Salivary nitrate-reduction capacity exhibits age-dependent dynamics and sex differences in adults. Salivary nitrite production is undetectable or minimal in newborns, with infants exhibiting significantly lower nitrite concentrations and oral nitrate-reductase capacity compared to adults (Timby et al., 2020). Nitrate-reduction activity peaks during middle age before declining in older adults (Ahmed et al., 2021). No sex-based differences in salivary nitrate/nitrite levels have been observed at 4–12 months of age (Timby et al., 2020). In contrast, despite comparable oral microbiome structures between sexes, female adults demonstrate higher post-nitrate-supplementation nitrite levels in saliva, plasma, and urine than males (Kapil et al., 2018). This divergence may be influenced by body mass index, lifestyle factors (e.g., diet and smoking), or sex hormones—factors previously linked to NOS activity regulation (Weiner et al., 1994).

Increased dietary nitrate intake, as a selective pressure for nitrate-reducing bacteria, may alter the oral microbiome, especially leading to the abundance of nitrate-reducing bacteria (Moran et al., 2024). Tongue samples of rats exhibited increased relative abundances of Streptococcus and Haemophilus (especially H. parainfluenzae) after nitrate supplementation (Hyde et al., 2014b). In healthy participants, dietary nitrate supplementation selectively regulated the composition of oral microbiota, characterized by a significant increase in the relative abundance of nitrate-reducing genera Neisseria (including N. flavescens, N. subflava) and Rothia (e.g., R. mucilaginosa), alongside a marked decrease in that of Prevotella (e.g., P. melaninogenica), Actinomyces (e.g., A. hyovaginalis), and Veillonella (Velmurugan et al., 2016; Vanhatalo et al., 2018; Burleigh et al., 2019; Rosier et al., 2020a; Moran et al., 2024; Zhang et al., 2025). The detailed study protocols are summarized in Table 1 . Denitrifying species, such as Neisseria and Rothia, are associated with increased systemic NO• levels, whereas DNRA organisms such as Prevotella and Veillonella are associated with low NO• levels (Morou-Bermúdez et al., 2022). Veillonella, which is the most abundant nitrate-reducing genus detected in tongue scrapings (Doel et al., 2005; Bryan et al., 2017), possesses the capacity for powerful nitrate reduction. After nitrate intake, the population of Veillonella has been reported to decreased ( Table 1 ). This discrepancy may be due to a change in oral pH (Rocha and Laranjinha, 2020).

Nitrite production on the tongue of adult humans is greatly reduced after administration of broad-spectrum antibacterial agents (Li et al., 1997). CHX and other antibacterial-containing mouthwashes abolish the effect of sodium nitrate supplementation (Govoni et al., 2008; Kapil et al., 2013; Pinheiro et al., 2016). CHX suppressed bacterial growth by binding and perforating cell membranes and inhibiting bacterial chemotaxis, fiagellar assembly, and lipopolysaccharide (LPS) biosynthesis and has been reported to preferentially target gram-negative bacteria because LPS is their major cell membrane component (Liu et al., 2023b). Short-term treatment with CHX decreased the relative abundance of Prevotella, Fusobacterium, and Selenomonas in hospitalized patients (Liu et al., 2023b). Haemophilus and Aggregatibacter were almost eliminated from the tongues of CHX-treated animals (Hyde et al., 2014b). CHX, as a potent antimicrobial, does not eradicate viable bacteria on the tongue or cause large-scale changes in the microbiome community structure, which would result in a significant reduction in bacterial viability (Tribble et al., 2019). The viability of nitrate-reducing bacteria and other conditional pathogenic bacteria decreased simultaneously after the usage of CHX or similar mouthwash products that do not target specific bacteria. Thus, it is necessary to produce a personalized antibacterial mouthwash that can effectively distinguish different functional bacteria according to different needs of patients and considering various factors.

Nitrate supplementation can increase the oral pH from 7.0 to 7.5 (Hohensinn et al., 2016), and pH 8 is optimal for nitrate reductase activity (vanMaanen et al., 1996). Nitrite can be reduced to ammonium (NH4⁺) and protons, which are consumed in the ANR and DNR pathways, resulting in an increase in the local pH. Additionally, lactic acid can act as an electron donor and carbon source in these pathways, which further modifies the pH (Rosier et al., 2022). In addition, NO• production from nitrite is pH-dependent and is increased at pH values below 5 (Rosier et al., 2022). Generally, an alkaline pH promotes DNRA over denitrification (Morou-Bermúdez et al., 2022). An acidic pH of 6 stimulates the reduction of nitrite more than that under pH 7 or pH 7.5 by the denitrification-related species Rothia in vitro (Rosier et al., 2020b). Furthermore, under acidic conditions, the levels of N₂O (production of NO reduction in the respiratory denitrification pathway) are two-fold higher than those of NO•, suggesting that the reduction of NO• is also pH-dependent (Schreiber et al., 2010). Under low pH, due to the high levels of nitrate or nitrite, certain bacteria and microbial communities capable of nitrate reduction may preferentially survive or expand (Koopman et al., 2016).

Areas with high nitrate reductase activity, such as the tongue dorsum and subgingival plaque, have a low oxygen partial pressure (pO₂, 8–13 mmHg, ~1%–2%) (Koch et al., 2017). The average oxygen concentration detected in the anterior aspect of the tongue is higher than that detected in the posterior portion ( Figure 5 ) (Eskow and Loesche, 1971). The majority of oral nitrate-reducing bacteria are facultative anaerobes that prefer aerobic respiration, but they can grow under anoxic or oxygen-limiting conditions by utilizing the respiratory nitrate reductive pathway. In DNRA, a strictly anaerobic process, Nap expression is highest under low oxygen and nitrate conditions (Stewart, 1994). Respiratory denitrification in dental biofilms occurs under aerobic conditions (Schreiber et al., 2010); however, in an oxygen-limiting environment, Nar expression is only upregulated under high nitrate concentrations (Sparacino-Watkins et al., 2014). The important determining factors of bacterial respiration at specific locations are oxygen tension and nitrate concentration (Koch et al., 2017).

Smoking compromises oral nitrate metabolism, as demonstrated in the study of Bailey et al. (2016), who found that nitrate supplementation could not reduce blood pressure in smokers. These findings can be explained by the cyanide in cigarette smoke, which is enzymatically converted to thiocyanate, leading to elevations in circulating thiocyanate levels (serum/saliva) proportional to smoking intensity (Degiampietro et al., 1987; Bailey et al., 2016). Compared with non-smokers, the concentrations of nitrate are increased and decreased, respectively, in the plasma and saliva of smokers, while higher levels of thiocyanate exist in both plasma and saliva (Bailey et al., 2016). Thiocyanate has the potential to impede the reduction of nitrate to nitrite, or to catalyze nitrite degradation, rather than interfering with salivary nitrate re-concentration (Dewhurst-Trigg et al., 2018). Concurrently, nitrate reductase activity is suppressed by >80% in smokers, directly impairing enzymatic conversion (Ahmed et al., 2017). In addition, unstimulated salivary pH is more acidic in cigarette smokers than in non-smokers, which may have effect on nitrate reduction. Smoking reduces the overall nitrate-reducing capacity (denitrification) and aerobic taxa abundance (Antonello et al., 2023). Jia et al. collected saliva samples from 316 healthy subjects (150 subjects who had never smoked, and 166 smokers), and found that smoking significantly altered the oral microbial composition, characterized by the increased relative abundance of Actinomyces and Veillonella alongside the decreased abundance of Neisseria and Haemophilus (Jia et al., 2021). Thus, the collective impairment arises from thiocyanate, enzymatic suppression, pH alteration, and microbiota imbalance induced by smoking; however, their mechanism remains incompletely resolved.

The subgingival plaque is associated with aerobic or facultative anaerobic bacteria capable of nitrate reduction, including Streptococcus, Rothia, Neisseria, Actinomyces, and Veillonella (Rosier et al., 2022; Kunath et al., 2024). In dental plaque, nitrate can be converted to N₂ via respiratory denitrification under aerobic conditions in a pH-dependent manner (Schreiber et al., 2010), and the DNRA pathway is active in anaerobic environments. Nitrite concentration is increased in the saliva and gingival crevicular fluid of patients with periodontal disease (Reher et al., 2007; Parwani et al., 2012; Sanchez et al., 2014; Topcu Ali et al., 2014). The potential mechanism underlying this phenomenon may involve iNOS activity (Batista et al., 2002; Oner et al., 2024), which is related to disease severity, and increased levels of NO• in gingival tissues and its subsequent oxidation to nitrate and nitrite.

The abundance of Rothia and Neisseria, two representative nitrate-reducing bacteria, is decreased in the subgingival plaque of patients with periodontitis (Wang et al., 2013; Kirst et al., 2015; Feres et al., 2021; Chen et al., 2022) and is negatively correlated with gingival inflammation (Huang et al., 2021; Rosier et al., 2022). Clinical studies have shown that nitrate supplementation attenuates chronic gingivitis by inhibiting gingival inflammation, resulting in an increase in the relative abundances of Rothia and Neisseria in subgingival plaque (Jockel-Schneider et al., 2016; Rosier et al., 2020a; Jockel-Schneider et al., 2021). The abundance of Prevotella, a major pathogen involved in periodontitis, decreases after increased nitrate intake (Vanhatalo et al., 2018; Burleigh et al., 2019). However, the relationship between nitrate-reducing bacterial abundance and periodontitis development remains unclear.

The salivary nitrate concentration is significantly lower in patients with caries, and it is negatively correlated with the severity of caries (Zhang et al., 2021b). Typically, caries occurs at pH < 5.5, and the reduction of nitrite to NO• typically occurs under similar acidic conditions. Ammonium production during DNRA, along with lactic acid and hydrogen sulfide (as electron donors) consumption, contributes to acid prevention (Wicaksono et al., 2020; Rosier et al., 2022; Feng et al., 2023).The abundances of nitrate-reducing bacterial genera, such as Neisseria, Actinomyces, Rothia, Propionibacterium, Haemophilus, Selenomonas, and Granulicatella, and representative nitrate-reducing species such as R. dentocariosa, Selenomonas noxia, Kingella oralis, V. dispar, and other Selenomonas sp. were decreased in patients with caries (Aas et al., 2008; Tanner et al., 2011; Luo et al., 2012; Jiang et al., 2018; Xu et al., 2018; Celik et al., 2021; Yang et al., 2021). Interestingly, Veillonella plays an essential role in the development of caries and closely interacts with caries-associated bacteria in bacterial adhesion, co-aggregation, and biofilm formation (Feng et al., 2023). The relative abundance and prevalence of Veillonella are similar or higher in the oral cavity of patients with caries compared with those in the oral cavity of caries-free individuals (Tanner et al., 2011; Jiang et al., 2018; Qudeimat et al., 2021). Thus, nitrate and nitrate-reducing bacteria can prevent the development of caries by regulating the pH, reducing the accumulation of lactic acid, and increasing denitrification (Li et al., 2007; Rosier et al., 2021). Further studies are required to explore the distribution, prevalence, abundance, interactions, and effects of nitrate-reducing bacteria on caries progression.

Salivary gland disorders, such as Sjogren’s syndrome (SS) or xerostomia, induce a decrease in salivary flow and acidification of the oral cavity’s pH, which affects microorganism colonization (Bustos-Lobato et al., 2023). The oral microbiome of patients with SS significantly differs from that of healthy individuals (Kim et al., 2022; Bustos-Lobato et al., 2023). Salivary gland dysfunction leads to a significant decrease in salivary nitrate concentration and an increase in urinary excretion, leading to changes in enterosalivary circulation (Xia et al., 2003a, Xia et al., 2003b). Changes in salivary nitrate levels may cause an increase in the conversion of nitrite and NO• by nitrate-inducing bacteria. Higher abundances of Veillonella, Neisseria, and Streptococcus have been observed in patients with SS compared with those in patients without SS (Kim et al., 2022), particularly V. parvula in subgingival biofilms (Singh et al., 2021). Interestingly, the abundances of other representative nitrate-reducing species, such as H. parainfluenzae, were significantly lower in SS than those in the controls (Tseng et al., 2021). While there has been no conclusive evidence of a link, the correlations between the nitrate-reducing microbiome composition and salivary gland dysfunction offer a potentially novel avenue for future investigations.

Nitrite and intragastric nitrogen oxides can affect physiological processes in the gastrointestinal tract, such as gastric mucosal blood flow and mucus formation (Bjorne et al., 2004; Petersson et al., 2007; Lundberg et al., 2008; Petersson et al., 2009). A small increase in intragastric NO• can be attributed to gastric or intestinal bacteria that may reduce nitrate to nitrite and NO• (Brittain et al., 1992). The acidic environment of the stomach, which has a pH between 1.5 and 3.5, is a natural barrier for most oral bacteria. Thus, only acid-resistant oral bacteria, such as Streptococcus spp., Veillonella spp., and Prevotella spp., are commonly found in the stomach, but their relative abundances differ (Kunath et al., 2024). Helicobacter, Stenotrophomonas, Haemophilus, Streptococcus, Veillonella, Rothia, Actinomyces, and Prevotella are the major genera in the stomach, as determined by pyrosequencing (Jo et al., 2016). Importantly, the pathogen Helicobacter pylori can neutralize gastric acidity by generating ammonium from urea using urease, enabling its survival and growth in acidic environments. H. pylori infection causes inflammation and alters stomach pH, ultimately reducing acidity, blocking NOS2 expression, and decreasing NO• production (Gobert and Wilson, 2016; Koch et al., 2017; Stewart et al., 2020). After co-culturing with H. pylori, nitrate-reducing bacteria increase the inflammation and atrophy of monocytic cells by modulating cytokine levels (Ojima et al., 2022).

Nitrate supplementation results in a 20% increase in the thickness of the firmly adherent mucus layer; this increase was absent in rats treated with antiseptic mouth spray (Petersson et al., 2009). Another study found that bilateral parotid and submandibular gland duct ligature (BPSDL) completely blocked the enterosalivary circulation of nitrate and significantly decreased the levels of gastric nitrate, nitrite, and luminal NO• in the stomach of rats. The animals in the BPSDL group displayed more severe gastric ulcers than normal rats, and nitrate administration successfully reduced the percentage of deep ulcers (Jin et al., 2013). However, the association between oral/gastrointestinal nitrate-reducing bacteria and gastric homeostasis remains unclear; therefore, the role of oral nitrate-reducing microbiota in gastric disorders warrants further investigation.

Compared with that of healthy individuals, the concentration of nitrate in the plasma of patients with gastroenteritis is high and similar to that in patients with inflammatory bowel disease (IBD) (Dykhuizen et al., 1996). Similarly, rectal NO• concentrations are significantly higher in patients with active IBD (Reinders et al., 2007) compared with that in normal controls. In a previous study, nitrite and nitrate concentrations exhibited variations that were not always in line with the disease activity index (DAI) of a dextran sodium sulfate (DSS)-induced colitis model, ranging from systemic drops to marked increases, indicating the complexity of NO• metabolism in the process of IBD (Saijo et al., 2010). Veillonella (including V. parvula and V. dispar), an important nitrate-reducing bacterium, is commonly enriched in the intestines of patients with IBD (Schirmer et al., 2018; Rojas-Tapias et al., 2022). Nitrate supplementation significantly alleviated epithelial cell necrosis, intestinal permeability, and disruption of tight junctions to prevent hypoxia-induced small intestinal injury (Xu et al., 2024). In aged mice, nitrate supplementation for 6 months via drinking water enhanced the integrity of the colon epithelial barrier and increased the relative abundance of some intestinal probiotics, such as Blautia, Alloprevotella, Butyricicoccus, and Ruminococcaceae (Wang et al., 2024). Gastrointestinal diseases are closely related to abnormal nitrate and NO metabolism, in which nitrate-reducing bacteria play an important role.

Nitrate can alter bacterial communities in the gut; however, the specific interactions between nitrate and gut bacteria remain unknown. Inorganic nitrate supplementation for 1 week or 3 weeks does not affect the gut microbial communities (Conley et al., 2017; Rocha et al., 2019). Previously, our group reported that inorganic dietary nitrate increases the abundance of Lactobacillus and prevents colon epithelial injury induced by total body irradiation (Wang et al., 2020). Oral nitrite supplementation prevents inflammation in DSS-induced colitis by supplying NO• (Ohtake et al., 2010). Similarly, our group previously reported that oral administration of nitrate alleviates DSS-induced colitis by regulating the microbiome in the colon by increasing the abundance of Lactobacillus (regulate intestinal immune response), Ruminococcaceae_UCG-014 (related to short chain fatty acids production), and Prevotellaceae_UCG-001 (Hu et al., 2020), suggesting that nitrate may modulate inflammatory and immune responses in IBD by reshaping the gut bacterial phenotype. Overall, these results show that the beneficial biological effects of nitrate are partially due to its ability to regulate the gut microbiome and that complex nitrate reduction in the gut microbiome needs further exploration.

Type 2 diabetes mellitus (T2DM) results in impaired NO• bioavailability (Bahadoran et al., 2021a). Dietary nitrate supplementation reverses metabolic syndrome features (including hypertension, dyslipidemia, insulin resistance, and visceral adiposity) in aged eNOS-deficient mice (Carlström et al., 2010). Nitrate supplementation in diabetic rats/mice ameliorated glycemic parameters, including gluconeogenesis, fasting glucose, insulin, lipid profiles, and insulin resistance (Li et al., 2016; Gheibi et al., 2018; Khorasani et al., 2019). Previous animal studies have demonstrated that nitrate/nitrite reduce oxidative stress, promote adipose tissue browning, and enhance insulin secretion, thus nitrate has been used in drugs to manage diabetes (Ghasemi and Jeddi, 2017). A 12-month study of high-fat/sucrose-fed mice revealed that nitrate does not improve metabolic dysfunction and exacerbates cholesterol dysregulation, cardiac fibrosis, steatotic liver disease, and hepatocellular carcinoma progression (Sowton et al., 2025). Clinical studies have reported conflicting outcomes regarding the therapeutic efficacy of dietary nitrate in T2DM, attributed to variations in intervention duration, dosage protocols, and patient-specific vascular dysfunction, as mentioned in Table 4 . In human clinical trials, nitrate supplementation for 4 days to 24 weeks did not improve insulin sensitivity or glycemic and lipid parameters in patients with T2DM (Gilchrist et al., 2013; Shepherd et al., 2015; Bahadoran et al., 2021b). Plasma glucose levels decreased following acute nitrate intake (Cermak et al., 2015), while exercise performance improved after chronic supplementation (Bock et al., 2022). Furthermore, a high intake of green leafy vegetables was associated with a 14% reduction in the risk of T2DM development (Carter et al., 2010). The effect and mechanism of action of nitrate in T2DM remain unknown, and the interaction of nitrate with the host microbiota may be central to the underlying mechanism (Liu et al., 2023a).

The beneficial effects of nitrate are absent in germ-free mice, resulting in abnormal glucose tolerance and increased fat content (Cordero-Herrera et al., 2019), indicating that nitrate-reducing bacteria play important roles in the development of diabetes. In the oral cavity, nitrate-reducing bacteria are negatively associated with blood glucose levels and insulin resistance (Goh et al., 2019). DNRA activity is inversely associated with insulin resistance, fasting blood glucose, and 2-h glucose (Morou-Bermúdez et al., 2025). Specifically, a high relative abundance of H. parainfluenzae (nitrate-reducing bacteria) and low abundance of N. flavescens (nitrite-reducing bacteria) are correlated with improved insulin resistance (Bahadoran et al., 2021a). Nitrate supplementation reduces the abundance of glucose metabolism-linked genera like Prevotella and Veillonella, with Prevotella copri strongly associated with insulin resistance and impaired glucose tolerance (Pedersen et al., 2016; Wei et al., 2020). In a previous study of 945 overweight/obese individuals (22% of participants used mouthwash ≥ twice daily), researchers evaluated the association between mouthwash use and the development of pre-diabetes/diabetes over 3 years. Using mouthwash ≥ twice daily was associated with a significantly increased risk of pre-diabetes/diabetes (Joshipura et al., 2017). Collectively, diminished oral nitrate-reducing capacity in T2DM may exacerbate metabolic dysfunction, while nitrate supplementation may partially improve glucose homeostasis by modulating nitrate-associated microbial dysbiosis.

Nitrate has emerged as a potential therapeutic dietary supplement for obesity and related conditions, including metabolic syndrome and metabolic dysfunction-associated steatotic liver disease (MASLD). Dietary nitrate can prevent metabolic syndrome and liver steatosis induced by a high-fat diet (HFD) (Liu et al., 2021). An HFD-induces hyperlipidemia and insulin resistance in mice, but these are alleviated by dietary nitrate supplementation (Li et al., 2016). Dietary nitrate attenuated HFD-induced pathological features, including developed increased myocardial fibrosis, glucose intolerance, and adipose inflammation, in HFD-fed mice (Petrick et al., 2023). Moreover, an HFD can alter intestinal microbial community composition (Petrick et al., 2023) and the bioavailability of oxygen and nitrate to gut bacteria (Yoo et al., 2021). Our group found that nitrate, nitrite, and cGMP levels increased after nitrate loading, and the abundances of Bacteroidales S24–7 and Alistipes were increased in an obesity model (Ma et al., 2020). These findings demonstrate the central role of the microbiome in the bioactivation of nitrate in metabolic syndromes; however, the specific influence of nitrate-reducing bacteria on metabolic activity requires further study.