海洋抑菌放线菌Y15的筛选、鉴定及其代谢产物理化性质

Screening, identification and characterization of marine antimicrobial actinomycetes Y15

摘要:

【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 rRNA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 rRNA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/mL，在168?216 h保持平衡为320 AU/mL。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pH 7.0?10.0抑菌活性稳定，在pH 2.0?6.0和pH 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。

Abstract:

[Objective] This paper aims at screening marine actinomycetes with broad-spectrum and producing efficient antimicrobial active substance, to lay the foundation for the development of new antibiotics. [Methods] Using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Listeria monocytogenes as indicator bacteria, target bacteria from sea mud samples was primarily screened with agar block method, and further screened with punching diffusion method; twenty indicator bacteria were used to examine the antimicrobial spectrum of the new isolates Y10, Y11, Y15, Y16 and Y21; taxonomic status of the strain Y15 was determined by morphologic observation and 16S rRNA gene sequence analysis; mode of action of antimicrobial active substance to Listeria monocytogenes was studied according to indicator bacteria OD600 value; the antimicrobial activity was used as an index to study the physical and chemical properties of antimicrobial active substances produced by Y15. [Results] A total of 12 strains showing antimicrobial activities to antagonistic strains were isolated; wherein Y15 had the widest spectrum, and could be against 18 of the 20 kinds of indicator strains, and on four media it could produce antimicrobial active substances. The 16S rRNA gene sequence analysis showed that Y15 had closest relationship with Micromonospora endolithica. Activity of Y15 antimicrobial metabolites reached a maximum of 480 AU/mL in 144 h, maintained 320 AU/mL from 168 h to 216 h. The action mode of Y15 antimicrobial active substance to Listeria monocytogenes was sterilization. Antimicrobial activity of Y15 antimicrobial active substances remained stable from ?20 °C to 60 °C, and gradually declined from 80 °C to 120 °C, but still retained 37.5% at 120 °C for 30 min; antimicrobial activity remained stable from pH 7.0 to 10.0, and from pH 2.0 to 6.0 and pH 11.0 to 12.0 antimicrobial activity declined; Y15 antimicrobial substances processed in the UV and four kinds of enzymes (Proteinase K, Trypsin, Papain and α-Amylase) were stable, there were non-protein and non-peptide antimicrobial substances. [Conclusion] The active substances produced by Y15 showed good antimicrobial spectrum and activity, and were relatively stable, indicating a high application value in the future.