Introduction

Dental caries is a multifactorial disease influenced by host defenses, dietary carbohydrates, dental plaque bacteria, and the duration of acid challenge to enamel1,2,3. Nutrition and oral hygiene are key determinants of caries risk. Dental caries is now recognized as a microbial biofilm–mediated disease driven by a diverse plaque microbiota whose composition and activity are strongly influenced by the frequency and availability of fermentable carbohydrates4. Dental plaque is a complex biofilm that adheres to tooth surfaces and, with frequent sugar exposure, its microbiome shifts toward acidogenic and aciduric bacteria that ferment carbohydrates into organic acids, lowering plaque pH and promoting enamel demineralization5,6. Following food intake, sugar fermentation rapidly lowers plaque pH which subsequently recovers through salivary buffering and clearance to allow remineralization7,8. The cariogenic microbiome is characterized by dysbiosis and includes Streptococcus mutans and other caries-associated species such as Actinomyces, Bifidobacterium, Lactobacillus, Veillonella, and Scardovia9,10,11,12.

To prevent caries, current strategies aim to reduce plaque biofilm mass, viability, acid production, and metabolic activity13. Common approaches include mechanical plaque removal, fluoride application, antimicrobial mouth rinses, and diet modification13. Dietary efforts have largely focused on reducing sugar intake and snacking frequency14. However, the potential of functional foods - those providing health benefits beyond basic nutrition - for oral health remains underexplored15, despite the established link between oral and systemic health16,17. Our group, and others have shown that specific foods or food components exhibit caries-protective properties by counteracting dietary sugars18,19,20, potentially by may targeting cariogenic bacterial virulence, including growth, metabolism, gene expression, and biofilm integrity21,22,23,24,25. Accordingly, virulence-targeted anti-caries strategies increasingly emphasized dietary natural products over broad-spectrum antimicrobials15,20,26,27.

Dietary polyphenols, which are abundant in many edible plants, possess antimicrobial, antioxidant, anti-cancer, and anti-inflammatory properties27,28,29. In vitro studies show that they inhibit biofilm formation and acid production by cariogenic bacteria30,31,32. Plant-based beverages, such as tea, coffee, wine, and cranberry juice contain antimicrobial compounds that can suppress oral pathogens23,24,25,33,34,35,36,37. For example, black tea rinses have been shown to significantly reduce plaque acid production, plaque index, and subsequent regrowth compared with water21.

American cranberries (Vaccinium macrocarpum) are source rich in polyphenolic bioactives, particularly proanthocyanidins (PACs), which may benefit human health38,39. Cranberry PACs show antimicrobial and anti-adhesive activity, inhibiting uropathogenic E. coli adhesion40 with emerging evidence suggesting similar effect in the oral cavity36,41,42,43,44,45,46,47,48. These include inhibition of S. mutans GTF enzymes and glucan synthesis, disruption of biofilms22,26,36,45,46,47,49,50 and suppression of periodontal pathogens42,43,44. Our previous work demonstrated that cranberry extract and US-marketed 27% cranberry juice cocktails rapidly induce bacterial aggregation and inhibit the in vitro growth and biofilm formation of S. mutans and, in children supragingival plaque bacteria37,41. Cranberry extract has also been shown to inhibit periodontal pathogens such as Porphyromonas gingivalis, Fusobacterium nucleatum and a multi-species anaerobic biofilm41,48,51. Although cranberry polyphenols are not strongly bactericidal, recent in vitro studies suggest that they can beneficially modulate the microbial ecology of dental plaque and may help reverse the microbiome dysbiosis associated with dental caries46.

To assess the cariostatic effects of cranberry juice or PACs, in vitro biofilm models using S. mutans or multispecies oral bacteria are predominantly used52,53, but they do not fully capture the complexity of natural dental plaque in the oral environment. A comprehensive 2026 review by Manso et al. found that 82 of 93 eligible studies were conducted in vitro53. The limited number of controlled in vivo studies on cranberry products has primarily focused on salivary outcomes rather than dental plaque metabolic activity, and only a few have included pediatric population54,55,56,57,58. A 2024 systematic review by Padawe et al.59 identified seven in vivo randomized clinical trials published since 2004; however, only three evaluated the effects of cranberry mouthwash or juice on dental plaque, and these assessed changes in plaque bacterial counts alone, with none examined plaque acid production. Here, we used the U.S. Food and Drug Administration (FDA)-approved Plaque Glycolysis and Regrowth Method (PGRM, 21 FDA CFR Part 356, Vol. 68:103, 2003)60,61, a validated clinical assay of plaque acidogenicity and regrowth, on adults and children. By directly exposing natural dental plaque to topical formulations (e.g., via brushing or rinsing) prior to removal and testing in vitro, the PGRM offers sensitive and reproducible estimates of antimicrobial efficacy against bacterial growth and metabolism in dental plaque following in vivo application. To our knowledge, this study is the first to apply the PGRM method as an “efficacy screen” for evaluating the anti-plaque activity of natural foods and beverages.

The objective of the current study was to evaluate the effects of short-term in vivo exposure to U.S.-marketed 100% cranberry juice (CJ-100) and cranberry juice cocktail (CJC-27; 27% cranberry juice) on regrowth and acid production of natural dental plaque. The utility of the PGRM as an efficacy screening tool for food-based anticaries interventions was also assessed. According to the U.S. FDA, only products that are 100% fruit juice may be labeled as “fruit juice”; products containing less than 100% juice must declare the percentage of juice and use terms such as “drink,” “beverage,” or “cocktail.”

Results

Effect of short-term exposure to cranberry juice on adults’ dental plaque regrowth and acid production (glycolysis)

The regrowth of adult plaque bacteria before and after exposure to cranberry juice, cranberry juice cocktail, and water is shown in Supplementary Table 1. The average regrowth of the pre-exposed adult plaque bacteria in all three groups reached similar level at both 2 and 4 h. After exposure to water, the average regrowth of plaque bacteria was comparable to that of the pre-exposed plaque bacteria. The regrowth of plaque bacteria after exposure to CJC-27 was numerically lower than that of the pre-exposed plaque bacteria, however, the difference was not statistically significant (p > 0.05). In contrast, exposure to CJ-100 resulted in a significant reduction in regrowth at both 2 h (p < 0.001) and at 4 h (p = 0.009) compared to the pre-exposed plaque bacteria.

The effect on regrowth [(ODpost/ODpre) x 100%] in the three exposure groups was assessed for the three exposure groups. As shown in Fig. 1a, plaque regrowth after exposure to water was unaffected. Regrowth following exposure to CJC-27 decreased to 89.5% at 2 h and 75.2% at 4 h; however, these reductions were not statistically significant compared to the water group. In contrast, exposure to CJ-100 resulted in a significant reduction with regrowth decreasing to 66.8% at 2 h (p = 0.003) and 56.1% at 4 h (p = 0.005).

The effect of cranberry juice exposure on plaque acid production in adults was assessed. Initially, the average pH values of pre-exposed plaque across all three groups at 0 h ranged from 6.446 to 6.565 (Supplementary Table 2). Following incubation, acid production caused a decrease in pH to between 4.977 and 5.112 at 2 h, reaching a stable pH of approximately 4.7 at 4 h across all three exposure groups.

Exposure to either CJC-27 or water resulted in similar patterns of pH decline, with pH values at both 2 h and 4 h not significantly different from those of the pre-exposed dental plaque. In contrast, exposure to CJ-100 inhibited plaque acid production, resulting in significantly higher pH values at both 2 h (5.542, p < 0.001) and 4 h (5.005, p = 0.010) compared with the pre-exposed dental plaque.

Fig. 1
Fig. 1
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Effect of in vivo short-term exposure of cranberry juice on dental plaque regrowth and acid production in adults (n = 8) and in children (n = 14). (a): Regrowth of adults’ plaque bacteria; (b): Acid production of adults’ plaque bacteria; (c): Regrowth of children’s plaque bacteria; (d): Acid production of children’s plaque bacteria. Regrowth was expressed as (ODpost/ODpre) x 100%. Plaque pH change was expressed as ΔpH = ΔpHpost - ΔpHpre. The ΔpHPost = pHPost 2–4 h – pHPost 0 h; ΔpHPre = pHPre 2–4 h – pHPre 0 h. * Denotes significance difference between groups (P < 0.05, 1-way ANOVA).

Figure 1b illustrates the changes in pH (ΔpH) following exposure across the three groups. No change in ΔpH was observed in the water group at either 2–4 h. The ΔpH values for CJ-27 were 0.269 ± 0.349 at 2 h and 0.112 ± 0.186 at 4 h, which were not significantly different from those of the water group. In contrast, the ΔpH values for CJ-100 were 0.771 ± 0.391 at 2 h and 0.508 ± 0.397 at 4 h, both of which were significantly higher than those observed in the CJC-27 and water groups (p < 0.05), indicating that plaque acid production was significantly inhibited following exposure to CJ-100.

Effect of short-term exposure to cranberry juice on children’s dental plaque regrowth and acid production (glycolysis)

The average regrowth of children’s plaque bacteria before and after exposure to CJ-100, CJC-27, and water is presented in Supplementary Table 3. Regrowth of pre-exposed plaque bacteria in all three groups increased gradually, reaching similar levels at both 2 h and 4 h. Following exposure to water or CJC-27, no significant inhibition of plaque regrowth was observed compared with the pre-exposed plaque. In contrast, exposure to CJ-100 resulted in a significant inhibition of plaque regrowth at 2 h (p = 0.003) and 4 h (p = 0.001).

The change in children’s plaque regrowth [(ODpost/ODpre) x 100%] after exposure to CJ-100, CJC-27, or water is shown in Fig. 1c. Neither water nor CJC-27 inhibited regrowth. However, after exposure to CJ-100, inhibition of regrowth was noted with regrowth at 66.96 ± 23.88% after 4 h. This inhibition was significantly different (p < 0.05) compared to the water and CJC-27 groups.

The acid production and corresponding pH changes in children’s dental plaque bacteria before and after exposure to CJ-100, CJC-27, and water are presented in Supplementary Table 4. Initially, the average pH values of pre-exposed children’s plaque across all three groups at 0 h ranged from 6.328 to 6.406. Following incubation, a reduction in pH was observed in all groups, reaching similar values at both 2 h and 4 h. Exposure to CJC-27 or water resulted in average plaque pH values of approximately 5.2 at 2 h and 4.8 at 4 h; these values were not significantly different from those of the pre-exposed dental plaque. In contrast, exposure to CJ-100 inhibited plaque acid production, resulting in significantly higher pH values at both 2 h (5.569, p < 0.001) and 4 h (5.190, p = 0.009) compared with the pre-exposed dental plaque.

When comparing the ΔpH among the three exposure groups (Fig. 1d), no significant difference was observed between the CJC-27 and the water groups. In contrast, exposure to CJ-100 significantly inhibited acid production resulting in a smaller decrease in pH at 2 h (ΔpH = 0.357 ± 0.419) and at 4 h (ΔpH = 0.317 ± 0.509) compared with both the CJC-27 and the water groups (p < 0.05).

Discussion

Numerous in vitro and in vivo studies have investigated the potential role of cranberries in the prevention and treatment of oral diseases, though the predominant evidence is derived from in vitro studies and clinical studies directly assessing dental plaque remains limited46,53. The in vivo studies assessed changes in plaque bacterial counts while none examined plaque acid production. The present PGRM study addresses this gap and is, to our knowledge, the first to evaluate the effects of in vivo exposure of natural human dental plaque to cranberry juice on subsequent acid production and regrowth.

Acidogenicity and aciduricity are critical virulence traits of cariogenic biofilms, as they promote enamel demineralization and microbial dysbiosis62. In the present study, in vivo exposure of dental plaque biofilms from both children and adults to CJ-100 significantly inhibited subsequent acid production compared with unexposed plaque. This inhibition corresponded to a ΔpH of 0.77 in adults and 0.36 in children (Fig. 1b and d). A systematic review of in vitro studies by Castellanos et al.52 demonstrated that cranberry extract increased the pH of S. mutans cultures (from 5.2 to 5.6) and inhibited both acid production and acid tolerance, likely through suppression of F-ATPase activity63, without affecting bacterial viability31. While the magnitude of pH inhibition observed in our study was comparable to that reported in in vitro studies using single-species S. mutans biofilms52, it is important to emphasize that our model involved only brief exposure (30 s, repeated three times) of a natural, multispecies human dental plaque biofilm to cranberry juice. Though S. mutans is a key cariogenic organism and a common target of anti-caries interventions, it is not solely responsible for caries development, which can occur in the absence of detectable S. mutans. Other acidogenic members of the dental plaque biofilm also contribute to disease progression64,65. Consequently, findings from in vitro, single-species models should be interpreted with caution, as they do not fully capture the biological complexity of in vivo biofilms. Our results provided evidence that brief in vivo exposure of natural dental plaque to 100% cranberry juice can lead to reduction of acid production and enhanced acid-buffering capacity of the plaque biofilm.

With respect to plaque regrowth, our study data indicated that CJ-100 significantly reduced plaque regrowth in both adults and children compared with the water control (p < 0.05; Fig. 1a and c). In contrast, exposure of children’s plaque to CJC-27 did not inhibit bacterial regrowth relative to water (Fig. 1c). In adults, CJC-27 reduced plaque regrowth to 75.2% after 4 h; however, this reduction did not reach statistical significance when compared with the water-exposed group (Fig. 1a). The greater numerical reduction observed in adults may reflect age-related differences in plaque composition, leading to variable susceptibility to cranberry-derived antimicrobial compounds. Recent studies have demonstrated clear age-dependent differences in the composition, diversity, and abundance of the oral microbiome, with distinct microbial communities contributing to the caries process in adults versus children64,65,66. The superior efficacy of CJ-100 relative to CJC-27 is likely attributable to its higher PAC content. Supporting this interpretation, our recent unpublished data indicate that in vivo exposure to marketed 100% apple juice does not inhibit subsequent bacterial acid production or plaque regrowth.

Beyond its effects on acid production, cranberry juice may also beneficially modulate oral microbial ecology, although most existing studies have focused on saliva rather than dental plaque47,67. In vitro studies using saliva-derived polymicrobial biofilms have shown that cranberry extract significantly reduces the relative abundance of caries-associated species, such as Prevotella denticola and Streptococcus sobrinus, while increasing levels of the beneficial species Streptococcus sanguinis47. Similarly, a 10-day rinse trial demonstrated measurable shifts in the salivary microbiome following exposure to cranberry extract67. Consistent with these observations, our recent ex vivo study showed that treatment of saliva samples with CJ-100 decreased the relative abundance of potential oral pathogens while increasing commensal taxa, including Rothia, Corynebacterium, and Haemophilus68. Future plaque-based microbiome studies are needed to determine whether 100% cranberry juice exerts comparable ecological effects, including the promotion of commensal dominance or selective suppression of pathogenic species in the plaque biofilm.

Findings on the oral health effects of cranberry extracts have been inconsistent, largely due to variability in extraction methods, concentrations, and product forms, as well as the inherently variable, food-based nature of cranberry products, which complicates cross-study comparisons54,55,57. Many studies have used non-standardized, high–molecular weight non-dialyzable material (NDM) lacking defined PAC content—the primary anti-adhesion constituents—potentially resulting in subtherapeutic dosing52,53. Moreover, in vitro models fail to account for bioavailability, oral clearance and retention, which are critical determinants of anti-plaque efficacy. Using the FDA-approved PGRM in vivo assay60,61, the present study directly exposed natural dental plaque in vivo to cranberry juice and demonstrated that bioavailable cranberry components significantly reduced plaque virulence. Consistent with our prior work, the PGRM provides a translational platform for assessing the effects of foods, beverages and chewing gum on dental plaque biofilm metabolism under real-use conditions69,70,71.

A potential concern with cranberry juice is its acidity and sour taste. However, evidence linking acidic beverages to dental erosion remains inconsistent and largely in vitro72 with risk influenced by exposure frequency, duration and salivary protection. Pure cranberry juice is highly acidic (pH ~ 2.31) with cranberry juice cocktail slightly less (pH 2.56), whereas orange and apple juices are less acidic (pH ~ 3.8 and 3.6, respectively). Despite this acidity, an in vitro study reported that commercial cranberry juice reduced dentin erosion, possibly through inhibition of matrix metalloproteinases73. Cranberry juice contains substantially less sugar (4–5 g/8 oz) than orange or apple juice (20–28 g/8 oz), suggesting lower cariogenic potential; however, its tartness limits palatability, particularly in children. Future incorporation of non-cariogenic sweeteners may improve acceptability without compromising safety or efficacy.

In conclusion, pure cranberry juice reduced in vivo plaque acidogenicity without significantly affecting bacterial viability, supporting its potential as a functional oral health beverage. Study limitations include the small sample size and relatively short exposure period. Standardized protocols for the preparation and formulation of cranberry extracts are needed to enable consistent interpretation and comparison across studies. Future larger, longer-term in vivo and plaque-based microbiome studies are required to confirm these findings and to determine whether cranberry exposure can shift dental plaque from dysbiosis to eubiosis, consistent with the ecological plaque hypothesis. The PGRM assay provides a practical in vivo tool for screening foods and beverages based on their effects on plaque metabolism, underscoring diet-based interventions as a feasible strategy for preventing oral disease and supporting overall health.

Materials and methods

Study design and participants

Two independent randomized crossover trials were conducted: one in adults (18–64 years, N = 8) and one in children (7–12 years, N = 14). This study assessed the effect of cranberry juice on dental plaque bacteria regrowth and acid production in medically healthy children and adults. Employing a uniform methodology, both studies were reviewed and approved by the Institutional Review Board, The Office of the Protection of Research Subjects (OPRS) of the University of Illinois Chicago (UIC) for a pediatric cohort (UIC IRB Protocol #2018 − 0421) and for adults (UIC IRB Protocol #2011 − 0598). The OPRS ensures that regulations are adhered to for the protection and welfare of subjects, investigators, and the University. All investigators met the OPRS training requirements prior to the study. All methods were performed in accordance with the relevant guidelines and regulations. The recruitment of prospective participants was conducted at the UIC College of Dentistry, where individuals seeking dental care underwent eligibility assessments during their routine dental examinations.

For the pediatric cohort, the selection criteria included children aged 7 to 12 years who were in the mixed dentition developmental stage and had optimal gingival health, characterized by the absence of gingival swelling, inflammation, or bleeding. Since oral and dental factors may influence plaque composition, eligibility also required that participants had not used antimicrobial mouth rinses or systemic antibiotics, had not received prophylactic dental cleaning in the preceding two weeks, did not have orthodontic appliances or pre-formed pediatric metal crowns, and were free from any known medical conditions. Similarly, adult participants were chosen based on an age range of 18 to 64 years, with an inclusive approach to race and gender. Health criteria included being non-smokers, having good general health status, and having at least 20 natural teeth without visible gingival problems such as swelling, inflammation, or bleeding. Adults who had used antimicrobial mouth rinses or systemic antibiotics in the month leading up to the study, had undergone prophylactic dental cleaning within the last two weeks, wore dentures, had extensive restorations, fixed orthodontic or prosthodontic appliances were excluded from participation. A three-day washout period between testing phases was implemented, consistent with prior PGRM studies, to minimize potential carryover effects from the test beverages.

Test groups

The test beverages included commercially marketed 100% pure cranberry juice, CJ-100 (Ocean Spray® Unsweetened Pure 100% Cranberry Juice with 0 g added sugar, Middleborough, Mass., USA), and a 27% cranberry juice cocktail, CJC-27 (Ocean Spray® Cranberry Juice Cocktail Original 27% Juice with 32 g of added sugar per serving). Water served as the control. All test products were purchased from a local grocery store in Chicago, IL. All test beverages were obtained from the same production batch to ensure consistency and were stored at 4 ˚C under refrigeration to preserve shelf life and to minimize the risk of contamination.

Study protocol

The study protocol was identical for both adults and children (Fig. 2). Participants were asked to refrain from oral hygiene practices for at least 24 h prior to the study and were restricted from eating and drinking (except water) from 10 p.m. the evening before the day of testing. Adult participants read and signed informed consent forms prior to participation. For children, participants and parents/guardians completed informed consent forms (assent and consent). All subjects were given numerical identification numbers for record keeping throughout the study.

Fig. 2
Fig. 2
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Study protocol

Plaque sample collection, processing, and subsequent assays were conducted according to the validated methods originally described by White et al.60. On the day of testing, using a cotton swab (CONSTIX SC-9, CONTEC, Spartanburg, SC, USA), overnight supragingival dental plaque samples were collected from the buccal and lingual surfaces along the gingival margin in the maxillary and mandibular left quadrants60. No plaque-staining dyes were used. These non-exposed plaque samples were designated as “Pre-” samples (Fig. 3). Participants then consumed 30 ml of one of two test juices or water (Control) in a ‘hold and consume’ manner, where they held and swished 10 ml of the juice in their mouth for 30 s to allow contact with the plaque bacteria on tooth surfaces before swallowing. This step was repeated two more times, with a 30-second wait between each repetition. This method, based on a previous pilot study, was found to be more effective than consuming 30 ml of juice all at once and simulated the ‘sipping’ action typical when consuming a beverage. All participants were able to follow the instructions given and adhered to the protocol. Thirty minutes after consuming the test beverage, supragingival plaque samples from the maxillary and mandibular right quadrants were collected (designated as ‘Post-’ samples). No eating or drinking was permitted during the plaque sampling and collection period. All plaque samples were placed in glass tubes on ice until further laboratory processing60. After a three-day washout period, participants revisited the clinic and repeated this procedure with a different test juice. The order of test juice consumption was randomized with a flip of a coin.

Fig. 3
Fig. 3
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Schematic representation of Plaque Glycolysis and Regrowth Method (PGRM) for assessing the efficacy of in vivo cranberry juice exposure on human dental plaque regrowth and glycolysis.

Plaque glycolysis and regrowth method (PGRM)

All plaque samples, both before (Pre-) and after (Post-) exposure, were processed in vitro using the PGRM method60(Fig. 3). This method facilitates direct in vivo exposure of naturally formed dental plaque biofilms to test juices, as well as the natural clearance of these juices from the oral cavity after use. It provides highly sensitive and reproducible estimates of antimicrobial efficacy against bacterial growth and metabolism in plaque following in vivo application. The reproducibility of the method is ensured by comparing the responses of treated plaque samples to those of untreated dental plaque collected from the same individual’s mouth, after adjusting each plaque sample to a similar biomass optical density (OD) prior to incubation in growth/metabolism media. The PGRM method is routinely performed at Wu’s laboratory with consistent results.

All Pre- and Post- plaque samples were resuspended in 0.03% Tryptic Soy Broth (TSB, BD, Becton, Dickinson and Company, Sparks, MD, USA), vortexed for 30 s and adjusted to OD600 nm = 0.20. The normalized plaque samples were kept on ice prior to testing. For acid production, 950 µl of normalized plaque sample was mixed with 50 µl 40% sucrose, incubated at 37 °C in a Thermomixer (Thermo Scientific, FisherScientific) at 1,200 rpm. Acid production (pH) was measured at 0, 2 and 4 h using a micro-pH electrode (InLab Micro Combination pH Electrode, METTLER TOLEDO), which was calibrated prior to use. Changes in acid production (ΔpH) were defined as the pH difference between Post- and Pre-exposure, calculated using the following formulas: ΔpH = ΔpHPost - ΔpHPre; ΔpHPost 2–4 h = pHPost 2–4 h - pHPost 0 h; and ΔpHPre 2–4 h = pHPre 2–4 h - pHPre 0 h.

For regrowth assay, 300 µl of normalized plaque was mixed with 650 µl of 5% TSB and 50 µl of 40% sucrose. Regrowth (OD600 nm) was measured (Spectronic 20 Genesys, Spectronic instruments, UK) at 0, 2 and 4 h after incubation at 37 °C. For experimental blank, 300 µl of 0.03% TSB without plaque was mixed with 650 µl of 5% TSB and used. The effect of the test juices on the regrowth of exposed dental plaque bacteria was expressed as the ratio of ODpost/ODpre.

Statistical analysis

Statistical analysis was conducted using SPSS software (IBM Corp., Armonk, NY, USA). Normality of data was evaluated by visual inspection of Q-Q plots. ANOVA and paired t-tests were employed to determine significant differences among the test beverages, with significance set at a p-value of < 0.05. Differences among the groups were further explored using Tukey’s multiple comparison test. Data from each study was not pooled and were analyzed separately.