乳酸菌肽聚糖的提取纯化及其对河豚毒素的毒性消减效果研究

Extraction and purification of peptidoglycan from lactic acid bacteria and its toxicity elimination effect to tetrodotoxin

乳酸菌中提取的肽聚糖已被发现对河豚毒素具有消减作用。但肽聚糖提取工艺复杂、效率低下,阻碍了毒素消减研究。该研究以具有A3α,A1γ,A4α三种类型肽聚糖的乳酸菌为原料,通过对磷壁酸去除条件、脂质去除试剂和蛋白质的酶解工艺进行优化获得高纯度肽聚糖,并采用小鼠生物法评价了纯化后的3种肽聚糖对河豚毒素(tetrodotoxin, TTX)的毒性消减效果。溶菌酶及扫描电镜结果显示,采用100 g/L三氯乙酸去除磷壁酸、50 g/L十二烷基硫酸钠溶液去除脂质、胰蛋白酶与链霉蛋白酶联用去除蛋白质后,3种肽聚糖呈现完整的球状形态,表明优化后的提取工艺对肽聚糖的球形结构完整性影响小。酶联免疫吸附法和小鼠生物法结果显示,纯化后的3种肽聚糖可以去除78%以上的TTX含量,并降低87%以上的毒性,消减效果均优于灭活菌株。由此可见,优化后的肽聚糖提取纯化工艺不会破坏肽聚糖消减TTX的主要组分,并能显著提高效率,为肽聚糖的工业化应用和毒素控制研究提供了理论基础。

Peptidoglycan(PG)derived from lactic acid bacteria(LAB)has been found to able to eliminate tetrodotoxin(TTX).However,the extraction process of PG is complex and inefficient,which hinders the study of toxin removal.In this study,LAB with three types of PGs(A3α,A1γ,A4α)were used as materials.High purity PGs were obtained by optimizing the removal conditions of teichoic acid,removal reagents of lipid,and enzymatic hydrolysis of proteins.The toxicity removal effect of TTX by the purified PGs was assessed using a mouse biological method.The results of lysozyme experiment and SEM found that the three kinds of PGs showed complete spherical shape after the removal of teichoic acid by 100 g/L trichloroacetic acid,lipid removal by 50 g/L sodium dodecyl sulfate solution,and protein removal by trypsin and Streptomyces protease.It indicated that the optimized extraction process had a few effects on the spherical structure integrity of PGs.Enzyme-linked immunosorbent assay and mouse biological method results showed that the purified PGs could remove more than 78%of TTX amount and reduce more than 87%of toxicity.Moreover,the toxicity removal effect of PGs was better than that of inactivated strains.Hence,the optimized extraction and purification process did not destroy the main components for TTX elimination,but significantly enhanced efficiency.It provides a theoretical basis for the industrial application of PGs and toxin control research.