Proliferation inhibitor for periodontal disease bacterium and application for the same

Abstract

To provide a new active ingredient for inhibiting the proliferation of periodontal disease bacteria.SOLUTION: A proliferation inhibitor for periodontal disease bacteria of the present invention includes at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei. A proliferation inhibition method for periodontal disease bacteria of the present invention allows the proliferation inhibitor for periodontal disease bacteria of the present invention to coexist with periodontal disease bacteria. The periodontal disease bacteria are, for example, bacteria of the genus Fusobacterium, bacteria of the genus Porphyromonas, and bacteria of the genus Tannerella.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a proliferation inhibitor for periodontal disease bacteria and uses thereof. [Background technology]

[0002] Fusobacterium, Porphyromonas, Tannerella, and other bacteria are indigenous to the oral cavity and are known to be involved in periodontal disease. In recent years, it has also been reported that Fusobacterium is involved in colon cancer. Therefore, it is believed that inhibiting the proliferation of periodontal disease bacteria present in the body will lead to the prevention and treatment of diseases in which they are involved. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, an object of the present invention is to provide a new active ingredient that inhibits the proliferation of periodontal disease bacteria. [Means for solving the problem]

[0004] In order to achieve the above object, the proliferation inhibitor for periodontal disease bacteria of the present invention is characterized by containing at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei.

[0005] The method for inhibiting the proliferation of periodontal disease bacteria of the present invention is characterized in that the proliferation inhibitor for periodontal disease bacteria of the present invention is allowed to coexist with periodontal disease bacteria.

[0006] The drug for periodontal disease bacteria-related diseases of the present invention is characterized by containing the proliferation inhibitor for periodontal disease bacteria of the present invention as an active ingredient. Effect of the Invention

[0007] The present inventors found that membrane vesicles of Lactobacillus reuteri and Lactobacillus casei inhibit the proliferation of periodontal disease bacteria such as Fusobacterium bacteria, and thus established the present invention. According to the present invention, the proliferation of periodontal disease bacteria can be inhibited, and therefore, for example, the inhibition of the proliferation can improve diseases caused by periodontal disease bacteria. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a graph showing the results of particle size distribution of membrane vesicles derived from Lactobacillus reuteri prepared in Example 1. [Diagram 2] FIG. 2 is a graph showing the results of particle size distribution of membrane vesicles derived from Lactobacillus casei prepared in Example 1. [Diagram 3] FIG. 3 is a photograph showing the culture state of Fusobacterium bacteria cultured in the presence of membrane vesicles derived from Lactobacillus reuteri or Lactobacillus casei in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] [1] A growth inhibitor for periodontal disease bacteria, comprising at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei. [2] The proliferation inhibitor described in [1], wherein the periodontal disease bacteria is at least one selected from the group consisting of Fusobacterium bacteria, Porphyromonas bacteria, and Tannerella bacteria. [3] The proliferation inhibitor described in [2], wherein the Fusobacterium bacterium is Fusobacterium nucleatum. [4] The proliferation inhibitor described in [2], wherein the Porphyromonas bacterium is Porphyromonas gingivalis. [5] The proliferation inhibitor described in [2], wherein the Tannerella bacterium is Tannerella forsythensis. [6] The proliferation inhibitor described in any one of [1] to [5], wherein the membrane vesicles are membrane vesicles isolated from a culture of Lactobacillus reuteri. [7] The proliferation inhibitor described in any one of [1] to [5], wherein the membrane vesicles are membrane vesicles isolated from a culture of Lactobacillus casei. [8] A method for inhibiting the proliferation of periodontal bacteria, comprising coexisting a proliferation inhibitor for periodontal bacteria described in any one of [1] to [7] with periodontal bacteria. [9] The proliferation-inhibiting method described in [8], wherein the coexistence is in vivo or in vitro.

[10] The proliferation-inhibiting method described in [8] or [9], wherein the coexistence is coexistence in the body of a human or non-human animal.

[11] A drug for periodontal disease bacteria-related diseases, comprising as an active ingredient a growth inhibitor for periodontal disease bacteria described in any one of [1] to [7].

[12] A drug for periodontal disease associated with bacteria described in

[11] , further comprising an excipient.

[13] The drug for periodontal bacteria-associated diseases described in

[11] or

[12] , wherein the periodontal bacteria-associated disease is myocardial infarction, cerebral infarction, oral disease, or digestive system disease.

[14] The drug for periodontal bacteria-related diseases described in

[13] , wherein the oral disease is periodontal disease or tooth decay.

[15] The drug for periodontal disease bacteria-related diseases described in

[13] , wherein the digestive system disease is a colon disease.

[16] The drug for periodontal disease bacteria-related diseases described in

[15] , wherein the colon disease is colitis or colon cancer.

[0010] Terms used in this specification can be used in the same manner as commonly used in the art unless otherwise specified.

[0011] As used herein, a "membrane vesicle" is a sac-like body released from the biomembrane of a bacterium, and is also called MV (Membrane Vesicle; MV).

[0012] As used herein, "inhibition of growth" refers to, for example, a reduction in the extent of growth of a target bacterium in the presence of an active ingredient compared to the extent of growth of the same target bacterium in the absence of the active ingredient. "Inhibition of growth" may also refer to, for example, the death of a target cell upon contact with the active ingredient.

[0013] As used herein, "treatment" may mean, for example, the suppression of worsening, alleviation (improvement), remission, or cure (complete recovery) of symptoms of a target disease. Furthermore, as used herein, "treatment" may include, for example, "prevention" in a broad sense. As used herein, "prevention" includes, for example, the suppression of onset of a target disease, delay of onset, and the like. As used herein, for example, when "treatment" is used in a narrow sense, "treatment" as used herein may be read as, for example, prevention, and the present invention may include a treatment method and a prevention method.

[0014] The present invention will be described below with reference to specific examples, but is not limited to these examples. In addition, the examples in each invention can be mutually incorporated.

[0015] (1) Antiproliferative agents As described above, the growth inhibitor for periodontal disease bacteria of the present invention is characterized by containing at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei. Hereinafter, the growth inhibitor for periodontal disease bacteria of the present invention is also referred to as a "growth inhibitor."

[0016] The bacteria targeted by the proliferation inhibitor of the present invention are periodontal disease bacteria. Specific examples of the periodontal disease bacteria include Fusobacterium, Porphyromonas, Tannerella, etc., which are anaerobic gram-negative bacteria that are indigenous to the oral cavity. Specific examples of Fusobacterium bacteria include F. nucleatum, F. necrophorum, F. varium, F. mortiferum, etc. Specific examples of Porphyromonas bacteria include Porphyromonas gingivalis, etc. Specific examples of Tannerella bacteria include Tannerella forsythensis, etc. The periodontal disease bacteria targeted by the present invention are not limited to these examples, and include Treponema bacteria such as Treponema denticola, etc.

[0017] The proliferation inhibitor of the present invention contains at least one membrane vesicle of Lactobacillus reuteri and Lactobacillus casei as an active ingredient, and may contain, for example, only membrane vesicles of Lactobacillus reuteri out of both, or may contain only membrane vesicles of Lactobacillus casei out of both. The proliferation inhibitor of the present invention may further contain, for example, membrane vesicles other than membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei as membrane vesicles.

[0018] In the proliferation inhibitor of the present invention, the content of the membrane vesicles is not particularly limited and can be appropriately set depending on, for example, the purpose of use, and specific examples will be described later.

[0019] Lactobacillus reuteri is classified as a Bacillus genus, has the scientific name Lactobacillus reuteri, and is a Gram-positive, anaerobic lactic acid bacillus. The strain of Lactobacillus reuteri in the present invention is not particularly limited, and examples thereof include BAA-2837, DSM 17938, and ATCC PTA 6475.

[0020] Lactobacillus casei is classified into the genus Bacillus, has the scientific name Lactobacillus casei, and is a gram-positive, anaerobic lactic acid bacillus. The strain of Lactobacillus casei in the present invention is not particularly limited, and examples thereof include K-1, Shirota, BC-90, SBR1202, and NY1301.

[0021] As described above, Lactobacillus reuteri and Lactobacillus casei are Gram-positive bacteria, and the membrane vesicles derived from these bacteria can be considered to be, for example, sac-like bodies composed of a lipid bilayer membrane, which is a biological membrane, specifically, spherical structures.

[0022] Membrane vesicles of Lactobacillus reuteri and Lactobacillus casei can be produced, for example, by culturing at least one of Lactobacillus reuteri and Lactobacillus casei as a raw material bacterium. A method for culturing the raw material bacterium is exemplified below. In the following examples, the raw material bacterium may be, for example, both Lactobacillus reuteri and Lactobacillus casei, or only Lactobacillus reuteri or only Lactobacillus casei.

[0023] The culture can be carried out by seeding the raw material bacteria in a medium and incubating. By culturing the raw material bacteria, membrane vesicles secreted from the raw material bacteria are released into the medium. The MV culture conditions are not particularly limited, and general culture conditions for lactic acid bacteria (preferably Lactobacillus bacteria) can be selected.

[0024] The medium is not particularly limited, and examples thereof include de Man-Rogosa-Sharpe (MRS) medium, arginine-containing MRS medium, Brain Heart Infusion (BHI) medium, and L-glutamine-containing Advanced RPMI1640 (final concentration of L-glutamine: 10 mM) medium. From the viewpoint of recovering the membrane vesicles, the medium is preferably a liquid medium. The medium may contain additives such as amino acids and glucose. The additives can refer to, for example, the types and concentrations of amino acids and glucose contained in RPMI1640 medium and DMEM medium.

[0025] The culture temperature is not particularly limited, and is, for example, the optimum temperature for lactic acid bacteria (preferably Lactobacillus bacteria), and specific examples thereof are 28 to 40°C, 29 to 38°C, and 30 to 37°C. The culture time is not particularly limited, and may be appropriately determined, for example, depending on the culture scale, the type of raw material bacteria, and the like, and may be cultured until confluence is reached. Confluence can be judged, for example, by the optical density (OD) of the culture medium, and as a specific example, confluence can be judged when the OD600 value reaches a range of about 2.5 to 3.5. The culture time can be, for example, 12 to 72 hours, 12 to 24 hours, 24 to 48 hours, and 48 to 72 hours.

[0026] The membrane vesicles can be separated from the culture medium, for example, by removing the cultured bacterial cells from the culture medium and recovering a fraction (for example, culture supernatant) containing the membrane vesicles. The method of separation is not particularly limited, and examples thereof include centrifugation of the culture medium. When the cultured bacterial cells and the culture supernatant are separated by centrifugation, the conditions are not particularly limited, and examples thereof are 4,000 to 6,000×g and 10 to 15 minutes.

[0027] In the present invention, the membrane vesicles may be, for example, the culture solution, but preferably, a fraction (the culture supernatant) obtained by removing cultured bacteria from the culture solution may be used as is, or a purified product obtained by removing impurities from the culture supernatant may be used, or a concentrate obtained by further concentrating the membrane vesicles contained in the culture supernatant may be used, or a concentrated purified product obtained by removing impurities and concentrating the membrane vesicles may be used. The impurities may be confirmed, for example, using a nanoparticle analysis system or the like.

[0028] Examples of purification methods for removing the impurities include standing, centrifugation such as ultracentrifugation, filtration, chromatography, electrophoresis, and the like.

[0029] In the case of standing still, for example, the culture supernatant is stirred and then allowed to stand to precipitate impurities, and a liquid fraction containing the membrane vesicles is collected. The conditions for standing still are not particularly limited, and are, for example, 5 to 60 minutes.

[0030] The centrifugation may be, for example, a method of precipitating impurities from the culture supernatant and recovering a liquid fraction containing the membrane vesicles, or a method of precipitating the membrane vesicles and removing the liquid fraction containing impurities, or the latter method may be performed after the former method. When precipitating the impurities, the conditions of the centrifugation are not particularly limited, and are, for example, 400 to 3000×g and 5 to 30 minutes, and are also called crude centrifugation. When precipitating the membrane vesicles, ultracentrifugation is preferable, and the conditions are, for example, 50,000 to 150,000×g and 50 to 140 minutes. The precipitated fraction containing the membrane vesicles may be suspended in a liquid solvent, and the suspension may be, for example, refrigerated or frozen.

[0031] The filtration is, for example, filtration using a filter such as a filter, and a specific example is ultrafiltration. The filter may be, for example, a filter through which the membrane vesicles pass, or a filter that the membrane vesicles cannot pass. In the former case, for example, the remaining matter of the filter may be removed as impurities, and a liquid fraction (filtrate) containing the membrane vesicles may be collected, and the pore size of the filter may be, for example, a pore through which the membrane vesicles can pass. In the latter case, for example, the filtrate containing impurities may be removed, and the membrane vesicles captured by the filter may be collected, and the pore size of the filter may be, for example, a pore through which the membrane vesicles cannot pass. In the latter case, the membrane vesicles captured by the filter may be, for example, suspended in the liquid solvent.

[0032] Examples of the chromatography include gel filtration chromatography, and examples of the electrophoresis include free-flow electrophoresis and capillary electrophoresis.

[0033] The liquid solvent is not particularly limited, and examples thereof include aqueous solvents such as water, buffer solutions, physiological saline, and physiological buffer solutions.

[0034] The method for concentrating the membrane vesicles includes, for example, ultracentrifugation, filtration, drying such as freeze-drying, etc. In the case of ultracentrifugation, for example, the membrane vesicles are precipitated and the obtained precipitate fraction is suspended in a desired amount of the liquid solvent, and in the case of filtration, for example, the membrane vesicles captured on a filter medium are suspended in a desired amount of the liquid solvent. In addition, in the case of freeze-drying, a liquid containing the membrane vesicles (for example, the culture supernatant, the liquid fraction, the suspension, etc.) is dried and the obtained dry matter is suspended in a desired amount of the liquid solvent.

[0035] The separation, purification, and concentration of the membrane vesicles from the culture medium can be carried out, for example, under refrigerated conditions to room temperature conditions (e.g., 4°C to 37°C), and centrifugation, filtration, chromatography, etc. are preferably carried out at 4°C ± 2°C.

[0036] The size of the membrane vesicles derived from Lactobacillus reuteri used in the present invention can be confirmed, for example, by particle size distribution, and the size can be expressed, for example, by at least one of the mode, the average value, and SD. The mode can also be referred to, for example, as a peak in the particle size distribution. For example, the membrane vesicles derived from Lactobacillus reuteri can have the following sizes.

[0037] (Membrane vesicles derived from Lactobacillus reuteri) Mode of particle size in particle size distribution Peak 1: For example, 60 to 80 nm, 71 ± 10, 71 ± 5 (specific example: 71 nm) Peak 2: For example, 85 nm to 110 nm, 93 ± 10 nm, 93 ± 5 nm (specific example: 93 nm) Peak 3: For example, 150-170 nm, 160±10, 160±5 (specific example: 160 nm) The average particle size in the particle size distribution (mean): For example, 121±20 nm, 121±10 nm, 121±5 nm (specific example: 121.4 nm)

[0038] Membrane vesicles derived from Lactobacillus reuteri may have, for example, one peak, two peaks, three peaks, or four or more peaks in the particle size distribution. Membrane vesicles derived from Lactobacillus reuteri preferably have, for example, a peak that falls within any of the ranges exemplified above, and specifically may have any of peak 1, peak 2, and peak 3, any two peaks (for example, peak 1 and peak 2), or three peaks.

[0039] The size of the membrane vesicles derived from Lactobacillus casei used in the present invention can be confirmed, for example, by particle size distribution, and the size can be expressed, for example, by at least one of the mode, the average value, and SD. The mode can also be, for example, the peak in the particle size distribution. For example, the membrane vesicles derived from Lactobacillus casei can have the following sizes.

[0040] (Membrane vesicles derived from Lactobacillus casei) Mode of particle size in particle size distribution Peak 1: For example, 90-110 nm, 99±10 nm, 99±5 nm (specific example: 99 nm) Peak 2: For example, 120 nm to 140 nm, 136 ± 10 nm, 136 ± 5 nm (specific example: 136 nm) The average particle size in the particle size distribution (mean): For example, 134±20 nm, 134±10 nm, 134±5 nm (specific example: 134.4 nm)

[0041] The membrane vesicles derived from Lactobacillus casei may have, for example, one peak, two peaks, or three or more peaks in the particle size distribution. The membrane vesicles derived from Lactobacillus casei preferably have, for example, a peak that falls within any of the ranges exemplified above, and specifically may have either peak 1 or peak 2, or both (for example, peak 1 and peak 2).

[0042] The method for measuring the particle size distribution of the membrane vesicles is not particularly limited, and may be, for example, a laser diffraction method, a light scattering method such as a dynamic light scattering method, a particle trajectory analysis method, a nanotracking analysis method, etc. For the measurement, a commercially available device such as NanoSight (product name, Quantum Design Co., Ltd.) may be used.

[0043] The proliferation inhibitor of the present invention may contain only an active ingredient that inhibits the proliferation of periodontal disease bacteria, or may contain the active ingredient and other additive ingredients. In the present invention, the active ingredient may contain at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei, as described above, and may be in a form that contains membrane vesicles of Lactobacillus reuteri but does not contain membrane vesicles of Lactobacillus casei, or in a form that contains membrane vesicles of Lactobacillus casei but does not contain membrane vesicles of Lactobacillus reuteri, or in a form that contains both membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei. In addition, the present invention may contain, as the active ingredient, a component that inhibits the proliferation of periodontal disease bacteria in addition to the membrane vesicles of Lactobacillus reuteri or the membrane vesicles of Lactobacillus casei.

[0044] In the proliferation inhibitor of the present invention, the other additive components are not particularly limited and can be appropriately selected depending on, for example, the field in which the proliferation inhibitor of the present invention is applied.

[0045] The growth inhibitor of the present invention may contain components other than the membrane vesicles contained in the culture solution, for example, as described above, since the membrane vesicles can be prepared from the culture solution of the raw material bacteria. In addition, as described above, the growth inhibitor of the present invention preferably uses, as the membrane vesicles, a fraction containing the membrane vesicles obtained by removing cultured bacteria from the culture solution (the culture supernatant), or a fraction obtained by further purifying and / or concentrating the membrane vesicles from the culture supernatant. For this reason, it is preferable that the growth inhibitor of the present invention does not substantially contain, for example, the bacterial bodies of Lactobacillus reuteri and Lactobacillus casei, which are the raw material bacteria. "Substantially not containing bacterial bodies" means, for example, that even when the growth inhibitor of the present invention is subjected to a microbiological detection method, live or dead bacteria of Lactobacillus reuteri and Lactobacillus casei are below the detection limit.

[0046] The proliferation inhibitor of the present invention can be used, for example, in vivo or in vitro depending on the purpose.

[0047] When the proliferation inhibitor of the present invention is used in vivo, the proliferation inhibitor of the present invention may be, for example, a pharmaceutical composition or a food composition.

[0048] When the proliferation inhibitor of the present invention is a pharmaceutical composition, it can be used, for example, to inhibit proliferation of periodontal disease bacteria in a living body, to treat diseases caused by periodontal disease bacteria (hereinafter referred to as periodontal disease bacteria-related diseases), etc. In this case, the pharmaceutical composition of the present invention may be administered to a target living body. The living body may be, for example, a human or a non-human animal, and the non-human animal may be, for example, a mammal such as a mouse, a rat, a dog, a cat, a monkey, a rabbit, a cow, a goat, or a camel.

[0049] The dosage of the pharmaceutical composition of the present invention is not particularly limited, and is preferably administered in a pharmacologic effective amount. The pharmacologic effective amount can be determined, for example, according to the administration route, the type of disease, the presence or absence of onset, the severity, age, etc. In addition, the content of the membrane vesicle in the pharmaceutical composition of the present invention is not particularly limited, and is preferably, for example, administered in a pharmacologic effective amount.

[0050] The administration route is not particularly limited and may be, for example, parenteral or oral. Examples of the parenteral route include percutaneous, subcutaneous, intravenous, intraarterial, intraperitoneal, intranasal, oral, inhalation, digestive tract such as the intestine, and topical.

[0051] As a specific example, when the pharmaceutical composition of the present invention is for parenteral use, the daily dosage of the membrane vesicle is, for example, 100 to 1,000 mg, and the number of times of administration per day is, for example, 1 to 3 times, 1 to 2 times, or 1 time. When the pharmaceutical composition of the present invention is for oral use, the daily dosage of the membrane vesicle is, for example, 10 to 100 mg, and the number of times of administration per day is not particularly limited, for example, 1 to 3 times, 1 to 2 times, or 1 time. The content of the membrane vesicle in the pharmaceutical composition of the present invention is not particularly limited as described above, and can be formulated to meet, for example, the dosage exemplified.

[0052] The pharmaceutical composition of the present invention may contain, for example, the active ingredient and other additive ingredients, as described above. The additive ingredients include, for example, pharma- ceutically acceptable ingredients. Specific examples include, for example, excipients and carriers (base materials). The excipients and carriers include, for example, aqueous solvents such as water, saline, and buffer solutions; fats and oils such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes; and the like. In addition, the additive ingredients include, for example, binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, wetting agents, thickeners, stabilizers, UV shielding agents, preservatives, preservatives, vitamins, minerals, colorants, and the like.

[0053] The dosage form of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected according to the administration route.Specific examples of the dosage form include injections, oral agents (internal medicines), and external agents (external medicines), and the oral agent form includes liquids, emulsions, gels, sols, granules, pills, tablets, capsules, tablets, and the like.The external agent includes transdermal agents such as skin medicines, nasal drops, eye drops, ear drops, oral medicines, suppositories, and the like.The external agent form includes liquids, emulsions, gels, sols, ointments, granules, pills, tablets, capsules, and the like.

[0054] The pharmaceutical composition of the present invention may be, for example, a drug, a quasi-drug, or a health product.

[0055] When the proliferation inhibitor of the present invention is a food and drink composition, it is, for example, a food and drink that contains the membrane vesicle of the active ingredient.The food and drink composition of the present invention may be, for example, a food or a drink.The food and drink composition of the present invention may be, for example, a functional health food composition.

[0056] The intake amount of the food and drink composition of the present invention is not particularly limited, and the intake amount of the membrane vesicle per day is, for example, 100 to 1,000 mg, etc., and the number of intakes per day is, for example, 1 to 3 times, 1 to 2 times, or 1 time. As described above, the content of the membrane vesicle in the food and drink composition of the present invention is not particularly limited, and can be formulated to satisfy, for example, the intake amount exemplified.

[0057] The food and drink composition of the present invention may contain the active ingredient and other additive ingredients, for example, as described above. Examples of the additive ingredients include aqueous solvents such as water, physiological saline, and buffer solutions, fats and oils such as soybean oil, alcohols such as ethanol, sugars such as glucose, sugar alcohols such as xylitol, emulsifiers, antioxidants, thickeners, organic acids, pH regulators, stabilizers, preservatives, vitamins, minerals, flavors, colorants, fillers, and carbonation agents.

[0058] (2) Growth inhibition method As described above, the proliferation inhibition method of the present invention is characterized by coexisting the proliferation inhibitor for periodontal disease bacteria of the present invention with periodontal disease bacteria. The proliferation inhibition method of the present invention can be applied to the above-mentioned description of the proliferation inhibitor of the present invention. The periodontal disease bacteria targeted in the present invention are not particularly limited, and examples thereof include the above-mentioned Fusobacterium bacteria, Porphyromonas bacteria, and Tannerella bacteria.

[0059] In the proliferation inhibition method of the present invention, the step of causing the proliferation inhibitor and periodontal disease bacteria to coexist (coexistence step) can be said to be, for example, a step of exposing periodontal disease bacteria to the proliferation inhibitor. The step may be, for example, in vivo or in vitro depending on the purpose.

[0060] When the proliferation inhibitory method of the present invention is carried out in vivo, the coexistence step is, for example, a step of administering the proliferation inhibitor to a target, and the target is a living body, i.e., an animal. The living body is, for example, a human or non-human animal as described above.

[0061] According to this embodiment, for example, the proliferation of periodontal disease bacteria in a living body can be suppressed. Also, according to this embodiment, for example, by suppressing the proliferation of periodontal disease bacteria, diseases associated with periodontal disease bacteria can be treated. Examples of periodontal disease bacteria-associated diseases will be described later.

[0062] The route of administration of the proliferation inhibitor may be, for example, parenteral or oral, as described above, and the dosage of the proliferation inhibitor is not particularly limited, and the above examples can be used.

[0063] When the proliferation inhibition method of the present invention is carried out in vitro, the coexistence step is, for example, a step of causing the proliferation inhibitor and periodontal disease bacteria to coexist in a medium. As a specific example, the coexistence step is a step of culturing periodontal disease bacteria in the medium containing the proliferation inhibitor. According to this embodiment, the proliferation of periodontal disease bacteria can be inhibited.

[0064] The culture conditions for the periodontal disease bacteria are not particularly limited. The type of the medium that can be used is, for example, ABHK medium, HK agar medium, etc. Examples of the medium include an agar medium and a liquid medium. The culture temperature is not particularly limited, and is, for example, the optimal temperature for the periodontal disease bacteria, specifically, 28 to 40°C, 29 to 38°C, or 30 to 37°C. The culture may be, for example, anaerobic culture or aerobic culture.

[0065] The ratio of the growth inhibitor to the periodontal disease bacteria is not particularly limited. As a specific example, the ratio of the growth inhibitor to be added is, for example, 100 to 1,000 membrane vesicles of the active ingredient per 1 cell of periodontal disease bacteria. In addition, the concentration of the membrane vesicles of the active ingredient in the medium is, for example, 50 to 100 μg / mL.

[0066] (3) Drugs and treatment methods for periodontal disease The drug for periodontal disease-related diseases (hereinafter referred to as the drug for diseases) of the present invention is characterized by containing the growth inhibitor for periodontal disease bacteria of the present invention as an active ingredient. The drug for diseases of the present invention is, for example, a drug for a living body, and may be a medicine, a quasi-drug, or a care product.

[0067] For the disease drug of the present invention, the description of the proliferation inhibitor of the present invention in (1) above can be applied, specifically, the description of the pharmaceutical composition in (1) above can be applied.

[0068] The method for treating periodontal disease-related diseases of the present invention is characterized by comprising a step of administering to a patient the growth inhibitor for periodontal disease bacteria of the present invention. In addition, in the method for treating periodontal disease-related diseases of the present invention, the administration step can be restated as, for example, a step of administering to a patient at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei.

[0069] In the present invention, a periodontal disease bacteria-related disease is a disease associated with periodontal disease bacteria. The disease is, for example, myocardial infarction, cerebral infarction, digestive system disease, or oral disease. The digestive system disease is, for example, a colon disease, specific examples of which include colitis or colon cancer. The oral disease is, for example, periodontal disease or dental caries. These diseases are associated with periodontal disease bacteria. Therefore, by administering membrane vesicles, which are the active ingredient in the proliferation inhibitor of the present invention, the proliferation of periodontal disease bacteria is inhibited, and as a result, the disease can be treated.

[0070] The method of administration of the proliferation inhibitor is not particularly limited, and may be, for example, parenteral or oral administration, as described above. The dosage of the proliferation inhibitor is not particularly limited, and for example, the above-mentioned examples can be used.

[0071] When the target disease is myocardial infarction, the administration of the proliferation inhibitor is preferably, for example, oral administration, intravenous administration, or the like.

[0072] When the target disease is cerebral infarction, the antiproliferative agent is preferably administered orally, by nasal drip, or intravenously, for example.

[0073] When the target disease is a digestive system disease, the antiproliferative agent is preferably administered orally, intravenously, or as a suppository, for example.

[0074] When the target disease is an oral disease, the proliferation inhibitor is preferably administered, for example, intraorally. In the case of intraorally administered, examples of the proliferation inhibitor include a mouthwash, an oral application agent, and a preparation that dissolves or disintegrates in the oral cavity (for example, a troche or a patch).

[0075] (4)Applications The present invention relates to membrane vesicles of Lactobacillus reuteri or Lactobacillus casei for use in inhibiting the proliferation of periodontal disease bacteria.

[0076] The present invention relates to membrane vesicles of Lactobacillus reuteri or Lactobacillus casei for use in the treatment of periodontal disease associated diseases. The present invention relates to membrane vesicles of Lactobacillus reuteri or Lactobacillus casei for use in the production of a medicament for periodontal disease associated diseases.

[0077] For the membrane vesicles of Lactobacillus reuteri and Lactobacillus casei in the present invention, the description in (1) above can be applied.

[0078] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. EXAMPLES

[0079] [Example 1] Membrane vesicles were collected from Lactobacillus reuteri and Lactobacillus casei, respectively.

[0080] As the raw material bacteria, Lactobacillus reuteri BAA-2837 strain (ATCC) was used as Lactobacillus reuteri, and Lactobacillus casei YA921 strain isolated from a commercial preparation (general name: Lactobacillus casei preparation powder, product name: Biolactis (registered trademark) powder) was used as Lactobacillus casei. The raw material bacteria were cultured in BHI medium (2% yeast extract, 0.2% cysteine) under the conditions of 37°C, 5% CO2 concentration, and anaerobic conditions.

[0081] The raw material bacteria (Lactobacillus reuteri or Lactobacillus casei) were allowed to form colonies on an agar medium, and precultured for 48 hours under the above culture conditions. The precultured colonies were then transferred to 1 L of fresh BHI liquid medium, and main culture was carried out for 24 hours under the above culture conditions until the colonies became subconfluent (OD600=2.5-3.5). The main culture liquid was subjected to centrifugation (6,000xg, 15 minutes, 4°C) and the supernatant was collected. The supernatant was filtered through a filter with a pore size of 0.22 μm, and the filtrate was collected. The filtrate was then subjected to ultracentrifugation (100,000g, 70 minutes, 4°C) and the precipitated fraction (pellet) was collected. 1 g of the pellet was suspended in 10 mL of physiological saline and collected as a membrane vesicle (MV) sample. The MV sample was subjected to a nanoparticle analysis system (product name NanoSight, LM10, laser 405 nm, Malvern) to confirm the particle size distribution of the membrane vesicles contained in the MV sample. The analysis software used was NTA3.4 (Malvern panalytical).

[0082] The particle size distribution results for the MV sample derived from Lactobacillus reuteri (pellet 1 g / 10 mL) are shown below. Membrane vesicle concentration: 9.35×10 10 particles / mL Mean: 121.4 nm Mode: 93nm SD: 52.1nm Median diameter: D10 71.4nm D50 104.7nm D90 208.8nm

[0083] The particle size distribution results for the MV sample (pellet 1 g / 10 mL) derived from Lactobacillus casei are shown below. Membrane vesicle concentration: 1.5×10 10 particles / mL Mean: 134.3 nm Mode: 98.6nm SD: 49.5nm Median diameter: D10 92.3nm D50 120.0nm D90 210.2nm

[0084] [Example 2] The ability of MV derived from Lactobacillus reuteri and MV derived from Lactobacillus caseinus to inhibit the growth of periodontal disease bacteria was evaluated.

[0085] (1) Fusobacterium nucleatum The periodontal disease bacteria used was Fusobacterium nucleatum KWIK-STIK strain (ATCC 25586). The membrane vesicles used were the Lactobacillus reuteri MV sample and the Lactobacillus casei MV sample prepared in Example 1. The MV samples were diluted with sterilized water as necessary.

[0086] The Lactobacillus reuteri MV sample or the Lactobacillus casei MV sample was added to a sterilized liquid ABHK agar medium to a predetermined concentration (0%, 1%, 5%), and the ABKH agar medium was solidified on a plate. A loopful of Fusobacterium bacteria was taken and suspended in 8 mL of ABHK liquid medium not containing agar. The suspended bacteria were then seeded on the plate and cultured at 37°C under anerobic conditions for 24 hours. After the culture, the plates were visually inspected for Fusobacterium bacteria and the CFU (colony forming units) were counted.

[0087] A photograph of the plate with Fusobacterium bacteria is shown in Figure 3, and the CFU results are shown in Table 1. Figure 3 is a photograph showing the culture state of Fusobacterium bacteria in the presence of MV, with the upper row showing the results in the presence of Lactobacillus reuteri MV and the lower row showing the results in the presence of Lactobacillus casei, and both the upper and lower rows showing the results for MV concentrations of 0%, 1%, and 5% from the left.

[0088] [Table 1]

[0089] As shown in Figure 3 and Table 1, whether the Lactobacillus reuteri MV or the Lactobacillus casei MV was used, the addition of MV reduced the viable cell count of Fusobacterium bacteria, and the reduction in viable cell count depended on the MV concentration. These results confirmed that the Lactobacillus reuteri MV and the Lactobacillus casei MV could inhibit the proliferation of Fusobacterium bacteria.

[0090] (2) Aggregatibacter actinomycetemcomitans As periodontal disease bacteria, Aggregatibacter actinomycetemcomitans (standard strain LYFO DISC, ATCC 29522) and Porphyromonas gingivalis (standard strain KWIK-STIK, ATCC 33277) were used. As membrane vesicles, Lactobacillus reuteri MV samples and Lactobacillus casei MV samples prepared in Example 1 were used. The MV samples were diluted with sterilized water as necessary.

[0091] Except for using A. actinomycetemcomitans and P. gingivalis, respectively, as periodontal disease bacteria, the periodontal disease bacteria were cultured in the presence of the Lactobacillus reuteri MV sample or the Lactobacillus casei MV sample in the same manner as in (1) above, and the culture conditions were confirmed.

[0092] Table 2 shows the results of CFU of A. actinomycetemcomitans, and Table 3 shows the results of CFU of P. gingivalis. As shown in Tables 2 and 3, when either Lactobacillus reuteri MV or Lactobacillus casei MV was used, the viable cell count of periodontal disease bacteria was reduced by the addition of MV, and further, the viable cell count was reduced depending on the concentration of MV. From these results, it was confirmed that the Lactobacillus reuteri MV and Lactobacillus casei MV can suppress the growth of other periodontal disease bacteria, regardless of whether they are Fusobacterium bacteria.

[0093] [Table 2] [Table 3]

[0094] [Example 3] We confirmed that the proliferation-suppressing ability was improved by isolating membrane vesicles.

[0095] Lactobacillus reuteri was cultured, filtered, and ultracentrifuged in the same manner as in Example 1. During this process, at the end of the main culture of Lactobacillus reuteri, the culture medium of the main culture was sampled (Sample 1: culture stock sample). In addition, a precipitated fraction containing MV and a supernatant were separated by the ultracentrifugation, and the supernatant was sampled (Sample 2: sample after MV removal). The precipitated fraction containing MV was suspended in the same amount of physiological saline as the medium used in the main culture to prepare a membrane vesicle (MV) sample (Sample 3). The particle size distribution of the MV sample prepared from the precipitated fraction was similar to that shown in FIG. 1 in Example 1.

[0096] Then, in the same manner as in Example 2(1), Fusobacterium bacteria were cultured in the presence of 1 mL of each sample, and the culture state was confirmed. Moreover, 1 mL of physiological saline was added instead of the sample, and the culture state was also confirmed for the untreated control. Table 4 shows the results of CFU of Fusobacterium bacteria. Also shown are the relative values ​​of CFU when each sample was used, assuming that the CFU of the untreated control is "1". The smaller the relative value, the more inhibited the proliferation.

[0097] [Table 4]

[0098] As shown in Table 4, the culture stock solution containing Lactobacillus reuteri itself (Sample 1) also showed growth inhibition compared to the untreated control, but the MV sample (Sample 3) recovered from the culture stock solution significantly inhibited the growth of Fusobacterium bacteria. This result shows that growth can be more effectively inhibited by isolating the secreted membrane vesicles from the bacterial cells, rather than using a culture solution containing the bacterial cells themselves. In addition, since the volume of Sample 3 was the same as the volume of the culture solution used to recover EVs, it can be said that the effect is not due to the EV concentration, but due to the isolated EVs.

[0099] Although the present invention has been described above with reference to the embodiment, the present invention is not limited to the above embodiment. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention. [Industrial Applicability]

[0100] According to the present invention, the proliferation of periodontal disease bacteria can be inhibited, and thus, for example, the inhibition of the proliferation makes it possible to improve diseases caused by periodontal disease bacteria.

Claims

1. A growth inhibitor for periodontal disease bacteria, comprising at least one of membrane vesicles of Lactobacillus reuteri and membrane vesicles of Lactobacillus casei.

2. The growth inhibitor according to claim 1, wherein the periodontal disease bacteria is at least one selected from the group consisting of bacteria of the genus Fusobacterium, bacteria of the genus Porphyromonas, and bacteria of the genus Tannerella.

3. The growth inhibitor according to claim 2, wherein the Fusobacterium bacterium is Fusobacterium nucleatum.

4. The growth inhibitor according to claim 2, wherein the Porphyromonas bacterium is Porphyromonas gingivalis.

5. The growth inhibitor according to claim 2, wherein the Tannerella bacterium is Tannerella forsythensis.

6. The growth inhibitor according to claim 1 or 2, wherein the membrane vesicles are membrane vesicles isolated from a culture of Lactobacillus reuteri.

7. The growth inhibitor according to claim 1 or 2, wherein the membrane vesicles are membrane vesicles isolated from a culture of Lactobacillus casei.

8. A method for inhibiting the proliferation of periodontal disease bacteria, which comprises allowing the growth inhibitor for periodontal disease bacteria of claim 1 or 2 to coexist with periodontal disease bacteria.

9. The method for inhibiting proliferation according to claim 8 , wherein the coexistence is in vivo or in vitro.

10. The method for inhibiting proliferation according to claim 8 , wherein the coexistence is in the living body of a human or non-human animal.

11. A drug for periodontal disease bacteria-related diseases, comprising the growth inhibitor for periodontal disease bacteria according to claim 1 or 2 as an active ingredient.

12. The pharmaceutical agent for periodontal disease bacteria-associated diseases according to claim 11, further comprising an excipient.

13. The drug for periodontal disease bacteria-associated diseases according to claim 11, wherein the periodontal disease bacteria-associated disease is myocardial infarction, cerebral infarction, oral disease, or digestive system disease.

14. The drug for periodontal disease bacteria-associated diseases according to claim 13, wherein the oral disease is periodontal disease or dental caries.

15. The drug for periodontal disease bacteria-associated diseases according to claim 13, wherein the digestive system disease is a colon disease.

16. The drug for periodontal disease bacteria-associated diseases according to claim 15, wherein the colon disease is colitis or colon cancer.

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