Food composition for preventing or improving alveolar bone resorption in periodontal disease, feed composition, preventive or therapeutic agent for alveolar bone resorption in periodontal disease, food composition for inhibiting osteoclast differentiation, feed composition, and osteoclast differentiation inhibitor

Abstract

To provide a food composition and pharmaceutical product capable of treating, preventing and improving, bone resorption diseases such as a periodontal disease and osteoporosis and the like.SOLUTION: There are provided: a food composition for preventing or improving a periodontal disease which is characterized in which, as an active ingredient, a microalga derived substance belonging to the genus Coccomyxa is included; a prevention or treatment agent for a periodontal disease; an antibacterial agent for periodontal disease bacteria; a food composition for preventing or improving a bone resorption disease; a prevention or treatment agent for a bone resorption disease; a food composition for reinforcing teeth; a food composition for bone resorption suppression; a bone resorption suppression agent and an osteoclastic differentiation inhibitor. As the microalga derived substance belonging to the genus Coccomyxa is an alga body of a microalga belonging to the genus Coccomyxa or dried powder thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a food composition for preventing or ameliorating periodontal disease, a preventive or therapeutic agent for periodontal disease, an antibacterial agent against periodontal disease bacteria, a food composition for preventing or ameliorating bone resorption diseases, a preventive or therapeutic agent for bone resorption diseases, a food composition for strengthening teeth, a food composition for inhibiting bone resorption, a bone resorption inhibitor, and an osteoclast differentiation inhibitor. [Background technology]

[0002] Healthy teeth and bones are extremely important from the perspective of quality of life in an aging society. Periodontal disease is an inflammatory disease caused by infection with periodontal bacteria. When periodontal bacteria stagnate at the junction between the teeth and gums, dental plaque accumulates, causing inflammation at the gum line. As periodontal disease progresses, the alveolar bone that supports the teeth dissolves. Periodontal bacteria have also been suggested to have various adverse effects on the body, worsening diabetes and, in recent years, have also been linked to dementia. Therefore, because periodontal disease does not simply cause tooth loss but is involved in the onset of serious diseases, effective methods for early diagnosis, treatment, and prevention are desired.

[0003] Furthermore, in recent years, the rise in various bone diseases, such as osteoporosis among middle-aged and elderly people and an increase in fracture rates among young people, has become a social issue. Femoral neck fractures and spinal compression fractures caused by osteoporosis can lead to bedridden conditions. Strengthening bones and increasing bone mass are important from the perspective of preventing the onset of osteoporosis, which occurs due to a decrease in bone density with age.

[0004] For example, Patent Document 1 describes an osteoclast formation inhibitor that can be used to prevent and treat diseases caused by inflammatory bone destruction, such as rheumatoid arthritis and periodontal disease, and metabolic bone diseases, such as osteoporosis, and that contains a sulfated glycosaminoglycan or a salt thereof as an active ingredient. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-210715 Summary of the Invention [Problem to be solved by the invention]

[0006] Bone resorption diseases such as periodontal disease and osteoporosis are expected to become increasingly serious social problems in the future, and there is a need for treatment, prevention, and improvement of these symptoms.

[0007] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide food compositions, pharmaceuticals, etc. that can treat, prevent, and improve bone resorption diseases such as periodontal disease and osteoporosis. [Means for solving the problem]

[0008] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that dried powder (algal bodies) of microalgae of the genus Coccomyxa exhibits excellent growth inhibitory effects against periodontal disease bacteria and also inhibits the differentiation of precursor cells into osteoclasts. Based primarily on the above results and considerations, the following inventions are provided. [1] A method for producing a microalgae-containing microorganism comprising the microalgae belonging to the genus Coccomyxa as an active ingredient, the microalgae being Coccomyxa sp. KJ strain. (Accession number: FERM BP-22254) The active ingredient is Coccomyxa sp. KJ KK 1. A food composition for preventing or improving alveolar bone resorption in periodontal disease, comprising at least one selected from the group consisting of algae bodies of the above algae and dried powders thereof. [2] A method for producing a microalgae-containing microorganism comprising the microalgae belonging to the genus Coccomyxa as an active ingredient, the microalgae being Coccomyxa sp. KJ strain. (Accession number: FERM BP-22254) The active ingredient is Coccomyxa sp. KJ KK A feed composition for preventing or improving alveolar bone resorption in periodontal disease, characterized by comprising at least one selected from the group consisting of algae bodies of the above algae and dried powder thereof. [3] A method for treating a bacterial infection comprising, as an active ingredient, microalgae belonging to the genus Coccomyxa, wherein the microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain. (Accession number: FERM BP-22254) The active ingredient is Coccomyxa sp. KJ KK 1. A preventive or therapeutic agent for alveolar bone resorption in periodontal disease, comprising at least one selected from the group consisting of algae bodies of the above algae and dried powders thereof. [4] A method for producing a microalgae-containing microorganism comprising the microalgae belonging to the genus Coccomyxa as an active ingredient, the microalgae being Coccomyxa sp. KJ strain. (Accession number: FERM BP-22254) The active ingredient is Coccomyxa sp. KJ KK and at least one selected from the group consisting of algae bodies and dry powders thereof. anti-inflammatory food product composition. [5] A method for producing a microalgae-containing microorganism comprising the microalgae belonging to the genus Coccomyxa as an active ingredient, the microalgae being Coccomyxa sp. KJ strain. (Accession number: FERM BP-22254) The active ingredient is Coccomyxa sp. KJ KK and at least one selected from the group consisting of algae bodies and dry powders thereof. control feeding Food composition. [6] A method for producing a microalgae-containing microorganism comprising the microalgae belonging to the genus Coccomyxa as an active ingredient, the microalgae being Coccomyxa sp. KJ strain. (Accession number: FERM BP-22254) The active ingredient is Coccomyxa sp. KJ KK 1. An osteoclast differentiation inhibitor comprising at least one selected from the group consisting of algae bodies and dried powders thereof. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide food compositions, medicines, etc. that contain microalgae belonging to the genus Coccomyxa, which are also used as food, as an active ingredient and are capable of treating, preventing, and ameliorating bone resorption diseases such as periodontal disease and osteoporosis. [Brief explanation of the drawings]

[0010] [Figure 1]1 is a graph showing the results of an osteoclast differentiation inhibition test (Test 1). [Figure 2] 1 shows images of TRAP stained cells, showing the results of an osteoclast differentiation inhibition test (Test 1). [Figure 3] This is the schedule for test 2. [Figure 4] 1 is a graph showing the results of measuring the Pg bacterial count in the gums. [Figure 5] These are the results of measuring the amount of alveolar bone resorption. [Figure 6] These are the results of periodontal disease-related bacteria (Test 3). [Figure 7] These are the results of subjective symptoms (Test 3). [Figure 8] These are the results of the dental examination (Test 3). DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment of the present invention will be described with reference to FIGS. 1 to 8 and Tables 1 and 2. The present embodiment relates to a food composition for preventing or ameliorating periodontal disease, a preventive or therapeutic agent for periodontal disease, an antibacterial agent against periodontal disease bacteria, a food composition for preventing or ameliorating bone resorption diseases, a preventive or therapeutic agent for bone resorption diseases, a food composition for strengthening teeth, a food composition for inhibiting bone resorption, a bone resorption inhibitor, and an osteoclast differentiation inhibitor, each containing a substance derived from microalgae belonging to the genus Coccomyxa as an active ingredient.

[0012] <Periodontal disease> Periodontal disease, also known as periodontal disease, is broadly divided into gingival lesions and periodontitis. Periodontal disease, excluding non-plaque-related gingival disease, is an infectious inflammatory disease caused by periodontal pathogenic bacteria and refers to a disease that occurs in the periodontal tissues consisting of the gingiva, cementum, periodontal ligament, and alveolar bone. Periodontal disease causes symptoms such as bad breath, swollen gums, gum pain, gingival fever, sneezing, and runny nose. Further progression can lead to periodontal ligament disease, the development of gingival pockets, and alveolar bone loss, resulting in tooth mobility and loss. Periodontal disease occurs when plaque and tartar are left untreated.

[0013] Tartar is a calcified substance formed when minerals such as calcium and phosphorus are deposited on dental plaque, and it adheres very firmly to the surface of the teeth. Tartar is also considered to be a factor in increasing dental plaque, promoting the adhesion of dental plaque and making it difficult to remove. When large amounts of dental plaque and dental tartar are deposited in this way, and periodontal disease occurs when periodontal disease bacteria such as Porphyromonas gingivalis proliferate chronically within them.

[0014] Furthermore, as described below, periodontal disease is a type of bone resorption disease, and therefore, in order to treat, prevent, improve, etc. periodontal disease, it is effective to inhibit the proliferation of periodontal disease bacteria and prevent bone resorption (inhibit osteoclast differentiation).

[0015] (Porphyromonas gingivalis) There are more than 10 species of bacteria that cause periodontal disease, but the most important pathogen is Porphyromonas gingivalis. Porphyromonas gingivalis is a non-motile, gram-negative, anaerobic, pathogenic bacterium belonging to the Bacteroidetes phylum. It is known to induce the expression of degradative enzymes such as collagenase and hyaluronidase in the gingival crevice (also known as the periodontal pocket), and to cause inflammation through the production of lipopolysaccharides derived from the bacterium. Porphyromonas gingivalis penetrates dental plaque and releases enzymes such as proteases for its own survival. These enzymes cause inflammation of the gums, leading to gingivitis, the onset of periodontal disease.

[0016] Substances derived from microalgae belonging to the genus Coccomyxa exhibit growth inhibitory effects against periodontal pathogens such as Porphyromonas gingivalis, making it possible to treat, prevent, and improve periodontal disease.

[0017] <Bone resorption by osteoclasts> Bone tissue dynamically remodels by regulating bone and calcium metabolism through repeated bone formation by osteoblasts and bone resorption by osteoclasts. Normally, bone metabolism is carried out by maintaining a balance between bone formation and bone resorption. When the functional balance between osteoblasts and osteoclasts becomes abnormal, the dynamic equilibrium is disrupted, resulting in a decrease in bone density or an excessive increase in bone density, leading to metabolic bone diseases such as osteoporosis and osteopetrosis.

[0018] In osteoporosis, bone resorption far exceeds bone formation, resulting in a decrease in bone mass. Therefore, inhibiting bone resorption is important in the treatment of osteoporosis. Furthermore, bone resorption also occurs in rheumatoid arthritis, periodontal disease, and other conditions associated with inflammatory responses, so inhibiting bone resorption is also necessary. Furthermore, alveolar bone resorption occurs after tooth extraction, and in both diseases, activated osteoclasts promote bone resorption at the site of bone resorption. Therefore, from the perspective of preventing and treating bone-resorbing diseases, including periodontal disease, it is important to inhibit osteoclast differentiation (i.e., osteoclast formation) to prevent bone resorption.

[0019] As used herein, "inhibition of osteoclast differentiation" means inhibition of differentiation from precursor cells into osteoclasts. Because substances derived from microalgae belonging to the genus Coccomyxa have the effect of inhibiting osteoclast differentiation, they can be used for the treatment, prevention, amelioration, etc. of bone resorption diseases.

[0020] <Bone resorption disease> As used herein, the term "bone resorption disease" refers to a disease, symptom, or condition resulting from bone resorption exceeding bone formation, and examples include alveolar bone resorption in periodontal disease (periodontal disease), alveolar bone resorption after tooth extraction, bone resorption after alveolar bone augmentation surgery, osteoporosis, osteopenia, inflammatory bone resorption in rheumatoid arthritis, Paget's disease of bone, bone resorption in multiple myeloma, and a combination of two or more thereof.

[0021] Substances derived from microalgae belonging to the genus Coccomyxa are more preferably applied to bone resorption diseases such as alveolar bone resorption in periodontal disease (periodontal disease), alveolar bone resorption after tooth extraction, bone resorption after alveolar bone augmentation surgery, osteoporosis, and rheumatoid arthritis, and are particularly preferably applied to alveolar bone resorption in periodontal disease (periodontal disease).

[0022] [About the active ingredients] In the present invention, the active ingredient used is at least one selected from algal bodies of microalgae belonging to the genus Coccomyxa or dry powder thereof.

[0023] <Microalgae of the genus Coccomyxa> In the present invention, the microalgae of the genus Coccomyxa used as an active ingredient is not particularly limited, but preferred examples include the Coccomyxa sp. KJ strain or a mutant thereof. The Coccomyxa sp. KJ strain (KJ Denso) was deposited on June 4, 2013, at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession number FERM P-22254, and was transferred to international deposit under the provisions of the Petroleum Exploration Treaty under accession number FERM BP-22254 on June 2, 2015. In addition, the former genus Pseudococcomyxa has been included in the genus Coccomyxa in the latest classification (Reference: PLoS One. 2015 Jun 16;10(6):e0127838. doi: 10.1371 / journal.pone.0127838. eCollection 2015.).

[0024] Mutant strains of Coccomyxa sp. KJ strain can be obtained by irradiation with ultraviolet rays, X-rays, gamma rays, etc., mutagen treatment, heavy beam irradiation, genetic manipulation (introduction of foreign genes, gene disruption, gene modification by genome editing, etc.), etc. The method for obtaining mutant strains that exhibit activity as active ingredients, their characteristics, etc. are not particularly limited.

[0025] The method for culturing microalgae of the genus Coccomyxa is not particularly limited. The medium for culturing microalgae of the genus Coccomyxa may be one commonly used for culturing microalgae, and for example, any of known media for freshwater microalgae and marine microalgae containing various nutrients, trace metal salts, vitamins, etc. may be used. Examples of media include AF6 medium. The composition of AF6 medium (per 100 ml) is as follows: NaNO 314mg NH4NO3 2.2mg MgSO4·7H2O 3mg KH2PO4 1mg K2HPO40.5mg CaCl2·2H2O 1mg CaCO31mg Fe-citrate 0.2mg Citric acid 0.2mg Biotin 0.2μg Thiamine HCl 1 μg Vitamin B6 0.1μg Vitamin B 12 0.1 μg Trace metals 0.5mL Distilled water 99.5mL

[0026] Nutrients include nitrogen sources such as NaNO3, KNO3, NH4Cl, and urea, and phosphorus sources such as K2HPO4, KH2PO4, and sodium glycerophosphate. Trace metals include iron, magnesium, manganese, calcium, and zinc. Vitamins include vitamin B1 and vitamin B 12 etc.

[0027] The culture method may be performed under aeration conditions with agitation and carbon dioxide supply. The culture is performed under a fluorescent light with a light / dark cycle, such as 12 hours of light and 12 hours of darkness, or under continuous light. The culture conditions are not particularly limited as long as they do not adversely affect the growth of the Coccomyxa microalgae. For example, the pH of the culture solution should be 3 to 9, and the culture temperature should be 10 to 35°C.

[0028] For information on the method for culturing the Kokkomykusa sp. KJ strain, see, for example, JP 2015-15918 A, WO 2015 / 190116 A1, and Satoh, A. et al., Characterization of the Lipid Accumulation in a New Microalgal Species, Pseudochoricystis ellipsoidea (Trebouxiophyceae) J. Jpn. Inst. Energy (2010) 89:909-913.

[0029] As an active ingredient, algal cells or a dry powder thereof (first embodiment) is used. The embodiments are explained below. Usually, either the algal cells or the dry powder thereof is the active ingredient, but this does not preclude the use of both as active ingredients.

[0030] <Algae or its dried powder> In this embodiment, algal cells or their dried powder are used as the active ingredient. The dried powder can be prepared by subjecting the collected algal cells to a drying process and a crushing (pulverizing) process. For example, drum drying, spray drying, freeze drying, etc. can be used as the drying process. For the crushing process, a bead-type crusher, homogenizer, French press, mixer / blender, fine grinder, etc. can be used. The order of the drying process and the crushing process does not matter. Alternatively, the drying process and the crushing process can be performed simultaneously using a device equipped with drying and crushing functions.

[0031] The particle size of the dry powder is not particularly limited, and the dry powder has an average particle size of, for example, 0.2 μm to 2 mm, preferably 0.4 μm to 400 μm.

[0032] [Uses of substances derived from microalgae belonging to the genus Coccomyxa] Substances derived from microalgae belonging to the genus Coccomyxa can be used as active ingredients in food compositions for preventing or ameliorating periodontal disease, preventive or therapeutic agents for periodontal disease, antibacterial agents against periodontal disease bacteria, food compositions for preventing or ameliorating bone resorption diseases, preventive or therapeutic agents for bone resorption diseases, food compositions for strengthening teeth, food compositions for inhibiting bone resorption, bone resorption inhibitors, and osteoclast differentiation inhibitors.

[0033] <Preventive or therapeutic agent for periodontal disease> The preventive or therapeutic agent for periodontal disease according to this embodiment is a preventive or therapeutic agent for periodontal disease that contains a substance derived from microalgae belonging to the genus Coccomyxa as an active ingredient. The preventive or therapeutic agent for periodontal disease according to this embodiment prevents, improves, or treats periodontal disease by inhibiting the proliferation of periodontal disease bacteria such as Porphyromonas gingivalis and / or inhibiting osteoclast differentiation, which the substance derived from microalgae belonging to the genus Coccomyxa possesses.

[0034] <Preventive or therapeutic agent for bone resorption diseases> The "prophylactic or therapeutic agent for bone resorption diseases" refers to an agent for preventing, ameliorating, or treating osteoporosis. The prophylactic or therapeutic agent for bone resorption diseases according to this embodiment prevents, ameliorate, or treats bone resorption diseases by inhibiting osteoclast differentiation and bone resorption due to the substance derived from microalgae belonging to the genus Coccomyxa.

[0035] <Osteoclast differentiation inhibitor> The osteoclast differentiation inhibitor according to this embodiment is an osteoclast differentiation inhibitor containing a substance derived from microalgae belonging to the genus Coccomyxa as an active ingredient. The osteoclast differentiation inhibitor according to this embodiment inhibits the differentiation of precursor cells into osteoclasts.

[0036] <Bone resorption inhibitor> "Inhibition of bone resorption" means reducing or stopping the rate of bone resorption. The bone resorption inhibitor of this embodiment is a bone resorption inhibitor containing, as an active ingredient, a substance derived from microalgae belonging to the genus Coccomyxa. The bone resorption inhibitor of this embodiment inhibits the differentiation of precursor cells into osteoclasts, thereby inhibiting bone resorption.

[0037] <Teeth strengthener> "Tooth strengthening agent" refers to an agent that strengthens teeth. Teeth strengthening can be evaluated, for example, by indicators such as tooth strength and tooth quality, but the evaluation criteria are not limited to these indicators. The tooth strengthening agent according to this embodiment strengthens teeth by inhibiting osteoclast differentiation and bone resorption with a substance derived from microalgae belonging to the genus Coccomyxa.

[0038] [Composition containing a substance derived from microalgae belonging to the genus Coccomyxa] The preventive or therapeutic agent for periodontal disease, antibacterial agent against periodontal disease bacteria, preventive or therapeutic agent for bone resorption disease, tooth strengthening agent, bone resorption inhibitor, and osteoclast differentiation inhibitor containing a substance derived from microalgae belonging to the genus Coccomyxa according to this embodiment as an active ingredient can be used as a food composition, oral composition, pharmaceutical composition, feed composition, or other composition by adding pharmacologically acceptable additives.

[0039] (Food compositions, food additives and feed compositions) The substance derived from a microalga belonging to the genus Coccomyxa of this embodiment can be used as a food composition or a food additive. The food composition of this embodiment can be provided by incorporating the substance derived from a microalga belonging to the genus Coccomyxa as a food ingredient in an amount sufficient to effectively exert the effects of preventing or improving periodontal disease, antibacterial activity, preventing or improving bone resorption diseases, strengthening teeth, inhibiting bone resorption, and inhibiting osteoclast differentiation into various foods.

[0040] That is, in the fields of food and feed (feed), the present invention can provide food compositions and feed compositions labeled for bone strengthening, etc. Examples of such food compositions include general foods, as well as foods for specified health uses, nutritional supplements (supplements, nutritional drinks, etc.), foods with functional claims, foods for hospital patients, supplements, etc. Furthermore, substances derived from microalgae belonging to the genus Coccomyxa can also be used as food additives and feed compositions (livestock feed, pet food, companion animal food, and companion animal nutritional supplements).

[0041] Examples of such food compositions include seasonings, processed meat products, processed agricultural products, beverages (soft drinks, alcoholic beverages, carbonated beverages, dairy drinks, fruit juice drinks, tea, coffee, energy drinks, etc.), powdered beverages (powdered juice, powdered soup, etc.), concentrated beverages, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chocolate, etc.), bread, cereals, etc. Furthermore, in the case of foods for specified health uses, foods with nutrient functions, foods with functional claims, etc., they may be in the form of capsules, chewable tablets, lozenges, syrups, granules, powders, etc. By providing the active ingredient of the present invention in the form of a food composition, it becomes easier to ingest the active ingredient on a daily basis or continuously.

[0042] Here, a food for specified health uses is a food containing functional health ingredients that affect physiological functions, etc., and can be labeled as suitable for specified health uses with the permission of the Commissioner of the Consumer Affairs Agency. In the present invention, the specified health uses are foods that are sold with labels such as "inhibits the growth of bacteria associated with periodontal disease," "has the effect of maintaining healthy gums," "makes teeth strong and healthy," "helps maintain healthy bones by supporting bone metabolism," "helps maintain bone health by supporting bone components," "helps maintain bone components," and "supports the maintenance of healthy bones by supporting bone metabolism."

[0043] In addition, a food with nutrient functions is a food used to supplement nutritional components (vitamins, minerals), and displays the function of the nutritional components. In order to be sold as a food with nutrient functions, the amount of nutritional components contained in the recommended daily intake must be within the range of the specified upper and lower limits, and in addition to displaying nutritional functions, warning labels must also be displayed.

[0044] Foods with functional claims are foods that display scientifically based functionality at the responsibility of the business operator, and information on the basis of safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale.

[0045] The food compositions, food additives, and feed compositions according to the present embodiments can contain, in addition to the substance derived from a microalga belonging to the genus Coccomyxa, one or more components that can be used in conventional food compositions, such as seasonings, preservatives, emulsifiers, stabilizers, flavorings, colorants, antiseptics, and pH adjusters.

[0046] In addition to the substance derived from microalgae belonging to the genus Coccomyxa, the food composition of this embodiment may also contain one or more substances known to have preventive or therapeutic effects on periodontal disease, antibacterial effects, preventive or therapeutic effects on bone resorption diseases, tooth strengthening effects, bone resorption inhibitory effects, and osteoclast differentiation inhibitory effects.

[0047] (Pharmaceutical composition) The substance derived from a microalga belonging to the genus Coccomyxa of this embodiment can be used as a pharmaceutical composition. The pharmaceutical composition of this embodiment is provided by combining a substance derived from a microalga belonging to the genus Coccomyxa in an amount sufficient to effectively exert the effects of preventing or treating periodontal disease, antibacterial activity against periodontal disease bacteria, preventing or treating bone resorption diseases, strengthening teeth, inhibiting bone resorption, and inhibiting osteoclast differentiation, together with pharmaceutically acceptable carriers and additives. The pharmaceutical composition may be a drug or a quasi-drug.

[0048] The pharmaceutical composition may be applied internally or externally, and the dosage form when formulated is not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, topical preparations (ointments, creams, lotions, liquids, gels, poultices, plasters, tapes, aerosols, etc.), and suppositories. Depending on the dosage form, the drug may be administered to the subject by oral or parenteral administration (local injection into the affected area, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.). Systemic administration and local administration may also be indicated depending on the subject. These administration routes are not mutually exclusive, and any combination of two or more may be used.

[0049] The pharmaceutical composition of this embodiment may contain one or more pharmaceutically acceptable additives. Pharmaceutical formulations can be prepared according to conventional methods. Pharmaceutically acceptable additives (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be added during formulation. Examples of excipients that can be used include lactose, starch, sorbitol, D-mannitol, sucrose, calcium stearate, magnesium stearate, dextrin, and maltitol. Examples of disintegrating agents that can be used include starch, carboxymethylcellulose, and calcium carbonate. Examples of buffering agents that can be used include phosphates, citrates, and acetates. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents that can be used include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. As a soothing agent, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As a stabilizer, propylene glycol, ascorbic acid, etc. can be used. As a preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. can be used. As an antiseptic, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

[0050] In addition to the substance derived from microalgae belonging to the genus Coccomyxa, the pharmaceutical composition of this embodiment may also contain one or more substances known to have preventive or therapeutic effects on periodontal disease, antibacterial effects, preventive or therapeutic effects on bone resorption diseases, tooth strengthening effects, bone resorption inhibitory effects, and osteoclast differentiation inhibitory effects.

[0051] (Oral composition) The substance derived from a microalga belonging to the genus Coccomyxa of this embodiment can be suitably used as an oral composition, and can be suitably prepared as, for example, toothpaste, liquid toothpaste, moisturizing toothpaste, mouthwash, gargling tablets, oral paste, lozenges, candies, gums, etc.

[0052] In addition to the essential components, the oral composition of the present embodiment may contain other optional additives depending on the formulation. In the case of dentifrices, for example, various active ingredients such as abrasives, binders, thickeners, surfactants, sweeteners, preservatives, flavorings, colorants, and other antibacterial agents may be blended, and the oral composition may be produced by mixing these ingredients with water.

[0053] <Dosage and administration> The preventive or therapeutic agent for periodontal disease, antibacterial agent against periodontal bacteria, preventive or therapeutic agent for bone resorption disease, tooth strengthening agent, bone resorption inhibitor, and osteoclast differentiation inhibitor of this embodiment may be administered orally, for example, in the form of powder, capsules, tablets, granules, liquid, or syrup.

[0054] The medicament, composition, etc. of the present invention contains an active ingredient in an amount necessary to obtain the expected effect (i.e., a therapeutically or prophylactically effective amount). The amount of the active ingredient in the present invention generally varies depending on the dosage form, but is set, for example, within the range of about 0.1% by weight to about 99% by weight so as to achieve the desired dosage.

[0055] The dosage of the pharmaceuticals, compositions, etc. of the present invention is set so as to obtain the expected effect. When determining a therapeutically or prophylactically effective dosage, the symptoms, age, sex, and body weight of the patient are generally taken into consideration. Those skilled in the art will be able to determine an appropriate dosage taking these factors into account. As an example of dosage, for an adult (body weight approximately 60 kg), the dosage can be set so that the amount of active ingredient per day is 1 mg to 20 mg, preferably 2 mg to 10 mg. The dosage schedule can be, for example, once to several times a day, once every two days, or once every three days. When creating the dosage schedule, the patient's condition and the duration of effect of the active ingredient can be taken into consideration.

[0056] As is clear from the above description, the present application also provides a method for treating, ameliorating, or preventing periodontal disease or bone resorption diseases, which comprises administering to a subject suffering from or at risk of suffering from periodontal disease or bone resorption diseases a therapeutically, ameliorating, or prophylactically effective amount of a pharmaceutical or composition comprising the preventive or therapeutic agent for periodontal disease, the antibacterial agent against periodontal pathogens, the preventive or therapeutic agent for bone resorption diseases, the tooth strengthening agent, the bone resorption inhibitor, and the osteoclast differentiation inhibitor of the present invention. The subject for treatment, amelioration, or prevention is typically humans, but the method may also be applied to mammals other than humans (e.g., monkeys, cows, pigs, sheep, cats, rabbits, dolphins, etc.), birds, reptiles, etc.

[0057] Furthermore, to women who are reported to have a high incidence of periodontal disease, particularly women during adolescence, pregnancy and childbirth, menopause, and postmenopause, when the balance of female hormones, which contributes to the worsening of periodontal disease, changes significantly, it is possible to administer or ingest medicines or compositions containing a substance derived from microalgae belonging to the genus Coccomyxa as an active ingredient, an antibacterial agent against periodontal bacteria, a preventive or therapeutic agent for bone resorption diseases, a tooth strengthening agent, a bone resorption inhibitor, and an osteoclast differentiation inhibitor.

[0058] Furthermore, in postmenopausal women, the decrease in female hormones due to menopause promotes bone resorption, which leads to bone resorption diseases such as osteoporosis. Therefore, substances derived from microalgae belonging to the genus Coccomyxa have the ability to inhibit bone resorption and osteoclast differentiation, thereby strengthening bones and increasing bone density, thereby preventing bone resorption diseases.

[0059] Furthermore, pharmaceuticals and compositions containing the present invention's preventive or therapeutic agent for periodontal disease, antibacterial agent against periodontal disease bacteria, preventive or therapeutic agent for bone resorption diseases, tooth strengthening agent, bone resorption inhibitor, and osteoclast differentiation inhibitor, which contain a substance derived from microalgae belonging to the genus Coccomyxa as an active ingredient, can be administered to people aged 40 or over, particularly elderly people aged 60 or 65 or over. People aged 40 or over, particularly elderly people aged 60 or 65 or over, tend to be susceptible to periodontal disease and bone resorption diseases as they age, but the various effects of the substance derived from microalgae belonging to the genus Coccomyxa can prevent them from contracting periodontal disease and bone resorption diseases. [Example]

[0060] The present invention will be described in detail below based on specific examples, but the present invention is not limited to these. In the following examples, dried algal powder (algal bodies) of microalgae belonging to the genus Coccomyxa was used as a substance derived from microalgae belonging to the genus Coccomyxa, and antibacterial tests, osteoclast differentiation inhibition tests, and tests on the effects on the oral environment of healthy individuals were conducted.

[0061] <Example 1: Preparation of dried powder (algal bodies) of microalgae of the genus Coccomyxa> Coccomyxa sp. KJ strain was cultured according to a previously reported method. Specifically, Coccomyxa sp. KJ strain was inoculated onto AF6 medium and cultured at room temperature (25°C) for 48 hours under 2% CO2 (v / v) and light (300 μmol / m2 / s). The algae were collected from the culture medium by centrifugation. The collected algae were dried in a drum dryer and then crushed in a fine grinder to produce a powder (dried algae powder).

[0062] <Test 1: Osteoclast differentiation inhibition test> (Test Method) To evaluate the suppression of bone resorption in vitro, we performed an inhibition test of early osteoclast differentiation from the RAW264.7 cell line (mouse macrophage cells, source: ATCC, catalog number: TIB-71). The osteoclast assay was evaluated by measuring tartrate-resistant acid phosphatase (TRAP) activity, an early differentiation marker, and by TRAP staining. RAW264.7 cells were cultured in a T75 flask using growth medium (DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) and cultured in a CO2 incubator (5% CO2, 37°C, humidified conditions; hereafter, the CO2 incubator conditions are the same). The medium was changed every two days. Culture was terminated when the cells reached 80% confluence. They were then detached using a cell scraper, collected, and centrifuged (180g, 5 minutes, room temperature). They were then suspended in fresh growth medium and seeded into a 96-well plate at 5,000 cells / 0.1 mL / well. Culture was then continued in a CO2 incubator. The next day, the medium was replaced with RANKL-added differentiation medium alone, differentiation medium containing the test substance plus RANKL, or differentiation medium containing heparin plus RANKL as a positive control (differentiation medium: MEMα containing 10% charcoal-stripped FBS, 1% penicillin-streptomycin, and 50 ng / mL RANKL). After culturing for 3 days in a CO2 incubator, TRAP staining and TRAP activity quantification were performed. The test substance was a dried powder of microalgae from the genus Coccomyxa.

[0063] (TRAP staining) The medium was removed from each well, and the cells were fixed with 100 μL of 10% neutral buffered formalin solution for 5 minutes. Next, each well was washed once with 100 μL of ultrapure water and then replaced with 50 μL of TRAP staining solution (prepared by adding 5 mL of buffer to 1 vial of chromogenic substrate from the TRAP staining kit). The wells were then incubated at 37°C for 40 minutes for TRAP staining. After washing once with 100 μL of ultrapure water, bright-field photographs were taken of the center of each well. TRAP-positive multinucleated (three or more nuclei) osteoclasts were counted over a certain area of ​​the field of view, and the number of osteoclasts was quantified.

[0064] (TRAP activity measurement) Each well was washed once with 100 μL of 0.9% sodium chloride solution, then the solution was replaced with 50 μL of TRAP reaction substrate solution (prepared by adding 5 mL of acid phosphatase buffer to 1 vial of pNPP substrate (supplied with the TRAP & ALP activity measurement kit) and dissolving it, followed by the addition of 0.5 mL of tartaric acid solution), and incubated at 37°C for 30 minutes. The reaction was stopped by adding 50 μL of 0.5 N sodium hydroxide solution to each well, and the absorbance (measurement wavelength: 405 nm) was measured using a plate reader, and the relative absorbance was used as the relative TRAP activity.

[0065] (Test 1 results) The results are shown in Figures 1 and 2. The results of the TRAP activity measurement are shown in Figure 1. The TRAP activity when only RANKL (a promoter) was added was set at 100, and a relative comparison was made. As a result, KJ dry powder (Example 1), which is a dry powder of microalgae of the genus Coccomyxa, inhibited osteoclast differentiation. In the stained image in Figure 2, giant osteoclasts were observed when only the promoter was added, but when the dry powder (Example 1) was added, osteoclastogenesis was strongly inhibited, confirming that the addition of the promoter exhibited a good inhibitory effect on osteoclast differentiation that was comparable to that of heparin, a known inhibitor of osteoclast differentiation.

[0066] <Summary of Exam 1> The results of Test 1 revealed that algae belonging to the genus Coccomyxa exhibit an inhibitory effect on osteoclast differentiation. Therefore, it was found that substances derived from microalgae belonging to the genus Coccomyxa can be used to treat, prevent, and improve periodontal disease and bone resorption diseases.

[0067] <Test 2: Periodontal disease alleviation effect confirmation test using periodontal disease model mice> In Experiment 2, mice were administered with a dry powder of Coccomyxa microalgae (Example 1) in advance, and then inoculated with periodontal bacteria. Then, periodontal tissues were collected and the reduction of Pg bacteria and osteoclast activity was examined. In this experiment, a mouse model in which periodontal disease was induced by maxillary molar ligation and Pg bacteria application was used.

[0068] Materials and Methods Test animals: C57BL / 6J male mice (approximately 6 weeks old, Japan SLC) Test substance: KJ dry powder, which is a dry powder of microalgae of the genus Coccomyxa (Example 1) Dosage: 15 mg / day (150 mg KJ / ml - 0.1 ml of 0.2% carboxymethylcellulose solution) Basal feed: MF (Oriental Yeast) The bacterial strain used: Porphyromonas gingivalis strain 381 (Pg strain, provided by the Department of Bacteriology, Nihon University School of Dentistry) was cultured in anaerobic GAM broth (Nissui Pharmaceutical) until plateau. The cells were harvested by centrifugation (3,000 rpm x 5 min) and then diluted to 1 x 10 in GAM broth supplemented with 5% CMC. 10 The bacteria were resuspended to a concentration of CFU / mL. CFU measurements were performed once in advance using GAM agar medium. Subsequent applications of the bacterial solution were performed under the same culture conditions. The number of bacteria was measured daily using a microscope.

[0069] (Test setup) The test groups were: (1) a control group in which sterile solution was applied and a placebo was applied (HC group, 8 animals); (2) a control group in which Pg solution was applied and a placebo was applied (IC group, 10 animals); (3) a group in which Pg solution was applied and the test substance was applied (IO group, 10 animals); and (4) a group in which Pg solution was applied and the test substance was administered orally by force (IG group, 11 animals).

[0070] (Breeding) The schedule is shown in Figure 3. Six-week-old mice were randomly assigned to four cages and housed in plastic cages. They were acclimated to the facility and diet for four days. After acclimation, mice in groups (2) IC, (3) IO, and (4) IG were anesthetized and ligated with 5-0 silk sutures around the maxillary second molars. After confirming that the sutures had not come loose, the mice were regrouped into groups with similar average weights: IC (10 mice), IO (10 mice), and IG (11 mice). For the IO group, 0.1 mL of a 0.15 g / mL suspension of the test substance in 2% carboxymethylcellulose (CMC) aqueous solution was applied around the periodontal tissue. For the IG group, 0.1 mL of the test substance was administered intragastrically via a stomach tube. For the HC group and IC group, 0.1 mL of the base material (2% CMC aqueous solution) was applied in the same manner. The application or administration was once a day, and continued until the day of autopsy at 10:00 every morning.

[0071] The test substance was applied or administered for 7 days, and then Pg bacterial solution was applied for 6 consecutive days. 10 The suspension was made to a concentration of CFU / mL immediately before use, and 100 μL was applied at approximately 11:00 every day. The application was evenly applied to the upper jaw on both sides.

[0072] (autopsy) After the 6-day Pg bacterial solution application, the test substance application continued for 22 days, and all animals were necropsied the following day. The animals were sacrificed by exsanguination under a triple anesthesia regimen (Domitor, Dormicum, and Betorfal), and the maxilla was collected. The right upper jaw was fixed in formalin. The gums of the left maxilla were collected, immersed in RNA-later, and stored refrigerated. To confirm bone resorption, the left maxilla was boiled for 10 minutes and then immersed overnight in 3% hydrogen peroxide. After removing the soft tissue, the left maxilla was immersed in 1% methylene blue and stained. The buccal side of the methylene blue-stained left maxilla was photographed and used to evaluate alveolar bone resorption.

[0073] Pg count: Pg count was measured using real-time PCR using extracted and transcribed cDNA. The measurement method was based on Ashimoto et al. (1996). Pg detection results were scored as 2 for individuals in which Pg was detected within the calibration curve range (the lower limit of the calibration curve is 19,000 cells / g), 1 for individuals outside the range, and 0 for individuals in which Pg was not detected.

[0074] Evaluation of alveolar bone resorption: The buccal side of the left maxilla was photographed after staining with methylene blue, and alveolar bone resorption was measured. The distance from the cementum-enamel junction to the alveolar bone crest of the molars was measured at three locations for the first molar and at two locations for the second and third molars, and the average value was calculated and used to evaluate alveolar bone resorption. The amount of alveolar bone resorption (μm) was defined as the distance between the cementoenamel junction and the crest of the alveolar bone. In addition, the alveolar bone resorption inhibition rate (%) for the group to which the test substance was applied or administered (IO group or IG group) was calculated as [(absorption amount of IC group) - (IO group or IG group)] / [(absorption amount of IC group) - (absorption amount of HC group)] × 100.

[0075] (Test 2 results) Figure 4 shows the results of measuring Pg counts in the gums. The Pg detection score was calculated as 2 for individuals with Pg detected within the calibration curve, 1 for individuals outside the calibration curve, and 0 for individuals where Pg was not detected. The average detection scores were 0.38 for the (1) HC group, 1.20 for the (2) IC group, 0.50 for the (3) IO group, and 0.36 for the (4) IG group. The (2) IC group had the highest score, followed by the (3) IO group, the HC group, and the IG group, with the IC group showing a significantly higher score than the IG group. The scores for the IO and IG groups were similar to those of the HC group, which did not receive Pg application, demonstrating that the test substance contributed to the reduction of Pg bacteria regardless of whether it was applied to the peri-periodontal tissue or administered intragastrically.

[0076] Table 1 shows the average amount of alveolar bone resorption. Figure 5 shows the results of a group comparison of the amount of alveolar bone resorption for the first, second, and third molars. For the second and third molars, the (3) IO group had significantly lower levels than the (2) IC group. However, no significant difference was observed between the (2) IC group and the (4) IG group (Figure 5). This suggests that the inhibitory effect on alveolar bone resorption observed after intraoral application of the test substance was not mediated by the digestive system, but rather was brought about by the test substance acting directly on the periodontal tissues.

[0077] [Table 1]

[0078] Furthermore, for the second and third molars, the extent to which the intake of the test substance contributed to the inhibition of alveolar bone resorption was calculated as the alveolar bone resorption inhibition rate, based on the (1) HC group. Table 2 shows the results showing the inhibition rate of alveolar bone resorption (second molar, third molar).

[0079] [Table 2]

[0080] The (3)IO group showed the highest rate of alveolar bone resorption inhibition for both the second and third molars. This suggests that ingesting the test substance may be effective in preventing periodontal disease, and that even greater effectiveness could be expected if the substance were administered in a manner that allows it to act directly on periodontal tissues, such as in the form of candy or chewable tablets. The reason that periodontal disease occurred not only in the second molars ligated with silk thread, but also in the third molars is thought to be that compared to the first molars, which are larger and less mobile, the third molars are significantly smaller. Therefore, horizontal force from the silk thread between them and the second molars was applied, causing tooth movement and making them more susceptible to Pg bacteria.

[0081] <Production Example 1: Chewable tablets> Chewable tablets containing the following ingredients were manufactured by conventional methods (recommended daily intake: 2 tablets). Dry powder of Coccomyxa microalgae (Example 1) Lactoferrin + lactoperoxidase Tea extract (contains catechin) Vitamin C Xylitol Acidulant: Malic acid Flavoring: Lemon juice Lubricant: calcium stearate) Sweetener: Isomaltulose

[0082] <Study 3: Effects of test food intake on the oral environment in healthy subjects: Open-label study> In Test 3, healthy Japanese adult men and women were given a test food containing dried powder of Coccomyxa microalgae (Example 1, Coccomyxa KJ) for six weeks to examine the effects on the oral environment. (intervention) (1) Test food Test food group: Food containing Kokkomykusa KJ The composition of the test food (tablet) (per 1,000 mg tablet) was 100 mg of Kokkomykusa powder (Example 1), 500 mg of dextrin, 385 mg of reduced maltose syrup, and 15 mg of calcium stearate.

[0083] (2) Exam period Intervention period: 6 weeks (3) Usage and administration Test food group: One tablet was taken once a day before going to bed (or after brushing teeth if brushing is used), by licking (without chewing) the tablet to allow it to dissolve slowly in the mouth.

[0084] (4) Basis for determining intake amount, intake method, and intake period The results of Study 1 clearly showed that Coccomyxa sp. KJ strain (Coccomyxa KJ) exhibited an inhibitory effect on osteoclast differentiation in vitro, so it was formulated as a chewable tablet that could be slowly dissolved in the oral cavity. Furthermore, because it takes 4 to 6 weeks for changes in the condition of the gums, which is an indicator of the effectiveness of improving the oral environment, the test food intake period was set at 6 weeks.

[0085] (Measurement items) (1) Periodontal disease-related bacteria Procedure: Saliva was collected from each study participant. ·Investigation item: P. gingivalis Evaluation method: Measurements were taken at screening and pre-intake tests and at 6-week post-intake tests.

[0086] (2) Subjective symptoms - Implementation details: Subjective symptoms were assessed using the Likert scale method. Survey items: "My mouth feels sticky when I wake up in the morning," "My mouth bleeds when I brush my teeth," "I'm concerned about bad breath or have other people told me about it," "My gums are itchy or painful," "My gums are red and swollen," "It's difficult to chew hard foods," "My teeth feel longer," "Gaps have appeared between my teeth or food gets stuck," "I'm concerned about tongue coating" Evaluation method: Screening and pre-intake test, 4-week post-intake test, and 6-week post-intake test. The 4-week post-intake test was conducted at home.

[0087] (3) Dental examination Survey items: Gingival Index (GI), Plaque Index (PI), bleeding on probing, periodontal pocket depth Evaluation method: GI is calculated by dividing the marginal gingiva of each tooth into four sections: buccal, lingual, mesial, and distal, and rating it on a four-point scale: 0 (normal), 1 (mild redness: mild inflammation), 2 (redness, swelling: moderate inflammation), and 3 (severe inflammation: severe inflammation). GI was calculated using the following formula: GI = sum of scores for each tooth surface / number of teeth tested

[0088] PlI is divided into four tooth surfaces similar to GI, and each tooth surface is scored on a four-point scale: 0 (no plaque), 1 (a thin film of plaque is attached to the free gingival margin and interdental areas on the tooth surface. Plaque can be seen using a stain or probe), 2 (a moderate accumulation of soft deposits visible to the naked eye), and 3 (a large amount of soft deposits in the gingival sulcus, on the tooth surface, and at the gingival margin). The index for that tooth was calculated by adding up the indexes for the four tooth surfaces and dividing by 4.

[0089] Bleeding during probing was indicated as absent (-) or present (+). As with GI, each tooth was divided into four tooth surfaces, and measurements of gingival sulcus depth were also performed using a probe. Bleeding confirmed before probing was indicated as (++). Measurements were taken at the screening / pre-intake test and at the test 6 weeks after intake.

[0090] (Outcome: efficacy evaluation item) (1) Primary outcome Actual values ​​of P. gingivalis after 6 weeks of ingestion Logarithmic transformation of P. gingivalis after 6 weeks of ingestion

[0091] (2) Secondary outcomes Actual measurements after 4 and 6 weeks of intake: "My mouth feels sticky when I wake up in the morning," "I bleed when I brush my teeth," "I'm concerned about bad breath or have other people pointed out that I have bad breath," "My gums are itchy or painful," "My gums are red and swollen," "It's difficult to chew hard foods," "My teeth feel longer," "Gaps have appeared between my teeth or food gets stuck," and "I'm concerned about tongue coating" Actual GI and PI values ​​after 6 weeks of intake Actual bleeding measurements at probing 6 weeks after ingestion -Measurement of periodontal pocket depth after 6 weeks of intake

[0092] (Sample size) Number of screening cases: 18 Target number of cases: 12 Number of cases: 14 Rationale for sample size design: The target number of cases was 12, the maximum number that could be achieved within the study budget. In addition, to account for dropouts during the study period, two more people were allowed to participate, making the total number of cases 14.

[0093] (Test results) The test results are shown below. (1) Study participants Study 3 involved healthy Japanese adult men and women. Of the 20 people who agreed to participate in the study, 14 who met the eligibility criteria were enrolled in the study. All completed the study, and there were no participants who violated the compliance rules or whose intake rate of the test food was less than 90%. The analysis dataset consisted of the per protocol set and safety analysis population, which were determined at the time of analysis planning, and consisted of a total of 14 people (5 men, 9 women, 52.6 ± 9.9 years old).

[0094] (2) Periodontal disease-related bacteria The mean (Mean) and standard deviation (SD) for each item of periodontal disease-related bacteria, as well as the results of statistical analysis, are shown in Figure 6. In Figure 6, each item is shown as the mean (Mean) and standard deviation (SD), and intragroup comparisons were performed using a paired t-test. Logarithmic values ​​indicate significant differences (P<0.001), while real values ​​indicate significant differences (P<0.01). Significant differences were observed for P. gingivalis (real values) (Mean: 4241.4 → 1716.6 copies / 10μL, P<0.010) and P. gingivalis (logarithmic values) (Mean: 3.4 → 3.0 Log copies / 10μL, P<0.001).

[0095] (3) Subjective symptoms The median and interquartile range (Q1: 1st quartile, Q3: 3rd quartile) of each subjective symptom item, as well as the results of statistical analysis, are shown in Figure 7. In Figure 7, the median, 1st quartile (Q1), and 3rd quartile (Q3) are shown, and intragroup comparisons were performed using the Wilcoxon signed-rank test. 1: Not at all applicable, 2: Very little applicable, 3: Not very applicable, 4: Somewhat applicable, 5: Quite applicable, and 6: Very applicable. Before intake (Scr) vs. 6 weeks after intake (6w) The item for which a significant difference was observed (P<0.01) was "When I wake up in the morning, my mouth feels sticky" (Median: 4.0 → 3.0, Q1: 4.0 → 3.0, Q3: 5.0 → 4.0, P=0.008).

[0096] (4) Dental examination The mean and SD of each dental examination item and the results of statistical analysis are shown in Figure 8. In Figure 8, the mean and standard deviation are shown, and intra-group comparisons were performed using a paired t-test. Regarding the gingival index (GI), the item in which a significant difference was observed was GI (Mean: 3.9 → 1.9, P<0.001). Regarding the plaque index (PlI), significant differences were observed in the following items: right mandibular central incisor (Mean: 1.1 → 0.8, P<0.010), right mandibular second premolar (Mean: 1.1 → 0.8, P=0.018), right mandibular first molar (Mean: 1.2 → 0.9, P=0.020), left mandibular central incisor (Mean: 1.1 → 0.8, P=0.025), and left mandibular first molar (Mean: 1.2 → 0.8, P=0.012). Regarding bleeding during probing, the item in which a significant difference was observed was bleeding before probing (Mean: 4.9 → 0.4 sides, P = 0.035).

[0097] (Consideration) The primary outcome in Study 3, the actual measured values ​​of P. gingivalis 6 weeks after intake and the logarithmically transformed values ​​of P. gingivalis 6 weeks after intake, were significantly lower than those before intake (actual measured values ​​of P. gingivalis, P<0.010; logarithmically transformed values ​​of P. gingivalis, P<0.001, Figure 6).

[0098] In this study, the subjective symptom of "my mouth feels sticky when I wake up in the morning" was significantly lower after six weeks of intake than before (P=0.008, Figure 7). It has been reported that saliva viscosity is related to the pathological condition of periodontal disease patients, and it is known that viscosity increases as symptoms worsen. The test food may have reduced oral viscosity by improving the condition of periodontal tissues through a reduction in P. gingivalis. In other words, food compositions such as chewable tablets and oral compositions containing dried powder of Coccomyxa microalgae can be used to reduce the sticky feeling in the mouth when you wake up in the morning.

[0099] Furthermore, the gingival index (GI) and plaque index (PlI) for each dental examination item in this study were confirmed to be significantly lower after 6 weeks of intake than before (GI, P<0.001; PlI, P<0.010, P=0.018, P=0.020, P=0.025, P=0.012, P=0.035, Figure 8). Previous studies on gingivalitis patients and healthy individuals have reported that the number of P. gingivalis bacteria showed a positive correlation with GI and PlI. Therefore, it is possible that Coccomyxa KJ improved GI and PlI by reducing P. gingivalis.

[0100] The results of this study confirmed a reduction in P. gingivalis, a periodontal disease-related bacterium, and improvements in GI and PI, suggesting that intake of Kokkomykusa KJ contributed to improving the oral environment of healthy individuals.

[0101] The test food may also contribute to increasing or maintaining bone mineral density. Gram-negative bacteria such as P. gingivalis produce lipopolysaccharide (LPS) in the oral cavity, which is found in the outer membrane of their cell walls, causing the expression of inflammatory cytokines. LPS is known to activate NF-κB, which contributes to the production of inflammatory cytokines, thereby regulating the differentiation of osteoclasts, which contribute to bone resorption. Osteoclasts are known to increase alveolar bone resorption.

[0102] In Study 2, in which Kokkomyxa KJ was administered intragastrically to periodontal disease model mice, it was revealed that Kokkomyxa KJ inhibits alveolar bone resorption, and in Study 1 it was also shown to inhibit osteoclast differentiation. Therefore, it is entirely possible that intake of Kokkomyxa KJ may affect bone metabolism.

[0103] (summary) In Study 3, healthy Japanese adult men and women were given the test food for six weeks to examine its effects on the oral environment. A significant reduction in P. gingivalis and improvements in GI and PI were confirmed with the test food, suggesting that oral ingestion of the test food contributes to improving the oral environment.

Claims

1. Contains microalgae belonging to the genus Coccomyxa as an active ingredient, The microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain (accession number: FERM BP-22254), A food composition for preventing or improving alveolar bone resorption in periodontal disease, characterized in that the active ingredient is at least one selected from the algae bodies of the Coccomyxa sp. KJ strain or dried powder thereof.

2. Contains microalgae belonging to the genus Coccomyxa as an active ingredient, The microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain (accession number: FERM BP-22254), A feed composition for preventing or improving alveolar bone resorption in periodontal disease, characterized in that the active ingredient is at least one selected from the algae of the Coccomyxa sp. KJ strain or its dried powder.

3. Contains microalgae belonging to the genus Coccomyxa as an active ingredient, The microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain (accession number: FERM BP-22254), A preventive or therapeutic agent for alveolar bone resorption in periodontal disease, characterized in that the active ingredient is at least one selected from the algae of the Coccomyxa sp. KJ strain or a dried powder thereof.

4. Contains microalgae belonging to the genus Coccomyxa as an active ingredient, The microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain (accession number: FERM BP-22254), 1. A food composition for inhibiting osteoclast differentiation, wherein the active ingredient is at least one selected from the algae of the Coccomyxa sp. KJ strain or a dried powder thereof.

5. Contains microalgae belonging to the genus Coccomyxa as an active ingredient, The microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain (accession number: FERM BP-22254), A feed composition for inhibiting osteoclast differentiation, wherein the active ingredient is at least one selected from the algae of the Coccomyxa sp. KJ strain or a dried powder thereof.

6. Contains microalgae belonging to the genus Coccomyxa as an active ingredient, The microalgae belonging to the genus Coccomyxa are Coccomyxa sp. KJ strain (accession number: FERM BP-22254), An osteoclast differentiation inhibitor, characterized in that the active ingredient is at least one selected from the algae of the Coccomyxa sp. KJ strain or a dried powder thereof.

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