乳酸乳球菌乳亚种NCU036018细菌素的分离纯化及其抗菌机制

从自然发酵泡菜中筛选到1 株对金黄色葡萄球菌具有抑制作用的乳酸菌，经16S rDNA和生理生化实验鉴定其为乳酸乳球菌乳亚种（Lactococcus lactis subsp. lactis）并命名为NCU036018。NCU036018发酵上清液中的抑菌物质对胰蛋白酶、木瓜蛋白酶、蛋白酶K、α-糜蛋白酶敏感，而对胃蛋白酶、脂肪酶、淀粉酶以及过氧化氢酶不敏感，在pH 2.0～10.0、温度50～100 ℃的范围内分别保留了70%和90%以上的抑菌活性。随后通过硫酸铵盐析、超滤、阳离子交换层析对发酵上清液抑菌成分进行分离纯化，将得到的细菌素命名为乳球菌素036018，其分子质量在14.4～20 kDa之间，最小抑菌浓度为0.50 mg/mL。乳球菌素036018能有效降低金黄色葡萄球菌生物膜的形成率，损坏细胞表面形态、破坏细胞膜的通透性，从而抑制甚至杀死细胞。将乳球菌素036018添加至牛奶中，发现其具有显著抑制金黄色葡萄球菌生长的效果，具有良好的食品防腐保鲜的应用前景。

Screening of Bacteriocin-producing Lactic Acid Bacteria: Isolation and Purification of and Antibacterial Mechanism of Bacteriocin

A strain of lactic acid bacteria (LAB) with an inhibitory effect on Staphylococcus aureus was isolated from naturally fermented pickle. The strain was identified as Lactococcus lactis subsp. lactis and named NCU036018 by 16S rDNA gene sequence analysis and physiological and biochemical experiments. The antibacterial substance in the fermentation supernatant of NCU036018 was sensitive to trypsin, papain, protease K and α-chymotrypsin, but not to pepsine, lipase, amylase or catalase, and retained more than 70% and 90% of its activity at pH 2.0–10.0 and 50–100 ℃, respectively. Then, the antibacterial substance was separated and purified by successive steps of ammonium sulfate salting-out, ultrafiltration and cation exchange chromatography. The obtained bacteriocin was named lactococcin 036018, whose molecular mass and minimum inhibitory concentration (MIC) were 14.4-20 kDa and 0.50 mg/mL, respectively. Lactococcin 036018 could effectively reduce the biofilm formation rate of S. aureus, damage the morphology of the cell surface, reduce the permeability of the cell membrane and consequently inhibit or even killing the cells. The bacteriocin had a significant inhibitory effect on the growth of S. aureus in milk, thereby showing great promise as a food preservative.