KR20220033009A - composition for anti-aging comprising extracellular vesicle derived from lactic acid bacteria - Google Patents

Abstract

The present invention relates to a composition for anti-aging which comprises a lactic acid bacteria-derived extracellular vesicle. The composition according to one aspect comprises the extracellular vesicle isolated from lactic acid bacteria lysate to have the excellent effects of alleviating skin wrinkles and alleviating skin elasticity and thus can be usefully used as a composition for anti-aging.

Description

Composition for anti-aging comprising extracellular vesicle derived from lactic acid bacteria

The present invention relates to an anti-aging composition comprising lactic acid bacteria-derived extracellular vesicles.

Probiotics is a compound word of Pro and Biotics. Pro means 'for' and 'Biotics' means life. The World Health Organization (WHO) defines probiotics as live microorganisms that have beneficial effects on health. Most probiotics known to date are lactic acid bacteria, which are generally consumed as food, health functional food, or medicine. In the cosmetic industry, there have been attempts to improve the skin condition by using a culture solution obtained by culturing probiotic microorganisms. However, since the culture medium of the strain contains various proteins, cytokines, growth factors, etc. secreted by the strain, it also contains components such as waste products secreted by the strain as it grows, so there is a possibility of exposure to various risks when used on the skin. . Therefore, in order to use the strain culture, a separate process for removing wastes or various dangerous components is required, and accordingly, the cost is high.

Extracellular ER is a generic term for membrane-structured endoplasmic reticulum secreted by cells for information exchange between cells. Exosomes, ectosomes, microvesicles, microparticles, and apoptosis apoptotic body) and the like. Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers made up of a phospholipid double membrane identical to the structure of the cell membrane, and contain cytokines, growth factors, miRNA, DNA, proteins, etc. It reflects the specific genetic material and bioactive factors according to the nature and condition of the derived cell.

Exosomes can be isolated from various types of body fluids, for example, saliva, urine, plasma, serum, and amniotic fluid, and can also be isolated from various types of cell culture supernatants. Although a functional cosmetic composition comprising exosomes isolated from human stem cell culture supernatant has been developed (Korean Patent Application Laid-Open No. 10-2016-0086253), exosomes and/or cells derived from cell lysates of probiotic microorganisms such as lactic acid bacteria Studies on the anti-aging use of the endoplasmic reticulum are insignificant.

One aspect is to provide a cosmetic composition for anti-aging comprising lactic acid bacteria-derived extracellular vesicles as an active ingredient.

Another aspect is to provide an anti-aging composition for external application for skin comprising lactic acid bacteria-derived extracellular vesicles as an active ingredient.

One aspect provides a cosmetic composition for anti-aging comprising lactic acid bacteria-derived extracellular vesicles as an active ingredient.

The term “extracellular vesicle” refers to vesicles secreted from cells and released into the extracellular space. and plasma membrane protein, nucleic acid, and cytoplasmic components, and may mean a smaller size than the original cell, but is not limited thereto. As the ER or multivesicular body formed after collection bursts, the membrane or the composition of the active ingredient inside may include the reconstituted ER. The extracellular endoplasmic reticulum binds to other cells and delivers membrane components, mRNAs, miRNAs, etc., and delivers these transporters to recipient cells, thereby acting as an extracellular transporter mediating cell-cell communication.

The term “exosome-like vesicle” refers to a nano-sized extracellular vesicle, including nano-sized exosomes, as well as endoplasmic reticulum having a nano-sized ER structure and composition similar to exosomes. It may be the broadest concept that includes.

In one embodiment, the lactic acid bacteria-derived extracellular vesicles may include lactic acid bacteria-derived exosomes and/or exosome-like endoplasmic reticulum.

The “lactic acid bacteria” may be an isolated strain or a variety of commercially available lactic acid bacteria that can be used without limitation.

In one embodiment, the lactic acid bacteria is Bifidobacterium ( Bifidobacterium spp. ), Lactobacillus ( Latobacillus spp. ), Streptococcus spp .), Enterococcus ( Enterococcus spp .), Leuconostoc spp . ) and Lactococcus ( Lactococcus spp .) may be any one selected from the group consisting of strains, specifically Lactobacillus ( Latobacillus spp .) It may be a sp. strain, for example, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus plantarum ( Lactobacillus plantarum ) may be.

In one embodiment, the extracellular vesicles may be separated from the lysate of the lactic acid bacteria culture. The lysate of the lactic acid bacteria culture may be a lysate of the lactic acid bacteria.

The term “culture solution” refers to the entire medium containing the strain, its metabolites, and extra nutrients, obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain can grow and survive in vitro. can

The term “culture supernatant” refers to only the liquid in the upper layer excluding the part that has sunk to the bottom by leaving the culture medium still for a certain period of time, or centrifugation of the culture medium to mean only the liquid in the upper layer except for the sediment at the bottom.

The term “lysate” may refer to a product obtained by crushing with chemical or physical force, and the term “lysate” may be used interchangeably with the term “cell lysate”. The lysate of the lactic acid bacteria culture solution may be a lysate of the lactic acid bacteria cell mass obtained by centrifuging the strain culture medium to remove the culture supernatant and suspending the recovered cells in an isotonic solution. When the extracellular ER is separated from the lysate of the lactic acid bacteria, the yield of the exosome-like ER can be increased compared to the case of separation from the culture supernatant, and the exosome-like ER with excellent functional activity in anti-aging can be isolated Bar, the composition comprising the exosome-like endoplasmic reticulum isolated from the lysate of the cell can be used as an anti-aging composition.

The term “anti-aging” may refer to any action that prevents or inhibits skin aging. Skin aging includes intrinsic aging over time and extrinsic aging caused by external environments. The skin aging may include skin wrinkles, blemishes, blemishes, and the like. The skin wrinkles may be fine wrinkles caused by the deterioration of the skin. The skin wrinkles may be due to photoaging, age, facial expression, lack of moisture, or a combination thereof. The photoaging may be skin aging caused by exposure to ultraviolet rays (including UVA, UVB, and UVC). The term “skin wrinkle improvement” may refer to suppressing or inhibiting the formation of wrinkles on the skin, or alleviating the already generated wrinkles.

In one embodiment, the composition may enhance fibrilin expression.

In one embodiment, the composition may be to promote elastin (Elastin) expression.

In one embodiment, the composition may promote collagen (collagen type I α1) expression.

The term "improvement" may refer to any action that at least reduces the severity of a parameter, eg, a symptom, associated with alleviation or treatment of a condition.

In one embodiment, the extracellular vesicles may be separated by ultrafiltration with a molecular weight cutoff (MWCO) filter of 20 kDa to 500 kDa. Specifically, the extracellular vesicles may be separated by ultrafiltration from the lysate of lactic acid bacteria, and more specifically, the lysate of the lactic acid bacteria is centrifuged to remove cell-related debris, and the obtained supernatant is filtered with a 0.45 μm filter. It may be separated by ultrafiltration from the filtered sample.

In one embodiment, the extracellular vesicles may have a diameter of 20 nm to 400 nm. For example, 20 nm to 350 nm, 20 nm to 300 nm, 20 nm to 250 nm, 20 nm to 200 nm, 50 nm to 400 nm, 50 nm to 350 nm, 50 nm to 300 nm, 50 nm to 250 nm, 50 nm to 200 nm, 100 nm to 400 nm, 100 nm to 350 nm, 100 nm to 300 nm, 100 nm to 250 nm, 100 nm to 200 nm, 100 nm to 150 nm, 150 nm to 400 nm, 150 nm to 350 nm, 150 nm to 300 nm, 150 nm to 250 nm, 150 nm to 200 nm, 200 nm to 400 nm, 200 nm to 350 nm, 200 nm to 300 nm, 200 nm to 250 nm, 250 nm to 400 nm, 250 nm to 350 nm, 250 nm to 300 nm, 300 nm to 400 nm, or 300 nm to 350 nm, specifically, 20 nm to 200 nm.

The composition comprises 0.000000001 wt% to 80 wt%, for example, 0.0001 wt% to 60 wt%, 0.0001 wt% to 40 wt%, 0.0001 wt% to 30 wt%, 0.0001 wt% to 20 wt% with respect to the total weight of the composition %, 0.0001% to 10%, 0.0001% to 5%, 0.001% to 80%, 0.001% to 60%, 0.001% to 40%, 0.001% to 30%, 0.001% to 20% by weight, 0.001% to 10% by weight, 0.001% to 5% by weight, 0.01% to 80% by weight, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% by weight % to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% to 10% by weight, 0.1% to 5% by weight of extracellular vesicles.

In one embodiment, the composition comprises 10 ⁶ cells/ml to 10 ¹² cells/ml, for example, 10 ⁶ cells/ml to 10 ¹¹ cells/ml, 10 ⁶ cells/ml to 10 ¹⁰ cells/ml. /ml, 10 ⁶ /ml to 10 ⁹ /ml, 10 ⁶ /ml to 10 ⁸ /ml, 10 ⁶ /ml to 10 ⁷ /ml, 10 ⁷ /ml to 10 ¹² /ml , 10 ⁷ /ml to 10 ¹¹ /ml, 10 ⁷ /ml to 10 ¹⁰ /ml, 10 ⁷ /ml to 10 ⁹ /ml, 10 ⁷ /ml to 10 ⁸ /ml, 10 ⁸ /ml to 10 ¹² /ml, 10 ⁸ /ml to 10 ¹¹ /ml, 10 ⁸ /ml to 10 ¹⁰ /ml, 10 ⁸ /ml to 10 ⁹ /ml, 10 ⁹ /ml to ^{10 12} pcs./ml, 10 ⁹ pcs./ml to 10 ¹¹ pcs./ml, 10 ⁹ pcs./ml to 10 10 pcs./ml, 10 ¹⁰ pcs./ml to 10 ¹² pcs./ml, 10 ¹⁰ pcs./ml It may be included at a concentration of 10 to ¹¹ pieces/ml, 10 ¹¹ pieces/ml to 10 ¹² pieces/ml. When the extracellular vesicles are included in less than 10 ⁶ / ml in the composition, a significant anti-aging effect may not appear, and when included in more than 10 ¹² / ml, formulation stability may be reduced.

The term "included as an active ingredient" means that the extracellular vesicles of the present specification are added to an extent capable of exhibiting the above-mentioned effects, and various components are added as subcomponents for delivery and stabilization, etc. It may mean that it includes formulation).

The cosmetic composition may have a cosmetic formulation of, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a lotion, an essence, a pack, a gel, an ampoule, or a skin adhesion type.

Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include

Another aspect provides an anti-aging composition for external application for skin comprising lactic acid bacteria-derived extracellular vesicles as an active ingredient. In the composition for external application for skin, lactic acid bacteria, extracellular vesicles, anti-aging, and the composition are as described above.

The external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal-blocking agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

The lactic acid bacteria-derived extracellular vesicles (extracellular vesicle) comprising the steps of (a) obtaining a lysate of the lactic acid bacteria culture; (b) filtering the lysate; And (c) ultra-filtering the filtered lysate; may be prepared by a method comprising.

In one embodiment, the lysate of the lactic acid bacteria culture in step (a) may be a lysate of the cells recovered by centrifuging the lactic acid bacteria culture to remove the culture supernatant.

In one embodiment, the crushing in step (a) may be to physically crush the cells by applying pressure. Specifically, the shredding may be breaking the cell membrane or breaking the multivesicular body existing inside the cell, and the cell membrane ruptures to collect the active ingredient inside and then forms the ER, or the multivesicular body ruptures the membrane or inside It may be to reform the endoplasmic reticulum with a different composition. Therefore, the lysate may include an extracellular vesicle artificially generated by a physical force, such as cell membrane or ER, in addition to the extracellular ER that occurs naturally in the metabolic process of lactic acid bacteria. In one embodiment, the physical crushing may be to use a high pressure homogenizer, for example, the physical crushing is to crush 1 to 10 times using a microfluidizer. may be, for example, 1 to 8 times, 1 to 6 times, 1 to 5 times, 1 to 4 times, but 1 to 3 times may be appropriate. If cell disruption is not performed, it may be difficult to obtain an extracellular vesicle with improved functional activity in improving skin condition, and when the number of disruptions is more than 3 times, the ER membrane is destroyed due to external turgor of the extracellular vesicle, and the yield is reduced. Problems can arise.

In the case of the chemical disruption method, it is not suitable for cell disruption in large quantities, and the step of removing chemical components is added to prevent chemical modification of membrane components or adverse effects caused by chemical components, and contamination may occur during reagent processing. there is. In the case of physical crushing methods other than the pressure method, for example, in the case of a sonicator using a high frequency, mass production is difficult due to a low yield, and heat may be generated to cause degeneration of the extracellular vesicles. In the case of the grinding method using a mortar and pestle, homogenization may not be uniform, and in the case of crushing using glass beads and vortexing, a problem of not homogenizing may occur.

In one embodiment, the pressure may be 700 to 2000 bar, for example, 700 to 1900 bar, 700 to 1800 bar, 700 to 1700 bar, 700 to 1600 bar, 700 to 1500 bar, 700 to 1400 bar , 700 to 1300 bar, 700 to 1200 bar, 700 to 1100 bar, 700 to 1000 bar, 800 to 2000 bar, 800 to 1900 bar, 800 to 1800 bar, 800 to 1700 bar, 800 to 1600 bar, 800 to 1500 bar , 800 to 1400 bar, 800 to 1300 bar, 800 to 1200 bar, 800 to 1100 bar, 800 to 1000 bar, 900 to 2000 bar, 900 to 1900 bar, 900 to 1800 bar, 900 to 1700 bar, 900 to 1600 bar , 900 to 1500 bar, 900 to 1400 bar, 900 to 1300 bar, 900 to 1200 bar, 900 to 1100 bar, 900 to 1000 bar. Specifically, the pressure may be 700 to 2000 bar. When the pressure exceeds 2000 bar, the desired exosome may be destroyed, and when the pressure is less than 700 bar, cell disruption may not occur smoothly.

In one embodiment, step (b) may be to centrifuge the lysate obtained in step (a) to remove debris and filter the recovered supernatant.

In one embodiment, the centrifugation may be performed at 8,000 to 100,000 × g for 20 minutes to 2 hours. For example, 8,000 to 80,000 xg, 8,000 to 60,000 xg, 8,000 to 40,000 xg, 8,000 to 30,000 xg, 8,000 to 20,000 xg, 8,000 to 10,000 xg, 9,000 to 100,000 xg, 9,000 to 80,000 xg g, 9,000 to 60,000 xg, 9,000 to 40,000 xg, 9,000 to 30,000 xg, 9,000 to 20,000 xg, 9,000 to 10,000 xg, 10,000 to 100,000 xg, 10,000 to 80,000 xg, 10,000 to 60,000 xg, 10,000 to 40,000 × g, 10,000 to 30,000 × g, 20 minutes to 2 hours, 20 minutes to 1 hour 30 minutes, 20 minutes to 1 hour, 20 minutes to 40 minutes, 20 minutes to 30 minutes at 10,000 to 20,000 × g; 30 minutes to 2 hours, 30 minutes to 1 hour and 30 minutes, 30 minutes to 1 hour, 30 minutes to 40 minutes may be performed, but is not limited thereto, and a person of ordinary skill in the art can appropriately select or modify it Available. When the gravitational acceleration is more than 100,000 × g, exosomes and debris of a target size are removed together, which may cause a problem in that the yield is lowered. If the gravitational acceleration is less than 8,000 × g, there may be a problem in that the cell disruption debris remains in the supernatant, and there may be a problem in that the efficiency is lowered to purify the high-purity exosomes.

In one embodiment, the step (b) may be filtering with a 0.3 to 1.0 μm filter, for example, 0.3 to 0.9 μm, 0.3 to 0.8 μm, 0.3 to 0.7 μm, 0.3 to 0.6 μm, 0.3 to 0.5 μm, 0.4 to 1.0 μm, 0.4 to 0.9 μm, 0.4 to 0.8 μm, 0.4 to 0.7 μm, 0.4 to 0.6 μm, 0.4 to 0.5 μm. If the size of the filter is more than 1.0 μm, cell disruption debris other than exosomes may be introduced to cause a problem that exosomes cannot be obtained in high purity, and if the size of the filter is less than 0.3 μm, there may be a problem of lowering the yield can

In one embodiment, the step (c) may be to use a MWCO (molecular weight cutoff) filter of 20 kDa to 500 kDa. For example, using a molecular weight cutoff (MWCO) filter of 20 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 100 kDa or more, 200 kDa or more, 300 kDa or more, 400 kDa or more, 500 kDa The above samples may be obtained and concentrated. When the size of the MWCO filter exceeds 500 kDa, substances other than exosomes are introduced, which may cause a problem that exosomes cannot be obtained with high purity, and when the size of the MWCO filter is less than 20 kDa, the introduction of exosomes of a target size This blocking problem may occur. In step (c), the cell lysate is concentrated through the membrane filter, and the material larger than the pore size is caught by the filter, and the small material that has passed through the membrane filter may be removed by diffusion. Specifically, when a 100 kDa MWCO (molecular weight cutoff) filter is used, organelles are removed and the extracellular vesicles containing exosomes and/or exosome-like vesicles of 100 kDa or more are highly concentrated while being caught in the filter.

In one embodiment, the method is a 0.01 to 0.3 μm filter, for example, 0.01 to 0.25 μm, 0.01 to 0.2 μm, 0.1 to 0.3 μm, 0.1 to 0.25 of the ultra-filtered lysate after step (c). It may further include the step of sterilizing and filtering with a filter of μm, 0.1 to 0.2 μm, 0.2 to 0.3 μm, or 0.2 to 0.25 μm. When the size of the filter is more than 0.3 μm, there may be a problem that sterilization filtration is not performed, and when the size of the filter is less than 0.01 μm, there may be a problem that the efficiency is lowered.

The composition according to one aspect contains extracellular vesicles isolated from lactic acid bacteria lysate and has excellent skin wrinkle improvement and skin elasticity improvement effects, and thus can be usefully used as an anti-aging composition.

1 is a flowchart showing a method for separating extracellular vesicles derived from lactic acid bacteria cell lysate according to an embodiment.
Figure 2 is a photograph observing the change in turbidity of the lysate sample according to the repetition of the cell disruption and debris removal process.
Figure 3 is a graph measuring the number of particles according to the size of the extracellular vesicles separated by the method of Example 1 by nano-sight.
4A and 4B are results of comparative analysis of fibrillin and elastin gene expression ability after treating HS68 cells with extracellular vesicles isolated from the cell lysate of Example 1, respectively.
5 is a result of comparative analysis of collagen type I α 1 gene expression ability after treating the extracellular vesicles isolated from the cell lysate of Example 1 in HDF cells.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Isolation of extracellular vesicles derived from lactic acid bacteria lysate (LR(L))

The extracellular vesicles were isolated from the Lactobacillus rhamnosus cell lysate. Lactobacillus rhamnosus ( Lactobacillus rhamnosus, KCCM 32405) was used after receiving the strain deposited in the Korea Center for Microorganisms (KCCM).

Specifically, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) After culturing at 37 ° C. in MRS medium for 24 hours, the obtained cell mixture was centrifuged at 4 ° C. at 10,000 × g for 30 minutes to remove the culture supernatant and recover the cells. The recovered cells were suspended in 500 mL of tertiary distilled water or isotonic solution (eg, saccharide or PBS, etc.) to prepare a cell sample (hereinafter, referred to as a lysate sample). Cell disruption was performed on the obtained lysate sample at a pressure of 1000 bar using a high-pressure homogenizer (Microfludizer, MN600P-300, Picomax, Seoul, Republc of Korea). The obtained cell lysate was centrifuged at 10,000 × g at 4° C. for 30 minutes to remove cell-related debris and a supernatant was obtained. The cell disruption and debris removal process was repeated three times. Figure 2 is a photograph observing the change in turbidity of the lysate sample according to the repetition of the cell disruption and debris removal process. As a result, as shown in FIG. 2 , it was confirmed that most of the debris caused by cell disruption was removed. The obtained supernatant was filtered through a 0.45 μm sterile filter to further remove residual cell debris. The eluted sample was subjected to ultrafiltration (ultrafiltration, Pellicon ^® 2 and 3 Mini Holder, Merck, Darmstadt, Germany) with a MWCO (molecular weight cutoff) 100 kDa filter to separate extracellular vesicles over 100 kDa. The obtained solution was sterilized and filtered through a 0.2 μm sterile filter, and after freeze-drying, it was stored at -20°C and used for the following test.

Experimental Example 1. Identification of lactic acid bacteria-derived extracellular vesicles

Nanoparticle tracking analysis was performed using nano-sight NS300 (Malvern Panalytical, Almelo, Nederland) to measure the size and concentration of the extracellular vesicles isolated by the methods of Examples and Comparative Examples.

Specifically, the lactic acid bacteria-derived extracellular vesicle sample was diluted with PBS or tertiary distilled water so that the number of particles was 1 to 4x10 ⁸ , and the number of frames during measurement was 20 to 30. 1mL of the sample was prepared and loaded into the instrument chamber. After focusing on the particle, adjusting the camera level, measurement was started. The capture duration was set between 30 and 60 seconds, and measurements were repeated 3 times. After the measurement, the detection threshold was adjusted, and the values measured three times were analyzed. In this case, it was considered appropriate if the pattern of the results repeated three times showed a similar pattern. Figure 3 is a graph measuring the number of particles according to the size of the extracellular vesicles separated by the method of Example 1 by nano-sight.

As a result, as shown in FIG. 3, it was confirmed that most of the endoplasmic reticulum diameter of Example 1 was 20 to 200 nm.

Experimental Example 2. Confirmation of anti-aging effect of lactic acid bacteria-derived extracellular vesicles

(1) Evaluation of fibrillin and elastin gene expression ability

In order to confirm the anti-aging effect of the lactic acid bacteria lysate-derived extracellular vesicles isolated in Example 1, human dermal fibroblasts (HS68) were treated with the extracellular vesicles of Example 1 and then fibrilin, elastin (Elastin) gene expression ability was evaluated.

Specifically, after culturing so that the density of HS68 cells is about 80%, the sample of Example 1 was treated for each concentration of 0.000000001 and 0.00000001% (v/v). The positive control group was treated with EGCG. After incubation for 24 hours after treatment, the supernatant is removed, mRNA is extracted using trizol, and cDNA is synthesized using reverse transcriptase. The synthesized cDNA is mixed with a primer, dNTP, and taq polimerase, and then PCR is performed. Confirmed. 4A and 4B are results of comparative analysis of fibrillin and elastin gene expression ability after treating HS68 cells with extracellular vesicles isolated from the cell lysate of Example 1, respectively.

As a result, as shown in FIGS. 4A and 4B , it was confirmed that the extracellular vesicles isolated in Example 1 significantly increased fibrillin and elastin expression in a concentration-dependent manner. This means that the extracellular vesicles having excellent anti-aging activity such as skin wrinkles and elasticity improvement were isolated according to the manufacturing method of the present invention for performing cell disruption.

(2) Collagen gene expression ability evaluation

In order to confirm the anti-aging effect of the lactic acid bacteria-derived extracellular vesicles isolated in Example 1, human skin fibroblasts (HDFs) were treated with the extracellular vesicles of Example 1 and then the gene expression ability of collagen type I α 1 was evaluated. evaluated.

Specifically, after culturing so that the density of HDF cells is about 80%, the sample of Example 1 was treated at a concentration of 0.00001% (v/v). After incubation for 48 hours after treatment, the supernatant was removed and mRNA was extracted according to the experimental method of the kit (TaKaRa MiniBEST Universal RNA Extraction Kit, Takara, Shiga, Japan), and then synthesized into cDNA using reverse transcriptase, and the synthesized cDNA was SYBR After mixing with green master mix (LightCycler® 480 SYBR Green I Master, Roche, Basel, Switzerland), realtime-PCR was performed to confirm gene expression in real time. 5 is a result of comparative analysis of collagen type I α 1 gene expression ability after treating the extracellular vesicles isolated from the cell lysate of Example 1 in HDF cells.

As a result, as shown in FIG. 5 , it was confirmed that the extracellular vesicles isolated in Example 1 significantly increased collagen type I α 1 expression. This means that the extracellular vesicles having excellent anti-aging activity, such as skin wrinkle improvement, were isolated according to the manufacturing method of the present invention for performing crushing.

Claims (11)

A cosmetic composition for anti-aging comprising lactic acid bacteria-derived extracellular vesicles as an active ingredient. The method according to claim 1, wherein the Bifidobacterium ( Bifidobacterium spp. ), Lactobacillus ( Latobacillus spp. ), Streptococcus spp ., Enterococcus spp., Enterococcus spp ., Leuconostoc spp . Caucus ( Lactococcus spp .) The cosmetic composition of any one selected from the group consisting of strains. The cosmetic composition according to claim 2, wherein the Lactobacillus sp. strain is Lactobacillus rhamnosus . The cosmetic composition according to claim 1, wherein the extracellular vesicles are isolated from the lysate of the lactic acid bacteria culture. The cosmetic composition of claim 4, wherein the lysate of the lactic acid bacteria culture solution is a lysate of the lactic acid bacteria. The cosmetic composition of claim 1, wherein the extracellular vesicles are separated by ultrafiltration with a molecular weight cutoff (MWCO) filter of 20 kDa to 500 kDa. The cosmetic composition of claim 1, wherein the extracellular vesicles have a diameter of 20 to 400 nm. The cosmetic composition according to claim 1, wherein the composition enhances the expression of fibrilin. The cosmetic composition of claim 1, wherein the composition enhances elastin expression. The cosmetic composition according to claim 1, wherein the composition promotes collagen (collagen type I α1) expression. A composition for external application for skin for anti-aging comprising lactic acid bacteria-derived extracellular vesicles as an active ingredient.

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