WO2021060657A1 - Staphylococcus cohnii st-3 strain and use thereof for improving skin condition - Google Patents

Abstract

The present invention relates to a novel microorganism, a lysate thereof, a culture thereof, an extract from the culture, and a use thereof for improving a skin condition.

Description

Staphylococcus cornii ST-3 strain and its use for improving skin condition

It relates to novel microorganisms, their lysates, cultures, extracts of cultures, and uses thereof to improve skin conditions.

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

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

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

One aspect is to provide a Staphylococcus cohnii ST-3 strain belonging to the genus Staphylococcus sp.

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

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

Another aspect is to provide a method for improving, preventing, or treating a skin condition comprising administering the strain, its lysate, a culture medium, and an extract of the culture medium to an individual.

One aspect provides a Staphylococcus cohnii ST-3 strain.

The Staphylococcus cohnii ST-3 strain may be a strain deposited under the accession number KCCM12548P.

The Staphylococcus cohnii ST-3 strain may be a strain containing 16s rRNA of SEQ ID NO: 1.

The strain may be one having an effect of improving skin beauty, for example, inhibiting skin aging, whitening skin, strengthening skin barrier, inhibiting skin wrinkles, or moisturizing skin.

The strain may be one that increases the expression of hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3) or filaggrin.

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

The strain is as described above.

In the present specification, the term "culture medium" may be used interchangeably with "culture supernatant", "conditional culture medium" or "conditioned medium", and nutrients so that Staphylococcus conii ST-3 strain can grow and survive in vitro. The strain obtained by culturing the strain for a certain period of time in a medium capable of supplying the strain, its metabolite, and may mean a whole medium including extra nutrients. In addition, the culture solution may mean a culture solution in which cells are removed from the cell culture solution obtained by culturing the strain. On the other hand, the liquid from which the cells have been removed from the 　 culture solution is also referred to as the "supernatant solution", and the culture solution is left for a certain period of time to take only the liquid in the upper layer excluding the submerged part, or remove the cells through filtration, or It can be obtained by removing the precipitation of and taking only the upper liquid. The "bacterial body" refers to the 　 strain 　 itself of the present invention, and includes the 　 strain 　 itself or 　 separated from the culture solution by culturing the 　 strain selected by separating from skin samples and the like. The cells can be obtained by taking the part that has settled in the lower layer by centrifuging the culture solution, or by allowing it to stand still for a certain period of time and then removing the upper liquid because it sinks into the lower layer of the culture solution by gravity.

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

The culture medium is by culturing the Staphylococcus conii ST-3 strain in a medium (eg, 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. It may be obtained.

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

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

The culture medium and culture conditions for culturing the Staphylococcus conii ST-3 strain may be appropriately selected or modified by those of ordinary skill in the art.

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

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

Another aspect provides the use of the strain, a lysate of the strain, a culture solution, or an extract of the culture solution. Specifically, it provides a composition comprising a Staphylococcus conii ST-3 strain, a lysate, a culture solution, or an extract of a culture solution thereof.

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

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

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

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

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

In the present specification, the term "wrinkle" refers to a state in which the elasticity of the skin is lost and loosened, and for example, the skin may be folded. Pigmentation refers to a condition in which the amount of pigment in the body is abnormal or where the pigment appears, and may be, for example, spots and freckles. The "skin wrinkle prevention or improvement" may mean 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 brightening skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones, or genetics.

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

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

The term “anti-inflammatory” may be used interchangeably with “inhibitory or ameliorating inflammation”, and may mean any action in which the immune response is alleviated and NO production is suppressed.

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

The damage to the skin barrier function may mean all changes that appear on the skin due to a decrease or damage to the function of the skin barrier. 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, eczema skin disease, dry eczema, erythema, urticaria, psoriasis, weak rash, and acne.

In another embodiment, the strain may be used together with a strain belonging to the genus Staphylococcus other strains having a skin improvement effect to exhibit a synergistic effect. Examples of strains belonging to the genus Staphylococcus are S. argenteus , S. aureus , S. schweitzeri , S. simiae, S. auricularis, S. carnosus , S. condimenti , S. debuckii , S. massiliensis , S. piscifermentans , S. simulans, S. capitis , S. caprae , S. epidermidis , S. saccharolyticus, S. devriesei , S. haemolyticus , S. hominis, S. hyicus-intermedius , S. agnetis , S. chromogenes , S. cornubiensis , S. felis , S. delphini , S. hyicus , S. intermedius , S. lutrae , S. microti , S. muscae , S. pseudintermedius , S. rostri , S. schleiferi, S. lugdunensis, S. arlettae , S. caeli , S. cohnii , S. equorum , S. gallinarum , S. kloosii , S. leei , S. nepalensis , S. saprophyticus , S. succinus , S. xylosus, S. fleurettii , S. lentus , S. sciuri , S. stepanovicii , S. vitulinus, S. simulans , S. pasteuri , or S. warneri . More specifically, examples of strains belonging to the genus Staphylococcus include Staphylococcus capitis ST-1, Staphylococcus lentus ST-2, Staphylococcus lentus ST-2, and Staphylococcus cornii. ST-3 ( Staphylococcus cohnii ST-3), Staphylococcus gallinarum ST-4, Staphylococcus saprophyticus ST-5, Staphylococcus saprophyticus ST-5, Staphylococcus epi Staphylococcus epidermidis ST-6, Staphylococcus sciuri ST-7, Staphylococcus haemolyticus ST-8, Staphylococcus haemolyticus ST-8, Staphylococcus simul Staphylococcus simulans ST-9, Staphylococcus xylosus ST-10, Staphylococcus intermedius ST-11, Staphylococcus intermedius ST-11, and Staphylococcus Reneri ST-12 ( Staphylococcus warneri ST-12), or Staphylococcus schleiferi ST-13 (Staphylococcus schleiferi ST-13).

The composition is from 0.001% to 80% by weight based on the total weight of the composition, for example, from 0.01% to 60% by weight, from 0.01% to 40% by weight, from 0.01% to 30% by weight, from 0.01% to 20% by weight %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20%, 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 the 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 strain of the present specification, its lysate, culture medium, or extract of the culture medium thereof is added to the extent that the above-mentioned effect can be exhibited, and various It means that the ingredients are added as an auxiliary ingredient to be formulated in various forms.

The composition may be a cosmetic composition.

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

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

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

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

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

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

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

The composition may be a health functional food composition.

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

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

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

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

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

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

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

In one aspect, according to the novel Staphylococcus conii ST-3 strain, there is an effect that can be usefully used in the prevention, improvement, or treatment of a skin-related condition or an inflammation-related condition.

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

Figure 2 is a graph showing the effect of the strain on the expression of HAS3 according to an embodiment.

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

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

Example. Isolation and identification of strains

A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy woman with sterile distilled water was inoculated into R2A medium (Becton Dickinson, Cockeysville, MD). After culturing in a 28°C incubator for 48 hours after inoculation, 100 colonies formed were separated and cultured with pure water, and cultivated in a 28°C incubator for 48 hours. The cultured colonies were subjected to 16s rRNA gene sequence identification. The primers used at this time were designed to amplify in response to only bacteria (SEQ ID NOs: 2 and 3). PCR amplification was carried out in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 75°C for 1 minute and 30 seconds, and finally treated at 72°C for 8 minutes and then stored at 4°C. After the PCR reaction was over, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA). The nucleotide sequence of the 16S rRNA site determined among the isolated and cultured microbial colonies was compared with other strains registered with the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI), when compared with 98% homology. Only novel species of were selected and used, and among them, a novel microorganism Staphylococcus cohnii ST-3 strain (hereinafter referred to as "ST-3") with 98% or less homology was selected. The selected ST-3 strain was deposited with the Korea Microbial Conservation Center on June 11, 2019, and was given the accession number KCCM12548P, and the ST-3 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Experimental example. Strain activity

In this experimental example, the activity of the identified strain was confirmed, and as a control strain, KCCM 41467 Staphylococcus cornii strain (sold in Korea Microbial Conservation Center) (hereinafter referred to as “Type Strain”) was used.

Anti-aging and anti-wrinkle activity analysis

The effect of the strain culture solution on factors related to aging skin aging and wrinkle formation by ultraviolet (UV) irradiation was analyzed.

Specifically, the human fibroblast cell line (Human dermal fibroblast, Hs68) ^{was dispensed into a 6-well plate at 3.5×10 5} , and then cultured for 24 hours in an incubator under conditions of 37° C. and 5% CO _2. After removing the medium and adding DPBS, 12 mJ/cm ² of UVB was irradiated or not. Immediately after UVB irradiation, DPBS was removed and replaced with a medium without FBS, and then the culture solution (1% (w/w)) of the ST-3 strain was treated and further cultured for 24 hours. As a negative control group, UV-treated and non-treated strain culture solution were used. After that, RNA was isolated from the cells of each sample using trizol (RNA iso, DAKARA, Japan), and RNA was quantified at 260 nm with nanodrops, and then cDNA was synthesized in an amplifier using 2 μg of each RNA. (C1000 Thermal Cycler, Bio-Rad, USA). A real-time PCR machine (Step One Plus, Applied Biosystems, USA) by adding cybergreen (SYBR Green supermix, Applied Biosystems, USA) together with primers and cDNA to the target gene COL1A1 using the synthesized cDNA as a template. Real-time polymerase chain reaction was performed at. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIG. 1.

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

These results mean that the ST-3 strain according to an embodiment has an effect of improving aging (eg, photoaging).

Skin barrier strengthening and skin moisturizing activity analysis

The effect of the strain culture solution on skin barrier strengthening and skin moisturizing activity was analyzed.

Specifically, HaCaT cells, a human keratinocyte, were cultured in DMEM medium (Dulbecco'smodified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were 37°C 5 % CO _{2 in an} incubator. The cultured cell line was treated with the culture solution (1% (w/w)) of the ST-3 strain, and further cultured for 24 hours. The expression level of HAS3 and filaggrin was measured in the same manner as in the above experimental example, except that primers for HAS3 (hyaluronic acid synthase) and filaggrin, which are factors related to skin barrier strengthening and moisturizing, were used. I did. The results are shown in FIGS. 2 and 3, respectively.

As shown in Figures 2 and 3, it was found that the strain according to one embodiment significantly increased both the expression levels of HAS3 and filaggrin.

These results mean that the ST-3 strain according to an embodiment can be usefully used for strengthening the skin barrier and moisturizing the skin.

Claims (4)

1. Staphylococcus cohnii ST-3 strain belonging to Staphylococcus, deposited under the accession number KCCM12548P.
2. The lysate or culture medium of the strain of claim 1.
3. A cosmetic composition comprising the strain of claim 1, a lysate, a culture solution, an extract of the culture solution, or a mixture thereof.
4. The cosmetic composition according to claim 3, which is for improving skin beauty.

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