松油烯-4-醇对荧光假单胞菌抑菌能力及作用机理

Antibacterial Activity and Mechanism of Terpinene-4-ol against Pseudomonas fluorescens

本实验通过测定最小抑菌剂量(minimum inhibitory concentration,MIC)及生长曲线,评价松油烯-4-醇对荧光假单胞菌的抑菌能力;随后通过碱性磷酸酶活力、电导率测定,二乙酸荧光素染色实验分析以及扫描电子显微镜观察评价松油烯-4-醇对细胞壁及细胞膜通透性的影响;最后通过测定DNA、总蛋白与胞内钠、钾离子三磷酸腺苷(adenosine triphosphate,ATP)酶活力,来进一步探究松油烯-4-醇对细胞内大分子及细胞代谢的影响。结果表明:松油烯-4-醇对荧光假单胞菌的MIC为2μL/mL;松油烯-4-醇可有效破坏荧光假单胞菌的细胞壁,破坏程度随剂量的升高而增强;松油烯-4-醇会使细胞膜的通透性增强,导致细胞内离子的外渗;扫描电子显微镜观察结果表明松油烯-4-醇能对细胞膜产生不可逆的破坏;凝胶阻滞电泳分析结果表明,松油烯-4-醇会使大分子的DNA泄露;通过测定总蛋白含量与Na+、K+-ATP酶活力可知,松油烯-4-醇能降低细胞内蛋白质的含量并能阻滞蛋白质的表达与ATP酶的合成,造成细胞凋亡。综上,松油烯-4-醇有望成为新型水产品保鲜剂,本实验可为拓展天然防腐剂在水产品的应用提供理论依据。

The antibacterial activity of terpinene-4-ol against Pseudomonas fluorescens was investigated by measuring the minimum inhibitory concentration(MIC)and growth curve.Its effect on cell wall and cell membrane permeability were evaluated by determination of alkaline phosphatase activity and electrical conductivity,fluorescein diacetate(FDA)staining and scanning electron microscope(SEM)observation,and its effect on intracellular macromolecules and cellular metabolism was further explored by determining DNA,total protein and Na+,K+-ATPase activity.The results showed that the MIC was determined to be 2μL/mL.Terpinen-4-ol could effectively destroy the cell wall of P.fluorescens in a dose-dependent manner,and enhance cell membrane permeability,leading to the leakage of intracellular ions.The results of SEM showed that terpinen-4-ol could damage the cell membrane irreversibly.Gel retardation electrophoresis analysis revealed that terpinene-4-ol could cause leakage of macromolecular DNA.Additionally,terpinen-4-ol reduced intracellular protein content and blocked the expression of protein and the synthesis of ATPase,thus resulting in cell apoptosis.Therefore,terpinen-4-ol has the potential to be used as a new preservative for aquatic products.