S. mutans ATCC 25175 and S. sobrinus ATCC 33478 were used in this study and cultivated by brain heart infusion broth (BHI)(BD bioscience, Sparks, MD, USA). In order to form biofilm, S. mutans and S. sobrinus were cultivated in BHI broth containing 2% sucrose according to the methods of Lee et al. [3].

Two types of EW were used in this study and in order to investigate its antibacterial activity. EW was generated from an electrolyzing distilled water containing 0.05% and 0.15% sodium chloride in an undivided anode chamber and cathode chamber. Also, the platinum electrodes were placed in the chamber horizontally. The pH and oxidation reduction potential (ORP) level of the EW was measured by a pH meter (Thermo Fisher Scientific, Waltham, MA, USA). The free available chlorine species (HClO and ClO-) were determined using N,N-diethyl-p-phenylenediamine (Sigma-Aldrich Co. St. Louis, MO, USA) [20]. The free chlorine, pH and ORP of the EW were analyzed within 10 min after electrolysis.

S. mutans and S. sobrinus were cultivated in BHI broth for 36 h and counted by a bacterial counting chamber (Marienfeld, Lauda-Konigshofen, Germany). The bacterial density was adjusted to 1 × 10⁷ cells/mL with BHI broth. Each bacterial suspensions (1 mL) was harvested by centrifugation at 4,000 × g for 10 min and then washed by phosphate buffered saline (PBS). The bacterial pellets were treated with 1 mL of the EW and commercial gargle solutions (alcohol- or fluoride-containing gargle) for 30 sec or 1 min, and then immediately added to 1 mL of BHI broth. The samples were diluted serially 10 fold to 105, each diluted suspension was spread on an agar plate and incubated at 37℃ until the colonies could be counted.

For biofilm formation, a plastic coverslip (SPL bioscience, Gyoengi-do) was coated with saliva according to the methods of Lee et al. [21]. S. mutans and S. sobrinus were inoculated into BHI broth including 2% sucrose and dispensed on the saliva-coated cover slip in a 12-well polystyrene plate (SPL bioscience). The plates were incubated at 37℃ for 72 h, and the media were changed with fresh BHI broth including 2% sucrose each day. The biofilms were washed twice with PBS to remove planktonic bacteria and treated with the EW or commercial gargle solutions for various durations. The EW and commercial gargle solution were immediately aspirated by suction pump. Each biofilm was stained with 0.5% crystal violet and washed three times with PBS. Absolute alcohol was added to dissolve the crystal violet on the bacteria in biofilm, and the supernatant was transferred into a 96-well polystyrene plate (SPL bioscience). The optical density of the supernatant was measured at a wavelength of 590 nm by a microplate reader (Biotek. Winooski, VT, USA). In order to analyze the viable bacteria in the biofilm, electrolyzed water-treated biofilms were disrupted mechanically, and weak sonication was performed (output 3W) to homogenize the bacteria. The bacterial suspensions were serially diluted from 103 to 106 with BHI broth, and the diluted suspensions (50 μL) were plated on Mitis-salivarius agar (BD bioscience). The plates were incubated at 37℃ for 48 h, and the colony forming units of each bacteria on each plate were counted.

The differences between the control and the samples were analyzed by Krusical-wallis and Mann-Whitney test using SPSS 21 (IBM, Armonk, NY, USA). P-values less than 0.05 were considered statistically significant.

When distilled water in the presence or absence of sodium chloride at the concentration of 0.05% and 0.15% was by an passed electric current through the platinum electrode, the level of pH was neutral, and did not change. However, the concentration of free chlorine was increased in a dose-dependent manner (Table 1). The concentration of free chlorine indicated hypochlorous acid (HOCl) and hypochlorite ion (OCl-).

The antimicrobial activity of the generated EW was compared with two commercial gargle solutions, including an alcohol-containing gargle for adults and a fluoride-containing gargle for children. The EW from the water including sodium chloride showed significant antimicrobial activity against S. mutans and S. sobrinus (Fig. 1). Furthermore, the sodium chloride-containing EW has more antimicrobial activity compared to the gargle solution for children.

Mature biofilm of S. mutans and S. sobrinus was formed and treated with the EW and commercial gargle solutions. Fluoride-containing gargle as a gargle for children did not affect the biofilm of S. mutans and S. sobrinus. However, the EW from the water including sodium chloride and the alcohol-containing gargle significantly disrupted the biofilm (Fig. 2). Furthermore, the count of S. mutans and S. sobrinus in the biofilm was decreased by both the EW and the alcohol-containing gargle (Fig. 3). Interestingly, the EW disrupted more the biofilm and killed more the cariogenic bacteria in the biofilm than the alcohol-containing gargle.