KR102095339B1 - Novel lactobacillus reuteri and composition for preventing or treating periodontal diseases comprising the same - Google Patents

Abstract

The present invention relates to a composition for preventing or treating periodontal disease comprising Lactobacillus luterian AN417 strain and the same as an active ingredient. The Lactobacillus luterian AN417 strain according to the present invention exhibits excellent anti-life and growth inhibitory activity against co-bacteria, decaying causative bacteria and periodontal disease causative bacteria, compared to conventional strains known to inhibit periodontal disease. In addition, as a result of determining the genomic sequence, the strain is considered to be a new strain with 99.67% homology to the standard strain and 16S rRNA gene sequence. Therefore, the composition comprising the Lactobacillus luterian AN417 strain or a culture medium thereof as an active ingredient can be usefully used for the prevention or treatment of periodontal disease.

Description

Novel LACTOBACILLUS REUTERI AND COMPOSITION FOR PREVENTING OR TREATING PERIODONTAL DISEASES COMPRISING THE SAME}

The present invention relates to the identification of a new Lactobacillus lutery strain and its use.

Recently, due to incorrect eating habits and aging, immunity decreases and nutritional and metabolic disorders increase, and the number of patients with periodontal disease is increasing. The direct cause of periodontal disease is plaque, a bacterial membrane that is continuously formed on the teeth and plaque formed by hardening without being removed. When plaque and tartar accumulate, the gums come off the teeth, causing inflammation between the teeth and the gums.

Periodontal disease is divided into gingivitis and periodontitis according to the degree of inflammation. Gingivitis causes redness of the gums and signs of bleeding, but can be recovered to some extent by careful brushing. In periodontitis, inflammation is more advanced in gingivitis. In the case of periodontitis, bad breath occurs, pus comes out between the teeth and gums, and discomfort occurs during mastication. In addition, symptoms of shaking teeth may appear.

The most basic way to prevent and treat periodontal disease is to remove bacterial plaque and plaque in the oral cavity through brushing and scaling to avoid creating a favorable environment for bacterial growth. In addition, there are medications such as gum treatment medicines, ferns, special medications placed between gums and teeth, and antibiotics.

On the other hand, health functional foods and therapeutic agents having the effect of preventing or treating periodontal disease have been developed. In particular, recently, it has been reported that lactic acid bacteria are effective in preventing periodontal disease by inhibiting the causative agent of periodontal disease in the oral cavity (KR 10-1690090 B1), and strains showing better anti-living properties against periodontal disease bacteria have been developed.

Republic of Korea Registered Patent 10-1690090

Thus, the present inventors identified a novel Lactobacillus bacterium in a pig small intestine while studying a novel strain that exhibits superior anti-bioactivity compared to a strain that has been used for the treatment of periodontal disease. The novel Lactobacillus reuteri strain was found to have better antibacterial activity against periodontal disease causative bacteria than KCTC 3594 and KCTC 3678, which are known to inhibit periodontal disease bacteria, and completed the present invention.

An object of the present invention is to provide a novel L. reuteri strain and treatment for periodontal disease using the same.

In order to achieve the above object, the present invention provides an L. reuteri AN417 strain deposited with accession number KCTC13231BP.

In order to achieve the other object, the present invention provides a composition for preventing or treating periodontal disease comprising L. reuteri AN417 strain or a culture medium thereof as an active ingredient.

The L. reuteri AN417 strain according to the present invention exhibits better anti-life and growth inhibitory activity against co-bacteria, caries-causing bacteria, and periodontal disease-causing bacteria than foreign strains known to inhibit periodontal disease bacteria. In addition, the 16S rRNA sequence was judged as a novel strain showing 99.67% homology with the standard strain. Therefore, the composition comprising the L. reuteri AN417 strain or a culture medium thereof as an active ingredient may be usefully used for the prevention or treatment of periodontal disease.

Figure 1a is Escherichia cultured in a plate medium coli ( E. coli ) It shows the antibacterial activity of L. reuteri standard strain and L. reuteri AN417 strain against bacteria.
Figure 1b is Pseudomonas cultured in a plate medium shows the antimicrobial activity of the L. reuteri strains and L. reuteri strain AN417 on aeruginasa (P. aeruginasa) bacteria.
Figure 1c shows the antibacterial activity of L. reuteri standard strain and L. reuteri AN417 strain against Streptococcus mutans ( S. mutans ) bacteria cultured in plate medium.
Figure 2a using a spectrophotometer (spectrophotometer), the periodontal disease causative agent, Porphyromonas Gingivalis ( Porphyromonas) gingivalis ) and the antibacterial activity of L. reuteri standard strain and L. reuteri AN417 strain.
Figure 2b using a spectrophotometer (spectrophotometer), the periodontal disease causative agent, Tannerella posidensis ( Tannerella forsythensis ) and the antibacterial activity of the L. reuteri standard strain and L. reuteri AN417 strain.
Figure 3 shows the viability of L. reuteri standard strain and L. reuteri AN417 strain in acidic medium (pH 2 and 3) and 0.5% bile acid medium.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides an L. reuteri AN417 strain deposited with accession number KCTC13231BP.

L. reuteri is a genus Lactobacillus found primarily in the gastrointestinal tract and small intestine of animals. It is also often found in the birth channels, breast milk, and mouths of healthy animals, including humans. L. reuteri is known to have antibiotic activity. The present invention provides a novel strain L. reuteri AN417 from pigs. The deposit number of L. reuteri AN417 is KCTC13231BP.

In addition, one aspect of the present invention provides an antimicrobial composition comprising the strain or a culture medium thereof as an active ingredient.

In the present invention, "culture medium" may be a medium obtained after culturing the L. reuteri AN417 strain. The collection time of the culture medium may be an exponential growth phase or a stagnation phase among the microbial growth curves, but is not limited thereto. In addition, the culture medium may be filtered and used.

The antimicrobial composition of the present invention is E. coli , P. aeruginasa , S. mutans , P. gingivalis , Tannerella It may be a composition having an antibacterial effect against various microorganisms such as forsythensis , and in particular, P. gingivalis , Tannerella It may be a composition having an antimicrobial effect against microorganisms causing periodontal diseases such as forsythensis .

In the present specification, "active ingredient" refers to a component that can exhibit the desired activity alone or itself can exhibit activity with an inactive carrier.

In addition, an aspect of the present invention is a deposit number Provided is a pharmaceutical composition for preventing or treating periodontal disease comprising L. reuteri AN417 strain deposited as No. KCTC13231BP or a culture medium thereof as an active ingredient.

In the present invention, "periodontal disease" is also referred to as qi, and may be a disease occurring in the periodontal tissue including gingivitis (gingival), periodontal ligament, bone tissue, such as gingivitis and periodontitis.

The "pharmaceutical composition" of the present invention includes pharmaceutically acceptable carriers, excipients, etc., in addition to its active ingredients, topical formulations such as creams, lotions, ointments (semi-solid external preparations), microemulsions, gels, pastes, Transdermal preparations (TTS) (eg, patch, bandages, etc.), sprays, gargles, and the like.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, and derivatives thereof (such as sodium carboxymethyl cellulose, ethyl cellulose, etc.), malt, gelatin, talc, Solid lubricants (such as stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, etc.), alginic acid, and the like. Depending on the formulation of the pharmaceutical composition of the present invention, one or more suitable ones may be selected and used.

The “pharmaceutical composition for preventing or treating periodontal disease” of the present invention may include Lactobacillus luterian AN417 strain, which is an active ingredient thereof, or a culture solution thereof in any amount (effective amount) as long as it can exhibit periodontal disease prevention or treatment activity. However, a typical effective amount will be determined within the range of 0.001% to 99.900% by weight based on the total weight of the composition. Here, the “effective amount” refers to the amount of active ingredients that can prevent, improve, treat, or delay the onset of these symptoms. Such an effective amount can be determined empirically within the ordinary skill in the art.

Terms that are not specifically defined in this specification follow the meanings commonly used in the art or in the Korean dictionary.

The "antibacterial composition" and "pharmaceutical composition for preventing or treating periodontal disease" (hereinafter "the composition of the present invention") of the present invention is in a specific aspect, the formulation is toothpaste, liquid mouthwash, dietary It includes all of several formulations such as film, mouse spray or chewing gum, and each composition can be prepared in a conventional manner.

When the pharmaceutical composition for preventing or treating periodontal disease of the present invention is formulated as a toothpaste, the toothpaste of the present invention may include a L. reuteri AN417 strain or a culture medium thereof. In addition, wetting agents, thickeners, foaming agents, preservatives, sweeteners, fragrances and other ingredients may be further included.

In addition, the present invention provides a food composition for preventing or improving periodontal disease comprising L. reuteri AN417 strain or a culture medium thereof as an active ingredient.

In the specific aspect of the "food composition for preventing or improving periodontal disease" (hereinafter "the composition of the present invention") of the present invention, the formulation is a health functional food, nutritional supplements, health supplements, food additives or functional beverages, etc. All formulations are included, and each composition can be prepared in a conventional manner.

When the composition of the present invention is identified as a food composition, beverages such as juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, Chinese medicine, breads, sweets, cotton, foods, powders, tablets, etc. , Capsules, and the like.

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

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the present invention is not limited to these.

Example  1. Sampling

Small intestine was collected from pigs at a slaughterhouse near Jeongeup and placed in an anaerobic jar to maintain a specific anaerobic condition.

Example  2. L. reuteri  Isolation of strain

L. reuteri from the pig intestine of Example 1 above To isolate the strain, an absolute anaerobic MRS medium was prepared and used. At this time, the anaerobic MRS medium using N ₂ MRS medium It was prepared by removing the oxygen present therein and sterilizing.

1 g of pig small intestine collected in Example 1 and maintained in an anaerobic state was suspended in 100 ml of MRS medium, and shake cultured at 37 ° C. for 1 hour. Then, serial dilution was performed. 50 μl of the diluted solution was plated on the MRS plate medium, followed by anaerobic culture at 37 ° C. for 2 days, followed by pure separation of 200 L. reuteri strains from the medium. The isolated strain was suspended in 20% (v / v) skim milk, and stored at -70 ° C for use in the following experiment.

Example  3. Separated L. reuteri Identification of strain

In Example 2 above Separated L. reuteri For molecular biological identification of strains, 27F (5'-AGA GTT TGA TCM TGG CTC A-3 ': SEQ ID NO: 1) and 1492R (5'-TAC GGY TAC CTT GTT ACG) are universal primers targeting the 16S rRNA gene. ACT T-3 ': SEQ ID NO: 2) was used. After obtaining the product using direct colony PCR technique, refer to Macrogen for L. reuteri The sequencing of the 16S rRNA gene of the strains was performed. At this time, the base sequence of the 16S rRNA gene of L. reuteri AN417 strain is shown in SEQ ID NO: 1.

EZbiocloud (http://www.ezbiocloud.net/) To search for the base sequences obtained by the above analysis. After that, securedL. reuteri The genome sequence analysis of the strain was commissioned to Chun Lab,L. reuteri The secured sequence of the strain has been previously reported.L. reuteri The strain was compared to the sequence. Analysis,L. reuteri It was confirmed that AN417 (Lre417) is a novel strain. Table 1 shows the results.

ANIb SD2112 I5007 DSM20016 ATCC53608 Lre401 Lre417 ZLR003 SD2112 - 95.33 95.34 95.21 99.98 95.3 95.47 I5007 95.25 - 96.15 99.18 95.27 99.09 99.29 DSM20016 95.43 96.38 - 95.68 95.43 96.35 95.71 ATCC53608 95.4 99.25 95.65 - 95.4 99.38 99.53 Lre401 99.99 95.43 95.39 95.22 - 95.4 95.56 Lre417 95.15 99.14 96.07 99.26 95.16 - 99.19 ZLR003 95.31 99.28 95.71 99.35 95.39 99.03 -

As shown in Table 1, isolated L. reuteri The AN417 strain (Lre417) was identified as a novel strain showing a significant difference from the previously reported L. reuteri standard strain with an ANI (average nucleotide identity) value of 95.15 to 99.26.

Experimental Example  1. For various pathogens L. reuteri Antibacterial activity analysis of AN417 strain

New L. reuteri secured in Example 3 above To analyze the antimicrobial activity of strain AN417, an excellent antimicrobial activity against periodontal disease bacteria reported L. reuteri KCTC 3594 and L. reuteri KCTC 3678 L. reuteri including As a standard strain, antibacterial activity against various pathogens was performed.

E. coli and P. aeruginasa widely used as co- bacterial And 10. ⁵ S. mutans , the causative agent, were plated on Nutrient agar plate medium (Difico). Subsequently, a disk absorbing the culture medium of the L. reuteri standard strain and the L. reuteri strain isolated thereon was placed on a plate medium on which the pathogen was plated, and the size of the growth inhibition of the strain culture medium was compared. The results are shown in FIGS. 1A to 1C.

As shown in Figures 1a to 1c, L. reuteri AN417 strain compared to other L. reuteri strains E. coli , P. aeruginasa and S. mutans It was confirmed that it shows the best antibacterial activity against the bacteria.

Experimental Example  2. About the causative agent of periodontal disease L. reuteri Antibacterial activity analysis of AN417 strain

L. reuteri To analyze the antibacterial activity of the AN417 strain, L. reuteri The standard strains L. reuteri KCTC 3594 and L. reuteri KCTC 3678 were used as controls to perform antibacterial activity against periodontal disease causative bacteria.

L. reuteri standard strain and L. reuteri Porphyromonas, a causative agent of periodontal disease, is removed from the culture solution of the AN417 strain with a filter having a 0.25 um pore. gingivalis KCTC 5352 and Tannerella forsythensis KCTC 5666 was inoculated at a concentration of 10 ⁵ cells / ml. Then, L. reuteri standard strain and L. reuteri inoculated into the periodontal disease causative bacteria at 600 nm using a spectrophotometer The growth inhibition effect of periodontal disease causative bacteria by AN417 strain was measured. The results are shown in FIGS. 2A and 2B.

2A and 2B, L. reuteri AN417 strain is the cause of periodontal disease compared to L. reuteri standard strain Porphyromonas It was confirmed that the antibacterial activity of inhibiting the growth of gingivalis and Tannerella forsythensis is excellent.

Experimental Example  3.  L. reuteri Acid condition of AN417 strain and Bile acids  Stability evaluation

L. reuteri To confirm the stability of the AN417 strain as probiotics, L. reuteri in acidic medium and 0.5% bile acid medium The viability of the AN417 strain was evaluated.

The culture medium was subjected to acidic conditions of pH 2 and 3, and the final concentration of bile acid to be added was adjusted to 0.5%. Later, L. reuteri Standard strains (KCTC 3594 and KCTC 3678) and L. reuteri The AN417 strains were inoculated into the acid medium and the medium to which 0.5% bile acid was added, respectively. After 12 hours, the viability of each strain was evaluated. The results are shown in FIG. 3.

As shown in Figure 3, L. reuteri Standard strain and L. reuteri It was confirmed that both AN417 strains survived in acidic conditions and 0.5% bile acid medium.

<110> Korea Research Institute of Bioscience and Biotechnology          Dr.s Medi Co., Ltd. <120> NOVEL LACTOBACILLUS REUTERI AND COMPOSITION FOR PREVENTING OR          TREATING PERIODONTAL DISEASES COMPRISING THE SAME <130> FPD / 201703-0001 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1499 <212> RNA <213> Artificial Sequence <220> <223> 16S rRNA of L. reuteri AN417 <400> 1 gatgaacgcc ggcggtgtgc ctaatacatg caagtcgtac gcactggccc aactgattga 60 tggtgcttgc acctgattga cgatggatca ccagtgagtg gcggacgggt gagtaacacg 120 taggtaacct gccccggagc gggggataac atttggaaac agatgctaat accgcataac 180 aacaaaagcc acatggcttt tgtttgaaag atggctttgg ctatcactct gggatggacc 240 tgcggtgcat tagctagttg gtaaggtaac ggcttaccaa ggcgatgatg catagccgag 300 ttgagagact gatcggccac aatggaactg agacacggtc catactccta cgggaggcag 360 cagtagggaa tcttccacaa tgggcgcaag cctgatggag caacaccgcg tgagtgaaga 420 agggtttcgg ctcgtaaagc tctgttgttg gagaagaacg tgcgtgagag taactgttca 480 cgcagtgacg gtatccaacc agaaagtcac ggctaactac gtgccagcag ccgcggtaat 540 acgtaggtgg caagcgttat ccggatttat tgggcgtaaa gcgagcgcag gcggttgctt 600 aggtctgatg tgaaagcctt cggcttaacc gaagaagtgc atcggaaacc gggcracttg 660 agtgcagaag aggacagtgg aactccatgt gtagcggtgg aatgcgtaga tatatggaag 720 aacaccagtg gcgaaggcgg ctgtctggtc tgcaactgac gctgaggctc gaaagcatgg 780 gtagcgaaca ggattagata ccctggtagt ccatgccgta aacgatgagt gctaggtgtt 840 ggagggtttc cgcccttcag tgccggagct aacgcattaa gcactccgcc tggggagtac 900 gaccgcaagg ttgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 960 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatcttgcg ctaaccttag 1020 agataaggcg ttcccttcgg ggacgcaatg acaggtggtg catggtcgtc gtcagctcgt 1080 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgttacta gttgccagca 1140 ttaagttggg cactctagtg agactgccgg tgacaaaccg gaggaaggtg gggacgacgt 1200 cagatcatca tgccccttat gacctgggct acacacgtgc tacaatggac ggtacaacga 1260 gtcgcaagct cgcgagagta agctaatctc ttaaagccgt tctcagttcg gactgtaggc 1320 tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1380 atacgttccc gggccttgta cacaccgccc gtcacaccat ggggagtttg taacgcccaa 1440 agtcggtggc ctaaccatta tggagggagc cgcctaaggc gggacagatg actggggtg 1499

Claims (9)

Lactobacillus reuteri AN417 strain deposited with accession No. KCTC13231BP. As a composition for antibacterial comprising the strain according to claim 1 or a culture medium thereof as an active ingredient,
The antimicrobial composition is characterized in that it has antibacterial activity to E. coli, P. aeruginasa, S. mutans , P. gingivalis , or Tannerella forsythensis strains. delete A pharmaceutical composition for preventing or treating periodontal disease, comprising the Lactobacillus reuteri AN417 strain according to claim 1 or a culture medium thereof as an active ingredient. The method of claim 4,
The periodontal disease is characterized in that any one selected from the group consisting of gingivitis and periodontitis, pharmaceutical composition. The method of claim 4 or 5,
The formulation of the composition is characterized in that any one selected from the group consisting of toothpaste, mouthwash, dietary film, mouse spray and chewing gum, pharmaceutical composition. Food composition for preventing or improving periodontal disease, comprising the Lactobacillus reuteri AN417 strain according to claim 1 or a culture medium thereof as an active ingredient. The method of claim 7,
The periodontal disease is characterized in that any one selected from the group consisting of gingivitis and periodontitis, food composition. The method of claim 7 or 8,
The formulation of the composition is characterized in that any one selected from the group consisting of functional foods, nutritional supplements, health foods, food additives, and combinations thereof, food composition.

Images