Compositions containing nitrate-rich foods and extracts for altering oral microbiome

Abstract

The present disclosure relates to oral compositions and / or confectionery compositions comprising an effective amount of a nitrate salt, e.g., a dietary nitrate salt derived from a nitrate-rich food and / or a nitrate-rich extract, for reducing volatile sulfur compounds (VSCs) in the oral cavity and freshening breath.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application Serial No. 63 / 454,457, filed March 24, 2023, the disclosure of which is incorporated herein by reference as if it were set forth in its entirety. Background Technology

[0002] This disclosure relates to oral compositions comprising nitrates and their uses. Specifically, this disclosure relates to confectionery compositions comprising sodium nitrate or potassium nitrate and dietary nitrates derived from nitrate-rich foods and / or nitrate-rich extracts, which produce a breath-freshening effect upon consumption, while exhibiting oral care effects against volatile sulfur compounds (VSCs) and enhancing heart health.

[0003] The term "bad breath" as used in this article refers to halitosis caused by physiological (transient or temporary) and / or pathological conditions. Physiological causes of halitosis include: halitosis caused by bad habits, morning halitosis, and dry mouth. Pathological causes of halitosis include: secondary or oral tissue conditions associated with gingival and periodontal disease, acute necrotizing ulcerative gingivitis, postoperative residual blood, debris under dental instruments, oral ulcerative lesions, tongue coating, dry mouth, salivary gland diseases, and tonsillolithiasis (tonsil stone).

[0004] The production of halitosis is primarily due to the breakdown of proteins into individual amino acids, some of which are then further broken down to produce detectable foul-smelling gases. The oral cavity provides a suitable environment for the growth of Gram-negative anaerobic bacteria, which metabolize proteins as an energy source by breaking down protein substrates from impacted food particles and shed oral cell debris. Typically, inflammation causes these cells to shed at a rate faster than saliva can clean them. Bacterial action then hydrolyzes the proteins into amino acids; and amino acids containing sulfur functional groups (such as methionine and cysteine) serve as precursors to volatile organic compounds (VSCs).

[0005] These gaseous VSCs contribute to oral malodor and are primarily composed of hydrogen sulfide (H2S), methanethiol (CH3SH), and dimethyl sulfide [(CH3)2S]. For example, in the presence of thiol-positive microorganisms, the amino acids methionine and cysteine ​​are reduced to hydrogen sulfide and methanethiol, respectively. Methanethiol has been found to be the main component of malodorous odor on the dorsum of the tongue in patients with periodontal disease, while hydrogen sulfide is dominant in oral-healthy subjects.

[0006] While halitosis represents a source of embarrassment or annoyance primarily, the VSCs that are primarily responsible for halitosis can also damage tissues in the oral cavity and can lead to periodontitis (inflammation of the gums and ligaments that support the teeth). Specifically, VSCs have been found to destroy collagen and proteoglycan components in connective tissue by breaking disulfide bonds. This disaggregation of the extracellular matrix allows microorganisms to penetrate the oral mucosa. As bacteria further accumulate in pockets formed next to the teeth, periodontal disease, as well as halitosis, also develops. If periodontal disease worsens significantly, it can compromise overall systemic health; for example, periodontal bacterial byproducts can enter the bloodstream and can lead to heart disease, stroke, and low birth weight babies.

[0007] For the treatment of oral malodor, the public has increasingly turned to commercially available oral freshening products. As chronic oral malodor patients experience personal discomfort and social embarrassment, the market for these products has been consistently growing.

[0008] Various compounds (such as chlorine dioxide, sodium chlorite) and metal salts (such as zinc and copper salts) have been used as VSC neutralizers in various oral compositions. Such compounds have been provided and are currently available as mouthwashes and mouth rinses for the prevention and / or treatment of oral malodor. Generally, many of the oral mouth rinses currently available are used to prevent and / or treat oral malodor or halitosis, but many mouth rinses provide little or no help for chronic halitosis.

[0009] While compounds (such as chlorine dioxide, sodium chlorite) and metal salts (such as zinc and copper salts) are effective against oral malodor, they impart a strong, unpleasant flavor and odor, thereby negatively impacting mouth feel and deterring use. Generally, mouth rinses incorporating these compounds can also cause some generalized irritation to the oral cavity, such as peeling, ulceration, and inflammation. In addition, oral compositions (other than mouthwashes and mouth rinses) incorporating these compounds, such as chewing gums, mints, and lozenges, remain in the oral cavity for a longer period of time, further enhancing irritation to the oral cavity when these compounds are used.

[0010] Recent research has shown that antibacterial mouthwash treatments not only kill harmful oral bacteria, but also kill benign microorganisms and disrupt the oral microbiota. For example, a mouthwash containing 0.12% chlorhexidine reduced both VSC-producing bacteria and nitric oxide-producing bacteria in the gut. It also simultaneously reduced oral and plasma nitrite levels in healthy volunteers and was associated with a sustained rise in systolic and diastolic blood pressure. This study suggests that oral nitrate-reducing bacteria can contribute to host nitrite levels and thus to NO levels, with measurable physiologic effects. In healthy volunteers, antibacterial compounds including chlorhexidine, chlorine dioxide, sodium chlorite, etc. can severely suppress the oral microbiota and affect systemic nitrite levels, thereby affecting blood pressure.

[0011] In view of the foregoing challenges, there is a continuing need to develop an effective treatment for oral malodor caused by physiological and / or pathological conditions. SUMMARY

[0012] In one aspect, the present disclosure relates to an oral composition or a confectionery composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity.

[0013] In another aspect, the present disclosure relates to an oral composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity; and an effective amount of ascorbic acid or a suitable salt thereof.

[0014] In another aspect, the present disclosure relates to a method of freshening breath comprising consuming a confectionery composition comprising an effective amount of a nitrate salt.

[0015] In some embodiments of the foregoing aspect, the nitrate salt comprises sodium nitrate or potassium nitrate.

[0016] In some embodiments of the foregoing aspect, which can be combined with any of the foregoing embodiments, the nitrate salt comprises dietary nitrate derived from a nitrate- rich food, a nitrate-rich extract, or a combination thereof.

[0017] In some embodiments of the foregoing aspect, which can be combined with any of the foregoing embodiments, the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the confectionery composition.

[0018] In some embodiments of the foregoing aspect, which can be combined with any of the foregoing embodiments, the nitrate-rich food is selected from beetroot, kale, cress, watercress, spinach, parsley, water dropwort, anise, Chinese cabbage, bok choy, chard, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, chicory leaf, bean sprout, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or a combination thereof.

[0019] In some embodiments of the foregoing aspect, which can be combined with any of the foregoing embodiments, the nitrate-rich extract is selected from beetroot extract, celery extract, or a combination thereof.

[0020] In some embodiments of the foregoing aspects, which can be combined with any of the preceding embodiments, the confectionery composition is in the form of a breath freshening mint, a low boiled candy, a chewing gum, a chewy candy, a hard boiled candy, an enrobed candy, a lozenge, a syrup, a pressed mint, a throat drop, or a chocolate.

[0021] In some embodiments of the foregoing aspects, which can be combined with any of the preceding embodiments, the chewing gum is a sugar-free chewing gum.

[0022] In some embodiments of the foregoing aspects, which can be combined with any of the preceding embodiments, the confectionery composition further comprises ascorbic acid or a suitable salt thereof.

[0023] In some embodiments of the foregoing aspects, which can be combined with any of the preceding embodiments, the ascorbic acid or suitable salt thereof is present in an amount of about 0.01% to about 5% by weight of the confectionery composition. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 An exemplary calibration curve generated from nitrate / nitrite testing assays based on the modified Griess assay disclosed herein is shown.

[0025] Figure 2 H2S production in vitro by saliva mixed with nitrate, saliva mixed with cysteine, and saliva mixed with a combination of cysteine and nitrate is shown.

[0026] Figure 3 Nitrite production in vitro by saliva alone, saliva mixed with cysteine, saliva mixed with nitrate, and saliva mixed with a combination of nitrate and cysteine is shown.

[0027] Figure 4A and 4B Nitrite production in vitro in saliva from two subjects when mixed with cysteine and / or nitrate compared to saliva alone or a control mixture containing water, nitrate, and cysteine is shown.

[0028] Figure 5 Nitrate reductase activity following treatment with chlorhexidine (CHX) is shown. A. actinomycetemcomitans A.a. ) following treatment with chlorhexidine (CHX).

[0029] Figure 6 Nitrate reductase activity following treatment with chlorhexidine (CHX) is shown. V. atypical V.a. ) following treatment with chlorhexidine (CHX). ​​

[0030] Figure 7 Nitrate reductase activity after treatment with Magnolia Bark Extract (MBE) is shown. A.a.

[0031] Nitrate reductase activity after treatment with MBE is shown. Figure 8 V.a. DETAILED DESCRIPTION

[0032] The present disclosure relates to oral compositions comprising an effective amount of nitrate salt and methods of use thereof.

[0033] Nitrate salts (e.g., sodium nitrate or potassium nitrate) and dietary nitrates derived from nitrate-rich foods and extracts can be actively absorbed by saliva. Salivary nitrates are metabolized to nitrites by nitrate reductase produced by anaerobic oral bacteria under anaerobic conditions. Nitrites act as a precursor to nitric oxide, which has been linked to various health benefits such as lowering blood pressure and overall cardiovascular and systemic health.

[0034] Surprisingly, it has been discovered that in the absence of nitrate salts, oral bacteria utilize sulfur-containing proteins as a primary food source and break down these proteins to generate volatile sulfur compounds (VSCs) that cause bad breath. It has further been discovered that in the presence of nitrate-rich foods, oral bacteria preferentially utilize the nitrate salts as a food source and generate nitric oxide, which has been proven to lower blood pressure and benefit cardiovascular and systemic health. As a result, fewer VSCs are generated, which cause oral malodor as well as damage to tooth soft and hard tissues.

[0035] Without being bound by theory, when dietary nitrates are added to oral compositions, such as confectionery compositions, they can help shift the oral microbiome and significantly reduce the production of VSCs, thereby achieving a breath freshening effect. They can further be converted to nitric oxide, thereby helping to lower systolic and diastolic blood pressure to enhance heart health.

[0036] ​​In various aspects, the present disclosure provides an oral composition comprising a nitrate salt in an effective amount for reducing volatile sulfur compounds in a user’s oral cavity. In various aspects, the oral composition can be in any form suitable for application to the oral surfaces of a human, dog, cat, or other animal, and provide a cosmetic prophylactic or therapeutic benefit within or originating from the oral cavity. In various aspects, the oral composition can include a dentifrice, such as a powder or paste; an edible or bioadhesive film; a confectionery composition, including but not limited to breath freshening mints, low temperature boiled sweets, chewing gums (such as sugarless chewing gums), chewy candies, hard boiled sweets, coated sweets, dragees, syrups, pressed mints, throat lozenges, and chocolates; pet foods, chews, or biscuits, and the like. In certain embodiments, the consumption or chewing of the oral composition can be repeated periodically.

[0037] As used herein, the term “effective amount” refers to a level, amount, dosage, or percentage that produces or is capable of producing a desired effect. All percentages and ratios used herein are by weight of the total composition.

[0038] In various aspects, suitable nitrate salts that can be included in the oral compositions of the present disclosure can be derived from any source of nitrate ions, including but not limited to nitrate salts, such as sodium nitrate or potassium nitrate; or dietary nitrate derived from nitrate-rich foods and / or extracts. In various aspects, the nitrate salt can be derived from nitrate-rich foods, including but not limited to beetroot, kale, cress, arugula, spinach, parsley, watercress, anise, Chinese cabbage, bok choy, leek, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, chicory leaf, bean sprouts, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or combinations thereof. In other aspects, the nitrate salt can be derived from nitrate-rich extracts, including but not limited to beetroot extract, celery extract, or combinations thereof.

[0039] In another aspect, the present disclosure provides an oral composition comprising a nitrate salt in an effective amount for reducing volatile sulfur compounds in a user’s oral cavity and an effective amount of ascorbic acid or a suitable salt thereof. Without being bound by theory, by combining the nitrate salt with ascorbic acid, the risk of forming potentially carcinogenic nitrosamines can be reduced.

[0040] In various aspects, the nitrate salt can be present in the oral composition in any amount effective to reduce volatile sulfur compounds in the oral cavity. In some aspects, the nitrate salt is present in an amount of from about 0.01% to about 5% by weight of the oral composition, or about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight of the oral composition, or from about 0.1% to about 3% by weight of the oral composition, or from about 1% to about 5% by weight of the oral composition, or from about 3% to about 5% by weight of the oral composition, or any percentage in between any of these values. In further aspects, the ascorbic acid or suitable salt thereof can be present in the oral composition in any amount effective to reduce the risk of nitrosamine formation. In some aspects, the ascorbic acid or suitable salt thereof is present in an amount of from about 0.01% to about 5% by weight of the oral composition, or about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight of the oral composition, or from about 0.1% to about 3% by weight of the oral composition, or from about 1% to about 5% by weight of the oral composition, or any percentage in between any of these values.

[0041] In various aspects, the oral compositions of the present disclosure contain a nitrate salt in an amount effective to reduce VSCs in combination with a suitable carrier. Suitable carriers can be food acceptable or food contact acceptable materials in which the nitrate salt can be incorporated or dispersed without adverse effects. Suitable carriers can include water-soluble solids or chewable solids, such as confectionery compositions. Another suitable carrier can be a dentifrice, such as a paste or powder. Other suitable carriers for cats, dogs, and other animals include, but are not limited to, chews, biscuits, kibbles (dry) and canned (wet / soft) pet foods.

[0042] As used herein, the term "confectionery composition" includes chewing gums and orally dissolvable tablets, beads, and lozenges. Saliva dissolves the lozenge or chewing gum product and facilitates prolonged contact with the surfaces of the oral cavity, such that delivery of the VSC reducing agent in the form of a lozenge tablet, bead, or chewing gum ensures that a sufficient dose of active ingredient is delivered to the surfaces of the oral cavity while the product is in use. Alternatively, the confectionery composition can be in the form of a coating, shell, film, syrup, or suspension.

[0043] In one aspect, the oral compositions of the present disclosure can be chewing gum compositions suitable for chewing and comprising 2% or more elastomer by weight of the composition. Typically, chewing gum compositions are chewed or chewed up by the consumer, through which process food is mashed and crushed by the teeth. Such chewing gum compositions can take a variety of shapes and forms, such as pellets, gum balls, squares, strips, and the like, and can be coated individually or in any combination with a variety of materials including, but not limited to, sugar, polyol, chocolate, syrup, film, and the like. Natural or artificial colors and combinations thereof, high intensity sweeteners, and flavoring agents can also be added to the coating solution. For pellets or coated chewing gum, zinc salts can be incorporated into the coating or center.

[0044] Suitable chewing gums can include sugar-free chewing gums comprising an amount of nitrate salt effective to reduce VSCs. In addition to the chewing gum base, the chewing gum formulation can contain one or more plasticizers, at least one sweetener, and at least one flavoring agent.

[0045] In one aspect, a chewing gum is provided comprising an amount of nitrate salt from about 0.01% to about 5% by weight of the chewing gum, or about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight of the chewing gum, or from about 0.1% to about 3% by weight of the chewing gum, or from about 1% to about 5% by weight of the chewing gum, or any percentage in between any of these values.

[0046] In another aspect, a mint is provided comprising an amount of nitrate salt from about 0.05% to about 2% by weight of the mint, or about 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% by weight of the mint, or from about 0.1% to about 1% by weight of the mint, or from about 0.5% to about 2% by weight of the mint, or any percentage in between any of these values.

[0047] In various aspects, optional coatings can also be applied to any of the oral compositions disclosed herein. Coating materials understood by one of skill in the art can include, but are not limited to, wax, shellac, polyol, carboxymethylcellulose, polyethylene / maleic anhydride copolymer, or kappa-carrageenan.

[0048] In another aspect, the present disclosure provides a method for freshening breath comprising consuming an oral composition or confectionery composition of the present disclosure.

[0049] In various aspects, the method can involve consuming an oral composition comprising a nitrate salt in an effective amount for reducing volatile sulfur compounds in the oral cavity of a user. In some aspects, the method can involve consuming an oral composition comprising (1) a nitrate salt in an effective amount for reducing volatile sulfur compounds in the oral cavity of a user, and (2) an effective amount of ascorbic acid or a suitable salt thereof. In other aspects, the method can involve consuming a confectionery composition comprising a nitrate salt in an effective amount for reducing volatile sulfur compounds in the oral cavity of a user.

[0050] In various aspects, the method can involve consuming an oral composition that is present in any form suitable for application to the oral surfaces of a human, dog, cat, or other animal, and that provides a cosmetic prophylactic or therapeutic benefit within or derived from the oral cavity.

[0051] EMBODIMENT The presently disclosed subject matter will be better understood by reference to the following examples, which are offered by way of illustration and not limitation.

[0052] Example 1: Nitrate / nitrite determination of dietary vegetables In view of the importance of dietary nitrate intake, a number of analytical methods have been developed and published. To date, based on the Griess test and ELISA kits, several commercial test kits are available for the detection of nitrate and nitrite compounds. This test allows for routine analysis of nitrate / nitrite in human blood, air and water, and in environmental soil. In order to analyze nitrate and nitrite concentrations in food and saliva quickly and accurately, a nitrate / nitrite test method based on a modified Griess test was developed. This method is capable of detecting nitrate or nitrite concentrations in any food extract and saliva within one minute, with a detection limit of 0.1 ppm. It was found that many nitrate-rich foods contain 0.1-1% nitrate.

[0053] A detailed test procedure is provided below: Step 1. Preparation of potassium nitrite calibration curve 1. Dissolve 0.5 g of modified Griess reagent (a mixture of 0.2% N-(1-naphthyl) ethylenediamine dihydrochloride and 2% sulfanilamide in 5% phosphoric acid and an inert carrier) in 100 mL D.I. water to make a 0.5% Griess working solution.

[0054] 2. Dissolve 1 g of KNO2 in 100 mL D.I. water to make a 1.0% KNO2 solution. Further dilute this solution with 100x water to make a 0.01% KNO2 solution.

[0055] 3. Add 1 mL of 0.5% modified Griess reagent prepared from Procedure 1 and mix with 0, 5, 10, 15 and 20 μL of 100 ppm (0.01%) KNO2 solution from Procedure 2 to make a series of solutions containing 0, 0.5, 1, 1.5 and 2 ppm of KNO2. 2的校准溶液。

[0056] 4. Vortex each vial briefly and measure the absorbance at 540 nm by spectrophotometer.

[0057] The resulting calibration curve is shown in Figure 1

[0058] Step 2. Measurement of saliva nitrate levels and after addition of KNO3.

[0059] Collect 5 mL of stimulated or unstimulated saliva and add 5 mL of D.I. water for 50% dilution. Vortex the diluted saliva briefly and pipette 0.05 mL of the 50% saliva solution into 1 mL of 0.5% Griess working solution (step-1 above). Shake the solution briefly and measure the absorbance at 540 nm by spectrophotometer. This solution serves as a representative of the background NO2 levels in saliva. - 2.

[0060] Step 3. Measurement of NO2 production by saliva - 2. 1. Prepare a 0.5% L-cysteine solution by dissolving 0.5 g of L-cysteine in 100 mL of D.I. water.

[0061] 2. Mix 0.5 mL of 50% diluted saliva and 0.5 mL of D.I. water in a 5 mL vial. Add 0.1 mL of 30% KNO3 and vortex the resulting solution. Prepare a 1 mL solution containing 25% saliva and 3% KNO3 (solution A).

[0062] 3. Pipette 0.5 mL of 50% diluted saliva and 0.5 mL of 0.5% cysteine into a 5 mL vial. Prepare a 1 mL solution containing 25% saliva and 0.25% L-cysteine (solution B).

[0063] 4. Mix and vortex 0.5 mL of 50% diluted saliva, 0.5 mL of 0.5% cysteine and 0.1 mL of 30% KNO3. Prepare a 1 mL solution containing 25% diluted saliva, 0.25% L-cysteine and 3% KNO3 (solution C).

[0064] ​5. Pipette 0.5 ml of D.I. water, 0.5 ml of 0.5% cysteine, and 0.1 ml of 30% KNO3 and vortex. Prepare a 1 mL solution containing 0.25% L-cysteine and 3% KNO3 (solution D).

[0065] 6. Every 15 minutes, sample solutions A, B, C, and D by pipetting 50 μΐ of each solution into 1 mL of 0.5% modified Griess reagent and vortex. Measure the absorbance at 540 nm by spectrophotometer.

[0066] It was observed that healthy human volunteers' saliva contains 1 ppm - 20 ppm of nitrite. When dietary nitrate is added, the nitrite level increases by 5 - 20 times. Furthermore, in the absence of saliva containing nitric oxide producing bacteria, no appreciable amount of nitrite is produced.

[0067] The top 5 nitrate-rich vegetables were found to be beetroot (0.5 - 1% nitrate), rocket (0.5%), mung bean sprouts (0.39 - 0.45%), kale, spinach, and celery (0.24 - 0.4%). Other vegetables, including lettuce, bok choy, and cabbage, also contain large amounts of nitrate (0.1 - 0.3%). Furthermore, cured red meats, including hot dogs, ham, bacon, etc., contain large amounts of nitrite, from 20 ppm to 140 ppm, as KNO2 or NaNO2 is often added to meat products for preservation.

[0068] Example 2: Nitrate-rich food for reducing oral malodor and enhancing breath freshening It was found that in the absence of nitrate, oral bacteria will utilize sulfur-containing proteins as the primary food source and break down the protein food into cystine and cysteine. Cystine and cysteine are then further broken down by anaerobic oral bacteria to generate hydrogen sulfide, methanethiol, and dimethyl sulfide, i.e., VSCs, and produce malodor. It was further found that in the presence of nitrate-rich food, oral bacteria will preferentially utilize nitrate as the food source and generate nitric oxide, which is absorbed by the human circulatory system through sublingual absorption. This results in the generation of less VSCs, which cause oral malodor as well as potential damage to teeth and soft / hard tissues.

[0069] For example, as shown in Figure 2 20 ppm of H2S was generated in the headspace air tested by gas chromatograph equipped with a chemiluminescent sulfur detector when saliva was mixed with the sulfur-containing amino acid cysteine. However, less than 5 ppm of H2S was generated when saliva was mixed with cysteine in the presence of potassium nitrate or beetroot powder. Furthermore, as shown in Figure 3As shown, when dietary nitrate was added to saliva, more than 50 ppm of nitrite was observed in the headspace vials. It was concluded that when dietary nitrate is added to oral compositions, it can significantly reduce volatile sulfur compounds that cause oral malodor, while converting nitrate to nitrite, which can bring the benefits of lowering systolic and diastolic blood pressure and enhancing heart health.

[0070] Example 3: Testing of nitrate reductase (NR) activity of oral bacteria General methods Test bacteria, growth and nitrate reductase assay Gram-negative anaerobic oral bacteria that are often associated with the dorsum of the tongue and halitosis were tested. These bacteria include: Nucleate Fusobacterium (ATCC 10953). Porphyromonas gingivalis (ATCC 33277), Actinobacillus naeslundii AN19 and MG1, Enterococcus faecalis (ATCC 29212), Streptococcus gordonii , Streptococcus mutans UA159, Streptococcus sanguis , Streptococcus sobrinus and V. atypical (ATCC 17744) Effect of natural and chemical antimicrobials on nitrite production ).

[0071] To assess the ability of oral bacteria to produce nitric oxide, overnight cultures of the test bacteria were washed once and transferred to media without any nitrate or nitrite compounds. 0.5% (or 1%) KNO3 was added to the media and further incubated anaerobically at 37°C for 24-48 hours. The concentration of nitrite was determined by the Griess assay over the desired time period (due to the reduction of nitrate by nitrate reductase).

[0072] Effect of cysteine addition on NR activity of salivary bacteria To examine the effect of natural and chemical antimicrobial agents on NO production, the test bacteria were pre-exposed to Magnolia biondii extract (MBE), a natural bactericidal compound, peppermint oil, and chlorhexidine (CHX). The nitrate reductase activity of the oral bacteria was determined by the Griess assay as described above. Untreated controls were included for each test strain. If the test antimicrobial compound affects nitrate reductase activity, the production of nitrite is reduced. Dose response curves were prepared.

[0073] Effect of nitrate reductase on volatile sulphur compound (VSC) production Human stimulated saliva was collected from two subjects and tested for NR activity in the presence of nitrate, cysteine, and a combination of nitrate and cysteine. The Griess assay was used for nitrite production.

[0074] A.a. Test bacteria In vitro and V.a were incubated with nitrate, cysteine or nitrate / cysteine and the production of VSCs was measured using a portable Oral Chroma. This instrument detects three volatile sulphur compounds. The effect of nitrate reductase activity on the test bacteria Methods was studied.

[0075] Example 4: Nitrate reductase (NR) activity of human saliva bacteria Figure 4A Collection of stimulated saliva In this study, two subjects were evaluated. The subjects avoided oral cleaning and eating the night before the test and on the morning of the test and their stimulated saliva was collected after chewing 1 g of gum base for 5 minutes. The saliva samples were immediately stored on ice, diluted 1 : 1 with D.I. H2O and used as the source of bacteria for the nitrate reductase (NR) activity test under anaerobic conditions.

[0076] Test protocol The content in each reaction mixture included (mL): The end product of NR and nitrite were detected by Griess reagent at OD 550nm = 540 nm.

[0077] As shown in Figures 1 and 2, saliva samples from two subjects showed similar results, i.e. nitrate (KNO3, 3%) induced NR activity of the saliva bacteria to produce nitrite. When the reaction mixture was assayed in the presence of cysteine (0.25%) in addition to nitrate, a decrease in NR activity was noted. Figure 4B Test bacteria: Actinobacillus naeslundii The same test was further performed on pooled saliva from 4 subjects and adjusted to different concentrations (OD 600nm = 0.7, 1.0 and 1.5) and similarly it was found that the addition of cysteine decreased NR activity by 23-49%.

[0078] Example 5: Nitrate reductase (NR) activity of selected test oral bacteria

[0079] Table 1 A.n. Table 2 A. actinomycetemcomitans Table 3 A.a. Fusobacterium nucleatum polymorphum Table 4 Porphyromonas gingivalis Table 5 ​​P.g. (ATCC 33277 and w83) Streptococcus gordonii (S.g.) ) Streptococcus mutans S.m.) (UA159) V. atypical Growth medium (ATCC 17744) (V.a) A. actinomycetemcomitans : A.a. ( Porphyromonas gingivalis ) Anaerobically grown in THB broth supplemented with 1% yeast extract and 0.001% hematin and 0.0001% vitamin K.

[0080] Fusobacterium nucleatum and Actinobacillus naeslundii Anaerobically grown in THB broth supplemented with 0.001% hematin and 0.0001% vitamin K.

[0081] Streptococcus gordonii , Streptococcus mutans , V. atypical Anaerobically grown in BHI broth for 24 hours.

[0082] NR activity measured in reaction mixtures containing KNO3 in the bacterial growth medium Grown in RCM medium without (NH4)2SO4(to avoid NH4impact on Griess reagent) and without agar (0.05%, to avoid impact on OD measurement).

[0083] A. actinomycetemcomitans Nitrate source KNO3(final 0.1%) was added to the respective growth medium of each test bacteria and incubated for 24-48 hours, after which 20 μΐ of the culture broth was taken and tested for nitrite with Griess reagent.

[0084] No NR activity was detected, i.e. no nitrite was produced, when the KNO3source was not present in the growth medium of the test bacteria.

[0085] Bacteria with positive NR activity included A.a. ( Actinobacillus naeslundii ), (ATCC 33277 and w83) A.n. ( V. atypical ) and V.a. A.a. (UA159). NR activity was also detected for A.a. under aerobic incubation. V. atypical NR activity using harvested cells (24-48 hours) of test bacteria in a buffer system (PBS) and Test bacteria were selected for further testing.

[0086] A. actinomycetemcomitans ​ A.a. : V. atypical ( Actinobacillus naeslundii ), A.n. (Va) and A. actinomycetemcomitans (AN19 & MG1) A.a. ) The test bacteria were anaerobically grown in the appropriate culture medium for 48 hours. The cells were centrifuged and resuspended in buffer or fresh culture medium for NR activity testing.

[0087] V. atypical ( Actinobacillus naeslundii NR activity was detected in both PBS and growth medium. However, NR activity was less stable and reproducible in PBS than in the growth medium.

[0088] A.n. (Va): NR activity was detected in PBS and reductive transport buffer. In PBS, NR activity was unstable and low. In reductive transport buffer, the activity was reproducible.

[0089] Actinobacillus naeslundii (AN19 and MG1) A.a. Both strains showed positive NR activity. CHX against In AN19, NR can be induced in both PBS and BHI media. Strain MG1 showed the best performance in BHI medium, while NR was barely detectable in the PBS system.

[0090] Example 6: Effect of chlorhexidine (CHX) on the activity of oral bacteria NR. Effect of CHX on A.a. A.a. NR activity​ A.a. Aa's short-term lethality Harvest freshly grown cells and adjust them to OD. 550nm =0.5, then mixed with CHX (75-1,000 μg / ml). At different time points, aliquots of cells were removed and mixed with (or not with) a stop reagent (0.5% Tween 80 and 0.07% lecithin) to terminate the CHX reaction on the cells. The treated cells were centrifuged to remove the supernatant containing CHX, and the cell pellet was spotted on BHI agar (supplemented with 0.001% heme chloride and 0.0001% vitamin K) to determine cell viability.

[0091] When a stop agent was added to the cell / CHX mixture after treatment, limited viability was observed after two minutes of treatment with CHX at concentrations ranging from 75 to 1,000 μg / ml, as shown in Table 1 below (0.1%) (+: viable colonies present; -: no viable colonies).

[0092] Table 1 NR activity of CHX treated A.a. NR activity was inhibited with 100 μg / ml for 10 minutes NR activity was completely inhibited with 1,000 μg / ml (0.1% present in CHX mouthwash) for 1 minute Figure 5

[0093] These results are shown in Tables 2 and 3 below: Table 2: Values represent V. atypical OD at 550 nm (NR activity test) NR activity after CHX treatment of V. a Short term time kill of CHX against V.a is further shown in A.a. .

[0094] CHX pair A.a. (V.a) Effect of NR activity Figure 6 Short term time kill experiments of CHX on V. a were performed as described for A. actinomycetemcomitans .

[0095] NR activity of CHX treated V. a The method used was the same as Short term time kill of MBE against A.a , except that the V. a cell concentration was adjusted to OD 550nm = 0.7 (10 8 CFR / ml) in the reduction transport buffer. NR activity was also measured in the same buffer system. The results are shown in Table 3 below.

[0096] Table 3 NR activity after CHX treatment of V. a A.a. is further shown in

[0097] Example 7: Effect of Magnolia Bark Extract (MBE) on NR activity of test oral bacteria MBE pair A.a. (A.a) Effect on NR activity A.a. Freshly grown cells were harvested and mixed with MBE for a short period of time and the viability of the treated cells was assessed. The treated cells were washed with 10% DMSO and the MBE was removed from the assay system.

[0098] ​​Immediately after treatment, add 10% DMSO to the cell mixture. Then centrifuge the cell mixture and wash again with 10% DMSO. Spot cells treated with MBE at different times onto agar plates, add 10% DMSO, and record the development of surviving colonies.

[0099] As shown in Table 4 below, for treatment with 100 and 250 μg / ml MBE A.a. It was noted that only a few colonies were observed on the agar plate. No growth was detected after treatment with ≥500 μg / ml MBE.

[0100] Table 4 After processing with MBE A.a. The NR activity is shown in Tables 5 and 6 below: Table 5: Treatment with MBE for 1 minute Figure 7 (NR activity at 2 or 3 hours) (OD) Table 6: Treatment with MBE for 10 minutes V. atypical (NR activity at 2 or 3 hours) After exposure to 100 μg / mL MBE for 1 minute Short term time kill of MBE against V.a The NR activity remained unchanged. At a treatment time of 50 μg / ml for 10 minutes, >90% of the NR activity was retained. Longer treatment times at 100 μg / ml for 10 minutes inhibited NR activity.

[0101] The nitrate reductase activity after MBE treatment of Aa was further demonstrated. V. atypical middle.

[0102] MBE pair Figure 8 (V.a) Effect of NR activity Before testing the effect of MBE on NR activity, its MIC and short-term toxic concentration against amino acids (Aa) were first determined. MBE was dissolved in 100% EtOH. The MIC of MBE against amino acids (Va) was 25 μg / ml.

[0103] A.a. It was found that for the viability assay system, Va requires a special transfer buffer instead of PBS.

[0104] After processing with MBE V.a. The NR activity is shown in Tables 7 and 8 below.

[0105] Table 7: Va (NR activity at 2 or 3 hours) after 1 minute of MBE treatment (OD) Table 8: V.a treated with MBE for 10 minutes (NR activity at 2 or 3 hours) A 50-75 μg / ml MBE treatment for 1 minute with the termination reagent noted less than 10% inhibition of NR activity. Similarly, a 10 minute treatment of V.a with 50 μg / ml MBE did not show significant inhibition of NR activity.

[0106] NR activity following MBE treatment of V.a is further shown in A.a. .

[0107] Example 8: Effect of Peppermint Oil on NR Activity Porphyromonas gingivalis and Fusobacterium nucleatum NR activity The MIC of peppermint oil for V.a. , Effect of peppermint oil on A.a NR activity and Effect of peppermint oil on V.a NR activity were all > 5 μL / ml. The MIC for V. atypical was 1.25 μL / ml.

[0108] Effect of cysteine on A.a NR activity Test bacterial cells were incubated with peppermint oil (1 μL / ml and 10 μL / ml concentrations prepared from stock samples) in growth media and 0.3% nitrate source. NR activity was measured for up to 3 hours and the results are shown in Table 9 below. The control contained no peppermint oil.

[0109] Table 9 NR activity of A.a was not affected when incubated in the presence of 1 μL / ml or 1000 ppm peppermint oil and nitrate.

[0110] Effect of cysteine on V.a NR activity After 3 hours of incubation, 1 μL / ml peppermint inhibited about 20% of NR activity. Example 9: Effect of Cysteine on NR Activity of Test Bacteria A. actinomycetemcomitans

[0111] Test bacterial cells were incubated with 1% cysteine in growth media and 0.3% nitrate source. NR activity was measured for up to 3 hours and the results are shown in Tables 18 and 19. The control contained no cysteine. Actinobacillus naeslundii Test bacterial cells were incubated with 1% cysteine in growth media and 0.3% nitrate source. NR activity was measured for up to 3 hours and the results are shown in Tables 18 and 19. The control contained no cysteine.

[0112] Table 11: NR activity of A. a with cysteine present The addition of cysteine did not significantly affect the NR activity of A. a.

[0113] V. atypical Table 12: NR activity of V. a with cysteine present The results of these examples indicate that salivary bacteria, in particular Sugar free gum A and B 、 Double layer peppermint gum A and B and ​ ​ are the major oral bacteria producing nitrite. Streptococcus does not significantly contribute to the generation of salivary nitrite. Treating A. actinomycetemcomitans and V. disporillum with natural bactericidal MBE (50-150 pg / ml) or mint flavor oil (at the dose typically incorporated in chewing gums and mints) does not significantly affect the activity of nitrate reductase and, therefore, the production of nitrite by these products and its impact on cardiovascular risk. On the other hand, treating these oral bacteria with chlorhexidine (>10 pg / ml) significantly inhibits the activity of oral bacterial nitrate reductase and, therefore, increases the risk of cardiovascular disease.

[0114] Example 10: Sugarless chewing gum containing dietary nitrate ​ For each of chewing gums A and B, the gum base was heated to 165°F and placed in a mixer. The high intensity sweetener, flavor, and other ingredients were added to the mixer, followed by the dietary nitrate. The mixture was mixed for 7-12 minutes to ensure homogeneity. The mixture was then pressed into tablets and cut, with a tablet weight of 2.75 g. The composition of chewing gums A and B is shown in Table 13 below.

[0115] Table 13 Example 11: Double-layer pressed mints containing dietary nitrate ​ For each of double-layer mints A and B, all ingredients were mixed using a Hobart mixer. 0.5 g of each layer was loaded into a press. The layers were pressed to form double-layer pressed mints. The hardness was adjusted to 15-18 kPa, and the tablet weight was adjusted to 1.0 g. The composition of double-layer mints A and B is shown in Table 14 below. This written description uses examples to disclose the technology, including the best mode, and also to enable any person skilled in the art to practice the technology, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the technology is defined by the claims, and can include other examples that occur to those skilled in the art. Such other examples are intended to fall within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent CLAIM (modified pursuant to Article 19 of the Treaty) 1. An oral composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity; wherein the nitrate salt comprises a combination of: sodium or potassium nitrate; and a nitrate salt-rich food. 2. The oral composition of claim 1, wherein the nitrate salt further comprises a nitrate salt-rich extract. 3. The oral composition of claim 1, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the oral composition. 4. The oral composition of claim 1, wherein the nitrate salt-rich food is selected from the group consisting of beetroot, kale, cress, mustard, spinach, parsley, watercress, fennel, Chinese cabbage, bok choy, chard, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, chicory leaf, bean sprout, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or combinations thereof. 5. The oral composition of claim 2, wherein the nitrate salt-rich extract is selected from the group consisting of beetroot extract, celery extract, or combinations thereof. 6. The oral composition of claim 1, wherein the composition is in the form of a confectionery composition selected from the group consisting of breath freshening mints, cool boiled sweets, chewing gum, chewy sweets, hard boiled sweets, coated sweets, pastilles, drops, compressed mints, throat lozenges, or chocolate. 7. The oral composition of claim 6, wherein the chewing gum is sugar-free chewing gum. 8. An oral composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity; wherein the nitrate salt comprises a combination of: sodium or potassium nitrate; and a nitrate salt-rich food; and an effective amount of ascorbic acid or a suitable salt thereof. 9. The oral composition of claim 8, wherein the nitrate salt further comprises a nitrate salt-rich extract. 10. The oral composition of claim 8, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the oral composition. 11. The oral composition of claim 8, wherein the ascorbic acid or suitable salt thereof is present in an amount of about 0.01% to about 5% by weight of the oral composition. 12. The oral composition of claim 8, wherein the nitrate salt-rich food is selected from beetroot, kale, cress, watercress, spinach, parsley, watercress, fennel, Chinese cabbage, bok choy, leek, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, endive leaf, bean sprout, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or combinations thereof. 13. The oral composition of claim 9, wherein the nitrate salt-rich extract is selected from beetroot extract, celery extract, or combinations thereof. 14. The oral composition of claim 8, wherein the composition is in the form of a confectionery composition selected from breath freshening mints, low temperature boiled sweets, chewing gum, chewy sweets, hard boiled sweets, coated sweets, pastilles, toffees, compressed mints, throat lozenges, or chocolate. 15. The oral composition of claim 14, wherein the chewing gum is sugar-free chewing gum. 16. A confectionery composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in an oral cavity; wherein the nitrate salt comprises a combination of: sodium nitrate or potassium nitrate; and a nitrate salt-rich food. 17. The confectionery composition of claim 16, wherein the nitrate salt further comprises a nitrate salt-rich extract. 18. The confectionery composition of claim 16, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the confectionery composition. 19. The confectionery composition of claim 16, wherein the nitrate salt-rich food is selected from beetroot, kale, cress, watercress, spinach, parsley, watercress, fennel, Chinese cabbage, bok choy, leek, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, endive leaf, bean sprout, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or combinations thereof. 20. The confectionery composition of claim 17, wherein the nitrate-rich extract is selected from beetroot extract, celery extract, or a combination thereof. 21. The confectionery composition of claim 16, wherein the confectionery composition is in the form of a breath freshening mint, a cold boiled candy, a chewing gum, a chewy candy, a hard boiled candy, a coated candy, a lozenge, a syrup, a pressed mint, a throat drop, or a chocolate. 22. The confectionery composition of claim 21, wherein the chewing gum is a sugarless chewing gum. 23. The confectionery composition of claim 16, wherein the composition further comprises ascorbic acid or a suitable salt thereof. 24. The confectionery composition of claim 23, wherein the ascorbic acid or a suitable salt thereof is present in an amount of about 0.01% to about 5% by weight of the confectionery composition. 25. A method for freshening breath, the method comprising consuming a confectionery composition comprising an effective amount of a nitrate; wherein the nitrate comprises a combination of: sodium or potassium nitrate; and a nitrate-rich food. 26. The method of claim 25, wherein the nitrate further comprises a nitrate-rich extract. 27. The method of claim 25, wherein the nitrate is present in an amount of about 0.01% to about 5% by weight of the confectionery composition. 28. The method of claim 25, wherein the nitrate-rich food is selected from beetroot, kale, cress, mustard, spinach, parsley, watercress, fennel, Chinese cabbage, bok choy, chard, chives, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, endive, bean sprouts, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or a combination thereof. 29. The method of claim 26, wherein the nitrate-rich extract is selected from beetroot extract, celery extract, or a combination thereof. 30. The method of claim 25, wherein the confectionery composition is in the form of a breath freshening mint, a cold boiled candy, a chewing gum, a chewy candy, a hard boiled candy, a coated candy, a lozenge, a syrup, a pressed mint, a throat drop, or a chocolate. 31. The method of claim 30, wherein the chewing gum is a sugarless chewing gum. 32. The method of claim 25, wherein the confectionery composition further comprises ascorbic acid or a suitable salt thereof. 33. The method of claim 32, wherein the ascorbic acid or a suitable salt thereof is present in an amount of from about 0.01% to about 5% by weight of the confectionery composition.

Claims

1. An oral composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity.

2. The oral composition of claim 1, wherein the nitrate salt comprises sodium nitrate or potassium nitrate.

3. The oral composition of claim 1, wherein the nitrate salt comprises a dietary nitrate derived from a nitrate-rich food, a nitrate-rich extract, or a combination thereof.

4. The oral composition of claim 1, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the oral composition.

5. The oral composition of claim 3, wherein the nitrate-rich food is selected from beetroot, kale, cress, watercress, spinach, parsley, water dropwort, anise, Chinese cabbage, bok choy, leek, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, endive leaf, bean sprout, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or a combination thereof.

6. The oral composition of claim 3, wherein the nitrate-rich extract is selected from beetroot extract, celery extract, or a combination thereof.

7. The oral composition of claim 1, wherein the composition is in the form of a confectionery composition selected from a breath freshening mint, a low temperature boiled candy, a chewing gum, a chewy candy, a hard boiled candy, a coated candy, a lozenge, a syrup, a pressed mint, a throat drop, or a chocolate.

8. The oral composition of claim 7, wherein the chewing gum is a sugarless chewing gum.

9. An oral composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity; and an effective amount of ascorbic acid or a suitable salt thereof.

10. The oral composition of claim 9, wherein the nitrate salt comprises sodium nitrate or potassium nitrate.

11. The oral composition of claim 9, wherein the nitrate salt comprises a dietary nitrate derived from a nitrate-rich food, a nitrate-rich extract, or a combination thereof.

12. The oral composition of claim 9, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the oral composition.

13. The oral composition of claim 9, wherein the ascorbic acid or a suitable salt thereof is present in an amount of about 0.01% to about 5% by weight of the oral composition.

14. The oral composition of claim 11, wherein the nitrate-rich food is selected from beetroot, kale, cress, watercress, spinach, parsley, water dropwort, anise, Chinese cabbage, bok choy, leek, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, endive leaf, bean sprout, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or a combination thereof.

15. The oral composition of claim 11, wherein the nitrate-rich extract is selected from beetroot extract, celery extract, or a combination thereof. ​ ​ 16. The oral composition of claim 9, wherein the composition is in the form of a confectionery composition selected from the group consisting of breath freshening mints, low temperature boiled candies, chewing gums, chewy candies, hard boiled candies, coated candies, lozenges, syrups, pressed mints, throat lozenges, or chocolates.

17. The oral composition of claim 16, wherein the chewing gum is a sugarless chewing gum.

18. A confectionery composition comprising an effective amount of a nitrate salt for reducing volatile sulfur compounds in the oral cavity.

19. The confectionery composition of claim 18, wherein the nitrate salt comprises sodium nitrate or potassium nitrate.

20. The confectionery composition of claim 18, wherein the nitrate salt comprises a dietary nitrate derived from a nitrate-rich food, a nitrate-rich extract, or a combination thereof.

21. The confectionery composition of claim 15, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the confectionery composition.

22. The confectionery composition of claim 20, wherein the nitrate-rich food is selected from the group consisting of beetroot, kale, cress, arugula, spinach, parsley, watercress, anise, Chinese cabbage, bok choy, chard, endive, lettuce, celery, radish, turnip, rocket, beet leaf, mustard, carrot, onion, garlic, kohlrabi, chicory leaf, bean sprouts, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or a combination thereof.

23. The confectionery composition of claim 20, wherein the nitrate-rich extract is selected from the group consisting of beetroot extract, celery extract, or a combination thereof.

24. The confectionery composition of claim 18, wherein the confectionery composition is in the form of a breath freshening mint, a low temperature boiled candy, a chewing gum, a chewy candy, a hard boiled candy, a coated candy, a lozenge, a syrup, a pressed mint, a throat lozenge, or a chocolate.

25. The confectionery composition of claim 24, wherein the chewing gum is a sugarless chewing gum.

26. The confectionery composition of claim 18, wherein the composition further comprises ascorbic acid or a suitable salt thereof.

27. The confectionery composition of claim 26, wherein the ascorbic acid or a suitable salt thereof is present in an amount of about 0.01% to about 5% by weight of the confectionery composition.

28. A method for freshening breath, the method comprising consuming a confectionery composition comprising an effective amount of a nitrate salt.

29. The method of claim 28, wherein the nitrate salt comprises sodium nitrate or potassium nitrate.

30. The method of claim 28, wherein the nitrate salt comprises a dietary nitrate derived from a nitrate-rich food, a nitrate-rich extract, or a combination thereof.

31. The method of claim 28, wherein the nitrate salt is present in an amount of about 0.01% to about 5% by weight of the confectionery composition.

32. The method of claim 30, wherein the nitrate-rich food is selected from beetroot, kale, cress, mustard, spinach, parsley, watercress, fennel, Chinese cabbage, bok choy, leek, lettuce, celery, radish, turnip, rocket, chard, mustard, carrot, onion, garlic, kohlrabi, chicory leaves, bean sprouts, watermelon, kiwi, apple, pomegranate, banana, orange, strawberry, peach, pear, grape, chocolate, or combinations thereof.

33. The method of claim 30, wherein the nitrate-rich extract is selected from beetroot extract, celery extract, or combinations thereof.

34. The method of claim 28, wherein the confectionery composition is in the form of a breath freshening mint, a cold boiled candy, a chewing gum, a chewy candy, a hard boiled candy, a coated candy, a lozenge, a syrup, a pressed mint, a throat drop, or a chocolate.

35. The method of claim 34, wherein the chewing gum is a sugarless chewing gum.

36. The method of claim 28, wherein the confectionery composition further comprises ascorbic acid or a suitable salt thereof.

37. The method of claim 36, wherein the ascorbic acid or a suitable salt thereof is present in an amount of about 0.01% to about 5% by weight of the confectionery composition.

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