KR20210109434A - NASA bacteria and improving skin condition uses of thereof - Google Patents

Abstract

The present invention relates to a novel microorganisms, and a lysate, a culture medium, and a culture medium extract thereof, and a skin condition improving use thereof. The Deinococcus radiodurans of the present invention can be usefully used for preventing, alleviating, or treating skin related conditions or inflammation related conditions.

Description

Nasa bacteria and improving skin condition uses thereof {NASA bacteria and improving skin condition uses of thereof}

It relates to a novel microorganism, its lysate, a culture solution, an extract of the culture solution, and their use for improving skin condition.

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

In the field of functional cosmetics, interest in products that help improve skin wrinkles by delaying skin aging, products that protect the skin from UV rays and the external environment, and products that help skin whitening are very popular. Among them, as we enter an aging society, efforts to develop wrinkle-improving cosmetics to delay skin aging are continuing. In general, wrinkles, reduced skin elasticity, sagging skin, and dryness that appear in aging skin are mostly due to changes in matrix protein present in the dermis. Since the dermis plays a role in supporting the strength and shape of the skin within the skin, when a morphological change occurs in this area as aging progresses, it plays a decisive role in the generation of wrinkles and sagging of the skin. Matrix metalloproteinases (MMPs) are representative enzymes that decompose matrix proteins, and MMPs activity in the skin is increased even after a single UV irradiation, and it is a major factor in promoting skin aging by remarkably decomposing collagen and other matrix proteins in the skin. works

Among skin aging, photoaging is considered as the biggest aging factor of the skin. Accordingly, many products to prevent photoaging are being released, but photoaging prevention technology using natural materials is limited in use and various problems occur during efficacy verification. There is a limit to the fact that there are many cases.

Accordingly, there is a need for the development of novel strains that can be usefully used for skin-related conditions.

One aspect provides a Deinococcus genus ( Deinococcus sp. ) belonging to Deinococcus radiodurans ( Deinococcus radiodurans ) strain.

Another aspect provides a lysate of the strain, a culture solution, an extract of the culture solution, or a mixture thereof.

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

One aspect provides a Deinococcus radiodurans ( Deinococcus radiodurans ) strain.

The deinococcus strain may be a strain deposited with accession number KCCM12643P.

The deinococcus strain may be a strain comprising 16s rRNA of SEQ ID NO: 1.

The strain may have a skin beauty improvement effect, for example, an antioxidant effect, skin aging suppression, skin wrinkle suppression, skin barrier strengthening, skin whitening, or skin moisturizing effect. In addition, the strain may have an effect of improving pruritus or skin inflammation.

The strain may increase (eg, COL1A1, or elastin (ELN)), or decrease (MMP-1) the expression of anti-aging and anti-wrinkle factors. The strain may increase the expression of hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3) or filaggrin related to skin barrier and skin moisturizing. The strain may be one that suppresses the expression of inflammatory cytokines (eg, IL-1β), or reduces the expression of factors related to atopy and itch (eg, TSLP). The strain may be to reduce skin whitening-related melanin synthesis. The strain may have antioxidant activity due to free radical scavenging ability.

Another aspect provides a lysate of the strain, a culture solution, an extract of the culture solution, or a mixture thereof.

The strain is as described above.

As used herein, the term "culture medium" can be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and can supply nutrients to allow the Deinococcus strain to grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain in a medium for a certain period of time, its metabolites, and extra nutrients. In addition, the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution. On the other hand, the liquid from which the cells have been removed from the 　 culture solution 　 is also called a “supernatant”, and the 　 culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the lower layer, remove the cells through filtration, or 　 centrifuge the culture solution to the lower part It can be obtained by removing the precipitation of and taking only the liquid at the top. The "cell" of the present invention refers to the strain itself of the present invention, and includes the strain isolated and selected from a skin sample, or the strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the 　 culture solution to take the part that has sunk to the lower layer, or by removing the liquid from the upper layer after leaving it still for a certain period of time because it sinks into the lower layer of the culturing solution by gravity.

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

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

In one embodiment, the culture medium may be a culture medium in MRS (De Man, Rogosa and Sharpe) medium or BCY medium. Specifically, the culture medium may be obtained by culturing the Deinococcus strain in MRS medium or BCY medium at any temperature of more than 10 ° C or less than 40 ° C for a certain period of time, for example, 4 to 50 hours.

MRS medium is any one selected from the group consisting of peptone, beef extract, yeast extract, glucose, sodium acetate, polysorbate 80, dipotassium hydrogen phosphate, ammonium citrate, magnesium sulfate, manganese sulfate, agar and distilled water, It may be a combination of two or more, or a combination of all of them. The concentration of the components in the above-mentioned medium can be appropriately changed, and the MRS medium may be a concept including all commercially available medium called MRS.

BCY medium contains sodium acetate, magnesium sulfate, manganese sulfate, dipotassium phosphate, pancreatic digest of casein, papaic digest of soybean (Peptone S), dextrose, and Any one selected from the group consisting of sodium chloride, a combination of two or more thereof, or may include all of them. The concentrations of the components in the above-mentioned medium can be suitably changed.

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

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

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

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

As used herein, the term "culture solution extract" means extraction from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and a fraction obtained by fractionating it. 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 a Deinococcus strain, a lysate thereof, a culture medium, an extract of the culture medium, or a mixture thereof.

Uses of the strain may include improvement of skin condition, improvement of skin beauty, prevention, improvement or treatment of skin diseases, prevention, improvement or treatment of skin inflammatory diseases, and the like.

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

As used herein, the term "skin aging" refers to the tangible and intangible changes that appear on the skin with age, for example, a phenomenon in which the thickness of the epidermis becomes thinner, the number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound healing, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and thermoregulation.

The strain or its culture medium may be for improving skin aging caused by extrinsic factors or intrinsic factors. The extrinsic factor refers to various external factors, such as ultraviolet light (light), and the intrinsic factor is also referred to as a chronological factor, and refers to a factor mainly caused by the passage of time. That is, the skin aging specifically refers to not only the premature aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemical substances, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with aging. It is a concept that includes all of wrinkles, loss of elasticity, sagging skin, and dryness. In addition, wrinkles include those that cause wrinkles by changing the components constituting the skin tissue by stimulation by changes in internal and external factors.

The aging may be photoaging. The term “photoaging” is a phenomenon induced by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cleavage of matrix proteins, and abnormal cross-linking, and repetition of these mechanisms leads to apparent skin aging.

As used herein, 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. Pigmentation refers to a condition in which the amount of pigment in the body is abnormal or there is an abnormality in the place where the pigment appears, and may be, for example, spots and freckles. The "prevention or improvement of skin wrinkles" may refer to any action of preventing or improving wrinkles by inhibiting the expression of factors related to wrinkles, or increasing the total amount of collagen.

The "skin whitening" may mean not only to brighten skin tone by inhibiting the synthesis of melanin pigment, but also to improve skin hyperpigmentation such as spots or freckles caused by ultraviolet rays, hormones, or heredity.

The "skin barrier strengthening" may refer to any action of enhancing the function of the skin barrier that is located at the outermost part of the skin and prevents loss of moisture and nutrients.

The "skin moisturizing" may refer to any action that maintains skin moisture or prevents moisture loss.

The term, “anti-inflammatory” may be used interchangeably with “inhibiting or improving inflammation”, and may refer to any action in which an immune response is alleviated to suppress NO production.

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

The damage to the skin barrier function may refer to any change that appears in the skin as the function of the skin barrier is reduced or damaged. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.

The skin inflammatory disease may be any one selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, weak rash, and acne.

The composition comprises 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, based on the total weight of the composition %, 0.00001% to 10% by weight, 0.00001% 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 % to 10% by weight, or 0.1% to 5% by weight of a strain, a lysate thereof, a culture solution, or an extract of a culture solution thereof.

The term, "included as an active ingredient" means that the extract of the strain of the present specification, its lysate, culture, or its culture is added to the extent that it can exhibit the above-mentioned effects, and various for drug delivery and stabilization, etc. It is meant to include being formulated in various forms by adding the component as a sub-component.

The composition may be a cosmetic composition.

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

Components included in the cosmetic composition may include components 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. may include.

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

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component normally used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

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

The pharmaceutical composition may further 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, 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 carboxymethyl cellulose calcium, 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. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, 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 other foods or food ingredients of the strain or its culture, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of 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 type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as additional components, like a conventional beverage. 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 the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition may also be added to 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 the carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.

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

The subject's condition may be a condition related to skin or a condition related to inflammation.

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

Administration may be administered by methods 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. can The administration may be 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 whitening, skin moisturizing, skin barrier strengthening, skin inflammation suppression, pruritus suppression, skin wrinkle improvement effect.

The administration is 0.00001 mg to 1,000 mg per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 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 prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site, and total daily or at intervals of 2 to 5 days. The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of humans per kg, or the above dose is converted, for example, by the volume ratio (for example, the average value) of the target animal and the organ (heart, etc.) One dose can be administered.

According to the novel Deinococcus strain according to an aspect, there is an effect that can be usefully used for the prevention, improvement, or treatment of skin-related conditions or inflammation-related conditions.

1 is a graph showing the effect of a strain according to an embodiment on the expression of COL1A1. None: UV untreated and strain culture untreated group; (-): UV-treated and strain culture untreated group; BCY: BCY medium treatment group without UV treatment and strain culture solution; BCY NASA: 10% (w/w) treatment group of Nasa bacteria culture medium using BCY medium.
2 is a graph showing the effect of a strain according to an embodiment on the expression of COL1A1. None: UV untreated and strain culture untreated group; (-): UV-treated and strain culture untreated group; Deinococcus radiodurans: 1% (w/w) treatment group of bacterial cultures using TSB medium.
3 is a graph showing the effect of a strain according to an embodiment on the expression of ELN. None: UV untreated and strain culture untreated group; (-): UV-treated and strain culture untreated group; BCY: BCY medium treatment group without UV treatment and strain culture solution; BCY NASA: 10% (w/w) treatment group of Nasa bacteria culture medium using BCY medium.
4 is a graph showing the effect of a strain according to an embodiment on the expression of ELN. None: UV untreated and strain culture untreated group; (-): UV-treated and strain culture untreated group; Deinococcus radiodurans: 1% (w/w) treatment group of bacterial cultures using TSB medium.
5 is a graph showing the effect of the strain according to one embodiment on the expression of MMP-1. None: UV untreated and strain culture untreated group; (-): UV-treated and strain culture untreated group; Deinococcus radiodurans: 1% (w/w) treatment group of bacterial cultures using TSB medium.
6 is a graph showing the effect of the strain according to one embodiment on the expression of HAS3. None: untreated group; MRS control: MRS medium treatment group without strain culture; MRS NASA: 10% (w / w) treatment group of Nasa bacteria culture using MRS medium; BCY control: BCY medium treatment group without strain culture solution; BCY NASA: 10% (w/w) treatment group of Nasa bacteria culture medium using BCY medium; RA: positive control treated with retinoic acid 1 uM.
7 is a graph showing the effect of the strain according to one embodiment on the expression of HAS3. None: untreated group; RA: positive control treated with retinoic acid 1 uM; Deinococcus radiodurans: 1% (w/w) treatment group of bacterial cultures using TSB medium.
8 is a graph showing the effect of a strain according to an embodiment on the expression of filaggrin. None: untreated group; Deinococcus radiodurans: 1% (w/w) treatment group of bacterial cultures using TSB medium.
9 is a graph showing the effect of the strain according to one embodiment on the expression of inflammatory cytokines. None: untreated group; (+): Poly I:C and IL-4 treatment, and the non-treated strain culture; MRS control: Poly I:C and IL-4 treatment, and strain culture medium-free MRS medium treatment group; MRS NASA: Poly I:C and IL-4 treatment, and 10% (w/w) treatment group of Nasa bacteria culture using MRS medium; BCY control: Poly I:C and IL-4 treatment, and BCY medium treatment group without strain culture; BCY NASA: Poly I:C and IL-4 treatment, and 10% (w/w) treatment group of Nasa bacteria culture medium using BCY medium; Dex: Poly I:C and IL-4 treatment, and a positive control group treated with dexamethasone 1 uM.
10 is a graph showing the effect of the strain according to one embodiment on the expression of inflammatory cytokines. None: untreated group; (-): Poly I:C and IL-4 treatment, and the non-treated strain culture; Deinococcus radiodurans: Poly I:C and IL-4 treatment, and 1% (w/w) treatment group of bacterial culture using TSB medium.
11 is a graph showing the effect of the strain according to one embodiment on the expression of TSLP, which is a factor related to atopy and itch. None: untreated group; (-): Poly I:C and IL-4 treatment, and the non-treated strain culture; Deinococcus radiodurans: Poly I:C and IL-4 treatment, and 1% (w/w) treatment group of bacterial culture using TSB medium.
12 is a graph showing the melanin synthesis inhibitory activity of the strain according to one embodiment. None: untreated group; Veh.: α-MSH treated group; Deinococcus radiodurans: α-MSH treatment and 1% (w/w) treatment group of bacterial culture using TSB medium.
13 is a graph showing the antioxidant activity of the strain according to one embodiment. None: untreated group; AA: ascorbic acid treatment group; Deinococcus radiodurans: 1% (w/w) treatment group of bacterial cultures using TSB medium.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of L. bacteria

In order to isolate and identify Nasa bacteria, 500ml of water from the upper Han River near Yeouido Park was collected in a sterilized bottle. Thereafter, 5 kGy gamma rays were irradiated, and the sample after gamma irradiation was diluted by a serial dilution method, and the suspension was spread on R2A agar (Difco) medium and cultured at 27° C. for 3 days to select bacteria. PCR amplification was performed on the cultured colonies, and the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies was transferred to another registered BLAST program provided on the website of the National Center for Biotechnology Information (NCBI). When compared with the strains, the homology of 99% or less Deinococcus radiodurans ( Deinococcus radiodurans ) strains (hereinafter referred to as 'nasal bacteria') were selected. The selected Nasa bacteria were deposited with the Korea Microbial Conservation Center on December 18, 2019 and were given an accession number KCCM12643P, and the Nasa bacteria has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Example 2. Preparation of a bacterial culture medium

The screw bacteria of Example 1 were cultured in various types of media to prepare a screw bacteria culture solution.

(1) Preparation of bacterial culture solution using BCY medium

As a culture medium, BCY medium (components: sodium acetate, magnesium sulphate, manganese sulphate, dipotassium phosphate, pancreatic digest of casein, papaya of soybean) Digest (Papaic digest of soybean), dextrose (Dextrose), sodium chloride (containing sodium chloride) was used to prepare a bacterial culture broth. The prepared Nasa bacteria culture medium was named BCY NASA.

(2) Preparation of bacterial culture solution using MRS medium

MRS medium (De Man, Rogosa and Sharpe; Purchased from: HiMedia Laboratories; Catalog No.: M369-500G; Product name: Lactobacillus MRS Broth; Ingredients: Sodium acetate, Magnesium sulphate, Manganese sulfate ( Manganese sulphate), dipotassium phosphate (Dipotassium phosphate)) was used to prepare a bacterial culture solution. The prepared Nasa bacteria culture medium was named MRS NASA.

(3) Preparation of bacterial culture solution using TSB medium

Using TSB (tryptic soy broth; BC, USA) medium as a culture medium, the bacteria were cultured in a 50 ml tube to prepare a bacterial culture medium. At this time, the enrichment culture was cultured by suspending the strain at 100 rpm/min, the temperature during the incubation period was about 25° C., and the culture was terminated after culturing for about 72 hours. Thereafter, the culture solution was subjected to filter sterilization using a 0.45 filter. The prepared Nasa bacteria culture medium was named TSB NASA.

Experimental Example 1. Analysis of anti-aging and anti-wrinkle activity

The skin aging and anti-wrinkle activity of the Nasa bacteria culture solution prepared in Example was analyzed.

Specifically, a human dermal fibroblast cell line (Human dermal fibroblast, Hs68) ^{was aliquoted at 3.5x10 5} in a 6-well plate, and then cultured for 24 hours at 37° C. in _{an incubator under 5% CO 2 conditions.} After removing the medium and adding DPBS (Dulbecco's phosphate-buffered saline), 12 mJ/cm ² of UVB was irradiated or not. Immediately after UVB irradiation, DPBS was removed and replaced with a medium without fetal bovine serum (FBS), treated with BCY NASA 10% (w/w) or TSB NASA 1% (w/w) of Example 2 for 24 hours Further incubation.

As a control group, a UV-treated and non-treated strain culture group, and a UV-treated and strain culture-free medium-treated group were used. After that, RNA was isolated from the cells of each sample using trizol (RNA iso, DAKARA, Japan), and RNA was quantified at 260 nm with nanodrops, and cDNA was synthesized in an amplifier using 2 μg of each RNA. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, the target genes COL1A1 (collagen, type I, alpha 1), elastin (ELN), and MMP-1 (Matrix metalloproteinase-1) were treated with cybergreen (SYBR Green supermix, Applied Biosystems, USA). A real-time polymerase chain reaction was performed in a real-time PCR machine (Step One Plus, Applied Biosystems, USA) by addition with primers and cDNA. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIGS. 1 to 5 .

1 is a graph showing the effect of a strain according to an embodiment on the expression of COL1A1.

2 is a graph showing the effect of a strain according to an embodiment on the expression of COL1A1.

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

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

5 is a graph showing the effect of the strain according to one embodiment on the expression of MMP-1.

1 and 2, the strain according to one embodiment was found to significantly increase the expression of COL1A1 reduced by ultraviolet light.

3 and 4, the strain according to one embodiment was found to significantly increase the expression of ELN reduced by ultraviolet irradiation.

As shown in FIG. 5 , it was found that the strain according to one embodiment significantly reduced the expression of MMP-1 increased by UV irradiation.

These results mean that the strain according to one embodiment has the effect of improving aging (eg, photoaging) and wrinkles.

Experimental Example 2. Skin barrier strengthening and skin moisturizing activity analysis

The skin barrier strengthening and skin moisturizing activity of the Nasa bacteria culture solution prepared in Examples were analyzed.

Specifically, HaCaT cells, which are human keratinocytes, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were cultured at 37°C and 5%. It was performed in a _{CO 2 incubator.} MRS NASA 10% (w / w), BCY NASA 10% (w / w), or TSB NASA 1% (w / w) of Example 2 was treated in the cultured cell line, respectively, and further cultured for 24 hours. As a positive control, 1 uM of retinoic acid (RA) was used. The expression levels of HAS3 and filaggrin were measured in the same manner as in Experimental Example 1, except that primers for HAS3 (hyaluronic acid synthase) and filaggrin, factors related to skin barrier strengthening and moisturizing, were used. The results are shown in FIGS. 6 to 8, respectively.

6 is a graph showing the effect of the strain according to one embodiment on the expression of HAS3.

7 is a graph showing the effect of the strain according to one embodiment on the expression of HAS3.

8 is a graph showing the effect of a strain according to an embodiment on the expression of filaggrin.

6 to 8 , it was found that the strain according to one embodiment significantly increased the expression levels of HAS3 and filaggrin.

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

Experimental Example 3. Analysis of anti-inflammatory, anti-atopic and anti-pruritic activity

The anti-inflammatory, anti-atopic and anti-pruritic activities of the Nasa bacteria culture solution prepared in Examples were analyzed.

Specifically, HaCaT cells, which are human keratinocytes, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were cultured at 37°C and 5%. It was performed in a _{CO 2 incubator.} The medium was changed every 3 to 4 days, and subculture was performed when the cells were excessively proliferated. Thereafter, the cells were aliquoted at 5x10 ⁵ /well, and after 24 hours of incubation, they were washed with phosphate buffered saline solution (PBS). Cells were aliquoted into each well containing DMEM medium without FBS, and Poly I:C 10 μg/ml, and IL-4 10 ng/ml were added. Next, each of MRS NASA 10% (w / w), BCY NASA 10% (w / w), or TSB NASA 1% (w / w) of Example 2 was treated and further cultured for 4 hours. As a positive control, 1 uM of dexamethasone was used. The expression levels of IL-1β and TSLP were analyzed in the same manner as in Experimental Example 1 except that primers for IL-1β and TSLP (Thymic stromal lymphopoietin) were used, and the results are shown in FIGS. 9 to 11 . It was.

9 is a graph showing the effect of the strain according to one embodiment on the expression of inflammatory cytokines.

10 is a graph showing the effect of the strain according to one embodiment on the expression of inflammatory cytokines.

11 is a graph showing the effect of the strain according to one embodiment on the expression of TSLP, which is a factor related to atopy and itch.

As shown in FIGS. 9 and 10 , it was found that the strain according to one embodiment significantly reduced the expression of IL-1β, an inflammatory cytokine, compared to the untreated control group.

In addition, as shown in FIG. 11 , it was found that the strain according to one embodiment reduced the expression of TSLP, a factor related to atopy and itch, compared to the untreated control group.

These results show that the L. bacteria according to one embodiment can be usefully used for the prevention, improvement, or treatment of skin inflammatory diseases, in particular, the prevention, improvement, or treatment of atopy, or prevention, improvement, or treatment of pruritus (itchiness), or It means that it can be usefully used for treatment.

Experimental Example 4. Analysis of skin whitening activity

The skin whitening activity of the Nasa bacteria culture solution prepared in Example was analyzed.

에서 1시간 건조시켜 10% DMSO가 포함된 1M NaOH용액 100 ㎕을 넣어 세포 내의 멜라닌을 얻었다. 이 멜라닌 액을 Microplate reader로 490nm에서 흡광도를 측정하여 멜라닌 함량을 평가하였고, 그 결과를 도 12에 나타내었다. Specifically, the melanin production inhibitory effect of the TSB NASA culture medium of Example 2 was measured using mouse pigment cells (B16 melanoma cells). First, murine melanoma (B16 F10) cells were suspended in DMEM containing 10% FBS ^{, inoculated in a 6-well plate at 1 Х 10 5} cells/well, and cultured until adhered to the bottom of the well. In order to induce melanin production, 100 nM α-MSH was treated, and then 1% (w/w) of TSB NASA of Example 2 was added and cultured for 3 days. The cultured cells were washed with phosphate buffered saline (PBS) and recovered with trypsin. The recovered cells were counted with a hematocytometer, ^{collected at 1Х10 6} cells/mL to obtain the same number of cells, and centrifuged at 1000 rpm for 10 minutes to obtain only cells. 60 recovered cells

After drying for 1 hour, 100 μl of 1M NaOH solution containing 10% DMSO was added to obtain melanin in the cells. The melanin content was evaluated by measuring the absorbance of this melanin solution at 490 nm with a Microplate reader, and the results are shown in FIG. 12 .

12 is a graph showing the melanin synthesis inhibitory activity of the strain according to one embodiment.

As shown in Figure 12, it was found that the increased melanin synthesis due to α-MSH was significantly reduced by the treatment of the culture solution of Nasa bacteria.

This result means that the culture solution of the strain according to one embodiment has skin whitening effect.

Experimental Example 5. Antioxidant activity analysis

The antioxidant activity of the bacterial cultures prepared in Examples were analyzed.

Specifically, free radical scavenging activity was measured using DPPH. DPPH was purchased from Sigma Co., Ltd. (USA) and used. First, a standard DPPH ethanol solution of 1.5 mM (0.06 mg/mL) was prepared. Next, ethanol was added to each of the TSB NASA 1% (w/w) and ascorbic acid (5 ppm) as a positive control to prepare a sample. Then, the sample and the standard DPPH solution were added in the same ratio, stirred well, reacted at 37° C. for 30 minutes, and absorbance was measured at 520 nm. In this case, as the control group, ethanol was added instead of the sample. Compared to the free radical scavenging ability of the no-addition control group, the free radical scavenging ability of the strain was calculated, and the results are shown in FIG. 13 .

13 is a graph showing the antioxidant activity of the strain according to one embodiment.

As shown in FIG. 13 , it was found that the strain according to one embodiment had antioxidant activity at a level similar to that of ascorbic acid, a positive control.

Korea Microorganism Conservation Center (Overseas) KCCM12643P 20191218

SEQUENCE LISTING <110> Cosmax, INC. HAVE&BE CO., Ltd. <120> NASA bacteria and improving skin condition uses of its <130> PN132115 <150> KR 10-2020-0024629 <151> 2020-02-27 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 1282 <212> DNA <213> Deinococcus radiodurans <400> 1 ctgacctacc cagaagtcac gaataactgg ccgaaaggtc cgctaatacg tgatgtggtg 60 atgcaccgtg gtgcatcact aaagatttat cgcttctgga tggggttgcg ttccatcagc 120 tggttggtgg ggtaaaggcc taccaaggcg acgacggata gccggcctga gagggtggcc 180 ggccacaggg gcactgagac acgggtccca ctcctacggg aggcagcagt taggaatctt 240 ccacaatggg cgcaagcctg atggagcgac gccgcgtgag ggatgaaggt tttcggatcg 300 taaacctctg aatctgggac gaaagagcct tagggcagat gacggtacca gagtaatagc 360 accggctaac tccgtgccag cagccgcggt aatacggagg gtgcaagcgt tacccggaat 420 cactgggcgt aaagggcgtg taggcggaaa tttaagtctg gttttaaaga ccggggctca 480 acctcgggga tggactggat actggatttc ttgacctctg gagaggtaac tggaattcct 540 ggtgtagcgg tggaatgcgt agataccagg aggaacacca atggcgaagg caagttactg 600 gacagaaggt gacgctgagg cgcgaaagtg tggggagcaa accggattag atacccgggt 660 agtccacacc ctaaacgatg tacgttggct aagcgcagga tgctgtgctt ggcgaagcta 720 acgcgataaa cgtaccgcct gggaagtacg gccgcaaggt tgaaactcaa aggaattgac 780 gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 840 caggtcttga catgctagga actttgcaga gatgcagagg tgcccttcgg ggaacctaga 900 cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 960 cgagcgcaac ccttgccttt agttgtcagc attcagttgg acactctaga gggactgcct 1020 atgaaagtag gaggaaggcg gggatgacgt ctagtcagca tggtccttac gtcctgggcg 1080 acacacgtgc tacaatgggt aggacaacgc gcagcaaacc cgcgagggta agcgaatcgc 1140 taaaacctat ccccagttca gatcggagtc tgcaactcga ctccgtgaag ttggaatcgc 1200 tagtaatcgc gggtcagcat accgcggtga atacgttccc gggccttgta cacaccgccc 1260 gtcacgccat gggattagat tt 1282

Claims (7)

Deposited with accession number KCCM12643P, Deinococcus genus ( Deinococcus sp. ) Deinococcus radiodurans belonging to ( Deinococcus radiodurans ) strain. The lysate of the strain of claim 1, or a culture solution. A cosmetic composition comprising the strain of claim 1, a lysate thereof, a culture solution, or a mixture thereof. The cosmetic composition of claim 3, wherein the cosmetic composition is for skin condition improvement, skin whitening, or moisturizing. The cosmetic composition according to claim 4, wherein the skin condition is wrinkles, skin barrier, pruritus, or skin inflammation. A pharmaceutical composition for preventing or treating skin inflammatory diseases comprising the strain of claim 1, a lysate thereof, a culture medium, or a mixture thereof. The method according to claim 6, wherein the skin inflammatory disease is any one selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, rash, and acne. enemy composition.

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