母乳婴儿源产细菌素双歧杆菌的分离鉴定

Separation and identification of bacteriocin-producing Bifidobacterium from breast-fed infants faeces

目的:以从母乳婴儿粪便分离得到的44株双歧杆菌为试材,筛选产细菌素双歧杆菌优良菌株。方法:采用双层琼脂平板扩散法初筛,通过排除有机酸、H2O2和菌体细胞干扰,检测所产细菌素蛋白敏感性实验对初筛得到的菌株进行复筛,并鉴定筛选得到的优良菌株。结果:初筛得到产细菌素双歧杆菌优良菌株BB04和BL-8,排除有机酸、H2O2和菌体细胞干扰后两株菌的发酵上清液仍表现较强抑菌效果,进一步硫酸铵盐析、透析后抑菌活性明显增强,蛋白酶K、胃蛋白酶和胰蛋白酶处理后活性消失,说明起抑菌作用的是细菌素,且其本质是蛋白质。抑菌活性实验结果显示,菌株BB04和BL-8所产细菌素抑菌谱较宽,对受试的李斯特菌、金黄色葡萄球菌、凝结芽孢杆菌等革兰氏阳性菌和革兰氏阴性菌大肠杆菌均表现出较强抑菌活性。最后,通过形态学观察、API 20A生理生化反应和16S rDNA序列分析,确定菌株BB04为动物双歧杆菌(Bifidobacterium animalis),BL-8为青春双歧杆菌(Bifidobacterium adolescentis)。结论:从母乳婴儿粪便中分离出2株产广谱细菌素的双歧杆菌优良菌株BB04和BL-8,经鉴定其分别为动物双歧杆菌和青春双歧杆菌。

Objective： Good bacteriocin- producing Bifidobacterium were obtained from 44 strains from breast- fedbabies faeces. Method： These strains were primarily screened by agar diffusion test and screened again byeliminating the disturbances of the organic acids,hydrogen peroxides and strain cells and proteinase assay.Goodstrains obtained were identified.Results ：The antimicrobial activity of the fermentation broth of good strains of BB04and BL-8 remained strong after eliminating the disturbances of the organic acids, hydrogen peroxides and straincells. In addition ,the activity was significantly enhanced after ammonium sulphate precipitation and dialysis and thebacteriocin lost activities after treated with proteinase K, pepsin and trypsin. The results indicated that thebacteriocin produced by strain BB04 and BL-8 worked as antibacterial substance whose nature was protein.What＇smore,the bacteriocin produced from strain BB04 and BL-8 had a broad inhibitory spectrum.They could both inhibitListeria monocytogenes, Staphylococcusaereu, Bacillu coagulans and Escherichia coll. Finally, based onmorphological, physiological and biochemical characteristics of API 20A anaerobic appraisal system and 16S rDNAgene sequence,strain BB04 was identified as Bifidobacterium animalis and BL-8 was identified as Bifidobacteriumadolescentis.Conclusion. Good Bifidobacterium strains BB04 and BL-8 producing broad spectrum bacteriocinwere separated from breast-fed infants faeces and identified as Bifidobacterium animalis and Bifidobacteriumadolescentis.