Composition containing extracellular vesicles derived from microalgae

Abstract

To provide a composition containing extracellular vesicles derived from microalgae.SOLUTION: In one embodiment of the present invention, a composition contains: (A) extracellular vesicles derived from microalgae; and one or more chemicals selected from the group consisting of (B-1) to (B-3), where (B-1) is one or more chemicals selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) is one or more chemicals selected from the group consisting of organic acids and salts thereof, and (B-3) is a chelating agent.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] In many cell types, vesicles having a lipid bilayer are secreted extracellularly. Such vesicles are called extracellular vesicles and typically contain functional molecules such as proteins and nucleic acids within the lipid bilayer. Extracellular vesicles are involved in the transfer of functional molecules in vivo and have recently been shown to be involved in various biological phenomena and diseases.

[0003] For example, it is known that extracellular vesicles produced by lactic acid bacteria of a specific bacterial species are used for preventing hair loss and promoting hair growth (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, there are still many unknown aspects regarding extracellular vesicles, and there is not enough reporting especially about extracellular vesicles derived from microalgae themselves and their applications.

[0006] Therefore, an object of the present invention is to provide a composition containing extracellular vesicles derived from microalgae and having excellent stability of extracellular vesicles.

Means for Solving the Problems

[0007] As a result of intensive studies by the present inventors to solve the above problems, it has been found that a composition containing extracellular vesicles derived from microalgae is superior in stability compared to extracellular vesicles derived from other sources, and also has good properties when combined with a base, thus completing the present invention.

[0008] That is, the present invention provides a composition containing (A) extracellular vesicles derived from microalgae.

[0009] One aspect of the present invention is as follows. [1] A composition containing (A) extracellular vesicles derived from microalgae.

Effects of the Invention

[0010] According to the present invention, a composition containing extracellular vesicles derived from microalgae and having excellent stability can be obtained.

Modes for Carrying Out the Invention

[0011] [Composition Containing Extracellular Vesicles] The composition of the present invention contains (A) extracellular vesicles derived from microalgae. Further, it may contain one or more selected from the group consisting of (B-1) to (B-3). (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) Chelating agents

[0012] (Extracellular Vesicles) The extracellular vesicles derived from microalgae in the present specification are typically those contained in the culture supernatant of microalgae. When preparing a composition containing extracellular vesicles, the culture supernatant can be used as it is, or it can be used after being roughly purified or purified from the culture supernatant. The composition may be a preparation obtained from the culture supernatant and may contain secretions obtained by culturing in addition to extracellular vesicles.

[0013] (Culture supernatant of microalgae) In this specification, microalgae refers to organisms that perform oxygen-generating photosynthesis, excluding moss plants, fern plants, and seed plants among them, and having a cell size of 0.1 μm to 1000 μm in diameter. Also included are labyrinthulids, which are protists closely related to microalgae.

[0014] Microalgae include, for example, prokaryotic cyanobacteria, as well as eukaryotic glaucophytes, rhodophytes (red algae), chlorophytes, cryptophytes (cryptomonads), haptophytes, heterokontophytes, dinophytes (dinoflagellates), euglenids, and organisms of the phylum Chlorarachniophyta.

[0015] Examples of microalgae include species of the genus Pavlova, Euglena (Japanese name: Midorimushi, protozoa / green algae), Spirulina (cyanobacteria), Chlorella (green algae), Dunaliella (green algae), Nannochloropsis (eustigmatophytes), Ichthyodinium (green algae), Botryococcus (green algae), microalgae belonging to labyrinthulids, and microalgae belonging to the genus Slagsthokitrid. The microalgae exemplified above may be used alone or in combination of two or more species.

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

[0017] Pavlova belongs to the phylum Haptophyta. Examples of microalgae of the genus Pavlova include, for example, P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, P. salina, etc., and P. granifera and / or P. gyrans are preferred.

[0018] Examples of microalgae of the genus Euglena include, for example, 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, E. cyclopicola, etc., and E. gracilis and / or E. longa are preferred.

[0019] Examples of microalgae of the genus Spirulina may be, for example, the genus Arthrospira which has been renamed from the genus Spirulina, and include S. platensis (A. platensis), S. maxima (A. maxima), etc., and S. platensis is preferred.

[0020] Examples of microalgae of the genus Chlorella include, for example, C. vulgaris, C. saccharophila, C. ellipsoidea, C. pyrenoidosa, C. sorokiniana, C. lobophora, etc., and C. vulgaris is preferred.

[0021] The microalgae exemplified above are widely distributed in seawater, fresh water such as ponds and marshes, and brackish water, and they may be separated and used therefrom, or any microalgae that have already been isolated may also be used.

[0022] The microalgae exemplified above include related species and mutant strains as long as the effects of the present invention are achieved. Examples of mutant strains include those obtained by genetic methods such as genetic recombination, transduction, and transformation.

[0023] In this specification, the culture supernatant of microalgae refers to the supernatant of the culture solution after culturing microalgae. For example, it includes the supernatant of the culture solution obtained by culturing microalgae by a known culture method and removing the microalgae by known separation means such as centrifugation.

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

[0025] (Culture conditions) The culture of microalgae can be carried out using a culture solution. A carbon source is added to the culture solution as a nutrient source, and inorganic carbon sources (such as CO2, NaHCO3, Na2CO3, etc.) and organic carbon sources (such as glucose, etc.) are used as the carbon source. The culture solution 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 such media include culture solutions to which nutrient salts such as a nitrogen source, a phosphorus source, and minerals are added, such as Cramer-Myers medium and modified Cramer-Myers medium.

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

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

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

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

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

[0031] In one embodiment, when culturing marine microalgae such as Pavlova, as long as the effects of the present invention are achieved, it is possible to use a known method. For example, Seawater concentration: 50% seawater Medium concentration: 2-fold concentration of IMK medium Volume of culture solution: 800 mL Light source: Side: Fluorescent lamp (100 - 150 μmol) Light-dark cycle: 12 hours each for light and darkness pH: Approximately 7.4 at the start of each culture Aeration: Conducted Mechanical stirring: None Culture period: Conducted with a target of about 10 days Culture temperature: 25°C to 28°C It is possible to culture under conditions satisfying at least one of the above.

[0032] (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. However, it is not particularly limited as long as it is a method capable of removing or substantially removing microalgae and appropriately recovering the culture supernatant. In an exemplary embodiment, the centrifugation performed to obtain the culture supernatant may be carried out 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, 1,500 to 10,000×g, etc. for 30 to 60 minutes. At this time, the centrifugation may be carried out stepwise by changing the speed or time. For example, after centrifuging at a low speed of 1,500 to 2,000×g to separate as a culture supernatant, it may be centrifuged at a high speed of 10,000 to 20,000×g to further remove cells or cell-related debris and residues. The supernatant thus obtained is recovered and subjected to the next step.

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

[0034] The cumulative 10% value (D10) of the particle size in extracellular vesicles derived from Porphyra yezoensis is not particularly limited as long as the effects of the present invention are achieved. For example, it may be 50 to 120 nm, 60 to 110 nm, 65 to 100 nm, etc. Further, the cumulative 50% value (D50) of the particle size may be, for example, 100 to 180 nm, 110 to 170 nm, 115 to 160 nm, etc. Further, the cumulative 90% value (D90) of the particle size may be, for example, 180 to 300 nm, 190 to 290 nm, 200 to 280 nm, etc. In this specification, the cumulative 10% value (D10) of the particle size in extracellular vesicles means the diameter at which 10% of the total extracellular vesicles are below this value. The cumulative 50% value (D50) of the particle size in extracellular vesicles means the diameter at which 50% of the total extracellular vesicles are below this value, and is synonymous with the median diameter. The cumulative 90% value (D90) of the particle size in extracellular vesicles means the diameter at which 90% of the total extracellular vesicles are below this value.

[0035] The cumulative 10% value (D10) of the particle size in extracellular vesicles derived from Spirulina is not particularly limited as long as the effects of the present invention are achieved. For example, it may be 50 to 110 nm, 60 to 100 nm, 70 to 90 nm, etc. Further, the cumulative 50% value (D50) of the particle size may be, for example, 100 to 160 nm, 110 to 150 nm, 120 to 140 nm, etc. Further, the cumulative 90% value (D90) of the particle size may be, for example, 190 to 250 nm, 200 to 240 nm, 210 to 230 nm, etc.

[0036] The cumulative 10% value (D10) of the particle size in extracellular vesicles derived from Chlorella is not particularly limited as long as the effects of the present invention are achieved. For example, it may be 90 to 150 nm, 100 to 140 nm, 110 to 130 nm, etc. Further, the cumulative 50% value (D50) of the particle size may be, for example, 180 to 240 nm, 190 to 230 nm, 200 to 220 nm, etc. Further, the cumulative 90% value (D90) of the particle size may be, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc.

[0037] The average particle number of extracellular vesicles of microalgae is, for example, if the purified product is in a liquid state, 1.0×10 3 or more, 1.0×10 4 or more, 1.0×10 5 or more, 1.0×10 6 or more, 1.0×10 7 or more, 1.0×10 8 or more, 1.0×10 9 or more, etc. are included, and 1.0×10 15 or less, 1.0×10 14 or less, 1.0×10 13 or less, 1.0×10 12 or less, 1.0×10 11 or less, etc. are included. The average particle number in the extracellular vesicles of microalgae is, for example, 1.0×10 4 ~1.0×10 15 particles, 1.0×10 4 ~1.0×10 14 particles, 1.0×10 4 ~1.0×10 13 particles, 1.0×10 4 ~1.0×10 12 particles, 1.0×10 4 ~1.0×10 11 particles, 1.0×10 5 ~1.0×10 15 particles, 1.0×10 5 ~1.0×10 14 particles, 1.0×10 5 ~1.0×10 13 particles, 1.0×10 5 ~1.0×10 12 particles, 1.0×10 5 ~1.0×10 11 particles, 1.0×10 6 ~1.0×10 15 particles, 1.0×10 6 ~1.0×10 14 particles, 1.0×10 6 ~1.0×10 13 particles, 1.0×10 6 ~1.0×10 12 particles, 1.0×10 6 ~1.0×10 11 particles, 1.0×10 7 ~1.0×1015 cells, 1.0×10 7 ~1.0×10 14 cells, 1.0×10 7 ~1.0×10 13 cells, 1.0×10 7 ~1.0×10 12 cells, 1.0×10 7 ~1.0×10 11 cells, 1.0×10 8 ~1.0×10 15 cells, 1.0×10 8 ~1.0×10 14 cells, 1.0×10 8 ~1.0×10 13 cells, 1.0×10 8 ~1.0×10 12 cells, 1.0×10 8 ~1.0×10 11 cells, 1.0×10 9 ~1.0×10 15 cells, 1.0×10 9 ~1.0×10 14 cells, 1.0×10 9 ~1.0×10 13 cells,, 1.0×10 9 ~1.0×10 12 cells, 1.0×10 9 ~1.0×10 11 cells, etc. may be mentioned. Regarding the number of extracellular vesicles, NanoSight (Malvern Panalytical, NanoSight L10) etc. based on nanoparticle tracking analysis (Nanoparticle Tracking Analysis: NTA) which is based on the analysis of both light scattering and Brownian motion can be used.

[0038] (Method for obtaining extracellular vesicles) As a method for obtaining the extracellular vesicles of the present invention, a method of preparing from the culture supernatant of algae can be mentioned.

[0039] In one embodiment, the extracellular vesicles of the present invention are obtained by heating the culture supernatant of microalgae.

[0040] The extracellular vesicles of the present invention can also be obtained by heating the culture supernatant of microalgae under temperature conditions of, but not limited to, for example, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, or 100°C or higher, and 200°C or lower, 150°C or lower, 120°C or lower, for a time condition of 30 seconds or longer, 1 minute or longer, 2 minutes or longer, 3 minutes or longer, or 4 minutes or longer, and 30 minutes or shorter, 20 minutes or shorter, 10 minutes or shorter.

[0041] Alternatively, in another embodiment, the extracellular vesicles of the present invention can also be obtained by treating the culture supernatant of microalgae by filter filtration.

[0042] The membrane pore size of the filter used for filter 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.

[0043] In yet another embodiment, as a method for obtaining the extracellular vesicles of the present invention, a purification step may also be included. Specific embodiments of the purification step include purification by ultrafiltration (for example, tangential flow filtration, etc.), ultracentrifugation, and affinity purification using an antibody. Among these, purification by ultrafiltration is preferred, but not limited to this.

[0044] When using purification by ultrafiltration, it can be a mode of filtration from the inside to the outside of the yarn using a hollow fiber membrane, a mode of using a spiral membrane including a filtration membrane and a support membrane, a mode of using a tubular membrane on a hollow cylinder, or a mode of using a flat membrane. Among these, it is preferable to include a step of performing filtration using a hollow fiber membrane and obtaining a liquid containing extracellular vesicles inside the hollow fiber membrane in a concentrated form, but not limited to this.

[0045] As the cut-off molecular weight, it is preferable to use a cut-off membrane with a molecular weight cut-off of 10 kDa to 1,000 kDa, 50 kDa to 1,000 kDa, or 100 kDa to 1,000 kDa, and more preferably a cut-off membrane with a molecular weight cut-off of 100 kDa to 500 kDa. A cut-off membrane with a molecular weight cut-off of 10 kDa or more, 50 kDa or more, 100 kDa or more, or 150 kDa or more is preferable, and a cut-off membrane with a molecular weight cut-off of 2,000 kDa or less, 1,500 kDa or less, 1,000 kDa or less, 750 kDa or less, 500 kDa or less, etc. is also preferable.

[0046] The extracellular vesicles of the present invention can be 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 filter filtration treatment, or a combination of a heat treatment step, a purification step, and a filter filtration treatment, etc.

[0047] The extracellular vesicles can be in the form of a purified solution of the culture supernatant, but can also be a solid or semi-solid obtained by drying the purified solution.

[0048] The extracellular vesicles can optionally be further purified to obtain a highly pure population of extracellular vesicles.

[0049] The extracellular vesicles can be in the form of the purified solution of the culture supernatant itself, but can also be a solid or semi-solid obtained by drying the purified solution.

[0050] (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.

[0051] In such a composition, the average number of particles of extracellular vesicles of microalgae is, for example, 1.0×10 3 or more, 1.0×10 4 or more, 1.0×10 5 or more, 1.0×10 6 or more, etc., and 1.0×10 16 or less, 1.0×10 15or less, 1.0×10 14 or less, 1.0×10 13 or less, 1.0×10 12 or less, 1.0×10 11 or less, 1.0×10 10 or less, 1.0×10 9 or less, 1.0×10 8 or less, 1.0×10 7 or less, 1×10 6 or less, etc. The average particle number of extracellular vesicles of microalgae is, for example, 1.0×10 3 ~1.0×10 14 or less, 1.0×10 3 ~1.0×10 13 or less, 1.0×10 3 ~1.0×10 12 or less, 1.0×10 3 ~1.0×10 11 or less, 1.0×10 3 ~1.0×10 10 or less, 1.0×10 3 ~1.0×10 9 or less, 1.0×10 3 ~1.0×10 8 or less, 1.0×10 3 ~1.0×10 7 or less, 1.0×10 4 ~1.0×10 14 or less, 1.0×10 4 ~1.0×10 13 or less, 1.0×10 4 ~1.0×10 12 or less, 1.0×10 4 ~1.0×10 11 or less, 1.0×10 4 ~1.0×10 10 or less, 1.0×10 4 ~1.0×10 9 or less, 1.0×10 4 ~1.0×10 8 or less, 1.0×10 4 ~1.0×10 7 or less, 1.0×10 5 ~1.0×10 14 or less, 1.0×10 5 ~1.0×10 13 or less, 1.0×10 5~1.0×10 12 pieces, 1.0×10 5 ~1.0×10 11 pieces, 1.0×10 5 ~1.0×10 10 pieces, 1.0×10 5 ~1.0×10 9 pieces, 1.0×10 5 ~1.0×10 8 pieces, 1.0×10 5 ~1.0×10 7 pieces, 1.0×10 6 ~1.0×10 14 pieces, 1.0×10 6 ~1.0×10 13 pieces, 1.0×10 6 ~1.0×10 12 pieces, 1.0×10 6 ~1.0×10 11 pieces, 1.0×10 6 ~1.0×10 10 pieces, 1.0×10 6 ~1.0×10 9 pieces, 1.0×10 6 ~1.0×10 8 pieces, 1.0×10 6 ~1.0×10 7 pieces, 1.0×10 7 ~1.0×10 14 pieces, 1.0×10 7 ~1.0×10 13 pieces, 1.0×10 7 ~1.0×10 12 pieces, 1.0×10 7 ~1.0×10 11 pieces, 1.0×10 7 ~1.0×10 10 pieces, 1.0×10 8 ~1.0×10 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 ~1.0×1014 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 pieces, etc. can be mentioned.

[0052] The average particle diameter of extracellular vesicles of microalgae is not particularly limited as long as the effects of the present invention are achieved. For example, 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, etc. can be mentioned, 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, etc. can be mentioned. Further, the average particle diameter of extracellular vesicles of microalgae is, for example, 10~700 nm, 10~600 nm, 10~500 nm, 10~400 nm, 10~300 nm, 10~250 nm, 30~700 nm, 30~600 nm, 30~500 nm, 30~400 nm, 30~300 nm, 30~250 nm, 50~700 nm, 50~600 nm, 50~500 nm, 50~400 nm, 50~300 nm, 50~250 nm, 70~700 nm, 70~600 nm, 70~500 nm, 70~400 nm, 70~300 nm, 70~250 nm, 100~700 nm, 100~600 nm, 100~500 nm, 100~400 nm, 100~300 nm, 100~250 nm, etc. can be mentioned.

[0053] The content of extracellular vesicles of microalgae in the composition can be appropriately adjusted according to the types and amounts of other components, dosage forms, etc., and is not limited. For example, in the state of the purified solution, it can be 0.001% by mass or more based on the total amount of the composition, and can also be 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. Further, the content of extracellular vesicles of microalgae can be, for example, 50% by mass or less based on the total amount of the composition, and examples include 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, etc. Also, the content of extracellular vesicles of microalgae is, for example, 0.001 to 50% by mass, 0.001 to 40% by mass, 0.001 to 30% by mass, 0.001 to 20% by mass, 0.001 to 15% by mass, 0.01 to 50% by mass, 0.01 to 40% by mass, 0.01 to 30% by mass, 0.01 to 20% by mass, 0.01 to 15% by mass, 0.1 to 50% by mass, 0.1 to 40% by mass, 0.1 to 30% by mass, 0.1 to 20% by mass, 0.1 to 15% by mass, 1 to 50% by mass, 1 to 40% by mass, 1 to 30% by mass, 1 to 20% by mass, 1 to 15% by mass, etc.

[0054] The content of extracellular vesicles of microalgae can be appropriately adjusted according to the types and amounts of other components, dosage forms, etc., and is not limited. For example, as the protein amount, it can be 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, 1 μg / mL or more based on the total amount of the composition. Also, examples include 100 μg / mL or less, 10 μg / mL or less, 1 μg / mL, 0.1 μg / mL or less, 0.01 μg / mL or less, etc.

[0055] ((B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols) The component (B-1) of the present invention is at least one selected from the group consisting of primary alcohols and polyhydric alcohols.

[0056] The primary alcohols that can be used in the present invention are not limited as long as they are used in the fields of pharmaceuticals, cosmetics, and optionally food. From the viewpoint of significantly exhibiting the effects of the present invention, the primary alcohol preferably has 1 to 3 carbon atoms. Examples of such alcohols include methanol, ethanol, n-propanol, isopropanol, etc. Further, the primary alcohol used in the present invention preferably contains at least ethanol, and more preferably is ethanol. In the present invention, the primary alcohol can be used alone or in combination of two or more.

[0057] The polyhydric alcohols that can be used in the present invention are not limited as long as they are used in the fields of pharmaceuticals, cosmetics, and optionally food. In this specification, the term "polyhydric alcohol" refers to an alcohol containing two or more hydroxy (-OH) groups in one molecule. Although not limited, from the viewpoint of achieving the effects of the invention, dihydric to hexahydric alcohols are preferably used as the polyhydric alcohol, more preferably dihydric to pentahydric alcohols, and even more preferably dihydric to trihydric alcohols. Here, for example, dihydric alcohols include dipropylene glycol, 1,3-butylene glycol, 1,3-propanediol, 3-methyl-1,3-butanediol, propylene glycol, pentanediol, hexanediol, octanediol, or polyethylene glycol, etc.; trihydric alcohols include glycerin, etc.; tetrahydric alcohols include pentaerythritol, etc.; pentahydric alcohols include diglycerin, xylitol, etc.; hexahydric alcohols include sorbitol, inositol, etc. The polyhydric alcohol can be used alone or in combination of two or more. As the polyhydric alcohol, one or more selected from the group consisting of glycerin, dipropylene glycol, 1,3-butylene glycol, 1,3-propanediol, pentanediol, and polyethylene glycol are preferred, and glycerin, 1,3-butylene glycol, 1,3-propanediol, and polyethylene glycol are particularly preferred.

[0058] Examples of polyethylene glycols include Macrogol 200 (liquid), Macrogol 300 (liquid), Macrogol 1540 (paste), Macrogol 400 (liquid), Macrogol 1500 (paste), Macrogol 4000 (solid) of the pharmaceutical additive standard, polyethylene glycol 200 (liquid), polyethylene glycol 300 (liquid), polyethylene glycol 400 (liquid), polyethylene glycol 600 (liquid), polyethylene glycol 1000 (wax), polyethylene glycol 1500 (paste), polyethylene glycol 1540 (wax), polyethylene glycol 2000 (wax), polyethylene glycol 4000 (solid) of the raw material standard for quasi-drugs in 2006, etc. As described above, the properties of polyethylene glycol vary depending on the molecular weight. In the present invention, it is preferable to use liquid, paste, or wax-like polyethylene glycol, more preferably liquid or paste, and even more preferably liquid.

[0059] The total content of the component (B-1) with respect to the total amount of the composition of the present invention is preferably 1 to 60% by mass, more preferably 3 to 50% by mass, and even more preferably about 5 to 40% by mass.

[0060] The content of the primary alcohol with respect to the total amount of the composition of the present invention is preferably 0.01 to 30% by mass, more preferably 1 to 25% by mass, and even more preferably about 3 to 20% by mass.

[0061] The total content of the polyhydric alcohol with respect to the total amount of the composition of the present invention is preferably 0.5 to 50% by mass, more preferably 2 to 40% by mass, and even more preferably about 3 to 30% by mass.

[0062] In the composition of the present invention, the number of the primary alcohol of the component (A) per 1% by mass is preferably 1×10 to 1×10 10 pieces, and 1×10 2 to 1×10 9Individuals are more preferred, 1×10 3 ~1×10 8 Individuals are even more preferred, 1×10 4 ~1×10 7 Individuals are even more preferably.

[0063] In the composition of the present invention, the number of (A) component polyhydric alcohols per 1% by mass is preferably 1×10 to 1×10 10 Individuals are preferred, 1×10 2 ~1×10 9 Individuals are more preferred, 1×10 3 ~1×10 8 Individuals are even more preferred, 1×10 4 ~1×10 7 Individuals are even more preferably.

[0064] ((B-2) One or more selected from the group consisting of organic acids and their salts) Component (B-2) of the present invention is an organic acid or a salt thereof. In the present invention, the organic acid is an acid of an organic compound having an organic group, with a molecular weight of 1000 or less, preferably 700 or less, more preferably 500 or less, still more preferably 350 or less, and particularly preferably 300 or less. The organic acid is preferably a water-soluble organic acid. The organic acid may be a carboxylic acid having a carboxyl group, a sulfonic acid having a sulfo group, or a phosphate ester having a phosphate group. More specifically, but not limited thereto, examples of the organic acid include ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, tartaric acid, malic acid, succinic acid, oxalic acid, gluconic acid, fumaric acid, aspartic acid, pyrrolidonecarboxylic acid, ε-aminocaproic acid, glutamic acid, aminoethylsulfonic acid, ellagic acid, kojic acid, glycyrrhizic acid, glycyrrhetinic acid, phytic acid, ferulic acid, glycolic acid, azelaic acid, etc. Among them, at least one selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconic acid, aspartic acid, pyrrolidonecarboxylic acid, ε-aminocaproic acid, glutamic acid, aminoethylsulfonic acid, ellagic acid, kojic acid, glycyrrhizic acid, glycyrrhetinic acid, phytic acid, ferulic acid, glycolic acid, azelaic acid is exemplified. Among these, at least one selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconic acid, pyrrolidonecarboxylic acid, ε-aminocaproic acid, glutamic acid, kojic acid, glycyrrhizic acid, glycyrrhetinic acid, phytic acid, ferulic acid, glycolic acid, azelaic acid is preferred, and at least one selected from the group consisting of ascorbic acid, tranexamic acid, citric acid, salicylic acid, lactic acid, succinic acid, tartaric acid, gluconic acid, kojic acid, phytic acid, glycolic acid, glycyrrhizic acid is more preferred, and at least one selected from citric acid and succinic acid is even more preferred.

[0065] In the present invention, the organic acid salt refers to a salt of such an organic acid. The salt forming the organic acid salt is a pharmaceutically acceptable salt. Although not limited, for example, salts with organic bases (e.g., salts with tertiary amines such as trimethylamine salt, triethylamine salt, monoethanolamine salt, triethanolamine salt, pyridine salt; basic ammonium salts such as arginine or lysine salt, etc.), or salts with inorganic bases (e.g., inorganic acid salts such as hydrochloride, sulfate, phosphate; ammonium salt; alkali metal salts such as sodium salt, potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt; or zinc salt, aluminum salt, etc.) and the like can be mentioned. Among them, preferred organic acid salts are triethanolamine salt, monoammonium salt, sodium salt, potassium salt, dipotassium salt, magnesium salt, or zinc salt, and particularly preferred organic acid salts are sodium salt, dipotassium salt, or magnesium salt.

[0066] In the present invention, preferred organic acid salts include sodium ascorbate, tranexamic acid hydrochloride, calcium tranexamate, sodium salicylate, calcium salicylate, magnesium salicylate, potassium salicylate, sodium lactate, sodium tartrate, sodium citrate, disodium citrate, trisodium citrate, potassium citrate, sodium succinate, disodium succinate, sodium oxalate, calcium gluconate, zinc gluconate, sodium pyrrolidonecarboxylate, zinc pyrrolidonecarboxylate, sodium glutamate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, sodium phytate, sodium glycolate, ammonium glycolate, etc. Among them, at least one selected from the group consisting of sodium ascorbate, tranexamic acid hydrochloride, calcium tranexamate, sodium salicylate, sodium lactate, sodium tartrate, sodium citrate, disodium citrate, trisodium citrate, sodium succinate, disodium succinate, calcium gluconate, zinc gluconate, zinc pyrrolidonecarboxylate, sodium pyrrolidonecarboxylate, sodium glutamate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, sodium phytate, sodium glycolate, ammonium glycolate, and sodium succinate, disodium succinate is preferred, and at least one selected from the group consisting of sodium ascorbate, sodium salicylate, sodium lactate, sodium citrate, disodium citrate, trisodium citrate, zinc gluconate, sodium pyrrolidonecarboxylate, sodium glutamate, sodium succinate, and disodium succinate is more preferred.

[0067] The total content of the organic acid or its salt relative to the total amount of the composition of the present invention is preferably 0.0001 to 30% by mass, more preferably 0.001 to 10% by mass, and even more preferably about 0.01 to 5% by mass.

[0068] In the composition of the present invention, the number of the organic acid or its salt of component (A) per 1% by mass is 1×10 to 1×10 16is preferably from 1×10 2 to 1×10 10 more preferably from 1×10 3 to 1×10 8 even more preferably from 1×10 4 to 1×10 7 and even more preferably so.

[0069] ((B-3) Chelating agent) The (B-3) component of the present invention is a chelating agent. Examples of the chelating agent used in the present invention include ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetate (sodium salt (sodium edetate: Japanese Pharmacopoeia, EDTA-2Na, etc.), potassium salt, etc.), phytic acid, gluconic acid, polyphosphoric acid, metaphosphoric acid, and the like. Among them, disodium edetate is preferred.

[0070] The total content of the chelating agent relative to the total amount of the composition of the present invention is preferably from 0.0001 to 2% by mass, more preferably from 0.001 to 1% by mass, and even more preferably about from 0.01 to 0.5% by mass.

[0071] In the composition of the present invention, the number of the chelating agent in the (A) component per 1% by mass is preferably from 1×10 to 1×10 16 more preferably from 1×10 2 to 1×10 10 even more preferably from 1×10 3 to 1×10 8 and even more preferably from 1×10 4 to 1×10 7 and even more preferably so.

[0072] ((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, 90% by mass or more, and 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, 40% by mass or less.

[0073] In the composition of the present invention, the number of the (A) component per 1% by mass of water is preferably 1×10 to 1×10 16 pieces, more preferably 1×10 2 to 1×10 14 pieces, still more preferably 1×10 3 to 1×10 12 pieces, and even more preferably 1×10 4 to 1×10 11 pieces. Further, in certain embodiments, the number of the (A) component per 1% by mass of water is preferably 1×10 to 1×10 10 pieces, more preferably 1×10 2 to 1×10 9 pieces, still more preferably 1×10 3 to 1×10 8 pieces, and even more preferably 1×10 4 to 1×10 7 pieces.

[0074] In the composition of the present invention, the pH may be 2.5 or more, 3.5 or more, 4.0 or more, 5.0 or more, 6.0 or more, or may be 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less. The pH of the composition may be pH 2.5 to 12, pH 3.5 to 10, or pH 5 to 8.

[0075] (Nonionic surfactant) The composition of the present invention may contain a nonionic surfactant in addition to the above (A) component and (B) component, 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 having an HLB of less than 15, more preferably a surfactant having an HLB of 14.5 or less, and still more preferably a surfactant having 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.

[0076] Note that the hydrophilic-lipophilic balance (HLB) value in the present invention refers to the value calculated by Kawakami's formula (the following formula (1)) using the molecular weight of the hydrophilic group (Mw) and the molecular weight of the lipophilic group (MO) of the surfactant molecule. HLB value = 7 + 11.7 log Mw / Mo (Formula 1)

[0077] 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) glycerol monostearate (denoted as "POE(5)". The same applies hereinafter), polyoxyethylene (15) glycerol monostearate (HLB value 13.5), polyoxyethylene (5) glycerol monooleate (HLB value 9.5), and polyoxyethylene (15) glycerol 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), POE(20) sorbitan trioleate (HLB value 11.0); Polyoxyethylene sorbit fatty acid esters such as POE(60) sorbitol tetrastearate (HLB value 13.0), POE(30) sorbitol tetraoleate (HLB value 11.5), POE(40) sorbitol tetraoleate (HLB value 12.5), POE(60) sorbitol tetraoleate (HLB value 14.0); Polyoxyethylene lanolin, lanolin alcohol, 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), POE(20) sorbit wax (HLB value 9.5); Polyoxyethylene castor oil and hydrogenated castor oil 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), POE(60) hydrogenated castor oil (HLB value 14.0); Polyoxyethylene sterol and hydrogenated sterol such as POE(5) phytosterol (HLB value 9.5), POE(10) phytosterol (HLB value 12.5), 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; Polyoxypropylene 1 mole 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), POE(30)POP(6) decyltetradecyl ether (HLB value 12.0) and other polyoxyethylene polyoxypropylene alkyl ethers. Polyethylene glycol 10 mole addition of monolauric acid (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), PEG diisostearate (HLB value 9.5) and other polyethylene glycol fatty acid esters; Polyoxyethylene glyceryl isostearates such as polyoxyethylene (8) glyceryl isostearate (HLB value 10.0), polyoxyethylene (10) glyceryl isostearate (HLB value 10.0), polyoxyethylene (15) glyceryl isostearate (HLB value 12.0), polyoxyethylene (20) glyceryl isostearate (HLB value 13.0), polyoxyethylene (25) glyceryl isostearate (HLB value 14.0), polyoxyethylene (30) glyceryl isostearate; are exemplified.

[0078] 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% by mass, more preferably 0.1 to 4% by mass, and even more preferably about 0.5 to 3% by mass.

[0079] (Additives) The composition of the present invention can also contain various additives other than those described above as long as the effects of the present invention are not impaired. Examples of additives include antioxidants, thickeners, pH adjusters, inorganic salts, stabilizers, ultraviolet absorbers or ultraviolet scattering agents, irritation reducers, coloring agents, fragrances, and the like. One or more additives can be used.

[0080] Here, as the inorganic salt, sodium chloride, disodium hydrogen phosphate, potassium chloride, or potassium dihydrogen phosphate is preferable.

[0081] [Uses] In one embodiment, the composition containing extracellular vesicles of the present invention is preferably used as an oral or external preparation.

[0082] The composition of the present invention can be added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feeds, or pet foods and used. Alternatively, it can be used as it is as a pharmaceutical, quasi-drug, cosmetic, food, beverage, feed, or pet food, etc. Furthermore, it can also be a raw material or a so-called premix product for preparing such products.

[0083] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the composition of the present invention is preferably a skin care cosmetic (e.g., lotion, cream, facial cream, facial lotion, emulsion, pack, liquid facial wash, soap, beauty essence), a makeup cosmetic (e.g., eyeliner, eyebrow pencil), a scalp and hair cosmetic (e.g., scalp lotion, scalp cream, shampoo, hair conditioner, hair treatment, hair essence, hair mist, hair tonic), more preferably a lotion, cream, facial cream, facial lotion, emulsion, pack, facial wash, soap, beauty essence, and particularly preferably a lotion, facial lotion, beauty essence, but is not limited thereto.

[0084] Thus, 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 aerosol preparations. Among them, solutions, suspensions, emulsions, lotions, and aerosol preparations are preferred, and solutions, lotions, and aerosol preparations are more preferred. Further, the premix product can be, for example, a liquid obtained by mixing extracellular vesicles, water, glycols, preservatives, excipients, solubilizers, pH adjusters, and the like.

[0085] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily dosage of extracellular vesicles of microalgae for an adult can be appropriately determined according to the individual's condition, body weight, gender, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, 200 μg or more, etc. Also, the daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, 100 μg or less, etc. Also, the daily dosage of extracellular vesicles of microalgae for an adult can be, for example, 1 - 1000 μg, 1 - 900 μg, 1 - 800 μg, 1 - 700 μg, 1 - 600 μg, 1 - 500 μg, 1 - 400 μg, 1 - 300 μg, 1 - 200 μg, 1 - 100 μg, 5 - 1000 μg, 5 - 900 μg, 5 - 800 μg, 5 - 700 μg, 5 - 600 μg, 5 - 500 μg, 5 - 400 μg, 5 - 300 μg, 5 - 200 μg, 5 - 100 μg, 10 - 1000 μg, 10 - 900 μg, 10 - 800 μg, 10 - 700 μg, 10 - 600 μg, 10 - 500 μg, 10 - 400 μg, 10 - 300 μg, 10 - 200 μg, 10 - 100 μg, 20 - 1000 μg, 20 - 900 μg, 20 - 800 μg, 20 - 700 μg, 20 - 600 μg, 20 - 500 μg, 20 - 400 μg, 20 - 300 μg, 20 - 200 μg, 20 - 100 μg, etc. Also, although the dosage of extracellular vesicles of microalgae varies depending on various factors such as the condition and age of the subject, when based on an adult, generally, the composition is 1 μg / kg - 200 mg / kg, and on the other hand, 50 μg / kg - 50 mg / kg can be administered in 1 - 3 divided doses per day, and the said dosage does not limit the scope of this specification by any method.

[0086] When used in so-called external preparations such as pharmaceuticals, quasi-drugs, and cosmetics, the daily dosage of extracellular vesicles of microalgae for adults can be appropriately determined according to the individual's condition, body weight, gender, age, activity of the material, intake or administration route, intake or administration schedule, dosage form, or other factors. The daily dosage of extracellular vesicles of microalgae for adults, as the amount of protein, can be, for example, 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, 200 μg or more, etc. Also, the daily dosage of extracellular vesicles of microalgae 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, 100 μg or less, etc. Also, the daily dosage of extracellular vesicles of microalgae for adults can be, for example, 1 - 1000 μg, 1 - 900 μg, 1 - 800 μg, 1 - 700 μg, 1 - 600 μg, 1 - 500 μg, 1 - 400 μg, 1 - 300 μg, 1 - 200 μg, 1 - 100 μg, 5 - 1000 μg, 5 - 900 μg, 5 - 800 μg, 5 - 700 μg, 5 - 600 μg, 5 - 500 μg, 5 - 400 μg, 5 - 300 μg, 5 - 200 μg, 5 - 100 μg, 10 - 1000 μg, 10 - 900 μg, 10 - 800 μg, 10 - 700 μg, 10 - 600 μg, 10 - 500 μg, 10 - 400 μg, 10 - 300 μg, 10 - 200 μg, 10 - 100 μg, 20 - 1000 μg, 20 - 900 μg, 20 - 800 μg, 20 - 700 μg, 20 - 600 μg, 20 - 500 μg, 20 - 400 μg, 20 - 300 μg, 20 - 200 μg, 20 - 100 μg, etc. Also, the dosage of extracellular vesicles of microalgae varies depending on various factors such as the condition and age of the subject, but when based on adults, the composition can be administered at 1 μg / kg - 200 mg / kg, and in another aspect, 50 μg / kg - 50 mg / kg can be divided into 1 - 3 times a day for administration. The above dosage does not limit the scope of this specification by any method.

[0087] In another embodiment, the daily dosage of extracellular vesicles of microalgae, in terms of the number, is, for example, 1×10 or more, 1×10 2 or more, 1×10 3 or more, 1×10 4 or more, etc. In addition, the daily dosage of extracellular vesicles of microalgae, in terms of the number, is, for example, 1×10 to 1×10 10 , 1×10 to 1×10 9 , 1×10 to 1×10 8 , 1×10 to 1×10 7 , 1×10 2 to 1×10 10 , 1×10 2 to 1×10 9 , 1×10 2 to 1×10 8 , 1×10 2 to 1×10 7 , 1×10 3 to 1×10 10 , 1×10 3 to 1×10 9 , 1×10 3 to 1×10 8 , 1×10 3 to 1×10 7 , 1×10 4 to 1×10 10 , 1×10 4 to 1×10 9 , 1×10 4 to 1×10 8 , 1×10 4 to 1×10 7 or the like.

[0088] The composition of the present invention can be taken or administered once to several times a day, usually 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 2 times a day is preferred.

[0089] When the composition of the present invention is added to or mixed with food, beverages, feed, or pet food, the composition of the present invention can also be used as food and beverages, namely, health food, foods with functional claims, foods for patients, and foods for specified health use. It can also be used as so-called doctor's supplements recommended or presented by doctors in internal medicine, orthopedics, veterinary hospitals, etc. in hospitals and / or clinics.

[0090] Health food, foods with functional claims, foods for patients, and foods for specified health use can specifically be used in various dosage forms such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, lozenges, etc.), liquid preparations (syrups, suspensions), enteral nutrition, etc. The food in dosage form can be manufactured in the same manner as known pharmaceutical preparations. After mixing an active ingredient and a carrier acceptable as food, such as a suitable excipient, etc., it can be manufactured using conventional means. Although not limited to the dosage form, from the viewpoint of significantly exhibiting the effects of the present invention, it is preferably an orally disintegrating tablet, chewable tablet, lozenge, granule, powder, or liquid preparation.

[0091] In addition, the composition of the present invention can be manufactured as liquid beverages such as soups, juices, fruit juice beverages, milk, milk beverages, whey beverages, lactic acid bacteria beverages, tea beverages, alcoholic beverages, coffee beverages, carbonated beverages, soft drinks, water beverages, cocoa beverages, jelly-like beverages, sports beverages, diet beverages, etc., semi-solid foods such as puddings and yogurts, noodles such as pasta, ramen, udon, and soba, confectioneries, spreads, etc.

[0092] The composition of the present invention can be applied in forms such as for oral use and for internal use. When used as a pharmaceutical composition, it may be used therapeutically or non-therapeutically.

[0093] The oral intake or dosage of extracellular vesicles of microalgae per day for an adult can be appropriately determined according to the individual's condition, body weight, gender, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The oral intake or dosage of extracellular vesicles of microalgae per day for an adult can be, for example, 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, 20 μg or more, 30 μg or more, 40 μg or more, 50 μg or more, 70 μg or more, 100 μg or more, 150 μg or more, 200 μg or more, etc. Also, the oral intake or dosage of extracellular vesicles of microalgae per day for an adult can be, for example, 5 mg or less, 3 mg or less, 1 mg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, 500 μg or less, 400 μg or less, 300 μg or less, 200 μg or less, 100 μg or less, etc. Further, the oral intake or dosage of extracellular vesicles of microalgae per day for an adult can be, for example, 1 - 1000 μg, 1 - 900 μg, 1 - 800 μg, 1 - 700 μg, 1 - 600 μg, 1 - 500 μg, 1 - 400 μg, 1 - 300 μg, 1 - 200 μg, 1 - 100 μg, 5 - 1000 μg, 5 - 900 μg, 5 - 800 μg, 5 - 700 μg, 5 - 600 μg, 5 - 500 μg, 5 - 400 μg, 5 - 300 μg, 5 - 200 μg, 5 - 100 μg, 10 - 1000 μg, 10 - 900 μg, 10 - 800 μg, 10 - 700 μg, 10 - 600 μg, 10 - 500 μg, 10 - 400 μg, 10 - 300 μg, 10 - 200 μg, 10 - 100 μg, 20 - 1000 μg, 20 - 900 μg, 20 - 800 μg, 20 - 700 μg, 20 - 600 μg, 20 - 500 μg, 20 - 400 μg, 20 - 300 μg, 20 - 200 μg, 20 - 100 μg, etc.

[0094] Also, in another embodiment, the oral intake or dosage of extracellular vesicles of microalgae per day for an adult, in terms of number conversion, in terms of number conversion, for example, is 1×10 or more, 1×10 2 or more, 1×10 3 or more, 1×10 4 or more, etc. In addition, the oral intake or dosage of extracellular vesicles of microalgae per day for an adult, in terms of the number of particles, is, for example, 1×10 9 or less, 1×10 8 or less, 1×10 7 or less, 1×10 6 or less, 1×10 5 or less, etc. In addition, the oral intake or dosage of extracellular vesicles of microalgae per day for an adult, in terms of the number of particles, is, for example, 1×10 to 1×10 9 particles, 1×10 to 1×10 8 particles, 1×10 to 1×10 7 particles, 1×10 to 1×10 6 particles, 1×10 2 to 1×10 9 particles, 1×10 2 to 1×10 8 particles, 1×10 2 to 1×10 7 particles, 1×10 2 to 1×10 6 particles, 1×10 3 to 1×10 9 particles, 1×10 3 to 1×10 8 particles, 1×10 3 to 1×10 7 particles, 1×10 3 to 1×10 6 particles, 1×10 4 to 1×10 9 particles, 1×10 4 to 1×10 8 particles, 1×10 4 to 1×10 7 particles, 1×10 4 to 1×10 6 particles, etc.

[0095] Note that the oral intake or dosage per day for an adult may be divided and taken, for example, as 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules, depending on the dosage form.

[0096] The composition of the present invention can be taken or administered once to several times a day, usually once to six times a day, once to three times a day, once to two times a day, or at any period and interval, but once a day is preferred.

[0097] When the present invention is used as feed, pet food, etc., the target organism is not particularly limited, but is preferably a mammal, reptile, amphibian, bird, or fish, and more preferably a mammal excluding humans.

[0098] When the present invention is used as feed, pet food, etc., it may be given several times a day by adding it to the staple food, etc., or it may be given at any time as a snack.

[0099] [Method for improving stability] The present invention relates to a method for improving the stability of extracellular vesicles derived from microalgae by co-existing one or more selected from the group consisting of (B-1) to (B-3) in (A) extracellular vesicles derived from microalgae. (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) Chelating agents. The present invention also relates to a composition with improved stability containing (A) extracellular vesicles derived from microalgae. The present invention also relates to a composition with improved stability of the extracellular vesicles derived from microalgae, containing (A) extracellular vesicles derived from microalgae and one or more selected from the group consisting of (B-1) to (B-3). (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) Chelating agents. Here, the improvement in stability means, for example, that the variation in the number of particles and / or size of the extracellular vesicles is small before and after storing the composition containing the extracellular vesicles. Extracellular vesicles are expected to have various bioactive functions, and maintaining their quantity and physical properties leads to the maintenance of the properties of the composition containing them.

[0100] The present invention includes the following aspects. [1] (A) A composition containing extracellular vesicles derived from microalgae. [2] (A) Extracellular vesicles derived from microalgae; and (B-1) A composition containing one or more selected from the group consisting of (B-1) to (B-3): (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) A chelating agent. [3] (A) A composition (excluding the anti-aging composition) containing extracellular vesicles derived from microalgae. [4] Furthermore, the composition according to any one of [1] to [3], further containing water. [5] The composition according to any one of [1] to [4], wherein the microalgae belong to microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella. [6] The composition according to any one of [1] to [5], which is for oral or external use. [7] (A) A method for improving the stability of extracellular vesicles derived from microalgae by coexisting one or more selected from the group consisting of (B-1) to (B-3) with the extracellular vesicles derived from microalgae: (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) A chelating agent.

Example

[0101] Next, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

[0102] (Preparation Example) Using a commercially available medium for algae, Pavlova sp., Arthrospira platensis, or Chlorella vulgaris was cultured by a conventional method to obtain a culture supernatant. Each culture supernatant was heat-treated at 105 °C for 4 minutes. After heating, 3000 mL of the supernatant obtained by centrifugation at 3000 × g for 60 minutes at room temperature was used for 0.2 μm filter filtration, and then concentrated to a volume of 150 mL or less by the tangential flow method using a 300 kDa hollow fiber membrane filter (MiniKros sampler S02-E300-05-N: manufactured by Repligen). Then, concentration washing was performed using 10000 mL of PBS, and finally 100 mL was recovered with PBS. Thereafter, each concentrated sample was filter-sterilized using a 0.22 μm filter, and the number of particles and the like were measured using NanoSight.

[0103] Each of the above samples was appropriately diluted with PBS(-), and using a nanoparticle analysis system NanoSight LM10 (manufactured by Malvern Panalytical), a number distribution graph (horizontal axis: particle diameter, vertical axis: particle number concentration) was created according to the attached software. The measurement of the particle diameter and particle size was performed 3 times, and the average value was taken as the average particle diameter and average particle size of each sample.

[0104] The following samples were obtained as described above. · Ultrafiltration sample of Pavlova culture supernatant (protein concentration 574 μg / mL) · Ultrafiltration sample of Arthrospira platensis culture supernatant (protein concentration 29.6 μg / mL) · Ultrafiltration sample of Chlorella vulgaris culture supernatant (protein concentration 10.4 μg / mL)

[0105] In the ultrafiltration sample of the Pavlova culture supernatant, extracellular vesicles derived from Pavlova had a particle number of 1.79×1012 per mL, with an average particle size of 146 nm, a cumulative 10% particle size value (D10) of 75 nm, a cumulative 50% particle size value (D50) of 135.3 nm, and a cumulative 90% particle size value (D90) of 229.3 nm.

[0106] In the ultrafiltration sample of the Spirulina culture supernatant, extracellular vesicles derived from Spirulina had a particle count of 2.88×10 11 per mL, with an average particle size of 138.7 nm, a cumulative 10% particle size value (D10) of 72.2 nm, a cumulative 50% particle size value (D50) of 125.9 nm, and a cumulative 90% particle size value (D90) of 217.8 nm.

[0107] In the ultrafiltration sample of the Chlorella culture supernatant, extracellular vesicles derived from Chlorella had a particle count of 8.18×10 9 per mL, with an average particle size of 227.7 nm, a cumulative 10% particle size value (D10) of 121.4 nm, a cumulative 50% particle size value (D50) of 215.8 nm, and a cumulative 90% particle size value (D90) of 348.5 nm.

[0108] (Examples and Comparative Examples) Compositions with the compositions shown in Tables 1 to 3 were prepared. Each raw material component was mixed to prepare the composition. The pH of the prepared composition was 3.8 - 6.9.

[0109] The main raw materials used in the examples are as follows. The sample prepared in the production example of the extracellular vesicle-containing solution derived from Pavlova was used as it was. Extracellular vesicle-containing solution derived from human stem cells (product name RemyStem-JP, manufacturer Anti-Aging Co., Ltd.) (7.28x10 8 particles / mL) Extracellular vesicle-containing solution derived from lactic acid bacteria (Extracellular Vesicles derived from Lactobacillus paracasei 180913-R1 strain, manufacturer Cosmo Bio Co., Ltd.) (3.9x10 10 particles / mL)

[0110] [Evaluation of Particle Size and Particle Number Stability] For each composition, the state immediately after preparation and after standing at 40 °C for one week was evaluated. That is, the number of extracellular vesicles and the particle size in the composition were measured. NanoSight was used to measure the particle size, and each composition was appropriately diluted with purified water for measurement. For the obtained number of particles and particle size, the particle number reduction rate and the particle size fluctuation rate were calculated using the following formulas. Particle number reduction rate = (Number of particles immediately after preparation - Number of particles after storage at 40 °C) / (Number of particles immediately after preparation) × 100 Particle size fluctuation rate = [(Particle size immediately after preparation - Particle size after storage at 40 °C) / (Particle size immediately after preparation) × 100] Note that [ ] represents the absolute value.

[0111] The evaluation results of the compositions of the examples and comparative examples are shown in Tables 1 to 3. Note that the numerical values in the tables represent mass %. The evaluation results are shown as follows.

[0112] <Particle number reduction rate> 20% or more × 14% or more and less than 20% △ 5% or more and less than 14% ○ Less than 5% ◎

[0113] <Particle size fluctuation rate> Absolute value 20% or more × Absolute value 10% or more and less than 20% △ Absolute value less than 10% ○ [Table 1] [Table 2] [Table 3]

[0114] As shown in the table, the extracellular vesicles derived from Pabroba showed little change in the number of particles and particle size before and after storage. Even when components serving as a base were added, the extracellular vesicles derived from Pabroba were stable. On the other hand, fluctuations in the number of particles and particle size were observed in the extracellular vesicles derived from humans and lactic acid bacteria before and after storage.

[0115] Furthermore, all of the compositions of the examples had a smooth texture, were excellent in moisturizing feeling, and had a good usability.

Claims

1. (A) Extracellular vesicles derived from microalgae; and (B-1) A composition containing one or more selected from the group consisting of (B-1) to (B-3): (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) Chelating agents. 】

2. (A) A composition (excluding the anti-aging composition) containing extracellular vesicles derived from microalgae. 】

3. The composition according to claim 1 or 2, further containing water. 】

4. The composition according to claim 1 or 2, wherein the microalgae belong to microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella. 】

5. The composition according to claim 1 or 2, which is for internal or external use. 】

6. (A) A method for improving the stability of extracellular vesicles derived from microalgae by co-existing one or more selected from the group consisting of (B-1) to (B-3) in the extracellular vesicles derived from microalgae: (B-1) One or more selected from the group consisting of primary alcohols and polyhydric alcohols, (B-2) One or more selected from the group consisting of organic acids and their salts, and (B-3) Chelating agents.

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