柠檬烯对荧光假单胞菌抑菌活性及机理研究

Antibacterial Activity and Mechanism of Limonene against Pseudomonas fluorescens

为研究柠檬烯对荧光假单胞菌的抑制活性及其抑菌机理,通过最小抑菌浓度(Minimal inhibition concentration,MIC)和生长曲线的测定研究柠檬烯对荧光假单胞菌细胞生长的影响;通过扫描电镜观察细胞结构,测定胞内蛋白含量和二乙酸荧光素(Fluorescein diacetate,FDA)染色实验研究柠檬烯对荧光假单胞菌细胞膜通透性的影响;通过测定膜电位、胞内ATP含量和琥珀酸脱氢酶(Succinic acid dehydrogenase,SDH)活性研究其对荧光假单胞菌能量代谢和三羧酸循环的影响。结果表明,柠檬烯对荧光假单胞菌具有较好的抑菌效果,其MIC值为20 mL/L,添加1×MIC和2×MIC浓度的柠檬烯3 h后膜电位分别下降至34.41和7.33 AU;12 h后SDH酶活性分别减小至11.12和22.69 U/mg。柠檬烯通过破坏荧光假单胞菌的细胞形态和结构,使细胞出现严重的破裂、塌陷和孔洞,从而引起细菌胞内蛋白含量降低,膜电位下降以及胞内ATP含量下降和SDH酶活性减小,从而发挥其抑菌作用。

The antibacterial activity and mechanism of limonene against Pseudomonas fluorescens were studied. The minimal inhibition concentration(MIC)and growth curve were determined to characterize the effects of growth. The scanning electron microscopy,intracellular protein content and fluorescein diacetate(FDA)staining experiment were studied to characterize the morphological structure and cell membrane permeability. The effects of membrane potential,intracellular ATP content and SDH enzyme activity on the energy metabolism and tricarboxylic acid cycle of Pseudomonas fluorescens were determined. The results showed that the antibacterial effect of limonene against Pseudomonas fluorescens was good and the MIC value was 20 mL/L. After added 1×MIC and 2×MIC limonene for 3 h,the membrane potential decreased to 34.41 and 7.33 AU;after 12 h,the SDH enzyme activity decreased to 11.12 and 22.69 U/mg,respectively. The antibacterial effect was exerted by destroyed cell morphology and structure,caused severe rupture,collapse and pores in the cells,leading to the decrease of intracellular protein content,membrane potential,intracellular ATP content and SDH enzyme activity.