马尾松毛虫肠道产细菌素细菌的筛选及抑菌特性

【背景】细菌素是一类由细菌产生的抗菌肽活性物质,对多种致病菌有抑制和杀灭作用。昆虫是世界上种类最多、肠道菌群资源最为丰富且多样的动物类群之一。昆虫肠道很可能蕴含着丰富的产细菌素菌种资源。【目的】以马尾松毛虫(Dendrolimus punctatu)幼虫为研究材料,对其肠道细菌进行分离、培养和鉴定,获得对典型致病菌有明显抑制作用的产细菌素细菌,推测其抑菌物质,并对产高活性细菌素细菌的抑菌特性进行研究,进一步丰富产细菌素细菌的资源。【方法】采用传统细菌纯培养方法分离细菌,利用牛津杯法检测抑菌物质活性,结合菌落形态特征及16SrRNA基因序列确定产细菌素细菌种类;进一步排除有机酸和过氧化氢的干扰,初步证实抑菌物质有类蛋白性质。【结果】从马尾松毛虫肠道中筛选得到13株产细菌素细菌,隶属于芽孢杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)、嗜冷杆菌属(Psychrobacte)和肠杆菌属(Enterobacter)。抑菌实验结果显示:13株产细菌素细菌对金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)两种重要病原指示菌有一定程度的抑制效果,尤其是解淀粉芽孢杆菌(Bacillusamyloliquefaciens)MW-1产生的细菌素经不同温度和酸碱性处理后仍保持较好的抑菌活性,而且经单因素方差分析发现在pH 7.0和37℃时抑菌活性最佳。【结论】马尾松毛虫肠道内存在多种产细菌素细菌且对重要病原菌有抑制作用。菌株MW-1细菌素具有高效的病原菌抑菌能力,所产细菌素对热和酸碱处理稳定性较好,显示了替代抗生素的应用潜力。

Screening and antibacterial activity of bacteriocin-producing bacteria in intestine of Dendrolimus punctatus

Background] Bacteriocin is a group of antibacterial-peptide substance produced by bacteria, with an inhibition and killing effect on various pathogenic bacteria. Insect, as the most abundant animal groups, is one of animals containing the most abundant and diverse intestinal microbial community in the world. Insect intestines may contain a rich bacteriocin-producing bacteria resource. Objective] Sampling Dendrolimus punctatus larvae as experimental materials, its intestinal bacteria were isolated, and these bacteria were further cultured and identified. Bacteriostatic bacteria with obvious inhibitory effect on typical pathogens were obtained. Bacteriostatic substance was preliminarily speculated, and bacteriocin-producing bacteria with high bacteriostatic activity were investigated to further enrich resources of the bacteriocin-producing bacteria. Methods] The bacteria were isolated by traditional-pure culture method, activity of the bacteriostatic substance was detected by using Oxford cup method, and bacteriocin-producing species were determined by combining morphological characteristics and 16S rRNA gene sequences of colony; interference of organic acid and hydrogen peroxide was further eliminated, and the bacteriostatic substance was preliminarily demonstrated with protein-like properties. Results] Thirteen strains of the bacteriocin-producing bacteria were screened from intestine of the D. punctatus larvae, which belong to genera Bacillus, Staphylococcus, Psychrobacter and Enterobacter. Results of bacteriostatic experiments showed that these 13 strains have a certain degree of inhibitory effects on two species of typically pathogenic indicator bacteria, Staphylococcus aureus and Escherichia coli. In particular, bacteriocin produced by Bacillus amyloliquefaciens MW-1 maintained a good antibacterial activity after treating with different temperature and pH conditions, and an optimum bacteriostatic activity was obtained under pH 7.0 and 37 °C. Conclusion] Many species of the bacteriocins-producing bacteria in the intestine of the D. punctatus larvae present antibacterial effects on important pathogens. The strain MW-1 with high bacteriostatic activity for pathogen shows potential to replace antibiotic, bacteriocin of which exhibited a good stability under heat, acids and alkaline treatments.