植物乳杆菌抑菌蛋白的分离鉴定及抑菌特性研究

doi:10.16431/j.cnki.1671-7236.2022.08.042

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (8): 3263-3272.doi: 10.16431/j.cnki.1671-7236.2022.08.042

• 基础兽医 • 上一篇

植物乳杆菌抑菌蛋白的分离鉴定及抑菌特性研究

李小宁¹, 李军¹, 尹杨燕², 李常挺¹, 马春霞¹, 陶立¹, 龚俞³, 钟舒红¹, 白慧丽¹, 彭昊¹, 廖玉英¹

1. 广西壮族自治区兽医研究所, 广西兽医生物技术重点实验室, 南宁 530001;
2. 广西大学动物科学技术学院, 南宁 530004;
3. 贵州省畜禽遗传资源管理站, 贵阳 550001

收稿日期:2022-01-05 出版日期:2022-08-05 发布日期:2022-07-21
通讯作者: 彭昊, 廖玉英 E-mail:hpeng2006@163.com;315951610@qq.com
作者简介:李小宁,E-mail:15602160653@163.com。
基金资助:
广西重点研发计划(AB21238003、AB21220005);广西基本科研业务费(桂科专项22-5、21-6、22-6);国家水禽产业技术体系建设专项(CARS-42);国家现代农业产业体系广西肉鸡产业创新团队建设专项(nycytxgxcxtd);南宁市重点研发计划(20202106、20212023);良庆区重点研发计划(202109);良庆区重大科技专项(202118);防城港市重点研发计划(AB21014016);武鸣区重点研发(20210102);贵州省农业动植物育种项目(黔农育专字[2018]016号)

Isolation,Identification and Antibacterial Property of Antibacterial Proteins from Lactobacillus plantarum

LI Xiaoning¹, LI Jun¹, YIN Yangyan², LI Changting¹, MA Chunxia¹, TAO Li¹, GONG Yu³, ZHONG Shuhong¹, BAI Huili¹, PENG Hao¹, LIAO Yuying¹

1. Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China;
2. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
3. Guizhou Livestock and Poultry Genetic Resources Management Station, Guiyang 550001, China

Received:2022-01-05 Online:2022-08-05 Published:2022-07-21

摘要/Abstract

摘要： 【目的】筛选新型广谱有效的抑菌蛋白,为全面解析植物乳杆菌抑菌机制和开发新型抗菌制剂奠定基础。【方法】以植物乳杆菌GX20200417-1为研究对象,采用牛津杯法测定其抑菌活性,以低温离心和超滤法初步分离获得胞外蛋白并测定其抑菌活性,对胞外蛋白进行不同温度、pH及蛋白酶处理,研究抑菌蛋白理化特性,利用液相色谱串联质谱(LC-MS/MS)进一步分析鉴定植物乳杆菌代谢产物中的抑菌成分。【结果】植物乳杆菌GX20200417-1对沙门氏菌、金黄色葡萄球菌和大肠杆菌均具有良好的抑菌作用;发酵上清液在发酵24 h的抑菌能力最强;胞外蛋白的抑菌能力与发酵上清液相近;植物乳杆菌抑菌蛋白具有较好的耐热特性,与对照组相比,在20~80 ℃时抑菌活性均无显著变化(P＜0.05),在pH 6.0~8.0时抑菌活性最佳,对蛋白酶敏感;经LC-MS/MS鉴定分析,检测出5种可信度较高且与抑菌作用相关的蛋白质,分别是片球菌素pediocin PA-1、溶菌素(lysin)、聚酮合酶(polyketide synthase)、辅助蛋白(accessory protein)和LysM peptidoglycan-binding domain-containing protein,分子质量分别为5.348、7.348、8.348、6.348和19.662 ku;蛋白质功能分析结果表明,片球菌素pediocin PA-1与溶菌素主要通过破坏细菌细胞壁与细胞膜,从而达到抑菌效果。LysM peptidoglycan-binding domain-containing protein可识别含有N-乙酰氨基葡萄糖(GlcNAc)残基的肽聚糖,上调抗菌肽的表达;辅助蛋白主要参与细菌素的合成,聚酮合酶主要参与抗生素的合成,二者通过参与抑菌物质的合成间接发挥抑菌作用。【结论】本研究成功分离并鉴定植物乳杆菌GX20200417-1的5种抑菌蛋白;植物乳杆菌GX20200417-1可能通过其代谢产物中的多种抑菌蛋白协同发挥抗菌作用。

关键词: 植物乳杆菌; 抑菌蛋白; 超滤法; 液相色谱串联质谱; 抑菌机制

Abstract: 【Objective】 This study was aimed to screen broader-spectrum and effective antibacterial proteins so as to lay the foundation for analyzing antibacterial mechanism of Lactobacillus plantarum thoroughly and exploring new antibacterial agents.【Method】 Lactobacillus plantarum GX20200417-1 was chosen to be the object of this study.Oxford cup method was selected to determine the antimicrobial activity.Proteins were extracted from Lactobacillus plantarum GX20200417-1 by the means of centrifugation at low temperature and ultrafiltration and their antibacterial activities were measured.The extracellular proteins were treated with different temperatures,pH and protease to study the physicochemical properties of antibacterial proteins.Antimicrobial components from Lactobacillus plantarum metabolites were further analyzed by LC-MS/MS.【Result】 Lactobacillus plantarum GX20200417-1 had strong antibacterial effect on Salmonella,Staphylococcus aureus and Escherichia coli.The antimicrobial activity of fermentation was tested at different time which was the strongest at 24 h with the antimicrobial diameter.The proteins extracted from fermentation had the similar antimicrobial activity with the fermentation.Lactobacillus plantarum antibacterial protein exhibited a high stability towards the temperatures. Compared with control group, there was no significant change in antibacterial activity at 20-80 ℃ (P<0.05),the best antibacterial activity at pH 6.0-8.0 and sensitive to protease.By GC-MS/MS analysis,five kinds of proteins were obtained which were not only corrected with bacteriostasis,but also had much more credibility.They were pediocin PA-1,lysin,polyketide synthase,accessory protein and LysM peptidoglycan-binding domain-containing protein and the molecular weight were 5.348,7.348,8.348,6.348 and 19.662 ku respectively.Pediocin PA-1 and lysin achieve bacteriostatic effect mainly by destroying bacterial cell wall and cell membrane.LysM peptidoglycan-binding domain-containing protein could recognize peptidoglycans including N-acetylglucosamine (GlcNAc) residue and upregulate the expression of antibacterial peptides.Accessory protein participated in the synthesis of bacteriocin,polyketide synthase participated in the synthesis of antibiotics,both of which played an indirect bacteriostatic role by participating in the synthesis of antimicrobial substances.【Conclusion】 In this study,five antimicrobial proteins of Lactobacillus plantarum GX20200417-1 were successfully identified,which laid a foundation for protein separation and purification.Its speculated that Lactobacillus plantarum GX20200417-1 could play an antibacterial role by the coordination of antibacterial proteins in metabolites.

Key words: Lactobacillus plantarum; antibacterial proteins; ultrafiltration; LC-MS/MS; antibacterial mechanism

中图分类号:

S816.73

李小宁, 李军, 尹杨燕, 李常挺, 马春霞, 陶立, 龚俞, 钟舒红, 白慧丽, 彭昊, 廖玉英. 植物乳杆菌抑菌蛋白的分离鉴定及抑菌特性研究[J]. 中国畜牧兽医, 2022, 49(8): 3263-3272.

LI Xiaoning, LI Jun, YIN Yangyan, LI Changting, MA Chunxia, TAO Li, GONG Yu, ZHONG Shuhong, BAI Huili, PENG Hao, LIAO Yuying. Isolation,Identification and Antibacterial Property of Antibacterial Proteins from Lactobacillus plantarum[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3263-3272.

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植物乳杆菌抑菌蛋白的分离鉴定及抑菌特性研究

Isolation,Identification and Antibacterial Property of Antibacterial Proteins from Lactobacillus plantarum

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