Oral microbiota improver

Abstract

To provide a material effective for enhancing the content ratio of bacteria belonging to the genus Neisseria and the genus Actinomyces in the oral cavity.SOLUTION: An oral microbiota improver comprises, as active ingredients, one or more salt compounds selected from organic acid salts, bicarbonates, and carbonates, and a nitrate ion-supplying compound.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an oral flora-improving agent that contributes to the prevention or improvement of oral diseases and the like. [Background technology]

[0002] In the human body, microorganisms form resident flora based on complex interactions and inhabit various parts of the body. The composition of the resident flora varies between individuals, and in recent years, many studies have pointed out the relationship between these individual differences and the health status of the host, attracting attention. Like the intestine, the oral cavity is a microbial ecosystem in which numerous types of bacteria coexist. Several pathogenic bacteria have been identified as responsible for oral infectious diseases such as dental caries and periodontal disease, but these bacteria also coexist in the oral cavity as part of the normal ecosystem. Therefore, it is speculated that interactions with other resident bacteria play a major role in the development of disease. While the traditional goal in preventing and treating oral disease has been to eliminate pathogenic bacteria that directly cause disease, it is believed that inducing a resident flora that makes it difficult for pathogenic bacteria to thrive could also be an approach to preventing oral disease.

[0003] Bacteria of the genus Neisseria and Actinomyces are known to exist as non-pathogenic normal bacteria in the oral cavity (Non-Patent Document 1). Neisseria mucosa, a type of Neisseria, or its culture has been found to have the effect of suppressing infection of gingival cells by periodontal pathogens, and these Neisseria are useful for the prevention or treatment of periodontal disease (Patent Document 1). In addition, Neisseria and Actinomyces possess nitrate reductase and nitrite reductase, which convert nitrate ions (NO3 - ) to nitrite ions (NO2 -It has been reported that nitrate ions have the ability to reduce nitrate to nitrite and nitric oxide (NO) (Non-Patent Documents 2 and 3). When nitrate ions are orally ingested, a portion of the nitrate ions absorbed into the body migrates from the blood into saliva at high concentrations, where they are reduced to nitrite by nitrate-reducing bacteria present in the oral cavity. Nitric oxide (NO) is then produced enzymatically or non-enzymatically in the stomach and various tissues. Nitric oxide is an important compound that exerts various functions throughout the body, including intercellular signaling, antibacterial activity, biological defense against pathogens, vasodilatory activity, and muscle contractility. It is particularly expected to have antibacterial activity against periodontal pathogens, blood pressure reduction due to improved vascular function, and motor function improvement due to improved muscle contractility (Non-Patent Documents 4-7). In addition, Actinomyces bacteria have urease activity, which hydrolyzes urea to produce ammonia (Non-Patent Document 8). Urease activity is thought to inhibit caries by suppressing acidification in the oral cavity, and it has been reported that people without caries have high urease activity (Non-Patent Document 9). Therefore, it is useful to increase the oral content of Neisseria and Actinomyces.

[0004] On the other hand, salts such as sodium citrate, sodium bicarbonate, sodium carbonate, sodium gluconate, sodium succinate, sodium fumarate, sodium lactate, sodium malate, and sodium tartrate are incorporated into oral compositions as pH adjusters, and potassium ions have the effect of reducing the hypersensitivity of the dental pulp nerve, and potassium nitrate and the like are known to be effective against tooth hypersensitivity (Non-Patent Document 10). However, the effects of salt compounds and potassium nitrate on Neisseria and Actinomyces bacteria are completely unknown. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent application 2020-200517 [Non-patent literature]

[0006] [Non-licensed document 1] PLoS One. 2018 Jul 6;13(7):e0200337. doi: 10.1371 / journal.pone.0200337 https: / / pubmed.ncbi.nlm.nih.gov / 29979786 / [Non-licensed document 2] Barth KR et al., Microbiology (2009),155:4093-4103 [Non-licensed document 3] Eur J Oral Sci. 2005 Feb;113(1):14-9. doi: 10.1111 / j.1600-0722.2004.00184.x. https: / / pubmed.ncbi.nlm.nih.gov / 15693824 /

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0007] The present invention relates to providing a material useful for increasing the content of Neisseria and Actinomyces bacteria in the oral cavity. [Means for solving the problem]

[0008] The inventors have discovered that by combining a specific salt compound with a nitrate ion supplying compound, the content of Neisseria and Actinomyces bacteria in the oral cavity can be increased, and this is useful for preventing or treating periodontal disease and increasing nitric oxide.

[0009] That is, the present invention relates to the following 1) to 4). 1) An oral flora improving agent containing, as active ingredients, one or more salt compounds selected from organic acid salts, bicarbonates and carbonates, and a nitrate ion supplying compound. 2) A food for improving oral flora, containing as active ingredients one or more salt compounds selected from organic acid salts, bicarbonates and carbonates, and a nitrate ion supplying compound. 3) An oral flora improving agent containing one or more salt compounds selected from citrates, bicarbonates and carbonates as an active ingredient. 4) A food for improving oral flora, containing as an active ingredient one or more salt compounds selected from citrates, bicarbonates and carbonates. [Effects of the Invention]

[0010] According to the present invention, the content of Neisseria and Actinomyces bacteria in the oral cavity can be increased, thereby enabling the prevention or treatment of periodontal disease and dental caries. Furthermore, the supply of nitric oxide improves vascular function and skeletal muscle contractility, thereby enabling the prevention or amelioration of hypertension and the improvement or enhancement of motor function. [Brief explanation of the drawings]

[0011] [Figure 1] Effects of salt compounds (citrates) and their combination with potassium nitrate on Neisseria bacteria in dental plaque biofilm flora. [Figure 2] The effects of salt compounds (bicarbonate, carbonate) and their combination with potassium nitrate on Neisseria bacteria in dental plaque biofilm flora. [Figure 3] Effects of salt compounds (gluconate, succinate, fumarate, lactate, malate, tartrate) and their combination with potassium nitrate on Neisseria species in dental plaque biofilm flora. [Figure 4] Effects of salt compounds (citrates) and their combination with potassium nitrate on Actinomyces bacteria in dental plaque biofilm flora. [Figure 5] The effects of salt compounds (bicarbonate, carbonate) and the combination of these salt compounds with potassium nitrate on Actinomyces bacteria in dental plaque biofilm flora. [Figure 6] The effects of salt compounds (gluconate, succinate, fumarate, lactate, malate, tartrate) and their combination with potassium nitrate on Actinomyces in dental plaque biofilm flora. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the present invention, the salt compound used is at least one selected from organic acid salts, bicarbonates, and carbonates. Examples of the salt include metal salts such as sodium, potassium, magnesium, aluminum, and calcium, with sodium salts and potassium salts being preferred. Examples of organic acid salts include citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate. It is preferable to use one or more selected from citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate, and it is more preferable to use citrate, succinate, or malate. From the viewpoint of improving oral bacterial flora, preferred salt compounds are sodium citrate, sodium bicarbonate, and sodium carbonate, and more preferred is sodium citrate.

[0013] In the present invention, the above salt compounds are preferably used in combination with a nitrate ion-supplying compound, which exhibits a more excellent effect of improving oral flora. However, when citrate, bicarbonate, or carbonate is used alone, it also exhibits an excellent effect of improving oral flora. Nitrate ion-donating compounds include compounds capable of generating nitrate ions upon contact with water, with nitrates being preferred. Nitrates include, for example, alkali metal nitrates and alkaline earth metal nitrates, with specific examples including sodium nitrate, potassium nitrate, and calcium nitrate, with potassium nitrate being preferred. While synthetic nitrates are preferred, natural nitrates may also be used, including inorganic nitrate salts and concentrates, extracts, and juices of nitrate-containing plants. Nitrate-containing plants may be any plant that contains nitrate, including, for example, sweet potato leaves, kale, barley, celery, Japanese mustard spinach, parsley, spinach, radish, beets, broccoli, burdock, and aloe, with one or more selected from sweet potato leaves, kale, celery, Japanese mustard spinach, and radish being preferred.

[0014] When a salt compound and a nitrate ion-supplying compound are used in combination, the form may be a combination drug in which effective amounts of each compound are formulated in a single dosage form at an appropriate blend ratio, or a kit in which drugs containing effective amounts of each compound are formulated individually and can be used simultaneously or separately at intervals. A combination drug form is preferred.

[0015] In the present invention, the ratio of the salt compound and the nitrate ion donating compound in combination can be appropriately selected, but for example, the nitrate ion donating compound is preferably 0.1 part by mass or more, more preferably 0.5 part by mass or more, more preferably 1 part by mass or more, and preferably 20 parts by mass or less, more preferably 10 parts by mass or less, more preferably 5 parts by mass or less, per 1 part by mass of the salt compound. Also, the ratio is preferably 0.1 to 20 parts by mass, more preferably 0.5 to 10 parts by mass, more preferably 1 to 5 parts by mass.

[0016] As shown in the Examples below, a dental plaque suspension containing dental plaque collected from a healthy subject was cultured in a medium containing potassium nitrate and a salt compound selected from sodium citrate, sodium gluconate, sodium succinate, sodium fumarate, sodium lactate, sodium malate, sodium tartrate, sodium bicarbonate, and sodium carbonate to prepare a dental plaque biofilm, and the contents of Neisseria and Actinomyces were calculated. A significant increase in the contents of Neisseria and Actinomyces was observed compared to the control group. Furthermore, when sodium citrate, sodium bicarbonate, and sodium carbonate were used alone without potassium nitrate, a significant increase in the contents of Neisseria and Actinomyces was observed compared to the control group. Therefore, a combination of one or more salt compounds selected from organic acid salts, bicarbonates, and carbonates with a nitrate ion-donating compound, or one or more salt compounds selected from citrate, bicarbonate, and carbonate, can serve as an oral flora-improving agent that increases oral Neisseria and / or Actinomyces bacteria, and can be used to produce such an oral flora-improving agent. Furthermore, a combination of one or more salt compounds selected from organic acid salts, bicarbonates, and carbonates with a nitrate ion-donating compound, or one or more salt compounds selected from citrate, bicarbonate, and carbonate, can be used to improve oral flora that increases oral Neisseria and / or Actinomyces bacteria. The use may involve administration to humans or non-human animals, or use in samples derived therefrom, and may be therapeutic or non-therapeutic. Here, non-human animals include non-human mammals such as apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, hamsters, and companion animals.

[0017] In the present invention, the genus Neisseria refers to non-pathogenic oral bacteria belonging to the genus Neisseria in the family Neisseria, including Neisseria mucosa, Neisseria sicca, Neisseria flava, Neisseria subflava, Neisseria flavescens, and Neisseria elongata.

[0018] Actinomyces refers to non-pathogenic oral bacteria belonging to the genus Actinomyces in the family Actinomycetaceae, including Actinomyces odontolyticus, Actinomyces oris, and Actinomyces naeslundii.

[0019] In the present invention, "increase in oral Neisseria bacteria" and "increase in oral Actinomyces bacteria" mean that the content of Neisseria bacteria or Actinomyces bacteria in the oral cavity increases by activating the metabolism of oral Neisseria bacteria or oral Actinomyces bacteria, thereby causing a change in the oral bacterial flora. Furthermore, "improvement of oral flora" refers to an increase in the content of Neisseria and / or Actinomyces in the oral cavity, which allows the physiological functions of Neisseria and Actinomyces to be exerted more effectively in the oral cavity, resulting in a change in the oral flora that is favorable for the host.

[0020] The content of Neisseria or Actinomyces can be calculated by measuring the copy number of the rRNA16S gene by real-time PCR using a probe that specifically detects the rRNA16S gene of all Neisseria or Actinomyces species, and then calculating the ratio to the copy number of the rRNA16S gene of all bacteria.

[0021] As mentioned above, Neisseria mucosa, a type of bacterium of the genus Neisseria, is useful for preventing or treating periodontal disease because its bacterial cells or cultures thereof suppress infection of gingival cells by periodontal pathogens and suppress gingival inflammation (Patent Document 1). Periodontal pathogens refer to bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum that infect periodontal tissues and cause gingivitis and even periodontal tissue destruction. In addition, Neisseria and Actinomyces bacteria produce nitrate ions (NO3 - ) to nitrite ions (NO2 - Since nitrate ion-supplying compounds have the ability to reduce nitrate ions to nitrite and nitric oxide (NO) (Non-Patent Documents 2 and 3, cited above), it is thought that these compounds are reduced by Neisseria bacteria to nitrite and then converted to nitrogen oxides, including nitric oxide, which exert a blood pressure lowering effect due to improved vascular function and an exercise function improving effect due to improved skeletal muscle contractility (Non-Patent Documents 4-7, cited above). In addition, Actinomyces bacteria are thought to inhibit dental caries by suppressing acidification in the oral cavity through urease activity (Non-Patent Documents 8 and 9 mentioned above). Therefore, the oral bacterial flora improving agent of the present invention serves as an agent for suppressing periodontal pathogen infection, an agent for preventing or improving periodontal disease, and an agent for preventing or improving dental caries, and can be used for suppressing periodontal pathogen infection, preventing or improving periodontal disease, and preventing or improving dental caries. In addition, in an embodiment in which a salt compound and a nitrate ion supplying compound are used in combination, the oral flora improving agent further functions as a nitric oxide increaser, an agent for preventing or improving high blood pressure, and an agent for improving motor function, and can also be used for increasing nitric oxide, improving or enhancing motor function, and preventing or improving high blood pressure.

[0022] Here, "periodontal disease" means an inflammatory disease in which inflammation of the gums is caused by bacteria in dental plaque, and includes gingivitis and periodontitis. "Increasing nitric oxide" means increasing the natural concentration of nitric oxide in the body. Increasing nitric oxide can prevent or improve vascular dysfunction caused by nitric oxide deficiency (e.g., vascular endothelial dysfunction, arteriosclerosis, etc.) and associated pathologies (hypertension, myocardial infarction, cerebral infarction, etc.). Increasing nitric oxide can also improve skeletal muscle contractility, thereby improving or enhancing motor function. The term "prevention" refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of an individual developing a disease or symptom. "Amelioration" means improvement of a disease, symptom or condition, prevention or delay of worsening of a disease, symptom or condition, or reversal, prevention or delay of progression of a disease or symptom, and includes the concept of so-called "treatment." "Improvement" means that physical functions, etc. move in a better or superior state from the current state.

[0023] The oral flora improving agent of the present invention can itself be used as a pharmaceutical, quasi-drug, food, or oral composition for improving oral flora, suppressing periodontal pathogen infection, preventing or improving periodontal disease, preventing or improving dental caries, increasing nitric oxide, preventing or improving high blood pressure, or improving or enhancing motor function, or can be used as a material or preparation to be incorporated into such pharmaceuticals, quasi-drugs, foods, or oral compositions. The foods include foods based on the concept of improving oral flora, suppressing periodontal pathogen infection, preventing or improving periodontal disease, preventing or improving dental caries, increasing nitric oxide, preventing or improving high blood pressure, improving or enhancing motor function, etc., and are labeled as such as functional foods, foods for specified health uses, foods for the sick, and supplements.

[0024] When used as a pharmaceutical or quasi-drug, the formulation is preferably an oral administration form, and examples of the dosage form include liquid preparations; solid preparations such as tablets, granules, fine granules, powders, and tablets; and various forms such as capsules, oral sprays, and troches containing the liquid preparations or solid preparations.

[0025] When used as a food, the forms include beverages such as fruit or vegetable juice drinks, carbonated drinks, tea drinks, dairy drinks, fermented milk, fermented fruit juice, fermented vegetable juice, alcoholic drinks, and soft drinks, as well as various foods such as jelly foods, various snacks, baked goods, cakes, chocolate, jam, bread, gum, candy, soups, pickles, and tsukudani (foods boiled in soy sauce), as well as supplements in the same form as the oral administration formulations described above (tablets, capsules, syrup, etc.).

[0026] Specific forms for use as oral compositions include mouthwash, mouthwash, toothpaste, powder toothpaste, dentifrice, oral ointment, gel, tablet, granule, fine granule, gummy jelly, troche, tablet, capsule, candy, chewing gum, etc., and preferred are toothpaste, mouthwash, gummy jelly, and troche.

[0027] The pharmaceuticals, quasi-drugs, foods, and oral compositions can be prepared according to standard methods by appropriately combining the salt compounds and nitrate ion supply compounds, or salt compounds, of the present invention with other active ingredients, or additives acceptable for pharmaceuticals, foods, etc., or any food ingredients, etc.

[0028] The content of the salt compound and nitrate ion supplying compound, or salt compound in the above-mentioned pharmaceuticals, quasi-drugs, foods, and oral compositions is not particularly limited and may be adjusted appropriately according to the daily dosage, etc.

[0029] In the above-mentioned pharmaceuticals, quasi-drugs, foods, and oral compositions, the intake or administration amount of the active ingredient can be determined appropriately depending on various conditions such as the patient's weight, age, sex, and symptoms. For an adult, the daily intake or administration amount of salt compounds is 10 mg or more, preferably 50 mg or more, more preferably 80 mg or more, and 6000 mg or less, preferably 3000 mg or less, and more preferably 500 mg or less. For nitrate ion supply compounds, the daily intake or administration amount is 10 mg or more, preferably 200 mg or more, more preferably 600 mg or more, and 6000 mg or less, preferably 3000 mg or less, and more preferably 2000 mg or less.

[0030] Preferred target subjects for the oral bacterial flora improving agent of the present invention include humans infected with periodontal pathogens, humans with gingival inflammation such as gingivitis or periodontitis, humans wishing to prevent the onset of gingival inflammation, humans with hypertension, humans wishing to lower their blood pressure, and humans wishing to improve or enhance motor function.

[0031] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> An oral flora improving agent containing, as active ingredients, one or more salt compounds selected from organic acid salts, bicarbonates and carbonates, and a nitrate ion supplying compound. <2> A food for improving oral flora, which contains as active ingredients one or more salt compounds selected from organic acid salts, bicarbonates and carbonates, and a nitrate ion supplying compound. <3> The organic acid salt is one or more selected from citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate; <1> or <2> Food. <4> The improvement of oral flora is an increase in the content of oral Neisseria and / or Actinomyces. <1> or <3> or <2> or <3> Food. <5> the nitrate ion-donating compound is nitrate; <1> or <3> or <2> or <3> Food. <6> the nitrate is sodium nitrate or potassium nitrate; <5> Agents or foods. <7> Used to suppress periodontal pathogen infections, <1> or <3> or <2> or <3> Food. <8> Used to prevent or improve periodontal disease, <1> or <3> or <2> or <3> Food. <9> Used to prevent or improve dental caries, <1> or <3> or <2> or <3> Food. <10> Used to increase nitric oxide <1> or <3> or <2> or <3> Food. <11> Used to prevent or improve high blood pressure, <1> or <3> or <2> or <3> Food. <12> Used to improve or enhance motor function, <1> or <3> or <2> or <3> Food. <13> An oral flora improving agent containing one or more salt compounds selected from citrates, bicarbonates and carbonates as an active ingredient. <14> A food for improving oral flora, containing one or more salt compounds selected from citrates, bicarbonates and carbonates as active ingredients. <15> The improvement of oral flora is an increase in the content of oral Neisseria and / or Actinomyces. <13> or <14> Food.

[0032] <16> 1. Use of a combination of one or more salt compounds selected from organic acid salts, bicarbonates and carbonates with a nitrate ion-supplying compound for producing an oral flora improving agent or oral flora improving food. <17> 1. Use of one or more salt compounds selected from citrates, bicarbonates, and carbonates for producing an oral flora improving agent or an oral flora improving food. <18> The organic acid salt is one or more selected from citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate; <16> Use of. <19> The improvement of oral flora is an increase in the content of oral Neisseria and / or Actinomyces. <16> or <17> Use of. <20> the nitrate ion-donating compound is nitrate; <16> Use of. <21> the nitrate is sodium nitrate or potassium nitrate; <20> Use of. <22> Improving oral flora is done to suppress periodontal pathogen infection. <16> or <17> Use of. <23> Oral flora improvement is performed to prevent or improve periodontal disease, <16> or <17> Use of. <24> Oral flora improvement is performed to prevent or improve dental caries, <16> or <17> Use of. <25> Oral flora improvement is performed to increase nitric oxide. <16> Use of. <26> Oral flora improvement is performed to prevent or improve hypertension. <16> Use of. <27> Oral flora improvement is performed to improve or enhance motor function, <16> Use of.

[0033] <28> A combination of one or more salt compounds selected from organic acid salts, bicarbonates and carbonates with a nitrate ion-supplying compound for use in improving oral flora. <29> Use of one or more salt compounds selected from citrates, bicarbonates, and carbonates for use in improving oral flora. <30> The organic acid salt is one or more selected from citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate; <28> Use of. <31> The improvement of oral flora is an increase in the content of oral Neisseria and / or Actinomyces. <28> or <29> Use of. <32> the nitrate ion-donating compound is nitrate; <31> Use of. <33> the nitrate is sodium nitrate or potassium nitrate; <32> Use of. <34> Improving oral flora is done to suppress periodontal pathogen infection. <28> or <29> Use of. <35> Oral flora improvement is performed to prevent or improve periodontal disease. <28> or <29> Use of. <36> Oral flora improvement is performed to prevent or improve dental caries, <28> or <29> Use of. <37> Oral flora improvement is performed to increase nitric oxide. <28> Use of. <38> Oral flora improvement is performed to prevent or improve hypertension. <28> Use of. <39> Oral flora improvement is performed to improve or enhance motor function, <28> Use of.

[0034] <40> A non-therapeutic use of a combination of one or more salt compounds selected from organic acid salts, bicarbonates and carbonates with a nitrate ion-supplying compound for improving oral flora. <41> 1. Non-therapeutic use of one or more salt compounds selected from citrates, bicarbonates and carbonates for improving oral flora. <42> The organic acid salt is one or more selected from citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate; <40> Non-therapeutic use of. <43> The improvement of oral flora is an increase in the content of oral Neisseria and / or Actinomyces. <40> or <41> Non-therapeutic use of. <44> the nitrate ion-donating compound is nitrate; <40> Non-therapeutic use of. <45> the nitrate is sodium nitrate or potassium nitrate; <44> Non-therapeutic use of. <46> Improving oral flora is done to suppress periodontal pathogen infection. <40> or <41> Non-therapeutic use of. <47> Oral flora improvement is performed to prevent or improve periodontal disease. <40> or <41> Non-therapeutic use of. <48> Oral flora improvement is performed to prevent or improve dental caries, <40> or <41> Non-therapeutic use of. <49> Oral flora improvement is performed to increase nitric oxide. <40> Non-therapeutic use of. <50> Oral flora improvement is performed to prevent or improve hypertension. <40> Non-therapeutic use of. <51> Oral flora improvement is performed to improve or enhance motor function, <40> Non-therapeutic use of.

[0035] <52> A method for improving oral flora, comprising administering or ingesting to a subject in need thereof effective amounts of one or more salt compounds selected from organic acid salts, bicarbonates, and carbonates, and a nitrate ion supplying compound. <53> A method for improving oral flora, comprising administering or ingesting an effective amount of one or more salt compounds selected from citrates, bicarbonates, and carbonates to a subject in need thereof. <54> The organic acid salt is one or more selected from citrate, gluconate, succinate, fumarate, lactate, malate, and tartrate; <52> How to do it. <55> The improvement of oral flora is an increase in the content of oral Neisseria and / or Actinomyces. <52> or <53> How to do it. <56> the nitrate ion-donating compound is nitrate; <52> How to do it. <57> the nitrate is sodium nitrate or potassium nitrate; <56> How to do it. <58> Improving oral flora is done to suppress periodontal pathogen infection. <52> or <53> How to do it. <59> Oral flora improvement is performed to prevent or improve periodontal disease, <52> or <53> How to do it. <60> Oral flora improvement is performed to prevent or improve dental caries, <52> or <53> How to do it. <61> Oral flora improvement is performed to increase nitric oxide. <52> How to do it. <62> Oral flora improvement is performed to prevent or improve hypertension. <52> How to do it. <63> Oral flora improvement is performed to improve or enhance motor function, <52> How to do it.

[0036] <64> <1> , <2> , <16> , <28> , <40> , <52> In the above, the ratio of the combination of the salt compound and the nitrate ion supply compound is preferably 0.1 part by mass or more, more preferably 0.5 part by mass or more, more preferably 1 part by mass or more, and preferably 20 parts by mass or less, more preferably 10 parts by mass or less, more preferably 5 parts by mass or less, or preferably 0.1 to 20 parts by mass, more preferably 0.5 to 10 parts by mass, more preferably 1 to 5 parts by mass, of the nitrate ion supply compound per 1 part by mass of the salt compound. <65> <1> ~ <63> In this case, the intake or administration amount of the active ingredient is, per adult per day, 10 mg or more, preferably 50 mg or more, more preferably 80 mg or more, and 6000 mg or less, preferably 3000 mg or less, more preferably 500 mg or less, as salt compounds, and 10 mg or more, preferably 200 mg or more, more preferably 600 mg or more, and 6000 mg or less, preferably 3000 mg or less, more preferably 2000 mg or less, as nitrate ion supply compounds. [Example]

[0037] Addition of salt compounds alone and in combination with nitrate increases the content of Neisseria and Actinomyces in dental plaque biofilm flora (1) Plaque collection and pretreatment Plaque adhering to the tooth surface of a healthy male was collected using a dental scaler, placed in a 1.5 mL tube, and washed twice with PBS(-) by centrifugation at 7,000 g for 2 minutes at room temperature. The pellet containing plaque bacteria was suspended in 300 μL of 15% glycerol solution, and the prepared plaque suspension was stored at −80°C until use. A portion of this plaque suspension was used to measure the copy number of the total bacterial rRNA16S gene in the plaque suspension using the DNA extraction method and real-time PCR method described below.

[0038] (2) Preparation of a dental plaque biofilm model using dental plaque suspension culture The total bacterial rRNA 16S gene copy number of the dental plaque suspension was measured using Brain Heart Infusion (BHI) medium (BD Japan) containing the test compounds sodium citrate (Fujifilm Wako Pure Chemical), sodium bicarbonate (Sigma-Aldrich), sodium carbonate (Fujifilm Wako Pure Chemical), potassium nitrate (Fujifilm Wako Pure Chemical), sodium gluconate (Fujifilm Wako Pure Chemical), sodium succinate (Fujifilm Wako Pure Chemical), sodium fumarate (Fujifilm Wako Pure Chemical), sodium lactate (Fujifilm Wako Pure Chemical), sodium malate (Fujifilm Wako Pure Chemical), and sodium tartrate (Fujifilm Wako Pure Chemical). 8 A culture medium containing dental plaque bacteria was prepared by diluting the culture medium to 100 copies / mL. One mL of the culture medium was added to a Poly-L-Lysine Coated 24-Well Microplate (AGC TECHNO GLASS) and cultured at 37°C under microaerobic conditions using Anaeropack Bikouki (Mitsubishi Gas Chemical) for 48 hours to produce dental plaque biofilms. After 48 hours of culture, the supernatant was carefully aspirated without disturbing the dental plaque biofilm on the bottom, and a dental plaque biofilm sample was prepared.

[0039] (3) Extraction of genomic DNA from dental plaque biofilm A 20 mg / mL lysozyme (Fujifilm Wako Pure Chemical Industries) solution (containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 1.2% Triton X-100) was added to the dental plaque biofilm sample attached to the bottom of the plate at 200 μL / well and incubated at 37°C for 60 minutes with shaking (160 rpm). Genomic DNA was extracted using the Dneasy Blood & Tissue kit (QIAGEN) according to the kit's standard protocol. Finally, the sample was eluted with 200 μL of elution solution to obtain a genomic DNA solution. This was further diluted 21-fold with the elution solution to prepare a genomic DNA sample for real-time PCR.

[0040] (4) Quantification of Neisseria and Actinomyces in genomic DNA samples Quantification of Neisseria and Actinomyces was performed by real-time PCR using TaqMan probes. To specifically detect Neisseria and Actinomyces, TaqMan probes and primers were prepared to specifically detect the rRNA 16S genes of all Neisseria and Actinomyces species (Table 1). TaqMan probes and primers to detect the rRNA 16S genes of all bacteria were prepared using sequences previously described (Nadkarni, MA, et al. Microbiology (2002) 148, 257-266) (Table 1). The composition of the reaction solution and reaction conditions for real-time PCR are shown in Table 2.

[0041] [Table 1]

[0042] [Table 2]

[0043] A calibration curve was prepared using a plasmid DNA containing the target gene sequence with a known copy number as a standard, and the copy numbers of the rRNA16S gene of Neisseria and Actinomyces, as well as the copy numbers of the rRNA16S gene of all bacteria, were calculated. The Neisseria content and Actinomyces content were then calculated using the following formula.

[0044]

number

[0045] (5) Results The effects of adding sodium citrate, sodium bicarbonate, or sodium carbonate alone and in combination with nitrate on increasing the content of Neisseria and Actinomyces in dental plaque biofilm flora are shown in Figures 1, 2, 4, and 5. The effect of adding sodium gluconate, sodium succinate, sodium fumarate, sodium lactate, sodium malate, and sodium tartrate in combination with nitrates on increasing the content of Neisseria and Actinomyces in dental plaque biofilm flora is shown in Figures 3 and 6.

[0046] As shown in Figures 1 and 2, the addition of sodium citrate, sodium bicarbonate, or sodium carbonate alone significantly increased the Neisseria content compared to the control group, and the addition of sodium citrate, sodium bicarbonate, or sodium carbonate in combination with 0.1% or 0.5% potassium nitrate showed an even stronger increase in the Neisseria content. As shown in FIG. 3, the combined addition of sodium gluconate, sodium succinate, sodium malate and 0.5% potassium nitrate increased the Neisseria content compared to the control group. As shown in Figures 4 and 5, when sodium citrate, sodium bicarbonate, or sodium carbonate was added alone, the Actinomyces content significantly increased compared to the control group, and the combined addition of sodium citrate, sodium bicarbonate, or sodium carbonate with 0.1% or 0.5% potassium nitrate also had the effect of increasing the Actinomyces content. As shown in Figure 6, the combined addition of sodium gluconate, sodium succinate, sodium fumarate, sodium lactate, sodium malate, and sodium tartrate with 0.5% potassium nitrate increased the Neisseria content compared to the control group.

Claims

1. An oral flora improving agent comprising, as active ingredients, one or more salt compounds selected from organic acid salts, bicarbonates and carbonates, and a nitrate ion supplying compound.

2. A food for improving oral flora, comprising as active ingredients one or more salt compounds selected from organic acid salts, bicarbonates and carbonates, and a nitrate ion supplying compound.

3. 3. The agent according to claim 1, or the food according to claim 2, wherein the organic acid salt is one or more selected from the group consisting of citrate, gluconate, succinate, fumarate, lactate, malate and tartrate.

4. 3. The agent according to claim 1, or the food according to claim 2, wherein the improvement of the oral flora is an increase in the content of Neisseria and / or Actinomyces in the oral cavity.

5. 3. The agent according to claim 1, or the food according to claim 2, wherein the nitrate ion-supplying compound is a nitrate salt.

6. 6. The agent or food according to claim 5, wherein the nitrate is sodium nitrate or potassium nitrate.

7. The agent according to claim 1 or the food according to claim 2, which is used for inhibiting periodontal pathogen infection.

8. The agent according to claim 1 or the food according to claim 2, which is used for preventing or ameliorating periodontal disease.

9. 10. The agent according to claim 1 or the food according to claim 2, which is used for preventing or improving dental caries.

10. The agent according to claim 1 or the food according to claim 2, which is used for increasing nitric oxide.

11. 10. The agent according to claim 1 or the food according to claim 2, which is used for preventing or improving hypertension.

12. The agent according to claim 1 or the food according to claim 2, which is used for improving or enhancing motor function.

13. An oral flora improving agent containing, as an active ingredient, one or more salt compounds selected from citrates, bicarbonates and carbonates.

14. A food for improving oral flora, comprising one or more salt compounds selected from citrates, bicarbonates and carbonates as an active ingredient.

15. The agent according to claim 13 or the food according to claim 14, wherein the improvement of the oral flora is an increase in the content of Neisseria and / or Actinomyces in the oral cavity.

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