白黄链霉菌T D-1产帕马霉素对黄曲霉2219等霉菌的抑制作用

Inhibition Effect of Pamamycin Produced by Streptomyces alboflavus T D-1 on Aspergillus flavus 2219 and Other Fungus

为研究微生物及其次级代谢产物对黄曲霉2219等霉菌的抑制机理,以分离于土壤中的白黄链霉菌(Streptomyces alboflavus)T D-1为研究对象,利用薄层色谱、硅胶柱层析、高效液相色谱和液相-质谱联用技术对其代谢产生的活性物质——帕马霉素进行分离。利用扫描电子显微镜、荧光显微镜和高效液相色谱方法研究帕马霉素对黄曲霉2219等霉菌生长的抑制效果及抑制机理。结果表明,白黄链霉菌T D-1上清液和菌丝体中活性物质的分子式预测为C_(35)H_(61)NO_(7),为帕马霉素及其同系物,相对分子质量为593、607、621、635、649。通过扫描电子显微镜观察帕马霉素处理后的霉菌细胞形态,其孢子和菌丝体会发生不同程度畸变。帕马霉素对桔青霉的最小抑菌浓度(minimal inhibitory concentration,MIC)为0.125 mg/mL,对灰霉的MIC为0.500 mg/mL,对黄曲霉2219、黑曲霉、哈茨木霉和米曲霉的MIC均为1.000 mg/mL。帕马霉素对霉菌的作用靶点为其细胞膜的麦角甾醇,抑制机制是破坏细胞壁的完整性,改变细胞膜的通透性,影响线粒体的能量代谢,抑制菌丝细胞膜中麦角甾醇的生物合成。帕马霉素对黄曲霉2219产生黄曲霉毒素B 1的抑制率达到98.3%。因此,可利用白黄链霉菌T D-1及其代谢产物帕马霉素对食品原料中的产毒真菌污染进行生物防治。

To study the inhibitory mechanism of microorganisms and their secondary metabolite against fungus such as Aspergillus flavus 2219,the active substance pamamycin produced by metabolism of Streptomyces alboflavus T D-1 screened from soil was isolated using thin layer chromatography,silica gel column chromatography,high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry.The inhibitory effect and mechanism of pamamycin against Aspergillus flavus 2219 and other fungus were determined by scanning electron microscope(SEM),fluorescence microscope and HPLC.Results showed that the molecular formula of active substances in the supernatant and mycelium of streptomyces alboflavus T D-1 was predicted to be C_(35)H_(61)NO_(7),pamamycin and its homologue,and the relative molecular weight were 593,607,621,635,649.According to SEM observation,the morphology of fungus cells treated with pamamycin was distorted in different extent.The minimum inhibitory concentration(MIC)of pamamycin for Penicillium citrus was 0.125 mg/mL,for Botrytis cinerea was 0.500 mg/mL,and for Aspergillus flavus 2219,Aspergillus niger,Trichoderma harzianum and Aspergillus oryzae was 1.000 mg/mL.The action target of pamamycin was ergosterol on fungal cell membrane.The inhibition mechanism was to destroy the integrity of cell wall,change the permeability of cell membrane,affect the energy metabolism of mitochondria,inhibit the ergosterol biosynthesis in mycelial membrane.Pamamycin could reduce the synthesis rate of aflatoxin B 1 by 98.3%.Therefore,Streptomyces alboflavus T D-1 and pamamycin could be used for biological control of toxigenic fungi in raw food materials.