CN113122459A

CN113122459A - Novel Rhodosporidium toruloides strain and cosmetic composition comprising strain culture solution thereof

Info

Publication number: CN113122459A
Application number: CN202110055382.8A
Authority: CN
Inventors: 李垠姃; 韩相佑; 金荷荣; 权大仁; 李敬银; 丁栋镇; 柳正镇; 崔在焕; 姜承贤; 朴明三; 金谁永; 柳贞均; 姜丞蕙; 姜相旼; 李艺瑟; 金彩娟; 金志雄; 金娜琳; 金莹洙
Original assignee: Cosmax Inc
Current assignee: Cosmax Inc
Priority date: 2020-01-15
Filing date: 2021-01-15
Publication date: 2021-07-16
Anticipated expiration: 2041-01-15
Also published as: CN113122459B; KR102167388B1

Abstract

The invention relates to a cosmetic composition, which comprises a novel Rhodosporidium toruloides strain and a fermentation liquid, a crushing liquid, an extracting solution or a culture solution thereof. According to one aspect, according to a composition comprising a Rhodosporidium toruloides strain or a fermentation broth, a disrupted solution, an extract or a culture solution thereof, it can significantly reduce the expression amount of MMP-1 and inhibit the expression of IL-6 as an inflammatory cytokine, and inhibit the production of NO, and at the same time, can increase the expression of silk fibroin to exhibit a dermatitis-improving effect, a wrinkle-improving effect, a skin irritation-mitigating effect, and a skin barrier-enhancing function, and thus can be effectively used in cosmetic compositions for improving the skin, compositions for external preparations for the skin, pharmaceutical compositions, health functional foods, and the like.

Description

Novel Rhodosporidium toruloides strain and cosmetic composition comprising strain culture solution thereof

Cross Reference to Related Applications

The present application claims priority and benefit from korean patent application No. 10-2020-.

Technical Field

The present invention relates to a cosmetic composition, a composition for skin external preparations, a pharmaceutical composition for preventing or treating skin diseases, and a health-care functional food composition, which comprises a novel Rhodosporidium toruloides strain and a fermentation broth, a broken solution, an extraction solution, or a culture solution thereof.

Background

Skin aging is affected by genetics, exposure to the environment (ultraviolet, heat), hormonal changes, and metabolic processes (reactive oxygen species, highly reactive compounds such as sugars and aldehydes). These factors result in cumulative changes in the structure, function and appearance of the skin. Aging of human skin is roughly classified into internal aging that occurs over time according to biological processes and external aging (photoaging) caused by environments such as continuous exposure to light, pressure, and smoking.

In dermal cells and tissues, collagen imparts strength and tension to the skin, thereby protecting the skin from external stimuli and external forces, and occupies 90% of the dermal layer, so that the reduction of collagen is closely associated with skin aging and wrinkle formation. Decomposition of collagen proceeds with aging, but when exposed to stimulation such as ultraviolet rays, biosynthesis of collagen lyase (MMPs) increases, and synthesis of collagen decreases, thereby causing formation of wrinkles. Therefore, there is a need to develop a novel wrinkle-improving material that is safe to the human body and has better functions of collagen lyase (MMP-1) inhibition and collagen synthesis than existing materials.

Keratinocytes (keratinocytes) form mainly the basal layer of the epidermis of the skin (stratum basal) and their main function is to form a barrier to prevent damage from outside the skin. In normal skin, keratinocytes express structural proteins (e.g., keratin (keratin) or filaggrin (filaggrin)) that are essential for maintaining the barrier function of the skin through a differentiation process, thereby protecting the skin from water loss and various environmental insults. The moisturizing and skin barrier change according to changes in the external environment and changes in lifestyle, for reasons including sudden changes in temperature, irritation due to environmental pollution, various stresses, excessive cleansing, and natural aging due to aging. As an effort to improve the skin barrier of such damage, the skin barrier can be improved and a moisturizing feeling can be imparted by a cosmetic composition. To date, despite active research on cosmetics for improving skin barrier, there is still a need to develop cosmetics that immediately and directly affect skin barrier improvement and moisturization after being applied to the skin.

Since the present fermentates and bioconversion techniques using microorganisms are actively used in the field of cosmetic materials, there is a need to find a novel microbial strain using such techniques as a cosmetic composition, which has good functions of collagen lyase (MMP-1) inhibition and collagen synthesis, and immediately and directly affects skin barrier improvement and moisturization upon application to the skin.

Disclosure of Invention

In one aspect, there is provided a Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain.

The Rhodosporidium toruloides (Rhodosporidium toruloides) is one of yeasts, which can be isolated from soil in Hongkuan city of the Korean Jianghuan. The strain can be isolated from food, soil, sea, etc. Also, the extract from the fermented product obtained by fermentation with the addition of the strain can significantly reduce the expression amount of collagen lyase (MMP-1) due to ultraviolet and heat irradiation, and increase the synthesis of collagen precursor. In addition, the fermented extract according to an aspect may increase the expression level of moisturizing factors in skin keratinocytes or fibroblasts and decrease anti-inflammatory factors, so that it may inhibit photoaging caused by heat or ultraviolet rays, improve skin barrier, and be used for skin moisturizing and calming purposes.

Another aspect provides an oil from Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain.

The oil derived from the strain may refer to, for example, an oil derived from a fermentation broth obtained by fermenting the strain, a disruption solution of the strain, an extract solution extracted from the strain, or a culture solution obtained by culturing the strain, and in the present specification, the oil derived from Rhodosporidium toruloides described in the present specification may be used interchangeably with pink oil (pink oil). The term "fermentation product or broth" in the present specification means that it is prepared by adding and fermenting the yeast. The oil from the strain may include fatty acids produced by the rhodosporidium toruloides yeast. The fatty acids are similar to the fatty acid components naturally present in human keratinocytes and thus may have an immediate barrier-improving effect when applied to the skin.

In another aspect, there is provided a cosmetic composition, a composition for skin external preparations, a pharmaceutical composition for preventing or treating skin diseases, and a health functional food composition, which comprises Rhodosporidium toruloides KCCM12644P strain, its fermentation broth, disruption solution, extraction solution, or culture solution.

The culture solution may be a culture solution obtained by culturing the Rhodosporidium toruloides KCCM12644P strain, a culture supernatant obtained by removing the strain therefrom or a concentrate of the culture supernatant, or a freeze-dried product.

The culture solution can be obtained by culturing Rhodosporidium toruloides KCCM12644P strain in a culture medium, wherein the culture medium comprises: at least one carbon source selected from the group consisting of glucose, fructose, mannose, galactose, xylose, ribose, maltose, sucrose, dextrin, glycerol and inulin; and at least one nitrogen source selected from potassium nitrate, ammonium sulfate, ammonium oxalate and diammonium phosphate. The carbon source or nitrogen source used in the medium may include, for example, about 0.1 to 30 w/v%, 1 to 25 w/v%, 1 to 20 w/v%, 1 to 10 w/v%, 1 to 5 w/v%, or 1 to 1.5 w/v%. The medium may include salts commonly used in a medium of a strain, the salts including, for example, at least one salt selected from potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate, and the salts may include, for example, about 0.01 to 10 w/v%, 0.1 to 7 w/v%, or 0.5 to 5 w/v%.

Further, the medium may include, for example, about 0.01 to 10 w/v%, 0.05 to 5 w/v%, or 0.1 to 2 w/v% of yeast autolysate, and the medium may have a pH of 2.0 to 7.0, but is not limited thereto.

In addition, the culture solution can be obtained by culturing rhodosporidium toruloides at a temperature of, for example, about 20 ℃ to 42 ℃, about 25 ℃ to 35 ℃, or about 27 ℃ to 30 ℃ for, for example, about 24 hours to 144 hours, 48 hours to 120 hours, or 15 hours to 35 hours. Also, the culture conditions of the strain may include a step of adding and fermenting the Rhodosporidium toruloides, and the fermentation step may use a general fermentation method, for example, the method may use fed-batch fermentation.

The rhodosporidium toruloides KCCM12644P strain, its fermentation broth, disruption solution, extraction solution, or culture solution may include an amount, for example, of 1 to 99.99 wt%, for example, 1.5 to 99.99 wt% or 2 to 99.99 wt%, based on the total weight of the composition. The Rhodosporidium toruloides KCCM12644P strain, its fermentation broth, disruption solution, extraction solution or culture solution may include equal to or more than 2% based on the total weight of the composition in terms of significantly reducing the expression amount of Matrix metalloproteinase-1 (MMP-1) and inhibiting the expression of Interleukin-6 (Interleukin-6; IL-6) as an inflammatory cytokine, and inhibiting the production of Nitric Oxide (NO) while increasing the expression of silk fibroin, thereby exhibiting a dermatitis-improving effect, a wrinkle-improving effect, a skin irritation-mitigating effect and a skin barrier-enhancing function.

The composition according to an embodiment may include the Rhodosporidium toruloides KCCM12644P strain, its fermentation broth, disruption solution, extraction solution or culture solution in an effective amount or as an effective ingredient. The effective amount may be appropriately selected depending on the individual. The effective amount may be determined based on factors including the following, as well as other factors well known in the physiological or medical arts: the severity of the disease or condition, the age, weight, health, sex of the individual, the sensitivity of the individual to the enzyme agent, the time of administration, the route and rate of excretion, the period of administration, other compositions mixed or used simultaneously with the composition.

The composition according to an embodiment may be a cosmetic composition, a composition for external preparation for skin, a pharmaceutical composition for preventing or treating skin diseases, or a health functional food composition. The cosmetic composition may be used in particular for moisturizing skin, for preventing or ameliorating skin damage, for enhancing the skin barrier, for preventing or ameliorating aging or for improving the skin condition. The aging may be aging caused by ultraviolet light or heat.

Thus, the composition may further comprise a cosmetically, dietetically or pharmaceutically acceptable excipient or carrier. The composition may further include various known additives according to the dosage form of the composition, and according to an embodiment, may further include an additive selected from the group consisting of a carrier, an emulsifier, a humectant, a surfactant, a chelating agent, an antioxidant, a bactericide, a stabilizer, and any combination thereof.

The carrier may include, for example, animal fibers, plant fibers, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicon, bentonite, silicon dioxide, talc, zinc oxide, lactose, aluminum hydroxide, calcium silicate, polyamide powder, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butyl glycol oil, glycerol fatty acid esters, liquid phase diluents, ethoxylated isostearyl alcohols, suspending agents such as polyoxyethylene sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, agar, sodium fatty alcohol sulfate, sodium fatty alcohol ether sulfate, sulfosuccinic acid monoesters, isethionates, imidazoline derivatives, methyl taurines, sarcosines, fatty acid amide ether sulfates, sodium alkyl sulfates, fatty alcohols, glycerol fatty acid esters, fatty acid diethanolamides, sodium salts of fatty acids, sodium salts of sodium sulfosuccinic acid, isethionates, methyl taurates, sarcosines, fatty acid amide ether sulfates, alkyl sulfates, fatty alcohols, fatty acid esters, fatty, Vegetable oils, lanolin derivatives, or ethoxylated glycerin fatty acid esters.

The emulsifier may include, for example, liquid paraffin, cetyl oleate, stearic acid, and the like.

The humectant may include a polyol selected from the group consisting of glycerin, butylene glycol, propylene glycol, dipropylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, sorbitol, and any combination thereof. Specifically, the polyol may contain the following amounts based on the total weight of the composition: 5 to 50 wt%, 5 to 30 wt%, 10 to 30 wt%, or 5 to 10 wt%. At this time, in terms of maintaining the moisturizing durability, the polyol may be included at 5% by weight or more, and in terms of suppressing the decrease in the use feeling due to the increase in stickiness and the wet feeling, the polyol may be included at 50% by weight or less.

The chelating agent may include ethylene diamine tetra acetic acid (EDTA), alpha-hydroxy fatty acid, lactoferrin, alpha-hydroxy acid, citric acid, lactic acid, malic acid, bilirubin, biliverdin, and the like.

The antioxidant may include butylated hydroxyanisole, dibutyl hydroxytoluene, propyl gallate, or the like.

In the cosmetic composition, Rhodosporidium toruloides KCCM12644P strain, its fermentation broth, disruption solution, extract or culture solution may be prepared into a dosage form comprising: lotions (skin lotions), emollients, lotions, astringents (astringets), lotions, milk lotions, moisturizing lotions, nutritional lotions, massage creams, nutritional creams, moisturizing creams, hand creams, foundations, essences, nutritional essences, films, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body washes, body cleansing liquids, suspensions, gels, powders, pastes (pastes), masks or sheets, masks or spray compositions. Compositions of such dosage forms may be manufactured according to conventional methods in the art. The mixing amount of the other ingredients such as the humectant and the like can be easily selected by those skilled in the art within a range not impairing the object and effect of the present invention, and the mixing amount thereof may be 0.001 to 5% by weight, specifically, 0.01 to 3% by weight, based on the total weight of the composition.

Any additional ingredients and/or amounts thereof can be selected by those skilled in the art such that the advantageous properties of the composition according to the present invention are not adversely affected or substantially unaffected by the intended addition. The mixing amount of the additional ingredient can be easily selected by those skilled in the art within a range not to impair the object and effect of the present invention, and can be about 0.001 to about 30% by weight, specifically about 0.01 to 25% by weight, based on the total weight of the composition.

In addition, in the cosmetic composition of each dosage form, appropriate ingredients may be selected and compounded according to the dosage form or the purpose of use of the cosmetic. Since the mixing components and method may be according to conventional techniques, a detailed description thereof will be omitted herein.

The term "treating or ameliorating" can refer to or include alleviating a disease, disorder, condition, or condition or one of the symptoms thereof and inhibiting or preventing the progression thereof, and the "active ingredient" or "pharmaceutically effective amount" can refer to any amount of the composition used in practicing the invention provided herein that is sufficient to alleviate a disease, disorder, condition, or condition or one of the symptoms thereof and inhibit or prevent the progression thereof. The effective amount (or amount administered) can be 0.0001mg to 10000mg, 0.001mg to 1000mg, 1.0mg to 100mg, 0.01mg to 1000mg, 0.01mg to 100mg, 0.01mg to 10mg, or 0.01mg to 1 mg.

Examples of the skin condition or skin-related condition may include skin aging, skin photoaging, wounds, dermatitis, atopic dermatitis, pruritus, eczematous dermatosis, eczema sicca, erythema, urticaria, psoriasis, rash or acne, papulosquamous dermatosis, insect-and parasite-mediated diseases, superficial dermatomycosis, bacterial infection diseases, viral diseases, adult diseases, autoimmune blistering diseases, connective tissue diseases, dyschromatosis, scleroderma pigmentosum, and the like.

The skin damage may include damage of skin tissue or cells from a clinical and cosmetic standpoint, which includes external physical damage, penetration by chemicals, bacteria, fungi, viruses, etc., exposure to ultraviolet rays, skin moisture loss, wrinkles caused by aging, etc., oxidation caused by free radicals (active oxygen), etc., skin pigmentation, dermatitis, seborrheic dermatitis, redness (redness, erythema), swelling, lichenification, eczema, itching (itching), atopic dermatitis, etc.

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

The composition may be formulated for parenteral administration. The parenteral administration dosage form may be an injection or a skin external preparation. The skin external preparation may be appropriately mixed with ingredients of skin external preparations generally used in cosmetics, pharmaceuticals, and the like, for example, aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, perfumes, colorants, various skin nutrients, and the like, as necessary.

The skin external preparation can be mixed with appropriate amount of disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, metal chelating agent such as gluconic acid, caffeine, tannin, verapamil, Glycyrrhrizae radix extract, glabridin, hot water extract of Cirsium japonicum fruit, various crude drugs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, and saccharides such as vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

In the present specification, the external preparation for skin may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a medicated bandage, an emulsion, or a combination thereof. The skin external preparation can be prepared into ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. The external preparation for skin may be appropriately mixed with ingredients of external preparations for skin generally used in cosmetics, pharmaceuticals, etc., for example, aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, perfumes, colorants, and various skin nutrients, or a combination thereof, as necessary. The skin external preparation can be mixed with appropriate amount of disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, metal chelating agent such as gluconic acid, caffeine, tannin, verapamil, Glycyrrhrizae radix extract, glabridin, hot water extract of Cirsium japonicum fruit, various crude drugs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, and saccharides such as vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

Furthermore, the composition is a pharmaceutical composition for external use.

The term "medical supplies" refers to a fiber, a rubber product or an article like the same for treating, alleviating, treating or preventing human or animal diseases, an article having little or no direct effect on the human body and being not a tool or a machine, an article other than a tool, a machine or a device among articles for diagnosing, treating, alleviating, treating or preventing human or animal conditions or diseases, among articles corresponding to one kind of a preparation used in sterilization, disinsection and use like the same for preventing infection, and an article other than a tool, a machine or a device among articles for pharmacologically affecting the structure and the function of human or animal, and may include skin external preparations and personal hygiene products.

In the pharmaceutical composition for preventing or treating skin diseases, skin diseases include, for example, skin injury, and specifically, may include, for example, skin aging, skin photoaging, wounds, dermatitis, atopic dermatitis, pruritus, eczematous dermatosis, dry eczema, erythema, urticaria, psoriasis, rash or acne, papulosquamous dermatosis, insect-and parasite-mediated diseases, superficial dermatomycosis, bacterial infection diseases, viral diseases, adult diseases, autoimmune vesiculosis, connective tissue diseases, dyschromatosis, scleroderma pigmentosum, but are not limited thereto.

The pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose or mannitol, and the lubricant may be magnesium stearate, talc or a combination thereof. The carrier may be an excipient, disintegrant, binder, lubricant, or combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium hydroxymethylcellulose, sodium starch glycolate, anhydrous dibasic calcium phosphate, or a combination thereof. The binding agent may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated in a dosage form for oral or parenteral administration. The oral administration dosage form may be granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof. The parenteral administration form may be an injection.

The health functional food composition may be a strain of Rhodosporidium toruloides KCCM12644P, its fermentation broth, disruption solution, extraction solution or culture solution used alone or together with other foods or food ingredients, and may be suitably used according to conventional methods. The mixing amount of the effective ingredient may be appropriately determined depending on the purpose of use (preventive, health or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present specification may be added in an amount of equal to or less than 15 parts by weight based on the raw materials. The kind of the health functional food is not particularly limited. In the type of the health functional food, the beverage composition may contain various flavors or natural carbohydrates as additional ingredients. The natural carbohydrate may be a monosaccharide such as glucose and fructose, a disaccharide such as maltose and sucrose, a polysaccharide such as dextrin and cyclodextrin, and a sugar alcohol such as xylitol, sorbitol, erythritol, etc. As the sweetener, natural sweeteners such as Thaumatin (Thaumatin) and stevia extract, or synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health functional food composition may further comprise nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acids and salts thereof, alginic acids and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents for carbonated beverages, or combinations thereof. The health functional food composition may further comprise pulp or a combination thereof for manufacturing natural fruit juice, fruit juice beverage and vegetable beverage.

Another aspect provides a method for obtaining oil from Rhodosporidium toruloides KCCM12644P strain, which comprises obtaining a fermented product from Rhodosporidium toruloides KCCM12644P strain, and extracting oil from the obtained fermented product.

The step of obtaining the fermentation product may comprise culturing rhodosporidium toruloides KCCM12644P in a culture medium, wherein the culture medium comprises: at least one carbon source selected from the group consisting of glucose, fructose, mannose, galactose, xylose, ribose, maltose, sucrose, dextrin, glycerol and inulin; and at least one nitrogen source selected from potassium nitrate, ammonium sulfate, ammonium oxalate and diammonium phosphate. The carbon source or nitrogen source used in the medium may include, for example, about 0.1 to 30 w/v%, 1 to 25 w/v%, 1 to 20 w/v%, 1 to 10 w/v%, 1 to 5 w/v%, or 1 to 1.5 w/v%. The medium may include salts commonly used in a medium of a strain, the salts including, for example, at least one salt selected from potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate, and the salts may include, for example, about 0.01 to 10 w/v%, 0.1 to 7 w/v%, or 0.5 to 5 w/v%.

In addition, the culture solution can be obtained by culturing rhodosporidium toruloides at a temperature of, for example, about 20 ℃ to 42 ℃, about 25 ℃ to 35 ℃, or about 27 ℃ to 30 ℃ for, for example, about 24 hours to 144 hours, 48 hours to 120 hours, or 15 hours to 35 hours. Also, the step of culturing the yeast may use a general fermentation method, for example, the method may use fed-batch fermentation.

The extraction solvent used in extracting oil from the obtained fermented product may be hexane, ethyl acetate, dodecane, n-decane, diesel oil, alcohol, and a combination thereof, the extraction method may be, for example, one of ultrasonic extraction, microwave extraction, agitation extraction, and heating extraction, and the extraction solvent may be used in an amount of about 5 to 30 times by weight of the extracted oil obtained in the entire fermentation process to extract an oil component.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the inventive concept.

FIG. 1 is a graph showing the results of confirming the fatty acid composition in an extract oil from Rhodosporidium toruloides.

FIG. 2 is a graph showing confirmation of MMP-1 expression level in fibroblasts when cells are contacted with an extract oil from Rhodosporidium toruloides after exposure of the fibroblast cell lines to ultraviolet rays.

FIG. 3 is a graph showing confirmation of MMP-1 expression level in keratinocytes when cells were contacted with an extract oil from Rhodosporidium toruloides after exposure to heat on a HaCaT keratinocyte cell line.

FIG. 4 is a graph showing confirmation of the expression level of IL-6 in keratinocytes when the cells were contacted with an extract oil from Rhodosporidium toruloides after exposure to heat on a HaCaT keratinocyte cell line.

FIG. 5 is a graph showing confirmation of the expression level of nitric oxide when an extract oil from Rhodosporidium toruloides was brought into contact with cells after an inflammation-inducing substance was treated on mouse mononuclear macrophage leukemia cells (RAW 264.7).

FIG. 6 is a graph showing confirmation of the expression level of filaggrin when an extract oil from Rhodosporidium toruloides was brought into contact with cells on a HaCat keratinocyte cell line.

Biological material preservation information

Rhodosporidium toruloides LAB-07 of the present invention has been deposited in Korea microbial culture center (KCCM) at 19.12.2019, accession number: 120-: KCCM12644P, culture name Rhodosporidium toruloides, taxonomic name Rhodosporidium toruloides.

Detailed Description

Hereinafter, examples and test examples will be described in detail to specifically describe the present disclosure. However, the examples and test examples according to the present invention have various modifications and variations, and the scope of the present invention should not be construed as being limited to the following examples and test examples. Examples and test examples of the present invention are provided to more fully describe the invention to those of ordinary skill in the art.

Example 1 isolation, identification and culture of Yeast

After soil from hong chuan city, a main river in korea was crushed and homogenized, 1.0g of the soil was suspended in 9.0ml of sterilized physiological saline (0.9% sodium chloride (NaCl)) and gradually diluted to be coated on YM growth medium. After culturing at 30 ℃ for 24 to 48 hours, strains having a state of population different from each other were selected and subjected to pure isolation by several pure cultures. The pure isolated strains were smeared on YM multiplication medium and cultured at 30 ℃ for 24 hours, and the pure isolated population was cultured in YM liquid medium and cultured at 1: 1 was mixed with 40% (v/v) glycerol (glycerol) and stored in an ultra low temperature refrigerator at-80 ℃.

To identify an isolated strain, after analysis of an Internal Transcribed Spacer (ITS) base sequence and a base sequence of 26S rRNA was performed, the strain was identified by searching the NCBI Basic Local Alignment Search Tool (BLAST). According to the analysis result of the base sequence, the strain was identified as Rhodosporidium toruloides (Rhodosporidium toruloides). Thus, the above-isolated identified strain was named Rhodosporidium toruloides LAB-07, and it was deposited at 19.12.2019 in the Korean culture Collection of microorganisms under accession No. KCCM 12644P.

Suitable culture conditions for the newly identified yeast are identified as 24 hours at 200rpm at 30 ℃. In addition, in order to find the optimum production conditions of the oil extractable by the yeast, cultivation was carried out in a medium with carbon source, nitrogen source and inorganic salt in various proportions, and it was determined that the fatty acid content in the oil derived from yeast was at most 34.5% under the optimum cultivation conditions.

Example 2 production of extracted oil from identified Yeast

Since the newly identified yeast can produce intracellular oil by fermentation, the yeast is cultured and oil including fatty acids extracted therefrom is obtained. Thereafter, in order to analyze the efficacy of the extract, the yeast was cultured in the medium composition identified in example 1, and the resulting fermentation was homogenized at a 20 volume ratio of ethanol (1 g in 20ml of solvent mixture). After dispersion, the whole mixture was extracted by ultrasonic extraction (sonication) for 3 hours. The extract was centrifuged at 4000rpm (10 minutes) to recover only the supernatant. After filtering the supernatant using a 0.2 μm filter, the lipid-containing supernatant was evaporated in a rotary evaporator under vacuum and then dissolved in dimethyl sulfoxide (DMSO) at an appropriate concentration to produce a final fermentation extract, and analysis for confirming a specific fatty acid composition of the produced extract was performed, the analysis results being shown in table 1 and fig. 1. Thereafter, the final fermented extract was used in the following examples.

[ TABLE 1 ]

Example 3 cellular evaluation of photoaging inhibition of oil from Rhodosporidium toruloides

Human immortalized keratinocyte (HaCaT) cell line from the fermentation extract of said example 2 was cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1X penicillin/streptomycin (P/S), after counting the cells, at 6cm²Diluted to 2X 10 on plate⁵Cells/well were inoculated and cultured in an incubator with 5% carbon dioxide at 37 ℃ for 24 hours.

Fibroblast cell lines, another cell, were cultured in FBM medium containing 10% FBS and 1X penicillin/streptomycin (P/S), and after counting the cells, diluted to 3 × 10 on 24-well plates⁴Cells/well were inoculated and cultured in an incubator with 5% carbon dioxide at 37 ℃ for 24 hours.

The supernatant of cultured HaCaT cells was removed, washed with Phosphate Buffered Saline (PBS), and then 1ml of PBS was added thereto and washed with 15mJ/cm²Ultraviolet rays are irradiated. PBS was removed and DMEM medium containing 1% FBS was replaced to perform a starvation (standing) step, followed by culture in an incubator of 5% carbon dioxide and 37 ℃ for 24 hours. The supernatant of HaCaT cells irradiated with ultraviolet rays and the supernatant of HaCaT cells not irradiated with ultraviolet rays were transferred to 50ml tubes, respectively, and set based on a non-toxic concentration range, and then diluted using the supernatant of HaCaT cells.

The supernatant of the cultured cell fibroblasts was removed. Samples made using HaCaT supernatant were treated according to concentration on cultured cell fibroblasts and then cultured in an incubator with 5% carbon dioxide at 37 ℃ for 24 hours. The supernatant of cultured cell fibroblasts was added to a 96-well plate at 150. mu.l per plate. The amount of MMP-1 present in the fibroblast supernatant was quantified using a Human matrix metalloproteinase-1 detection kit (Human Total MMP-1ELISA kit) (DY 901).

In order to confirm the wrinkle-improving effect of the oil derived from the fermented extract, whether the expression level of MMP-1 as collagen lyase was reduced or not was confirmed by treating the sample according to the concentration, and the results are shown in fig. 2.

As shown in FIG. 2, from the results of measuring MMP-1 activity, it was confirmed that when oil from Rhodosporidium toruloides was added to cells, MMP-1 activity was inhibited in proportion to the treatment concentration. It was confirmed that MMP-1 activity was significantly inhibited from a concentration of 75ppm or more, particularly that MMP-1 activity was inhibited by 85% at a concentration of 150ppm, as compared with the negative control group, and it was confirmed that the oil derived from Rhodosporidium toruloides could effectively inhibit photoaging.

Example 4 evaluation of cells for thermal aging inhibition of oil from Rhodosporidium toruloides

The oil from the fermentation extract of example 2 was diluted in the medium to concentrations of 1, 10 and 100 μ g/ml and used in the following experiments.

Specifically, the test was carried out in human keratinocytes (HaCaT keratinocytes (p19)) under the following conditions. First, keratinocytes were cultured at 3X 10⁵The strains were aliquoted into 6-well plates and then cultured in an incubator with conditions of 37 ℃ and 5% carbon dioxide for 24 hours. Then, the medium was removed and washed with DPBS, and thereafter, the remaining cell groups other than the control group were irradiated with thermal infrared rays for 15 minutes, and oil from the fermentation product was added according to the concentration to further culture for 24 hours.

Thereafter, RNA was isolated from the cells of each sample using triazole (RNA iso, DAKARA, Japan), and then the RNA was quantified at 260nm using an ultraviolet spectrophotometer (nanodrop), and cDNA was synthesized in an amplifier using 2. mu.g of each RNA (C1000 thermocycler, Bio-Rad, USA). The synthesized cDNA was subjected to real-time Polymerase Chain Reaction (PCR) using a mixture to which MMP-1 primer as a target protein and Cyberrin as a cyanine dye were added in a real-time PCR apparatus to finally confirm the expression level of MMP-1 gene. The expression level of the gene was finally analyzed by correcting the β -actin (β -actin) gene, and the analysis results are shown in fig. 3. In addition, primers for confirming gene expression are shown in table 2.

[ TABLE 2 ]

As shown in FIG. 3, in contrast to the increase in MMP-1 expression in the group of thermal infrared rays in the negative control group, it was confirmed that the group treated with the oil derived from the fermentation extract significantly reduced the expression level of MMP-1 gene at treatment concentrations of 1, 10, and 100. mu.g/ml.

Therefore, it can be known that the oil from the fermented extract exhibits wrinkle-improving effects and anti-aging effects by reducing the expression of MMP-1 increased by heat.

Example 5 evaluation of Interleukin-6 (IL-6) inhibitory Effect of oil from Rhodosporidium toruloides fermentation extract Estimation of

To confirm the IL-6 inhibitory effect of the oil from the fermented extract, the experiment was performed under the same conditions using the same cells as described in the example 4. However, in the final real-time polymerase chain reaction, IL-6 primer was used instead of MMP-1 primer. The expression level of the gene was finally analyzed by correcting the β -actin gene, and the analysis results are shown in fig. 4. In addition, primers for confirming gene expression are shown in table 3.

[ TABLE 3 ]

As shown in FIG. 4, it was confirmed that the treated groups of the oil derived from the fermentation extract significantly reduced the IL-6 gene expression level at the treatment concentrations of 1, 10 and 100. mu.g/ml. In general, it was confirmed that the treatment group of the oil derived from the fermentation extract significantly and effectively reduced the IL-6 gene expression amount in a concentration-dependent manner. Therefore, it was found that the oil derived from the fermented extract exhibited dermatitis-improving effects, wrinkle-improving effects, and skin irritation-alleviating effects by reducing the expression of IL-6, which is an inflammatory cytokine.

Example 6 anti-inflammatory Effect of oils from Rhodosporidium toruloides fermentation extract cell evaluation

After counting RAW264.7 cells, diluted to 2.5 × 105 cells/well, 2.5 × 105 cells/well were seeded into 48-well plates and cultured in an incubator with 5% carbon dioxide at 37 ℃ for 24 hours. After cultivation, serum-free medium was replaced, followed by a starvation step, and oil samples from rhodosporidium toruloides were diluted to each concentration and treated. Dexamethasone used as a positive control group was cultured in an incubator at 37 ℃ and 5% carbon dioxide for 24 hours after being treated with 1ppm, and was treated so that LPS became 1. mu.g/ml, followed by culturing in an incubator at 37 ℃ and 5% carbon dioxide for 24 hours. 100 μ l of the culture medium including synthetic Nitric Oxide (NO) was transferred to a 96-well plate, and treated in the 96-well plate according to each concentration using 500 μ M of NaNO2 as a standard, and then reacted at room temperature for 20 minutes. To measure absorbance, after performing multiskan GO program, absorbance was set to 540nm, a measurement area was selected on the plate, and absorbance was measured. The anti-inflammatory effect of the measured NO production amount was confirmed by calculating the value of each synthesized nitric oxide using the standard curve and calculating the result value from the average value thereof, and the result is shown in fig. 5.

As shown in FIG. 5, it was confirmed that the amount of NO produced was decreased in a concentration-dependent manner when the oil derived from Rhodosporidium toruloides was treated, and that the amount of NO produced was significantly decreased at all the treatment concentrations as compared with the negative control group, thereby confirming a direct anti-inflammatory effect. In particular, it was confirmed that when the extract was 150ppm, the expression of NO was inhibited by about 58% as compared with the negative control group, which effectively reduced the amount of NO production at a level similar to that of the positive control group. Therefore, it can be known that the oil derived from the fermented extract exhibits a dermatitis-improving effect, a wrinkle-improving effect, and a skin irritation-alleviating effect by effectively reducing the expression of NO.

Example 7 cellular evaluation of moisturizing Effect of oils from Rhodosporidium toruloides fermentation extract

To evaluate the skin moisturizing effect of the oil from fermented extract of example 2, the following test was performed. Human keratinocytes HaCaT cells (purchased from Korea Cell Line Bank) were cultured at 3X 10⁵Was seeded in 6-well cell culture dishes and cultured in an incubator at 37 ℃ and 5% carbon dioxide for 24 hours. Thereafter, 1, 10 and 100. mu.g/ml of the composition of example 1 were added to the medium, followed by further culturing for 24 hours, and the cells were recovered and cDNA was synthesized in the same manner as in the evaluation of the heat aging-inhibited cells from the oil of the fermentation extract. The synthesized cDNA was subjected to real-time polymerase chain reaction in a real-time PCR instrument by using a mixture to which silk fibroin primers and Sayborglin (SYBR Green supermix, applied biosystems, USA) were added. A 40 cycle real-time PCR reaction was performed under the following conditions: after activating the polymerase at 94 ℃ for 5 minutes, 30 seconds at 95 ℃, 1 minute at 54 ℃ and 1 minute at 72 ℃. The primer sequences are shown in table 4 below. The expression level of the gene was analyzed by correcting the β -actin gene, and the results are shown in fig. 6.

[ TABLE 4 ]

As shown in FIG. 6, it was confirmed that the expression level of filaggrin gene was significantly increased in the treatment group of oil derived from fermentation extract in a concentration-dependent manner. Therefore, it was confirmed in particular that the oil derived from the fermented extract can exhibit a moisturizing effect by a barrier-improving effect by increasing the expression of the filaggrin.

[ DENSATION NUMBER ]

The name of the depository institution: korea center for preservation of microorganisms (foreign)

Accession No. KCCM12644P

Storage date 20191219

According to one aspect, according to a composition comprising a Rhodosporidium toruloides strain and a fermentation broth, a disrupted solution, an extract or a culture thereof, it can significantly reduce the expression amount of matrix metalloproteinase-1 (MMP-1) and inhibit the expression of interleukin-6 (IL-6) as an inflammatory cytokine, and inhibit the production rate of Nitric Oxide (NO), and at the same time, can increase the expression of silk fibroin, thereby exhibiting a dermatitis-improving effect, a wrinkle-improving effect, a skin irritation-mitigating effect, and a skin barrier-enhancing function, and thus can be effectively used in cosmetic compositions for improving skin, compositions for external skin preparations, pharmaceutical compositions, health functional foods, and the like.

While certain exemplary embodiments and implementations have been described herein, other embodiments and modifications will be apparent from this description. The inventive concept is therefore not limited to the embodiments but is to be accorded the widest scope consistent with the claims appended hereto, and various modifications and equivalent arrangements will be apparent to those skilled in the art.

Sequence listing

<110> kesi meishi co

<120> novel Rhodosporidium toruloides strain and cosmetic composition comprising strain culture solution thereof

<130> P21131434W

<150> 10-2020-0005607

<151> 2020-01-15

<160> 8

<170> PatentIn version 3.5

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<213> Artificial Sequence

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<223> MMP-1 Forward primer sequence

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cgaatttgcc gacagagatg a 21

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<223> MMP-1 reverse primer sequence

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<223> beta-actin forward primer sequence

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ttttctgcca gtgcctcttt 20

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<223> Silk polymerization protein Forward primer sequence

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agtgcactca gggggctcac a 21

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Claims

1. Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain.

2. An oil derived from Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain.

3. A cosmetic composition comprises Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain, its fermentation broth, disruption solution, extract or culture solution.

4. The cosmetic composition according to claim 3, wherein the culture solution is a culture solution obtained by culturing Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain, a culture supernatant obtained by removing the strain therefrom, or a concentrate or freeze-dried product of the culture supernatant.

5. The cosmetic composition according to claim 3, wherein the culture solution is obtained by culturing Rhodosporidium toruloides LAB-07 (accession No: KCCM12644P) strain in a culture medium at a temperature of 20 ℃ to 42 ℃ for 24 hours to 144 hours, wherein the culture medium comprises:

at least one carbon source selected from the group consisting of glucose, fructose, mannose, galactose, xylose, ribose, maltose, sucrose, dextrin, glycerol and inulin; and

at least one nitrogen source selected from the group consisting of potassium nitrate, ammonium sulfate, ammonium oxalate and diammonium phosphate.

6. The cosmetic composition according to claim 3, wherein the cosmetic composition is used for moisturizing skin, enhancing skin barrier, preventing or improving skin damage, preventing or improving aging, or preventing or improving skin condition.

7. The cosmetic composition according to claim 6, wherein the aging is aging caused by ultraviolet light or heat.

8. The cosmetic composition of claim 3, wherein the Rhodosporidium toruloides LB-07 (accession No: KCCM12644P) strain, its fermentation broth, disruption solution, extraction solution or culture solution is included in an amount of 1 to 99.99% by weight, based on the total weight of the composition.

9. A composition for skin external agent comprises Rhodosporidium toruloides LB-07 (accession No: KCCM12644P) strain, its fermentation liquid, broken liquid, extract or culture liquid.

10. A health functional food composition comprises Rhodosporidium toruloides LB-07 (preservation number: KCCM12644P) strain, its fermentation liquid, broken liquid, extractive solution or culture solution.

11. A method for obtaining oil from Rhodosporidium toruloides LB-07 (accession No: KCCM12644P) strain, comprising:

obtaining a fermented product from Rhodosporidium toruloides LB-07 (accession No: KCCM12644P) strain; and

extracting oil from the obtained fermented product.

12. The process according to claim 11, wherein the extraction solvent used in extracting oil from the obtained fermentate is hexane, ethyl acetate, dodecane, n-decane, diesel, alcohols and combinations thereof.

13. The method according to claim 11, wherein the extraction method used in extracting oil from the obtained fermented product is one of ultrasonic extraction, microwave extraction, agitation extraction and heating extraction.