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枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化

钱荣 续晓琪 许宗奇 徐虹 李莎 徐铮 罗正山

钱荣,续晓琪,许宗奇,等. 枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化[J]. 食品工业科技,2022,43(15):123−131. doi:  10.13386/j.issn1002-0306.2021100269
引用本文: 钱荣,续晓琪,许宗奇,等. 枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化[J]. 食品工业科技,2022,43(15):123−131. doi:  10.13386/j.issn1002-0306.2021100269
QIAN Rong, XU Xiaoqi, XU Zongqi, et al. Isolation and Identification of Antibacterial Lpopeptides from Bacillus subtilis KC-WQ Fermentation Broth and Optimization of Fermentation Conditions[J]. Science and Technology of Food Industry, 2022, 43(15): 123−131. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021100269
Citation: QIAN Rong, XU Xiaoqi, XU Zongqi, et al. Isolation and Identification of Antibacterial Lpopeptides from Bacillus subtilis KC-WQ Fermentation Broth and Optimization of Fermentation Conditions[J]. Science and Technology of Food Industry, 2022, 43(15): 123−131. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021100269

枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化

doi: 10.13386/j.issn1002-0306.2021100269
基金项目: 国家自然科学基金(31801496);江苏省自然科学基金面上项目(BK20201358);中国博士后基金面上项目(2021M690191)山东省博士后创新项目(202102056)。
详细信息
    作者简介:

    钱荣(1997−),男,硕士研究生,研究方向:食品微生物,E-mail:297888741@qq.com

    通讯作者:

    续晓琪(1990−),女,博士,副教授,研究方向:食品微生物与营养支持,E-mail:xiaoqi_xu@njtech.edu.cn

  • 中图分类号: TS201.3

Isolation and Identification of Antibacterial Lpopeptides from Bacillus subtilis KC-WQ Fermentation Broth and Optimization of Fermentation Conditions

  • 摘要: 本文研究了一株枯草芽孢杆菌KC-WQ的胞外分泌物的抑菌效果及功效成分。实验发现枯草芽孢杆菌KC-WQ对大肠杆菌(Escherichia coli)、沙门氏菌(Salmonella enterica subsp. enterica)和嗜水气单胞菌(Aeromonas hydrophila)均有明显的抑菌效果,其中对沙门氏菌的抑制效果最为显著。在此基础上,采用酸沉淀法分离抗菌成分,通过薄层色谱法(TLC)、高效液相色谱法(HPLC)、基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析结构发现,该菌株分泌抗菌物质为脂肽类,分子量分布在1030.5~1491.4 Da之间,主要由伊枯草菌素(Iturin)、表面活性素(Surfactin)和芬荠素(Fengycin)组成,最小抑菌浓度为0.8 mg/mL。该脂肽成分经热处理(105 ℃,20 min)后,仍保持良好的抗菌效果。采用响应面法对KC-WQ的发酵条件进行优化,得到最佳发酵条件为:初始pH为6.0,发酵温度为36 ℃,摇床转速为208 r/min,预测粗产物产量为1.311 g/L,后经实验验证,实际产量可达1.236 g/L。上述结果表明,枯草芽孢杆菌KC-WQ具有良好的抗菌性能和加工性能,可作为抑菌成分,在食品保藏和抗菌包装材料开发中具有应用前景。
  • 图  1  枯草芽孢杆菌KC-WQ胞外发酵液抑菌图

    Figure  1.  Antibacterial diagram of extracellular fermentation broth of Bacillus subtilis KC-WQ

    注:a为枯草芽孢杆菌胞外发酵液抑制沙门氏菌,b为枯草芽孢杆菌胞外发酵液抑制大肠杆菌,c为枯草芽孢杆菌胞外发酵液抑制嗜水气单胞菌;(Ⅰ)为对照组,50 μL LB液体培养基,(Ⅱ)组添加50 μL发酵液,(Ⅲ)组添加100 μL发酵液,(Ⅳ)组添加150 μL发酵液。

    图  2  脂肽粗提物TLC结果

    Figure  2.  TLC results of crude lipopeptide extract

    注:A、B为平行组。

    图  3  脂肽粗提物与标准品HPLC保留时间对比

    Figure  3.  Comparison of HPLC retention time between crude lipopeptide extract and standard product

    图  4  脂肽粗提物MALDI-TOF结果

    Figure  4.  MALDI-TOF analysis results of crude lipopeptide extract

    注:a:Iturin族和Surfactin族;b:Fengycin族。

    图  5  抗菌脂肽耐热效果评价

    Figure  5.  Evaluation of the heat resistance of lipopeptides

    注:对照组样品为未经热处理的粗脂肽。

    图  6  碳源(a)、氮源(b)优化对脂肽粗产量及OD600 nm的影响

    Figure  6.  The influence of carbon source (a) and nitrogen source (b) optimization on the crude lipopeptide yield and OD600 nm

    图  7  摇床发酵条件优化结果

    Figure  7.  Optimized results of shaker fermentation conditions

    图  8  脂肽粗产量的响应面曲线图及等高线图

    Figure  8.  Response surface curve and contour plot of crude lipopeptide yield

    表  1  各变量及其高低水平设计

    Table  1.   Variables and their high and low level design

    编号因素−10+1
    X1初始pH678
    X2发酵温度(℃)333537
    X3摇床转速(r/min)150200250
    下载: 导出CSV

    表  2  枯草芽孢杆菌KC-WQ抑菌圈直径

    Table  2.   Inhibition zone diameter of Bacillus subtilis KC-WQ

    抑菌圈直径(mm)
    沙门氏菌0.0011.00±1.00a14.83±2.47b16.50±2.18c
    大肠杆菌0.0011.33±1.53a12.00±2.65b12.83±2.37c
    嗜水气单胞菌0.0010.00±2.00a14.40±1.04b15.77±1.57c
    注:具有显著性差异的组用不同的字母标记。
    下载: 导出CSV

    表  3  脂肽粗提物样品HPLC保留时间及面积

    Table  3.   HPLC retention time and area of crude lipopeptide extract sample

    保留时间(峰尖,min)面积峰面积占比(%)
    19.977836.2504.275
    210.402299.3841.530
    314.1492971.64115.191
    415.3011701.1978.697
    515.859841.1694.300
    617.189241.5111.235
    719.5081170.9285.986
    820.897873.0004.463
    下载: 导出CSV

    表  4  MS结果分析

    Table  4.   MS result analysis

    Familym/z(M+H+(M+Na+(M+K+
    IturinC13~C161030.4771052.4471068.357
    1044.4821066.3561082.319
    1058.4861080.3641096.326
    1072.4941094.3651110.332
    C13~C15 Bacillomycin D1031.4781053.3121069.408
    1045.4831067.3531083.316
    1059.4861081.3221097.322
    SurfactinC12~C15 Surfactin1016.4721032.437
    1030.4771046.441
    1044.4821060.443
    1058.4861074.449
    FengycinC15~C18 Fengycin A1471.3661487.338
    1485.3861501.355
    1499.3941515.385
    1513.4061529.373
    下载: 导出CSV

    表  5  不同浓度的KC-WQ抗菌脂肽对沙门氏菌的抑菌效果

    Table  5.   Inhibition of different concentrations of KC-WQ crude lipopeptide against Salmonella

    抗菌脂肽质量浓度(mg·mL−1抑菌圈直径(mm)
    0.4
    0.810.0±0.1
    1.611.0±0.1
    3.111.5±0.3
    6.212.6±0.3
    12.214.7±0.3
    2516.7±0.2
    下载: 导出CSV

    表  6  响应值试验及结果

    Table  6.   Response value test and results

    实验号因素脂肽粗产量(g/L)
    X1X2X3
    17352001.256±0.06
    27372501.224±0.06
    36352501.021±0.05
    46372000.976±0.04
    57352001.324±0.06
    68372000.889±0.04
    77371501.132±0.05
    88351501.024±0.05
    97352001.256±0.06
    108332000.932±0.04
    117352001.256±0.06
    127352001.256±0.06
    137331501.004±0.05
    146351500.952±0.04
    157332501.136±0.05
    168352501.044±0.05
    176332000.951±0.04
    下载: 导出CSV

    表  7  二次多项回归模型方差结果

    Table  7.   Quadratic polynomial regression model variance results

    方差来源平方和自由度均方FP显著性
    模型0.3190.03415.150.0008***
    X10.07110.07131.680.0008***
    X20.05310.05323.840.0018**
    X33.433E-00313.433E-0031.530.2560不显著
    X1X22.712E-00312.712E-0031.210.3080不显著
    X1X31.211E-00311.211E-0030.540.4865不显著
    X2X34.000E-00414.000E-0040.180.6855不显著
    X120.2010.2087.94<0.0001***
    X220.05010.05022.470.0021**
    X325.510E-00315.510E-0032.460.1611不显著
    残差0.01672.244E-003
    失拟项0.01234.002E-0034.330.0954不显著
    纯误差3.699E-00349.248E-003
    总和0.3216
    注:R2=0.9512,R2Adj=0.8884,*P<0.05,**P<0.01,*** P<0.001。
    下载: 导出CSV
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    HE L, WANG L Y, ZHU Q, QIAO J Y, WANG W J. Comparison of two methods for drawing the growth curve of Bacillus subtilis and Escherichia coli[J]. Tianjin Agricultural Sciences, 2017, 23(5): 14-18. doi:  10.3969/j.issn.1006-6500.2017.05.004
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出版历程
  • 收稿日期:  2021-10-26
  • 网络出版日期:  2022-06-19
  • 刊出日期:  2022-08-03

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