基于抑菌活性的ε-聚赖氨酸的微孔板生物检测法

A microplate bioassay to determine ε-polylysine based on antimicrobial activity

摘要:

基于ε-聚赖氨酸的抑菌活性, 以96微孔板为平台, 建立适合大规模样品快速分析的微孔板生物检测法。结果表明, 藤黄微球菌(Micrococcus luteus)为最适敏感指示菌, 当敏感指示菌初始浓度为107?108 CFU/mL, 培养时间为4 h时, ε-聚赖氨酸浓度在100.00?500.00 mg/L范围内与抑菌率呈较显著的线性关系(R2=0.997 5)。该方法不仅表现出良好的精密度和准确度, 与HPLC和甲基橙法的实验结果对比, 微孔板生物检测法的相对标准偏差(RSD)和相对偏差(RD)分别小于3.5%和3.0%, 说明该方法是一种与HPLC法有类似分析精度, 但能直接反映样品的抑菌活性, 且更适合大规模样品快速检测的方法, 可用于发酵过程中ε-聚赖氨酸产量的检测和突变株的高通量筛选。

Abstract:

Based on the antimicrobial activity of ε-Polylysine, a 96-well microplate bioassay was established to rapidly determine large numbers of ε-Polylysine samples. The results showed that a high correlation coefficient (R2=0.997 5) was obtained for linear regression in the ε-polylysine concentration range of 100.00?500.00 mg/L with 107?108 CFU/mL indicator strain (Micrococcus luteus) and the incubation time of 4 h. Using these parameters, the microplate bioassay has showed good precision and accuracy, and the relative standard derivation (RSD) and relative derivation (RD) were lower than 3.5% and 3.0% respectively. Compared with the results by Itzhaki method and HPLC assay, the microplate bioassay shows no considerable difference with HPLC, but can directly indicate the inhibition activity and more suitable for rapid determination of numerous samples, which can be used for ε-polylysine quantification in fermentation process and high throughput screening from large numbers of mutants.