2.6. Time-Kill Assay

To evaluate the effect of RR9 on planktonic oral streptococci, the time-killing assay was used [22]. First, 200 μL of RR9 solution and 800 μL of BHI broth were mixed with 1 mL of bacterial culture (5 × 10⁷ CFU mL⁻¹), yielding 2 mL of the final volume with 100%, 200%, or 400% of the MBC final peptide concentrations. After 0, 0.25, 0.5, 1, 2, 4, 24, and 48 h of incubation at 37 °C, 10 μL of each suspension was pipetted, serially diluted 10 times, and finally transferred to a BHI agar plate for CFU determination. The time–kill kinetic curves were obtained by recording log(CFU mL⁻¹) as a function of incubation time over 6 h.