Introduction

Hydrogen-rich water (HW), characterized by an abundance of dissolved hydrogen gas, has been reported to have various effects on human organs and tissues, including the skin, hair, and mucosa [1-3]. These effects are presumed to be due to the antioxidative effects of the hydrogen molecules in HW, as oxidative stress is a pathological factor in aging and many diseases, including metabolic syndrome, inflammatory disorders, and cancer [4-6]. Recently, Kim et al. [7] reported inhibitory effects of HW on streptococcal biofilms. Their study also showed that HW significantly decreased the gene expression of glucosyltransferases and glucan-binding proteins in Streptococcus mutans. The inhibitory effects of HW on streptococcal biofilm formation may be important for the prevention of tooth caries.

To effectively prevent tooth caries, the following points related to the cariogenicity of the oral microbe S. mutans should be considered: (1) the formation of cohesive-adherent plaques, (2) the generation of an acidic environment, and (3) the number of acid-base-producing bacteria in addition to S. mutans. In the early stages of caries formation, controlling biofilm formation and acidification initiated by S. mutans is more effective in preventing caries than eliminating whole cariogenic bacteria with respect to the preservation of normal bacterial flora. Thus, the inhibitory effects of HW on streptococcal biofilms are useful for daily mouthwash [7]. HW contains a high concentration of molecular hydrogen (>1 ppm) and is commonly generated by compressing hydrogen into drinkable water. HW is generally sold in aluminum-coated cans or plastic containers [8]. However, hydrogen gas can be generated at the cathode during water electrolysis [9]. With the development of new technologies, it has become possible to generate hydrogen-rich electrolyzed water (HEW), in which >1 ppm of hydrogen molecules is generated from tap water (TW) at home using an electrolysis device [8]. HW and HEW exhibit different electrochemical characteristics depending on the manufacturing method used. For example, the hydrogen or oxygen concentration, pH, chloride ion content, and accessory ingredients of electrolyzed water (EW) vary according to the parameters related to the electrolyzing chamber, electrodes, and electrolyte [10]. A well-known benefit of EW is its antimicrobial effects against bacteria, viruses, spores, and fungi [10]. Oxidative EW has also shown various antibacterial effects against oral bacteria without environmental toxicity [11,12]. In particular, weakly acidic EW causes no irritation to the hands, skin, or mucous membranes; therefore, clinical applications have been assessed [13]. However, to the best of our knowledge, studies on the antibacterial and antibiofilm effects of HEW, characterized by neutral pH and high hydrogen concentration, have not been conducted. Considering the inhibitory effect of HW on streptococcal biofilms, there is a need for studies on HEW, as it can be easily produced at home and is not toxic to the human body or environment [14]. Therefore, we tested the antibacterial and antibiofilm effects of HEW against S. mutans both in vitro and in vivo. In an in vivo trial, we examined the effect of HEW on the maturity of tooth plaques using quantitative light-induced fluorescence (QLF) imaging in which porphyrin could be detected [15]. Porphyrin is produced by some cariogenic oral microbes in mature plaques and exhibits red fluorescence that is visible under violet light in the range of 400 nm to 420 nm. Because an increase in plaque maturation represents cariogenicity of the oral environment, QLF images are useful for measuring the effectiveness of HEW as a mouthwash.

Methods

Ethics statement: The study protocol was approved by the Institutional Review Board (IRB) of Kyungpook National University (IRB No: KNU 2019-020). All participants were recruited, and informed consent was obtained after explaining the study protocol.

1. Bacterial culture

S. mutans (KCOM 1054) was obtained from the Korean Collection for Oral Microbiology (KCOM, Korea), maintained in brain heart infusion (BHI; Difco Laboratories, Sparks, MD, USA) medium, and cultured under aerobic conditions (5% CO₂, 37°C).

2. Preparation of hydrogen-rich water

The HEW was prepared by subjecting TW to an electrolytic water generator (Natural Gargle Plus, EBIOMW-100, Ebioteco, Korea) for 5 minutes. The concentration of dissolved hydrogen and pH of the HEW were 1.2 to 1.3 ppm and 7.1, respectively.

3. Exposure of Streptococcus mutans to hydrogen-rich water

S. mutans was inoculated into BHI broth and cultured for 6 hours until the exponential phase of bacterial growth was obtained (optical density at 600 nm, 0.2; 10⁷–10⁸ cells/mL). The bacterial broth was centrifuged at 7,500 revolutions/min (rpm) for 15 minutes, and the resulting bacterial pellets were washed three times in phosphate-buffered saline (PBS). The pellets were then exposed to 5 mL of HEW or TW for 1 minute to mimic typical clinical usage conditions, in which mouthwashes are used for short durations (approximately 1–2 minutes). Immediately after exposure, 45 mL of PBS was added, and the pellets were centrifuged at 7,500 rpm for 15 minutes. After discarding the supernatant, 50 mL BHI-0.3% sucrose medium was added for biofilm formation only.

4. Bacterial growth and biofilm formation: in vitro tests

After exposure to HEW or TW, the suspended bacterial pellets were serially diluted in PBS at dilutions of 10^–1 to 10^–7. The diluted suspensions (150 μL) were spread on Mitis Salivarius Agar-Bacitracin medium and cultured for 48 hours under aerobic conditions (5% CO₂, 37°C) to determine counting colony-forming units (CFUs). To evaluate the effect of HEW on bacterial biofilm formation, 2 mL of the bacterial suspension that had been exposed to HEW or TW was cultured in 6-well polystyrene plates coated with artificial saliva (Xerova solution, Kolmar Korea, Sejong, Korea) for 24 hours. After gently discarding the supernatants, the wells were washed three times with PBS, and the residual biofilm was stained for 30 minutes with 0.2% crystal violet dissolved in 10% ethanol. Finally, the stained crystal violet was extracted with an acidic solvent, and the absorbance was measured at 590 nm. All experiments were repeated three times.

5. Gene expression of gtfB and gtfC in Streptococcus mutans

After exposure to HEW or TW, 8 mL of the suspended bacterial pellet was cultured for 8 hours under aerobic conditions (5% CO₂, 37°C). After the bacterial pellets were washed with PBS, total RNA was extracted using QIAzol solution (QIAGEN, Germantown, MD, USA). Finally, complementary DNA was synthesized from 1 µg of isolated RNA using an Omniscript RT kit (QIAGEN) and amplified by real-time polymerase chain reaction on an ABI Prism 7500 thermocycler (Applied Biosystems, Foster City, CA, USA), according to the protocol described in a previous study [7]. The primers for 16S ribosomal RNA (rRNA), gtfB, and gtfC were described in a previous study [16]. Differences in gene expression levels were calculated using the delta-delta threshold cycle method and normalized to those determined for 16S rRNA according to a previous study [17]. All experiments were repeated twice.

6. Quantification of salivary streptococci and plaque formation after oral rinsing with hydrogen-rich electrolyzed water or tap water: in vivo tests

All participants were healthy and aged between 20 and 23 years. Those who had orthodontic appliances on their teeth, untreated caries, extensive calculus, or had taken antibiotics within the previous 2 weeks were excluded from the study. A single-blind randomized crossover trial was designed using two groups to which participants were randomly assigned, and the order of the test solutions was HEW-TW or TW-HEW.

All participants were instructed to gargle with 30 mL of the test solution for 60 seconds twice daily for 2 weeks. A washout period of 1 week was included between the gargling periods of each test solution. Research assistants contacted the participants once daily to ensure complete adherence with the study protocol during the gargling period. To gargle with HEW, the patients received an electrolytic water generator and were instructed on its operation. All participants received the same electrolytic water generator model to ensure the same experimental conditions. In addition, the generators were installed at each participant’s residence and detailed user instructions were provided to standardize their use. For example, details about the generator operation time and single-batch yield of HW were provided.

Stimulated saliva was collected, and QLF images were captured of the buccal surface of the left upper and lower second molars using a Qraypen C (AIOBIO, Seoul, Korea) on the final day of each gargling period. A CRT bacteria kit (Ivoclar Vivadent AG, Schaan, Liechtenstein) was used to examine the number of S. mutans in the saliva; this value was categorized into four levels according to the manufacturer’s instructions (bacterial count: level 0, <10⁴ CFU/mL; level 1, ≥10⁴ and <10⁵ CFU/mL; level 2, ≥10⁵ and <10⁶ CFU/mL; and level 3, ≥10⁶ CFU/mL). To assess the maturity of tooth plaques, the average red/green fluorescence (R/G) ratio of the QLF images of the buccal surface of the two molars was calculated as a representative value for each participant. The R/G ratio was measured using ImageJ software [18].

7. Statistical analysis

Statistical analyses were performed using IBM SPSS ver. 23.0 for Windows (IBM Corp., Armonk, NY, USA). Nonparametric tests were used to analyze the differences in CFU counts, biofilm formation, and messenger RNA (mRNA) expression of genes in vitro among the test solutions. For differences between pairs of data on S. mutans from the CRT bacteria kit and tooth plaque assessment of QLF images in vivo, a linear mixed-effects model was used, with a p-value <0.05 set as the significance level.

Results

1. Effect of hydrogen-rich electrolyzed water on in vitro bacterial growth and biofilm formation

After exposure of bacterial pellets to HEW or TW for 1 minute, the CFU counts were 8.8±1.4×10⁷ and 6.5±1.4×10⁷, respectively, which were not significantly different (Fig. 1A). However, the absorbance at 590 nm from the crystal violet-stained biofilms in the HEW and TW groups was 0.19±0.11 and 0.80±0.07, respectively (Fig. 1B), indicating that bacteria exposed to HEW had significantly decreased biofilm formation (p<0.001).

2. Effect of hydrogen-rich electrolyzed water on gtfB and gtfC gene expression

After exposing the bacterial pellets to HEW or TW for 1 minute, the suspended bacterial pellets were cultured for 8 hours. The normalized fold induction values of gtfB mRNA in the HEW and TW groups were 0.65±0.09 and 1.01±0.16, respectively, indicating significant downregulation in the HEW group (p<0.05) (Fig. 2) [19]. For the gtfC gene, the normalized mRNA fold induction showed a significant difference between the HEW and TW groups, at 0.63±0.04 and 1.00±0.09 (p<0.01), respectively (Fig. 2).

3. Effect of oral rinse with hydrogen-rich electrolyzed water on salivary streptococcus counts and dental plaque formation

None of the participants dropped out or complained of any side effects from gargling with HEW. After gargling HEW or TW twice per day for 2 weeks, the mean values for each group as calculated with the CRT bacteria kit using stimulated saliva were 0.8±0.8 and 1.3±1.0, respectively (Fig. 3A). The CRT bacteria kit level for HEW gargling was significantly lower than that for TW gargling (p<0.01) (Fig. 3B). No significant differences were observed according to the period or sequence of HEW and TW gargling. This indicates that HEW gargling significantly reduced salivary S. mutans counts more than TW gargling did. The average R/G ratios of the HEW and TW groups were 1.34±0.24 and 1.58±038, respectively (Fig. 3C). When comparing each participant’s R/G ratio following HEW and TW gargling, the R/G ratio for HEW was significantly lower than that for TW (p<0.01) (Fig. 3D). Furthermore, there was no significant difference according to the sequence of HEW and TW gargling. However, the R/G ratio was significantly lower for the second period than for the first period (p<0.01). A low R/G ratio indicates low red fluorescence in the QLF images and minimal plaque maturation [15]. Therefore, gargling with HEW significantly inhibited the growth of cariogenic bacteria in plaques compared to gargling with TW.

Discussion

Mechanical tooth brushing is the primary method used to prevent tooth caries; however, auxiliary mechanisms such as using dental floss, interdental toothbrushes, and mouthwash are also useful for rigorous plaque removal [20,21]. Daily mouth washing is particularly helpful for children, older adults, and those who are disabled because they are vulnerable to oral health problems due to difficulties in performing elaborate hand movements [22]. However, many mouthwashes focus on bactericidal activity to prevent caries, which may affect the balance of the bacterial flora required to maintain a healthy oral environment [23]. Therefore, it is recommended that chlorhexidine be used continuously for only 2 weeks [24]. Additionally, alcohol-based mouthwashes can enhance the penetration of certain ingredients into soft tissues, which may cause side effects with prolonged use [25]. Although some mouthwashes, such as those containing essential oils, are regarded as safe, there is still a need for a promising mouthwash with high cost-effectiveness, no possibility of latent side effects, and no environmental impact [26,27]. In addition, given the benefits of preserving the normal oral microflora, mouthwash strategies can change from bacterial removal to dental biofilm reduction [28]. Based on our results, HEW is proposed as a promising mouthwash.

In this study, HEW inhibited streptococcal biofilm formation without exerting antibacterial activity, similar to the results of a previous study by Kim et al [7]. Interestingly, the mRNA expression levels of gtfB and gtfC were lower in HEW-exposed bacteria than in TW-exposed bacteria. These genes are involved in the synthesis of glucosyltransferases, enzymes that synthesize insoluble extracellular polysaccharides in S. mutans [29]. TW-based EW with neutral pH has been reported to have a low antibacterial effect because of the low concentration of chloride ions in TW, which agrees with our results [30]. An in vivo mouthwash trial showed significant reductions in salivary S. mutans and tooth plaque when participants rinsed their mouths with HEW for 2 weeks. The decrease in salivary S. mutans indicated that continuous biofilm growth could be reduced by inhibition of the initial dental biofilm. In addition, a decrease in plaque maturity represents the possibility of inhibiting dental caries owing to a decrease in cariogenic bacteria [31].

Despite the different electrochemical characteristics of HW and HEW, the most common factor contributing to the inhibition of biofilm formation is the presence of hydrogen molecules. Although the molecular mechanism underlying the inhibitory effect on biofilm formation is unclear, some studies have suggested that hydrogen molecules affect gene expression, including nuclear factor-kappa B–regulated gene expression in rodents and Ca²⁺-dependent gene expression in humans [32,33]. In addition, the possibility of using hydrogen as an antitumor agent through the regulation of cancer-related gene expression has been suggested [34]. In the present study, hydrogen molecules in HEW reduced the attachment of S. mutans to tooth surfaces by regulating the expression of genes encoding glucosyltransferases, which are important for the growth of cariogenic biofilms [35].

In summary, the reduction in streptococcal biofilms and salivary S. mutans and plaque maturation without antibacterial activity by HEW is considered a good strategy for caries prevention. HEW has a neutral pH and is easily produced from TW using an electrolysis device. Therefore, it is suitable for daily use regardless of an individual’s salivary conditions. According to a previous study, no adverse effects, such as mutagenicity, genotoxicity, or oral toxicity, were found in cells and rats exposed to HEW (dissolved hydrogen, 0.90–1.14 ppm) [19]. In addition, HEW reverts to normal water over time, making it environmentally safe. In conclusion, HEW can be used as a daily mouthwash for men and women of all ages, with many advantages, including its colorless, tasteless, and odorless characteristics, cost-effectiveness, and the other advantages described above.

Although the key factor influencing the biological mechanism of HEW is assumed to be the hydrogen molecule, the minimum concentration of hydrogen required to exert an antibiofilm effect is not known. In the current in vivo experiment, we instructed the participants to gargle as soon as possible after generating HEW to avoid losing volatile hydrogen molecules. Therefore, we assumed the hydrogen concentration was almost the same as that quoted by the HEW manufacturer (1.2–1.3 ppm).

However, this study had some limitations. First, this was a preliminary study with a small sample size. Therefore, future studies should include diverse populations encompassing various age groups, sexes, and oral health statuses to enhance the external validity of the findings. Second, while the current study employed only TW as a control to assess the antibiofilm effects of HEW, future studies should incorporate comparisons with commercially available mouthwashes, such as chlorhexidine and essential oil-based products. This multi-arm study design will enable a more comprehensive evaluation of HEW’s relative efficacy and mechanism of action. Third, the cost-effectiveness of at-home HEW production makes it accessible for daily oral care applications. These unique characteristics indicate that HEW may offer improved biofilm inhibition and enhanced oral hygiene benefits compared to conventional HW. However, follow-up studies are necessary to precisely evaluate and compare the efficacies of HEW and HW as mouthwashes.

Therefore, additional studies are needed to determine criteria for HEW to be used as a mouthwash, such as the minimum required concentration of hydrogen molecules. Additionally, large-scale clinical studies on the long-term effects of HEW as mouthwash should be conducted.

Article information

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Funding

None.

Author contributions

Conceptualization: HYY, JHK, SHH, EKK; Formal analysis: MJC, SHH, EKK; Project administration: EKK, SHH; Investigation: HYY, JHK, MJC; Supervision: SHH, EKK; Writing-original draft: HYY, JHK; Writing-review & editing: HYY, JHK, EKK, SHH.

Fig. 1.

Effect of hydrogen-rich electrolyzed water (HEW) on bacterial growth and biofilm formation. Streptococcus mutans is exposed to HEW or tap water (TW) for 1 minute. (A) The bacterial broth is serially diluted in brain heart infusion-0.3% sucrose and smeared on Mitis Salivarius Agar-Bacitracin-coated plates. The colony-forming units (CFUs) are then counted after 48 hours. (B) Each bacterial suspension is inoculated in polystyrene plates coated with artificial saliva and cultured for 24 hours. To quantify biofilm formation, the attached bacteria are stained with 0.2% crystal violet for 30 minutes (upper image). After washing five times, crystal violet is extracted with an acid solvent, and absorbance is measured at 590 nm. The absorbance of test samples is normalized to that of TW (^***p<0.001). All experiments are repeated thrice, and bars represent the standard error.

Fig. 2.

Effect of hydrogen-rich electrolyzed water (HEW) on the expression of gtfB and gtfC. After Streptococcus mutans is exposed to HEW or tap water (TW) for 1 minute, the bacteria are cultured in brain heart infusion-0.3% sucrose broth for 8 hours. The expression of gtfB and gtfC in S. mutans was measured using quantitative polymerase chain reaction (^*p<0.05, ^**p<0.01). All experiments are repeated three times, and bars represent the standard error.

Fig. 3.

Effect of hydrogen-rich electrolyzed water (HEW) on bacterial growth and plaque formation in vivo. A total of 24 participants had gargled with HEW or tap water (TW) twice daily for 2 weeks, with a 1-week wash-out period. To evaluate Streptococcus mutans and tooth plaques, the CRT bacteria kit and quantitative light-induced fluorescence (QLF) images of the two first molars (#27, #37) are used. The CRT bacteria kit is used to selectively culture S. mutans. QLF images indicate mature tooth plaques as the red/green fluorescence (R/G) ratio. (A) Comparison of the bacterial colonies cultured using the CRT bacteria kit between the two groups. The counts of S. mutans colonies assessed with the CRT bacteria kit are categorized into four levels (bacterial count: level 0, <10⁴ CFU/mL; level 1, ≥10⁴ and <10⁵ CFU/mL; level 2, ≥10⁵ and <10⁶ CFU/mL; and level 3, ≥10⁶ CFU/mL). (B) The levels of bacterial colonies determined using the CRT bacteria kit of each participant. (C) Comparisons of the R/G ratio in QLF images between groups. (D) R/G ratio in QLF image of each participant (^**p<0.01). Standard errors are represented as bars. CRT, Caries Risk Test (Ivoclar Vivadent AG, Schaan, Liechtenstein); CFU, colony-forming units.

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Figure & Data

References

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