关键环境因子对ε-聚赖氨酸抑菌活性的影响

Effects of Critical Environmental Factors on the Antimicrobial Activity of ε-Poly-L-lysine

摘要:

【目的】探究关键环境因子［水分活度（a_w）、pH及温度］对ε-聚赖氨酸（ε-PL）抑菌活性的影响，为其应用提供科学依据。【方法】以金黄色葡萄球菌（Staphylococcus aureus）为指示菌，采用微量稀释法、琼脂稀释法和抑菌动力学评价ε-PL的抑菌活性，通过蛋白质泄漏分析ε-PL对细胞膜活性的影响，通过Live/Dead荧光染色分析ε-PL对细胞膜通透性的影响。【结果】ε-PL对金黄色葡萄球菌的最小抑菌质量浓度（MIC）为40.0 μg/mL。琼脂稀释法结果显示，ε-PL抑菌活性随a_w升高而增强；当pH为5.0~7.0时，ε-PL抑菌活性较强；当pH为4.0时，有一定抑菌活性；当pH为8.0时，有微弱抑菌活性；当pH为9.0时，丧失抑菌活性；在中性条件下，当温度为30 ℃时，ε-PL有最大抑菌活性。抑菌动力学结果显示，ε-PL在不同pH条件下的抑菌活性与琼脂稀释法结果相近；在中性条件下，温度为25 ℃与30 ℃时，ε-PL的抑制作用强烈。在弱酸性及中性（pH 5.0~7.0）条件下，ε-PL对金黄色葡萄球菌细胞膜有很强破坏作用；在较低温度（25~30 ℃）下，ε-PL对细胞膜的破坏作用较强；而在较高温度（30~35 ℃）下，ε-PL对细胞膜的破坏作用变弱。Live/Dead荧光染色结果表明，当pH为4.0时，ε-PL可提高细胞膜的通透性；当pH为9.0时，失去破坏作用。【结论】a_w、pH及温度对ε-PL抑菌活性有显著影响。

Abstract:

[Objective]This study explored the effects of critical environmental factors [water activity (a_w), pH, and temperature] on the antimicrobial activity of ε-poly-L-lysine (ε-PL), aiming to provide a basis for the application of ε-PL.[Method]With Staphylococcus aureus as an indicator strain, the micro-broth dilution and agar dilution methods as well as antimicrobial growth kinetics were utilized to determine the antimicrobial activity of ε-PL. The protein leakage assay was employed to evaluate the bacterial membrane disruption activity of ε-PL. Live/Dead fluorescence staining was performed to observe the bacterial membrane permeability.[Result]The minimum inhibitory mass concentration (MIC) of ε-PL against Staphylococcus aureus was 40.0 μg/mL. The agar dilution method results indicated that the antimicrobial activity of ε-PL increased with the increase in a_w. Additionally, ε-PL demonstrated strong antimicrobial activity at pH 5.0~7.0, moderate antimicrobial activity at pH 4.0, weak antimicrobial activity at pH 8.0, and no antimicrobial activity at pH 9.0,while the maximum antimicrobial activity of ε-PL occurred at 30 ℃ under neutral conditions. The antimicrobial kinetic results showed that the antimicrobial efficacy of ε-PL under different pH conditions was consistent with the agar dilution method results, with strong inhibitory effects at 25 ℃ and 30 ℃ under neutral conditions. ε-PL had strong membrane disruption activity on Staphylococcus aureus under weakly acidic to neutral conditions (pH 5.0~7.0), and the membrane disruption activity of ε-PL was more pronounced at low temperatures (25~30 ℃) and weak at high temperatures (30~35 ℃). The Live/Dead fluorescence staining showed that the bacterial membrane permeability was enhanced by ε-PL at pH 4.0, while little effect was observed at pH 9.0.[Conclusion]The environmental factors a_w, pH, and temperature demonstrate significant influences on the antimicrobial activity of ε-PL.