KR102210092B1 - Lactobacillus reuteri MG505 having anticariogenic activities and composition comprising the same - Google Patents

Abstract

The present invention is an antibacterial composition for inhibiting caries or periodontitis including Lactobacillus salivarius and Lactobacillus luteri derived from human body and various fermented foods, dental caries or periodontal disease prevention, treatment, pharmaceutical composition for improvement, health functional food composition and quasi-drugs It relates to the composition. The Lactobacillus luteri MG505 strain of the present invention has excellent inhibitory activity of Streptococcus mutans or Porilomonas gingivalins, and is suitable for probiotics because of its excellent acid resistance, bile resistance and self-aggregation ability, so it is a pharmaceutical for preventing, treating or improving tooth decay. , Food, quasi-drugs, etc. can be used in various ways.

Description

Composition comprising Lactobacillus reuteri MG505 having anti-caries activity {Lactobacillus reuteri MG505 having anticariogenic activities and composition comprising the same}

The present invention relates to an antibacterial composition for inhibiting tooth decay or periodontitis including Lactobacillus luteri derived from the human body and various fermented foods, dental caries or periodontal disease prevention, treatment, pharmaceutical composition for improvement, health functional food composition and quasi-drug composition.

Lactic acid bacteria have a special physiological activity and are generally regarded as safe bacteria (GRAS). Lactic acid bacteria are not only used in the production of various fermented foods, but also are widely used in dairy products and fermented fruit products having functional and probiotic properties. Recently, as consumers' demand for natural supplements to replace chemical supplements increases, it has emerged as an alternative method. Probiotics are living microorganisms that have a very beneficial effect on the health of the host animal, and improve the balance of intestinal microorganisms and enhance the absorption of nutrients. In addition, probiotics have the properties of showing the antibacterial action of pathogenic microorganisms in the intestinal environment. Probiotics contain various microorganisms, but Lactobacillus In and Bifidobacterium (Bifidobacterium) Genus occupies the most distribution. In particular, the genus Lactobacillus is generally used in the fermentation process of dairy products, meat, fruits and vegetables and cereal products.

On the other hand, more than 400 kinds of bacteria are propagating in human oral cavity, and the number of bacteria reaches 10 billion, and it is known that bacteria at the level of 10 ⁸ to 10 ⁹ CFU/ml are present in saliva. The teeth are perforated with a large number of microscopic holes, so bacteria that cause tooth decay enter the perforated hole and make the tooth sick. This is caries. These cavities are one of the most common diseases that can be seen in teeth and are also called dental caries. 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 the interaction of food with microorganisms in plaque. Among microorganisms, Streptococcus mutans is the main causative agent of tartar, and this microorganism is insoluble glucan from sucrose in food by glucosyltransferase (GTase), which is produced after attaching to the surface of the tooth. To synthesize. Synthesized glucan increases the binding between microorganisms that proliferate on the tooth surface, and S. mutans attached to the tooth surface is known to cause tooth decay by decalcification of tooth enamel by generating organic acids such as lactic acid during sugar metabolism. have. In Korea, the morbidity of permanent teeth reaches about 80%, and it is reported that one person has an average of 2-3 cavities.

Conventional methods for preventing dental caries or periodontal disease include reducing sugar intake through brushing and an appropriate diet, or preventing the production of acid by caries or periodontal disease-causing bacteria using xylitol. It is a situation in which the fundamental effect of preventing tooth decay is not obtained through the death of the cavities-causing bacteria or periodontal disease-causing bacteria.

Therefore, there is a need for a new method for preventing tooth decay that is harmless to the human body using lactic acid bacteria derived from the human body or food, which is harmless to the human body, is easy to ingest, and directly inhibits 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 inhibitory effect of lactobacilli against Streptococcus mutans and Streptococcus sobrinus and probiotics efficacy of yogurt (Kor J Oral Maxillofac Pathol 2009;33(3):167-174)

Accordingly, the present inventors were studying lactic acid bacteria that are effective in preventing caries among lactic acid bacteria derived from human body and food, Lactobacillus luteri MG505 (accession number: KCTC 13523BP) strain is streptococcus mutans and periodontitis, which are caries-causing bacteria. It has excellent antibacterial effect against Porilomonas Jinjivalin, and has excellent acid resistance, bile resistance, and self-aggregation ability, confirming that it is useful as a probiotic and completed the present invention.

Accordingly, an object of the present invention is an antibacterial composition for inhibiting caries or periodontitis including a Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain, a culture medium thereof or a cell-free supernatant thereof, and prevention, treatment, and improvement of dental caries or periodontal disease using the same It is to provide a composition for use.

In order to achieve the above object, the present invention provides an antibacterial composition for inhibiting caries and periodontitis, comprising a Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain, a culture solution thereof or a cell-free supernatant thereof.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dental caries and periodontal disease, comprising a Lactobacillus luteri MG505 (accession number: KCTC 13523BP) strain, a culture medium thereof, or a cell-free supernatant thereof.

In addition, the present invention provides a health functional food composition for preventing or improving dental caries and periodontal disease, including the Lactobacillus luteri MG505 (Accession Number: KCTC 13523BP) strain, a culture medium thereof, or a cell-free supernatant thereof.

In addition, the present invention provides a quasi-drug composition for preventing or improving dental caries and periodontal disease, including the Lactobacillus luteri MG505 (Accession Number: KCTC 13523BP) strain, a culture medium thereof, or a cell-free supernatant thereof.

The Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain of the present invention has excellent inhibitory activity against Streptococcus mutans and Porilomonas gingivalins, and has excellent acid resistance, bile resistance, and self-aggregation ability, so it is suitable for probiotics, so dental caries Or it can be variously used as medicines, foods, quasi-drugs, etc. for the prevention, treatment or improvement of periodontal disease.

1 is Lactobacillus raised bariwooseu MG242, and MG505 ruteri Lactobacillus, Lactobacillus raised bariwooseu MG4293, MG4527 bariwooseu Lactobacillus salicylate, salicylate Lactobacillus culture broth of MG5183 strain bariwooseu (Cell mixture), the cell-free supernatant (CFS mixture) of S. mutans It is a diagram showing the results of confirming the inhibitory activity.
Figure 2 is a diagram showing the results of confirming the self-aggregation ability of Lactobacillus salivaryus MG242 and Lactobacillus lutheri MG505, Lactobacillus salivarian MG4293, Lactobacillus salivarian MG4527, Lactobacillus salivarian MG5183 strain.
FIG. 3 is a S. mutans of Lactobacillus salivarius MG242, Lactobacillus luteri MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, Lactobacillus salivarius MG5183 strain mixed CFS, cell mixed culture solution, and CFS and cell mixed culture solution. It is a diagram showing the inhibitory effect.

The present invention provides an antibacterial composition for inhibiting caries and periodontitis, comprising a Lactobacillus luteri MG505 (accession number: KCTC 13523BP) strain, a culture medium thereof, or a cell-free supernatant thereof.

The Lactobacillus luteri MG505 strain of the present invention has excellent inhibitory activity of Streptococcus mutans and/or Porilomonas gingivalins, and has excellent acid resistance, bile resistance, and self-aggregation ability.

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 fermenting saccharides widely distributed in nature to obtain energy, and morphologically, it represents a polymorphism as Gram-positive aspore bacilli. Lactobacillus salivarius MG4293 and Lactobacillus salivarius MG4527 strains, which have excellent inhibitory activity of Streptococcus mutans and/or Porilomonas gingivalins according to the present invention, are excellent in acid resistance, bile resistance, and self-aggregation ability, respectively, are Korean Biotechnology. It was deposited with the Research Institute and was given the accession numbers KCTC 13727BP and KCTC 13728BP.

In addition, the composition of the present invention is a group consisting of Lactobacillus salivarius MG242 and Lactobacillus salivarius MG5183, which are another strains having Streptococcus mutans and/or Porilomonas gingivalins inhibitory activity, bile resistance and self-aggregation ability. It may further include one or more strains selected from, a culture medium thereof, or a cell-free supernatant thereof, and Lactobacillus salivarius MG242 and Lactobacillus salivarius MG5183 are each deposited with the Korea Research Institute of Bioscience and Biotechnology to give accession numbers KCTC 18554P and KCTC 13729BP. received.

In the present invention, the Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, and Lactobacillus luteri MG505 strains not only exhibit excellent antimicrobial activity against all of the representative caries bacteria Streptococcus mutans and Porilomonas gingivalins. It is characterized by having 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 a strong acid condition of pH 2 to 4, more preferably a gastric acid environment of pH 2.

In addition, Lactobacillus salivaryus MG242 and Lactobacillus luteri MG505, Lactobacillus salivarian MG4293, Lactobacillus salivarian MG4527, Lactobacillus salivarius MG5183 strains of the present invention have bile resistance against bile salts similar to the intestinal environment, and from 0.1 to It can exhibit excellent resistance even in 1.5% bile salt, 0.3 to 1% bile salt, and preferably 1% bile salt.

Lactobacillus salivarius MG242 and Lactobacillus luteri MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, Lactobacillus salivarius MG5183 strains of the present invention exhibit excellent autoaggregation ability of the intestine due to probiotic spasm. Removal can be prevented, and in particular, Lactobacillus salivarius MG4293 and Lactobacillus salivarian MG4527 strains can exhibit very good self-aggregation ability.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dental caries or periodontal disease, comprising a Lactobacillus luteri MG505 strain, a culture medium thereof or a cell-free supernatant thereof, a health functional food composition, a food composition, a quasi-drug composition.

The pharmaceutical composition, health functional food composition, food composition, quasi-drug composition may contain Lactobacillus lutheri MG505 strain, a culture medium thereof or a cell-free supernatant thereof as an active ingredient, and Lactobacillus salivarian strain MG4293 (Accession No.: KCTC 13727BP ), Lactobacillus salivarius MG4527 strain (Accession No.: KCTC 13728BP), Lactobacillus salivarius MG242 (Accession No.: KCTC 18554P) and Lactobacillus salivarius MG5183 (Accession No.: KCTC 13729BP) at least one strain selected from the group consisting of , It may further include a culture medium 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 based on a total of 100 parts by weight of the pharmaceutical composition, more preferably 1 to 80 parts by weight. If the content is less than 0.01 parts by weight, the dosage efficiency may be deteriorated, and if it exceeds 95 parts by weight, there may be difficulties in formulation.

Lactobacillus salivarius MG242, Lactobacillus luteri MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, and Lactobacillus salivarius MG5183 strains according to the present invention have excellent antibacterial activity against caries-causing bacteria and periodontitis. , Bile resistance and self-cohesiveness are excellent, so it can be used directly as a composition itself having a preventive, ameliorating or therapeutic effect of various diseases caused by caries bacteria and periodontitis, and can also be used as an adjuvant.

The pharmaceutical composition according to the present invention comprises a pharmaceutically effective amount of Lactobacillus salivarius MG242, Lactobacillus luteri MG505, Lactobacillus salivarius MG4293, Lactobacillus salivarius MG4527, or Lactobacillus salivarius MG5183 strain alone or one The above pharmaceutically acceptable carriers, excipients, or diluents may be included.

Dental caries and periodontal disease, dental caries is both an infectious bacterial disease is Streptococcus mutans (S. mutans), Streptococcus on Queenie's (S. sanguinis), Streptococcus small debris Augustine (S. sobrinus), Streptococcus Latina ( S. ratti ), S. criceti , S. anginosus , or S. gordonii . Periodontal disease is a bacterium that causes periodontitis. Porphyromonas gingivalis gingivalis ), Prevotella intermedia , Actinobacillus actinomycetemcomitans , Treponema denticola and Fusobacterium nucliatum nucleatum ), etc. The pharmaceutical composition according to the present invention is very effective in preventing or treating dental caries or periodontal disease because it can effectively inhibit representative dental caries and periodontal disease-causing bacteria.

Therefore, the term "pharmaceutically effective amount" above, for example, inhibits the growth of Streptococcus mutans and Porilomonas gingivalins, which are bacteria that cause tooth decay and periodontitis, thereby preventing dental caries or periodontal disease that may occur, It refers to an amount sufficient to exert an improvement or therapeutic effect. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions.

In addition, the pharmaceutical composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods. I can. Suitable formulations known in the art are preferably 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, gum acacia, 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 a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, and surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the composition, such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like 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 conceived by a researcher, veterinarian, doctor or other clinician, that is, the symptom of the disease or disorder being treated. It can be administered in a therapeutically effective amount, which is an amount that induces remission. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary according to the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. It can be adjusted according to various factors including condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and drugs used simultaneously. For a desirable effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, and may be administered once a day or several times. It can also be administered separately.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

The pharmaceutical composition according to the present invention may be used alone, but according to the choice of a person skilled in the art, other appropriate treatment methods (e.g., surgery, radiation therapy, hormone therapy, chemical treatment) that have a therapeutic enhancing effect for dental caries or periodontal disease It can be used in combination with treatment and biological response modifiers).

In the present invention, the term "prevention" may mean any action of inhibiting or delaying the onset of dental caries or periodontal disease by administering the 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" may mean any action to improve or benefit the symptoms of dental caries or periodontal disease-related diseases by administering the composition according to the present invention to an individual suspected of dental caries or periodontal disease.

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

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

In the food composition according to the present invention, the type of 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 include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , Drinks, alcoholic beverages, and vitamin complexes. When using the composition as a food additive, the composition may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

In the present specification, the term "food" refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through some degree of processing, and is a common meaning, The food composition is intended to include all foods, food additives, health functional foods and beverages.

Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gum, candy, tea, vitamin complexes, and functional foods. In addition, food in the present invention includes special nutritional foods (e.g., formula, infant food, etc.), processed meat products, fish meat products, tofu, muk, noodles (e.g., ramen, noodles, etc.), health supplement food, seasoning food ( Example, soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (eg, snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruit, 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.

In the health functional food composition according to the present invention, the term "health functional food" refers to food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, and mixing specific ingredients as raw materials for the purpose of supplementing health. It refers to foods designed and processed to sufficiently exert biological control functions such as biological defense, biological rhythm control, and disease prevention and recovery by the above ingredients, and related to the prevention of diseases or recovery of health, etc. It refers to something that can perform a function.

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

In addition, there is no particular limitation on the kind of health functional food in which the strain according to the present invention can be used. For example, ramen, other noodles, beverages, tea, drinks, alcoholic beverages, various soups, meat, sausage, bread, chocolate, candy, confectionery, pizza, gum, dairy products including ice cream, or vitamin complexes. In particular, the strain according to the present invention is excellent in viability against gastric acid or bile salts in the digestive tract, intracellular adhesion, and inhibitory effect against caries or periodontitis, so it is suitable to be prepared as a variety of 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, kimchi, and the like. The fermented milk or fermented product can be easily manufactured 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 may be mixed with known additives and other suitable auxiliary ingredients that may be normally contained in health functional foods 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 present 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 may be used together with other quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may 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 in formulations such as toothpaste, oral cleansers, oral cleansers, and oral sprays as oral compositions.

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

Example 1. Isolation of used strain and preparation of used sample

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 Experiments were carried out using MG5212. Lactobacilli MRS (Difco, MI, USA) culture medium was used to cultivate lactic acid strains at 37°C, and centrifugation (4,000 × g, 4℃, 5 minutes) After that, the sterilized supernatant (CFS, cell free supernatant) obtained using a 0.22 μm filter was used in the experiment. In order to measure the antibacterial activity, Streptococcus mutans KCTC3065 and Porphyromonas gingivalis KCTC5352 standard strains purchased from 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 cultured anaerobicly at 37°C.

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

The antimicrobial 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. The tooth decay-causing bacteria were cultured in each liquid culture medium for 24 hours before, and then suspended with McFarland 0.5 in a new solid culture medium and plated. 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 culturing at 37°C, the inhibitory ring was confirmed, and the results of confirming the antibacterial effect according to the inhibitory ring are shown in Table 1. .

[Table 1]

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

The antimicrobial effect on the cell-free supernatant and culture medium against S. mutans was additionally confirmed by using lactic acid strains that showed inhibitory rings on S. mutans and P. gingivalis . The antibacterial experiment through the culture filtrate was performed by adding 10% of the cell-free supernatant (CFS), which is the sterilized supernatant of the lactic acid strain, to the BHI liquid medium, and then inoculating S. mutans at 10 ⁵ CFU/mL. Antimicrobial experiments through mixed culture consisted of lactic acid strains and S. mutans in a mixed medium (MRS liquid medium and BHI liquid medium were mixed at 1:1 ( v/v )) so that the concentration of bacteria was finally 10 ⁵ CFU/mL. It was carried out by inoculation. Each antimicrobial effect confirmation experiment was carried out in a manner of confirming the number of viable bacteria of S. mutans by anaerobic cultivation at 37°C for 8 hours and then spreading on BHI solid medium coated with erythromycin (0.3 μL/mL). The results are shown in FIG. 1.

1, Lactobacillus salivarius MG242, L. reuteri MG505, L. salivarius MG4293, L. salivarius MG4527, L. salivarius MG5183 5 species were S. mutans even when cell-free supernatant and culture were inoculated. It was confirmed that high activity was shown. Strains exhibiting excellent activity as described above were deposited with the Korea Institute of Bioscience and Biotechnology, and Lactobacillus salivarius MG242 accession number: KCTC 18554P, L. reuteri MG505 accession number: KCTC 13523BP, L. salivarius MG4293 accession number: KCTC 13727BP, L. salivarius MG4527 No.: KCTC 13728BP, L. salivarius MG5183 Deposit No.: KCTC 13729BP has been assigned a deposit number.

Example 3. Of the selected strain Acid resistance And Bile resistance Confirm

In order for lactic acid bacteria to be used as probiotics, it must have safety and stability. In particular, when lactic acid bacteria are administered orally, they must pass through the stomach where gastric juice and various enzymes are present and reach the intestine, which is the final target site, through the duodenum where bile is present.Therefore, the acid resistance and bile resistance of lactic acid bacteria Sex is an important indicator for use as probiotics. Therefore, an experiment was conducted to determine whether the five lactic acid strains selected in Example 2 have acid resistance and bile resistance to acidic and bile salts such as the intestinal environment and maintain viability. Specifically, after culturing the lactic acid strain for 18 hours, centrifugation (4,000 × g, 4° C., 5 minutes) and washed twice with phosphate-buffered saline (PBS, pH 7), and adjusted to OD ₆₀₀ 1.0 to each acidic It was used in the test of resistance to bile salts. To confirm the resistance to acidity, 1 mL of a diluted solution of lactic acid bacteria was added to 9 mL PBS (pH 2 and 7), shaken, and incubated at 37°C for 3 hours, and the number of viable bacteria was checked. Bile salt tolerance is confirmed through the method of inoculating 1% lactic acid bacteria diluted solution in MRS medium containing 0~1% oxgall (Difco, MI, USA) and incubating at 37°C for 24 hours, and then checking the number of viable cells. It is shown in 2.

[Table 2]

As shown in Table 2, as a result of the acid resistance test, all strains at pH 2 corresponding to the gastric juice environment showed a number of viable cells of 10 ² CFU/mL or more, and MG505 (5.86 log CFU/mL) was the highest number of viable cells. It was confirmed that it showed. The pH of the gastric juice in the body is maintained at approximately 3.0, and in general, the time for microorganisms to pass through the stomach is 3 hours. Therefore, it can be determined that a high resistance to acidity is exhibited if the number of viable cells is maintained at pH 3 for 3 hours or more. 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 in the case of the lactic acid strains selected in this study, MG505, MG4293, MG4527 in particular have excellent resistance to acid. 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 concentration of 0.3% bile salt to confirm resistance to bile salts, the five strains of the present invention have a higher 1% bile salt concentration. Also showed a very high viable cell count of 8.01 log CFU / mL on average, it can be seen that showing very excellent resistance to bile salts.

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

In order to indirectly confirm the intestinal cell adhesion, an experiment was performed to confirm autoaggregation. The culture solution in which the lactic acid bacteria were pre-cultured in the MRS medium was adjusted to an OD _{600 of} 1.0, and 1% was inoculated in a new 5 mL MRS medium, and cultured for 18 hours was used for the experiment. The cultured lactic acid bacteria were centrifuged (4,000 × g, 4°C, 5 minutes), washed twice in PBS, adjusted to OD ₆₀₀ 1.0, shaken 5 mL of the strain suspension for 10 seconds, and left for 5 hours to self-aggregate. The ability was confirmed. Immediately after the start of the experiment (A0) and 5 hours later (A), 0.1 mL of the supernatant was taken, mixed with 0.9 mL PBS, and the absorbance was measured at 600 nm (A0, A), and the self-aggregation ability was calculated according to the following calculation formula. And the results are shown in FIG. 2.

Self-aggregation ability (%) = (A0A) / A0 × 100

As shown in FIG. 2, the MG4293 and 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 five strains was 61.64%. Since it is reported that the in vitro self-aggregation ability of lactic acid bacteria is also related to the in vivo attachment ability, 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 on

Lactobacillus salivarius MG242, L. reuteri MG505, L. salivarius MG4293, L. salivarius MG4527, L. salivarius MG5183 Cell-free supernatant (CFS, cell free supernatant) of five mixed strains and S. mutans using a culture solution The antibacterial effect on was confirmed.

Cell-free supernatant (CFS) is prepared by culturing the strains in MRS liquid medium for 24 hours, centrifuging the culture solution at 4,000 × g, 4° C., 5 minutes, and filtering it with a 0.22 μm filter. I did. In addition, in order to confirm the antibacterial effect of the cell mixture, ⁵ ml of the culture solution of each of the ^five strains (10 ⁵ CFU/mL) were mixed to prepare a mixed culture solution. The experimental groups used in the experiment to confirm the antibacterial effect of the mixed strain are as follows.

Experimental group 1 (mixed cell-free supernatant): Cell-free supernatant of 5 strains was mixed at the same volume ratio

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

Experimental group 3 (cell-free supernatant and culture solution mixture): Cell-free supernatant and cell culture solution of each of the 5 strains were mixed at the same volume ratio

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

As shown in Fig. 3, in all of the experimental groups treated with the mixed cell-free supernatant, the mixed culture solution and the mixture thereof compared to the untreated control group, S. mutans, which is effective compared to the control group The inhibitory effect was confirmed. Based on these results, all five strains of Lactobacillus salivarius MG242, L. reuteri MG505, L. salivarius MG4293, L. salivarius MG4527, L. salivarius MG5183 of the present invention were S. mutans. It showed an excellent antibacterial effect against, and it was confirmed that particularly, when a mixed culture solution of these strains was used, an excellent antibacterial effect was exhibited.

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 (10)

Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain, a culture medium thereof or a cell-free supernatant thereof, and the Lactobacillus luteri MG505 is acid-resistant at pH 2 to pH 4 and resistant to 0.1 to 1.5% (w/v) bile salt. An antibacterial composition for inhibiting tooth decay and periodontitis, characterized in that it has biliary properties.
The antimicrobial composition of claim 1, wherein the caries bacteria are Streptococcus mutans, which inhibits caries and periodontitis.
The antibacterial composition of claim 1, wherein the periodontitis is Porphyromonas gingivalis .
delete delete The antibacterial composition of claim 1, wherein the strain has an autoaggregation ability.
According to claim 1, characterized in that it comprises a culture medium of Lactobacillus luteri MG505 (accession number: KCTC 13523BP) strain, caries and periodontitis antibacterial composition for inhibiting.
Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain, a culture medium thereof or a cell-free supernatant thereof, and the Lactobacillus luteri MG505 is acid-resistant at pH 2 to pH 4 and resistant to 0.1 to 1.5% (w/v) bile salt. A pharmaceutical composition for preventing or treating dental caries and periodontal disease, characterized in that it has biliary properties.
Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain, a culture medium thereof or a cell-free supernatant thereof, and the Lactobacillus luteri MG505 is acid-resistant at pH 2 to pH 4 and resistant to 0.1 to 1.5% (w/v) bile salt. A health functional food composition for preventing or improving dental caries and periodontal disease, characterized in that it has biliary properties.
Lactobacillus luteri MG505 (Accession No.: KCTC 13523BP) strain, a culture medium thereof or a cell-free supernatant thereof, and the Lactobacillus luteri MG505 is acid-resistant at pH 2 to pH 4 and resistant to 0.1 to 1.5% (w/v) bile salt. A quasi-drug composition for preventing or improving dental caries and periodontal disease, characterized in that it has biliary properties.

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