Yeast-derived extracellular vesicles and a topical skin preparation containing the same

Abstract

To find a novel agent that protects the skin from external stimuli (stress) such as ultraviolet rays and heat and has the effect of repairing and improving damaged skin; and to provide external formulations for skin containing the same.SOLUTION: There are provided an extracellular membrane vesicle with an average particle size of 100 to 200 nm, which is derived from a heat-shocked yeast, and an external formulation for skin containing the same.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to extracellular membrane vesicles derived from yeast, and more particularly to extracellular membrane vesicles derived from heat-shocked yeast and a skin external preparation containing the same.

Background Art

[0002] The skin is a tissue located on the outermost layer of the human body and is constantly directly affected by stresses such as heat, sunlight, chemical substances, heavy metals, tobacco smoke, and exhaust gas. In order to protect the human body from such external stimuli (stresses), it is extremely important to maintain and improve the barrier function of the skin.

[0003] When cells are exposed to strong stresses such as heat, sunlight, chemical substances, heavy metals, tobacco smoke, and exhaust gas, they produce heat shock proteins (HSPs) to counteract them and have a system to defend tissues from damage caused by stresses. HSPs are special proteins produced by cells and organisms in response to various stresses as well as heat stimulation. It is known that when the synthesis of HSPs increases, the resistance to stresses such as heat and sunlight in the skin is improved. However, repeated exposure to stresses reduces the barrier function of the skin, causing swelling, dryness, or cracking. As one method for this, attempts have been made to prevent skin aging by incorporating yeast HSPs into skin external preparations (see Patent Document 1).

[0004] In addition, for protecting the skin from damage caused by heat stress, a cosmetic care method of locally applying a composition containing Artemia salina extract together with a physiologically acceptable medium to at least a part of the skin of the body or face (see Patent Document 2), and a skin external preparation containing a heat shock protein derived from yeast and an extract of germinated seeds of rye as a heat shock protein production promoter (Patent Document 3) have been reported.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-331602 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-502050 [Patent Document 3] Japanese Patent No. 4562536 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] According to the method of the prior art, although a certain effect of repairing and improving the skin damaged by the action of HSP is expected, since HSP itself is added to the skin external preparation, it is likely to cause protein degradation and denaturation, etc., and there is also a limit to the skin protection effect.

[0007] Therefore, an object of the present invention is to find a novel agent having an effect of protecting the skin from external stimuli (stress) such as ultraviolet rays and heat, and repairing and improving the damaged skin, and to provide a skin external preparation containing the same. [Means for Solving the Problems]

[0008] The inventors of the present invention have found that the particle diameter of extracellular vesicles produced by yeast changes depending on the culture temperature, and that extracellular vesicles derived from heat-shocked yeast have a promoting effect on the proliferation of epidermal keratinocytes, and thus completed the present invention.

[0009] That is, the present invention includes the following embodiments. (1) Extracellular vesicles derived from heat-shocked yeast, having an average particle diameter of 100 to 200 nm. (2) The extracellular vesicles according to (1), wherein the yeast belongs to the genus Pichia. (3) The yeast is Pichia nakazawae isolated from the sap of Camellia japonica. イAn extracellular vesicle according to (1) or (2), which is (Pichia naganishii) and has an average particle size of 170 nm or more. An extracellular vesicle according to (1) or (2), wherein the yeast is Pichia membranaefaciens derived from apple and has an average particle size of 180 nm or more. An extracellular vesicle according to (1) or (2), wherein the yeast is Pichia veronae isolated from the sap of oak and has an average particle size of 120 nm or more. An external preparation for protecting the skin from stress, containing the extracellular vesicle according to any one of (1) to (5). A method for producing an extracellular vesicle having a skin stress-protecting effect, comprising culturing Pichia naganishii isolated from the sap of camellia at a temperature of 36 to 38 °C to obtain a culture solution, removing the cells from the culture solution to recover the culture supernatant, and recovering the extracellular vesicle from the culture supernatant. イ A method for producing an extracellular vesicle, comprising culturing Pichia naganishii isolated from the sap of camellia at a temperature of 36 to 38 °C to obtain a culture solution, removing the cells from the culture solution to recover the culture supernatant, and recovering the extracellular vesicle from the culture supernatant.

Effect of the Invention

[0010] According to the present invention, it is possible to provide a novel agent having an effect of protecting the skin from external stimuli (stress) such as ultraviolet rays and heat, and repairing and improving damaged skin, and a skin external preparation containing the same.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0012] (Definition) As used herein, "heat shock" refers to exposing cells to stress such as high temperature. Heat shock proteins (HSPs) that are expressed intracellularly are known to maintain cell homeostasis and survival against heat shock. Members of this multigene superfamily are characterized by their molecular size and related functions, and include, for example, HSP110, HSP90, HSP70, HSP60, HSP40, and small heat shock proteins. HSPs function as molecular chaperones that assist in the refolding of misfolded proteins and the restoration of cellular functions, or lead to proteasome-mediated degradation of damaged proteins. HSP expression is rapidly induced by heat shock transcription factors (HSFs), particularly HSF1. HSF activates the transcription of HSP genes through binding to the heat shock element (SHE) sequence of the genes to be regulated.

[0013] "Extracellular vesicles" refer to particles with a size of about several tens to several thousand nanometers, covered with a membrane mainly composed of phospholipids and released from cells. Extracellular vesicles include exosomes, microvesicles, apoptotic bodies, etc. In many cases, biomolecules are present in extracellular vesicles. For example, exosomes or microvesicles contain at least one kind of biomolecule selected from polypeptides and nucleic acids (RNAs such as mRNA, miRNA, non-coding RNA, etc.). For example, apoptotic bodies contain at least one kind selected from fragmented nuclei and organelles. Extracellular vesicles preferably contain at least one kind of biomolecule selected from polypeptides and RNAs. Here, a polypeptide refers to a compound in which a plurality of amino acids are bonded by peptide bonds, and includes proteins with a relatively large molecular weight and peptides with a relatively small molecular weight. The size of extracellular vesicles can be measured, for example, by methods based on the Brownian motion of extracellular vesicles, light scattering methods, and electrical resistance methods.

[0014] "Average particle size" means the average value of the values obtained by measuring the particle size of extracellular vesicles by any measurement method. As measurement methods, for example, there are wet measurement methods for observing extracellular vesicles in a solution state using the electrical resistance nanopulse method (qNANO) and nanoparticle tracking method, and dry measurement methods for observing the morphology of extracellular vesicles while maintaining their form using a transmission electron microscope.

[0015] (Extracellular vesicles derived from heat-shocked yeast) In one embodiment of the present invention, extracellular vesicles having an average particle size of 100 to 200 nm and derived from heat-shocked yeast (hereinafter sometimes referred to as "extracellular vesicles of this embodiment") are provided. Yeast can be used without limitation, including wild yeast, isolated strains, or commercially available yeast. Wild yeast is yeast existing in nature and is widely distributed, such as on the surface of fruits, sap, nectaries of flowers, soil, seawater, etc. Isolated strains are clones from basically the same cells isolated from nature and are considered to be genetically homogeneous populations.

[0016] In a preferred embodiment, the yeast is isolated and identified from nature and may include one or more selected from, for example, the genus Pichia sp., the genus Saccharomyces sp., the genus Schizosaccharomyces sp., and the genus Neurospora sp. The genus Pichia is a genus of yeasts in the family Saccharomycetaceae. More than 100 species of this genus are known. Particularly well-known species include Pichia Pastoris, Pichia guilliermondii, Pichia norvegensis, Pichia ohmeri, Pichia naganishii イ (Pichia naganishii), Pichia membranaefaciens, Pichia veronae, Pichia anomala, etc.

[0017] A typical heat shock method for yeast includes, for example, culturing at a temperature above the optimal growth temperature of the yeast. Here, the "optimal growth temperature of the yeast" means the temperature at which the growth rate of the target yeast is maximized. The temperature conditions above the optimal growth temperature of the yeast vary depending on the type of yeast used. For example, it is generally a temperature 5°C or more higher than the optimal growth temperature, and a range of 25°C to 40°C is preferred. A temperature above 25°C is preferred, and a temperature of 35°C or more is more preferred from the viewpoint of further promoting the expression of heat shock proteins.

[0018] Although not bound by any theory, extracellular vesicles recovered from heat-shocked yeast have an increased average particle size compared to when cultured at lower temperatures, suggesting an increased content of polypeptides and nucleic acids encapsulated within the extracellular vesicles. Proteomic and genomic analyses of exosomes from various cell types have revealed that exosomes are cell-type specific and contain a wide range of signaling factors that are regulated based on the environment of the secreting cell (Webber, J., et al., Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan) platform. Mol Cell Proteomics, 2014. 13(4): p. 1050-64.). HSP70 has previously been shown to be a component of the exosome cargo (Clayton, A., et al., Induction of heat shock proteins in B-cell exosomes. J Cell Sci, 2005. 118(Pt16): p. 3631-8.). The genetic information contained within exosomes can affect or direct the fate of target cells, for example, by inducing activation, migration, proliferation, differentiation or dedifferentiation of the target cells, or by promoting apoptosis or necrosis.

[0019] The extracellular vesicles of this embodiment have an average particle size of 100 to 200 nm, although the diameter of individual particles may be in the range of about 20 to 500 nm. In a preferred embodiment, the lower limit of the average particle size of the extracellular vesicles is 110 nm or more, more preferably 115 nm or more. Also, in a preferred embodiment, the upper limit of the average particle size of the extracellular vesicles may be 190 nm or less.

[0020] The average particle size of these extracellular vesicles may vary depending on the type of yeast. For example, Pichia nakazawae isolated from the sap of Camellia イThe origin extracellular vesicles have a diameter of about 100 nm to 650 nm, and their average particle size is about 170 nm or more. On the other hand, in the case of yeast Pichia membranifaciens derived from apples, it has a diameter of about 100 nm to 400 nm, and its average particle size is about 180 nm or more. In contrast, yeast Pichia veronae derived from the sap of birch is slightly smaller, having a diameter of about 100 nm to 300 nm, and its average particle size is about 120 nm or more.

[0021] (Production method) In other embodiments, the extracellular vesicles include a step of culturing yeast isolated from nature at a temperature above the optimum growth temperature to obtain a culture solution, a step of removing the cells from the culture solution to recover the culture supernatant, and a step of recovering the extracellular vesicles from the culture supernatant. The method for culturing yeast is not particularly limited as long as it is a method commonly used for yeast, and can follow known techniques (for example, there are references such as The Yeasts. A Taxonomic Study, 4th ed. Elsevier Science BV, Amsterdam. 240 - 241 (1998), etc.). For example, shaking culture, aeration culture, static culture, stirring culture, etc. can be mentioned. Also, either a batch type or a continuous type may be used. These can be carried out alone or in combination of two or more. The culture conditions of Pichia yeast in a preferred embodiment (for example, aerobic, anaerobic, pH in the medium, dissolved oxygen, culture temperature, culture time, etc.) are not particularly limited and can be appropriately selected according to the purpose.

[0022] After culturing the yeast obtained in this way, the cells can be removed from the culture broth by a conventional method such as filtration or centrifugation to recover the culture supernatant. As an exemplary embodiment, centrifugation can be carried out at 1000 - 20000×g, or 1500 - 20000×g, or 1500 - 15000×g, or 1500 - 10000×g for 30 - 60 minutes. At this time, the centrifugation speed or time may be changed and carried out step by step. For example, after centrifuging at a low speed of 1500 - 2000×g to separate and remove the yeast cells from the culture broth and separate the cells from the supernatant culture broth, centrifugation may be carried out at a high speed of 10000 - 20000×g to further remove cells or cell-related debris and residues. Alternatively, after removing the cells, filtration may be carried out with a filter having a size of 0.3 - 0.5 μm. The purity of the centrifuged culture broth can be increased by this filtration step.

[0023] Subsequently, the culture supernatant of the yeast obtained in this way may be produced by sedimentation by ultracentrifugation at 100000×g or more, specifically, 100000 - 200000×g, or 100000 - 150000×g, or 150000 - 200000×g for 1 - 5 hours.

[0024] In one embodiment, the extracellular vesicles of this embodiment may be separated using one or more methods selected from the group consisting of centrifugation, ultracentrifugation, differential centrifugation, equilibrium density gradient centrifugation, density gradient, filtration, dialysis, and free flow electrophoresis, but the separation method of the extracellular vesicles of this embodiment is not limited thereto.

[0025] Density gradient is the most commonly used method for separating substances with different densities. Specific examples of this method can be implemented using density gradient separation materials such as Ficoll, glycerol, sucrose, cesium chloride, iodixanol, etc., but are not limited thereto. On one hand, density gradient may be used together with ultracentrifugation or the like. On another hand, gel filtration or ultrafiltration may be used to select extracellular vesicles. On still another hand, dialysis may be used instead of filtration to remove small molecules. On still another hand, free flow electrophoresis may be used.

[0026] In a specific embodiment, a method for producing extracellular vesicles having a stress-protecting effect on the skin comprises the steps of culturing Pichia nakazawae isolated from camellia sap at a temperature of 36 to 38 °C to obtain a culture solution, removing bacterial cells from the culture solution to recover a culture supernatant, and recovering extracellular vesicles from the culture supernatant. イ

[0027] (External preparation for skin) On another hand, the extracellular vesicles of this embodiment can be used as external preparations such as cosmetics, external pharmaceuticals, quasi-drugs, etc. The extracellular vesicles obtained by the above-described method are prepared into the form of a general external preparation for skin using a pharmaceutically acceptable appropriate formulation carrier and used to protect the skin from stress.

[0028] ​In one embodiment, the above-mentioned topical skin preparation is a cosmetic composition. The cosmetic composition may further contain, in addition to the extracellular vesicles of the present embodiment, functional additives and components contained in general cosmetic compositions. The functional additives may include components selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts. Other compounding components included (preferably components compounded to such an extent that the structure of the extracellular vesicles is not broken) include oil and fat components, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like.

[0029] In the present embodiment, the cosmetic composition is preferably produced in the form of lotion, lotion, cream, oil, powder, gel, etc. to such an extent that the structure of the extracellular vesicles is not broken. For this purpose, the composition of the present invention may further contain a solvent, a suitable carrier, excipient or diluent usually used in the production of cosmetic compositions.

[0030] The type of solvent further added to the cosmetic composition of the present invention is not particularly limited. For example, water, saline, DMSO or a combination thereof can preferably be used to such an extent that the structure of the extracellular vesicles is not broken. Carriers, excipients or diluents include purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, etc., but are not limited thereto. Further, if necessary, whitening agents, humectants, vitamins, ultraviolet blockers, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents can be included.

[0031] As the oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm kernel oil, jojoba oil, avocado oil are used, and as the wax, beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin are used.

[0032] In addition, preferably, a humectant, thickening agent, antioxidant, etc., which are widely known in the cosmetic field, can be included to such an extent that the structure of the extracellular vesicles is not broken, and the types and amounts of these are based on those known in the art.

[0033] In other embodiments, the above external preparation for skin may be a pharmaceutical composition. This pharmaceutical composition may further contain, in addition to the extracellular vesicles of the present embodiment, pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents, or salts and / or buffers for osmotic pressure adjustment, and other therapeutically useful substances, and may be formulated in the form of various parenteral dosage forms by ordinary methods. Examples of the form of the parenteral dosage form include transdermal dosage forms, and for example, dosage forms such as injections, drip infusions, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches, etc. may be used, but are not limited thereto.

[0034] The determination of the dosage of the active ingredient is within the technical level of those skilled in the art, and the daily dosage of the drug varies depending on various factors such as the degree of progression of the symptoms of the subject to be administered, the onset time, age, health status, and complications. However, when based on adults, generally, the composition is 1 μg / kg to 200 mg / kg, and on the other hand, 50 μg / kg to 50 mg / kg may be administered in 1 to 3 divided doses per day, and the above dosage does not limit the scope of this specification by any method.

[0035] Next, examples will be given to explain the present invention in more detail, but the present invention is not limited to these examples in any way. In the following examples, the unit % of the numerical values indicating the addition amounts of various components means mass %. Also, the OD value of the yeast culture solution is the absorbance at a wavelength of 600 nm when the yeast culture solution is measured with a spectrophotometer.

Example

[0036] (Experiment 1: Confirmation of the action effect of extracellular vesicles derived from heat-shocked yeast) The experimental contents shown in FIGS. 1 to 4 are described in this Experiment 1.

[0037] (Preparation of Yeast Culture Solution) To collect (prepare) extracellular vesicles, first, a yeast culture solution derived from camellia, a yeast culture solution derived from apple, and a yeast culture solution derived from oak were prepared. These yeast culture solutions were yeast culture solutions containing a yeast count such that the OD value was 2.5 or more (solution with a pH of approximately 5.0 to 6.5).

[0038] As the yeast to be contained in the yeast culture solution derived from camellia, Pichia naganishii (AOK - Y1918) isolated from the sap of camellia, as the yeast to be contained in the yeast culture solution derived from apple, Pichia membranaefaciens (AOK - Y1238), and as the yeast to be contained in the yeast culture solution derived from oak, Pichia veronae (AOK - Y1915) isolated from the sap of oak were each prepared as stored strains smeared on a slant medium. The storage of the strains was carried out using a slant medium prepared by dissolving yeast extract 0.3% (Wako Pure Chemical 398 - 02151), malt extract 0.3% (Becton Dickinson 218630), glucose 1.0% (Wako Pure Chemical 049 - 31165), peptone 0.5% (Wako Pure Chemical 398 - 02117) in distilled water, adjusting the pH to 6.0, adding agar 1.5% (Wako Pure Chemical Industries 010 - 15815), autoclaving at 121°C for 20 minutes, and then dispensing and solidifying. These yeasts are products of the applicant, Akita Konno Shoten Co., Ltd.

[0039] The yeast culture solution derived from camellia was inoculated from the above - stored strain AOK - Y1918, the yeast culture solution derived from apple was inoculated from the above - stored strain AOK - Y1238, and the yeast culture solution derived from oak was inoculated from the above - stored strain AOK - Y1915 into a test tube containing 10 mL of a preculture solution with the composition shown in Table 1 below using an inoculation loop, and shake - cultured at 25°C for 1 day at 120 rpm.

[0040]

Table 1

[0041] Subsequently, 0.5 mL of the preculture medium with an OD value of 2.0 or higher was added to a flask containing 50 mL of the main culture medium with the composition described in Table 1, and subculture was performed for 2 days while shaking at 120 rpm. At this time, the culture temperature was 25 °C and 37 °C for yeast derived from camellia, 20 °C and 25 °C for yeast derived from apple, and 20 °C and 25 °C for yeast derived from oak for the main culture. After culturing for 2 days, the OD value of all yeasts became 2.5 or higher.

[0042] (Method for recovering extracellular vesicles from yeast culture) 50 mL of the yeast culture (OD value of 2.5 or higher) was centrifuged at 3000×g and 4 °C for 5 minutes to precipitate the cells. The supernatant after this centrifugation was collected and filtered through 0.45 μm and 0.22 μm filters. The filtrate was subjected to centrifugal filtration at 2000×g and 4 °C for 30 minutes using an ultrafiltration centrifuge with a fractionation membrane having a molecular weight cut-off of 100,000. This supernatant was collected with 10 mL of PBS(-).

[0043] The collected supernatant was ultracentrifuged at 100,000×g and 4 °C for 3 hours using an ultracentrifuge. Then, the supernatant was removed, and the pellet was suspended in 5 mL of PBS(-). The collected suspension was ultracentrifuged again under the same conditions, the pellet was suspended in 5 mL of PBS(-), and after further washing with 2 mL of PBS(-), it was subjected to centrifugal filtration at 2000×g and 4 °C for 30 minutes using an ultrafiltration centrifuge with a fractionation membrane having a molecular weight cut-off of 100,000. The supernatant was collected twice using 200 μL of PBS(-) to obtain an extracellular vesicle sample. The obtained sample was observed for particle size with qNANO.

[0044] The results of measuring the particle size distribution of the extracellular vesicle sample recovered from the yeast culture solution derived from apples using a qNANO nanoparticle multi-analyzer (manufactured by IZON) are shown in Figure 1. Figure 1(A) is a sample recovered from yeast cultured at 20°C in the method described above, with an average particle size of 125 nm (standard deviation 30.2 nm), a maximum particle size of 312 nm, and a minimum particle size of 95 nm. On the other hand, Figure 1(B) is a sample recovered from yeast cultured at 25°C, with an average particle size of 188 nm (standard deviation 34.6 nm), a maximum particle size of 348 nm, and a minimum particle size of 127 nm. From these results, it is considered that Pichia membranifaciens derived from apples undergoes heat shock at a culture temperature of 25°C, resulting in a significant increase in the average particle size. For yeast Pichia nakano-shii derived from camellia and yeast Pichia veronae derived from oak, a tendency for an increase in the average particle size was also observed when they were subjected to heat shock at culture temperatures of 37°C and 25°C, respectively.

[0045] (Evaluation of changes in cell growth ability due to heat stress) Normal human epidermal keratinocytes (NHEK) were purchased from Kurabo and cultured in a carbon dioxide incubator at 37°C and 5% CO2 in KBM-Gold TM basal medium (manufactured by Lonza). On the first day of the experiment, NHEK cells at 2×10 4 cells / mL were seeded in a 24-well plate. After culturing overnight at 37°C and 5% CO2, on the second day, the culture solution in the 24-well plate was replaced with KBM-Gold TM basal medium without EGF and BPE. The extracellular vesicles of yeast recovered above were diluted to various concentrations and added to each well. For the blank, PBS without extracellular vesicles was added. Three hours after adding the sample, the culture plate was transferred to a constant temperature bath at 45°C and held for 1 hour (stress treatment). This stress treatment operation was performed once a day for 3 consecutive days, and the cells were recovered 72 hours after adding the sample. On the fifth day after the start of the experiment, the cell growth ability of the recovered cells was measured using Cell Counting Kit-8 (manufactured by Dojindo Laboratories). Cells to which the blank was added and not subjected to stress treatment were used as the untreated control.

[0046] The results are shown in FIGS. 2 to 4. An explanation will be given regarding the horizontal axis of the graph. "Control normal" means an untreated control group in which cells (NHEK) to which PBS (blank) not containing extracellular vesicles is added are not stress-treated. "blank" means a group in which cells (NHEK) are stress-treated but yeast-derived extracellular vesicles are not added. The number of particles (per mL) means the number of yeast-derived extracellular vesicles cultured at a predetermined temperature. Also, the cell growth rate (%) shown on the vertical axis of the graph is represented as a relative value (%) when the number of cells when each sample is added is compared with Control normal (set as 100%). FIG. 2(A) shows the measurement results using extracellular vesicles recovered from a culture solution obtained by culturing yeast derived from camellia at 25°C. FIG. 2(B) shows the measurement results using extracellular vesicles recovered from a culture solution obtained by culturing the same yeast at 37°C. Compared with the untreated control group, the cell growth rate significantly decreased in the blank in which normal human epidermal keratinocytes were stress-treated, but when yeast-derived extracellular vesicles cultured at 37°C were added, the cell growth rate significantly recovered in a concentration-dependent manner. On the other hand, no significant difference was observed when yeast-derived extracellular vesicles cultured at 25°C were added. FIGS. 3 and 4 show the results of performing the same evaluation with the type of yeast changed to yeast derived from apple and oak yeast, respectively. For any yeast, when extracellular vesicles cultured at 25°C, which is a heat shock condition, were added, the cell growth rate of normal human epidermal keratinocytes significantly recovered. From these results, it was suggested that extracellular vesicles derived from heat-shocked yeast have an effect of protecting normal human epidermal keratinocytes from stress.

[0047] (Experiment 2: Confirmation of the action effect of extracellular vesicles derived from yeast produced using koji extract medium) In this Experiment 2, the action effect of extracellular vesicles derived from yeast produced using koji extract medium was confirmed. The confirmation results will be described below.

[0048] (Preparation of yeast culture solution) To collect (produce) extracellular vesicles, first, a yeast culture solution derived from apples was prepared. These yeast culture solutions were yeast culture solutions (solutions with a pH of 8.0 at the start of culture) containing a yeast count such that the OD value was 2.5 or higher.

[0049] As the yeast to be contained in the apple-derived yeast culture solution, Pichia membranaefaciens (AOK-Y1238) was prepared as a stored strain smeared on a slant medium (slant).

[0050] The yeast of the stored strain was inoculated into a test tube containing 10 mL of the medium with the composition described in Table 2 below using an inoculation loop, and shake culture was performed at 25°C for 1 day at 120 rpm.

[0051]

Table 2

[0052] The koji extract described in Table 2 was produced as follows using the koji starter "No. 5" (strain: Aspergillus oryzae) manufactured by Akita Imakino Shoten and rice produced in Akita Prefecture with a milling ratio of 70%. First, the rice produced in Akita Prefecture was washed, absorbed with water, steamed for 1 hour, and then allowed to cool. The cooled rice was inoculated with the koji starter, and cultured for 5 days while maintaining the temperature around 35°C and the humidity around 90% using a koji lid. Koji was obtained through this culture. Four times the amount of purified water was added to the koji, and it was decocted in hot water at 60°C for 6 hours. After this decoction, the decocted material was filtered, further boiled and concentrated, and then filtered again to obtain a clear koji extract (koji extract).

[0053] Subsequently, 0.5 mL of the preculture solution with an OD value of 2.0 or higher was added to a flask containing 50 mL of the medium with the composition described in Table 2, and subculture was performed for 2 days while shaking at 120 rpm. At this time, for the apple-derived yeast, the main culture was performed at 20°C or 25°C. This main culture at 25°C is a culture under conditions that give heat shock to the yeast, different from this main culture at 20°C. After culturing for 2 days, the OD value of both yeasts became 2.5 or higher.

[0054] (Method for recovering extracellular vesicles from yeast culture broth) 50 mL of yeast culture broth (OD value of 2.5 or more) was centrifuged at 3000×g and 4°C for 5 minutes to precipitate the cells. The supernatant after this centrifugation was collected and filtered through 0.45 μm and 0.22 μm filters. The filtrate was ultrafiltration centrifuged at 2000×g and 4°C for 30 minutes using an ultrafiltration centrifuge with a fractionation membrane having a molecular weight cut-off of 100,000. This supernatant was collected with 10 mL of PBS(-).

[0055] The collected supernatant was ultracentrifuged at 100000×g and 4°C for 3 hours using an ultracentrifuge. Thereafter, the supernatant was removed, and the pellet was suspended in 5 mL of PBS(-). The collected suspension was ultracentrifuged again under the same conditions, the pellet was suspended in 5 mL of PBS(-), and after further washing with 2 mL of PBS(-), it was ultrafiltration centrifuged at 2000×g and 4°C for 30 minutes using an ultrafiltration centrifuge with a fractionation membrane having a molecular weight cut-off of 100,000. The supernatant was collected twice using 200 μL of PBS(-) to obtain an extracellular vesicle sample.

[0056] (Evaluation of changes in cell proliferation ability due to heat stress) Normal human epidermal keratinocytes (NHEK) were purchased from Kurabo and cultured in a carbon dioxide incubator at 37°C and 5% CO2 in KBM-Gold TM basal medium (manufactured by Lonza). On the first day of the experiment, 2×10 4 NHEK cells per mL were seeded in a 24-well plate. After culturing overnight at 37°C and 5% CO2, on the second day, the culture medium in the 24-well plate was replaced with KBM-Gold TM basal medium without EGF and BPE. The extracellular vesicles of yeast recovered above were added to each well to a final concentration (1×10 6 particles / mL), and the following experimental groups were set up. The experimental groups and stress treatments are described below.

[0057] · Group 1: A group in which the extracellular vesicles were not added (only PBS was added), and no stress treatment was performed below. Only normal culture was carried out at 37°C under 5% CO2 conditions. · Group 2: A group in which the extracellular vesicles were not added (only PBS was added), and the following stress treatment was performed. · Group 3: A group in which the extracellular vesicles (prepared from yeast prepared by the main culture at 20°C according to Table 2) were added to a final concentration of (1×10 6 particles / mL), and the following stress treatment was performed. · Group 4: A group in which the extracellular vesicles (prepared from yeast prepared by the main culture at 25°C (conditions for giving heat shock to yeast) according to Table 2) were added to a final concentration of (1×10 6 particles / mL), and the following stress treatment was performed. · Stress treatment: The culture plate was transferred to a constant temperature bath at 45°C and held for 1 hour. This treatment operation was performed once a day for 3 consecutive days.

[0058] The cultures of Groups 1 to 4 were cultured for 72 hours after the addition of the sample (cultured in a carbon dioxide incubator at 37°C and 5% CO2 conditions except for the stress treatment). After the culture, the cells of each group were collected. On the 5th day after the start of the experiment, the cell count (cell proliferation ability) of the collected cells was measured using Cell Counting Kit-8 (Dojindo Laboratories).

[0059]

Table 3

[0060] The results are shown in Table 3. The measurement results describe the average value (n = 4) for each experimental group, with the value for Group 1 described as 100. “**” in Table 3 indicates a significant difference (Student's t-test, p < 0.01) compared to the measurement results of Group 2. Compared to Group 2, at least in the group (Group 4) supplemented with extracellular vesicles (prepared from yeast prepared by culturing according to Table 2 under this main culture at 25°C (conditions for subjecting yeast to heat shock)), it was confirmed that the cell proliferation ability of normal human epidermal keratinocytes was high (the cell proliferation ability was restored). From the results of this Experiment 2, it was also suggested that “extracellular vesicles derived from a predetermined yeast have an effect of protecting normal human epidermal keratinocytes from stress”.

Industrial Applicability

[0061] The plant-derived extracellular vesicles provided by the present invention are excellent in skin permeability, and not only have excellent skin improvement effects such as skin moisturizing, skin whitening, wrinkle improvement and anti-aging during absorption, but also are safe without irritation or side effects on the skin.

Claims

1. Extracellular vesicles with an average particle size of 100 to 200 nm, derived from heat-shocked yeast, wherein the yeast is Pichia naniganshii isolated from the sap of Camellia japonica, and the average particle size is 170 nm or more.

2. Extracellular vesicles with an average particle size of 100 to 200 nm, derived from heat-shocked yeast, wherein the yeast is Pichia membranaefaciens derived from apple, and the average particle size is 180 nm or more.

3. Extracellular vesicles with an average particle size of 100 to 200 nm, derived from heat-shocked yeast, wherein the yeast is Pichia veronae isolated from the sap of Quercus mongolica, and the average particle size is 120 nm or more.

4. An external preparation for protecting the skin from stress, containing the extracellular vesicles according to any one of Claims 1 to 3.

5. A method for producing extracellular vesicles having a skin stress-protecting effect, comprising: culturing Pichia naniganshii isolated from the sap of Camellia japonica at a temperature of 36 to 38 °C to obtain a culture solution, or culturing Pichia membranaefaciens derived from apple or Pichia veronae isolated from the sap of Quercus mongolica at a temperature of 25 °C to obtain a culture solution; removing the cells from the culture solution to recover the culture supernatant; recovering extracellular vesicles from the culture supernatant; A method for producing extracellular vesicles, comprising the above steps.

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