KR102197174B1 - Cutibacterium granulosum strain and skin condition improving uses of thereof - Google Patents

Abstract

The present invention relates to a novel microorganism having a 16s rRNA sequence represented by SEQ ID NO: 1, to lysate thereof, to a culture medium, to an extract of the culture medium, and to a use thereof for improving skin conditions. A Cutibacterium granulosum strain is useful for preventing, ameliorating, or treating skin-related conditions.

Description

Cutibacterium granulosum strain and skin condition improving uses of thereof

It relates to novel microorganisms, their lysate, a culture medium, an extract of the culture medium, and their use for improving skin conditions.

The skin's ecosystem provides a variety of habitats for microorganisms, and a wide range of microorganisms live there. Humans, as hosts, have a symbiotic relationship with them, and are known to have many positive effects on the host. The skin constitutes a variety of habitats, such as indentations and specialized crevices, and helps a wide distribution of microorganisms to grow. Basically, the skin forms a physical film and helps to defend against potential hazards and toxic substances from the outside. The skin becomes a point of connection with the external environment and is also a collection of various microorganisms (fungi, bacteria, viruses and small larvae). In accordance with the choice of physical and chemical functions, microorganisms prepare habitats by adapting to specialized niches. In general, the skin is cold, acidic, and remains dry. Structurally, the epidermis forms the skin barrier, blocks the penetration of microorganisms and toxins, and plays an important role in maintaining moisture. The uppermost layer of the epidermis is composed of the stratum corneum. The epidermis has a shape called'brick and mortar structure', and the skin tissue goes through a continuous self-healing process, and the scales that have gone through the end of the differentiation process are constantly being removed from the skin tissue.

Probiotics is a generic term for microorganisms that have beneficial effects on the human body, and refers to microorganisms that provide benefits to our body. Most of the probiotics known to date are known as lactobacilli. Probiotics have been reported to produce effective efficacy through various beneficial effects on the human body, but studies on the relationship between skin flora and skin are insufficient.

Accordingly, there is a need for development of dermatophytes that can be usefully used in skin-related conditions.

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S Corvec. Clinical and Biological Features of Cutibacterium (Formerly Propionibacterium) avidum, an Underrecognized Microorganism. Clinical Microbiology Reviews May 2018, 31 (3) e00064-17.

One aspect is to provide a Cutibacterium granulosum strain belonging to the Cutibacterium sp.

Another aspect is to provide a lysate of the strain, a culture medium, or an extract of the culture medium.

Another aspect is to provide a composition comprising the strain, a lysate thereof, a culture medium, and an extract of the culture medium.

One aspect provides a Cutibacterium granulosum strain.

The Cutibacterium granulosum strain may be a strain deposited with accession number KCCM12681P.

The Cutibacterium granulosum strain may be a strain containing 16s rRNA of SEQ ID NO: 1.

The strain may have an effect of improving skin beauty or skin condition, for example, suppressing skin aging, suppressing skin wrinkles, moisturizing skin, strengthening skin barrier, or soothing skin.

The strain increases (e.g., COL1A1 or elastin (ELN)) or decreases (MMP-1) the expression of anti-aging and anti-wrinkle factors, or skin moisturizing and skin barrier-related hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3), aquaporin 3 (AQP3: Aquaporin 3), or may increase the expression of pilagrin.

Another aspect provides a lysate of the strain, a culture medium, or an extract of the culture medium.

Details of the strain are as described above.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditional culture medium" or "conditioned medium", and provides nutrients so that the Cutibacterium granulosum strain can grow and survive in vitro. It may mean a whole medium including the strain, its metabolites, and extra nutrients obtained by culturing the strain for a certain period of time in a possible medium. In addition, the culture solution may mean a culture solution in which cells are removed from the cell culture solution obtained by culturing the strain. On the other hand, the liquid from which the cells have been removed from the 　 culture solution is also referred to as the "supernatant solution", and the culture solution is left for a certain period of time to take only the liquid in the upper layer excluding the sinking part in the lower layer, or remove the cells through filtration, or It can be obtained by removing the precipitation of and taking only the upper liquid. The "cell" refers to the 　 strain 　 itself of the present invention, and includes the 　 strain 　 itself or 　 separated from the culture solution by culturing the 　 strain selected by separating from skin samples. The cells can be obtained by taking the part that has settled in the lower layer by centrifuging the culture solution, or by allowing it to stand still for a certain period of time and then removing the upper liquid because it sinks into the lower layer of the culture solution by gravity.

The culture solution may include a culture solution itself obtained by culturing the strain, a concentrate thereof, or a culture supernatant obtained by removing the strain from the lyophilisate or the culture solution, a concentrate thereof, or a lyophilisate.

The culture medium is a culture medium (e.g., R2A medium, RCM medium, or TSA medium) in a medium (e.g., R2A medium, RCM medium, or TSA medium) at any temperature of more than 10 °C or less than 40 °C for a certain time, for example 4 to 50 hours. It may be obtained by culturing.

In one embodiment, the culture supernatant of the strain may be obtained by removing the strain by centrifuging or filtering the strain culture solution.

In another embodiment, the concentrate may be obtained by concentrating the obtained supernatant after filtering the strain culture solution itself or the culture solution using a centrifuge or filter.

The culture medium and culture conditions for culturing the Cutibacterium granulosum strain may be appropriately selected or modified by those of ordinary skill in the art.

In the present specification, the term "lysis solution" may refer to a product obtained by crushing the cell wall of the strain itself by chemical or physical force.

In the present specification, the term "culture solution extract" refers to extraction from the culture solution or its concentrate, and includes an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or these preparations or purified products, and a fraction obtained by fractionating them. I can.

Another aspect provides the use of the strain, a lysate of the strain, a culture solution, or an extract of the culture solution. Specifically, it provides a composition comprising an extract of the Cutibacterium granulosum strain, its lysate, a culture solution, or a culture solution thereof.

The use of the strain may include improving skin conditions, improving skin beauty, preventing, improving or treating skin diseases.

The skin condition improvement or skin beauty improvement may be suppressing or improving skin aging, anti-wrinkle, strengthening the skin barrier, or moisturizing the skin.

In the present specification, the term "skin aging" refers to the morphological and intangible changes that appear in the skin as age goes on, such as the phenomenon of thinning the thickness of the epidermis, the number of cells or blood vessels in the dermis, the ability to repair DNA damage, the cell replacement cycle, It refers to wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage protection, chemical removal ability, immune response, sensory function, decrease in body temperature regulation.

The strain or a culture medium thereof may be for improving skin aging caused by an exogenous factor or an endogenous factor. The exogenous factor refers to various external factors, such as ultraviolet (light), and the endogenous factor is also referred to as a chronological factor, and refers mainly to a factor caused by the passage of time. Specifically, the skin aging is not only a symptom of premature aging induced by external stimuli caused by ultraviolet rays, pollution, tobacco smoke, chemical substances, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It includes wrinkles, elasticity reduction, skin sagging, and dryness. In addition, wrinkles include the stimulus caused by changes in internal and external factors to change the components constituting the skin tissue, causing wrinkles.

The aging may be photoaging. The term "photoaging" is a phenomenon caused by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteases, chain cleavage of matrix proteins, and abnormal cross-linking, and repetition of this mechanism results in apparent skin aging.

In the present specification, the term "wrinkle" refers to a state in which the elasticity of the skin is lost and loosened, and for example, the skin may be folded. The "skin wrinkle prevention or improvement" may mean any action of preventing or improving wrinkles or increasing the total amount of collagen by inhibiting the expression of factors related to wrinkles.

The "skin barrier strengthening" may refer to any action that enhances the function of the skin barrier that is located on the outermost side of the skin and prevents loss of moisture and nutrition.

The “skin moisturizing” may mean any action of maintaining skin moisture or preventing moisture loss.

The "skin disease" may be a disease caused by impaired skin barrier function, skin aging, skin wounds, skin scars, or skin inflammation. The term “prevention” includes inhibiting the occurrence of a disease. The term "treatment" includes inhibition, alleviation, or elimination of the development of a disease.

The damage to the skin barrier function may mean all changes that appear on the skin due to a decrease or damage to the skin barrier function. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.

In another embodiment, the strain may be used together with a strain belonging to the genus Cutibacterium having a skin improvement effect to exhibit a synergistic effect. Strains belonging to the genus Cutibacterium are, for example, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium namnetense, Cutibacterium acnes subsp. acnes, Cutibacterium acnes subsp. defendens, or Cutibacterium acnes subsp. elongatum, etc.

The composition is from 0.001% to 80% by weight based on the total weight of the composition, for example, from 0.01% to 60% by weight, from 0.01% to 40% by weight, from 0.01% to 30% by weight, from 0.01% to 20% by weight %, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% % To 10% by weight, or 0.1% to 5% by weight of the strain, a lysate thereof, a culture solution, or an extract of a culture solution thereof.

In the present specification, the term "included as an active ingredient" means that the strain of the present specification, its lysate, culture medium, or extract of the culture medium thereof is added to the extent that it can exhibit the above-mentioned effect, drug delivery and stabilization, etc. For this purpose, it is meant to include formulations in various forms by adding various ingredients as auxiliary ingredients.

The composition may be a cosmetic composition.

The cosmetic composition may be, for example, a softening lotion, a nutritional lotion, a massage cream, a nutritional cream, an essence, a pack, a gel, an ampoule, or a cosmetic formulation of a skin adhesion type.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient. For example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances It may include.

In addition, the composition may be a composition for external application for skin.

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The above skin external preparations are ingredients commonly used in external preparations for skin such as cosmetics and pharmaceuticals, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances. , Colorants, various skin nutrients, or combinations thereof and may be appropriately formulated according to need. The external preparations for skin include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glavidine, and caline. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytilithic acid, tranexamic acid and derivatives or salts thereof, vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately blended.

In another embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and as a lubricant, magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. The oral dosage form may be a granule, powder, liquid, tablet, capsule, dry syrup, or a combination thereof. The parenteral dosage form may be an injection.

The composition may be a health functional food composition.

The health functional food composition may be used alone or in combination with the strain or a culture solution thereof or other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the kind of the health functional food. Among the types of health functional foods, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional beverage. The natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The health food composition may also be used in nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages. Carbonating agents used, or combinations thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, pulp for the production of vegetable beverages, or a combination thereof.

Another aspect also provides a method of preventing, ameliorating, or treating a condition in an individual comprising the step of treating or administering to an individual in need thereof an effective amount of the above composition.

The condition of the individual may be a condition related to skin.

As used herein, the terms "administering", "introducing", and "implanting" are used interchangeably and into a subject by a method or route that results in at least partial localization of a composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to one embodiment of.

Administration may be administered by a method known in the art. Administration can be administered directly to a subject by any means, for example by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. I can. The administration can be administered systemically or locally.

The subject may be a mammal, for example a human, cow, horse, pig, dog, sheep, goat, or cat. The individual may be an individual in need of improving skin beauty, for example, moisturizing the skin, strengthening the skin barrier, suppressing skin inflammation, and improving skin wrinkles.

The administration of the composition according to an embodiment is 0.1 mg to 1,000 mg per individual per day, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be variously prescribed according to factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. Taking these factors into account, the dosage can be appropriately adjusted. The number of administration can be once a day or two or more times within the range of clinically acceptable side effects, and the administration site can be administered at one or two or more locations, daily or at intervals of 2 to 5 days. The number of days of administration can be administered from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate time period. For non-human animals, the same dose per kg as human or, for example, the volume ratio (e.g., average value) of the target animal and the human organ (heart, etc.) One amount can be administered.

According to the novel Cutibacterium granulosum strain according to an aspect, it can be usefully used in the prevention, improvement, or treatment of skin-related conditions.

1 is a graph showing the effect of the strain on the expression of COL1A1 according to an embodiment.
Figure 2 is a graph showing the effect of the strain on the expression of MMP-1 according to an embodiment.
3 is a graph showing the effect of strains according to an embodiment on the expression of Filaggrin.
4 is a graph showing the effect of the strain on the expression of HAS3 according to an embodiment.
5 is a graph showing the effect of the strain on the expression of AQP3 according to an embodiment.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Isolation and identification of strains

A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy woman with sterile distilled water was inoculated into RCM (Reinforced Clostridium Medium) medium (BD). After culturing in a 28°C incubator for 48 hours after inoculation, 100 colonies formed were separated and cultured with pure water, and cultivated in a 28°C incubator for 48 hours. The cultured colonies were subjected to 16s rRNA gene sequence identification. The primers used at this time were designed to amplify by reacting only to bacteria (SEQ ID NOs: 2 and 3). PCR amplification was carried out in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 75°C for 1 minute and 30 seconds, and finally treated at 72°C for 8 minutes and then stored at 4°C. After the PCR reaction, the DNA sequence of the isolated and cultured species was determined using ABI-3730XL (ABI, USA). Less than 99% of homology when compared with other strains registered with the BLAST program provided on the National Center for Biotechnology Information (NCBI) website for the nucleotide sequence of the 16S rRNA site determined among the isolated and cultured microbial colonies The new microorganism of Cutibacterium granulosum ( Cutibacterium granulosum ) strain (hereinafter referred to as "YM-2") was selected. The selected YM-2 strain was deposited with the Korean Microbial Conservation Center on March 27, 2020, and was given the accession number KCCM12681P, and the YM-2 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

[Experimental Example]

Anti-aging and anti-wrinkle activity analysis

The effect of the YM-2 strain culture medium isolated in Example 1 on factors related to aging skin aging and wrinkle formation by ultraviolet (UV) irradiation was analyzed.

Specifically, the human fibroblast cell line (Human dermal fibroblast, Hs68) was dispensed into a 6-well plate at 3.5×10 ⁵ , and then cultured for 24 hours in an incubator at 37° C. and 5% CO ₂ . After removing the medium and adding DPBS, 12 mJ/cm ² of UVB was irradiated or not. Immediately after UVB irradiation, DPBS was removed and replaced with a medium without FBS, and the culture solution (1% (w/w)) of the YM-2 strain was treated and further cultured for 24 hours. As a negative control group, UV-treated and non-treated strain culture solution were used. After that, RNA was isolated from the cells of each sample using trizol (RNA iso, DAKARA, Japan), and then RNA was quantified at 260 nm with nanodrops, and cDNA was synthesized in an amplifier using 2 μg of RNA each. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, cybergreen (SYBR Green supermix, Applied Biosystems, USA) was added to the target genes COL1A1, ELN (elastin) and MMP-1 together with primers and cDNA, and a real-time PCR machine ( Step One Plus, Applied Biosystems, USA) carried out real-time polymerase chain reaction. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIGS. 1 and 2.

As shown in FIG. 1, it was confirmed that the strain according to one embodiment significantly increased the expression of COL1A1 reduced by ultraviolet rays.

As shown in Figure 2, it was confirmed that the strain according to one embodiment significantly reduced the expression of MMP-1 increased by ultraviolet rays.

These results mean that the YM-2 strain according to an embodiment is effective in improving aging (eg, photoaging).

Skin barrier strengthening and skin moisturizing activity analysis

The effect of the YM-2 strain culture solution isolated in Example 1 on skin barrier strengthening and skin moisturizing activity was analyzed.

Specifically, HaCaT cells, a human keratinocyte, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were 37°C 5%. It was carried out in a CO ₂ incubator. The cultured cell line was treated with the culture solution (1% (w/w)) of the YM-2 strain and further cultured for 24 hours. Retinoic acid (RA) 1 uM was used as a positive control. Filaggrin, HAS3 and Filagreen, HAS3 and the same method as in the above Experimental Example, except that primers for the factors related to skin barrier strengthening and moisturizing, such as Filaggrin, HAS3 (hyaluronic acid synthase), and AQP3 (aquaporin) were used The expression level of AQP3 was measured. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIGS. 3 to 5, respectively.

3 is a graph showing the effect of the strain on the expression of pilagrin according to an embodiment.

4 is a graph showing the effect of the strain on the expression of HAS3 according to an embodiment.

5 is a graph showing the effect of the strain on the expression of AQP3 according to an embodiment.

As shown in Figures 3 to 5, it was confirmed that the strain according to one embodiment significantly increased the expression levels of pilagrin, HAS3 and AQP3.

These results mean that the YM-2 strain according to an embodiment can be usefully used for strengthening the skin barrier and moisturizing the skin.

Korea Microorganism Conservation Center (overseas) KCCM12681P 20200327

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Claims (7)

Cutibacterium granulosum (Cutibacterium granulosum) strain, which belongs to Cutibacterium sp., deposited with accession number KCCM12681P, which has a 16s rRNA sequence represented by SEQ ID NO: 1. The strain according to claim 1, wherein the strain has an ability to increase COL1A1 expression, and an ability to inhibit MMP-1 expression. As a cosmetic composition for improving skin aging and skin wrinkles comprising the strain of claim 1, its lysate, a culture solution, an extract of a culture solution, or a mixture thereof,
The cosmetic composition that the skin aging is aging due to ultraviolet rays (UV). A cosmetic composition for strengthening the skin barrier and moisturizing the skin, comprising the strain of claim 1, its lysate, a culture solution, an extract of the culture solution, or a mixture thereof. delete delete delete

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