Topical prebiotic nitrate: can extending the ‘hang-time’ in the mouth improve oral-vascular health outcomes?

Both oral and systemic health outcomes may improve by simply extending the residence or ‘ hang time ’ of prebiotic nitrate formulations in the oral cavity. This may be particularly helpful in individuals where salivary ﬂ ow of nitrate is limited, as in the case of dry mouth, or those with cardiometabolic complications that exhibit dysbiosis and low nitric oxide (NO) bioavailability because of poor dietary nitrate compliance. The thoughtful in vitro studies of Mazurel et al. and Rosier et al. shows that nitrate admixed with saliva from periodontal and healthy subjects shifts the microbiome away from disease-associated to health-associated nitric oxide-promoting microbiota. Contemporaneously, a slow-release nitrate formulated chewing gum or lozenge in healthy subjects reinforced the in vitro bio ﬁ lm observations and the bene ﬁ cial microbiome shift that appears to occur acutely and at low dosing with prebiotic nitrate.

Both oral and systemic health outcomes may improve by simply extending the residence or 'hang time' of prebiotic nitrate formulations in the oral cavity.This may be particularly helpful in individuals where salivary flow of nitrate is limited, as in the case of dry mouth, or those with cardiometabolic complications that exhibit dysbiosis and low nitric oxide (NO) bioavailability because of poor dietary nitrate compliance.
The thoughtful in vitro studies of Mazurel et al. and Rosier et al. shows that nitrate admixed with saliva from periodontal and healthy subjects shifts the microbiome away from disease-associated to healthassociated nitric oxide-promoting microbiota.Contemporaneously, a slow-release nitrate formulated chewing gum or lozenge in healthy subjects reinforced the in vitro biofilm observations and the beneficial microbiome shift that appears to occur acutely and at low dosing with prebiotic nitrate.
Dietary nitrate (NO 3 ), rich in leafy greens and the DASH Diet, is a vital micronutrient that sustains systemic NO bioavailability.NO, an ephemeral lipophilic gas, is produced by a tightly regulated family of L-arginine-dependent nitric oxide synthases found in nearly every cell type.From regulating vascular tonality and oxygen delivery to serving as an effector molecule during an immune response, NO plays a multitude of roles in human health and disease1 .Unfortunately, Nitric Oxide Synthase (NOS) activity, especially in endothelial cells, wanes with age.Such a loss is believed to contribute to chronic disease [1][2][3] .
However, a redundant NO pathway exists, the entrosalivary loop, which is primary driven by NO 3 -rich dietary sources.Here, NO is restored through various redox reactions beginning with the reduction of dietary NO 3 to nitrite (NO 2 ) to NO as well as a mix of nitrogen oxide intermediates, such as long-lived S-nitrosothiols, a storage pool for NO [1][2][3] .
A requisite step in the bioconversion of NO 3 to NO 2 is catalyzed by a yet-to-be fully understood community of nitrate-reducing bacteria in the mouth 3 .The systemic importance of this NO-promoting, nitrate-reducing community is best highlighted with an increase in blood pressure after the oral cavity is exposed to mouthwash resulting in dysbiosis with a concomitant decrease in NO bioavailability and elevated blood pressure 2 .
Several clinical studies have demonstrated the anti-hypertensive effects of NO through the ingestion of 4-11 mM NO 3 -rich vegetable juice or encapsulated nitrate salt, such as potassium nitrate 4 .Upon swallowing the nitrate-containing juices or capsules, NO 3 is absorbed in the gut and approximately 1/4 of the total NO 3 circulates back to the mouth where NO 3 is selectively concentrated in the salivary glands and subsequently secreted into the mouth affording both systemic and oral benefit.As to the oral benefit, a significant reduction in caries and counts of Streptococcus mutans and Lactobacillus spp. was found in patients with high salivary NO 3 5 .Likely through the local production of NO, the reduced-NO 2 in the mouth beginning with the bioconversion from dietary NO 3 , inhibits growth of S. mutans, Lactobacillus sp., Actinomyces naeslundii, as well as, periodontal disease-associated pathobiont Fusobacterium sp., Eikenella corrodens and Porphyromonas gingivalis 6,7 .Reportedly, the increase in salivary NO 3 contributes to the overall protective effect against periodontal-associated pathobiont affecting both hard and soft oral tissues 8 .Sulfate-reducing bacteria, Desulfovibrio spp., implicated in bad breath was also found to be inhibited by NO 3 9 .Mazurel et al. reported that 5 mM NO 3 decreased the biofilm quantity and dysbiosis index in an in vitro biofilm assay consisting of subgingival plaque derived from periodontal patients 10 .A small synbiotic response was also observed with the addition of the nitrate-reducing probiotic, Rothia aeria, in the presence of 5 mM NO 3 resulting in further elevation of the health promoting NO precursor, NO 2 .And the addition of 50 mM NO 3 , in the absence of nitrate-reducing probiotic, further decreased periodontitisassociated species such as Fusobacterium nucleatum, Tannerella forsythia, among other pathogenic species.
Using the same in vitro biofilm assay, Rosier, Mira and colleagues demonstrated that the addition of 6.5 mM NO 3 to saliva of healthy subjects increased the oral-health-associated NO 3 -reducing genera Neisseria and Rothia with a lowering of periodontitis and halitosis-associated Porphyromonas, Fusobacterium, Leptotrichia, Avoprevotella, Tannerella and Treponema, and the caries-associated Oribacterium 11 .As such, NO 3 has emerged as a prebiotic in reversing oral dysbiosis.Saliva from a mixed population of healthy subjects were assessed for the relative abundance of nitrate reducers in comparison to an inflammation-associated bacteria cluster.As shown in Fig. 1, the nitratereducing community correlated negatively with inflammation-associated cluster (r = −0.65,N = 23, p < 0.01; see list of communities for nitratereducers and disease-associated bacteria in figure legend).A subset of 9 healthy individuals from this cohort were subsequently identified as low nitrate-reducers as determined by the absence or <20 μM salivary NO 2 and consented to chewing a prebiotic NO 3 -ascorbic acid formulated chewing gum.
Figure 2A, shows the difference in the saliva microbiome of pre-and post-chewing of the selected bacteria species with >1% relative abundance of the pre-chewing gum sampling and with a prior knowledge of nitratereducing capacity.Pre-and post-chewing saliva samples were collected for metagenomic sequencing of all the DNA present in the saliva samples and mapped to a database of functional annotated genes.Individuals chewed a 0.62 mM prebiotic NO 3 formulated gum for 5-10 min per session.This was repeated throughout day for a total of 3 chewing sessions.Each chewing session was separated by ~2.5 h for a total of 1.86 mM NO 3 for the day.Postsaliva samples for metagenomic sequencing were collected 5-6 h after exposure to the first gum.
After intermittent exposure to NO 3 gum, a significant increase was observed in the oral health-associated nitrate-reducing Neisseria flavescens, Neisseria subflava (both p < 0.05), Haemophilus parainfluenzaea, and Rothia mucilaginosa.A significant decrease in Prevotella melaninogenica and Prevotella histicola (both p < 0.05) was also observed with an insignificant decrease in both Veillonella sp.within 1 day.Periodontitis-disease-associated species were also searched for, albeit this community was well less than 1% abundance in pre-saliva sampling.Nonetheless, Fig. 2B shows that periodontitis-associated bacterial, to include Porphyromonas gingivalis, Fusobacterium nucleatum, and Tannerella Forsythia were sensitive to the prebiotic NO 3 chewing gum resulting in a reduction during this short period.
These initial observations suggest that intermittent chewing of a dietary NO 3 source over the course of hours begins to shift the oral microbiome to a nitric oxide favorable microbiota with a concomitant decrease in disease-associated bacteria with as little as 1.86 mM NO 3 within 1 day.These observations are the basis of a larger clinical study examining the effects of intermittent chewing of a low-dose prebiotic NO 3 -ascorbic acid formulation in a slow-release chewing gum for 21 days on gingival inflammation and systemic endothelial function in periodontal patients that has received ethical approval at the University Maryland Dental School, Baltimore, MD.
Previous studies have shown that a daily bolus ingestion of drinking a beetroot or lettuce juice concentrate consisting of 3-12 mM NO 3 /day over the course of 10 days to 6 weeks enriched a NO-favorable microbiome and improved NO-mediated oral-systemic health 4 .Vanhatalo et al. showed that Fig. 1 | Scoring of nitrate-reducing bacteria and gum inflammation-associated bacteria communities from saliva of periodontally healthy individuals.Shotgun metagenomic sequencing was used to look at all the DNA present in the saliva microbiome samples mapped to a database of functional annotated genes.Saliva was collected and tested according to the methodology by Bristle Oral Health measuring the abundance of these bacteria using an in-house bioinformatics pipeline and then compared those abundances across thousands of samples.The scoring method is based on a priori knowledge of selected bacteria in context of functional annotated genes and takes into consideration the relative abundance of each bacterial strain to compute a nitrate-reducing and risk score for gum disease-associated bacteria where 0 is the lowest and 10 is the highest abundance.The nitrate-reducing community consisted of Rothia mucilaginosa, Neisseria flavescens, Veillonella dispar, Veillonella atypica, Haemophilus parainfluenzae and the gum inflammation-associated cluster included Tannerella forsythia, Treponema socranskii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium peridoncum, Fusobacterium nucleatum, Parvimonas micra.Fig. 2 | Change in the relative abundance of pre and post topical administration of a prebiotic nitrate formulated chewing gum in healthy subject with low pregum salivary nitrite, as determined by the absence or <20 μM salivary nitrite (Saliva Nitric Oxide Test Strips by MyFitStrips).A The change of the saliva microbiome of pre-and post-chewing of selected bacteria species with >1% relative abundance of the pre-chewing gum sampling and with a prior knowledge of nitratereducing capacity.B The change or difference in abundance of pre-and post-chewing in healthy subjects pre-selected with a prior knowledge of diseaseassociated attributes, albeit this community was well less than 1% abundance in presaliva sampling.Whole genome shotgun metagenomic sequencing was used to looks at all DNA present in the saliva microbiome samples from pre-and postchewing.Saliva was collected and tested according to the methodology by Bristle Oral Health.*p < 0.05 between pre-and post-chewing after 4-5 h from the initial piece of gum of a total of 3 dispersed over this time. https://doi.org/10.1038/s41522-024-00527-3

Matters arising
npj Biofilms and Microbiomes | (2024) 10:57 10 days of drinking 6.2 mM NO 3 /day increased the abundance of Rothia spp (+127%) and Neisseria spp (+351%) with a corresponding decrease in Veillonella spp (−60%) and Prevotella melaninogenica (−60%) with an increase in NO 2 and reduced systemic blood pressure 12 .After 6 weeks of drinking 6.7 mM NO 3 beet juice daily, Velmurugan et al. observed improvement in endothelial function with a corresponding increase in Rothia mucilaginosa and Neisseria flavescens 13 .And Jockel-Schneider et al. observed in periodontal recall patients a marked decrease of gingival inflammation with a corresponding increase in Rothia spp and Neisseria spp after consuming 3 mM of NO 3 -rich lettuce juice daily for 2 weeks 14 .
By simply extending the residence or 'hang time' of prebiotic NO 3 formulations in the oral cavity may acutely improve both oral and cardiovascular health outcomes.As such, hypertensives with poor dietary NO 3 compliance or where salivary flow of NO 3 is limited, as in the case of Xerostomia patient, may benefit the most.Restoring a NO-favorable oral microbiome may emerge to be equally beneficial to smokers, ex-smokers and those with cardiometabolic complications who all exhibit dysbiosis and low NO bioavailability.The latter is reinforced by Goh et al. showing a causal relationship between elevated Neisseria flavescens and Haemophilus parainfluenzae with lower insulin resistance and blood pressure 15 .Extended chewing-time of a prebiotic nitrate-ascorbic acid-polyphenol formularies delivered through a functional chewing gum, lozenge, or slowly munching a whole food plant-based source, such as a rocket-beet-citrus salad, may emerge to be more effective and durable in NO-mediated oral-systemic health outcomes than simply drinking NO 3 -rich beet juices or swallowing a NO 3 pill.At a minimum, by enhancing a nitric oxide favorable oral microbiome with prebiotic nitrate formulated chewing gums or slow-release lozenge will potentiate heart healthy foods and diets that improve nitric oxide bioavailability 16 .