解淀粉芽孢杆菌AF1发酵液的抗菌活性与抗菌机理

摘要

This study aims to measure the antimicrobial spectrum of fermentation broth of Bacillus amyloliquefaciens AF1 and to explore the activity stability and mechanism of anti-MRSA,for providing an experimental basis in seeking new anti-MRSA compounds. The drug-resistant S. aureus SA46 was used as indicating pathogen,the rest antimicrobial activities of AF1 fermentation broth were detected after it was dealed with different pH,temperatures and proteases. The antmicrobial mechanism was studied preliminarily by observing the morphologic change of SA46 tread with AF1 fermentation broth under both optical microscope and transmission electron microscope,and by detecting the protein and DNA leakage of treated SA46 with AF1 fermentation broth with SDS-PAGE and agarose gel electrophoresis. The results showed that AF1 fermentation broth had a broad antimicrobial spectrum. It presented a strong inhibitory ability to all tested clinical drug-resistant Staphylococcus aureus,common clinical Escherichia coli and S. aureus;also it inhibited the most of the tested phytopathogen. The antimicrobial activity of AF1 fermentation broth remained more than 95% in pH 2.0 - 9.0 for 120 min from 4 to 80℃,and it was stable when adding protease K, pepsin and trypsin into the broth respectively. The cell walls of SA46 treated by AF1 fermentation broth(MIC)were destroyed at varied level while observed under both optical microscope and transmission electron microscope. Furthermore,the leakage of protein and DNA of treated SA46 with AF1 fermentation broth were checked by SDS-PAGE and agarose gel electrophoresis. The electrophoretic bands of experimental groups were clear,while there was no bands in control groups on both DNA and protein electrophorograms. Thus,a preliminary deduction was that the cell walls of MRSA were damaged by AF1 fermentation broth. In conclusion,B. amyloliquefaciens AF1 might be developed as a potential new source for the novel anti-MRSA medicines.%测定解淀粉芽孢杆菌AF1发酵液的抗菌谱,对发酵液抗MRSA的活性稳定性及抗MRSA机制进行初步探究,为寻找新的抗MRSA化合物提供实验基础.以耐药金葡菌SA46为病原指示菌,不同pH、温度和蛋白酶处理后,考察AF1发酵液剩余抗菌活性.用显微镜和透射电镜观察经发酵液处理后的SA46形态变化,并用凝胶电泳分析SA46的蛋白质和DNA渗漏情况,初步探讨其抗菌机制.结果显示,AF1发酵液具有广谱抗菌活性:不仅对临床耐药金葡菌、一般临床大肠杆菌和金葡菌有很强抑菌作用,同时对大部分试验的植物病原菌也有较好抑菌作用.发酵液在pH 2.0-9.0,4-80℃,120 min后抗菌活性均保持95%以上;并且对蛋白酶K、胃蛋白酶和胰蛋白酶稳定.SA46经发酵液(MIC)处理后,显微镜和透射电镜观察发现其细胞壁受到不同程度破坏;在DNA和蛋白质电泳图上,试验组SA46的DNA和蛋白质条带很清晰,即SA46蛋白质和DNA出现渗漏,而对照组均没有条带出现,初步推断AF1对MRSA的抗菌机理是破坏病原菌的细胞壁来抑制细胞生长的.菌株AF1可作为抗MRSA药物研发的潜在新来源.