Composition for improving skin condition, novel sphingomonas oleosphingomonas strain, and lysate, culture solution and extract of novel sphingomonas oleosphingomonas strain

Abstract

The present invention relates to the use of Sphingomonas oleii for improving skin conditions, in which a composition comprising the Sphingomonas oleii or a lysate, a culture medium or an extract thereof as an active ingredient accelerates the repair of damaged epidermis, increases the expression of skin barrier-related factors, reduces the expression of inflammatory factors, and improves the skin conditions. Therefore, the composition is effectively used for improving skin conditions.

Description

Technical Field

[0001] The present invention relates to the use of Sphingomonas olei for improving skin conditions. More specifically, the present invention relates to a composition comprising Sphingomonas olei or a lysate, culture fluid, or extract thereof as an active ingredient, for improving skin conditions by accelerating the repair of damaged epidermis, increasing the expression of skin barrier-related factors, and reducing the expression of inflammatory factors. Background Art

[0002] Because the human skin surface has a large amount of metabolic waste such as keratin, sweat, and sebum, various microorganisms inhabit and form a skin microbiome (skin microbiome), that is, the skin's resident flora. In the skin's resident flora, beneficial bacteria and harmful bacteria coexist. The healthier the skin, the more beneficial bacteria are distributed, while in the case of atopic skin or acne skin, the more harmful bacteria are distributed. Therefore, it is well known that maintaining the balance of the resident bacteria present in the skin helps maintain a healthy skin state. However, there is still little research on the effects of these skin-resident bacteria on the skin.

[0003] Normally, skin-resident bacteria, which reside on the skin, form a skin barrier, preventing the invasion of more harmful microorganisms (pathogens) and contributing to the skin's immune function. Since beneficial skin bacteria do not cause disease or stimulate the immune system, if beneficial skin bacteria could be used in the production of pharmaceuticals or cosmetics, they could be used safely without side effects such as skin irritation.

[0004] When a wound or infection develops on the skin, the skin tissue becomes damaged or inflamed. If the damage is not severe, the skin typically heals on its own. The present invention was completed through research aimed at developing skin-resident bacteria that contribute to skin regeneration and repair, as well as the healing process of inflammation. This led to the identification of beneficial skin bacteria that can improve skin conditions through skin regeneration and inflammation healing. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] Based on this, the present invention provides a cosmetic material composition for improving skin condition, which contains Sphingomonas oleifera or its lysate, culture solution or extract as an active ingredient.

[0007] The present invention provides a quasi-drug composition for improving skin condition, comprising Sphingomonas oleifera or its lysate, culture solution or extract as an active ingredient.

[0008] The present invention provides a skin external preparation composition for improving skin condition, which contains Sphingomonas oleifera or its lysate, culture solution or extract as an active ingredient.

[0009] The present invention provides a health functional food composition for improving skin condition, which comprises Sphingomonas oleifera or its lysate, culture solution or extract as an effective ingredient.

[0010] The present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases or treating or improving skin wounds, comprising Sphingomonas oleifera or its lysate, culture fluid or extract and a pharmaceutically acceptable carrier.

[0011] The invention provides a Sphingomonas oleifera strain preserved with the preservation number KACC 81169BP.

[0012] The invention provides a lysate, a culture solution, an extract or a mixture thereof of a Sphingomonas oleifera strain.

[0013] Means used to solve problems

[0014] In one embodiment, the present invention provides a cosmetic material composition for improving skin condition, comprising Sphingomonas oleifera or a lysate, culture solution, or extract thereof as an active ingredient.

[0015] In the present invention, Sphingomonas oleifera may be a live bacterium (alive bacteria) or a dead bacterium (dead bacteria). More specifically, the dead bacterium may be a bacterium killed by heat treatment.

[0016] In one embodiment, the Sphingomonas oleae may be a Sphingomonas oleae strain deposited under the accession number KACC 81169BP.

[0017] As used herein, the term "lysate" refers to a solution or suspension in an aqueous culture medium of cells of a microorganism, such as lysed Sphingomonas oleifera. Cell lysates include macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids, and / or small molecules such as amino acids, sugars, fatty acids, or their fractions. In addition, lysates also include cell debris, which may be smooth or granular in structure.

[0018] As methods for achieving cell lysis of microorganisms, various well-known methods can be used, and any method capable of achieving cell lysis of microorganisms can be used. For example, the opening / destruction of cells can be performed enzymatically, chemically, or physically. Non-limiting examples of enzymes and enzyme mixtures include proteases such as proteinase K, lipases, or glycosidases; non-limiting examples of chemical substances include ion carriers, detergents such as sodium lauryl sulfate, acids, or bases; non-limiting examples of physical means include high pressure such as French pressing, osmotic pressure, and temperatures such as heat or cold. In addition, methods using enzymes other than proteolytic enzymes, acids, and bases can also be appropriately combined.

[0019] The term "culture fluid" as used herein may be used interchangeably with "culture supernatant," "conditioned culture fluid," or "conditioned medium," and may refer to a whole culture medium comprising microorganisms, metabolites, and residual nutrients obtained by culturing Sphingomonas oleifera in a culture medium for a period of time, wherein the culture medium for culturing Sphingomonas oleifera is a culture medium that can provide nutrients so that Sphingomonas oleifera can grow and survive in vitro. In addition, the culture fluid may also refer to a culture fluid obtained by culturing microorganisms, wherein the cells are removed from the cell culture fluid obtained by culturing the microorganisms. On the other hand, the liquid from which the cells are removed from the culture medium is also referred to as the "supernatant," which may be obtained by taking only the supernatant liquid excluding the portion that sinks to the lower layer by allowing the culture fluid to stand for a period of time; or removing the cells by filtration; or removing the lower sediment by centrifuging the culture fluid and then taking only the supernatant liquid. The "cells" refer to the microbial bodies of the present invention, including the microbial bodies screened out by separation from skin samples, etc., or microorganisms separated from the culture fluid after culturing the microorganisms. The bacteria can be obtained by taking the portion that sinks to the lower layer after centrifugation; or, since the bacteria will sink to the lower layer of the culture solution due to gravity, they can be obtained by standing for a period of time and then removing the upper liquid.

[0020] In one embodiment, the culture of Sphingomonas oleifera of the present invention can use a culture medium that can be easily selected by those skilled in the art from a culture medium for culturing microorganisms according to the purpose. Specifically, a culture medium for culturing Sphingomonas can be used, such as Reinforced Clostridium Medium (RCM), Tryptic Soy Broth (TSB), or Brain Heart Infusion (BHI) medium, but is not limited thereto. According to one embodiment, the culture of Sphingomonas oleifera of the present invention is prepared by inoculating Sphingomonas oleifera into a microbial culture medium and using a microbial culture method known in the art (e.g., static culture, etc.).

[0021] The above-mentioned culture solution may include a culture solution obtained by culturing microorganisms, a concentrate or a lyophilized product thereof, or a culture supernatant obtained by removing microorganisms from a culture solution, a concentrate or a lyophilized product thereof.

[0022] The culture solution can be obtained by culturing Sphingomonas oleae in a suitable culture medium (eg, R2A medium or TSB medium) at any temperature between 10°C and 40°C for a certain period of time (eg, 4 to 50 hours).

[0023] In one embodiment, the culture supernatant of the microorganisms can be obtained by centrifuging or filtering the microorganism culture solution to remove the microorganisms.

[0024] In another embodiment, the concentrate can be obtained by concentrating a microorganism culture solution or a supernatant obtained by centrifuging or filtering the culture solution.

[0025] The culture medium and culture conditions for culturing the Sphingomonas oleifera can be appropriately selected or modified by those skilled in the art.

[0026] The term "extract" used herein means a substance extracted from a lysate, a culture solution or a concentrate thereof, and may be an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a crude extract or purified product of these, or a fraction obtained by fractionating them.

[0027] Sphingomonas oleifera or its lysate, culture fluid or extract has a skin condition improving effect, for example, one or more skin condition improving effects selected from the group consisting of improving skin barrier, skin moisturizing, improving skin immunity, enhancing skin resistance, inhibiting skin inflammation, calming skin and skin regeneration.

[0028] The term "improving skin barrier" as used herein refers to the meaning including strengthening skin barrier or protective function, wherein, skin barrier (skin barrier) is the outermost layer of the epidermis, i.e., stratum corneum (stratum corneum), which is mainly composed of anucleated flat keratinocytes (corneocyte). The multilayer lipid membrane (multilamella lipid layer) formed by the intercellular lipids such as ceramide, cholesterol and fatty acids synthesized by the keratinocytes of the skin barrier maintained by the division and differentiation process of normal epidermal cells plays the role of a protective barrier to prevent skin moisture evaporation. This improving skin barrier means alleviating the skin diseases (such as psoriasis, contact dermatitis, eczematous dermatitis, photosensitive dermatitis, seborrheic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus, pyoderma gangrenosum, pemphigus, epidermolysis bullosa, systemic sclerosis or leprosy) caused by weakening skin barrier function, or improving all effects of damaged skin barrier function.

[0029] As used herein, the term "skin moisturizing" refers to all actions that retain moisture in the skin or prevent moisture loss.

[0030] The term "skin regeneration" as used herein means all cases where the skin is regenerated, including not only regeneration of the skin due to aging but also cases where the epidermis damaged by scratches or the like is healed.

[0031] The skin inflammation is any one selected from the group consisting of dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin diseases, pruritus, eczematous skin diseases, dry eczema, erythema, urticaria, psoriasis, drug eruption and acne.

[0032] Sphingomonas oleifera or its lysate, culture fluid or extract promotes the migration of keratinocytes on the surface of damaged skin to accelerate the repair of damaged epidermis.

[0033] Sphingomonas oleifera or its lysate, culture fluid or extract can increase the expression of skin barrier-related factors (such as loricrin, a protein required for keratin formation).

[0034] In addition, Sphingomonas oleifera or its lysate, culture medium or extract can reduce the expression of inflammatory factors, such as atopy and pruritus-related factors (such as thymicstromal lymphopoietin (TSLP)) and proinflammatory cytokines (such as IL-6, IL-8 or IL-1α).

[0035] In one embodiment, Sphingomonas oleifera or its lysate, culture fluid or extract can promote the migration of keratinocytes, or increase the expression of loricrin, or reduce the expression of one or more selected from the group consisting of TSLP, IL-6, IL-8 and IL-1α.

[0036] Relative to the total weight, the present invention may include 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%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to % to 40wt%, 0.05wt% to 30wt%, 0.05wt% to 20wt%, 0.05wt% to 10wt%, 0.05wt% to 5wt%, 0.1wt% to 60wt%, 0.1wt% to 40wt%, 0.1wt% to 30wt%, 0.1wt% to 20wt%, 0.1wt% to 10wt% or 0.1wt% to 5wt% of microorganisms, or their lysates, culture solutions or extracts.

[0037] The term "comprising... as an effective ingredient" as used herein means adding a microorganism or its lysate, culture fluid or extract to an extent that can exhibit the effect, and also includes various forms of formulations in which various ingredients are added as auxiliary ingredients for drug delivery and stabilization, etc.

[0038] The above-mentioned cosmetic material composition can be prepared into the dosage form including lotion (skin lotion), emollient (skin), skin softener (skin softener), skin toner (skin toner), astringent (astringent), lotion (lotion), milk lotion (milk lotion), moisturizing lotion (moisture lotion), nutrient emulsion, massage cream, nutrient cream, moisturizing cream, hand cream, hand cleanser, foundation, essence, nutrient essence, facial mask (pack), soap, cleansing foam, cleansing lotion, cleansing cream, body milk, shower gel, suspension, gel, powder, paste, patch-type facial mask (mask pack) and tablet. The composition of this dosage form can be prepared according to conventional methods in this area. Those skilled in the art can easily select the mixing amount of additional ingredients such as the above-mentioned wetting agent within the scope of not damaging the purpose and effect of the present invention.

[0039] In addition to the active ingredients disclosed herein, the cosmetic material composition may further include functional additives and ingredients found in conventional cosmetic material compositions, including commonly used purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, fragrances, or combinations thereof. The functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular-weight peptides, high-molecular-weight polysaccharides, sphingolipids, and seaweed extracts. Examples of carriers include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, UV scatterers, UV absorbers, colorants, and fragrances. Compounds / compositions used for the alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, UV scatterers, UV absorbers, colorants, and fragrances are well known in the art, and those skilled in the art can select and use appropriate corresponding substances / compositions. In addition, the cosmetic material composition may also contain, as needed, UV blockers, antioxidants (butylated hydroxyanisole, propyl gallate, isoascorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben, propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, disodium hydrogen phosphate, etc.), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methyl gluceth-20, etc.), lubricants, etc.

[0040] In addition, in various dosage forms of cosmetic material compositions, appropriate ingredients can be selected and mixed according to the dosage form or purpose of use of the cosmetic material. Since the mixing ingredients and methods can follow conventional techniques, their detailed description will be omitted in the present invention.

[0041] In another embodiment, the present invention provides a quasi-drug composition for improving skin condition, comprising Sphingomonas oleifera or a lysate, culture solution or extract thereof as an active ingredient.

[0042] Among them, Sphingomonas oleifera or its lysate, culture fluid or extract, and its skin condition improving effect are the same as described above.

[0043] As used herein, the term "quasi-drug" refers to articles used to diagnose, treat, ameliorate, alleviate, cure, or prevent human or animal diseases that have milder effects than pharmaceuticals. For example, under the Pharmaceutical Affairs Act, quasi-drugs include articles other than pharmaceuticals, including fiber / rubber products used to treat or prevent human and animal diseases, articles that have minimal or no direct effect on the human body and are not instruments or machines, and the like, as well as disinfectants and pesticides used to prevent infectious diseases.

[0044] The type or dosage form of the quasi-drug composition of the present invention is not particularly limited, but it can be a bandage, gauze, cotton wool, adhesive bandage, disinfectant cleanser, bath foam, mouthwash, wet wipes, laundry soap, hand soap, humidifier refill, face mask or filter refill, etc.

[0045] When the composition of the present invention is incorporated into a quasi-drug for the purpose of improving skin conditions, the composition itself may be incorporated into the quasi-drug or used in combination with other quasi-drug ingredients. These may be used appropriately according to conventional methods. The amount of active ingredient incorporated can be appropriately determined depending on the intended use. The quasi-drug composition of the present invention may contain 0.01 wt% to 20 wt% of a microorganism, its lysate, culture fluid, or mixture thereof, relative to the total weight of the composition.

[0046] In another embodiment, the present invention provides a skin external application composition for improving skin condition, comprising Sphingomonas oleifera or a lysate, culture solution or extract thereof as an active ingredient.

[0047] Among them, Sphingomonas oleifera or its dissolved product, culture fluid or extract and its skin condition improving effect are the same as described above.

[0048] The skin preparation for external use may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, an emulsion, or a combination thereof. The skin preparation for external use may be mixed as needed with ingredients used in skin preparations for external use such as common cosmetics or pharmaceuticals (e.g., aqueous ingredients, oily ingredients, powdered ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or a combination thereof). The skin external preparation can also be appropriately mixed with metal blocking agents (disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.), drugs (caffeine, tannin, verapamil, licorice extract, glycyrrhizin, hot water extract of Cephalotaxus fasciculatus fruit, various crude drugs, tocopheryl acetate, glycyrrhizic acid, tranexamic acid and its derivatives or their salts, etc.), sugars (vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.), etc.

[0049] The external skin preparation composition according to the present invention may contain 0.00001 wt % to 80 wt % of the microorganism, a lysate thereof, a culture solution thereof, or a mixture thereof, relative to the total weight of the composition.

[0050] The aforementioned skin includes all skin areas of the body including the face, hands, arms, legs, feet, chest, abdomen, back, buttocks and scalp.

[0051] In another embodiment, the present invention provides a health functional food composition for improving skin condition, comprising Sphingomonas oleifera or its lysate, culture solution or extract as an active ingredient.

[0052] Among them, Sphingomonas oleifera or its dissolved product, culture fluid or extract and its skin condition improving effect are the same as described above.

[0053] The above-mentioned health functional food composition can use Sphingomonas oleifera or its culture solution alone, or can be used together with other foods or food ingredients, or can be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health care or therapeutic treatment). Usually, when manufacturing food or beverages, the composition of the present application specification sheet below 15 parts by weight can be added relative to the raw materials.

[0054] There are no particular restrictions on the types of health functional foods described above. Examples of foods to which the above substances can be added include dosage forms selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, tea extracts, and health beverages, and include all conventional health foods. Like conventional beverages, beverage compositions within the health functional food category may contain various flavorings or natural carbohydrates as additional ingredients.

[0055] The above-mentioned natural carbohydrates can be 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 sweeteners, natural sweeteners (such as thaumatin and stevia extract) and synthetic sweeteners (such as saccharin and aspartame) can be used. The health food composition may also contain: nutrients, vitamins, electrolytes, flavorings, colorants, pectin acid and its salts; alginic acid and its salts; organic acids; protective colloid thickeners; pH regulators; stabilizers; preservatives; glycerol; alcohol; carbonating agents for carbonated beverages, or combinations thereof. The health functional food composition may also contain fruit pulp used to make natural fruit juices, fruit juice beverages, or vegetable beverages, or combinations thereof.

[0056] In another embodiment, the present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases or treating or improving skin wounds, comprising Sphingomonas oleifera or its lysate, culture fluid or extract and a pharmaceutically acceptable carrier as active ingredients.

[0057] The descriptions of Sphingomonas oleifera or its lysate, culture medium and extract are the same as those described above.

[0058] The inflammatory skin disease may be any one 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, drug eruption and acne.

[0059] Skin wounds are also called "wounds," meaning injuries to a living body. These injuries can be caused by, but are not limited to, external factors (e.g., physical damage, radiation, irritation from chemicals, microbial proliferation, etc.) or internal factors (e.g., pressure). These wounds can include all types of wounds, including incisions, puncture wounds, cuts, contusions, lacerations, gunshot wounds, and bite wounds. "Wound treatment" can refer to a series of biological reactions that repair wounds. "Wound improvement" can refer to any action that alleviates or reduces the severity of wounds on the face or body.

[0060] As used herein, the term "pharmaceutical composition" may refer to a molecule or compound that confers some beneficial effect when administered to a subject. Beneficial effects can include: enabling a diagnostic decision; amelioration of a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally managing a disease, symptom, disorder, or condition.

[0061] As used herein, the term "prevention" refers to partially or completely delaying or preventing the onset or recurrence of a condition, disorder, or its attendant symptoms; preventing the acquisition or reacquisition of a condition or disorder; or reducing the risk of acquiring a condition or disorder. For example, the above-mentioned prevention refers to all actions that inhibit or delay the occurrence of skin damage or symptoms by administering a composition according to the present invention.

[0062] As used herein, the term "treating" includes inhibiting, alleviating or eliminating the progression of a disease.

[0063] As used herein, the term "improvement" may mean all actions that at least reduce a parameter associated with the alleviation or treatment of a condition (eg, the extent of a symptom).

[0064] The above-mentioned pharmaceutical composition can be administered in a non-oral manner during clinical administration and can be used in the form of conventional pharmaceutical preparations. Non-oral administration can mean administration by a route other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal (Nasal), inhalation, ocular and subcutaneous administration. When the pharmaceutical composition of the present invention is used for pharmaceuticals, it can further include one or more active ingredients with the same or similar functions.

[0065] The above-mentioned pharmaceutical composition can further comprise one or more pharmaceutically acceptable carriers to prepare.Pharmaceutically acceptable carriers can be mixed with one or more of saline, sterile water, Ringer's solution, buffered saline, glucose solution (dextrose solution), maltodextrin solution, glycerol, ethanol and these compositions, and other conventional additives (such as antioxidants, buffer, antibacterial agents, etc.) can be added as needed. In addition, diluents, dispersants, surfactants, adhesives and lubricants can also be added to be formulated into injection dosage forms (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules or tablets. Further, it is possible to preferably carry out formulation according to each disease or composition by the appropriate method in this area.

[0066] When the above-mentioned pharmaceutical composition is prepared, diluents or excipients such as commonly used fillers, extenders, adhesives, wetting agents, disintegrants, surfactants can be utilized to modulate. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable esters (such as ethyl oleate) etc. can be used as non-aqueous solvents and suspensions. As the matrix of suppositories, semi-synthetic fatty acid esters (Witepsol), polyethylene glycol (Macrogol), Tween 61, cocoa butter, lauric glyceride (Laurin oil), glycerin gelatin etc. can be used.

[0067] In order to improve the stability and absorbability of the above-mentioned pharmaceutical composition, carbohydrates (such as glucose, sucrose or dextrose), antioxidants (such as ascorbic acid or glutathione), chelating agents and low molecular weight proteins or other stabilizers can be used as agents.

[0068] In another embodiment, the present invention provides a method for preventing, improving or treating a condition in an individual, comprising the step of administering the pharmaceutical composition to the individual.

[0069] The above-mentioned individual condition may be a skin-related condition or an inflammation-related condition.

[0070] Administration can be carried out by methods known in the art. It can be directly applied to an individual by any means, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The above-mentioned administration can be systemic or local administration.

[0071] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat or cat. The subject may be a subject in need of improving skin condition or treating inflammatory skin diseases.

[0072] The above-mentioned administration may be to administer 0.00001 mg to 1000 mg of the composition according to the present invention to each individual 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 1000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg or 10 mg to 25 mg of the composition according to the present invention may be administered. However, the dosage can be prescribed in various ways according to factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art can appropriately adjust the dosage in consideration of these factors. The number of administrations may be once a day, or more than twice within the range of clinically acceptable side effects; in terms of the administration site, the administration may be performed at 1 or more than 2 sites; in terms of the number of administration days, the administration may be performed every day or at intervals of 2 to 5 days, within the range of 1 to 30 days. If necessary, the same treatment may be repeated after an appropriate time. For animals other than humans, the administration may be performed at the same dosage per kg as for humans, or the administration may be performed at a converted dosage, such as an amount converted based on the volume ratio (such as the average value) of the target animal's organ (heart, etc.) to that of a human.

[0073] In another embodiment, the present invention provides a Sphingomonas oleifera strain deposited under the accession number KACC 81169BP.

[0074] The Sphingomonas oleae strain may be a strain comprising the 16S rRNA of SEQ ID NO: 3.

[0075] The above strains may have skin condition improving effects, for example, having one or more skin condition improving effects selected from the group consisting of improving skin barrier, skin moisturizing, improving skin immunity, enhancing skin resistance, inhibiting skin inflammation, calming skin and skin regeneration.

[0076] The above strains can promote the migration of keratinocytes on the surface of damaged skin, thereby accelerating the repair of damaged epidermis. The above strains can increase the expression of skin barrier-related factors (such as loricrin). In addition, the above strains can also reduce the expression of inflammatory factors, such as atopy and itch-related factors (such as TSLP) and proinflammatory cytokines (such as IL-6, IL-8 or IL-1α).

[0077] In another embodiment, the present invention provides a lysate, culture solution, extract or a mixture thereof of a Sphingomonas oleifera strain.

[0078] The descriptions of the Sphingomonas oleifera strain, its lysate, culture solution and extract are the same as those described above.

[0079] Effects of the Invention

[0080] According to one embodiment of the present invention, the composition comprising Sphingomonas oleifera, its dissolved product, culture fluid or extract as an active ingredient has the effects of accelerating the repair of damaged epidermis, increasing the expression of skin barrier-related factors, and reducing the expression of inflammatory factors, and can therefore be effectively used to improve skin condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The graph shows the results of evaluating the viability of human keratinocytes treated with the culture medium of the strain according to one embodiment of the present invention using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis.

[0082] Figure 2 The results are as follows: evaluation of the migration of human keratinocytes treated with the culture medium of the strain according to one embodiment of the present invention (N-CNT (negative control group) - negative control group (0% FBS); P-CNT (positive control group) - positive control group (EGF 10 ng / mL); Media CNT (control group), TSB medium).

[0083] Figure 3 : is a graph showing the results of measuring the changes in loricrin mRNA expression in human keratinocytes treated with the culture medium of the strain of one embodiment of the present invention by real-time PCR (relative to the blank group (None) (Poly I:C + IL-4 untreated group) is #; relative to the Poly I:C + IL-4 treated group is *; #, *p<0.05; ##, **p<0.001; ###, ***p<0.0001; significant negative values ​​(decrease) or significant positive values ​​(increase) are marked in the Figure 3 ).

[0084] Figure 4 Graph showing the results of real-time PCR measurement of TSLP mRNA expression changes in human keratinocytes treated with a culture medium containing a strain according to an embodiment of the present invention (# relative to the blank group (None) (Poly I:C + IL-4 untreated group); * relative to the Poly I:C + IL-4 treated group (negative control); #, *p<0.05; ##, **p<0.001; ###, ***p<0.0001; significant negative values ​​(decrease) or significant positive values ​​(increase) are indicated in the Figure 4 ).

[0085] Figure 5 The graph shows the results of real-time PCR measurement of changes in IL-6, IL-8, and IL-1α mRNA expression in human keratinocytes treated with a culture medium containing the strain of one example of the present invention. Figure 5 a is a graph showing the results of IL-6 mRNA expression, Figure 5 b is a graph showing the results of IL-8 mRNA expression, Figure 5 Figure c shows the results of IL-1α mRNA expression (# relative to the blank group (Poly I:C + IL-4 untreated group); * relative to the Poly I:C + IL-4 treated group; #, *p<0.05; ##, **p<0.001; ###, ***p<0.0001; significant negative (decrease) or significant positive (increase) values ​​are marked in Figure 5 ). DETAILED DESCRIPTION

[0086] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only used to exemplify the present invention, and the scope of the present invention is not limited to these examples.

[0087] Example 1. Isolation and identification of strains

[0088] Skin samples were collected from healthy Korean individuals, mixed in phosphate-buffered saline (PBS), and then inoculated into R2A (Reasoner's 2A agar) medium via serial dilution. Bacterial colonies formed on plates after 139 hours of culture at 30°C were screened and purified using the streaking method using the same medium and conditions. To confirm the molecular phylogenetics of the cultured strains, 16S rRNA gene base sequence analysis was performed. PCR (polymerase chain reaction) amplification was performed by repeating 32 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 45 seconds, followed by a final treatment at 72°C for 5 minutes. Primers designed to specifically target common bacterial genes (SEQ ID NOs: 1 and 2) were used. The PCR amplification product was purified and the base sequence was confirmed (Macrogen, South Korea), and the sequence was analyzed using the BLAST program of the National Center for Biotechnology Information (NCBI). Compared with other previously registered strains, species with more than 99% base sequence homology were identified as closely related species. The strain isolated in this embodiment has a 16S rRNA sequence of SEQ ID NO:3 (complementary DNA), which is identified as Sphingomonas olei. The inventors named the strain Sphingomonas olei CBN003 (Sphingomonas olei CBN003), which was deposited in the Agricultural Genetic Resources Center on September 10, 2021, and the deposit number obtained is KACC 81169BP.

[0089] Example 2: Cultivation of strains

[0090] The strain isolated in Example 1 was cultured in Tryptic Soy Broth (TSB) in a 34°C incubator. The culture fluid and bacterial cells were collected at the final time point of the division phase (logarithmic phase) on the growth curve. The culture fluid was prepared by centrifugation at 6000 rpm for 30 minutes, followed by filtration and sterilization using a 0.2 μm filter. When used for evaluation and activity analysis, evaluation and activity analysis were performed no later than one day after preparation.

[0091] Experimental Example 1: Cytotoxicity Evaluation

[0092] It was confirmed whether the strain isolated in Example 1 was toxic to cells.

[0093] Specifically, first, human epidermal keratinocyte cell line (HaCaT) was cultured in a 37°C 5% CO2 incubator using Dubelco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL penicillin.

[0094] The cytotoxicity of the culture medium of the strain was evaluated by MTT assay. 3 / well were divided into 96-well plates, and then after culturing for 24 hours under the same conditions, the culture fluid of the above strains was treated with 0 (treated with TSB medium only), 1, 2, 5, and 10% (v / v) concentrations, and continued to be cultured for 24 hours under the same conditions. At this time, the 0% treatment group was used as a negative control group, and the total volume of each well was 100 μL. Subsequently, 20 μL of 5 mg / mL MTT was added to each cell culture well, and after continuing to culture for 4 hours under the same conditions, 100 μL of DMSO was added, and the absorbance at 590 nm was measured, and the results are shown in Figure 1 middle.

[0095] Figure 1 This figure shows the results of evaluating the viability of human keratinocytes treated with the culture medium of the strain isolated in Example 1 by MTT analysis.

[0096] like Figure 1 As shown, when the culture fluid of the strain was treated at 1, 2, 5, and 10% (v / v), there was no significant difference in the viability of human keratinocytes compared to the negative control group that was not treated with the culture fluid of the strain, indicating that the culture fluid of the strain was non-cytotoxic. This result indicates that the culture fluid of the strain is safe for application to the skin.

[0097] Experimental Example 2: Analysis of skin regeneration activity

[0098] The effects of the strains isolated in Example 1 on skin regeneration function were analyzed.

[0099] Specifically, first, human epidermal keratinocyte cell line (HaCaT) was cultured in Dubelco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL penicillin in a 5% CO2 incubator at 37°C. The cultured cell line was cultured at 5×10 5 / well were distributed in 6-well plates, and then cultured under the same conditions for 24 hours in order to allow the cells to attach. After a straight scratch was made on the monolayer of cells, serum-free DMEM medium (Dubelcco's modified eagle medium) containing the culture solution of the strain at 0 (TSB medium only), 1, 2, 5, 10% (v / v) was used to culture in a 5% CO2 incubator at 37°C for 24 hours. As a positive control group, epidermal growth factor (EGF) was treated with 10 ng / mL and cultured in the same way. The scratch interval of 0 hours and the interval of 24 hours were observed and compared under a microscope, and the results are shown in Figure 2 middle.

[0100] Figure 2 The figure shows the results of evaluating the migration of human keratinocytes treated with the culture medium of the strain isolated in Example 1 (N-CNT, negative control group (0% FBS); P-CNT, positive control group (EGF 10 ng / mL); Media CNT, TSB medium).

[0101] like Figure 2 As shown, when treated with the culture medium of the strain isolated in Example 1, it was confirmed that the migration of keratinocytes was generally active after 24 hours of culture, compared to the scratch interval of 0 hours, resulting in a significant shortening of the scratch interval. These results indicate that this strain can be effectively used for skin regeneration and wound treatment.

[0102] Experimental Example 3: Analysis of skin barrier enhancement and moisturizing activity

[0103] The effects of the strain isolated in Example 1 on skin barrier strengthening and skin moisturizing activity were analyzed.

[0104] Specifically, the expression of loricrin, a marker of skin barrier function, was analyzed. First, human epidermal keratinocyte cell line (HaCaT) was differentiated. The cell line was cultured at 5×105 / well was divided into 6-well plates and cultured in a serum-free medium containing 0.09mM low concentration calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) in a 37°C 5% CO2 incubator for 2 days to induce differentiation. Afterwards, a serum-free medium containing 2.8mM high concentration calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) was used to culture in a 37°C 5% CO2 incubator for 7 days, and the culture medium was changed every 2 to 3 days. Poly I: C (10μg / mL) and IL-4 (50ng / mL) were added to the cell lines differentiated by the above process to induce an inflammatory response. After culturing in an incubator under the same conditions for 24 hours, the strain culture solution was treated with 0 (TSB medium only) and 10% (v / v) and cultured for another 24 hours. As a negative control group, an untreated group (only treated with Poly I: C + IL-4) was used, and as a positive control group, a 1 μM dexamethasone-treated group was used. Subsequently, TRIZOL (Invitrogen, USA) was used to extract RNA from cells treated with each sample, and RNA was quantified using a nanospectrophotometer, and then 1 μg of RNA was used to synthesize cDNA in an amplification instrument. Using the synthesized cDNA as a template, SYBR Green (Thermo Fisher Scientific, USA) was added to the target gene together with primers for real-time PCR. At this time, the target gene used was the loricrin gene, a marker of skin barrier function, and finally the expression level of the gene was analyzed by correction of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene, and the results are shown in Figure 3 middle.

[0105] Figure 3 It is a graph showing the results of measuring changes in loricrin (LOR) mRNA expression in human keratinocytes treated with the culture medium of the strain of one embodiment of the present invention by real-time PCR (# relative to the blank group (Poly I:C + IL-4 untreated group); * relative to the Poly I:C + IL-4 treated group; #, *p<0.05; ##, **p<0.001; ###, ***p<0.0001; red, significantly negative value (decrease); blue, significantly positive value (increase)).

[0106] like Figure 3As shown, when the strain isolated in Example 1 was treated with 10% culture medium, the expression of loricrin was significantly increased compared to the negative control group. These results indicate that the strain can be effectively used for strengthening the skin barrier and moisturizing the skin.

[0107] Experimental Example 4: Anti-inflammatory activity analysis

[0108] In order to analyze the effects of the strains isolated in Example 1 on anti-inflammatory activity, the expression of TSLP and the expression of proinflammatory cytokines IL-6, IL-8, and IL-1α were analyzed.

[0109] Specifically, first, human epidermal keratinocyte cell line (HaCaT) was differentiated. 5 / well was divided into 6-well plates and cultured in a serum-free medium containing 0.09mM low concentration calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) in a 37°C 5% CO2 incubator for 2 days to induce differentiation. Afterwards, a serum-free medium containing 2.8mM high concentration calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) was used to culture in a 37°C 5% CO2 incubator for 7 days, and the culture medium was changed every 2 to 3 days. Poly I: C (10μg / mL) and IL-4 (50ng / mL) were added to the cell lines differentiated by the above process to induce an inflammatory response. After culturing in an incubator under the same conditions for 24 hours, the strain culture solution was treated with 0 (TSB medium only) and 10% (v / v) and cultured for another 24 hours. As a negative control group, an untreated group (only treated with Poly I: C + IL-4) was used, and as a positive control group, a 1 μM dexamethasone-treated group was used. Subsequently, TRIZOL (Invitrogen, USA) was used to extract RNA from cells treated with each sample, and RNA was quantified using a nanospectrophotometer, and then 1 μg of RNA was used to synthesize cDNA in an amplifier. Using the synthesized cDNA as a template, SYBR Green (Thermo Fisher Scientific, USA) was added to the target gene together with primers for real-time PCR. At this time, the target genes used were the TSLP gene and the proinflammatory cytokines IL-6, IL-8, and IL-1α genes. Finally, the expression levels of the genes were analyzed by correction of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene, and the results are shown in Figure 4 and Figure 5 middle.

[0110] Figure 4 Graph showing the results of real-time PCR measurement of changes in TSLP mRNA expression in human keratinocytes treated with the culture medium of the strain isolated in Example 1 (# relative to the blank group (Poly I:C + IL-4 untreated group); * relative to the Poly I:C + IL-4-treated group (negative control); #, *p<0.05; ##, **p<0.001; ###, ***p<0.0001; red, significantly negative value (decrease); blue, significantly positive value (increase)).

[0111] Figure 5The graph shows the results of measuring changes in IL-6, IL-8, and IL-1α mRNA expression in human keratinocytes treated with the culture medium of the strain isolated in Example 1 by real-time PCR. Figure 5 a is a graph showing the results of IL-6 mRNA expression, Figure 5 b is a graph showing the results of IL-8 mRNA expression, Figure 5 c is a graph showing the results of IL-1α mRNA expression (# relative to the blank group (Poly I:C+IL-4 untreated group); * relative to the Poly I:C+IL-4 treated group; #, *p<0.05; ##, **p<0.001; ###, ***p<0.0001; red, significantly negative value (decrease); blue, significantly positive value (increase)).

[0112] like Figure 4 As shown, when the strain isolated in Example 1 was used for 10% culture solution treatment, the expression level of TSLP was significantly reduced compared with the negative control.

[0113] In addition, if Figure 5 As shown, when the strain isolated by Example 1 was treated with 10% culture medium, the expression levels of IL-6, IL-8 and IL-1α were significantly reduced compared with the negative control group. In particular, IL-6 and IL-1α showed significantly reduced expression at a level similar to that of the positive control group.

[0114] The above results indicate that the above strains can be effectively used to prevent, improve and treat inflammatory skin diseases.

[0115] [Accession number]

[0116] Name of depository institution: Agricultural Genetic Resources Center (Overseas)

[0117] Accession number: KACC 81169BP

[0118] Date of deposit: September 10, 2021

Claims

1. A cosmetic material composition for improving skin condition, wherein: The cosmetic material composition contains Sphingomonas oleifera, a lysate, a culture solution or an extract thereof as an effective ingredient.

2. The cosmetic material composition for improving skin condition according to claim 1, wherein: The improvement of skin condition is one or more selected from the group consisting of improving skin barrier, skin moisturizing, improving skin immunity, enhancing skin resistance, inhibiting skin inflammation, calming skin and skin regeneration.

3. The cosmetic material composition for improving skin condition according to claim 1, wherein: The oleaginous bacteria, its lysate, culture fluid or extract can promote the migration of keratinocytes, or increase the expression of loricrin, or reduce the expression of one or more selected from the group consisting of TSLP, IL-6, IL-8 and IL-1α.

4. The cosmetic material composition for improving skin condition according to claim 1, wherein: The Sphingomonas oleifera is a Sphingomonas oleifera strain deposited under the deposit number KACC 81169BP.

5. A quasi-drug composition for improving skin condition, wherein: The quasi-drug composition contains Sphingomonas oleifera, its lysate, culture solution or extract as an active ingredient.

6. A composition for external use on skin for improving skin condition, wherein: The skin external application composition contains Sphingomonas oleifera, a lysate, a culture solution or an extract thereof as an active ingredient.

7. A health functional food composition for improving skin condition, wherein: The health functional food composition comprises Sphingomonas oleifera, its lysate, culture solution or extract as an effective ingredient.

8. A pharmaceutical composition for preventing or treating inflammatory skin diseases or for treating or improving skin wounds, wherein: The pharmaceutical composition comprises Sphingomonas oleifera, its lysate, culture solution or extract, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition for preventing or treating inflammatory skin diseases or for treating or improving skin wounds according to claim 8, wherein: The inflammatory skin disease is any one 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, drug eruption and acne.

10. A Sphingomonas oleifera strain deposited under the accession number KACC 81169BP.

11. A lysate, culture broth or extract of the strain according to claim 10.

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