Extracellular vesicle contents, extracellular vesicles, and preparation method thereof

Abstract

To provide a method for preparing extracellular vesicles from culture medium of microalgae, and to provide a composition containing extracellular vesicles obtained by the method.SOLUTION: A preparation method including filtering a culture supernatant of microalgae, and a composition obtained by such a preparation method are provided. Such an extracellular vesicle-containing composition can be used as a composition for maintaining or increasing a fibrous structural protein.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to extracellular vesicle contents or extracellular vesicles derived from microalgae, and a method for preparing the same. [Background technology]

[0002] Many cell types secrete vesicles with lipid bilayer membranes to the outside of the cell. Such vesicles are called extracellular vesicles, and typically contain functional molecules such as proteins and nucleic acids within the lipid bilayer membrane. Extracellular vesicles are involved in the delivery of functional molecules in the body, and in recent years, they have been found to be involved in various life phenomena and diseases.

[0003] For example, it has been disclosed that a population prepared by culturing stem cells that produce cell-derived vesicles under specific conditions can promote angiogenesis (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2019-501179 Summary of the Invention [Problem to be solved by the invention]

[0005] However, much remains unknown about extracellular vesicles, and there have been few reports on their functions or methods for obtaining them, particularly for extracellular vesicles derived from microalgae.

[0006] Therefore, the present invention aims to provide a method for obtaining extracellular vesicle-containing matter or extracellular vesicles derived from microalgae, and a product obtained by such a method. [Means for solving the problem]

[0007] In order to solve the above problems, the inventors conducted intensive research and discovered that by filtering and purifying the culture supernatant of microalgae under specific conditions, it is possible to obtain extracellular vesicle-containing material or extracellular vesicles with excellent properties, thereby completing the present invention.

[0008] That is, a preferred embodiment of the present invention provides the extracellular vesicle-containing material or extracellular vesicle described below.

[0009] [1] Extracellular vesicles or a substance containing extracellular vesicles, obtained by a method comprising purifying a culture supernatant of microalgae by filtration through a filter having a membrane pore size of 0.001 to 0.2 μm. [2] Extracellular vesicles or a substance containing extracellular vesicles, obtained by a method comprising purifying a culture supernatant of a microalgae by filtration through a filter having a molecular weight cutoff of 10 KDa to 1000 KDa. [3] A material containing extracellular vesicles or extracellular vesicles obtained by heating a culture or culture supernatant of microalgae at 40°C or higher. [4] Item 4. The extracellular vesicles or the composition containing extracellular vesicles according to any one of Items 1 to 3, wherein the particle diameter of the extracellular vesicles is 50 nm or more and 700 nm or less. [5] Item 5. The extracellular vesicle or extracellular vesicle-containing material according to any one of Items 1 to 4, for maintaining or increasing a fibrous structural protein. [6] Item 6. The extracellular vesicle or the material containing extracellular vesicles according to any one of items 1 to 5, wherein the fibrous structural protein is collagen or elastin. [7] Item 7. The extracellular vesicles or the extracellular vesicle-containing composition according to any one of Items 1 to 6, wherein the amount of the fibrous structural protein produced is increased compared to that of the extracellular vesicles or the extracellular vesicle-containing composition obtained by using ultracentrifugation instead of the filter filtration. [8] Item 8. The extracellular vesicles or the extracellular vesicle-containing material according to any one of Items 1 to 7, wherein the number of extracellular vesicle particles is increased compared to the extracellular vesicles or the extracellular vesicle-containing material obtained by using ultracentrifugation instead of the filter filtration. [9] 9. The extracellular vesicles or the material containing extracellular vesicles according to claim 1, wherein the microalgae are microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[10] A method for preparing extracellular vesicles or a material containing extracellular vesicles, comprising purifying a culture supernatant of microalgae by filtration through a filter having a membrane pore size of 0.001 to 0.2 μm.

[11] A method for preparing extracellular vesicles or a material containing extracellular vesicles, comprising purifying a culture supernatant of microalgae by filtration through a filter having a molecular weight cutoff of 10 KDa to 1000 KDa.

[12] Item 12. The method for preparing extracellular vesicles according to Item 10 or 11, further comprising heating the culture or culture supernatant of the microalgae at 40°C to 120°C. Effect of the Invention

[0010] According to the present invention, extracellular vesicle-containing matter or extracellular vesicles derived from microalgae can be easily prepared (or purified). [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the results of nanoparticle analysis of an ultrafiltration sample of Pavlova culture supernatant in Test Example 1. [Diagram 2] FIG. 2 is a graph showing the results of nanoparticle analysis of the Spirulina culture supernatant ultrafiltration sample in Test Example 1. [Diagram 3] FIG. 3 is a graph showing the results of nanoparticle analysis of the ultrafiltration sample of Chlorella culture supernatant in Test Example 1. [Figure 4] FIG. 4 is a graph showing the results of nanoparticle analysis of a sample of Pavlova culture supernatant subjected to ultracentrifugation in Test Example 3. [Diagram 5]FIG. 5 is a graph showing the results of nanoparticle analysis of a sample of Pavlova culture supernatant subjected to heating and ultrafiltration in Test Example 5. [Figure 6] 6A is a graph showing some of the test results of adding a substance containing extracellular vesicles of microalgae and / or extracellular vesicles to human fibroblasts in Test Example 2. FIG 6B is a graph showing some of the test results of adding a substance containing extracellular vesicles of microalgae and / or extracellular vesicles to human fibroblasts in Test Example 4. [Figure 7] 7A is a graph showing some of the test results of adding a substance containing extracellular vesicles of microalgae and / or extracellular vesicles to human fibroblasts in Test Example 2. FIG. 7B is a graph showing some of the test results of adding a substance containing extracellular vesicles of microalgae and / or extracellular vesicles to human fibroblasts in Test Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] [Extracellular vesicles or extracellular vesicle contents] In one embodiment, the extracellular vesicle-containing composition of the present invention is obtained by filtering a culture supernatant of a microalga. Alternatively, in another embodiment, the extracellular vesicle-containing composition of the present invention is obtained by heating a culture or culture supernatant of a microalga.

[0013] (Microalgae culture supernatant) In this specification, microalgae refers to organisms that perform oxygen-producing photosynthesis, excluding mosses, ferns, and seed plants, and have a cell size of 0.1 μm to 1000 μm in diameter.

[0014] Microalgae include, for example, prokaryotic cyanobacteria, as well as eukaryotic organisms of the Glaucophyta, Rhodophyta (red algae), Chlorophyta, Cryptophyta (cryptophyta), Haptophyta (haptophyta), Heterokontophyta, Dinophyta (dinoflagellates), Euglenida, and Chlorarachniophyta.

[0015] Examples of microalgae include microalgae belonging to the genus Pavlova, Euglena (Japanese name: Midorimushi, protozoa / green algae), Spirulina (cyanophyte), Chlorella (green algae), Dunaliella (green algae), Nannochloropsis (true eyespot algae), Lipodria (green algae), or Botryococcus (green algae). The above-listed microalgae may be used alone or in combination of two or more.

[0016] Among microalgae, from the viewpoint of significantly achieving the effects of the present invention, microalgae belonging to at least one species selected from the group consisting of the genera Pavlova, Euglena, Spirulina, and Chlorella are preferred, microalgae belonging to at least one species selected from the group consisting of the genera Pavlova, Euglena, and Spirulina are more preferred, microalgae belonging to at least one species selected from the group consisting of the genera Pavlova and Euglena are even more preferred, and microalgae belonging to the genus Pavlova are particularly preferred.

[0017] Pavlova is included in the haptophyte division, and examples of microalgae in the genus Pavlova include P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, P. salina, etc., with P. granifera and / or P. gyrans being preferred.

[0018] Examples of microalgae of the genus Euglena include E. gracilis, E. longa, E. caudata, E. oxyuris, E. tripteris, E. proxima, E. viridis, E. sociabilis, E. ehrenbergii, E. deses, E. pisciformis, E. spirogyra, E. acus, E. geniculata, E. intermedia, E. mutabilis, E. sanguinea, E. stellata, E. terricola, E. klebsi, E. rubra, and E. cyclopicola, with E. gracilis and / or E. longa being preferred.

[0019] Examples of microalgae of the genus Spirulina include the genus Altrospira, whose scientific name has been changed from Spirulina, and include S. platensis (A. platensis) and S. maxima (A. maxima), with S. platensis being preferred.

[0020] Examples of microalgae belonging to the genus Chlorella include C. vulgaris, C. saccharophila, C. Ellipsoidea, C. pyrenoidosa, C. sorokiniana, and C. lobophora, with C. vulgaris being preferred.

[0021] The microalgae exemplified above are widely distributed in seawater, fresh water such as ponds and swamps, and brackish water, and may be separated from these and used, or any microalgae that has already been isolated may be used.

[0022] The microalgae exemplified above include closely related species and mutants thereof, as long as they provide the effects of the present invention. Mutants include those obtained by genetic methods such as gene recombination, transduction, and transformation.

[0023] In this specification, the culture supernatant of microalgae refers to the supernatant of a culture solution after culturing microalgae. For example, the culture supernatant may be obtained by culturing microalgae by a known culture method and removing the microalgae by a known separation means such as centrifugation. Here, the culture supernatant contains extracellular vesicles derived from microalgae.

[0024] The culture conditions for preparing the culture supernatant of microalgae are not particularly limited as long as the effects of the present invention are achieved, and known methods can be used. Examples of the culture conditions include the following.

[0025] (Culture conditions) Microalgae can be cultured using a culture medium. A carbon source is added to the culture medium as a nutrient source, and the carbon source is an inorganic carbon source (CO 2 , NaHCO 3 , Na 2 CO 3 The culture medium is preferably an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source, although there is no limitation thereon. For example, a culture medium containing a nitrogen source, a phosphorus source, a mineral, or other nutrient salts, such as Cramer-Myers medium or modified Cramer-Myers medium, may be used.

[0026] Depending on the type of microalgae, when seawater is used, it is possible to use a culture medium for marine microalgae or artificial seawater, or it is also possible to use commercially available products such as IMK medium.

[0027] The pH of the culture medium is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 2 to 8, 3 to 8, 4 to 8, 5 to 8, 2 to 7.5, 3 to 7.5, 4 to 7.5, and 5 to 7.5.

[0028] The culture temperature is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 15 to 40°C, 20 to 34°C, and 23 to 28°C.

[0029] The culture period is not particularly limited as long as the effects of the present invention are achieved, and examples include 4 to 30 days, 4 to 20 days, and 5 to 15 days.

[0030] Depending on the type of microalgae, the type of light source, the presence or absence of light and darkness, aerobic or anaerobic conditions, etc. can be appropriately selected.

[0031] In one embodiment, when culturing marine microalgae such as pavlova, a known method can be used as long as the effects of the present invention are achieved. For example, Seawater concentration: 50% seawater Medium concentration: IMK medium 2x concentration Culture solution volume: 800mL Light source: Side: Fluorescent lamp (100~150μmol) Light / dark cycle: 12 hours light and 12 hours dark pH: pH approximately 7.4 at the start of each culture Aeration: Implementation Mechanical agitation: None Cultivation period: Approximately 10 days Culture temperature: 25℃~28℃ The cells can be cultured under conditions that satisfy at least one of the following:

[0032] (Collection of culture supernatant) After culturing the microalgae under the culture conditions exemplified above, the culture supernatant is collected using a known separation means such as centrifugation, but the method is not particularly limited as long as it can remove or substantially remove the microalgae and appropriately collect the culture supernatant. In an exemplary embodiment, the centrifugation to obtain the culture supernatant may be performed for 40 to 80 minutes or 30 to 60 minutes at a centrifugal force of, for example, 1,000 to 20,000 x g, 1,500 to 20,000 x g, 1,500 to 15,000 x g, or 1,500 to 10,000 x g. At this time, the centrifugation may be performed stepwise by changing the speed or time. For example, after separating the culture supernatant by centrifugation at a low speed of 1,500 to 2,000 x g, it may be centrifuged at a high speed of 10,000 to 20,000 x g to further remove cells or cell-related debris and residual matter. The supernatant thus obtained is collected and subjected to the next step.

[0033] (Extracellular Vesicles) In the present specification, extracellular vesicles refer to vesicles secreted from microalgae, which usually have an average diameter of 10 nm to 1 μm. Such an average diameter is also referred to as an average particle size in the present specification. Although not limited, it is preferable that the extracellular vesicle-containing material of the present invention does not contain the microalgae cells themselves. Here, the term diameter or particle size in specifying the size of the extracellular vesicles, as used herein, refers to the maximum dimension of the extracellular vesicles, since the extracellular vesicles are not necessarily spherical. The diameter or particle size can be measured using microscopy techniques, particularly techniques for measuring nanoparticle size by transmission electron microscopy. In addition, it can also be measured using nanoparticle tracking analysis (NTA), which is based on the analysis of both light scattering and Brownian motion. In the present invention, the diameter or particle size of the extracellular vesicles is measured using a NanoSight (Malvern Panalytical, NanoSight L10) for extracellular vesicles with a particle size of 50 nm or more, and using a transmission electron microscope for extracellular vesicles with a particle size of less than 50 nm.

[0034] The cumulative 10% value (D10) of the particle diameter of the Pavlova-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are exhibited, and examples thereof include 80 to 140 nm, 90 to 130 nm, and 100 to 120 nm. In addition, the cumulative 50% value (D50) of the particle diameter is, for example, 120 to 210 nm, 130 to 200 nm, and 140 to 190 nm. In addition, the cumulative 90% value (D90) of the particle diameter is, for example, 200 to 320 nm, 210 to 310 nm, and 220 to 300 nm. In this specification, the cumulative 10% value (D10) of the particle diameter of the extracellular vesicles means a diameter below which 10% of the whole extracellular vesicles are. The cumulative 50% value (D50) of the particle diameter of the extracellular vesicles means a diameter below which 50% of the whole extracellular vesicles are, and is synonymous with the median diameter. The cumulative 90% particle diameter (D90) of extracellular vesicles means the diameter below which 90% of all extracellular vesicles fall.

[0035] The cumulative 10% value (D10) of the particle diameter of the Spirulina-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 50 to 110 nm, 60 to 100 nm, 70 to 90 nm, etc. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 100 to 160 nm, 110 to 150 nm, 120 to 140 nm, etc. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 190 to 250 nm, 200 to 240 nm, 210 to 230 nm, etc.

[0036] The particle diameter of the Chlorella-derived extracellular vesicles is not particularly limited as long as it exhibits the effects of the present invention, and may be, for example, 90 to 150 nm, 100 to 140 nm, 110 to 130 nm, etc. The particle diameter is, for example, 180 to 240 nm, 190 to 230 nm, 200 to 220 nm, etc. The particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc. The particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc.

[0037] (Extracellular vesicle content) In this specification, the term "material containing extracellular vesicles" refers to a concentrate or purified material obtained by filtering the culture supernatant containing extracellular vesicles obtained from the culture supernatant of microalgae. In addition to the extracellular vesicles, the material may also contain secretions obtained by the culture.

[0038] In the extracellular vesicle-containing composition of the present invention, the average particle number of the extracellular vesicles of the microalgae is, for example, 1.0 × 10 per ml when the purified product is in a liquid state. 3 pcs or more, 1.0×10 4 pcs or more, 1.0×10 5 pcs or more, 1.0×10 6 pcs or more, 1.0×10 7 pcs or more, 1.0×10 8 pcs or more, 1.0×10 9 1.0×10 15 pcs or less, 1.0×10 14 pcs or less, 1.0×10 13 pcs or less, 1.0×10 12 pcs or less, 1.0×10 11 The average number of particles in the extracellular vesicle content of microalgae is, for example, 1.0 × 10 4 ~1.0×10 15 pieces, 1.0×10 4 ~1.0×10 14 The number of extracellular vesicles can be measured using an exosome measurement system (ExoCounter, JVC Kenwood Corporation) that uses a method of sandwich detection specific to surface antigens using antibodies on disks and nanobeads.

[0039] (Method for Obtaining Extracellular Vesicles or Extracellular Vesicle Contents) As a method for obtaining the extracellular vesicle-containing material or the extracellular vesicles of the present invention, a method for filtering the culture supernatant can be used. The membrane pore size of the filter used for filtering is preferably 0.001 to 0.2 μm, more preferably 0.005 to 0.1 μm, and even more preferably 0.01 to 0.05 μm.

[0040] Here, the filter may be a hollow fiber membrane that filters from the outside to the inside of the fiber. Alternatively, the filter may be a spiral membrane including a filtration membrane and a support membrane, a tubular membrane on a hollow cylinder, or a flat membrane. Among these, it is preferable to use a hollow fiber membrane.

[0041] A hollow fiber membrane is a type of permeable membrane, and refers to a membrane formed in a thin straw-like capillary. A hollow fiber membrane module is a hollow fiber membrane bundle in which a number of hollow fiber membranes are bundled together and stored in a sealed state in a housing container formed of a vinyl chloride synthetic resin or a metal such as aluminum. In general, the diameter (inner diameter) of each hollow fiber membrane is about 100 μm to several mm, but in order to efficiently circulate liquid, it is preferably 100 μm to 800 μm, and more preferably 200 μm to 600 μm. In addition, the length of each hollow fiber membrane can be adjusted depending on the application, but if it is too long, the pressure for flowing liquid increases, so that the length excluding the fixing parts at both ends in the form of a hollow fiber membrane bundle, that is, the so-called effective length cm, is preferably formed to be 120 mm to 450 mm, more preferably 100 mm to 300 mm, and even more preferably 150 mm to 250 mm. From the viewpoint of industrialization, the thickness is preferably 250 mm to 1000 mm, and more preferably 400 mm to 800 mm. The membrane thickness of the hollow fiber membrane is generally about 10 μm to 100 μm, but is preferably 20 μm to 60 μm, more preferably 20 μm to 50 μm, and even more preferably 30 μm to 50 μm. In the present invention, it is preferable to use a hollow fiber membrane module (the hollow fiber membrane constitutes a hollow fiber membrane module) from the viewpoint of purification efficiency.

[0042] The membrane area of ​​the hollow fiber membrane in the hollow fiber membrane module used in the present invention can be the converted value at the middle part of the thickness, i.e., the average value of the outer surface area and the inner surface area, taking into account the difference in diameter between the inside and outside of the hollow fiber membrane. Since the membrane area of ​​the hollow fiber membrane and the processing speed are proportional to each other, using a hollow fiber membrane module with a larger membrane area will increase the processing speed, shorten the processing time, and reduce damage to extracellular vesicles. On the other hand, since the membrane area of ​​the hollow fiber module and the dead volume (amount remaining in the flow path) during purification are also proportional to each other, using a hollow fiber module with a larger membrane area will increase the loss accordingly. Therefore, it is preferable to select a hollow fiber membrane module with an appropriate membrane area according to the purpose. The membrane area of ​​the hollow fiber membrane in the hollow fiber membrane module used in the present invention is 1 cm 2 ~3,000cm 2 For example, if the purpose is to concentrate the solution to a small volume, a 20cm diameter is selected. 2 It is preferable to use a module with a small membrane area of ​​about 115 cm. In addition, when the purpose is concentration, filtration, removal of impurities, etc. of cell culture supernatant, a module with a membrane area of ​​about 115 cm is used according to the amount of processing. 2 Approximately 1,000cm 2 Approximately 1,600cm 2 From the viewpoint of industrialization, it is preferable to use a hollow fiber membrane module having an area of ​​about 1,000 cm. 2 ~100,000cm 2 is preferred, and 10,000 cm 2 ~60,000cm 2 is more preferred.

[0043] The pore size of the hollow fiber membrane used in the present invention (including the hollow fiber membrane provided in the hollow fiber membrane module) can be selected according to the purpose, but for example, when removing impurities larger than the desired extracellular vesicles, a pore size that does not allow only the impurities to pass through is selected. Such a pore size is preferably 0.001 to 0.2 μm, more preferably 0.005 μm to 0.1 μm, and even more preferably 0.01 μm to 0.05 μm. It may also be 0.0001 μm or more, 0.0005 μm or more, 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, 0.015 μm or more, 0.2 μm or less, 0.15 μm or less, 0.1 μm or less, 0.05 μm or less, 0.04 μm or less, or 0.03 μm or less.

[0044] The cutoff molecular weight is preferably 10 kDa to 1,000 kDa, 50 kDa to 1000 kDa, or 100 kDa to 1,000 kDa, and more preferably 100 kDa to 500 kDa. Cutoff membranes of 10 kDa or more, 50 kDa or more, 100 kDa or more, or 150 kDa or more are preferred, and cutoff membranes of 2000 kDa or less, 1500 kDa or less, 1000 kDa or less, 750 Dka or less, or 500 DKa or less are also preferred.

[0045] After culturing microalgae, the culture supernatant after removing the cells can be successively diluted and concentrated to remove unnecessary substances and at the same time exchange the solvent to obtain the required extracellular vesicles.

[0046] The material of the filter used in the present invention, particularly the hollow fiber membrane provided in the hollow fiber membrane module, is not particularly limited as long as it is a material that can be used for the purpose of appropriately separating extracellular vesicles derived from cells, and examples thereof include polyethersulfone (PES), modified polyethersulfone (mPES), polyacrylonitrile (PAN), etc. Among these, from the viewpoints of high hydrophilicity and low protein adsorption, polyethersulfone (PES) and modified polyethersulfone (mPES) are preferred, and modified polyethersulfone (mPES) is more preferred.

[0047] The extracellular vesicles or the material containing extracellular vesicles of the present invention can also be obtained by heating a culture or culture supernatant of a microalgae under temperature conditions, but not limited to, for example, 40° C. or higher, 50° C. or higher, 60° C. or higher, 70° C. or higher, 80° C. or higher, 90° C. or higher, or 100° C. or higher, 200° C. or lower, 150° C. or lower, or 120° C. or lower, for 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, or 4 minutes or more, or 30 minutes or less, 20 minutes or less, or 10 minutes or less.

[0048] The extracellular vesicle-containing material of the present invention can also be obtained by a method comprising heating a culture or culture supernatant of a microalgae and concentrating or purifying the culture supernatant obtained after heating by filtering.

[0049] The material containing extracellular vesicles may be in the form of a purified liquid of culture supernatant, but may also be a solid or semi-solid obtained by drying the purified liquid.

[0050] The extracellular vesicle content can optionally be further purified to provide a pure population of extracellular vesicles.

[0051] The material containing extracellular vesicles may be in the form of a purified liquid of the culture supernatant itself, but may also be a solid or semi-solid obtained by drying the purified liquid.

[0052] (Extracellular vesicle-containing material or composition comprising extracellular vesicles) In one aspect of the present invention, it is possible to prepare a composition containing either the extracellular vesicle contents or the extracellular vesicles thus obtained.

[0053] In such compositions, the average particle number of microalgae extracellular vesicles is, for example, 1.0×10 3 pcs or more, 1.0×10 4 pcs or more, 1.0×10 5 pcs or more, 1.0×10 6 pcs or more, 1.0×10 7 pcs or more, 1.0×10 8 pcs or more, 1.0×10 91.0×10 14 pcs or less, 1.0×10 13 pcs or less, 1.0×10 12 pcs or less, 1.0×10 11 pcs or less, 1.0×10 10 The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 4 ~1.0×10 14 pieces, 1.0×10 4 ~1.0×10 13 pieces, 1.0×10 4 ~1.0×10 12 pieces, 1.0×10 4 ~1.0×10 11 pieces, 1.0×10 4 ~1.0×10 10 pieces, 1.0×10 5 ~1.0×10 14 pieces, 1.0×10 5 ~1.0×10 13 pieces, 1.0×10 5 ~1.0×10 12 pieces, 1.0×10 5 ~1.0×10 11 pieces, 1.0×10 5 ~1.0×10 10 pieces, 1.0×10 6 ~1.0×10 14 pieces, 1.0×10 6 ~1.0×10 13 pieces, 1.0×10 6 ~1.0×10 12 pieces, 1.0×10 6 ~1.0×10 11 pieces, 1.0×10 6 ~1.0×10 10 pieces, 1.0×10 7 ~1.0×10 14 pieces, 1.0×10 7 ~1.0×10 13 pieces, 1.0×10 7 ~1.0×10 12 pieces, 1.0×10 7 ~1.0×10 11 pieces, 1.0×10 7 ~1.0×10 10 pieces, 1.0×10 8 ~1.0×1014 pieces, 1.0×10 8 ~1.0×10 13 pieces, 1.0×10 8 ~1.0×10 12 pieces, 1.0×10 8 ~1.0×10 11 pieces, 1.0×10 8 ~1.0×10 10 pieces, 1.0×10 9 ~1.0×10 14 pieces, 1.0×10 9 ~1.0×10 13 pieces, 1.0×10 9 ~1.0×10 12 pieces, 1.0×10 9 ~1.0×10 11 pieces, 1.0×10 9 ~1.0×10 10 Examples include:

[0054] The average particle size of the extracellular vesicles of microalgae is not particularly limited as long as the effects of the present invention are achieved, and examples include 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, and the like; and 700 nm or less, 600 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, and the like. The average particle size of the extracellular vesicles of microalgae is, for example, 10 to 700 nm, 10 to 600 nm, 10 to 500 nm, 10 to 400 nm, 10 to 300 nm, 10 to 250 nm, 30 to 700 nm, 30 to 600 nm, 30 to 500 nm, 30 to 400 nm, 30 to 300 nm, 30 to 250 nm, 50 to 700 nm, 50 to 600 nm, 50 to Examples of the thickness include 500 nm, 50 to 400 nm, 50 to 300 nm, 50 to 250 nm, 70 to 700 nm, 70 to 600 nm, 70 to 500 nm, 70 to 400 nm, 70 to 300 nm, 70 to 250 nm, 100 to 700 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm, 100 to 300 nm, and 100 to 250 nm.

[0055] The content of microalgal extracellular vesicles in the composition is appropriately adjusted depending on the type and amount of other components, dosage form, etc., and is not limited, but can be, for example, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 1% by mass or more, 5% by mass or more, 10% by mass or more, etc., relative to the total amount of the composition. The content of microalgal extracellular vesicles can be, for example, 50% by mass or less, such as 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, etc., relative to the total amount of the composition. The content of microalgal extracellular vesicles can be, for example, 0.001 to 0.1% by mass (for example, 2×10 particle number) relative to the total amount of the composition. 11 pieces / mL~4×10 11 0.001~50% by mass, 0.001~40% by mass, 0.001~30% by mass, 0.001~20% by mass, 0.001 ~15% by mass, 0.01-50% by mass, 0.01-40% by mass, 0.01-30% by mass, 0.01-20% by mass, 0.01-15% by mass, 0.1-5 Examples include 0% by mass, 0.1-40% by mass, 0.1-30% by mass, 0.1-20% by mass, 0.1-15% by mass, 1-50% by mass, 1-40% by mass, 1-30% by mass, 1-20% by mass, 1-15% by mass, 5-50% by mass, 5-40% by mass, 5-30% by mass, 5-20% by mass, or 5-15% by mass.

[0056] The content of microalgal extracellular vesicles is appropriately adjusted depending on the type and amount of other components, dosage form, etc., and is not limited, and can be, for example, 0.01 μg / mL or more, 0.1 μg / mL or more, 1 μg / mL or more, 10 μg / mL or more, or 100 μg / mL or more in terms of protein amount relative to the total amount of the composition. Examples of the content include 100,000 μg / mL or less, 10,000 μg / mL or less, 1000 μg / mL, 100 μg / mL or less, and 10 μg / mL or less.

[0057] [Application] In one embodiment, the extracellular vesicle-containing material or extracellular vesicle of the present invention, or a composition containing the same, is preferably used for maintaining or increasing fibrous structural proteins. In this specification, fibrous structural proteins refer to fibrous components in the body or components involved in the tissue structure. Although not limited thereto, the fibrous structural protein is preferably a protein associated with the skin, more preferably a protein associated with the epidermis or dermis, and even more preferably a protein associated with the dermis. In another embodiment, the fibrous structural protein is preferably a protein associated with the elasticity of the skin.

[0058] The skin is roughly divided into the epidermis and the dermis, and the constituents of the dermis are classified into interstitial components related to fibrous tissue and cellular components. The interstitial components related to fibrous tissue are mostly collagen fibers such as type I and type III collagen (also simply referred to as collagen in this specification), and also include elastic fibers composed of elastin, fibrillin, etc., which are collectively referred to as the extracellular matrix (ECM).

[0059] Representative examples of fibrous structural proteins include collagen and elastin.

[0060] There are about 28 subtypes of collagen, and it is known that about 80% of the collagen that constitutes the dermis is type I. Type I collagen contributes to the elasticity of the skin, and type III collagen contributes to the recovery of skin damage. Collagen is important as a supporting tissue to maintain the strength of the skin, as it has high resistance to tension acting especially along the course of the fibers and poor extensibility. Elastic fibers are composed of elastin and fibrillin, and are involved in the elasticity of the skin and the extensibility of arteries and tendons by being interposed between the layered structure created by collagen.

[0061] When the enzymes that degrade fibrous structural proteins are activated, the amount of the fibrous structural proteins can decrease. For example, matrix metalloproteinases (MMPS), especially matrix metalloproteinase 1 (MMP-1), are responsible for degrading collagen and elastin fibers in the skin.

[0062] Examples of genes encoding fibrous structural proteins, particularly in skin collagen, include type I collagen-related genes and type III collagen-related genes, such as COL1A1 (collagen type I alpha 1 chain) and COL3A1 (collagen type III alpha 1 chain).

[0063] Therefore, if a component is capable of suppressing fibrous structural protein degrading enzymes, activating fibrous structural protein synthases, or enhancing gene expression of fibrous structural proteins, it will lead to maintaining fibrous structural proteins by suppressing their decrease, or to increasing fibrous structural proteins.

[0064] In the examples described below, it has been confirmed that the extracellular vesicle-containing material and / or the extracellular vesicles of microalgae enhances the expression of elastin genes and COL3A1 genes. These results are expected to contribute to the maintenance or increase of fibrous structural proteins, leading to anti-aging of the skin, specifically, improvement of skin elasticity, firmness, wrinkles, texture, moisture, etc., and suppression of dryness and roughness of the skin.

[0065] The composition of the present invention can be used by adding to or mixing with medicines, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods, or can be used as it is as medicines, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods.

[0066] When the composition of the present invention is used as an addition to or mixture with so-called external preparations such as medicines, quasi-drugs, and cosmetics, the composition of the present invention can be used as a medicine, quasi-drug, or cosmetic that explicitly or implicitly specifies, as an efficacy, effect, or function, improvement of skin elasticity, skin firmness, wrinkles, texture, moisture, dryness, roughness, and the like.

[0067] When the above functionality is not clearly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those who want skin elasticity, those who want skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those who want moisturized skin, those who are concerned about dry skin, those who are concerned about rough skin, those who are concerned about pores, those who are concerned about skin fluctuations, those who are concerned about pores, etc., or a similar indication.

[0068] When used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the composition of the present invention is preferably prepared as an external preparation for skin beautification, an external preparation for anti-aging, etc. Among these, particularly preferred applications of the composition of the present invention include skin care cosmetics (e.g., lotions, creams, facial creams, facial lotions, milky lotions, packs, liquid facial cleansers, soaps, beauty essences), makeup cosmetics (e.g., eyeliners, eyebrows), and scalp and hair cosmetics (e.g., scalp lotions, scalp creams, shampoos, hair conditioners, hair treatments, hair essences, hair mist, and hair tonics), and most preferably, may be lotions, creams, facial creams, facial lotions, milky lotions, packs, facial cleansers, soaps, and beauty essences, but are not limited thereto.

[0069] Thus, when used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the dosage form of the composition of the present invention is not particularly limited, and examples thereof include liquids, suspensions, emulsions, creams, gels, liniments, lotions, ointments, and aerosols, etc. Among these, liquids, suspensions, emulsions, lotions, and aerosols are preferred, and liquids, lotions, and aerosols are more preferred.

[0070] When used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the daily dose of microalgal extracellular vesicles for adults can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, ingestion or administration route, ingestion or administration schedule, formulation form, or other factors. Examples of the daily dose of microalgal extracellular vesicles for adults include 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, and 200 μg or more. In addition, the daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, or 100 μg or less. In addition, the daily dosage of microalgae extracellular vesicles for adults is, for example, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 300 μg, 1 to 200 μg, 1 to 100 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 100 μg. g, 10 to 1000 μg, 10 to 900 μg, 10 to 800 μg, 10 to 700 μg, 10 to 600 μg, 10 to 500 μg, 10 to 400 μg, 10 to 300 μg, 10 to 200 μg, 10 to 100 μg, 20 to 1000 μg, 20 to 900 μg, 20 to 800 μg, 20 to 700 μg, 20 to 600 μg, 20 to 500 μg, 20 to 400 μg, 20 to 300 μg, 20 to 200 μg, and 20 to 100 μg. The dosage of microalgal extracellular vesicles varies depending on various factors such as the condition and age of the subject, but based on an adult, generally, 1 μg / kg to 200 mg / kg of the composition, and in another aspect, 50 μg / kg to 50 mg / kg, may be administered 1 to 3 times a day, and the dosage is not intended to limit the scope of the present specification in any way.

[0071] When used in so-called external preparations such as medicines, quasi-drugs, and cosmetics, the daily dose of microalgal extracellular vesicles for adults can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, ingestion or administration route, ingestion or administration schedule, formulation form, or other factors. The daily dose of microalgal extracellular vesicles for adults can be, for example, 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, or 200 μg or more in terms of protein amount. In addition, the daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, or 100 μg or less. In addition, the daily dosage of microalgae extracellular vesicles for adults is, for example, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 300 μg, 1 to 200 μg, 1 to 100 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 100 μg. g, 10 to 1000 μg, 10 to 900 μg, 10 to 800 μg, 10 to 700 μg, 10 to 600 μg, 10 to 500 μg, 10 to 400 μg, 10 to 300 μg, 10 to 200 μg, 10 to 100 μg, 20 to 1000 μg, 20 to 900 μg, 20 to 800 μg, 20 to 700 μg, 20 to 600 μg, 20 to 500 μg, 20 to 400 μg, 20 to 300 μg, 20 to 200 μg, and 20 to 100 μg. The dosage of microalgal extracellular vesicles varies depending on various factors such as the condition and age of the subject, but based on an adult, the composition may be administered at 1 μg / kg to 200 mg / kg, or in another aspect, 50 μg / kg to 50 mg / kg, 1 to 3 times a day, and the dosage is not intended to limit the scope of the present specification in any way.

[0072] In another embodiment, the daily dose of microalgal extracellular vesicles for an adult is, for example, 1 × 10 7 pcs or more, 1×10 8 pcs or more, 1×10 9 The daily dose of microalgae extracellular vesicles for adults is, for example, 1 × 10 14 pcs or less, 1×10 13 pcs or less, 1×10 12 pcs or less, 1×10 11 pcs or less, 1×10 10 The daily dose of microalgae extracellular vesicles for adults is, for example, 1 × 10 7 ~1×10 14 pieces, 1×10 7 ~1×10 13 pieces, 1×10 7 ~1×10 12 pieces, 1×10 7 ~1×10 11 pieces, 1×10 8 ~1×10 14 pieces, 1×10 8 ~1×10 13 pieces, 1×10 8 ~1×10 12 pieces, 1×10 8 ~1×10 11 pieces, 1×10 9 ~1×10 14 pieces, 1×10 9 ~1×10 13 pieces, 1×10 9 ~1×10 12 pieces, 1×10 9 ~1×10 11 Examples include:

[0073] The daily dose of the microalgae culture supernatant for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, administration schedule, formulation form, or other factors. The daily dose of the microalgae culture supernatant for an adult can be, for example, the amount of microalgae extracellular vesicles contained in the above-mentioned intake or administration amount.

[0074] The composition of the present invention can be ingested or administered once or several times a day, typically 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any period and interval, but once a day is preferred.

[0075] When the composition of the present invention is added to or mixed with food, beverage, feed, or pet food, the composition of the present invention can be used as a food or beverage that expressly or implies that it has the functionality of improving skin elasticity, skin firmness, wrinkles, texture, moisture, dryness, roughness, etc., i.e., health food, functional food, food for sick people, and food for specified health uses. In addition, even if the functionality is not expressly or implied, it can be used as a so-called doctor's supplement recommended or presented by a doctor in the internal medicine, orthopedics, veterinary hospital, etc. of a hospital and / or clinic.

[0076] When the above functionality is not clearly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those who want skin elasticity, those who want skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those who want moisturized skin, those who are concerned about dry skin, those who are concerned about rough skin, those who are concerned about pores, those who are concerned about skin fluctuations, those who are concerned about pores, etc., or a similar indication.

[0077] Health foods, functional foods, foods for the sick, and foods for specified health uses are specifically solid preparations. The food can be used in various formulations such as tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candies, etc.), liquids (syrups, suspensions), and liquid foods. The food in the formulation form can be produced in the same manner as known pharmaceutical preparations, and can be produced by mixing the active ingredient with a carrier acceptable for food, such as a suitable excipient, and then using conventional means. The formulation form is not limited, but is preferably an orally disintegrating tablet, a chewable tablet, a candy, a granule, a powder, or a liquid from the viewpoint of significantly exhibiting the effects of the present invention.

[0078] The composition of the present invention can also be manufactured as liquid beverages such as soups, juices, fruit juice drinks, milk, dairy drinks, whey drinks, lactic acid bacteria drinks, tea drinks, alcoholic drinks, coffee drinks, carbonated drinks, soft drinks, water drinks, cocoa drinks, jelly drinks, sports drinks, and diet drinks; semi-solid foods such as pudding and yogurt; noodles such as pasta, ramen, udon, and soba; confectioneries; and spreads.

[0079] The composition of the present invention can be applied in the form of oral administration, internal administration, etc. When used as a pharmaceutical composition, it may be used therapeutically or non-therapeutically.

[0080] The daily oral intake or administration amount of microalgal extracellular vesicles for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. Examples of the daily oral intake or administration amount of microalgal extracellular vesicles for an adult include 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, and 200 μg or more. In addition, the daily oral intake or administration amount of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, 100 μg or less, etc. The daily oral intake or administration amount of microalgal extracellular vesicles for an adult is, for example, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 300 μg, 1 to 200 μg, 1 to 100 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 5 ... ~100μg, 10~1000μg, 10~900μg, 10~800μg, 10~700μg, 10~600μg, 10~500μg, 10~400μg, 10~300μg, 10~200μg, 10~100μg, 20~1000μg, 20~900μg, 20~800μg, 20~700μg, 20~600μg, 20~500μg, 20~400μg, 20~300μg, 20~200μg, 20~100μg, etc.

[0081] In another embodiment, the daily oral intake or administration amount of microalgal extracellular vesicles for an adult is, for example, 1 × 10 7 pcs or more, 1×10 8 pcs or more, 1×10 9 The daily oral intake or administration amount of microalgae extracellular vesicles for an adult is, for example, 1×10 14pcs or less, 1×10 13 pcs or less, 1×10 12 pcs or less, 1×10 11 pcs or less, 1×10 10 The daily oral intake or administration amount of microalgae extracellular vesicles for an adult is, for example, 1 × 10 7 ~1×10 14 pieces, 1×10 7 ~1×10 13 pieces, 1×10 7 ~1×10 12 pieces, 1×10 7 ~1×10 11 pieces, 1×10 8 ~1×10 14 pieces, 1×10 8 ~1×10 13 pieces, 1×10 8 ~1×10 12 pieces, 1×10 8 ~1×10 11 pieces, 1×10 9 ~1×10 14 pieces, 1×10 9 ~1×10 13 pieces, 1×10 9 ~1×10 12 pieces, 1×10 9 ~1×10 11 Examples include:

[0082] The daily oral intake or administration amount of the microalgal culture supernatant for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or administration amount of the microalgal culture supernatant for an adult can be, for example, the amount of microalgal extracellular vesicles contained in the above-mentioned intake or administration amount.

[0083] The daily oral intake or dosage for an adult may be divided into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules depending on the dosage form.

[0084] The composition of the present invention can be ingested or administered once or several times a day, typically 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any period and interval, but once a day is preferred.

[0085] When the present invention is used as feed, pet food, or the like, the target organism is not particularly limited, but is preferably a mammal, reptile, amphibian, bird, or fish, and more preferably a mammal other than humans, such as a platypus, echidna, opossum, quoll, kangaroo, aardvark, rock badger, elephant, armadillo, sloth, anteater, tree shrew, flying lemur, chimpanzee, rabbit, degu, dormouse, squirrel, raccoon, mouse, hedgehog, chinchilla, ferret, camel, wild boar, giraffe, deer, cow, goat, hippopotamus, whale, dolphin, horse, rhinoceros, tapir, bat, monkey, tiger, wolf, weasel, bear, seal, dog, cat, parakeet, parrot, finch, owl, owl, etc., and more preferably a dog or cat.

[0086] When the present invention is used as feed, pet food, or the like, it may be given to the pet in divided portions several times a day by adding it to the staple food, or it may be given to the pet as a snack at any time. EXAMPLES

[0087] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0088] [Test Example 1 in the Examples: Preparation and Evaluation of Extracellular Vesicle-containing Material and / or Extracellular Vesicles of Microalgae 1] Pavlova sp., Spirulina (Arthrospira platensis), or Chlorella (Chlorella vulgaris) was cultured in a commercially available algae medium by a conventional method, and a culture supernatant was obtained. 500 mL of each culture supernatant was used and concentrated to a volume of 5 mL or less using a 200K ultrafiltration membrane (manufactured by ADVANTEC). Then, concentration washing was performed three times using 100 mL of PBS, and finally, the sample was collected with 50 mL of PBS. Then, each concentrated sample was sterilized using a 0.22 μm filter, and the particle number, etc. were measured using NanoSight.

[0089] Each of the above samples was appropriately diluted with PBS(-), and a number distribution graph (horizontal axis: particle diameter, vertical axis: particle number concentration) was created using a nanoparticle analysis system NanoSight LM10 (Malvern Panalytical) in accordance with the attached software.

[0090] The above gave the following samples: Pavlova culture supernatant ultrafiltration sample (protein concentration 11.9 μg / mL, Pavlova EVs1) Spirulina culture supernatant ultrafiltration sample (protein concentration 29.6 μg / mL) · Chlorella culture supernatant ultrafiltration sample (protein concentration 10.4 μg / mL)

[0091] The results of the Pavlova culture supernatant ultrafiltered sample are shown in Figure 1, the results of the Spirulina culture supernatant ultrafiltered sample are shown in Figure 2, and the results of the Chlorella culture supernatant ultrafiltered sample are shown in Figure 3.

[0092] As shown in Figure 1, in the ultrafiltrated sample of Pavlova culture supernatant, Pavlova-derived extracellular vesicles had a particle number of 3.32 × 10 11 The particle size was 162.8 nm, the cumulative 10% value of particle size (D10) was 105.4 nm, the cumulative 50% value of particle size (D50) was 151.9 nm, and the cumulative 90% value of particle size (D90) was 232.3 nm.

[0093] As shown in Figure 2, in the ultrafiltrated sample of Spirulina culture supernatant, Spirulina-derived extracellular vesicles had a particle number of 2.88 × 10 11The particle size was 138.7 nm, the cumulative 10% value of particle size (D10) was 72.2 nm, the cumulative 50% value of particle size (D50) was 125.9 nm, and the cumulative 90% value of particle size (D90) was 217.8 nm.

[0094] As shown in Figure 3, in the ultrafiltrated sample of Chlorella culture supernatant, Chlorella-derived extracellular vesicles had a particle number of 8.18 × 10 9 The particle size was 227.7 nm, the cumulative 10% value of particle size (D10) was 121.4 nm, the cumulative 50% value of particle size (D50) was 215.8 nm, and the cumulative 90% value of particle size (D90) was 348.5 nm.

[0095] [Test Example 2 in the Examples: Test of adding microalgae extracellular vesicle-containing material and / or extracellular vesicles to human fibroblast cells] Each ultrafiltration sample obtained in Test Example 1 was diluted 10-fold, 100-fold, or 10,000-fold (1.18 μg / mL, 0.118 μg / mL, 0.00118 μg / mL) and allowed to act on human fibroblast cells (CC-2509, manufactured by LONZA). Then, the samples were incubated at 37° C., 5% CO 2 The cells were cultured in an incubator for 48 hours.

[0096] After treating each sample for 48 hours with human fibroblasts, the medium was removed and the cells were washed with HBSS at 1 mL / well. QIAzol (QIAGEN) was added at 700 mL / well. The cells were collected into 1.5 mL tubes using a cell scraper and stored at -80°C. RNA was then extracted using the miRNeasy mini Kit (QIAGEN).

[0097] The RNA concentration of the RNA samples was measured using a NanoDrop ND-1000 (Thermo Fisher Scientific), after which the samples were treated with DNase and converted to cDNA by reverse transcription. After the reverse transcription, the cDNA sample was diluted 5-fold with RNase-free water and stored at -30°C.

[0098] Gene expression analysis of elastin (also referred to as ELN) and COL3A1 (collagen type III alpha 1 chain) was performed by Real-Time PCR using TaqMan probe with QuantStudio®3 Real-Time PCR System (Thermo Fisher Scientific). The control was a sample without any addition of each sample (a sample with only PBS added), and the internal standard was β-actin (also referred to as ACTB). Data analysis was performed using the ΔΔCT method. The gene expression results of COL3A1 are shown in Figure 6A, and the gene expression results of elastin are shown in Figure 7A.

[0099] It was confirmed that the act of microalgal extracellular vesicle-containing material and / or extracellular vesicles on human fibroblasts enhanced the expression of elastin genes and COL3A1 genes in human fibroblasts. Therefore, microalgal extracellular vesicle-containing material and / or extracellular vesicles can be suitably used for maintaining or increasing fibrous structural proteins, and can be suitably used for anti-aging of the skin, specifically, for improving skin elasticity, skin firmness, wrinkles, texture, moisture, etc.

[0100] [Test Example 3 in Comparative Examples: Preparation and Evaluation of Extracellular Vesicle-containing Material and / or Extracellular Vesicles of Microalgae 2] purification Pavlova sp., Spirulina (Arthrospira platensis), or Chlorella (Chlorella vulgaris) was cultured in a commercially available algae medium in a conventional manner, and then subjected to ultracentrifugation as described below. Each culture supernatant was transferred to an ultracentrifuge clear tube at 11 mL per tube. Ultracentrifugation was performed using an Optima XE-90 Ultracentrifuge (BECKMAN COULTER) at 35,000 rpm, 70 minutes, and 4°C. The supernatant was removed and the precipitate was suspended in PBS(-). Ultracentrifugation was performed again at 35,000 rpm, 70 minutes, and 4°C, and the supernatant was removed and the precipitate was suspended in PBS(-). The obtained samples were stored at 4°C.

[0101] Each of the above samples was appropriately diluted with PBS(-), and a number distribution graph (horizontal axis: particle diameter, vertical axis: particle number concentration) was created using a nanoparticle analysis system NanoSight LM10 (Malvern Panalytical) in accordance with the attached software.

[0102] The above resulted in the following samples (Figure 4).

[0103] In the ultracentrifuged sample of the Pavlova culture supernatant, the Pavlova-derived extracellular vesicles had a particle number of 1.15 × 10 10 particles / mL, mean particle size 196.0 nm, cumulative 10% value of particle size (D10) 119.4 nm, cumulative 50% value of particle size (D50) 158.9 nm, cumulative 90% value of particle size (D90) 316.4 nm.

[0104] In the ultracentrifuged sample of Spirulina culture supernatant, Spirulina-derived extracellular vesicles had a particle number of 4.19 × 10 9 The particle size was 212.4 nm, the cumulative 10% value of particle size (D10) was 119.4 nm, the cumulative 50% value of particle size (D50) was 205.4 nm, and the cumulative 90% value of particle size (D90) was 321.4 nm.

[0105] In the ultracentrifuged sample of the Chlorella culture supernatant, the Chlorella-derived extracellular vesicles had a particle number of 2.37 × 10 9 The particle size was 161.7 nm, the cumulative 10% value of particle size (D10) was 100.9 nm, the cumulative 50% value of particle size (D50) was 170.5 nm, and the cumulative 90% value of particle size (D90) was 214.9 nm.

[0106] In all cases, the ultrafiltrated samples obtained from the same amount of Pavlova culture supernatant, Spirulina culture supernatant, or Chlorella culture supernatant had a higher particle count than the samples obtained by ultracentrifugation.

[0107] [Comparative Example Test Example 4. Test of adding microalgae extracellular vesicle-containing material and / or extracellular vesicles to human fibroblasts] Each ultracentrifugation sample obtained in Test Example 3 was diluted to 10 μg / mL and allowed to act on human fibroblasts (CC-2509, manufactured by Lonza). Then, the cells were incubated at 37° C., 5% CO 2 The cells were cultured in an incubator for 48 hours.

[0108] After treating each sample for 48 hours with human fibroblasts, the medium was removed and the cells were washed with HBSS at 1 mL / well. QIAzol (QIAGEN) was added at 700 mL / well. The cells were collected into 1.5 mL tubes using a cell scraper and stored at -80°C. RNA was then extracted using the miRNeasy mini Kit (QIAGEN).

[0109] The RNA concentration of the RNA samples was measured using a NanoDrop ND-1000 (Thermo Fisher Scientific), after which the samples were treated with DNase and converted to cDNA by reverse transcription. After the reverse transcription, the cDNA sample was diluted 5-fold with RNase-free water and stored at -30°C.

[0110] Gene expression analysis of elastin (also referred to as ELN) and COL3A1 (collagen type III alpha 1 chain) was performed by Real-Time PCR using TaqMan probe with QuantStudio®3 Real-Time PCR System (Thermo Fisher Scientific). The control was a sample without any addition (a sample with only PBS added), and the internal standard was β-actin (also referred to as ACTB). Data analysis was performed using the ΔΔCT method. The gene expression results of COL3A1 are shown in Figure 6B, and the gene expression results of elastin are shown in Figure 7B.

[0111] By applying the extracellular vesicle contents and / or extracellular vesicles of microalgae to human fibroblasts, the enhancement of elastin gene expression and the enhancement of COL3A1 gene expression in human fibroblasts were confirmed. However, the degree of these activities was lower than that of the ultrafiltration sample in Test Example 2, and the amount of protein required for the same gene expression was different, about 10 to 100 times higher.

[0112] [Test Example 5 in the Examples: Preparation and Evaluation of Extracellular Vesicle-containing Materials and / or Extracellular Vesicles of Microalgae] Pavlova sp. and Spirulina (Arthrospira platensis) were cultured in a commercially available algae medium by a conventional method to obtain cultures. The culture was heated at 105°C for 4 minutes, and after heating, centrifuged to obtain a culture supernatant. 500 mL of the culture supernatant was purified to a volume of 5 mL or less using a 200K ultrafiltration membrane (manufactured by ADVANTEC). Then, purification washing was performed three times using 100 mL of PBS, and finally, the sample was collected with 50 mL of PBS. Then, each purified sample was filter sterilized using a 0.22 μm filter, and the particle number, etc. were measured using NanoSight.

[0113] Each of the above samples was appropriately diluted with PBS(-), and a number distribution graph (horizontal axis: particle diameter, vertical axis: particle number concentration) was created using a nanoparticle analysis system NanoSight LM10 (Malvern Panalytical) in accordance with the attached software.

[0114] The above gave the following samples: Pavlova culture supernatant ultrafiltration sample (protein concentration 201.5 μg / mL)

[0115] The results of the Pavlova culture supernatant ultrafiltration samples are shown in FIG.

[0116] As shown in Figure 5, in the sample (PavlovaEVs2) obtained by ultrafiltration and heat treatment of the Pavlova culture supernatant, the Pavlova-derived extracellular vesicles had a particle number of 8.34 × 10 11The protein content was 17 times higher in the heat-treated sample (PavlovaEVs2) than in the non-heat-treated sample (PavlovaEVs1).

[0117] As shown in the test examples, it was found that the microalgal extracellular vesicle-containing material and / or extracellular vesicles obtained by ultrafiltration or a combination of ultrafiltration and heating have a higher particle concentration and a higher protein concentration than the extracellular vesicle-containing material and / or extracellular vesicles obtained by other means such as ultracentrifugation. The number of particles obtained from the same amount of starting material was greater in the sample obtained by ultrafiltration than in the sample obtained by ultracentrifugation, and was even greater in the sample obtained by ultrafiltration plus a heating step. Furthermore, the extracellular vesicle-containing material and / or extracellular vesicles of microalgae obtained by ultrafiltration have a better effect of maintaining or increasing fibrous structural proteins than those obtained by ultracentrifugation, and can be suitably used for anti-aging of the skin, specifically, for improving skin elasticity, skin firmness, wrinkles, texture, moisture, etc.

Claims

1. Extracellular vesicles or a substance containing extracellular vesicles, obtained by a method comprising purifying a culture supernatant of a microalgae by filtration through a filter having a membrane pore size of 0.001 to 0.2 μm.

2. Extracellular vesicles or a substance containing extracellular vesicles, obtained by a method comprising purifying a culture supernatant of a microalgae by filtration through a filter having a molecular weight cutoff of 10 KDa to 1000 KDa.

3. A material containing extracellular vesicles or extracellular vesicles obtained by heating a culture or culture supernatant of microalgae at 40°C or higher.

4. The extracellular vesicles or the material containing extracellular vesicles according to claim 1, wherein the particle diameter of the extracellular vesicles is 50 nm or more and 700 nm or less.

5. The extracellular vesicle or extracellular vesicle-containing material described in claim 1, for maintaining or increasing fibrous structural proteins.

6. The extracellular vesicle or extracellular vesicle content of claim 1, wherein the fibrous structural protein is collagen or elastin.

7. The extracellular vesicles or extracellular vesicle-containing compositions of claim 1, wherein the amount of fibrous structural protein produced is increased compared to extracellular vesicles or extracellular vesicle-containing compositions obtained using ultracentrifugation instead of filter filtration.

8. The extracellular vesicles or extracellular vesicle-containing composition of claim 1, wherein the number of extracellular vesicles is increased compared to extracellular vesicles or extracellular vesicle-containing composition obtained by using ultracentrifugation instead of filter filtration.

9. The extracellular vesicles or extracellular vesicle-containing material according to any one of claims 1 to 8, wherein the microalgae are microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

10. A method for preparing extracellular vesicles, comprising purifying a culture supernatant of microalgae by filtration through a filter having a membrane pore size of 0.001 to 0.2 μm.

11. A method for preparing extracellular vesicles or a material containing extracellular vesicles, comprising purifying a culture supernatant of microalgae by filtration through a filter having a molecular weight cutoff of 10 KDa to 1,000 KDa.

12. The method for preparing extracellular vesicles described in claim 11, further comprising heating the culture or culture supernatant at 40°C or higher.

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