长寿老人源产细菌素乳酸菌的筛选与分子生物学鉴定

Screening and identification of bacteriocin-producing lactic acid bacteria from centenarians′ intestine

本研究以124株我国广西巴马百岁以上长寿老人源乳酸菌菌株为试材,采用双层琼脂平板扩散法筛选产细菌素的优良菌株。在排除有机酸、H2O2等的干扰后,菌株B02、B03、B04、B07、B11、B25、B24和B78的发酵上清液对受试的大肠埃希菌、金黄色葡萄球菌等5株指示菌都表现出很强的抑制作用;进一步硫酸铵沉淀、透析及浓缩处理后,其抑菌活性显著增强,同时蛋白酶敏感性试验显示其具有蛋白质性质,这些结果共同确定其为乳酸菌细菌素。最后,通过16S rRNA序列分析鉴定后确定B02为副干酪乳杆菌;B03为植物乳杆菌;B04为动物双歧杆菌;B07为干酪乳杆菌;B11为德氏乳杆菌保加利亚亚种;B24为鼠李糖乳杆菌;B25为粪肠球菌;B78为植物乳杆菌。

124 strains of lactic acid bacteria （LAB） isolated from centenarians＇ intestine were examined for production of bacteriecin by agar diffusion method. After eliminating the effects of organic acid and H2O2 ,cell-free supematants from 8 strains （B02,B03,B04,B07,B11 ,B25,B24 and B78） exhibited inhibitory activity against Escherichia coil,Staphylococcus aureus,Bacillus coagulans,B, cereus and Clostridium butyricum tested. The activity was enhanced after ammonium sulphate precipitation,dialysis and concentration, and these substances were sensitive to proteinase. The protein properties and antimicrobial activity indicated they belong to baeteriocins. With 16S rRNA sequence analysis, the above 8 bacteriecinproducing strains were identified as LactobaciUus paracasei （ B02 ）, L. plantarum （ B03,B78 ）, Bifidobacterium animals （ B04 ）, L casei（ B07 ）, L. delbrueckii subsp, bulgaricus （ B 11 ）, L. rhamnosus （B24） and Enterococcus faecail （ B25 ）.