Isolation of a novel bacteriocin-producing lactic acid bacteria and its use in food preservation

一株新型产细菌素乳酸菌的分离及其在食品保鲜中的应用

• 批准号: 10450311
• 负责人: SHIOYA Suteaki
• 金额: $ 7.04万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10450311/
• 关键词: enterocin A; bacteriocin; shuttle vector; E. faecium; immunity for enterocin A; 発酵食品; 乳酸菌; Lactobacillus plantarum; Enterococcus faecium

Enterococcus faecium N15 was isolated from nuka (Japanese rice bran paste), utilized as starter for fermented vegetables, and was found to produce a bacteriocin which exhibited a broad spectrum of activity including against Listeria monocytogenes and Bacillus circulans JCM2504. The characteristics of the bacteriocin suggest that bacteriocin N15 belongs to class IIa bacteriocins. Then, a 2-kbp DNA fragment was amplified by PCR with degenerated PCR primers, which were designed based on the conserved amino acid sequence of class IIa bacteriocins. It was found that E. faecium N15 produced enterocin A and entI gene encoding immunity protein for enterocin A was obtained.Enterococcus faecium N15 produced enterocin A and was found to have at least three plasmids, pEFNP1, pEFNP2, and pEFNP3. The nucleotide sequence of the smallest plasmid, pEFNP1, was determined. pEFNP1 was used to construct shuttle vectors that could replicate in Escherichia coli and E. faecium. The chimeric plasmid pKU201, composed of pUC19 and pEFNP1, was constructed. The shuttle vector plasmid pKU203 was then obtained by inserting the erythromycin (Em) resistance gene into pKU201. pKU203 was transformed in E. faecium N15 and its replication was confirmed. However, pKU203 was not stable in strain N15. Next, the entAI operon of E. faecium N15 was ligated into pKU203 and the plasmid pKU204 was constructed. pKU204 was also introduced into E. faecium N15 and E. faecalis JCM8726 and E. faecium EFN204 as a result of which the transformants E. faecalis ECL204 were obtained. pKU204 was stably maintained in E. faecium strain EFN204 without Em, suggesting that the entI gene improved the plasmid stability. In the case of E. faecalis strain ECL204, pKU204 was considerably more stable under the enterocin A pressure than without selection pressure. These findings indicated that the entI gene could be utilized as a selection marker gene.

屎肠球菌N15是从用作发酵蔬菜的发酵剂的nuka（日本米糠糊）中分离的，并且被发现产生细菌素，该细菌素表现出广谱活性，包括抗单核细胞增多性李斯特菌和环状芽孢杆菌JCM 2504。细菌素N15属于Ⅱ a类细菌素。根据Ⅱ a类细菌素的保守氨基酸序列设计简并引物，通过PCR扩增出2kbp的DNA片段。结果表明，E.屎肠球菌（Enterococcus faecium）N15能产生肠菌素A，并含有至少3个质粒pEFNP 1、pEFNP 2和pEFNP 3。测定了最小质粒pEFNP 1的核苷酸序列。利用pEFNP 1构建穿梭载体，并在大肠杆菌和大肠杆菌中进行复制。屎室构建了pUC 19与pEFNP 1的嵌合质粒pKU 201。将红霉素（Em）抗性基因插入pKU201中，得到穿梭载体pKU203。pKU203转化E. faecium N15的克隆和复制。然而，pKU203在菌株N15中不稳定。其次，对E.将N15连接到pKU203中，构建了pKU204质粒。pKU204也被导入E. faecium N15和E. faecalis JCM 8726和E. faecium EFN 204的转化子E. faecalis ECL 204。pKU204在E. faecium菌株EFN204中没有Em，表明entI基因提高了质粒稳定性。在E. pKU204在肠菌素A压力下比在无选择压力下稳定得多。这些结果表明，entI基因可以用作选择标记基因。