柠檬醛缓释制剂的稳定性及其对黄曲霉菌的抑制作用

夏诗琪; 王培玲; 陈尚钘; 范国荣; 廖圣良; 王宗德

doi:10.13386/j.issn1002-0306.2021070371

柠檬醛缓释制剂的稳定性及其对黄曲霉菌的抑制作用

Stability of Citral Sustained-release Preparation and Its Inhibitory Effect on Aspergillus flavus

摘要

摘要: 本文旨在研究柠檬醛脂质体（Citral Liposome，CL）和柠檬醛-壳聚糖复合脂质体(Citral Liposome-Chitosan，CL-CS)这两种柠檬醛缓释制剂的稳定性及对黄曲霉菌（Aspergillus flavus）的抑菌效果、抑菌机理，为后期开发绿色、高效、安全的柠檬醛缓释抑菌剂提供数据支撑。通过设定不同的温度及pH条件，以外观、粒径、Zeta电位和保留率为指标探究储藏稳定性；用孢子计数法测定柠檬醛缓释制剂的瞬时抑菌率，并分析抑菌长效性；根据细胞通透性变化揭示柠檬醛缓释制剂的抑菌机理。结果表明，两种柠檬醛缓释制剂均具有纳米级粒径且粒径均一，经壳聚糖修饰后，体系Zeta电位由−16.63±1.67变为35.72±3.29。4 ℃条件下储藏28 d后粒径和Zeta电位变化较小，保留率更高，且CL-CS比CL更稳定；pH为4~6时，两种柠檬醛缓释制剂更稳定，CL和CL-CS的粒径别在150和200 nm左右。CL、CL-CS在48 h内对黄曲霉菌的瞬时抑菌EC₅₀分别为77.88和68.20 mg·L⁻¹，继续培养48 h后CL和CL-CS的抑制率分别为67.69%和82.89%，而柠檬醛只有30.26%，表明CL-CS具有良好的瞬时抑菌效果和长效抑菌效果。这两种柠檬醛缓释制剂处理后黄曲霉菌的胞外电导率和核酸含量均升高，说明细胞膜通透性增加，且所需要的作用时间比游离柠檬醛更短。综上，两种柠檬醛缓释制剂具有良好的稳定性，能通过改变细胞通透性、使内溶物渗出，从而抑制黄曲霉菌的生长，且具有长效抑菌能力，CL-CS比CL性能更好，具有开发成植物源防霉剂的潜能。

Abstract: To provide data support for the development of green, efficient and safe sustained-release bacteriostatic agent of citral, the stability of the two kinds of sustained-release preparation of citral liposome (CL) and citral liposome-chitosan (CL-CS) and the bacteriostatic effect and mechanism of Aspergillus flavus were investigated. Under the condition of different temperature and pH, the appearance, particle size, Zeta potential and retention rate were studied to explore the storage stability. The instantaneous bacteriostasis rate and long-term bacteriostasis rate of citral sustained-release were determined by spore counting method. The bacteriostatic mechanism of citral sustained-release preparations was revealed according to the changes of cell permeability. The results showed that the two citral sustained-release preparations had uniform nano-sized particle sizes. After chitosan modification, the Zeta potential of the system changed from −16.63±1.67 to 35.72±3.29. After 28 days of storage at 4 ℃, the change of particle size and Zeta potential was small, and the retention rate was higher, CL-CS was more stable than CL. At pH4~6, the two sustained-release preparations were more stable, and the particle sizes of CL and CL-CS were about 150 and 200 nm. The instantaneous inhibitory EC₅₀ of CL and CL-CS against Aspergillus flavus within 48 h were 77.88 and 68.20 mg·L⁻¹, respectively. After 48 h of culture, the inhibitory rates of CL and CL-CS were 67.69% and 82.89%, respectively, while citral was only 30.26%. The results showed that CL-CS had good bacteriostatic effect and long-term bacteriostatic effect. The extracellular conductivity and nucleic acid content of Aspergillus flavus both increased after treatment with the two kinds of sustained release preparations, indicating that the permeability of cell membrane increased and the action time was shorter than that of free citral. In conclusion, the two kinds of sustained-release preparations have good stability. They inhibited the growth of Aspergillus flavus by the change of cell permeability and the leak of insoluble substance. The properties of CL-CS was better than CL, and it would have the potential to be developed as plant source mildew inhibitor.