苯乳酸对荧光假单胞菌基于细胞膜损伤和DNA破坏的双靶位抑菌机制

doi:10.7506/spkx1002-6630-20200406-072

食品科学 ›› 2021, Vol. 42 ›› Issue (7): 60-67.doi: 10.7506/spkx1002-6630-20200406-072

苯乳酸对荧光假单胞菌基于细胞膜损伤和DNA破坏的双靶位抑菌机制

宁亚维，侯琳琳，李明蕊，杨正，马梦戈，王志新，王世杰，贾英民

（1.河北科技大学食品与生物学院，河北石家庄 050018；2.北京工商大学食品与健康学院，北京 100048）

出版日期:2021-04-15 发布日期:2021-05-17
基金资助:
“十三五”国家重点研发计划重点专项（2018YFC1604305）；河北省重点研发计划项目（19222805D；19227130D）

Dual-Target Antibacterial Mechanism of Phenyllactic Acid against Pseudomonas fluorescens Based on Cell Membrane and DNA Damage

NING Yawei, HOU Linlin, LI Mingrui, YANG Zheng, MA Mengge, WANG Zhixin, WANG Shijie, JIA Yingmin

(1. College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang 050018, China; 2. School of Food and Health, Beijing Technology and Business University, Beijing 100048, China)

Online:2021-04-15 Published:2021-05-17

摘要/Abstract

摘要： 荧光假单胞菌是导致冷藏食品腐败变质常见的嗜冷菌，抑制荧光假单胞菌的生长繁殖对延长冷藏食品货架期和提高食品安全性具有重要意义。本实验通过测定最小抑菌浓度（minimum inhibitory concentration，MIC）和时间-抑菌曲线考察苯乳酸对荧光假单胞菌的抑菌活性，从细胞膜电势、细胞膜渗透性和完整性、细胞超微结构、蛋白质表达和DNA结构等方面研究苯乳酸对荧光假单胞菌的抑菌机制。结果表明：苯乳酸对荧光假单胞菌的MIC为1.25 mg/mL；苯乳酸可导致细胞膜电势消散，且消散程度呈剂量依赖性；苯乳酸可导致细胞内钾离子显著泄漏（P＜0.05），增加细胞膜的渗透性；MIC苯乳酸处理菌体0.5 h后，流式细胞术分析结果显示，碘化丙啶沾染率为57.6%，扫描电子显微镜结果显示，部分菌体破裂，表明苯乳酸可以破坏细胞膜完整性；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果表明苯乳酸对蛋白质表达无明显影响；凝胶阻滞电泳与荧光光谱结果显示苯乳酸可以破坏DNA结构。结论：苯乳酸可以通过损伤细胞膜和破坏DNA发挥双靶位抑菌作用，苯乳酸对荧光假单胞菌抑菌机制的阐释结果可为冷藏食品中嗜冷菌的控制以及苯乳酸在食品中的应用提供理论依据。

关键词: 苯乳酸；荧光假单胞菌；基因组DNA；抑菌机制

Abstract: Pseudomonas fluorescens is one of the most common psychrophilic bacteria causing spoilage of refrigerated foods. Inhibiting the growth and reproduction of P. fluorescens is of great significance for extending the shelf life of refrigerated foods and improving food safety. In this study, the antibacterial activity of phenyllactic acid (PLA) against P. fluorescens was evaluated by determining the minimum inhibitory concentration (MIC) and time-inhibition curve. The inhibitory mechanism was explored from different perspectives: membrane potential, membrane permeability and integrity, cell ultrastructure, protein expression and DNA structure. The results showed that the MIC of PLA was 1.25 mg/mL. PLA could depolarize the cell membrane in a dose-dependent manner, and cause significant leakage of intracellular potassium ion (P < 0.05), indicating increased permeability of the cell membrane. After treatment for 0.5 h by PLA at MIC concentration, the propidium iodide staining rate of cells was 57.6% as determined by flow cytometry, and ruptured cells were observed by scanning electron microscopy (SEM), suggesting that PLA could damage the membrane integrity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that PLA had no significant effect on protein expression. Gel retardation electrophoresis and fluorescence spectroscopy showed that PLA could destroy the structure of DNA. Therefore, PLA can exert a bacteriostatic effect by destroying both the cell membrane and DNA, which will provide theoretical evidence for the control of psychrophilic spoilage bacteria in refrigerated foods and the application of PLA in foods.

Key words: phenyllactic acid; Pseudomonas fluorescens; genomic DNA; bacteriostatic mechanism

中图分类号:

TS202.3

宁亚维，侯琳琳，李明蕊，杨正，马梦戈，王志新，王世杰，贾英民. 苯乳酸对荧光假单胞菌基于细胞膜损伤和DNA破坏的双靶位抑菌机制[J]. 食品科学, 2021, 42(7): 60-67.

NING Yawei, HOU Linlin, LI Mingrui, YANG Zheng, MA Mengge, WANG Zhixin, WANG Shijie, JIA Yingmin. Dual-Target Antibacterial Mechanism of Phenyllactic Acid against Pseudomonas fluorescens Based on Cell Membrane and DNA Damage[J]. FOOD SCIENCE, 2021, 42(7): 60-67.

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苯乳酸对荧光假单胞菌基于细胞膜损伤和DNA破坏的双靶位抑菌机制

Dual-Target Antibacterial Mechanism of Phenyllactic Acid against Pseudomonas fluorescens Based on Cell Membrane and DNA Damage

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