4.6. Antimicrobial and Cytotoxicity Assays

Peptides were tested for antimicrobial activity against the gram-negative species Escherichia coli (ATCC 25922) and the gram-positive species Staphylococcus aureus (ATCC 25923) and for cytotoxic activity towards intestinal epithelial cells using the human cancerous colonic cells (Caco-2) monolayer model. All assays were performed in triplicate. Antimicrobial activity against bacteria was measured by assessing the lowest peptide concentration at which no growth was detected in a series of twofold dilutions (minimum inhibitory concentration, MIC). Bacterial cultures (5 × 10⁵ colony forming units per ml) were prepared in Müller-Hinton broth and transferred to serial dilutions of peptides ranging from 512 µM down to 1 µM in 96-well polypropylene plates. After incubation at 37 °C for 18 h, growth of the cultures was visually checked.

The cytotoxic activity of peptides against Caco-2 cells was measured by quantifying the fraction of surviving cells after peptide treatment using a neutral red assay. Caco-2 cells were seeded at an initial concentration of 5 × 10⁵ cells/mL in Hank’s balanced salt solution medium with Mg²⁺ and Ca²⁺ (HBSS+) in polystyrene flat-bottom 96-well plates and incubated for 21 days (at 37 °C, 5% CO₂ and 95% humidity) to grow morphologically differentiated monolayers of interconnected cells. Cytotoxic activity of peptides was verified by exposing the resulting monolayers for 15 min to a range of peptide concentrations between 512 and 0.5 µM. Five wells with cell medium only (HBSS+) and five wells containing 1% Triton-X solution were used as negative and positive controls, respectively. After 15 min, peptide solutions were removed and 200 µL neutral red solution (33 µg/mL) was added to each well. After two hours of incubation at 37 °C, the dye solution was removed and cells were rinsed three times with 200 µL HBSS+ solution. Wells were then incubated with 200 µL glacial acetic acid solution (glacial acetic acid: ethanol: water = 1:50:49) for 10 min in darkness. Finally, 150 µL medium from each well was transferred to an optically clear ELISA well-plate, after which the optical density (OD) was measured at 550 nm using a multiskan microplate photometer/ELISA reader (Thermo Scientific, Waltham, MA, USA). The cytotoxic activity for each peptide concentration, expressed as a percentage, was calculated as 100 × (OD_obs − OD₀)/(OD₁₀₀ − OD₀), where OD_obs is the OD measured for the peptide concentration, OD₀ is the average OD for the negative controls (0% cytotoxicity) and OD₁₀₀ is the average OD after treatment with Triton-X (100% cytotoxicity).