Principle Investigation and Method Standardization of Inhibition Zone Assay Based on Antimicrobial Peptides Extracted...

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.