KR102647633B1 - Novel Lacticaseibacillus rhamnosus DM163 strain, and use thereof - Google Patents

Abstract

The present invention provides a Lacticaseibacillus rhamnosus DM163 strain derived from the oral cavity of infants and children, and a composition containing the same with periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects. The strain according to the present invention suppresses the production of inflammatory factors and osteoclasts in immune cells and promotes the growth of beneficial bacteria, while suppressing the growth of harmful bacteria. Therefore, the Lacticaseibacillus rhamnosus DM163 strain according to the present invention or a composition containing it as an active ingredient can be usefully used for preventing periodontitis, inhibiting osteoclasts, proliferating harmful bacteria, and inhibiting harmful bacteria.

Description

Novel Lacticaseibacillus rhamnosus DM163 strain, and use thereof}

The present invention relates to a novel Lactica J Bacillus rhamnosus DM163 strain and its use. More specifically, it is derived from the oral cavity of infants and young children, inhibits the production of inflammatory factors and osteoclasts in immune cells, and promotes the growth of beneficial bacteria. The present invention relates to a novel Lactica J Bacillus rhamnosus DM163 strain that has various effects on inhibiting the growth of periodontitis-causing bacteria and its uses.

Lactic acid bacteria are used in the production of various fermented foods, and as the demand for pharmaceuticals and functional foods made from natural ingredients increases, the importance of probiotics with various functions is being highlighted. Probiotics are microorganisms that improve the balance of intestinal microorganisms and enhance the absorption of nutrients. Lacticaseibacillus rhamnosus has recently been known to have various effects on digestive diseases, periodontal diseases, and anti-inflammatory diseases, and is being studied as a raw material for health functional foods and pharmaceutical compositions.

Republic of Korea Patent No. 10-2502109 relates to “Compositions and health functional foods containing Lactica J Bacillus rhamnosus IDCC 3201 with excellent vitamin B1 production ability”. In a composition with a vitamin B1 production function, the vitamin B1 production ability is Eggplant discloses a composition and health functional food containing Lactica J Bacillus rhamnosus IDCC 3201 [Accession number: ATCC BAA-2836]. The composition and health functional food containing the Lactica J Bacillus rhamnosus IDCC 3201 have the advantage of having excellent vitamin B1 production ability, but the problem is that they do not have various effects such as periodontitis, osteoclast inhibition, prebiotics, and antibacterial effect. There was this.

In addition, Republic of Korea Patent No. 10-2546196 relates to “Lactobacillus reuteri strain with periodontitis treatment effect and use thereof,” deposited under the deposit number (KCTC 14507BP) containing the 16s rDNA sequence represented by SEQ ID NO: 1. Prevention of oral diseases, comprising at least one selected from the group consisting of Lactobacillus reuteri MG5346 strain, its culture medium, and its cell-free supernatant, and characterized by inhibiting oral inflammation and differentiation or activity of osteoclasts. Or a therapeutic composition is disclosed. The Lactobacillus reuteri strain and its use, which have the effect of treating periodontitis, have the advantage of suppressing osteoclast formation and expression of inflammation-related factors and having oral disease prevention and treatment effects, but prebiotics, culture ability, and antibacterial efficacy There was a problem with not having it.

The problem to be solved in the present invention is the novel Lactica J Bacillus rhamnosus, which is derived from the oral cavity of infants and children and has the effect of effectively preventing periodontitis, inhibiting osteoclast formation, promoting the growth of beneficial bacteria, and inhibiting the growth of periodontitis-causing bacteria. ( Lacticaseibacillus rhamnosus ) DM163 strain and a composition containing it that has periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects.

The present invention relates to the Lacticaseibacillus rhamnosus DM163 strain deposited with deposit number KCTC15333BP.

Here, the strain is characterized as originating from the oral cavity of infants and young children.

The strain is characterized by having a periodontitis prevention effect by suppressing inflammatory factors produced in immune cells by Porphyromonas gingivalis lipopolysaccharide.

The strain is characterized by excellent culturing ability.

The strain is characterized by its ability to suppress inflammatory gene expression.

The strain is characterized by having an osteoclastogenesis inhibitory effect.

The strain is characterized by having a probiotic effect that can promote the growth of beneficial bacteria.

The strain is characterized by having antibacterial efficacy.

In addition, the present invention relates to pharmaceutical compositions and food compositions containing the Lacticaseibacillus rhamnosus DM163 strain deposited under deposit number KCTC15333BP as an active ingredient.

The present invention is a novel Lacticaseibacillus rhamnosus that has excellent efficacy in preventing periodontitis and inhibiting osteoclast formation, has excellent culturing ability, promotes the growth of beneficial bacteria, and has the effect of effectively inhibiting the growth of periodontitis-causing bacteria. ) The invention has the effect of providing a DM163 strain and a composition containing it that has periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects.

Figure 1 shows the relative ratio (%) of the 540 nm absorbance for each of the 168 lactic acid bacteria strains when the 540 nm absorbance when treated with Porphyromonas gingivalis lipopolysaccharide alone is set as 100%. This is a graph showing .
Figure 2 is a graph showing the culture ability for each of 11 lactic acid bacteria strains with high periodontitis prevention efficacy among 168 lactic acid bacteria strains.
Figure 3 is a graph showing the efficacy of suppressing mRNA expression of IL-1β and IL-6 genes by the Lacticaseibacillus rhamnosus DM163 strain and the LGG strain.
Figure 4 is a graph showing the efficacy of suppressing osteoclastogenesis by the Lacticaseibacillus rhamnosus DM163 strain and the LGG strain.
Figure 5 is a photograph showing that the Lacticaseibacillus rhamnosus DM163 strain has a prebiotic effect that can promote the growth of the beneficial bacteria, the Limosilactobacillus fermentum DM075 strain.
Figure 6 is a photograph showing that the Lacticaseibacillus rhamnosus DM163 strain does not promote the growth of the harmful Streptococcus mutans KCOM1054 strain.
Figure 7 shows the antibacterial activity of Lacticaseibacillus rhamnosus DM163 strain and Lacticaseibacillus rhamnosus LGG strain against Porphyromonas gingivalis W83 strain cultured on plate medium. This is a photo showing.

The above-described objects, features and advantages of the present invention will become more apparent by describing the preferred embodiments of the present invention in detail and referring to the drawings. The detailed description that follows is not intended to be limiting, and the scope of the present invention is limited only by the appended claims, together with all equivalents to what the claims assert.

The present invention relates to the Lacticaseibacillus rhamnosus DM163 strain deposited with the International Depository on February 22, 2023. Lacticaseibacillus rhamnosus strain is a strain of the genus Lacticaseibacillus. The present invention provides a novel Lacticaseibacillus rhamnosus DM163 strain derived from the oral cavity of infants and young children. The accession number of the Lacticaseibacillus rhamnosus DM163 strain is KCTC15333BP.

In addition, another aspect of the present invention provides a composition having periodontitis, osteoclast inhibition, prebiotics, and antibacterial effects containing the strain as an active ingredient. The composition having periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects of the present invention may be a composition that prevents periodontitis, inhibits osteoclast formation, promotes the growth of beneficial bacteria, but inhibits the growth of periodontitis-causing bacteria. . In this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In addition, another aspect of the present invention is a novel Lacticaseibacillus rhamnosus DM163 strain derived from the oral cavity of infants and young children deposited under the accession number KCTC15333BP, or a periodontitis, osteoclast inhibition, prebiotic and antibacterial agent containing it as an active ingredient. A pharmaceutical composition having efficacy is provided. The “pharmaceutical composition” of the present invention contains, in addition to the active ingredient, a pharmaceutically acceptable carrier or excipient, and is used in topical formulations such as creams, lotions, ointments (semi-solid external medicines), microemulsions, gels, pastes, It can be manufactured as a transdermal formulation (TTS) (e.g. patches, bandages, etc.), sprays, gargles, etc.

Examples of the pharmaceutically acceptable carrier include lactose, glucose, sucrose, starch (e.g. corn starch, potato starch, etc.), cellulose, its derivatives (e.g. sodium carboxymethyl cellulose, ethylcellulose, etc.), malt, gelatin, talc. , solid lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oil (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, etc., One or more suitable carriers may be selected and used depending on the formulation of the pharmaceutical composition of the present invention.

The "pharmaceutical composition with periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects" according to the present invention is a periodontitis, osteoclast inhibition, and prebiotic treatment of the active ingredient, Lacticaseibacillus rhamnosus DM163 strain. Tix may be included in any amount (effective amount) as long as it can exhibit antibacterial efficacy, and a typical effective amount will be determined within the range of 0.001% by weight to 99.900% by weight based on the total weight of the composition. Here, “effective amount” refers to the amount of an active ingredient that can prevent, improve, treat symptoms, or induce a delay in the onset of these symptoms. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art. Terms not specifically defined in this specification follow the meaning of the Korean dictionary or the meaning generally accepted in the art.

The "composition having periodontitis, osteoclast inhibition, prebiotics, and antibacterial effects" and "pharmaceutical composition having periodontitis, osteoclast inhibition, prebiotics, and antibacterial effects" of the present invention include, in a specific embodiment, the formulation. It includes various formulations such as toothpaste, liquid mouthwash, dietary film, mouth spray, or chewing gum, and each composition can be prepared by conventional methods.

When the pharmaceutical composition having periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects of the present invention is formulated into a toothpaste, the toothpaste of the present invention may contain the Lacticaseibacillus rhamnosus DM163 strain. there is. In addition, it may additionally contain wetting agents, thickeners, foaming agents, preservatives, sweeteners, flavorings and other ingredients.

In addition, the present invention provides a food composition containing Lacticaseibacillus rhamnosus DM163 strain or the same as an active ingredient and having periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects.

In a specific embodiment, the "food composition with periodontitis, osteoclast inhibition, prebiotic, and antibacterial effects" of the present invention is used in various forms such as health functional foods, nutritional supplements, health supplements, food additives, or functional beverages. All formulations are included, and each composition can be prepared by conventional methods.

When the composition of the present invention is considered a food composition, it can be used in beverages such as juice, carbonated beverages, and isotonic drinks, processed oils such as milk and yogurt, foods such as gums, rice cakes, Korean snacks, breads, confectionery, and noodles, powders, and tablets. It can be food preparations in the form of capsules, etc.

The food composition of the present invention may contain sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.

Hereinafter, the present invention will be described in more detail with reference to the attached drawings and examples.

Example

Collection of samples and preparation of strains

Example 1. Sample collection

Tongue samples were collected from 20 infants and 100 adults who visited Ilsan Apple Tree Dental Hospital, placed in an anaerobic jar, and maintained under a specific anaerobic condition.

Example 2. Isolation of lactic acid bacteria

To isolate lactic acid bacteria from the tongue sample of Example 1, an obligately anaerobic LBS (Lactobacillus Selective) medium was prepared and used. At this time, the anaerobic LBS medium was prepared by removing oxygen present in the LBS medium using N ₂ . 200 μl of the tongue sample collected in Example 1 and maintained in an anaerobic state was suspended in 2 ml of LBS liquid medium, and then 100 μl each was plated on LBS plate medium and cultured anaerobically at 37°C for 2 days. A total of 168 lactic acid bacteria strains, 39 and 129, respectively, were isolated from infant and adult tongue coating, suspended in LBS liquid medium containing 20% (v/v) glycerol, and stored at -70°C for the following experiments. used.

Example 3. Identification of lactic acid bacteria

To identify the 168 lactic acid bacteria strains of Example 2, the whole genomic DNA of each of the 168 lactic acid bacteria strains was extracted, and 785F (GGATTAGATACCCTGGTA) and 907R ( CCGTCAATTCMTTTRAGTTT) primers and PCR equipment were used. The results were entered into BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov) software to identify the species names of all 168 lactic acid bacteria strains.

Analysis

1. Identification results of 168 lactic acid bacteria strains

As a result of identifying all 168 lactic acid bacteria strains in Example 3, they were classified into 14 species. The results are shown in Table 1 below.

Table 1 above shows the species names and numbers of 168 beneficial bacteria isolated and identified from the oral cavity of infants, children, and adults. As can be seen in Table 1 above, 39 isolates from infant tongue coating were classified into 7 species, and 129 isolates from adult tongue coating were classified into 12 species.

2. Periodontitis prevention efficacy of 168 lactic acid bacteria strains

Among 168 lactic acid bacteria strains, those with high efficacy in preventing periodontitis were screened. RAW 264.7 cells, a mouse macrophage cell line, were cultured in a 96 well culture dish using DMEM medium containing 10% FBS for 2 days. Each of the 168 lactic acid bacteria strains cultured anaerobically for 1 day at 37°C was treated at 100°C for 30 minutes and then treated with RAW 264.7 cells at 5×10 ⁷ cells each. Two hours later , Porphyromonas gingivalis lipopolysaccharide was treated at a concentration of 1 μg/ml for 24 hours. 50 μl of RAW 264.7 cell culture was reacted with 50 μl of a 1:1 mixture of 1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, and the absorbance at 540 nm was measured 30 minutes later.

Figure 1 shows the relative ratio (%) of the 540 nm absorbance for each of the 168 lactic acid bacteria strains when the 540 nm absorbance when treated with Porphyromonas gingivalis lipopolysaccharide alone is set as 100%. This is a graph showing . As shown in Figure 1, among the 168 lactic acid bacteria strains, 11 lactic acid strains indicated in a red graph reduced the absorbance at 540 nm to less than 60% compared to treatment with Porphyromonas gingivalis lipopolysaccharide alone. I was able to confirm what I ordered. These results showed that 11 of 168 lactic acid bacteria strains were effective in preventing periodontitis.

3. Cultivation ability of 11 lactic acid bacteria strains with high efficacy in preventing periodontitis

Among the 168 lactic acid bacteria strains, the culture ability was measured for each of the 11 lactic acid bacteria strains with high periodontitis prevention efficacy. The results of confirming the cultivation ability of 11 lactic acid bacteria strains using the production facilities of Mediogen Co., Ltd., which is well equipped with a manufacturing process for commercialization of lactic acid bacteria, are shown graphically in Figure 2.

Figure 2 is a graph showing the culture ability for each of 11 lactic acid bacteria strains with high periodontitis prevention efficacy among 168 lactic acid bacteria strains. As shown in Figure 1, it was confirmed that the culture capacity of the DM163 strain was 9.8 billion/ml, which was the highest compared to other lactic acid bacteria strains. DM163 is a Lacticaseibacillus rhamnosus strain derived from the oral cavity of infants and young children according to the present invention.

4. Lactica J Bacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Inhibitory efficacy of inflammatory gene expression in DM163 strain

The efficacy of the Lacticaseibacillus rhamnosus DM163 strain according to the present invention to suppress inflammatory gene expression was confirmed. RAW 264.7 cells, a mouse macrophage cell line, were cultured in a 12 well culture dish using DMEM medium containing 10% FBS for 2 days. In this experiment, the LGG strain, known to have excellent efficacy in suppressing inflammatory gene expression, was used as a comparison group for the Lacticaseibacillus rhamnosus DM163 strain according to the present invention. Lacticaseibacillus rhamnosus DM163 strain and Lacticaseibacillus rhamnosus LGG strain, cultured anaerobically at 37°C for 1 day, were each treated at 100°C for 30 minutes, then 5×10 cells were added to RAW 264.7 cells. ^Eight pieces were processed each. Two hours later , Porphyromonas gingivalis lipopolysaccharide was treated at a concentration of 1 μg/ml for 24 hours. The mRNA expression levels for each of the representative inflammatory genes, IL-1β and IL-6, are shown in Figure 3.

Figure 3 is a graph showing the efficacy of suppressing mRNA expression of IL-1β and IL-6 genes by the Lacticaseibacillus rhamnosus DM163 strain and the LGG strain. As shown in Figure 3, when the case of treatment with Porphyromonas gingivalis lipopolysaccharide alone is set to 1, both IL-1β and IL-6 genes are produced more by the DM163 strain than by the LGG strain. It was confirmed that mRNA expression was significantly reduced. In the case of IL-1β gene mRNA expression, the LGG strain reduced it by 96% and the DM163 strain reduced it by 99%, and in the case of IL-6 gene mRNA expression, the LGG strain reduced it by 20% and the DM163 strain reduced it by 60%.

5. Lactica J Bacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Osteoclastogenesis inhibitory effect of DM163 strain

The efficacy of the Lacticaseibacillus rhamnosus DM163 strain according to the present invention to inhibit osteoclastogenesis was confirmed. To differentiate RAW 264.7 cells, a mouse macrophage cell line, into osteoclasts, they were cultured in a 96 well culture dish for 2 days using DMEM medium containing 10% FBS, 50 ng/ml M-CSF, and 100 ng/ml RANKL. . In this experiment, the LGG strain, known to have excellent osteoclastogenesis inhibition efficacy, was used as a comparison group for the Lacticaseibacillus rhamnosus DM163 strain according to the present invention. At this time, the Lacticaseibacillus rhamnosus DM163 strain and the LGG strain Lacticaseibacillus rhamnosus , which were cultured anaerobically for 1 day at 37°C, were each treated at 100°C for 30 minutes, and then 5 to RAW 264.7 cells. ×10 ⁷ pieces were processed together. To measure the activity of tartrate-resistant acid phosphatase (TRAP), an osteoclast biomarker, 4-nitrophenyl phosphate disodium (pNPP) was used as a substrate. The results are shown in Figure 4.

Figure 4 is a graph showing the efficacy of suppressing osteoclastogenesis by the Lacticaseibacillus rhamnosus DM163 strain and the LGG strain. As shown in Figure 4, when TRAP activity (PBS RANKL) when treated with RANKL alone was set to 100%, the LGG strain (LGG RANKL) reduced it by 52% and the DM163 strain (DM 163 RANKL) reduced it by 84%. I was able to confirm.

6. Lactica J Bacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Prebiotic efficacy of DM163 strain

It was confirmed whether the Lacticaseibacillus rhamnosus DM163 strain has a prebiotic effect that can promote the growth of beneficial bacteria. Nutritionally normal medium mixed with 10% sugar and 1% yeast extract, nutrient-depleted medium mixed with 10% sugar and 0.05% yeast extract, and 0.2% DM163 strain (treated at 100°C for 30 minutes) added to the nutrient-depleted medium. One badge was produced. The results of inoculating three different media with different numbers of domestically patented strains of Limosilactobacillus fermentum DM075 (No. 10-2500747) and Streptococcus mutans KCOM1054. They are shown in Figures 5 and 6, respectively.

Figure 5 is a photograph showing that the Lacticaseibacillus rhamnosus DM163 strain has a prebiotic effect that can promote the growth of the beneficial bacteria, the Limosilactobacillus fermentum DM075 strain. As shown in Figure 5, compared to the nutrient-normal medium, no more than 10,000 beneficial bacteria were cultured in the nutrient-depleted medium, but 0.2% of Lacticaseibacillus rhamnosus DM163 strain was added to the nutrient-depleted medium. It was confirmed that less than 10,000 beneficial bacteria grew well again.

Figure 6 is a photograph showing that the Lacticaseibacillus rhamnosus DM163 strain does not promote the growth of the harmful Streptococcus mutans KCOM1054 strain. As shown in Figure 6, compared to the nutrient-normal medium, no more than 10,000 harmful bacteria were cultured in the nutrient-depleted medium, and 0.2% of the Lacticaseibacillus rhamnosus DM163 strain was added to the nutrient-depleted medium. It was confirmed that adding less than 10,000 harmful bacteria did not grow again. Rather, by adding 0.2% of the Lacticaseibacillus rhamnosus DM163 strain, it was confirmed that not even 100,000 harmful bacteria grew.

7. Lactica J Bacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Antibacterial efficacy of DM163 strain

It was confirmed whether the Lacticaseibacillus rhamnosus DM163 strain has antibacterial activity against the Porphyromonas gingivalis strain. Porphyromonas gingivalis ( Porphyromonas gingivalis ) W83 strain 10 ⁶ were plated on BHI (brain heart infusion) plate medium, and Lacticaseibacillus rhamnosus was cultured anaerobically in MRS liquid medium at 37°C for 1 day. ) 3㎕ of DM163 strain was dropped onto the plate medium on which the pathogen was smeared. By culturing anaerobically at 37°C for 3 days, it was confirmed that the Lacticaseibacillus rhamnosus DM163 strain inhibited the growth of the Porphyromonas gingivalis strain. The results are shown in Figure 7.

Figure 7 shows the antibacterial activity of Lacticaseibacillus rhamnosus DM163 strain and Lacticaseibacillus rhamnosus LGG strain against Porphyromonas gingivalis W83 strain cultured on plate medium. This is a photo showing. As shown in Figure 7, it was confirmed that the growth of the Porphyromonas gingivalis W83 strain was suppressed around the Lacticaseibacillus rhamnosus DM163 strain, creating a transparent ring. On the other hand, the LGG strain of Lacticaseibacillus rhamnosus was previously known to have a good inhibitory effect on the strain of Porphyromonas gingivalis, but in this experiment, it was better than the strain of Lacticaseibacillus rhamnosus DM163. It was confirmed that the inhibitory effect against Porphyromonas gingivalis strain was weak.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present application can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it will be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of protection of this application should be construed as including all changes or modified forms that can be derived from the meaning and scope of the patent claims and their equivalent concepts, which are described later, rather than the detailed description described above, as being included in the scope of rights of this application.

Korea Biological Resources Center KCTC15333BP 20230222

Claims (10)

Lacticaseibacillus rhamnosus DM163 strain deposited with accession number KCTC15333BP. The Lacticaseibacillus rhamnosus DM163 strain according to claim 1, wherein the strain is derived from the oral cavity of infants and young children. The method of claim 1, wherein the strain is Lactica J Bacillus rhamno, characterized in that it has a periodontitis prevention effect by suppressing inflammatory factors produced in immune cells by Porphyromonas gingivalis lipopolysaccharide. sus ( Lacticaseibacillus rhamnosus ) DM163 strain. The Lacticaseibacillus rhamnosus DM163 strain according to claim 1, wherein the strain has excellent culturing ability. The Lacticaseibacillus rhamnosus DM163 strain according to claim 1, wherein the strain has the effect of suppressing inflammatory gene expression. The Lacticaseibacillus rhamnosus DM163 strain according to claim 1, wherein the strain has an osteoclastogenesis inhibitory effect. The Lacticaseibacillus rhamnosus DM163 strain according to claim 1, wherein the strain has a probiotic effect capable of promoting the growth of beneficial bacteria. The Lacticaseibacillus rhamnosus DM163 strain according to claim 1, wherein the strain has antibacterial efficacy. delete A food composition containing the Lacticaseibacillus rhamnosus DM163 strain deposited with deposit number KCTC15333BP as an active ingredient.