
    14−11004.08
    1510−104.48
    16100−17.85
    17010−15.06
    1810016.32
    19−10−105.47
    2001015.93
    210−10−14.17
    2211007.93
    23−1−1004.02
    240−1−105.81
    251−1004.75
    2610106.21
    2700−1−16.04
    2801−103.26
    2900007.04
    下载: 导出CSV

    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
    MS
    F
    F value
    P
    P value
    显著性
    Significance
    模型
    Model
    4378.3014312.745.8700.0011**
    A885.801885.8016.6260.0011**
    B185.651185.653.4850.0830
    C125.451125.452.3550.1472
    D9.5419.540.1790.6786
    A × B243.361243.364.5680.0507
    A × C392.041392.047.3590.0168*
    A × D0.0610.060.0010.9732
    B × C540.561540.5610.1460.0066**
    B × D0.5610.560.0110.9196
    C × D0.7210.720.0140.9089
    A2211.791211.793.9750.0660
    B21244.5511244.5523.3600.0003**
    C21096.5011096.5020.5810.0005**
    D268.06168.061.2770.2774
    残差 Residual745.871453.28
    失拟项 Lack of fit715.281071.539.3540.0226
    纯误差 Pure error30.5947.65
    总误差 Total error5124.1728
    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.
    下载: 导出CSV

    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 a6.10 ± 0.18 a90.9 a
    2 号基础培养基 No.2 basic medium 215.3 ± 17.2 b3.30 ± 0.17 b65.2 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.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-10-22
  • 录用日期:  2021-11-05
  • 网络出版日期:  2022-01-29
  • 刊出日期:  2022-06-10

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