Composition containing extracellular vesicles derived from microalgae

Abstract

An object of the present invention is to provide a composition comprising extracellular vesicles derived from microalgae.SOLUTION: In one embodiment of the present invention, a composition for external or internal use containing (A) microalgae-derived extracellular vesicles and one or more selected from the group consisting of (B-1) and (B-2), and (B-1) one or more selected from the group consisting of a tetrahydric to decahydric polyhydric alcohol, (B-2) a high-intensity sweetener and a saccharide is prepared.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition containing extracellular vesicles derived from microalgae. [Background technology]

[0002] Many cell types secrete lipid bilayer membrane-containing vesicles. These vesicles are called extracellular vesicles (ECVs), and typically contain functional molecules such as proteins and nucleic acids within the lipid bilayer membrane. ECVs are involved in the delivery of functional molecules within the body, and in recent years, they have been shown to be involved in various biological phenomena and diseases.

[0003] For example, it is known that extracellular vesicles produced by specific species of lactic acid bacteria are used to prevent hair loss and promote hair growth (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2018-529720 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, particularly about microalgae-derived extracellular vesicles themselves and their applications.

[0006] Therefore, an object of the present invention is to provide a composition containing extracellular vesicles derived from microalgae, in which the extracellular vesicles have excellent stability. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors conducted extensive research and found that compositions containing extracellular vesicles derived from microalgae are more stable than extracellular vesicles of other origins and also have good properties when combined with a base. Furthermore, the present inventors found that compositions containing extracellular vesicles derived from microalgae and one or more selected from the group consisting of tetrahydric to decahydric polyalcohols, high-intensity sweeteners, and saccharides are even more stable, leading to the completion of the present invention.

[0008] That is, the present invention provides a composition containing extracellular vesicles derived from microalgae as described below and a method for improving the stability of extracellular vesicles derived from microalgae. [1] (A) Extracellular vesicles derived from microalgae, and a composition for external or internal use containing one or more compounds selected from the group consisting of (B-1) and (B-2); (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars. [2] The composition according to [1], wherein the component (B-1) is a sugar alcohol. [3] The composition according to [1], wherein the component (B-1) is polyglycerin. [4] The composition according to [1], wherein the component (B-1) is a pentahydric to hexahydric polyhydric alcohol. [5] The composition according to [1], wherein the high-intensity sweetener of component (B-2) is one or more selected from the group consisting of aspartame, acesulfame potassium, sucralose, steviol glycosides, and saccharin. [6] The composition according to [1], wherein the sugar of the component (B-2) is one or more selected from the group consisting of glucose, fructose, sucrose, and lactose. [7] The composition according to [1], wherein the component (B-2) is one or more selected from the group consisting of acesulfame potassium, aspartame, and sucralose. [8] The composition described in [1], wherein the microalgae contain chloroplasts or thylakoids. [9] The composition according to [1], wherein the microalgae is one or more species selected from the group consisting of Pavlova, Chlorella, Euglena, and Spirulina.

[10] (A) A method for improving the stability of microalgae-derived extracellular vesicles by adding one or more members selected from the group consisting of (B-1) and (B-2) to the microalgae-derived extracellular vesicles; (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars. [Effects of the Invention]

[0009] According to the present invention, a composition having superior stability can be obtained by further comprising one or more members selected from the group consisting of tetrahydric to decahydric polyhydric alcohols, high-intensity sweeteners, and sugars in addition to extracellular vesicles derived from microalgae. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Composition containing extracellular vesicles] The composition of the present invention may contain (A) extracellular vesicles derived from microalgae, and one or more species selected from the group consisting of (B-1) and (B-2). (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars.

[0011] (extracellular vesicles) The extracellular vesicles derived from microalgae in this specification are typically contained in the culture supernatant of the microalgae, and when preparing a composition containing extracellular vesicles, the culture supernatant can be used as is, or can be crudely or purified from the culture supernatant before use. The composition is a preparation obtained from the culture supernatant, and may contain secretions obtained by culture in addition to the extracellular vesicles.

[0012] (Microalgae culture supernatant) In this specification, microalgae refers 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.

[0013] Microalgae include, for example, prokaryotic Cyanobacteria, as well as eukaryotic Glaucophyta, Rhodophyta (red algae), Chlorophyta, Cryptophyta, Haptophyta, Heterokontophyta, Dinophyta, Euglenida, and Chlorarachniophyta.

[0014] 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 (eyespot algae), Livistona (green algae), and Botryococcus (green algae). The above-listed microalgae may be used singly or in combination of two or more.

[0015] Examples of microalgae include microalgae containing chloroplasts or thylakoids. From the viewpoint of significantly achieving the effects of the present invention, microalgae containing chloroplasts or thylakoids are preferred, and microalgae containing chloroplasts are more preferred.

[0016] Examples of microalgae containing chloroplasts include Prokaryotic Chlorophyceae, Glaucophyceae, Rhodophyceae, Prasinophyceae, Ulvaphyceae, Chlorophyceae, Treboxiophyceae, Charophyceae, Cryptophyceae, Chlorarachniophyceae, Euglenophyceae, Dinophyceae, Chrysophyceae, Raphidophyceae, Euglenoplastphyceae, Xanthophyceae, Phaeophyceae, Bacillariophyceae, Dictyophyceae, Pelagophyceae, and Haptophyceae. The above-listed microalgae may be used singly or in combination of two or more.

[0017] Examples of microalgae containing thylakoids include microalgae belonging to the order Cyanobacteria. More specifically, examples include microalgae belonging to the orders Chlorococcales, Necrocapitales, Oscillatoriales, Prochlorococcales, Synechocystisales, and Plebydosphaerales. The above-listed microalgae may be used singly or in combination of two or more.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] Examples of microalgae of the genus Spirulina include the genus Altrospira, whose scientific name was changed from Spirulina, and include S. platensis (A. platensis) and S. maxima (A. maxima), with S. platensis being preferred.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] As used herein, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained after culturing microalgae. For example, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained by culturing microalgae using a known culture method and removing the microalgae using a known separation means such as centrifugation.

[0026] 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.

[0027] (Culture conditions) Microalgae can be cultured using a culture medium. A carbon source is added to the culture medium as a nutrient source, and examples of the carbon source include inorganic carbon sources (CO2, NaHCO3, Na2CO3, etc.) and organic carbon sources (glucose, etc.). The culture medium is not limited, but it is preferable to use an autotrophic medium that does not contain organic carbon sources such as glucose as a nutrient source. Examples of such media include culture media supplemented with nutrient salts such as nitrogen sources, phosphorus sources, and minerals, such as Cramer-Myers medium and modified Cramer-Myers medium.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] In one embodiment, when culturing marine microalgae such as pavlova, known methods 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 (100-150 μmol) Light / dark cycle: 12 hours light and 12 hours dark pH: pH approximately 7.4 at the start of each culture Aeration: Implementation Mechanical stirring: 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:

[0034] (Collection of culture supernatant) After culturing microalgae under the culture conditions exemplified above, the culture supernatant is recovered using a known separation method such as centrifugation. However, the method is not particularly limited as long as it removes or substantially removes the microalgae and allows the culture supernatant to be appropriately recovered. In an exemplary embodiment, the centrifugation to obtain the culture supernatant may be carried out for 30 to 60 minutes at a centrifugal force of, for example, 1,000 to 20,000 × g, 1,500 to 20,000 × g, 1,500 to 15,000 × g, or 1,500 to 10,000 × 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 × g, and then further centrifuged at a high speed of 10,000 to 20,000 × g to remove cells or cell-related debris and residues. The supernatant thus obtained is recovered and subjected to the next step.

[0035] (A) Extracellular vesicles derived from microalgae As used herein, extracellular vesicles refer to vesicles secreted by microalgae, typically having an average diameter of 10 nm to 1 μm. This average diameter is also referred to as the average particle size. Although not limited thereto, it is preferred that the compositions containing the extracellular vesicles of the present invention do not contain microalgae cells themselves. Here, the term "diameter" or "particle size" used herein to specify the size of extracellular vesicles refers to the maximum dimension of the extracellular vesicles, since extracellular vesicles are not necessarily spherical. Diameter or particle size can be measured using microscopy techniques, such as techniques for measuring nanoparticle size using a transmission electron microscope. Furthermore, measurement can also be performed using particle tracking analysis (PTA), which is based on the analysis of bright spots of particles undergoing Brownian motion. In the present invention, the diameter or particle size of extracellular vesicles is measured using a Viewsizer (ViewSizer 3000, manufactured by HORIBA Scientific).

[0036] The cumulative 10% value (D10) of 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 50 to 120 nm, 60 to 110 nm, and 65 to 100 nm. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 100 to 180 nm, 110 to 170 nm, and 115 to 160 nm. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 180 to 360 nm, 190 to 350 nm, and 200 to 340 nm. As used herein, the cumulative 10% value (D10) of particle diameter of extracellular vesicles refers to the diameter below which 10% of all extracellular vesicles are. The cumulative 50% value (D50) of particle diameter of extracellular vesicles refers to the diameter below which 50% of all 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.

[0037] 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.

[0038] The cumulative 10% value (D10) of particle diameter of 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.

[0039] The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 per ml if the purified product is in liquid form. 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 vesicles of microalgae is, for example, 1.0 × 10 4 ~1.0×10 15 pieces, 1.0×10 4 ~1.0×10 14 pieces, 1.0×10 4 ~1.0×10 131.0×10 4 ~1.0×10 12 1.0×10 4 ~1.0×10 11 1.0×10 5 ~1.0×10 15 1.0×10 5 ~1.0×10 14 1.0×10 5 ~1.0×10 13 1.0×10 5 ~1.0×10 12 1.0×10 5 ~1.0×10 11 1.0×10 6 ~1.0×10 15 1.0×10 6 ~1.0×10 14 1.0×10 6 ~1.0×10 13 1.0×10 6 ~1.0×10 12 1.0×10 6 ~1.0×10 11 1.0×10 7 ~1.0×10 15 1.0×10 7 ~1.0×10 14 1.0×10 7 ~1.0×10 13 1.0×10 7 ~1.0×10 12 1.0×10 7 ~1.0×10 11 1.0×10 8 ~1.0×10 15 1.0×10 8 ~1.0×10 14 1.0×10 8 ~1.0×10 13 1.0×10 8 ~1.0×10 12 1.0×10 8 ~1.0×10 11 1.0×10 9 ~1.0×10 15 1.0×10 9 ~1.0×10 14 1.0×109 ~1.0×10 13 pieces,, 1.0×10 9 ~1.0×10 12 pieces, 1.0×10 9 ~1.0×10 11 The number of extracellular vesicles can be measured using a particle tracking analysis (PTA) tool such as Viewsizer (ViewSizer3000, manufactured by HORIBA Scientific), which is based on the analysis of bright spots of particles undergoing Brownian motion.

[0040] (Method for obtaining extracellular vesicles) Methods for obtaining the extracellular vesicles of the present invention include preparation from the culture supernatant of algae.

[0041] In one embodiment, the extracellular vesicles of the present invention are obtained by heating a culture supernatant of a microalga.

[0042] The extracellular vesicles of the present invention can also be obtained by heating the culture supernatant 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, or 100°C or higher, 200°C or lower, 150°C or lower, or 120°C or lower, for a time period of 30 seconds or longer, 1 minute or longer, 2 minutes or longer, 3 minutes or longer, or 4 minutes or longer, 30 minutes or shorter, 20 minutes or shorter, or 10 minutes or shorter.

[0043] Alternatively, in another embodiment, the extracellular vesicles of the present invention can be obtained by filtering the culture supernatant of microalgae.

[0044] The membrane pore size of the filter used for filtration is preferably 0.001 to 0.5 μm, more preferably 0.005 μm to 0.3 μm, and even more preferably 0.01 μm to 0.25 μm.

[0045] In yet another embodiment, the method for obtaining extracellular vesicles of the present invention can also include a purification step. Specific embodiments of the purification step include purification by ultrafiltration (e.g., tangential flow filtration, etc.), ultracentrifugation, and affinity purification using antibodies. Of these, purification by ultrafiltration is preferred, but is not limited to this.

[0046] When purification by ultrafiltration is used, it may be performed using a hollow fiber membrane to filter from the inside to the outside of the fiber, using a spiral membrane including a filtration membrane and a support membrane, using a tubular membrane on a hollow cylinder, or using a flat membrane. Of these, but not limited to, it is preferable to perform filtration using a hollow fiber membrane to obtain a concentrated liquid containing extracellular vesicles on the inside of the hollow fiber membrane.

[0047] The molecular weight cutoff of a membrane is preferably 10 kDa to 1000 kDa, 50 kDa to 1000 kDa, or 100 kDa to 1000 kDa, and more preferably 100 kDa to 500 kDa. A membrane with a cutoff of 10 kDa or more, 50 kDa or more, 100 kDa or more, or 150 kDa or more is preferred, and a membrane with a cutoff of 2000 kDa or less, 1500 kDa or less, 1000 kDa or less, 750 Dka or less, or 500 Dka or less is also preferred.

[0048] The extracellular vesicles of the present invention may be prepared by one or a combination of two or more of these preparation methods, for example, but not limited to, a combination of a heat treatment step and a purification step, a combination of a purification step and a filtration step, or a combination of a heat treatment step, a purification step, and a filtration step.

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

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

[0051] (Composition containing extracellular vesicles) In one aspect of the present invention, it is possible to prepare a composition containing any of the extracellular vesicles thus obtained.

[0052] In such a composition, the average particle number of the extracellular vesicles of the microalgae is, for example, 1.0 × 10 per 100 g. 3 pcs or more, 1.0×10 4 pcs or more, 1.0×10 5 pcs or more, 1.0×10 6 1.0×10 16 pcs or less, 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 pcs or less, 1.0×10 10 pcs or less, 1.0×10 9 pcs or less, 1.0×10 8 pcs or less, 1.0×10 7 pcs or less, 1.0×10 6 The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 3 ~1.0×10 14 pieces, 1.0×10 3 ~1.0×10 13 pieces, 1.0×10 3 ~1.0×10 12 pieces, 1.0×10 3 ~1.0×10 11 pieces, 1.0×10 3 ~1.0×10 10 pieces, 1.0×10 3 ~1.0×10 9 pieces, 1.0×10 3 ~1.0×10 8 pieces, 1.0×10 3 ~1.0×10 7 pieces, 1.0×10 4 ~1.0×10 14 pieces, 1.0×10 4 ~1.0×10 13 pieces, 1.0×10 4 ~1.0×10 121.0×10 4 ~1.0×10 11 1.0×10 4 ~1.0×10 10 1.0×10 4 ~1.0×10 9 1.0×10 4 ~1.0×10 8 1.0×10 4 ~1.0×10 7 1.0×10 5 ~1.0×10 14 1.0×10 5 ~1.0×10 13 1.0×10 5 ~1.0×10 12 1.0×10 5 ~1.0×10 11 1.0×10 5 ~1.0×10 10 1.0×10 5 ~1.0×10 9 1.0×10 5 ~1.0×10 8 1.0×10 5 ~1.0×10 7 1.0×10 6 ~1.0×10 14 1.0×10 6 ~1.0×10 13 1.0×10 6 ~1.0×10 12 1.0×10 6 ~1.0×10 11 1.0×10 6 ~1.0×10 10 1.0×10 6 ~1.0×10 9 1.0×10 6 ~1.0×10 8 1.0×10 6 ~1.0×10 7 1.0×10 7 ~1.0×10 14 1.0×10 7 ~1.0×10 13 1.0×10 7 ~1.0×10 12 1.0×107 ~1.0×10 11 pieces, 1.0×10 7 ~1.0×10 10 pieces, 1.0×10 8 ~1.0×10 14 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:

[0053] 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.

[0054] The content of microalgal extracellular vesicles in the composition is adjusted appropriately 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. in the state of a purified liquid relative to the total amount of the composition. Furthermore, 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 extracellular vesicles of microalgae may be, for example, 0.001 to 50 mass%, 0.001 to 40 mass%, 0.001 to 30 mass%, 0.001 to 20 mass%, 0.001 to 15 mass%, 0.01 to 50 mass%, 0.01 to 40 mass%, 0.01 to 30 mass%, 0.01 to 20 mass%, 0.01 to 15 mass%, 0.1 to 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%, or 1 to 15 mass%, relative to the total mass of the composition.

[0055] 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, 0.00001 μg / mL or more, 0.0001 μg / mL or more, 0.001 μg / mL or more, 0.01 μg / mL or more, 0.1 μg / mL or more, or 1 μg / mL or more in terms of protein amount relative to the total amount of the composition. Other examples include 100 μg / mL or less, 10 μg / mL or less, 1 μg / mL, 0.1 μg / mL or less, and 0.01 μg / mL or less.

[0056] ((B-1) Tetrahydric to decahydric polyalcohols) The component (B-1) of the present invention is a tetrahydric to decahydric polyalcohol.

[0057] The tetrahydric to decahydric polyhydric alcohols that can be used in the present invention are not limited as long as they are used in the fields of pharmaceuticals or cosmetics, and in some cases, food. In this specification, "tetrahydric to decahydric polyhydric alcohol" refers to an alcohol containing 4 to 10 hydroxy (-OH) groups per molecule. Examples of tetrahydric alcohols include diglycerin and pentaerythritol; examples of pentahydric alcohols include xylitol; examples of hexahydric alcohols include sorbitol and inositol; and examples of nonahydric alcohols include maltitol. From the perspective of significantly achieving the effects of the present invention, the tetrahydric to decahydric polyhydric alcohol is preferably a sugar alcohol or polyglycerin. Polyglycerin refers to a compound having a structure in which two or more glycerins are polymerized. Examples of sugar alcohols or polyglycerins include pentaerythritol, xylitol, sorbitol, inositol, maltitol, reduced maltose syrup, and diglycerin. The tetrahydric to decahydric polyhydric alcohol used in the present invention is preferably a pentahydric to hexahydric polyhydric alcohol. Examples of such alcohols include sorbitol, diglycerin, etc. In the present invention, the tetrahydric to decahydric polyhydric alcohols may be used alone or in combination of two or more kinds.

[0058] The content of the tetrahydric to decahydric polyhydric alcohol relative to the total amount of the composition of the present invention is preferably 0.01 to 30 mass %, more preferably 1 to 25 mass %, and even more preferably about 3 to 20 mass %.

[0059] In the composition of the present invention, the number of molecules per 1% by mass of the tetrahydric to decahydric polyhydric alcohol of component (A) is 1.0×10 to 1.0×10 15 1.0 x 10 is preferred 2 ~1.0×10 14 1.0 x 10 is more preferable. 3 ~1.0×10 13 1.0×10 is more preferable, 4 ~1.0×10 12 is even more preferred.

[0060] ((B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars) The component (B-2) that can be used in the present invention is one or more selected from the group consisting of high-intensity sweeteners and sugars. In this specification, "high-intensity sweetener" refers to a synthetic (artificial) sweetener or natural sweetener that exhibits a sweetness several tens to several hundreds times that of sucrose. The high-intensity sweetener is not limited as long as it is used in the fields of pharmaceuticals, cosmetics, or food. Examples of high-intensity sweeteners include aspartame, acesulfame potassium, sucralose, steviol glycosides, saccharin, neotame, and advantame. Among these, at least one selected from the group consisting of aspartame, acesulfame potassium, sucralose, steviol glycosides, and saccharin is exemplified. Of these, from the viewpoint of significantly achieving the effects of the present invention, the high-intensity sweetener is preferably at least one selected from the group consisting of aspartame, acesulfame potassium, sucralose, steviol glycosides, and saccharin, and more preferably at least one selected from the group consisting of acesulfame potassium, aspartame, and sucralose.

[0061] The steviol glycoside is preferably at least one selected from the group consisting of stevioside, dulcoside A, rebaudioside A, rebaudioside C, rebaudioside D, and rebaudioside M, which are stevia sweeteners.

[0062] In the present invention, "saccharides" refers to monosaccharides, oligosaccharides, and / or polysaccharides. The saccharides are not limited as long as they are used in the fields of pharmaceuticals, cosmetics, or foods. For example, monosaccharides include glucose (grape sugar) and fructose (fruit sugar). Oligosaccharides include sucrose (cane sugar), lactose (milk sugar), maltose, lactulose, trehalose, etc. Polysaccharides include starch, glycogen, cellulose, alginic acid, hyaluronic acid, etc. Among these, from the viewpoint of significantly achieving the effects of the present invention, at least one saccharide selected from the group consisting of glucose (grape sugar), fructose (fruit sugar), sucrose (cane sugar), and lactose (milk sugar) is more preferred.

[0063] When the (B-2) component is a high-intensity sweetener, the total content of the (B-2) component relative to the total amount of the composition of the present invention is preferably 0.0000001 to 30% by mass, more preferably 0.000001 to 15% by mass, and even more preferably about 0.00001 to 10% by mass, and may be 0.0001 to 1% by mass, 0.001 to 1% by mass, 0.01 to 1% by mass, or the like.

[0064] When the component (B-2) is a sugar, the total content of the component (B-2) relative to the total amount of the composition of the present invention is preferably 0.001 to 99% by mass, more preferably 0.01% to 90% by mass, even more preferably 0.1 to 85% by mass, and even more preferably 1 to 80% by mass, or may be 0.1 to 50% by mass, 1 to 40% by mass, 5 to 30% by mass, etc.

[0065] In the composition of the present invention, the number of components (A) per 1% by mass of one or more selected from the group consisting of high-intensity sweeteners and saccharides is 1.0 × 10 to 1.0 × 10 20 1.0 x 10 is preferred 2 ~1.0×10 19 1.0 x 10 is more preferable. 3 ~1.0×10 18 1.0×10 is more preferable, 4 ~1.0×1017 In addition to the above, 1.0×10 5 ~1.0×10 20 pieces, 1.0×10 6 ~1.0×10 19 pieces, 1.0×10 7 ~1.0×10 18 The number may be, for example, pieces.

[0066] The total content of components (B-1) and (B-2) relative to the total amount of the composition of the present invention is preferably 0.0000001 to 99% by mass, more preferably 0.00001 to 85% by mass, and even more preferably about 0.001 to 80% by mass, and may be 0.001 to 50% by mass, 0.001 to 30% by mass, 0.01 to 10% by mass, etc.

[0067] In the composition of the present invention, the number of components (A) per 1% by mass of the total of components (B-1) and (B-2) is 1.0×10 to 1.0×10 20 1.0 x 10 is preferred 2 ~1.0×10 19 1.0 x 10 is more preferable. 3 ~1.0×10 18 1.0×10 is more preferable, 4 ~1.0×10 17 In addition to the above, 1.0×10 5 ~1.0×10 20 pieces, 1.0×10 6 ~1.0×10 19 pieces, 1.0×10 7 ~1.0×10 18 The number may be, for example, pieces.

[0068] ((C)Water) The composition of the present invention may also contain water. The total content of water relative to the total amount of the composition of the present invention is preferably 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, 80% by mass or more, or 90% by mass or more, and is preferably 99% 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, or 40% by mass or less.

[0069] In the composition of the present invention, the number of components (A) per 1% by mass of water is 1.0×10 to 1.0×10 16 1.0 x 10 is preferred 2 ~1.0×10 14 1.0 x 10 is more preferable. 3 ~1.0×10 12 1.0×10 is more preferable, 4 ~1.0×10 11 In one embodiment, the number of components (A) per 1% by mass of water is 1.0×10 to 1.0×10 10 1.0 x 10 is preferred 2 ~1.0×10 9 1.0 x 10 is more preferable. 3 ~1.0×10 8 1.0×10 is more preferable, 4 ~1.0×10 7 is even more preferred.

[0070] The pH of the composition of the present invention may be 2.5 or more, 3.5 or more, 4.0 or more, 5.0 or more, or 6.0 or more, or 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9.0 or less, or 8.0 or less. The pH of the composition may be pH 2.5 to 12, pH 3.5 to 10, or pH 5 to 8.0.

[0071] (nonionic surfactant) The composition of the present invention may contain a nonionic surfactant in addition to the components (A) and (B) described above, as long as the effects of the present invention are not impaired. The nonionic surfactant that can be used in the present invention is not limited, but is preferably a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) of less than 15, more preferably a surfactant with an HLB of 14.5 or less, and even more preferably a surfactant with an HLB of 14 or less. The HLB of the nonionic surfactant may be 6 to 14.5, preferably 8 to 14.5, and more preferably 10 to 14.5.

[0072] The HLB value in the present invention refers to a value calculated by the following formula 1 using the hydrophilic group molecular weight (Mw) and lipophilic group molecular weight (MO) of the surfactant molecule. HLB value = 7 + 11.7 log Mw / Mo (Equation 1)

[0073] Examples of nonionic surfactants include: Polyglycerin fatty acid esters such as hexaglyceryl monolaurate (HLB value 14.5), hexaglyceryl monomyristate (HLB value 11), hexaglyceryl monostearate (HLB value 9.0), hexaglyceryl monooleate (HLB value 9.0), decaglyceryl monomyristate (HLB value 14.0), decaglyceryl monostearate (HLB value 12.0), decaglyceryl monoisostearate (HLB value 12.0), decaglyceryl monooleate (HLB value 12.0), decaglyceryl distearate (HLB value 9.5), and decaglyceryl diisostearate (HLB value 10.0); Polyoxyethylene glycerin fatty acid esters such as polyoxyethylene 5-mol-added monostearate (referred to as "POE(5)"; hereinafter the same) glyceryl (HLB value 9.5), POE(15) glyceryl monostearate (HLB value 13.5), POE(5) glyceryl monooleate (HLB value 9.5), and POE(15) glyceryl monooleate (HLB value 14.5); Polyoxyethylene sorbitan fatty acid esters such as POE (20) sorbitan monostearate (HLB value 14.9), POE (6) sorbitan monostearate (HLB value 9.5), POE (20) sorbitan tristearate (HLB value 10.5), POE (20) sorbitan monoisostearate (HLB value 15.0), POE (6) sorbitan monooleate (HLB value 10.0), and POE (20) sorbitan trioleate (HLB value 11.0); Polyoxyethylene sorbitan fatty acid esters such as POE(60) sorbitan tetrastearate (HLB value 13.0), POE(30) sorbitan tetraoleate (HLB value 11.5), POE(40) sorbitan tetraoleate (HLB value 12.5), and POE(60) sorbitan tetraoleate (HLB value 14.0); Polyoxyethylene lanolin, lanolin alcohol, and beeswax derivatives such as POE(10) lanolin (HLB value 12.0), POE(20) lanolin (HLB value 13.0), POE(5) lanolin alcohol (HLB value 12.5), and POE(20) sorbitol beeswax (HLB value 9.5); Polyoxyethylene castor oils and hydrogenated castor oils such as POE(20) castor oil (HLB value 10.5), POE(40) castor oil (HLB value 12.5), POE(50) castor oil (HLB value 14.0), POE(60) castor oil (HLB value 14.0), POE(20) hydrogenated castor oil (HLB value 10.5), POE(30) hydrogenated castor oil (HLB value 11.0), POE(40) hydrogenated castor oil (HLB value 13.5), and POE(60) hydrogenated castor oil (HLB value 14.0); Polyoxyethylene sterols and hydrogenated sterols such as POE(5) phytosterol (HLB value 9.5), POE(10) phytosterol (HLB value 12.5), and POE(25) phytostanol (HLB value 14.5); POE (2) lauryl ether (HLB value 9.5), POE (4.2) lauryl ether (HLB value 11.5), POE (9) lauryl ether (HLB value 14.5), POE (5.5) cetyl ether (HLB value 10.5), POE (7) cetyl ether (HLB value 11.5), POE (10) cetyl ether (HLB value 13.5), POE (4) stearyl ether (HLB value 9.0), POE (7) oleyl ether (HLB value 10.5), POE (10) oleyl ether (HLB value 14.5), POE (10) behenyl ether (HLB value 10.0), POE (2) (C12-15) alkyl ether (HLB value 9.0), POE (4) (C12-15) alkyl ether (HLB value 10.5), POE (5) secondary alkyl ether (HLB value 10.5), POE (7) secondary alkyl ether (HLB value 12.0), POE (9) alkyl ether (HLB value 13.5), POE (12) alkyl ether (HLB value 14.5), and other polyoxyethylene alkyl ethers; Polyoxyethylene polyoxypropylene alkyl ethers such as polyoxypropylene 1-mol-addition (referred to as "POP(1)"; the same applies hereinafter) POP(4) cetyl ether (HLB value 9.5), POE(10)POP(4) cetyl ether (HLB value 10.5), POE(20)POP(8) cetyl ether (HLB value 12.5), POE(20)POP(6) decyltetradecyl ether (HLB value 11.0), and POE(30)POP(6) decyltetradecyl ether (HLB value 12.0); polyethylene glycol fatty acid esters such as 10-mol polyethylene glycol monolaurate (referred to as "PEG(10)"; the same applies hereinafter) (HLB value 12.5), PEG(10) monostearate (HLB value 11.0), PEG(10) monooleate (HLB value 11.0), and PEG diisostearate (HLB value 9.5); Polyoxyethylene glyceryl isostearates such as PEG (8) glyceryl isostearate (HLB value 10.0), PEG (10) glyceryl isostearate (HLB value 10.0), PEG (15) glyceryl isostearate (HLB value 12.0), PEG (20) glyceryl isostearate (HLB value 13.0), PEG (25) glyceryl isostearate (HLB value 14.0), and PEG glyceryl (30) isostearate; is exemplified.

[0074] The total content of nonionic surfactants with an HLB of less than 15 relative to the total amount of the composition of the present invention is preferably 0.01 to 5 mass%, more preferably 0.1 to 4 mass%, and even more preferably about 0.5 to 3 mass%.

[0075] (Additives) The composition of the present invention may contain various additives other than those described above, provided that the effects of the present invention are not impaired. Examples of additives include antioxidants, thickeners, pH adjusters, inorganic salts, stabilizers, UV absorbers or UV scatterers, irritation reducers, colorants, and fragrances. One or more additives may be used.

[0076] Here, the inorganic salt is preferably sodium chloride, disodium hydrogen phosphate, potassium chloride, or potassium dihydrogen phosphate.

[0077] [Application] In one embodiment, the composition comprising the extracellular vesicles of the present invention is preferably used as an external preparation.

[0078] The composition of the present invention can be added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods. Alternatively, it can be used as is as pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet foods. Furthermore, it can also be used as a raw material for preparing such products or as a so-called premix product.

[0079] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the composition of the present invention is preferably exemplified by skin care cosmetics (e.g., lotions, creams, facial creams, facial lotions, emulsions, packs, liquid facial cleansers, soaps, and serums), makeup cosmetics (e.g., eyeliners and eyebrow makeup), and scalp and hair cosmetics (e.g., scalp lotions, scalp creams, shampoos, hair conditioners, hair treatments, hair essences, hair mists, and hair tonics), more preferably lotions, creams, facial creams, facial lotions, emulsions, packs, facial cleansers, soaps, and serums, and particularly preferably lotions, facial lotions, and serums, but is not limited to these.

[0080] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the dosage form of the composition of the present invention is not particularly limited, and examples thereof include solutions, suspensions, emulsions, creams, gels, liniments, lotions, ointments, and aerosols. Among these, solutions, suspensions, emulsions, lotions, and aerosols are preferred, and solutions, lotions, and aerosols are more preferred. In addition, premix products may be liquids containing, for example, extracellular vesicles, water, glycol, preservatives, excipients, solubilizers, pH adjusters, and the like.

[0081] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily adult dosage of microalgae extracellular vesicles can be appropriately determined depending on the individual's condition, body weight, age, activity of the material, formulation form, and other factors. Examples of daily adult dosages of microalgae extracellular vesicles 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. Examples of daily adult dosages of microalgae extracellular vesicles include 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, and 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.

[0082] 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, age, activity of the material, ingestion or administration route, formulation, 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 adults 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.

[0083] In another embodiment, the daily dose of microalgal extracellular vesicles for an adult is, in terms of number, for example, 1.0 × 10 or more, 1.0 × 10 2 pcs or more, 1.0×10 3 pcs or more, 1.0×10 4 The daily dose of microalgae extracellular vesicles for an adult is, for example, 1.0 × 10 to 1.0 × 10 10 pieces, 1.0×10~1.0×109 pieces, 1.0×10~1.0×10 8 pieces, 1.0×10~1.0×10 7 pieces, 1.0×10 2 ~1.0×10 10 pieces, 1.0×10 2 ~1.0×10 9 pieces, 1.0×10 2 ~1.0×10 8 pieces, 1.0×10 2 ~1.0×10 7 pieces, 1.0×10 3 ~1.0×10 10 pieces, 1.0×10 3 ~1.0×10 9 pieces, 1.0×10 3 ~1.0×10 8 pieces, 1.0×10 3 ~1.0×10 7 pieces, 1.0×10 4 ~1.0×10 10 pieces, 1.0×10 4 ~1.0×10 9 pieces, 1.0×10 4 ~1.0×10 8 pieces, 1.0×10 4 ~1.0×10 7 Examples include:

[0084] The composition of the present invention can be taken 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 twice a day is preferred.

[0085] 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, i.e., a health food, a functional food, a food for patients, or a food for specified health uses.It can also be used as a so-called doctor's supplement recommended or presented by a doctor in the internal medicine department, orthopedics department, veterinary hospital, etc. in a hospital and / or clinic.

[0086] Health foods, functional foods, foods for patients, and foods for specified health uses can be used in various dosage forms, such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candy, etc.), liquid preparations (syrups, suspensions), and liquid foods. Food preparations can be manufactured in the same manner as known pharmaceutical preparations, by mixing the active ingredient with a food-acceptable carrier, such as a suitable excipient, and then manufacturing the preparation using conventional means. The dosage form is not limited, but is preferably an orally disintegrating tablet, chewable tablet, candy, granules, powder, or liquid preparation, from the viewpoint of significantly achieving the effects of the present invention.

[0087] 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 such as gummies, candies, and snacks; spreads, and the like.

[0088] 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.

[0089] 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. In addition, 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. Furthermore, the oral intake or administration amount of microalgal extracellular vesicles per day 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 500 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 500 μg, 5 to 6 ... Examples include 10 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. 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 this specification, regardless of the method used.

[0090] 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, 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 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. Furthermore, the oral intake or administration amount of microalgal extracellular vesicles per day 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 500 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 500 μg, 5 to 6 ... Examples include 10 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. 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 this specification, regardless of the method used.

[0091] In another embodiment, the daily oral intake or administration amount of microalgal extracellular vesicles for an adult is, in terms of number of particles, for example, 1.0 × 10 or more, 1.0 × 10 or more, 2 pcs or more, 1.0×10 3 pcs or more, 1.0×10 4 The daily oral intake or administration amount of extracellular vesicles of microalgae for an adult is, for example, 1.0 × 10 9 pcs or less, 1.0×10 8 pcs or less, 1.0×10 7 pcs or less, 1.0×10 6 pcs or less, 1.0×10 5 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, 1.0 × 10 to 1.0 × 10 9 pieces, 1.0×10~1.0×10 8 pieces, 1.0×10~1.0×10 7 pieces, 1.0×10~1.0×10 6 pieces, 1.0×10 2 ~1.0×10 9 pieces, 1.0×10 2 ~1.0×10 8 pieces, 1.0×10 2 ~1.0×10 7 pieces, 1.0×10 2 ~1.0×10 6 pieces, 1.0×10 3 ~1.0×10 9 pieces, 1.0×10 3 ~1.0×10 8 pieces, 1.0×10 3 ~1.0×10 7 pieces, 1.0×10 3 ~1.0×10 6 pieces, 1.0×10 4 ~1.0×10 9 pieces, 1.0×10 4 ~1.0×10 8 pieces, 1.0×10 4 ~1.0×10 7 pieces, 1.0×10 4 ~1.0×10 6 Examples include pieces, etc.

[0092] 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.

[0093] The composition of the present invention can be taken 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.

[0094] When the present invention is used as feed, pet food, etc., the target organisms are not particularly limited, but are preferably mammals, reptiles, amphibians, birds, and fish, and more preferably mammals other than humans.

[0095] When the present invention is used as feed, pet food, or the like, it may be given to the pet in divided doses several times a day by adding it to the staple food, or it may be given as a snack at any time.

[0096] [How to improve stability] The present invention relates to a method for improving the stability of extracellular vesicles derived from microalgae (A) by further adding one or more species selected from the group consisting of (B-1) and (B-2) to the extracellular vesicles derived from microalgae. (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars.

[0097] The present invention also relates to a composition having improved stability of extracellular vesicles derived from microalgae, which further comprises one or more components selected from the group consisting of components (B-1) and (B-2) in addition to extracellular vesicles derived from microalgae (A). (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars. Here, improved stability refers to, for example, little change in the particle number and / or average particle diameter (size) of the extracellular vesicles before and after storage of a composition containing the extracellular vesicles, and / or less likelihood of aggregates forming. Extracellular vesicles are expected to have various physiologically active functions, and maintaining their number and physical properties will lead to the maintenance of the properties of the composition containing them. [Example]

[0098] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0099] Examples and Comparative Examples The main raw materials used in the examples and comparative examples are as follows.

[0100] (Preparation of pavlova-derived extracellular vesicle-containing solution) Pavlova sp. was cultured in a commercially available algae medium by standard methods to obtain a culture supernatant. The culture supernatant was heated at 105°C for 4 minutes. After heating, 3000 mL of the supernatant was centrifuged at 3000 × g at room temperature for 60 minutes. It was filtered through a 0.2 μm filter and 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: Repligen). The concentrate was then concentrated and washed with 10,000 mL of PBS, and finally recovered with 100 mL of PBS. The concentrated sample was then filter-sterilized using a 0.22 μm filter, and a Pavlova-derived extracellular vesicle-containing solution (1.28 × 10 11 particles / mL).

[0101] As a comparative example, a solution containing extracellular vesicles derived from human stem cells (product name: RemyStem-JP, manufacturer: Anti-Aging Co., Ltd.) (5.55 × 10 7 particles / mL) was used.

[0102] [Stability evaluation test of extracellular vesicle-containing composition] The components described in Tables 1 and 2 were stirred at room temperature, and the stirred composition was filled into 20-ml glass vials and then stored statically at 40°C for 10 days. The number of particles and the average particle diameter of each composition after static storage at 40°C for 10 days were measured. For the measurement, Viewsizer (manufactured by HORIBA Scientific, ViewSizer 3000) was used, and each composition was appropriately diluted with PBS for measurement.

[0103] Based on the numerical values of the number of particles and the average particle diameter of each composition immediately after preparation, the variation rates of the number of particles and the average particle diameter of each composition after static storage at 40°C for 10 days were calculated by the following formulas. Particle number variation rate = [(Number of particles immediately after preparation - Number of particles after static storage at 40°C) / (Number of particles immediately after preparation) × 100] Average particle diameter variation rate = [(Particle diameter immediately after preparation - Particle diameter after static storage at 40°C) / (Particle diameter immediately after preparation) × 100] Note that [ ] represents the absolute value.

[0104] Also, the measurement conditions of Viewsizer 3000 are as follows.

[0105] <Measurement conditions of ViewSizer 3000> Measurement temperature: 22°C Methods: Exosomes [[ID=第23号]] Videos count: 25 times (for Table 1), 10 times (for Table 2)

[0106] Also, in Comparative Example 1 and Examples 1 and 2, each composition after static storage at 40°C for 10 days was visually observed to confirm the presence or absence of aggregates in each composition.

[0107] The evaluation results of the compositions of the examples and comparative examples are shown in Tables 1 and 2. Note that the numerical values in the tables represent mass%.

[0108] The evaluation results of the stability of the examples and comparative examples were evaluated based on the following stability evaluation criteria.

[0109] <Particle number variation rate> Absolute value less than 30%: 〇 Absolute value 30% or more: × No data:-

[0110] <Average particle size variation rate> Absolute value 15% or more: × Absolute value 10% or more and less than 15%: △ Absolute value 5% or more but less than 10%: 〇 Absolute value less than 5%: ◎

[0111] [Table 1]

[0112] As shown in Table 1, Examples 1 and 2, which used pavlova-derived extracellular vesicles, had lower particle number fluctuation rates and average particle size fluctuation rates and were more stable than Comparative Example 1, which used human-derived extracellular vesicles. Furthermore, in Comparative Example 1, which was a composition containing human-derived extracellular vesicles in the presence of sorbitol, white aggregates were visually observed after storage at 40°C for 10 days. On the other hand, in Examples 1 and 2, no aggregates were visually observed even under the same conditions.

[0113] [Table 2]

[0114] As shown in Table 2, the average particle size of Comparative Example 2 after 10 days of static storage at 40° C. fluctuated significantly by 15% or more. On the other hand, the average particle size fluctuation rate of Examples 5 to 7 was small even under the same conditions, confirming good stability.

[0115] Furthermore, all of the compositions of the Examples had a smooth feel on the skin, were excellent in moisturizing properties, and provided a good feeling when used.

Claims

1. (A) Extracellular vesicles derived from microalgae, and A composition for external or internal use containing one or more selected from the group consisting of (B-1) and (B-2); (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars.

2. The composition according to claim 1, wherein the component (B-1) is a sugar alcohol.

3. 2. The composition according to claim 1, wherein the component (B-1) is polyglycerin.

4. 2. The composition according to claim 1, wherein the high-intensity sweetener of component (B-2) is one or more selected from the group consisting of aspartame, acesulfame potassium, sucralose, steviol glycosides, and saccharin.

5. The composition according to claim 1, wherein the sugar of the component (B-2) is one or more selected from the group consisting of glucose, fructose, sucrose, and lactose.

6. 2. The composition according to claim 1, wherein the component (B-2) is one or more selected from the group consisting of acesulfame potassium, aspartame, and sucralose.

7. The composition of claim 1 , wherein the microalgae comprises chloroplasts or thylakoids.

8. 2. The composition according to claim 1, wherein the microalgae is one or more species selected from the group consisting of Pavlova, Chlorella, Euglena, and Spirulina.

9. (A) A method for improving the stability of microalgae-derived extracellular vesicles by adding one or more compounds selected from the group consisting of (B-1) and (B-2) to the microalgae-derived extracellular vesicles; (B-1) tetrahydric to decahydric polyalcohols, (B-2) One or more selected from the group consisting of high-intensity sweeteners and sugars.

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