KR20200141199A - NOVEL STRAIN OF Lactobacillus plantarum AND USE THEREOF - Google Patents

Abstract

The present invention relates to: a Lactobacillus plantarum strain (accession number: KCCM 12499P) having antimicrobial activity on Streptococcus mutans and an ability to inhibit biofilm formation; and a probiotic composition, a composition for preventing, alleviating or treating dental caries or periodontal disease, and a composition for oral cavity, all of which contain the Lactobacillus plantarum strain as an active component.

Description

A novel Lactobacillus plantarum strain and its use {NOVEL STRAIN OF Lactobacillus plantarum AND USE THEREOF}

The present invention is antimicrobial activity against Streptococcus mutans ; And a novel Lactobacillus plantarum strain having biofilm formation inhibitory ability and its use.

Dental caries or periodontal disease is an oral disease caused by oral bacteria that have many experiences of onset regardless of age or sex. One of its major etiological factors is the proliferation of oral bacteria on the tooth surface. In addition, the biofilm is composed of oral bacteria and its metabolites, causing dental caries or periodontal disease.The microbial community inside the biofilm has strong resistance to antimicrobial factors, and close contact between microorganisms is a possibility of interaction. Increase This specific interaction has the problem of enhancing the pathogenic potential of oral bacteria.

At this time, Streptococcus mutans is a representative oral bacteria, and has a great influence on the formation of a biofilm in which microbial communities in the oral cavity proliferate inside. Streptococcus mutans uses sucrose as a substrate to produce glucosyltransferases (GTFs) that synthesize intracellular and extracellular polysaccharides (EPS). The insoluble glucans formed from GTFs have very strong adhesion to the oral surface, providing a binding site to other oral bacteria with poor adhesion and facilitating the formation of biofilms. By suppressing Streptococcus mutans in the cluster, the volume of the biofilm can be greatly reduced and the expansion of the biofilm can be slowed down.

On the other hand, probiotics settle as a part of the intestinal microflora when ingested and provide beneficial activities for the physiological activity of the host.Also, while acting as probiotics, studies on strains that are effective for oral diseases such as dental caries or periodontal disease have been studied. It is a necessary situation.

The present invention is antimicrobial activity against Streptococcus mutans ; And it is intended to provide a Lactobacillus plantarum strain (KCCM 12499P) having an ability to inhibit formation of a biofilm.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention is antimicrobial activity against Streptococcus mutans ; And it provides a Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P) having a biofilm formation inhibitory ability.

The biofilm may contain insoluble glucan.

The insoluble glucan may be formed from glucosyltransferases (GTFs) produced by Streptococcus mutans .

The strain may additionally have acid resistance, bile resistance or intestinal adhesion.

The strain may include the 16S rRNA nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, it provides a probiotic composition comprising a Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

The strain may be prepared as a culture medium or a culture filtrate.

In another embodiment of the present invention, it provides a pharmaceutical composition for the prevention or treatment of dental caries or periodontal disease comprising the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

In another embodiment of the present invention, it provides a health functional food for preventing or improving dental caries or periodontal disease, comprising the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

In another embodiment of the present invention, it provides an oral composition comprising the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

Lactobacillus plantarum according to the present invention ( Lactobacillus plantarum ) strain (KCCM 12499P) has excellent probiotic properties, while having excellent probiotic properties, antimicrobial activity against Streptococcus mutans ; And it has the ability to inhibit the formation of a biofilm, it can be usefully used as a composition for preventing, improving or treating dental caries or periodontal disease or a composition for oral cavity.

1 is a schematic diagram of the strain Lactobacillus plantarum 200661 isolated in Example 1.
2(a) is a graph showing the ability to inhibit biofilm formation in the case of the sediment and supernatant of the Lactobacillus plantarum 200661 strain isolated in Example 1, and FIG. 2(b) is a sediment and supernatant of the Lactobacillus rhamnosus LGG strain known as a commercial probiotic. In the case of, it is a graph showing the biofilm formation inhibition ability.
3 is a supernatant (□) of the Lactobacillus plantarum 200661 strain isolated in Example 1 and the supernatant (■) of the Lactobacillus rhamnosus LGG strain known as a commercial probiotic, biofilm formation related genes ( gtf A, gtf B, gtf D and ftf ) is a graph showing the expression level.
4 is a graph showing insoluble glucan-forming ability in the case of the supernatant (□) of the Lactobacillus plantarum 200661 strain isolated in Example 1 and the supernatant (■) of the Lactobacillus rhamnosus LGG strain known as a commercial probiotic.

The present inventors Lactobacillus having probiotic properties plantarum Among strains, antibacterial activity against Streptococcus mutans ; And Lactobacillus plantarum having inhibitory ability to form biofilm 200661 The present invention was completed by isolating and identifying strains and evaluating their efficacy.

In the present specification, the term "biofilm" is a structure formed in the oral cavity, and is also referred to as plaque. It is composed of oral bacteria and metabolites formed by the proliferation of oral bacteria, and acts as a major cause of dental caries or periodontal disease, and conventionally, physical methods such as brushing were required for effective removal thereof.

Hereinafter, the present invention will be described in detail.

The present invention is antimicrobial activity against Streptococcus mutans ; And it provides a Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P) having a biofilm formation inhibitory ability.

The strain (KCCM 12499P) can be seen as a subspecies of Lactobacillus plantarum. Lactobacillus plantarum is a Gram-positive bacillus, slightly aerobic, and is one of the most widely distributed lactic acid bacteria in nature. Lactobacillus plantarum undergoes normal lactic acid fermentation and produces lactic acid by fermenting arabinose, glucose, fructose, galactose, maltose, sucrose, and dextran as representative kimchi lactic acid bacteria. It is mainly separated from dairy products, pickles, chives such as kimchi, tomatoes, etc. In particular, when kimchi is fermented a lot in the late fermentation of kimchi and has a sour taste, it mainly grows and becomes dominant, thereby inhibiting the growth of other species. It is known to have excellent resistance to dampness.

The schematic diagram of the strain (KCCM 12499P) is as shown in FIG. 1, and may be isolated from kimchi. As a result of analyzing the 16S rRNA sequence to identify the isolated strain, the strain (KCCM 12499P) was confirmed to have the 16S rRNA nucleotide sequence of SEQ ID NO: 1, and deposited with the Korea Microbiology Conservation Center on April 19, 2019. Thus, it was assigned the accession number KCCM 12499P.

In addition, the strain (KCCM 12499P) is characterized by excellent antibacterial activity against oral bacteria, particularly, Streptococcus mutans , among the Lactobacillus plantarum strain. At this time, Streptococcus mutans is a major oral bacterium that causes dental caries or periodontal disease, and decomposes organic matter to discharge acids and causes teeth to be demineralized by these acids. Specifically, the strain (KCCM 12499P) is characterized by superior antibacterial effect against Streptococcus mutans compared to the Lactobacillus rhamnosus LGG strain known as a commercial probiotic.

In addition, the strain (KCCM 12499P) is characterized by having a biofilm formation inhibitory ability. At this time, the biofilm may contain insoluble glucan, but using sucrose as a substrate, it may be formed from glucosyltransferases (GTFs) produced by Streptococcus mutans . Accordingly, it may be effective in preventing, improving or treating dental caries or periodontal disease. Such biofilm formation inhibitory ability can be confirmed through changes in expression of biofilm formation-related genes ( gtf A, gtf B, gtf D and ftf, etc.) using PCR.

In addition, the strain (KCCM 12499P) may additionally have acid resistance, bile resistance, or intestinal adhesion. Specifically, the acid resistance may have a survival rate of 90% or more (preferably, 100% or more) when reacting for 3 hours at a pH of 2.5 conditions, and bile resistance is 0.3% oxgall when reacting for 24 hours, the survival rate of the strain This may be 90% or more (preferably 10% or more).

In addition, the present invention provides a probiotic composition comprising the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

The strain (KCCM 12499P) is as described above, and the strain (KCCM 12499P) may be prepared as a culture medium or a culture filtrate, and is preferably prepared as a culture filtrate, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dental caries or periodontal disease, including the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

The strain (KCCM 12499P) is as described above, and the strain (KCCM 12499P) may be prepared as a culture medium or a culture filtrate, and is preferably prepared as a culture filtrate, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dental caries or periodontal disease, including the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

The strain (KCCM 12499P) is as described above, and the strain (KCCM 12499P) may be prepared as a culture medium or a culture filtrate, and is preferably prepared as a culture filtrate, but is not limited thereto.

The term "dental caries" in the present specification refers to a disease such as tooth decay in which hemorrhoids are destroyed by acids made by oral bacteria attached to the tooth surface, and "periodontal disease" in the present specification refers to a disease surrounding the tooth. It refers to diseases appearing in tissues around teeth such as gingival, periodontal ligaments, and alveolar bone, and all of these diseases are caused by the formation of biofilm together with oral bacteria.

The pharmaceutical composition according to the present invention is formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. For formulation, it may contain a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

As the carrier or excipient or diluent, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided And various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In the case of formulation, it can be prepared using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration may be prepared by mixing the strain (KCCM 12499P) with at least one excipient, for example, starch, calcium bonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be 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, sweeteners, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, 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 paper, glycerol gelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day, or may be divided several times. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition according to the present invention can be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a health functional food for preventing or improving dental caries or periodontal disease comprising the Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

The strain (KCCM 12499P) is as described above, and the strain (KCCM 12499P) may be prepared as a culture medium or a culture filtrate, and is preferably prepared as a culture filtrate, but is not limited thereto.

In the health functional food according to the present invention, when the strain (KCCM 12499P) is used as an additive of a health functional food, it can be added as it is or used with other foods or food ingredients, and can be used appropriately according to a conventional method. have. The mixing amount of the active ingredient can be appropriately determined according to each purpose of use, such as prevention, health or treatment.

Formulations of health functional foods may be in the form of powders, granules, pills, tablets, capsules, as well as general foods or beverages.

The type of food is not particularly limited, and examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream. , Various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and may include all foods in the usual sense.

In general, in the manufacture of food or beverage, the strain (KCCM 12499P) may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on 100 parts by weight of raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.

Among the health functional foods according to the present invention, the beverage may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional beverage. The natural carbohydrates described above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin. , Alcohols, carbonated beverages may contain carbonation agents. In addition, the health functional food according to the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These ingredients may be used independently or in combination. Although the ratio of these additives is not limited, it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food according to the present invention.

In addition, the present invention provides a composition for oral cavity including Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

The strain (KCCM 12499P) is as described above, and the strain (KCCM 12499P) may be prepared as a culture medium or a culture filtrate, and is preferably prepared as a culture filtrate, but is not limited thereto.

In the oral composition according to the present invention, it may be formulated in the form of a powder, granule, tablet, capsule, cream, transparent gel or translucent gel. That is, the oral composition is characterized in that it is not formulated in a liquid form, such as in the form of a solution, suspension, or emulsion, and has an advantage of minimizing side effects of existing oil pooling. For the formulation, suitable abrasives, wetting agents, binders, foaming agents, sweetening agents, preservatives, medicinal agents, flavoring agents, acidity modifiers, and brighteners that are commonly used in the preparation of pharmaceutical compositions may additionally contain one or more additives selected from the group consisting of. have. In this case, the additive is preferably included in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the oral composition, but is not limited thereto.

The abrasive may be at least one selected from the group consisting of calcium monohydrogen phosphate, precipitated silica, calcium carbonate, hydrous alumina, kaolin, and sodium bicarbonate, and preferably calcium monohydrogen phosphate or precipitated silica.

The humectant may be at least one selected from the group consisting of glycerin, sorbitol, amorphous sorbitol solution, propylene glycol, polyethylene glycol, and xylitol, and preferably sorbitol.

As the binder, sodium carboxymethylcellulose, carrageenan, xanthan gum, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, guar gum, gellan gum, carbomer, pectin, polyvinylpyrrolidone, carboxyvinylpoly And, it may be at least one selected from the group consisting of sodium alginate and laponite, preferably carboxymethyl cellulose sodium.

The foaming agent is a nonionic surfactant group consisting of anionic surfactants such as sodium lauryl sulfate and sodium lauryl sarcosine, and sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene-polyoxypropylene copolymer, etc. Or, it may be at least one selected from the group of amphoteric surfactants such as cocoidopropylbetaine, and preferably sodium lauryl sulfate.

The sweetener may be at least one selected from the group consisting of sodium saccharin, sucralose, maltitol, aspartame, erythritol and licorice, and preferably sodium saccharin.

The preservative may be at least one selected from the group consisting of paraoxybenzoic acid ester and sodium benzoate, preferably paraoxybenzoic acid ester.

As the medicinal agents, sodium fluoride, sodium fluoride phosphate, one stone fluoride, amine fluoride, chlorhexidine, aminocaproic acid, dipotassium glycyrrhizin, tranexamic acid, allotoin, capronic acid, polyphosphates, enzymes and herbal extracts It may be one or more selected from the group consisting of, preferably sodium fluoride.

As the perfume, an appropriate amount of natural perfumes such as peppermint oil and spearmint oil and synthetic flavoring ingredients such as menthol and carbon are mixed in an appropriate amount, and an appropriate amount of anise oil may be mixed with a perfume obtained by mixing them in a certain ratio.

In addition, the present invention can provide a starter for fermentation, a seed composition, or a fermented food comprising a Lactobacillus plantarum strain (KCCM 12499P).

As described above, the Lactobacillus plantarum strain (KCCM 12499P) according to the present invention has excellent probiotic properties, while antimicrobial activity against Streptococcus mutans ; And it has the ability to inhibit the formation of a biofilm, it can be usefully used as a composition for preventing, improving or treating dental caries or periodontal disease or a composition for oral cavity.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

< Example >

Example One: Lactobacillus plantarum 200661 strain isolation and identification

In order to isolate a new strain, the cabbage kimchi broth made at home was sampled and used as a sample. The cabbage kimchi broth was spread on LBS agar medium using a 10-fold dilution method, streaked several times, and cultured, and then the colony shape was observed to isolate a new strain. 16S rRNA sequencing was requested to Bionics (Seoul, Korea) for identification of the isolated new strain. Specifically, 16S rRNA sequencing was performed, and PCR was performed on the 16S rRNA gene using 27F and 1492R primers. Based on the results of nucleotide sequence analysis, the BLAST program was used to compare homology with other standard strains registered in Genbank's database. The new strain was identified as Lactobacillus plantarum 200661 strain, and the similarity was 99%. In addition, homology was analyzed using Mega 7 program and a schematic diagram was obtained. At this time, the Lactobacillus plantarum 200661 strain was deposited with the Korea Microbial Conservation Center on April 19, 2019, and was given the accession number KCCM 12499P.

Later, Lactobacillus plantarum The 200661 strain was cultured in lactobacilli MRS broth at 37°C for 12 hours, the initial number of bacteria was adjusted to 10 ⁷ CFU/mL, and then centrifuged (5,000 Хg, 10 minutes) to separate the precipitate (culture solution) and the supernatant (culture filtrate). .

Comparative example One: Lactobacillus rhamnosus LGG Strain preparation

Lactobacillus rhamnosus LGG strain known as commercial probiotics was prepared. Culture conditions are Lactobacillus plantarum 200661 strain was the same.

Experimental example 1: strain of Probiotic Property evaluation

Lactobacillus plantarum isolated in Example 1 As probiotic properties of the 200661 strain, acid resistance, bile resistance, and intestinal adhesion were evaluated.

(One) Acid resistance And Bile resistance evaluation

Lactobacillus plantarum isolated in Example 1 In order to evaluate the acid resistance and diarrhea resistance of the 200661 strain, the pH was adjusted to 2.5, the strain was inoculated in MRS liquid medium containing 0.3% pepsin, and the number of bacteria after incubation at 25° C. for 3 hours was compared with the initial inoculated bacteria number, The results are shown in Table 1 below. In addition, the number of bacteria after inoculating the strain in MRS liquid medium containing 0.3% oxgall and culturing at 25° C. for 24 hours was compared with the number of initial inoculated bacteria, and the results are shown in Table 1 below.

process Number of bacteria (Log CFU/mL) Example 1 Comparative Example 1 Initial inoculation 9.07 ± 0.03 9.26 ± 0.02 0.3% pepsin, pH 2.5, 3 hours 9.07 ± 0.03 9.18 ± 0.02 0.3% oxgall, 24 hours 9.13 ± 0.09 9.25 ± 0.02

As shown in Table 1, Lactobacillus plantarum isolated in Example 1 The acid resistance of the 200661 strain was about 100%, and the bile resistance was about 100.66%, which was found to show higher levels than the Lactobacillus rhamnosus LGG strain known as a commercial probiotic.

(2) Attachment ability evaluation

Lactobacillus plantarum isolated in Example 1 In order to evaluate the intestinal adhesion ability of the 200661 strain, human-derived intestinal epithelial cells, HT-29 cell line, were inoculated into a 24-well plate at a concentration of 2 × 10 ⁵ cells/well and cultured until a monolayer was formed. Inoculated at a concentration of 100 μL (1Х10 ⁸ CFU/mL) and incubated at 37°C for 2 hours. Wash three times with PBS to remove the strain that does not adhere to the cells, dissolve the adhered cells with 1% Triton X-100 solution, and plate the lysate diluted with an appropriate multiple on MRS agar medium and spread at 25°C. Incubated for 24 hours and the number of attached bacteria was measured to evaluate the intestinal adhesion ability as follows, and the results are shown in Table 2 below:

Intestinal adhesion (%) = (number of attached bacteria (Log CFU/mL)/number of inoculated bacteria (Log CFU/mL)) × 100.

process Example 1 Comparative Example 1 Book attachment ability (%) 13.24 ± 4.20 18.03 ± 5.81

As shown in Table 2, the intestinal adhesion ability of the Lactobacillus plantarum 200661 strain isolated in Example 1 was about 13.24%, and it was confirmed that it adhered well to the intestinal epithelium at the same level as the Lactobacillus rhamnosus LGG strain known as a commercial probiotic.

Thus, Lactobacillus plantarum isolated in Example 1 The 200661 strain has excellent probiotic properties such as acid resistance, dystrophy resistance, and intestinal adhesion, so it can be viewed as a beneficial lactic acid bacterium.

Experimental example 2: Evaluation of the antibacterial activity of the strain

Lactobacillus plantarum isolated in Example 1 In order to evaluate the antimicrobial activity of 200661 strain against Streptococcus mutans, a minimum inhibitory concentration was used. In a 96 well plate, three Streptococcus mutans were diluted to a concentration of 1 Х10 ⁵ CFU/mL, and a sample (supernatant of strain) was diluted to a concentration of 3.125% to 50.0%, and 50 μL each was added. At this time, the control group was put in the same amount of sodium ampicillin instead of the sample. After incubation for 2 hours, absorbance was measured at 570 nm to confirm the degree of inhibition of growth of the three Streptococcus mutans, and the first concentration that inhibited the growth of the three Streptococcus mutans was the minimum inhibitory concentration. It is shown in Table 3.

process Number of bacteria (Log CFU/mL) Example 1 Comparative Example 1 S. mutans KCTC 5124 6.25 12.5 S. mutans KCTC 5458 12.5 25.0 S. mutans KCTC 5316 12.5 25.0

As shown in Table 3, Lactobacillus plantarum isolated in Example 1 The minimum inhibitory concentration for the three Streptococcus mutans of the 200661 strain is 6.25 to 12.5%, and it is confirmed that the antibacterial activity against all three Streptococcus mutans is superior compared to the Lactobacillus rhamnosus LGG strain known as a commercial probiotic. .

Experimental example 3: Biofilm formation of strain Inhibitory ability evaluation

Lactobacillus plantarum isolated in Example 1 The biofilm formation inhibitory ability of the 200661 strain was evaluated. 900 μL of BHI broth containing 3% (w/v) sucrose was added to a 24 well plate. After incubating Streptococcus mutans KCTC 5124 in BHI broth, washed twice with PBS buffer, and then adjusted to a concentration of 2 Х10 ⁶ CFU/mL in BHI broth (culture solution and supernatant )) 100 μL, 200 μL, 500 μL and 1,000 μL were inoculated into a well plate. At this time, the control group was put in the same amount of physiological saline instead of the sample. After incubation at 37° C. for 24 hours, 200 μL of a 0.1% (w/v) crystal violet solution was added to each well, dyed for 10 minutes, washed three times with distilled water, and dried. After decoloring by putting 100 μL of an acid-alcohol solution, it was transferred to a new 96 well plate and absorbance was measured at 570 nm, and the biofilm formation inhibitory ability was calculated as follows, and the results are shown in FIGS. 2(a) and (b). Indicated:

Biofilm formation inhibitory ability (%) = {1-(absorbance of treatment group/absorbance of control group)} Х 100.

As shown in Figure 2 (a), Lactobacillus plantarum isolated in Example 1 In the case of the sediment (> 100 μL) and the supernatant (> 200 μL) of the 200661 strain, it was confirmed that the inhibitory ability of biofilm formation according to Streptococcus mutans was 72.46% and 98.09% or more, respectively. On the other hand, in the case of the supernatant (100 μL) of the strain Lactobacillus plantarum 200661 isolated in Example 1, it was confirmed that the ability to inhibit biofilm formation according to Streptococcus mutans was only insignificant.

On the other hand, as shown in Figure 2 (b), in the case of the precipitate and supernatant of the Lactobacillus rhamnosus LGG strain known as commercial probiotics, compared to the precipitate and the supernatant of the Lactobacillus plantarum 200661 strain isolated in Example 1, according to Streptococcus mutans. It was confirmed that the biofilm formation inhibitory ability was not effective.

Experimental example 4: PCR Biofilm formation of strains through Inhibitory ability evaluation

The biofilm formation inhibitory ability of the Lactobacillus plantarum 200661 strain isolated in Example 1 was evaluated using PCR. Inoculate Streptococcus mutans KCTC 5124, inoculate new BHI broth at a concentration of 5Х10 ⁵ CFU/mL into each test tube, incubate at 37°C for 24 hours, and inoculate a sample (supernatant of strain) for 12 hours. After culturing, RNA is extracted and converted into cDNA, and then the expression changes of the genes associated with biofilm formation according to Table 4 below using real time PCR It was confirmed, and the results are shown in FIG. 3.

gene Forward primer (5'→3') Reverse primer (3'→5') Sucrose phosphorylase ( gtfA ) AGGAAGTGAAGCGGCCAGT TCAATACGGCCATCCAAATCA Glucosyltransferase B ( gtfB ) AGCAATGCAGCCAATCTAAAT ACGAACTTTGCCGTTATTGTCA Glucosyltransferase-1 ( gtfD ) ACAGCAGACAGCAGCCAAGA ACTGGGTTTGCTGCGTTTG Fructosyltransferase( ftf ) AAATATGAAGGCGGCTACAACG CTTCACCAGTCTTAGCATCCTGAA 16S rRNA, normalizing internal standard (16S rRNA) CCTACGGGAGGCAGCAGTAG CAACACGAGCTTTACGATCCGAAA

As shown in Figure 3, the expression of gtf A, gtf B, gtf D, and ftf of the strain Lactobacillus plantarum 200661 isolated in Example 1 was 10.79%, 8.22%, 11.68% and 22.85%, respectively, known as commercial probiotics. Compared to the Lactobacillus rhamnosus LGG strain, it was found that the degree of inhibition of the expression of genes related to biofilm formation such as gtf A, gtf B, gtf D and ftf was all higher.

Experimental example 5: strain of Glucosyltransferase ( GTFs ) Insoluble glucan formation through inhibition of activity Inhibitory ability

The ability of Lactobacillus plantarum 200661 strain isolated in Example 1 to inhibit the formation of insoluble glucans through inhibition of glucosyltransferase (GTFs) activity was evaluated.

After incubation of Streptococcus mutans KCTC 5124 in BHI broth, centrifugation at 4° C., 500 Хg for 30 minutes to obtain a supernatant. After filtering the supernatant with a 0.2 μm syringe filter, a crude enzyme solution was prepared using an Amicon Ultra Centrifugal filter (MWCO 30 kDa, Millipore). GTFs activity was measured using the absorbance of insoluble glucans produced using sucrose as a substrate. Substrate solution (12.5 g of sucrose and 0.25 g of NaN ₃ dissolved in 0.0625 M potassium phosphate buffer (pH 6.5), 1 L) 800 μL, 20 μL of coenzyme solution, 180 μL of sample (supernatant of strain) were added. The final volume was made to be 1 mL. At this time, the control group was put in the same amount of distilled water instead of the sample. The test tube was inclined by 30° and reacted in an incubator at 37° C. for 30 hours. After the reaction, the supernatant was discarded by centrifugation, 4 mL of distilled water was added, and the insoluble glucan formed by sonication for 10 minutes was dispersed and the absorbance was measured at 550 nm, and the insoluble glucan formation ability was calculated as follows. Is shown in Figure 4:

Insoluble glucan formation ability (%) = (absorbance of treatment group/absorbance of control group) Х 100.

As shown in FIG. 4, the Lactobacillus plantarum 200661 strain isolated in Example 1 had an insoluble glucan formation ability of 52.97% by GTFs, compared to the Lactobacillus rhamnosus LGG strain known as a commercial probiotic, the degree of inhibition of insoluble glucan formation by GTFs All are confirmed to be high.

Hereinafter, an example of the preparation of a composition comprising the strain of the present invention as an active ingredient will be described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation Example 1: Preparation of powder

Lactobacillus plantarum 200661 strain 20 mg

100 mg lactose hydrate

10 mg of talc

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2: Preparation of tablets

Lactobacillus plantarum 200661 Strain 10 mg

100 mg corn starch

100 mg lactose hydrate

2 mg of magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3: Preparation of capsules

Lactobacillus plantarum 200661 strain 10 mg

Microcrystalline cellulose 3 mg

Lactose Hydrate 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above ingredients, the capsules were prepared by filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4: Preparation of injection

Lactobacillus plantarum 200661 Strain 10 mg

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Sodium monohydrogen phosphate 26 mg

After mixing the above ingredients, it was prepared in the amount of the above ingredients per ampoule (2 mL) according to a conventional method for preparing injections.

Formulation Example 5: Preparation of liquid formulation

Lactobacillus plantarum 200661 Strain 10 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

Lemon flavor appropriate amount

The above ingredients are dissolved by adding each ingredient to purified water according to a conventional manufacturing method, added an appropriate amount of lemon flavor, adjusted to 100 mL by adding purified water, sterilized and filled in a brown bottle to prepare a liquid formulation.

Formulation Example 6: Preparation of health functional food

Lactobacillus plantarum 200661 strain 10 mg

Vitamin mixture right amount

Vitamin A acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

Nicotinic acid amide 1.7 mg

Folic acid 50 ㎍

Calcium pantothenate 0.5 mg

Mineral mixture Appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for the health functional food, but it is possible to arbitrarily modify the composition, and the above ingredients are mixed according to the general health functional food manufacturing method. Then, granules are prepared, and can be used in the manufacture of health functional foods according to a conventional method.

Formulation Example 7: Preparation of health drink

Lactobacillus plantarum 200661 strain 10 mg

Vitamin C 15 g

100 g of vitamin E (powder)

19.75 g of iron lactate

Zinc oxide 3.5 g

3.5 g of nicotinic acid amide

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g vitamin B2

Purified water quantification

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used in the manufacture of the health beverage composition of the invention.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

Korea Microorganism Conservation Center (overseas) KCCM12499P 20190419

<110> Konkuk University Industrial Cooperation Corp <120> NOVEL STRAIN OF Lactobacillus plantarum AND USE THEREOF <130> 1065771 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1482 <212> RNA <213> Lactobacillus plantarum <400> 1 gcaatggcta gcgccaagta tgcagttcga acgaactctg gtattgatgt ggtgcttgca 60 tcatgattta catttgagtg agtggcgaac tggtgagtaa cacgtgggaa acctgcccag 120 aagcggggga taacacctgg aaacagatgc taataccgca taacaacttg gaccgcatgg 180 tccgagtttg aaagatggct tcggctatca cttttggatg gtcccgcggc gtattagcta 240 gatggtgggg taacggctca ccatggcaat gatacgtagc cgacctgaga gggtaatcgg 300 ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc 360 acaatggacg aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta 420 aaactctgtt gttaaagaag aacatatctg agagtaactg ttcaggtatt gacggtattt 480 aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540 ttgtccggat ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag 600 ccttcggctc aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca gaagaggaca 660 gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag 720 gcggctgtct ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta 780 gataccctgg tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct 840 tcagtgctgc agctaacgca ttaagcattc cgcctgggga gtacggccgc aaggctgaaa 900 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta 960 cgcgaagaac cttaccaggt cttgacatac tatgcaaatc taagagatta gacgttccct 1020 tcggggacat ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg caacgagcgc aacccttatt atcagttgcc agcattaagt tgggcactct 1140 ggtgagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200 ttatgacctg ggctacacac gtgctacaat ggatggtaca acgagttgcg aactcgcgag 1260 agtaagctaa tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat 1320 gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccatgagaag tttgtaacaa cccaaaaagt cggtggggta 1440 aacctattag gaaccagccg cgctaataga tgtcagagtt cg 1482

Claims (10)

Antibacterial activity against Streptococcus mutans ; And Lactobacillus plantarum strain (KCCM 12499P) having the ability to inhibit biofilm formation.
The method of claim 1,
The biofilm contains insoluble glucan, Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P).
The method of claim 2,
The insoluble glucan is formed from glucosyltransferases (GTFs) produced by Streptococcus mutans , Lactobacillus plantarum strain (KCCM 12499P).
The method of claim 1,
The strain further has acid resistance, bile resistance or intestinal adhesion ability, Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P).
The method of claim 1,
The strain comprises the 16S rRNA nucleotide sequence of SEQ ID NO: 1, Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P).
A probiotic composition comprising Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.
The method of claim 6,
The strain is prepared as a culture medium or culture filtrate, a probiotic composition.
Lactobacillus plantarum ( Lactobacillus plantarum ) a pharmaceutical composition for preventing or treating dental caries or periodontal disease comprising a strain (KCCM 12499P) as an active ingredient.
Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P) containing as an active ingredient dental caries or periodontal disease prevention or improvement health functional food.
A composition for oral cavity containing Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

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