<i>Cutibacterium granulosum </i>strain, and composition comprising such strain or culture thereof for preventing or treating acne

Abstract

The present invention relates to Cutibacterium granulosum GENSC02 strain (KCTC 13597BP). 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 acne, atopic dermatitis or skin inflammation by fine dust.

Claims

1. A method of inhibiting, improving or treating acne, atopic dermatitis or inflammatory skin diseases, enhancing a skin moisturizing, or removing or reducing body odor, comprising: administering to a subject in need thereof a therapeutically effective amount of Cutibacterium granulosum GENSC02 strain or its culture.

2. The method according to claim 1, wherein the Cutibacterium granulosum GENSC02 strain or its culture has an antibacterial activity against Staphylococcus aureus or Cutibacterium acnes.

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

4. 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 granulosum GENSC02 strain (KCTC 13597BP) or its culture.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the 16s rRNA sequence of Cutibacterium granulosum GENSCO2 strain (Accession number: KCTC 13597BP) of the present invention.

(2) FIG. 2 shows the color change according to the use of glycerol by each C. granulosum strain (color change at days 0, 3 and 6).

(3) FIG. 3 shows the result of smearing the Cutibacterium granulosum GENSCO2 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.

(4) FIG. 4 shows the result of inhibiting the growth for Staphylococcus aureus KCTC 1621 by Cutibacterium granulosum GENSCO2 strain.

(5) FIG. 5 shows the result of inhibiting the growth for Cutibacterium acnes ATCC 6919 by Cutibacterium granulosum GENSCO2 strain.

(6) FIG. 6 shows the result of confirming the expression of IL-6 by strain, and IL-6 expression of GENSCO2 was observed as the lowest.

(7) FIG. 7 is the result showing the inhibitory activity of IL-6 expression of the fermented filtrates of GENSCO2 strain by concentration.

(8) FIG. 8 is the result showing the inhibitory activity of IL-8 expression of the fermented filtrates of GENSCO2 strain by concentration.

(9) FIG. 9 is the result showing the inhibitory activity of TNF-alpha expression of the fermented filtrates of GENSCO2 by concentration.

(10) FIG. 10 shows the result of cytotoxicity test of the fermented filtrates of GENSCO2 strain of the present invention.

(11) FIG. 11 shows the result of enhanced filaggrin expression by treatment of the fermented filtrates of GENSCO2 strain.

(12) FIG. 12 shows the result of enhanced claudin 1 expression by treatment of the fermented filtrates of GENSCO2 strain.

(13) FIG. 13 shows the result of enhanced HAS3 (Hyaluronic acid Synthase) expression by treatment of the fermented filtrates of GENSCO2 strain.

(14) FIG. 14 shows the result of enhanced aquaporin expression by treatment of the fermented filtrates of GENSCO2 strain.

(15) FIG. 15 is the result showing that the biofilm formation-inhibiting effect was excellent by treatment of the fermented filtrates of GENSCO2 strain.

(16) FIG. 16 is the result showing the reduction of TSLP expression by the fermented filtrates of Cutibacterium granulosum GENSCO2 strain.

(17) FIG. 17 shows the inhibitory activity of Cutibacterium acnes ATCC 6919 lipase by the fermented filtrates of Cutibacterium granulosum GENSCO2 strain.

(18) FIG. 18 is the result of confirming that the fermented filtrates of Cutibacterium granulosum GENSCO2 strain is effective in reduction of inflammation by fine dust.

DETAILED DESCRIPTION

(19) 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 granulosum GENSC02

(20) 1-1. Isolation of Strain

(21) 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 steaked on an RCM agar plate by picking it with a platinum loop, and this procedure was repeated 3-4 times to separate pure colonies.

(22) 1-2. Identification of Strain

(23) 1) Biological Identification Using API Kit

(24) 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.

(25) 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 showed the same biochemical properties as Propionibacterium (=Cutibacterium) granulosum.

(26) The API 20A reading result was shown in the following Table 1.

(27) TABLE-US-00001 TABLE 1 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 +

(28) 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. Based on the above result, the strain was named “Cutibacterium granulosum GENSC02” 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 KCTC13597BP.

(29) 1-3. Classification of Strains by Glycerol Use

(30) In order to confirm differences from other strains of the same kind with the deposited strain, the glycerol use was confirmed.

(31) To confirm the glycerol use, a medium was prepared by adding 2% glycerol to rich media (10 g/L yeast extract, 5 g/L TSB media, 2.5 g/L K.sub.2HPO.sub.4 and 1.5 g/l KH.sub.2PO.sub.4). In addition, when the inoculated medium used glycerol, the color of the medium changed from red to yellow, and therefore, 0.001% (w/v) of phenol red was added to visibly determine the use. Each of bacteria was inoculated in an RCM medium at 10.sup.5 CFU/ml through the active state and was anaerobically cultured at 37° C., and the medium inoculated with none was stored under the same conditions to clearly check the color change. The aspect of the change was observed at the same time for 6 days.

(32) As a result, from the 3rd day of culture, the color began to change in NSM13-4, CSM1-3 and CSM5-1, and the red color disappeared as time progressed, indicating perfect yellow color after 4-5 days. On the other hand, GENSC02 was slightly reddish compared to that before incubation, but the color difference was low compared to the control. Thus, it was confirmed that GENSC02 had a characteristic of little or no use of glycerol under rich media, and this means that the ability of using glycerol differs depending on the culture media such as rich media, API 20A media and the like.

(33) Table 2 shows the glycerol use result by each strain. As it turns yellow, it was marked as −/+/++/+++. In addition, the color change depending on glycerol use by each C. granulosum strain was shown in FIG. 2. (color change at day 1. 3 and 6). Thus, it was confirmed that GENSC02 has a difference from other stains in the ability of using glycerol.

(34) TABLE-US-00002 TABLE 2 Strain Species 0 day 1 day 2 days 3 days 4 days 5 days 6 days a control − − − − − − − b GENSC02 C. granulosum − − − − − − − c NSM13-4 C. granulosum − − − + ++ ++ ++ d CSM1-3 C. granulosum − − − ++ ++ +++ +++ e CSM5-1 C. granulosum − − − + ++ ++ ++

[Example 2] Hemolysis Test of Cutibacteria granulosum GENSC02

(35) A considerable number of Cutibacterium granulosum strains have hemolytic toxicity and are harmful to the human body depending on strains. To confirm the safety of Cutibacterium granulosum GENSC02, the presence or absence of hemolytic toxicity was confirmed. The Cutibacterium granulosum GENSC02 purely cultured in a liquid medium was collected by a platinum loop and it was streaked 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 granulosum GENSC02 had no hemolysis for sheep blood and therefore it was not harmful to the human body, as could be seen in FIG. 3.

[Example 3] Growth Inhibition on Staphylococcus aureus KCTC 1621 and Cutibacterium acnes ATCC 6919 (Overlay Clear Zone Test)

(36) 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 granulosum GENSC02 could not grow and they became transparent, if Cutibacterium granulosum GENSC02 had the effect of inhibiting the growth of bacteria. The Cutibacterium granulosum GENSC02 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 granulosum GENSC02 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 granulosum GENSC02 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 granulosum GENSC02. 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 granulosum GENSC02 was observed, and the transparency was reduced with distance from Cutibacterium granulosum GENSC02, thereby confirming that the Cutibacterium granulosum GENSC02 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 GENSC02 treatment. The result was shown in FIG. 4.

(37) In addition, as could be seen in FIG. 5, in the result of inoculating C. acnes, the transparent area around the Cutibacterium granulosum GENSC02 was observed, and the transparency was reduced with distance from Cutibacterium granulosum GENSC02, thereby confirming that the Cutibacterium granulosum GENSC02 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 GENSC02 treatment.

(38) Through these results, it was confirmed that the Cutibacterium granulosum GENSC02 of the present invention could provide an effect of improving, preventing or treating acne by inhibiting an acne-causing bacterium, C. acnes.

[Example 4] Preparation of Fermented Filtrates of the Strain of the Present Invention

(39) Cutibacterium granulosum GENSC02 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 5] Anti-Inflammatory Efficacy Evaluation (Selection of Beneficial Bacteria)

(40) At first, fermented filtrates of C. granulosum strain 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 one of inflammation response cytokine factors, IL-6 was confirmed by real-time PCR.

(41) In the result of FIG. 6, it could be seen that inflammation was caused by increases of expression of IL-6 by C. acnes. In the comparative experiment with various strains, it was shown that the expression of IL-6 by GENSCO2 fermented filtrates was reduced, and it was confirmed that GENSCO2 had a more excellent anti-inflammatory efficacy than other strains. FIG. 6 shows the result of confirming the expression of IL-6 by strain, and the IL-6 expression of GENSCO2 was observed as the lowest.

(42) Through the result of FIG. 6, in order to investigate the anti-inflammatory efficacy depending on the content of fermented filtrates of GENSCO2, the same experimental procedure as the above experiment was progressed, and the anti-inflammatory efficacy was reproduced again by treating the GENSCO2 fermented filtrates to be pre-treated at a concentration of 0.01, 0.1, and 1%, respectively.

(43) In the result of FIG. 7 to FIG. 9, it could be seen that the inflammation by C. acnes was caused by increases of expression of IL-6, IL-8, and TNF-α. It was confirmed that the expression of IL-6 was gradually reduced depending on the GENSCO2 fermented filtrates and the expression of IL-8 and TNF-alpha had an excellent anti-inflammatory efficacy in the low content of GENSCO2 fermented filtrates.

[Example 6] Cytotoxicity Confirmation

(44) To evaluate the cytotoxicity of fermented filtrates prepared in the Example 4, 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-yl)-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.

(45) The cytotoxicity test result of the fermented filtrates was shown in FIG. 10. As could be seen in FIG. 10, the treatment of GENSCO2 did not significantly affect the survival rate of HaCaT cell lines. Thus, it can be seen that GENSCO2 has no cytotoxicity.

[Example 7] Confirmation of Skin Barrier Function Enhancement Efficacy

(46) In order to investigate the GENSCO2 fermented filtrates had a function of skin barrier enhancement for the HaCaT cell line, in addition to the inflammation response, the fermented filtrates of GENSCO2 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.

(47) In the results of FIG. 11 and FIG. 12, it was confirmed that the expression of filaggrin and claudin-1 was increased to 1.5 times by the positive control group, retinoic acid (RA′, 1 μM), but on the other hand, the expression of claudin was increased 2 times or more from 0.1% of the GENSCO2 fermented filtrates, and the expression of filaggrin was increased from 0.01% of the GENSCO2 fermented filtrates.

(48) Specifically, as can be seen in FIG. 11, it can be seen that GENSCO2 has an excellent filaggrin expression effect compared than retinoic acid (positive control group) known to have an excellent filaggrin expression effect. Through this, GENSCO2 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.

(49) As can be seen in FIG. 12, it can be seen that GENSCO2 has an excellent claudin expression effect compared than retinoic acid (positive control group) known to have an excellent filaggrin expression effect. Through this, GENSCO2 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.

[Example 8] Skin Moisturizing Function Enhancement Efficacy Confirmation

(50) To investigate a function for a skin moisturizing effect by GENSCO2 fermented filtrates, it was confirmed that the fermented filtrates of GENSCO2 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 HAS3 (hyaluronic acid synthase) and aquaporin which were markers of the skin moisturizing function on RNA.

(51) In the results of FIG. 13 and FIG. 14, it was confirmed that the expression of HAS3 (hyaluronic acid synthase) and aquaporin was increased to 2˜4 times by the positive control group, retinoic acid (RA′, 1 μM), and the expression of HAS3 (hyaluronic acid synthase) and aquaporin was increased to 2 times or more compared to the negative control group (− group) by the fermented filtrates. Based on the results of FIG. 13 and FIG. 14, it was confirmed that GENSCO2 enhanced the function of skin moisturizing.

[Example 9] Measurement of Inhibitory Effect of Formation of Biofilm

(52) 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.

(53) As FIG. 15, it can be seen that GENSCO2 strain has a better biofilm formation inhibitory ability than the positive control group.

[Example 10] Confirmation of Itchiness Alleviation Efficacy

(54) The expression of TSLP, the cytokine acting as one of causes of atopic dermatitis was confirmed on RNA, by treating fermented filtrates of GENSCO2 to the HaCaT cell line by % and then reacting for 4 hours. As shown in FIG. 16, it was confirmed that the TSLP expression degree of fermented filtrates was reduced. Thus, it was confirmed that the GENSCO2 culture of the present invention inhibited the TSLP expression and solved skin itchiness, thereby having an effect in improvement of atopic dermatitis.

[Example 11] Cutibacterium acnes ATCC 6919 Lipase Inhibitory Activity (4-Methyl Umbelliferyl Oleate (MUO) Assay)

(55) The lipase inhibitory activity was evaluated using 4-methylumbelliferyl oleate as a substrate. In other words, the positive control group, ketoconazole (2.5 μg/ml), the negative group, and the fermented filtrates were treated to C. acnes in RCM broth (OD.sub.600=1) as 1:1, respectively, and they were anaerobically cultured at 37° C. The 100 ul of supernatant obtained by centrifuging them and 100 ul of 4-MU oleate (0.2 mg/ml in DMSO) were mixed in black 96-well microtiter plates, and they were reacted at 37° C. for 24 hours. The 4-methylumbelliferone released by lipase was measured with a fluorometric microplate reader.

(56) In the result of FIG. 17, it was confirmed that the similar effect to the positive control group was obtained when treating 1% of fermented filtrates, and the Cutibacterium acnes ATCC 6919 lipase inhibitory activity was shown in a concentration dependent manner.

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

(57) 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 GENSCO2 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. 18. It was confirmed that the expression of IL-6 was significantly reduced by the GENSCO2 culture filtrates and it was further reduced in case of treatment for 2 hours.

INDUSTRIAL APPLICABILITY

(58) The present invention provides a new Cutibacterium granulosum strain or culture thereof which can be used as a cosmetic composition.

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

(60) [Accession Number]

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

(62) Accession number: KCTC13597BP

(63) Deposit date: 20180724

(64) 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).