基于琼脂扩散法的抗菌肽定量测定方法优化  

Optimization of Quantitative Determination of Antimicrobial Peptides by Agar Diffusion Method

作　　者：魏东东[1] 刘杨柳 王志新[3] 郭润芳[1] 贾英民 于宏伟[1] WEI Dong-dong;LIU Yang-liu;WANG Zhi-xin;GUO Run-fang;JIA Ying-min;YU Hong-wei(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,Hebei,China;School of Food Science and Technology,Beijing Technology and Business University,Beijing 100048,China;College of Bioscience and Bioengineering,Hebei University of Science and Technology,Shijiazhuang 050018,Hebei,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]北京工商大学食品学院,北京100048 [3]河北科技大学生物科学与工程学院,河北石家庄050018

出　　处：《食品研究与开发》2018年第17期105-110,共6页Food Research and Development

基　　金：国家重点研发计划项目(2016YFF0202300);河北省科技计划项目(16227506D)

摘　　要：抗菌肽在试验室研究、生产实践以及安全性评价过程中,存在定量测定方法不统一,结果重复性差,缺乏比较依据等问题。以硫酸多粘菌素B为代表,以铜绿假单胞菌为指示菌,采用琼脂扩散法建立抗菌肽浓度对数值与抑菌圈直径之间的线性关系,以重复性和精密度为指标,对指示菌菌龄、菌浓、琼脂浓度和培养基厚度等影响因素进行优化,确定琼脂扩散法测定抗菌肽效价的最佳测定条件为:指示菌的最佳培养时期为稳定期,浓度为10~6 cfu/mL,培养基厚度为3.15 mm(Φ90 mm,20 mL),琼脂浓度为1.5%。在此条件下,选用其它抗菌肽和指示菌进行验证,结果重复性好,精密度高(R^2=0.997),该研究将为抗菌肽在试验和生产过程中的定量测定提供参考和依据。In the research, production and safety evaluation for antimicrobial peptides, there are a lot of problems, such as non-uniform in the quantitative estimation, poor reproducibility in the study result, lack basis for comparison etc. Polymyxin B sulfate as the representative, Pseudomonas aeruginosa as indicator bacteria, agar diffusion method was used to establish a linear relationship between the antibacterial peptide concentration and inhibition zone diameter. The indicator bacteria age, bacteria concentration, agar concentration and media thickness and other influencing factors are optimized with repeatability and precision as indicators. Results showed that the optimal conditions for quantitative estimation of antibacterial peptide by the agar diffusion method were indicator bacteria with stable culture period and 10G cfu/mL, medium thickness with 3.15 mm （Ф 90 mm,20 mL）and the agar concentration with 1.5 %. Under these conditions, other antibacterial peptides and indicator bacteria were selected for validation. The results showed good repeatability and high precision （R2 = 0.997）. It would provide a reference and basis for the quantitative estimation of antimicrobial peptides in the process of research and production.

关 键 词：琼脂扩散法 抗菌肽 定量测定 效价 硫酸多粘菌素B 

分 类 号：TQ465[化学工程—制药化工]