Effect of Kangen and reverse osmosis water on dental plaque, salivary pH and salivary Streptococcus mutans counts: a randomized-controlled trial (A preliminary study)

Our body consists of over 70% water, and all biological functions, including circulation, digestion, absorption and excretion, rely on it. Water is also essential for the blood, the lymphatic system, and for healthy skin and muscles (Goyal et al. 2017). The human body tends to maintain a tightly controlled pH range of approximately 7.35–7.45 in the extracellular fluid, through respiratory excretion of carbon dioxide and renal excretion of a non-carbonic (i.e., non-volatile) acid or base. Everyday metabolism produces acid as non-volatile sulfate from amino-acid catabolism, non-metabolized organic acids, and phosphoric and other acids. The kidney reabsorbs all of the filtered bicarbonate (HCO₃⁻) and generates new HCO₃⁻ in the collecting duct. Under normal steady-state conditions, the net quantity of acid secreted and the consequent renal generation of new HCO₃⁻ equals the rate of metabolic proton generation, thereby preserving pH balance (Mousa 2016).

Dental caries is caused by acids produced by commensal microbes within oral biofilms known as plaque. These acids include organic acids, such as acetic, lactic and propionic acids, which are produced by the metabolism of sugar by oral bacteria such as Streptococcus mutans and Streptococcus sobrinus. The inorganic component of the tooth enamel, i.e., hydroxyapatite, begins to dissolve under acid conditions of pH < 5.5, leading to the breakdown of tooth surface and subsequent cavity formation (Kianoush et al. 2014; Kondo et al. 2022). Dental caries is also regulated by saliva as salivary pH variations influence the pathogenesis of dental caries. Higher salivary pH values reduce the risk of demineralization and vice versa (Rusu et al. 2022; Braine et al. 2023; Giacaman et al. 2023).

Similarly, acid erosion is an increasingly common dental condition, caused by acids such as citric acid, malic acid and tartaric acid, which are all naturally occurring acids found in foods and beverages (Kondo et al. 2022). Various functional water products have recently been investigated, including several types of electrolytically generated hypochlorite water, ozonated water and ozone ultrafine bubble water (Kondo et al. 2022).

Alkaline beverages can be used to quickly neutralize the oral cavity that has been acidified by acidic food; however, few such beverages have been confirmed to be safe to drink. Alkaline water raises the pH of our body, allowing the body to get rid of toxins (Goyal et al. 2017). Tanaka et al. reported that alkaline electrolyzed water produced by electrolysis of purified tap water is effective in improving gastrointestinal symptoms, and confirmed in a large-scale clinical study that alkaline electrolyzed water is suitable for drinking (Pasiga & Akbar 2019).

Kangen water is produced by Enagic's alkaline ionizer and water filtration machines. There is minimal loss of minerals during the purification process. It has a negative oxidation-reduction potential. Molecular hydrogen acts as an efficient antioxidant that diffuses rapidly across cell membranes and reduces free radicals, ultimately suppressing oxidative stress. A pH range of 8.5 and 9.5 is considered perfect for drinking (Enagic, Europe).

Available literature shows studies conducted on the use of alkaline water as a mouthwash for plaque reduction, but there is little information available about the effect of drinking alkaline water on plaque reduction (Pasiga & Akbar 2019; Kondo et al. 2022). Therefore, this preliminary study was conducted with the objective of evaluating and comparing the effect of alkaline Kangen water and RO water on dental plaque, salivary pH and salivary Streptococcus mutans count. The null hypothesis tested was that there was no difference in dental plaque, salivary pH and salivary Streptococcus mutans counts after drinking Kangen RO water.

This randomized control trial was conducted for a period of 14 days on 24 randomly selected participants from a pool of undergraduate dental students. This preliminary study was approved by the Institutional Ethical Committee (Dean/2021/EC2716). The participants were enrolled after explaining the objectives of the study and obtaining written consent. Since this was a preliminary study, 12 participants were taken in each group (Julious 2005).

• Mild to moderate plaque index score (Silness and Loe Plaque Index).

• Decay-missing-filled tooth (DMFT) score <2.

• Normal Body Mass Index (BMI).

• With fixed or removable orthodontic appliances or removable prosthesis.

• Subjects with any salivary gland disease or systemic diseases.

• History of oral prophylaxis at least 3 months prior to study.

• History of antibiotic therapy or anti-inflammatory drug in the subjects within the previous 3 months.

Plaque level was checked using the Silness and Loe Plaque Index (Karimi et al. 2012) by a trained and calibrated examiner. The examiner was trained for plaque assessment at the Dept. of Public Health Dentistry under a professor. Intra-examiner reliability using an intraclass correlation coefficient was assessed on 10 volunteers who were not part of the main study. The value of ICC was 0.84, which indicated almost perfect agreement.

After that, participants were asked to close their mouths and tilt their heads a little downward to get the saliva collected on the floor of the mouth and spit it into a universal container. This process was done for 5 min. The collected saliva was first checked for pH by a pre-calibrated digital pH meter (KERRO pH meter) then the sample was sent to the Multidisciplinary Research Unit (MRU), Department of Microbiology, IMS, BHU for evaluation of Streptococcus mutans count (CFU).

The saliva sample was transferred into an Eppendorf and then centrifuged (TARSON SPINWIN MC-03 Micro Centrifuge) at 10,000 RPM for 10 min. The upper portion of the saliva, free from sediments, was collected and transferred to another Eppendorf and centrifuged again. This process was repeated three times. 100 μL of the saliva was then added to 900 μL of normal saline (pH 7.2) and shaken for 15 s in a vortex. A 100 μL aliquot was obtained after the first dilution and added to a second tube with 900 μL of normal saline. This process was repeated once again, resulting in 1:10, 1:100 and 1:1,000 dilutions of the saliva sample. 100 μL of each of the diluted saliva samples was then plated in BHI agar plates using a micropipette (Grez et al. 2013). All processes were performed under sterile conditions inside a laminar chamber (1300 series A2). The plates were kept in an anaerobic chamber (Thermo Scientific) at CO₂ 5% and 37 °C for 48 h. The plates were checked for translucent colonies. Suspected colonies of Streptococcus mutans were counted using a magnifying glass. The confirmation of the colonies was done by biochemical tests, including oxidase, catalase and fermentation of glucose, fructose, galactose and mannitol. Finally, gram staining was done.

The data were entered and compiled in MS Excel and subjected to statistical analysis using SPSS version 26. The level of significance was set at 5%. Data were subjected to an assessment of normal distribution using the Shapiro–Wilk test, which showed that the data were normally distributed. Intragroup analysis was performed using repeated measures of analysis of variance, followed by the Bonferroni test for adjustment for multiple comparisons. Intergroup analysis was done using the independent samples t-test for comparing between groups. Baseline imbalances, if any, were adjusted using analysis of covariance.

Table 1 shows the results of the repeated measures analysis of variance. In the Kangen water group, plaque score showed a reduction from baseline to 7 days and an increase from 7 to 14 days. There was a significant difference between the follow-ups with a p-value of 0.001. The pH score increased from the baseline value and the highest pH value was seen after 7 days follow-up. Similarly, the CFU count showed a reduction from baseline to 7 days and an increase from 7 to 14 days. Pairwise comparison in Kangen water showed a significant difference between baseline to 7 days and baseline to 14 days in all three outcome measures. In the RO water group, there was a non-significant difference in the change in each outcome variable from baseline to 14 days.

Table 2 shows the comparison of plaque score, pH and CFU count between two groups at follow-up intervals before and after adjusting for baseline covariates. After the 7 days follow-up, the mean plaque score of the RO water group was greater than the Kangen water group, and there was a significant difference (p = 0.033) between the two groups. The results of the plaque score showed a significant difference after adjusting for baseline plaque values in both groups (p = 0.001). After 14 days follow-up, the mean plaque score of the RO water group was higher than the Kangen water group; however, there was no difference between the two groups (p = 0.042). Two groups did not differ in terms of pH at any interval. CFU count in the Kangen water group was less than that of the RO water group after 7 and 14 days follow-up period. But, the CFU count showed a significant difference between the two groups only after adjusting for the baseline CFU score.

Table 3 shows the change in plaque score categories during different follow-ups as per Silness and Loe Plaque Index. In the Kangen water group, at baseline, 7 (58.3%) subjects had good scores of plaque index, after 7 days of follow-up, 11 (91.6%) subjects scored good, and after 14 days follow-up, all the subjects had good scores. While in the RO water group, at baseline, 7 (58.3%) subjects had good scores of plaque index, after 7 days of follow-up, 8 (66.66%) subjects had good scores and it remained the same till the end of the study.

Rinsing the mouth after consuming meals is critical for maintaining oral health. Modern lifestyles require preventive measures that are easy, require less time and effort. Water is an acceptable and easily available vehicle (Mosallam & Mohamed 2019). Therefore, a randomized-controlled trial was conducted to assess the effect of drinking Kangen water and RO water on salivary plaque level, salivary pH and salivary Streptococcus mutans.

In a healthy state, the pH of the human body ranges from 7.35 to 7.45, with the average being 7.40. A slightly basic pH is ideal for different biological processes, one of the most important being oxygenation of the blood (Hopkins et al. 2023). In the oral cavity, the pH is maintained nearly between 6.7 and 7.3 by the buffering action of saliva. But when the oral cavity's pH decreases below 5.5, demineralization of the enamel surface begins. The lower the pH of ingested substances like food or water, the higher the dental erosion occurs. A study conducted by Sato et al. (2021) showed that alkaline ionized water is useful in preventing dental erosion caused by acidic beverages.

Kangen water is, at its most basic level, hydrogen water. Regular tap water is passed through a machine that filters out impurities and then separates the hydrogen and oxygen in the water (electrolysis). This results in an ‘alkaline, antioxidant, mineral-rich and healthy drinking water’ (Bass 2020). Kangen water ranges from 2.5 (strongly acidic) water to 11.5 (strongly alkaline) water, with a pH range of 8.5–9.5 indicated for drinking (Bass 2020). This electrolytically reduced, hydrogen-rich water works to restore the body to a more alkaline state, which is optimal for good health. The four major potential benefits are increased hydration, balancing the body's pH, increased blood oxygenation and neutralizing free radicals.

In the present study, the intervention group which received Kangen water showed a significant decrease in plaque score and CFUs of Streptococcus mutans after 1 week of intervention, and there was a significant increase in the pH of saliva after 1 week of the intervention. The decrease in plaque score from baseline to 1-week follow-up was 32.4%. Similar results were seen in a study done by Pasiga & Akbar (2019), in which there were four treatment groups with different alkaline ionized waters of pH range 7, 9, 11.5 and a control group. All the participants gargled with the respective water for 7 days. The highest percentage of dental plaque reduction before and after 7 days of treatment for the alkaline ionized water group with pH 9 was 26.6%. The pH change in the present study from baseline to 1-week follow-up was 2.9%, while in the study done by Pasiga & Akbar (2019), the pH change was 15%. The mean of the pH after treatment in both studies remained above 7, consequently inhibiting bacterial growth (Pasiga & Akbar 2019).

The mean change of Streptococcus mutans count followed by drinking Kangen water from baseline to 1 week was 33.8%. In a study done by Goyal et al. (2017), in which the participants were given magnetized water as a mouthwash for 2 weeks, the mean change from baseline value to 1 week was 28.9%. Water with the alkaline property always has oxygen inside, which provides energy to the cells and prevents the proliferation of anaerobic bacteria. Since Streptococcus mutans is an anaerobic bacterium, therefore, the bacterial growth was hampered and the count decreased after the intervention with Kangen water (Goyal et al. 2017). After the intervention was stopped, there was a slight increase in the plaque score and CFU, while the pH score slightly increased, but the differences were non-significant. This might be due to the residual effect of the Kangen water.

The null hypothesis was rejected, and an obvious finding from this study was that there was a statistically significant decrease in plaque level and Streptococcus mutans count after drinking Kangen water for 1 week.

This study suggests that using alkaline Kangen water for drinking purposes on a regular basis can be considered a practical recommendation for dental caries prevention. The strength of the study is that this is the only study that compared Kangen water with RO water for plaque, pH and bacterial count when used for drinking. The generalization of the result is debatable since the study subjects were dental students. Therefore, further studies are recommended to be conducted on larger samples with wider geographical representations.

It can be concluded that regular drinking of alkaline Kangen water with pH 9 effectively reduces plaque and salivary Streptococcus mutans count compared to drinking RO water, which can be useful for maintaining general oral health. However, larger studies with longer time frames are advisable before validating the results.

The Streptococcus mutans count was conducted at the Multidisciplinary Research Unit (MRU), Department of Microbiology, IMS, BHU, under the guidance of Dr Gopal Nath. The authors would like to thank Dr Alka Shukla for her assistance during the laboratory work.

All the authors contributed to the study conception and design. The first draft of the manuscript was written by A. K. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

The author(s) received no financial support for the research, authorship and/or publication of this article.

Data cannot be made publicly available; readers should contact the corresponding author for details.

The authors declare there is no conflict.