为研究从人体肠道分离、筛选的乳酸菌(lactic acid bacteria,LAB),其体外降解3-甲基吲哚的功能。采用MRS培养基从3~8岁健康儿童肠道中筛选乳酸菌,提取细菌总DNA,扩增16S rRNA序列,构建系统发育树。在以3-甲基吲哚为唯一碳源、氮源的无机盐培养基中胁迫培养乳酸菌,紫外分光光度计测定吸光度值,测定乳酸菌在高浓度3-甲基吲哚环境中的生长曲线;使用GC-MS动态检测3-甲基吲哚残留量,测定乳酸菌对3-甲基吲哚的降解能力。结果显示,筛选获得8株乳酸菌,其中LAB1为干酪乳杆菌属(Lactobacillus casei)、LAB2为类肠膜魏斯氏菌属(Weissella paramesenteroides)、LAB3、LAB5、LAB6、LAB7、LAB8均归属为肠膜明串珠菌属(Leuconostoc mesenteroides)、LAB4为格氏乳球菌属(Lactococcus garvieae)。在添加3-甲基吲哚的无机盐培养基中培养8株细菌,其降解3-甲基吲哚的能力均较强。LAB1~LAB8对3-甲基吲哚的体外降解率在168 h分别达到62.21%、60.45%、52.50%、62.19%、61.91%、61.94%、55.34%、58.73%,其中LAB1(干酪乳杆菌)对3-甲基吲哚的降解率最高,证实来源于儿童肠道的乳酸菌具有降解3-甲基吲哚的功能。该实验结果丰富了降解肠道毒素的微生物资源,同时也为今后扩展乳酸菌的应用范围提供了切实可行的理论依据。
The purpose of this study was to investigate the in vitro degradation of 3-methylindole by lactic acid bacteria(LAB) isolated from human intestine. MRS medium were used to screen LAB from the intestine of healthy children (aged 3 to 8 years), and then, total bacterial DNA was extracted, 16S rRNA sequence was amplified and the phylogenetic tree was constructed. LABs were cultured under stress in inorganic salt medium using 3-methylindole as sole carbon source and nitrogen source. The absorbance of LAB in high concentration skatole environment was measured by UV photometer. Then the degradation of 3-methylindole by LAB was determined by dynamic determination of 3-methylindole residue by GC-MS. The results showed that eight strains were all LAB, among which LAB1 belonged to Lactobacillus casei, LAB2 belonged to Weissella paramesenteroides, LAB3, LAB5, LAB6, LAB7, LAB8 belonged to Leuconostoc mesenteroides, and LAB4 belonged to Lactococcus garvieae. The eight strains grew slowly in the inorganic salt medium supplemented with 3-methylindole, but had strong ability to degrade 3-methylindole. The in vitro degradation rates of LAB1-LAB8 to 3-methylindole were 62.21%, 60.45%, 52.50%, 62.19%, 61.91%, 61.94%, 55.34% and 58.73%, respectively at 168 h, and the degradation rate of LAB1 (Lactobacillus casei) was the highest. It was proved that the LAB from children's intestines could degrade 3-methylindole. The results of our study provide a new microbial resource for degrading enterotoxin, and also provide a feasible theoretical basis for expanding the application of LAB in the future.
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