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【目的】筛选产广谱、高效抑菌活性物质的海洋放线菌,为新型抗生素的开发奠定基础。【方法】采用琼脂块法初筛,打孔扩散法复筛,以金黄色葡萄球菌、大肠埃希氏菌、枯草芽孢杆菌和单核细胞增生李斯特菌为指示菌从海泥样品中筛选目标菌株;利用20种指示菌株考查Y10、Y11、Y15、Y16和Y21的抑菌谱;通过形态观察和16S r RNA基因序列分析确定菌株分类地位;以单核细胞增生李斯特菌的OD600值为指标探讨Y15抑菌活性物质对该菌的作用方式;以抑菌活性为指标研究Y15抑菌活性物质的理化性质。【结果】共分离12株具抑菌活性的海洋放线菌,其中Y15抑菌谱最广,对20种指示菌株中的18种具有抑菌活性,并且在4种培养基中均能产生抑菌活性物质。16S r RNA基因序列分析表明Y15属于小单孢菌属,并与Micromonospora endolithica亲缘关系最近。Y15抑菌活性物质在144 h达到最高值为480 AU/m L,在168-216 h保持平衡为320 AU/m L。Y15抑菌活性物质对单核细胞李斯特菌作用方式为杀菌。Y15抑菌活性物质在-20-60°C抑菌活性保持稳定,在80-120°C活性逐渐下降,但在120°C处理30 min仍保留37.5%的活性;在p H 7.0-10.0抑菌活性稳定,在p H 2.0-6.0和p H 11.0-12.0抑菌活性均有所损失;Y15抑菌活性物质对紫外和4种酶(蛋白酶K、胰蛋白酶、木瓜蛋白酶和α-淀粉酶)处理均保持稳定,表明活性物质为非蛋白和非多肽类的抑菌物质。【结论】Y15产生的活性物质具有良好的抑菌谱和抑菌活性,且较为稳定,具有较高的应用价值。
【Objective】 Screening marine actinomycetes producing broad-spectrum and highly effective antibacterial active substances laid the foundation for the development of new antibiotics. 【Method】 The target was screened from the samples of sea mud using the method of agar block screening and puncturing and diffusion screening, and Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Listeria monocytogenes as indicator bacteria Strain; using 20 kinds of indicator strains to check Y10, Y11, Y15, Y16 and Y21 antibacterial spectrum; by morphological observation and 16S rRNA gene sequence analysis to determine the strain classification; Listeria monocytogenes OD600 value as an indicator To investigate the mode of action of the antibacterial substance Y15 on the bacteria and to study the physicochemical properties of the antibacterial substance Y15 with antibacterial activity as an index. 【Result】 Twelve strains of actinomycetes with antibacterial activities were isolated. Among them, Y15 had the most extensive antibacterial spectrum and had antibacterial activity against 18 of the 20 indicated strains, Bacteria active substance. Sequence analysis of the 16S rRNA gene showed that Y15 belongs to the genus Micromonospora and has the closest genetic relationship with Micromonospora endolithica. The highest inhibitory activity of Y15 was reached at 480 AU / m L at 144 h and 320 AU / m L at 168-216 h. Y15 antibacterial activity of Listeria monocytogenes mode of action for sterilization. Y15 antibacterial activity at -20-60 ° C antibacterial activity remained stable at 80-120 ° C activity decreased gradually, but at 120 ° C for 30 min still retained 37.5% activity; p H 7.0-10.0 inhibition The activity of the bacterium was stable, and the antibacterial activity was lost at p H 2.0-6.0 and p 11.0-12.0. The antibacterial activity of Y15 against UV and four enzymes (protease K, trypsin, papain and alpha-amylase) The treatment was stable, indicating that the active substance is a non-protein and non-peptide antibacterial substances. 【Conclusion】 The active substance produced by Y15 has good antibacterial spectrum and antibacterial activity, is stable and has high application value.