Composition for skin improvement

Abstract

The present invention relates to a cosmetic composition for skin improvement, comprising Lactobacillus plantarum-derived extracellular vesicles. The cosmetic composition for skin improvement, of the present invention, exhibits the effects of skin elasticity improvement, skin wrinkle reduction, skin moisturization, skin soothing or skin regeneration, and thus can be used for skin condition improvement.

Description

Composition for skin improvement

[0001] The present invention relates to a cosmetic composition comprising an artificial exosome derived from Lactobacillus plantarum and a method for producing the same.

[0002] Extracellular vesicles (EVs) are small membrane-enclosed sacs released by cells. They play a crucial role in cell-to-cell communication by transporting various biomolecules, such as proteins, nucleic acids, and lipids, between cells. EVs can deliver their own material to recipient cells, thereby regulating various cellular processes, including cell signaling, immune response regulation, and gene regulation. EVs can either directly fuse with the cell membrane or be taken up by recipient cells through endocytosis. EVs have also attracted significant attention in biomedical research and clinical applications. They have been found in various bodily fluids, including blood, urine, and cerebrospinal fluid, and can be used as diagnostic biomarkers for various diseases, including cancer, neurodegenerative diseases, and cardiovascular diseases. Furthermore, EVs also have therapeutic potential as drug delivery vehicles and a source of regenerative molecules.

[0003] There are several types of extracellular vesicles, including exosomes, microvesicles, and apoptotic bodies. Exosomes are the smallest type, measuring 30 to 150 nanometers. They are intraluminal vesicles formed when the endosomal membrane of multivesicular endosomes inwardly in the maturation process, and are secreted when multivesicular endosomes fuse with the cell surface. Exosomes contain various biologically active substances, such as proteins, lipids, nucleic acids, and metabolites, and reflect the state of the cells they originate from. Exosomes function as intercellular signaling molecules secreted by cells, and are known to play a role in cell-to-cell communication because they contain nuclear components as well as receptors and proteins. Furthermore, because exosomes contain bioactive molecules, they can easily induce physiologically active effects in target cells by fusion with them and delivering these molecules. Furthermore, because exosomes are derived from cells, they have low immunogenicity and possess a membrane topology identical to that of cells, allowing them to readily attach to target cells. These characteristics make exosomes useful in a variety of applications, including cancer diagnosis, treatment, and drug delivery. Their potential for tissue regeneration and recovery is also contributing to the growing use of exosomes in cosmetics.

[0004] However, existing exosomes require specialized culture systems to promote exosome production and specialized processes for extracting exosomes from cells. Furthermore, expensive, specialized processes for concentrating and isolating exosomes are required, resulting in low reproducibility and yield. Therefore, the development of artificial exosomes that address these issues is urgently needed.

[0005] To address the above-described issues, the inventors of the present invention have conducted extensive research efforts to produce artificial exosomes with high yields without requiring a special culture system or exosome purification process. As a result, the present inventors isolated and identified a Lactobacillus plantarum strain suitable for artificial exosome production, isolated and produced Lactobacillus plantarum-derived extracellular vesicles through high-pressure homogenization, and used them as artificial exosomes for skin improvement, thereby completing the present invention.

[0006] The purpose of the present invention is to provide a Lactobacillus plantarum OD11 strain deposited under the accession number KCTC16121BP.

[0007] Another object of the present invention is to provide a cosmetic composition for improving skin, comprising as an active ingredient Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP; and a culture, a pulverized product or an extract thereof.

[0008] Another object of the present invention is to provide a cosmetic composition comprising extracellular vesicles manufactured by including a step of subjecting a sample including a Lactobacillus plantarum strain to high-pressure homogenization.

[0009] Another object of the present invention relates to the use of extracellular vesicles derived from Lactobacillus plantarum for improving skin.

[0010] Another object of the present invention is to provide a use of a composition comprising Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP for the manufacture of a cosmetic composition for improving skin; and a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

[0011] Another object of the present invention is to provide a skin improvement method comprising a step of applying a skin improvement cosmetic composition comprising an effective amount of Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP; and a culture, a pulverized product or an extract thereof as an effective ingredient, to the skin of a subject in need thereof.

[0012] Another object of the present invention is to provide a method for improving skin, comprising a step of applying a cosmetic composition comprising extracellular vesicles prepared by subjecting a sample containing an effective amount of a Lactobacillus plantarum strain to the skin of a subject in need thereof, including a step of subjecting the sample to high-pressure homogenization.

[0013] Hereinafter, the configuration of the present invention will be described in detail.

[0014] One aspect of the present invention is the Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP.

[0015] In the present invention, “Lactobacillus plantarum” is a Gram-positive bacterium belonging to the Lactic Acid Bacteria (LAB) family, and is isolated from fermented foods such as kimchi, soybean paste, yogurt, cheese, and sauerkraut. It is a Gram-positive bacterium with a rod shape (bacillus), has facultative anaerobic characteristics that allow it to survive even in oxygen-free conditions, mainly obtains energy by fermenting sugars, and can perform both homolactic fermentation and heterolactic fermentation. It grows well at 30 to 37°C and can survive up to 45°C. It shows strong resistance to an acidic environment (pH 3.2 to 4.0). It produces enzymes that can break down various carbohydrates, and some strains have been shown to metabolize complex polysaccharides, particularly starch, cellulose, and xylan. Its high tolerance to bile salts and gastric acid increases its potential as a probiotic, reaching the intestines alive. It grows well on Man, Rogosa, Sharpe (MRS) medium, is non-motile, lacks flagella, and does not form spores.

[0016] In the present invention, the Lactobacillus plantarum OD11 strain may have the 16S rRNA base sequence of sequence number 1, but is not limited thereto.

[0017] In the present invention, Lactobacillus plantarum OD11 may exist as live cells or dead cells, and may also exist in a dried or lyophilized form. In addition, a culture of the Lactobacillus plantarum OD11 strain may be an active ingredient, and the culture may include a live cell culture solution or a dead cell supernatant. The form and formulation method of lactic acid bacteria suitable for inclusion in various compositions are well known to those skilled in the art. For example, the Lactobacillus plantarum OD11 strain may be a culture obtained by culturing in a known liquid medium or solid medium, a fermented product obtained by culturing the strain together with an additional component, an extract obtained by extracting the strain with an organic solvent, a lysate (or lysate) obtained by dissolving, disrupting, or homogenizing the cell membrane of the strain, etc., but is not limited thereto.

[0018] According to the following examples, artificial exosomes derived from Lactobacillus plantarum OD11 showed high efficacy in improving wrinkles, synthesizing hyaluronic acid, anti-inflammation, wound healing, and cell penetration.

[0019] Another aspect of the present invention is a cosmetic composition for improving skin, comprising as an active ingredient Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP; and a culture thereof, a lysate thereof, an extract thereof, or an extracellular vesicle thereof.

[0020] In the present invention, the extracellular vesicles may be naturally secreted from Lactobacillus plantarum or artificially manufactured, but are not limited thereto.

[0021] In the present invention, the extracellular vesicles may be isolated from a Lactobacillus plantarum culture, but are not limited thereto.

[0022] In the present invention, extracellular vesicles can be isolated using at least one method selected from the group consisting of high pressure treatment, centrifugation, ultra-high speed centrifugation, heat treatment, extrusion, sonication, cell lysis, homogenization, freeze-thawing, electroporation, mechanical dissociation, chemical treatment, filtration by filter, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis of a culture containing bacterial cells. In addition, the method may further include a process such as washing to remove impurities and concentrating the obtained extracellular vesicles.

[0023] The term “culture” in this specification may mean the entire medium including the strain, its metabolites, and extra nutrients obtained by culturing the strain for a certain period of time in a medium that can supply nutrients so that the strain can grow and survive in a test tube.

[0024] The term “extracellular vesicle (EV)” in this specification is used to encompass membrane vesicles, exosomes, ectosomes, shedding vesicles, microparticles, microvesicles, or equivalents thereof. Depending on the separation environment, conditions, and method, an extracellular vesicle may have the same meaning as an exosome, or may include nanovesicles that are the same or similar in size to an exosome but do not have the composition of an exosome, or may mean something similar in composition to an exosome but do not have the size of an exosome.

[0025] The term “exosome” used herein refers to a small vesicle or vesicle secreted from a cell and released into the extracellular space. It is divided into an inside and an outside by a lipid bilayer membrane, and contains membrane lipids, membrane proteins, nucleic acids, cytoplasmic components, etc., so it can indirectly determine the properties and state of a cell. Exosomes act as messengers that mediate cell-to-cell communication by delivering membrane components, mRNAs, miRNAs, proteins, etc. to recipient cells.

[0026] In one specific embodiment of the present invention, extracellular vesicles may exhibit an effect in improving wrinkles. As shown in Example 2-1 of the present invention, it was confirmed that extracellular vesicles, when treated with photoaging-induced fibroblasts, increased the expression of COL1A1 and ELN, and decreased the expression of MMP1.

[0027] In one specific embodiment of the present invention, extracellular vesicles can exhibit hyaluronic acid synthesis efficacy. As shown in Example 2-2 of the present invention, it was confirmed that extracellular vesicles enhance hyaluronic acid synthesis capacity in keratinocytes.

[0028] In one specific embodiment of the present invention, extracellular vesicles may exhibit anti-inflammatory effects. As shown in Examples 2-3 of the present invention, it was confirmed that extracellular vesicles significantly reduced the expression of inflammatory cytokines IL-6 and IL-1α.

[0029] In one specific embodiment of the present invention, extracellular vesicles may exhibit wound healing efficacy. As shown in Example 2-4 of the present invention, it was confirmed that extracellular vesicles significantly reduced wounds by promoting the proliferation of fibroblasts.

[0030] In one specific embodiment of the present invention, extracellular vesicles can exhibit the efficacy of increasing cell permeability. As shown in Example 2-5 of the present invention, it can be confirmed that the extracellular vesicles exhibit increased cell permeability compared to the control group (a group not treated with extracellular vesicles).

[0031] In one specific embodiment of the present invention, extracellular vesicles can exhibit skin improvement effects in human efficacy evaluations. As shown in Example 3 of the present invention, extracellular vesicles were found to exhibit improved effects in wrinkle improvement, moisturizing rate, and pore area reduction.

[0032] In one specific embodiment of the present invention, extracellular vesicles may exhibit collagen synthesis-promoting properties and boosting effects. As shown in Example 4 of the present invention, it was confirmed that extracellular vesicles exhibited a collagen synthesis-enhancing effect when treated with adenosine and HSA (10-hyroxystearic acid) compared to when treated alone.

[0033] As used herein, “improvement” means any action that reduces a parameter related to the condition being treated, for example, the severity of a symptom.

[0034] In this specification, “prevention” refers to any act of inhibiting or delaying the occurrence of a disease by administering or prescribing a composition according to the present invention.

[0035] In the present invention, improvement in skin condition may include improvement in skin elasticity, improvement in skin wrinkles, skin moisturizing, skin soothing, or skin regeneration, but is not limited thereto.

[0036] In the present invention, skin soothing may be soothing the skin from one or more inflammatory diseases selected from the group consisting of atopic dermatitis, contact dermatitis, allergic dermatitis, acne, eczema, rosacea, oily skin, psoriasis, eczema, pruritus, itching, urticaria, idiopathic chronic urticaria, scleroderma, vitiligo, intractable melasma, erythema, scleroderma, Behcet's disease, and contact dermatitis, but is not limited thereto.

[0037] In the present invention, skin regeneration may mean reducing wounds by inducing the promotion of proliferation of fibroblasts.

[0038] In the present invention, the extracellular vesicle may have a size of 30 to 500 nm, 150 to 400 nm, for example, 250 to 400 nm, but is not limited thereto.

[0039] In the present invention, the extracellular vesicles are 10 per unit volume of 1 ml of the composition. 9 10 inland 15 , for example, 10 10 10 inland 15 It may include, but is not limited to, the contents of.

[0040] In the present invention, the cosmetic composition may be manufactured in any formulation commonly manufactured in the technical field to which the present invention pertains. For example, it may be formulated as a softening toner, a nourishing toner, an emulsion, a lotion, a cream, a paste, a gel, a solution, a suspension, an oil, a wax, a pack, a lyophilization, a powder, a foundation, a spray, a surfactant-containing cleanser, etc., but is not limited thereto. More specifically, it may be manufactured in the form of a skin lotion, a skin softener, a skin toner, a nourishing cream, a massage cream, a milk lotion, a powder, an essence, an eye cream, a sun lotion, a sunscreen, a makeup primer, a makeup base, a BB cream, a powder foundation, an emulsion foundation, a cleansing cream, a cleansing foam, a cleansing water, a soap, a pack, a stick product, a balm-type product, a spray, a lyophilization, or a powder.

[0041] The above cosmetic composition may further comprise any conventional cosmetic ingredient selected from additional ingredients commonly used in cosmetics, such as thickeners, dispersants, fragrances, fillers, preservatives, antiseptics, neutralizers, sweeteners, vitamins, free-radical scavengers, metal ion sequestrants, functional ingredients, and mixtures thereof. A person skilled in the art can select any additional ingredient and / or its amount such that the advantageous properties of the composition according to the present disclosure are not adversely affected or substantially not affected by the anticipated addition.

[0042] When the above cosmetic composition is a surfactant-containing cleansing formulation, it may further include, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkylamidobetaine, an aliphatic alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a linolenic derivative, or an ethoxylated glycerol fatty acid ester.

[0043] When the cosmetic composition according to the present invention is in the form of a cream or gel, it may further include animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, hydrogel, bentonite, silica, talc, or zinc oxide as a carrier component.

[0044] When the above cosmetic composition is in the form of a solution or emulsion, it may further include a solvent, solvating agent or emulsifying agent, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, propylene glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0045] If the above cosmetic composition is in the form of a suspension formulation, it may further include a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth as a carrier component.

[0046] If the above cosmetic composition is in a powder or spray form, it may further include lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as a carrier component, and particularly if it is in a spray form, it may further include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0047] If the above cosmetic composition is a freeze-dried formulation, it may further include purified water, methionine, mannitol, etc. as carrier components.

[0048] The above cosmetic composition may be applied alone or in combination, or may be applied in combination with other cosmetic compositions other than those of the present invention. Furthermore, the cosmetic composition according to the present invention may be used according to conventional methods of use or in conjunction with medical devices or skin treatment methods / devices, and the frequency of use may vary depending on the user's skin condition or preference.

[0049] Another aspect of the present invention is a cosmetic composition comprising extracellular vesicles manufactured by including a step of subjecting a sample including a Lactobacillus plantarum strain to high-pressure homogenization.

[0050] In the present invention, Lactobacillus plantarum may be Lactobacillus plantarum OD11 deposited under accession number KCTC16121BP.

[0051] In the present invention, improvement in skin condition may include improvement in skin elasticity, improvement in skin wrinkles, skin moisturizing, or skin soothing, but is not limited thereto.

[0052] In the present invention, the high-pressure homogenization treatment step may be performed at a pressure of 1000 to 3000 bar, 1500 to 3000 bar, 2000 to 3000 bar, for example, 2000 bar, but is not limited thereto.

[0053] In the present invention, the high-pressure homogenization treatment step may be performed 1 to 7 times, for example, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3 times, but is not limited thereto.

[0054] In the present invention, high-pressure homogenization treatment can be performed under continuous pressure.

[0055] In the present invention, the high-pressure homogenization treatment step is performed at a set pressure for a certain amount of material, and may vary depending on the amount injected.

[0056] Another aspect of the present invention is the use of extracellular vesicles derived from Lactobacillus plantarum for skin improvement.

[0057] Another aspect of the present invention is the use of a composition comprising, as an active ingredient, Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP for the manufacture of a cosmetic composition for improving skin; and a culture, a pulverized product or an extract thereof.

[0058] In the present invention, skin improvement may be, but is not limited to, improvement in skin elasticity, improvement in skin wrinkles, skin moisturizing, or skin soothing.

[0059] Another aspect of the present invention is a method for improving skin, comprising the step of applying a cosmetic composition for improving skin, which comprises, as an effective ingredient, an effective amount of Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP; and a culture, a pulverized product or an extract thereof, to the skin of a subject in need thereof.

[0060] Another aspect of the present invention is a method for improving skin, comprising the step of applying a cosmetic composition comprising extracellular vesicles prepared by subjecting a sample containing an effective amount of a Lactobacillus plantarum strain to the skin of a subject in need thereof, including the step of subjecting the sample to high-pressure homogenization.

[0061] The present invention relates to a cosmetic composition for improving skin, comprising extracellular vesicles derived from Lactobacillus plantarum. The cosmetic composition for improving skin, according to the present invention, exhibits effects of improving skin elasticity, improving wrinkles, moisturizing, soothing, or regenerating skin, and can thus be utilized for improving skin condition.

[0062] FIG. 1 is a graph and diagram showing the results of measuring the physical properties of an artificial exosome according to an example of the present invention.

[0063] Figure 2 is a graph showing the results of evaluating the wound healing ability of fibroblasts treated with artificial exosomes according to an example of the present invention.

[0064] Figure 3 is a diagram showing the results of evaluating the cell permeability of artificial exosomes according to an example of the present invention.

[0065] Figure 4 is a diagram showing the results of evaluating the degree of wrinkle improvement after prescribing artificial exosomes according to an example of the present invention.

[0066] FIG. 5 is a diagram showing the results of evaluating the degree of wrinkle improvement after prescribing artificial exosomes according to an example of the present invention.

[0067] Figure 6 is a diagram showing the results of evaluating the pore area improvement rate after artificial exosome prescription according to an example of the present invention.

[0068] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0069] Manufacturing Example 1. Isolation and identification of strains

[0070] Organic mulberries grown in Buan, Jeollanam-do, were fermented with brown sugar in an earthenware jar at 25℃ for 7 days. The fermented mulberries were then ground in a blender. The ground material was then filtered through sterilized gauze, spread on MRS-agar medium, and cultured at 37℃ for 48 hours. The strain that formed colonies on the medium was subcultured and purified into a single strain. The 16S rRNA gene sequencing analysis of the isolated strain confirmed the 16S rRNA base sequence of sequence number 1. The strain showed 99.87% homology to Lactobacillus plantarum. It was named Lactobacillus plantarum OD11 and deposited with the Korea Research Institute of Bioscience and Biotechnology on October 31, 2024 (Accession number: KCTC1612BP).

[0071]

[0072] Manufacturing Example 2. Production of artificial exosomes using a high-pressure homogenizer and optimization of conditions.

[0073] 2-1. Cultivation of strains

[0074] To prepare the OD11 culture of Lactobacillus plantarum (L. plantarum), two stages of cultivation were performed. The pre-culture stage was a preliminary culture, in which 1 ml of the L. plantarum OD11 strain was inoculated into 100 ml of sterilized MRS medium and cultured at 30°C for 18 hours with shaking at 180 rpm. Then, in the main culture stage, 1 ml of the preliminary culture was inoculated into 100 to 400 ml of MRS medium and cultured at 30°C for 18 hours with shaking at 180 rpm.

[0075] As a result of this culture, the culture showed a pH of 3.86 to 3.97, and the number of microorganisms in the culture was 1.1 X 109 8.5 X 10 9 CFU / ml was shown.

[0076] 2-2. Manufacturing of artificial exosomes

[0077] Artificial exosomes were prepared from the culture through the 2-1 culture process using a high-pressure homogenizer (microfluidizer, MF).

[0078] Specifically, 50 ml of lactic acid bacteria culture was centrifuged at 1500 rpm, 25 °C, and pelleted for 5 minutes. The pellet was then redispersed at a ratio of 9:1, 4:1, or 2:1, and then treated using a high-pressure homogenizer (MN400BF, Micronox) at a pressure of 2000 bar for cycles of 3 and 5. Subsequently, the mixture was filtered using a vacuum pump (Corning®500 mL Bottle Top Vacuum Filter, 0.40 μm Pore 33.2 cm² PES Membrane, Fits 45 mm Diameter Necks, Sterile), and the physical properties were analyzed by DLS (Dynamic Light Scattering).

[0079] Additionally, the concentration of exosomes according to MF cycle variables was measured using nanoparticle tracking analysis (NTA).

[0080] The DLS analysis results confirmed that the size was 250 nm ± 30 and the PDI value was 0.24 ± 0.06. In addition, the NTA analysis results showed that the particle concentration was 10 9 10 inland 11 It was confirmed that the number / ml range was within.

[0081]

[0082] Example 1. Evaluation of changes in the physical properties of artificial exosomes

[0083] The physical properties of artificial exosomes manufactured using a high-pressure homogenizer of Manufacturing Example 1 (Example) and artificial exosomes manufactured using an extruder (Comparative Example) were analyzed using DLS (Dynamic light scattering, size, PDI measurement) and NTA (Nanoparticle Tracking Analysis, number measurement) equipment, and the results are shown in Table 1 and Figure 1 below.

[0084] Comparative Example Example Size (nm) 200~300 200~400 PDI 0.18~0.3 0.18~0.3 Number 10 9 ~ 10 11 10 9 ~ 10 11

[0085]

[0086] Experimental results showed that the size of the example was slightly larger than that of the comparative example. In addition, TEM image analysis results confirmed that, unlike cell debris, the particles were formed into spherical shapes (Fig. 1).

[0087]

[0088] Example 2. Efficacy evaluation of artificial exosomes derived from Lactobacillus plantarum

[0089] 2-1. Evaluation of wrinkle improvement efficacy

[0090] To evaluate the wrinkle improvement efficacy of artificial exosomes derived from Lactobacillus plantarum, the expression of major genes related to wrinkle improvement, COL1A1 (Collagen Type I Alpha 1 Chain), MMP1 (Matrix Metalloproteinase 1), and ELN (Elastin), was confirmed.

[0091] ​Specifically, fibroblasts (HS68) were seeded in 12-well plates and cultured for 24 h in an incubator maintained at 37°C and 5% CO2 with DMEM (Dulbecco's Modified Eagle Medium, Gibco, Waltham, MA, USA) medium. The cells were washed once with PBS (Phosphate Buffered Saline, Gibco, Waltham, MA, USA), and 500 μl PBS was added. Then, UVB 25 mj / cm was irradiated using a UV irradiator. 2 Photoaging was induced by investigating. Then, PBS was removed, and DMEM containing artificial exosomes or lysates according to Table 2 was added to the cells. After 24 hours, RNA was extracted, and cDNA was synthesized using this RNA as a template. Then, the expression level of each gene in the cDNA was analyzed by RT-qPCR, and the results are shown in Table 2 below.

[0092] UV-treated sample COL1A1MMP1ELN Untreated group X-1.001.001.00 Comparative group O-0.301.230.53 Example 1O Artificial exosome 100 ppm0.590.671.01 Comparative example 2O Lysate 100 ppm0.510.890.72

[0093]

[0094] [qPCR experimental conditions]

[0095] 95℃ for 45 seconds, 60℃ for 1 minute, 72℃ for 45 seconds, 30 cycles

[0096]

[0097] As a result of the experiment, it was confirmed that when artificial exosomes were treated, the expression levels of COL1A1 and ELN increased more than in the control group, and the expression level of MMP1 decreased more, indicating that it was effective in improving wrinkles.

[0098] 2-2. Evaluation of hyaluronic acid synthesis efficacy

[0099] After setting the culture conditions to DMEM (Dulbecco's Modified Eagle Medium, Gibco, Waltham, MA, USA) medium and an incubator maintained at 37°C and 5% CO2, HaCat cells (human keratinocytes, American type culture collection, Manassas, VA, USA) were seeded in 24-well plates and cultured for 24 hours. Then, the medium was replaced with serum-free DMEM, and artificial exosomes were treated at the concentrations listed in Table 3, and cultured for an additional 48 hours. Next, the hyaluronic acid concentration was measured using a hyaluronic acid ELISA kit (R&D Systems, catalog number DHYAL0), and the percentage increase in the hyaluronic acid production rate of the sample compared to the control group (HaCat cells cultured in DMEM containing 0% FBS) was calculated, and the results are shown in Table 3 below (positive control group: 0.3 ppm Retinoic acid (RA)).

[0100] HA synthesis ability [%] Untreated group 0 Positive control group (RA 0.3 ppm) 14.61 ppm 0.710 ppm 1.21000 ppm 6.2

[0101]

[0102] Experimental results confirmed that artificial exosomes enhance the hyaluronic acid synthesis ability of keratinocytes.

[0103]

[0104] 2-3. Anti-inflammatory efficacy evaluation

[0105] After setting the culture conditions to DMEM medium and an incubator maintained at 37℃ and 5% CO2, HaCat cells were seeded in 24-well plates and cultured for 24 hours. Then, the medium was replaced with serum-free DMEM, and artificial exosomes were treated at the concentrations according to Table 6, and cultured for an additional 48 hours. Then, IL-6 and IL-1a concentrations were measured using an ELISA kit (R&D Systems) to quantify IL-6 and IL-1a, and the percentage increase in the secretion of inflammatory factors compared to the control group (HaCat cells cultured in DMEM containing 0% FBS) was calculated, and the results are shown in Table 4 below (positive control: Dexamethasone 0.2 ppm).

[0106] IL-6 secretion amount [%] IL-1α Secretion amount [%] Untreated group 100100 Positive control group (Dexamethasone 0.2 ppm) 43.0580.571 ppm 68.3170.5310 ppm 81.2169.50100 ppm 78.0272.15

[0107]

[0108] Experimental results confirmed that artificial exosomes significantly reduced the expression of inflammatory cytokines from keratinocytes.

[0109]

[0110] 2-4. Wound healing ability assessment

[0111] Fibroblasts (HS68) were seeded in 12-well plates and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2 using DMEM (Dulbecco's Modified Eagle Medium, Gibco, Waltham, MA, USA). The culture conditions were as follows: 12-well plates were seeded with DMEM (Dulbecco's Modified Eagle Medium, Gibco, Waltham, MA, USA) medium and 5% CO2 at 37°C. Fibroblasts (HS68) were seeded in 12-well plates and cultured for 24 hours. The medium was then removed, and evenly wounds were created using a small scratcher (SPLScar™, SPL) in a cell confluence state. Then, serum-free DMEM containing artificial exosomes was treated according to the concentrations listed in Table 5, and after 24 hours, the results were photographed under an optical microscope. The reduction in the wound area between the yellow lines was measured and calculated, and the results are shown in Table 5 below.

[0112] Wound area [%] Untreated group 100 1 ppm 85 10 ppm 89 100 ppm 61 1000 ppm 53

[0113]

[0114] Experimental results confirmed that artificial exosomes can significantly reduce wounds by promoting the proliferation of fibroblasts.

[0115]

[0116] 2-5. Cell penetration evaluation of artificial exosomes

[0117] During the MF treatment process of the artificial exosome manufacturing process, the cell pellet was redispersed in an aqueous FITC (fluorescein isothiocyanate) solution (10 to 200 mM) to prepare artificial exosomes loaded with FITC. Next, after setting the culture conditions to DMEM medium and an incubator maintained at 37°C and 5% CO2, fibroblasts were seeded in a 12-well plate and cultured for 24 hours. Then, the medium was removed and washed three times with PBS. Next, the artificial exosomes containing FITC and FITC of the same fluorescence intensity (based on the highest concentration) were diluted in DMEM and treated to the fibroblasts, followed by microscopic observation, and the results are shown in Fig. 3.

[0118] As shown in Figure 3, the artificial exosome (100 ppm) treatment group showed more FITC fluorescence within the cells compared to the untreated group. This confirmed that the FITC-loaded artificial exosomes had penetrated into the cells.

[0119] In addition, to evaluate the skin permeability of artificial exosomes, 200 μl of artificial exosomes containing FITC and FITC (final concentration standard) with the same fluorescence intensity were applied to the upper part (stratum corneum side) of pig skin (Apures, 2.5 cm X 2.5 cm). Then, pig skin was attached to a Franz diffusion cell device and maintained in an incubation at 37°C for 24 hours. At this time, PBS was used as the preservation solution. Then, after 24 hours, the indicator substance was dissolved from pig skin using PBS and the fluorescence intensity was measured using a fluorometer (spectrometer, Exitation 488 / Emission 512). The results are shown in Table 6 below.

[0120] Unit: ng Total amount Permeation efficiency Free-FITC 2.27 100% Artificial exosomes (1X) 2.88 127% Artificial exosomes (0.5X) 2.75 121%

[0121]

[0122] The experimental results confirmed that the group treated with artificial exosomes could penetrate the skin.

[0123]

[0124] Example 3. Evaluation of human efficacy of artificial exosomes

[0125] The human efficacy evaluation was conducted on a total of 10 subjects aged between 40 and 50 years. Prior to participating in the study, the subjects were informed of the possible side effects and consent was obtained.

[0126] Specifically, subjects applied a toner containing 5% artificial exosomes to their entire face twice daily. The toner was formulated to contain EDTA-3Na, 1,2-hexanediol, 1,3-butylene glycol, hydrous citric acid, sodium citrate, and purified water.

[0127] Before the measurement, the subjects' faces were thoroughly washed and allowed to adapt for 20 minutes in an air-conditioned room (temperature 22 ± 2 °C, relative humidity 50 ± 10%). Facial wrinkles, pigmentation, skin texture, and pores were measured with Antera 3D (Miravex, Dublin, Ireland). Full-face images were captured with Janus-1 (PIE, Seoul, South Korea). Skin redness was measured using a colorimeter (CR-400, Konica Minolta, Osaka, Japan), skin moisture was measured using a corneometer (MPA580, Courage - Khazaka Electronic, Cologne, Germany), and wrinkles under the eyes, around the eyes, and around the nasolabial folds were measured using Antera 3D (Miravex, Dublin, Ireland). The results are shown in Figures 4 to 6 and Table 7.

[0128] Evaluation Results After 8 weeks (Unit: %) Moisturizing Improvement Rate 9.30 Redness Improvement Rate 7.18 Skin Texture Improvement Rate 11.79 Pore Area Improvement Rate 6.81

[0129]

[0130] As a result of the experiment, the Antera analysis showed an average wrinkle improvement rate of about 15% (Figs. 4 and 5). As a result of the corneometer measurement, it was confirmed that the moisturizing improvement rate increased to about 9.30% after 8 weeks. In addition, as a result of comparing the erythema values ​​before and after the experiment using a chromameter, it was confirmed that the redness was improved by about 7.18%. In addition, the Ra value calculation result of the forehead area showed an improvement of about 11.79%, and the average pore area calculation result of the cheek area showed an improvement of about 6.81% (Fig. 6).

[0131]

[0132] Example 4. Evaluation of the boosting effect of collagen synthesis-promoting ingredients.

[0133] We evaluated whether artificial exosome components exhibited a synergistic effect with other collagen synthesis-promoting components. Adenosine and 10-hydroxystearic acid (HSA) were used as evaluation substances.

[0134] Fibroblasts (HS68) were seeded in 12-well plates and cultured for 24 h in an incubator maintained at 37°C and 5% CO2 in DMEM (Dulbecco's Modified Eagle Medium, Gibco, Waltham, MA, USA) medium. The cells were washed once with PBS (Phosphate Buffered Saline, Gibco, Waltham, MA, USA), and 500 μl of PBS was added. Then, UVB irradiation was performed using a UV irradiator at 25 mJ / cm 2Photoaging was induced by investigating. Then, PBS was removed, and DMEM containing artificial exosomes, adenosine, or HSA according to the conditions described in Table 8 was added to the cells and cultured for an additional 48 hours. Then, the concentration of the produced collagen was measured using the Human pro-collagen 1 alpha 1 (COL1A1) ELISA kit (R&D Systems), and the relative increase compared to the untreated group was calculated, and the results are shown in Table 8 below.

[0135] Sample COL1A1 Untreated group 1.00 Artificial exosome 100 ppm 1.30 Adenosine 50 ppm 1.56 Artificial exosome 100 ppm + adenosine 50 ppm 1.95 Adenosine 100 ppm 1.56 Artificial exosome 100 ppm + adenosine 100 ppm 1.98 HAS 1 ppm 1.60 Artificial exosome 100 ppm + HAS 1 ppm 2.01 HAS 5 ppm 1.50 Artificial exosome 100 ppm + HAS 5 ppm 2.15

[0136]

[0137]

[0138] As a result of the experiment, it was confirmed that a greater collagen synthesis effect was observed when artificial exosomes, adenosine, and HSA were treated together than when treated alone.

[0139] 16S rRNA sequence of Lactobacillus plantarum OD11 InfoATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACA ACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTA

[0140]

[0141] [Accession number]

[0142] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0143] Accession number: KCTC16121BP

[0144] Date of acceptance: 20241031

[0145]

Claims

1. Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP.

2. A cosmetic composition for improving skin, comprising Lactobacillus plantarum OD11 strain deposited under accession number KCTC16121BP; and a culture thereof, a lysate thereof, an extract thereof, or an extracellular vesicle thereof as an active ingredient.

3. In paragraph 2, A cosmetic composition for improving skin, wherein the extracellular vesicles are naturally secreted or artificially manufactured from Lactobacillus plantarum.

4. In paragraph 2, A cosmetic composition for improving skin, wherein the extracellular vesicles are prepared from a Lactobacillus plantarum culture.

5. In paragraph 2, A cosmetic composition for improving skin, wherein the improvement in skin condition is improvement in skin elasticity, improvement in skin wrinkles, skin moisturizing, skin soothing or skin regeneration.

6. In paragraph 5, A cosmetic composition for improving skin, wherein the above skin soothing soothes the skin from at least one inflammatory disease selected from the group consisting of atopic dermatitis, contact dermatitis, allergic dermatitis, acne, eczema, rosacea, oily skin, psoriasis, eczema, pruritis, itchy skin, urticaria, idiopathic chronic urticaria, scleroderma, vitiligo, intractable melasma, flushing, erythema, scleroderma, Behcet's disease, and contact dermatitis.

7. In paragraph 2, A cosmetic composition for improving skin, wherein the extracellular vesicles have a diameter of 30 to 500 nm.

8. In paragraph 2, The above extracellular vesicles are 10 per unit volume of 1 ml of the composition. 8 10 inland 15 A cosmetic composition for improving skin, comprising the following content:

9. A cosmetic composition comprising extracellular vesicles manufactured by including a step of high-pressure homogenization treatment on a sample including a Lactobacillus plantarum strain.

10. In paragraph 9, A cosmetic composition, wherein the above Lactobacillus plantarum is Lactobacillus plantarum OD11 deposited under accession number KCTC16121BP.

11. In paragraph 9, A cosmetic composition wherein the improvement in the above skin condition is improvement in skin elasticity, improvement in skin wrinkles, skin moisturizing or skin soothing.

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