对单增李斯特菌有抑制作用的乳酸菌的筛选鉴定及其细菌素的研究

Screening of Lactic acid bacteria for Production of Anti-listeria Bacteriocin

摘要:

本文采用双层平板法从中国东北地区发酵酸黄瓜中进行产抑单核增生李斯特氏菌细菌素的乳酸菌的筛选和鉴定，对其产生的细菌素进行了分离纯化、分子量测定及稳定性研究。在排除有机酸和过氧化氢的影响后，从东北发酵酸黄瓜中筛选到一株对单核增生李斯特氏菌仍有抑制作用的乳酸菌ZLG85，酶解实验发现该抑菌物质可完全被胃蛋白酶、胰蛋白酶和中性蛋白酶降解，确定该抑菌物质为细菌素。经形态和生理生化实验初步鉴定该菌株为明串株菌属；16S rDNA序列同源性分析将乳酸菌ZLG85鉴定为肠膜明串珠菌肠膜亚种。理化分析表明该细菌素的热稳定性很强，121 ℃处理30 min后相对抗菌活性仍为93.77%，pH值在2.0~10.0范围内处理4 h后相对抗菌活性在80%以上。N-羟甲基甲基甘氨酸-SDS-PAGE电泳确定肠膜明串珠菌素ZLG85的分子质量约为2.5 kDa。因此，肠膜明串珠菌素ZLG85是一种稳定性强的新型抗李斯特氏菌的乳酸菌细菌素。

Abstract:

By double agar diffusion methods, the strain producing bacteriocin with inhibitory activity against Listeria monocytogenes was screened and identified from Chinese northeast fermented cucumber. And the bacteriocin was separated and purified and its molecular mass and stability were researched. After eliminating the effects of organic acids and hydrogen peroxide, the strain of Lactobacillus spp. ZLG85 which still had activity against Listeria monocytogenes was screened. The inhibitory activity was degraded sharply by pepsin, trypsin and neutral protease in enzymatic hydrolysis, which was confirmed that ZLG85 can produce bacteriocin. By appearance detection, physiological and biochemical characteristics analysis and 16S rDNA gene sequence homology analysis, ZLG85 was identified as Leuconstoc mesenteroides subsp.mesenteroides. Physical and chemical analysis showed that the mesentericin ZLG85 was heat-stable. Its relative inhibitory activity was 93.77% after incubation at 121 ℃ for 30 min, and above 80% after incubation 4 h within a wide pH range from 2.0 to 10.0. The molecular weight of mesentericin ZLG85 was 2.5 kDa conformed by tricine-SDS-PAGE electrophoresis. Mesentericin ZLG85 is a new kind of bacteriocin with strong stability against Listeria monocytogenes.