{"title":"Principle Investigation and Method Standardization of Inhibition Zone Assay Based on Antimicrobial Peptides Extracted from Black Soldier Fly Larvae","authors":"Wenyue Shen, Ranxia Xue, Yanxia Liu, Shibo Sun, Xi Chen, Dongye Sun, Ouyang Han, Yuxin Li, Jianqiang Xu, Xiaoying Dong, Fengyun Ji, Weiping Xu","doi":"10.3390/biotech13030031","DOIUrl":null,"url":null,"abstract":"The black soldier fly is a valuable resource insect capable of transforming organic waste while producing antimicrobial peptides (AMPs). The inhibition zone assay (IZA) is a method used to demonstrate the antimicrobial activity of AMPs. This study aimed to examine the experimental principles and establish a standardized IZA method. Results indicated that the AMPs extract consisted of proteins ranging in molecular weights from 0 to 40 kDa. The AMPs diffused radially on an agar plate through an Oxford cup. The diffusion radius was influenced by the concentration and volume of the AMPs but ultimately determined by the mass of the AMPs. The swabbing method was found to be effective for inoculating bacteria on the agar plate. Among the conditions tested, the plate nutrient concentration was the most sensitive factor for the IZA, followed by bacterial concentration and incubation time. Optimal conditions for the IZA included a nutrient plate of 0.5× TSA, a bacterial concentration of 106 CFU/mL, and an incubation time of 12 h, with oxytetracycline (OTC) at 0.01 mg/mL serving as the positive control. The antimicrobial-specific activity of AMPs could be standardized by the ratio of inhibition zone diameters between AMPs and OTC. These findings contribute to the standardization of the IZA method for profiling the antimicrobial activity of AMPs.","PeriodicalId":2,"journal":{"name":"ACS Applied Bio Materials","volume":"9 24","pages":""},"PeriodicalIF":4.6000,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Bio Materials","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/biotech13030031","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MATERIALS SCIENCE, BIOMATERIALS","Score":null,"Total":0}
引用次数: 0
Abstract
The black soldier fly is a valuable resource insect capable of transforming organic waste while producing antimicrobial peptides (AMPs). The inhibition zone assay (IZA) is a method used to demonstrate the antimicrobial activity of AMPs. This study aimed to examine the experimental principles and establish a standardized IZA method. Results indicated that the AMPs extract consisted of proteins ranging in molecular weights from 0 to 40 kDa. The AMPs diffused radially on an agar plate through an Oxford cup. The diffusion radius was influenced by the concentration and volume of the AMPs but ultimately determined by the mass of the AMPs. The swabbing method was found to be effective for inoculating bacteria on the agar plate. Among the conditions tested, the plate nutrient concentration was the most sensitive factor for the IZA, followed by bacterial concentration and incubation time. Optimal conditions for the IZA included a nutrient plate of 0.5× TSA, a bacterial concentration of 106 CFU/mL, and an incubation time of 12 h, with oxytetracycline (OTC) at 0.01 mg/mL serving as the positive control. The antimicrobial-specific activity of AMPs could be standardized by the ratio of inhibition zone diameters between AMPs and OTC. These findings contribute to the standardization of the IZA method for profiling the antimicrobial activity of AMPs.
期刊介绍:
ACS Applied Bio Materials is an interdisciplinary journal publishing original research covering all aspects of biomaterials and biointerfaces including and beyond the traditional biosensing, biomedical and therapeutic applications.
The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrates knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important bio applications. The journal is specifically interested in work that addresses the relationship between structure and function and assesses the stability and degradation of materials under relevant environmental and biological conditions.