CN113122459B - Novel rhodosporidium toruloides strain and cosmetic composition comprising strain culture solution thereof - Google Patents

Novel rhodosporidium toruloides strain and cosmetic composition comprising strain culture solution thereof Download PDF

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CN113122459B
CN113122459B CN202110055382.8A CN202110055382A CN113122459B CN 113122459 B CN113122459 B CN 113122459B CN 202110055382 A CN202110055382 A CN 202110055382A CN 113122459 B CN113122459 B CN 113122459B
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rhodosporidium toruloides
skin
cosmetic composition
culture
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CN113122459A (en
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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
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Cosmax Inc
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Abstract

The invention relates to a cosmetic composition, which comprises a novel rhodosporidium toruloides strain and fermentation liquor, crushing liquor, extracting liquor or culture liquor thereof. According to one aspect, according to a composition comprising a rhodosporidium toruloides strain or a fermentation broth, a crushing liquid, an extract or a culture liquid thereof, it is possible to remarkably reduce the expression amount of MMP-1 and inhibit the expression of IL-6 as an inflammatory cytokine, and inhibit the production of NO, while it is possible to increase the expression of silk fibroin, thereby exhibiting a dermatitis improving effect, a wrinkle improving effect, a skin irritation-relieving effect and a skin barrier-enhancing function, and thus can be effectively used in a cosmetic composition for improving skin, a composition for external preparations of skin, a pharmaceutical composition, a health functional food, 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.10-2020-0005607 filed on 15 months 01 in 2020, which is incorporated herein by reference for all purposes as if fully set forth herein.
Technical Field
The invention relates to a cosmetic composition, a composition for external skin preparation, a pharmaceutical composition for preventing or treating skin diseases and a health-care functional food composition, which comprise a novel rhodosporidium toruloides strain and fermentation liquor, crushing liquor, extracting liquor or culture liquor thereof.
Background
Skin aging is affected by inheritance, exposure to the environment (ultraviolet light, heat), hormonal changes, and metabolic processes (reactive oxygen species, highly reactive compounds such as sugars and aldehydes). These factors lead to cumulative changes in the structure, function and appearance of the skin. Aging of human skin is roughly classified into internal aging occurring over time according to biological processes and external aging (photoaging) caused by environments such as continuous exposure to light, pressure, smoking, and the like.
In dermal cells and tissues, collagen imparts strength and tension to the skin, thereby protecting the skin from external stimulus and external force, and occupies 90% of the dermis layer, so reduction of collagen is closely related to skin aging and wrinkle formation. Collagen degradation proceeds with aging, but when exposed to a stimulus such as ultraviolet rays, biosynthesis of collagen lyase (MMPs) increases, while collagen synthesis decreases, resulting in formation of wrinkles. Therefore, there is a need to develop a novel wrinkle-improving material which is safe to the human body and has better functions of collagen lyase (MMP-1) inhibition and collagen synthesis than existing materials.
Keratinocytes (keratinocytes) mainly form the basal layer of the epidermis of the skin (stratum basale), and their main function is to form a barrier to prevent damage from the outside of the skin. In normal skin, keratinocytes express structural proteins (e.g., keratin (keratin) or silk-aggregation protein (filaggrin)) that are critical for maintaining skin barrier function through a differentiation process, thereby protecting the skin from water division and various environmental attacks. The moisture retention and skin barrier change according to changes in external environment and changes in lifestyle, including abrupt changes in temperature, irritation due to environmental pollution, various pressures, excessive cleansing of the face, and natural aging due to aging. As an effort to improve such damaged skin barrier, it is possible to improve skin barrier and impart moisturizing feel through cosmetic compositions. To date, although research on cosmetics for improving skin barrier is active, there is still a need to develop cosmetics that affect skin barrier improvement and moisture immediately and directly after application to the skin.
Since fermentations and bioconversion technologies using microorganisms are now actively used in the field of cosmetic materials, it is required to find a new microorganism strain using this technology as a cosmetic composition which has good functions of collagen lyase (MMP-1) inhibition and collagen synthesis and affects skin barrier improvement and moisture immediately and directly after application to the skin.
Disclosure of Invention
In one aspect, a rhodosporidium toruloides (Rhodosporidium toruloides) LAB-07 (accession number: KCCM 12644P) strain is provided.
The "rhodosporidium toruloides (Rhodosporidium toruloides)" is one type of yeast, which can be isolated from the soil of the korean Jiang Yuandao Hong Chuan market. The strain may be isolated from food, soil, sea, etc. Also, the extract from the fermented product obtained by adding the strain for fermentation can significantly reduce the expression level of collagen lyase (MMP-1) due to ultraviolet light and heat irradiation and increase the synthesis of collagen precursor. Furthermore, the fermented extract according to an aspect can increase the expression level of a moisturizing factor in skin keratinocytes or fibroblasts and decrease anti-inflammatory factors, so that photoaging caused by heat or ultraviolet rays can be suppressed, skin barrier can be improved, and skin moisturizing and sedative uses can be enhanced.
In another aspect, an oil is provided from the rhodosporidium toruloides LAB-07 (accession number: KCCM 12644P) strain.
The oil derived from a strain may refer to, for example, an oil derived from a fermentation broth obtained by fermenting the strain, a disrupted broth of the strain, an extract extracted from the strain, or a culture broth obtained by culturing the strain, and in this specification, an oil derived from rhodosporidium toruloides described in this specification may be used interchangeably with pink oil (pink oil). The term "fermentation or broth" in this specification means that it is made by adding and fermenting the yeast. The oil from the strain may include fatty acids produced by the rhodosporidium toruloides yeast. The fatty acid is similar to the fatty acid component naturally occurring in human keratinocytes, and thus can have an immediate barrier improvement effect when applied to the skin.
In another aspect, there is provided a cosmetic composition, a composition for external skin preparations, a pharmaceutical composition for preventing or treating skin diseases, and a health functional food composition, which comprises rhodosporidium toruloides KCCM12644P strain, a fermentation broth, a crushing broth, an extraction broth, or a culture broth thereof.
The culture solution may be a culture solution obtained by culturing 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 the following components: at least one carbon source selected from 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. The carbon or nitrogen source used in the medium may include, for example, about 0.1w/v% to 30w/v%, 1w/v% to 25w/v%, 1w/v% to 20w/v%, 1w/v% to 10w/v%, 1w/v% to 5w/v%, or 1w/v% to 1.5w/v%. The medium may include salts commonly used in the culture medium of the strain, the salts including, for example, salts of at least one selected from potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate, and the salts may include, for example, about 0.01w/v% to 10w/v%, 0.1w/v% to 7w/v%, or 0.5w/v% to 5w/v%.
Further, the medium may include, for example, about 0.01w/v% to 10w/v%, 0.05w/v% to 5w/v%, or 0.1w/v% to 2w/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 may 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 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 usual fermentation method, for example, the method may use fed-batch (fed-batch) fermentation.
The rhodosporidium toruloides KCCM12644P strain, fermentation broth, crushing broth, extract or culture broth thereof may comprise, for example, 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, a fermentation broth, a crushing liquid, an extract or a culture liquid thereof may include an amount 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 (Matrix metalloproteinase-1; MMP-1) and inhibiting the expression of Interleukin-6 (Interleukin-6; IL-6) as inflammatory cytokines, and inhibiting the production of Nitric Oxide (NO) while increasing the expression of silk fibroin, thereby exhibiting dermatitis improving effect, wrinkle improving effect, skin irritation alleviation effect and skin barrier enhancing function.
The composition according to an embodiment may include the rhodosporidium toruloides KCCM12644P strain, a fermentation broth, a crushing broth, an extraction broth or a culture broth thereof in an effective amount or as an effective ingredient. The effective amount may be appropriately selected according to the individual. The effective amount may be determined based on factors including the following and other factors well known in the physiological or medical arts: the severity of the disease or condition, the age, weight, health, sex, sensitivity of the individual to the enzyme agent, the time of administration, the route of administration and rate of excretion, the period of administration, other compositions mixed with or used simultaneously with the composition.
The composition according to an embodiment may be a cosmetic composition, a composition for external skin preparations, a pharmaceutical composition for preventing or treating skin diseases, or a health functional food composition. The cosmetic composition may be used in particular for skin moisturization, for preventing or improving skin damage, for enhancing skin barrier, for preventing or improving aging or for improving skin condition. The aging may be aging caused by ultraviolet light or heat.
Thus, the composition may further comprise a cosmetically, pharmaceutically or pharmaceutically acceptable excipient or carrier. Depending on the dosage form of the composition, the composition may further comprise various known additives, and according to an embodiment, may further comprise additives selected from the group consisting of carriers, emulsifiers, humectants, surfactants, chelating agents, antioxidants, bactericides, stabilizers, and any combination thereof.
The carrier may include, for example, animal fibers, vegetable fibers, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide, lactose, aluminum hydroxide, calcium silicate, polyamide powders, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylglycol oil, fatty acid glycerides, liquid phase diluents, ethoxylated isostearyl alcohols, suspending agents such as polyoxyethylene sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, agar-agar, sodium fatty alcohol sulfate, sodium fatty alcohol ether sulfate, sulfosuccinic acid monoesters, isethionates, imidazoline derivatives, methyl taurine, sarcosine, fatty acid amide ether sulfates, sodium alkyl sulfates, fatty alcohols, fatty acid glycerides, fatty acid diethanolamines, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters, and the like.
The emulsifier may include, for example, liquid paraffin, cetyl esters of fatty acids, stearic acid, and the like.
The humectant may include a polyhydric alcohol 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, the polyol may be included in an amount of 5% by weight or more in terms of maintaining the moisturizing endurance, and the polyol may be included in an amount of 50% by weight or less in terms of suppressing the decrease in the sense of use due to the increase in the tackiness and the wet feeling.
The chelating agent may include ethylenediamine tetraacetic acid (ethylene diamine tetraacetic acid; EDTA), alpha-hydroxy fatty acids, lactoferrin, alpha-hydroxy acids, citric acid, lactic acid, malic acid, bilirubin, biliverdin (biliverdin), and the like.
The antioxidant may include butylated hydroxyanisole, dibutylhydroxytoluene, propyl gallate, or the like.
In the cosmetic composition, rhodosporidium toruloides KCCM12644P strain, fermentation broth, crushing liquid, extracting liquid or culture liquid thereof can be manufactured into a dosage form comprising: lotions, astringent (Astringent), emulsion, milk emulsion, moisturizing emulsion, nutritional emulsion, massage cream, nutritional cream, moisturizing cream, hand cream, foundation, essence, nutritional essence, film, soap, facial foam, facial cleansing emulsion, facial cleansing cream, body lotion, body cleansing liquid, suspension, gel, powder, stick (paste), mask or sheet pack or spray composition. Compositions of such dosage forms may be manufactured according to methods conventional in the art. The mixing amount of other components such as the humectant and the like 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 may be 0.001 to 5% by weight, specifically, 0.01 to 3% by weight, based on the total weight of the composition.
The person skilled in the art can choose any additional ingredients and/or amounts thereof such that the advantageous properties of the composition according to the invention are not adversely affected or substantially not affected by the intended addition. The mixing amount of the additional ingredient may be easily selected by those skilled in the art within a range not to impair the object and effect of the present invention, and may 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 blended according to the dosage form or purpose of use of the cosmetic. Since the mixing ingredients and methods may be according to conventional techniques, detailed descriptions thereof will be omitted herein.
The term "treating or ameliorating" may refer to or include alleviating a disease, disorder, condition or symptom or one of the symptoms thereof and inhibiting or preventing the progression thereof, and "active ingredient" or "pharmaceutically effective amount" may refer to any amount of the composition used in practicing the invention provided herein that is sufficient to alleviate a disease, disorder, condition or symptom or one of the symptoms thereof and inhibit or prevent the progression thereof. The effective amount (or amount of administration) may 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 1mg.
Examples of the skin condition or skin-related condition may include skin aging, skin photoaging, wounds, dermatitis, atopic dermatitis, itching, eczema skin disorders, dry eczema, erythema, urticaria, psoriasis, rash or acne, papulosquamous skin disorders, insect and parasite mediated diseases, superficial skin mycoses, bacterial infection diseases, viral diseases, adult diseases, autoimmune vesicular diseases, connective tissue diseases, pigment abnormalities, pigmented scleroderma, and the like.
The skin lesions may include lesions of skin tissue or cells in clinical and cosmetic viewpoints, which include external physical lesions, wrinkles caused by penetration of chemicals, bacteria, fungi, viruses, etc., exposure to ultraviolet rays, loss of skin moisture, aging, etc., oxidation caused by free radicals (reactive oxygen species) 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, abdomen, 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 external skin preparation may be appropriately mixed with components of external skin preparations which are usually used for cosmetics, medicines, etc., for example, aqueous components, oily components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, etc., as needed.
The external preparation for skin may be suitably mixed with a metal chelating agent such as disodium ethylenediamine tetraacetate, trisodium ethylenediamine tetraacetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, valerian, licorice extract, glabridin, hot water extract of Japanese thistle fruit, various crude drugs, tocopheryl acetate, glycyrrhizic acid, tranexamic acid or derivatives or salts thereof, and a saccharide such as vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose.
In the present specification, the skin external agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, medicated bandage, emulsion, or a combination thereof. The skin external preparation can be made into ointment, emulsion, spray, patch, cream, powder, suspension, gel or gel state. The external skin preparation may be appropriately mixed with components of external skin preparations which are usually used for cosmetics, medicines, etc., for example, aqueous components, oily components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients or combinations thereof, as required. The external preparation for skin may be suitably mixed with a metal chelating agent such as disodium ethylenediamine tetraacetate, trisodium ethylenediamine tetraacetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, valerian, licorice extract, glabridin, hot water extract of Japanese thistle fruit, various crude drugs, tocopheryl acetate, glycyrrhizic acid, tranexamic acid or derivatives or salts thereof, and a saccharide such as vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose.
Furthermore, the composition is a pharmaceutical external composition.
The term "medical external product" refers to a fiber, a rubber product or the like for treating, alleviating, managing or preventing a disease of a human or animal, an article having little or no direct effect on the human body, and an article other than a tool, a machine or an equipment among articles corresponding to one kind of agents used in sterilization, disinsection and the like for preventing infection, among articles for diagnosing, treating, alleviating, managing or preventing a condition or disease of a human or animal, and articles other than tools, machines or equipment among articles for pharmacologically affecting the structure and functions of a human or animal, and may include skin external preparations and personal hygienic products.
In the pharmaceutical composition for preventing or treating skin diseases, skin diseases include, for example, skin lesions, and in particular, may include, for example, skin aging, skin photoaging, wounds, dermatitis, atopic dermatitis, itching, eczema skin diseases, dry eczema, erythema, urticaria, psoriasis, rash or acne, papulosquamous skin diseases, insect and parasite-mediated diseases, superficial skin mycoses, bacterial infection diseases, viral diseases, adult diseases, autoimmune vesicular diseases, connective tissue diseases, pigment abnormality, pigmentation scleroderma, 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 a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxy cellulose, or a combination thereof. The disintegrant may be calcium hydroxymethyl cellulose, sodium starch glycolate, anhydrous dibasic calcium phosphate, or a combination thereof. The binding agent may be polyvinylpyrrolidone, low substituted hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silica, talc or a combination thereof.
The pharmaceutical composition may be formulated for oral or parenteral administration. The oral administration dosage form may be a granule, powder, liquid, tablet, capsule, dry syrup, or a combination thereof. The parenteral dosage form may be an injection.
The health functional food composition may be used alone, with a fermentation broth, a crushing broth, an extraction broth or a culture broth of rhodosporidium toruloides KCCM12644P strain or with other foods or food ingredients, and may be suitably used according to conventional methods. In general, when manufacturing a food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw materials. The kind of the health functional food is not particularly limited. In the category of health functional foods, the beverage composition may contain various flavors or natural carbohydrates or the like as additional ingredients. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, erythritol, and the like. As the sweetener, natural sweeteners such as Thaumatin (Thaumatin) and stevia extract or synthetic sweeteners such as saccharin and aspartame, etc. can be used. The health functional food composition may further contain a nutritional agent, a vitamin, an electrolyte, a flavoring agent, a coloring agent, pectic acid and its salts, alginic acid and its salts, an organic acid, a protective colloid thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent for carbonated beverages, or a combination thereof. The health functional food composition may further contain pulp or a combination thereof for producing natural fruit juice, fruit juice beverage and vegetable beverage.
In another aspect, a method of obtaining oil from a rhodosporidium strain KCCM12644P is provided, comprising obtaining a fermentation from a rhodosporidium strain KCCM12644P, and extracting oil from the obtained fermentation.
The step of obtaining the ferment may comprise culturing rhodosporidium toruloides KCCM12644P in a medium, wherein the medium comprises: at least one carbon source selected from 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. The carbon or nitrogen source used in the medium may include, for example, about 0.1w/v% to 30w/v%, 1w/v% to 25w/v%, 1w/v% to 20w/v%, 1w/v% to 10w/v%, 1w/v% to 5w/v%, or 1w/v% to 1.5w/v%. The medium may include salts commonly used in the culture medium of the strain, the salts including, for example, salts of at least one selected from potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate, and the salts may include, for example, about 0.01w/v% to 10w/v%, 0.1w/v% to 7w/v%, or 0.5w/v% to 5w/v%.
Further, the medium may include, for example, about 0.01w/v% to 10w/v%, 0.05w/v% to 5w/v%, or 0.1w/v% to 2w/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 may 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 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 usual fermentation method, for example, the method may use fed-batch (fed-batch) fermentation.
The extraction solvent used in extracting the oil from the obtained fermented product may be hexane, ethyl acetate, dodecane, n-decane, diesel oil, alcohol, and combinations thereof, the extraction method may be, for example, one of ultrasonic extraction, microwave extraction, stirring 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 whole fermentation process to extract the 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 principles of the invention.
Fig. 1 is a graph showing the results of confirming the fatty acid composition in the extract oil from rhodosporidium toruloides.
FIG. 2 is a graph showing the amounts of MMP-1 expressed in fibroblasts when an extract oil from Rhodosporidium toruloides was brought into contact with the cells after irradiation of ultraviolet rays on the fibroblast cell lines.
FIG. 3 is a graph showing the amounts of MMP-1 expressed in keratinocytes when an extract oil from Rhodosporidium toruloides was contacted with cells after heat was irradiated on a HaCaT keratinocyte cell line.
FIG. 4 is a graph showing the expression level of IL-6 in keratinocytes when an extract oil from Rhodosporidium toruloides was contacted with cells after heat was irradiated on a HaCaT keratinocyte cell line.
FIG. 5 is a graph showing the expression level of nitric oxide when an extract oil from Rhodosporidium toruloides was contacted with cells after treating an inflammation-inducing substance on mouse mononuclear macrophage leukemia cells (RAW 264.7).
FIG. 6 is a graph showing the expression level of silk fibroin when an extract oil from Rhodosporidium toruloides was contacted with cells on a HaCat keratinocyte cell line.
Biological material preservation information
The rhodosporidium toruloides LAB-07 of the present invention has been deposited in the Korean microbiological culture center (KCCM) at 2019, 12 months and 19 days, the deposit address: the first 120-861 of the republic of korea, the gate-district, hongji tunnel 2 street, willow building 45, with the preservation number: KCCM12644P, culture name is Rhodosporidium toruloides, classification name is Rhodosporidium toruloides.
Detailed Description
Hereinafter, examples and test examples will be described in detail to specifically describe the present disclosure. However, the embodiments 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 embodiments and test examples. Examples and test examples of the present invention are provided to more fully describe the present invention to those of ordinary skill in the art.
Example 1 isolation, identification and cultivation of Yeast
After the soil from the korean original river Hong Chuan was crushed and homogenized, 1.0g was suspended in 9.0ml of sterilized physiological saline (0.9% sodium chloride (NaCl)) and gradually diluted to paint on YM proliferation medium, respectively. After culturing at 30℃for 24 to 48 hours, strains having different states of the population from each other are selected and pure isolation is performed by several pure cultures (pure cultures). Pure isolated strains were smeared on YM proliferation medium and incubated at 30 ℃ for 24 hours, pure isolated populations were incubated in YM liquid medium and at 1:1 is mixed with 40% (v/v) glycerol (glycidol) and stored in an ultra-low temperature refrigerator at-80 ℃.
To identify the isolated strain, after analysis of the base sequence of the transcribed spacer (Internal Transcribed Spacer; ITS) and the base sequence of 26S rRNA, the strain was identified by searching the NCBI Basic Local Alignment Search Tool (BLAST). Based on the results of the analysis of the base sequence, the strain was identified as rhodosporidium toruloides (Rhodosporidium toruloides). Thus, the above-isolated identification bacteria was named as rhodosporidium toruloides LAB-07 and was deposited at the Korean microorganism deposit center at 12.19.2019 with accession number KCCM12644P.
Suitable culture conditions for the newly identified yeasts were confirmed to be cultured at 200rpm for 24 hours at 30 ℃. In addition, in order to find the optimal production conditions of the oil that can be extracted by the yeast, cultivation was performed in a medium with different proportions of carbon source, nitrogen source and inorganic salt, and it was determined that the fatty acid content in the oil from yeast was at most 34.5% under the optimal cultivation conditions.
Example 2 production of extract oil from identified Yeast
Since the newly identified yeast can produce intracellular oil by fermentation, the yeast is cultured and an 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 confirmed in example 1, and the resultant fermentation was homogenized at a volume ratio of 20% ethanol (1 g in 20ml of solvent mixture). After dispersion, the whole mixture was extracted by ultrasonic extraction (sound) 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 (dimethyl sulfoxide; DMSO) at an appropriate concentration to manufacture a final fermented extract, and analysis for confirming the specific fatty acid composition of the manufactured extract was performed, the analysis results are shown in table 1 and fig. 1. Thereafter, the final fermented extract was used in the following examples.
[ Table 1 ]
Example 3 photoaging suppressor cell assessment of oils from rhodosporidium toruloides
Will come from theThe human immortalized keratinocyte (HaCaT) cell line of the fermentation extract of example 2 was cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1X penicillin/streptomycin (P/S) and after counting the cells, it was cultured in 6cm 2 On-board dilution to 2X 10 5 Cells/wells were seeded in parallel and incubated in a incubator at 37℃with 5% carbon dioxide for 24 hours.
Fibroblast cell line as another cell was cultured in FBM medium containing 10% FBS and 1X penicillin/streptomycin (P/S), and after counting the cells, diluted to 3X 10 on 24-well plates 4 Cells/wells were seeded in parallel and incubated in a incubator at 37℃with 5% carbon dioxide for 24 hours.
The supernatant of the cultured HaCaT cells was removed, washed with Phosphate Buffered Saline (PBS), then 1ml of PBS was added, and 15mJ/cm was used 2 Irradiating ultraviolet rays. PBS was removed and DMEM medium containing 1% fbs was replaced to perform starvation (station) step, and then cultured in a incubator at 5% carbon dioxide and 37 ℃ for 24 hours. The supernatant of the ultraviolet-irradiated HaCaT cells and the supernatant of the non-ultraviolet-irradiated HaCaT cells were transferred to 50ml tubes, respectively, and set based on a nontoxic concentration range, and then diluted with the supernatant of the 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 a incubator at 5% carbon dioxide and 37 ℃ for 24 hours. The supernatant of cultured cell fibroblasts was added to a 96-well plate at 150. Mu.l each. The amount of MMP-1 present in the fibroblast supernatant was quantified using a Human matrix metalloproteinase-1 detection kit (Human Total MMP-1 ELISA kit) (DY 901).
In order to confirm the wrinkle-improving effect of the oil derived from the fermented extract, it was confirmed whether the amount of MMP-1 expressed as a collagen lyase was reduced by treating the sample according to 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 MMP-1 activity was inhibited in proportion to the treatment concentration when the oil from Rhodosporidium toruloides was added to the cells. It was confirmed that MMP-1 activity was significantly inhibited from a concentration of 75ppm or more, in particular, that MMP-1 activity was inhibited by about 85% at a concentration of 150ppm, as compared with the negative control group, so that it was confirmed that the oil derived from Rhodosporidium toruloides was able to effectively inhibit photoaging.
Example 4 evaluation of Heat aging-inhibited cells of oils from rhodosporidium toruloides
The oil from the fermentation extract of example 2 was diluted in the medium to concentrations of 1, 10 and 100 μg/ml and used in the following experiments.
Specifically, the test was performed in human keratinocytes (HaCaT keratinocytes (p 19)) under the following conditions. First, keratinocytes were cultured at 3X 10 5 The cells were separated into 6-well plates and then cultured in an incubator with 37℃and 5% carbon dioxide for 24 hours. Then, the medium was removed and washed with DPBS, after which the remaining cell groups other than the control group were irradiated with thermal infrared rays for 15 minutes, and further cultured for 24 hours by adding oil from the fermented product according to concentration.
Thereafter, RNA was isolated from the cells of each sample using triazole (RNAiso, DAKARA, japan), and then 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 in a real-time PCR apparatus using a mixture of MMP-1 primer as a target protein and Cyperus (SYBR Green supermix, applied biosystems, USA) as a cyanine dye to finally confirm the degree of expression of MMP-1 gene. The expression amount of the gene was finally analyzed by correcting the beta-actin (beta-actin) gene, and the analysis result is shown in fig. 3. In addition, the primers used for confirming the gene expression are shown in Table 2.
[ Table 2 ]
As shown in FIG. 3, it was confirmed that the treated groups of the oil derived from the fermentation extract significantly reduced the expression level of MMP-1 gene at the treatment concentrations of 1, 10 and 100. Mu.g/ml, contrary to the increase of MMP-1 expression in the thermal infrared ray group in the negative control group.
Thus, it is known that the oil from the fermented extract exhibits the wrinkle-improving effect and the aging-preventing effect by reducing the increased expression of MMP-1 due to heat.
EXAMPLE 5 evaluation of the 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 test was performed under the same conditions using the same cells as described in example 4. However, IL-6 primers were used instead of MMP-1 primers in the final run of the real-time polymerase chain reaction. 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, the primers used for confirming the gene expression are shown in Table 3.
[ Table 3 ]
As shown in FIG. 4, it was confirmed that the treated groups of oils derived from fermented extracts significantly reduced the IL-6 gene expression levels at treatment concentrations of 1, 10 and 100. Mu.g/ml. In general, it was confirmed that the treated group of oils from fermented extracts 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 effect, wrinkle improving effect and skin irritation alleviating effect by reducing the expression of IL-6 as an inflammatory cytokine.
Example 6 anti-inflammatory Effect of oil from Rhodosporidium toruloides fermentation extract cell evaluation fruit
After counting RAW264.7 cells, diluted to 2.5×105 cells/well, 2.5×105 cells/well were inoculated into a 48-well plate, and cultured in a incubator at 37 ℃ with 5% carbon dioxide for 24 hours. After culturing, the medium was replaced with serum-free medium, then starvation step was performed, and oil samples from rhodosporidium toruloides were diluted to each concentration and treated. Dexamethasone used as a positive control group was treated with 1ppm and then cultured in a incubator at 5% carbon dioxide and 37℃for 24 hours, and the treatment was performed so that LPS became 1. Mu.g/ml, followed by culturing in a incubator at 5% carbon dioxide and 37℃for 24 hours. 100 μl of medium including synthetic Nitric Oxide (NO) was transferred into a 96-well plate, and treated in the 96-well plate according to each concentration using 500 μM NaNO2 as a standard, and then reacted at room temperature for 20 minutes. To measure absorbance, after performing multiskan GO procedure, absorbance was set to 540nm, a measurement area was selected on the plate, and absorbance was measured. The anti-inflammatory effect of the amount of NO produced was confirmed by calculating the value of each synthesized nitric oxide using a standard curve and obtaining 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 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 NO expression was inhibited by about 58% compared to 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 is known that the oil derived from the fermented extract exhibits dermatitis improving effect, wrinkle improving effect and skin irritation alleviating effect by effectively reducing the expression of NO.
EXAMPLE 7 cell evaluation of moisturizing Effect of oil from Rhodosporidium toruloides fermentation extract
To evaluate the skin moisturizing effect of the oil from the fermented extract of example 2, the following test was performed. Human keratinocyte HaCaT cells (purchased from Korean cell line Bank (Korea Cell Line Bank)) were cultured at 3×10 5 Is inoculated in 6-well cell culture dishes and cultured at 37℃and 5% carbon dioxideThe cells were incubated for 24 hours. Thereafter, 1, 10 and 100. Mu.g/ml of the composition of example 1 was added to the medium, followed by culturing for another 24 hours, and cells were recovered and cDNA was synthesized in the same manner as in the evaluation of the heat aging-inhibiting cells from the oil of the fermentation extract. The synthesized cDNA was subjected to real-time polymerase chain reaction in a real-time PCR apparatus by using a mixture of silk-polymer protein primer and Siboglin (SYBR Green supermix, applied biosystems, USA). The 40-cycle real-time PCR reaction was performed under the following conditions: after 5 minutes of activation of the polymerase at 94 ℃, 30 seconds at 95 ℃,1 minute at 54 ℃,1 minute at 72 ℃. Primer sequences are shown in table 4 below. The expression level of the gene was finally analyzed by correcting the β -actin gene, and the result is shown in fig. 6.
[ Table 4 ]
As shown in FIG. 6, it was confirmed that the silk fibroin gene expression level was increased significantly and in a concentration-dependent manner in the treated group of oils derived from the fermented extract. Therefore, it was particularly confirmed that the oil from the fermentation extract can exhibit a moisturizing effect by increasing the expression of silk fibroin, which is an effect of improving barrier.
[ PREPARATION METHOD ]
Preservation agency name: korean microorganism collection center (foreign)
Accession number KCCM12644P
Date of preservation 20191219
According to one aspect, according to a composition comprising a rhodosporidium toruloides strain and a fermentation broth, a crushing liquid, an extract or a culture liquid thereof, it is possible to remarkably 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), while it is possible to increase the expression of silk-polymerized protein, thereby exhibiting dermatitis improving effect, wrinkle improving effect, skin irritation-relieving effect and skin barrier-enhancing function, and thus can be effectively used in cosmetic compositions for improving skin, compositions for external preparations for skin, pharmaceutical compositions, health functional foods, and the like.
Although certain exemplary embodiments and implementations have been described herein, other embodiments and modifications will be apparent from this description. Accordingly, the present inventive concept is not limited to these embodiments, but is to be limited to the broader scope of the appended claims and various obvious modifications and equivalent arrangements will be apparent to those skilled in the art.
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Claims (15)

1. A rhodosporidium toruloides (Rhodosporidium toruloides) LAB-07 strain characterized by a deposit number: KCCM12644P.
2. An oil from the rhodosporidium toruloides LAB-07 strain of claim 1.
3. Cosmetic composition comprising the rhodosporidium toruloides LAB-07 strain, its crushed liquid, extract or culture liquid according to claim 1.
4. A cosmetic composition according to claim 3, wherein the culture broth is a fermentation broth.
5. A cosmetic composition according to claim 3, wherein the culture broth is a culture broth obtained by culturing the rhodosporidium toruloides LAB-07 strain, a culture supernatant obtained by removing the strain therefrom, or a concentrate or a freeze-dried product of the culture supernatant.
6. A cosmetic composition according to claim 3, wherein the culture broth is obtained by culturing the rhodosporidium toruloides LAB-07 strain in a medium at a temperature of 20 ℃ to 42 ℃ for 24 hours to 144 hours, wherein the medium comprises:
at least one carbon source selected from 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.
7. A cosmetic composition according to claim 3, wherein the cosmetic composition is for preventing or improving a skin condition.
8. The cosmetic composition of claim 7, wherein the cosmetic composition is used for moisturizing skin, enhancing skin barrier, preventing or ameliorating skin damage, or preventing or ameliorating aging.
9. The cosmetic composition of claim 8, wherein the aging is aging caused by ultraviolet light or heat.
10. A cosmetic composition according to claim 3, wherein it comprises 1 to 99.99% by weight of the rhodosporidium toruloides LB-07 strain, a crushed solution, an extract solution or a culture solution thereof, based on the total weight of the composition.
11. A composition for external skin preparations comprising the rhodosporidium toruloides LB-07 strain, a crushed solution, an extract solution or a culture solution thereof according to claim 1.
12. The composition for external skin preparations according to claim 11, wherein the culture solution is a fermentation solution.
13. A method of obtaining oil from rhodosporidium toruloides LB-07 strain according to claim 1, comprising:
obtaining a fermentation product from the rhodosporidium toruloides LB-07 strain; and
extracting oil from the obtained fermented product.
14. The method of claim 13, wherein the extraction solvent used in extracting the oil from the resulting fermentation is hexane, ethyl acetate, dodecane, n-decane, diesel, alcohol, or a combination thereof.
15. The method according to claim 13, wherein the extraction method used in extracting oil from the obtained fermented product is one of ultrasonic extraction, microwave extraction, stirring extraction and heating extraction.
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CN102834509A (en) * 2009-11-19 2012-12-19 奥克西雷恩英国有限公司 Yeast strains producing mammalian-like complex N-glycans
CN103328646A (en) * 2010-09-29 2013-09-25 奥克西雷恩英国有限公司 Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
WO2016039685A1 (en) * 2014-09-08 2016-03-17 Temasek Life Sciences Laboratory Limited Methods for efficient production of polyunsaturated fatty acids (pufa) in rhodosporidium and rhodotorula species
WO2016099401A1 (en) * 2014-12-15 2016-06-23 Temasek Life Sciences Laboratory Limited Methods for tuning carotenoid production levels and compositions in rhodosporidium and rhodotorula genera

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KR20090032584A (en) * 2007-09-28 2009-04-01 주식회사 코리아나화장품 Cosmetic composition for screening uv-rays and preventing skin-aging comprising carotenoid of colorless as active ingredients
JP2016034240A (en) * 2014-08-01 2016-03-17 国立研究開発法人科学技術振興機構 Oil and fat-producing yeast, and method of producing oil and fat

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Publication number Priority date Publication date Assignee Title
CN102834509A (en) * 2009-11-19 2012-12-19 奥克西雷恩英国有限公司 Yeast strains producing mammalian-like complex N-glycans
CN103328646A (en) * 2010-09-29 2013-09-25 奥克西雷恩英国有限公司 Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
WO2016039685A1 (en) * 2014-09-08 2016-03-17 Temasek Life Sciences Laboratory Limited Methods for efficient production of polyunsaturated fatty acids (pufa) in rhodosporidium and rhodotorula species
WO2016099401A1 (en) * 2014-12-15 2016-06-23 Temasek Life Sciences Laboratory Limited Methods for tuning carotenoid production levels and compositions in rhodosporidium and rhodotorula genera

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