一株拮抗单增李斯特菌的蒙氏肠球菌A9-1A的筛选及其细菌素抗菌活性分析

Enterococcus mundtii A9-1A with antagonistic effect on Listeria monocytogenes: screening and characterization of antimicrobial activity of its bacteriocin

摘要:

【背景】单核细胞增生李斯特菌(Listeria monocytogenes)，简称单增李斯特菌，是一种重要的食源性致病菌，易感人群感染后致死率可达20%−30%。【目的】筛选高效拮抗单增李斯特菌的乳酸菌菌株，并探究其细菌素的理化特性及对单增李斯特菌的杀伤作用。【方法】采用牛津杯法筛选高效拮抗单增李斯特菌的产细菌素乳酸菌菌株；通过形态特征、16S rRNA基因序列分析及平均核苷酸一致性分析进行菌株分类学鉴定；并进一步分析其细菌素的理化特性；利用激光共聚焦显微镜和扫描电子显微镜表征细菌素粗提物对单增李斯特菌的拮抗作用。【结果】筛选获得一株高效拮抗单增李斯特菌的菌株A9-1A，其抑菌圈大小为37.56 mm，仅对李斯特菌属(Listeria)和粪肠球菌(Enterococcus faecalis)具有拮抗作用，经形态特征、16S rRNA基因序列分析和平均核苷酸一致性分析鉴定为蒙氏肠球菌(Enterococcus mundtii)；其产生的细菌素粗提物能够耐受强酸强碱(pH 2.0−11.0)、高温(37−121 ℃)环境，对淀粉酶和脂肪酶不敏感，但对蛋白酶敏感，最低抑菌浓度为16 μg/mL，激光共聚焦显微镜和扫描电子显微镜结果表明细菌素粗提物通过损伤细胞的细胞膜并形成孔洞，造成细胞内容物泄漏，从而杀死单增李斯特菌。【结论】分离筛选到一株高效拮抗单增李斯特菌的产细菌素蒙氏肠球菌A9-1A，其细菌素粗提物具有耐受强酸强碱和高温的特性，具有在食品生产过程中高效防控单增李斯特菌污染的潜在应用前景。

Abstract:

[Background] Listeria monocytogenes is a major food-borne pathogen, causing the mortality rate of 20%–30% in susceptible populations. [Objective] To screen the lactic acid bacteria with antagonistic effect on L.monocytogenes and explore the physicochemical properties and killing effect of the bacteriocin produced by the isolate on L.monocytogenes. [Methods] The bacteriocin-producing lactic acid bacteria with antagonistic effect on L.monocytogenes were screened by the Oxford cup method. The strain screened out was identified based on morphological characteristics, 16S rRNA gene sequencing, and average nucleotide identity (ANI). The physicochemical properties of the bacteriocin produced by the strain were analyzed and the antagonistic effect of the bacteriocin crude extract on L.monocytogenes was characterized by laser confocal microscopy and scanning electron microscopy. [Results] A strain A9-1A with strong antagonistic effect on L.monocytogenes was obtained, with the inhibition zone diameter of 37.56 mm and antagonistic effects on Listeria and Enterococcus faecalis. It was identified as Enterococcus mundtii. The bacteriocin produced by this strain could tolerate strong acids and alkali (pH 2.0–11.0) and high temperatures (37–121 ℃). It was not sensitive to amylase and lipase but sensitive to proteases. The minimum inhibitory concentration of the bacteriocin crude extract was 16 μg/mL. The results of confocal laser scanning microscopy and scanning electron microscopy showed that the bacteriocin crude extract damaged the cell membrane and led to the formation of holes, causing the leakage of cell contents and thereby killing L.monocytogenes. [Conclusion] A bacteriocin-producing strain E. mundtii A9-1A with strong antagonistic effect on L.monocytogenes was screened out. Its bacteriocin could tolerate strong acids and alkali and high temperatures, demonstrating the application potential in the prevention and control of L.monocytogenes contamination in food production.