4.7. Antibacterial Studies

Antibacterial activity of recombinant C–L was assayed by the agar diffusion method with S. aureus ATCC25923 as indicator strain. A dilution of the strain was spread on MH plates, cylinders were placed on the agar surface, and 100 μL of purified recombinant SUMO–C–L (100 μg/mL) and C–L (20 μg/mL) was added to each cylinder. The same volume of PBS was used as negative control. The inhibition zone was measured after incubation overnight at 37 °C. Additionally, the minimal inhibitory concentrations (MICs) of C, L, and C–L were also determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines using S. aureus ATCC 25923, E. coli CVCC245, and L. mono. CVCC1599 as indicator strains. The indicator strains were cultured at 37 °C to logarithmic stage and diluted to the concentration of 1 × 10⁶ cfu/mL with Mueller–Hinton (MH) broth medium, and 180 µL culture was dispensed into per well of 96-well microtiter plate. Peptides were then serially diluted, and dilutions were added into the wells (final volume 200 µL). Each assay was performed in triplicate. After incubation at 37 °C for 12 h, the plate was assessed by measuring the OD₆₀₀. The MIC was defined as the lowest concentration (at which there was no change in optical density) required to prevent the growth of bacteria [2].