KIMCHI LACTIC ACID BACTERIA LACTOBACILLUS SAKEI WIKIM0109 HAVING EFFICACY FOR RELIEF OF ARTHRITIS

Abstract

The present disclosure relates to novel Lactobacillus sakei WIKIM0109 isolated from kimchi and a composition containing the same. The Lactobacillus sakei WIKIM0109 according to the present disclosure is a lactic acid bacterium having an activity of inhibiting IgG and IgG2a production in blood and an activity of inhibiting IFN-γ, IL-17 and TNF-α production, and can be used in various applications, such as for preventing and ameliorating arthritis or improving intestinal regulation in human or animals.

Claims

1. Lactobacillus sakei WIKIM0109 with an accession number KCTC13818BP.

2. A pharmaceutical composition for preventing or treating inflammatory arthritis, comprising Lactobacillus sakei WIKIM0109 (accession number: KCTC13818BP), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

3. The pharmaceutical composition according to claim 2, wherein the inflammatory arthritis is rheumatoid arthritis, ankylosing spondylitis, osteoarthritis or psoriatic arthritis.

4. The pharmaceutical composition according to claim 2, wherein the composition reduces hypersensitive inflammatory response by inhibiting IgG and IgG2a production in blood.

5. The pharmaceutical composition according to claim 2, wherein the composition reduces hypersensitive inflammatory response by inhibiting collagen antigen-specific IFN-γ, IL-17 and TNF-α production.

6. The pharmaceutical composition according to claim 2, wherein the composition is a composition for oral administration.

7. A food composition for preventing and ameliorating inflammatory arthritis, comprising Lactobacillus sakei WIKIM0109 (accession number: KCTC13818BP), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

8. The food composition according to claim 7, wherein the inflammatory arthritis is rheumatoid arthritis, ankylosing spondylitis, osteoarthritis or psoriatic arthritis.

9. The food composition according to claim 7, wherein the composition reduces hypersensitive inflammatory response by inhibiting IgG and IgG2a production in blood.

10. The food composition according to claim 7, wherein the composition reduces hypersensitive inflammatory response by inhibiting collagen antigen-specific IFN-γ, IL-17 and TNF-α production.

11. The food composition according to claim 7, wherein the food is a functional health food.

12. The food composition according to claim 7, wherein the food is a beverage, a bar or a fermented milk.

13. A composition for enhancing immunity, comprising Lactobacillus sakei WIKIM0109 of claim 1 (accession number: KCTC13818BP), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

14. A composition for intestinal regulation, comprising Lactobacillus sakei WIKIM0109 of claim 1 (accession number: KCTC13818BP), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

15. A lactic acid bacterium starter for fermenting a food or a feed, comprising Lactobacillus sakei WIKIM0109 of claim 1 (accession number: KCTC13818BP), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

16. A feed or feed additive composition, comprising Lactobacillus sakei WIKIM0109 of claim 1 (accession number: KCTC13818BP), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1 shows the incidence of arthritis and arthritic score in a mouse model to which the strain of the present disclosure was administered orally.

[0043] FIG. 2 shows a result of measuring the paw thickness of an arthritis-induced mouse model to which the strain of the present disclosure was administered orally.

[0044] FIG. 3 shows a result of measuring IgG and collagen-specific IgG (CII-IgG) production in blood.

[0045] FIG. 4 shows a result of measuring IgG2a and collagen-specific IgG2a (CII-IgG2a) production in blood.

[0046] FIG. 5 shows a result of measuring collagen antigen-specific IFN-γ, IL-17 and TNF-α production.

[0047] FIG. 6 shows a result of comparing immunoregulatory capacity with LS35 and LS41, which are Lactobacillus sakei strains different from WIKIM0109.

BEST MODE

[0048] Hereinafter, the present disclosure is described in detail through examples. The following examples are for illustrative purposes only and the scope of the present disclosure is not limited by the examples.

EXAMPLES

Example 1

Isolation and Identification of Strain

[0049] A bacterial single colony obtained by smearing a crude liquid extract of kimchi in MRS culture medium was collected and cultured in MRS broth. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany). The extracted DNA was identified using 1% agarose gel. PCR was conducted using the extracted genomic DNA as a template to amplify the 16S rRNA gene. The PCR condition was 30 cycles of denaturation at 95° C. for 1 minute, annealing at 45° C. for 1 minute and extension at 72° C. for 1 minute 30 seconds. The sequence of the obtained PCR product was analyzed by Macrogen (Seoul, Korea). Bacterial identification was performed by similarity analysis of the 16S rRNA sequence using the Basic Local Alignment Search Tool (BLAST) search engine of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).

[0050] As a result of the 16S rRNA base sequence analysis for identification of the microorganism, the strain isolated in this example was found to have a nucleic acid sequence of SEQ ID NO: 1.

[0051] The microorganism of the present disclosure was named Lactobacillus sakei WIKIM0109 and deposited on Mar. 7, 2019 in the Korean Collection for Type Cultures (accession number: KCTC13818BP).

Example 2

Confirmation of Arthritis-Ameliorating Effect of Lactobacillus sakei WIKIM0109

[0052] The Lactobacillus sakei WIKIM0109 strain isolated in Example 1 was cultured in MRS medium at 30° C. for 24 hours, and the cultured bacteria were centrifuged at 8,000 rpm for 5 minutes and then washed with PBS to remove the remaining medium components. Then, the number of the bacteria was quantified to be 1×10.sup.10 CFU/mL using PBS, and 0.1 mL (1×10.sup.9 CFU) was orally administered to a collagen-induced arthritis mouse model using a sonde, five times a week. Sterile PBS was administered to negative and positive control groups.

[0053] 1) Measurement of Incidence of Arthritis and Arthritic Score in Collagen-Induced Arthritis Mouse Model

[0054] Arthritic score and incidence of arthritis for 12 weeks during which the Lactobacillus sakei WIKIM0109 strain was orally administered to the collagen-induced arthritis mouse model are shown in FIG. 1.

[0055] As a result, the collagen-induced arthritis control group (CIA-No LAB) showed the highest incidence of arthritis and arthritic score, and the incidence of arthritis and arthritic score were decreased significantly in the Lactobacillus sakei WIKIM0109 treatment group (WIKIM0109).

[0056] 2) Sensory Evaluation and Measurement of Paw Thickness

[0057] After orally administering the Lactobacillus sakei WIKIM0109 strain to experimental animals for 4 weeks, the condition of arthritis was evaluated by clinical visual evaluation and paw thickness was measured.

[0058] As shown in FIG. 2, symptoms such as erythema, swelling, etc. were observed in the joint and paw of the collagen-induced arthritis control group (CIA-No LAB) in contrast to the normal group (Naive). Also, it was confirmed that the erythema and joint swelling were ameliorated and the paw thickness was decreased in the Lactobacillus sakei WIKIM0109 treatment group (WIKIM0109) as compared to the control group.

[0059] 3) Measurement of Concentrations of IgG and IgG2a in Blood

[0060] After taking blood from the experimental animals, the concentrations of IgG, collagen antigen-specific IgG, IgG2a and collagen antigen-specific IgG2a in blood were measured. FIG. 3 shows a result of measuring the concentrations of IgG and collagen antigen-specific IgG (CII-IgG), and FIG. 4 shows a result of measuring the concentrations of IgG2a and collagen antigen-specific IgG2a (CII-IgG2a).

TABLE-US-00001 Before administration After administration inhibition(%) IgG 6.52 ± 0.81 mg/ml 4.796 ± 1.08 mg/ml 26.5 CII-IgG 14.04 ± 1.51 μg/ml 3.77 ± 2.21 μg/ml 73.2 IgG2a 118.2 ± 19.55 μg/ml 57.65 ± 9.02 μg/ml 51.3 CII-IgG2a 16.17 ± 0.88 μg/ml 11.26 ± 2.34 μg/ml 30.4

[0061] As seen from FIG. 3 and Table 1, the production of IgG and IgG2a in blood was induced by the arthritis induction as compared to the normal group. It was also confirmed that the administration of the Lactobacillus sakei WIKIM0109 to the experimental animals resulted in decreased concentrations of IgG and IgG2a. In particular, the production of collagen antigen-specific IgG2a (CII-IgG2a) was significantly inhibited by about 30.4% as compared to the control group.

[0062] 4) Measurement of Production of Collagen Antigen-Specific IFN-γ, IL-17 and TNF-α

[0063] After obtaining single cells from the lymph nodes around the joint of the experimental animals, 5×10.sup.5 cells were dispensed onto a 96-well plate. After treating with collagen (50 μg/mL) for 48 hours, the concentrations of antigen-specific IFN-γ, IL-17 and TNF-α were measured from the supernatant. As seen from FIG. 5, the production of antigen-specific IFN-γ, IL-17 and TNF-α was increased in the arthritis-induced group as compared to the normal group. It was also confirmed that the production of antigen-specific IFN-γ, IL-17 and TNF-α was significantly decreased when the Lactobacillus sakei WIKIM0109 was administered to experimental animals.

[0064] 5) Comparative Experiments of WIKIM0109 with Other Strains of Lactobacillus sakei

[0065] In order to investigate whether the effect of the Lactobacillus sakei WIKIM0109 is common to Lactobacillus sakei, (1) the increase in the production of the immune-suppressing cytokine IL-10 and (2) the decrease in the production of the immune-enhancing cytokine IL-17 were compared with Lactobacillus sakei LS35 and LS41 acquired from the World Institute of Kimchi, an annex of the Korea Food Research Institute.

[0066] Lactobacillus sakei LS35 was isolated from watery kimchi and Lactobacillus sakei LS41 were isolated from pa-gat kimchi. They were statically cultured in a 30° C. incubator for 24 hours using Lactobacilli MRS (BD Difco) liquid medium. After sequencing of the gene encoding 16S rRNA, they were identified as Lactobacillus sakei through homology analysis using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

[0067] OTII mouse having OVA-specific T cells were purchased from Jackson Laboratory and used after breeding. 5×10.sup.5 single splenocytes of the OTII mouse were dispensed onto a 96-well plate and cultured at 37° C. for 72 hours after treating with OVA peptide (OVA323-339) and kimchi lactic acid bacteria at MOI 1. The amount of IL-17 and IL-10 present in the culture medium was measured using the Cytometric Bead Assay (CBA) mouse Th1/Th2/Th17 kits purchased from BD Bioscience. The production of the cytokines was analyzed based on standard curves by using FACSCanto II (BD Bioscience).

[0068] As a result, it was confirmed that the WIKIM0109 strain resulted in the highest production of IL-10, the lowest production of IL-17 and the highest IL-10/IL-17 ratio among the different strains of Lactobacillus sakei (FIG. 6).

[0069] Although the LS41 strain also increased the production of the immune-suppressing cytokine IL-10, the WIKIM0109 strain showed about 40% or more production of IL-10. The production by the LS35 strain was only about ⅓ of that of the WIKIM0109 strain.

[0070] For the immune-enhancing cytokine IL-17, although the LS41 strain did not show significant decrease in production, the WIKIM0109 strain significantly decreased the production of IL-17. On the contrary, the LS35 strain increased the production of IL-17.

[0071] Although the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are merely preferred exemplary embodiments and the scope of the present disclosure is not limited thereto. Accordingly, it is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

[0072] [Deposition Number]

[0073] Depository agency: Korean Collection for Type Cultures (international).

[0074] Accession number: KCTC13818BP.

[0075] Date of accession: Mar. 7, 2019.