琼脂扩散法定量测定多黏菌素对霉菌的抑菌活力

Quantitative Determination of the Antifungal Activity of Polymyxin against Mold Fungi by Agar Infusion Method

摘要:

本文研究抗菌肽抑制霉菌的活力定量测定方法，选用多黏菌素B和产黄青霉作为研究对象，采用琼脂扩散法优化指示菌浓度、抗菌肽添加量、培养时间等影响活力定量测定的条件，进一步以黑曲霉为指示菌进行方法验证；通过建立抗菌肽浓度对数值与抑菌圈直径之间的线性方程进行抗菌肽抑菌活力的计算。研究结果显示，琼脂扩散法测定多黏菌素B抑制霉菌活力的最佳条件：90 mm平板添加PDA培养基15 mL，指示菌孢子浓度（1~5）×106个/mL，多黏菌素B添加量100 μL，4 ℃预扩散时间6~10 h，28 ℃培养时间24 h。以黑曲霉为指示菌进行方法验证时发现方法重复性好、精密度高。在此条件下，多黏菌素B对产黄青霉和黑曲霉的抑菌活性分别为524.81 U/mg和605.76 U/mg。该研究为抗菌肽在试验和生产过程中的活力测定提供参考和依据。

Abstract:

The quantitative determination method of antimicrobial peptides against mold fungi was studied in this paper. The antifungal activity of polymyxin B against Penicillium chrysogenum was determined by agar infusion method, and the determination conditions of indicator concentration, peptide addition amount and culture time were then optimized. Furthermore, the quantitative determination method was verified with Aspergillus niger. The antifungal activity was calculated by establishing a linear equation between the antimicrobial peptide concentration and inhibition zone diameter. The results showed that the optimal conditions for quantitatively testing the activities of polymyxin B against mold fungi were as follows: The amount of PDA in 90 mm petri dish was 15 mL, the spore concentration was (1~5)×106 spores/mL, the addition of polymyxin B was 100 μL, the pre-diffusion time was 6~10 h at 4 ℃ and the culture time was 24 h at 28 ℃. The quantitative determination method was verified with Aspergillus niger, and the results indicated that this determination method had good repeatability and high precision. Under these conditions, the antifungal activities of polymyxin B against Penicillium chrysogenum and Aspergillus niger were 524.81 U/mg and 605.76 U/mg, respectively. This study could provide a reference and basis for the activity determination of antimicrobial peptides in the process of research and production.