Lacticaseibacillus paracasei ok strain and use thereof

Abstract

The present invention relates to a Lacticaseibacillus paracasei OK strain and a use thereof. The strain according to the present invention has an excellent ability to suppress harmful oral bacteria, and thus has excellent effects in alleviating, preventing, and treating oral diseases including bad breath, tooth decay, and periodontal disease.

Description

Lacticazeibacillus paracasei OK strain and its use

[0001] The present invention relates to a Lacticaseibacillus paracasei OK strain and its use, and more particularly, to a composition for improving, preventing, or treating oral diseases, comprising the Lacticaseibacillus paracasei OK strain.

[0002] Halitosis is defined as a foul odor coming from the mouth or nasal cavity, or an unpleasant odor from the mouth. It is known that the main cause of halitosis is volatile sulfur compounds (VSC), such as hydrogen sulfide (H2S) and methyl mercaptan (CH3SH), which are produced by the decomposition of sloughed epithelial cells and white blood cells by oral anaerobic bacteria.

[0003] Volatile sulfur compounds are reported to be produced from sulfur-containing amino acids such as cysteine ​​and methionine. Bacteria known to produce hydrogen sulfide from cysteine ​​include Peptostreptococcus, Eubacterium, Selenomonas, Centipeda, Bacteroides, Fusobacterium, and Prevotella. Bacteria known to produce methyl mercaptan from methionine include Fusobacterium, Bacteroides, Porphyromonas, and Eubacterium.

[0004] There have been numerous reports that zinc ions, chlorhexidine (CHX), cetylpyridinium chloride (CPC), and metal salts are effective in preventing bad breath by inhibiting the growth of bad breath-causing bacteria or reducing the production of volatile sulfur compounds. However, research on the inhibition of bad breath-causing bacteria using oral lactic acid bacteria is minimal. Therefore, there is a need for the development of a technology for preventing bad breath that is effective, safe, and practical using oral lactic acid bacteria.

[0005] Meanwhile, dental caries is a disease in which the enamel of teeth and dentine is eroded by acids produced by bacteria attached to the tooth surface. Various species of oral bacteria are known to cause caries, with Streptococcus spp. being the most representative, and various studies have been conducted on methods to inhibit their growth.

[0006] Antibiotics such as penicillin, erythromycin, and tetracycline have been used to prevent tooth decay. However, long-term use has limitations, such as the development of antibiotic resistance. Furthermore, research has been conducted on using natural substances to inhibit oral pathogens or the activity of glucosyltransferase (GTase), an enzyme that causes tooth decay. However, these methods either do not show lasting effects or suppress not only harmful oral bacteria but also normal oral microflora. Therefore, there is a pressing need to develop effective, safe, and practical tooth decay prevention technologies that can maintain oral health.

[0007] The purpose of the present invention is to provide a Lacticazeibacillus paracasei OK strain.

[0008] Another object of the present invention relates to the use of the Lacticazeibacillus paracasei OK strain, and more specifically, to provide a composition for improving, preventing or treating oral diseases, comprising the Lacticazeibacillus paracasei OK strain.

[0009] The present invention provides Lacticaseibacillusparacasei OK strain (KCCM13365P).

[0010] At this time, the strain has an effect of inhibiting the growth of oral harmful bacteria, and the oral harmful bacteria may be, for example, at least one selected from among Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, and Fusobacterium nucleatum.

[0011] Meanwhile, the present invention provides a pharmaceutical composition for preventing or treating oral diseases comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

[0012] In the pharmaceutical composition of the present invention, the oral disease may be, for example, any one selected from bad breath, caries, and periodontal disease. In this case, the bad breath may preferably be caused by Fusobacterium nucleatum, the caries may preferably be caused by Streptococcus mutans, and the periodontal disease may preferably be caused by any one selected from Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia.

[0013] At this time, the pharmaceutical composition may preferably contain any one selected from live cells, dead cells, or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

[0014] Meanwhile, the present invention provides a food composition for improving oral diseases comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

[0015] At this time, the food composition may preferably contain any one selected from live cells, dead cells, or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

[0016] Meanwhile, the present invention provides a composition for suppressing bad breath comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

[0017] At this time, the composition for suppressing bad breath may preferably contain any one selected from live cells, dead cells, or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

[0018] The strain according to the present invention has excellent oral harmful bacteria inhibition ability, and thus has excellent effects in improving, preventing, and treating oral diseases including bad breath, tooth decay, and periodontal disease.

[0019] Figure 1 is a graph showing the inhibitory ability of a candidate strain for improving oral health according to the present invention against Porphyromonas gingivalis, a harmful oral bacterium.

[0020] Figure 2 is a graph showing the inhibitory ability of a candidate strain for improving oral health according to the present invention against Prevotella intermedia, a harmful oral bacterium.

[0021] Figure 3 is a graph showing the inhibitory ability of the oral health improvement candidate strain according to the present invention against Streptococcus mutans, a harmful oral bacteria.

[0022] Figure 4 is a graph showing the inhibitory ability of the oral health improvement candidate strain according to the present invention against Fusobacterium nucleatum, a harmful oral bacterium.

[0023] Figure 5 is a genealogy result for the 16S rRNA base sequence of the strain Lacticaseibacillusparacasei OK (KCCM13365P) of the present invention.

[0024] Figure 6 shows the results of confirming the ability of Lacticaseibacillus paracasei OK of the present invention to inhibit the adhesion of Streptococcus mutans to KB cells, which are oral epithelial cells.

[0025] Figure 7 shows the results of confirming the ability of Lacticaseibacillus paracasei OK of the present invention to inhibit the attachment of Fusobacterium nucleatum to KB cells, which are oral epithelial cells.

[0026] Figure 8 shows the results of confirming the ability of Lacticaseibacillus paracasei OK of the present invention to inhibit the adhesion of Porphyromonas gingivalis to KB cells, which are oral epithelial cells.

[0027] Figure 9 shows the results of confirming the ability of Lacticaseibacillus paracasei OK of the present invention to inhibit the adhesion of Prevotella intermedia to KB cells, which are oral epithelial cells.

[0028] To avoid confusion due to overlapping content, the description of redundant content has been omitted below. In other words, the content of the invention is not limited to the content described below, and should be interpreted based on the overall scope of the invention.

[0029] The present inventors have discovered a lactic acid bacteria material derived from human feces and have confirmed its excellent ability to inhibit the growth of oral harmful bacteria and the attachment of oral harmful bacteria to oral epithelial cells, thereby providing the material.

[0030] Accordingly, the present invention provides Lacticaseibacillusparacasei OK strain (KCCM13365P).

[0031] At this time, the strain has an effect of inhibiting the growth of oral harmful bacteria, and the oral harmful bacteria may be, for example, at least one selected from among Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, and Fusobacterium nucleatum.

[0032] In one embodiment of the present invention, it was confirmed that the strain according to the present invention has excellent efficacy in inhibiting the growth of the oral harmful bacteria exemplified above and in inhibiting the attachment of these oral harmful bacteria to oral epithelial cells.

[0033] Accordingly, in the present invention, the strain was named 'Lacticaseibacillus paracasei OK' and deposited at the Korea Center for Microorganism Conservation (KCCM), and was assigned the accession number KCCM13365P on June 30, 2023.

[0034]

[0035] The present invention provides a pharmaceutical composition for preventing or treating oral diseases comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

[0036] In the pharmaceutical composition of the present invention, the oral disease may be, for example, any one selected from bad breath, caries, and periodontal disease. In this case, the bad breath may preferably be caused by Fusobacterium nucleatum, the caries may preferably be caused by Streptococcus mutans, and the periodontal disease may preferably be caused by any one selected from Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia.

[0037] In one embodiment of the present invention, it was confirmed that the strain according to the present invention has excellent efficacy in inhibiting the growth of the oral harmful bacteria exemplified above and in inhibiting the attachment of these oral harmful bacteria to oral epithelial cells.

[0038] The above Fusobacterium nucleatum is a major anaerobic bacterium that produces hydrogen sulfide or methyl mercaptan. It is a Gram-negative bacterium that normally exists in the oral cavity, causes periodontitis, and produces volatile sulfur compounds that cause bad breath.

[0039] Streptococcus mutans is a mesophilic bacterium that grows at temperatures between 18 and 40°C. It ferments sugars in the mouth, producing acids. It inhabits not only the oral cavity but also the pharynx and intestines. Its ability to adhere to enamel and produce acidic byproducts plays a key role in caries formation. Streptococcus mutans obtains energy by breaking down sugars, which produces lactic acid, creating an acidic environment that removes salts from the tooth surface. This action dissolves calcium and contributes to tooth decay.

[0040] Porphyromonas gingivalis is a Gram-negative, obligate anaerobic bacterium that is part of the human oral microbiota. It inhabits periodontal pockets and the grooves between the teeth and gums. While it is primarily found in the oral cavity and is associated with periodontal disease, it is also found in the upper gastrointestinal tract, respiratory tract, and large intestine, and has been linked to bacterial vaginosis in women. In humans, Porphyromonas gingivalis is most commonly associated with the subgingival region of the oral cavity, where it forms part of the biofilm that forms dental plaque. This causes painful inflammation in periodontal disease. This oral disease is estimated to affect 10-15% of adults worldwide.

[0041] Prevotella intermedia is a Gram-negative, obligate anaerobic pathogenic bacterium implicated in periodontal infections, including gingivitis and periodontitis, and is frequently found in acute necrotizing ulcerative gingivitis. Known as a major causative agent of periodontal disease, Prevotella intermedia thrives by producing various enzymes, including proteolytic enzymes, to decompose surrounding organic matter. When these organic substances are collagen, which forms the gums, periodontal disease develops.

[0042] The above pharmaceutical composition may preferably contain any one selected from live cells, dead cells or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

[0043] The term “prevention” used in the present invention means any act of suppressing or delaying the onset of an oral disease by administering a pharmaceutical composition according to the present invention.

[0044] The term "treatment" used in the present invention means any action in which symptoms of an oral disease are improved or beneficially changed by administration of a pharmaceutical composition according to the present invention.

[0045] The strain of the present invention may contain one or more effective ingredients exhibiting the same or similar function as the above components.

[0046] The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier in addition to the strain according to the present invention.

[0047] The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the relevant technical field can be used. Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. These may be used alone or in combination of two or more.

[0048] In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as antioxidants, excipients, diluents, buffers or bacteriostatic agents, if necessary, and may be used by additionally adding surfactants, binders, fillers, bulking agents, wetting agents, disintegrants, dispersants or lubricants.

[0049] In the pharmaceutical composition of the present invention, the strain according to the present invention may be included in an amount of 0.00001 wt% to 99.99 wt% based on the total weight of the pharmaceutical composition, preferably 0.1 wt% to 90 wt%, more preferably 0.1 wt% to 70 wt%, and even more preferably 0.1 wt% to 50 wt%, but is not limited thereto and may vary depending on the condition of the administration subject, the type and degree of specific symptoms, etc. If necessary, it may also be included in the total content of the pharmaceutical composition.

[0050] That is, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, body weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment. For example, the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1,000 mg / kg, preferably 0.1 to 100 mg / kg, and may be administered once or several times a day in divided doses.

[0051] The pharmaceutical composition of the present invention may be administered once daily or divided into several doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, the pharmaceutical composition may be administered in an amount that achieves maximum efficacy with minimal side effects, as can be readily determined by those skilled in the art.

[0052] The pharmaceutical composition of the present invention can be additionally used in combination with various methods such as hormone therapy and drug therapy to prevent or treat the above-mentioned diseases.

[0053] The term "administration" used in the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and any route and method of administration may be followed without particular limitation as long as the pharmaceutical composition can reach the desired site.

[0054] The pharmaceutical composition described above may be administered orally or parenterally, and may be formulated into a variety of suitable dosage forms for oral or parenteral administration. Preferably, the pharmaceutical composition may be formulated into one or more dosage forms selected from, but not limited to, toothpaste, mouthwash, oral spray, and oral ointment.

[0055] Non-limiting examples of oral administration preparations using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs.

[0056] In order to formulate the pharmaceutical composition of the present invention for oral administration, binders such as sorbitol, mannitol, starch, amylopectin, cellulose, lactose, saccharose or gelatin; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; and fragrances, syrups, sweeteners, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be additionally used.

[0057] As a parenteral administration method of the pharmaceutical composition of the present invention, intramuscular administration, transdermal administration, or subcutaneous administration to the diseased area may be used, and a method of applying or spraying the composition to the diseased area may also be used, but is not limited thereto.

[0058] Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injections, ointments, powders for application, oils, aerosols for spraying, creams, etc.

[0059] In order to formulate the pharmaceutical composition of the present invention for parenteral administration, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, etc. can be used. As the non-aqueous solvent and suspension, vegetable oils such as olive oil, propylene glycol, polyethylene glycol, injectable esters such as ethyl oleate, etc. can be used.

[0060] When the pharmaceutical composition of the present invention is formulated as an injection, the pharmaceutical composition of the present invention may be prepared as a solution or suspension by mixing it in water with a stabilizer or buffer, and this may be formulated for unit dose in an ampoule or vial.

[0061] When the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like can be mixed with additives to disperse the water-dispersed concentrate or wet powder.

[0062] When the pharmaceutical composition of the present invention is formulated into an ointment, oil, cream, powder for application, external skin preparation, etc., it can be formulated using animal oil, vegetable oil, wax, paraffin, polyethylene glycol, silicone, bentonite, silica, talc, starch, tragacanth, cellulose derivative, zinc oxide, etc. as a carrier.

[0063]

[0064] Meanwhile, the present invention provides a food composition for improving oral diseases comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

[0065] At this time, the food composition may preferably contain any one selected from live cells, dead cells, or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

[0066] In one embodiment of the present invention, it was confirmed that the strain according to the present invention has excellent efficacy in inhibiting the growth of the oral harmful bacteria exemplified above and in inhibiting the attachment of these oral harmful bacteria to oral epithelial cells.

[0067] In the food composition of the present invention, the content of the strain is not particularly limited and may vary depending on the condition of the subject of administration, the type and extent of the specific disease, etc. If necessary, it may also be included as the total content of the food.

[0068] The food composition of the present invention may be, for example, any one selected from among noodles, gum, dairy products, ice cream, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, alcoholic beverages, alcohol, vitamin complexes, and other health supplements. Preferably, the food composition may have at least one formulation selected from among gum, candy, and tablets, but is not necessarily limited thereto.

[0069] When the food composition of the present invention is used in the form of a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

[0070] In addition, the "food composition" of the present invention encompasses the meaning of including a health functional food, and the "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and "functionality" refers to consuming for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0071]

[0072] Meanwhile, the present invention provides a composition for suppressing bad breath comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

[0073] At this time, the composition for suppressing bad breath may preferably contain any one selected from live cells, dead cells, or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

[0074] In the present invention, the composition for suppressing bad breath may preferably be formulated as a toothpaste, mouthwash or mouthwash, but there is no limitation on the formulation as long as it is a desirable formulation depending on the method of use.

[0075] For example, when the oral composition is formulated as a toothpaste, it may contain an abrasive, a humectant, a foaming agent, a thickening agent, a preservative, a sweetener, a flavoring agent, and other additives.

[0076] The above abrasive is a component for removing plaque, tartar, etc. attached to teeth, and colloidal silicon dioxide, calcium dihydrogen phosphate, calcium carbonate, aluminum hydroxide, precipitated silica, hydrous silicic acid, etc. can be used.

[0077] The above humectant is a component that suppresses evaporation of water in the toothpaste composition and prevents the composition from solidifying when exposed to air, and one or two or more types of polyhydric alcohols such as glycerin, sorbitol solution, non-crystalline sorbitol solution, polyethylene glycol, and propylene glycol can be selected and used.

[0078] The foaming agent is a component that emulsifies water-soluble components and oil-soluble components in a toothpaste composition and has a cleansing effect in the oral cavity, and may be used alone or in combination of two or more anionic and nonionic surfactants such as sodium lauryl sulfate, sodium N-lauroyl sarcosylate, N-long-chain acylglutamate, sucrose fatty acid ester, polyoxyethylene hydrogenated castor oil, sorbitan fatty acid ester, and polyoxyethylene polyoxypropylene copolymer.

[0079] The above thickener is a component that binds insoluble inorganic powder components and liquid components in the toothpaste composition to prevent phase separation over time and provides viscosity to the composition, and may be sodium carboxymethyl cellulose, carrageenan, xanthan gum, polyvinyl pyrrolidone, sodium alginate, or other gums.

[0080] The above preservative is an ingredient for extending the shelf life of the toothpaste composition, and may include parahydroxybenzoic acid ester, benzoic acid, sodium benzoate, etc.

[0081] The above sweeteners and flavoring agents are ingredients that improve the taste when brushing teeth and provide a refreshing aftertaste after brushing teeth. As sweeteners, sodium saccharin, aspartame, stevioside, licorice acid, etc. can be used, and as flavoring agents, peppermint, spearmint oil, menthol, carvone, anethole, eugenol, etc. can be mixed and used.

[0082] Other additives include whitening agents such as titanium dioxide and food colorings such as Blue No. 1, which can be used as coloring agents.

[0083] For example, when the oral composition is formulated as a mouthwash, it may contain a humectant, a sweetener, a foaming agent, a solubilizer, an astringent, a preservative, a coloring agent, a flavoring agent, a solvent, etc.

[0084] The ingredients of the above oral cleanser may be the same as those of the respective ingredients in the toothpaste composition, except that zinc chloride may be used as an astringent and ethanol and purified water may be used as solvents.

[0085] For example, when the oral composition is formulated as a mouthwash, it may contain a foaming agent, a desiccant, a foaming agent, a binder, a lubricant, a fragrance, a solvent, etc.

[0086] The above foaming agent is an ingredient for oral cleaning, and at least one selected from the group consisting of sodium bicarbonate, ammonium bicarbonate, potassium bicarbonate, and iron carbonate can be used.

[0087] The above-mentioned desiccant is an ingredient for formulating an oral cleanser into an anhydrous tablet, and sodium carbonate may be used.

[0088] A water-soluble polymer can be used as the above binder, and preferably carboxycellulose or polyvinyl pyrrolidone can be used.

[0089] As the above-mentioned lubricant, one selected from the group consisting of magnesium stearate, stearic acid, stearyl alcohol, sucrose fatty acid ester, talc, light anhydrous silicic acid, and sodium benzoate can be used, and sodium benzoate can be preferably used.

[0090]

[0091] Hereinafter, the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical concepts equivalent thereto.

[0092] [Example 1: Selection and identification of Lacticaseibacillus paracasei OK strain]

[0093] 1. Selection of Lacticaseibacillus paracasei OK strain

[0094] In this example, we aimed to select a novel strain with excellent oral health improvement, prevention, or treatment effects.

[0095] Specifically, among human fecal-derived strains, six candidate strains for oral health improvement were selected as lactic acid bacteria that inhibit the growth of four representative oral pathogens (Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Fusobacterium nucleatum). The six selected lactic acid bacteria were named 'LP OK', 'HMD', 'GUT10', 'HMU', 'CD4', and 'LF OK', respectively, using the last alphabet and number of the strain name.

[0096] The oral health improvement candidate strains and oral harmful bacteria used in this experiment are specifically shown in Table 1 below.

[0097] Antibacterial active bacteria Candidate bacteriaHarmful bacteriaLacticaseibacillusparacaseiOK (LP OK)Lactobacillus gasseriHHuMIN D (HMD)Lactobacillus helveticusGUT10 (GUT10)Lactobacillus salivariusHHuMin-U (HMU)Lactobacillus salivariusCD4 (CD4)Lactobacillus fermentumOK (LF OK)Porphyromonas gingivalisKCTC 5352Prevotella intermediaKCTC 15693Fusobacteriumnucleatumsubsp.nucleatumKCTC 2640Streptococcus mutansKCTC 3065

[0098]

[0099] Meanwhile, to evaluate antibacterial activity, each oral health improvement candidate strain selected above was inoculated into an optimal medium and cultured at 37°C until reaching a stationary phase. The culture supernatant that reached the stationary phase was collected, and solids and bacterial cells were removed using a 0.2 μm filter, and then used as a sample to confirm antibacterial activity.

[0100] 3% (v / v) or 5% (v / v) of the above candidate strain culture supernatant was dispensed into a 96-well plate (SPL Life Sciences Co., Ltd. Pocheon, Korea), and MRS medium adjusted to pH 5.5 was used as the control group.

[0101] The culture medium inoculated with 1% (w / v) of activated harmful strains (3% (w / v) for P. gingivalis) was dispensed into a 96-well plate containing the above candidate strain culture supernatant or control, to a final volume of 200 μl, and cultured anaerobically at 37°C. Each well was measured at 600 nm using a microplate reader (Epoch2, BioTek, USA) every hour.

[0102] As a result of the experiment, the strain 'Lacticaseibacillus paracasei OK (hereinafter referred to as LP OK)' was finally selected as a candidate strain that showed growth inhibition activity on all harmful bacteria compared to the control group, and particularly showed excellent inhibition activity on two types of harmful bacteria (Porphyromonas gingivalis and Prevotella intermedia) compared to other candidate strains for improving oral health, and the growth inhibition rate (antibacterial activity) of the strain against harmful bacteria is shown in Table 2 below (Figs. 1 to 4).

[0103] Figures 1 to 4 are graphs showing the inhibitory ability of the oral health improvement candidate strain according to the present invention against oral harmful bacteria such as Porphyromonas gingivalis (Figure 1), Prevotella intermedia (Figure 2), Streptococcus mutans (Figure 3), and Fusobacterium nucleatum (Figure 4).

[0104] StrainsInhibition level(%)Porphyromonas gingivalisKCTC 535234±7Prevotella intermediaKCTC 1569326±12Fusobacterium nucleatumKCTC 264021±0Streptococcus mutansKCTC 306528±9

[0105]

[0106] As shown in Table 2, the LP OK strain showed the highest inhibition rate of 34±7% against Porphyromonas gingivalis, 26±12% against Prevotella intermedia, 21% against Fusobacterium nucleatum, and 28±9% against Streptococcus mutans, confirming that it has excellent antibacterial activity against oral pathogens.

[0107]

[0108] 2. Identification of Lacticaseibacillus paracasei OK strain

[0109] In order to genetically identify the 'LP OK' strain finally selected above, 16S rRNA sequencing was performed, and then Molecular Phylogenetic Status was performed in parallel based on the base sequence (Fig. 5). Fig. 5 shows the genealogy results for the 16S rRNA base sequence of the strain Lacticaseibacillus paracasei OK (KCCM13365P) of the present invention.

[0110] The 16S rRNA base sequence of the 'LP OK' strain was multiple aligned with the 16S rRNA sequence of Lacticaseibacillus sp. listed in GeneBank to obtain a similarity matrix and create a genealogy. As a result, the range of 16S rRNA homology was found to be 100% in all cases.

[0111] That is, the 'LP OK' strain showed a high homology of 100% with Lacticaseibacillusparacasei and was finally named 'Lacticaseibacillusparacasei OK'.

[0112] Accordingly, the Lacticaseibacillus paracasei OK strain of the present invention was deposited at the Korea Center for Microorganism Conservation (KCCM, 3F, Yulim Building, 45 Hongje-nae 2-ga-gil, Seodaemun-gu, Seoul (Hongje-dong)) on June 30, 2023, and was assigned the accession number KCCM13365P.

[0113]

[0114] [Example 2: Evaluation of the ability of Lacticaseibacillus paracasei OK to inhibit oral pathogenic bacteria adhesion to oral epithelial cells]

[0115] To evaluate the inhibition ability of Lacticaseibacillus paracasei OK strain on oral pathogenic bacteria cell adhesion, KB cells (human mouth epithelial cells; Korean Cell Line Bank 10017) were used.

[0116] Specifically, KB cells were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640, Welgene, Daegu, Korea) medium containing 300 mg / L L-glutamine, 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL penicillin at 5% CO2 and 37°C.

[0117] The LP OK strain and oral pathogens were obtained by centrifuging the culture broth of the strain at 4,000 rpm for 10 minutes, and the pellet was incubated at 1.0×10 using RPMI 1640. 8 It was used after diluting to a concentration of CFU / ㎖.

[0118] KB cells were seeded at 3.0 × 10 in a 24-well plate (SPL Life Sciences Co., Ltd. Pocheon, Korea). 5 After adding cells / well, it was cultured under conditions of 5% CO2 and 37℃ until a cell monolayer was formed. Afterwards, the supernatant of each well was removed and the cell monolayer was inoculated with LP OK strain and oral pathogen dilution solution (1.0×10 8 2 ml of a mixture containing equal amounts of 10 CFU / ml was added to each plate. Afterwards, the plate was incubated at 5% CO2 and 37°C for 1 hour to allow attachment of oral pathogenic bacteria.

[0119] The supernatant from each well was removed, washed twice with PBS, and cells and attached bacteria were collected by treating with 200 μl trypsin-EDTA (Welgene, Daegu, Korea) for 5 min. The number of bacteria attached to each KB cell was counted by real-time PCR.

[0120] Specifically, the MagMax Microbiome Ultra Nucleic Acid Isolation Kit (Thermo Fisher Scientific, St. Peters, MO, USA) was used to extract the genomic DNA of attached bacteria from the harvested pellet. To quantify the number of bacteria, 1.0 × 10 8 The genomic DNA of the bacteria was extracted from CFU and diluted using the serial dilution method to create a standard curve.

[0121] Meanwhile, the positive control group was treated with oral pathogen dilution (1.0×10) on KB cell monolayers. 8 The test was conducted in the same manner as above, except that 2 ml of (CFU / ml) was added. The adhesion inhibition effect was confirmed by the ratio of the experimental group to the positive control group, and the results are shown in Table 3 below.

[0122] StrainsExperimental GroupsNumber of bacteria (Total cells)Inhibitory rate (%)S. mutansS. mutansKCTC 3065799563 ± 9530730.99 ± 2.74S.mutansKCTC 3065 + LP OK551793 ± 21895F. nucleatumF.nucleatumKCTC 2640619282 ± 14776216.77 ± 16.49F.nucleatumKCTC 2640 + LP OK515447 ± 102127P. gingivalisP.gingivalisKCTC 53521035773 ± 14443323.16 ± 4.25P.gingivalisKCTC 5352 + LP OK795904 ± 43994P. intermediaP.intermediaKCTC 156933601149 ± 26555088.25 ± 0.04P.intermediaKCTC 15693 + LP OK423306 ± 1446 ***

[0123]

[0124] As can be seen in Table 3, the LP OK strain showed the highest inhibitory effect on oral pathogenic bacteria adhesion to oral epithelial cells against Prevotella intermedia KCTC15693 at 88.25±0.04%. In addition, it showed the inhibitory effect on oral pathogenic bacteria adhesion in the following order: 30.99±2.74% against Streptococcus mutans KCTC3065, 23.16±4.25% against Porphyromonas gingivalis KCTC5352, and 16.77±16.49% against Fusobacterium nucleatum KCTC2640 (Figs. 6 to 9).

[0125] Figures 6 to 9 show the results of confirming the adhesion inhibition ability of Streptococcus mutans (Figure 6), Fusobacterium nucleatum (Figure 7), Porphyromonas gingivalis (Figure 8), and Prevotella intermedia (Figure 9) by Lacticaseibacillus paracasei OK of the present invention in KB cells, which are oral epithelial cells.

[0126]

Claims

1. Lacticaseibacillus paracasei OK strain (KCCM13365P).

2. In paragraph 1, The above strains are, A strain having the effect of inhibiting the growth of oral pathogenic bacteria.

3. In paragraph 2, The above oral harmful bacteria are, A strain selected from at least one of Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, and Fusobacterium nucleatum.

4. A pharmaceutical composition for preventing or treating oral diseases containing Lacticaseibacillus paracasei OK strain (KCCM13365P).

5. In paragraph 4, The above pharmaceutical composition, A pharmaceutical composition containing any one selected from live cells, dead cells or a culture solution thereof of Lacticaseibacillus paracasei OK strain.

6. In paragraph 4, The above oral diseases are, A pharmaceutical composition, wherein any one of the following is selected from the group consisting of bad breath, dental caries and periodontal disease.

7. In paragraph 5, The above bad breath is, A pharmaceutical composition caused by Fusobacterium nucleatum.

8. In paragraph 5, The above caries, A pharmaceutical composition caused by Streptococcus mutans.

9. In paragraph 5, The above periodontal disease is, A pharmaceutical composition caused by any one selected from Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia.

10. Food composition for improving oral disease containing Lacticaseibacillus paracasei OK strain (KCCM13365P).

11. In paragraph 10, The above food composition, A food composition containing any one selected from live cells, dead cells or a culture solution thereof of Lacticaseibacillus paracasei OK strain.

12. A composition for suppressing bad breath, comprising Lacticaseibacillus paracasei OK strain (KCCM13365P).

13. In paragraph 12, The above composition for suppressing bad breath is, A composition for suppressing bad breath, comprising any one selected from live cells, dead cells or a culture solution thereof of the Lacticaseibacillus paracasei OK strain.

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