KR20200070081A - Lactobacillus salivarius having anticariogenic activities and composition comprising the same - Google Patents

Abstract

The present invention relates to an antibacterial composition for inhibiting Streptococcus mutans or Porphyromonas gingivalis, which includes Lactobacillus salivarius and Lactobacillus reuteri derived from the human body and various fermented foods; and a pharmaceutical composition, a health functional food composition and a quasi-drug composition for preventing, treating and ameliorating dental carries or periodontal disease. According to the present invention, Lactobacillus salivarius MG242 strains (accession number: KCTC 18554P) or Lactobacillus salivarius MG242 strains (accession number: KCTC 13729BP) have excellent inhibitory activity of Streptococcus mutans or Porphyromonas gingivalis, and are suitable for probiotics due to excellent bile acid resistance and self-agglutinating capacity, and thus can be variously utilized as a medicine, food, quasi-drugs, etc., for preventing, treating or ameliorating tooth decay.

Description

Composition comprising Lactobacillus salivarius having anticariogenic activities and composition comprising the same}

The present invention relates to an antibacterial composition for inhibiting dental caries or periodontitis, including a human body and various fermented food-derived Lactobacillus salivarius, a pharmaceutical composition for preventing, treating, and improving dental caries or periodontal disease, a health functional food composition, and a quasi-drug composition .

Lactobacillus has a special physiological activity, and is generally treated as a generally regarded as safe bacteria (GRAS). Lactic acid bacteria are not only used in the production of various fermented foods, but are also widely used in dairy products and fermented fruit products with functional and probiotic properties. Recently, as consumers' demand for natural supplements to replace chemical supplements has increased, it has emerged as an alternative. Probiotics are living microorganisms that have a very beneficial effect on the health of the host animal, improving the balance of intestinal microorganisms and promoting absorption of nutrients. In addition, probiotics have antimicrobial activity of pathogenic microorganisms in the intestinal environment. Probiotics include a variety of microorganisms, but Lactobacillus In and Bifidobacterium (Bifidobacterium) The genus has the largest distribution. In particular, the genus Lactobacillus is generally used in the fermentation process of dairy products, meat, vegetables and cereal products.

On the other hand, it is known that more than 400 types of bacteria are propagated in the human oral cavity, the number of bacteria reaches 10 billion, and bacteria in the level of 10 ⁸ to 10 ⁹ CFU/ml exist in saliva. The teeth have a number of microscopic holes, so bacteria that cause tooth decay go into the perforated holes and make the teeth sick, which is tooth decay. These cavities are also known as dental caries as one of the most common diseases seen in teeth. Tooth decay is an infectious bacterial disease in which food in the oral cavity is decayed by acid-resistant streptococcus and lactic acid bacteria, and the components of teeth are destroyed by acid. It is a multifactorial disease caused by interaction of microorganisms and food in plaque. Among microorganisms, Streptococcus mutans is the main causative agent of calculus, and these microorganisms are insoluble glucans from sucrose in food by glucosyltransferase (GTase), which is produced after attaching to the coating of the tooth surface. Synthesizes. Synthetic glucan increases binding between microorganisms that proliferate on the tooth surface, and S. mutans attached to the tooth surface are known to induce tooth decay by decalcification of tooth enamel by producing organic acids such as lactic acid during carbohydrate metabolism. have. In Korea, the morbidity rate of permanent teeth reaches about 80%, and one person has been reported to have on average 2-3 cavities.

Conventional methods for preventing dental caries or periodontal disease include reducing sugar intake through brushing and proper diet, or using xylitol to prevent the production of acid by tooth decay or periodontal disease-causing bacteria. It is a situation that does not have a fundamental effect of preventing tooth decay through the death of tooth decay-causing bacteria or periodontal disease-causing bacteria.

Therefore, there is a need for a new method of preventing tooth decay, which is harmless to the human body and easy to ingest using lactic acid bacteria derived from the human body or food that is harmless to the human body, and directly suppresses the growth of tooth decay-causing bacteria or periodontal disease-causing bacteria.

KR 10-2017-0029106 A KR 10-2016-0144248 A KR 10-2018-0093356 A

Growth inhibition effect of lactic acid bacteria and probiotics efficacy of yogurt against dental caries-induced Streptococcus mutans and Streptococcus sobrinus (Kor J Oral Maxillofac Pathol 2009;33(3):167-174)

Accordingly, the present inventors were studying Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP) ) Has excellent antimicrobial effect against cavities-causing bacteria, Streptococcus mutans and periodontitis, Porolimonas gingivalis, and has excellent bile resistance and self-cohesiveness, confirming its usefulness as a probiotic and completing the present invention.

Therefore, an object of the present invention is to inhibit the cavities or periodontitis bacteria including Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), a culture solution thereof, or a cell-free supernatant thereof. It provides an antibacterial composition and a composition for preventing, treating, and improving dental caries or periodontal disease using the same.

In order to achieve the above object, the present invention comprises Lactobacillus salivarius MG242 strain (accession number: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (accession number: KCTC 13729BP), a culture thereof or a cell-free supernatant thereof And it provides an antibacterial composition for inhibiting periodontal infection.

In addition, the present invention includes a Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or a Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), a culture medium thereof, or a cell-free supernatant thereof, to prevent dental caries and periodontal disease. Provided is a pharmaceutical composition for treatment.

In addition, the present invention includes a Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or a Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), a culture medium thereof, or a cell-free supernatant thereof, to prevent dental caries and periodontal disease. Provides a health functional food composition for improvement.

In addition, the present invention includes a Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or a Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), a culture medium thereof, or a cell-free supernatant thereof, to prevent dental caries and periodontal disease. Provided is a quasi-drug composition for improvement.

The Lactobacillus salivarius MG242 strain of the present invention (Accession No.: KCTC 18554P) or the Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP) has excellent inhibitory activity against Streptococcus mutans and Porolimonas gingivalis, and bile bile. Since it has excellent sex and self-cohesiveness and is suitable for probiotics, it can be used in various ways as medicine, food, quasi-drugs, etc. for preventing, treating or improving dental caries or periodontal disease.

Figure 1 is Lactobacillus salivarius MG242 and Lactobacillus saluterius MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, Lactobacillus salivarius MG5183 strain culture (Cell mixture), cell-free supernatant (CFS mixture) S. mutan It is the figure which showed the result which confirmed the inhibitory activity.
Figure 2 is a diagram showing the results confirming the self-cohesive ability of Lactobacillus salivarius MG242 and Lactobacillus saluterius MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, Lactobacillus salivarius MG5183 strain.
FIG. 3 shows S. mutans of mixed CFS, cell mixture culture and CFS and cell mixture culture of Lactobacillus salivarius MG242, Lactobacillus salivarius MG505, Lactobacillus salivarius MG4227, Lactobacillus salivarius MG4527, Lactobacillus salivarius MG5183 strain It is a diagram showing the inhibitory effect.

The present invention is a Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), an antimicrobial composition for inhibiting dental caries and periodontitis, comprising a culture solution thereof or a cell-free supernatant thereof Gives

The Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or the Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP) of the present invention has excellent inhibitory activity against Streptococcus mutans and/or Porolimonas gincilvalins, It has excellent bile resistance and self-cohesiveness.

Hereinafter, the present invention will be described in more detail.

In the present invention, the term "lactobacillus" is a bacterium that generates a large amount of lactic acid by obtaining energy by fermenting sugars widely distributed in nature, and morphologically refers to polymorphism as Gram-positive apoptotic bacilli. The Lactobacillus salivarius MG4293 and Lactobacillus salivarius MG4527 strains, each of which has excellent Streptococcus mutans and/or Porolimonas anti-Givalins inhibitory activity, excellent acid resistance, bile resistance, and self-cohesiveness, respectively, Korean Biotechnology Deposited with the researcher, the accession numbers KCTC 13727BP and KCTC 13728BP were assigned.

In addition, the composition of the present invention is Streptococcus mutans and / or Poroliromonas gibbalis inhibitory activity, acid resistance, bile resistance and other strains having self-cohesive ability, Lactobacillus salivarius MG242 and Lactobacillus salivarius MG5183 It may include one or more strains selected from the group consisting of, a culture medium thereof or a cell-free supernatant thereof, and Lactobacillus salivarius MG242 and Lactobacillus salivarius MG5183 were deposited with the Korea Research Institute of Bioscience and Biotechnology, respectively, and were granted KCTC 18554P and KCTC 13729BP. .

In the present invention, the Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527 and Lactobacillus luteri MG505 strains not only exhibit excellent antimicrobial activity against both the representative caries bacteria, Streptococcus mutans and Porolimonas gincivalines. , Excellent acid resistance and bile resistance. More specifically, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527 and Lactobacillus luteri MG505 strains can maintain a very high survival rate in strongly acidic conditions pH 2 to 4, more preferably in a gastric acid environment pH2.

In addition, Lactobacillus salivarius MG242 and Lactobacillus salvarius MG505, Lactobacillus salivarius MG4527, Lactobacillus salivarius MG5183 strains have bile salt resistance to bile salts similar to the intestinal environment, 0.1 to 0.1 1.5% bile salts, 0.3 to 1% of bile salts, preferably 1% of bile salts may exhibit excellent resistance.

The Lactobacillus salivarius MG242 and Lactobacillus salvarius MG505, Lactobacillus salivarius MG4527, and Lactobacillus salivarius MG5183 strains of the present invention exhibit excellent autoaggregation ability, and probiotics by intestinal spasticity Removal can be prevented. In particular, Lactobacillus salivarius MG4293 and Lactobacillus salivarius MG4527 strains can exhibit excellent self-aggregation ability.

In addition, the present invention prevents or treats dental caries or periodontal disease, including Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), a culture solution thereof, or a cell-free supernatant thereof. It provides a pharmaceutical composition, health functional food composition, food composition, quasi-drug composition.

The pharmaceutical composition, health functional food composition, food composition, quasi-drug composition is Lactobacillus salivarius MG242 strain (accession number: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (accession number: KCTC 13729BP), a culture solution thereof, or It may contain a cell-free supernatant, Lactobacillus salivarius MG4293 strain (Accession No.: KCTC 13727BP), Lactobacillus salivarius MG4527 strain (Accession No.: KCTC 13728BP) and Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) One or more strains selected from the group consisting of, may further include a culture solution or a cell-free supernatant thereof.

In the case of a pharmaceutical composition, the strains are preferably included in an amount of 0.01 to 95 parts by weight, more preferably 1 to 80 parts by weight based on 100 parts by weight of the pharmaceutical composition. If the content is less than 0.01 part by weight, the taking efficiency may be reduced, and when it exceeds 95 parts by weight, there may be difficulty in formulation.

Lactobacillus salivarius MG242, Lactobacillus saluterius MG505, Lactobacillus salivarius MG4527, and Lactobacillus salivarius MG5183 strains according to the present invention have excellent antimicrobial activity against caries-causing bacteria and periodontitis , Since it has excellent bile resistance and self-cohesiveness, it can be used directly as a composition itself having a prevention, improvement, or treatment effect of various diseases caused by cavities and periodontitis, and can also be used as an adjuvant.

The pharmaceutical composition according to the present invention alone or contains a pharmaceutically effective amount of Lactobacillus salivarius MG242, Lactobacillus saluterius MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, or Lactobacillus salivarius MG5183 The above may include a pharmaceutically acceptable carrier, excipient or diluent.

Both dental caries and periodontal disease are bacterial infectious diseases. Dental caries are S. mutans , S. sanguinis , S. sobrinus , S. sobrinus , and S. sobrinus . by bacteria such as S. ratti), Streptococcus Cri Shetty (S. criceti), Streptococcus not Gino Versus (S. anginosus) or Streptococcus highly Needle (S. gordonii), periodontal disease is the bacteria that causes periodontal disease Porphyromonas gingivalis ), Prevotella intermedia , Actinobacillus actinomycetemcomitans , Treponema denticola and Fusobacterium nuclitum nucleatum ). Since the pharmaceutical composition according to the present invention can effectively suppress representative dental caries and periodontal disease-causing bacteria, it is very effective in preventing or treating dental caries or periodontal disease.

Therefore, in the above, "a pharmaceutically effective amount" means, for example, prevention of dental caries or periodontal disease, which may be caused by inhibiting the growth of cavities-causing and periodontitis-inducing bacteria Streptococcus mutans and Porolimonas gingivalis, It is an amount sufficient to exert an improvement or treatment effect. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness or similar reaction when administered to humans.

In addition, the pharmaceutical composition according to the present invention is formulated in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injection solution according to a conventional method. Can be. Suitable formulations known in the art are preferred to use those disclosed in Remington's Pharmaceutical Science, recently Mack Publishing Company, Easton PA. Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like. When formulating the pharmaceutical composition, it is prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the composition, such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition of the present invention is an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, i.e., the symptoms of the disease or disorder being treated. It can be administered in a therapeutically effective amount, an amount that induces relief. It is apparent to those skilled in the art that the therapeutically effective dose and the number of administrations to the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, the type of disease, the severity of the disease, the content of active ingredients and other ingredients in the composition, the type of formulation, and the patient's age, weight, general health It can be adjusted according to various factors including condition, sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used simultaneously. For a desired effect, the pharmaceutical composition of the present invention can be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, or once a day, several times It can also be administered in divided doses.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. Any mode of administration can be expected, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrathecal dura or cerebral vascular injection.

The pharmaceutical composition according to the present invention may be used alone, but other suitable treatment methods (e.g., surgery, radiation treatment, hormone treatment, chemistry) that generally enhance the treatment of dental caries or periodontal disease according to the choice of a person skilled in the art Treatment and biological response modifiers, etc.).

In the present invention, the term "prevention" may mean all actions to suppress or delay the onset of dental caries or periodontal disease by administering a composition for preventing or treating dental caries or periodontal disease according to the present invention to an individual.

In the present invention, the term, "treatment", by administering the composition according to the present invention to a subject suspected of dental caries or periodontal disease may mean all actions that improve or benefit the symptoms of dental caries or periodontal disease-related diseases.

In the present invention, the term, "improvement" may mean any action that at least reduces the severity of symptoms associated with the condition being treated, for example.

In the present invention, the term, "individual", can mean all animals, including humans who have had or are likely to develop dental caries or periodontal disease.

In the food composition according to the present invention, the type of the food is not particularly limited, and may include all foods in a conventional sense. Non-limiting examples of foods to which the above substances can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, tea , Drinks, alcoholic beverages and vitamin complexes. When the composition is used as a food additive, the composition may be added as it is or used with other foods or food ingredients, and may be suitably used according to conventional methods.

The term food in the present specification means a natural product or a processed product containing one or more nutrients, and preferably means a state that can be directly eaten through a certain processing process, and is a common meaning, The food composition is intended to include all food, food additives, health functional food and beverages.

Foods to which the composition of the present invention can be added include, for example, various foods, beverages, chewing gum, candy, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food products include special nutritional foods (e.g., prepared dairy products, English, baby foods, etc.), processed meat products, fish meat products, tofus, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( Yes, soy sauce, miso, red pepper paste, mixed sauce, etc., sauces, confectionery (e.g., snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), drinks ( Examples include, but are not limited to, fruits, vegetable beverages, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements. The food, beverage or food additive may be prepared by a conventional manufacturing method.

"Health functional food" in the health functional food composition according to the present invention is a food prepared or processed by a method such as extracting, concentrating, refining, or mixing a specific component as a raw material or a specific component contained in a food raw material for the purpose of health supplementation. Refers to foods designed and processed to sufficiently exert bioregulatory functions on the living body, such as bio-defense, control of biorhythms, prevention and recovery of diseases, and related to prevention of disease or recovery of health, etc. It means that you can perform a function.

When the strain according to the present invention is used as a health functional food, it can be added as it is or used with other foods or food ingredients, which can be selected and used as appropriate. In addition, the added strains may be used alone or in combination, and in this case, the mixing amount may be appropriately determined according to the purpose of use.

In addition, there is no particular limitation on the type of health functional food to which the strain according to the present invention can be used. Examples include ramen, other noodles, beverages, teas, drinks, alcoholic beverages, various soups, meat, sausages, bread, chocolate, candy, confectionery, pizza, gums, dairy products including ice cream, or vitamin complexes. In particular, the strain according to the present invention is excellent in viability of gastric acid or bile salts in the digestive tract, adhesion in cells and inhibitory effect on cavities or periodontitis, and is suitable for manufacturing various health functional foods. Fermented milk health functional foods that may contain the strain of the present invention include, for example, yogurt, calpis, cheese, butter, and the like, and fermented products include tofu, miso, cheonggukjang, jelly, and kimchi. The fermented milk or fermented product can be easily prepared by replacing only the strain with the strain according to the present invention in a conventional manufacturing method.

In addition, the strain according to the present invention can be mixed with other suitable components and known additives, which may be contained in the dietary supplement, according to the choice of a person skilled in the art. The known additives also include other microorganisms that can be used with the strain according to the invention.

In addition, the strain of the present invention may be included in the quasi-drug composition for the purpose of preventing or improving dental caries or periodontal disease.

When the strain of the present invention is used as a quasi-drug, the strain may be added as it is or used with other quasi-drug components, and may be suitably used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). When the present invention is used as a quasi-drug composition, it can be prepared and used as a formulation such as toothpaste, oral cleansers, mouthwashes, and the like, which are oral compositions.

Hereinafter, preferred examples and formulation examples are provided to help understanding of the present invention. However, the following examples and formulation examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited thereby.

Example 1. Separation of used strains and preparation of used samples

Lactobacillus derived from human body and food Lactobacillus salivarius (L. salivarius) MG242, Lactobacillus ruteri (L. reuteri) MG505, Lactobacillus ruteri (L. reuteri) MG422 4, L. salivarius MG4265, L. salivarius MG4293, L. salivarius MG4525, L. salivarius MG4526, L. salivarius MG4527, L. salivarius MG5183, L. reuteri MG5149, L. salivarius MG5207 And L. salivarius The experiment was performed using MG5212. Lactobacilli MRS (Difco, MI, USA) medium was used to culture the lactic acid bacteria at 37°C, and centrifuged the culture solution of the lactic acid bacteria on the MRS liquid medium for 24 hours (4,000 × g, 4°C, 5 minutes). After that, the sterilized supernatant (CFS, cell free supernatant) obtained using a 0.22 μm filter was used for the experiment. In order to measure the antibacterial activity, Streptococcus mutans KCTC3065 and Porphyromonas gingivalis KCTC5352 standard strains supplied by the Korean Collection for Type Cultures (KCTC) were used. S. mutans was cultured using BHI (Difco, MI, USA) medium, and P. gingivalis was inoculated in sheep blood medium and anaerobicly cultured at 37°C.

Example 2. S. mutans And P. gingivalis Antibacterial effect against

The antibacterial activity against S. mutans and P. gingivalis was confirmed through a paper disc diffusion assay method using three Lactobacillus reuteri and nine Lactobacillus salivarius of Example 1. Caries-causing bacteria were cultured 24 hours before each in the liquid culture medium, and then suspended and plated in a new solid culture medium with McFarland 0.5. After spotting 10 μL of the culture solution of the lactic acid bacteria cultured in MRS medium for 15 hours on the smeared solid culture medium and incubating at 37° C., the inhibition rings were identified, and the results of the antibacterial effect confirmation according to the inhibition rings are shown in Table 1. .

[Table 1]

As shown in Table 1 above, all L. reuteri and L. salivarius It was confirmed that it does not show inhibitory activity against S. mutans and P. gingivalis , and strains showing inhibitory effects on both cavities and periodontitis are MG242, MG505, MG4265, MG4293, MG4526, MG4527, MG5183, MG5207, MG5212 strains A total of 9 species were identified.

Lactobacillus strains showing inhibitory rings on S. mutans and P. gingivalis were additionally used to confirm antibacterial effects on cell-free supernatant and culture on S. mutans . The antibacterial experiment through the culture filtrate was performed by adding 10% of the cell-free supernatant (CFS), which is the sterile supernatant of the lactic acid bacteria, to the BHI liquid medium and inoculating S. mutans to 10 ⁵ CFU/mL. Antibacterial experiments through mixed cultures were performed so that the concentration of bacteria in the mixed medium (MRS liquid medium and BHI liquid medium mixed 1:1 ( v/v )) of lactic acid bacteria and S. mutans was finally 10 ⁵ CFU/mL. Inoculation was performed. Each antimicrobial effect confirmation experiment was performed by statically incubating at 37° C. for 8 hours, and then plating the BHI solid medium coated with erythromycin (0.3 μL/mL) to confirm the number of live bacteria of S. mutans . The results are shown in FIG. 1.

As shown in FIG. 1, 5 species of Lactobacillus salivarius MG242, L. reuteri MG505, L. salivarius MG4293, L. salivarius MG4527, and L. salivarius MG5183 were also inoculated with S. mutans even when inoculated with cell-free supernatant and culture. It was confirmed to show a high activity. Strains showing excellent activity as described above were deposited with the Korea Institute of Bioscience and Biotechnology, Lactobacillus salivarius MG242 Accession number: KCTC 18554P, L. reuteri MG505 Accession number: KCTC 13523BP, L. salivarius MG4293 Accession number: KCTC 13727BP, L. salivarius MG4527 Number: KCTC 13728BP, L. salivarius MG5183 Accession number: KCTC 13729BP was assigned an accession number.

Example 3. Screening strains Acid resistance And Bile resistance Confirm

In order for lactic acid bacteria to be used as probiotics, it must have safety and stability. Particularly, when the lactic acid bacteria are administered orally, the functional effect can be exhibited only when the gastric juice and various enzymes pass through the stomach and the bile exists in the duodenum to reach the final target site, the functional effect. Gender is an important indicator for use as probiotics. Accordingly, an experiment was conducted to confirm whether the five lactic acid bacteria selected in Example 2 had acid resistance and bile salt resistance to acidic and bile salts, such as the intestinal environment, to maintain viability. Specifically, after lactic acid bacteria were incubated for 18 hours, centrifuged (4,000 × g, 4°C, 5 minutes), washed twice with phosphate-buffered saline (PBS, pH 7), adjusted to OD ₆₀₀ 1.0, and acidic, respectively. It was used in the experiment of resistance to bile salts. To confirm the resistance to acidity, 1 mL of lactic acid bacteria dilution solution was added to 9 mL PBS (pH 2 and 7), shaken, and incubated at 37° C. for 3 hours to confirm the number of live cells. Bile salt resistance is confirmed by a method of confirming the number of live bacteria after inoculating 1% of lactic acid bacteria in MRS medium containing 0-1% oxgall (Difco, MI, USA) and incubating at 37°C for 24 hours. It is shown in 2.

[Table 2]

As shown in Table 2, as a result of the acid resistance test, all strains showed a viable cell count of 10 ² CFU/mL or higher at pH 2 corresponding to the gastric juice environment, and the highest viable cell count of MG505 (5.86 log CFU/mL). It confirmed that it showed. The pH of gastric juice in the body is maintained at approximately 3.0, and in general, since the time for microorganisms to pass through the stomach is 3 hours, maintaining a viable cell count at pH 3 for 3 hours or more can be judged to show high resistance to acidity. It is known that several strains used as probiotics generally show a viable cell count of 10 ⁴ CFU/mL or less at pH 2, and MG505, MG4293, and MG4527 are particularly resistant to acidity in the case of lactic acid bacteria selected in this study. You can confirm that there is.

In addition, unlike conventional lactic acid bacteria are sensitive to bile salts at 4 log CFU/mL or less at a bile salt concentration of 0.3% for confirming resistance to bile salts, 5 strains of the present invention have a higher 1% bile salt concentration. Also shows a very high viable cell count of an average of 8.01 log CFU/mL, and it can be seen that it shows very good resistance to bile salts.

Example 4. Selected Lactic acid bacteria Self-aggregation ability Confirm

In order to indirectly confirm the adhesion capacity in the intestinal cells, an experiment was performed to confirm autoaggregation. The culture medium in which the lactic acid bacteria were pre-cultured in MRS medium was adjusted to OD ₆₀₀ 1.0, inoculated 1% in fresh 5 mL MRS medium, and cultured for 18 hours to be used in the experiment. The cultured lactic acid bacteria were centrifuged (4,000 × g, 4° C., 5 minutes), washed twice in PBS, and adjusted to OD ₆₀₀ 1.0 to shake 5 mL of the strain suspension for 10 seconds and then self-aggregate while standing for 5 hours. Ability was confirmed. Immediately after the experiment (A0) and after 5 hours (A), 0.1 mL of supernatant was taken and mixed with 0.9 mL PBS, and absorbance was measured at 600 nm (A0, A). And the results are shown in FIG. 2.

Self-cohesive capacity (%) = (A0A) / A0 × 100

As shown in Figure 2, the MG4293, MG4527 strains showed the highest self-aggregation ability at 74.14 ± 2.18% and 65.78 ± 1.39%, respectively, and the average of the self-aggregation ability for the selected 5 strains was 61.64%. Since the self-aggregation ability of lactic acid bacteria in vitro is also reported to be related to the adhesion ability in vivo, it was confirmed that all five strains of the present invention have excellent self-aggregation ability and adhesion ability.

Example 5 Mixed strains S. mutans Antibacterial effect against

Lactobacillus salivarius MG242, L. reuteri MG505, L. salivarius MG4293, L. salivarius MG4527, L. salivarius MG5183 Cell free supernatant (CFS) of 5 mixed strains and S. mutans using culture medium Antibacterial effect was confirmed.

Cell-free supernatant (CFS) is prepared by statically incubating the strains in MRS liquid medium for 24 hours, and then centrifuging the culture solution under conditions of 4,000 × g, 4° C., 5 minutes, and filtering it with a 0.22 μm filter. Did. In addition, in order to confirm the antibacterial effect of the mixed culture (cell mixture), ⁵ ml of each of the ⁵ strains (10 ⁵ CFU/mL) was mixed to prepare a mixed culture. The experimental group used in the experiment to confirm the antibacterial effect of the mixed strain is as follows.

Experimental group 1 (mixed cell-free supernatant): Mix cell-free supernatant of 5 strains in equal volume ratio, respectively

Experimental group 2 (mixed culture solution): Mixing the cell culture solution of each of the 5 strains in the same volume ratio

Experimental group 3 (mixed cell-free supernatant and culture): Mix cell-free supernatant and cell culture of each of 5 strains in the same volume ratio

The mixed cell-free supernatant or the mixed culture of the above five mixed strains and S. mutans were mixed in a mixed medium (MRS liquid medium and BHI liquid medium in 1:1 ( v/v )), and the concentration of the bacteria was finally 10 ⁵ CFU. /mL each was inoculated. To confirm the antibacterial effect, after incubating for 8 hours at 37°C, S. mutans was smeared on BHI solid medium coated with 0.3 μL/mL of erythromycin. Was counted, and the results are shown in FIG. 3.

As shown in FIG. 3, S. mutans effective in comparison to the control group in all of the experimental groups treated with the mixed cell-free supernatant, the mixed culture solution, and a mixture thereof compared to the untreated control group The inhibitory effect was confirmed. Lactobacillus salivarius of the present invention from these results MG242, MG505 L. reuteri, L. salivarius MG4293, MG4527 L. salivarius, L. salivarius MG5183 5 strains are all S. mutans It shows excellent antimicrobial effect, especially when using a mixed culture of these strains.

Korea Research Institute of Bioscience and Biotechnology KCTC13523BP 20180509 Korea Research Institute of Bioscience and Biotechnology KCTC13727BP 20181121 Korea Research Institute of Bioscience and Biotechnology KCTC13728BP 20181121 Korea Research Institute of Bioscience and Biotechnology KCTC13729BP 20181121 Korea Research Institute of Bioscience and Biotechnology KCTC18554P 20170222

Claims (9)

Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), antimicrobial composition for inhibiting dental caries and periodontitis, comprising a culture thereof or a cell-free supernatant thereof.
According to claim 1, wherein the cavities are Streptococcus mutans ( Streptococcus mutans) , antibacterial composition for inhibiting cavities and periodontitis.
According to claim 1, wherein the periodontitis bacteria is Poroliromonas gingivalis ( Porphyromonas gingivalis ), antibacterial composition for inhibiting dental caries and periodontitis.
According to claim 1, wherein the Lactobacillus salivarius MG242 strain or Lactobacillus salivarius MG5183 strain is 0.1 to 1.5% (w / v) having bile salt resistance, antimicrobial composition for inhibiting cavities and periodontitis.
According to claim 1, The strain is characterized in that it has an autoaggregation ability (autoaggregation ability), antibacterial composition for inhibiting dental caries and periodontitis.
According to claim 1, Lactobacillus salivarius MG242 strain or characterized in that it comprises a culture medium of Lactobacillus salivarius MG5183 strain, antibacterial composition for inhibiting dental caries and periodontitis.
Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), a pharmaceutical solution for the prevention or treatment of dental caries and periodontal disease, comprising a culture medium thereof or a cell-free supernatant thereof Composition.
Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), its culture medium or its cell-free supernatant, for preventing or improving dental caries and periodontal disease Food composition.
Lactobacillus salivarius MG242 strain (Accession No.: KCTC 18554P) or Lactobacillus salivarius MG5183 strain (Accession No.: KCTC 13729BP), comprising a culture medium or a cell-free supernatant thereof, for preventing or improving dental caries and periodontal disease .

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