Enhanced dominance of nitrate-reducing bacteria using a combination of nitrate and erythritol in in vitro cultured oral biofilm

ABSTRACT

Background

Oral nitrate-reducing bacteria are associated with good oral health, with inorganic nitrate specifically promoting the growth of these beneficial bacteria. Sugar alcohols affect the composition of oral microbiota, potentially impacting oral health. The present study aimed to investigate the combined effects of nitrate and sugar alcohols on nitrate-reducing bacteria and nitrate metabolism in oral microbiota cultured in vitro.

Methods

Species-level microbial analysis using 16S rRNA gene sequencing of DNA extracted from the supragingival plaque-derived biofilm cultured under micro-aerobic conditions for 48 h with nitrate and/or sugar alcohols was conducted. Nitrate metabolites, lactate, and pH in culture supernatants were also measured.

Results

The combined addition of nitrate and erythritol, but not xylitol or sorbitol, significantly increased the relative abundance of Haemophilus parainfluenzae and Neisseria subflava, which are nitrate-reducing bacteria. This shift was accompanied by a corresponding decrease in Streptococcus oralis, which simultaneously induced an increase in the nitrate-reducing capacity and a decrease in lactate production and acidification from sugar metabolism.

Conclusions

The combination of nitrate and erythritol serve as a preventive and therapeutic approach for periodontitis or dental caries by promoting the growth of oral commensal nitrate-reducing bacteria. However, human clinical studies are required to clarify these beneficial effects.

KEY MESSAGES

• The combined addition of nitrate and erythritol significantly increases both the relative abundance of nitrate-reducing bacteria and the nitrate-reducing capacity.

• This combination also significantly reduces the relative abundance of Streptococcus oralis and the lactate production and acidification resulting from sugar metabolism.

• The nitrate-erythritol combination may serve as a preventive and therapeutic strategy for periodontitis or dental caries.

Introduction

The oral cavity houses diverse bacterial microbiota, containing approximately 700 different microorganisms [1–3]. Prevention and treatment of oral diseases, such as dental caries and periodontal disease, have primarily focused on reducing pathogenic bacteria in dental plaque using antibacterial agents, such as cetylpyridinium chloride (CPC), chlorhexidine, and triclosan [4–6], along with physical disruption through tooth brushing. However, in recent years, it has become evident that modifying the composition of the commensal oral microbiota can enhance oral health [7,8]. Notably, oral microbiota plays a role in maintaining not only oral health but also systemic health, including blood pressure regulation [9–11].

In the last decade, nitrate-reducing bacterial genera within the commensal microbiota, such as Actinomyces, Corynebacterium, Rothia, Haemophilus, Neisseria, and Veillonella, have attracted considerable attention for their role in nitrate-to-nitrite conversion in the oral cavity [12–17]. Nitrate-to-nitrite conversion by nitrate-reducing bacteria is an important step in nitric oxide (NO) production [13–17]. NO is a signaling molecule with antimicrobial properties [18] and plays an important role in regulating physiological functions, including vasodilation, nervous transmission, and host defense [13,15]. Although NO was initially considered to be synthesized from L-arginine via the three isoforms of NO synthetases (NOS) [19], it can also be produced from inorganic nitrate via an NOS-independent pathway by nitrate-reducing bacteria [20,21]. Two pathways of NO production are illustrated in Supplementary Figure S1. Nitrite, produced by oral nitrate-reducing bacteria, is chemically reduced to NO in acidic or hypoxic environments, such as those found in the oral cavity or gastrointestinal tract [14,15]. In addition, nitrite can be further enzymatically reduced to NO by specific nitrate-reducing bacteria in the oral cavity [13]. Notably, an enhanced abundance of nitrate-reducing bacteria in the oral cavity may suppress dental caries and periodontal disease [20,22–25]. Therefore, modifying the oral microbiota to increase the composition of nitrate-reducing bacteria could represent a novel and effective preventive and therapeutic approach for oral diseases such as dental caries and periodontal disease.

The composition of oral commensal microbiota, including nitrate-reducing bacteria, is influenced by various factors, such as diet, smoking, alcohol consumption, antimicrobial agent use, and pregnancy [26–29]. For example, inorganic nitrate, which is found in vegetables such as spinach, beets, and lettuce [30], increases the relative abundance of health-associated nitrate-reducing bacteria, including Rothia and Neisseria, in in vitro culture systems of human dental plaque or saliva [22,31]. In humans, the consumption of nitrate-rich lettuce juice for two weeks decreases gingival inflammation, increases the abundance of periodontal health-associated Rothia and Neisseria, and reduces the abundance of periodontitis-associated Prevotella, Fretibacterium, and Treponema in subgingival plaque [32]. In addition, xylitol alters the oral microbiota and increases the relative abundance of Neisseria, as demonstrated by in vitro culture systems using human saliva [33].

Sugar alcohols, such as xylitol, erythritol, and sorbitol, are commonly used as sugar substitutes in many foods and beverages [34], owing to their low risk of causing dental caries [34]. In human clinical studies, the consumption of erythritol or xylitol, but not sorbitol, was found to decrease Streptococcus mutans, a dental caries-associated bacterium, in dental plaque and saliva [35,36]. Nevertheless, the effects of sugar alcohol consumption on oral nitrate-reducing bacteria remain unclear [37]. Moreover, while nitrate and sugar alcohols increase the relative abundance of nitrate-reducing bacteria in vitro, their combined effects require further investigation.

In the present study, we aimed to determine the combined effects of nitrate and sugar alcohols on nitrate-reducing bacteria in vitro. Figure 1 provides a schematic overview of the experimental protocol used in our study with supragingival plaque-derived biofilms cultured in vitro.

Figure 1.

 Schematic diagram of the overall experimental protocol performed in our study.

Methods

Human supragingival plaque sampling and preparation of plaque solution

Supragingival plaque samples were collected from 12 orally and systemically healthy Japanese adults (8 men and 4 women) aged 25–55 years. Exclusion criteria were as follows: having systemic diseases such as diabetes, heart disease, kidney disease, liver disease, or congenital heart disease; receiving medical treatment for oral diseases; undergoing orthodontic treatment; wearing dentures; being pregnant; taking prescribed medications for illness; and using mouth rinses containing antibiotics. Participants were instructed to use only specified toothpaste without antibiotics (e.g. chlorhexidine, CPC) for oral care one week prior to plaque collection. On the morning of the collection, they were asked to refrain from oral care until after plaque collection, and after breakfast, they were required to abstain from eating or drinking for at least 1 h before plaque collection. All available supragingival plaque was collected using sterile dental curettes before lunch. The plaque collected from each participant was washed twice with sterile phosphate-buffered saline (PBS). After washing with PBS, the collected plaque was vigorously agitated and suspended in 300 µL of brain heart infusion (BHI) broth (BD Biosciences, Franklin Lakes, NJ, USA) containing 15% glycerol (Abcam, Cambridge, UK). To avoid repeated freeze-thaw cycles, each plaque solution was divided into several aliquots and stored at −80°C until use.

DNA extraction from the plaque solution and quantification of total bacterial 16S rRNA gene copies using quantitative polymerase chain reaction (qPCR)

A total of 200 μL of Enzyme Lysis buffer, containing 20 mM Tris-HCl, pH8.0 (Nippon Gene, Tokyo, Japan), 2 mM EDTA (Nippon Gene), 1.2% Triton-X 100 (Cayman Chemical, Ann Arbor, MI, USA) and 20 mg/mL lysozyme (Fujifilm Wako Pure Chemical, Tokyo, Japan) was added to 20 μL of thawed plaque solution, followed by incubation at 37 °C for 60 min. DNA extraction was performed using enzyme treatment followed by the DNeasy Blood & Tissue Kits (QIAGEN, Hilden, Germany), following the manufacturer’s instructions.

Quantification of total bacterial 16S rRNA gene copies in the extracted DNA was performed using a TaqMan® real-time qPCR kit, the bacterial universal primers (including the forward primer 5′-TCCTACGGGAGGCAGCAGT-3′ and the reverse primer 5′-GGACTACCAGGGTATCTAATCCTGTT-3′), and the probe (FAM)-5′-CGTATTACCGCG- GCTGCTGGCAC-3′-(TAMRA) [38]. The reaction mixture (10 μL) contained 0.9 μM of each primer, 0.25 μM of the probe, 4 μL of the DNA template, and 5 μL of the universal qPCR mix (TaqMan® Fast Universal PCR Master Mix 2×; Applied Biosystems, Foster City, CA, USA), and was amplified in Fast Optical 96-Well reaction plates (Applied Biosystems) sealed with optical adhesive film (Applied Biosystems). Thermal cycling (95°C for 20 s followed by 40 cycles at 95°C for 3 s and 60°C for 45 s) was performed using a 7500 Fast Real-Time PCR machine. The bacterial gene copy number standard, consisting of the 16S rRNA gene of Neisseria mucosa inserted into the cloning vector pUC18 (Stratagene, San Diego, CA, USA), was supplied by GenScript (GenScript Japan, Tokyo, Japan). A standard curve for quantifying bacterial gene copy number was generated using a range of 2 × 10⁴ to 2 × 10⁸ copies per qPCR reaction.

Preparation of the nitrate and sugar alcohol solution and in vitro oral biofilm culture using supragingival plaque solution

Each reagent solution containing potassium nitrate (KNO₃), erythritol, xylitol, and sorbitol (all purchased from Fujifilm Wako Pure Chemical) was prepared by dissolving each in BHI broth containing 5% glucose and sterilizing the solutions using 0.22 µm membrane filters (Millex-GP; Millipore Co, Burlington, MA, USA). The final concentrations of nitrate (50 mM) [22] and sugar alcohols (10%) [39] used in our study were selected based on their strong inhibitory effects on the plaque or oral bacteria-derived biofilm mass. In addition, the commercially available xylitol gum used in a previously published human study (Epic Spearmint; 1.5 g/pellet) contains 70% xylitol (1.05 g/pellet) [37]. Eight groups were prepared as follows: A: Control; B: 10% erythritol; C: 10% xylitol; D: 10% sorbitol; E: 50 mM nitrate; F: 50 mM nitrate + 10% erythritol; G: 50 mM nitrate + 10% xylitol, and H: 50 mM nitrate + 10% sorbitol. In addition, the media of all eight groups contained 0.3% D-glucose, because BHI broth used in our study contains 0.3% dextrose (D-glucose) as one of its components. Each group was prepared by combining a prepared nitrate solution (100 mM) with a sugar alcohol solution [20%(W/V) erythritol, 20%(W/V) xylitol, and 20%(W/V) sorbitol]. An inoculum plaque solution (10 μL) containing 10⁷ copies of the total bacterial 16S rRNA gene was prepared by diluting defrosted plaque solution with BHI broth, based on the quantification of total bacterial 16S rRNA gene copies in the plaque solution, as calculated in the previous section. The inoculum plaque solution (10 μL) was added to 1 mL of a BHI broth containing each reagent, and the mixture was aliquoted in triplicate into 24-well poly-L-lysine coated culture plates (Iwaki Glass, Funabashi, Japan) to support biofilm formation in vitro [40,41]. The plates were incubated without shaking at 37 °C for 48 h under micro-aerobic conditions using the AnaeroPack™ MicroAero system (Mitsubishi Gas Chemical Inc., Tokyo, Japan), which maintains micro-aerobic conditions of 6–12% O₂ and 5–8% CO₂ [42]. After cultivation for 48 h, the culture supernatant was carefully transferred to a new 1.5 mL tube from each well of the culture plate and centrifuged at 10,000 × g for 5 min to remove most of the bacteria. A 200 μL of the culture supernatant was used for pH measurement. The remaining culture supernatant was stored at −80°C until subsequent analyses of lactate, nitrate, nitrite, and ammonium. After removing the supernatant, the remaining biofilm was used for DNA extraction.

pH measurement

The pH of the undiluted culture supernatant was measured using a compact pH meter (LAQUAtwin, Horiba, Kyoto, Japan).

DNA extraction from in vitro cultivated biofilm

DNA extraction from in vitro cultivated biofilm was performed using enzyme treatment and DNeasy Blood & Tissue Kits, as described above. A total of 200 μL of Enzyme Lysis buffer containing 20 mM Tris-HCl (pH8.0), 2 mM EDTA, 1.2% Triton-X 100, and 20 mg/mL lysozyme was added to the cultured biofilm in a plate well and incubated at 37 °C for 60 min. DNA extraction was performed following the manufacturer’s instructions. The extracted DNA was divided into two microtubes and stored at −80°C until further use for the quantification of total bacterial DNA content and 16S rRNA gene sequencing.

Quantification of total bacterial DNA content in the cultivated biofilm

Quantification of total bacterial DNA extracted from the cultivated biofilm was performed using Qubit dsDNA HS Assay Kits and Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions.

16S rRNA gene sequencing and data processing

he microbial composition of the supragingival plaque inoculum and in vitro cultivated biofilm was assessed using high-throughput sequencing of the V3-V4 region of the 16S rRNA gene using the Illumina MiSeq Platform (Illumina Inc.) at the Genome-lead incorporation (Kagawa, Japan). The V3-V4 regions of the 16S rRNA genes from each sample were amplified using the following primers: 341F (5′- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNCCTACGGGNGGCWGCAG-3′) and 806 R (5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGN GACTACHVGGG TATCTAATCC-3′), which can amplify approximately 460 bp of the V3–V4 region. The amplicon PCR products were purified using AMPure XP (Beckman Coulter, Brea, CA, USA) after confirming amplification via agarose gel electrophoresis. Fifty microliters of AMPure XP were used for each sample, purified following the reagent protocol, and eluted into 50 μL of 10 mM Tris-HCl (pH 8.5). Index PCR was performed using the Nextera XT Index Kit (Illumina) to incorporate bound barcode identifiers. The Index PCR product concentration was determined using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and pooled for library preparation. The pooled library was quantified using a Qubit Fluorometer 2.0 (Life Technologies, Carlsbad, CA, USA) and diluted to a final concentration of 10 pM, with 40% phiX Control v3 (Illumina) included as an internal control. Next-generation sequencing was performed using the MiSeq platform (MiSeq Reagent V3, 600 cycles; Illumina). The read length was 301 bp with paired-end sequencing. The obtained reads exhibited a cluster density of 769 K/mm², a passing filter of 95.05%, and an average Q30 of 86.77%.

All 16S rRNA raw sequence data were analyzed using the QIIME2 pipeline (version 2021.2) [43]. DADA2 plugin [44] was used to join paired-end reads, filter sequencing reads, and construct amplicon sequence variants. The sequence reads were rarified to 12,057 reads per sample before further analysis. Exact amplicon sequence variants (ASVs) in each sample were identified, and an ASV table was constructed. Each ASV was taxonomically classified by performing a BLAST search against 319 oral bacterial 16S rRNA gene sequences (16S rRNA RefSeq version 16.01) in the Expanded Human Oral Microbiome Database (eHOMD) [45]. The nearest-neighbor species, defined as those over 99.5% identity, were selected as candidates for each ASV. Alpha diversity (Chao1 and Shannon indices) analysis was performed using the phyloseq package in R software. Beta diversity was assessed using principal component analysis based on nonmetric multidimensional scaling (NMDS) ordination with Bray – Curtis distances, derived from abundance tables of microbial taxa at the species level. Differential abundance analysis was performed using Analysis of Compositions of Microbiomes with Bias Correction 2 (ANCOM-BC2) to account for sample- and taxon-specific bias [46].

Lactate, nitrate, nitrite, and ammonium measurements

After thawing at room temperature, the lactate concentration in the culture supernatant was diluted 1:121 with ultrapure water and measured using the Lactate Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer’s instructions. The nitrate and nitrite concentrations in the culture supernatant, also diluted 1:121 with ultrapure water, were measured using the NO₂/NO₃ Assay kit-FX (Fluorometric) 2,3-Diaminonaphthalene Kit (Dojindo Laboratories), which is based on the Griess reaction, following the manufacturer’s instructions. The ammonium concentration in the culture supernatant was diluted 1:242 with ultrapure water and measured using the Ammonia Assay Kit for 200FL (Sigma Aldrich, MO, USA), following the manufacturer’s instructions.

Statistical analyses

Alpha diversity across the nine groups containing inoculum was compared using p-values adjusted with the Wilcoxon rank sum test, followed by Bonferroni correction for multiple tests. For differences in beta diversity between the nine groups containing inoculum, a permutational multivariate analysis of variance (PERMANOVA) was performed. The log-fold changes in microbiome composition were performed using ANCOM-BC2 with P-values adjusted using the Benjamini – Hochberg correction method. All statistical analyses, except for the alpha/beta diversity and the log-fold changes in microbial composition, were performed using the IBM^TM SPSS^TM Statistics version 28 software for Windows (Redmond, WA, USA). Numerical data are expressed as mean ± standard deviation (SD). Comparisons among various groups were performed using the Kruskal – Wallis test, followed by Bonferroni correction for multiple tests. Correlations among parameters of the cultured supernatant fluid and between microbiota and parameters of the cultured supernatant fluid were analyzed using Spearman’s correlation test. Spearman’s correlation analysis was performed for four groups containing 50 mM nitrate or all eight groups to evaluate the influence of nitrate addition, with r values of 0.7–1.0, 0.5–0.699, and 0.2–0.499 indicating strong, moderate, and weak correlations, respectively. p < 0.05 was considered statistically significant.

Results

Total bacterial DNA content is unaffected by the single or combination addition of nitrate and sugar alcohols

In the Kruskal – Wallis test with post-hoc Bonferroni’s correction among the eight groups, the amount of total bacterial DNA in the supragingival plaque-derived biofilm after 48 h of culturing was not influenced by the single or combined addition of nitrate and any sugar alcohol compared to the control group. However, the difference between the nitrate and nitrate + sorbitol groups was statistically significant (Figure 2).

Figure 2.

 Amount of total bacterial DNA of supragingival plaque-derived biofilm after cultivation for 48 h with or without either combined or single additions of nitrate and sugar alcohols. Bar plots indicate means and standard deviations of the amount of total bacterial DNA in culture biofilm from 12 donors. A comparison among eight groups was performed using the Kruskal–Wallis test, followed by Bonferroni correction for multiple tests. *p < 0.05.

Effects of the combined addition of nitrate and erythritol on microbial diversity

The microbiota of the inoculum exhibited significantly greater alpha diversity compared to all eight groups cultured in vitro (Figure 3a). The microbiota of the nitrate + erythritol group demonstrated significantly greater alpha diversity in Shannon indices (p < 0.01) than that of the control group (Figure 3a). In contrast, the microbial alpha diversity of the nitrate + xylitol and nitrate + sorbitol groups was not significantly different from that of the control group (Figure 3a).

Figure 3.

 Microbial diversity analysis. (a) Box plots of alpha diversity (Chao1 and Shannon indices) at the species level showing the richness and diversity of species in the samples. Comparisons of alpha diversity across the nine groups containing inoculum were performed with p-values adjusted using the Wilcoxon rank sum test, followed by Bonferroni correction for multiple tests. *p < 0.05; **P  < 0.01. (b) Comparisons of microbial community structure across the nine groups containing inoculum were performed using NMDS analyses based on the Bray–Curtis distances at the species level. The accompanying table indicates the p-value from the PERMANOVA, highlighting the significant differences between groups in the multivariate analyses.

In addition, a beta diversity analysis based on the Bray – Curtis distance was performed using NMDS ordinations. The results indicated that the microbial community structures of the nitrate + erythritol group were significantly different (PERMANOVA; Bray – Curtis dissimilarity; F model = 26.762; R² = 0.549; p = 0.0036) from those of the control group (Figure 3b). In contrast, the microbial community structures of the nitrate + xylitol and nitrate + sorbitol groups were not significantly different (F model = 2.748; R² = 0.111; p = 1 and F model = 0.171; R² = 0.008; p = 1, respectively) from those of the control group (Figure 3b).

Combined addition of nitrate and erythritol significantly influences microbiota composition

The species with a mean relative abundance exceeding 1% in dental plaque before culture were, in descending order of relative abundance, as follows: Streptococcus oralis (10.0%), Veillonella parvula (9.1%), Haemophilus parainfluenzae (7.9%), Fusobacterium polymorphum (6.8%), Streptococcus sanguinis (3.3%), Porphyromonas pasteri (3.1%), Leptotrichia hongkongensis (3.0%), Campylobacter gracilis (2.5%), Neisseria subflava (2.3%), Rothia dentocariosa (2.2%), Neisseria mucosa (2.1%), Neisseria elongata (2.0%), Actinomyces oris (2.0%), Cardiobacterium hominis (1.7%), Corynebacterium matruchotii (1.7%), Granulicatella adiacens (1.6%), Prevotella nigrescens (1.6%), Leptotrichia hofstadii (1.5%), Kingella oralis (1.4%), Capnocytophaga leadbetteri (1.3%), Streptococcus gordonii (1.3%), Capnocytophaga gingivalis (1.2%), Corynebacterium durum (1.2%), Capnocytophaga sputigena (1.1%), Rothia aeria (1.1%), Lautropia mirabilis (1.1%), and Streptococcus lactarius (1.0%) (Figure 4). In contrast, in the control group incubated in BHI broth at 37 °C for 48 h under micro-aerobic conditions, five species, namely S. oralis (68.8%), V. parvula (8.3%), H. parainfluenzae (1.0%), G. adiacens (5.1%), and S. lactarius (3.7%), had a mean relative abundance above 1% (Figure 4). As indicated by the results presented in Figure 4, the combination of nitrate and sugar alcohols influenced the abundance of nitrate-reducing bacteria. Consequently, we conducted ANCOM-BC2 analysis to assess the log-fold changes in the differential abundances of bacteria in four groups containing nitrate – nitrate, nitrate + erythritol, nitrate + xylitol, and nitrate + sorbitol – compared to the control (Figure 5). This revealed significant differences in the nitrate + erythritol group, including a decrease in S. oralis and increases in H. parainfluenzae and N. subflava (Figure 5). However, the single addition of nitrate or the combined addition of nitrate and xylitol or sorbitol did not significantly affect the relative abundance of H. parainfluenzae, N. subflava, or S. oralis (Figure 5).

Figure 4.

 Bacterial composition of the oral bacterial. Composition of the top 27 species, constituting over 1%, and other species, constituting less than 1% of the oral bacteria in the plaque inoculum.

Figure 5.

 A heatmap of log-fold changes in bacterial differential abundance of the four assessed groups containing nitrate compared to the control based on ANCOM-BC2 analysis. Red and blue colors indicate increased and decreased abundance in the comparison group compared to the control, respectively. After cultivation for 48 h in the presence of nitrate alone or combined with a sugar alcohol, only species with a mean relative abundance greater than 0.5% of the total oral bacterial community were analyzed. Statistical significance for multiple comparisons versus the control was determined following Benjamini – Hochberg correction. *p < 0.05; **P  < 0.01.

Effects of the combined addition of nitrate and erythritol on acidification and lactate concentration

The culture supernatant fluid of the control exhibited low pH (pH 5.1 ± 0.3) and high lactate concentration (20.9 ± 6.0 mM) (Figure 6a,b), despite the initial pH of the BHI broth before cultivation being between 7.2–7.4, and the lactate concentration in the BHI broth was approximately 2 mM (data not shown). In contrast, when nitrate was added in combination with erythritol, the pH of the culture supernatant fluid significantly increased (p < 0.05), whereas lactate significantly decreased (p < 0.05), compared to those of the control (Figure 6a,b). In the erythritol group, the lactate concentration significantly decreased compared to that of the control group, whereas the pH did not significantly change. In addition, when nitrate was added in combination with xylitol or sorbitol, neither the pH nor lactate concentration significantly changed compared to those of the control group (Figure 6a,b).

Figure 6.

 Effects of combining nitrate and sugar alcohols on (a) pH and (b) lactate levels of the culture supernatant. Bar plots indicate means and standard deviations of pH and lactate levels in culture supernatant from 12 donors. A comparison among eight groups was performed using the Kruskal–Wallis test, followed by Bonferroni correction for multiple tests. *p < 0.05; **p < 0.01.

Combined addition of nitrate and erythritol increases nitrate metabolism

As nitrate metabolism depends on the amount of nitrate added, the effect of sugar alcohols on nitrate metabolism was compared across the four groups containing nitrate. The nitrate concentration of the culture supernatant fluid in the nitrate group was 39.7 ± 18.1 mM. Upon adding nitrate to the culture with erythritol, the nitrate concentration of the culture supernatant fluid significantly decreased (p < 0.05) compared to that in the nitrate-only group (Figure 7a). In contrast, the concentration of nitrate in the culture supernatant fluid in the nitrate + xylitol and nitrate + sorbitol groups was not significantly different from that in the nitrate group (Figure 7a). There was a significant difference (p < 0.05) in the amount of nitrite in the culture supernatant between the nitrate + erythritol and nitrate + sorbitol groups (Figure 7b). When nitrate was added in combination with erythritol, the ammonium concentration of the culture supernatant significantly increased (p < 0.05) compared to that in the nitrate + xylitol group. However, this increase was not significant compared to that in the nitrate group (Figure 7c).

Figure 7.

 Effects of combining nitrate and sugar alcohols on (a) nitrate, (b) nitrite, and (c) ammonium levels in the culture supernatant. Bar plots indicate means and standard deviations of nitrate, nitrite, and ammonium levels in the culture supernatant from 12 donors. Comparison among four groups containing nitrate was performed using the Kruskal – Wallis test followed by Bonferroni correction for multiple tests. *p < 0.05, **p < 0.01.

Correlations among pH, lactate, nitrate, nitrite, ammonium, and microbiota

To evaluate the influence of nitrate addition, Spearman’s correlation analysis was conducted to assess the relationship between pH, lactate, nitrate, nitrite, and ammonium levels in cultures, either for all eight groups or specifically for the four groups of cultures containing nitrate. Correlation analysis of all eight groups revealed a strong negative correlation between pH and lactate (p < 0.01), suggesting that lactate production is the driver of acidification. In contrast, no correlation between pH and nitrate was observed (Table 1). The correlation analysis performed only for the four groups containing nitrate revealed a strong negative correlation between pH and lactate (p < 0.01), strong negative correlation (suggesting that lower nitrate levels are associated with more alkaline pH values) between pH and nitrate (p < 0.01), and strong positive correlation between lactate and nitrate (p < 0.01; Table 2).

Table 1.

Spearman’s correlation analysis of the correlations among physiological parameters (pH, lactate, nitrate, nitrite, and ammonium) in all eight groups.

	pH	Lactate	Nitrate	Nitrite	Ammonium
pH	1.000	−0.753**	−0.098	0.573**	0.498**
Lactate		1.000	0.293**	−0.416**	−0.221*
Nitrate			1.000	0.291**	−0.213*
Nitrite				1.000	0.165
Ammonium					1.000

Spearman’s r values < 0 indicate a negative correlation, while r values > 0 indicate a positive correlation. Strong correlations with r values > 0.7 are indicated in bold.

**p < 0.01, *p < 0.05.

Table 2.

Spearman’s correlation analysis of the correlations among physiological parameters (pH, lactate, nitrate, nitrite, and ammonium) in the four groups containing nitrate.

	pH	Lactate	Nitrate	Nitrite	Ammonium
pH	1.000	−0.892**	−0.881**	0.593**	0.610**
Lactate		1.000	0.952**	−0.655**	−0.405**
Nitrate			1.000	−0.648**	−0.374**
Nitrite				1.000	0.202
Ammonium					1.000

Spearman’s r values < 0 indicate a negative correlation, while r values > 0 indicate a positive correlation. Strong correlations with r values > 0.7 are indicated in bold. **p < 0.01.

Spearman’s correlation analysis between the parameters of the cultured supernatant (pH, lactate, nitrate, nitrite, and ammonium) and the microbiota in the four groups containing nitrate was performed (Supplementary Table S1). In the correlation analysis with nitrate levels, S. oralis exhibited the strongest positive correlation (p < 0.01). In contrast, H. parainfluenzae had the strongest negative correlation (p < 0.01), followed by N. subflava (p < 0.01) and V. parvula (p < 0.01) (Supplementary Table S1). In the correlation analysis with nitrite levels, V. parvula exhibited the strongest positive correlation (p < 0.01), followed by H. parainfluenzae (p < 0.01) (Supplementary Table S1).

Discussion

The present study suggests that the combined addition of nitrate and erythritol significantly increased the relative abundance of H. parainfluenzae and N. subflava, which are nitrate-reducing bacteria, with a significant corresponding decrease in S. oralis.

In a previous study, Mazurel et al. [22] reported that after 12 h of growth under anaerobic conditions in an in vitro subgingival plaque culture system, the addition of 50 mM nitrate significantly increases the relative abundance of the nitrate-reducing bacteria a Neisseria sp. and a Rothia sp. compared to the control. In our study, compared to the control, the single addition of nitrate did not significantly increase the relative abundance of N. subflava, N. flavescens, or R. dentocariosa after 48 h of growth under micro-aerobic conditions in an in vitro plaque culture system using frozen supragingival plaque. This inconsistency may be attributed to the reduced growth of nitrate-reducing bacteria resulting from the use of frozen plaque in our study. However, we demonstrated that the combined addition of nitrate and erythritol significantly increased the relative abundances of H. parainfluenzae and N. subflava, and along with a corresponding decrease in S. oralis. These results suggest that the combined addition of nitrate and erythritol significantly enhances their individual effects on specific nitrate-reducing bacteria and S. oralis. In addition, alpha and beta diversity analyses indicated that the combined addition of nitrate and erythritol significantly altered the microbial community diversity compared to the control. Notably, the combined addition of nitrate and either xylitol or sorbitol had no significant effect on the relative abundances of H. parainfluenzae, N. subflava, and S. oralis, nor the diversity of the microbial community.

Most oral bacteria are neutrophilic, with an optimal growth pH of approximately 7. However, many Streptococcus spp. are resistant to low pH and can grow well even under acidic conditions [47]. In our study, BHI broth containing 0.3% glucose caused the pH to drop below 6 after 48 h of cultivation. Under low pH conditions, the growth of several bacterial species, including H. parainfluenzae and N. subflava, may be inhibited. In addition, the single addition of erythritol, but not xylitol or sorbitol, significantly reduced lactate levels. However, it did not significantly affect pH after 48 h of cultivation. Our findings suggest that erythritol may maintain an elevated pH by reducing lactate levels through the growth inhibition of S. oralis, thereby supporting the growth of low-pH-sensitive bacteria, such as H. parainfluenzae and N. subflava. Collectively, our results suggest that, when combined with erythritol, nitrate may exert strong effects on H. parainfluenzae and N. subflava by maintaining an elevated pH level.

Many oral Streptococcus spp., including S. oralis, S. salivarius, S. gordonii, S. mitis, and S. mutans, produce lactate to varying degrees [48–52]. Lactate, acetate, formate, and pyruvate are the primary factors that lower the pH of the oral cavity and contribute to the development of dental caries [53]. In our study, the correlation analysis between all eight groups revealed no correlation between pH and nitrate and a weak positive correlation between lactate and nitrate. Notably, when correlation analysis was performed for only the four groups containing nitrate, we observed a strong negative correlation between pH and nitrate and a strong positive correlation between lactate and nitrate. These results are consistent with those of a previous report in which 6.5 mM nitrate alone was added to saliva-derived biofilm in vitro [31]. Our results suggest that the decrease in lactate and the increase in pH are associated with nitrate metabolism. Human saliva contains a large amount of concentrated nitrate released from plasma, which is then metabolized to nitrite by nitrate-reducing bacteria in the oral cavity [54,55]. Nitrite is further metabolized by oral bacteria through two main pathways: conversion to NO through the denitrification pathway and conversion to ammonium through the dissimilatory nitrate reduction to the ammonium pathway [56]. Lactate is consumed during nitrate metabolism by nitrate-reducing bacteria [57]. Although nitrate, in combination with erythritol, may promote the growth of nitrate-reducing bacteria by maintaining an elevated pH level, as mentioned above, this increase in nitrate-reducing bacteria could also lead to lactate consumption, potentially raising the pH further. Notably, Neisseria, which increased by the combined addition of nitrate and erythritol in our study, is more abundant in children without caries than in those with early childhood caries [58,59]. Thus, the combined addition of nitrate and erythritol may increase pH and induce lactate consumption, potentially preventing caries. However, human clinical studies are needed to confirm these effects.

In our study, the combined addition of nitrate and erythritol significantly increased the relative abundance of specific nitrate-reducing bacteria, which, in turn, substantially enhanced nitrate metabolism. Mazurel et al. [22] reported that the addition of 50 mM nitrate decreased nitrate concentration by approximately 40% after 12 h under anaerobic conditions in an in vitro subgingival plaque culture compared to the initial concentration. In contrast, in our study, although the samples from some donors exhibited decreased nitrate levels in the nitrate group, this overall decrease was not significant. This inconsistency may be attributed to differences in inoculum and culture conditions. Notably, the combined addition of nitrate and erythritol decreased nitrate concentration by approximately 60% compared to the initial nitrate concentration. This significant decrease in nitrate metabolism was accompanied by an increase in nitrate-reducing bacteria, including H. parainfluenzae and N. subflava. NO produced from nitrite denitrification is a free radical known for its extensive antibacterial activities [18,60]. NO is also effective against periodontal disease-associated Porphyromonas gingivalis in a study using NO-releasing materials [61,62]. In our study, the nitrate concentration in the culture supernatant after the combined addition of nitrate and erythritol was significantly lower than that after the addition of nitrate alone. In the correlation analysis of the four groups containing nitrate, S. oralis exhibited the strongest positive correlation with nitrate levels. In contrast, H. parainfluenzae demonstrated the highest negative correlation with nitrate levels, followed by N. subflava. Given that H. parainfluenzae and N. subflava are also the species whose abundance increased the most when nitrate was added in combination with erythritol, it is likely that nitrate metabolism is primarily driven by these species. Notably, unlike other oral nitrate-reducing bacteria, such as V. parvula and R. dentocariosa, both H. parainfluenzae and N. subflava possess the nirK gene that enables metabolism of nitrite to NO [13]. However, in our study, we only measured nitrite and ammonium, not NO, as nitrate metabolites. Moreover, V. parvula exhibited a strong positive correlation with nitrite levels, whereas H. parainfluenzae and N. subflava demonstrated moderate or no positive correlations. These results suggest that H. parainfluenzae and N. subflava, but not V. parvula, can metabolize nitrite to NO.

The supragingival-derived biofilm culture system used in our study has some limitations. In the control group, which was incubated in BHI broth containing 0.3% glucose for 48 h under micro-aerobic conditions, the mean relative abundance of S. oralis was 68.8%, despite being 10.0% in the plaque inoculum. Streptococcus spp. are known to overgrow in vitro in the presence of glucose or sucrose in growth broth media [33,63]. Similarly, in our study, cultivation in BHI broth containing 0.3% glucose also induced the overgrowth of S. oralis and subsequent acidification, which may have reduced the viability of nitrate-reducing bacteria and nitrate-reducing capacity. In addition, the use of frozen plaque inoculum in our study may also have contributed to the observed decrease in nitrate-reducing bacterial viability and nitrate-reducing capacity.

Conclusions

The present study on oral biofilm cultivation using supragingival plaque in vitro demonstrated that the combined addition of nitrate and erythritol significantly increased the relative abundance of H. parainfluenzae and N. subflava. This combination simultaneously decreased the abundance of S. oralis and induced a shift to a nitrate-reducing bacteria-dominant microbiota. As a result, the nitrate-reducing capacity increased, whereas the lactate levels and acidification decreased. These results suggest that the combined addition of nitrate and erythritol may promote an oral cavity environment that is less conducive to the development of dental caries. In addition, the increased abundance of H. parainfluenzae and N. subflava, which possess genes that metabolize nitrite to NO, suggests that NO production may also be enhanced, potentially preventing periodontal disease. Collectively, the combination of nitrate and erythritol may be a preventive or therapeutic option for dental caries and periodontal disease, although human clinical studies are needed to clarify its effects.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Sequencing data are available in the National Center for Biotechnology Information sequence read archive (NCBI SRA) under BioProject PRJNA1218920. All other datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics approval

This study was approved by the Ethics Committee of Kao Corporation (approval number: K0089–2203) and was performed in accordance with the principles of the Declaration of Helsinki. All participants provided written informed consent after receiving a sufficient explanation of the purpose and content of the study.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/20002297.2025.2526069