Extracellular vesicles or extracellular vesicle contents

Abstract

The present invention aims to obtain extracellular vesicles or extracellular vesicle-containing materials obtained from a culture medium of microalgae, which are highly stable, particularly after storage at high temperatures. [Solution] Extracellular vesicles or extracellular vesicle-containing materials obtained from a culture medium of microalgae are prepared, which contain glyceroglycolipids and / or betaine lipids. In one embodiment, the content of SQDG in the total amount of glyceroglycolipids is 30 to 80%, or the content of DGDG in the total amount of glyceroglycolipids is 2 to 20%. In another embodiment, the content of DGCC in the total amount of betaine lipids is 25 to 60%, or the content of MGTA in the total amount of betaine lipids is 8 to 30%.

Description

[Technical Field]

[0001] The present invention relates to extracellular vesicles or extracellular vesicle contents 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 has been disclosed that a population prepared by culturing under specific conditions stem cells that produce cell-derived vesicles can promote angiogenesis (Patent Document 1). [Prior art documents] [Patent documents]

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

[0005] There are many unknowns about extracellular vesicles, and in particular, there are insufficient reports on the composition and function of extracellular vesicles derived from microalgae. Furthermore, the stability and absorbability of extracellular vesicles generally vary depending on their membrane structure, and their lipid distribution can change depending on the manufacturing process and the properties of the starting materials.

[0006] The lipids that make up the membrane of extracellular vesicles contribute greatly to the stability of the extracellular vesicles themselves.

[0007] Therefore, an object of the present invention is to provide extracellular vesicles or extracellular vesicle-containing materials derived from microalgae that have a characteristic composition and are more stable. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors conducted extensive research and found that extracellular vesicles or extracellular vesicle-containing materials containing a certain proportion of glyceroglycolipids and betaine lipids are highly stable even when stored at high temperatures, leading to the completion of the present invention.

[0009] That is, the present invention provides the following extracellular vesicles or extracellular vesicle-containing materials.

[0010] [1] An extracellular vesicle or extracellular vesicle-containing material derived from microalgae, which contains glyceroglycolipids, and the content of SQDG in the total amount of the glyceroglycolipids is 30 to 80%. [2] An extracellular vesicle or extracellular vesicle-containing material derived from microalgae, which contains glyceroglycolipids, and the content of DGDG in the total amount of the glyceroglycolipids is 2 to 20%. [3] Extracellular vesicles or extracellular vesicle-containing material derived from microalgae, which contain betaine lipids and have a DGCC content of 25 to 60% of the total amount of the betaine lipids. [4] Extracellular vesicles or extracellular vesicle-containing material derived from microalgae, which contain betaine lipids, and the content of MGTA in the total amount of the betaine lipids is 8 to 30%. [5] Extracellular vesicles or a substance containing extracellular vesicles, which contain betaine lipids, and the content of DGTA in the total amount of the betaine lipids is 25 to 30%. [6] The extracellular vesicles or the material containing extracellular vesicles according to any one of [1] to [5], wherein the microalgae contain chloroplasts or thylakoids. [7] The extracellular vesicles or extracellular vesicle-containing material according to [6], wherein the microalgae are microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella. [8] Extracellular vesicles or extracellular vesicle-containing substances derived from microalgae, which contain glyceroglycolipids, and the content of SQDG in the total amount of SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG and their stereoisomers is 30 to 80%. [9] Extracellular vesicles or an extracellular vesicle-containing composition derived from microalgae, which contain glyceroglycolipids, and the content of DGDG in the total amount of SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG and their stereoisomers is 2 to 20%.

[10] Extracellular vesicles or extracellular vesicle-containing matter derived from microalgae, which contain betaine lipids and in which the content of DGCC in the total amount of DGTA, DGTS, DGCC, MGTA, MGCC and their stereoisomers is 25 to 60%.

[11] Extracellular vesicles or extracellular vesicle-containing matter containing betaine lipid, wherein the content of MGTA in the total amount of DGTA, DGTS, DGCC, MGTA, MGCC and their stereoisomers is 8 to 30%.

[12] Extracellular vesicles or extracellular vesicle-containing matter containing betaine lipid, wherein the content of DGTA in the total amount of DGTA, DGTS, DGCC, MGTA, MGCC and their stereoisomers is 25 to 30%. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide extracellular vesicles or extracellular vesicle-containing materials derived from microalgae that are highly stable when stored at high temperatures. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Extracellular vesicles or extracellular vesicle contents] The present invention relates to microalgae-derived extracellular vesicles or extracellular vesicle-containing materials containing glyceroglycolipids and / or betaine lipids. Here, the extracellular vesicle-containing materials may be the culture supernatant of microalgae itself, or may be a culture supernatant-derived material obtained by crude purification of the culture supernatant.

[0013] (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.

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

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

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] (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 culture medium include culture mediums supplemented with nutrient salts such as nitrogen sources, phosphorus sources, and minerals, such as Cramer-Myers medium and modified Cramer-Myers medium.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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:

[0035] (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. 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, followed by centrifugation 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.

[0036] (extracellular vesicles) 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 extracellular vesicle-containing material of the present invention does 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. The diameter or particle size can be measured using microscopy techniques, particularly techniques for measuring nanoparticle size using a transmission electron microscope. Furthermore, it can also be measured 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 (HORIBA). The measurements were taken using a Scientific ViewSizer 3000.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] (Extracellular vesicle content) As used herein, the term "extracellular vesicle-containing material" refers to a concentrate or purified product containing extracellular vesicles obtained from the culture supernatant of microalgae and obtained by filtering the culture supernatant. In addition to the extracellular vesicles, the material may also contain secreted products obtained by the culture.

[0042] 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 13 pieces, 1.0×10 4 ~1.0×10 12 pieces, 1.0×10 4 ~1.0×10 11 pieces, 1.0×10 5 ~1.0×10 15 pieces, 1.0×10 5 ~1.0×10 14 pieces, 1.0×10 5 ~1.0×10 13 pieces, 1.0×10 5 ~1.0×10 12 pieces, 1.0×10 5 ~1.0×10 11 pieces, 1.0×10 6 ~1.0×10 15 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 7 ~1.0×10 15 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 8 ~1.0×10 15 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 9 ~1.0×10 15 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 The number of extracellular vesicles can be measured using a Viewsizer (HORIBA Scientific, ViewSizer3000) or other particle tracking analysis (PTA) based on the analysis of bright spots of particles undergoing Brownian motion.

[0043] (Method for Obtaining Extracellular Vesicles or Extracellular Vesicle Contents) Methods for obtaining the extracellular vesicles or extracellular vesicle-containing materials of the present invention include preparation from the culture supernatant of algae.

[0044] In one embodiment, the extracellular vesicles or extracellular vesicle-containing material of the present invention are obtained by filtering a culture supernatant of microalgae.

[0045] In one embodiment, the extracellular vesicles or extracellular vesicle-containing material of the present invention are obtained by heating a culture of microalgae and recovering the culture supernatant. In another embodiment, the extracellular vesicles or extracellular vesicle-containing material of the present invention are obtained by heating a culture of microalgae, collecting the supernatant, and then purifying it. The purification may be crude purification.

[0046] The extracellular vesicles or extracellular vesicle-containing materials of the present invention can be obtained by heating a culture of microalgae at a temperature, for example, but not limited to, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 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.

[0047] In one embodiment, the extracellular vesicles or extracellular vesicle-containing material of the present invention can be obtained by filtering the culture supernatant of microalgae.

[0048] 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.

[0049] The method for obtaining extracellular vesicles or extracellular vesicle contents 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), ultracentrifugation, and affinity purification using antibodies. Of these, purification by ultrafiltration is preferred, although not limited thereto.

[0050] 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.

[0051] The molecular weight cutoff of a membrane is preferably 10 kDa to 1,000 kDa, 50 kDa to 1,000 kDa, or 100 kDa to 1,000 kDa, and more preferably 100 kDa to 500 kDa. Cutoff membranes of 10 kDa or more, 50 kDa or more, 100 kDa or more, or 150 kDa or more are preferred, and cutoff membranes of 2,000 kDa or less, 1,500 kDa or less, 1,000 kDa or less, 750 Dka or less, or 500 Dka or less are also preferred.

[0052] The extracellular vesicles or extracellular vesicle-containing materials 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, or a combination of a heat treatment step, a purification step, and a filter filtration step.

[0053] The extracellular vesicles or the material containing extracellular vesicles may be in the form of a purified liquid of the culture supernatant, or may be a solid or semi-solid obtained by drying the purified liquid.

[0054] The extracellular vesicles or extracellular vesicle contents can optionally be subjected to further purification steps to produce a highly purified population of extracellular vesicles.

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

[0056] (Lipid analysis of extracellular vesicles or extracellular vesicle contents) The extracellular vesicles or extracellular vesicle contents of the present invention contain glyceroglycolipids and / or betaine lipids as lipids.

[0057] Here, the method for analyzing lipids conforms to the analytical method specifically described in the Examples.

[0058] As used herein, "glyceroglycolipid" refers to a compound in which a fatty acid diester of glycerin is glycosylated. The hydroxyl group to which the sugar chain is covalently bonded is a hydroxyl group that is not ester-bonded to a fatty acid. Examples of glyceroglycolipids include, but are not limited to, one or more selected from the group consisting of sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride. More specifically, sulfoquinovosyldiacylglycerol (SQDG), monoglycosyldiacylglycerol and / or monogalactosyldiacylglycerol (MGDG), diglycosyldiacylglycerol and / or digalactosyldiacylglycerol (DGDG), diglycosylmonoacylglycerol and / or digalactosylmonoacylglycerol (DGMG), glucuronosyldiacylglycerol (GADG), GADG hexose adducts (Hex The glycosylglycerol may be one or more selected from the group consisting of glycosylglycerol and / or galactosyldiacylglycerol (Gal-DAG), glycosylmonoacylglycerol and / or monogalactosylmonoacylglycerol (MGMG), gluconosylmonoacylglycerol and / or galactosylmonoacylglycerol (GAMG), monoglycosyldiacylglycerol and / or monogalactosyldiacylglycerol (MGDG), sulfoquinovosylmonoacylglycerol (SQMG), acylsulfoquinovosyldiacylglycerol (ASQDG), acylmonoglycosyldiacylglycerol and / or acylmonogalactosyldiacylglycerol (AMGDG), and acyldiglycosyldiacylglycerol and / or acyldigalactosyldiacylglycerol (ADGDG). That is, in this specification, these lipids may have either a glycosyl group or a galactosyl group, and both are represented by the same abbreviation. Furthermore, these lipids may have oxidized glycerol carbon chains. The extracellular vesicles or extracellular vesicle-containing materials of the present invention may contain one or more of these.Preferably, the extracellular vesicles or extracellular vesicle contents of the present invention contain at least sulfoquinovosyldiacylglycerol (SQDG) as a glyceroglycolipid.

[0059] In one embodiment of the present invention, the detection peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 30 to 80%, more preferably 40 to 80%, and even more preferably 50 to 75% of the total detection peak area of ​​glyceroglycolipids detected in the extracellular vesicles or extracellular vesicle-containing materials. This percentage is the percentage relative to the total amount of all glyceroglycolipids contained in the extracellular vesicles or extracellular vesicle-containing materials.

[0060] In another embodiment of the present invention, the detected peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 30 to 80%, more preferably 40 to 80%, and even more preferably 50 to 75% of the total detected peak area of ​​SQDG, MGDG, DGDG, GADG, HexGADG, MGMG, DGMG, GAMG, SQMG, ASQDG, SQDG[sn2_0], AMGDG, MGDG[sn2+00], DGDG[0], DGMG[sn2_0], MGMG[0], and ADGDG detected in the extracellular vesicles or extracellular vesicle-containing materials. Some components listed above may not be included.

[0061] In another embodiment of the present invention, the detected peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 30 to 80%, more preferably 40 to 80%, and even more preferably 50 to 75% of the total detected peak area of ​​SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG, and their stereoisomers. Some of the above-listed components may not be present.

[0062] In another embodiment of the present invention, the detected peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 30 to 80%, more preferably 40 to 80%, and even more preferably 50 to 75% of the total detected peak area of ​​SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, and ADGDG. Some of the above-listed components may not be present.

[0063] In another embodiment of the present invention, the detection peak area of ​​DGDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 2 to 20%, more preferably 2.5 to 15%, and even more preferably 3 to 10% of the total detection peak area of ​​SQDG, MGDG, DGDG, GADG, HexGADG, MGMG, DGMG, GAMG, SQMG, ASQDG, SQDG[sn2_O], AMGDG, MGDG[sn2+00], DGDG[O], DGMG[sn2_O], MGMG[O], and ADGDG detected in the extracellular vesicles or extracellular vesicle-containing materials. Some components listed above may not be included.

[0064] In another embodiment of the present invention, the detection peak area of ​​DGDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 2 to 20%, more preferably 2.5 to 15%, and even more preferably 3 to 10% of the total detection peak area of ​​SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG, and their stereoisomers. There may be some components not included among the above-listed components.

[0065] In another embodiment of the present invention, the detection peak area of ​​DGDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 2 to 20%, more preferably 2.5 to 15%, and even more preferably 3 to 10%, of the total detection peak area of ​​SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, and ADGDG. Some components listed above may not be present.

[0066] In this specification, the term "betaine lipid" refers to a lipid having a diacylglycerol moiety as a basic skeleton, in which a methylated hydroxyamino acid and the diacylglycerol moiety are ether-bonded.

[0067] Examples of betaine lipids include, but are not limited to, one or more selected from the group consisting of diacylglyceryl hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA), diacylglyceryl hydroxymethylcholine (DGCC), diacylglyceryl 3-O-4-(N,N-trimethyl)homoserine (DGTS), monoacylglyceryl hydroxymethyl-N,N,N-trimethyl-β-alanine (MGTA), monoacylglyceryl 3-O-carboxyhydroxymethylcholine (MGCC), and monoacylglyceryl 3-O-4-(N,N-trimethyl)homoserine (MGTS). The extracellular vesicles or extracellular vesicle-containing materials of the present invention may contain one or more of these. Other components may also be present. Preferably, the extracellular vesicles or extracellular vesicle-containing materials of the present invention contain one or more betaine lipids selected from the group consisting of DGTA, DGTS, DGCC, MGTA, and MGCC. Alternatively, the extracellular vesicles or extracellular vesicle-containing materials of the present invention contain all of DGTA, DGTS, DGCC, MGTA, and MGCC as betaine lipids.

[0068] In one embodiment of the present invention, the detection peak area of ​​DGCC in extracellular vesicles or extracellular vesicle contents is preferably 25 to 60%, more preferably 30 to 55%, and even more preferably 35 to 50%, of the total detection peak area of ​​betaine lipids detected in the extracellular vesicles or extracellular vesicle contents.

[0069] In another aspect of the present invention, the detection peak area of ​​DGCC in extracellular vesicles or extracellular vesicle-containing materials is preferably 25 to 60%, more preferably 30 to 55%, and even more preferably 35 to 50% of the total detection peak area of ​​DGTA, DGCC, MGTA, MGCC, DGTS, and their stereoisomers detected in the extracellular vesicles or extracellular vesicle-containing materials.

[0070] In another aspect of the present invention, the detection peak area of ​​DGCC in extracellular vesicles or extracellular vesicle-containing materials is preferably 25 to 60%, more preferably 30 to 55%, and even more preferably 35 to 50% of the total detection peak area of ​​DGTA, DGCC, MGTA, MGCC, and DGTS detected in the extracellular vesicles or extracellular vesicle-containing materials.

[0071] In one embodiment of the present invention, the detection peak area of ​​MGTA in extracellular vesicles or extracellular vesicle contents is preferably 8 to 30%, more preferably 10 to 28%, and even more preferably 13 to 25% of the total detection peak area of ​​betaine lipids detected in the extracellular vesicles or extracellular vesicle contents.

[0072] In another aspect of the present invention, the detection peak area of ​​MGTA in extracellular vesicles or extracellular vesicle contents is 8 to 30%, more preferably 10 to 28%, and even more preferably 13 to 25%, of the total detection peak area of ​​DGTA, DGTS, DGCC, MGTA, MGCC, and their stereoisomers.

[0073] In another aspect of the present invention, the detection peak area of ​​MGTA in extracellular vesicles or extracellular vesicle-containing matter is 8 to 30% of the total detection peak area of ​​DGTA, DGTS, DGCC, MGTA, and MGCC, more preferably 10 to 28%, and even more preferably 13 to 25%.

[0074] In one embodiment of the present invention, the detection peak area of ​​DGTA in extracellular vesicles or extracellular vesicle contents is preferably 5 to 30%, more preferably 10 to 30%, and even more preferably 15 to 25% of the total detection peak area of ​​betaine lipids detected in the extracellular vesicles or extracellular vesicle contents.

[0075] In another aspect of the present invention, the detection peak area of ​​DGTA in extracellular vesicles or extracellular vesicle-containing materials is preferably 5 to 30%, more preferably 10 to 30%, and even more preferably 15 to 25% of the total detection peak area of ​​DGTA, DGCC, MGTA, MGCC, DGTS, and their stereoisomers detected in the extracellular vesicles or extracellular vesicle-containing materials.

[0076] In another aspect of the present invention, the detection peak area of ​​DGTA in extracellular vesicles or extracellular vesicle-containing materials is preferably 5 to 30%, more preferably 10 to 30%, and even more preferably 15 to 25% of the total detection peak area of ​​DGTA, DGCC, MGTA, MGCC, and DGTS detected in the extracellular vesicles or extracellular vesicle-containing materials.

[0077] In one embodiment of the present invention, the ratio of the total detected peak area of ​​glyceroglycolipids to the total detected peak area of ​​betaine lipids in extracellular vesicles or extracellular vesicle contents is preferably 0.001 to 9, more preferably 0.01 to 7, and even more preferably 0.1 to 5.

[0078] In other embodiments, the extracellular vesicles or extracellular vesicle contents of the present invention comprise, as lipids, glyceroglycolipids, betaine lipids, fatty acids, sterol metabolites, acylhexoses, sphingolipids, neutral glycerolipids, and / or glycosphingolipids.

[0079] Examples of fatty acids include carbon chains having 16 to 32 carbon atoms. From the viewpoint of achieving the effects of the present invention, the proportion of fatty acids having 16 to 18 carbon atoms in the fatty acids is preferably 10 to 60%, more preferably 15 to 55%, and even more preferably 20 to 50%. From another viewpoint, the proportion of unsaturated fatty acids having 16 to 18 carbon atoms in the total fatty acids having 16 to 18 carbon atoms is preferably 50 to 90%, more preferably 55 to 85%, and even more preferably 60 to 80%.

[0080] Sterol metabolites include, for example, hydroxycampestanol sulfate, hydroxystigmastanol sulfate, G1 stigmasterol, G4 stigmasterol, G3 stigmasterol, G5 stigmasterol, brassicasterol sulfate, sitosterol sulfate, G2 stigmasterol, stigmasterol, stigmasterol sulfate, G6 stigmasterol, β-sitosterol, G7 stigmasterol, and / or cholesterol sulfate.

[0081] Examples of acylhexoses include diacylhexoses and / or monoacylhexoses.

[0082] Sphingolipids include, for example, ceramides and / or sulfonoceramides.

[0083] Neutral glycerolipids include, for example, triacylglycerols and / or diacylglycerols.

[0084] An example of glycosphingolipids is glucosylceramide.

[0085] (Compositions comprising extracellular vesicles or extracellular vesicle contents) In one aspect of the present invention, a composition containing either the extracellular vesicles or the contents of the extracellular vesicles thus obtained can be prepared.

[0086] [Application] The composition containing the extracellular vesicles or extracellular vesicle-containing material of the present invention can be added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet food. Alternatively, the extracellular vesicles or extracellular vesicle-containing material can be used as is as pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet food.

[0087] When the composition containing the extracellular vesicles or extracellular vesicle-containing material of the present invention is added to or mixed with so-called topical preparations such as pharmaceuticals, quasi-drugs, and cosmetics, it can be used as pharmaceuticals, quasi-drugs, and cosmetics that explicitly or implicitly claim to improve skin elasticity, firmness, wrinkles, texture, moisture, dryness, roughness, etc. as efficacy, effects, or functions.

[0088] When the composition containing the extracellular vesicles or extracellular vesicle-containing material of the present invention is added to or mixed with food, beverage, feed, or pet food, it can be used as a food or beverage that explicitly or implicitly claims to have the functionality of improving skin elasticity, firmness, wrinkles, texture, moisture, dryness, roughness, etc., i.e., as a health food, functional food, food for patients, or food for specified health uses. Furthermore, even if the above functionality is not explicitly or implicitly claimed, it can be used as a so-called doctor's supplement recommended or suggested by doctors in internal medicine, orthopedic surgery, veterinary clinics, etc. in hospitals and / or clinics.

[0089] When the above functionality is not explicitly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those who seek skin elasticity, those who seek skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those who seek skin moisture, those who are concerned about dry skin, those who are concerned about rough skin, those who are concerned about pores, those who are concerned about skin fluctuations, those who are concerned about pores, or similar indications.

[0090] Health foods, functional foods, foods for the sick, and foods for specified health uses are specifically solid preparations. The composition can be used in various dosage forms such as tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candy, etc.), liquids (syrups, suspensions), and liquid foods. Food preparations can be produced 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 producing the food using conventional means. The dosage form is not limited, but is preferably an orally disintegrating tablet, chewable tablet, candy, granules, powder, or liquid from the viewpoint of significantly exhibiting the effects of the present invention. [Example]

[0091] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0092] Example 1 Pavlova sp. was cultured in a commercially available algae medium by a conventional method, and the culture was heated to 90°C and then centrifuged at 3000 xg at room temperature for 60 minutes to obtain a culture supernatant.

[0093] 3000 mL of the resulting culture supernatant was centrifuged, and 3000 mL of the resulting supernatant was filtered through a 0.22 μm filter. The supernatant was then concentrated to a volume of 150 mL or less using a 300 kDa hollow fiber membrane filter (MiniKros sampler S04-E300-05-N: Repligen) by tangential flow. The concentrate was then washed with 10,000 mL of PBS, and 100 mL was finally recovered with PBS. Each concentrated sample was then sterilized using a 0.20 μm filter to obtain the extracellular vesicle-containing product.

[0094] Example 2 The extracellular vesicle-containing material obtained in Example 1 was diluted 10 times with purified water to prepare a solution.

[0095] (Comparative Example 1) Pavlova sp. was cultured in a commercially available algae medium by a conventional method, and the culture supernatant was obtained by centrifugation at 3000×g at room temperature for 60 minutes.

[0096] 3000 mL of the resulting culture supernatant was centrifuged, and 3000 mL of the resulting supernatant was filtered through a 0.22 μm filter. The supernatant was then concentrated to a volume of 150 mL or less using a 300 kDa hollow fiber membrane filter (MiniKros sampler S04-E300-05-N: Repligen) by tangential flow. The concentrate was then washed with 10,000 mL of PBS, and 100 mL was finally recovered with PBS. Each concentrated sample was then sterilized using a 0.20 μm filter to obtain the extracellular vesicle-containing product.

[0097] The extracellular vesicle-containing materials of Example 1 and Comparative Example 1 were appropriately diluted with PBS(-), and the particle number, average particle size, and mode particle size were measured using a Viewsizer (ViewSizer3000, manufactured by HORIBA Scientific).

[0098] Extracellular vesicles of Example 1: 3.221 × 10 11 Particles / mL, average particle size 180.76nm, mode particle size 129.53nm Extracellular vesicles of Comparative Example 1: 3.253 × 10 11 Particles / mL, average particle size 168.22nm, mode particle size 131.19nm

[0099] (Comparative Example 2) The extracellular vesicle-containing material obtained in Comparative Example 1 was diluted 10 times with purified water to prepare a solution.

[0100] [Lipid analysis of extracellular vesicles and / or extracellular vesicle contents of microalgae] <Extraction operation> The extracellular vesicle contents of Examples 1-2 and Comparative Examples 1-2 were each subjected to lipid analysis. As an extract, 225 μL of an extract containing an internal standard substance (CHCl3:MeOH = 1:2) was added. This extract was prepared by mixing 4 μL of Equi SPLASH, 500 μL of CHCl3, and 996 μL of MeOH. The added extract was subjected to ultrasonic treatment for 5 seconds.

[0101] Furthermore, 225 μL of the extract (CHCl3:MeOH = 1:2) was added again. This extract was prepared by mixing 500 μL of CHCl3 and 1000 μL of MeOH. Then, ultrasonic treatment was performed again for 30 seconds.

[0102] <Centrifugation> Centrifugation was performed at 750 rpm for 20 minutes at 20 °C. By this operation, the solid components in the sample were separated. Next, 30 μL of water was added to homogenize the sample. Centrifugation was performed again at 750 rpm for 20 minutes at 20 °C. Further clarification of the sample was performed in this step.

[0103] Next, centrifugation was performed at 1670 G for 10 minutes at 20 °C. By this centrifugation, fine solid components were completely removed.

[0104] <Sample collection> Finally, 150 μL aliquots of the supernatant were collected twice from a total of 480 μL of the solution. Thereby, samples for analysis were obtained. <We000465> <LC-MS analysis> The collected samples were injected into LC vials. The injection volume was 3 μL in negative ion mode and 1.5 μL in positive ion mode. The instruments used were UPLC (Waters) and TripleTOF 6600 (SCIEX). Ionization method: ESI Measurement mode: Negative-ion mode measurement, Positive-ion mode Ionization conditions: [Negative-ion mode] (Source / Gas)Ion Source:DuoSplay Ion Source Ion Source Gas 1:50 Ion Source Gas 2:50 Curtain Gas:30 Temperature: 300 Ion Splay Voltage Floating:-4500 (Compound) Declustering Potential:-80 Collision Energy:-5 [Positive-ion mode] (Source / Gas) Ion Source:DuoSplay Ion Source Ion Source Gas 1:40 Ion Source Gas 2:80 Curtain Gas:30 Temperature: 250 Ion Splay Voltage Floating:5500 (Compound) Declustering Potential:80 Collision Energy: 5 Mobile phase: A: MeOH: ACN: Water (=1:1:3) *B: Isopropanol* (*Contains 5 mM ammonium acetate / 10 nM EDTA.) Analysis conditions: Gradient analysis and DDA were used. “B_0% to 64%(0-6.5min) → 64% to 76.5%(6.5-13.5min)→ 76.5% to 98%(13.5-18min) → 98% to 98%(19-20min)“ Flow rate: 0.3 mL / min Column temperature: 45℃ MS range: 140-1700 MS / MS range: 70-1700 MS2 cleavage method: CID

[0106] <Analysis> For the analysis, qualitative and quantitative analyses were performed using in-house lipid screening (Kazusa Genome Technologies, Inc.).

[0107] <Stability evaluation> The extracellular vesicle-containing materials of the Examples and Comparative Examples were each filled into 20 ml light-shielding glass vials and stored at 60° C., 50° C., and 40° C. The particle diameter and particle size were measured after one week had passed. The measurement conditions are as follows: Equipment used: Viewsizerhttps: / / www.horiba.com / jpn / scientific / products / detail / action / show / Product / viewsizer3000-1862 / Measurement temperature: 22℃ Methods: Exosomes Video count: 10 times

[0108] Average particle size fluctuation rate = |(d_mean_after 1 week / d_mean_immediately after preparation) × 100| Mode particle size fluctuation rate = |(d_mode_1 week later / d_mode_immediately after preparation) × 100| Particle number fluctuation rate = |(n_1 week later / n_immediately after preparation) × 100| Note that || indicates an absolute value. Improvement rate (%) = (Comparative example - Example) / Comparative example × 100

[0109] The results of lipid analysis of the extracellular vesicle contents in Example 1 and Comparative Example 1 are shown in Tables 1 and 2, and the results of the stability tests in the Examples and Comparative Examples are shown in Tables 3 and 4. Table 1 shows the results of the peak area ratios of betaine lipids obtained by the method described in the <LC-MS analysis> section. The peak area ratio was calculated as the relative ratio of each betaine lipid in the detected betaine lipid species by normalizing the detected peak area of each betaine lipid with the peak area of the internal standard. As the internal standard, 15:0~18:1(d7)PC among the standard substances contained in Equi SPLASH was used. Table 2 shows the results of the peak area ratios of glyceroglycolipids obtained by the method described in the <LC-MS analysis> section. The peak area ratio was calculated as the relative ratio of each glyceroglycolipid in the detected glyceroglycolipid species by normalizing the detected peak area of each glyceroglycolipid with the peak area of the internal standard. As the internal standard, 15:0~18:1(d7)DAG among the standard substances contained in Equi SPLASH was used.

[0110]

Table 1

[0111]

Table 2

[0112] The extracellular vesicle contents in the Examples contain a high proportion of SQDG and MGDG among glyceroglycolipids, and furthermore contain a large amount of betaine lipids.

[0113]

Table 3

[0114]

Table 4

Claims

1. An extracellular vesicle or extracellular vesicle-containing material derived from microalgae, which contains glyceroglycolipids, and the content of SQDG in the total amount of the glyceroglycolipids is 30 to 80%.

2. An extracellular vesicle or extracellular vesicle-containing material derived from microalgae, which contains glyceroglycolipids, and the content of DGDG in the total amount of the glyceroglycolipids is 2 to 20%.

3. An extracellular vesicle or extracellular vesicle-containing material derived from microalgae, which contains betaine lipids and has a DGCC content of 25 to 60% of the total amount of the betaine lipids.

4. An extracellular vesicle or an extracellular vesicle-containing substance, which contains betaine lipid, and the content of MGTA in the total amount of the betaine lipid is 8 to 30%.

5. The extracellular vesicles or extracellular vesicle-containing material according to any one of claims 1 to 4, wherein the microalgae contain chloroplasts or thylakoids.

6. The extracellular vesicles or extracellular vesicle-containing material according to claim 5, wherein the microalgae are microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

7. Extracellular vesicles or extracellular vesicle-containing materials derived from microalgae, which contain glyceroglycolipids, and in which the content of SQDG is 30 to 80% of the total amount of SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG, and stereoisomers thereof.

8. A microalgae-derived extracellular vesicle or extracellular vesicle-containing composition comprising glyceroglycolipids, in which the content of DGDG is 2 to 20% of the total amount of SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG and stereoisomers thereof.

9. An extracellular vesicle or extracellular vesicle-containing material derived from microalgae, which contains betaine lipid and has a DGCC content of 25 to 60% in the total amount of DGTA, DGTS, DGCC, MGTA, and MGCC and their stereoisomers.

10. Extracellular vesicles or extracellular vesicle contents containing betaine lipid, wherein the content of MGTA in the total amount of DGTA, DGTS, DGCC, MGTA, and MGCC and their stereoisomers is 8 to 30%.

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