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枯草芽孢杆菌PW2挥发性产物对黄曲霉的抑制作用
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TS 210.1；Q 939.92

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湖南省自然科学基金项目（2019JJ40541）；湖南省教育厅重点项目（20A513）


Inhibition of Aspergillus flavus by Volatile Organic Compounds Produced by Bacillus subtilis PW2
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    摘要:

    为探寻安全、高效的黄曲霉新型抑制物，采用顶空固相微萃取-气质联用(HS-SPME-GC-MS)技术对枯草芽孢杆菌（Bacillus subtilis）PW2所产具有抗霉活性的挥发性有机化合物（volatile organic compounds, VOCs）进行了成分鉴定，并利用平板对扣法从鉴定出的成分中筛选具有强抗霉活性的目标化合物，通过平板对扣法和96孔板梯度稀释法测定目标化合物对黄曲霉的最小抑菌体积分数（MIC)，使用黄曲霉毒素B1（aflatoxin B1, AFB1）ELISA检测试剂盒分析了目标化合物对黄曲霉产AFB1的抑制作用，使用扫描电子显微镜(scanning electron microscope, SEM)观察受试黄曲霉孢子形态的变化和采用分光光度法测定受试黄曲霉细胞膜麦角甾醇相对含量，以探讨抗霉机理。结果显示，从PW2所产VOCs中共鉴定出41种组分，并从中筛选出异辛醇为目标化合物；平板对扣法和96孔板梯度稀释法测得异辛醇对黄曲霉的MIC分别为0.169 μL/mL和6.25 μL/mL；PDB培养基中异辛醇体积分数分别为3.13、6.25、12.50 μL/mL时，均能有效抑制AFB1的产生，且随异辛醇体积分数的升高，抑制效果增强；异辛醇处理可导致黄曲霉孢子表面凹陷和孢子破裂，并且影响细胞膜中麦角甾醇的相对含量，造成细胞膜受损。该研究可为开发以异辛醇为功能成分的黄曲霉抑制剂提供理论参考。

    Abstract:

    In order to investigate safe and efficient novel inhibitors of Aspergillus flavus (A. flavus), the compounds of the antifungal volatile organic compounds (VOCs) produced by Bacillus subtilis PW2 were identified by headspace solid-phase microextraction gas chromatography-mass spectrometer (HS-SPME-GC-MS) technique. The target compounds with strong antifungal activity were screened from the identified components by double petri dish method. The minimum inhibitory concentration (MIC) of the target compound against A. flavus was determined by double petri dish method and 96-well plate gradient dilution method. The inhibitory effect of the target compound on aflatoxin B1(AFB1) production by A. flavus was analyzed using an AFB1 enzyme-linked immunosorbent assay(ELISA) kit. The morphological changes of the tested A. flavus spores were observed using scanning electron microscope (SEM), and the ergosterol relative content in the tested A. flavus cell membrane was analyzed using spectrophotometry method to explore the antifungal mechanisms. As a result, a total of 41 components were identified from the VOCs produced by PW2, and among them, 2-ethyl-1-hexanol was selected as the target compound. The MIC values of 2-ethyl-1-hexanol against A. flavus determined by the double petri dish method and 96-well plate gradient dilution method were 0.169 μL/mL and 6.25 μL/mL, respectively. The AFB1 were effectively inhibited by 2-ethyl-1-hexanol at the volume fractions of 3.13, 6.25 μL/mL and 12.50 μL/mL in PDB medium, and the inhibition effect was enhanced as the volume fraction of 2-ethyl-1-hexanol increased. Treatment with 2-ethyl-1-hexanol resulted in surface depression and the rupture of A. flavus spores, as well as alteration in the relative content of ergosterol in the cell membrane, leading to cell membrane damage. This study can provide a theoretical reference for the development of aflatoxin inhibitors with 2-ethyl-1-hexanol as a functional component.

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张凯歌,魏 琛,余 璐,杜明艳,李晓冉,林亲录,王青云.枯草芽孢杆菌PW2挥发性产物对黄曲霉的抑制作用[J].食品与生物技术学报,2024,43(5):122-129.

ZHANG Kai-ge, WEI Chen, YU Lu, DU Ming-yan, LI Xiao-ran, LIN Qin-lu, WANG Qing-yun. Inhibition of Aspergillus flavus by Volatile Organic Compounds Produced by Bacillus subtilis PW2[J]. Journal of Food Science and Biotechnology,2024,43(5):122-129.

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