Cutibacterium avidum strain, and composition for preventing or treating atopic dermatitis, comprising strain or cultured product thereof

Abstract

The present invention relates to Cutibacterium avidum GENSC01 strain (KCTC 13596BP). The present invention also relates to a composition comprising the strain or its culture, and use thereof. The present invention is effective in improvement, prevention or treatment of atopic dermatitis, acne or skin inflammation by fine dust.

Claims

1. A method of inhibiting, improving or treating atopic dermatitis or inflammatory skin diseases, comprising: administering a subject in need thereof a therapeutically effective amount of Cutibacterium avidum GENSCO1 strain (KCTC 13596BP) or its culture.

2. The method according to claim 1, wherein the Cutibacterium avidum GENSCO1 strain or its culture has an antibacterial activity against Staphylococcus aureus or Cutibacterium acnes, or has an activity of removal or formation-inhibiting of biofilm produced by Staphylococcus aureus or Cutibacterium acnes.

3. The method according to claim 1, wherein the inflammatory skin diseases include skin inflammation by fine dust.

4. The method according to claim 1, wherein Cutibacterium avidum GENSCO1 strain (KCTC 13596BP) or its culture is administered with rosmarinic acid.

5. The method according to claim 1, wherein the Cutibacterium avidum GENSCO1 strain or its culture, and rosmarinic acid are administered at a weight ratio of 1:0.000001 to 1:0.01.

6. A method of inhibiting the growth of Staphylococcus aureus or Cutibacterium acnes of skin of a subject, comprising: administering to the subject in need thereof a therapeutically effective amount of Cutibacterium avidum GENSCO1 strain (KCTC 13596BP) or its culture.

7. The method according to claim 6, wherein Cutibacterium avidum GENSCO1 strain (KCTC 13596BP) or its culture is administered with rosmarinic acid.

8. A composition comprising Cutibacterium avidum GENSCO1 strain (KCTC 13596BP) or its culture, at least one excipient, and rosmarinic acid.

9. The composition according to claim 8, wherein the composition comprises the Cutibacterium avidum GENSCO1 strain or its culture, and rosmarinic acid at a weight ratio of 1:0.000001 to 1:0.01.

10. The composition according to claim 8, which is a pharmaceutical composition.

11. The composition according to claim 8, which is a cosmetic composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the 16s rRNA sequence of Cutibacterium avidum GENSC01 strain (Accession number: KCTC 13596BP) of the present invention.

(2) FIG. 2 shows the result of smearing the Cutibacterium avidum GENSC01 strain of the present invention on sheep blood agar and culturing it. No transparent ring was found around the microbial cell, and thereby it can be seen that it is not harmful to the human body because there is no hemolysis.

(3) FIG. 3 is the result of comparing the inhibitory effects of formation of biofilm between strains of different each other.

(4) FIG. 4 shows the result of inhibiting the growth for Staphylococcus aureus KCTC 1621 by Cutibacterium avidum GENSC01 strain.

(5) FIG. 5 shows the result of inhibiting the growth for Cutibacterium acnes ATCC 6919 by Cutibacterium avidum GENSC01 strain.

(6) FIG. 6 shows the result of cytotoxicity test of fermented filtrates of Cutibacterium avidum GENSC01 strain.

(7) FIG. 7 shows the efficacy of facilitating filaggrin expression of fermented filtrates of Cutibacterium avidum GENSC01 strain.

(8) FIG. 8 shows the efficacy of facilitating claudin 1 expression of fermented filtrates of Cutibacterium avidum GENSC01 strain.

(9) FIG. 9 shows the reduction of TSLP expression of fermented filtrates of Cutibacterium avidum GENSC01 strain.

(10) FIG. 10 shows the result which shows that the inhibitory effect of formation of biofilm was excellent when treating fermented filtrates of Cutibacterium avidum GENSC01 strain and rosmarinic acid together, than the case of treating each of them alone.

(11) FIG. 11 is the result of confirming the IL-8 expression rate when treating fermented filtrates of Cutibacterium avidum GENSC01 strain and rosmarinic acid respectively or together.

(12) FIG. 12 is the result of confirming the IL-6 expression rate when treating fermented filtrates of Cutibacterium avidum GENSC01 strain and rosmarinic acid respectively or together.

(13) FIG. 13 is the result of confirming that fermented filtrates of Cutibacterium avidum GENSC01 strain are effective in reducing inflammation by fine dust.

DETAILED DESCRIPTION

(14) Hereinafter, the present invention will be described by the following examples and the like in order to described it more specifically. However, the examples according to the present invention may be modified to various other forms, and the scope of the present invention should not be construed as being limited to the examples described below. The examples of the present invention are illustratively provided in order to facilitate a specific understanding of the present invention.

[Example 1] Isolation and Identification of Cutibacteria avidum GENSC01

(15) 1-1. Isolation of Strain

(16) Skin-derived bacteria isolation was carried out from adults who have never had skin diseases such as atopy, psoriasis or acne and the like, or who have not had a history of treatment related to it in the past 6 months. To collect skin samples, unwashed both cheeks and ala nasi were rubbed with a sterile swab dampened with sterilized water by applying a force. The swab was immediately sealed in a test tube containing Reinforced Clostridial Medium (RCM), and the test tube was filled with nitrogen and incubated at 37° C. for 48 to 72 hours. The medium of the test tube containing the cultured swab was streaked on an RCM agar plate by picking it with a platinum loop, and this procedure was repeated 3˜4 times to separate pure colonies.

(17) 1-2. Identification of Strain

(18) 1) Biological Identification Using API Kit

(19) As a method for biochemically identifying an isolated strain, an anaerobic bacterium API 20A kit (biomerieux Co., France) was used. After culturing at 37° C. for 24 hours in a RCM liquid medium of 10 ml and then centrifuging, the medium was removed. After washing with PBS 2˜3 times and then OD.sub.600=3 resuspending with a medium comprised in a kit according to the protocol provided by the manufacturer, it was aliquoted in an appropriate amount to each well of API 20A kit and was anaerobically cultured at 37° C. for 24 hours and then was read.

(20) The final result was identified in a program for identification, API web, and the result was shown in the following Table 1. As the result of identification of API 20A, it was identified as Propionibacterium (=Cutibacterium) propionicum/avidum, and as the result of API ID32, it showed the same biochemical properties as Propionibacterium avidum.

(21) TABLE-US-00001 TABLE 1 API 20A reading result No Carbohydrates Utilized 0 L-tryptophane − 1 urea − 2 D-glucose + 3 D-mannitol − 4 D-lactose (bovine origin) − 5 D-saccharose (sucrose) + 6 D-maltose + 7 salicin − 8 D-xylose − 9 L-arabinose − 10 gelatin (bovine origin) + 11 esculin ferric citrate − 12 glycerol + 13 D-cellobiose − 14 D-mannose + 15 D-melezitose + 16 D-raffinose − 17 D-sorbitol − 18 L-rhamnose − 19 D-trehalose +

(22) 2) A 16s RNA gene sequence was determined by collecting 1 ml of pure culture solution of the identified and isolated strain through a 16s rRNA gene sequence and requesting to Macrogen. Primers for PCR were universal primers of 16s rRNA gene, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′), and 785F (5 ‘-GGATTAGATACCCTGGTA-3’) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) were used for sequencing. The 16s rRNA sequence of the isolated strain was shown in FIG. 1 and SEQ ID NO: 1, and this strain showed the 99% homology to Cutibacterium avidum. Based on the above result, the strain was named “Cutibacterium avidum GENSC01” strain, and it was deposited to Korea Research Institute of Bioscience & Biotechnology Korean Collection for Type Cultures (KCTC) on Jul. 24, 2018 and received Accession number KCTC 13596BP.

[Example 2] Hemolysis Test of Cutibacteria Avidum GENSC01

(23) A considerable number of Cutibacterium avidum strains have hemolytic toxicity and are harmful to the human body depending on strains. To confirm the safety of Cutibacterium avidum GENSC01, the presence or absence of hemolytic toxicity was confirmed. The Cutibacterium avidum GENSC01 purely cultured in a liquid medium was collected by a platinum loop and it was steaked on a sheep blood agar and it was anaerobically cultured at 37° C. for 48 hours. The hemolysis was determined by the presence of transparent rings around microbial cells, and it was determined that Cutibacterium avidum GENSC01 had no hemolysis for sheep blood and therefore it was not harmful to the human body, as could be seen in FIG. 2.

[Example 3] Preparation of Fermented Filtrates of the Strain of the Present Invention

(24) Cutibacterium avidum GENSC01 strain was anaerobically cultured on a RCM agar plate at 37° C. for 72 hours. Single colony shown in a solid medium was subcultured in an RCM liquid medium of 10 ml and was cultured under the same condition. 72 hours later, 0.1% was inoculated to the same liquid medium, and it was cultured for 72 hours under the same condition, and the supernatant was centrifuged and filtrated with a 0.22 um pore size filter.

[Example 4] Measurement of Inhibitory Effect of Formation of Biofilm (Selection of Beneficial Bacteria)

(25) A Staphylococcus aureus strain (Staphylococcus aureus KCTC 1621) was liquid cultured in a titration medium (TSB+0.2% glucose) for 16 to 24 hours. After adding TSB with 0.2% glucose on a 6-well plate (polystyrene), a test group was added to each well in an approximately 5-10% volume. Then, the cultured bacterial solution was inoculated to each well so that the final strain concentration was to be 2×10.sup.6 CFU/well. Then, it was under static culturing in a 37° C. incubator for 24 hours. After culturing, the culture solution was eliminated and each well was washed twice using sterile PBS of 1˜2 ml. After washing, PBS of 2 ml was added and the biofilm was scraped out with a scraper and was suspended, and then the absorbance was measured at 600 nm. The absorbance measurement was conducted using BioPhotometer D30. Untreated wells were used as the negative control group and wells inoculated with baicalein (25 μm/ml) were used as the positive control group to calculate biofilm formation inhibitory ability.

(26) FIG. 3 shows the biofilm formation inhibitory result compared between strains. As can be seen in FIG. 3, it can be found that the GENSC01 strain has the excellent biofilm formation inhibitory ability than other strains.

(27) In other words, compared to the same kind of microorganisms, the GENSC01 of the present invention has the excellent biofilm formation inhibitory effect than other strains. Thus, the strain or its culture of the present invention can exhibit an excellent effect for treatment, improvement or prevention of atopic dermatitis, acne or inflammatory skin disease, than using other strains.

[Example 5] Growth Inhibition on Staphylococcus aureus KCTC 1621 and Cutibacterium acnes ATCC 6919 (Overlay Clear Zone Test)

(28) The effect of growth inhibition of S. aureus and C. acnes was confirmed by observing formation of a clear zone. When these bacteria are inoculated on an agar medium, bacteria grow in a light color, and therefore the color of the medium becomes cloudy, and it looks transparent if it does not grow. The experiment was progressed, expecting that S. aureus and C. acnes around Cutibacterium avidum GENSC01 could not grow and they became transparent, if Cutibacterium avidum GENSC01 had the effect of inhibiting the growth of bacteria. The Cutibacterium avidum GENSC01 culture broth was collected with a platinum loop and was anaerobically cultured in a thin RCM agar plate at 37° C. for 72 hours by drawing a line about 2.5 cm. After confirming that Cutibacterium avidum GENSC01 sufficiently grew, S. aureus and C. acnes strains adjusted to 10.sup.4 cfu/ml were inoculated in 10 ml RCM agar at about 45° C. which was not yet solidified, and they were well suspended before the medium was hardened, and they were evenly solidified by pouring them on the agar plate in which Cutibacterium avidum GENSC01 grew. In the solidified agar plate, S. aureus and C. acnes were further cultured anaerobically at 37° C. for about 40 hours and about 72 hours, respectively, to observe the size of the clear zone appearing around Cutibacterium avidum GENSC01. As the negative control group, phosphate-buffered saline (PBS) was used, and as the positive control group, triclosan was used, and S. aureus and C. acnes were treated at an amount of 10 mg/ml and 200 mg/ml, respectively. As a result, as FIG. 4, the transparent area around the Cutibacterium avidum GENSC01 was observed, and the transparency was reduced with distance from Cutibacterium avidum GENSC01, thereby confirming that the Cutibacterium avidum GENSC01 had an ability of inhibiting growth of S. aureus. It could be seen that when treating the negative control group, PBS, the clear zone was not observed, and when treating a bactericide, triclosan as the positive control group, the clear zone was observed, but it had much smaller area than GENSC01 treatment. The result was shown in FIG. 4.

(29) In addition, as could be seen in FIG. 5, in the result of inoculating C. acnes, the transparent area around the Cutibacterium avidum GENSC01 was observed, and the transparency was reduced with distance from Cutibacterium avidum GENSC01, thereby confirming that the Cutibacterium avidum GENSC01 had an ability of inhibiting growth of C. acnes. Also, it could be seen that when treating the negative control group, PBS, the clear zone was not observed, and when treating a bactericide, triclosan as the positive control group, the clear zone was observed, but it had much smaller area than GENSC01 treatment.

(30) Through these results, it was confirmed that the Cutibacterium avidum GENSC01 of the present invention could provide an effect of improving, preventing or treating acne by inhibiting an acne-causing bacterium, C. acnes.

[Example 6] Cytotoxicity Confirmation

(31) To evaluate the cytotoxicity of fermented filtrates prepared in the Example 3, the following experiment was progressed. HaCaT cells were attached on a 96-well cell culture plate for 24 hours at 5×10.sup.3 cells/well each and then the test group samples were added by concentration to culture it under the condition of 5% CO.sub.2 and 37° C. for 48 hours. 48 hours later, the cultured cell medium was removed, and 0.5 mg/ml dimethylthiazol-2-y1)-2-5-diphenyltetrazolium bromide (MTT) formazan solution was treated to cells, and they were reacted for 4 hours, and after the time passage, all the cell medium was removed and the formazan solution was dissolved by DMSO and then the absorbance was measured at 570 nm with SpectraMax M2. Then, the survival rate of the HaCaT cell line was calculated by converting with the equation of O.D.sub.sample/O.D.sub.control×100.

(32) As could be seen in FIG. 6, the treatment of GENSC01 did not affect the survival rate of the HaCaT cell line significantly. Thus, it could be confirmed that GENSC01 had no cytotoxicity.

(33) On the other hand, in case of the experimental group in which rosmarinic acid was added, interestingly, it was confirmed that the cell survival rate could be increased. In other words, it was confirmed that the effect of cytotoxicity reduction could be expected by the addition of rosmarinic acid.

[Example 7] Confirmation of Skin Barrier Function Enhancement Efficacy

(34) In order to investigate the GENSC01 fermented filtrates had a function of skin barrier enhancement for the HaCaT cell line, in addition to the efficacy of inhibiting biofilm of S. aureus, the fermented filtrates of GENSC01 were treated to the HaCaT cell line by % to the cell culture solution, and then were reacted for 24 hours to confirm the expression of filaggrin and claudin-1 which were markers of the skin barrier function on RNA.

(35) 7-1. Confirmation of Expression Facilitating Efficacy of Filaggrin

(36) As shown in FIG. 7, it was confirmed that the degree of expression of filaggrin of fermented filtrates increased compared to the positive control group, retinoic acid (RA′, 1 μM). FIG. 7 shows the filaggrin expression facilitating efficacy of fermented filtrates. GENSC01 0.01%, 0.1% and 1% were treated, respectively.

(37) As can be seen in FIG. 7, it can be seen that GENSC01 has an excellent filaggrin expression effect compared than retinoic acid (positive control group) known to have an excellent filaggrin expression effect. Through this, GENSC01 can provide an atopic dermatitis improvement effect, an acne improvement effect, a skin barrier enhancement effect, and a skin moisture content maintenance or increase effect by increasing filaggrin expression.

(38) 7-2. Confirmation of Expression Facilitating Efficacy of Claudin-1

(39) As shown in FIG. 8, it was confirmed that the claudin expression degree of the fermented filtrates increased in proportion to the concentration, compared to the positive control group, retinoic acid (RA′, 1 μM). FIG. 8 shows the claudin expression facilitating efficacy of fermented filtrates.

(40) As can be seen in FIG. 8, it can be seen that GENSC01 has an excellent claudin expression effect compared than retinoic acid (positive control group) known to have an excellent claudin expression effect. Through this, GENSC01 can provide an atopic dermatitis improvement effect, an acne improvement effect, a skin barrier enhancement effect, and a skin moisture content maintenance or increase effect by increasing claudin expression.

[Example 8] Confirmation of Itchiness Alleviation Efficacy

(41) The expression of TSLP, the cytokine acting as one of causes of atopic dermatitis was confirmed on RNA, by treating fermented filtrates of GENSC01 to the HaCaT cell line by % and then reacting for 4 hours. As shown in FIG. 9, it was confirmed that the TSLP expression degree of fermented filtrates was reduced. Thus, it was confirmed that the GENSC01 culture of the present invention inhibited the TSLP expression and solved skin itchiness, thereby having an effect in improvement of atopic dermatitis.

[Example 9] Deodorization Effect of Rosmarinic Acid

(42) 9-1. Preparation of Preparations

(43) At first, using the fermented filtrates and rosmarinic acid, preparations shown in Table 2 (Comparison 1, Samples 1˜3) were prepared and the deodorization function was investigated. The sensory test was carried out by 57 panels, and the test result was shown as an average value after conducting on the basis of fermented filtrates (5%).

(44) TABLE-US-00002 TABLE 2 Comparison 1 Sample 1 Sample 2 Sample 3 GENSC01 fermented 5 parts by weight filtrates Rosmarinic acid 0 0.0001 part 0.001 part 0.01 part (Sigma-aldrich, USA) by weight by weight by weight Water to 100 parts by weight

(45) 9-2. Evaluation result

(46) [Evaluation Criteria]

(47) 1: No odor.

(48) 2: A little odor remains.

(49) 3: Odor remains.

(50) 4: Most odor remains.

(51) 5: No change.

(52) TABLE-US-00003 TABLE 3 Sensory test result Comparison 1 5 Sample 1 3.1 Sample 2 2.8 Sample 3 2.8

(53) As shown in the Table 3, the case of treating rosmarinic acid together was effective for unpleasant odor removal, compared to single treatment of GENSC01 fermented filtrates.

[Example 10] Measurement of Biofilm Formation-Inhibiting Effect (Containing Rosmarinic Acid)

(54) The experiment method was same as the method of Example 4, the positive combination effect of the biofilm formation-inhibiting ability of rosmarinic acid and the biofilm formation-inhibiting ability of the fermented filtrates was to be measured.

(55) When treating the rosmarinic acid aqueous solution and fermented filtrates respectively, the effect of inhibiting biofilm formation of the Staphylococcus aureus strain of about 35% was shown, compared to the untreated group. As the baicalein treatment group as the positive control showed about 30% inhibitory ability, the result that the effect of rosmarinic acid and fermented filtrates had the similar or better inhibitory ability than the positive control was shown. In addition, in the experimental groups in which the fermented filtrates and rosmarinic acid aqueous solution were mixed (containing rosmarinic acid of A=0.01, B=0.1, C=0.5 mg/ml), compared to the blank group, 50%, 57% and 62% of biofilm formation-inhibiting effect was shown, respectively.

(56) As shown in FIG. 10, it was confirmed that it inhibited the biofilm formation ability of Staphylococcus aureus strain more effectively than the cases of treating the rosmarinic acid aqueous solution and fermented filtrates separately.

[Example 11] Anti-Inflammatory Efficacy Evaluation

(57) At first, GENSCO1 fermented filtrates were pre-treated for 1 hour to cells in which 2×10.sup.5 of HaCaT human keratinocyte lines were attached in a 6-well plate, respectively, by culturing them in a 37° C. and 5% CO.sub.2 incubator for 24 hours. Then, the heat-treated C. acnes (100MOI) was treated and was reacted for 4 hours. Then, after extracting RNA for each sample, the RNA expression for the inflammation response cytokine factors, IL-6 and IL-8 was confirmed by real-time PCR.

(58) 1. IL-8 Expression Rate Confirmation

(59) As a result, as shown in FIG. 11, it was demonstrated that the inflammation response was reduced in the group treated by the culture filtrates and rosmarinic acid aqueous solution respectively, compared to the control group induced to C. acnes. In addition, it was shown that the inflammation response did not occur in the mixed composition of the culture filtrates and rosmarinic acid aqueous solution, compared to single treatment.

(60) 2. IL-6 Expression Rate Confirmation

(61) The result was shown in FIG. 12. It was confirmed that the expression of IL-6 was reduced at a similar level in the GENSC01 culture filtrates, rosmarinic acid aqueous solution and the mixed composition of the culture filtrates and rosmarinic acid aqueous solution.

[Example 12] Evaluation of Anti-Inflammation Efficacy Against Fine Dust

(62) At first, inflammation was induced by treating fine dust(Aldrich) 50 ug/ml to cells for 2 hours in which 6.5×10.sup.5 of HaCaT human keratinocyte lines were attached in a 6-well plate, respectively, by culturing them in a 37° C. and 5% CO.sub.2 incubator for 24 hours, and then it was washed out 3 times. Then, after treating the GENSCO1 fermented filtrates for 1 hour and 2 hours and then extracting RNA for each sample, the RNA expression for the inflammation response cytokine factor, IL-6, was confirmed by real-time PCR. The result was shown in FIG. 13. It was confirmed that the expression of IL-6 was significantly reduced by the GENSCO1 culture filtrates and it was further reduced in case of treatment for 2 hours.

INDUSTRIAL APPLICABILITY

(63) The present invention provides a new Cutibacterium avidum strain or its culture which can be used as a cosmetic composition.

(64) The strain or culture of the present invention can be used as a cosmetic composition.

(65) [Accession Number]

(66) Depository institution: Korea Research Institute of Bioscience & Biotechnology

(67) Accession number: KCTC13596BP

(68) Deposit date: 20180724

(69) This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e).