Method for producing exosomes with improved yield using an high pressure homogenizer

Abstract

The present invention relates to a method for producing exosomes with improved yield using a high-pressure nanohomogenizer, and more specifically, to a method for producing brewer's yeast exosomes comprising the steps of: culturing brewer's yeast; grinding the culture solution at 30,000 to 40,000 psi using a high-pressure nanohomogenizer; centrifuging the ground solution; and ultrafiltering the supernatant after centrifugation. Through the above steps, brewer's yeast exosomes are manufactured economically and efficiently with high yield, and are particularly effective in that they are non-cytotoxic and have a high cell proliferation rate. In addition, they have excellent antioxidant capacity and whitening effects.

Description

Technology Field

[0001] The present invention relates to a method for producing exosomes with improved yield using a high-pressure nanohomogenizer, and more specifically, to a method for producing brewer's yeast exosomes comprising the steps of: culturing brewer's yeast (Saccharomyces cerevisiae) at room temperature to 43°C for 15 to 60 minutes; grinding the culture medium at 30,000 to 40,000 psi using a high-pressure nanohomogenizer; centrifuging the ground medium; and ultrafiltering the supernatant after centrifugation. Background Technology

[0002] With recent economic growth and rising interest in quality of life, interest in health and beauty is also increasing. Consequently, the cosmetics industry as a whole is showing robust growth compared to other manufacturing sectors. The Korean cosmetics market is growing day by day, and there is active movement toward entering the global market.

[0003] As customers' overall knowledge and demands regarding cosmetics increase, the importance of cosmetic ingredients is also rising, and active research is being conducted on this topic.

[0004] In the cosmetics industry, numerous raw materials have been developed using plant and animal resources. When classifying these developed substances chronologically, the first generation (Extract) is dominated by extracts utilizing water-containing solvents. The second generation (Fermentation) refers to the fermentation of substances obtained from the first generation using various microorganisms. The third generation (Bioconversion) is not simple fermentation but a process that uses specific microorganisms to alter only the specific structure desired by the developer. The fourth generation (Plant Stem Cell) is a method that utilizes plant calluses—marketed as "plant stem cells"—to cultivate natural substances in large quantities in a laboratory that are difficult to harvest from nature or are expensive. To date, plant-based cosmetic materials utilizing technologies from the first to the fourth generations have constituted the mainstream.

[0005] However, although exosomes were first discovered in reticulocytes, the immature red blood cells of mammals, by Professor Philip Stahl's team in 1983, they did not receive much attention because their biological significance or relationship with disease was unknown. It was not until 1987, when Professor Rose M. Johnstone's team studied their various mechanisms of action, that the aforementioned name came into use. It was revealed that during the maturation process of red blood cells, exosomes play a role in removing intracellular proteins. This is referred to as the fifth generation.

[0006] Starting from this point, the field of research on exosomes has expanded since 2010, and active research is being conducted in medical fields such as biological phenomena, disease diagnosis, bioinformatics, and drug delivery systems.

[0007] Exosomes are information-exchanging substances generated within a cell and released outside the cell. They range in size from 50 to 150 nm and are contained within vesicles; they are transferred to target cells through endocytosis via exocytosis on the cell surface. Therefore, the term exosome is a compound of EXO and Some, an abbreviation of Extra + Cellular + Vesicle, meaning a spherical particle that emerges from the cell.

[0008] Exosomes are important substances involved in the information, structure, and creation of stem cells, and are also called the smallest messengers for skin cells.

[0009] In the past, the prevailing view in the scientific community was that exosomes were cellular waste or cellular feces. For this reason, exosomes were considered worthless substances; however, in recent years, after it was confirmed that they contain large amounts of cell growth factors and genetic material, which are components related to life-sustaining mechanisms, they have begun to be applied in the cosmetics industry.

[0010] It is evident that when using substances such as lactic acid bacteria and yeast in cosmetics, exosomes, which are in the nanometer range, are much more easily absorbed than these substances, which are in the micrometer range. The easiest way for active ingredients in cosmetics to be absorbed into the skin is through pores. Generally, the diameter of pores is known to be 20,000 to 50,000 nm, and since this is thousands of times smaller than the typical size of exosomes, which is 50 to 150 nm, exosomes have a size that facilitates absorption.

[0011] While stem cell culture media can be used in cosmetics, stem cells themselves cannot be used due to various potential side effects. However, it has been discovered that culture media containing cultured stem cells also possess various effects similar to those of the stem cells themselves, leading to the active use of stem cell culture media in cosmetics recently. It is reported that this effect occurs because exosomes migrate from one stem cell to another and remain in the culture medium.

[0012] Conventional technologies for isolating such exosomes include exosome isolation kits, ultrafiltration, centrifugation, density gradient centrifugation, polymer-based precipitation, and size exclusion chromatography.

[0013] However, to date, the development of various exosomes for application in the cosmetics industry is still minimal, and due to the very complex separation and purification processes and low yields required to obtain cell-derived exosomes, exosome materials are currently sold at very high prices. Therefore, in order to utilize exosomes more actively in the cosmetics industry, it is time for innovative manufacturing methods different from existing ones. Prior art literature

[0014] Korean Registered Patent No. 10-1895916 (2018.08.31.) Korean Published Patent No. 10-2018-0003344 (2018.01.09.) Korean Registered Patent No. 10-1807081 (2017.12.04.) Korean Registered Patent No. 10-2125567 (2020.06.16.) Korean Registered Patent No. 10-2008667 (2019.08.02.) Korean Registered Patent No. 10-1927701 (2018.12.05.)

[0015] Ahn In-sook et al., “Inhibition of Melanin Production by L-cysteine ​​in B16F10 Mouse Melanoma Cells”, Journal of the Korean Society of Cosmetology and Dermatology, Vol. 5, No. 2, pp. 239–246, 2007. The problem to be solved

[0016] The objective of the present invention is to provide a method for economically and efficiently isolating exosomes from brewer's yeast with a high yield.

[0017] Another objective of the present invention is to provide a cosmetic composition comprising the above-mentioned beer yeast exosomes. means of solving the problem

[0018] The objective of the present invention is achieved by providing a method for producing high-yield brewer's yeast exosomes, comprising the steps of: culturing brewer's yeast at room temperature to 43°C for 15 to 60 minutes; grinding the culture medium at 30,000 to 40,000 psi using a high-pressure nanohomogenizer; centrifuging the ground medium; and ultrafiltering the supernatant after centrifugation.

[0019] The present invention is characterized by repeating the grinding step three or more times.

[0020] The present invention is characterized by selecting Saccharomyces cerevisiae as the beer yeast.

[0021] In addition, the present invention provides brewer's yeast exosomes having a particle size of 100 to 150 nm in diameter, manufactured by the above manufacturing method.

[0022] The above brewer's yeast exosomes provide a cosmetic composition for skin regeneration characterized by increased survival rates of keratinocytes and fibroblasts in the WST-1 assay.

[0023] The above-mentioned beer yeast exosomes provide an antioxidant cosmetic composition characterized by a concentration-dependent increase in the free radical scavenging rate in a DPPH assay.

[0024] The above brewer's yeast exosomes provide a cosmetic composition for skin whitening characterized by a concentration-dependent increase in the inhibition rate of melanin formation.

[0025] The present invention comprises 1.0 to 5.0 weight percent of the brewer's yeast exosome based on the total weight of the cosmetic composition. Effects of the invention

[0026] According to the present invention, the method for producing exosomes using an ultra-high pressure nanohomogenizer has the advantage of efficiently separating exosomes in high concentrations at an economical and low cost.

[0027] In addition, according to the method of the present invention, exosomes with excellent antioxidant and skin whitening effects compared to conventional separation methods and cosmetic compositions containing such exosomes can be produced in large quantities. In particular, it exhibits an excellent effect of providing exosomes that are non-cytotoxic and have a high cell proliferation rate. Brief explanation of the drawing

[0028] Figure 1 shows microscopic images of brewer's yeast exosomes according to the present invention identified as Olympus CX3 according to pressure size. Figure 2 shows the average particle size of brewer's yeast exosomes analyzed by Nanoparticle Tracking Analysis (NTA, NanoSight NS300) according to the present invention. Figure 3 is a graph showing the cytotoxicity evaluation of brewer's yeast extract and exosomes prepared through an example of the present invention, measured according to concentration. Figure 4 is a graph showing the antioxidant activity measured by DPPH Assay of Brewer's yeast extract and exosomes prepared through an example of the present invention. Figure 5 is a graph showing the melanin synthesis inhibition (Melanin contents assay) effect of brewer's yeast extract and exosomes prepared through an example of the present invention, measured according to concentration. Specific details for implementing the invention

[0029] Preferred embodiments and experimental examples of the present invention are described in detail below. This description is intended to enable a person skilled in the art to easily practice the invention, and does not imply that the technical scope and concept of the present invention are limited thereby.

[0030] Exosomes are known to exist in the cells of living animals and plants as well as in saliva. However, in the present invention, exosomes were isolated from beer yeast cells that are discarded after beer fermentation.

[0031] The Saccharomyces yeast used in the embodiments of the present invention, which is a byproduct of beer fermentation, is described as being limited to Saccharomyces cerevisiae, but is not limited thereto. This is obtained by separating the yeast separately and drying it during the process of making beer.

[0032] Accordingly, the yeast of the genus Saccharomyces used in the present invention may be any one selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces euvarum, Saccharomyces ellipsoideus, Saccharomyces carlosbergensis, Saccharomyces sake, Saccharomyces coreanus, Saccharomyces repolitica, Saccharomyces boulardii, and Saccharomyces pastorianus, or a mixture of two or more types.

[0033] The aforementioned brewer's yeast (Saccharomyces yeast) contains protein, B vitamins, and minerals such as chromium and selenium; in particular, the B vitamins abundant in brewer's yeast help maintain healthy skin. The same applies to selenium, chromium, and protein.

[0034] Here, the Vitamin B complex aids in cell replication, while chromium helps skin cells obtain energy from fats, carbohydrates, and proteins. In particular, it is highly effective in preventing hair loss as it contains biotin—a Vitamin B group essential for protein metabolism in hair growth—as well as cystine and methionine, which constitute hair protein. Furthermore, it helps boost immunity and prevent aging by promoting the production of immune cells. Therefore, it can be usefully applied in the cosmetics industry where antioxidants are required.

[0035] Meanwhile, the Saccharomyces yeast decomposition extract of the present invention can generally be extracted using water or proteolytic enzymes such as trypsin or protease, but is not limited thereto.

[0036] In the method for preparing a Saccharomyces genus yeast decomposition extract of the present invention, culture is performed at room temperature up to a maximum of 43°C, most preferably at 43°C for 15 to 60 minutes, and may be extended up to 72 hours when performed at room temperature. Above, if the culture temperature exceeds 43°C, it is undesirable as there is a concern about denaturation of protein enzymes, and the culture time is determined according to the culture temperature. When the culture temperature is 43°C, the culture is performed for 15 to 60 minutes, more preferably for 30 to 60 minutes, and the active ingredient derived from Saccharomyces genus dried yeast can be extracted at a high concentration.

[0037] Accordingly, the culture time is determined in an inverse relationship with the above culture temperature, and is determined by considering extraction efficiency so that the active ingredient can be extracted at a high concentration within a short period of time during the culture step.

[0038] Because exosomes are so small that it is difficult to weigh them, their separation and purification are time-consuming, cumbersome, and costly.

[0039] Although the method using existing exosome isolation kits is very simple, there are difficulties in commercialization due to the use of expensive single-use kits. In other words, it is a method of isolating exosomes by adding a substance that can bind to exosomes, but there are limitations to industrialization due to the inconvenience of having to remove the binding substance after obtaining the exosomes, as well as the increase in production costs resulting from obtaining a small amount of exosomes (up to 100 ml) and producing them in small quantities.

[0040] Furthermore, ultrafiltration is a method for separating fine substances by size, and allows for relatively mass production compared to the aforementioned separation kit. Situated in the intermediate range between microfiltration and reverse osmosis, it is a method that separates specific substances based on the size difference between the membrane pores and the solute. However, this requires very expensive equipment, and since the membrane filters are disposable, it necessitates both initial investment and continuous investment. Additionally, there are disadvantages, such as the type of membrane filter varying depending on the type of cell to be separated, and a limited capacity for separation.

[0041] Currently, centrifugation is the most widely used method, which is a technique that separates particles by weight by increasing the rotation speed of the centrifuge in the order of 300G (low speed), 2,000G (medium speed), 10,000G (high speed), and 100,000G (ultra-high speed). However, centrifuges at high speed or higher can process less than 3kg at a time, and since centrifugation must be performed repeatedly, this also has limitations in obtaining large quantities of exosomes.

[0042] In addition, a combination of centrifugation and ultrafiltration is used to improve yield.

[0043] Accordingly, the present invention used an ultra-high pressure nano homogenizer to improve high yield and manufactured exosomes by combining centrifugation and ultrafiltration methods.

[0044] A method for producing exosomes according to the present invention comprises: a step of culturing brewer's yeast; a step of grinding the culture solution at 10,000 to 40,000 psi using a high-pressure nanohomogenizer; a step of centrifuging the ground solution; and a step of ultrafiltration of the supernatant after centrifugation. At this time, it is most preferable to perform the grinding step at a pressure of 30,000 to 40,000 psi. Furthermore, the grinding step is characterized by being repeated three or more times.

[0045] Although the cell walls of microorganisms begin to break down gradually even at 10,000 to 15,000 psi, if they are crushed at ultra-high pressure of 30,000 to 40,000 psi, both exosomes inside and outside the cell can be obtained. In this process, unnecessary bacteria and fungi are also sterilized, and compared to existing technology, it has the advantage of higher productivity and yield and reduced working time.

[0046] In other words, due to the technical configuration that includes a step of grinding in this ultra-high pressure range, it was possible to obtain high-yield exosomes that could not be obtained previously.

[0047] In a manufacturing method according to one example of the present invention, when the manufactured exosomes were subjected to a WST-1 assay using keratinocytes and fibroblasts, the survival rate of both keratinocytes and fibroblasts decreased when brewer's yeast extract was used at a concentration of 10.0% or higher. However, brewer's yeast exosomes showed results in which cells did not die and proliferated within the experimental design concentration. Through this, it can be inferred that brewer's yeast exosomes have no cytotoxicity and possess excellent cell proliferation effects, indicating that they have cell regeneration capabilities.

[0048] In a manufacturing method according to one example of the present invention, the antioxidant capacity (DPPH assay) of the prepared brewer's yeast extract and brewer's yeast exosomes was compared and evaluated, and the antioxidant capacity of the exosomes was superior to that of the extract at all concentrations. That is, it means that the antioxidant effect increases as the free radical scavenging rate increases.

[0049] In a manufacturing method of one example of the present invention, the melanin synthesis inhibitory ability of the prepared brewer's yeast extract and brewer's yeast exosomes was compared and evaluated, and it was confirmed that the melanin synthesis inhibitory effect of the exosomes was superior to that of the extract, and that the exosomes had a higher whitening effect.

[0050] Therefore, it can be applied in the field of cosmetics where antioxidant or anti-inflammatory effects are required, or for skin whitening and skin regeneration effects.

[0051] Another embodiment of the present invention may be prepared using the manufacturing method described above, comprising an extract and exosomes produced from brewer's yeast and a cosmetic composition containing the same. Here, the cosmetic composition preferably contains about 0.1 to 10.0 weight%, preferably 1.0 to 5.0 weight%, of the brewer's yeast exosomes based on the total weight.

[0052] In addition to exosomes, the above cosmetic composition may further include functional additives and ingredients included in general cosmetic compositions. The above functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts. Other ingredients included in the formulation may include oil components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohols, colorants, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

[0053] The above cosmetic composition is not specifically limited in its formulation and can be appropriately selected according to the purpose. For example, it may be manufactured in one or more formulations selected from the group consisting of skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto.

[0054] The present invention will be explained in more detail below through examples.

[0055] Example 1. Exosomes according to the present invention

[0056] Exosomes were isolated from beer fermented with Saccharomyces cerevisiae (Leibar yeast). Specifically, active ingredients were extracted at a high concentration after culturing in synthetic complete medium with shaking at 43°C for 45 minutes.

[0057] The above-mentioned extract culture solution was ground at 10,000 psi at least three times using a high-pressure nanohomogenizer (also called a nanogenizer). The experimental results were observed using a microscope (OLYMPUS, CX-33) to examine the particle state of the brewer's yeast (see Fig. 1). After grinding, the solution was first centrifuged at 10,000 rpm for 30 minutes (Hanil Science Industrial, Continent 512R Plus), and the supernatant was ultrafiltered to collect brewer's yeast exosomes.

[0058] Example 2. Exosomes according to the present invention

[0059] The procedure was performed in the same manner as Example 1, except that the pressure of the ultra-high pressure nanohomogenizer of Example 1 was treated three times at 15,000 psi.

[0060] Example 3. Exosomes according to the present invention

[0061] The procedure was performed in the same manner as Example 1, except that the pressure of the ultra-high pressure nanohomogenizer of Example 1 was treated three times at 20,000 psi.

[0062] Example 4. Exosomes according to the present invention

[0063] The procedure was performed in the same manner as Example 1, except that the pressure of the ultra-high pressure nanohomogenizer of Example 1 was treated three times at 25,000 psi.

[0064] Example 5. Exosomes according to the present invention

[0065] The procedure was performed in the same manner as Example 1, except that the pressure of the ultra-high pressure nanohomogenizer of Example 1 was treated three times at 30,000 psi.

[0066] Example 6. Exosomes according to the present invention

[0067] The procedure was performed in the same manner as Example 1, except that the pressure of the ultra-high pressure nanohomogenizer of Example 1 was treated three times at 35,000 psi.

[0068] Looking at the micrograph in Figure 1, it can be seen that the cell walls of brewer's yeast are gradually destroyed at 15,000 psi, and above 30,000 psi, a large number of cell walls are destroyed compared to other pressures. In particular, when treated three times at 35,000 psi, it was confirmed that all brewer's yeast cells except for one were destroyed. This pressure condition shows the most desirable particle state.

[0069] That is, the brewer's yeast exosomes collected according to Example 6 (35,000 psi) have a transparent liquid form, a pH of 6.60 ± 0.2, an average particle size of 101.5 nm, and a obtained concentration of 420,000,000,000 particles / ml, which is 24 times higher than the concentration of 17,400,000,000 particles / ml of exosomes collected according to Example 2 (15,000 psi). Specific experimental results for this are shown in Table 1 and Figure 2.

[0070] division High pressure range (psi) Average particle size (nm) Exosome concentration (particles / ml) Example 1 10,000 147.8 18,100,000,000 Example 2 15,000 119.8 17,400,000,000 Example 3 20,000 102.6 112,000,000,000 Example 4 25,000 107.0 226,000,000,000 Example 5 30,000 107.2 334,000,000,000 Example 6 35,000 101.5 420,000,000,000

[0072] Comparative Example 1. Beer yeast extract according to the conventional method

[0073] This relates to a simple extract of brewer's yeast, and a high-pressure homogenizer was not used. Specifically, the same concentration as the yeast used to prepare the exosomes in the above example was mixed with purified water and extracted at 70–75°C for 3 hours. The extract obtained in this way was subjected to centrifugation and ultrafiltration to remove undissolved solids, thereby finally producing a brewer's yeast extract.

[0075] Experimental Example 1. Cytotoxicity Evaluation (WST-1 assay)

[0076] A WST-1 assay was performed to confirm the cell proliferation and cell viability of the brewer's yeast exosomes prepared in Example 6 of the present invention and the brewer's yeast extract prepared in Comparative Example 1. 200 μL of 1 × 10⁴ cells / well was dispensed into each well of a 96-well plate and cultured for 24 hours in an incubator at 37°C and 5% CO₂. After culture, DMEM medium without FBS was added to Example 6 and Comparative Example 1 at various concentrations and reacted for 24 hours. 20 μL of WST-1 solution (Cell Proliferation Reagent WST-1) was added to each well to make up 10% of the total volume, and after reacting for 2 hours in a cell incubator at 37°C and 5% CO₂, absorbance was measured at 450 nm and 620 nm using a microplate spectrophotometer. Cell viability was calculated based on the control group.

[0077] As a result, brewer's yeast extract showed an effect of proliferating both keratinocytes and fibroblasts up to a concentration of 10%, but from that concentration onwards, it showed a rapid decrease in both keratinocytes and fibroblasts. However, brewer's yeast exosomes showed results of concentration-dependent proliferation of both keratinocytes and fibroblasts (see Fig. 3).

[0078] Experimental Example 2. Antioxidant effect (DPPH Assay)

[0079] The DPPH method measures the antioxidant effect due to reducing power using a free radical called DPPH (2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl) free radical. In Example 6 and Comparative Example 1, the degree to which the absorbance decreases due to the reduction of DPPH is compared with the absorbance of the blank solution to measure the free radical scavenging rate (%) at a wavelength of 560 nm. As the reagent used, 61.88 mg of a 0.1 mM solution of 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl) free radical (Sigma Aldrich, MW=618.76) was dissolved in ethanol to make 100 mL. 100 μl (0.4 mM) of DPPH solution dissolved in ethanol and equal amounts of the exosomes of Example 6 and the extract of Comparative Example 1 at concentrations of 0.0% to 100.0% were added to a 96-well microplate and reacted at 37°C for 30 minutes. After the reaction, DPPH radical scavenging activity was measured by measuring the absorbance at 560 nm using an enzyme-linked immunosorbent assay (ELISA).

[0080] The result of the free radical scavenging rate (%) was calculated using the following formula.

[0081] Free radical scavenging rate (%) = [1 - (St - S0) / (Bt - B0)] × 100

[0082] St: Absorbance at 560 nm after free radical scavenging of the sample solution

[0083] Bt: Absorbance at 560 nm of the blank solution after free radical scavenging

[0084] S0: Absorbance at 560 nm before reaction when free radicals are not added to the sample solution

[0085] B0: Absorbance at 560 nm before reaction of the blank solution without free radical addition

[0086] As a result, the antioxidant effect of the beer yeast exosome of Example 6 was confirmed to be superior to that of the beer yeast extract of Comparative Example 1 at all concentrations (see Fig. 4).

[0087] Experimental Example 3. Melanin formation inhibitory effect (Melanin contents assay)

[0088] The exosomes of Example 6 and the extract of Comparative Example 1 were added to the culture medium of B16F10 mouse melanoma cells to test the whitening effect at the cellular level. Brewer's yeast extract and brewer's yeast exosomes were added to the culture medium of B16F10 mouse melanoma at concentrations of 1.0%, 5.0%, and 10.0%, and cultured for 24 hours. Afterward, attached and growing cells were treated with trypsin-EDTA solution to detach them from the culture vessel, centrifuged, and the generated melanin was extracted. The melanin was dissolved by adding 1 mL of 1N NaOH solution to the extracts and exosomes and boiling for 10 minutes. After cooling to room temperature, the absorbance was measured at 400 nm using a spectrophotometer, and the amount of generated melanin was expressed as absorbance per unit cell (104 cells). The relative amount of melanin produced relative to the control group (β-arbutin, 1.0%) was calculated as an inhibition rate, and the results are shown in Figure 5.

[0089] As a result, brewer's yeast exosomes showed a lower inhibitory effect on melanin production at the same concentration compared to β-arbutin, a single component used as a control and a notified ingredient for whitening function, but showed a higher inhibitory effect on melanin synthesis at the same concentration compared to brewer's yeast extract prepared by a general extraction method. In other words, the whitening effect of brewer's yeast exosomes can be considered to be 2 to 4 times superior.

[0091] Accordingly, the method for producing brewer's yeast exosomes with improved yield according to the present invention utilizes an ultra-high pressure nano homogenizer, thereby obtaining both exosomes inside the cell that have not yet been released and exosomes that have already been released outside the cell, thus increasing productivity and yield, and providing exosomes with almost no skin toxicity due to the effect of sterilizing all unnecessary bacteria, etc.

[0092] Furthermore, compared to beer yeast extracts used in the industry, it exhibited no cytotoxicity and demonstrated excellent concentration-dependent cell proliferation ability. Through this, it provides the regenerative capacity of damaged keratinocytes, as well as superior antioxidant and whitening effects compared to beer yeast extracts.

[0093] Although the present invention has been described in detail above with reference to embodiments, it is obvious to those skilled in the art that various modifications and variations are possible within the scope of the technical spirit of the invention, and such modifications and variations are naturally included in the appended claims.

Claims

Claim 1 A method for producing high-yield exosomes having an average particle size of 101 to 107 nm in diameter, comprising: a step of culturing brewer's yeast at room temperature to 43°C for 15 to 60 minutes; a step of grinding the culture medium at 30,000 to 35,000 psi using a high-pressure nanohomogenizer; a step of centrifuging the ground liquid; and a step of ultrafiltering the supernatant after centrifugation; wherein the grinding step is repeated three or more times, and the brewer's yeast is Saccharomyces cerevisiae. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 A cosmetic composition for skin regeneration characterized by containing 1.0 to 5.0 weight% of brewer's yeast exosomes produced by the method of claim 1 as an active ingredient, and increasing the survival rate of keratinocytes and fibroblasts in a WST-1 assay. Claim 10 An antioxidant cosmetic composition comprising 1.0 to 5.0 weight% of brewer's yeast exosomes prepared by the method of claim 1 as an active ingredient, characterized by a concentration-dependent increase in the free radical scavenging rate in a DPPH assay. Claim 11 A cosmetic composition for skin whitening characterized by containing 1.0 to 5.0 weight% of brewer's yeast exosomes produced by the method of claim 1 as an active ingredient, and having a melanin formation inhibition rate that increases in a concentration-dependent manner.

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