Determination of Minimum Inhibitory Concentration of Peptides

The minimum inhibitory concentration (MIC) of HPLC purified peptides was evaluated by using microtiter plate dilution assay. The peptide concentration was estimated using BCA protein assay (Thermo Fisher Scientific, United States) method as described by manufacturer. Actively growing test strains (5 × 10⁵ CFU/ml) were inoculated in a 96-well plate with different concentrations of the peptides and plates were incubated for 24–48 h at 37°C (final volume in each well is 200 μl). OD was measured at 600 nm after 24 and 48 h for bacteria and yeast, respectively, using ELISA microplate reader (Thermo, United States). MIC against phytopathogenic fungi was determined using 96-well microtiter plate assay (Kaur et al., 2016). In 96-well microplates, 100 μl of fungal spore suspension (1 × 10⁵ spores/ml) prepared in potato dextrose broth (PDB, Hi-media, India) was mixed with100 μl of fresh PDB containing different concentrations of lipopeptide. Control well contained 100 μl of fungal spore suspension and 100 μl of only medium. The microplates were incubated at 25°C and readings were recorded at 600 nm after 48 h. The lowest concentration showing no growth was considered as MIC. Each test was done in triplicate and optical density (OD₆₀₀) of peptide treated wells were compared with uninoculated wells (blank medium control).