Minimal inhibitory concentration determination

The 50 AMPs were tested for antimicrobial activity using the broth microdilution method¹²⁹. MIC values were considered as the concentration of the peptides that killed 100% of cells after 24 h of incubation at 37°C. First, peptides diluted in water were added to untreated flat-bottom polystyrene microtiter 96-well plates in two-fold dilutions ranging from 64 to 1 μmol·L⁻¹, and then peptides were exposed to an inoculum of 2·10⁶ cells in LB or BHI broth, for pathogens and gut commensals, respectively. After the incubation time, the absorbance of each well representing each of the conditions was analyzed using a spectrophotometer at 600 nm. The assays were conducted in three biological replicates to ensure statistical reliability.