Composition for preventing, improving, or treating inflammatory skin diseases comprising a Cutibacterium strain

Abstract

The present application relates to the use of a Cutibacterium strain, a culture of said strain, or a combination thereof for preventing, improving, and / or treating inflammatory skin diseases, and a composition containing a Cutibacterium strain, a culture of said strain, or a combination thereof does not exhibit cytotoxicity, inhibits the proliferation of immune cells that induce skin inflammation, inhibits immune responses, has antibacterial effects against acne-inducing bacteria, and promotes the regeneration of skin cells, thereby providing excellent effects in preventing, improving, and / or treating inflammatory skin diseases.

Description

[Technical Field]

[0001] The present application relates to the use of a Cutibacterium strain, a culture of said strain, or a combination thereof, for the prevention, amelioration, or treatment of inflammatory skin diseases. [Background technology]

[0002] Skin protects the body from various oxidants, air pollution, heavy metals, and ultraviolet radiation (UV), and acts as a protective barrier to prevent bacteria, fungi, and viruses from invading the skin and preventing moisture loss. Skin is broadly divided into three layers: the epidermis, dermis, and subcutaneous fat. The dermis, which accounts for 90% of the skin, is composed of collagen, elastin, hyaluronic acid, and glycoproteins. Collagen, the main component, is an important fibrous protein that accounts for approximately 25% of the total protein mass in the human body and makes up the majority of connective tissue, fulfilling important functions such as acting as an intercellular matrix, providing structural support, inducing cell division and differentiation, and providing tensile strength to the skin.

[0003] Furthermore, it is well known that sebum components, sweat components, and fatty acids, higher alcohols, and proteins in cosmetic ingredients excreted from the body are broken down into highly toxic substances by the resident bacteria on the skin, which can induce skin inflammation. It is also well known that secondary infection exposure by pathogenic microorganisms due to damage to the skin barrier caused by ultraviolet rays from the sun can induce various skin diseases such as acne, itching, dermatitis, and allergies.

[0004] In order to protect the skin from such various harmful environments, it is necessary to discover substances that effectively inhibit the growth of harmful bacteria on the skin without causing side effects, and to develop medicines and / or cosmetics that use such substances. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent No. 10-2032546 Summary of the Invention [Problem to be solved by the invention]

[0006] The purpose of this application is to (a) an Lrp / AsnC family transcriptional regulator or a gene encoding the same; and (b) To provide a Cutibacterium strain containing a lactococcin 972 family bacteriocin or a gene encoding the same.

[0007] Another object of the present application is to provide a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, comprising said strain, a culture of said strain, or a combination thereof.

[0008] Another object of the present application is to provide an antibacterial composition against inflammatory skin disease-causing bacteria, comprising said strain, a culture of said strain, or a combination thereof.

[0009] Another object of the present application is to provide a food composition for preventing or ameliorating inflammatory skin diseases, comprising said strain, a culture of said strain, or a combination thereof.

[0010] Another object of the present application is to provide a feed composition for preventing or ameliorating inflammatory skin diseases, comprising said strain, a culture of said strain, or a combination thereof. [Means for solving the problem]

[0011] The present applicant has made extensive efforts to develop a composition that is effective in preventing, improving, and / or treating inflammatory skin diseases such as acne and rosacea, and has found that a composition containing a Cutibacterium strain, a culture of said strain, or a combination thereof is effective in preventing, improving, and / or treating inflammatory skin diseases, thereby completing the present application.

[0012] Thus, the present application provides a novel Cutibacterium strain.

[0013] The present application provides the use of a Cutibacterium strain, a culture of said strain, or a combination thereof for the prevention, amelioration, and / or treatment of inflammatory skin diseases.

[0014] One example of the present application provides a composition for preventing, ameliorating, and / or treating an inflammatory skin disease, comprising a Cutibacterium strain, a culture of said strain, or a combination thereof.

[0015] In one example, the composition may be a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition.

[0016] Another example provides a pharmaceutical composition for preventing, ameliorating, and / or treating an inflammatory skin disease, comprising a Cutibacterium strain, a culture of said strain, or a combination thereof.

[0017] Another example provides a food composition for preventing and / or improving inflammatory skin diseases, comprising a Cutibacterium strain, a culture of the strain, or a combination thereof. The food composition may be a food additive or a functional health food.

[0018] Another example provides a feed composition for preventing and / or improving inflammatory skin diseases, comprising a Cutibacterium strain, a culture of the strain, or a combination thereof. The feed composition may be a feed additive.

[0019] Another example provides a cosmetic composition for preventing and / or improving inflammatory skin diseases or for improving skin, which comprises a Cutibacterium strain, a culture of said strain, or a combination thereof.

[0020] Another example provides a method for preventing, ameliorating, and / or treating an inflammatory skin disease, comprising administering an effective amount of a Cutibacterium strain, a culture of said strain, or a combination thereof to a subject in need of such prevention, amelioration, and / or treatment.

[0021] Another example provides the use of a Cutibacterium strain, a culture of said strain, or a combination thereof, for the manufacture of a composition (e.g., a pharmaceutical composition, cosmetic composition, food composition, or feed composition) for the prevention, amelioration, and / or treatment of inflammatory skin diseases.

[0022] Another example provides the use of a Cutibacterium strain, a culture of said strain, or a combination thereof, for inhibiting inflammatory skin disease-causing bacteria.

[0023] Another example provides an antibacterial composition against inflammatory skin disease-causing bacteria, which comprises a Cutibacterium strain, a culture of said strain, or a combination thereof.

[0024] Another example provides a method for inhibiting inflammatory skin disease-causing bacteria, comprising applying an effective amount of a Cutibacterium strain, a culture of said strain, or a combination thereof to a subject in need of antibacterial treatment against inflammatory skin disease-causing bacteria.

[0025] Another example provides the use of a Cutibacterium strain, a culture of said strain, or a combination thereof for the manufacture of an antibacterial composition against inflammatory skin disease-causing bacteria.

[0026] Another example provides a disinfectant comprising a Cutibacterium strain, a culture of said strain, or a combination thereof as an active ingredient.

[0027] Other examples provide uses of the Cutibacterium strain, a culture of the strain, or a combination thereof for the manufacture of a disinfectant.

[0028] Another example provides a method of disinfection comprising applying the disinfectant to an object in need of disinfection.

[0029] Another example provides a cleaning agent comprising a Cutibacterium strain, a culture of said strain, or a combination thereof as an active ingredient.

[0030] Another example provides the use of a Cutibacterium strain, a culture of said strain, or a combination thereof, for use in the manufacture of a cleaning agent.

[0031] Another example provides a method of cleansing comprising applying the cleanser to an object in need of cleansing.

[0032] The present application is described in more detail below.

[0033] Cutibacterium sp. strains and cultures thereof

[0034] One embodiment provides a Cutibacterium strain.

[0035] As used herein, the term "Cutibacterium strain" may include all strains of the genus Cutibacterium, and may be, but is not limited to, one or more species (one, two, or three) selected from the group consisting of Cutibacterium acnes (C. acnes), Cutibacterium avidum (C. avidum), and Cutibacterium granulosum (C. granulosum).

[0036] In one example, the Cutibacterium strain may be, but is not limited to, C. acnes subsp. acnes (Type I), C. acnes subsp. defendens (Type II), or C. acnes subsp. elongatum (Type III). In one example, the Cutibacterium strain may be C. acnes subsp. defendens (Type II).

[0037] In one example, the Cutibacterium strain may have a ribotype of, but is not limited to, RT1, RT2, RT3, RT4, RT5, RT6, RT7, RT8, RT9, or RT10. In one example, the Cutibacterium strain may be RT2.

[0038] In one example, the Cutibacterium strain may have a sequence type of 69 or 153.

[0039] In one example, the Cutibacterium strain may have a clonal complex (CC) of CC72 (Type II).

[0040] In one example, the Cutibacterium sp. strain is (a) an Lrp / AsnC family transcriptional regulator (NCBI Reference Sequence: WP_002531510.1) and / or a gene encoding the same; and (b) containing one or more (one or two) selected from the group consisting of lactococcin 972 family bacteriocin (NCBI Reference Sequence: WP_070651130.1) and / or genes encoding the same, and / or It may not contain one or more (one, two, three, four, five, or six) proteins and / or genes encoding the same selected from the group consisting of the following (1) to (6): (1) beta-glucuronidase (NCBI Reference Sequence: WP_002518535.1) or a gene encoding the same; (2) 2-isopropylmalate synthase (NCBI Reference Sequence: WP_142263178.1) or a gene encoding the same; (3) 3-isopropylmalate dehydratase (NCBI Reference Sequence: WP_002513330.1) or a gene encoding the same; (4) Type IE CRISPR-associated protein Cse1 / CasA (NCBI Reference Sequence: WP_002514606.1) or a gene encoding the same; (5) Type IE CRISPR-associated protein Cas7 / Cse4 / CasC (NCBI Reference Sequence: WP_002514608.1) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 (NCBI Reference Sequence: WP_002514605.1) or a gene encoding the same.

[0041] In one example, the Cutibacterium sp. strain is (a) Lrp / AsnC family transcriptional regulator (NCBI Reference Sequence: WP_002531510.1) and / or a gene encoding the same; and (b) containing one or more (one or two) selected from the group consisting of lactococcin 972 family bacteriocin (NCBI Reference Sequence: WP_070651130.1) and / or genes encoding the same; It may not contain one or more (one, two, three, four, five, or six) proteins and / or genes encoding the same selected from the group consisting of the following (1) to (6): (1) beta-glucuronidase (NCBI Reference Sequence: WP_002518535.1) or a gene encoding the same; (2) 2-isopropylmalate synthase (NCBI Reference Sequence: WP_142263178.1) or a gene encoding the same; (3) 3-isopropylmalate dehydratase (NCBI Reference Sequence: WP_002513330.1) or a gene encoding the same; (4) Type IE CRISPR-associated protein Cse1 / CasA (NCBI Reference Sequence: WP_002514606.1) or a gene encoding the same; (5) Type IE CRISPR-associated protein Cas7 / Cse4 / CasC (NCBI Reference Sequence: WP_002514608.1) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 (NCBI Reference Sequence: WP_002514605.1) or a gene encoding the same.

[0042] In one example, the Cutibacterium sp. strain is (a) an Lrp / AsnC family transcriptional regulator (NCBI Reference Sequence: WP_002531510.1) or a gene encoding the same; and (b) lactococcin 972 family bacteriocin (NCBI Reference Sequence: WP_070651130.1) or a gene encoding the same; It may not contain any of the following proteins (1) to (6) or the genes encoding them: (1) beta-glucuronidase (NCBI Reference Sequence: WP_002518535.1) or a gene encoding the same; (2) 2-isopropylmalate synthase (NCBI Reference Sequence: WP_142263178.1) or a gene encoding the same; (3) 3-isopropylmalate dehydratase (NCBI Reference Sequence: WP_002513330.1) or a gene encoding the same; (4) Type IE CRISPR-associated protein Cse1 / CasA (NCBI Reference Sequence: WP_002514606.1) or a gene encoding the same; (5) Type IE CRISPR-associated protein Cas7 / Cse4 / CasC (NCBI Reference Sequence: WP_002514608.1) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 (NCBI Reference Sequence: WP_002514605.1) or a gene encoding the same.

[0043] In one example, the Cutibacterium strain may contain a 16S rRNA gene consisting of SEQ ID NO:8.

[0044] In one example, the Cutibacterium strain may be selected from the group consisting of: CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656.

[0045] The strains selected from the group consisting of CJRS-10651 strain, CJRS-10652 strain, CJRS-10653 strain, CJRS-10654 strain, CJRS-10655 strain, and CJRS-10656 strain may commonly contain the 16S rRNA sequence of SEQ ID NO: 8.

[0046] The CJRS-10651 strain may be that having deposit number KCCM13032P (designated "Cutibacterium acnes CJRS-10651" by the depositor).

[0047] The CJRS-10652 strain may be that having deposit number KCCM13033P (designated "Cutibacterium acnes CJRS-10652" by the depositor).

[0048] The CJRS-10653 strain may be that having deposit number KCCM13034P (designated "Cutibacterium acnes CJRS-10653" by the depositor).

[0049] The CJRS-10654 strain may be that having deposit number KCCM13035P (designated "Cutibacterium acnes CJRS-10654" by the depositor).

[0050] The CJRS-10655 strain may be that having deposit number KCCM13036P (designated "Cutibacterium acnes CJRS-10655" by the depositor).

[0051] The CJRS-10656 strain may be that having deposit number KCCM13037P (designated "Cutibacterium acnes CJRS-10656" by the depositor).

[0052] As used herein, a "culture" of a Cutibacterium strain may refer to a product obtained by culturing a Cutibacterium strain. The Cutibacterium strain culture may be in a form in which the bacterial cells have been removed or in a form in which the bacterial cells have not been removed. The Cutibacterium strain culture in a form in which the bacterial cells have not been removed may contain a Cutibacterium strain within the culture.

[0053] The culture may be a whole culture of a Cutibacterium strain, a dilution thereof, a concentrate, a dried product (e.g., a freeze-dried product), a disrupted product, and / or a fraction thereof, and the concentrate may be obtained by centrifuging or evaporating the culture, the dried product may be obtained by drying the culture using a dryer, etc., the freeze-dried product may be obtained by freeze-drying the culture using a freeze-dryer, etc., the disrupted product may be obtained by physically or ultrasonically treating the strain or culture, and the fraction may be obtained by subjecting the culture, disrupted product, etc. to a method such as centrifugation or chromatography.

[0054] The culture may be, but is not limited to, solid phase (solid, eg, dry), liquid phase (liquid), or fluid phase.

[0055] In one example, the culture may refer to a whole medium containing a cultured strain of the genus Cutibacterium obtained by culturing the strain for a certain period of time, its metabolites, and / or additional nutrients.

[0056] In one example, the culture may be a cell-free culture obtained by culturing a Cutibacterium strain in a medium and then removing the bacterial cells (strain). The bacterial cells may be removed by a conventional method such as separation (e.g., centrifugation) or filtration, for example, by centrifugation. In one specific example, the cell-free culture may be a supernatant obtained by centrifuging a culture obtained by culturing a Cutibacterium strain in a medium.

[0057] The culture may also be a liquid (or culture filtrate) remaining after filtering a culture obtained by culturing a Cutibacterium strain in a medium or a cell-free culture from which the strain has been removed.

[0058] In one example, the culture may be one in which the molecular weight of a component contained in the culture satisfies a specific numerical range. The culture may be in a strain-free form or a strain-free form.

[0059] For example, the culture may be one in which the molecular weight of the components contained in the culture is 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, 3 kDa or less, 2.5 kDa or less, 2 kDa or less, 1.5 kDa or less, 1 kDa or less, or 0.5 kDa or less, but is not limited thereto. As used herein, kDa refers to kilodalton, and dalton refers to atomic mass unit (g / mol). Also, as used herein, "or less" should be interpreted as including a range exceeding 0. For example, "3 kDa or less" can refer to a numerical range of more than 0 to 3 kDa or less.

[0060] More specifically, the culture contains components with molecular weights of 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, 3 kDa or less, 2.5 kDa or less, 2 kDa or less, 1.5 kDa or less, 1 kDa or less, 0.5 kDa or less, or 0.5~4kDa、0.5~3.75kDa、0.5~3.5kDa、0.5~3.25kDa、0.5~3kDa、0.5~2.75kDa、0.5~2.5kDa、0.5~2.25kDa、0.5~2kDa、0.5~1.75kDa、0.5~1.5kDa、0.5~ 1.25kDa、0.5~1kDa、0.5~0.75kDa、0.75~4kDa、0.75~3.75kDa、0.75~3.5kDa、0.75~3.25kDa、0.75~3kDa、0.75~2.75kDa、0.75~2.5kDa、0.75~2.25kDa、 0.75~2kDa、0.75~1.75kDa、0.75~1.5kDa、0.75~1.25kDa、0.75~1kDa、1~4kDa、1~3.75kDa、1~3.5kDa、1~3.25kDa、1~3kDa、1~2.75kDa、1~2.5kDa、1~2.2 5kDa、1~2kDa、1~1.75kDa、1~1.5kDa、1~1.25kDa、1.25~4kDa、1.25~3.75kDa、1.25~3.5kDa、1.25~3.25kDa、1.25~3kDa、1.25~2.75kDa、1.25~2.5kDa、1 .25~2.25kDa、1.25~2kDa、1.25~1.75kDa、1.25~1.5kDa、1.5~4kDa、1.5~3.75kDa、1.5~3.5kDa、1.5~3.25kDa、1.5~3kDa、1.5~2.75kDa、1.5~2.5kDa、1 .5~2.25kDa、1.5~2kDa、1.5~1.75kDa、1.75~4kDa、1.75~3.75kDa、1.75~3.5kDa、1.75~3.25kDa、1.75~3kDa、1.75~2.75kDa、1.75~2.5kDa、1.75~2.25k Da、1.75~2kDa、2~4kDa、2~3.75kDa、2~3.5kDa、2~3.25kDa、2~3kDa、2~2.75kDa、2~2.5kDa、2~2.25kDa、2.25~4kDa、2.25~3.75kDa、2.25~3.5kDa、2.25~ 3.25kDa、2.25~3kDa、2.25~2.75kDa、2.25~2.5kDa、2.5~4kDa、2.5~3.75kDa、2.5~3.5kDa、2.5~3.25kDa、2.5~3kDa、2.5~2.75kDa、2.75~4kDa、2.75~3.The range may be, but is not limited to, 75 kDa, 2.75 to 3.5 kDa, 2.75 to 3.25 kDa, 2.75 to 3 kDa, 3 to 4 kDa, 3 to 3.75 kDa, 3 to 3.5 kDa, 3 to 3.25 kDa, 3.25 to 4 kDa, 3.25 to 3.75 kDa, 3.25 to 3.5 kDa, 3.5 to 4 kDa, 3.5 to 3.75 kDa, or 3.75 to 4 kDa.

[0061] For example, appropriate means, procedures, processes, etc. may be performed to ensure that the molecular weight of the component contained in the culture satisfies the above numerical range, such as filtration, separation, etc. As used herein, "filtration" may be used interchangeably with "separation" and may refer to any type of process performed to obtain a component contained in a culture with a size or molecular weight within a specific numerical range.

[0062] For example, the culture may be obtained by filtering a culture of a Cutibacterium strain through a filter to a particle size of 0.3 μm or less, 0.29 μm or less, 0.28 μm or less, 0.27 μm or less, 0.26 μm or less, 0.25 μm or less, 0.24 μm or less, 0.23 μm or less, 0.22 μm or less, 0.21 μm or less, or 0.2 μm or less, but is not limited thereto. In a specific example, the culture may be obtained by filtering a culture of a Cutibacterium strain through a filter to a particle size of 0.22 μm. As used herein, "or less" should be interpreted as including a range exceeding 0 (e.g., "0.3 μm or less" means a numerical range of greater than 0 to 0.3 μm or less). Any filter commonly used for filtering cultures can be used without limitation. The culture may be in a form in which the Cutibacterium strain has been removed or not.

[0063] For example, the culture may be a filtrate obtained by filtering a culture of a Cutibacterium strain through a filter to a molecular weight of 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, 3 kDa or less, 2.5 kDa or less, 2 kDa or less, 1.5 kDa or less, 1 kDa or less, or 0.5 kDa or less, but is not limited thereto. In another specific example, the culture may be a culture obtained by filtering a culture of a Cutibacterium strain through a filter to a molecular weight of 3 kDa or less. Any filter commonly used for filtering cultures may be used without limitation.

[0064] As used herein, the term "culture obtained by filtration to a specific number of kDa or less" can be used interchangeably with the term "isolate obtained by separation to a specific number of kDa or less." That is, a culture obtained by filtration to a specific number of kDa (or an isolate obtained by separation) can mean that the substances contained in the culture have a molecular weight of a specific number of kDa or less.

[0065] For example, a culture obtained by filtration to 3 kDa or less may mean that the substances contained in the culture have a molecular weight of 3 kDa or less. That is, a culture obtained by filtration within a specific range (kDa) herein may mean that the molecular weights of all substances contained in the culture satisfy the specific range (kDa).

[0066] More specifically, the culture is a culture of a Cutibacterium strain, which is filtered using a filter to filter the sieve size to 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, 3 kDa or less, 2.5 kDa or less, 2 kDa or less, 1.5 kDa or less, 1 kDa or less, 0.5 kDa or less, or 0.5~4kDa、0.5~3.75kDa、0.5~3.5kDa、0.5~3.25kDa、0.5~3kDa、0.5~2.75kDa、0.5~2.5kDa、0.5~2.25kDa、0.5~2kDa、0.5~1.75kDa、0.5~1.5kDa、0.5~ 1.25kDa、0.5~1kDa、0.5~0.75kDa、0.75~4kDa、0.75~3.75kDa、0.75~3.5kDa、0.75~3.25kDa、0.75~3kDa、0.75~2.75kDa、0.75~2.5kDa、0.75~2.25kDa、 0.75~2kDa、0.75~1.75kDa、0.75~1.5kDa、0.75~1.25kDa、0.75~1kDa、1~4kDa、1~3.75kDa、1~3.5kDa、1~3.25kDa、1~3kDa、1~2.75kDa、1~2.5kDa、1~2.2 5kDa、1~2kDa、1~1.75kDa、1~1.5kDa、1~1.25kDa、1.25~4kDa、1.25~3.75kDa、1.25~3.5kDa、1.25~3.25kDa、1.25~3kDa、1.25~2.75kDa、1.25~2.5kDa、1 .25~2.25kDa、1.25~2kDa、1.25~1.75kDa、1.25~1.5kDa、1.5~4kDa、1.5~3.75kDa、1.5~3.5kDa、1.5~3.25kDa、1.5~3kDa、1.5~2.75kDa、1.5~2.5kDa、1 .5~2.25kDa、1.5~2kDa、1.5~1.75kDa、1.75~4kDa、1.75~3.75kDa、1.75~3.5kDa、1.75~3.25kDa、1.75~3kDa、1.75~2.75kDa、1.75~2.5kDa、1.75~2.25k Da、1.75~2kDa、2~4kDa、2~3.75kDa、2~3.5kDa、2~3.25kDa、2~3kDa、2~2.75kDa、2~2.5kDa、2~2.25kDa、2.25~4kDa、2.25~3.75kDa、2.25~3.5kDa、2.25~ 3.25kDa、2.25~3kDa、2.25~2.75kDa、2.25~2.5kDa、2.5~4kDa、2.5~3.75kDa、2.5~3.5kDa、2.5~3.25kDa、2.5~3kDa、2.5~2.75kDa、2.75~4kDa、2.75~3.The culture may be obtained by filtration through a sieve of 75 kDa, 2.75 to 3.5 kDa, 2.75 to 3.25 kDa, 2.75 to 3 kDa, 3 to 4 kDa, 3 to 3.75 kDa, 3 to 3.5 kDa, 3 to 3.25 kDa, 3.25 to 4 kDa, 3.25 to 3.75 kDa, 3.25 to 3.5 kDa, 3.5 to 4 kDa, 3.5 to 3.75 kDa, or 3.75 to 4 kDa, but is not limited thereto.

[0067] In one example, the culture obtained by filtration within the above range may have excellent (1) preventive, ameliorative, and / or therapeutic effects on inflammatory skin diseases, and / or (2) antibacterial effects against acne-causing bacteria.

[0068] For example, the culture of a Cutibacterium strain may be a culture obtained by culturing a Cutibacterium strain in a medium for a certain period of time, or may be a culture containing killed cells of a Cutibacterium strain obtained by heat-treating (or sterilizing (e.g., autoclaving or hot air drying)) the culture.

[0069] As used herein, "cultivation" refers to a series of actions to grow microorganisms under appropriately artificially controlled environmental conditions. Any culture conditions and culture methods known in the art can be used for the culture. For example, the culture can be continuously conducted using known batch culture methods, continuous culture methods, fed-batch culture methods, batch processes, fed-batch processes, or repeated fed-batch processes. The culture conditions are not particularly limited, but can be adjusted to an appropriate pH (e.g., pH 5-9, pH 6-8, or pH 6.8) using basic compounds (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compounds (e.g., phosphoric acid or sulfuric acid). For example, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester, or aerobic conditions can be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture.

[0070] For example, the culture temperature may be 20 to 45°C, 25 to 40°C, 30 to 40°C, 30 to 35°C, or 35 to 40°C, the culture conditions may be 100 to 500 rpm, 150 to 300 rpm, 150 to 250 rpm, or 200 rpm, and the culture time (duration) may be 1 to 160 hours, 10 to 100 hours, 12 to 72 hours, 24 to 72 hours, 30 to 60 hours, 40 to 50 hours, 45 to 50 hours, or 48 hours. For example, the culture may be performed at a culture temperature within the above ranges for a culture time within the above ranges.

[0071] The medium used for the culture should meet the requirements of the specific strain in an appropriate manner, and those skilled in the art can use the appropriate medium according to known methods.

[0072] To culture the Cutibacterium strain, the survival requirements of the specific strain can be met in an appropriate manner by adjusting the temperature, pH, etc. under anaerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used in the medium include, but are not limited to, sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid), either individually or in combination. Nitrogen sources include, but are not limited to, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal powder, and urea) or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), which may be used individually or in combination. Phosphorus sources include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts. The culture medium may also contain other metal salts (e.g., magnesium sulfate or iron sulfate) necessary for growth, or essential growth substances such as amino acids and vitamins, but are not limited to these.

[0073] Effects of Cutibacterium sp. strains and / or their cultures

[0074] For example, the Cutibacterium strain and / or a culture thereof may have one or more (one, two, three, four, or all five) activities selected from the group consisting of anti-inflammatory activity, antibacterial activity, skin regenerating activity, skin moisturizing activity, and activity for preventing, improving, and / or treating inflammatory skin diseases, or the activities may be enhanced. The enhanced activity may include exhibiting an activity that was not originally possessed, or exhibiting an activity that is improved compared to the intrinsic activity or the activity before transformation.

[0075] As used herein, the term "anti-inflammatory activity" may refer to any activity that reduces inflammation, such as inhibiting the occurrence of inflammation or reducing the severity of inflammation. For example, the anti-inflammatory activity may refer to an activity such as inhibiting the proliferation of inflammatory cells (e.g., macrophages) that induce inflammation, or inhibiting the gene transcription or protein translation of inflammatory substances (e.g., inflammatory cytokines, histamine, etc.) produced by inflammatory cells. For example, the anti-inflammatory activity may refer to a reduction in inflammatory substances produced by immune responses (e.g., inflammatory cytokines (e.g., interleukin-6), chemokine ligands (e.g., CCL2 (CC Motif Chemokine Ligand 2)), tumor necrosis factor (TNF-α), etc.). The inflammation may be caused by an inflammatory skin disease.

[0076] As used herein, the term "antibacterial activity" may refer to the activity of inhibiting the growth of and / or killing bacteria that induce inflammation. The bacteria that induce inflammation may be bacteria that induce inflammatory skin diseases.

[0077] In this specification, the term "skin" may include not only the face but also the scalp and the entire body. In this specification, the term "skin-improving activity" may include all of skin regeneration, skin moisturizing, skin whitening, improving skin elasticity, improving skin wrinkles, preventing skin aging, providing skin moisture, providing skin nutrients, improving skin tone, reducing pores, improving skin texture, exfoliating, improving skin volume, protecting the scalp, preventing scalp or skin sebum, preventing hair loss, providing hair moisture, providing hair nutrients, improving hair volume, protecting the skin (e.g., for alleviating or improving skin inflammation, improving skin itching, or improving atopy), or improving skin condition.

[0078] The skin improvement may be one or more selected from the group consisting of skin wrinkle improvement, skin elasticity improvement, skin anti-aging, skin moisturizing improvement, skin moisture supply, and skin nutrition supply.

[0079] The skin improvement may be one or more selected from the group consisting of scalp protection, hair loss prevention, hair moisture supply, and hair nutrition supply.

[0080] The skin improvement may refer to improvement of sensitive skin. The improvement of sensitive skin may be improvement of skin reactions (e.g., skin troubles, immune reactions, inflammatory reactions, etc.) caused by temperature (e.g., high or low temperature), wind, harmful substances (e.g., fine dust, sebum, oil, bacteria, etc.), or chemicals (e.g., hormones, etc.).

[0081] As used herein, the terms "skin regenerating activity" and "skin moisturizing activity" may refer to any activity that maintains healthy skin or improves or promotes skin conditions that have deteriorated due to disease. The "skin regenerating activity" and "skin moisturizing activity" may refer to promoting or inducing the proliferation of cells present in the epidermis, dermis, and / or subcutaneous tissue of the skin. The disease that worsens the skin condition may be an inflammatory skin disease. In one example, the "skin regenerating activity" and "skin moisturizing activity" may refer to increasing the expression level of a skin regeneration-related marker (e.g., a protein or gene such as Src or MMP2 (metalloproteinase-2)) or increasing the expression level of a skin moisturizing-related marker (e.g., a protein or gene such as HAS3 (hyaluronic acid synthase 3) or AQP3 (aquaporin 3)).

[0082] In one example, the Cutibacterium sp. strain and / or a culture of the strain (a) an Lrp / AsnC family transcriptional regulator (NCBI Reference Sequence: WP_002531510.1) or a gene encoding the same; and (b) does not contain one or more (one or two) selected from the group consisting of lactococcin 972 family bacteriocin (NCBI Reference Sequence: WP_070651130.1) or genes encoding the same, and / or The activity may be superior to that of a strain (e.g., a strain of the genus Cutibacterium) and / or a culture thereof containing one or more (one, two, three, four, five, or six) proteins selected from the group consisting of the following (1) to (6) or genes encoding the same: (1) beta-glucuronidase (NCBI Reference Sequence: WP_002518535.1) or a gene encoding the same; (2) 2-isopropylmalate synthase (NCBI Reference Sequence: WP_142263178.1) or a gene encoding the same; (3) 3-isopropylmalate dehydratase (NCBI Reference Sequence: WP_002513330.1) or a gene encoding the same; (4) Type IE CRISPR-associated protein Cse1 / CasA (NCBI Reference Sequence: WP_002514606.1) or a gene encoding the same; (5) Type IE CRISPR-associated protein Cas7 / Cse4 / CasC (NCBI Reference Sequence: WP_002514608.1) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 (NCBI Reference Sequence: WP_002514605.1) or a gene encoding the same.

[0083] Pharmaceutical compositions containing Cutibacterium sp. strains and / or cultures thereof

[0084] Another embodiment provides a pharmaceutical composition for preventing, ameliorating, and / or treating an inflammatory skin disease, comprising a Cutibacterium strain, a culture of said strain, or a combination thereof. Another example provides a method for preventing, ameliorating, and / or treating an inflammatory skin disease, comprising administering an effective amount of a Cutibacterium strain, a culture of said strain, or a combination thereof to a subject in need thereof.

[0085] In this application, "prevention" refers to any action that suppresses or delays the onset of a disease (illness) by administering a composition according to an embodiment, "treatment" refers to any action that improves or favorably changes the suspicion of a disease and the symptoms of an affected individual by administering a composition according to an embodiment, and "amelioration" refers to any action that at least reduces a parameter related to the state of the disease being treated, for example, the severity of symptoms, by administering a composition according to an embodiment. The disease may refer to an inflammatory skin disease.

[0086] As used herein, "inflammatory skin disease" may be one or more selected from the group consisting of alopecia, rosacea, redness, bumps, blisters, hives, itching, peeling skin, rash, allergic drug rash, non-allergic drug rash (e.g., drug rash caused by antipsychotics, tetracycline, sulfa antibiotics, hydrochlorothiazide, or nonsteroidal anti-inflammatory drugs (NSAIDs)), Stevens-Johnson syndrome, toxic epidermolysis, erythema nodosum, erythema multiforme, keratosis pilaris, psoriasis, eczema, bacterial dermatitis (e.g., dermatitis caused by Cutibacterium spp. (e.g., Cutibacterium acnes, Cutibacterium avidum, and / or Cutibacterium granulosum)), viral dermatitis, atopic dermatitis, injection dermatitis, and acne.

[0087] The pharmaceutical composition according to one embodiment may be administered by any method as long as it can reach the target tissue. For example, it may be administered parenterally, such as intraperitoneally, intraperitoneally, intraarticularly, intra-arterially, intravenously, transdermally (e.g., applied to the scalp or skin), transdermal injection, topically, intramuscularly, or intraperitoneally, or it may be administered orally, but is not limited thereto. Furthermore, the pharmaceutical composition may be administered by any device that can transport the active substance to the target cells.

[0088] An exemplary pharmaceutical composition may further include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. Specifically, the pharmaceutical compositions may be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injections by conventional methods. Examples of carriers, excipients, and diluents included in the pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, may be used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid formulations are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. can be used.

[0089] The pharmaceutical composition according to an embodiment can be administered in a pharmaceutically effective amount. In the present application, a "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level can be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, and sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present application used, the duration of treatment, drugs used in combination with or co-administered with the composition of the present application, and other factors well known in the medical field. The pharmaceutical composition according to an embodiment can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect at the minimum dose without side effects.

[0090] The dosage of the pharmaceutical composition (based on the dry weight of the culture of the Cutibacterium sp. strain) for one day to a mammal is, for example, about 0.0001 to 10,000 mg / kg, 0.0001 to 5,000 mg / kg, 0.0001 to 1,000 mg / kg, 0.0001 to 900 mg / kg, 0.0001 to 800 mg / kg, 0.0001 to 700 mg / kg, 0.0001 to 600 mg / kg, 0.0001 to 500 mg / kg, 0.0001 to 400 mg / kg kg, 0.0001~300mg / kg, 0.0001~200mg / kg, 1~1000mg / kg, 1~900mg / kg, 1~800mg / kg, 1~700mg / kg, 1~600mg / kg, 1~500mg / kg , 1~450mg / kg, 1~400mg / kg, 1~350mg / kg, 1~300mg / kg, 1~250mg / kg, 10~1000mg / kg, 10~900mg / kg, 10~800mg / kg, 10~700mg / kg kg, 10~600mg / kg, 10~500mg / kg, 10~450mg / kg, 10~400mg / kg, 10~350mg / kg, 10~300mg / kg, 10~250mg / kg, 100~1000mg / kg, 100~900mg / kg, 100~800mg / kg, 100~700mg / kg, 100~600mg / kg, 100~500mg / kg, 100~450mg / kg, 100~400mg / kg, 100~350mg / The pharmaceutical composition of the present application may be administered at a dose of, but not limited to, 100-300 mg / kg, 100-250 mg / kg, 200-1000 mg / kg, 200-900 mg / kg, 200-800 mg / kg, 200-700 mg / kg, 200-600 mg / kg, 200-500 mg / kg, 200-450 mg / kg, 200-400 mg / kg, 200-350 mg / kg, 200-300 mg / kg, or 200-250 mg / kg. The frequency of administration of the pharmaceutical composition of the present application is not particularly limited, but may be administered once a day or in divided doses administered several times a day. The above dosages do not limit the scope of the present application in any way.

[0091] The subjects to which the pharmaceutical compositions provided herein are administered may be mammals including humans, dogs, cats, horses, cows, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures thereof isolated therefrom. In one example, the subject may be an individual (a mammal such as a human) in need of prevention, amelioration, and / or treatment of an inflammatory skin disease as described above, or an individual having an inflammatory skin disease, or cells, tissues, or cultures thereof isolated therefrom.

[0092] An example pharmaceutical composition includes the Cutibacterium strain, a culture of the strain, or a combination thereof in an amount of 1 to 80% by weight, 5 to 80% by weight, 5 to 75% by weight, 5 to 70% by weight, 5 to 65% by weight, 50 to 70% by weight, 55 to 65% by weight, 60 to 65% by weight, 10 to 60% by weight, 15 to 60% by weight, 20 to 60% by weight, 1 to 50% by weight, 5 to 50% by weight, 10 to 50% by weight , 15 to 50% by weight, 20 to 50% by weight, 1 to 40% by weight, 5 to 40% by weight, 10 to 40% by weight, 15 to 40% by weight, 20 to 40% by weight, 1 to 30% by weight, 5 to 30% by weight, 10 to 30% by weight, 15 to 30% by weight, 20 to 30% by weight, 1 to 25% by weight, 5 to 25% by weight, 10 to 25% by weight, 15 to 25% by weight, 20 to 25% by weight, or 23 to 25% by weight.

[0093] Food composition containing a strain of the genus Cutibacterium and / or a culture thereof

[0094] In yet another aspect, there is provided a food composition for preventing and / or improving inflammatory skin diseases, comprising a Cutibacterium strain, a culture of said strain, or a combination thereof. The food composition may be a food itself, a food additive added to food, or a functional health food.

[0095] Exemplary food compositions may refer to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, vitamin complexes, functional health foods, and health foods, and include all foods in the usual sense.

[0096] The term "functional food" refers to a food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrients. Here, "function" refers to the regulation of nutrients for the structure and function of the human body or the attainment of beneficial health effects, such as physiological effects. The food of the present application can be prepared by methods commonly used in the art, and can be prepared by adding raw materials and ingredients commonly used in the art. The food formulation can also be prepared without limitation as long as it is recognized as a food. The food composition of the present application can be prepared in various dosage forms. Unlike general medicines, it has the advantage of being made from food ingredients, eliminating side effects that can occur with long-term drug administration. It is also highly portable, and the food of the present application can be taken as an adjuvant to enhance the effects of preventing or improving inflammatory skin diseases.

[0097] The term "health food" refers to food that has more active health maintenance and promotion effects than general foods, and "health supplement food" refers to food for the purpose of supplementing health. In some cases, the terms "health functional food," "health food," and "health supplement" can be used interchangeably.

[0098] Specifically, the functional health food refers to a food product in which the composition according to the example is added to food ingredients such as beverages, teas, spices, gum, and confectioneries, or is made into capsules, powders, suspensions, etc., and which brings about specific health benefits when ingested. However, unlike general medicines, the functional health food has the advantage of being made from food ingredients and not having side effects that can occur when taking medicines for a long period of time.

[0099] The food composition according to the example can be taken on a daily basis, and is therefore expected to be highly effective in preventing or improving inflammatory skin diseases, making it highly useful.

[0100] The food composition may additionally contain a physiologically acceptable carrier, but the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

[0101] The food composition may also contain additional ingredients commonly used in food compositions to improve aroma, taste, and visual appearance. For example, vitamins A, C, D, E, B1, B2, B6, and B12, niacin, biotin, folate, and pantothenic acid may be included. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr) may also be included. Amino acids such as lysine, tryptophan, cysteine, and valine may also be included.

[0102] The food compositions may also contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium D-bitartrate, etc.), strengtheners, emulsifiers, thickeners (thickeners), coating agents, gum bases, foam inhibitors, solvents, and improvers. The additives can be selected according to the type of food and used in appropriate amounts.

[0103] The composition according to the present invention can be added directly or with other foods or food ingredients, and can be used appropriately in a conventional manner. The amount of the active ingredient to be added can be determined appropriately depending on the intended use (prevention, health, or therapeutic treatment). Generally, when producing a food or beverage, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, to the food or beverage. However, when taking the food or beverage for long-term health and hygiene purposes, the content can be less than the above range, and since there is no safety issue, the active ingredient can be used in an amount greater than the above range.

[0104] One example of the food composition is a health drink composition, which may contain various flavorings or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates per 100 mL of the health drink composition of the present application may generally be about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g.

[0105] In addition to the above, the health drink composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. Fruit pulp may also be included for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not critical, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

[0106] The food composition according to an example may be contained in various weight percentages as long as it can exhibit the effect of preventing or improving inflammatory skin diseases. For example, the food composition according to an example may be contained in an amount of 0.00001 to 100 wt % or 0.01 to 80 wt % of the total weight of the food composition, but is not limited thereto.

[0107] Feed composition containing a strain of the genus Cutibacterium and / or a culture thereof

[0108] Yet another aspect provides a feed composition for preventing and / or ameliorating inflammatory skin diseases, comprising a Cutibacterium strain, a culture of said strain, or a combination thereof.

[0109] As used herein, "feed" can mean any natural or artificial diet, meal, etc., or component of said meal, intended or suitable for eating, ingesting, and digestion by an animal.

[0110] The feed composition for preventing and / or improving inflammatory skin diseases, which comprises a Cutibacterium strain, a culture of said strain, or a combination thereof, as described herein, can be used in the feed itself or in the form of a feed additive that is added to the feed.

[0111] The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, bottle gourds, and grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, and food and drink. These can be used alone or in combination of two or more.

[0112] The feed composition may further include excipients, diluents, and / or additives. In addition to the above-mentioned components, the feed composition may include ingredients effective for promoting animal growth, nutritional ingredients, nutritional supplements, ingredients for enhancing storage stability, coating ingredients, amino acid supplements for disease prevention, vitamin supplements, enzymes, non-protein nitrogen compounds, silicate agents, buffers, extractants, probiotics, enzymes such as amylase and lipase, vitamins such as L-ascorbic acid, choline chloride, and inositol, minerals such as potassium chloride, iron citrate, magnesium oxide, and phosphates, amino acids such as lysine, alanine, and methionine, organic acids or salts thereof such as fumaric acid, butyric acid, and lactic acid, antioxidants such as vitamin C and vitamin E, mold inhibitors such as calcium propionate, emulsifiers such as lecithin and glycerin fatty acid esters, and / or pigments. Even if not described above, the feed composition according to the example may additionally include other nutritional ingredients within the scope of what one skilled in the art would expect.

[0113] There are no particular limitations on the individuals (animals) to which feed containing an example composition can be fed, but they may be mammals, fish, crustaceans and / or shellfish, such as pigs, cows, horses, goats, deer, sheep, chickens, ducks, geese, turkeys, dogs, cats, rabbits, and fish.

[0114] When the feed composition is used in the form of a feed additive, it can be prepared by adding the feed additive according to the example to a commercially available feed. The feed in which the feed additive according to the example can be used is preferably in the form of a powder or pellet, or a liquid phase, but is not limited thereto. In addition, the amount of the feed additive according to the present application added to the feed is not particularly limited.

[0115] The feed can be prepared by separately preparing the composition of the present application in the form of a feed additive and mixing it with the feed, or by adding it directly during feed preparation. The feed additive of the present application contained in the feed can be in a liquid or dry state, for example, in a dried powder form. The feed additive can be present in an amount of 0.005 to 10 wt%, 0.05 to 10 wt%, 0.1 to 10 wt%, 0.005 to 5 wt%, 0.05 to 5 wt%, 0.1 to 5 wt%, 0.005 to 2 wt%, 0.05 to 2 wt%, or 0.1 to 2 wt% of the total weight of the feed, but is not limited thereto. In addition to the feed additive, the feed can also contain conventional additives that can improve the shelf life of the feed.

[0116] The feed to which the feed additives can be added can be selected from the group consisting of commercially available feed, grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, bottle gourds, grain by-products, proteins, inorganic substances, minerals, single-cell proteins, zooplankton, leftover food and drink, etc., but is not limited thereto.

[0117] Antibacterial composition containing a strain of the genus Cutibacterium and / or a culture thereof

[0118] Another embodiment provides an antibacterial composition against inflammatory skin disease-causing bacteria, comprising a Cutibacterium strain, a culture of the strain, or a combination thereof. Another example provides a method for inhibiting inflammatory skin disease-causing bacteria, comprising applying an effective amount of a Cutibacterium strain, a culture of the strain, or a combination thereof to a subject in need of antibacterial treatment against inflammatory skin disease-causing bacteria. Another example provides use of a Cutibacterium strain, a culture of the strain, or a combination thereof for the manufacture of an antibacterial composition against inflammatory skin disease-causing bacteria.

[0119] As used herein, the term "antibacterial composition" may refer to a composition having antibacterial activity. The antibacterial activity is as described above and may refer to inhibiting the growth of and / or killing inflammatory skin disease-causing bacteria. The term "antibacterial activity" may be used interchangeably with "antibiotic activity," and the term "antibacterial composition" encompasses all forms of preparations that have the ability to inhibit the growth of and / or kill inflammatory skin disease-causing bacteria, and unless otherwise specified, may be used interchangeably with "antibiotic composition," "antibacterial agent," "antibiotic agent," "antiseptic," "bactericide," etc.

[0120] The bacteria that cause the inflammatory skin disease may be one or more (one, two, three, four, five, or all six) selected from the group consisting of Cutibacterium acnes (C. acnes), Cutibacterium avidum (C. avidum), Cutibacterium granulosum (C. granulosum), Staphylococcus aureus (S. aureus), Bacteroides fragilis (B. fragilis), and Staphylococcus epidermidis (S. epidermidis), but is not limited thereto.

[0121] The inflammatory skin disease is as described above and may be, for example, but not limited to, acne or rosacea.

[0122] Another example provides a disinfectant comprising a Cutibacterium strain, a culture of the strain, or a combination thereof as an active ingredient. Another example provides a use of the Cutibacterium strain, a culture of the strain, or a combination thereof for the manufacture of a disinfectant. Another example provides a disinfection method comprising applying the disinfectant to an object in need of disinfection. Another example provides a cleaning agent comprising a Cutibacterium strain, a culture of the strain, or a combination thereof as an active ingredient. Another example provides a use of the Cutibacterium strain, a culture of the strain, or a combination thereof for the manufacture of a cleaning agent. Another example provides a cleaning method comprising applying the cleaning agent to an object in need of cleaning.

[0123] The disinfectant is a general term for preparations for preventing infection by pathogenic bacteria, and can be used as a disinfectant for general daily life, a disinfectant for food and cooking areas and facilities, and a disinfectant for buildings such as poultry farms and livestock barns, livestock, drinking water, bedding, egg trays, transport vehicles, tableware, and various other growing supplies.

[0124] Since the antibacterial composition has an antibacterial effect against bacteria that cause inflammatory skin diseases, it can be used to cleanse the skin surface or various parts of the body of an individual that are exposed or may be exposed to bacteria that cause inflammatory skin diseases.

[0125] Cosmetic composition containing a strain of the genus Cutibacterium and / or a culture thereof

[0126] In yet another embodiment, there is provided a cosmetic composition for preventing and / or improving inflammatory skin diseases or for improving skin, comprising a Cutibacterium strain, a culture of said strain, or a combination thereof.

[0127] The cosmetic composition according to an example may be a cosmetic composition or a topical skin composition, and such a cosmetic composition or topical skin composition may have anti-inflammatory or skin-improving activity, such as promoting skin regeneration activity and / or enhancing skin moisturizing activity (function). The skin-improving activity is as described above.

[0128] The formulation of the topical skin composition or cosmetic composition provided herein is not particularly limited, and may be formulated into, for example, a solution, suspension (anhydrous and aqueous), emulsion, paste, gel, cream (oil-in-water type, water-in-oil type, multi-phase type, etc.), powder, ointment, patch, lotion, essence, gel, lotion, solution, anhydrous product (oil and glycol type), mask, pack, powder, or capsule (soft capsule, hard capsule) coated with gelatin or the like, or spray.

[0129] The cosmetic composition may be in any one dosage form selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a powder, an ointment, a patch, a lotion, a skin lotion, a gel, a lotion, a mask, a pack, a powder, a capsule, and a spray.

[0130] In this application, the concept of skin includes not only the face but also the scalp and the whole body. Skin topical compositions that can be applied to the scalp include shampoos, rinses, treatments, hair growth agents, etc., and can be manufactured in various forms for use as body cleansers that can be applied to the whole body.

[0131] For example, the composition may contain a conventional surfactant (emulsifier) ​​and may be emulsified or solubilized in the form of O / W (oil in water), W / O (water in oil), W / O / W, or O / W / O. The surfactant may be one or more surfactants selected from all surfactants commonly used in the preparation of topical skin compositions and / or cosmetic compositions, such as, but not limited to, one or more surfactants selected from the group consisting of nonionic surfactants and anionic surfactants. The nonionic surfactant may be one or more surfactants selected from the group consisting of polyoxyethylene surfactants, polyglycerin surfactants, sugar ester surfactants, etc., such as polyglyceryl-methylglucose distearate, polyglyceryl-methylglucose stearate, polyglyceryl-distearate (e.g., polyglyceryl-10 distearate), polyglyceryl stearate, hydrogenated lecithin, cetearyl olivate, sorbitan olivate, PEG (poly(ethylene glycol) dimethicone), PEG-10 distearate, ... glycol))-stearates (e.g., PEG-100 stearate, PEG-40 stearate, etc.), glyceryl stearate, glyceryl stearate ES, decyl glucoside, coco-glucoside, lauryl glucoside, caprylyl / capryl glucoside, arachidyl glucoside, C12-20 (linear or branched) alkyl glucoside, cetearyl glucoside, ceteareths (e.g., ceteareth-20, etc.), steareths (e.g., steareth- The polysorbate may be one or more selected from the group consisting of, but not limited to, polyethylene glycol ether of oleyl alcohol (e.g., oleth-21, etc.), polyethylene glycol ether of oleyl alcohol (e.g., oleth-20, etc.), methyl glucose sesquistearate, sorbitan stearate, sucrose cocoate, polysorbates (e.g., polysorbate 60, etc.), sorbitan sesquioleate, sorbitan olivate, oil palm oil, oil palm kernel oil, etc.The anionic surfactant may be one or more selected from the group consisting of sodium lauryl sulfate, ammonium lauryl sulfate, magnesium lauryl sulfate, triethanolamine lauryl sulfate, sodium polyoxyethylene lauryl sulfate, ammonium polyoxyethylene lauryl sulfate, sodium cocoamphoacetate, ammonium laureth sulfate, ammonium lauryl sulfate, cocamide MEA, disodium laureth sulfosuccinate, TEA-cocoyl glutamate, sodium cocoyl malic acid amino acid, cocamide DEA, sodium laureth sulfate, sodium lauryl sulfate, potassium cocoyl glycinate, sodium stearoyl glutamate, potassium cetyl phosphate, etc., but is not limited thereto.

[0132] The topical skin composition and / or cosmetic composition may contain, in addition to the Cutibacterium strain, a culture of the strain, or a combination thereof, one or more components selected from the group consisting of ingredients typically contained in compositions for application to the skin, oils, polymers, thickeners, additives, etc. The typically contained ingredients and their contents can be appropriately adjusted depending on the intended function, properties, etc. of the composition.

[0133] For example, the oil may be one selected from the group consisting of silicone oils, such as cyclopentasiloxane, dimethicone, dimethiconol, cyclomethicone, trisiloxane, amodimethicone, phenyltrimethicone, disiloxane, cyclohexasiloxane, PEG-dimethicones (e.g., PEG-12 dimethicone, PEG-10 dimethicone, PEG-7 dimethicone, etc.), polysilicones (e.g., polysilicone-11, etc.), simethicone, diphenyl dimethicone, PEG-9 polydimethylsiloxyethyl dimethicone, etc. or more thereof; non-silicone oils, for example, one or more selected from the group consisting of caprylic / capric triglyceride, butylene glycol dicaprylate / dicaprate, hydrogenated polyisobutene, isopropyl myristate, cetyl ethylhexanoate, hydrogenated polydecene, diethoxyethyl succinate, hexyl laurate, triethylhexanoin, isononyl isononanoate, polyisobutene, etc.; triglycerides; and waxes, etc. The polyol may be at least one selected from the group consisting of butylene glycol, glycerin, glycereth (e.g., glycereth-26), methylpropanediol, PEG (poly(ethylene glycol)) / PPG (poly(propylene glycol)) copolymers (e.g., PEG / PPG-17 / 6 copolymer), methyl gluceths (e.g., methyl gluceth-20), dipropylene glycol, propylene glycol, propanediol, caprylyl glycol, pentylene glycol, etc. The thickener may be at least one selected from the group consisting of polymer compounds such as carbomer, ammonium acryloyldimethyltaurate / VP copolymer, xanthan gum, sodium polyacrylate, hydroxyethyl cellulose, acrylate / C10-30 alkyl acrylate crosspolymer, sodium polyacrylate, sodium acrylate / sodium acryloyldimethyltaurate copolymer, magnesium aluminum silicate, and sodium carbomer.The additives may include, but are not limited to, one or more selected from the group consisting of sequestering agents, emulsion stabilizers, pH adjusters, skin conditioning agents, fragrances, preservatives, disinfectants, oxidation stabilizers, antioxidants, free radical scavengers, opacifying agents, stabilizers, emollients, vitamins, insect repellents, preservatives, anti-inflammatory agents, basifying or acidifying agents, colorants (pigments), solubilizers, carriers, and the like, which are commonly used in the manufacture of topical skin preparations and / or cosmetics.

[0134] The composition may contain a solvent commonly used in the manufacture of topical skin preparations and / or cosmetic compositions, and the solvent may be one or more selected from the group consisting of purified water, bedrock water, hot spring water, glacial water, seawater, deep ocean water, plant extract water, etc., but is not limited thereto.

[0135] In addition to the above-mentioned components, the composition may further contain an active ingredient that exhibits beneficial effects in skin care and / or hair / scalp care. For example, the beneficial effects may mean skin whitening, improving skin elasticity, reducing wrinkles, preventing skin aging, providing skin moisture, moisturizing skin, providing skin nutrients, reducing pores, improving skin texture, removing dead skin cells, improving skin volume, protecting the scalp, preventing scalp or skin sebum, preventing hair loss, providing hair moisture, providing hair nutrients, improving hair volume, etc., but are not limited thereto and may be any effect desired for the skin / hair / scalp to which the composition is applied. For example, the active ingredient may be one or more selected from the group consisting of various plant extracts (e.g., Anemone Rhizome extract, green tea extract, grape extract, lotus flower extract, tomato extract, rhododendron extract, camellia flower extract, seaweed extract, etc.), plant callus culture or culture extract, yeast extract, cell-containing or cell-free yeast culture or culture extract, vitamins, various peptides, lipids, or fatty acids (e.g., lipids, fatty acids, peptides, etc. derived from various fish and shellfish), hyaluronic acid, glycosides (e.g., arbutin), and various skin compounds other than these, but is not limited thereto. An appropriate active ingredient can be selected and used depending on the desired effect. For example, the active ingredient may be, but is not limited to, a mixture of hydrogenated polyisobutene and Anemone Rhizome extract.

[0136] Another example provides a product containing the topical skin composition or cosmetic composition. The product may be a skin, hair, and / or scalp care product, for example, any type of cosmetic and / or cleansing product to be applied to the skin, scalp, hair, etc., of the face, hands, feet, or entire body. In one example, the product may be one or more selected from the group consisting of cosmetics such as skin lotions, creams, essences, packs, foundations, tinted cosmetics, sun creams (UV protection agents), BB creams, two-way cakes, face powders, compacts, makeup bases, skin covers, eye shadows, lipsticks, lip glosses, lip fixes, eyebrow pencils, and lotions; and cleansing products such as shampoos, soaps, cleansing foams, and body cleansers.

[0137] The subjects to which the compositions (pharmaceutical compositions, cosmetic compositions, food compositions, feed compositions, and / or antibacterial compositions) and methods comprising the Cutibacterium strains, cultures of the strains, or combinations thereof provided herein are administered may be mammals including humans, dogs, cats, horses, cows, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures thereof isolated therefrom. In one example, the subject may be an individual (a mammal such as a human) in need of prevention, amelioration, and / or treatment of an inflammatory skin disease as described above, or an individual having an inflammatory skin disease, or cells, tissues, or cultures thereof isolated therefrom. [Effects of the Invention]

[0138] The present application relates to the use of a Cutibacterium strain, a culture of said strain, or a combination thereof for the prevention, improvement, and / or treatment of inflammatory skin diseases, and a composition containing said Cutibacterium strain, a culture of said strain, or a combination thereof has excellent effects in the prevention, improvement, and / or treatment of inflammatory skin diseases. [Brief explanation of the drawings]

[0139] [Figure 1]These are the results of a hemolytic test conducted to confirm the toxicity due to hemolytic activity of the six bacterial strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656 strains; hereinafter the same). [Figure 2a] 1 is a graph showing a phylogenetic tree to confirm common genetic characteristics of six strains with RT2 ribotype selected in the present application. [Figure 2b] 1 is a graph showing a phylogenetic tree to confirm common genetic characteristics of six strains with RT2 ribotype selected in the present application. [Figure 3] 1 is a graph showing immune responses (immune cell proliferation and expression levels of IL-6, CCL2, and TNF-α) in immune cells when treated with cultures of the six bacterial strains selected in this application, compared to a control group (cell only with stimuli; group in which only an inflammatory response was induced), a positive control group (group treated with Dex after induction of an inflammatory response), a negative control group (group treated with plant-based co-culture medium (broth) after induction of an inflammatory response), and comparison groups (groups treated with cultures of Type I (ATCC 6919), Type II (ATCC 11828), Type I (CJIN1-1), Type I (CJIN1-2), Type II (CJIN2-1), Type II (CJIN2-2), Type III (CJIN3-1), B. fragilis, and S. epidermidis after induction of an inflammatory response). [Figure 4]1 is a graph showing immune responses (immune cell proliferation and expression levels of IL-6, CCL2, and TNF-α) in immune cells when treated with cultures of the six bacterial strains selected in this application, compared to a control group (cell only with stimuli; group in which only an inflammatory response was induced), a positive control group (group treated with Dex after induction of an inflammatory response), a negative control group (group treated with plant-based co-medium (broth) after induction of an inflammatory response), and comparison groups (groups treated with cultures of Type I (ATCC 6919), Type II (ATCC 11828), Type I (CJIN1-1), Type I (CJIN1-2), Type II (CJIN2-1), Type II (CJIN2-2), Type III (CJIN3-1), B. fragilis, and S. epidermidis after induction of an inflammatory response). [Figure 5] This is a graph showing the antimicrobial activity of each of the cultures of the six bacterial strains selected in this application against the acne-causing bacterial strain C. acnes (ATCC 6919). (NC: untreated; Doxy: doxycycline-treated group as a positive control) [Figure 6] This is a graph showing the inhibitory effect of each of the cultures of the six strains selected in this application on the formation of a bacterial (S. epidermidis) biofilm (NC: untreated, Doxy: doxycycline-treated group as a positive control). [Figure 7] This is a graph showing the cytotoxic effect when cultures of the six strains selected in this application were treated at different concentrations (5% (v / v), 10% (v / v), and 20% (v / v)). [Figure 8a] These are figures and graphs confirming the skin efficacy (skin cell regeneration efficacy) when cultures of the six strains selected in this application were treated (0 hours, 4 hours, and 8 hours after wounding). [Figure 8b] These are figures and graphs confirming the skin efficacy (skin cell regeneration efficacy) when cultures of the six strains selected in this application were treated (0 hours, 4 hours, and 8 hours after wounding). [Figure 9] 1 is a graph showing the expression levels of skin regeneration-related markers Src and MMP2 (metalloproteinase-2), and the expression levels of skin moisturizing-related markers HAS3 (hyaluronic acid synthase 3) and AQP3 (aquaporin 3) when cultures of the six strains selected in this application were treated. [Figure 10] 1 is a graph showing the expression levels of skin regeneration-related markers Src and MMP2 (metalloproteinase-2), and the expression levels of skin moisturizing-related markers HAS3 (hyaluronic acid synthase 3) and AQP3 (aquaporin 3) when cultures of the six strains selected in this application were treated. [Figure 11] The six strains (live bacteria) selected in this application were co-cultured with Raw264.7 macrophages, and the secretion levels of the inflammatory cytokine (IL-6) and anti-inflammatory cytokine (IL-10) secreted by the Raw264.7 macrophages were calculated as the IL-10 / IL-6 ratio and shown in the graph. (The B. fragilis strain was used as a control.) [Figure 12] This is a graph showing the proliferation of immune cells and the expression levels of inflammatory cytokines (IL-6 and IL-8) when cultures of the two strains selected in this application (CJRS-10652 and CJRS-10653) were treated. (Positive control group: group treated with dexamethasone (Dex)) [Figure 13] This is a graph showing the proliferation of immune cells and the expression levels of inflammatory cytokines (IL-6 and IL-8) when cultures of the two strains selected in this application (CJRS-10652 and CJRS-10653) were treated. (Positive control group: group treated with dexamethasone (Dex)) [Figure 14a]These graphs and photographs show the degree of skin erythema, epithelial tissue hyperplasia, and skin inflammation when cultures of two strains selected in this application (CJRS-10652 and CJRS-10653) were intraperitoneally administered to mice with rosacea. (Vehicle: untreated control group; LL37: group in which rosacea lesions were induced by treatment with LL37 peptide; Doxy: positive control group in which doxycycline was intraperitoneally administered to mice with lesions induced.) [Figure 14b] These graphs and photographs show the degree of skin erythema, epithelial tissue hyperplasia, and skin inflammation when cultures of two strains selected in this application (CJRS-10652 and CJRS-10653) were intraperitoneally administered to mice with rosacea. (Vehicle: untreated control group; LL37: group in which rosacea lesions were induced by treatment with LL37 peptide; Doxy: positive control group in which doxycycline was intraperitoneally administered to mice with lesions induced.) [Figure 15] These graphs and photographs show the degree of skin erythema, epithelial tissue hyperplasia, and skin inflammation when cultures of two strains selected in this application (CJRS-10652 and CJRS-10653) were intraperitoneally administered to mice with rosacea. (Vehicle: untreated control group; LL37: group in which rosacea lesions were induced by treatment with LL37 peptide; Doxy: positive control group in which doxycycline was intraperitoneally administered to mice with lesions induced.) [Figure 16] The results show that the skin model T&R HuSkin was treated with the culture of the bacterial strain selected in this application, and the expression levels of filaggrin, an indicator of barrier improvement, and aquaporin-3, an indicator of moisturizing improvement, were confirmed. [Figure 17] The results show that the skin model T&R HuSkin was treated with the culture of the bacterial strain selected in this application, and the expression levels of filaggrin, an indicator of barrier improvement, and aquaporin-3, an indicator of moisturizing improvement, were confirmed. [Figure 18] The results show that human fibroblasts were treated with the culture of the strain selected in this application to check for cytotoxicity and collagen production function. [Figure 19] The results show that human fibroblasts were treated with the culture of the strain selected in this application to check for cytotoxicity and collagen production function. DETAILED DESCRIPTION OF THE INVENTION

[0140] The present invention will be described in more detail below with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention. [Example]

[0141] Experimental example 1. Obtaining bacterial strains

[0142] 1) Selection of test subjects The study was conducted on 73 healthy adult men and women aged 18 to 39 years old (C.A. Korea was selected as the institution entrusted with collecting human sebum samples, and was approved by the IRB (approval number: CRAIRB20-030502)).

[0143] 2) Skin sebum collection After gently washing the face with water, the nose was gently wiped with gauze moistened with sterile water. The sterile gauze moistened with warm water was then placed over the nose for approximately 5 minutes. A drop of instant adhesive (cyanoacrylate) was applied to the tape, and the patient was left waiting for approximately 10 seconds. After applying the tape to the nose, the patient was then left waiting for 1 minute before extracting sebum (SSB, Skin Surface Biopsy). The sebum-covered tape was placed in a 1.5ml e-tube (50% glycerol + 50% RCM (Reinforced Clostridial Medium, BD Difco)) to collect a skin sebum sample.

[0144] 3) Skin strain culture and acquisition The sebum samples were diluted in PBS (100 ~10 3 After streaking onto RCM agar plates, the samples were placed in anaerobic gas pack jars and cultured at 37°C for 96 hours. Ten to 20 single colonies per plate were placed in RCM broth and activated in an anaerobic gas pack jar at 37°C for 24 hours. After 24 hours, 1% of the sample was inoculated into fresh RCM broth and cultured in an anaerobic gas pack jar at 37°C for 48 hours, yielding a total of 1,735 human skin-derived strains. The isolated strains were subjected to 16S rRNA and RecA sequencing using the primers listed in Table 1 below, and 166 unique C. acnes strains were identified based on the sequencing results. Six unique C. acnes strains were identified through macrophage-based proliferation and inflammatory marker (IL-6, CCL2, TNF-α) inhibition tests.

[0145] The six strains selected in this application, CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656, were confirmed to commonly contain the 16S rRNA sequence of SEQ ID NO: 8. The sequence type, ribotype, and immunological phenotype of the identified strains were confirmed and are shown in Table 2 below.

[0146] [Table 1]

[0147] [Table 2]

[0148] The sequence type and ribotype of the isolated C. acnes strains were classified according to the eMLST analysis results in Example 2-2 below, and the immunological phenotype was classified according to the immune index analysis results in Example 3-3 below.

[0149] As shown in Table 2, the six strains selected in this application, CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656, were confirmed to share the following characteristics: sequence type is type II, ribotype is RT2, and immunological phenotype is effective.

[0150] Experimental Example 2: Obtaining cultures The bacterial strains listed in Table 2, as well as the B. fragilis (Bacteroides fragilis, KCTC5013) and S. epidermidis (Staphylococcus epidermidis, KCTC3958) strains used as controls, were cultured in RCM medium (BD Difco) or plant medium (the composition of the plant medium is shown in Table 3 below) for 48 hours, and then centrifuged (4000 g, 20 minutes, room temperature) to remove the bacterial strains and obtain a cell-free supernatant.

[0151] [Table 3]

[0152] The pH of the collected culture was adjusted to pH 7 using NaOH, and then the culture was filtered through a 0.22 μm pore size filter. The culture was filtered through a 3 kDa Amicon filter to obtain a culture of 3 kDa or less, which was used as the final test substance.

[0153] In the following examples, the culture of the C. acnes (ATCC 6919) strain in Table 2 was adjusted to pH 7 and filtered through a 0.22 μm pore size filter to prepare a culture for use as a substance for inducing an inflammatory response.

[0154] Example 1. Hemolytic activity test of skin-derived bacterial strains A hemolytic test was conducted to confirm the toxicity of the six skin-derived bacterial strains selected in this application (strain CJRS-10651, strain CJRS-10652, strain CJRS-10653, strain CJRS-10654, strain CJRS-10655, and strain CJRS-10656) due to their hemolytic activity.

[0155] The skin-derived strains activated in RCM broth were then smeared on sheep blood agar and cultured in an anaerobic chamber at 37°C for 48 hours. The formation of a clear zone around the colony cluster due to erythrocyte lysis was confirmed, and this is shown in Figure 1.

[0156] As shown in Figure 1, the test results showed that all of the bacterial strains selected in this application (CJRS-10651 strain, CJRS-10652 strain, CJRS-10653 strain, CJRS-10654 strain, CJRS-10655 strain, and CJRS-10656 strain) did not exhibit hemolysis, and the bacterial strains selected in this application were determined to be safe.

[0157] Example 2. Genetic analysis of selected strains

[0158] Example 2-1. Confirmation of phylogenetic features of strains. C. acnes is known to have significant intraspecies genomic variation. In fact, due to the large genomic differences between strains within a species, although they are currently classified as the same species, it has been proposed that type I should be reclassified as C. acnes subspecies acnes, type II as C. acnes subspecies defendens, and type III as C. acnes subspecies elongatum. These types also differ in 16S rDNA sequences and are known to be divided into various ribotypes (RT) based on their relative abundance in the skin metagenomes of various individuals.

[0159] The ribotype (RT) of the strains prepared in Experimental Example 1 was confirmed through whole genome sequencing (WGS) for each strain (the results are shown in Table 2 above). Using the whole genome data of six strains with RT2 ribotype selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656 Note) and 37 strains with RT2 ribotype (reference strains) obtained from the NCBI genome site, core genes and accessory genes were identified using the ROARY tool. After confirming phylogenetic features using the whole genome sequence, alignment was performed using the MUSCLE algorithm, and a tree structure was created using the maximum likelihood method, as shown in Figures 2a and 2b. Specifically, a phylogenetic tree was drawn using the core gene set, and the core gene and accessory gene set were included in a matrix, as shown in Figures 2a and 2b. The edge scores are confidence values ​​obtained after 10,000 bootstrap iterations. The six strains selected in this application are shown in Figure 2a in red text within a red box and indicated by a left-arrow, and the reference strain is shown in black.

[0160] In Figure 2b, the gene clusters contained in the strains are displayed in blue.

[0161] As can be seen in Figure 2b, the reference strains with ribotype RT2 (the remaining strains not included in the red box in Figure 2a) have specific gene clusters, but the six strains isolated in this application were confirmed to lack such specific gene clusters (these areas are indicated by upward arrows in Figure 2b).

[0162] Among the strains included in the red box in Figure 2a, strain JCM18920, shown in black, had an additional accessory gene compared to the six strains isolated in this application, as can be seen in Figure 2b. This indicates that strain JCM18920 has a different appearance from the six strains isolated in this application, confirming its difference from the six strains isolated in this application (this part is indicated by a down arrow in Figure 2b).

[0163] Example 2-2. Expanded Multi-locus Sequence Typing (eMLST) analysis of strains Additionally, eMLST analysis was performed using the eBURSTv3 program (http: / / eburst.mlst.net / default.asp) to compare the 37 ribotype RT2 strains (reference strains) obtained from the NCBI genome site with the six ribotype RT2 strains selected in this application (strains CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) (Dreno et al. 2018). eMLST analysis involves the separation of gene sequences conserved in C. acnes strains, which offers improved phylogenetic resolution compared to 16S rRNA sequences. Gene sequence variation led to the classification of specific sequence types (ST), which were then grouped into specific clonal complexes (CC) based on frequency, as shown in Table 4 below. ST is a classification method based on sequence variation, and CC is a method of classifying the classified STs into a single cluster using sequence differences. Table 4 below shows the sequence types (ST) and clonal complexes determined through eMLST analysis of the selected RT2 C. acnes strains.

[0164] [Table 4] TIFF0007753538000005.tif160168

[0165] As can be seen from Table 4 above, the six strains selected in this application were confirmed to have an ST (Sequence type) of 69 or 153 as a result of eMLST analysis (for strains CJRS-10651, CJRS-10652, CJRS-10654, CJRS-10655, and CJRS-10656, the ST was 69 and the CC (clonal complex) was CC72 (type II); for strain CJRS-10653, the ST was 153 and the CC was NA (not confirmed)).

[0166] The difference between ST 69 and ST 153 was confirmed to be a single nucleotide sequence difference in camp2, a hemolytic factor-related gene, and the difference was weak. Therefore, the CC of the CJRS-10653 strain, which was confirmed to be ST 153, can be determined to be similar to CC72 (type II), the CC of the CJRS-10651, CJRS-10652, CJRS-10654, CJRS-10655, and CJRS-10656 strains.

[0167] As a result of eMLST analysis, C. acnes JCM 18920 and C. acnes CA17 strains were identified as strains with eMLST distributions similar to those of the six strains selected in this application (strains CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656 Note). However, it was confirmed that the six strains selected in this application differ from C. acnes JCM 18920 and C. acnes CA17 strains for the following reasons.

[0168] As confirmed in Example 2-1 above, the C. acnes JCM 18920 strain was confirmed to have an additional accessory gene, unlike the six strains selected in this application, and was therefore different from the six strains selected in this application.

[0169] To confirm the differences between the six strains selected in this application and the C. acnes CA17 strain, we performed a genome comparison with the C. acnes CA17 strain. Through WGS and genome sequence alignment of the six strains selected in this application with the C. acnes CA17 strain, we confirmed that the plasmid region 1-3467 of scaffold 4 is uniquely present in the C. acnes CA17 strain. By matching this region with nucleotide sequence databases (GenBank, EMBL, DDBJ, PDB, and RefSeq) using BLAST, we confirmed that this region is a plasmid sequence derived from Salmonella enterica strains. This confirms that the C. acnes CA17 strain differs from the six strains selected in this application by containing a plasmid derived from Salmonella enterica strains.

[0170] Example 2-3. Orthologous Gene Analysis of Strains The orthologous genes of the six strains (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) selected in this application with ribotype RT2 were analyzed using Orthofinder.

[0171] BLASTP was used to select strain-specific genes based on specific thresholds (identity > 40% and coverage > 80%), and genes with poor annotations such as hypothetical proteins or family proteins were excluded. Strain-specific literature searches were also conducted, and genes were categorized based on those mentioned in literature related to C. acnes and immunophenotypes. Some of the results are shown in Table 5 below.

[0172] [Table 5]

[0173] (Selected ‡ : Six strains selected in this application whose ribotype is RT2 (strains CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656 Note); ATCC 11828: a strain whose existing ribotype is RT2 (reference)

[0174] As shown in Table 5 above, the six strains selected in this application with ribotype RT2 (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) were confirmed to be different from the previously identified strain with ribotype RT2 (control group, ATCC 11828).

[0175] That is, the six strains selected in this application with the ribotype RT2 were confirmed to contain, unlike the ATCC 11828 strain, an Lrp / AsnC family transcriptional regulator (NCBI Reference Sequence: WP_002531510.1) or a gene encoding the same, and a lactococcin 972 family bacteriocin (NCBI Reference Sequence: WP_070651130.1) or a gene encoding the same.

[0176] Furthermore, the six strains selected in this application with ribotype RT2 were confirmed to lack beta-glucuronidase (NCBI Reference Sequence: WP_002518535.1) or the gene encoding it, unlike the ATCC 11828 strain. Beta-glucuronidase is known to aggregate bile from the gallbladder, leading to the formation of stones and to induce colon cancer.

[0177] Furthermore, it was confirmed that the six strains selected in this application, which have the ribotype RT2, differ from the ATCC 11828 strain in that they do not contain 2-isopropylmalate synthase (NCBI Reference Sequence: WP_142263178.1) or the gene encoding it, and 3-isopropylmalate dehydratase (NCBI Reference Sequence: WP_002513330.1) or the gene encoding it (they lack the L-leucine pathway).

[0178] Furthermore, it was confirmed that the six strains selected in this application, which have the ribotype RT2, differ from the ATCC 11828 strain in that they do not contain type IE CRISPR-associated protein Cse1 / CasA (NCBI Reference Sequence: WP_002514606.1) or the gene encoding it, type IE CRISPR-associated protein Cas7 / Cse4 / CasC (NCBI Reference Sequence: WP_002514608.1) or the gene encoding it, or CRISPR-associated helicase / endonuclease Cas3 (NCBI Reference Sequence: WP_002514605.1) or the gene encoding it.

[0179] Example 3. Immune index analysis The immune indicators of immune cells (Raw264.7 macrophages) were confirmed for cultures of the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656).

[0180] Example 3-1. Immune cell culture The immune cells used in this experiment were Raw264.7 (KCLB 40071) macrophages. One week before the experiment, Raw264.7 macrophages were cultured and maintained in DMEM containing 10% (v / v) FBS. 18 hours before the experiment, 5 × 10 Raw264.7 macrophages were added to the culture medium. 4 1 / well into a flat bottom 96 well plate.

[0181] Example 3-2. Co-culture of bacterial strain culture with immune cells: To induce an inflammatory response in Raw264.7 macrophages, the cells were treated with 5 μl / well of the pathogenic C. acnes (ATCC 6919) culture prepared in Experimental Example 2, and 95 μl of RPMI Media (Gibco) was added, for a total of 100 μl, which was incubated in an incubator at 37°C for 1 hour. During the induction of the inflammatory response, all groups except for the normal cell group (far left in Figure 3, Cell only) were treated with 5 μl / well of the pathogenic C. acnes (ATCC 6919) culture to provide an inflammatory stimulus.

[0182] After 1 hour, the culture of the strain prepared in Experimental Example 2 (20 μl) was added to fresh RPMI Media (80 μl), and a total of 100 μl was added to the plates treated with a culture of the pathogenic C. acnes (ATCC 6919), an inflammation inducer (total 200 μl / well), and cultured in a 37°C incubator for a total of 24 hours.

[0183] That is, to induce an immune response in Raw264.7 macrophages, cultures of C. acnes (ATCC 6919) were treated, and then cultures of various bacterial strains were treated to check the immune indicators. The results are shown in Figures 3 and 4.

[0184] Dexamethasone (Sigma) (100 nM) (Dex in Figure 3) was used as a positive control. Normal cells (Cell only) in plant co-culture medium (Broth in Figure 3) or fresh RPMI Media (Gibco) were used as a negative control.

[0185] Example 3-3. Immune index analysis After 24 hours, the cultured supernatant was collected and the remaining Raw264.7 macrophages were treated with Cell Counting Kit 8 (Enzo, 5 μl / well) plus fresh RPMI media (95 μl) to check their viability and proliferation. After incubation in a 37°C incubator for 2 hours, the absorbance was measured at 450 nm.

[0186] The collected supernatant was used to measure the levels of inflammatory cytokines secreted by Raw264.7 macrophages (interleukin 6 (IL-6), CC Motif Chemokine Ligand 2 (CCL2), and tumor necrosis factor alpha (TNF-α)) via ELISA. Using Raw264.7 macrophages, the reduction in inflammatory cytokine levels and the degree of inhibition of Raw264.7 macrophage proliferation were determined for each strain, and the results are shown in Figures 3 and 4. Strains were labeled as effective or ineffective depending on their level of inflammation suppression (the effective / ineffective results are shown in Table 2 above).

[0187] As shown in the upper graph of Figure 3 and Figure 4, normal cells (Raw264.7 macrophages without any treatment; Cell only) did not secrete inflammatory cytokines, but when Raw264.7 macrophages were treated with a culture of the pathogenic C. acnes (ATCC 6919) strain to induce an inflammatory response (Cell only w / stimuli), the levels of inflammatory cytokines (IL-6, CCL2, TNF) significantly increased. As a positive control, Raw264.7 macrophages were treated with a culture of the pathogenic C. acnes (ATCC 6919) strain and then treated with dexamethasone (Dex), which confirmed that the levels of inflammatory cytokines were reduced. As a negative control, an inflammatory response was induced in Raw264.7 macrophages with a culture of the pathogenic bacteria C. acnes (ATCC 6919), and the inflammatory response was not reduced when treated with a plant-based co-culture medium (broth).

[0188] Treatment of cultures of the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) significantly reduced the secretion of inflammatory cytokines (IL-6, CCL2, and TNF) that increased with the induction of an inflammatory response in cultures of the pathogenic C. acnes (ATCC 6919). This result confirmed that the reduction in the secretion of inflammatory cytokines was equal to or greater than the effect of using dexamethasone (Dex), the positive control.

[0189] When cultures of other Cutibacterium strains (ATCC 6919, ATCC 11828, CJIN1-1, CJIN1-2, CJIN2-1, CJIN2-2, CJIN3-1) or other bacteria (B. fragilis or S. epidermidis) were treated, the secretion of inflammatory cytokines (IL-6, CCL2, TNF) increased due to the induction of an inflammatory response in cultures of the pathogenic C. acnes (ATCC 6919) strain was either unchanged or even significantly increased, in contrast to the treatment of cultures of the six strains selected in this application.

[0190] The lower graph in Figure 3 shows the percentage of proliferation of Raw264.7 macrophages that induce an inflammatory response compared to normal cells (Raw264.7 macrophages that were not treated with anything; Cell only). As shown in the lower graph in Figure 3, when Raw264.7 macrophages were treated with a culture of the pathogenic C. acnes (ATCC 6919) strain to induce an inflammatory response (Cell only with stimuli), the proliferation of Raw264.7 macrophages that induce an inflammatory response increased.

[0191] It was confirmed that when cultures of the six bacterial strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) were treated, the proliferation of Raw264.7 macrophages was reduced to a level equal to or less than that when the Raw264.7 macrophages were not treated with any agent.

[0192] Example 4. Growth Inhibitory Effect of Acne-Causing Strain C. acnes (ATCC 6919) The antibacterial activity of cultures of the six strains selected in this study (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) against C. acnes (ATCC 6919), a known acne-causing bacterium, was confirmed. Doxycycline (0.025 μg / ml) was used as a positive control.

[0193] The culture concentrate of the strain was diluted with RCM broth and added to a 96-well plate. C. acnes ATCC 6919 was then diluted to 10^6 CFU / mL with RCM broth and inoculated into the 96-well plate at 10% (v / v). After inoculation, the strain was cultured in an anaerobic chamber at 37°C for 48 hours, and the absorbance at 600 nm was measured. The results are shown in Figure 5.

[0194] FIG. 5 is a graph showing the growth inhibition effect (% of inhibition) of the ATCC 6919 strain in comparison to the untreated negative control (NC).

[0195] As shown in Figure 5, when doxycycline (Doxy) was used as a positive control, the growth of C. acnes ATCC 6919 was reduced. The growth of C. acnes ATCC 6919 was also reduced when cultures of the six strains selected in this application were treated, and these results were equal to or greater than the effect of using doxycycline (Doxy) as a positive control.

[0196] Example 5. Inhibition of biofilm formation Cultures of the six strains selected in this study (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) were confirmed to inhibit biofilm formation by S. epidermidis. Doxycycline (Doxy) (4 μg / ml) was used as a positive control.

[0197] Inoculate 1 x 10 S. epidermidis bacteria into a 96-well cell culture plate. 7 After 24 hours of incubation at 100 CFU / mL to allow attachment, the cells were either untreated (negative control; NC), treated with doxycycline (Doxy) as a positive control, or treated with cultures of the six strains selected in this study. These cells were then cultured in TSB (Tryptic Soy Broth, BD Difco) medium at 37°C for 24 hours. After 24 hours, the biofilms were stained with crystal violet (0.1% v / v) to measure the level of biofilm formation, and the OD was measured at 570 nm using a Tecan™. The biofilm formation inhibition rate was calculated using the formula: OD sample / OD control x 100.

[0198] Specifically, S. epidermidis bacteria stored in a deep freezer were removed and inoculated into TSB (Tryptic Soy Broth, BD Difco) at 3% (v / v) and cultured at 37°C for 16 hours. The culture medium was inoculated into fresh TSB medium at 1 x 10 7 The culture solution was diluted to CFU / mL. The freeze-dried culture solution in the experimental group was diluted with distilled water to match the concentration to prepare the sample.

[0199] Each well of a 96-well microtiter polystyrene plate was treated with 100 μl of biofilm-forming S. epidermidis bacteria, 10 μl of a 5% (v / v) culture of one of the six strains selected in this study (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656), and 90 μl of TSB medium, for a total volume of 200 μl. The plates were cultured for 24 hours in a 37°C incubator. After 24 hours of culture, the microbial strains were pretreated for staining with crystal violet solution. The supernatant, excluding the biofilm on the bottom of the plate, was removed by suction. Each well was washed once with 100 μl of 1×PBS solution. The washed biofilms were stained with 0.1% crystal violet solution for 20 minutes at room temperature in the dark. After staining, the wells were washed with 100 μl of 1×PBS solution. The stained biofilms were removed from the plate using 33% acetic acid solution. The formation / inhibition of the lysed biofilms was assessed using an optical density plate reader at OD 570 nm, and the results are shown in Figure 6.

[0200] As shown in Figure 6, when S. epidermidis bacteria were not treated with anything (negative control; NC), no inhibitory effect on biofilm formation was observed. When S. epidermidis bacteria were treated with doxycycline (Doxy) as a positive control, an inhibitory effect on biofilm formation was observed.

[0201] When cultures of the six strains of S. epidermidis selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) were treated with the compound, an inhibitory effect on biofilm formation was also observed. These results confirmed that the inhibitory effect on biofilm formation was equal to or greater than that of the positive control, doxycycline (Doxy).

[0202] Example 6. Toxicity test Cultures of the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) were confirmed to be non-cytotoxic.

[0203] HaCaT cells were grown in a 96-well cell culture plate at 2 x 10^4 cells / well for 24 hours. Concentrated broth (vegetative co-culture medium) was added (0% (v / v), 5% (v / v), 10% (v / v), or 20% (v / v)) (negative control; broth) or concentrated cultures of the six bacterial strains selected in this study were added (5% (v / v), 10% (v / v), or 20% (v / v)). The cells were cultured for 24 hours at 5% CO2 and 37°C. After 24 hours, the culture medium was removed, and the cells were treated with Cell Counting Kit-8 solution. After incubation for 2 hours, the absorbance was measured at 450 nm using a Tecan™. The survival rate (cell viability) of the HaCaT cell line was calculated using the formula OD sample / OD control x 100. The results are shown in Figure 7.

[0204] As shown in Figure 7, compared to untreated HaCaT cells, treatment with cultures of the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) did not affect the viability of HaCaT cells, but rather increased cell viability.

[0205] These results confirmed that the cultures of the six strains selected in this application are not cytotoxic, and rather, a cytotoxicity-reducing effect can be expected.

[0206] Example 7. Skin Efficacy Test The skin regeneration efficacy of cultures of the six strains selected in this application (CJRS-10651 strain, CJRS-10652 strain, CJRS-10653 strain, CJRS-10654 strain, CJRS-10655 strain, or CJRS-10656 strain) was confirmed.

[0207] HaCaT cells were seeded onto cell culture inserts (Ibidi) in 70 μl volumes at 1 × 10^6 / ml and cultured for 24 hours at 5% CO2 and 37°C. After 24 hours of cell culture, the inserts were removed using sterile forceps to create the wound conditions required for the wound healing assay, followed by two washes with PBS. The medium was replaced with FBS-free medium, and either no treatment was performed (cell only) or cultures of six strains selected in this study (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) were added at 15% (v / v) and cultured at 5% CO2 and 37°C. After culture treatment, cell photographs were taken using a microscope at 0, 4, and 8 hours, and the wound area was calculated using the cell profiler program. Wound closure of the HaCaT cell line was confirmed by converting the area at 0 hours to the area at n hours using the formula (area at 0 hours - area at n hours) / area at 0 hours × 100. The results are shown in Figure 8a and Figure 8b.

[0208] As shown in Figures 8a and 8b, compared to untreated HaCaT cells (cell only), treatment with cultures of the six bacterial strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) resulted in increased wound closure and demonstrated excellent skin cell regeneration efficacy.

[0209] In addition, cultures of the six strains selected in this application were added to HaCaT cell lines to confirm the expression of skin regeneration-related markers Src and MMP2 (metalloproteinase-2). Increased Src activity promotes the regeneration of epidermal epithelial cells, the dermis, and vascular endothelium, while MMP2 is known to help regenerate the extracellular matrix, which is important for tissue regeneration, and promote the migration of cells important for re-epithelialization, such as keratinocytes and fibroblasts.

[0210] HaCaT cells were seeded at 1 x 10^5 in 24-well cell culture plates and cultured for 24 hours at 5% CO2 and 37°C. After 24 hours of cell culture, the medium was replaced with FBS-free medium. Cells were either left untreated (cell only) or supplemented with 15% (v / v) culture of one of the six bacterial strains selected in this study (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656). The cells were incubated for 24 hours at 5% CO2 and 37°C. Expression of Src and MMP2 was confirmed on RNA samples. The results are shown in Figure 9.

[0211] As shown in Figure 9, compared to untreated HaCaT cells (cell only), treatment with cultures of the six bacterial strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) resulted in increased expression of Src and MMP2. These results demonstrate the skin regeneration efficacy of cultures of the six bacterial strains selected in this application.

[0212] To confirm the skin moisturizing effect, HaCaT cells were seeded onto a 24-well cell culture plate at a density of 1x10^5 and then cultured at 5% CO2 and 37°C for 24 hours. After 24 hours of cell culture, the medium was changed to one containing no FBS, and either no treatment was performed (cell only) or 15% (v / v) of a culture of one of the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) was added. The cells were then incubated for 24 hours at 5% CO2 and 37°C to confirm the expression of HAS3 (hyaluronic acid synthase 3) and AQP3 (aquaporin 3), which are markers of skin moisturizing function, on RNA. The results are shown in Figure 10.

[0213] As shown in Figure 10, compared to untreated HaCaT cells (cells only), treatment with cultures of the six bacterial strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) increased the expression levels of HAS3 and AQP3. These results demonstrate that the cultures of the six bacterial strains selected in this application have excellent efficacy in enhancing skin moisturizing function.

[0214] Example 8. Test to evaluate the efficacy of live bacteria in controlling inflammation The six strains selected for this study (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656) were cultured in plant medium (Table 3) and then centrifuged (4000g, 20 minutes, room temperature) to remove the bacterial culture and obtain viable bacteria. The obtained viable bacteria were co-cultured with immune cells (Raw264.7 macrophages) at a ratio of 5 x 10^4 / well (1:1) in a 96-well plate for 24 hours, and the inflammation-modulating effects of the strains were confirmed. A B. fragilis strain, known to be an inflammation-reducing strain, was used as a control. The amounts of inflammatory cytokine (IL-6) and anti-inflammatory cytokine (IL-10) secreted by Raw264.7 macrophages were measured by ELISA, and the values ​​were calculated as the IL-10 / IL-6 ratio, which is shown in Figure 11.

[0215] As can be seen from Figure 11, the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, and CJRS-10656 strains) reduced the secretion of pro-inflammatory cytokines (IL-6) and promoted the secretion of anti-inflammatory cytokines (IL-10) compared to B. fragilis strains, demonstrating superior overall anti-inflammatory activity.

[0216] Example 9. Primary immune cell culture and immune index analysis The immune indicators of immune cells were confirmed when cultures of the six strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656) were treated.

[0217] The immune cells used in this experiment were mouse marrow-derived macrophages (mouse BMDM, Hanlim Co., Ltd.) and human monocyte-derived macrophages (HMDM, ATCC). One week before the experiment, the cells were cultured under the following conditions: BMDM with 10 ng / ml of mouse M-CSF or HMDM with 50 ng / ml of human M-CSF in 10% RPMI media. Fresh RPMI media containing M-CSF was changed every three days. 18 hours before the experiment, 5 x 10 immune cells to be tested were cultured. 4 1 / well into a flat bottom 96 well plate.

[0218] Mouse BMDMs (Figure 12) or HMDMs (Figure 13) were seeded at 5x10^4 / well in M-CSF-containing medium onto a 96-well cell culture plate and cultured for 24 hours at 5% CO2 and 37°C. To induce an inflammatory response, immune cells were treated with 5μl / well of C. acnes (ATCC 6919) culture medium, and 95μl of RPMI Media was added. A total of 100μl was incubated in a 37°C incubator for 1 hour. To induce an inflammatory response, all groups except the normal cell (cell only) group were treated with C. acnes (ATCC 6919) culture medium to provide an inflammatory stimulus. After 1 hour, 20 μl of the culture of the selected CJRS-10652 or CJRS-10653 strain was added to 80 μl of fresh media, and a total of 100 μl was added to the inflammation inducer-treated plates (total 200 μl / well) and cultured in a 37°C incubator for 24 hours. Dexamethasone (Dex) (100 nM) was used as a positive control. Normal cells treated with fresh media (cell only) served as a negative control. After 24 hours of culture, the entire supernatant was collected and treated with Cell Counting Kit-8 (5 μl per well) plus fresh media (95 μl) to assess the proliferation of remaining immune cells. After incubation in a 37°C incubator for 2 hours, the absorbance was measured at 450 nm, and the degree of cell proliferation was expressed as a percentage relative to the cell only standard. The amount of cytokines (IL-6, IL-8) secreted from immune cells was measured by ELISA using the collected cell supernatant.

[0219] As can be seen in Figure 12, when mouse BMDM cells were not treated with anything (cell only), they did not secrete the inflammatory cytokine (IL-6). When cells with stimuli were used, the secretion of inflammatory cytokines (IL-6) increased significantly, and the proliferation of mouse BMDM cells, which are immune cells, increased significantly compared to when no treatment was used (cell only). When dexamethasone (Dex) was used as a positive control, it was confirmed that an inflammatory response was induced in mouse BMDM cells, significantly reducing the amount of increased IL-6 and the increased proliferation of mouse BMDM cells.

[0220] When cultures of the CJRS-10652 or CJRS-10653 strains selected in this application were treated, an inflammatory response was induced in mouse BMDM cells, and the increased amount of IL-6 and the increased proliferation of mouse BMDM cells were significantly reduced. These results were found to be equivalent to or superior to those obtained when dexamethasone was used as a positive control.

[0221] Figure 13 shows the results of examining the secretion of inflammatory cytokines (IL-6, IL-8) using HMDM cells instead of mouse BMDM cells. When HMDM cells were not treated with anything (Cell only), there was almost no secretion of inflammatory cytokines (IL-6, IL-8). When Cell w / stimuli was used, there was a significant increase in the secretion of inflammatory cytokines (IL-6, IL-8), but when dexamethasone (Dex) was used as a positive control, there was a significant decrease in the amount of IL-6 and IL-8 that had increased due to the induction of an inflammatory response in HMDM cells.

[0222] When cultures of the strains selected in this application (CJRS-10652 or CJRS-10653) were treated, the secretion of IL-6 and IL-8, which had increased due to the induction of an inflammatory response in HMDM cells, was significantly reduced.

[0223] Example 10. Efficacy evaluation of rosacea animal model For this animal efficacy study, 32 5-week-old female BALB / c mice were purchased from Orient Bio Co., Ltd., and were placed in a breeding room with a 12-hour artificial photoperiod for 1 week to acclimate them before use in the experiment.

[0224] To create a rosacea animal model, hair was removed from the back of mice using a depilator and hair removal cream. 24 hours after hair removal, 50 μl of 320 μM LL37 peptide (Peptron Co., Ltd.) was injected intradermally using an insulin syringe to induce rosacea lesions. LL37 injections were administered twice daily for two days (a total of four injections), and 150 μl of a culture of the strain selected in this study (CJRS-10652 or CJRS-10653) was administered intraperitoneally once daily for three days (a total of three injections) at the same time as the second LL37 injection. Doxycycline, a conventional rosacea treatment, was administered intraperitoneally at 1 mg / kg once daily for three days (a total of three injections).

[0225] 24 hours after the last administration of the bacterial culture, the size (area) of the erythema was measured using a caliper, and the animals were euthanized using CO2 gas. The lesions were then photographed and the extent of the erythema was visually observed. The tissue photographs and the extent of the inflammation were shown graphically in Figures 14a and 14b.

[0226] As shown in Figures 14a and 14b, when mice were treated with LL37 peptide to induce rosacea, significant erythema appeared on the skin compared to when the mice were not treated with anything (vehicle). When mice were treated with doxycycline (Doxy) as a positive control, some reduction in erythema was observed.

[0227] When mice were treated (intraperitoneally) with cultures of the strains selected in this application (CJRS-10652 strain or CJRS-10653 strain), it was confirmed that erythema on the skin was significantly reduced, and these results showed surprisingly excellent effects even when compared to the positive control group treated with doxycycline.

[0228] To confirm the pathological results of the animal model injected with the culture of the strains selected in this study (CJRS-10652 or CJRS-10653), skin samples were excised from the LL37 peptide injection site, fixed in 4% formaldehyde solution, and embedded in paraffin for histological staining. Sections were made from the paraffin-embedded tissue, stained with hematoxylin-eosin (H&E), and observed under a microscope to observe histological changes. Histological photographs are shown in Figure 15, and the histopathological scores are shown in Table 6 below.

[0229] [Table 6]

[0230] As shown in Figure 15, when mice were treated with the LL37 peptide to induce rosacea (LL37), the area of ​​inflammation significantly increased compared to when the mice were not treated with anything (vehicle). When mice were treated with doxycycline (Doxy) as a positive control, the area of ​​inflammation was slightly reduced. When mice were treated with cultures of the strains selected in this application (CJRS-10652 or CJRS-10653), the area of ​​inflammation significantly decreased, demonstrating a significantly better effect than when the mice were treated with doxycycline (the positive control).

[0231] Furthermore, as shown in Table 6, when mice were treated with LL37 peptide to induce rosacea (LL37), epithelial hyperplasia and dermal inflammation were significantly increased compared to mice that were not treated with anything (Vehicle). When mice were treated with doxycycline (Doxy) as a positive control, epithelial hyperplasia was reduced, but dermal inflammation could not be reduced.

[0232] However, when mice were treated with cultures of the strains selected in this application (CJRS-10652 or CJRS-10653), it was confirmed that the increased epithelial hyperplasia and dermal inflammation were significantly reduced.

[0233] These results confirmed that when cultures of the strains selected in this application were administered intraperitoneally to mice, erythema, epithelial hyperplasia, and dermal inflammation were all reduced.

[0234] Example 11. Confirmation of skin barrier improvement and moisturizing effects The main causes of skin wrinkles and aging include a breakdown of the dermal barrier, moisture loss, and decreased collagen production. Collagen, in particular, is the main protein that makes up the dermis and plays a role in maintaining skin structure and elasticity. Collagen deficiency is one of the causes of skin wrinkles, and as aging progresses, its production decreases and its degradation increases, leading to the collapse of the dermis and the development of skin wrinkles. In addition, moisture loss slows skin regeneration and reduces elasticity, causing fine wrinkles and accelerating skin aging. Therefore, wrinkle improvement and anti-aging effects can be confirmed by checking key indicators such as improvement of the skin barrier (filaggrin), collagen (procollagen type I) production level, and improvement of moisture (aquaporin-3).

[0235] Inflammation was induced using poly I:C in T&R HuSkin, a 3D skin mini-tissue (organoid) produced by T&R BIOFAB. The bacterial strain cultures selected in this study were then applied to examine changes in skin improvement indicators (barrier improvement and moisturization).

[0236] Test Method 1) T&R HuSkin is a full-thickness skin model consisting of the dermis and epidermis layers, and was produced using T&R BIOFAB's 3DXPrinter bioprinter. 2) To fabricate the dermis layer, a bioink containing a pig skin-derived extracellular matrix-based hydrogel and human fibroblasts (Lonza, Basel, Switzerland) at a concentration of 100 x 10^4 cells / ml was printed and allowed to gel in a 37°C incubator for 40 minutes. 3) Human-derived keratinocytes (Lonza) were then dispensed onto the gelled dermal layer at 55 x 10^4 cells / insert and cultured completely immersed in keratinocyte growth media (KGM; Lonza) for one day. 4) After 1 day, in order to provide the same culture conditions as skin, air-liquid interface (ALI) conditions were achieved, in which only the surface of the dermis layer where the keratinocytes were dispensed was exposed to air, and the tissue was organized for 7 days. 5) T&R HuSkin cells cultured under ALI conditions for 7 days were treated simultaneously with an inflammation inducer (poly I:C, 20 μg / mL) and a test substance (a culture of a bacterial strain selected in this application, 0.5x concentration, 5% (v / v)). After culturing the HuSkin cells for an additional 2 days, changes in skin improvement-related indicators were observed (N=2 per condition). 6) After obtaining the IF fluorescent photographs, the fluorescence intensity value of each photograph (area of ​​interest: entire photograph area) was quantified using the mean gray value of Image J.

[0237] Test results 1) Barrier Improvement The expression level of filaggrin, an indicator of barrier improvement, was comparatively analyzed using IF staining in T&R HuSkin, and the results are shown in FIG.

[0238] As can be seen from Figure 16, in all groups treated with cultures of the six bacterial strains selected in this application (CJRS-10651 strain, CJRS-10652 strain, CJRS-10653 strain, CJRS-10654 strain, CJRS-10655 strain, or CJRS-10656 strain), the expression level of filaggrin increased to a similar or greater extent than in the negative control group (treated with Poly I:C alone). Of these, it was confirmed that the expression level of filaggrin in the groups treated with the CJRS-10651, CJRS-10653, CJRS-10654, or CJRS-10655 strain cultures was restored to the same level as the normal control group (non-treated), and in the group treated with the CJRS-10656 strain culture, it was confirmed that the expression level of filaggrin was further increased compared to the normal control group (non-treated).

[0239] 2) Improved moisturizing The expression level of aquaporin (aquaporin-3), an indicator of moisturizing improvement, was comparatively analyzed using IF in T&R HuSkin, and the results are shown in Figure 17.

[0240] As can be seen from Figure 17, in all groups treated with cultures of the six bacterial strains selected in this application (CJRS-10651, CJRS-10652, CJRS-10653, CJRS-10654, CJRS-10655, or CJRS-10656), the expression level of aquaporin-3 increased to a level equal to or greater than that of the negative control group (treated with Poly I:C alone). Among these, the groups treated with cultures of CJRS-10654, CJRS-10655, or CJRS-10656 strains showed a recovery of aquaporin-3 expression to a level equal to that of the normal control group (non-treated).

[0241] Example 12. Collagen index confirmation. The culture of the strain selected in this application was treated with human fibroblasts to confirm the toxicity of the culture and the level of collagen production and secretion by the culture.

[0242] Experimental Method 1) Human fibroblast culture Human fibroblast cells (CCD-986sk cells) were used in this experiment. One week before the experiment, human fibroblast cells were cultured and maintained in DMEM containing 10% (v / v) FBS. 18 hours before the experiment, 1 × 10 human fibroblast cells were cultured and maintained in DMEM containing 10% (v / v) FBS. 4 1 / well into a flat bottom 96 well plate.

[0243] 2) Co-culture of bacterial cultures with human fibroblasts The bacterial culture stock solution (cell-free supernatant obtained by centrifuging the bacterial culture in Experimental Example 2 to remove the bacterial cells) and the bacterial culture filtered to 3 kDa or less prepared in Experimental Example 2 were prepared using FBS 10% (v / v) DMEM to concentrations of 10, 20, 30, and 40% (v / v). 100 μL of each solution was added to a plate containing 100 μL of human fibroblasts to give final concentrations of 5, 10, 15, and 20% (v / v) (total 200 μL / well), and the plate was cultured in a 37°C incubator for a total of 24 hours.

[0244] The effects of culture medium on the viability of human fibroblasts and the level of collagen production were examined, and the results are shown in Figures 18 and 19. Normal cells (cell only) supplemented with DMEM containing 10% (v / v) FBS were used as a negative control.

[0245] 3) Human fibroblast toxicity test After 24 hours, the cultured supernatant was collected and treated with Cell Counting Kit 8 (Enzo, 5 μL / well) and fresh RPMI media (95 μL) to confirm the viability of the remaining human fibroblasts. After incubation in a 37°C incubator for 2 hours, the absorbance was measured at 450 nm. The results are shown in Figure 18.

[0246] As can be seen from Figure 18, when the undiluted culture solution of the strain selected in this application and the culture filtered to 3 kDa or less were treated, there was no cytotoxicity in human fibroblasts at any concentration.

[0247] 4) Measurement of human fibroblast collagen production function The supernatant obtained in 3) above was used to examine the collagen synthesis function of procollagen type I C-peptide (PIP) secreted from human fibroblasts using an ELISA kit, and the results are shown in Figure 19.

[0248] As can be seen from Figure 19, when the undiluted culture solution of the strain selected in this application and the culture filtered to 3 kDa or less were treated, PIP secretion was found to be increased at all concentrations to a similar or greater extent than the control group (cell only).

[0249] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts thereof.

[0250] [Accession number] Depository institution: Korea Microorganism Collection Center Accession number: KCCM13032P Acceptance date: August 13, 2021 JPEG0007753538000008.jpg240169Depository: Korea Microorganism Collection Accession number: KCCM13033P Acceptance date: August 13, 2021 JPEG0007753538000009.jpg240169Depository: Korea Microorganism Collection Accession number: KCCM13034P Acceptance date: August 13, 2021 JPEG0007753538000010.jpg240169Depository: Korea Microorganism Collection Accession number: KCCM13035P Acceptance date: August 13, 2021 JPEG0007753538000011.jpg239169Depository: Korea Microorganism Collection Accession number: KCCM13036P Acceptance date: August 13, 2021 JPEG0007753538000012.jpg240169Depository: Korea Microorganism Collection Accession number: KCCM13037P Acceptance date: August 13, 2021 JPEG0007753538000013.jpg240169

Claims

1. a Cutibacterium strain, a culture of said strain, or a combination thereof; The Cutibacterium sp. strain is (a) an Lrp / AsnC family transcriptional regulator or a gene encoding the same; and (b) a lactococcin 972 family bacteriocin or a gene encoding the same; The Cutibacterium strain is selected from the group consisting of Cutibacterium acnes deposited under accession number KCCM13032P, Cutibacterium acnes deposited under accession number KCCM13033P, Cutibacterium acnes deposited under accession number KCCM13034P, Cutibacterium acnes deposited under accession number KCCM13035P, Cutibacterium acnes deposited under accession number KCCM13036P, and Cutibacterium acnes deposited under accession number KCCM13037P.

2. The pharmaceutical composition according to claim 1, wherein the Cutibacterium strain does not contain one or more proteins or genes encoding the same selected from the group consisting of the following (1) to (6): (1) beta-glucuronidase or a gene encoding the same; (2) 2-isopropylmalate synthase or a gene encoding the same; (3) 3-isopropylmalate dehydratase or a gene encoding the same; (4) Type I-E CRISPR-associated protein Cse1 / CasA or a gene encoding the same; (5) Type I-E CRISPR-associated protein Cas7 / Cse4 / CasC (type I-E CRISPR-associated protein Cas7 / Cse4 / CasC) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 or a gene encoding the same.

3. 3. The pharmaceutical composition according to claim 1, wherein the inflammatory skin disease is one or more selected from the group consisting of alopecia, rosacea, erythema nodosum, erythema multiforme, keratosis pilaris, psoriasis, eczema, atopic dermatitis, and acne.

4. a Cutibacterium strain, a culture of said strain, or a combination thereof; The Cutibacterium sp. strain is (a) an Lrp / AsnC family transcriptional regulator or a gene encoding the same; and (b) a lactococcin 972 family bacteriocin or a gene encoding the same; The antibacterial composition for use against inflammatory skin disease-causing bacteria, wherein the Cutibacterium strain is selected from the group consisting of Cutibacterium acnes deposited under accession number KCCM13032P, Cutibacterium acnes deposited under accession number KCCM13033P, Cutibacterium acnes deposited under accession number KCCM13034P, Cutibacterium acnes deposited under accession number KCCM13035P, Cutibacterium acnes deposited under accession number KCCM13036P, and Cutibacterium acnes deposited under accession number KCCM13037P.

5. The antibacterial composition according to claim 4, wherein the Cutibacterium strain does not contain one or more proteins selected from the group consisting of the following (1) to (6) or genes encoding the same: (1) beta-glucuronidase or a gene encoding the same; (2) 2-isopropylmalate synthase or a gene encoding the same; (3) 3-isopropylmalate dehydratase or a gene encoding the same; (4) Type I-E CRISPR-associated protein Cse1 / CasA or a gene encoding the same; (5) Type I-E CRISPR-associated protein Cas7 / Cse4 / CasC (type I-E CRISPR-associated protein Cas7 / Cse4 / CasC) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 or a gene encoding the same.

6. The antibacterial composition according to claim 4 or 5, wherein the inflammatory skin disease-inducing bacteria is one or more selected from the group consisting of Cutibacterium acnes (C. acnes), Cutibacterium avidum (C. avidum), Cutibacterium granulosum (C. granulosum), Staphylococcus aureus (S. aureus), and Staphylococcus epidermidis (S. epidermidis).

7. a Cutibacterium strain, a culture of said strain, or a combination thereof; The Cutibacterium sp. strain is (a) an Lrp / AsnC family transcriptional regulator or a gene encoding the same; and (b) a lactococcin 972 family bacteriocin or a gene encoding the same; The food composition for preventing or improving an inflammatory skin disease, wherein the Cutibacterium strain is selected from the group consisting of Cutibacterium acnes deposited under accession number KCCM13032P, Cutibacterium acnes deposited under accession number KCCM13033P, Cutibacterium acnes deposited under accession number KCCM13034P, Cutibacterium acnes deposited under accession number KCCM13035P, Cutibacterium acnes deposited under accession number KCCM13036P, and Cutibacterium acnes deposited under accession number KCCM13037P.

8. The food composition according to claim 7, wherein the Cutibacterium strain does not contain one or more proteins selected from the group consisting of the following (1) to (6) or genes encoding the same: (1) beta-glucuronidase or a gene encoding the same; (2) 2-isopropylmalate synthase or a gene encoding the same; (3) 3-isopropylmalate dehydratase or a gene encoding the same; (4) Type I-E CRISPR-associated protein Cse1 / CasA or a gene encoding the same; (5) Type I-E CRISPR-associated protein Cas7 / Cse4 / CasC (type I-E CRISPR-associated protein Cas7 / Cse4 / CasC) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 or a gene encoding the same.

9. The food composition according to claim 7 or 8, wherein the inflammatory skin disease is one or more selected from the group consisting of alopecia, rosacea, erythema nodosum, erythema multiforme, keratosis pilaris, psoriasis, eczema, atopic dermatitis, and acne.

10. a Cutibacterium strain, a culture of said strain, or a combination thereof; The Cutibacterium sp. strain is (a) an Lrp / AsnC family transcriptional regulator or a gene encoding the same; and (b) a lactococcin 972 family bacteriocin or a gene encoding the same; A feed composition for preventing or ameliorating an inflammatory skin disease, wherein the Cutibacterium strain is selected from the group consisting of Cutibacterium acnes deposited under accession number KCCM13032P, Cutibacterium acnes deposited under accession number KCCM13033P, Cutibacterium acnes deposited under accession number KCCM13034P, Cutibacterium acnes deposited under accession number KCCM13035P, Cutibacterium acnes deposited under accession number KCCM13036P, and Cutibacterium acnes deposited under accession number KCCM13037P.

11. The feed composition according to claim 10, wherein the Cutibacterium strain does not contain one or more proteins selected from the group consisting of the following (1) to (6) or genes encoding the same: (1) beta-glucuronidase or a gene encoding the same; (2) 2-isopropylmalate synthase or a gene encoding the same; (3) 3-isopropylmalate dehydratase or a gene encoding the same; (4) Type I-E CRISPR-associated protein Cse1 / CasA or a gene encoding the same; (5) Type I-E CRISPR-associated protein Cas7 / Cse4 / CasC (type I-E CRISPR-associated protein Cas7 / Cse4 / CasC) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 or a gene encoding the same.

12. The feed composition according to claim 10 or 11, wherein the inflammatory skin disease is one or more selected from the group consisting of alopecia, rosacea, erythema nodosum, erythema multiforme, keratosis pilaris, psoriasis, eczema, atopic dermatitis, and acne.

13. (a) an Lrp / AsnC family transcriptional regulator or a gene encoding the same; and (b) a lactococcin 972 family bacteriocin or a gene encoding the same; The Cutibacterium strains include Cutibacterium acnes deposited under accession number KCCM13032P, Cutibacterium acnes deposited under accession number KCCM13033P, Cutibacterium acnes deposited under accession number KCCM13034P, Cutibacterium acnes deposited under accession number KCCM13035P, Cutibacterium acnes deposited under accession number KCCM13036P, and Cutibacterium acnes deposited under accession number KCCM13037P. A Cutibacterium strain selected from the group consisting of: S. acnes.

14. The Cutibacterium strain according to claim 13, which does not contain one or more proteins selected from the group consisting of the following (1) to (6) or genes encoding the same: (1) beta-glucuronidase or a gene encoding the same; (2) 2-isopropylmalate synthase or a gene encoding the same; (3) 3-isopropylmalate dehydratase or a gene encoding the same; (4) Type I-E CRISPR-associated protein Cse1 / CasA or a gene encoding the same; (5) Type I-E CRISPR-associated protein Cas7 / Cse4 / CasC (type I-E CRISPR-associated protein Cas7 / Cse4 / CasC) or a gene encoding the same, and (6) CRISPR-associated helicase / endonuclease Cas3 or a gene encoding the same.

15. The Cutibacterium strain according to claim 13, wherein the Cutibacterium strain comprises a 16S rRNA gene of SEQ ID NO:

8.

16. The Cutibacterium strain according to any one of claims 13 to 15, wherein the strain has one or more activities selected from the group consisting of anti-inflammatory activity, antibacterial activity against inflammatory skin disease-causing bacteria, skin regenerating activity, skin moisturizing activity, and activity for preventing, improving, and / or treating inflammatory skin diseases.

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