KR102320379B1 - NOVEL STRAIN OF Lactobacillus plantarum AND USE THEREOF - Google Patents

Abstract

The present invention provides an antibacterial activity against Streptococcus mutans; And Lactobacillus plantarum having a biofilm formation inhibitory ability ( Lactobacillus plantarum ) strain (KCCM 12499P); a probiotic composition comprising it as an active ingredient; a composition for preventing, improving or treating dental caries or periodontal disease; and oral compositions.

Description

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

The present invention provides an antibacterial activity against Streptococcus mutans; And it relates to a novel Lactobacillus plantarum strain having the ability to inhibit biofilm formation and uses thereof.

Dental caries or periodontal disease is an oral disease caused by oral bacteria that is experienced by people of all ages. 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. to increase This specific interaction is problematic in enhancing the pathogenic potential of oral bacteria.

At this time, Streptococcus mutans is a representative oral bacterium, and has a great influence on the formation of a biofilm in which the microbial community in the oral cavity proliferates. Streptococcus mutans produces glucosyltransferases (GTFs) that synthesize intracellular polysaccharides and extracellular polysaccharides (EPS) using sucrose as a substrate. The insoluble glucan formed from GTFs has very strong adhesion to the surface of the oral cavity, providing a binding site for other oral bacteria with poor adhesion and facilitating the formation of biofilms. By suppressing Streptococcus mutans in the colony, 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 microbial flora when ingested and provide beneficial activities in the host's physiological activity. it is necessary.

The present invention provides an antibacterial activity against Streptococcus mutans; And Lactobacillus plantarum ( Lactobacillus plantarum ) strain (KCCM 12499P) and the like having a biofilm formation inhibitory ability.

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

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

The biofilm may include insoluble glucan.

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

The strain may further 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, Lactobacillus plantarum ( Lactobacillus plantarum ) It provides a probiotic composition comprising a strain (KCCM 12499P) as an active ingredient.

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

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

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

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

Lactobacillus plantarum according to the present invention ( Lactobacillus plantarum ) strain (KCCM 12499P) has excellent probiotic properties, Streptococcus mutans ( Streptococcus mutans ) Antibacterial activity against; And since it has the ability to inhibit biofilm formation, 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 shows a phylogenetic diagram of the Lactobacillus plantarum 200661 strain isolated in Example 1.
Figure 2 (a) is a graph showing the biofilm formation inhibitory ability in the case of the precipitate and the supernatant of the Lactobacillus plantarum 200661 strain isolated in Example 1, Figure 2 (b) is the precipitate and supernatant of the Lactobacillus rhamnosus LGG strain known as commercial probiotics In the case of , it is a graph showing the ability to inhibit biofilm formation.
3 shows the supernatant (□) of the Lactobacillus plantarum 200661 strain isolated in Example 1 and the supernatant (■) of the Lactobacillus rhamnosus LGG strain known as commercial probiotics, biofilm formation-related genes ( gtf A, gtf B, gtf D and ftf ) is a graph showing the expression level.
4 is a graph showing the insoluble glucan-forming ability of the supernatant (□) of the Lactobacillus plantarum 200661 strain isolated in Example 1 and the supernatant (■) of the Lactobacillus rhamnosus LGG strain known as commercial probiotics.

The present inventors have Lactobacillus with probiotic properties plantarum Among the strains, antibacterial activity against Streptococcus mutans; and Lactobacillus plantarum having a biofilm formation inhibitory ability 200661 By isolating and identifying the strain, and evaluating its efficacy, the present invention was completed.

As used herein, the term "biofilm" refers to a structure formed in the oral cavity, also referred to as plaque. It is composed of oral bacteria and metabolites formed due to the proliferation of oral bacteria, and acts as a major cause of dental caries or periodontal disease.

Hereinafter, the present invention will be described in detail.

The present invention provides an antibacterial 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, a slightly aerobic strain, and one of the most widely distributed lactic acid bacteria in nature. Lactobacillus plantarum is a typical kimchi lactic acid bacterium that undergoes normal lactic acid fermentation and produces lactic acid by fermenting arabinose, glucose, fructose, galactose, maltose, sucrose, and dextran. It is mainly isolated from dairy products, pickles, pickles, tomatoes, etc., and especially in the late stage of kimchi fermentation, when kimchi is fermented and sour, it mainly grows and dominates, inhibiting the growth of other species. It is known to have excellent anti-corrosion resistance.

The phylogenetic 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, it is confirmed that the strain (KCCM 12499P) has the 16S rRNA base sequence of SEQ ID NO: 1, deposited at the Korea Microorganism Conservation Center on April 19, 2019 Therefore, it was given accession number KCCM 12499P.

In addition, the strain (KCCM 12499P) is characterized by excellent antibacterial activity against oral bacteria, particularly, Streptococcus mutans among Lactobacillus plantarum strains. At this time, Streptococcus mutans ( Streptococcus mutans ) is a major oral bacterium that causes dental caries or periodontal disease, decomposes organic matter to discharge acid, and causes a phenomenon in which teeth are demineralized by this acid. Specifically, the strain (KCCM 12499P) is characterized in that it has an excellent antibacterial effect on Streptococcus mutans compared to the Lactobacillus rhamnosus LGG strain known as commercial probiotics.

In addition, the strain (KCCM 12499P) is characterized in that it has a biofilm formation inhibitory ability. In this case, the biofilm may include insoluble glucan, but using sucrose as a substrate, and 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. This biofilm formation inhibitory ability can be confirmed through the expression change 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 can be 90% or more (preferably, 100% or more) of the viability of the strain when reacting for 3 hours at pH 2.5, and the bile resistance is the viability of the strain when reacting for 24 hours under 0.3% oxgall conditions. It may be 90% or more (preferably, 10% or more).

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

As for the strain (KCCM 12499P) as described above, the strain (KCCM 12499P) may be prepared as a culture solution 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 comprising Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

As for the strain (KCCM 12499P) as described above, the strain (KCCM 12499P) may be prepared as a culture solution 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 comprising Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

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

As used herein, "dental caries" refers to diseases such as tooth decay in which hemorrhoids are destroyed by acid produced by oral bacteria attached to the tooth surface, and "periodontal disease" in the present specification refers to diseases surrounding the teeth. It refers to diseases that appear in the tissues surrounding the teeth, such as the gingiva, periodontal ligaments, and alveolar bone, and all these diseases are caused by the formation of biofilms together with oral bacteria.

The pharmaceutical composition according to the present invention may be formulated in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. and may include suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions for formulation.

The carrier or excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, unknown. 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 commonly used diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants.

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

Liquid formulations for oral use include suspensions, 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 preparations, suspensions, emulsions, lyophilized preparations, and suppositories. As a non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerol gelatin and the like can be used.

A preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, 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 administered in several divided doses. 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 rats, 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 Lactobacillus plantarum strain (KCCM 12499P) as an active ingredient.

As for the strain (KCCM 12499P) as described above, the strain (KCCM 12499P) may be prepared as a culture solution 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 for 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 may be appropriately determined according to each purpose of use, such as prevention, health or treatment.

The formulation of health functional food may be in the form of powder, granules, pills, tablets, and capsules, as well as in the form of general food or beverages.

There is no particular limitation on the type of food, and examples of food to which the substance 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, vitamin complexes, and the like, and may include all foods in a conventional sense.

In general, when preparing 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 the raw material. However, in the case of long-term ingestion for health and hygiene purposes or for health control purposes, the amount may be less than or equal to the above range.

Beverages among health functional foods according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like 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 its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, and carbonated beverages used in carbonated beverages. In addition, the health functional food according to the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the health functional food according to the present invention.

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

As for the strain (KCCM 12499P) as described above, the strain (KCCM 12499P) may be prepared as a culture solution 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, granules, tablets, capsules, creams, transparent gels or translucent gels. That is, the composition for oral use 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 the advantage of minimizing the side effects of conventional oil pulling. For the formulation, one or more additives selected from the group consisting of suitable abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, medicinal agents, fragrances, acidity regulators and brighteners commonly used in the preparation of pharmaceutical compositions may be additionally included. 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 composition for oral cavity, 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 wetting agent 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.

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

The foaming agent includes anionic surfactants such as sodium lauryl sulfate and sodium lauryl sarcosinate, and a group of nonionic surfactants consisting of sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene-polyoxypropylene copolymer, and the like. Or it may be one or more selected from the group of amphoteric surfactants such as cocoidopropyl betaine, preferably sodium lauryl sulfate.

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

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

The active agents include sodium fluoride, sodium fluorophosphate, ferrous fluoride, amine fluoride, chlorhexidine, aminocaproic acid, dipotassium glycyrrhizinate, tranexamic acid, allotoins, caproic acids, polyphosphates, enzymes and herbal extracts. It may be at least one selected from the group consisting of, preferably sodium fluoride.

As the fragrance, an appropriate amount of a natural fragrance such as peppermint oil and spearmint oil and a synthetic fragrance such as menthol and carbon is mixed in an appropriate amount.

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

As described above, the Lactobacillus plantarum strain (KCCM 12499P) according to the present invention has excellent probiotic properties, while Streptococcus mutans ( Streptococcus mutans ) Antibacterial activity; And since it has the ability to inhibit biofilm formation, 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, preferred examples are presented to help the understanding of the present invention. However, the following examples are only 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 Isolation and identification of strains

In order to isolate the new strain, the homemade cabbage kimchi broth was sampled and used as a sample. The cabbage kimchi broth was smeared on LBS agar medium using a 10-fold dilution method, and a new strain was isolated by observing the colony morphology after plated and cultured several times. 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 the Genbank database. The new strain was identified as Lactobacillus plantarum 200661 strain, and the similarity was 99%. In addition, using the Mega 7 program, homology was analyzed and a phylogenetic tree was obtained. At this time, the Lactobacillus plantarum 200661 strain was deposited with the Korea Microorganism Conservation Center on April 19, 2019 and was given an accession number KCCM 12499P.

Afterwards, 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 (5,000Хg, 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 It was the same as that of the 200661 strain.

Experimental example 1: of the strain probiotic characterization

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

(One) acid resistance and bile tolerance evaluation

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

process Bacteria (Log CFU/mL) Example 1 Comparative Example 1 Initial Inoculation Bacteria 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 is about 100%, and the bile resistance is about 100.66%, which is confirmed to be all higher than the Lactobacillus rhamnosus LGG strain known as commercial probiotics.

(2) tenon ability evaluation

Lactobacillus plantarum isolated in Example 1 To evaluate the intestinal adhesion ability of the 200661 strain, the HT-29 cell line, a human-derived intestinal epithelial cell, ^{was inoculated in a 24-well plate at a concentration of 2Х10 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 non-attached strains, lysate the adherent cells with 1% Triton X-100 solution, and spread the lysate diluted with an appropriate multiple on MRS agar medium, then plated at 25°C. By measuring the number of adherent bacteria by culturing for 24 hours, the intestinal adhesion ability was evaluated as follows, and the results are shown in Table 2 below:

Longitudinal adhesion (%) = (Number of adherent bacteria (Log CFU/mL) / Number of inoculated bacteria (Log CFU/mL)) × 100.

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

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

Therefore, Lactobacillus plantarum isolated in Example 1 The 200661 strain is excellent in all of the probiotic properties such as acid resistance, dryness resistance and intestinal adhesion ability, so it can be seen as a beneficial lactic acid bacteria for the human body.

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

Lactobacillus plantarum isolated in Example 1 In order to evaluate the antibacterial activity against Streptococcus mutans of the 200661 strain, the minimum inhibitory concentration was used. In a 96-well plate, three kinds of Streptococcus mutans ^{were diluted to a concentration of 1Х10 5} CFU/mL, and a sample (supernatant of the 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 culturing for 2 hours, absorbance was measured at 570 nm to confirm the degree of growth inhibition of three kinds of Streptococcus mutans, and the first concentration that inhibited the growth of three kinds of Streptococcus mutans was used as the minimum inhibitory concentration, and the results Table 3 shows.

process 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 of the 200661 strain on three kinds of Streptococcus mutans is 6.25 to 12.5%, and it is confirmed that the antibacterial activity against all three kinds of Streptococcus mutans is excellent compared to the Lactobacillus rhamnosus LGG strain known as commercial probiotics. .

Experimental example 3: Formation of biofilm of the strain deterrent 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. Streptococcus mutans KCTC 5124 was cultured in BHI broth, washed twice with PBS buffer ^{, and samples adjusted to a concentration of 2Х10 6} CFU/mL in BHI broth (culture solution) and supernatant (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 0.1% (w/v) crystal violet solution was added to each well, stained for 10 minutes, washed 3 times with distilled water, and dried. After decolorization by adding 100 μL of 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 inhibition 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 precipitate (> 100 μL) and the supernatant (> 200 μL) of the 200661 strain, it is confirmed that the biofilm formation inhibitory ability according to Streptococcus mutans is 72.46% and 98.09% or more, respectively. On the other hand, in the case of the supernatant (100 μL) of the Lactobacillus plantarum 200661 strain isolated in Example 1, it is confirmed that the biofilm formation inhibitory ability according to Streptococcus mutans is 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, Streptococcus mutans It is confirmed that the ability to inhibit biofilm formation is not effective.

Experimental example 4: PCR Formation of biofilms of strains through deterrent evaluation

The biofilm formation inhibitory ability of the Lactobacillus plantarum 200661 strain isolated in Example 1 was evaluated using PCR. Streptococcus mutans KCTC 5124 was inoculated and inoculated ^{into each test tube at a concentration of 5Х10 5} CFU/mL in fresh BHI broth and cultured for 24 hours at 37° C. After culturing, RNA was extracted and converted to cDNA, and the expression change of biofilm formation-related genes according to Table 4 below was measured using real-time PCR. 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

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

Experimental example 5: of the strain Glucosyltransferase ( GTFs ) formation of insoluble glucan through activity inhibition deterrent

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

Streptococcus mutans KCTC 5124 was cultured in BHI broth, then centrifuged at 4° C., 500Хg, 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 absorbance of insoluble glucan produced using sucrose as a substrate. 800 μL of 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), 20 μL of crude enzyme solution, and 180 μL of sample (supernatant of the strain) were added. The final volume was made to 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 about 30° and reacted for 30 hours in an incubator at 37°C. After the reaction, the supernatant was discarded by centrifugation, 4 mL of distilled water was added, the insoluble glucan formed by sonication for 10 minutes was dispersed, and the absorbance was measured at 550 nm, and the insoluble glucan forming ability was calculated as follows, and the result is shown in Figure 4:

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

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

Hereinafter, a formulation example 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 the present invention, but merely to describe it in detail.

Formulation Example 1: Preparation of powder

Lactobacillus plantarum 200661 strain 20 mg

Lactose hydrate 100 mg

talc 10 mg

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

Formulation Example 2: Preparation of tablets

Lactobacillus plantarum 200661 strain 10 mg

100 mg cornstarch

Lactose hydrate 100 mg

2 mg magnesium stearate

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

Formulation Example 3: Preparation of capsules

Lactobacillus plantarum 200661 strain 10 mg

Microcrystalline Cellulose 3 mg

Lactose hydrate 14.8 mg

0.2 mg magnesium stearate

After mixing the above ingredients, the capsules were prepared by filling in gelatin capsules according to a conventional manufacturing method of capsules.

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, the content of the above components per 1 ampoule (2 mL) was prepared according to a conventional method for preparing injections.

Formulation Example 5: Preparation of liquid formulation

Lactobacillus plantarum 200661 strain 10 mg

10 g isomerized sugar

5 g of mannitol

Purified water appropriate amount

Lemon flavored amount

Dissolve the above components by adding each component to purified water according to a conventional manufacturing method, add an appropriate amount of lemon flavor, add purified water to adjust the total volume to 100 mL, sterilize, and fill a brown bottle to prepare a solution.

Formulation Example 6: Preparation of health functional food

Lactobacillus plantarum 200661 strain 10 mg

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg folic acid

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 monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

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

Formulation Example 7: Preparation of health drinks

Lactobacillus plantarum 200661 strain 10 mg

15 g vitamin C

100 g vitamin E (powder)

19.75 g of iron lactate

3.5 g zinc oxide

3.5 g of nicotinic acid amide

0.2 g vitamin A

0.25 g of vitamin B1

Vitamin B2 0.3g

Purified water quantitative

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

Although the composition ratio is prepared by mixing ingredients suitable for comparatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and national preferences such as the demanding class, the demanding country, and the use.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms 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 restrictive.

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 having a biofilm formation inhibitory ability, additionally having acid resistance, bile resistance or intestinal adhesion ability, and Lactobacillus plantarum containing the 16S rRNA nucleotide sequence of SEQ ID NO: 1 200661 strain (KCCM 12499P).
According to claim 1,
The biofilm contains insoluble glucan, Lactobacillus plantarum ( Lactobacillus plantarum ) 200661 strain (KCCM 12499P).
3. The method of claim 2,
The insoluble glucan is Streptococcus mutans ( Streptococcus mutans ) glucosyltransferases (glucosyltransferases; GTFs) produced by, Lactobacillus plantarum ( Lactobacillus plantarum ) 200661 strain (KCCM 12499P).
delete delete A probiotic composition comprising the Lactobacillus plantarum 200661 strain (KCCM 12499P) comprising the 16S rRNA nucleotide sequence of SEQ ID NO: 1 as an active ingredient.
delete delete Health functional food for dental protection comprising the Lactobacillus plantarum 200661 strain (KCCM 12499P) containing the 16S rRNA base sequence of SEQ ID NO: 1 as an active ingredient.
An oral composition comprising the Lactobacillus plantarum 200661 strain (KCCM 12499P) comprising the 16S rRNA base sequence of SEQ ID NO: 1 as an active ingredient.

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