L.LactisKLDS4.0325细菌素理化性质的研究

Physicochemical Characterization of a Bacteriocin from Lactococcus lactis KLDS4.0325

该研究以分离自新疆牧民家庭自制酸马奶中的乳酸乳球菌KLDS4.0325为研究对象,以双层平板法为检测方法,以抑菌圈直径为检测指标,以大肠杆菌为指示菌,对该菌株所产细菌素进行提取、纯化及理化特性分析,首先无细胞发酵上清液通过60%饱和度硫酸铵溶液盐析得到细菌素粗提液,然后细菌素粗提液通过SP Sepharose Fast Flow阳离子交换层析法得到纯化的细菌素样品,其比活力可达51.02 IU/mg,较优化前提高了约18倍,并通过tricine-SDS-PAGE蛋白电泳测定分子量约为3.4 ku;最后,对细菌素样品的生物学特性进行研究,通过温度、pH和各种酶的敏感性试验得出,细菌素样品热稳定性强,在pH 2~10的范围抑菌圈直径变化不大,有较高的稳定性,经蛋白酶处理后,抑菌活性消失,但经淀粉酶处理后仍能保持较高的稳定性,这也说明该细菌素是蛋白类物质。

A bacteriocin produced by Lactococcus lactis KLDS4.0325 isolated by Xinjiang herdsmen from self-made koumiss was purified and characterized. The double-plate method was used for detection. The diameter of the inhibition zone（mm） was used as the evaluation index, and it was quantified against Escherichia coli ATCC 25922. The bacteriocin produced by this strain was extracted, purified, and subjected to physicochemical analysis. The bacteriocin crude extract was extracted from the cell-free fermentation supernatant by precipitation with 60% ammonium sulfate. This was followed by SP Sepharose FF cation exchange chromatography to obtain purified bacteriocin samples. The specific activity of the purified bacteriocin was estimated to be 51.02 IU/mg and the inhibitory activity was increased by 18 folds. The molecular mass of the purified bacteriocin was estimated using tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be approximately 3.4 ku. Additionally, the biological characteristics of the bacteriocin were studied by sensitivity experiments of temperature, pH, and various enzymes. The results showed that the bacteriocin was heat-stable and its inhibition zone diameter showed insignificant changes when pH of incubation was increased from 2.0 to 10.0, showing good stability. The inhibitory activity of the bacteriocin was found to be sensitive to proteolytic enzymes such as pepsin, trypsin, papain, α-chymotrypsin, and proteinase K,indicating that it is a type of protein.