<i>Lactobacillus curvatus </i>WIKIM55 having activity of promoting hair growth, and composition containing same

Abstract

The present disclosure relates to novel Lactobacillus curvatus WIKIM55 isolated from kimchi, and a composition containing the same. The Lactobacillus curvatus WIKIM55 according to the present disclosure is a probiotic having activities of promoting hair growth and can be diversely applied for intestinal regulation and promotion of hair growth in humans or animals.

Claims

1. A method for promoting hair growth, which comprises administering Lactobacillus curvatus WIKIM55 (accession number KCCM12011P) or a culture thereof to a subject, wherein Lactobacillus curvatus WIKIM55 (accession number KCCM12011P) is derived from kimchi and the Lactobacillus curvatus WIKIM55 (accession number KCCM12011P) or a culture thereof is administered orally.

2. The method according to claim 1, wherein the Lactobacillus curvatus WIKIM55 (accession number KCCM12011P) or a culture thereof promotes hair growth by increasing the number of hair follicles in the anagen phase.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a result of observing the hair growth pattern of mice fed with the strain of the present disclosure for 30 days with the naked eye.

(2) FIG. 2 shows the skin tissue of a mouse fed with the strain of the present disclosure and a result measuring the skin thickness.

(3) FIG. 3 shows a result of measuring the number of hair follicles in the subcutaneous tissue of a mouse fed with the strain of the present disclosure.

(4) FIG. 4 shows a result of observing the hair growth pattern of a mouse fed with the strain of the present disclosure for 20 weeks and identifying the anagen.

(5) FIG. 5 shows a result of confirming the distribution of follicular stem cells of a mouse fed with the strain of the present disclosure by FACS analysis.

Best Mode

(6) Hereinafter, the present disclosure will be described in detail through examples. However, 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

(7) 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 confirmed using 1% agarose gel. PCR was conducted using the extracted genomic DNA as a template to amplify the 16S rDNA 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 and 30 seconds. The sequence of the PCR product obtained was analyzed by Macrogen (Seoul, Korea). Bacterial identification was performed via similarity analysis of the Basic Local Alignment Search Tool (BLAST) search engine of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) of the 16S rDNA sequence.

(8) As a result of the 16S rDNA base sequence analysis for identification of microorganisms, the strain isolated in this example was found to have a nucleic acid sequence of SEQ ID NO: 1.

(9) The microorganism of the present disclosure was named Lactobacillus curvatus WIKIM55 and deposited on Apr. 7, 2017 in the Korea Culture Center of Microorganisms (accession number: KCCM12011P).

(10) TABLE-US-00001 16S rDNA sequence of Lactobacillus curvatus WIKIM55 SEQ ID NO: 1 ACATGCAAGTCGAACGCACTCTCGTTAGATTGAAGAAGCTTGCTTCTGAT TGATAACATTTGAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCT GCCCTAAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAA ACCTAGCACCGCATGGTGCAAGGTTGAAAGATGGTTTCGGCTATCACTTT AGGATGGACCCGCGGTGCATTAGTTAGTTGGTGAGGTAAAGGCTCACCAA GACCGTGATGCATAGCCGACCTGAGAGGGTAATCGGCCACACTGGGACTG AGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAA TGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGG ATCGTAAAACTCTGTTGTTGGAGAAGAACGTATTTGATAGTAACTGATCA GGTAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA GCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCTTCGGCTCAACC GAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGG AACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTG GCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGG GTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGT GCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAA GCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGA CGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCG AAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCT TTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGT GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTA GTTGCCAGCATTTAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCG GAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCT ACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTT AGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGC CTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAAGAGTTTG TAACACCCAAAGCCGGGTGAGGTAACCTTCGGGAGCCAGCCGTCTAAGG.

Example 2: Confirmation of Hair Growth Promoting Effect of Lactobacillus curvatus WIKIM55

(11) The Lactobacillus curvatus WIKIM55 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.19 CFU/mL using PBS, and 0.2 mL (2×10.sup.9 CFU) was orally administered to test animals, five times a week, using a sonde. Sterile PBS was administered to negative and positive control groups.

(12) 1) Sensory Evaluation

(13) 6-week-old mice (C57/BL6) ahead of the telogen phase were accustomed in the breeding room for one week and the hair on the back was removed using a hair removing apparatus for animals. Then, after orally administrating Lactobacillus curvatus WIKIM55 for 30 days, the hair growth pattern was evaluated by clinical visual evaluation.

(14) As a result, the group fed with the Lactobacillus curvatus WIKIM55 strain (WIKIM55) showed faster hair growth as compared with the control group fed with PBS (PBS), as shown in FIG. 1.

(15) 2) Measurement of Skin Thickness

(16) Hair repeats growth and shedding through the hair cycle. It is known that, during the anagen phase when hair grows, the skin layer becomes thicker and hair follicles exist primarily in the subcutaneous layer of the skin. Thus, the skin tissue and skin thickness were investigated 30 days after the hair of the mice was removed (FIG. 2).

(17) As a result, the skin thickness of the PBS-treated group was 393.28 μm, whereas the group fed with Lactobacillus curvatus WIKIM55 had a skin thickness of 670.95 μm. It was also confirmed that most of the hair follicles were present in the subcutaneous layer. Through this, it confirmed that most of the hair follicles of the group fed with Lactobacillus curvatus WIKIM55 were in the anagen phase.

(18) 3) Measurement of Number of Hair Follicles in Subcutaneous Tissue

(19) The number of hair follicles in the subcutaneous tissue was measured 30 days after the hair of the mice was removed.

(20) As a result, the number of hair follicles was significantly larger for the group fed with Lactobacillus curvatus WIKIM55 than the PBS-treated group, as shown in FIG. 3

Example 3: Confirmation of Hair Growth Promoting Effect of Lactobacillus curvatus WIKIM55

(21) The Lactobacillus curvatus WIKIM55 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.19 CFU/mL using PBS, and 0.2 mL (2×10.sup.9 CFU) was orally administered to test animals, five times a week, using a sonde. Sterile PBS was administered to negative and positive control groups.

(22) 1) Sensory Evaluation

(23) 6-week-old mice (C57/BL6) ahead of the telogen phase were accustomed in the breeding room for one week and the hair on the back was removed using a hair removing apparatus for animals. After orally administrating Lactobacillus curvatus WIKIM55 for 20 weeks, the hair growth pattern was evaluated by clinical visual evaluation.

(24) As shown in FIG. 4, whereas the control group (PBS) fed with PBS had the anagen phase after about 7 weeks of the resting phase, the group fed with the Lactobacillus curvatus WIKIM55 strain (WIKIM55) switched to the anagen phase after about 2-3 weeks of the resting phase, leading to faster hair growth.

(25) 2) Confirmation of Distribution of Hair Follicle Stem Cells

(26) In order to compare the distribution of hair follicle stem cells of the mice of the control group (PBS) and the group fed with Lactobacillus curvatus WIKIM55 for 20 weeks (WIKIM55), the skin tissue on the back was excised. After removing subcutaneous fat from the excised tissue and then washing with phosphate buffered saline (PBS), the tissue was placed in RPM11640 culture medium containing 4 mg/mL collagenase IV, 2 mg/mL hyaluronidase and 100 U/mL DNase I and hair follicle stem cells were isolated from the skin tissue using the gentleMACS dissociator (Miltenyibiotec, Germany). FACS (fluorescence-activated cell sorting) analysis was performed to investigate the distribution of the hair follicle stem cells. The verified antigens showed the immunological characteristics of CD45-CD34+CD49f+. 1×10.sup.5 cells isolated from the skin tissue were washed with PBS solution containing 2% FBS and were allowed to react at room temperature with antibodies for the respective antigens. The expression of the antigens was confirmed by flow cytometry.

(27) As can be seen from FIG. 5, it was confirmed that more hair follicle stem cells showing the immunological characteristics of CD34+ CD49f+ were distributed in the skin tissue of the in the group fed with WIKIM55 as compared to the control group (PBS, naive). Because the hair follicle stem cells are involved in the growth of new hair, it is thought that the intake of WIKIM55 will increase hair growth by producing hair follicle stem cells.