Composition for maintaining or increasing fibrous structural protein

Abstract

The purpose of the present invention is to provide a preparation that is effective for maintaining or increasing a hair fibrous structural protein. Provided are a composition for maintaining or increasing a fibrous structural protein, a composition for maintaining or increasing skin elasticity, a composition for improving hair quality, and a composition for promoting the production of collagen, elastin, or hyaluronic acid, wherein each of the compositions contains a culture supernatant of microalgae and / or extracellular vesicles of microalgae.

Description

Composition for maintaining or increasing fibrous structural proteins

[0001] The present invention relates to a composition for maintaining or increasing fibrous structural proteins.

[0002] The skin is broadly divided into the epidermis and the dermis. The reticular layer of the dermis occupies the majority of the dermis and contains fibrous and matrix materials known as the extracellular matrix (ECM). Fibrous structural proteins in the ECM include fibrous components such as collagen and elastin, and matrix components such as hyaluronic acid.

[0003] Elastin is composed of yellow random-coil fibrous proteins and is known for its strong elasticity, like rubber, which allows it to return to its original shape when relaxed even after being stretched nearly twice its original size. Elastin crosslinks collagen in connective tissues, supporting them like a spring, acting as the driving force behind expansion and contraction, and plays a major role in maintaining the elasticity and firmness of skin. In the body, elastin is widely distributed in organs that require elasticity, such as the dermis of the skin, ligaments, tendons, and blood vessel walls, and plays a very important role in providing elasticity to these organs.

[0004] However, it is known that the amount of elastin gradually decreases due to aging, ultraviolet rays, active oxygen, stress, etc. This decrease causes a gradual loss of elasticity, leading to wrinkles and sagging of the skin, and is known to be a major factor in aging. It has also been known that a deficiency of normal elastin can contribute to vascular diseases such as arteriosclerosis.

[0005] To date, several elastin production promoters have been proposed to improve conditions caused by elastin loss. For example, Patent Document 1 proposes an extract of Polygonatum plants as an elastin production promoter.

[0006] Furthermore, in the skin's dermal extracellular matrix, collagen forms mesh-like bundles to maintain tissue morphology. As collagen matures and proliferates, cross-linking progresses, forming thick, linear fiber bundles that provide appropriate firmness to young skin. However, in aging skin, the amount of collagen in the dermal extracellular matrix decreases significantly, resulting in a loss of the skin's inherent elastic firmness. As a result, wrinkles and sagging skin develop. A detailed study of photoaging-induced changes in collagen fiber bundle structure in hairless mice (Non-Patent Document 1) showed that UVB-irradiated hairless mice developed wrinkles, and that the collagen fiber bundle structure collapsed and skin elasticity decreased in line with the formation of wrinkles. Collagen is also known to have excellent moisture-retaining properties.

[0007] For example, Non-Patent Document 2 proposes retinoic acid as a collagen production promoter.

[0008] Hyaluronic acid is a mucopolysaccharide composed of glucuronic acid and N-acetylglucosamine, and is present in large quantities in the dermis as a bonding substance that fills the gaps between cells and fibers in the body. It has also been confirmed to be present in the epidermis, and has high water retention and viscoelasticity (Non-Patent Document 3).

[0009] Special Publication No. 2001-513509

[0010] Fragrance Journal, No. 4, p36-37, 1998R. Marks et al., British Journal of Dermatology, 122, 91-98, 1990 Fragrance Journal, No. 2, 30, 1990

[0011] As mentioned above, it is assumed that any ingredient that can maintain or increase fibrous structural proteins such as collagen, elastin, and hyaluronic acid will lead to the maintenance or improvement of skin condition, but there have not yet been sufficient reports on which ingredients are suitable in terms of maintaining or increasing hair fibrous structural proteins.

[0012] Therefore, an object of the present invention is to provide a composition that is effective in maintaining or increasing fibrous structural proteins.

[0013] In order to solve the above problems, the present inventors conducted extensive research and discovered that culture supernatants and / or extracellular vesicles of microalgae have an effect on maintaining or increasing fibrous structural proteins, leading to the completion of the present invention.

[0014] That is, a preferred embodiment of the present invention provides the following composition.

[0015] [1] A composition for maintaining or increasing fibrous structural proteins, comprising a culture supernatant and / or extracellular vesicles of microalgae.

[0016] According to the present invention, it is possible to maintain and increase fibrous structural proteins by using culture supernatant and / or extracellular vesicles of microalgae.

[0017] FIG. 1 is a graph showing the results of nanoparticle analysis of a Pavlova culture supernatant ultrafiltration sample in Test Example 1. FIG. 2 is a graph showing the results of nanoparticle analysis of a Spirulina culture supernatant ultrafiltration sample in Test Example 1. FIG. 3 is a graph showing the results of nanoparticle analysis of a Chlorella culture supernatant ultrafiltration sample in Test Example 1. FIG. 4 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 5 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 6 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 7 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 8 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 9 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 10 is a graph showing the results of a test in Test Example 2 in which microalgae culture supernatant and / or extracellular vesicles were added to human fibroblasts. FIG. 11 is a graph showing the results of a test in Test Example 3 in which microalgae culture supernatant and / or extracellular vesicles were added to three-dimensionally cultured skin. FIG. 12 is a graph showing the results of a test in Test Example 3 in which microalgae culture supernatant and / or extracellular vesicles were added to three-dimensionally cultured skin. FIG. 13 is a graph showing the results of a test in Test Example 3 in which microalgae culture supernatant and / or extracellular vesicles were added to three-dimensionally cultured skin.

[0018] [Composition for maintaining or increasing fibrous structural proteins] In one embodiment, the composition of the present invention for maintaining or increasing fibrous structural proteins contains culture supernatant and / or extracellular vesicles of microalgae.

[0019] (Culture supernatant and / or extracellular vesicles of microalgae) In this specification, microalgae refer to organisms that perform oxygen-producing photosynthesis, excluding mosses, ferns, and spermatophytes, and have a cell size of 0.1 μm to 1000 μm in diameter.

[0020] Microalgae include, for example, prokaryotic organisms of the phylum Cyanobacteria, as well as eukaryotic organisms of the phylum Glaucophyta (Glaucophyta), phylum Rhodophyta (red algae), phylum Chlorophyta (Chlorophyta), phylum Cryptophyta (Cryptophyta), phylum Haptophyta (Haptophyta), phylum Heterokontophyta (Heterokontophyta), phylum Dinophyta (Dinoflagellates), phylum Euglenida, and phylum Chlorarachniophyta.

[0021] Examples of microalgae include microalgae belonging to the genus Pavlova, Euglena (Japanese name: Midorimushi, protozoa / green algae), Spirulina (cyanophyceae), Chlorella (green algae), Dunaliella (green algae), Nannochloropsis (true eyespot algae), Licoricephala (green algae), or Botryococcus (green algae). The above-listed microalgae may be used singly or in combination of two or more.

[0022] 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 genus Pavlova, the genus Euglena, the genus Spirulina, and the genus Chlorella are preferred, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova, the genus Euglena, and the genus Spirulina are more preferred, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova and the genus Euglena are even more preferred, and microalgae belonging to the genus Pavlova are particularly preferred.

[0023] Pavlova is included in the haptophyte division, and examples of microalgae of the genus Pavlova include P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, and P. salina, with P. granifera and / or P. gyrans being preferred.

[0024] Examples of microalgae belonging to the genus Euglena include E. gracilis, E. longa, E. caudata, E. oxyuris, E. tripteris, E. proxima, E. Viridis, E. socialis, E. ehrenbergii, E. Deses, E. pisciformis, E. spirogyra, E. acus, E. geneculata, E. intermedia, E. mutabilis, E. sanguinea, E. stellata, E. terricola, E. Klebsi, E. rubra, E. Examples of suitable strains include E. cyclopicola, and E. gracilis and / or E. longa are preferred.

[0025] Examples of microalgae of the genus Spirulina include the genus Altrospira, which has a scientific name change from Spirulina, and include S. platensis (A. platensis) and S. maxima (A. maxima), with S. platensis being preferred.

[0026] Examples of microalgae of the genus Chlorella include C. vulgaris, C. saccharophila, C. ellipsoidea, C. pyrenoidosa, C. sorokiniana, and C. lobophora, with C. vulgaris being preferred.

[0027] The microalgae exemplified above are widely distributed in seawater, freshwater such as ponds and marshes, and brackish water, and may be separated from these and used, or any microalgae that has already been isolated may be used.

[0028] The microalgae exemplified above include closely related species and mutant strains thereof, as long as they exhibit the effects of the present invention. Mutant strains also include those obtained by genetic methods such as gene recombination, transduction, and transformation.

[0029] As used herein, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained after culturing microalgae. For example, the supernatant may be obtained by culturing microalgae using a known culture method and removing the microalgae using a known separation method such as centrifugation. Here, the culture supernatant preferably contains extracellular vesicles derived from the microalgae.

[0030] 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, including the following examples.

[0031] (Cultivation Conditions) Microalgae can be cultured using a culture medium. A carbon source is added to the culture medium as a nutrient source. The carbon source is an inorganic carbon source (CO ２ , NaHCO ３ , Na ２ CO ３ The culture medium is not limited, but it is preferable to use an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source. Examples of the culture medium include a culture medium supplemented with nutrient salts such as a nitrogen source, a phosphorus source, and minerals, such as Cramer-Myers medium and modified Cramer-Myers medium.

[0032] 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.

[0033] The pH of the culture medium is not particularly limited as long as the effects of the present invention are achieved, and examples 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.

[0034] The culture temperature is not particularly limited as long as the effects of the present invention are achieved, but examples include 15 to 40°C, 20 to 34°C, and 23 to 28°C.

[0035] The culture period is not particularly limited as long as the effects of the present invention are achieved, but examples include 4 to 30 days, 4 to 20 days, and 5 to 15 days.

[0036] Depending on the type of microalgae, the type of light source, the presence or absence of light and dark, aerobic conditions, anaerobic conditions, etc. can be appropriately selected.

[0037] In one embodiment, when culturing marine microalgae such as pavlova, any known method can be used as long as the effects of the present invention are achieved. For example, the culture can be carried out under conditions that satisfy at least one of the following: Seawater concentration: 50% seawater Culture medium concentration: 2x IMK medium Culture solution volume: 800 mL Light source: Side: fluorescent light (100 to 150 μmol) Light / dark cycle: 12 hours of light and 12 hours of dark pH: pH of about 7.4 at the start of each culture Aeration: performed Mechanical stirring: none Culture period: approximately 10 days Culture temperature: 25°C to 28°C

[0038] (Recovery of culture supernatant) After culturing microalgae under the culture conditions exemplified above, the culture supernatant is recovered using a known separation means such as centrifugation, but there are no particular limitations on the method as long as it can appropriately recover the culture supernatant of microalgae.

[0039] In the present invention, extracellular vesicles of microalgae can also be used. As used herein, extracellular vesicles refer to vesicles secreted from microalgae that typically have an average diameter of 10 nm to 1 μm. Although not limited thereto, when extracellular vesicles of microalgae are used in the composition of the present invention, it is preferable that the composition does not contain the microalgae cells themselves.

[0040] The method for separating extracellular vesicles can be any known separation means and is not particularly limited. For example, the method for separating extracellular vesicles can be, but is not limited to, a technique such as centrifugation.

[0041] In one embodiment, the method for producing extracellular vesicles includes, but is not limited to, (1) centrifuging a culture supernatant of microalgae to obtain a supernatant; and (2) further centrifuging the obtained product, if necessary, to obtain a precipitate. This method can produce microalgae-derived extracellular vesicles that exhibit effective activity.

[0042] In exemplary embodiments, the centrifugation in steps (1) and (2) may be carried out 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. In this case, the centrifugation may be carried out stepwise by varying the speed or time. For example, the culture supernatant may be separated by centrifugation at a low speed of 1,500 to 2,000 x g, and then further centrifuged at a high speed of 10,000 to 20,000 x g to remove cells or cell-related debris and residues.

[0043] A method for producing extracellular vesicles can also be used, in which a culture supernatant of microalgae is filtered through a filter. The pore size of the filter used for the filter filtration is, for example, preferably 0.001 μm to 0.2 μm, more preferably 0.005 μm to 0.1 μm, and even more preferably 0.01 μm to 0.05 μm.

[0044] 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. Of these, it is preferable to use a hollow fiber membrane.

[0045] A hollow fiber membrane is a type of permeable membrane and refers to a membrane formed into a thin, straw-like capillary tube. A hollow fiber membrane module is a hollow fiber membrane bundle, consisting of a large number of such hollow fiber membranes bundled together, hermetically housed in a housing container made of vinyl chloride synthetic resin or metal such as aluminum. The diameter (inner diameter) of each individual hollow fiber membrane is generally about 100 μm to several mm, but for efficient liquid flow, it is preferably 100 μm to 800 μm, and more preferably 200 μm to 600 μm. The length of each hollow fiber membrane can be adjusted depending on the application. However, if it is too long, the pressure required to flow the liquid increases. Therefore, the length of the hollow fiber membrane bundle, excluding the fixed portions at both ends, i.e., the effective length, is preferably 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, from the viewpoint of purification efficiency, it is preferable to use a hollow fiber membrane module (the hollow fiber membrane constitutes the hollow fiber membrane module).

[0046] The membrane area of ​​the hollow fiber membrane in the hollow fiber membrane module used in the present invention can be calculated as 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 increases the processing speed, shortens the processing time, and reduces 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 increases loss accordingly. Therefore, it is preferable to select a hollow fiber membrane module with an appropriate membrane area depending on the purpose. The membrane area of ​​the hollow fiber membrane in the hollow fiber membrane module used in the present invention is 1 cm ２~3,000 cm ２ For example, if the purpose is to concentrate the solution to a small volume, a 20 cm ２ It is preferable to use a module with a small membrane area of ​​about 115 cm. In addition, when the purpose is to concentrate, filter, or remove impurities from 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 of about 1,000 cm. ２ ~100,000 cm ２ is preferred, and 10,000 cm ２ ~60,000 cm ２ is more preferred.

[0047] The pore size of the hollow fiber membrane used in the present invention (including hollow fiber membranes provided in hollow fiber membrane modules) can be selected depending on the purpose. For example, when removing contaminants larger than the desired extracellular vesicles, a pore size that does not allow only the contaminants to pass through is selected. Such pore sizes are 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. Possible pore sizes include 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, and 0.03 μm or less.

[0048] The molecular weight cutoff of a cutoff membrane is preferably 10 kDa to 1,000 kDa, 50 kDa to 1,000 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 2,000 kDa or less, 1,500 kDa or less, 1,000 kDa or less, 750 Dka or less, or 500 Dka or less are also preferred.

[0049] After culturing microalgae, the culture supernatant after removing the cells can be continuously diluted and concentrated to remove unnecessary substances and simultaneously exchange the solvent, thereby obtaining the required extracellular vesicles.

[0050] The material of the filter, particularly the hollow fiber membrane provided in the hollow fiber membrane module, is not particularly limited as long as it can be used for the purpose of appropriately separating cell-derived extracellular vesicles, 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.

[0051] The extracellular vesicles of the present invention can also be obtained by heating a culture supernatant or culture of microalgae at a temperature, for example, but not limited to, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 200°C or lower, 150°C or lower, or 120°C or lower, for a time of 30 seconds or longer, 1 minute or longer, 2 minutes or longer, 3 minutes or longer, 4 minutes or longer, 30 minutes or shorter, 20 minutes or shorter, or 10 minutes or shorter.

[0052] The extracellular vesicles of the present invention can also be obtained by a method comprising heating a culture supernatant or culture of a microalga and concentrating or purifying the heated culture supernatant or culture by filtering.

[0053] The present invention includes extracellular vesicles obtained from the culture supernatant of microalgae, and can also be obtained as a concentrate or purified liquid (extracellular vesicle-containing material) obtained by filtering the culture supernatant. The extracellular vesicle-containing material can be in the form of a concentrate or purified liquid of the culture supernatant itself, or it can be a solid or semi-solid obtained by drying the concentrate or purified liquid.

[0054] The extracellular vesicles of the present invention can optionally be further purified to provide a highly pure population of extracellular vesicles.

[0055] The extracellular vesicles of the present invention may be in the form of a concentrated or purified culture supernatant liquid itself, or may be a solid or semi-solid obtained by drying the concentrated or purified liquid.

[0056] In the composition of the present invention, the average particle number of the extracellular vesicles of the microalgae 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 ９ or more, and 1.0 × 10 １４ Less than or equal to 1.0×10 １３ Less than or equal to 1.0×10 １２ Less than or equal to 1.0×10 １１ Less than or equal to 1.0×10 １０ The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 ４ ~1.0 x 10 １４ pieces, 1.0×10 ４ ~1.0 x 10 １３ pieces, 1.0×10 ４ ~1.0 x 10 １２ pieces, 1.0×10 ４ ~1.0 x 10 １１ pieces, 1.0×10 ４ ~1.0 x 10 １０ pieces, 1.0×10 ５ ~1.0 x 10 １４ pieces, 1.0×10 ５ ~1.0 x 10 １３ pieces, 1.0×10 ５ ~1.0 x 10 １２ pieces, 1.0×10 ５ ~1.0 x 10 １１ pieces, 1.0×10 ５ ~1.0 x 10 １０ pieces, 1.0×10 ６ ~1.0 x 10 １４ pieces, 1.0×10 ６ ~1.0 x 10 １３ pieces, 1.0×10 ６ ~1.0 x 10 １２ pieces, 1.0×10 ６~1.0 x 10 １１ pieces, 1.0×10 ６ ~1.0 x 10 １０ pieces, 1.0×10 ７ ~1.0 x 10 １４ pieces, 1.0×10 ７ ~1.0 x 10 １３ pieces, 1.0×10 ７ ~1.0 x 10 １２ pieces, 1.0×10 ７ ~1.0 x 10 １１ pieces, 1.0×10 ７ ~1.0 x 10 １０ pieces, 1.0×10 ８ ~1.0 x 10 １４ pieces, 1.0×10 ８ ~1.0 x 10 １３ pieces, 1.0×10 ８ ~1.0 x 10 １２ pieces, 1.0×10 ８ ~1.0 x 10 １１ pieces, 1.0×10 ８ ~1.0 x 10 １０ pieces, 1.0×10 ９ ~1.0 x 10 １４ pieces, 1.0×10 ９ ~1.0 x 10 １３ pieces, 1.0×10 ９ ~1.0 x 10 １２ pieces, 1.0×10 ９ ~1.0 x 10 １１ pieces, 1.0×10 ９ ~1.0 x 10 １０ The number of extracellular vesicles can be measured using an exosome measurement system (ExoCounter, JVC Kenwood Corporation) that uses a surface antigen-specific sandwich detection method using a disk and an antibody on nanobeads.

[0057] 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, but 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 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 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.

[0058] The cumulative 10% value (D10) of the particle diameter of pavlova-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 80 to 140 nm, 90 to 130 nm, and 100 to 120 nm. Furthermore, 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. Furthermore, 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. As used herein, the cumulative 10% value (D10) of the particle diameter of extracellular vesicles refers to the diameter below which 10% of all extracellular vesicles are located. The cumulative 50% value (D50) of the particle diameter of extracellular vesicles refers to the diameter below which 50% of all extracellular vesicles are located, 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.

[0059] The cumulative 10% value (D10) of particle diameter of 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.

[0060] The cumulative 10% value (D10) of particle diameter of the Chlorella-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 90 to 150 nm, 100 to 140 nm, 110 to 130 nm, etc. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 180 to 240 nm, 190 to 230 nm, 200 to 220 nm, etc. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc.

[0061] The content of the microalgae culture supernatant 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, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, etc. Furthermore, the content of the microalgae culture supernatant can be, for example, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 1% by mass or less, 0.1% by mass or less, etc. The content of the culture supernatant of microalgae is, for example, 0.001 to 0.1% by mass (for example, 2×10 particles) relative to the total amount of the composition. １１ pieces / mL ~ 4×10 １１0.01-100% by mass, 0.01-90% by mass, 0.01-50% by mass, 0.01-30% by mass, 0.01-10% by mass, 0.01-1% by mass, 0.1-100% by mass, 0.1-90% by mass, 0.1-50% by mass, 0.1-30% by mass, 0.1 -10% by mass, 0.1-1% by mass, 0.5-100% by mass, 0.5-90% by mass, 0.5-50% by mass, 0.5-30% by mass, 0.5-10% by mass, 0.5-1% by mass, 1-100% by mass, 1-90% by mass, 1-50% by mass, 1-30% by mass, 1-10% by mass, etc.

[0062] The content of microalgal extracellular vesicles is adjusted appropriately depending on the type and amount of other components, dosage form, etc., and is not limited, but may 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. Furthermore, the content of microalgal extracellular vesicles may be, for example, 50% by mass or less, relative to the total amount of the composition, examples of which include 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 1% by mass or less, 0.1% by mass or less, etc. Furthermore, the content of microalgal extracellular vesicles may 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 to 50% by mass, 0.001 to 40% by mass, 0.001 to 30% by mass, 0.001 to 20% by mass, 0.00 1-15% 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- 50 mass%, 0.1 to 40 mass%, 0.1 to 30 mass%, 0.1 to 20 mass%, 0.1 to 15 mass%, 1 to 50 mass%, 1 to 40 mass%, 1 to 30 mass%, 1 to 20 mass%, 1 to 15 mass%, 5 to 50 mass%, 5 to 40 mass%, 5 to 30 mass%, 5 to 20 mass%, 5 to 15 mass%, etc.

[0063] The content of microalgal extracellular vesicles is adjusted appropriately 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, 1,000 μg / mL, 100 μg / mL or less, and 10 μg / mL or less.

[0064] [Uses] In one embodiment, the composition of the present invention is suitable for use in maintaining or increasing fibrous structural proteins. As used herein, fibrous structural proteins refer to fibrous components in the body and components involved in their 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 skin elasticity.

[0065] As mentioned above, skin is broadly divided into the epidermis and the dermis, and the constituent components of the dermis are classified into interstitial components related to fibrous tissue and cellular components. The interstitial components related to fibrous tissue are mostly made up of 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., and matrices such as hyaluronic acid, which are collectively referred to as the extracellular matrix (ECM).

[0066] Examples of fibrous structural proteins include collagen, elastin, proteoglycans such as hyaluronic acid, glycosaminoglycans, laminin, and fibronectin.

[0067] There are approximately 28 subtypes of collagen, and approximately 80% of the collagen that makes up the dermis is known to be type I. Type I collagen contributes to skin elasticity, while type III collagen contributes to skin damage recovery. Collagen is important as a supportive tissue for maintaining skin strength because it has high resistance to tension, especially along the fiber axis, but poor extensibility. Elastic fibers, composed of elastin and fibrillin, are interposed between the collagen layers and contribute to skin elasticity and the extensibility of arteries and tendons. Hyaluronic acid, a type of proteoglycan, is present between fibrous components such as collagen and elastin and between cells, stabilizing the fibrous structure and retaining moisture. Glycoproteins such as laminin and fibronectin mediate adhesion between the ECM and cells.

[0068] When fibrous structural protein-degrading enzymes are activated, the above-mentioned fibrous structural proteins may be reduced. Examples of fibrous structural protein-degrading enzymes include matrix metalloproteinases (MMPS), and in particular, matrix metalloproteinase 1 (MMP-1) is responsible for the degradation of collagen and elastin fibers in the skin.

[0069] Furthermore, when the activity of the synthase of a fibrous structural protein or the gene expression of a fibrous structural protein is attenuated, the above-mentioned fibrous structural protein may be reduced. An example of a fibrous structural protein synthase is hyaluronic acid synthase. HAS-1 to -3 are known as hyaluronic acid synthases (also referred to as HAS herein), and these enzymes on the cell membrane directly synthesize hyaluronic acid from UDP (uridine diphosphate)-glucuronic acid (UDP-GlcA) and UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and supply it to the outside of the cell. In the epidermis, HAS-3 is the main contributor, while in the dermis, HAS-1 and HAS-2, especially HAS-2, contribute significantly.

[0070] 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).

[0071] Therefore, if a component is capable of inhibiting fibrous structural protein degrading enzymes, activating fibrous structural protein synthases, or enhancing gene expression of fibrous structural proteins, it will lead to maintaining or increasing fibrous structural proteins by suppressing their decrease.

[0072] In the Examples described below, it has been confirmed that microalgae culture supernatant and / or extracellular vesicles suppress matrix metalloproteinase 1 (MMP-1), enhance elastin gene expression, enhance COL1A1 gene expression, enhance COL3A1 gene expression, and activate hyaluronic acid synthase HAS-1 or hyaluronic acid synthase HAS-2. These results are expected to contribute to the maintenance or increase of fibrous structural proteins, leading to anti-aging of the skin, specifically, improvements in skin elasticity, firmness, wrinkles, texture, and moisture, as well as suppression of dryness and roughness of the skin.

[0073] The composition of the present invention can be added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods, or can be used as is as pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods.

[0074] When the composition of the present invention is added to or mixed with so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the composition of the present invention can be used as pharmaceuticals, quasi-drugs, and cosmetics that explicitly or implicitly claim to improve skin elasticity, firmness, wrinkles, texture, moisture, dryness, roughness, and the like as efficacy, effects, or functions.

[0075] When the above functionality is not explicitly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those who seek skin elasticity, those who seek skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those who seek skin moisture, 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, or similar indications.

[0076] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily dose 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, ingestion or administration route, ingestion or administration schedule, formulation form, and other factors. Examples of the daily dose 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 dosage of microalgal extracellular vesicles 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. The daily dose of microalgal 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 dosage is 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 in divided doses one to three times a day, and the dosage does not limit the scope of the present specification in any way.

[0077] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily adult dosage of microalgal extracellular vesicles 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, and other factors. Examples of the daily adult dosage of microalgal extracellular vesicles in terms of protein amount 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 microalgal extracellular vesicles 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. The daily dose of microalgal 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, in 1 to 3 divided doses per day, and the dosage does not limit the scope of the present specification in any way.

[0078] In another embodiment, the daily dose of microalgal extracellular vesicles for an adult is, for example, 1 x 10 ７ pcs or more, 1×10 ８ pcs or more, 1×10 ９ The daily dose of extracellular vesicles of microalgae for an adult is, for example, 1 x 10 １４ Less than or equal to 1 x 10 １３ Less than or equal to 1 x 10 １２ Less than or equal to 1 x 10 １１ Less than or equal to 1 x 10 １０ The daily dose of extracellular vesicles of microalgae for an adult is, for example, 1 x 10 ７ ~1 x 10 １４ pieces, 1×10 ７ ~1 x 10 １３ pieces, 1×10 ７ ~1 x 10 １２ pieces, 1×10 ７ ~1 x 10 １１ pieces, 1×10 ８ ~1 x 10 １４ pieces, 1×10 ８ ~1 x 10 １３ pieces, 1×10 ８ ~1 x 10 １２ pieces, 1×10 ８ ~1 x 10 １１ pieces, 1×10 ９ ~1 x 10 １４ pieces, 1×10 ９ ~1 x 10 １３ pieces, 1×10 ９ ~1 x 10 １２ pieces, 1×10 ９ ~1 x 10 １１ Examples include:

[0079] The daily dose of the microalgae culture supernatant for an adult can be determined appropriately 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.

[0080] The target population for the composition of the present invention is not particularly limited as long as it is a subject requiring the maintenance or increase of fibrous structural proteins, but it may be, for example, a young person in their 20s to 30s.

[0081] The composition of the present invention can be ingested or administered once to 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.

[0082] The composition of the present invention can be used as a food or drink that explicitly or implicitly claims to have the functionality of improving skin elasticity, firmness, wrinkles, texture, moisture, dryness, roughness, etc., i.e., health food, functional food, food for patients, and food for specified health uses. Furthermore, even if the above functionality is not explicitly or implicitly claimed, it can be used as a so-called doctor's supplement recommended or presented by doctors in internal medicine, orthopedic surgery, veterinary clinics, etc. in hospitals and / or clinics.

[0083] When the above functionality is not explicitly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those who seek skin elasticity, those who seek skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those who seek skin moisture, 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, or similar indications.

[0084] 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.

[0085] The daily oral intake or administration amount of microalgal extracellular vesicles for an adult can be determined appropriately 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. Furthermore, the daily oral intake or administration amount of microalgal extracellular vesicles 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. The daily oral intake or administration amount of microalgal 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 500 μg, 5 to 600 μg, 5 to 200 μg, 5 to 500 μg, 5 to 500 μg, 5 to 400 μg, 5 to 300 μg, 5 to 200 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 500 μg, 5 to 6 ... Examples include up to 100 μg, 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.

[0086] 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 extracellular vesicles of microalgae for an adult is, in terms of the number of particles, for example, 1 × 10 １４ Less than or equal to 1 x 10１３ Less than or equal to 1 x 10 １２ Less than or equal to 1 x 10 １１ Less than or equal to 1 x 10 １０ The daily oral intake or administration amount of extracellular vesicles of microalgae for an adult is, in terms of the number of particles, for example, 1 x 10 ７ ~1 x 10 １４ pieces, 1×10 ７ ~1 x 10 １３ pieces, 1×10 ７ ~1 x 10 １２ pieces, 1×10 ７ ~1 x 10 １１ pieces, 1×10 ８ ~1 x 10 １４ pieces, 1×10 ８ ~1 x 10 １３ pieces, 1×10 ８ ~1 x 10 １２ pieces, 1×10 ８ ~1 x 10 １１ pieces, 1×10 ９ ~1 x 10 １４ pieces, 1×10 ９ ~1 x 10 １３ pieces, 1×10 ９ ~1 x 10 １２ pieces, 1×10 ９ ~1 x 10 １１ Examples include:

[0087] The daily oral intake or administration amount of a microalgae culture supernatant for an adult can be determined appropriately 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 a 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.

[0088] The oral intake or dosage per day 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.

[0089] The composition of the present invention can be ingested or administered once to 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.

[0090] When the present invention is used as feed, pet food, or the like, the target organisms are not particularly limited, but are preferably mammals, reptiles, amphibians, birds, or fish, and more preferably mammals other than humans, such as 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, and horned owl, and more preferably dog ​​or cat.

[0091] When the present invention is used as a feed or pet food, it may be given to a pet in divided doses several times a day by adding it to the staple food, or it may be given to a pet as a snack at any time.

[0092] The composition of the present invention includes the following aspects: [1] A composition for maintaining or increasing fibrous structural proteins, comprising a culture supernatant and / or extracellular vesicles of microalgae.

[0093] [2] A composition for inhibiting a fibrous structural protein degrading enzyme, comprising a microalgae culture supernatant and / or extracellular vesicles. [3] A composition for activating a fibrous structural protein synthase, comprising a microalgae culture supernatant and / or extracellular vesicles. [4] A composition for enhancing gene expression of a fibrous structural protein, comprising a microalgae culture supernatant and / or extracellular vesicles. [5] The composition according to any one of [1] to [4], wherein the fibrous structural protein is collagen, elastin, or hyaluronic acid. [6] The composition according to [5], wherein the elastin is skin elastin. [7] A composition for maintaining or increasing skin elasticity, comprising a microalgae culture supernatant and / or extracellular vesicles. [8] A composition for promoting the production of collagen, elastin, or hyaluronic acid, comprising a microalgae culture supernatant and / or extracellular vesicles. [9] A composition for inhibiting matrix metalloproteinase 1 (MMP-1), comprising a microalgae culture supernatant and / or extracellular vesicles.

[10] A composition for enhancing elastin gene expression, comprising a microalgal culture supernatant and / or extracellular vesicles.

[11] A composition for enhancing COL1A1 gene expression, comprising a microalgal culture supernatant and / or extracellular vesicles.

[12] A composition for enhancing COL3A1 gene expression, comprising a microalgal culture supernatant and / or extracellular vesicles.

[13] A composition for activating hyaluronic acid synthase HAS-1, comprising a microalgal culture supernatant and / or extracellular vesicles.

[14] A composition for activating hyaluronic acid synthase HAS-2, comprising a microalgal culture supernatant and / or extracellular vesicles.

[15] The composition according to any of [1] to

[14] , wherein the microalgal culture supernatant is obtained by a method comprising purifying the culture supernatant by filtration through a filter having a membrane pore size of 0.001 μm to 0.2 μm.

[16] The composition according to any one of [1] to

[15] , wherein the molecular weight cutoff of the filter is 10 KDa to 1000 KDa.

[17] The composition according to any one of [1] to

[16] , wherein the particle size of the extracellular vesicles is 50 nm to 700 nm.

[18] The composition according to any one of [1] to

[17] , wherein a unit dosage form of the composition contains 0.001 to 0.1 mass% of microalgae culture supernatant and / or extracellular vesicles derived from the microalgae.

[19] A unit dosage form of the composition contains 1.0 x 10 microalgae-derived extracellular vesicles. ６ ~1.0 x 10 １１

[20] The composition according to any one of [1] to

[19] , wherein the microalgae belong to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[21] The composition according to any one of [1] to

[20] , wherein the microalgae culture supernatant and / or extracellular vesicles derived from the microalgae are provided as a food or drink, cosmetic, pharmaceutical, or quasi-drug.

[22] The composition according to any one of [1] to

[21] , wherein the microalgae culture supernatant and / or extracellular vesicles derived from the microalgae are provided for young people.

[0094] The present invention further includes the following aspects. More specific embodiments of each aspect are the same as those described above in the section [Composition for maintaining or increasing fibrous structural proteins]. [1] Use of microalgal culture supernatant and / or extracellular vesicles for producing a composition for maintaining or increasing fibrous structural proteins. [2] Use of microalgal culture supernatant and / or extracellular vesicles for producing a composition for inhibiting a fibrous structural protein-degrading enzyme. [3] Use of microalgal culture supernatant and / or extracellular vesicles for producing a composition for activating a fibrous structural protein synthase. [4] Use of microalgal culture supernatant and / or extracellular vesicles for producing a composition for enhancing gene expression of a fibrous structural protein. [5] The use according to any one of [1] to [4], wherein the fibrous structural protein is collagen, elastin, or hyaluronic acid. [6] The use according to [5], wherein the elastin is skin elastin. [7] Use of microalgae culture supernatant and / or extracellular vesicles for producing a composition for maintaining or increasing skin elasticity. [8] Use of microalgae culture supernatant and / or extracellular vesicles for producing a composition for promoting collagen, elastin, or hyaluronic acid production. [9] Use of microalgae culture supernatant and / or extracellular vesicles for producing a composition for inhibiting matrix metalloproteinase 1 (MMP-1).

[10] Use of microalgae culture supernatant and / or extracellular vesicles for producing a composition for enhancing elastin gene expression.

[11] Use of microalgae culture supernatant and / or extracellular vesicles for producing a composition for enhancing COL1A1 gene expression.

[12] Use of microalgae culture supernatant and / or extracellular vesicles for producing a composition for enhancing COL3A1 gene expression.

[13] Use of a culture supernatant and / or extracellular vesicles of a microalga for producing a composition for activating the hyaluronan synthase HAS-1.

[14] Use of a culture supernatant and / or extracellular vesicles of a microalga for producing a composition for activating the hyaluronan synthase HAS-2.

[15] The use according to any of [1] to

[14] , wherein the culture supernatant of the microalga is obtained by a method comprising purifying the culture supernatant by filtration through a filter having a membrane pore size of 0.001 μm to 0.2 μm.

[16] The use according to any one of [1] to

[15] , wherein the molecular weight cut-off of the filter is 10 KDa to 1000 KDa.

[17] The use according to any one of [1] to

[16] , wherein the particle size of the extracellular vesicles is 50 nm to 700 nm.

[18] The use according to any one of [1] to

[17] , wherein a unit dosage form of the composition contains 0.001 to 0.1 mass% of microalgal culture supernatant and / or extracellular vesicles derived from the microalgae.

[19] The unit dosage form of the composition contains 1.0 x 10 extracellular vesicles derived from the microalgae. ６ ~1.0 x 10 １１

[20] The use according to any one of [1] to

[19] , wherein the microalgae belong to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[21] The use according to any one of [1] to

[20] , wherein the microalgae culture supernatant and / or extracellular vesicles derived from the microalgae are provided as a food or drink, cosmetic, pharmaceutical, or quasi-drug.

[22] The use according to any one of [1] to

[21] , wherein the microalgae culture supernatant and / or extracellular vesicles derived from the microalgae are provided for young people.

[0095]

[23] A method for maintaining or increasing fibrous structural proteins, comprising administering or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[24] A method for inhibiting fibrous structural protein degrading enzymes, comprising administering or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[25] A method for activating fibrous structural protein synthases, comprising administering or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[26] A method for enhancing gene expression of fibrous structural proteins, comprising administering or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[27] The method according to any of

[23] to

[26] , wherein the fibrous structural protein is collagen, elastin, or hyaluronic acid.

[28] The method according to

[27] , wherein the elastin is skin elastin.

[29] A method for maintaining or increasing skin elasticity, comprising administering to or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[30] A method for promoting collagen, elastin, or hyaluronic acid production, comprising administering to or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[31] A method for suppressing matrix metalloproteinase 1 (MMP-1), comprising administering to or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[32] A method for enhancing elastin gene expression, comprising administering to or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[33] A method for enhancing COL1A1 gene expression, comprising administering to or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[34] A method for enhancing COL3A1 gene expression, comprising administering or ingesting an effective amount of a culture supernatant and / or extracellular vesicles of a microalga to a subject.

[35] A method for activating hyaluronic acid synthase HAS-1, comprising administering or ingesting an effective amount of a culture supernatant and / or extracellular vesicles of a microalga to a subject.

[36] A method for activating hyaluronic acid synthase HAS-2, comprising administering or ingesting an effective amount of microalgal culture supernatant and / or extracellular vesicles to a subject.

[37] The method according to any one of

[23] to

[36] , wherein the microalgal culture supernatant is obtained by a method comprising purifying the culture supernatant by filtration through a filter having a pore size of 0.001 μm to 0.2 μm.

[38] The method according to any one of

[23] to

[37] , wherein the molecular weight cutoff of the filter is 10 KDa to 1,000 KDa.

[39] The method according to any one of

[23] to

[38] , wherein the particle size of the extracellular vesicles is 50 nm to 700 nm.

[40] The method according to any one of

[23] to

[39] , wherein a unit dosage form of the composition contains 0.001 to 0.1% by mass of microalgal culture supernatant and / or extracellular vesicles derived from the microalgae.

[41] A unit dosage form of the composition contains 1.0 x 10 extracellular vesicles derived from the microalgae. ６ ~1.0 x 10 １１

[42] The method according to any one of

[23] to

[41] , wherein the microalgae belong to the genus Pavlova, Euglena, Spirulina, or Chlorella.

[43] The method according to any one of

[23] to

[42] , wherein the microalgae culture supernatant and / or extracellular vesicles derived from the microalgae are provided as a food or drink, cosmetic, pharmaceutical, or quasi-drug.

[44] The method according to any one of

[23] to

[43] , wherein the microalgae culture supernatant and / or extracellular vesicles derived from the microalgae are provided for young people.

[0096] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0097] Test Example 1. Preparation and Evaluation of Microalgae Culture Supernatant and / or Extracellular Vesicles Pavlova (Pavlova sp.), Spirulina (Arthrospira platensis), or Chlorella (Chlorella vulgaris) was cultured using commercially available algae medium by conventional methods to obtain culture supernatants. 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). Subsequently, concentration and washing were performed three times using 100 mL of PBS, and finally, the sample was collected using 50 mL of PBS. Each concentrated sample was then sterilized using a 0.22 μm filter, and particle counts and the like were measured using NanoSight.

[0098] 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 (manufactured by Malvern Panalytical) according to the attached software.

[0099] The following samples were obtained as described above. These samples were diluted 10-fold, 100-fold, 1000-fold, or 10,000-fold and added to cells in the following test examples. - Pavlova culture supernatant ultrafiltration sample (protein concentration 11.9 μg / mL, also referred to as "PavloEV" in the figure) - Spirulina culture supernatant ultrafiltration sample (protein concentration 29.6 μg / mL, also referred to as "SpirulEV" in the figure) - Chlorella culture supernatant ultrafiltration sample (protein concentration 10.4 μg / mL, also referred to as "ChloreEV" in the figure)

[0100] The results for the Pavlova culture supernatant ultrafiltration sample are shown in Figure 1, the results for the Spirulina culture supernatant ultrafiltration sample are shown in Figure 2, and the results for the Chlorella culture supernatant ultrafiltration sample are shown in Figure 3.

[0101] As shown in Figure 1, in the ultrafiltrated sample of pavlova culture supernatant, pavlova-derived extracellular vesicles had a particle number of 3.32 x 10 １１The 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.

[0102] 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 １１ The 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.

[0103] As shown in Figure 3, in the ultrafiltration sample of the Chlorella culture supernatant, Chlorella-derived extracellular vesicles had a particle number of 8.18 x 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.

[0104] Test Example 2: Test of adding culture supernatant and / or extracellular vesicles of microalgae to human fibroblasts Each ultrafiltrated sample obtained in Test Example 1 above was diluted 10-fold, 100-fold, 1000-fold, or 10,000-fold and allowed to act on human fibroblasts (CC-2509, manufactured by LONZA). Then, the diluted samples were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 48 hours.

[0105] After treating each sample for 48 hours with human fibroblasts, the medium was removed and the cells were washed once. 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).

[0106] The RNA concentration of the RNA sample was measured using a NanoDrop ND-1000 (Thermo Fisher Scientific), followed by DNase treatment and reverse transcription to produce cDNA. After reverse transcription, the cDNA sample was diluted 5-fold with RNase-free water and stored at -30°C.

[0107] Gene expression analysis of MMP1, elastin (also referred to as ELN), fibrillin 1 (also referred to as FBN-1), hyaluronan synthase 1 (also referred to as HAS-1), hyaluronan synthase 2 (also referred to as HAS-2), COL1A1 (collagen type I alpha 1 chain), and COL3A1 (collagen type III alpha 1 chain) was performed by real-time PCR using a TaqMan probe on a QuantStudio® 3 real-time PCR system (Thermo Fisher Scientific). Samples without any addition of each sample served as controls (samples with only PBS added), and β-actin (also referred to as ACTB) was used as an internal standard. Data analysis was performed using the ΔΔCT method. The gene expression results for MMP1 are shown in Figure 4, the gene expression results for elastin are shown in Figure 5, the gene expression results for fibrillin 1 are shown in Figure 6, the gene expression results for hyaluronic acid synthase 1 are shown in Figure 7, the gene expression results for hyaluronic acid synthase 2 are shown in Figure 8, the gene expression results for COL1A1 are shown in Figure 9, and the gene expression results for COL3A1 are shown in Figure 10.

[0108] 4 to 10 , it was confirmed that the action of microalgae culture supernatant and / or extracellular vesicles on human fibroblasts suppressed matrix metalloproteinase 1 (MMP-1), enhanced elastin gene expression, enhanced COL1A1 gene expression, enhanced COL3A1 gene expression, and activated the hyaluronic acid synthase HAS-1 or HAS-2 in the human fibroblasts. Therefore, microalgae culture supernatant and / or extracellular vesicles can be suitably used to maintain or increase fibrous structural proteins, and can be suitably used for skin anti-aging, specifically for improving skin elasticity, firmness, wrinkles, texture, moisture, etc.

[0109] Test Example 3: Test of adding microalgae culture supernatant and / or extracellular vesicles to three-dimensionally cultured skin (Preculture of three-dimensionally cultured skin) In this test example, three-dimensionally cultured skin (EFT-400, manufactured by Kurabo Industries, Ltd.) was used for evaluation. 2.5 ml of EFT-400-specific medium (EFT-400-ASY or EFT-400-MM, manufactured by Kurabo Industries, Ltd.) heated to 37°C was dispensed into each well of a sterilized 6-well receiver plate (hereinafter referred to as plate) in the EFT-400 kit, and it was confirmed that the surface of each well was uniformly covered with the medium.

[0110] Next, the three-dimensional cultured skin was transferred from the culture cup stored at 4°C to the above-mentioned plate supplemented with the medium. ２ The cells were left to stand in an incubator for about 16 to 18 hours for cultivation.

[0111] (Preparation of a composition containing pavlova-derived extracellular vesicles) Pavlova (Pavlova sp.) was cultured using a commercially available algae medium by a conventional method to obtain a culture supernatant. The culture supernatant was heat-treated at 105°C for 4 minutes. After heating, 3,000 mL of the supernatant was centrifuged at room temperature and filtered through a 0.2 μm filter. The supernatant was then concentrated to a volume of 150 mL or less by tangential flow using a 300 kDa hollow fiber membrane filter (MiniKros sampler S02-E300-05-N: manufactured by Repligen). The mixture was then concentrated and washed using 10,000 mL of PBS, and finally recovered using 100 mL of PBS. The concentrated sample was then filter-sterilized using a 0.22 μm filter to obtain a composition containing pavlova-derived extracellular vesicles (ultrafiltration sample). Pavlova-derived extracellular vesicles in the ultrafiltration sample: 2.7 x 10 １２ particles / mL, average particle size 158.8 nm, protein concentration 836 μg / ml

[0112] (Addition of Ultrafiltration Sample) After pre-culture of the three-dimensional cultured skin, the medium was replaced once. Then, the Pavlova-derived extracellular vesicle-containing composition (ultrafiltration sample) was added to the three-dimensional cultured skin so that the final concentrations were 0%, 0.001%, 0.01%, and 0.1%. Then, the three-dimensional cultured skin was incubated at 37°C and 5% CO ２The cells were cultured in an incubator for about 48 hours.

[0113] After carefully removing the medium and drugs from the wells, the cells were washed three times with PBS. The culture cups were removed from the wells, the membranes were peeled off, and the skin was transferred to a 10 cm dish (430167, Corning). The skin was cut in half with a high-quality double-edged stainless steel razor and further separated into epidermis and dermis.

[0114] The dermal tissue fragments were transferred to a 1.5 mL tube containing 300 μL of RLT solution (prepared by diluting 99% 2-mercaptetanol to 1% Buffer RLT Lysis buffer), and RNA was extracted according to the protocol of the RNeasy Fibrous Tissue Kit (74704, manufactured by QIAGEN). Specifically, the dermal tissue fragments were crushed, and then 590 μL of RNase-free water and 10 μL of proteinase K were added for proteinase K treatment, followed by incubation at 55°C for 10 minutes. After centrifugation at 10,000 × g for 3 minutes, 450 μL of 99.5% EtOH was added. The mixture was transferred to a spin column and centrifuged. Then, 350 μL of RW buffer was added and centrifuged again. Next, a mixture of 10 μL of DNase I solution dissolved in RNase-free water and 70 μL of RDD buffer was added to the membrane and incubated at room temperature for 15 minutes. Then, 350 μL of RW buffer was added again and centrifuged at 10,000 × g for 1 minute. 500 μL of RPE buffer was added and centrifuged twice at 10,000 × g (1 minute for the first time and 2 minutes for the second time), and finally centrifuged once more to dry. The column was replaced, 35 μL of RNase-free water was added, the RNA was eluted, and the RNA concentration was measured.

[0115] (cDNA synthesis) cDNA was synthesized according to the protocol of ReverTra Ace® qPCR RT Master Mix (FSQ-301, manufactured by Toyobo Co., Ltd.). Specifically, the amount of sample introduced was 300 ng, and 2 μL of 4x DN Master Mix was added to 6 μL of the sample mixed with RNase-free water according to the measured RNA concentration. DNase reaction was carried out at 37°C for 5 minutes and 4°C-hold. Subsequently, 2 μL of 5x RT Master Mix II was added, and reverse transcription reaction (RT reaction) was carried out in the following sequence: 37°C for 15 minutes, 50°C for 5 minutes, 98°C for 5 minutes, and 4°C-hold. The mixture was diluted 4-fold with RNase-free water.

[0116] (RT-PCR) Real-time PCR was performed essentially according to the protocol for TaqMan® Gene Expression Assays (Thermo Fisher Scientific). For each primer of COL1A1, COL3A1, and ELN, 5 μL of TaqMan fast Master Mix (2x), 0.5 μL of TaqMan Gene Expression Assay (20x), 3.5 μL of RNase-free water, and 1 μL of cDNA were prepared and added to a 384-well plate. Real-time PCR was performed using a Quant studio 3Flex system. The PCR cycle conditions were set as follows: 45 cycles of 50°C for 2 minutes, 95°C for 20 seconds, 95°C for 1 second, and 60°C for 20 seconds.

[0117] As shown in Figures 11 to 13, when microalgae culture supernatant and / or extracellular vesicles were applied to three-dimensional cultured skin, it was confirmed in the three-dimensional cultured skin that there was a tendency for the expression of elastin genes, COL1A1 genes, and COL3A1 genes to be increased in a concentration-dependent manner when the final concentration of the composition containing Pavlova-derived extracellular vesicles (ultrafiltration sample) was between 0.001% and 0.1%.

Claims

1. A composition for maintaining or increasing fibrous structural proteins, comprising culture supernatant and / or extracellular vesicles of microalgae.

2. The composition of claim 1, wherein the fibrous structural protein is collagen, elastin, or hyaluronic acid.

3. The composition of claim 2, wherein the elastin is skin elastin.

4. The composition according to claim 1, wherein the maintenance or increase of the fibrous structural protein is achieved by suppressing a fibrous structural protein degrading enzyme, activating a fibrous structural protein synthesizing enzyme, or enhancing gene expression of the fibrous structural protein.

5. The composition according to claim 4, wherein the fibrillar structural protein degrading enzyme is matrix metalloproteinase 1.

6. A composition for maintaining or increasing skin elasticity, comprising culture supernatant and / or extracellular vesicles of microalgae.

7. A composition for promoting the production of collagen, elastin, or hyaluronic acid, comprising culture supernatant and / or extracellular vesicles of microalgae.

8. The composition according to any one of claims 1 to 7, wherein the filter has a molecular weight cut-off of 10 KDa to 1,000 KDa.

9. A composition described in any one of claims 1 to 7, wherein the particle diameter of the extracellular vesicles is 50 nm to 700 nm.

10. The composition according to any one of claims 1 to 7, wherein the content of microalgae culture supernatant and / or extracellular vesicles derived from the microalgae is 0.001 to 0.1% by mass.

11. The content of extracellular vesicles derived from the microalgae is 1.0 x 10 ６ ~1.0 x 10 １１ The composition according to any one of claims 1 to 7, wherein the composition is in a concentration of 0.1 to 0.5% by weight per mL.

12. The composition according to any one of claims 1 to 7, wherein the microalgae is microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

13. The composition according to any one of claims 1 to 7, which is a food or drink, a cosmetic, a medicine, or a quasi-drug.

14. The composition according to any one of claims 1 to 7, which is intended for young people.

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