KR102361015B1 - Use of Sphingomonas olei to improve skin condition - Google Patents

Abstract

The present invention relates to a use of Sphingomonas olei capable of improving skin condition. A composition comprising Sphingomonas olei, a lysate thereof, a culture liquid, or a mixture thereof as an active ingredient improves recovery of damaged epidermis, increases expression of skin barrier related factors, and reduces expression of inflammation factors, thereby being effectively used for improving skin condition.

Description

Use of Sphingomonas olei to improve skin condition}

The present invention relates to the use of Sphingomonas olei for improving skin condition. More specifically, the present invention provides a composition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient to enhance the recovery of damaged epidermis, increase the expression of skin barrier-related factors, and increase inflammation It relates to use for improving skin condition by reducing factor expression.

Since there are many waste products such as dead skin cells, sweat, and sebum on the surface of human skin, various microorganisms inhabit and form the skin microbiome, which is the skin flora. In the skin flora, beneficial and harmful bacteria coexist, and healthy skin has a greater distribution of beneficial bacteria, and in the case of atopic or acne-prone skin, the distribution of harmful bacteria is greater. Therefore, it is known that maintaining the balance of the flora present in the skin helps to maintain a healthy skin condition (Patent Document 1). However, studies on the effect of these skin flora on the skin are still insignificant.

Skin flora, which normally resides on the skin, forms a skin barrier that prevents the penetration of other more harmful microorganisms (pathogens) and helps skin immunity. Since beneficial skin bacteria do not cause disease and do not stimulate the immune system, if medicines or cosmetics can be manufactured using beneficial skin bacteria, it can be safely used without side effects such as skin irritation.

When the skin is injured or infected, damage or inflammation of the skin tissue occurs, but if the degree is not severe, the skin usually heals on its own. In the present invention, as a result of research to develop skin flora that helps in skin regeneration and recovery and the healing process of inflammation, skin beneficial bacteria that can improve skin conditions through skin regeneration and inflammation healing were identified and invention was completed.

Korean Patent Publication No. 2297406 (published on: September 7, 2021)

Accordingly, the present invention is to provide a cosmetic composition for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

An object of the present invention is to provide a quasi-drug composition for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

An object of the present invention is to provide a composition for external application for skin for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

An object of the present invention is to provide a health functional food composition for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory skin diseases, or for treating or improving skin wounds, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

The present invention is to provide a Sphingomonas olei strain deposited with accession number KACC 81169BP.

The present invention is to provide a lysate of the Sphingomonas olei strain, a culture solution, an extract of the culture solution, or a mixture thereof.

One aspect of the present invention provides a cosmetic composition for improving skin condition, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof has a skin condition improvement effect, for example, skin barrier improvement, skin moisturizing, skin immunity improvement, skin defense enhancement, skin inflammation suppression, skin soothing and skin regeneration. It may have an effect of improving one or more skin conditions selected from the group.

Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof may promote the migration of keratinocytes from the damaged skin surface, thereby enhancing the recovery of the damaged epidermis.

Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof may increase the expression of a skin barrier-related factor (eg, Loricrin, a protein required for keratinogenesis).

In addition, Sphingomonas olei, a lysate thereof, a culture medium or a mixture thereof may contain inflammatory factors, such as atopy and itch-related factors (eg, Thymic stromal lymphopoietin (TSLP)) and pro-inflammatory cytokines (eg, IL-6, IL-8 or IL-1α).

In one embodiment, Sphingomonas olei may be live (live bacteria) or dead cells (dead bacteria). In more detail, the dead cells may be dead cells by heat treatment.

In one embodiment, Sphingomonas olei may be a Sphingomonas olei strain deposited with accession number KACC 81169BP.

As used herein, the term “lysate” refers to a solution or suspension in an aqueous medium of cells of a microorganism such as broken Sphingomonas olei. Cell lysates include, for example, macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids, etc. and/or micromolecules such as amino acids, sugars, fatty acids, etc., or fractions thereof. The lysate also contains cell debris, which may be smooth or granular in structure.

As a method for achieving cell lysis of the microorganism, various known methods may be used, and any method capable of achieving cell lysis of the microorganism may be used. For example, cell opening/destruction can be performed enzymatically, chemically or physically. Non-limiting examples of enzymes and enzyme mixtures are proteases such as proteinase K, lipases or glycosidases; Non-limiting examples of chemicals are ion permeable carriers, detergents such as sodium dodecyl sulfate, acids or bases; Non-limiting examples of physical means are high pressure such as French pressing, osmotic pressure, temperature such as heat or cold. In addition, a method using an appropriate combination of enzymes other than proteolytic enzymes, acids, bases, and the like can also be used.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and can provide nutrients so that Sphingomonas olei can grow and survive in vitro. The microorganism obtained by culturing Sphingomonas olei in a medium for a certain period of time may refer to the entire medium including the microorganism, its metabolites, and extra nutrients. In addition, the culture solution may refer to a culture solution obtained by culturing microorganisms in which cells are removed from the culture solution. On the other hand, the liquid from which the cells are removed from the culture medium is also called "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, or to 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" refers to the microorganism itself of the present invention, and includes the microorganism itself selected by separating it from a skin sample or the like, or a microorganism isolated from a culture solution by culturing the microorganism. The cells can be obtained by centrifuging the culture solution to take the part that has sunk to the lower layer, or it can be obtained by removing the upper liquid after leaving it for a certain period of time because it sinks to the lower layer of the culture solution by gravity.

According to one aspect, the culture of Sphingomonas olei of the present invention may use a medium easily selected by a person skilled in the art according to the purpose among the medium used for culturing microorganisms, specifically, a medium used for culturing Sphingomonas, such as RCM (Reinforced Clostridium Medium) medium, TSB (Tryptic soy broth) or BHI (Brain Heart Infusion) medium may be used, but is not limited thereto. According to one embodiment, the culture of Sphingomonas olei of the present invention is inoculated with Sphingomonas olei in the microbial culture medium, and prepared according to a microbial culture method known in the art (eg, stationary culture, etc.) can

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

The culture medium may be obtained by culturing Sphingomonas olei in an appropriate medium (eg, R2A medium or TSB medium) at any temperature of 10°C to 40°C for a certain period of time, for example, 4 to 50 hours. .

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

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

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

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 fractionation thereof. can

The skin condition improvement may be at least one selected from the group consisting of skin barrier improvement, skin moisturizing, skin immunity improvement, skin defense enhancement, skin inflammation suppression, skin soothing, and skin regeneration.

As used herein, the term "improving skin barrier" is meant to include both skin barrier strengthening or protective functions, where the skin barrier is the outermost layer of the epidermis, the stratum corneum, mainly composed of non-nucleated flat keratinocytes. It is made up of corneocytes. The multi-lamellar lipid layer formed of intercellular lipids such as ceramide, cholesterol, and fatty acids synthesized by keratinocytes of the skin barrier maintained through the division and differentiation process of normal epidermal cells is a protective barrier to prevent moisture in the skin from evaporating. plays a role The improvement of the skin barrier is psoriasis, contact dermatitis, eczematous dermatitis, photodermatitis, seborrheic dermatitis, dermatitis herpeticus, lichen planus, lichen planus, pyoderma gangrene, pyoderma gangrenosum, epidermis bullous It can refer to any action that relieves alopecia, systemic sclerosis or leprosy or improves the function of the damaged skin barrier.

As used herein, the term “skin moisturizing” may refer to any action that maintains skin moisture or prevents moisture loss.

In the present specification, the term "skin regeneration" means not only skin regeneration due to aging, but also all cases in which the skin is regenerated, such as when the epidermis damaged due to scratches or the like is healed.

The skin inflammation is selected from the group consisting of dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin disease, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, rash, and acne. may be one.

In one embodiment, Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof promotes the migration of stromal cells, increases the expression of loricrin, or TSLP, IL-6, IL-8 and IL It may be to reduce the expression of one or more selected from the group consisting of -1α.

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% with respect to 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 microorganism, a lysate thereof, a culture solution, or an extract of a culture solution thereof.

As used herein, the term "included as an active ingredient" means that the microorganism, its lysate, culture medium, or extract of its culture medium 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 cosmetic composition is a lotion (skin lotion), skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, hand sanitizer, foundation, essence, Nutrient essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, and may be prepared in a formulation including a sheet. The composition of such a formulation can be prepared according to a method conventional in the art. The blending amount of the additional ingredients such as the moisturizing agent can be easily selected by those skilled in the art within a range that does not impair the purpose and effect of the present invention.

The cosmetic composition may further include functional additives and components included in general cosmetic compositions in addition to the active ingredients disclosed herein, and commonly used purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, It may further include a fragrance or a combination thereof. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extract. Examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, color developer, fragrance, and the like. The alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, coloring agent, perfume, etc. are already known in the art. Therefore, those skilled in the art can select and use an appropriate material/composition. In addition, the cosmetic composition includes, if necessary, a sunscreen agent, an antioxidant (butylhydroxyanisole, propyl gallic acid, elisorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), a preservative (methylparaben, butylparaben, etc.) , propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorant, pH adjuster (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid , sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, etc.), humectants (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycerose-26, methyl glue Seth-20, etc.), lubricants, and the like may be further added.

In addition, in the cosmetic composition of each formulation, appropriate ingredients may be selected and formulated according to the formulation or purpose of use of the cosmetic. Since the compounding component and method may follow conventional techniques, a detailed description thereof will be omitted herein.

Another aspect of the present invention provides a quasi-drug composition for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

At this time, Sphingomonas olei, a lysate thereof, a culture medium or a mixture thereof, and the skin condition improvement effect thereof are the same as described above.

As used herein, the term "quasi-drug" refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs Textiles and rubber products used for the treatment or prevention of diseases in humans and animals, which do not act directly on the human body, are not instruments or machines, and are similar to those that are not used for pharmaceutical purposes. This includes disinfectants and pesticides to prevent

The type or formulation of the quasi-drug composition of the present invention is not particularly limited, but may be a bandage, gauze, cotton wool, bandage, disinfectant cleaner, shower foam, gargrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, or filter filler. have.

When the composition of the present invention is included in a quasi-drug for improving skin condition, the composition may be used as it is or used together with other quasi-drug ingredients, and may be used appropriately according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use, and the quasi-drug composition according to the present invention may contain 0.01 to 20% by weight of microorganisms, their lysates, cultured solutions, or mixtures thereof based on the total weight of the composition. have.

Another aspect of the present invention provides a composition for external application for skin for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

At this time, Sphingomonas olei, a lysate thereof, a culture medium or a mixture thereof, and the skin condition improvement effect thereof are the same as described above.

The external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, lotion, or a combination thereof. The external preparation for skin is a component 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 extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and 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.

The composition for external application for skin according to the present invention may contain 0.00001 wt% to 80 wt% of a microorganism, a lysate thereof, a culture solution, or a mixture thereof based on the total weight of the composition.

The skin includes all skin parts of the body including the face, hands, arms, legs, feet, chest, belly, back, buttocks, and scalp.

Another aspect of the present invention provides a health functional food composition for improving skin condition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

At this time, Sphingomonas olei, a lysate thereof, a culture medium or a mixture thereof, and the skin condition improvement effect thereof are the same as described above.

The health functional food composition may be used alone or in combination with other foods or food ingredients, Sphingomonas olei or its culture medium, 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. Examples of foods to which the above substances can be added include powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leached teas, and formulations selected from the group consisting of health drinks. It includes all health foods in the ordinary sense. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component 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 taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages 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 of the present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases, or for treating or improving skin wounds, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient.

In this case, the description of Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof is the same as described above.

The inflammatory skin disease is selected from the group consisting of dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin disease, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, weak rash, and acne It may be either one.

A skin wound is also called a "wound" and means damage to a living body. The damage may be due to external factors such as physical damage, radiation, stimulation by chemicals, proliferation of microorganisms, or internal factors such as stress, but is not limited thereto. The wounds may include all kinds of wounds, such as cleavage, puncture, cleavage, acne, fissure, bronchial, and school spear. "Wound healing" may refer to a series of biological reactions to repair a wound. "Improving wounds" may refer to any action that lightens or reduces the extent of wounds on the face or body.

As used herein, the term “pharmaceutical composition” may refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Advantageous effects include enabling diagnostic decisions; amelioration of a disease, symptom, disorder or condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and responding to a disease, symptom, disorder or condition in general.

As used herein, the term “prevention” refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or concomitant symptom thereof, preventing the acquisition or reacquisition of the disease or disorder, or preventing the disease or disorder from occurring. How to reduce the risk of acquisition. For example, the prevention refers to any action that suppresses or delays the occurrence of skin damage or symptoms by administration of the composition according to the present invention.

As used herein, the term “treatment” includes inhibiting, alleviating or eliminating the development of a disease.

As used herein, the term “improvement” may refer to any action that at least reduces a parameter related to alleviation or treatment of a condition, for example, the degree of a symptom.

The pharmaceutical composition may be administered parenterally during clinical administration and may be used in the form of general pharmaceutical formulations. Parenteral administration may refer to administration via a route other than oral administration, such as rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. When the pharmaceutical composition of the present invention is used as a pharmaceutical, it may further contain one or more active ingredients exhibiting the same or similar function.

The types of pharmaceutically active ingredients capable of delivering the active ingredient into an individual include anticancer agents, contrast agents (dyes), hormones, antihormones, vitamins, calcium agents, inorganic agents, saccharides, organic acid preparations, protein amino acid preparations, detoxifying agents, enzymes Agents, metabolic agents, diabetic combination agents, tissue revitalization agents, chlorophyll agents, pigment agents, tumor agents, tumor agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapies, antibiotic agents, antiviral agents, combined antibiotics, chemicals Therapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospirin serum, blood products, biologics, analgesics, immunogenic molecules, antihistamines, allergens, non-specific immunogenic agents, anesthetics, stimulants, psychoneurologic agents, small molecule compounds, nucleic acids, aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs and microRNAs, and the like.

The pharmaceutical composition may be prepared by additionally including one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. , and other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art.

When formulating the pharmaceutical composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, liulinji, glycerogelatin, etc. may be used.

The pharmaceutical composition may include carbohydrates such as glucose, sucrose or dextran, antioxidants such as Ascorbic acid or Glutathione, chelating agents, and low molecular weight proteins to increase stability or absorption. Alternatively, other stabilizers may be used as medicaments.

Another aspect of the present invention provides a method of preventing, ameliorating, or treating a condition in a subject, comprising administering the pharmaceutical composition to the subject.

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

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

Administration may be administered by methods known in the art. Administration may be administered directly to a subject by any means, for example, 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 improvement in skin condition or healing of an inflammatory skin disease.

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, A person skilled in the art can appropriately adjust the dosage 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 places for the site of administration, 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 a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.

Another aspect of the present invention provides a Sphingomonas olei strain deposited with accession number KACC 81169BP.

The Sphingomonas olei strain may be a strain comprising 16S rRNA of SEQ ID NO: 3.

The strain is a skin condition improvement effect, for example, to have one or more skin condition improvement effects selected from the group consisting of skin barrier improvement, skin moisturizing, skin immunity improvement, skin defense enhancement, skin inflammation suppression, skin soothing and skin regeneration can

The strain may promote the movement of keratinocytes on the damaged skin surface to promote recovery of the damaged epidermis. The strain may increase the expression of skin barrier-related factors (eg, loricrin). In addition, the strain reduces the expression of inflammatory factors, such as atopy and itch-related factors (eg, TSLP) and pro-inflammatory cytokines (eg, IL-6, IL-8 or IL-1α). it could be

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

Another aspect of the present invention provides a lysate of the Sphingomonas olei strain, a culture medium, an extract of the culture medium, or a mixture thereof.

Descriptions of the Sphingomonas olei strain, its lysate and culture are the same as described above.

A composition comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof according to an aspect of the present invention as an active ingredient enhances the recovery of damaged epidermis, increases the expression of skin barrier-related factors, and reduces the expression of inflammatory factors It can be usefully used to improve skin condition because

1 is a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis of the viability of human keratinocytes according to treatment with a culture solution of a strain according to an embodiment of the present invention. It is a graph showing the evaluation result.
2 is a result of evaluating the movement of human keratinocytes according to the treatment of the culture medium of the strain according to an embodiment of the present invention. (N-CNT (negative control) - negative control (0% FBS); P-CNT (positive control) - positive control (EGF 10 ng/mL); Media CNT (control), TSB medium)
3 is a graph showing the results of real-time PCR measurement of the change in loricrin mRNA expression in human keratinocytes according to the treatment of the culture medium of the strain according to an embodiment of the present invention. (None (Poly I:C+IL-4 untreated group) #; Poly I:C + IL-4 treated group *; #,* p<0.05;##,**p<0.001;###,***p<0.0001; red, significant negative values (decrease); blue, significant positive values (increase))
4 is a graph showing the results of real-time PCR measurement of changes in TSLP mRNA expression in human keratinocytes according to the treatment of the culture medium of the strain according to an embodiment of the present invention. (None (Poly I:C+IL-4 untreated group) #; Poly I:C + IL-4 treated group (negative control) *; #,* p<0.05;##,** p <0.001;###,***p<0.0001; red, significant negative values (decrease); blue, significant positive values (increase))
5 is a graph showing the results of real-time PCR measurement of changes in the expression of IL-6, IL-8 and IL-1α mRNA in human keratinocytes according to the treatment of the culture medium of the strain according to an embodiment of the present invention. Figure 5a is a graph showing the IL-6 mRNA expression results, Figure 5b is a graph showing the IL-8 mRNA expression results, Figure 5c is a graph showing the IL-1α mRNA expression results. (None (Poly I:C+IL-4 untreated group) #; Poly I:C + IL-4 treated group *; #,* p<0.05;##,**p<0.001;###,***p<0.0001; red, significant negative values (decrease); blue, significant positive values (increase))

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

Example 1. Isolation and identification of strains

A sample was collected from healthy Korean skin, put in a PBS (phosphate buffered saline) solution, mixed, and then inoculated into R2A (Reasoner's 2A agar) medium through serial dilution. Bacterial colonies formed on a plate were selected by culturing in an incubator at 30° C. for 139 hours, and pure separation was performed using a streaking method under the same medium and conditions. In order to confirm the molecular phylogenetic characteristics of the cultured strain, 16S rRNA gene sequencing was performed. PCR (Polymerase chain reaction) amplification was repeated 32 times at 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 1 minute and 45 seconds each, and finally, it was treated at 72° C. for 5 minutes. In this case, primers (SEQ ID NOs: 1 and 2) designed for a specific reaction of the bacterial common gene were used. The PCR amplification product was purified and the nucleotide sequence was confirmed (Macrogen, Korea), and the sequence was analyzed using the BLAST program of the National Center for Biotechnology Information (NCBI). When compared with other previously registered strains, a species having at least 99% nucleotide sequence homology was determined as a related species. The strain isolated in this example has a 16S rRNA sequence of SEQ ID NO: 3 (complementary DNA) and was identified as Sphingomonas olei . The present inventors named the strain Sphingomonas olei CBN003 and deposited it with the Agricultural Genetic Resources Center on September 10, 2021 and was given an accession number KACC 81169BP.

Example 2. Culture of the strain

The strain isolated in Example 1 was cultured in an incubator at 34° C. in TSB (Tryptic soy broth) medium, and the strain culture medium and cells were collected at the final time point of the division phase (Log phase) on the growth curve. The culture solution was prepared by centrifugation at 6,000 rpm for 30 minutes, followed by filtration sterilization using a 0.2 μm filter. When used for evaluation and activity analysis, it was carried out not more than 1 day after preparation.

Experimental Example 1. Cytotoxicity evaluation

It was confirmed whether the strain isolated in Example 1 was toxic to cells.

Specifically, first, a human epidermal keratinocyte cell line (HaCaT) was treated with DMEM medium (Dubelcco's modified eagle) containing 10% Fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin. medium) at 37 ° C. 5% CO ₂ Incubated in an incubator.

Cytotoxicity evaluation of the culture medium of the strain was performed through the MTT assay. The cultured cell line was aliquoted in a 96-well plate at 8Х10 ³ /well, and after culturing for 24 hours under the same conditions, the culture medium of the strain was 0 (TSB medium only), 1, 2, 5, 10% (v/ v) and further incubated for 24 hours under the same conditions. At this time, the 0% treatment group was used as a negative control, and the total volume was 100 μL per well. Thereafter, 20 μL of 5 mg/mL MTT was added to each cell culture well, and after additional incubation for 4 hours under the same conditions, 100 μL of DMSO was added to measure the absorbance at 590 nm, and the results are shown in FIG. indicated.

1 is a graph showing the results of evaluating the viability of human keratinocytes according to the treatment of the culture medium of the strain isolated in Example 1 by MTT analysis.

As shown in FIG. 1, when treated with 1, 2, 5, and 10% (v/v) strain culture, there is no significant difference in viability between the negative control and human keratinocytes not treated with the strain culture. It can be seen that the strain culture is not cytotoxic. These results mean that it is safe when the culture solution of the strain is applied to the skin.

Experimental Example 2. Analysis of skin regeneration efficacy activity

The effect of the strain isolated in Example 1 on skin regeneration efficacy was analyzed.

Specifically, first, a human epidermal keratinocyte cell line (HaCaT) was treated with DMEM medium (Dubelcco's modified eagle) containing 10% Fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin. medium) at 37 ° C. 5% CO ₂ Incubated in an incubator. The cultured cell line was aliquoted at 5Х10 ⁵ /well in a 6-well plate and cultured under the same conditions for 24 hours for cell adhesion. After making a straight scratch on the cell monolayer, the culture solution of the strain was treated with 0 (TSB medium only), 1, 2, 5, and 10% (v/v) in serum-free DMEM medium (Dubelcco's modified eagle medium) at 37 ° C. of 5% CO ₂ Incubated for 24 hours in an incubator. As a positive control, EGF (Epidermal growth factor) was treated with 10 ng/mL and cultured in the same manner. The scratch interval of 0 hours and the interval of 24 hours were observed under a microscope and comparatively analyzed, and the results are shown in FIG. 2 .

Figure 2 shows the results of evaluating the migration of human keratinocytes according to the treatment of the culture medium of the strain isolated in Example 1. (N-CNT, negative control (0% FBS); P-CNT, positive control (EGF 10 ng/mL); Media CNT, TSB medium)

As shown in FIG. 2, it was confirmed that the keratinocytes actively migrated and the scratch interval was remarkably reduced when cultured for 24 hours compared to the scratch interval of 0 hours as a whole when the culture solution of the strain isolated in Example 1 was treated. These results suggest that the strain can be usefully used for skin regeneration and wound healing.

Experimental Example 3. Skin barrier strengthening and moisturizing activity analysis

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

Specifically, the expression of loricrin, a marker of skin barrier function, was analyzed. First, a human epidermal keratinocyte cell line (HaCaT) was differentiated. Cell lines were aliquoted in a 6-well plate at 5Х10 ⁵ /well, and cultured in a serum-free medium containing low-concentration calcium 0.09 mM, epidermal growth factor (EGF), and bovine pituirary extract (BPE) at 37°C in 5% CO ₂ incubator for 2 days. to induce differentiation. Then, in a serum-free medium containing high concentration of calcium 2.8 mM, EGF (Epidermal growth factor), and BPE (Bovine pituirary extract), it was cultured for 7 days in a 5% CO ₂ incubator at 37°C, and the medium was replaced every 2-3 days. did. An inflammatory response was induced by adding Poly I:C 10 μg/mL and IL-4 50 ng/mL to the cell line differentiated through the above process. After culturing for 24 hours in an incubator under the same conditions, culture medium 0 (TSB medium only) and 10% (v/v) of the strain were treated and further cultured for 24 hours. An untreated group (only Poly I:C+IL-4 treatment) was used as a negative control group, and a 1 μM dexamethasone treatment group was used as a positive control group. Thereafter, RNA was extracted from each sample-treated cell using TRIZOL (Invitrogen, USA), and RNA was quantified using a nanospectrophotometer, and cDNA was synthesized in an amplifier using 1 μg of each RNA. Using the synthesized cDNA as a template, real-time PCR was performed by adding SYBR Green (Thermo Fisher Scientific, USA) together with a primer to the target gene. In this case, the target gene is a skin barrier function marker. Lorricrin gene was used, and the expression level of the gene was finally analyzed through correction for GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) gene, and the results are shown in FIG. 3 .

3 is a graph showing the results of real-time PCR measurement of changes in Loricrin (LOR) mRNA expression in human keratinocytes according to the treatment with the culture medium of the strain isolated in Example 1. FIG. (None (Poly I:C+IL-4 untreated group) #; Poly I:C + IL-4 treated group *; #,* p<0.05; ##,** p<0.001; # ##,*** p<0.0001; red, significant negative values (decrease); blue, significant positive values (increase))

As shown in FIG. 3 , when the 10% culture solution of the strain isolated in Example 1 was treated, the expression of loricrin was significantly increased compared to the negative control. These results suggest that the strain can be usefully used for strengthening the skin barrier and moisturizing the skin.

Experimental Example 4. Anti-inflammatory activity analysis

In order to analyze the effect of the strain isolated in Example 1 on the anti-inflammatory activity, the expression of TSLP and the pro-inflammatory cytokines IL-6, IL-8 and IL-1α were analyzed.

Specifically, a human epidermal keratinocyte cell line (HaCaT) was first differentiated. The cell line was aliquoted at 5Х10 ⁵ /well in a 6-well plate, and cultured in a serum-free medium containing low-concentration calcium 0.09 mM, EGF (Epidermal growth factor) and BPE (Bovine pituirary extract) at 37°C in 5% CO ₂ incubator for 2 days. to induce differentiation. Then, in a serum-free medium containing high concentration of calcium 2.8 mM, EGF (Epidermal growth factor), and BPE (Bovine pituirary extract), in a 5% CO ₂ incubator at 37° C. for 7 days, and the medium was replaced every 2-3 days. did. An inflammatory response was induced by adding Poly I:C 10 μg/mL and IL-4 50 ng/mL to the cell line differentiated through the above process. After culturing for 24 hours in an incubator under the same conditions, culture medium 0 (TSB medium only) and 10% (v/v) of the strain were treated and further cultured for 24 hours. An untreated group (only Poly I:C+IL-4 treatment) was used as a negative control, and a 1 μM dexamethasone treatment group was used as a positive control. Thereafter, RNA was extracted from each sample-treated cell using TRIZOL (Invitrogen, USA), and RNA was quantified using a nanospectrophotometer, and cDNA was synthesized in an amplifier using 1 μg of each RNA. Using the synthesized cDNA as a template, SYBR Green (Thermo Fisher Scientific, USA) was added with a primer to the target gene, and real-time PCR was performed. At this time, the target gene is the TSLP gene and the pro-inflammatory The cytokine IL-6, IL-8 and IL-1α genes were used, and the expression level of the gene was finally analyzed through correction for the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, and the results are shown in FIGS. 4 and 5 shown in

4 is a graph showing the results of real-time PCR measurement of changes in TSLP mRNA expression in human keratinocytes according to the treatment with the culture medium of the strain isolated in Example 1. FIG. (None (Poly I:C+IL-4 untreated group) #; Poly I:C + IL-4 treated group (negative control) *; #,* p<0.05; ##,** p <0.001; ###,*** p<0.0001; red, significant negative (decrease); blue, significant positive (increase))

5 is a graph showing the results of real-time PCR measurement of changes in the expression of IL-6, IL-8 and IL-1α mRNA in human keratinocytes according to the treatment with the culture solution of the strain isolated in Example 1. FIG. Figure 5a is a graph showing the IL-6 mRNA expression results, Figure 5b is a graph showing the IL-8 mRNA expression results, Figure 5c is a graph showing the IL-1α mRNA expression results. (None (Poly I:C+IL-4 untreated group) #; Poly I:C + IL-4 treated group *; #,* p<0.05; ##,** p<0.001; # ##,*** p<0.0001; red, significant negative values (decrease); blue, significant positive values (increase))

As shown in FIG. 4 , when the 10% culture solution of the strain isolated in Example 1 was treated, the expression level of TSLP was significantly reduced compared to the negative control.

In addition, as shown in FIG. 5, when the 10% culture solution of the strain isolated in Example 1 was treated, the expression levels of IL-6, IL-8 and IL-1α were significantly reduced compared to the negative control, and in particular, IL-6 and IL-1α showed a significant decrease in expression at a level similar to that of the positive control.

The above results suggest that the strain can be usefully used for the prevention, improvement and treatment of skin inflammatory diseases.

Agricultural Genetic Resource Center (Overseas) KACC81169BP 20210910

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Claims (12)

Sphingomonas olei ( Sphingomonas olei ), its lysate (lysate), a culture solution, or a cosmetic composition for improving skin condition comprising a mixture thereof as an active ingredient. The cosmetic composition for improving skin condition according to claim 1, wherein the skin condition improvement is at least one selected from the group consisting of skin barrier improvement, skin moisturizing, skin immunity improvement, skin defense enhancement, skin inflammation suppression, skin soothing and skin regeneration. The method according to claim 1, Sphingomonas olei, a lysate thereof, a culture medium or a mixture thereof promotes the migration of keratinocytes, increases the expression of Loricrin, or TSLP (Thymic stromal lymphopoietin); A cosmetic composition for improving skin condition, which reduces the expression of one or more selected from the group consisting of IL (interleukin)-6, IL-8 and IL-1α. The cosmetic composition for improving skin condition according to claim 1, wherein Sphingomonas olei is a Sphingomonas olei strain deposited with accession number KACC 81169BP. A quasi-drug composition for improving skin condition, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient. A composition for external application for skin for improving skin condition, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient. A health functional food composition for improving skin condition, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient. A pharmaceutical composition for preventing or treating inflammatory skin diseases, or for treating or improving skin wounds, comprising Sphingomonas olei, a lysate thereof, a culture medium, or a mixture thereof as an active ingredient. The method according to claim 8, wherein the inflammatory skin disease is dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin disease, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, rash, and acne. Any one selected from the group consisting of, the pharmaceutical composition. Sphingomonas olei strain deposited with accession number KACC 81169BP. A lysate of the strain according to claim 10 . The culture solution of the strain according to claim 10.

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