On-demand caries prevention: on-site generated slightly acidic electrolyzed water as a potent and biocompatible anti- Streptococcus mutans agent

Abstract

Background

Dental caries remains a prevalent public health disorder dominated by Streptococcus mutans. Novel biocompatible antibacterial agents are urgently required for caries control. Slightly acidic electrolyzed water (SAEW), rich in endogenous hypochlorous acid, boasts broad bactericidal effects and minimal cellular toxicity, yet rapid degradation during storage limits its reproducibility. Thus, this study adopted freshly prepared SAEW to systematically assess its anti-cariogenic performance for translational reference.

Methods

In vitro, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SAEW against planktonic S. mutans were determined, and bacterial growth was monitored. Morphology was examined by scanning electron microscopy (SEM). Biofilm structure, viability, and membrane integrity were assessed by confocal laser scanning microscopy (CLSM) with live/dead staining. The cytotoxic effects of SAEW on human gingival fibroblasts (HGFs) were evaluated using the CCK-8 assay. 25 Sprague-Dawley rats were randomized into five groups: blank control, caries model, 0.12% chlorhexidine, high-concentration SAEW, and low-concentration SAEW groups. All non-blank rats received cariogenic induction and daily topical intervention. Salivary streptococcal loads were quantified, and mandibular caries severity was graded via Keyes scoring combined with radiography.

Results

Freshly prepared SAEW exhibited definitive antibacterial activity against S. mutans Its MIC and MBC against planktonic bacteria were 6.25 ppm and 12.5 ppm, respectively. SAEW compromised bacterial cell membrane integrity (activity reduced to 94.5 ± 0.8% in the high-concentration group). At its effective antibacterial concentrations, SAEW showed no significant cytotoxicity to human oral cells (Cell viability exceeded 80%). In a rat caries model, SAEW significantly reduced oral streptococcal levels and mitigated the severity of carious lesions (caries scores at superficial, moderate, and deep dentinal levels were 10.2 ± 1.30, 6.60 ± 0.89, and 1.00 ± 0.71 in the high-concentration group; all P < 0.05). No obvious histopathological alterations or systemic toxicity were induced by SAEW.

Conclusions

Freshly prepared SAEW eliminates planktonic S. mutans primarily by disrupting cell membranes, with favorable cytocompatibility at working concentrations. It restrains cariogenic colonization and relieves caries progression in rodent models, marking a promising eco-friendly candidate for clinical caries prevention.

Background

Dental caries is a highly prevalent chronic infectious disease worldwide, posing a significant public health challenge and markedly affecting patients’ quality of life [1–3]. The pathogenesis of caries is fundamentally rooted in the ecological dysbiosis of the oral biofilm [4–6]. Under physiological conditions, a dynamic balance exists between the complex oral microbial community and the host, which collaboratively maintains local immune homeostasis and mucosal barrier integrity. However, when this equilibrium is disrupted by host factors, environmental changes, or microbial succession, specific pathogens or opportunistic pathogens, such as Streptococcus mutans, can over proliferate and form highly structured biofilms, thereby driving the initiation and progression of dental caries [7–10].

Topical fluoride application, primarily through remineralization, remains the cornerstone of caries prevention. However, its efficacy can be insufficient for high-risk populations or active carious lesions, necessitating adjunctive ecological modulation strategies in clinical practice [11, 12]. Chlorhexidine (CHX), a broad-spectrum antimicrobial agent, has long been regarded as the gold standard chemotherapeutic agent [13, 14]. Its cationic biguanide structure effectively disrupts microbial cell membranes and provides sustained antibacterial action through persistent adsorption onto oral hard and soft tissues. Nevertheless, its long-term use is associated with several limitations, including tooth staining, taste disturbance, potential cytotoxicity to oral mucosal cells, and the risk of inducing microbial adaptive tolerance, which collectively constrain its long-term safety profile [15–19].

Consequently, there is a pressing clinical need for novel agents that combine potent anti-caries efficacy with favorable biosafety. Slightly acidic electrolyzed water (SAEW) presents a promising alternative in this context. SAEW is produced by the electrolysis of dilute hydrochloric acid and sodium chloride solution. Its key active component, hypochlorous acid (HClO), is an endogenous antimicrobial effector molecule generated by activated neutrophils as part of the innate immune response [20–24]. HClO exerts its effect by nonspecifically damaging fundamental microbial structures via potent oxidation, and resistance development against this physicochemical mode of action is uncommon [25, 26]. As an exogenous agent, SAEW has demonstrated favorable efficacy and tissue compatibility in wound disinfection and the control of skin and ocular infections [27–29]. Its near-neutral pH, low corrosiveness, and absence of harmful residues post-application also contribute to its use in medical device disinfection, food safety, and agriculture [30].

Despite growing research on SAEW applications in other medical fields, a systematic, high-evidence-level evaluation of its role in caries management remains significantly lacking. Several critical questions remain unanswered: First, what is the comparative antibacterial efficacy of SAEW versus chlorhexidine (the clinical gold standard) against S. mutans? Second, and central to its safe use in the daily oral environment, what is its cytotoxicity towards key oral cells (gingival fibroblasts cells)? Finally, and most critical for translation, can topical application of HOCl effectively suppress the initiation and progression of dental caries on tooth surfaces in a validated in vivo animal model, and how does its overall anticaries efficacy compare with that of CHX? Currently, there is a paucity of integrated, controlled studies combining in vitro biofilm models, cytocompatibility assessments, and in vivo efficacy validation to directly address these questions.

To address these knowledge gaps, this study aims to conduct a systematic, head-to-head comparative evaluation of an electrolytically generated SAEW solution versus CHX. We designed a three-tiered, progressive experimental framework: (1) at the in vitro level, to quantitatively compare the antimicrobial efficacy of both agents against S. mutans; (2) at the biosafety level, to systematically evaluate and compare their cytotoxicity towards human gingival fibroblasts cells; (3) at the in vivo efficacy level, to directly compare their ultimate effectiveness in preventing smooth-surface and sulcal caries in a validated rat caries model through daily topical application.

The novelty of this work lies in its interdisciplinary, integrated perspective—merging microbiology, material biocompatibility assessment, and experimental pathology to provide, for the first time, a complete evidence chain from the molecular and cellular to the whole-animal level for HOCl’s application in caries management. Through this rigorous controlled study, we aim to provide crucial experimental evidence for optimizing caries management protocols and establishing a more favorable balance between high efficacy and safety.

Materials and methods

Materials and equipment

Dilute acetic acid solution (Harbin, China, Ruibiao Tech Co., Ltd.); Sodium thiosulfate standard solution (Guangdong, China, Xinyueyuan Tech Co., Ltd.); Potassium iodide standard solution (Guangdong, China, Xinyueyuan Tech Co., Ltd.); Soluble starch solution (Guangdong, China, Xinyueyuan Tech Co., Ltd.); Murexide (Shanghai, China, Yuanye Bio-Technology Co., Ltd.).Cell Counting Kit-8 (CCK-8) (Shanghai, China, Beyotime Biotechnology Co., Ltd.); Live/Dead Bacterial Viability Kit (Thermo Fisher Scientific, USA).Brain heart infusion agar (Qingdao, China, Hope Bio-Technology Co., Ltd.); Brain heart infusion broth (Qingdao, China, Hope Bio-Technology Co., Ltd.); Fetal bovine serum (Beijing, China, Thermo Fisher Scientific Co., Ltd.); Dulbecco’s Modified Eagle Medium (DMEM) (Beijing, China, Thermo Fisher Scientific Co., Ltd.).

SAEW generator and SAEW electrolyte (Hefei, China, Qiaoshi Health Technology Co., Ltd.); Optical microscope (Olympus, Japan); Laser scanning confocal microscope (Zeiss, Germany); Scanning electron microscope (Hitachi, Japan).

Preparation of standardized SAEW solution

Materials used in this study included an SAEW generator (integrated in the slightly acidic electrolyzed water bionic bactericidal cup, Hefei, China, Hefei Qiaoshi Health Technology Co., Ltd.) with available chlorine as the main component. The standard HOCl solution was prepared through two key steps: determination of core physicochemical properties (available chlorine concentration and pH value), with all operations conducted in a specific laboratory environment (temperature: 25 ± 1℃, relative humidity: 45%-65%) to ensure the accuracy of measurement results.

Available chlorine (ACC) determination

The available chlorine content (ACC) was determined via an optimized iodometric titration method, based on the Chinese National Standard GB/T 26373 − 2018. Specifically, a dilute acetic acid solution (1 mol/L, Harbin, China, Ruibiao Technology Co., Ltd.) was employed as the acidifying agent to mitigate the risk of chlorine gas generation associated with the use of strong acids. The required iodide ions were supplied by a 10% potassium iodide (KI) aqueous solution (KI purchased from Guangzhou, China, Xinchengyuan Technology Co., Ltd.). The titration was performed using a 0.01 mol/L sodium thiosulfate standard solution (Guangzhou, China, Xinchengyuan Technology Co., Ltd.), with a soluble starch solution (Guangzhou, China, Xinchengyuan Technology Co., Ltd.) as the indicator for endpoint detection. This optimized protocol, particularly in the choice of acid medium, ensures both operational safety and measurement accuracy.

pH value determination

The pH of the solutions was determined using a pH meter method. Each sample was first subjected to a preliminary test with universal pH test paper (pH range: 1–14, Shanghai, China, Sansaisi Reagent Co., Ltd.). Precise measurement was then performed using a pH meter (model: EB12-03). The instrument was calibrated with two buffer solutions: potassium hydrogen phthalate buffer (pH = 4.00, Tianjin, China, Huasheng Chemical Reagent Co., Ltd.) followed by a mixed phosphate buffer (pH = 6.86 ± 0.02, Guangzhou, China, Xinchengyuan Technology Co., Ltd.). The calibrated electrode was immersed in the sample, and the pH value was recorded after the reading stabilized. All measurements were conducted with two independent replicates, and the final pH was reported as the arithmetic mean.

In summary, both ACC and pH determination were independently repeated 3 times. Results were expressed as “X ± S” to ensure reliability. In this study, slightly acidic electrolyzed water (SAEW) was freshly prepared on-site. The solution was stored in sealed containers away from light and used within 30 min after preparation. All subsequent experimental operations were completed within the 30-minute validity period after preparation, effectively preventing changes in its physicochemical properties caused by prolonged storage. This standardized measurement protocol established a consistent and reliable physicochemical foundation for the follow-up animal experiments.

Antibacterial experiment of SAEW

This section focuses on evaluating the antibacterial efficacy of SAEW solution and 0.12% CHX (as positive control) against planktonic S. mutans. To systematically evaluate the antibacterial efficacy and mechanism of action of SAEW, the following experimental approaches were employed: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SAEW against planktonic S. mutans were determined using the broth microdilution method. The dynamic inhibitory effect of different SAEW concentrations on bacterial growth was analyzed via time-growth kinetic assays. Alterations in the bacterial surface ultrastructure following SAEW treatment were observed using scanning electron microscopy (SEM). Furthermore, the integrity of the bacterial cell membrane was quantitatively assessed by confocal laser scanning microscopy (CLSM) combined with live/dead fluorescent staining. Collectively, these methods—encompassing assessments of antibacterial potency, kinetic action, and morphological/functional mechanisms—constitute a comprehensive in vitro evaluation framework for the antibacterial activity of SAEW.

Bacterial strains

The strain used in this experiment was S. mutans standard strain (BNCC 336931, was sourced from the Anhui Provincial Key Laboratory of Oral Diseases Research, Anhui Medical University) was adopted in this study. After strain resuscitation and purity identification, the isolate was subcultured 2–3 times on BHI agar plates. A representative single colony was picked with a sterile inoculating loop and inoculated into BHI broth, followed by 24 h enrichment incubation at 37 °C under microaerophilic conditions with 5% CO₂. The harvested bacterial suspension was washed twice with PBS via centrifugation at 3000 rpm for 5 min to eliminate residual medium. Prior to all in vitro tests, the suspension was fully homogenized by vortexing, and its concentration was calibrated to 1 × 10⁶ CFU/mL using a microplate reader for subsequent antibacterial assays.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Slightly Acidic Electrolyzed Water against Planktonic S. mutans

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SAEW against S. mutans. were determined using a modified broth microdilution method. Briefly, a bacterial suspension at 1 × 10⁶ CFU/mL was centrifuged, and the bacterial pellet was resuspended in 1 mL of SAEW solution at a series of concentrations (ranging from 25 to 1.56 ppm). Negative control (sterile saline instead of SAEW), positive control (0.12% CHX), and blank control were included. The mixtures were incubated at 37 °C for 5 min under light-protected conditions. Subsequently, the treated suspensions were transferred to a 96-well plate, mixed with an equal volume (100 µL) of fresh BHI broth, and incubated at 37 °C for 24 h. The MIC was determined as the lowest concentration at which no visible turbidity was observed. To determine the MBC, 5 µL from each well was spotted onto BHI agar plates and incubated under the same conditions for an additional 24 h. The MBC was defined as the lowest concentration that resulted in no bacterial growth on the agar. All experiments were performed in triplicate.

Growth kinetics under different SAEW concentrations

To dynamically assess the effect of SAEW on S. mutans, growth kinetic curves were established in the presence of varying SAEW concentrations. A standardized bacterial suspension of S. mutans (1 × 10⁶ CFU/mL) was incubated anaerobically at 37 °C for 24 h with HClO at final concentrations of 0.5×, 1×, and 2× the minimum inhibitory concentration (MIC). Sterile normal saline and 0.12% CHX were included as negative and positive controls, respectively. Bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) at 2-h intervals using a microplate reader. Growth curves were plotted with incubation time on the x-axis and OD₆₀₀ on the y-axis to visualize the concentration-dependent suppression of bacterial biomass over time.

Microscopic observation

Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were employed to examine the morphological and viability changes of S. mutans under different treatments. Experimental groups included bacteria treated with SAEW at MIC and 2×MIC concentrations, with sterile normal saline and 0.12% CHX serving as the negative and positive controls, respectively. For SEM, samples were prepared using a standard protocol (fixation with 2.5% glutaraldehyde, dehydration through an ethanol gradient, and critical-point drying) prior to observation. CLSM was combined with Live/Dead fluorescent staining (SYTO 9 for viable bacteria, green; propidium iodide for dead bacteria, red) to assess bacterial viability. For quantitative analysis of bacterial viability, CLSM images were processed using ImageJ software. The SYTO 9 (green, total bacteria) and PI (red, dead bacteria) channels were separated and subjected to threshold segmentation. The ratio of the red fluorescence area to the total red-plus-green fluorescence area in each field of view was calculated and defined as the proportion of membrane-damaged bacteria. At least five random fields per experimental group were analyzed. Results are presented as mean ± standard deviation and statistically compared between groups.

Cell proliferation toxicity assay (CCK-8 Method)

CCK-8 assay evaluated the proliferative cytotoxicity of standardized HClO on human gingival fibroblast (HGF) cells (preserved by the Laboratory of Infectious Diseases, First Affiliated Hospital of Anhui Medical University)。.

Human gingival fibroblast (HGF) cells in the logarithmic growth phase were harvested using 0.25% trypsin, collected by centrifugation, and resuspended in complete medium. The cell density was adjusted to 5 × 10⁴ cells/mL. The cell suspension (100 µL) was seeded into the inner 60 wells of a 96-well plate. The peripheral wells were filled with 100 µL of PBS to minimize evaporation-related errors. The plate was then incubated at 37 °C under 5% CO₂ for 24 h to allow for cell attachment and stabilization.

After incubation, the culture medium was aspirated, and the cells were gently washed three times with PBS. Subsequently, 200 µL of treatment solution containing different concentrations of SAEW (MIC, 2×MIC, 4×MIC), 0.12% CHX (positive control), or PBS (negative control) was added to the respective wells. The cells were treated in the dark for 5 min. Following treatment, the solution in each well was carefully aspirated, and the cells were gently washed three times with PBS. Subsequently, 100 µL of complete culture medium was added to each well. In parallel, three additional wells containing only 100 µL of complete medium (without cells) were prepared as blank controls. Then, 10 µL of CCK-8 reagent was added to every well, followed by incubation in the dark for 2 h. The optical density (OD) of each well was finally measured at 450 nm using a microplate reader. The cell viability of the negative control group was defined as 100%, and the relative cell viability (%) of the treatment groups was calculated accordingly.

Animal experiments

A rat caries model was established using Sprague-Dawley (SD) rats to evaluate the in vivo anti-caries efficacy and safety of SAEW in comparison with the clinically used CHX. The experimental protocol was approved by the Animal Ethics Committee of Anhui Medical University (Approval No. LLSC20241689). Three-week-old male SD rats were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Jiangsu, China) and housed in the specific pathogen-free (SPF) barrier facility of the Experimental Animal Center at the First Affiliated Hospital of Anhui Medical University. All animals had ad libitum access to standard irradiated feed and drinking water.

Twenty-five 3-week-old, male, specific pathogen-free (SPF) SD rats were acclimatized for 3 days and then randomly divided into 5 groups (n = 5 per group): the normal control group (N), caries control group (A), SAEW low-concentration group (B, 6.25ppm), SAEW high-concentration group (C, 12.5ppm), and CHX positive control group (D, 0.12%).

After the start of the experiment, all rats were given antibiotic-containing drinking water for 3 consecutive days, with antibiotic concentrations of 200 mg/L penicillin and 1500 mg/L streptomycin to eliminate the original flora in the oral cavity. On the 7th day, the supply of antibiotic drinking water was stopped, and the rats were fasted for 2 h. Oral saliva samples of rats in each group were collected using sterile cotton swabs. The swabs with saliva were suspended in sterile normal saline, and bacterial count detection was performed using BHI agar medium. During the entire experiment, the rats were fed with Co60-irradiated disinfected food and water. The dental caries modeling was officially initiated when the BHI plate showed negative results for oral bacteria.

Except for Group N, which remained on a standard diet throughout, the other four groups (A, B, C, and D) were switched to a cariogenic diet (Keyes Diet 2000, containing 56% sucrose) and supplied with 5% sucrose water starting from the initiation of caries induction, and this regimen was maintained for the entire experimental period. Rats in Groups A, B, C, and D underwent active caries induction for 7 days following the method described by Beiraghi et al. [31]. The Keyes scoring and radiographic assessment for caries lesions were performed according to a standardized rat caries model protocol [32]0.1 mL of S. mutans bacterial solution (concentration of 10⁸ CFU/mL) was used to soak a sterile cotton swab, and the swab was pressed in each of the four quadrants of the rat’s oral cavity for 15 s to achieve oral colonization of S. mutans. On the 7th day of modeling, oral swab samples of rats were cultured on BHI agar plates to confirm the successful colonization of S. mutans in the oral cavity, indicating successful modeling.

After successful establishment of the caries model, each group received the intervention regimen outlined in Table 1. Schematic Diagram of the Experimental Protocols for Different Treatment Groups. Specifically, Group A (caries control) received topical application of sterile normal saline twice daily (1 ml per application); Groups B, C, and D received their respective solutions of HClO or CHX (1 ml per application). All interventions were administered for 2 consecutive weeks, followed by a 4-week observation period without intervention. During the 4-week follow-up period (weeks 5–8), all treatments were ceased, while cariogenic diet and 5% sucrose water were maintained ad libitum to sustain the cariogenic environment.

Table 1.

Schematic diagram of the experimental protocols for different treatment groups

Group	Week 1	Week 2	Week3-4	Week5-8
N	Days 1–3 acclimatized, Days4–6 Antibiotic watering.	Normal diet	Normal diet	Normal diet
A	Same as the normal group	Caries model construction	topical application of Sterile normal saline	Observations
B	Same as the normal group	Caries model construction	topical application of SAEW(6.25ppm)	Observations
C	Same as the normal group	Caries model construction	topical application of SAEW (12.5ppm)	Observations
D	Same as the normal group	Caries model construction	topical application of 0.12% CHX	Observations

Observation and microbiological analysis

The mortality, mental state, and coat luster of rats were observed and recorded daily, and the body weight of rats was measured and recorded weekly. The entire experimental period was 8 weeks, and samples were collected for detection on days 7, 14, 28, 42 and 56 respectively. The N group was tested in the first week only. The sample collection method was as follows: the occlusal surfaces of the molars in the oral cavity of rats in each group were wiped with a sterile cotton swab in a clockwise circular motion. Immediately after collection, the swab was placed in a centrifuge tube containing 1 mL of normal saline and soaked for 2 min. After serial dilution of the soaking solution, the appropriately diluted solution was inoculated on BHI plates for bacterial counting.

Caries scoring

At the end of the 8th week, all rats were subjected to body weight measurement. Subsequently, the rats were anesthetized via intraperitoneal injection of pentobarbital sodium (50 mg/kg) to induce deep anesthesia. Following blood collection from the abdominal aorta, each rat was placed in a transparent, sealed plastic chamber connected to a CO₂ gas supply line and continuously observed. CO₂ gas was introduced continuously into the sealed chamber until the cessation of movement and respiration, along with pupil dilation, was confirmed. The CO₂ supply was then terminated.

Prior to dissection, all surgical instruments were sterilized in an autoclave and subsequently dried in an oven for later use. Tissues including the mandible, oral mucosa, tongue mucosa, gingival mucosa, major organs, and blood were collected from the rats. The cleaned rat mandibles were stained with 0.4% murexide (ammonium purpurate) and then examined under a stereomicroscope for caries evaluation using the Keyes scoring system [33]. The scoring method involved visually assessing the linear extent of the lesion within a single plane and recording the penetration depth, categorized as: enamel only (E), slight dentinal involvement (Ds), moderate dentinal involvement (Dm), and extensive dentinal involvement (Dx). The specific scoring criteria are detailed in Table 2. Number of linear units assigned to each molar.

Table 2.

Number of linear units assigned to each molar

Caries site	Molars
	Maxillary			Mandibular
	1st	2nd	3rd	1st	2nd	3rd
Buccal	6	4	3	6	4	4
Lingual	6	4	3	6	4	4
Sulcal	5	3	2	7	5	2
Proximal	1	2	2	1	2	1

Safety evaluation

Whole blood samples were collected from the abdominal aorta of rats in the blank control group for an in vitro hemolysis assay. For all groups, serum was isolated from the whole blood, and the levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), and blood urea nitrogen (BUN) were measured to evaluate hepatic and renal functions. In addition, the heart, liver, spleen, lung, kidney, Tongue and Gingiva tissues were harvested from rats in each group, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological observation.

Statistical analysis

SPSS Statistics 26.0 was used for analysis. All quantitative experiments, including antibacterial assays and cytotoxicity evaluations, were independently repeated three times (n = 3) to ensure reproducibility, representing biological replicates. Within each independent experiment, three technical replicates (wells) were established for each treatment group. All data were assessed for normality using the Shapiro‑Wilk test and for homogeneity of variances using Levene’s test. Data that satisfied both normality and homoscedasticity are presented as the mean ± standard deviation (x̄ ± s). Comparisons among multiple groups were performed with one‑way analysis of variance (ANOVA), followed by the least significant difference (LSD) test for post‑hoc pairwise comparisons. Data that did not meet the assumptions of normality or homoscedasticity are expressed as the median with interquartile range [M (IQR)], and the Kruskal‑Wallis test was used for group comparisons. A two‑tailed p-value < 0.05 was considered statistically significant.

Results

Characterization of standard SAEW solution

The SAEW solution samples prepared in this study exhibited an available chlorine content of 25 ± 0.443 ppm and a pH value of 6.532 ± 0.002.

In vitro antibacterial activity and cytocompatibility

SAEW exhibited a clear and concentration-dependent inhibitory effect on the growth of planktonic S. mutans. Quantitative assessment of its antibacterial efficacy via in vitro susceptibility testing determined the MIC and MBC of SAEW against this bacterium to be 6.25 ppm (Fig. 1A) and 12.5 ppm (Fig. 1B), respectively. This effective concentration range indicates significant in vitro antimicrobial potential.

Fig. 1.

A Minimum inhibitory concentration of SAEW. B Minimum bactericidal concentration of SAEW

Dynamic monitoring via time-growth curves (Fig. 2) clearly delineated the mode by which SAEW interferes with the bacterial growth cycle. The negative control group displayed a typical growth curve, entering the logarithmic phase at approximately 4 h and reaching the stationary phase around 12 h. Treatment with SAEW at 1/2 MIC significantly delayed the bacterial proliferation rate, demonstrating that even at subinhibitory concentrations, SAEW can disrupt bacterial metabolism. When the SAEW concentration reached its MIC, bacterial growth was potently inhibited, with the OD600 value showing only a marginal increase over a 12-hour incubation period, indicating that the growth of the vast majority of bacteria was arrested. At a concentration of 2×MIC, the growth curve completely overlapped with that of the positive control (0.12% CHX), showing no increase in turbidity throughout the incubation, confirming that this concentration rapidly and thoroughly inhibited the growth of all bacteria, achieving an immediate bactericidal effect comparable to the classic disinfectant CHX.

Fig. 2.

Time-growth curves of S. mutans treated with different concentrations of SAEW

Morphological and functional evidence for the antimicrobial mechanism was further substantiated. Scanning electron microscopy (SEM) observation (Fig. 3) visually demonstrated characteristic ultrastructural damage in SAEW-treated bacteria, including shrinkage, depression, and rupture of the cell wall, as well as leakage of intracellular contents. Functional analysis via Confocal Laser Scanning Microscope (CLSM) combined with quantitative live/dead bacterial staining (Fig. 4) provided direct evidence for SAEW-induced membrane damage. The proportion of bacteria with compromised membranes (PI-positive) increased from 0.9 ± 0.1% in the negative control to 92.5 ± 1.1% and 94.5 ± 0.8% after treatment with MIC and 2×MIC SAEW, respectively (both p < 0.001 versus control). This dose-dependent increase in membrane permeability confirms that rapid disruption of cell membrane integrity is a core mechanism underlying the antibacterial action of SAEW. This mechanism effectively disrupts and terminates the normal growth cycle of planktonic S. mutans.

Fig. 3.

Morphological alterations of S. mutans induced by SAEW treatment, as observed by scanning electron microscopy (SEM). A Negative control group (normal saline): bacteria exhibited intact, smooth surfaces with typical coccoid morphology. B Low-concentration SAEW group (MIC): bacteria showed initial signs of damage, including surface roughness and slight shrinkage. C High-concentration SAEW group (2×MIC): More severe morphological disruption was observed, characterized by cell wall collapse, deep depressions, and content leakage. D Positive control group (0.12% CHX): Bacteria demonstrated the most severe shrinkage and extensive content leakage. Scale bar: 2 μm. The images illustrate the concentration-dependent damaging effect of SAEW on bacterial cellular integrity. Scale bars: 1 μm

Fig. 4.

Assessment of S. mutans cell membrane integrity by live/dead fluorescence staining. Bacterial cells were stained with SYTO 9 (green, intact membrane) and propidium iodide (PI, red, damaged membrane) following treatment. A Negative control (normal saline): predominantly green fluorescence, indicating intact cell membranes. B Low-concentration SAEW group (MIC): mixed green and red fluorescence, indicating initial membrane damage. C High-concentration SAEW group (2×MIC): increased red fluorescence, demonstrating enhanced membrane disruption. D Positive control (0.12% CHX): predominantly red fluorescence, indicating severe membrane damage. Scale bar: 300 μm. The images visually demonstrate the concentration-dependent compromise of cell membrane integrity induced by SAEW treatment

Following the determination of the effective antibacterial concentrations of SAEW against S. mutans, its biocompatibility with human gingival fibroblasts (HGFs) was further assessed using the CCK-8 assay. The results demonstrated favorable biocompatibility between SAEW and HGFs at its effective antibacterial concentrations (Fig. 5). Compared to the negative control, treatment with SAEW at the minimum inhibitory concentration (MIC) did not significantly reduce the viability of HGFs. A slight but statistically significant decrease in cell viability was observed only at higher concentrations (2×MIC and 4×MIC), yet it remained above 60%. In stark contrast, treatment with the positive control, 0.12% chlorhexidine (CHX), resulted in a severe loss of cell viability.

Fig. 5.

Effect of SAEW on the viability of HGFs cells. Cell viability was assessed by the CCK-8 assay after treatment with various concentrations of SAEW. Compared to the negative control (PBS), SAEW at its minimum inhibitory concentration (MIC) did not significantly reduce cell viability. In stark contrast, the positive control (0.12% CHX) caused a severe loss of cell viability. The data are expressed as the means ± SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

In summary, from the inhibition and eradication of planktonic bacteria and the elucidation of the cell membrane damage mechanism to its impact on biofilm activity, this series of experiments systematically confirms that SAEW, as a potential oral antimicrobial agent, possesses multi-faceted anti-S. mutans activity.

In vivo anti-caries efficacy of SAEW

SAEW significantly reduces the level of salivary streptococci in rats

Bacterial enumeration from the in vivo experiment demonstrated that SAEW significantly reduced the level of oral streptococci in rats (Fig. 6). Compared to the caries model group, the salivary streptococcal counts in both the high- and low-concentration SAEW treatment groups were significantly lower (P < 0.05). The antibacterial effect of the high-concentration SAEW was comparable to that of the chlorhexidine positive control group. These results indicate that SAEW can effectively inhibit the proliferation of cariogenic streptococci within the complex oral microenvironment, thereby reducing the microbial load associated with caries development.

Fig. 6.

Counts of S. mutans in the oral cavity of rats in different groups. Group A: Caries model control; Group B: Low-concentration SAEW (MIC); Group C: High-concentration SAEW (2×MIC); Group D: Positive control (0.12% Chlorhexidine)

Observation and scoring of the dental profile of rats

Stereomicroscopic observation (Fig. 7) revealed clearly demarcated spots or band-like stained areas on the occlusal surfaces and fissures of the molars in Group A (caries model group). These morphological alterations confirmed demineralization of the dental hard tissues, indicating the successful establishment of the caries animal model.

Fig. 7.

Inhibitory effect of SAEW on Caries development in rats representative images and scores from different treatment groups: A Caries model control; (B) Low-concentration SAEW (MIC); (C) High-concentration SAEW (2×MIC); (D) Positive control (0.12% Chlorhexidine)

Histological evaluation using the Keyes caries scoring system (Table 3) demonstrated that the severity of carious lesions on both smooth surfaces and fissures was significantly reduced in the low- and high-concentration SAEW treatment groups compared to the model group (P < 0.05). Specifically, the fissure caries scores for the high-concentration SAEW group at the superficial, moderate, and deep dentinal levels were (10.2 ± 1.30), (6.60 ± 0.89), and (1.00 ± 0.71), respectively, all of which were significantly lower than those in the model group (all P < 0.05).

Table 3.

Effect of different groups on caries development (incidence and severity) in rats

Groups	Caries level
	E1	Ds	Dm	Dx
A	30.4 ± 2.07a2	20.2 ± 1.92a	11.8 ± 1.64a	4.2 ± 1.48a
B	23.2 ± 2.59b2	12.4 ± 2.30b	8.2 ± 1.48b	1.6 ± 1.14b
C	18.8 ± 1.92c2	10.2 ± 1.30bc	6.6 ± 0.89b	1.0 ± 0.71b
D	18.6 ± 1.52c	8.4 ± 1.67c	6.0 ± 1.58b	0.8 ± 0.84b

¹E, enamel only; Ds, slight dentinal penetration; Dm, moderate dentinal penetration; Dx, extensive dentinal penetration

²Within the same caries category, groups marked with different letters differ significantly (p < 0.05).

Radiographic analysis further corroborated these findings. Radioparent bands were evident beneath the enamel in the model group, often extending into the deep dentin. In contrast, the teeth in the SAEW-treated groups showed more localized and shallower radiolucent areas. The radiographic appearance of the high-concentration SAEW group was similar to that of the chlorhexidine group, with lesions primarily confined to the enamel and the superficial to moderate dentin.

In vivo biocompatibility of SAEW in rats

Hemolysis assay results indicated that SAEW at various concentrations did not induce hemolytic activity. Histological examination with H&E staining showed no obvious pathological alterations in the oral mucosa or major organs following SAEW treatment, with normal cellular morphology and tissue architecture observed (Fig. 8). Regarding biochemical parameters, no statistically significant differences were found in the key indices of liver function or renal function (Fig. 9) between any SAEW-treated groups and the model control group (all P > 0.05). Throughout the experimental period, all rats remained healthy and active with no mortality. Body weight increased steadily in all groups, showing similar growth patterns among them (Fig. 10).

Fig. 8.

H&E staining of major organs and oral mucosa in rats from each group

Fig. 9.

Assessment of hepatic and renal function in rats across treatment groups. (The red dashed line denotes the normal reference range for serum biochemical parameters.)

Fig. 10.

Body weight of rats in different treatment groups. (No significant difference among groups, P > 0.05.)

Discussion

Pharmacological intervention remains a key strategy for caries control. Eco-friendly agents derived from the host’s innate immune mechanisms, characterized by their rapid, broad-spectrum antimicrobial action and favorable tissue compatibility, have emerged as an important research direction for ideal caries prevention strategies. Currently, a variety of approaches based on physicochemical antimicrobial principles, including photodynamic therapy, antimicrobial peptides and metal nanomaterials, have been developed as adjunctive treatments for oral infectious diseases, and their promising application prospects have been well documented [34–37].

Antimicrobial agents differ substantially in their working mechanisms. SAEW is produced by water electrolysis, with hypochlorous acid acting as the primary bactericidal component. Notably, hypochlorous acid is an endogenous effector molecule released by neutrophils during the host immune response. In the present study, we verified that SAEW inhibits S. mutans—the principal etiological bacterium of dental caries—in a concentration-dependent manner. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SAEW against planktonic S. mutans were determined as 6.25 ppm and 12.5 ppm, respectively. Consistent with previous mechanistic studies on electrolyzed water, SAEW exerts rapid bactericidal activity mainly by destroying the integrity of bacterial cell membranes. This study verified that freshly prepared slightly acidic electrolyzed water (SAEW) exhibits rapid bactericidal kinetics against planktonic S. mutans. After reaching the MIC, viable bacterial counts decline markedly within a short duration. Results from scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) provided direct morphological and functional evidence for this mode of action: SAEW treatment led to obvious shrinkage and rupture of bacterial bodies, increased cell membrane permeability, and a remarkable decline in the proportion of viable S. mutans cells. Notably, this study indicates that the antimicrobial mechanism of SAEW—rapid membrane damage—likely leads to swift bacterial death within the biofilm and indirectly compromises the stability of the extracellular matrix, thereby weakening the structural and functional integrity of the biofilm. This mode of action, involving the direct physicochemical disruption of the pathogen’s fundamental structures, also makes it less prone to inducing microbial resistance.

To contextualize our findings against current research on anti-cariogenic strategies targeting S. mutans, various antimicrobial approaches exert effects via distinct modes of action. One study evaluated the disinfection efficacy of common root canal antiseptics against Enterococcus faecalis, a persistent pathogen in endodontic treatment. The results revealed that different disinfectants presented varied performance in eliminating bacteria within dentinal tubules [38]. Another work established a stable ex vivo oral polymicrobial biofilm model and adopted this system to assess the inhibitory effect of natural extracts and classic antibacterial agents on oral biofilms at different growth stages [39]. Compared with these agents that primarily regulate bacterial metabolism or gene expression, SAEW relies on physical damage to bacterial structures. This direct physicochemical disruption of essential bacterial components not only achieves rapid bactericidal effects but also greatly reduces the risk of drug resistance development, which is a prominent advantage for long-term oral application.

Similar to tea tree oil (TTO), freshly prepared SAEW can also be categorized as a naturally derived antibacterial agent. Its core active component, hypochlorous acid, is an endogenous immune bactericide spontaneously produced in the human body, while TTO is extracted from natural tea tree plants. Both agents avoid the adverse side effects caused by the synthetic cationic disinfectant chlorhexidine (CHX) and exert satisfactory inhibitory effects on planktonic cariogenic bacteria. Nevertheless, striking differences exist between the two natural candidates: SAEW relies on small-molecule hypochlorous acid to rapidly penetrate mature biofilm matrices, whereas TTO merely exerts superficial bacteriostatic effects. In terms of biosafety, the cited study only verified that 12.5% TTO shows no cytotoxicity toward human gingival fibroblasts (HGFs), with cytotoxicity assessments at higher concentrations absent. As its authors noted, the long-term oral effects and potential oral and systemic risks of TTO remain unclear, rendering its overall safety questionable [40]. In contrast, the endogenous origin of hypochlorous acid endows SAEW with excellent biological selectivity for oral host cells, and our CCK-8 results further confirm that SAEW maintains high HGF viability at concentrations with full anti-cariogenic activity. Collectively, as a naturally derived anti-caries agent, SAEW addresses TTO’s safety limitations—untested cytotoxicity at elevated doses —and provides a promising alternative for the prevention of caries.

The broad-spectrum antimicrobial activity and biosafety profile of SAEW are supported by findings in other infection models. Studies have shown that low-concentration hypochlorous acid solutions inhibit periodontal pathogens, peri-implantitis-related microbiota, and various fungi, while exhibiting significantly lower toxicity to oral mucosal cells and fibroblasts compared to traditional chemical disinfectants like chlorhexidine [41]. The cytotoxicity results of this study are consistent with previous reports. Following a 5-minute treatment at effective antibacterial concentrations—mimicking acute mouthwash exposure—SAEW did not induce significant toxicity in human gingival fibroblasts, with cell viability remaining above 80%. These findings preliminarily confirm the favorable biocompatibility of SAEW under acute exposure conditions. However, given the limited duration of treatment, these results primarily reflect short-term safety.

In a rat caries model, topical application of SAEW significantly reduced oral streptococcal levels and effectively mitigated the severity of carious lesions on both smooth surfaces and fissures of molars, demonstrating a clear dose-dependent anti-caries effect. However, while the immediate efficacy of SAEW was significantly superior to the saline control, it did not fully reach the level achieved by 0.12% chlorhexidine. This discrepancy is partly attributed to the inherent physicochemical properties of the agents: unlike chlorhexidine, which exhibits strong substantivity and retains residual antibacterial activity due to mucosal adsorption, SAEW undergoes rapid degradation (as confirmed by our stability assays). Thus, the slightly lower performance of SAEW likely reflects a trade-off between potent, long-lasting antisepsis and favorable biosafety. It is worth noting that while CHX is not specifically licensed for routine clinical caries prevention, it serves as the gold-standard reference for antimicrobial potency in dental research, and its known drawbacks (e.g., staining, taste alteration) underscore the rationale for exploring SAEW as a safer alternative.

In summary, SAEW exerts its antimicrobial effect by disrupting bacterial cell membranes, is safe for host cells at effective concentrations, and has demonstrated anti-caries potential in an animal model. It represents a potential eco-friendly anti-caries agent with a defined mechanism of action, good biosafety, and in vivo efficacy. Nevertheless, several limitations of the present study should be acknowledged. This work exclusively focused on planktonic S. mutans without investigating biofilm-associated phenotypes, and the in vitro monoculture model cannot fully recapitulate the complex polymicrobial ecology of oral biofilms. The oral stability, functional duration, and long-term ecological impacts of SAEW on indigenous oral microbiota also remain to be clarified. Accordingly, our follow-up independent study will specifically address the anti-biofilm performance, penetration behavior, and clearance mechanisms of SAEW within complex biofilm matrices. Future translational research should also explore the synergistic effects of SAEW combined with remineralizing agents (e.g., fluoride) and conduct long-term preclinical and clinical evaluations to comprehensively validate its anti-caries efficacy and clinical application potential, thereby promoting its bench-to-bedside translation.

Conclusions

In conclusion, this study demonstrates that on-site generated slightly acidic electrolyzed water (SAEW) represents a highly promising topical agent for caries prevention. Our findings establish that SAEW exhibits potent, rapid bactericidal activity against the primary cariogenic pathogen Streptococcus mutans, with an efficacy comparable to the clinical gold standard, 0.12% chlorhexidine (CHX). Crucially, SAEW achieves this antimicrobial performance while presenting a vastly superior biocompatibility profile, causing significantly less cytotoxicity to human gingival fibroblasts. Although the in vivo anticaries efficacy in a rat model was slightly lower than that of 0.12% CHX, SAEW still produced a highly significant reduction in both caries incidence and lesion severity. The core innovation of this work lies not merely in the evaluation of a new antimicrobial solution, but in the validation of a practical, on-demand electrochemical generation system that delivers a stable, near-neutral pH agent. This effectively addresses the key limitations of traditional acidic electrolyzed water—instability and tissue irritation—while simultaneously overcoming the major clinical drawbacks of CHX, such as staining and cytotoxicity.

The significance of this research is twofold, spanning both scientific innovation and clinical translation. From a scientific perspective, it successfully bridges electrochemistry, microbiology, and preventive dentistry, providing a mechanistic and applied evaluation of an innate immunity-inspired oxidant. It offers robust evidence that a mild oxidant can achieve targeted antimicrobial action without proportional damage to host cells, a finding with relevance beyond dentistry to broader antimicrobial surface and wound care research. From a clinical and public health standpoint, this study is directly relevant to the urgent need for safer, long-term adjunctive caries prevention strategies. By introducing a protocol for generating an effective, biocompatible agent on demand, this work proposes a viable alternative for high-risk patients, in professional preventive regimens, or potentially in community-based public health initiatives. It shifts the paradigm from relying on pre-manufactured chemotherapeutic agents with side effects to a sustainable, on-site technology that balances efficacy with safety. Therefore, this research makes a substantive contribution to the field by presenting a translatable technology that has the potential to impact clinical practice and improve oral health outcomes.

Abbreviations

S. mutans

Streptococcus mutans

CHX

Chlorhexidine

SAEW

Slightly acidic electrolyzed water

HClO

Hypochlorous acid

FAC

Free available chlorine

BHI

Brain heart infusion

BHIs

BHI supplemented with 1% sucrose

CFU

Colony forming units

PBS

Phosphate buffer saline

HGF

Human Gingival Fibroblasts

SPF

Specific Pathogen Free。

Fig

Figure

g

Gram

L

Liter

OD

Optical density

pH

Hydrogen ion concentration

CLSM

Confocal laser scanning microscopy

SEM

Scanning Electron Microscope

Authors’ contributions

M.Y. Luan and X.Z. Gaocontributed equally to this work and share first authorship. M.Y. Luan and X.Z. Gao jointly conceived the study and developed the methodology. M.Y.Luan performed software implementation, formal analysis, investigation, and data curation. M.Y.Luan and X.Z. Gao collaborated on validation of theresults. N. Lv acquired funding, provided resources, supervised the project, and administered the project. M.Y. Luan wrote the original draft. N. Lv andQ.Q. Cao reviewed and edited the manuscript, with Q.Q. Cao contributing to data visualization. All authors read and approved the final manuscript.

Funding

This work was supported by the Anhui Provincial Department of Education University Scientific Research Program under Grant 2024AH04011.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

An in vivo rat caries model was established using Sprague-Dawley rats to evaluate the anticaries efficacy and safety of SAEW in comparison with chlorhexidine (CHX). All animal experiments were conducted in accordance with the ethical standards approved by the Animal Ethics Committee of Anhui Medical University (Approval No. LLSC20241689). This article does not contain any studies with human participants or client-owned animals performed by any of the authors.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.