The antibacterial properties of the products were assayed against S. mutans, A. naeslundii, and P. gingivalis. Each bacterium was procured from the American Type Culture Collection (Manassas, VA, USA) and frozen until further analysis. First, the frozen bacterial stocks were thawed and grown on brain heart infusion (BHI) medium (Pearlcore®, Eiken Chemical, Co., Ltd., Tokyo, Japan) supplemented with 0.1% antibiotic (0.05% gramicidin D and 0.05% bacitracin, FUJIFILM Wako Pure Chemical Corporation) and 1% sucrose (FUJIFILM Wako Pure Chemical Corporation).
In a 96-well plate, S. mutans (1.1 × 106 CFU/200 µL/well), A. naeslundii (1.0 × 106 CFU/200 µL/well), and P. gingivalis (2.8 × 109 CFU/200 µL/well) were incubated for 24 h at 37 °C under anaerobic conditions in the BHI medium supplemented with the product at various doses: 0 (ctrl), 0.001, 0.01, and 0.1 w/v%. After incubation, the metabolic activity of the bacteria (proportional to the number of living bacteria) was assayed using a microbial viability assay kit-WST (DOJINDO Laboratories, Mashiki, Japan) according to the manufacturer’s instructions, and the absorbance was measured at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA). The pH of each bacterial suspension was measured before and after incubation using a portable pH meter (LAQUA-PH-SE, HORIBA, Ltd., Kyoto, Japan).
In a 96-well plate, S. mutans (1.1 × 106 CFU/200 µL/well), A. naeslundii (1.0 × 106 CFU/200 µL/well), and P. gingivalis (2.8 × 109 CFU/200 µL/well) were incubated for 24 h at 37 °C under anaerobic conditions in the BHI medium supplemented with the product at various doses: 0 (ctrl), 0.001, 0.01, and 0.1 w/v%. After incubation, the metabolic activity of the bacteria (proportional to the number of living bacteria) was assayed using a microbial viability assay kit-WST (DOJINDO Laboratories, Mashiki, Japan) according to the manufacturer’s instructions, and the absorbance was measured at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA). The pH of each bacterial suspension was measured before and after incubation using a portable pH meter (LAQUA-PH-SE, HORIBA, Ltd., Kyoto, Japan).