KR102773839B1 - Composition for improving skin condition comprising probiotics extracellular vesicles and preparation method thereof - Google Patents

Abstract

The present invention provides culture conditions capable of increasing the secretion efficiency of extracellular vesicles from probiotics, and provides a process capable of highly efficiently separating them, thereby providing a material containing a high concentration of an effective substance, and further provides a composition for improving skin by encapsulating an effective ingredient containing the probiotic extracellular vesicles manufactured above.

Description

{COMPOSITION FOR IMPROVING SKIN CONDITION COMPRISING PROBIOTICS EXTRACELLULAR VESICLES AND PREPARATION METHOD THEREOF}

The present invention relates to a culture method and a separation method capable of obtaining extracellular vesicles from probiotics at a high yield, and a composition for improving skin containing probiotic extracellular vesicles.

All cells secrete various substances to exchange information with other cells or the external environment. Among the secreted substances of cells, extracellular vesicles (EV) are nano-sized (30–150 nm) vesicles surrounded by a lipid bilayer. Depending on their origin, size, and production process, they are classified into exosomes, microvesicles, and apoptotic bodies. Since they are not very different in appearance and have in common that they are vesicles secreted outside of cells, they are currently collectively called “extracellular vesicles.”

Extracellular vesicles contain various biologically active substances such as proteins, lipids, nucleic acids (mRNA, miRNA, etc.), and metabolites. These are called extracellular vesicle cargoes, and they contain a wide range of signaling factors. Through extracellular vesicle cargoes, they can fuse with other cells to deliver contents, or regulate the functions of other cells (activation, growth, migration, differentiation, apoptosis, necrosis, etc.).

Due to these characteristics, research is being conducted to utilize extracellular vesicles for purposes such as diagnosis and treatment. Research related to extracellular vesicles is mainly focused on extracellular vesicles derived from mammals including humans, and as interest in extracellular vesicles has increased recently, research on extracellular vesicles derived from various sources such as microorganisms, plants, and microalgae has begun.

Extracellular vesicles have been mainly used in the pharmaceutical field, but their scope of application is expanding as accessibility expands with the development of technology. Recently, research on skin and extracellular vesicles is being conducted in various fields, such as the effect of stem cell-derived extracellular vesicles on wound healing and regeneration of skin, and the role of extracellular vesicles secreted from skin cells.

In the future, extracellular vesicles are expected to become a material that receives more attention in the cosmetics field. Skin-related research using extracellular vesicles or their application to the cosmetics field is most actively being conducted in Korea. Extracellular vesicles as an effective material are most widely studied in human stem cell origin, and the scope is gradually expanding to microorganisms and plants.

Although research technology for extracellular vesicles is rapidly developing, there are many technical barriers to commercialization. Since small physiological changes within cells can affect the composition of extracellular vesicles, it is difficult to culture them stably, and it is economically and time-consuming to isolate a large amount of extracellular vesicles with high purity. Commercialized extracellular vesicle isolation kits are suitable for research, but they are difficult to apply to mass production due to cost and capacity. In order to commercialize extracellular vesicles, efficient technology must be secured for many stages from culture to separation and purification, mass production, and processing.

The technologies applied to the isolation step of extracellular vesicles to date include size exclusion chromatography, ion exchange chromatography, density gradient centrifugation, ultracentrifugation, ultrafiltration, immunoaffinity capture, microfluidics-based isolation, total exosome isolation kit, and polymer-based precipitation. Each of these isolation technologies has its own advantages and disadvantages in terms of cost, time, capacity, and purity, and it is necessary to select and optimize a technology suitable for the source cell of the extracellular vesicles.

Meanwhile, probiotics refer to living microorganisms that have a beneficial effect on human health. Research on probiotics has mainly been conducted as materials or means for fermentation of natural products, but recently, awareness of the role and importance of probiotics themselves, such as their involvement in regulating the intestinal microbiome, has been increasing. The human microbiome refers to 100 trillion microorganisms and their genetic information existing in the human body, including the mouth, nose, skin, and intestines, and plays an important role in providing information on the causes of human health and diseases. For this reason, research on the microbiome is increasing in the fields of pharmaceuticals, functional foods, and cosmetics.

Cosmetic materials using microorganisms have low skin absorption in the form of culture solutions, dissolved substances, and fermented products, so they have low effects on the actual skin. In addition, there is a disadvantage in that it is difficult to manufacture high concentrations of effective ingredients due to impurities such as culture media components and metabolites (toxins, etc.) produced by microorganisms in the culture solution. In addition, due to the nature of cosmetics, sensory factors such as sight, smell, and touch have a significant impact on consumer choice.

Microbial cultures have a unique odor that can adversely affect sensory factors, which limits their application as cosmetic raw materials. By using the characteristics of extracellular vesicles with small particle sizes to remove impurities during the filtration process, the effective ingredients can be increased while the skin absorption rate can be increased, maximizing the actual effect on skin cells.

Accordingly, the present invention seeks to develop a material containing a high concentration of an effective substance by finding culture conditions that can increase the secretion efficiency of extracellular vesicles from probiotics and developing a process that can separate them with high efficiency.

Korean Patent Registration No. 10-2253418 Korean Patent Publication No. 10-2019-0002079 Korean Patent Publication No. 10-2022-0033010

The present invention seeks to develop a material containing a high concentration of an effective substance by finding culture conditions that can increase the secretion efficiency of extracellular vesicles from probiotics and developing a process that can separate them with high efficiency.

In addition, the present invention aims to provide a composition for improving skin, which encapsulates an effective ingredient by containing the probiotic extracellular vesicles manufactured as described above.

It is preferable that the skin improvement composition according to the present invention contain probiotic extracellular vesicles derived from at least one of Saccharomyces cerevisiae and Lactobacillus plantarum.

It is more preferable that the skin improvement composition according to the present invention contains probiotic extracellular vesicles derived from Saccharomyces cerevisiae and Lactobacillus plantarum.

It is preferable that the skin improvement composition according to the present invention increases collagen synthesis, inhibits collagen decomposition enzymes, and regulates the microbiome.

The size of the above probiotic extracellular vesicles is preferably 30 to 150 nm.

As another specific embodiment of the present invention, a method for producing a probiotic extracellular vesicle according to the present invention preferably includes a step of culturing Saccharomyces cerevisiae or Lactobacillus plantarum in a medium containing trehalose (TR), ascorbic acid (AS), citric acid (CI), and malic acid (MA) in YM Broth Basal medium or MRS Broth Basal medium.

The method for producing probiotic extracellular vesicles according to the present invention preferably further includes a step of centrifuging the culture solution produced in the step of culturing in the medium and filtering the supernatant with 0.22 μm PES.

The method for producing probiotic extracellular vesicles according to the present invention preferably further includes a step of filtering through a membrane filter of 30 kDa to 0.22 ㎛.

It is preferable that the above skin improvement composition additionally contains at least one additive selected from butylene glycol, hexanediol, ethylhexylglycerin, and phenoxyethanol.

It is preferable that the above skin improvement composition be in a formulation selected from the group consisting of toner, nourishing cream, massage cream, essence, pack, gel, lotion, ointment, patch, and spray.

As another specific embodiment of the present invention, it is preferable to use the probiotic extracellular vesicles as a carrier for delivering the active ingredient into the skin or cells or extracellular vesicles.

As another specific example of the present invention, a method is preferable in which the effective ingredient is naturally encapsulated in an extracellular vesicle by using the extracellular vesicle delivery vehicle during microbial culture, or the effective ingredient is artificially encapsulated in an extracellular vesicle by sonication after being introduced into an extracellular vesicle during microbial culture.

The composition for improving skin, including the probiotic extracellular vesicles of the present invention, can be used in a cosmetic method for improving the condition of mammalian skin, excluding therapeutic purposes.

The above cosmetic method preferably comprises: (a) directly applying the skin-improving composition to the skin of a mammal, (b) contacting or attaching a patch, mask pack or mask sheet on which the skin-improving composition has been applied or deposited, to the skin of a mammal, or sequentially performing (a) and (b).

In addition, it is preferable to further include a step of (c) removing the patch, mask pack or mask sheet from the skin of the mammal after step (b) and applying the skin improvement composition to the skin of the mammal.

The present invention provides culture conditions capable of increasing the secretion efficiency of extracellular vesicles from probiotics, and provides a process capable of highly efficiently separating them, thereby providing a material containing a high concentration of an effective substance, and further provides a composition for improving skin by encapsulating an effective ingredient containing the probiotic extracellular vesicles manufactured above.

Figure 1 illustrates the 2DE analysis results of Examples 1-1 and 1-2.
Figure 2 illustrates the results of collagen biosynthesis evaluation of Example 1-2.
Figure 3 illustrates the results of the evaluation of collagen decomposition enzyme inhibition ability of Example 1-1.
Figure 4 illustrates the results of the evaluation of collagen decomposition enzyme inhibition ability of Example 1-2.
Figure 5 illustrates the results of evaluating the collagen decomposition enzyme inhibition ability of Examples 2-3 to 2-6.
Figure 6 illustrates the results of evaluating the skin barrier improvement rate of Example 2-3.
Figure 7 illustrates the results of the skin barrier improvement rate evaluation of Example 2-6.

Hereinafter, embodiments of the present invention will be described in detail so that those with ordinary knowledge in the technical field to which the present invention pertains can easily practice it. Embodiments of the present invention are provided so that those with average knowledge in the art can more completely explain the present invention. Therefore, embodiments of the present invention can be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below.

Throughout the specification of the present invention, when a part is said to “include” a certain component, this means that it further includes other components, rather than excluding other components, unless specifically stated otherwise.

Throughout the specification of the present invention, when a step is said to be located “on” or “before” another step, this may include the same rights as when a step has a direct time-series relationship with the other step, as well as when the two steps have an indirect time-series relationship where the time-series order may be changed, such as a mixing step after each step.

The terms “about,” “substantially,” etc., used throughout the specification of the present invention are used in a meaning close to or at the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to prevent unscrupulous infringers from unfairly using the disclosure contents in which exact or absolute values are mentioned to help understanding of the present invention. The terms “~ (doing) step” or “~ step” used throughout the specification of the present invention do not mean “~ step for.”

The present invention provides a composition for improving skin containing probiotic extracellular vesicles derived from at least one of Saccharomyces cerevisiae and Lactobacillus plantarum.

More preferably, the present invention provides a skin improvement composition containing probiotic extracellular vesicles derived from Saccharomyces cerevisiae and Lactobacillus plantarum.

As confirmed in the following examples and experimental examples, the composition for improving skin according to the present invention is preferably effective in increasing collagen synthesis, inhibiting collagen decomposition enzymes, and regulating the microbiome.

As another specific embodiment of the present invention, a method for producing a probiotic extracellular vesicle according to the present invention preferably includes a step of culturing Saccharomyces cerevisiae or Lactobacillus plantarum in a medium containing trehalose (TR), ascorbic acid (AS), citric acid (CI), and malic acid (MA) in YM Broth Basal medium or MRS Broth Basal medium.

Ultracentrifugation is mainly used as a method for producing extracellular vesicles, but when considering mass production, the separation method below is preferable.

The method for producing probiotic extracellular vesicles according to the present invention preferably further includes a step of centrifuging the culture solution produced in the step of culturing in the medium and filtering the supernatant with 0.22 μm PES.

The method for producing probiotic extracellular vesicles according to the present invention preferably further includes a step of filtering through a membrane filter of 30 kDa to 0.22 ㎛.

The extracellular vesicles separated in the present invention are 30 to 150 nm in size, and probiotic-derived extracellular vesicles can be considered natural nanostructures. Probiotic-derived extracellular vesicles themselves have the effect of preventing and improving skin wrinkles, so they can be utilized as products.

In addition, nano-sized structures have the characteristic of easily combining with bioactive substances that function to remove wrinkles or prevent aging. Nanocosmetics are being developed by applying these nano-structures as carriers to cosmetics, and since their size is much smaller than the gap between skin cells, they are easily absorbed into the skin. In other words, nano-structures can act as carriers that easily deliver the effective ingredients of cosmetics into the skin, into cells, or into extracellular vesicles.

By using these nanostructures as carriers and putting the active ingredient into microbial-derived extracellular vesicles, more products can be derived. For example, a method of naturally encapsulating the active ingredient by adding the active ingredient through a probiotic extracellular vesicle carrier during microbial culture, or a method of artificially encapsulating the active ingredient by inducing a structural change in the extracellular vesicle instantaneously through ultrasonic treatment can be used.

The above skin improvement composition may further include one or more additives selected from butylene glycol, hexanediol, ethylhexylglycerin, and phenoxyethanol. The additives are necessary for the formation of a subsequent formulation, but are not limited thereto.

The above skin improvement composition may be in a formulation selected from the group consisting of, but not limited to, a softening toner, a nourishing toner, a nourishing cream, a massage cream, an essence, a pack, a gel, a lotion, an ointment, a patch, and a spray.

In addition, the composition for improving skin, including the probiotic extracellular vesicles of the present invention, can be used in a cosmetic method for improving the condition of mammalian skin, excluding therapeutic purposes.

The above cosmetic method preferably comprises: (a) directly applying the skin-improving composition to the skin of a mammal, (b) contacting or attaching a patch, mask pack or mask sheet on which the skin-improving composition has been applied or deposited, to the skin of a mammal, or sequentially performing (a) and (b).

In addition, it is preferable to further include a step of (c) removing the patch, mask pack or mask sheet from the skin of the mammal after step (b) and applying the skin improvement composition to the skin of the mammal.

Hereinafter, the contents of the present invention will be explained in more detail through the following examples or experimental examples.

Example 1. Preparation of extracellular vesicles

Example 1-1. Yeast extracellular vesicles

A medium was prepared by adding 0 to 0.1% trehalose (TR), ascorbic acid (AS), citric acid (CI), or malic acid (MA) to YM Broth Basal medium, and the yeast strain was inoculated and cultured. The culture solution was centrifuged at 4,000 rpm at 4 °C for 20 minutes, and the supernatant was filtered through 0.22 μm PES. After reacting the total exosome isolation reagent, centrifugation was performed at 10,000 rpm at 4 °C for 1 hour, and the supernatant was removed to recover the extracellular vesicles. The particle distribution and concentration of the extracellular vesicles were analyzed using a nanoparticle tracking analyzer (NS300). The results of the analysis are disclosed in Table 1 below.

Comparative Example/Example Badge composition (%) suspension
condition Extracellular vesicle concentration
(Particles/ml) YM TR AS CI MA RPM Comparative Example 1-1-1 100 - - - - 0 1.75e+10 Comparative Example 1-1-2 100 - - - - 100 2.08e+10 Comparative Example 1-1-3 100 - - - - 200 1.37e+11 Comparative Example 1-1-4 99.9 0.1 - - 200 1.28e+11 Comparative Example 1-1-5 99.95 - 0.05 - 200 6.6e+10 Comparative Example 1-1-6 99.95 - - 0.05 200 8.28e+10 Example 1-1 99.95 - - - 0.05 200 6.44e+10

Example 1-2. Lactic acid bacteria extracellular vesicles

A medium was prepared by adding 0 to 0.1% trehalose (TR), ascorbic acid (AS), citric acid (CI), or malic acid (MA) to MRS Broth Basal medium, and the Lactobacillus plantarum lactic acid bacteria strain was inoculated and cultured. The culture solution was centrifuged at 4,000 rpm at 4 °C for 20 minutes, and the supernatant was filtered through 0.22 μm PES. After reacting the total exosome isolation reagent, centrifugation was performed at 10,000 rpm at 4 °C for 1 hour, and the supernatant was removed to recover the extracellular vesicles. The particle distribution and concentration of the extracellular vesicles were analyzed using a nanoparticle tracking analyzer (NS300). The results of the analysis are disclosed in Table 2 below.

Comparative Example/Example Badge composition (%) suspension
condition Extracellular vesicle concentration
(Particles/ml) MRS TR AS CI MA RPM Comparative Example 1-2-1 100 - - - - 0 2.45e+10 Comparative Example 1-2-2 100 - - - - 100 4.36e+10 Comparative Example 1-2-3 100 - - - - 200 2.52e+10 Comparative Example 1-2-4 99.9 0.1 - - 200 3.48e+10 Comparative Example 1-2-5 99.95 - 0.05 - 100 1.05e+10 Comparative Example 1-2-6 99.95 - - 0.05 200 3.31e+10 Example 1-2 99.95 - - - 0.05 200 3.43e+10

Experimental Example 1. Analysis of extracellular vesicle efficacy substances

The extracellular vesicles prepared in Examples 1-1 and 1-2 were treated with RIPA Buffer and reacted at low temperature for 30 minutes. After centrifugation at 4°C and 12,000 rpm for 30 minutes, the supernatant was collected and the effective substance was analyzed using 2DE/MS. The results of the analysis are disclosed in Fig. 1 and Table 3 below.

Example Effective substances (Protein hits) Example 1-1 gi|296178361 gi|226790 gi|171332 Example 1-2 gi|2073333746 gi|2172108857

Experimental Example 2. Evaluation of collagen synthesis ability

As skin aging progresses, the function of fibroblasts responsible for collagen and elastin expression decreases. As a result, wrinkles occur, elasticity decreases, and the function of the skin barrier deteriorates. In order to confirm the effect of the composition of the present invention on improving skin aging and barrier, the collagen synthesis ability by sample treatment in fibroblasts was evaluated.

Human dermal fibroblasts were seeded in a 24-well plate at a concentration of 6 x 10 ⁴ cells/ml and cultured for 24 hours. The samples were treated and cultured for an additional 24 hours. The medium was collected and the procollagen type I C-terminal peptide (PIP) secreted into the extracellular space was confirmed using the ELISA (Enzyme-linked immuosorbent assay) method to calculate the collagen synthesis rate.

As shown in Fig. 2, it was confirmed that the extracellular vesicles prepared in Example 1-2 were effective in increasing collagen synthesis in fibroblasts.

Experimental Example 3. Evaluation of collagen decomposition enzyme inhibition ability

As skin aging progresses, the secretion of collagenase increases in fibroblasts, which decomposes collagen and affects the occurrence of wrinkles, loss of elasticity, and weakening of the skin barrier function. In order to confirm the effect of the composition of the present invention on improving skin aging and barrier, the collagenase inhibition ability by sample treatment in fibroblasts was evaluated.

Human dermal fibroblasts were seeded in a 24-well plate at a concentration of 6 x 10 ⁴ cells/ml and cultured for 24 hours. The samples were treated and cultured for an additional 24 hours. The medium was recovered, and the extracellular Matrix metalloproteinase-1 (MMP-1) was confirmed by ELISA (Enzyme-linked immuosorbent assay), and the collagenase inhibition rate was calculated.

As shown in FIGS. 3 and 4, Examples 1-1 and 1-2 were confirmed to be effective in reducing the secretion of collagen-decomposing enzymes in fibroblasts.

Experimental Example 4. Evaluation of Microbiome Regulation Ability

We cultured strains of beneficial skin bacteria (Lactobacillus plantarum) and harmful skin bacteria (Staphylococcus aureus) and examined the growth changes of each microorganism before and after treating the sample. It was determined that the microbiome regulation effect was present when beneficial bacteria increased or harmful bacteria decreased compared to the control group.

As disclosed in Tables 4 and 5 below, Examples 1-1 and 1-2 were confirmed to be effective in regulating the microbiome by increasing beneficial skin bacteria and reducing harmful skin bacteria.

Comparative Example/Example density Lactobacillus plantarum growth changes
(% of Control) 8 hours 24 hours Unprocessed - 100.00 100.00 Example 1-1 0.2% 97.43 86.04 0.5% 82.16 110.17 Example 1-2 0.2% 108.92 90.14 0.5% 106.06 117.60

Comparative Example/Example density Staphylococcus aureus Growth changes
(% of Control) 8 hours 24 hours 48 hours Unprocessed - 100.00 100.00 100.00 Example 1-1 0.2% 99.51 102.35 75.26 0.5% 101.11 140.39 95.66 Example 1-2 0.2% 107.76 140.78 78.06 0.5% 115.64 77.65 72.45

Example 2. Establishment of conditions for isolating extracellular vesicles

A medium was prepared by adding 0.1% malic acid to MRS Broth Basal medium, inoculating the Lactobacillus plantarum lactic acid bacteria strain, and culturing it. The culture solution was centrifuged at 4000 rpm at 4°C for 20 minutes, and the supernatant was filtered through 0.22 μm PES.

After reacting the culture filtrate with the Total exosome isolation reagent, centrifugation was performed at 4°C, 10,000 rpm for 1 hour, and the supernatant was removed to recover extracellular vesicles, thereby producing Comparative Example 2-1. The culture filtrate was individually or stepwise injected into membrane filtration devices of 100 kDa, 30 kDa, 5 kDa, and 2 kDa, and then filtered to recover extracellular vesicles, thereby producing Comparative Examples 2-2 to 2-6 and Examples 2-1 to 2-2.

The particle distribution and concentration of extracellular vesicles were analyzed using a nanoparticle tracking analyzer (NS300) to calculate the extracellular vesicle recovery rate (%) for Comparative Example 2-1. The results are disclosed in Table 6 below.

Comparative Example/Example Strain Filter Conditions Extracellular vesicles
Recovery rate (%) Comparative Example 2-1 Lactobacillus - 100.0 Comparative Example 2-2 Lactobacillus < 0.22 ㎛ 109.8 Example 2-1 Lactobacillus 100 kDa < < 0.22 μm 168.8 Comparative Example 2-3 Lactobacillus < 100 kDa 79.5 Example 2-2 Lactobacillus 30 kDa < < 100 kDa 130.2 Comparative Example 2-4 Lactobacillus < 30 kDa 32.4 Comparative Example 2-5 Lactobacillus 5 kDa < < 30 kDa 42.9 Comparative Example 2-6 Lactobacillus < 5 kDa 10.2

Lactobacillus plantarum lactic acid bacteria strain and Saccharomyces cerevisiae yeast strain were inoculated into MRS Broth Basal medium containing 0.1% malic acid and YM Broth Basal medium containing 0.1% malic acid (MA), respectively, and cultured. The culture solution was centrifuged at 4,000 rpm at 4°C for 20 minutes, and the supernatant was filtered with 0.22 μm PES. The culture filtrate was individually or stepwise injected into a 100 kDa or 30 kDa membrane filtration device, and then filtered to recover extracellular vesicles to prepare Examples 2-3 to 2-6. The contents are disclosed in Table 7.

Example Strain Filter Conditions Example 2-3 Saccharomyces 30 kDa < < 100 kDa Example 2-4 Saccharomyces 100 kDa < < 0.22 μm Example 2-5 Lactobacillus 30 kDa < < 100 kDa Example 2-6 Lactobacillus 100 kDa < < 0.22 μm

Experimental Example 5. Evaluation of collagen decomposition enzyme inhibition ability

As skin aging progresses, the secretion of collagenase increases in fibroblasts, which decomposes collagen and affects the occurrence of wrinkles, loss of elasticity, and weakening of the skin barrier function. In order to confirm the effect of the composition of the present invention on improving skin aging and barrier, the collagenase inhibition ability by sample treatment in fibroblasts was evaluated.

Human dermal fibroblasts were seeded in a 24-well plate at a concentration of 6 x 10 ⁴ cells/ml and cultured for 24 hours. The samples were treated and cultured for an additional 24 hours. The medium was recovered, and the extracellular Matrix metalloproteinase-1 (MMP-1) was confirmed by ELISA (Enzyme-linked immuosorbent assay), and the collagenase inhibition rate was calculated.

As shown in Fig. 5, Examples 2-3, 2-4, 2-5, and 2-6 were confirmed to be effective in reducing the secretion of collagenase in fibroblasts.

Experimental Example 6. Evaluation of skin barrier improvement

For the study of 20 subjects, the skin was treated with SLS (sodium lauryl sulfate), and the water loss (g/h/ ^m2 ) of the same area of the skin before and after using the sample was measured using a transepidermal water loss meter (Tewameter: Courage + Khazaka electronic) to confirm the improvement in the skin barrier. If the water loss was reduced compared to the control group, it was judged to be effective in strengthening the skin barrier.

As shown in FIGS. 6 and 7, the extracellular vesicles manufactured in Examples 2-3 and 2-6 were confirmed to have the effect of improving damaged skin barriers.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible within a scope that does not depart from the technical spirit of the present invention described in the claims.

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Claims (14)

A composition for improving a skin barrier, comprising probiotic extracellular vesicles obtained by culturing Saccharomyces cerevisiae in a medium containing YM Broth Basal medium and malic acid, and culturing Lactobacillus plantarum in a medium containing MRS Broth Basal medium and malic acid (MA). delete A composition for improving the skin barrier, comprising probiotic extracellular vesicles characterized by increasing collagen synthesis, inhibiting collagen decomposition enzymes, and regulating the microbiome in claim 1. A composition for improving the skin barrier containing probiotic extracellular vesicles, characterized in that the size of the extracellular vesicles in claim 1 is 30 to 150 nm. delete A composition for improving a skin barrier containing probiotic extracellular vesicles, characterized in that the composition is obtained by additionally including a step of centrifuging the culture solution prepared in the step of culturing in the medium in claim 1 and filtering the supernatant with 0.22 ㎛ PES. delete A composition for improving a skin barrier containing probiotic extracellular vesicles, characterized in that in claim 1, the composition for improving a skin further comprises at least one additive selected from butylene glycol, hexanediol, ethylhexylglycerin, and phenoxyethanol. A composition for improving a skin barrier containing probiotic extracellular vesicles, characterized in that in claim 1, the skin improvement composition is in a formulation selected from the group consisting of a toner, a nourishing cream, a massage cream, an essence, a pack, a gel, a lotion, an ointment, a patch, and a spray. delete delete A cosmetic method for improving the condition of mammalian skin, excluding therapeutic use, by using a composition for improving skin barrier comprising probiotic extracellular vesicles according to Article 1. A cosmetic method comprising: (a) directly applying the composition for improving skin barrier to the skin of a mammal, (b) contacting or attaching a patch, mask pack or mask sheet on which the composition for improving skin barrier is applied or deposited, or performing (a) and (b) sequentially. A cosmetic method in claim 13, further comprising the step of (c) removing the patch, mask pack or mask sheet from the skin of the mammal after step (b) and applying the composition for improving the skin barrier to the skin of the mammal.