KR20240002756A - Novel Nocardioides microorgamism and uses thereof - Google Patents

Abstract

It relates to microorganisms of the genus Nocaldioides and has distinct phylogenetic and chemical taxonomic characteristics compared to strains of other or identical species.

Description

Novel Nocardioides microorgamism and uses thereof}

It is about microorganisms of the genus Nocaldioides.

The skin ecosystem provides various types of habitats for microorganisms, and a wide range of microorganisms live there. Human hosts have a symbiotic relationship with them, and they are known to have many positive effects on the host. The skin constitutes various types of habitats, including indentations and specialized crevices, and helps a wide range of microorganisms to grow. Basically, the skin forms a physical membrane and helps defend against potential hazards and toxic substances from the outside. The skin serves as a point of contact with the external environment and is also a collection site for various microorganisms (fungi, bacteria, viruses, and small larvae). Depending on the selection of physical and chemical functions, microorganisms adapt to specialized niches and establish habitats. Typically, the skin remains cool, acidic, and dry. Structurally, the epidermis forms the skin barrier and plays an important role in blocking the penetration of microorganisms and toxins and maintaining moisture. The uppermost layer of the epidermis is composed of the stratum corneum. The epidermis has a form called a 'brick and mortar structure'. The skin tissue goes through a continuous self-recovery process, and the scales (squames) that have completed the differentiation process constantly repeat the process of being shed from the skin tissue.

Probiotics are a general term for microorganisms that have beneficial effects on the human body. They refer to microorganisms that benefit our bodies. Most probiotics known to date are known as lactic acid bacteria. Probiotics have been reported to produce effective effects through various beneficial effects on the human body, but research on the relationship between skin flora and skin is insufficient.

The skin barrier is composed of dead keratinocytes and intercellular lipids, and plays a key role in skin health as a skin protective film that protects the skin from external stimuli and prevents moisture from evaporating from the skin. In other words, it prevents excessive release of moisture from the body and prevents harmful substances such as chemicals or microorganisms from entering our body. The keratinocyte envelope, which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. Keratinocytes differentiate and create a skin barrier through the keratinization process. The skin barrier function can be destroyed by aging or external factors, and damage to the skin barrier can reduce skin moisture and cause wrinkles.

Accordingly, the present inventors observed changes in the skin due to changes in skin flora and further studied whether changes in skin flora could lead to potential improvement in the skin environment. From this, strains of the Nocardioides genus were isolated and identified, and it was confirmed that they could be usefully used for skin-related conditions, and the present invention was completed.

One aspect is to provide a new Nocardioides epidermidis strain belonging to the genus Nocardioides ( Nocardioides sp.).

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

Another aspect provides a composition comprising a Nocardioides epidermidis strain, its lysate, a culture, an extract of the culture, or a mixture thereof.

One aspect provides a novel Nocardioides epidermidis strain belonging to the genus Nocardioides ( Nocardioides sp.).

Strains belonging to the genus Nocardioides ( Nocardioides sp.) may have any one or more of the following mycological properties;

(1) Rod-Shaped;

(2) Non-motile;

(3) Gram positive;

(4) non-spore producing (non-motile);

(5) The length of the cell is 0.9~2.0 ㎛ and the diameter is 0.4~0.6 ㎛;

(6) Colony shape is round and convex, catalase-positive;

(6) MK-8(H4) (tetrahydrogenated menaquinone with eight units) is the isoprenoid quinone detected;

(7) Polar lipids include diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), five phospholipids (PL), and two one or more selected from the group consisting of unidentified lipids (UL);

(8) The polyamine is at least one selected from the group consisting of Putrescine, 2-hydroxyputrescine, and Spermine; and

(9) DNA G+C content is 71.9 mol%.

The phylogenetic characteristics of strains belonging to the genus Nocardioides ( Nocardioides sp.) show clear differences from other species to which other strains belong, and the chemical taxonomic characteristics also show clear differences, so the new Nocardioides epidemiology It was identified as a Nocardioides epidermidis strain.

The strain may contain 16s rRNA containing the base sequence represented by SEQ ID NO: 1. In one embodiment, the strain has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity with SEQ ID NO. It may contain 16S rRNA having . Additionally, the strain may have been deposited under the deposit number KACC 22414.

The Nocardioides epidermidis strain may have one or more of the following mycological properties;

(1) aerobic;

(2) heterotrophic;

(3) positive catalase activity;

(4) Grows on R2A (Reasoner's 2A), TSB (Tryptic soy broth), and LB (Luria-Berani) agar, but does not grow on MA (Marine) and PDB (Potato dextrose broth) agar;

(5) Grows on R2A agar at 18°C to 37°C (optimum 30°C), but does not grow at 10°C or 40°C;

(6) grows at pH 5.5 to 9.0 and NaCl concentrations up to 4% (optimum is pH 6.5, 0% NaCl);

(7) Nitrate is not reduced to nitrite, nor is it reduced to nitric acid;

(8) Starch is degraded, DNA is slightly degraded, but casein, cellulose, and Tween 80 are not degraded;

(9) At least one selected from the group consisting of β-glucosidase, D-Glucose, D-Maltose, and Gluconate activities. assimilation positive;

(10) Indole, glucose, arginine, urease, protease, β-galactosidase, L-Arabinose, D-Mannose, D-Mannitol, N-Acetyl-D-glucosamine, Caprate, Adipate, Malate ( Negative for assimilation of Malate, Citrate and Phenyl-acetate;

(11) alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase ( acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-glucosidase, and β-glucosidase activities are present. ;

(12) Lipase (C14), valine arylamidase, crystine arylamidase, trypsin, α-chymotrypsin, α-gal Lactosidase (α-galatosidase), β-galactosidase (β-galatosidase), β-glucuronidase, N-acetyl-β-glucosaminidase (N-acetyl-β -glucosaminidase, α-mannosidase, and α-fucosidase activities are absent; and

(13) It has the characteristics shown in Tables 1 to 3.

Another aspect provides lysate, culture fluid, or extract of the culture fluid of the strain.

Specific details of the strain are as described above.

As used herein, the term “culture medium” may be used interchangeably with “culture supernatant,” “conditioned culture medium,” or “conditioned medium,” and refers to a medium that can supply nutrients for Dokdo strains to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time. Additionally, the culture medium may refer to a culture medium obtained by removing the bacterial cells from the bacterial culture medium obtained by culturing the strain. On the other hand, the liquid from which the bacteria have been removed from the culture medium is also called "supernatant", and the culture medium is left alone for a certain period of time and only the liquid in the upper layer excluding the part that has settled in the lower layer is taken, the bacteria are removed through filtration, or the culture medium is centrifuged and the lower layer is removed. It can be obtained by removing the precipitation and taking only the upper liquid. The "bacteria" refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a skin sample, etc., or a strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the part that sinks to the lower layer. Alternatively, since they sink to the lower layer of the culture medium by gravity, they can be obtained by leaving them still for a certain period of time and then removing the upper liquid.

The culture medium is the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating a strain belonging to the genus Nocardioides , or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product. may include.

The culture medium may be obtained by culturing the strain in a medium (e.g., R2A medium or TSA medium) at any temperature above 10°C or below 40°C for a certain period of time, for example, 4 to 50 hours.

In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture medium to remove the strain.

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

The culture medium and culture conditions for cultivating the Kera G14 strain belonging to the Nocardioides genus ( Nocardioides sp.) can be appropriately selected or modified by those skilled in the art.

As used herein, the term “lysate” may refer to a product obtained by breaking the cell wall of the strain itself using chemical or physical force.

As used herein, the term "culture extract" refers to an extract from the culture medium or its concentrate, and may include extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, crude or purified products thereof, and fractions thereof. You can.

Another aspect provides the use of the strain, a lysate of the strain, a culture medium, or an extract of the culture medium. In detail, a composition containing the Dokdo strain, its lysate, culture medium, or extract of the culture medium is provided.

Uses of the strain may include improving skin condition, improving skin beauty, preventing, improving or treating skin diseases, etc.

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

As used herein, the term "skin aging" refers to both tangible and intangible changes that occur in the skin as one ages, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and temperature regulation.

The strain or its culture medium may be used to improve skin aging caused by exogenous factors or endogenous factors. The exogenous factors refer to various external factors, such as ultraviolet rays (light), and the endogenous factors are also referred to as chronological factors and mainly refer to factors that occur due to the passage of time. In other words, the skin aging is not only a premature aging phenomenon induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging, and dryness. In addition, wrinkles include stimulation caused by changes in internal and external factors that change the components that make up skin tissue, causing wrinkles.

The aging may be photoaging. The term “photoaging” is a phenomenon caused by external environmental factors, the most representative factor being ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cutting of matrix proteins, and abnormal cross-linking, and repetition of this mechanism causes skin aging that is evident in appearance.

As used herein, the term “wrinkle” refers to a state in which the skin loses its elasticity and becomes loose, for example, the skin may be folded. The term “prevention or improvement of skin wrinkles” may refer to any action that prevents or improves wrinkles by suppressing the expression of factors related to wrinkles, or increases the total amount of collagen.

The “skin barrier strengthening” may refer to any action that improves the function of the skin barrier, which is located on the outermost layer of the skin and prevents moisture and nutrition loss.

The term “skin moisturizing” may refer to any action that maintains skin moisture or prevents moisture loss.

The “skin disease” may be a disease caused by damage to the skin barrier function, skin aging, skin wound, skin scar, or skin inflammation. The term “prevention” includes suppressing the occurrence of a disease. The term “treatment” includes inhibiting, alleviating, or eliminating the development of a disease.

The damage to the skin barrier function may mean any change that appears in the skin due to decreased or damaged skin barrier function. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, etc.

In another embodiment, the strain may be used together with another strain belonging to the genus Nocardioides ( Nocardioides sp.), which has a skin-improving effect, to show a synergistic effect. Strains belonging to the genus Nocaldioides include, for example, Nocardioides terrae , Nocardioides opuntiae, Nocardioides daecheongensis , and Nocardioides. Nocardioides panacihumi , Nocardioides marinus, Nocardioides baekrokdamisoli , Nocardioides agrisoli , Nocardioides maranoden It may include Nocardioides maradonensis , Nocardioides ginkgobilobae , Nocardioides sambongensis , etc.

The composition is 0.001% to 80% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% to 20% by weight, based on the total weight of the composition. %, 0.01% to 10% by weight, 0.01% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight It may include % to 10% by weight, or 0.1% to 5% by weight of the strain, its lysate, culture medium, or extract of the culture medium. At this time, if the content of the strain, its lysate, culture, or extract of the culture is less than the above range, the effect of improving skin condition, for example, inhibiting skin aging, whitening skin, strengthening the skin barrier, inhibiting skin wrinkles, or moisturizing the skin There is a problem that the effect is not sufficiently effective.

As used herein, 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 is added to the extent that it can exhibit the effects mentioned above, drug delivery, stabilization, etc. This means that it is formulated into various forms by adding various ingredients as sub-ingredients.

The composition may be a cosmetic composition.

The cosmetic composition may have, for example, an softening lotion, nourishing lotion, massage cream, nourishing cream, essence, pack, gel, ampoule, or skin-adhesive type cosmetic formulation.

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 auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances. may include.

Additionally, the composition may be a composition for external skin application.

In this specification, the external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and calin. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytylitinic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately mixed.

Additionally, 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 the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, disintegrant, binder, 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, calcium monohydrogen phosphate anhydride, 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. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrup, or combinations thereof. Parenteral dosage forms may be injectable.

Additionally, the composition may be a health functional food composition.

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

Additionally, another aspect provides a method of preventing, ameliorating, or treating a condition of a subject comprising treating or administering an effective amount of the composition described above to the subject in need thereof.

The condition of the subject may be a skin-related condition or an inflammation-related condition.

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

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

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of skin beauty improvement, for example, skin moisturizing, skin barrier strengthening, skin inflammation inhibition, and skin wrinkle improvement effects.

The administration of the composition according to one embodiment is 0.1 mg to 1,000 mg, 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 mg per day. 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. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The frequency of administration can be once a day or two or more times within the range of clinically acceptable side effects, and 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 from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg is the same as for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and human. One dose can be administered.

According to one aspect, a new strain of the Nocaldioides genus has phylogenetically and chemically distinct characteristics compared to strains of other species or of the same genus. Additionally, the strain or its culture medium can be usefully used to prevent, improve, or treat skin-related conditions.

Figure 1 is a diagram showing the results of lineage classification of Kera G14.
Figure 2 shows the results of confirming the morphological characteristics of Kera G14.
Figure 3 shows the results of confirming the polyamine of Kera G14.
Figure 4 shows the results of confirming the quinone of Kera G14.
Figure 5 shows the results of confirming the polar lipids of Kera G14.
Figure 6 shows the results showing the effect of Kera G14 strain on the expression of COL1A1.
Figure 7 shows the results showing the effect of Kera G14 strain on the expression of AQP-3.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Isolation of strains

A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy adult with sterile distilled water was inoculated into R2A (Reasoner's 2A) medium (Becton Dickinson, Cockeysville, MD). After inoculation, 100 colonies formed after culturing in an incubator at 28°C for 48 hours were isolated and cultured in pure form, and re-cultured in an incubator at 28°C for 48 hours. The 16s rRNA gene sequence was identified for the colonies whose culture was completed. The primers used at this time were designed to react and amplify only bacteria. PCR amplification was performed in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, and 75°C for 1 minute and 30 seconds, and was finally treated at 72°C for 8 minutes and stored at 4°C. After the PCR reaction was completed, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA). The base sequence of the 16s rRNA region determined among the isolated and cultured microbial colonies was compared and analyzed with other strains registered with the BLAST program provided on the US National Center for Biotechnology Information (NCBI) website. Only novel species with 97% or less homology were selected and used, and among them, a novel microorganism (hereinafter referred to as “Kera G14”) was selected. Kera G14 has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Example 2. Confirmation of taxonomic and mycological properties of new microorganisms

2-1. Taxonomic properties

Homology analysis for the 16s rRNA gene was calculated using the Ezbiocloud server (Yoon S. H et al., (2017)). Sequence data alignment and phylogenetic analysis were performed using MEGA version 11.0 (Tamura, K et al. (2021)). Phylogenetic trees were drawn using neighbor-joining analysis (Sitou and Nei (1987)), maximum-likelihood and maixmum-parsimony algorithms, and the stability of the phylogenetic trees was assessed using bootstrap analysis (1000 replications) (Felsenstein 1985). evaluated.

As a result, Kera G14 was confirmed to have 96.24% and 96.19% homology with Nocardioides terrae CGMCC 1.7056 and Nocardioides opuntiae OS1-21, respectively.

Figure 1 is a diagram showing the results of lineage classification of Kera G14.

As shown in Figure 1, Kera G14 was confirmed to be a new species belonging to Nocardioides that has not been reported so far.

2-2. mycological properties

2-2-1. Morphological properties

The morphological characteristics of Kera G14 were analyzed as follows.

First, the cell morphology of Kera G14 was cultured in R2A at 25°C for 1 day and then observed using a transmission electron microscope.

For observation under a transmission electron microscope, the sample was pretreated as follows. After inoculating Kera G14 on R2A solid medium and culturing for one day, the formed colonies were dissolved in 10㎕ distilled water. The grid was placed in distilled water with the bacteria dissolved in it for at least 1 drop to allow the cells to stick, and then moisture was removed as much as possible. The grid was stained in 1% PTA (Terephthalic acid) for 10 seconds, and moisture was removed. Afterwards, the grid was washed twice for 2 seconds with distilled water and observed using a transmission electron microscope (120kv, model name: LIBRA 120, country of manufacture: Germany, manufacturer Carl Zeiss), and the results are shown in Figure 2.

Gliding motility was assessed using the hanging-drop technique on fresh Kera G14 cells in R2A medium.

To determine Gram positivity, an experiment was conducted in the following manner. Specifically, one Kera G14 colony cultured well using R2A solid medium was collected and fixed on a glass slide. After completely drying the slide glass, it was stained with crystal violet for 1 to 2 minutes, washed in running water, and dried again. The dried slide was again stained with iodine-potassium iodide for 1 minute. Afterwards, the slide was decolorized using ethyl alcohol, washed with running water, and dried. To determine Gram positive/negative, secondary staining was performed with a red dye such as safranin for 1 to 3 minutes, washed in water, and observed under a microscope (Olympus microscope, GX71).

As a result, it was confirmed that Kera G14 had the following morphological characteristics.

(1) Rod-Shaped;

(2) non-motile;

(3) Gram positive;

(4) non-spore forming;

(5) The length of the cell is 0.9~2.0 ㎛ and the diameter is 0.4~0.6 ㎛; and

(6) The colony shape is round, convex, pale white, and has a size of 0.1 mm.

2-2-2. Culture and physiological characteristics

To confirm the culture conditions of Kera G14, the optimal growth temperature was set from 4℃ to 50℃ to confirm the optimal culture temperature, and the optimal salt concentration was determined by adding NaCl from 0% to 10% (w/v) to the medium at 25℃. It was confirmed by culturing for 3 days. pH was checked using the following buffer system; Sodium acetate/acetic acid (pH<6), Tris/HCl (pH 6 to 9), and glycine/sodium hydroxide (pH>9).

Additionally, catalase activity was determined by assessing bubble production in 3% (v/v) H ₂ O ₂ .

In addition, to check the growth medium, R2A (Reasoner's 2A), TSB (Tryptic soy broth), LB (Luria-Berani), MA (Marine), and PDB (Potato dextrose broth) medium (BD) was used and grown for 3 days at 25°C. It was confirmed by culturing for one day.

Additionally, API 20NE and API ZYM (biomeriuex, France) were used to confirm the material availability of Kera G14. The method of use was carried out according to the manual provided by the manufacturer. The API test was read after at least 48 hours of incubation at 25°C, and the API ZYM test was read after 4 hours of incubation at 25°C. The results of comparison with other species are shown in Table 1 below.

In addition, the anaerobic growth test was confirmed by culturing for 3 days at 25°C in an oxygen-free container with AnaeroPack (MGC).

In addition, tests on the decomposition ability of DNA, casein, starch, Tween 80, and carboxymethylcellulose were evaluated after culturing at 25°C for 7 days.

characteristic One 2 3 4 API-20NE test Nitrate reduction - + + - Reduction of nitrates to nitrogen - - Indole production - - - - Glucose Acidification - + - + Arginine dihydrolase - - - - Urease - - - - β-Glucosidase (esculin hydrolysis) + + - + Protease (gelatin hydrolysis) - - - - β-Galactosidase (PNPG) - + - - D-Glucose + + + + L-Arabinose - - - - D-Mannose - - - + D-Mannitol - + - - N-Acetyl-D-glucosamine - - - + D-Maltose + + - + Gluconate + + + - Caprate - - - - Adipate - - + - Malate - - - - Citrate - - - - Phenyl-acetate - + + - API-ZYM test Alkaline phosphatase + + + + Esterase (C4) + + + + Esterase Lipase (C8) + + + + Lipase (C14) - - - - Leucine arylamidase + + + - Valine arylamidase - - - - Cystine arylamidase - - - - Trypsin - - - - a-chymotrypsin - - - - Acid phosphatase + + + + Naphtol-AS-BI-phosphohydrolase + + + + α-galactosidase - - - - β-galactosidase - + - - β-glucuronidase - - - - α-glucosidase + + - + β-glucosidase + + - + N -acetyl -β-glucosaminidase - + - - a - mannosidase - - - - a-fucosidase - - - -

+: positive, -: negative

1: Kera G14

2: Nocardioides terrae CGMCC 1.7056)

3: Nocardioides insulae DS-51

4: Nocardioides opuntiae DSM 23990)

As a result, it was confirmed that Kera G14 had the following culture and physiological characteristics.

(1) aerobic;

(2) heterotrophic;

(3) positive catalase activity;

(4) Characteristics in Table 1;

(5) Grows on R2A (Reasoner's 2A), TSB (Tryptic soy broth), and LB (Luria-Berani) agar, but does not grow on MA (Marine) and PDB (Potato dextrose broth) agar;

(6) grows on R2A agar at 18°C to 37°C (optimum 30°C), but does not grow at 10°C or 40°C;

(7) Grow at pH 5.5 to 9.0 and NaCl concentrations up to 4% (optimum is pH 6.5, 0% NaCl);

(8) Nitrate is not reduced to nitrite, nor is it reduced to nitric acid;

(9) Starch is degraded, DNA is slightly degraded, but casein, cellulose, and Tween 80 are not degraded;

(10) At least one selected from the group consisting of β-glucosidase, D-Glucose, D-Maltose, and Gluconate activities. assimilation positive;

(11) Indole, glucose, arginine, urease, protease, β-galactosidase, L-Arabinose, D-Mannose, D-Mannitol, N-Acetyl-D-glucosamine, Caprate, Adipate, Malate ( Negative for assimilation of Malate, Citrate and Phenyl-acetate;

(12) alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase ( acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-glucosidase, and β-glucosidase activities are present. ; and

(13) Lipase (C14), valine arylamidase, crystine arylamidase, trypsin, α-chymotrypsin, α-gal Lactosidase (α-galatosidase), β-galactosidase (β-galatosidase), β-glucuronidase, N-acetyl-β-glucosaminidase (N-acetyl-β -glucosaminidase), α-mannosidase, and α-fucosidase activities do not exist.

2-2-3. biochemical properties

The G+C content of Kera G14's DNA was confirmed by whole genome sequencing using Illumina Hiseq equipment. In addition, the test methods and conditions for analysis of Cellular Fatty Acid composition, Quinone, Polar lipid, and Polyamine are as follows.

에서 10분간 가열한 뒤 급냉 하였다. 이후, 1.25 ㎖의 헥산/메틸-터트-부틸에테르(hexane/methyl-tert-butylether)(1:1, v/v)을 넣고 10분간 잘 섞어주었다. 실온에 정치한 후 반응액이 2개의 층으로 분리되면 하등액만을 제거하고 3 ㎖의 dilute NaOH(10.8g NaOH/900㎖ D.W)를 첨가하여 10분간 잘 섞어준 후 실온에 정치하였고, 상등액의 2/3 정도를 바이알(screw-capped sample vial)(12 X 32mm, Agilent technologies)로 옮겨 캡핑(capping)하여 시료로 사용하였다. 시료는 셜록 MIS 소프트웨어(Sherlock MIS Software)의 표준 프로토콜에 따라 사포닌화, 메틸화 및 추출되었다. 지방산은 아질런트 테크놀로지스 6890 가스 크로마토그래피(Agilent technologies 6890 Gas chromatography)와 A30m X 0.320mm X 0.25㎛ 가교 메틸 실록산 컬럼(Crosslinked Methyl siloxane column) (HP-1)을 사용하여 분석하였으며, MIS 패키지의 TSBA40 데이터베이스(MIDI, Version 4.5)를 사용하여 확인하였다. 모든 실험은 최소 3번 수행하였고, 그 결과를 하기 표 2에 나타내었다.First, the cellular fatty acid composition of the isolated strain was determined according to Miller's method. After transferring about 40 mg of cultured cells to a tube, 1 ml of a solution containing 15% NaOH in 50% methanol was added, heated at 100°C for 30 minutes, and cooled to room temperature. Add 2 ml of methanolic-HCl (a mixed solution of 325 ml of 6.0N HCl and 275 ml of methanol) and mix 80 ml.

It was heated for 10 minutes and then rapidly cooled. Afterwards, 1.25 ml of hexane/methyl-tert-butylether (1:1, v/v) was added and mixed well for 10 minutes. After standing at room temperature, when the reaction solution was separated into two layers, only the lower liquid was removed, 3 mL of dilute NaOH (10.8 g NaOH/900 mL DW) was added, mixed well for 10 minutes, and left at room temperature. 2% of the supernatant was added. About 3 was transferred to a screw-capped sample vial (12 Samples were saponified, methylated and extracted according to standard protocols in Sherlock MIS Software. Fatty acids were analyzed using Agilent technologies 6890 Gas chromatography and A30m This was confirmed using (MIDI, Version 4.5). All experiments were performed at least three times, and the results are shown in Table 2 below.

One 2 3 4 Saturated C _10:0 - - 0.39 0.16 C _12:0 - - 2.96 1.61 C _14:0 0.20 - 1.30 0.65 C _16:0 1.19 1.29 1.81 1.32 C _17:0 0.49 2.13 0.27 1.91 C _18:0 - 1.62 1.12 1.37 Unsaturated C _17:1 ω6c 12.8 6.87 2.01 5.51 C _17:1 ω8c 0.38 3.33 1.14 4.55 C _18:1 ω9c 0.86 1.24 9.03 1.68 Branched-chain fatty acid C _12:0 iso - - 0.15 0.42 C _13:0 iso - - - 0.21 C _14:0 iso 0.52 1.36 1.93 1.65 C _15:0 iso 1.77 3.54 0.70 4.07 C _15:0 iso 3OH 0.29 0.28 - - C _16:0 iso 31.98 46.44 61.89 42.88 C _16:0 iso 3OH 1.00 0.36 - - C _16:1 iso H 1.20 3.19 0.81 1.80 C _17:0 iso 5.50 2.07 1.48 2.55 C _17:0 iso 3OH 1.17 0.31 - - C _18:0 iso 2.59 0.67 3.20 0.61 C _19:1 iso I 0.25 - C _15:0 anteiso 0.53 1.06 0.29 0.75 C _16:0 anteiso 0.26 - - 0.23 C _17:0 anteiso 9.11 3.27 3.39 3.41 C _17:1 anteiso ω9c 0.95 1.06 - - C _17:0 10-methyl 4.42 8.24 1.49 12.03 C _18:0 10-methyl, TBSA 12.31 4.37 1.00 5.12 C _19:0 10-methyl - - - 0.23 Hydroxy fatty acids C _16:0 2OH 0.77 - - - C _16:0 3OH 0.27 - - - C _16:1 2OH 1.11 0.57 - 0.41 C _17:0 3OH - 0.33 - - Summed features Summed feature 3 1.40 1.43 2.21 1.05 Summed feature 6 0.50 - 0.55 - Summed feature 7 - - 0.13 - Summed feature 8 - - 0.22 - Summed feature 9 6.43 4.97 0.27 3.83

1: Kera G14

2: Nocardioides terrae CGMCC 1.7056)

3: Nocardioides insulae DS-51

4: Nocardioides opuntiae DSM 23990)

Summed Feature 3: C _16:1 ω7c/C _16:1 ω6c

Summed Feature 6: C _19:1 ω11c/C _19:1 ω9c

Summed Feature 7: C _19:1 ω6c/.846/19cy

6cSummed Feature 8: C _18:1 ω7c/C _18:1

6c

Summed Feature 9: C _17:1 iso ω6c/C _16:0 10-methyl

Quinone was analyzed as follows. After culturing the strain in R2A medium for 72 hours at 25°C, only the bacterial cells were collected and freeze-dried. The freeze-dried sample was extracted under the following conditions. A chloroform:methanol (2:1) solution was added and shaken for 3 to 4 hours. Cells were removed by filtering using filter paper (Whatman No. 2), and the filtered solution was concentrated. Afterwards, it was dissolved in 100 ㎕ of chloroform:methanol (8.5:1.5), centrifuged at 14,000 rpm for 5 minutes, and the supernatant was collected and subjected to HPLC analysis. HPLC conditions were as follows;

HPLC: YOUNG LIN YL9100 (YL9111 Binary pump)

Detector: YOUNG LIN YL9120 UV/Vis Detector

Chromatograph data system: YOUNG LIN Autochro-3000

Analysis column: Waters HPLC (2487 detector, 717 plus Autosampler, 1525 pump)

Analysis solvent: n-hexane: water (1:1, v/v).

Polar lipid analysis was performed after culturing the strain in R2A medium at 25°C for 72 hours. First, polar lipids from the strains were purified using Minnikin D.E. Extraction was performed according to the method described in et al (1984) (J Microbiol Methods 2, 233-241). Afterwards, polar lipids were identified using two-dimensional TLC (Komakata K et al., 1987, Methods Microbiol 19, 161-206).

Polyamines were analyzed as follows. After culturing the strain in R2A medium at 25°C for 72 hours, 200 mg of bacterial cells were collected and added to a sterile tube with 200 μl of cold 1.0M HClO ₄ . After mixing well, 200 ㎕ of cold 2M K ₂ CO ₃ was added and the tube cap was left open for 1 minute. After centrifugation at 13,000 rpm for 1 minute, the supernatant was discarded and washed twice with 0.5 ml of cold PBS. 200 ㎕ of cold 1.0M HClO ₄ was added and mixed well with the bacteria, and then 100 ㎕ of 2M K ₂ Cl ₃ was added. HPLC analysis was performed according to the method described by K Hamana et al (1992) (Critical Reviews in Microbiology, 18(4):26 1-283).

As a result, it was confirmed that Kera G14 has the following biochemical characteristics;

(1) DNA G+C content is 71.9 mol%;

(2) MK-8(H4) (tetrahydrogenated menaquinone with eight units) is the isoprenoid quinone detected;

(3) Polar lipids include diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), five phospholipids (PL), and two one or more selected from the group consisting of unidentified lipids (UL);

(4) The polyamine is at least one selected from the group consisting of Putrescine, 2-hydroxyputrescine, and Spermine; and

(5) Fatty acid composition in Table 2.

In addition, the characteristics with related species were finally compared and summarized and shown in Table 3 below.

One 2 3 4 cell shape Rods Short rods Rods Rods color Pale white Cream Ivory Moderate yellow Size (㎛) 0.4-0.6
0.9-2.0 0.2-0.3
0.1-1.0 0.6-1.0
1.3-6.0 0.5-0.8
1.2-3.1 major fatty acids C _16:0 iso, C _17:1 ω6c, C _18:0 10-methyl (TBSA) C _16:0 iso, C _18:0 10-methyl C _16:0 iso C _16:0 iso, C _18:0 10-methyl G+C mol % 71.9 71.6 71 73.7 Main quinones MK-8(H4) MK-8(H4) MK-8(H4) MK-8(H4)

1: Kera G14

2: Nocardioides terrae CGMCC 1.7056)

3: Nocardioides insulae DS-51

4: Nocardioides opuntiae DSM 23990)

As shown in Table 3, Kera G14 was confirmed to have the same characteristics as Table 3 above. In particular, key biochemical characteristics are different from related species, and the combination of these chemical classification results, together with the results of phylogenetic classification of the strain, proves that Kera G14 is a new species.

Specifically, Kera G14 has key biochemical properties that differ from its related species, as follows.

(1) Rod-Shaped;

(2) The colony shape is round and convex, pale white, and the size is 0.1 mm;

(3) The length of the cell is 0.9~2.0 ㎛ and the diameter is 0.4~0.6 ㎛;

6c 및 C_18:0 10-methyl(TBSA) 이다;(4) The main fatty acids are C _16:0 iso, C _17:1

6c and C _18:0 10-methyl(TBSA);

(5) DNA G+C content is 71.9 mol%; and

(6) MK-8(H4) (tetrahydrogenated menaquinone with eight units) is the isoprenoid quinone detected.

As a result of the above results, it was confirmed that Kera G14 isolated from human epidermal keratinocyte is a new genus of microorganism belonging to the genus Nocardioides ( Nocardioides sp.), and it was named epidermidis according to the nomenclature of the Society of Genealogy. It was named. In addition, the Nocardioides epidermidis Kera G14 strain belonging to the genus Nocardioides ( Nocardioides sp.) was transferred to the Korean Agricultural Culture Collection (KACC) on September 24, 2021. It was deposited and given the deposit number KACC 22414.

Example 3. Activity of strains

3-1. Confirmation of anti-aging activity

To analyze the effect of the culture medium of the Kera G14 strain isolated in Example 1 on factors related to skin aging and wrinkle formation in aging skin caused by ultraviolet (UV) irradiation, COL1A1 (Collagen, type I, alpha 1) Expression was confirmed.

Specifically, the human fibroblast cell line (Human dermal fibroblast, Hs68) was distributed at 3.5x10 ⁵ in a 6-well plate and then cultured in an incubator at 37°C and 5% CO ₂ conditions for 24 hours. Afterwards, the medium was removed, DPBS (Dulbecco's phosphate-buffered saline) was added, and 12 mJ/cm ² UVB was irradiated or not. Immediately after UVB irradiation, DPBS was removed and the medium was changed to FBS-free medium, and then treated with 0.1% (w/w) of the culture medium of the Kera G14 strain and further cultured for 24 hours. A group untreated with ultraviolet rays and strain culture was used as a negative control group, and EGCG was used as a positive control group. Afterwards, RNA was isolated from the cells of each sample using Trizol (RNA iso, DAKARA, Japan), RNA was quantified at 260 nm with a nanodrop, and 2 ㎍ of RNA was used in an amplifier. cDNA was synthesized (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 gene, COL1A1, along with primers and cDNA, using a real-time PCR machine (Step One Plus, Applied Biosystems, USA). Real-time polymerase chain reaction was performed. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in Figure 6.

Figure 6 shows the results showing the effect of Kera G14 strain on the expression of COL1A1.

As a result, as shown in Figure 6, the expression of COL1A1 was decreased by ultraviolet irradiation, and it was confirmed that COL1A1 was significantly increased by treating the strain culture medium.

That is, the Nocardioides epidermidis Kera G14 strain according to one embodiment prevents skin aging (e.g., photoaging) and prevents and improves skin wrinkles by increasing the expression of COL1A1, which is reduced by ultraviolet rays. It can be used.

3-2. Strengthening skin barrier and confirming moisturizing activity

The effect of the culture medium of the Kera G14 strain isolated in Example 1 on skin barrier strengthening and skin moisturizing activity was analyzed.

Specifically, HaCaT cells, a human keratinocyte cell line, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were performed at 37°C. It was performed in a % CO ₂ incubator. The cultured cell line was treated with the culture medium (0.1% (w/w)) of the Kera G14 strain and further cultured for 24 hours. As a positive control, 1 uM of retinoic acid (RA) was used. The expression level of AQP3 was measured in the same manner as above, except that a primer for AQP3 (aquaporin), a factor related to skin barrier strengthening and skin moisturization, was used. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in Figure 7.

Figure 7 shows the results showing the effect of Kera G14 strain on the expression of AQP-3.

As a result, as shown in Figure 7, it was confirmed that the Kera G14 strain culture medium significantly increased the expression of AQP-3.

That is, the Nocardioides epidermidis Kera G14 strain according to one embodiment can be usefully used to strengthen the skin barrier and improve skin moisturization by increasing the expression of AQP-3.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Rural Development Administration National Academy of Agricultural Sciences Microbial Bank (KACC) KACC22414 20210924

SEQUENCE LISTING <110> Cosmax, INC. <120> Novel Nocardioides microorgamism and uses it <130> PN144093 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 1473 <212> DNA <213> Artificial <220> <223> Kera G14 16s rRNA <400> 1 caggacgaac gctggcggcg tgcttaacac atgcaagtcg agcggaaag ccctttcggg 60 ggtactcgag cggcgaacgg gtgagtaaca cgtgagtaat ctgccctgca ctctgggata 120 gccttgggga actgagatta ataccggata tgacacatgc tcgcatgggt gtgtgtggaa 180 agttttttcg gtgcaggatg tgctcgcggc ctatcagctt gttggtgggg taatggccta 240 ccaaggcttc gacgggtagc cggcctgaga gggtgaccgg ccacactggg actgagacac 300 ggcccagact cctacgggag gcagcagtgg ggaatattgg acaatgggcg gaagcctgat 360 ccagcaacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc agcacagacg 420 aagcgcaagt gacggtatgt gcagaagaag gaccggccaa ctacgtgcca gcagccgcgg 480 taatacgtag ggtccgagcg ttgtccggaa ttattgggcg taaagggctc gtaggcggtt 540 tgttgcgtcg ggagtgaaaa cccagggctt aactctgggc ttgctttcga tacgggcaga 600 cttgaggcat tcaggggaga acggaattcc tggtgtagcg gtgaaatgcg cagatatcag 660 gaggaacacc ggtggcgaag gcggttctct gggaatgttc tgacgctgag gagcgaaagt 720 gtggggagcg aacaggatta gataccctgg tagtccacac cgtaaacgtt gggcgctagg 780 tgtgggacac attccacgtg ttccgtgccg cagctaacgc attaagcgcc ccgcctgggg 840 agtacggccg caaggctaaa actcaaagga attgacgggg gcccgcacaa gcggcggagc 900 atgcggatta attcgatgca acgcgaagaa ccttacctgg gtttgacata tacgggaagc 960 ccccagagat gggggtctct ttgatactcg tatacaggtg gtgcatggct gtcgtcagct 1020 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca accctcgtcc tatgttgcca 1080 gcacgtgatg gtggggactc ataggagact gccggggtca actcggagga aggtggggat 1140 gacgtcaagt catcatgccc cttatgtcca gggcttcacg catgctacaa tggccggtac 1200 aaaccgctgc gatcccgtga gggggagcga atcggaaaaaa gccggtctca gttcggattg 1260 gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta atcgcagatc agcaacgctg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggcaaca 1380 cccgaagccg gtggcctaac cccttgtggg gagggagccg tcgaaggtgg ggttggcgat 1440 tgggacgaat ctacaggagg ggcgcccaga aag 1473

Claims (11)

A novel Nocardioides epidermidis strain belonging to the genus Nocardioides ( Nocardioides sp.) and having the following mycological properties;
(1) Rod-Shaped;
(2) Non-motile;
(3) Gram positive; and
(4) Non-spore production (non-motile). The method according to claim 1, wherein the strain further has any one of the following mycological properties;
(1) DNA G+C content is 71.9 mol%;
(2) MK-8(H4) (tetrahydrogenated menaquinone with eight units) is the isoprenoid quinone detected;
(3) The major polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), five phospholipids (PL), and two At least one selected from the group consisting of unidentified lipids (UL); and
(4) The polyamine is at least one selected from the group consisting of Putrescine, 2-hydroxyputrescine, and Spermine. The method according to claim 1, wherein the strain is a strain having any one or more of the following mycological properties;
(1) The length of the cell is 0.9~2.0 ㎛ and the diameter is 0.4~0.6 ㎛;
(2) The colony shape is round, convex, and pale white;
(3) grown on R2A (Reasoner's 2A), TSB (Tryptic soy broth), and LB (Luria-Berani) agar;
(4) Grows on R2A agar at 18°C to 37°C (optimum 30°C), but does not grow at temperatures beyond this;
(5) grows at pH 5.5 to 9.0 and at a salinity (NaCl) of less than 4% (optimum is pH 6.5, 0% NaCl);
(6) Nitrate is not reduced to nitrite, nor is it reduced to nitric acid;
(7) Starch is degraded, DNA is slightly degraded, but casein, cellulose, and Tween 80 are not degraded; and
(8) It has the characteristics shown in Tables 1 to 3. The method according to claim 1, wherein the Nocardioides epidermidis ( Nocardioides epidermidis ) is a strain comprising a 16sRNA sequence having more than 96% sequence identity with the base sequence represented by SEQ ID NO: 1. The method according to claim 4, wherein the Nocardioides epidermidis ( Nocardioides epidermidis ) is a strain deposited under deposit number KACC 22414. A lysate, culture medium, or extract of the culture medium of the strain of claim 1. The culture medium according to claim 6, wherein the culture medium is the culture medium itself obtained by culturing the strain, or a concentrate or freeze-dried product thereof, or a culture supernatant obtained by removing the strain from the culture medium, an extract thereof, or a freeze-dried product. A cosmetic composition comprising Nocardioides epidermidis strain belonging to the genus Nocardioides (Nocardioides sp .), its lysate, culture medium, extract of the culture medium, or a mixture thereof. The cosmetic composition according to claim 8, which is used for strengthening the skin barrier, preventing or improving skin aging, moisturizing the skin, or improving skin elasticity. For the prevention, improvement or treatment of skin conditions, including the Nocardioides epidermidis strain belonging to the genus Nocardioides (Nocardioides sp .), its lysate, culture, extract of the culture, or mixtures thereof Composition for external application to the skin. A health functional food composition comprising Nocardioides epidermidis strain belonging to the genus Nocardioides (Nocardioides sp .), its lysate, culture medium, extract of the culture medium, or a mixture thereof.