NOVEL KAZACHSTANIA TURICENSIS CAU Y1706 AND COMPOSITION USING SAME

Abstract

The present invention relates to a novel Kazachstania turicensis CAU Y1706 and a composition using same, and more specifically, to a Kazachstania turicensis CAU Y1706 (Accession No.: KCTC13794BP) strain, and a pharmaceutical composition, a food composition, a cosmetic composition, and a probiotic composition for preventing or treating inflammatory diseases, comprising the strain or a culture thereof as an active ingredient.

Claims

1. Kazachstania turicensis CAU Y1706 (accession number: KCTC13794BP) strain.

2. The strain according to claim 1, wherein the Kazachstania turicensis has anti-inflammatory activity.

3. The strain according to claim 1, wherein the Kazachstania turicensis has acid resistance or bile resistance.

4. The strain according to claim 1, wherein the Kazachstania turicensis improves Th1/Th2 immune response imbalance.

5. A pharmaceutical composition for preventing or treating an inflammatory disease comprising the strain of claim 1 or a culture thereof as an active ingredient.

6. The composition according to claim 5, wherein the inflammatory disease is any one selected from the group consisting of acute or chronic organ transplant rejection, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory skin disease, multiple sclerosis, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, allergic conjunctivitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondyloarthropathy, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteric spondylitis, juvenile arthritis, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious Arthritis, Lou Gehrig's disease, polyarteritis nodosa, hypersensitivity vasculitis, granulomatosis Lou Gehrig's disease, polymyalgia rheumatica, articular cell arteritis, calcium crystallization arthropathy, pseudo gout, non-articular rheumatism, bursitis, tendinitis, epicondylitis, neuropathic joint Diseases (neuropathic joint disease or cheroot joint), hemarthrosis, allergic purpura, hypertrophic osteoarthropathy, multicentral reticulocytoma, scoliosis, hemochromatosis, hemoglobinopathy, hyperproteinemia, hypogammaglobulinemia, familial Mediterranean fever, Behcet's disease, systemic lupus erythematosus, recurrent fever, sepsis, septic shock, acute respiratory distress syndrome, multiple organ failure, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, broncho-pulmonary dysplasia, type 1 diabetes, type 2 diabetes, arteriosclerosis, Alzheimer's dementia, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, neonatal multisystem inflammatory disease, chronic infantile neurologic cutaneous articular syndrome, adult-onset Still's disease, contact dermatitis, hydatidiform mole, PAPA syndrome (syndrome of pyogenic arthritis, pyoderma gangrenosum and acne), hyperimmunoglobulin d syndrome, cryopyrin-associated periodic syndrome, keratitis, conjunctivitis, retinitis, retinal vasculitis, uveitis, blepharitis, allergic conjunctivitis, dry eye, progressive systemic sclerosis, polymyositis, autoimmune encephalomyelitis, myasthenia gravis, polyarteritis nodosa and fibromyalgia syndrome.

7. The composition according to claim 6, wherein the inflammatory skin disease is any one selected from the group consisting of psoriasis, atopic dermatitis, dermatitis eczema, contact dermatitis, seborrheic dermatitis, pneumoconiosis rosacea, lichen planus, vasculitis, pityriasis pilaris, cellulitis, folliculitis, pemphigus, pemphigus, bullous pemphigus, epidermal blistering, urticaria, angioedema, vasculitis, erythema and cutaneous eosinophilia.

8. A food composition for preventing or improving an inflammatory disease comprising the strain of claim 1 or a culture thereof as an active ingredient.

9. A cosmetic composition for preventing or improving an inflammatory disease comprising the strain of claim 1 or a culture thereof as an active ingredient.

10. A probiotic composition comprising the strain of claim 1 or a culture thereof as an active ingredient.

11. An enteral composition comprising the strain of claim 1 or a culture thereof as an active ingredient.

12. An immunomodulatory composition comprising the strain of claim 1 or a culture thereof as an active ingredient.

13. A food additive composition comprising the strain of claim 1 or a culture thereof as an active ingredient.

14. Use of the strain of claim 1 or a culture thereof for preparing an agent for the prevention or treatment of inflammatory diseases.

15. A method for treating an inflammatory disease comprising administering to a subject in need thereof an effective amount of a composition comprising the strain of claim 1 or a culture thereof as an active ingredient.

16. Use of the strain of claim 1 or a culture thereof for the production of a probiotic agent.

17. Use of the strain of claim 1 or a culture thereof for preparing an enteral agent.

18. Use of the strain of claim 1 or a culture thereof for preparing an immunomodulatory agent.

19. A method of immunomodulation comprising administering to a subject in need thereof an effective amount of a composition comprising the strain of claim 1 or a culture thereof as an active ingredient.

20. Use of the strain of claim 1 or a culture thereof for preparing a food additive agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0119] FIG. 1 is a diagram showing the experimental design and schedule of atopic dermatitis induction in OVA-sensitized mice for the experiment.

[0120] FIG. 2 is a scanning electron microscope (SEM) photograph of Kazachstania turicensis CAU Y1706.

[0121] FIGS. 3a to 3g are results confirming the effect of oral administration of Kazachstania turicensis CAU Y1706 on cytokine concentration. Serum was used to analyze the level, and the ELIZA kit was used. Significance indicates the difference between the ANOVA analysis value and the mean value of the positive group. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001.

[0122] FIG. 3a is a result confirming the effect on the IL-4 concentration.

[0123] FIG. 3b is a result confirming the effect on the IL-5 concentration.

[0124] FIG. 3c is a result confirming the effect on the IL-10 concentration.

[0125] FIG. 3d is a result confirming the effect on the IL-12 concentration.

[0126] FIG. 3e is a result confirming the effect on the IFN-γ concentration.

[0127] FIG. 3f is a result confirming the effect on the TNF-α concentration.

[0128] FIG. 3g is a result confirming the effect on the IL-113 concentration.

[0129] FIGS. 4a to 4e are results of confirming the effect of oral administration of Kazachstania turicensis CAU Y1706 on blood. Inflammation was confirmed by whole blood analysis. Significance indicates the difference between the ANOVA analysis value and the mean value of the positive group. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001.

[0130] FIG. 4a is a result of measuring the IgE level in serum by ELISA analysis to confirm an allergic reaction.

[0131] FIG. 4b is the result of confirming the eosinophil ratio.

[0132] FIG. 4c is a result of confirming the number of eosinophils.

[0133] FIG. 4d is the result of confirming the neutrophil ratio.

[0134] FIG. 4e is a result of confirming the basophil ratio.

[0135] FIGS. 5a to 5h are results confirming that oral administration of Kazachstania turicensis CAU Y1706 contributes to the abundance and diversity of the intestinal microbial population of atopic dermatitis-induced mice.

[0136] FIG. 5a is the result of measuring the Shannon-wiener index (defined as 97% similarity).

[0137] FIG. 5b is a result of measuring the Simpson index.

[0138] FIG. 5c is the result of estimating the difference in the intestinal microbial group with an Unweighted UniFrac-based (PCoA) 3D diagram (Blue label: negative control group; Red label: positive control group; Green label: K. turicensis CAU Y1706 administration group; Purple label: Betotastine besilate administration group).

[0139] FIG. 5d is a result showing the relative abundance ratio of units of the genus.

[0140] FIG. 5e is a result showing a scatter plot of Faecalibacterium.

[0141] FIG. 5f is a result showing a scatter plot of the genus Akkermansia.

[0142] FIG. 5g is a result showing a scatter plot of the genus Lactobacillus.

[0143] FIG. 5h is a result showing a scatter plot of the genus Ruminococcus.

[0144] A statistical difference was revealed by the nonparametric Kruskal-Wallis test in the comparison of the four groups of FIGS. 5e to 5h (p<0.05).

[0145] FIGS. 6a to 6d are results confirming the effect of oral administration of Kazachstania turicensis CAU Y1706 on atopic dermatitis skin lesions in mice. Significance indicates the difference between the ANOVA analysis value and the mean value of the positive group. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001.

[0146] FIG. 6a shows that atopic lesions of the skin such as a mouse were evaluated as a dermatitis index, and the dermatitis score was calculated as the sum of the scores of three symptoms: erythema, dryness, and scratching by the rat.

[0147] FIG. 6b is the result of measuring the number of mast cells using a microscope by staining the paraffin block of mouse back skin with toluidine blue (scale bar=100 μm).

[0148] FIG. 6c is the result of measuring the number of mast cells using a microscope by staining the paraffin block of the mouse ileum with toluidine blue (scale bar=100 μm).

[0149] FIG. 6d is a result of quantifying the number of eosinophils through microscopic analysis after staining mouse back skin tissue with Congo red.

MODE FOR CARRYING OUT INVENTION

[0150] Hereinafter, the present invention will be described in detail.

[0151] However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0152] Experimental materials and methods Isolation and identification of Kazachstania turicensis CAU Y1706 Kimchi samples were obtained from a traditional market in Seoul, Korea. All samples were chopped and mixed with 10 mL of peptone solution (0.85% mass/vol). After serial dilution, samples were plated on MRS (Man, Rogosa, and Sharpe; BD BBL, Spatks, Md. USA) solid medium, and was cultured for 2-3 days in a MIR-254-PK incubator (Panasonic, Osaka, Japan) at 30° C. For pure isolation, white colonies were selected and separated and cultured several times on a soybean casein digested agar medium (Tryptic Soy Agar-TSA; BD BBL), and all separations were performed at 30° C. for 24 hours. For isolation and identification of yeast, rRNA gene (18S, 26S, 5.8-ITS) sequencing method was used, and a 3730 automatic DNA sequencer (Applied Biosystems, Foster City, Calif., USA) was used for the analysis. The gene sequence was analyzed through NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cqj). After the selected yeast is cultured in TSA at 30° C., cell appearance was observed using an optical microscope, DM 1000 light (Leica, Wetzlar, Germany) and a scanning electron microscope, SIGMA field-emission scanning electron microscope (Carl Zeiss, Dresden, Germany). Biochemical properties were confirmed using API 20C AUX and API 50CH, and performed according to the manufacturers instructions (biomerieux, MarcyIl′É Toile, France). One of the isolates, K. turicensis CAU Y1706, was used in the study of atopic dermatitis.

[0153] The Kazachstania turicensis strain selected in this way was named CAU Y1706. The novel Kazachstania turicensis strain isolated and identified was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) on Jan. 22, 2019 (Accession number: KCTC13794BP).

[0154] Ethics and Experimental Design

[0155] Animal experiments were conducted in accordance with the guidelines of the Korea Food and Drug Administration. The rat blood and tissue samples were collected according to the guidelines of the Animal Bioethics Committee of Chung-Ang University. Five-week-old female BALB/c rats (40 rats) were sold from Central Experimental Animals (Seoul, Korea). The experimental design and schedule are shown in FIG. 1. All mice were randomly assigned to 4 groups of 10 each, negative control group (negative control, OVA non-sensitized+PBS), positive control group (positive control, OVA sensitized+PBS), K. turicensis CAU Y1706 group (OVA sensitized+K. turicensis CAU Y1706), bepotastine besilate group (OVA sensitization+bepotastine besilate). To induce atopic dermatitis through skin sensitization, all mice were depilated with a hair clipper and depilatory cream. An aqueous solution of OVA grade V (Sigma-Aldrich, St. Louis, Mich., USA) (50 mg/mL) and alum (Sigma-Aldrich) dissolved in phosphate buffered saline (PBS) was injected intraperitoneally on the 7th, 21st, 35th, and 49th days from the start of the experiment, and according to the procedure, the skin was directly sensitized for 8 weeks. Lyophilized K. turicensis CAU Y1706 (1×10.sup.10 CFU/mouse) was dissolved in 200 μl of phosphate buffered saline and then orally administered. The negative control group was administered the same dose of phosphate buffered saline for 8 weeks. Bepotastine besilate (Sigma-Aldrich) in the positive treatment control group was orally administered once a day (0.5 mg/kg). After 8 weeks, mice were sacrificed for analysis, and back skin, ileum, and blood were collected.

[0156] Blood and Serum Cytokine Analysis

[0157] Blood samples were obtained through orbital blood sampling using a capillary. Whole blood for eosinophil analysis and number confirmation was collected using an EDTA blood collection tube (Green Cross Laboratories, Yongin, Korea). Serum for IgE and cytokine (IL-4, IL-5, IL-10, IL-12, IL-1β, TNF-α, IFN-γ) analysis was coagulated at 4° C. for 1 hour and then was obtained after the thrombus was settled through a 5,000×g centrifugation process. Cytokines were identified using an ELISA kit, and was performed according to the manufacturer's instructions (R&D systems, Minneapolis, Minn. USA). To check the absorbance at 450 nm, an Infinite 200 PRO NanoQuant microplate reader (Tecan, Mannedorf, Switzerland) was used.

[0158] Gut Microbiome Analysis

[0159] The fecal sample collected from 16-week-old mice was placed in a sterile 2 mL tube and immediately stored on ice, and then stored at −80° C. DNA was extracted using FastDNA SPIN kit for bacterial DNA, and performed according to the manufacturer's instructions (MP Biomedicals, Santa Ana, Calif., USA). The V3-V4 region of the 16S rRNA gene was amplified through PCR, and analysis was performed by MiSeq-based high-throughput sequencing (Illumina, San Diego, Calif., USA). After sequencing, the adapter sequence was removed through the Scythe (v 0.994; https://github.com/vsbuffalo/scythe) and Sickle programs (https://github.com/vsbuffalo/scythe). Sequences that are too short (<36 bp), extra-long tails, chimeric reads, and noise sequences were cut out using CD-HIT-OTU (http://weizhong-lab.ucsd.edu/cd-hit-otu). (Li et al., 2012). Minimum quality scores and length were limited to >20, 300 bp, respectively. The remaining representative sequences were combined, and operational taxonomic units were defined with 97% similarity through CD-HIT-OTU. Intestinal microbiome analysis was performed using QIIME (Quantitative Insight Into Microbial Ecology; v.1.9.1) software.

[0160] Tissue Analysis

[0161] The skin tissue and ileum of the back of the sacrificed mice were used for tissue analysis. The tissue was fixed in a formalin solution diluted to 10% using phosphate buffered saline and then embedded in paraffin. Tissue samples were cut to a thickness of 4-5 μm using a microtome, and then were stained with hematoxylin, eosin, toluidine blue and Congo red, respectively, for the measurement of mast cells and eosinophils. All toluidine blue stained areas of ileal tissue samples for mast cell count were observed at 400 times magnification by DM 4000B microscopy. For the quantification of mast cells, 5 of the toluidine blue stained areas of the skin tissue of the back were randomly selected, and for the quantification of eosinophils, 20 of Congo red stained areas were randomly selected.

[0162] Statistics

[0163] GraphPad Prism (v.7.0) software was used for statistical analysis, and the data were expressed as mean±standard error. Comparisons between groups to identify relative differences in microbial classes and genera were based on statistically significant differences by nonparametric Kruskal-Wallis test. Significant differences in cytokine, blood sample, and tissue analysis were calculated by ANOVA analysis to compare two or more groups, and if the p value was less than 0.05, it was considered as significant.

Example 1: Isolation of Kazachstania turicensis CAU Y1706

[0164] K. turicensis CAU Y1706 was cultured for one day in TSA medium (Bacto) in an environment of 30° C. to form round glossy cream-colored colonies. Cells were observed to be ellipsoidal, about 1.6-2.3 μm in diameter and 1.6-2.3 μm in length (FIG. 2). It was confirmed that K. turicensis CAU Y1706 can utilize D-glucose, D-galactose, D-saccharose, D-trehalose, D-raffinose, D-fructose, D-mannose, 2-keto-D-gluconate as a carbon source. On the other hand, it was found that glycerol, L-arabinose, D-xylose, adonitol, xylitol, inositol, D-sorbitol, alpha-methyl-D-glucoside, N-acetyl-D-glucosamine, D-cellobiose, D-lactose, D-maltose, D-melezitose, D-ribose, D-mannitol, D-turanose and D-lyxose could not be used as carbon sources. After 100 μl treatment and reaction for 1 hour, absorbance was measured at 450 nm.

Example 2: Effects of Kazachstania turicensis CAU Y1706 on Cytokine Levels

[0165] The effect of K. turicensis CAU Y1706 on serum cytokine production was examined. When compared with the positive control group, the concentration of Th2 cytokines such as IL-4 and IL-5 in the serum of the K. turicensis CAU Y1706 administration group was significantly lower. Bepotastine besilate administered group also showed a low level (FIGS. 3a and 3b). Compared with the positive control group, Treg cytokine levels such as IL-10 and IL-1β in the serum were also lower in the K. turicensis CAU Y1706 administration group (FIGS. 3c and 3g).

[0166] On the other hand, it was confirmed that the level of Th1 cytokines such as IFN-γ, TNF-α, IL-12 was higher in the K. turicensis CAU Y1706 administration group compared to the positive control group (FIGS. 3d to 3f). These results support the therapeutic effect of K. turicensis CAU Y1706 on inflammatory diseases by promoting Treg-mediated Th2 immune response.

[0167] In addition, it was confirmed that the group treated with the strain of the present invention was significantly superior in IFN-γ, TNF-α, IL-12 production compared to the control group.

Example 3: Effects of Kazachstania turicensis CAU Y1706 on IgE Levels in Blood and Serum

[0168] Serum IgE levels were higher in the positive control group, and the K. turicensis CAU Y1706 administration group and the bepotastine besilate administration group showed significantly lower levels (FIG. 4a).

[0169] In addition, when comparing the positive control group, the group administered with bepotastine besilate and the group administered with K. turicensis CAU Y1706, it was confirmed that the number of eosinophils and the ratio of eosinophils, neutrophils, and basophils in the K. turicensis CAU Y1706 administration group were remarkably reduced (FIGS. 4b to 4e).

Example 4: Effects of Kazachstania turicensis CAU Y1706 on the Regulation of Gut Microbiota

[0170] To confirm the diversity of the intestinal microbiota, bacterial DNA was extracted from the fecal sample, and the 16S rRNA gene sequence was amplified through PCR. On average, 80.778 bacterial nucleotide sequences with an average length of 599 bp (±9.2 bp) were obtained per sample, a total of 3,231,137. The diversity and abundance of bacterial populations were statistically processed through operational taxonomic units (OTUs) (defined as 97%) and Shannon and Simpson indices (FIGS. 5a and 5b). Compared to the positive control group, the K. turicensis CAU Y1706 administration group showed superior abundance, uniformity, and diversity of intestinal microorganisms. Differences in the profiles of the gut microbiota at the genus level were evaluated through unweighted UniFrac-based 3D PCoA (FIG. 5c). These results show that the K. turicensis CAU Y1706 administration group had a significant difference compared to the positive control group.

[0171] A total of 11 bacterial phyla were found when analyzed at the phylum level, and the three most dominant types were Firmicutes, Bacteroidetes, and Verrucomicrobia (FIG. 5d).

[0172] A total of 94 genera were found in the three types of phyla, of which four were found abundantly in all experimental groups (FIGS. 5e to 5h).

[0173] Faecalibacterium included in the genus Firmicutes was found to be lower in the negative control group and the K. turicensis CAU Y1706 administration group than in the positive control group (p<0.0001). Conversely, Ruminococcus showed higher levels in the negative control group and the K. turicensis CAU Y1706 administration group than in the positive control group (p<0.05).

[0174] Akkermansia of the Verrucomicrobia phylum was abundant in the negative control group and the K. turicensis CAU Y1706 administration group compared to the positive control group (p<0.0001). These results show that there is a difference in the intestinal microbial group including the abundance of genus taxonomic units between the positive control group and the K. turicensis CAU Y1706 administration group.

[0175] The gut microbiota is known to produce short chain fatty acids (SCFAs). SOFA Butyrate plays an important role in innate immunity, and has functions such as protection of intestinal health, anti-inflammatory effect, and promotion of Treg cell differentiation. Also, Bacteroides, Ruminococcus, and Akkermansia are known to produce butyrate, and Lactobacillus is known to be related to the promotion of butyrate production.

[0176] Therefore, as a result of oral administration of the yeast of the present invention, it was found that a variety of and abundant intestinal microflora that produce or promote butyrate production compared to the positive control group were found, and it was confirmed that the yeast of the present invention can be usefully used as a bowel preparation agent.

Example 5: Effects of Kazachstania turicensis CAU Y1706 on Skin Inflammation and Intestines

[0177] To verify the efficacy of K. turicensis CAU Y1706 on inflammation, evaluation and pathological analysis using the dermatitis score of OVA-sensitized mice administered K. turicensis CAU Y1706 were performed.

[0178] It was observed that the dermatitis score of the K. turicensis CAU Y1706 administration group was dramatically lower than that of the positive control group (FIG. 6a). These results indicate that K. turicensis CAU Y1706 is effective in improving skin lesions in atopic dermatitis mice.

[0179] In addition, local infiltration of mast cells in back skin cells and ileal lesions was confirmed by toluidine blue staining. In OVA-sensitized mice administered with K. turicensis CAU Y1706, it was observed that mast cell accumulation in both lesions was suppressed (FIGS. 6b and 6c).

[0180] Also, it was confirmed that the number of eosinophils was significantly lower in OVA-sensitized mice administered K. turicensis CAU Y1706 compared with the positive control group. When observing the skin lesion tissue stained with Congo red, it was observed that the eosinophil infiltration level of the K. turicensis CAU Y1706 administration group was reduced as in the bepotastine besilate administration group (FIG. 6d). These results show that K. turicensis CAU Y1706 has the effect of reducing the infiltration of mast cells and eosinophils in the back skin and ileum of atopic dermatitis mice.

Example 6: Acid Resistance Evaluation of Kazachstania turicensis CAU Y1706

[0181] As an in vitro probiotic characterization test, acid resistance and bile resistance tests are widely used because it is necessary to have resistance to gastric juice and bile, which is necessary for a large number of bacteria to survive and reach the intestines stably in the human digestive system.

[0182] Therefore, in order to investigate the acid resistance of Kazachstania turicensis CAU Y1706, it was inoculated and cultured in TSB liquid medium titrated to pH 2.0 (Pancreatic digest of Casein 17 g/L, Papaic digest of Soybean 3 g/L, Dextrose 2.5 g/L, Sodium Chloride 5 g/L, Dipotassium Phosphate 2.5 g/L), and then the number of viable cells was measured. More specifically, after the Kazachstania turicensis CAU Y1706 strain was inoculated in TSB broth, cultured at 30° C. for 12 hours, and the cells were recovered by centrifugation. After adjusting the OD600 value to 1.0 with sterile water, 30 μl of the suspension was inoculated in a TSB liquid medium titrated to pH 2.0, and incubated for 0-120 minutes at 30° C., and then the number of viable cells was measured at A600 nm with Nanodrop (NanoQuant infinite M200, TECAN).

[0183] As shown in Table 1 of the experimental results, CAU Y1706 showed the ability to grow in MRS medium at pH 2.0 (same conditions as gastric juice). As for CAU Y1706, the number of bacteria increased at a significant rate as time passed, and as a result, it was confirmed that it was not inhibited under strong acid conditions. Therefore, it was confirmed that CAU Y1706 of the present invention has excellent acid resistance.

TABLE-US-00001 TABLE 1 Time Cell/ml (10.sup.6) 0  8.00 ± 0.00 15 12.43 ± 0.15 30 16.40 ± 0.26 60 20.07 ± 0.80 120 29.73 ± 0.11

Example 7: Evaluation of Bile Tolerance of Kazachstania turicensis CAU Y1706

[0184] To test the resistance properties of Kazachstania turicensis CAU Y1706 to bile acids, bile acid (ox-bile dry pure, Merck, Germany) was inoculated and cultured in TSB liquid medium to which 0, 0.2 and 0.4% (w/v) were added, and then the number of viable cells were measured. More specifically, after the Kazachstania turicensis CAU Y1706 strain was inoculated in MRS liquid medium, cultured at 30° C. for 12 hours, and the cells were recovered by centrifugation. After adjusting the OD600 value to 1.0 with sterile water, 30 μl of the suspension was inoculated in a TSB liquid medium containing 0, 0.2 and 0.4% bile acid and incubated for 0-12 hours at 30° C., and then the number of viable cells was measured at A600 nm with Nanodrop (NanoQuant infinite M200, TECAN).

[0185] As shown in Table 2 of the experimental results, Kazachstania turicensis CAU Y1706 showed the ability to grow in TSB medium supplemented with 0.2% or 0.4% bile, and the bacteria showed excellent growth ability in 0.2% and 0.4% bile. CAU Y1706 was confirmed to have high bile resistance (BTR) ability of 1.25 (0.2% bile condition) and 1.05 (0.4% bile condition) compared to the control group without the addition of bile. Therefore, through the above test, it was confirmed that the strain of the present invention can stably pass through the digestive tract, and a large number of bacteria can survive and reach the intestine.

TABLE-US-00002 TABLE 2 Cell counts as log incubation time (h) of Kazachstania turicensis CAU Y1706 Bile BTR concentration (Bile Tolerance (%) 3 h 6 h 9 h 12 h Rate) 0 4.13 ± 0.14 25.77 ± 0.06 44.60 ± 0.00 45.15 ± 0.05 1 0.2 7.27 ± 0.08 25.74 ± 0.06 46.13 ± 1.52 55.23 ± 0.06 1.25 0.4 5.57 ± 0.05 17.26 ± 0.42 46.20 ± 0.52 52.47 ± 0.06 1.05

[0186] [Accession Number]

[0187] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

[0188] accession number: KCTC13794BP

[0189] deposit date: 20190122

[0190] Depositary address: (56212) Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Republic of Korea