Composition for promoting production of extracellular matrix components and composition for promoting wound healing, containing components derived from bacteria and / or fungi

Abstract

To provide a preparation effective in promoting the production of any one of collagen, elastin, or hyaluronic acid, which are extracellular matrix components, or to provide a preparation effective in promoting epithelial wound healing.SOLUTION: Provided are: a composition for promoting the production of extracellular matrix components, the composition containing extracellular vesicles derived from bacteria and / or fungi, and the extracellular matrix components being at least one selected from the group consisting of collagen, elastin and hyaluronic acid; or a composition for promoting the expression of COL1A1, COL3A1, ELN, HAS1 or HAS2 genes; a composition for improving the physical properties or appearance of skin, eyeball or connective tissue; and a composition for promoting epithelial wound healing, containing extracellular vesicles derived from bacteria and / or fungi.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for promoting the production of extracellular matrix components, containing components derived from bacteria and / or fungi, and more specifically, a composition for promoting the production of at least one selected from the group consisting of collagen, elastin, and hyaluronic acid, and a composition for improving the physical properties or appearance of the skin, eyeball, or connective tissue. The present invention also relates to a composition for promoting wound healing and a cell activation composition, containing components derived from bacteria and / or fungi.

Background Art

[0002] Cytoskeletons such as collagen and elastin, and water-retaining components such as hyaluronic acid are components that constitute the extracellular matrix, which is abundantly contained in connective tissues such as the skin, eyeball, tendon, and teeth.

[0003] In the skin (dermis), these extracellular matrix components bring about physical properties such as elasticity, flexibility, stretchability, and firmness of the skin, and are known to be greatly involved in changes in texture, skin gloss, wrinkles, sagging, and appearance changes due to them. It is known that when the hyaluronic acid in the skin decreases, fine wrinkles are likely to form.

[0004] In the eyeball, these extracellular matrix components contribute to the maintenance of the normal structure of the eye. That is, the structure of collagen and water retention by hyaluronic acid maintain the structure of the vitreous body, and collagen and elastin maintain the elasticity and shape of the conjunctiva.

[0005] Furthermore, in connective tissues such as tendons and teeth, it is known that due to a deficiency of extracellular matrix components, their elasticity and flexibility decrease, resulting in a decrease in the mechanical strength and function of the tissue.

[0006] Collagen and elastin are broken and denatured by the action of external forces, light, chemical substances, etc., and decrease with aging. Hyaluronic acid also decreases with aging. As a result, in the skin, eyes, and connective tissues, it can lead to deterioration of physical properties such as a decrease in elasticity and firmness, deterioration of skin texture and gloss, the formation of wrinkles and sagging, etc.

[0007] Therefore, in order to improve the physical properties and appearance of the skin, eyes, and connective tissues, means to promote the production of collagen, elastin, and hyaluronic acid are required, and various means have been developed. For example, Patent Document 1 discloses a collagen production promoter and a hyaluronic acid production promoter characterized by containing an extract of chome and / or hondawara. Also, Patent Document 2 discloses an elastin production promoter having a lipocalin family protein and / or a lipocalin family protein degradation product as an active ingredient.

[0008] On the other hand, epithelia such as the skin, cornea, and mucosa are constantly exposed to the risk of wounds. In particular, the epidermis consisting of epidermal cells (keratinocytes) and the stratum corneum derived therefrom is located on the outermost side of the skin and is easily damaged by external forces, heat, etc. Also, the corneal epithelium is easily wounded by friction during blinking, wearing contact lenses, etc. It is known that epithelial wounds cause a decrease in the barrier function of the epithelium, leading to water loss and a decrease in the protective function against foreign pathogens and chemical substances.

[0009] Thus, since epithelial wounds can lead to further deterioration of the health condition, means to promote wound healing are required. For example, Patent Document 3 discloses that a skin cell activator containing a function inhibitor of ORAI3 protein or Orai3 gene in the skin has a wound healing promoting effect. Patent Document 4 describes that a peptide of specific 4 - 6 amino acid residues has a wound healing promoting effect.

[0010] In addition, the production of collagen and elastin also plays an important role in wound healing. During the wound healing process, the production of collagen at the wound site is promoted. Then, the deposition of collagen at the wound site increases the strength of the wound site and promotes healing. On the other hand, elastin is known to impart resilience and durability to tissues in wound healing and to induce cell activities such as cell migration and proliferation and extracellular matrix synthesis (see the abstract of Non-Patent Document 1). In addition, Patent Document 5 reports that tropoelastin, which is a precursor of elastin synthesized from the elastin gene (ELN), improves re-epithelialization of full-thickness skin wounds and promotes the wound repair process.

[0011] As also disclosed in Patent Documents 3 and 4 above, the wound healing promoting effect can be confirmed by a scratch assay using epithelial cells. In the scratch assay, first, an area (wound gap) where no cells of a specific width exist is formed by an artificial scratch on the cell monolayer membrane. Then, when the culture is continued after the scratch is formed, the rate at which the cells migrate or proliferate and the wound gap is filled can be quantified by the decrease in the area of the wound gap portion. Therefore, when the decrease in the wound gap after scratching is larger under the condition containing the component than under the condition not containing the specific component, it is shown that the component has a wound healing promoting effect.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0013] [Non-Patent Document 1] Birth Defects Research (Part C), 2012, Vol.96, pp.248-257 [Summary of the Invention] [Problems to be Solved by the Invention]

[0014] As described above, collagen, elastin, and hyaluronic acid are known to be effective in improving the physical properties and appearance of the skin, eyes, and connective tissues, promoting wound healing, and activating cells, and components that promote their production are also known. However, in order to meet various needs in beauty and medicine, new components that promote the production of collagen, elastin, or hyaluronic acid are awaited.

[0015] In addition, although it is known that cell-activating components that promote the proliferation and migration of epithelial cells exhibit a wound healing promoting effect, it is not clear which components are suitable as wound healing promoters.

[0016] Therefore, an object of the present invention is to provide a preparation effective in promoting the production of any of collagen, elastin, or hyaluronic acid, which are extracellular matrix components, or to provide a preparation effective in promoting epithelial wound healing. [Means for Solving the Problems]

[0017] As a result of intensive studies by the present inventors to solve the above problems, it has been found that extracellular vesicles derived from bacteria or fungi have an action of promoting the production of collagen, elastin, and hyaluronic acid, and also have an action of promoting epithelial wound healing and cell activation, leading to the completion of the present invention.

[0018] That is, the present invention includes, but is not limited to, the following aspects. [1] A composition for promoting the production of extracellular matrix components, comprising containing extracellular vesicles derived from bacteria and / or fungi, a composition, wherein the extracellular matrix component is at least one selected from the group consisting of collagen, elastin, and hyaluronic acid. [2] The composition according to [1], wherein the production promotion is production promotion in fibroblasts. [3] A composition for promoting the expression of the COL1A1, COL3A1, ELN, HAS1, or HAS2 gene, containing extracellular vesicles derived from bacteria and / or fungi. [4] A composition for improving the physical properties or appearance of the skin, eye, or connective tissue, containing extracellular vesicles derived from bacteria and / or fungi. [5] The composition according to [4], wherein the improvement of the physical properties or appearance of the skin is improvement of skin elasticity, skin flexibility, skin stretchability, skin wrinkles, skin sagging, skin moisture, skin protection, skin dryness, skin firmness, skin texture, skin gloss, or skin blemishes. [6] The composition according to any one of [1] to [5], wherein the bacteria are Gram-positive bacteria or Gram-negative bacteria. [7] The composition according to [6], wherein the Gram-positive bacteria are Weizmannia coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, or Tetragenococcus halophilus. [8] The composition according to [6], wherein the Gram-negative bacteria are Escherichia coli or Pantoea agglomerans. [9] The composition according to any one of [1] to [5], wherein the fungus is Saccharomyces cerevisiae or Aspergillus oryzae.

[10] The composition according to any one of [1] to [9], which is a pharmaceutical, quasi-drug, medical device, cosmetic or food or drink.

[11] A composition for promoting epithelial wound healing, containing extracellular vesicles derived from bacteria and / or fungi.

[12] A composition for cell activation, containing extracellular vesicles derived from bacteria and / or fungi.

[13] The composition according to

[12] , wherein the cell activation includes promotion of cell proliferation, promotion of migratory ability, or both.

[14] A composition for improving the barrier function of epithelium, containing extracellular vesicles derived from bacteria and / or fungi.

[15] The composition according to any one of

[11] to

[14] , wherein the bacteria are Gram-positive bacteria or Gram-negative bacteria.

[16] The composition according to

[15] , wherein the Gram-positive bacteria are Weizmannia coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, or Tetragenococcus halophilus.

[17] The composition according to

[15] , wherein the Gram-negative bacteria are Escherichia coli or Pantoea agglomerans.

[18] The composition according to any one of

[11] to

[14] , wherein the fungus is Saccharomyces cerevisiae or Aspergillus oryzae.

[19] The composition according to any one of

[11] to

[18] , which is a pharmaceutical, quasi-drug, medical device, cosmetic or food or drink.

[20] An external composition containing extracellular vesicles derived from bacteria and / or fungi.

[21] The composition according to

[20] , wherein the bacteria and / or fungi are Weizmannia coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, Escherichia coli, Pantoea agglomerans, Saccharomyces cerevisiae, or Aspergillus oryzae.

Advantages of the Invention

[0019] The composition of the present invention can promote the production of collagen, elastin and hyaluronic acid, which are extracellular matrix components, by containing extracellular vesicles derived from bacteria and / or fungi. In addition, the composition of the present invention can promote epithelial wound healing by containing extracellular vesicles derived from bacteria and / or fungi.

Modes for Carrying Out the Invention

[0020] As used herein, "improvement" of a disease, symptom, health condition, or aesthetic condition refers to the cure, improvement, or alleviation of the disease, symptom, health condition, or aesthetic condition; the prevention or delay of the deterioration of the disease, symptom, health condition, or aesthetic condition; or the reversal, prevention, or delay of the progression of the disease or symptom.

[0021] The composition of the present invention contains extracellular vesicles derived from bacteria and / or fungi.

[0022] As used herein, "extracellular vesicles" derived from microorganisms such as bacteria or fungi is a concept that includes membrane vesicles and exosomes (vesicles released from endosomes), and represents vesicles released from cells. Extracellular vesicles are bag-shaped structures released when the cell membrane (outer membrane) of a cell bulges outward and constricts in a budding-like form, and have a lipid bilayer structure. The diameter of extracellular vesicles is 10 nm to 5000 nm, more generally 30 to 1000 nm, and even more generally 50 to 700 nm. For example, extracellular vesicles of bacteria generally have a diameter of 20 to 300 nm. Extracellular vesicles can be distinguished from the living cells from which they are derived based on factors such as size (diameter), constituent components, membrane composition, and the presence or absence of cell activities (such as the presence or absence of division and proliferation).

[0023] As used herein, bacteria include eubacteria and archaea. In the composition of the present invention, the bacteria are selected from the group consisting of Gram-positive bacteria and Gram-negative bacteria, and Gram-positive bacteria are preferred.

[0024] Gram-positive bacteria include, for example, bacteria of the genus Weizmannia, Lactobacillus, Leuconostoc, Enterococcus, Tetragenococcus, Bifidobacterium, Lactococcus, Pediococcus, Weissella, or Streptococcus. In one embodiment, the gram-positive bacteria are of the genus Lactobacillus, Leuconostoc, Enterococcus, Tetragenococcus, or Weissella.

[0025] Weizmannia bacteria include Weizmannia coagulans (formerly Bacillus coagulans), Weizmannia acidiproducens (W. acidiproducens), Weizmannia ginsengihumi, etc. From the viewpoint of more significantly demonstrating the effects of the present invention, Weizmannia coagulans is preferred.

[0026] Bacteria of the genus Lactobacillus include Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus animalis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus kefir, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus crispatus, Lactobacillus sakei, etc. From the viewpoint of more significantly achieving the effects of the present invention, Lactobacillus paracasei is preferred.

[0027] Leuconostoc bacteria include Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc lactis, Leuconostoc fallax, Leuconostoc citreum, Leuconostoc pseudomesenteroides, etc. From the perspective of more significantly achieving the effects of the present invention, Leuconostoc mesenteroides is preferred.

[0028] Enterococcus bacteria include Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus lactis, Enterococcus olivae, etc. From the perspective of more significantly achieving the effects of the present invention, Enterococcus faecalis or Enterococcus faecium is preferred.

[0029] Tetragenococcus bacteria include Tetragenococcus halophilus, Tetragenococcus koreensis, Tetragenococcus muriaticus, Tetragenococcus osmophilus, Tetragenococcus solitarius, etc. From the perspective of more significantly achieving the effects of the present invention, Tetragenococcus halophilus is preferred.

[0030] Bifidobacterium bacteria include Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, etc.

[0031] Lactococcus bacteria include Lactococcus lactis, Lactococcus raffinolactis, Lactococcus plantarum, etc.

[0032] Pediococcus bacteria include Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus clausenii, Pediococcus damnosus, etc.

[0033] Bacteria of the Weissella genus include Weissella cibaria, Weissella confusa, Weissella halotolerans, Weissella hellenica, Weissella kandleri, Weissella kimchii, Weissella koreensis, Weissella minor, Weissella paramesenteroides, Weissella soli, Weissella thailandensis, Weissella viridescens, etc.

[0034] Bacteria of the Streptococcus genus include Streptococcus thermophilus, Streptococcus lactis, etc.

[0035] Gram-negative bacteria include, for example, bacteria of the genus Escherichia or Pantoea. From the viewpoint of more significantly exhibiting the effects of the present invention, Escherichia coli is preferred among the bacteria of the genus Escherichia. From the viewpoint of more significantly exhibiting the effects of the present invention, Pantoea agglomerans is preferred among the bacteria of the genus Pantoea.

[0036] In this specification, fungi include yeasts and Aspergillus. Examples of yeasts include organisms of the genus Saccharomyces, Schizosaccharomyces, Candida, Cryptococcus, Debaryomyces, Endomyces, Endomycopsis, Galactomyces, Hanseniaspora, Hansenula, Kluyveromyces, Pichia, Torulopsis, Tricosporon, or Yarrowia. Examples of Aspergillus include organisms of the genus Aspergillus. Among them, fungi are preferably organisms of the genus Saccharomyces, Schizosaccharomyces, Candida, Pichia, or Aspergillus, and more preferably organisms of the genus Saccharomyces or Aspergillus.

[0037] Examples of organisms of the genus Saccharomyces include S. cerevisiae and S. veronae. From the viewpoint of more significantly demonstrating the effects of the present invention, S. cerevisiae is preferred.

[0038] Examples of organisms of the genus Aspergillus include A. oryzae, A. sojae, A. luchuensis, and A. luchuensis mut. Kawachii. From the viewpoint of more significantly demonstrating the effects of the present invention, A. oryzae is preferred.

[0039] As the above-mentioned bacteria or fungi, for example, the strains described in Table 1 of this specification can be used as suitable strains in the corresponding species.

[0040] The bacteria or fungi exemplified above include their wild strains and mutant strains as long as they exhibit the effects of the present invention. In this specification, the "mutant strain" refers to a strain in which a mutation has occurred in the DNA possessed by the original strain of the biological species. The mutation is not particularly limited and can be, for example, a substitution, deletion, insertion, duplication, translocation, or inversion of bases in genomic DNA or a plasmid originally possessed by the strain. The mutation may be naturally occurring or artificially induced. The sequence identity of the genomic DNA between a mutant strain and its closest wild strain (e.g., a standard strain) can be, for example, 90% or more, 95% or more, 99% or more, 99.9% or more, or 99.99% or more. The sequence identity of the 16S rRNA gene between a mutant strain of bacteria and its closest wild strain (e.g., a standard strain) can be, for example, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more. The sequence identity of the ribosomal RNA ITS (Internal transcribed spacer) region or the D1 / D2 region of the 26S / 28S rRNA gene between a mutant strain of fungi and its closest wild strain (e.g., a standard strain) can each independently be, for example, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more.

[0041] In addition to genomic DNA, the cells of bacteria or fungi from which extracellular vesicles are derived may carry foreign DNA other than the plasmids originally possessed by the strain (e.g., plasmids, cosmids, artificial chromosomes, etc.).

[0042] The content of extracellular vesicles in the composition of the present invention is appropriately adjusted depending on the type and amount of other components, the use, the form of the composition, and the like. The content of extracellular vesicles in the composition of the present invention is, for example, 1×10 -9 % by mass or more, 1×10 -8 % by mass or more, 1×10 -7 % by mass or more, 1×10-6 % by mass or more, 1×10 -5 % by mass or more, 0.0001% by mass or more, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 1% by mass or more, 5% by mass or more, or 10% by mass or more, and, for example, with respect to the total amount of the composition, 50% by mass or less, 10% by mass or less, 1% by mass or less, 0.1% by mass or less, 0.01% by mass or less, 0.001% by mass or less, 0.0001% by mass or less, or 1×10 -5 % by mass or less. The content of extracellular vesicles in the composition of the present invention is, with respect to the total amount of the composition, for example, 1×10 -9 ~50% by mass, 1×10 -8 ~10% by mass, 1×10 -7 ~1% by mass, 1×10 -6 ~0.1% by mass, 1×10 -5 ~0.01% by mass, 0.0001 to 0.1% by mass, or 0.001 to 0.01% by mass.

[0043] The content of extracellular vesicles in the composition of the present invention is, in terms of protein equivalent, with respect to the total amount of the composition, for example, 0.1 pg / mL or more, 1 pg / mL or more, 10 pg / mL or more, 100 pg / mL or more, 1 ng / mL or more, 10 ng / mL or more, 100 ng / mL or more, 1 μg / mL or more, 10 μg / mL or more, 100 μg / mL or more, or 1 mg / mL or more, and may be 100,000 μg / mL or less, 50,000 μg / mL or less, 20,000 μg / mL or less, 10,000 μg / mL or less, 5000 μg / mL or less, 2000 μg / mL or less, 1000 μg / mL or less, 500 μg / mL or less, 200 μg / mL or less, 100 μg / mL or less, or 10 μg / mL or less. The extracellular vesicle content of the composition of the present invention is, in terms of protein equivalent, for example, 0.1 pg / mL to 100,000 μg / mL, 1 pg / mL to 50,000 μg / mL, 10 pg / mL to 20,000 μg / mL, 100 pg / mL to 10,000 μg / mL, 1 ng / mL to 5000 μg / mL, 10 ng / mL to 2000 μg / mL, 100 ng to 1000 μg / mL, 1 to 500 μg / mL or 10 to 200 μg / mL with respect to the total amount of the composition.

[0044] The average diameter of extracellular vesicles of bacteria or fungi is, for example, 5 nm or more, 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, 90 nm or more or 100 nm or more, and can be, for example, 1000 nm or less, 500 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less or 200 nm or less. The average diameter of extracellular vesicles of bacteria or fungi can be, for example, 5 to 1000 nm, 10 to 500 nm, 10 to 400 nm, 10 to 350 nm, 10 to 250 nm or 10 to 200 nm.

[0045] The particle density of extracellular vesicles of the composition of the present invention is, for example, 1.0×10 2 per mL or more, 1.0×10 3 per mL or more, 1.0×10 4 per mL or more, 1.0×10 5 per mL or more, 1.0×10 6 per mL or more, 1.0×10 7 per mL or more, 1.0×10 8 per mL or more or 1.0×10 9 per mL or more, and can be, for example, 1.0×10 14 per mL or less, 1.0×10 13 per mL or less, 1.0×10 12 per mL or less, 1.0×10 11 per mL or less or 1.0×10 10 per mL or less. The particle density of extracellular vesicles of the composition of the present invention is, for example, 1.0×10 2 to 1.0×1014 cells / mL, 1.0×10 3 ~1.0×10 13 cells / mL, 1.0×10 4 ~1.0×10 12 cells / mL, 1.0×10 5 ~1.0×10 11 cells / mL or 1.0×10 6 ~1.0×10 10 cells / mL may be used.

[0046] In this specification, for the counting of extracellular vesicles and the measurement of diameter or particle size, conventional techniques for measuring nanoparticle size such as microscopy techniques (e.g., transmission electron microscopy) or light scattering techniques can be used. Alternatively, nanoparticle tracking analysis (NTA) based on the analysis of both light scattering or light interference patterns and Brownian motion can also be used. Examples of those using light scattering include the NanoSight series from Malvern Panalytical, and an example of an instrument using a light interference pattern is VIDEO DROP from Maywa Focus. Alternatively, an electrical resistance nanopulse method can be used, in which a voltage is changed in a solution sandwiching a nanopore and the electrical resistance nanopulse when the nanopore passes through is measured. Examples of instruments using the electrical resistance nanopulse method include, for example, the nanoparticle multi-analyzer qNano, Exoid, etc. from Maywa Focus.

[0047] In this specification, for the extracellular vesicles according to the present invention, for extracellular vesicles with a particle size of 50 nm or more, NanoSight (Malvern Panalytical, NanoSight LM10) is used, and for extracellular vesicles with a particle size of less than 50 nm, the measurement is made using a transmission electron microscope. When using a transmission electron microscope, the diameter or particle size is measured as the maximum dimension of the extracellular vesicle of the particle. In addition, in this specification, the number of extracellular vesicles according to the present invention is assumed to be measured using NanoSight (Malvern Panalytical, NanoSight LM10). For the measurement of the number of extracellular vesicles, in addition to the methods exemplified above, an exosome measurement system (ExoCounter, JVC Kenwood Corporation) using a method of sandwich detection of surface antigens specifically with antibodies of disks and nanobeads can also be used. Regarding the measured values of the alternative measurement methods exemplified above, when they can be converted into the measured values of the measurement method specified above, the converted values shall be used.

[0048] The composition of the present invention may further contain, in addition to the above extracellular vesicles, for example, a base, a carrier, an additive, etc., depending on the use, form, etc. of the composition.

[0049] (Manufacturing method) The extracellular vesicles in this specification are generated, for example, by spontaneous budding or exocytosis of the cell membrane (outer membrane) of living cells, or by budding or exocytosis due to artificial operations (such as stimulation by heating), etc., where a part of the cell membrane is pinched off from the cell. Then, by separating the generated extracellular vesicles from the cells and optionally passing through steps such as concentration, the extracellular vesicles of the composition of the present invention can be prepared.

[0050] As the cells from which the extracellular vesicles are derived, for example, those cultured under the conditions usually used for culturing the cells can be used. Alternatively, the cells may be those cultured by changing the medium composition, temperature, pH, oxygen concentration, cell density, culture time, etc. from the normal culture conditions.

[0051] The culture temperature may be higher than normal culture conditions, or the culture containing cells may be transiently heated after culture and before the separation of extracellular vesicles. These operations are preferable in that they can promote the production of extracellular vesicles from living cells and increase the yield while suppressing the contamination and growth of miscellaneous bacteria. The heating temperature can be, for example, 40°C or higher, 50°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 90°C or higher, or 100°C or higher, and can be, for example, 121°C or lower, 100°C or lower, 90°C or lower, 80°C or lower, or 70°C or lower. The heating temperature can be, for example, 40 - 121°C, 50 - 121°C, 60 - 121°C, 65°C - 121°C, 70 - 100°C, or 80 - 90°C. In one embodiment, the extracellular vesicles can be extracellular vesicles separated through heat treatment of cells.

[0052] The culture after culture (for example, the culture medium, the culture supernatant, etc.) contains extracellular vesicles. The culture may be directly used in the extracellular vesicle separation step, or may go through the step of separating cells from the culture solution. In the steps after culture, the cells may be removed or killed, or the subsequent operations may be carried out in the co - existence of cells.

[0053] In one embodiment, the extracellular vesicles are separated from a culture containing living cells. This embodiment is preferable in that the yield of extracellular vesicles can be increased by performing heat treatment on the cells.

[0054] In one embodiment, the extracellular vesicles are separated from dead cells. This embodiment is preferable in that high - temperature heating is easy within the range that does not impair the effect of the active ingredient of the extracellular vesicles, thus enhancing storage stability and safety.

[0055] The culture may go through a concentration step, a drying step, a heating step, and a step of adding water or other solvents. When steps such as drying and heating are included, it is preferable in that contamination and growth of miscellaneous bacteria can be suppressed as described above.

[0056] As a method for separating extracellular vesicles from the cultured product after culturing, separation means utilizing the difference in size between extracellular vesicles and cells can be used. Such means include, for example, ultrafiltration, tangential flow filtration (TFF), centrifugation (e.g., ultracentrifugation, density gradient centrifugation, etc.), dialysis, size exclusion chromatography (SEC), etc., which can be used alone or in combination of two or more. Among them, ultrafiltration or tangential flow filtration is preferable, and tangential flow filtration using a hollow fiber membrane is more preferable in terms of the ability to continuously and massively separate extracellular vesicles. By using a hollow fiber membrane or a filter membrane with a cut-off value of 0.01 μm or more (e.g., 0.05 μm), for example, the extracellular vesicles according to the composition of the present invention can be separated and concentrated, and by using a membrane with a cut-off value of 0.2 μm, cells can be separated and removed from the extracellular vesicles. The extracellular vesicles of the composition of the present invention are preferably obtained by purifying the culture by filtration using a cut-off membrane of 0.001 to 0.2 μm (preferably 0.01 μm to 0.05 μm) or a cut-off membrane of 100 kDa to 1,000 kDa (preferably 100 kDa to 500 kDa).

[0057] Before, during, or after the above separation step, the extracellular vesicles may be concentrated and the dilution may be repeated at least once with another solution to remove or exchange the solvent, small molecules, etc. in the composition.

[0058] After the separation of extracellular vesicles, a further concentration step may be provided. The concentration step can be carried out by applying various means described in the above separation step using a membrane or centrifugation conditions suitable for concentrating extracellular vesicles. Alternatively, the solvent can be evaporated for concentration.

[0059] Furthermore, the method for producing the composition of the present invention may further include an addition step of a carrier, a purification step, a sterilization step, a freezing step, etc.

[0060] (Use) (1) Use for promoting the expression of genes related to extracellular matrix components As shown in the examples, the extracellular vesicles derived from the above bacteria or fungi may have an effect of promoting the expression of related genes of each extracellular matrix component of COL1A1, COL3A1, ELN, HAS1, and HAS2. Therefore, the composition of the present invention and the extracellular vesicles derived from the above bacteria or fungi are suitable for use in promoting the expression of at least one gene selected from the group consisting of COL1A1, COL3A1, ELN, HAS1, and HAS2.

[0061] The COL1A1 (Collagen Type I Alpha 1 chain) gene (NCBI Gene ID: 1277) encodes the pro-alpha1 chain of type I collagen. The COL3A1 (Collagen Type III Alpha 1 chain) gene (NCBI Gene ID: 1281) encodes the pro-alpha1 chain of type III collagen. The ELN gene (NCBI Gene ID: 2006) encodes elastin. HAS1 (NCBI Gene ID: 3036) and HAS2 (NCBI Gene ID: 3037) each encode different isoforms of hyaluronan synthase.

[0062] The promotion of the expression of each of the above genes more specifically includes the promotion of the expression of mRNA or protein, preferably including the promotion of the expression of mRNA.

[0063] In the use of promoting the expression of the COL1A1 gene, the origin of the extracellular vesicles of the composition of the present invention is preferably Weissmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, Escherichia coli, Pantoea agglomerans, or Saccharomyces cerevisiae, and more preferably Weissmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, or Saccharomyces cerevisiae.

[0064] In the use of promoting the expression of the COL3A1 gene, the extracellular vesicles of the composition of the present invention are preferably derived from Weissella coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, Pantoea agglomerans or Saccharomyces cerevisiae, more preferably from Weissella coagulans, Lactobacillus paracasei, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus or Saccharomyces cerevisiae, and even more preferably from Tetragenococcus halophilus.

[0065] In the use of promoting the expression of the ELN gene, the extracellular vesicles of the composition of the present invention are preferably derived from Weissella coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, Pantoea agglomerans or Saccharomyces cerevisiae, more preferably from Weissella coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus or Saccharomyces cerevisiae, and even more preferably from Lactobacillus paracasei, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus or Saccharomyces cerevisiae.

[0066] In the use for promoting the expression of the HAS1 gene, the origin of the extracellular vesicles of the composition of the present invention is preferably Weissmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pantoea agglomerans or Saccharomyces cerevisiae, more preferably Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Pantoea agglomerans or Saccharomyces cerevisiae, and still more preferably Lactobacillus paracasei or Saccharomyces cerevisiae.

[0067] In the use for promoting the expression of the HAS2 gene, the origin of the extracellular vesicles of the composition of the present invention is preferably Weissmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Escherichia coli, Pantoea agglomerans or Saccharomyces cerevisiae, and more preferably Pantoea agglomerans.

[0068] The cells targeted for the use of (1) are preferably fibroblasts, and more preferably skin fibroblasts.

[0069] <(Use for promoting the production of extracellular matrix components)> The composition of the present invention is also suitable for use in promoting the production of at least one extracellular matrix component selected from the group consisting of collagen, elastin and hyaluronic acid by the action of promoting the expression of the above genes. The preferred origin organisms of extracellular vesicles for the production of each extracellular matrix component are the same as those for the use of promoting the related genes of each extracellular matrix component described above. The cells targeted for the use of (2) are preferably fibroblasts, and more preferably skin fibroblasts.

[0070] <(Use for improving the physical properties or appearance of the skin, eyes, connective tissue, etc.)> As described in the Background Art section, collagen, elastin, and hyaluronic acid are involved in maintaining the physical properties or appearance of the skin, eyeballs, connective tissues, etc. Therefore, the composition of the present invention and the extracellular vesicles derived from the above-mentioned bacteria or fungi are also suitable for use in improving the physical properties or appearance of the skin, eyeballs, connective tissues, etc.

[0071] In one embodiment, the composition of the present invention is used for improving skin elasticity, skin flexibility, skin stretchability, skin wrinkles, skin sagging, skin moisture, skin protection, skin dryness, skin texture, skin gloss, skin firmness, or skin roughness. In a specific embodiment, the improvement of wrinkles is, for example, making fine wrinkles caused by dryness less noticeable, or improving wrinkle grades 1 to 3 or grades 3 to 5 of the "Evaluation Guidelines for Anti-Wrinkle Products for Obtaining New Efficacy" of the Japan Cosmetics Society (Journal of the Japan Cosmetics Society, 2006, Vol. 30, No. 4, pp. 316-332).

[0072] In one embodiment, the composition of the present invention is used for improving the sagging of the white part of the eye (conjunctiva) and the deformation of the vitreous body.

[0073] In one embodiment, the composition of the present invention is used for improving the function of tendons or ligaments. In one embodiment, the tissues targeted for each of the above uses (1) to (3) can be the skin, eyeballs, or connective tissues (for example, connective tissues such as tendons, ligaments, teeth, blood vessels, lungs, etc.). The tissues targeted for promoting the expression of each gene in the above (1) and the tissues targeted for promoting the production of each extracellular matrix component in the above (2) are preferably the skin or eyeballs, more preferably the skin, and even more preferably the dermis.

[0074] <(4) Use for promoting wound healing> As also shown in the examples, the extracellular vesicles derived from the above-mentioned bacteria or fungi have a wound healing promoting effect. In addition, since the extracellular vesicles derived from the above-mentioned bacteria or fungi promote the production of collagen and elastin, a wound healing promoting effect brought about by these components can be expected. Therefore, the composition of the present invention and the extracellular vesicles derived from the above-mentioned bacteria or fungi are suitable for use in promoting epithelial wound healing.

[0075] In this specification, "epithelium" refers to a layer of cells covering the surfaces inside and outside the living body. The epithelium is not particularly limited, and examples include the epithelium of the skin (epidermis), the epithelium of the eye (corneal epithelium, retinal pigment epithelium, conjunctival epithelium, etc.), or the mucosal epithelium covering body cavities and organs (for example, the oral cavity; the intestinal tract such as the small intestine and large intestine (colon, rectum); the stomach; the esophagus; the vagina, etc.).

[0076] In this specification, "wound" includes open wounds (a state where the continuity of the epithelium is disrupted) and non-open wounds (a state where the continuity of the epithelium is maintained). The cause of the injury is not particularly limited, and examples include external force, heat (burns, etc.), chemical substances, light, diseases (for example, inflammation, etc.). Among them, the composition of the present invention is preferably used for promoting the healing of open wounds.

[0077] In one embodiment, the composition of the present invention is used for improving diseases or disorders caused by skin wounds (for example, improving wounds, injuries, rough skin, cracks, red streaks, sensitive skin, and other skin diseases accompanied by wounds). In one embodiment, the composition of the present invention is used for improving diseases or disorders caused by corneal or conjunctival epithelial wounds (for example, improving corneal and conjunctival epithelial disorders, corneal wounds, conjunctival wounds, dry eye, and other corneal or conjunctival diseases accompanied by wounds).

[0078] <(5) Cell activation use> As shown by the scratch assay in the examples, the extracellular vesicles derived from the above-mentioned bacteria or fungi can promote the migration and proliferation of epithelial cells. Also, as shown in the examples, those extracellular vesicles promote the production of collagen and elastin, so promotion of cell migration, proliferation, extracellular matrix synthesis, etc. by the produced collagen and elastin can be expected. Therefore, the composition of the present invention and the extracellular vesicles derived from the above-mentioned bacteria or fungi have a cell activation effect. Therefore, the composition of the present invention and the above extracellular vesicles are suitable for cell activation uses. In one embodiment, the composition of the present invention and the above extracellular vesicles are applied to the use of epithelial cell activation.

[0079] In this specification, the activation of cells means that at least one selected from the group consisting of cell proliferation and migration is promoted. In the use of activating cells of the composition of the present invention, preferably both cell proliferation and migration are promoted.

[0080] The fact of having the epithelial wound healing promoting effect and cell activating effect described in the above (4) and (5) can be confirmed, for example, by the scratch assay described in the Examples.

[0081] <(6) Use for improving epithelial barrier function> The composition of the present invention can also be used for improving the barrier function of the epithelium. It is understood that the composition of the present invention can also improve the barrier function of blocking the influence from the outside world and preventing the loss of moisture, body fluids, etc. by promoting the healing of epithelial wounds.

[0082] In the uses of the above (4) to (6), the epithelium is preferably the skin or eye epithelium in each of the following uses, and more preferably the skin epithelium.

[0083] In the uses of the above (4) to (6), from the viewpoint of making the effects of the present invention more remarkable, the origin of the extracellular vesicles in the composition of the present invention is Weissmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, Escherichia coli, Pantoea agglomerans, or Saccharomyces cerevisiae, preferably Weissmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, or Pantoea agglomerans, more preferably Weissmania coagulans, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, or Pantoea agglomerans.

[0084] In one embodiment, in each of the above uses (1) to (6), the extracellular vesicles can be derived from organisms excluding Enterococcus faecalis, Enterococcus faecium, and Aspergillus oryzae among the options listed as suitable sources for each use, or from organisms excluding at least one selected from the group consisting of Enterococcus faecalis, Enterococcus faecium, and Aspergillus oryzae.

[0085] The reason why the extracellular vesicles derived from bacteria and / or fungi used in the present invention exhibit effects such as promoting the production of extracellular matrix components collagen, elastin, and hyaluronic acid, and promoting epithelial wound healing is not clear, but it is presumed that such functions are exerted by the cell wall components and the like derived from bacteria or fungi contained in the extracellular vesicles acting on the cells of a subject such as a human.

[0086] <Therapeutic use, non-therapeutic use> In each of the above uses (1) to (6), the composition of the present invention may be used for therapeutic purposes or for non-therapeutic purposes.

[0087] In this specification, "non-therapeutic use" refers to uses that do not include medical acts, for example, uses that do not include surgical, treatment, or diagnostic acts on a subject by a doctor or a person under the doctor's instruction (for example, uses related to health maintenance or promotion, or beauty).

[0088] (Subject) The subject to which the composition of the present invention is administered or ingested can be, for example, a human or a non-human animal [for example, non-human mammals (dogs, cats, hamsters, rabbits, ferrets, guinea pigs, cows, pigs, horses, goats, sheep, mice, rats, monkeys, etc.), reptiles, amphibians, birds, fish, etc.]. When the composition of the present invention is a pharmaceutical product, quasi-drug, medical device, cosmetic, or food or drink product, the subject is preferably a human. Also, when the composition of the present invention is a feed or pet food, the subject is preferably a non-human animal, more preferably a non-human mammal.

[0089] The age and gender of the subject are also not particularly limited.

[0090] When the subject is a human, in one embodiment, the subject is an adult, for example, an adult 40 years old or older, 50 years old or older, or 60 years old or older.

[0091] In each of the above uses (1) to (3), the subject is preferably (i) a person in whom a decrease in the gene expression of COL1A1, COL3A1, ELN, HAS1, or HAS2 is observed or who has a risk thereof, (ii) a person in whom a decrease in extracellular matrix components is observed or who has a risk thereof, or (iii) a person in whom a deterioration in the physical properties or appearance of the skin, eyeball, or connective tissue is observed or who has a risk thereof.

[0092] In each of the above uses (4) to (6), the subject is preferably (i) a person who has or is at risk of having an epithelial wound, (ii) a person who requires activation of epithelial cells (for example, a person who has or is at risk of having a decrease in epithelial cell function due to aging, malnutrition, epithelial diseases, etc.), or (iii) a person in whom the epithelial barrier function is decreased or who has a risk thereof.

[0093] The administration route of the composition of the present invention is not particularly limited, and examples thereof include transdermal administration, oral administration, ophthalmic administration, nasal administration, intradermal or subcutaneous administration, intravenous administration, intramuscular administration, enteral administration, vaginal administration, intraperitoneal administration, and inhalation administration. Transdermal administration, oral administration, or ophthalmic administration is preferred, and transdermal administration or oral administration is more preferred.

[0094] (Form of the composition) The composition of the present invention can be added to, for example, pharmaceuticals, quasi-drugs, medical devices (such as first aid bandages, contact lenses, etc.), cosmetics, food and drink products (such as health foods, supplements, foods with functional claims, foods for patients, foods for specified health uses, foods with nutritional functions, etc.), feeds, or pet foods and used for their production. Further, the composition of the present invention can be used as, for example, a pharmaceutical, a quasi-drug, a cosmetic, a food and drink product, a feed, or a pet food as it is.

[0095] The composition of the present invention can be, for example, a pharmaceutical product, quasi-drug, medical device, cosmetic, food or drink (health food, supplement, food with function claims, food for patients, food for specified health use, etc.), or feed, etc., which explicitly or implicitly indicates at least one of the uses (1) to (6) above as an efficacy, effect or function.

[0096] The composition of the present invention can be, for example, a pharmaceutical product, quasi-drug, medical device, cosmetic, food or drink (health food, supplement, food with function claims, food for patients, food for specified health use, etc.), or feed, etc., which explicitly or implicitly indicates a subject for which at least one of the uses (1) to (6) above is required or recommended.

[0097] As an indication that implies the efficacy, etc. related to the use (3) above, for example, those that express the efficacy, effect, etc. metaphorically (such as "plump skin", "for the skin of those in their teens and twenties", etc.); or Those that include words that imply application to abnormalities in the physical properties or appearance of the skin, eyeball or connective tissue such as loss of elasticity, firmness, flexibility or stretchability, formation of wrinkles, sagging, etc. (for example, "for the bothersome wrinkles", etc.); and the like.

[0098] As an indication that implies the efficacy, etc. related to the use (4) above, for example, those that include words that imply application to abnormalities in the epithelium accompanied by wounds (for example, "for wounds", "for those concerned about rough skin", etc.), and indications that show potential promotion of wound healing (for example, "enhance the skin's recovery ability", etc.). Here, among the abnormalities in the epithelium, the abnormalities in the skin epithelium include, for example, injuries, wounds, rough skin, cracks, red streaks, sensitive skin, and other skin diseases accompanied by wounds. The abnormalities in the corneal or conjunctival epithelium include, for example, corneal and conjunctival epithelial disorders, corneal wounds, conjunctival wounds, or dry eye, etc.

[0099] As an indication that implies the efficacy, etc. related to the use (5) above, for example, indications such as "make the epithelial tissue or epithelial cells look young", "activate", "stimulate", "energize", "activate", "revive".

[0100] The pharmaceutical product can be, for example, a topical pharmaceutical product, an oral preparation (capsule, tablet, chewable tablet, powder, granule, troche, liquid preparation, syrup, etc.), a transdermal absorbent, an injection, a suppository, an inhalant, etc.

[0101] When the composition of the present invention is a so-called topical preparation such as a topical pharmaceutical product, a quasi-drug, or a cosmetic, more specifically, basic cosmetics such as lotion, lotion, sunscreen cream, emulsion, cream, lotion, oil, and pack; makeup cosmetics such as foundation, lipstick, lip cream, mascara, eyeshadow, eyeliner, eyebrow pencil, and nail polish; cleansing agents such as facial wash, cleansing, and body wash; various topical compositions belonging to the fields of cosmetics, topical pharmaceutical products, or quasi-drugs such as antiperspirants, athlete's foot treatment agents, antipruritics, wound healing agents, cleansing agents, detergents, anti-inflammatory analgesics, acne treatment agents, hemorrhoid agents, bactericidal disinfectants, whitening agents, and ultraviolet protection agents. From the action effect on the skin, the present invention is preferably used for products applied to the skin such as topical skin preparations (preparations for the outer skin).

[0102] When the composition of the present invention is a topical preparation, its dosage form can be, for example, a liquid preparation, a suspension, an emulsion, a cream preparation, a gel preparation, a liniment preparation, a lotion preparation, an ointment, or an aerosol preparation. The dosage form of the composition of the present invention is preferably a liquid preparation, a suspension, an emulsion, a cream preparation, or a lotion preparation.

[0103] When the composition of the present invention is a food or drink, its form can be, for example, 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 pudding and yogurt, noodles such as pasta, ramen, udon, and soba, confectioneries, or spreads; or oral preparations (capsules, tablets, chewable tablets, powders, granules, troches, liquid preparations, syrups, etc.).

[0104] The composition of the present invention can be, for example, a preparation with the daily dosage or the dosage per administration shown below as one unit.

[0105] (Dosage) The daily dosage of the composition of the present invention can be appropriately determined according to the use, the state of the individual, body weight, gender, age, activity of the material, ingestion or administration route, ingestion or administration schedule, dosage form or other factors. Although not particularly limited, for each use, the dosages within the ranges exemplified below can be independently set.

[0106] In this specification, "dosage" represents the amount of the composition administered or ingested by an individual.

[0107] The daily dosage of the composition of the present invention can be, for example, 0.01 mg or more, 0.1 mg or more, 1 mg or more, 10 mg or more, or 100 mg or more per 1 kg of the subject's body weight, and can be, for example, 10 g or less, 5 g or less, 1 g or less, 500 mg or less, or 200 mg or less. The daily dosage of the composition of the present invention can be, for example, 0.01 mg to 10 g, 0.1 mg to 5 g, 1 mg to 1 g, 10 mg to 500 mg, or 100 mg to 200 mg per 1 kg of the subject's body weight.

[0108] The daily dosage of the composition of the present invention, in terms of the amount equivalent to protein, can be, for example, 0.1 pg or more, 1 pg or more, 10 pg or more, 100 pg or more, 1 ng or more, 10 ng or more, 100 ng or more, 1 μg or more, 10 μg or more, 100 μg or more, 1 mg or more, or 10 mg or more per 1 kg of the subject's body weight, and can be, for example, 1 g or less, 0.5 g or less, 100 mg or less, 50 mg or less, 20 mg or less, 10 mg or less, 5 mg or less, 2 mg or less, or 1 mg or less. The daily dosage of the composition of the present invention, in terms of the amount equivalent to protein, can be, for example, 0.1 pg to 1 g, 10 pg to 100 mg, 1 ng to 10 mg, or 100 ng to 1 mg per 1 kg of the subject's body weight.

[0109] The daily dosage of the composition of the present invention, in terms of the number of extracellular vesicles, can be, for example, 1×10 5 or more, 1×106 or more than 1 × 10 7 or more than 1 × 10 8 or more than 1 × 10 9 or more than 1 × 10 10 or more than 1 × 10, and for example, 1 × 10 16 or less than 1 × 10 15 or less than 1 × 10 14 or less than 1 × 10 13 or less than 1 × 10 12 or less than 1 × 10 11 or less than 1 × 10, and can be set as such. The daily dosage of the composition of the present invention, in terms of the number of extracellular vesicles, is, for example, 1 × 10 per 1 kg of the subject's body weight 5 ~1 × 10 16 or 1 × 10 6 ~1 × 10 15 or 1 × 10 7 ~1 × 10 14 or 1 × 10 8 ~1 × 10 13 or 1 × 10 9 ~1 × 10 12 or 1 × 10 10 ~1 × 10 11 or 1 × 10

[0110] When the subject is an adult human, in one embodiment, the daily dosage of the composition of the present invention per individual is, for example, determined based on the average body weight of an adult human being 60 kg. That is, in this case, the dosage per adult human individual can be calculated by multiplying the dosage per 1 kg of body weight by 60.

[0111] The frequency of ingestion or administration of the composition of the present invention can be appropriately determined according to the individual's condition, body weight, gender, age, activity of the active ingredient, ingestion or administration route, dosage, formulation form, or other factors. The frequency of ingestion or administration of the composition of the present invention can be, for example, once a month, once a week, once every three days, 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or any period and interval.

Example

[0112] Next, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

[0113] [Preparation of Extracellular Vesicles] The extracellular vesicles (EVs) shown in Table 1 were prepared and used in this test example.

[0114]

Table 1

[0115] EV1 to EV6: Commercially available products from Cosmo Bio Co., Ltd. (Catalog numbers: ECEV, LBEV01, LBEV-R2, LBEV-R1, YSEV-R5, LPSEV01) were used. EV7: Ten times the amount of purified water was added to dried baker's yeast (HB-P03; manufactured by Asahi Group Foods Co., Ltd.), and after extraction at 50°C for 2 hours, centrifugation was performed. The obtained supernatant was washed with 10 times the amount of purified water using a hollow fiber purification module and a hollow fiber membrane (C02-S05U-05-N: MWCO 0.05 μm, C02-P20U-05-N: MWCO 0.2 μm) (manufactured by Repligen). Then, it was powdered as extracellular vesicles. The extracellular vesicle powder was suspended in PBS to prepare a test sample. EV8, 9: 0.7 g of dead cells of Tetragenococcus halophilus (IBIRO1) (obtained from Ichibiki Co., Ltd.) was suspended in 25 mL of water, allowed to stand at 4°C overnight, and then centrifuged. The obtained supernatant was passed through a 0.22 μm syringe filter and then concentrated using a 100 kDa cut-off ultrafiltration membrane (Amicon Ultra-15). After washing three times with PBS, it was concentrated 10-fold to prepare a test sample. EV10: 0.7 g of heat-killed cells of Enterococcus faecium PTA-5844 WR strain (obtained from Wakamoto Pharmaceutical Co., Ltd.) was suspended in 25 mL of water, allowed to stand at room temperature for 1 hour, and then centrifuged. The obtained supernatant was passed through a 0.22-μm syringe filter and then concentrated using a 100-kDa cut-off ultrafiltration membrane (Amicon Ultra-15). Further, after washing 3 times with PBS, it was concentrated 10-fold to prepare a test sample. EV11: 0.7 g of heat-killed cells of Enterococcus faecium PTA-5844 WR strain was suspended in 25 mL of water, allowed to stand at 60°C for 1 hour, and then centrifuged. The obtained supernatant was passed through a 0.22-μm syringe filter and then concentrated using a 100-kDa cut-off ultrafiltration membrane (Amicon Ultra-15). Further, after washing 3 times with PBS, it was concentrated 10-fold to prepare a test sample. EV12: 19.7 mg of Aspergillus oryzae NK strain (obtained from Wakamoto Pharmaceutical Co., Ltd.) was aliquoted, distilled water was added to make it 1 mg / mL, shaken at room temperature for 90 minutes, centrifuged (2000 x g, 4°C, 10 minutes), the supernatant was collected, passed through a 0.22-μm syringe filter, and a test sample was prepared.

[0116] [Test Example 1. Confirmation Test of the Effect of Extracellular Vesicles Derived from Bacteria or Fungi on Promoting the Expression of Collagen and Elastin Genes] (Method) As shown below, first, normal human fibroblasts (derived from newborns) (HDF) were treated with various extracellular vesicles. The medium for HDF was DMEM (High Glucose) (manufactured by Gibco) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (×100). Next, the expression levels of collagen genes (COL1A1, COL3A1) and elastin gene (ELN) were measured by qPCR including a reverse transcription step for the mRNA amount. Then, the gene expression level relative to the control treated with phosphate-buffered saline (PBS) without extracellular vesicles was calculated. (1) In a 24-well plate, pre-cultured HDF was seeded at 1×10 5Seed to form cell / wells, and incubate in a CO 2 incubator at 37 °C for 3 days until semiconfluent (6.4×10 4 cells / well in the untreated group). (2) Add 1 / 100 volume (10 μL / well) of the stock solutions of EV1 - 11 in Table 1 to the medium, and culture for an additional 2 days. The test was performed using 3 wells (n = 3) for each extracellular vesicle. (3) Remove the medium from each well, wash, and then use the RNeasy Micro Kit (QIAGEN) to recover total RNA. (4) Using the obtained total RNA as a template, synthesize cDNA with the Prime Script TM RT reagent Kit (manufactured by Takara Bio Inc.). (5) Perform qPCR for the collagen gene and the elastin gene using the obtained cDNA. qPCR was performed using TB Green TM Premix Ex Taq TM II (Tli RNaseH Plus) (manufactured by Takara Bio Inc.). GAPDH was used as the internal standard. The ΔΔCt method was used to analyze the gene expression levels relative to the control. (6) Based on the relative expression values with the expression level of the control obtained for each extracellular vesicle set to 1, the expression promotion effect was evaluated as follows. The magnitude of the expression promotion effect is S > A > B > C > D. <Evaluation of expression promotion effect> S: Relative expression level is 2 or more A: Relative expression level is 1.5 or more and less than 2 B: Relative expression level is 1.3 or more and less than 1.5 C: Relative expression level is 1.1 or more and less than 1.3 D: Relative expression level is less than 1.1

[0117] (Results) The results of qPCR are shown in Table 2. It was found that EV1 - 11 promoted the expression of COL1A1. Also, it was found that EV2 - 4, 6 - 11 promoted the expression of COL3A1. EV2 - 11 was found to promote the expression of ELN.

[0118] [Table 2]

[0119] [Test Example 2. Confirmation Test on the Effect of Promoting the Expression of Hyaluronic Acid Synthase Gene in Extracellular Vesicles Derived from Bacteria or Fungi] (Method) The expression levels of hyaluronic acid synthase genes (HAS1, HAS2) were measured and analyzed. (i) As extracellular vesicles, EV1 - 6 and 10 - 11 were used for the expression analysis of HAS1 and HAS2. (ii) Tests were conducted with multiple stock solution addition amounts ranging from 1 / 10 to 1 / 10,000. (iii) Except that the cell density at the time of RNA recovery for each EV was 2×10 5 cells / well, the tests were carried out in the same manner as in Test Example 1.

[0120] (Results) The results of qPCR for HAS1 are shown in Table 3, and the results of qPCR for HAS2 are shown in Table 4. The EV dilution rate is the reciprocal of the addition ratio of the stock solution of extracellular vesicles to the medium. It was found that EV1 - 6 promoted the expression of both HAS1 and HAS2. EV10 and 11 were found to promote the expression of HAS1.

[0121] [Table 3]

[0122] [Table 4]

[0123] According to Test Examples 1 and 2, extracellular vesicles derived from bacteria or fungi such as EV1 to 11 were found to promote the expression of at least one gene selected from the group consisting of COL1A1, COL3A1, ELN, HAS1, and HAS2, which are genes involved in the production of extracellular matrix by fibroblasts. Therefore, by promoting the expression of these genes, extracellular vesicles derived from bacteria or fungi were shown to promote the production of extracellular matrix components such as collagen, elastin, and hyaluronic acid. Furthermore, it was suggested that they are suitable for improving the physical properties (elasticity, flexibility, stretchability, firmness, moisture, etc.) of the skin, eyes, and connective tissues, as well as improving the appearance (e.g., wrinkles, sagging, texture, gloss, rough skin, etc.).

[0124] [Test Example 4. Confirmation test of the wound healing promoting effect of extracellular vesicles derived from bacteria or fungi] (Scratch assay) For EV1 to 4, 6 to 8, 10 to 11, a scratch assay was performed using human epidermal cells (HaCaT) according to the following procedure. As the medium, DMEM (Low Glucose) (manufactured by Gibco) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (×100) was used. (1) Pre-cultured HaCaT was seeded in a 24-well plate at a density of 5×10 5 cells / well and cultured in a CO 2 incubator at 37°C for 6 days. (2) Using a 200 μL micropipette tip, a scratch of the same width was made in the center of the well on the obtained HaCaT monolayer. (3) After washing each well with 1 mL of phosphate buffered saline (PBS), a medium containing each EV stock solution at a volume of 1 / 1000 (v / v) or 1 / 10,000 was added and cultured for 24 hours. As a control, PBS was added to the medium instead of extracellular vesicles and the same treatment was performed. (4) After washing with 1 mL of PBS, it was fixed by treating with 4% paraformaldehyde overnight. After fixation, Giemsa staining solution (manufactured by Sigma-aldrich, code: 101.092040103) was added and incubated for 10 minutes, and then washed three times with distilled water. Thereafter, each well was dried. (5) Each well was photographed at three locations under a microscope (magnification: 4×), and the area of the unstained region (Open Area) was measured using image analysis software (ImageJ). For each group, when the average value of the area values at three locations of the unstained region immediately after scratch formation was set to 100, the scratch area (A Rel ) after culture was calculated. (6) According to the following formula, the reduction rate (ΔA Rel (%)) of the scratch area based on the control was determined and evaluated according to the following evaluation criteria. The magnitudes of the wound healing promotion effect, cell activation effect, and barrier function improvement effect are S > A > B > C > D. <ΔA Rel calculation formula> ΔA Rel (%) = {(A of the control Rel ) - (A of the treatment group Rel )} / (A of the control Rel ) × 100 <Evaluation criteria> S: ΔA Rel is 60 or more A: ΔA Rel is 40 or more and less than 60 B: ΔA Rel is 20 or more and less than 40 C: ΔA Rel is 10 or more and less than 20 D: ΔA Rel is less than 10

[0125] (Results) The results of each scratch assay are shown in Table 5.

[0126]

Table 5

[0127] In contrast, in the groups to which extracellular vesicles derived from bacteria or fungi such as EV1-4, 6-8, 10-11 were added, all showed lower values of A Rel and the evaluation of ΔΔ Rel was C or higher. Therefore, it was confirmed that extracellular vesicles derived from bacteria or fungi activate epithelial cells, promote proliferation or migration, and promote wound healing. And these extracellular vesicles were shown to have an effect of improving the epithelial barrier function based on the wound healing promoting effect and the cell activation effect.

[0128] [Test Example 5: Confirmation test of the effect of promoting the expression of elastin and collagen by extracellular vesicles derived from fungi] The expression levels of the collagen gene (COL1A1) and the elastin gene (ELN) of HDF treated with extracellular vesicles of fungi were measured and analyzed. (i) The point of using EV12 as extracellular vesicles, (ii) The point of adding extracellular vesicles so that there are 0, 10, 100 or 1000 extracellular vesicles per HDF1 cell, and the test was conducted with n = 2, (iii) The test was conducted in the same manner as Test Example 1 except that actin was used as the internal standard.

[0129] The results of qPCR are shown in Table 6. It was found that extracellular vesicles derived from fungi such as EV12 promote the expression of COL1A1 and ELN. Therefore, it was shown that by promoting the expression of these genes, extracellular vesicles derived from bacteria or fungi bring about the promotion of the production of extracellular matrix components such as collagen and elastin. Furthermore, it was suggested that it is suitable for improving the physical properties (elasticity, flexibility, stretchability, firmness, moisture, etc.) of the skin, eyes, and connective tissues, and improving the appearance (for example, wrinkles, sagging, texture, gloss, rough skin, etc.).

Table 6

Claims

1. A composition for promoting production of extracellular matrix components, comprising: Contains extracellular vesicles derived from bacteria and / or fungi, The composition, wherein the extracellular matrix component is at least one selected from the group consisting of collagen, elastin, and hyaluronic acid.

2. The composition according to claim 1 , wherein the enhanced production is in fibroblasts.

3. A composition for promoting expression of the COL1A1, COL3A1, ELN, HAS1 or HAS2 gene, comprising extracellular vesicles derived from bacteria and / or fungi.

4. A composition for improving the physical properties or appearance of skin, eye or connective tissue, comprising extracellular vesicles derived from bacteria and / or fungi.

5. 5. The composition according to claim 4, wherein the improvement in the physical properties or appearance of the skin is improvement in skin elasticity, skin softness, skin stretchability, skin wrinkles, skin sagging, skin moisture, skin protection, skin dryness, skin firmness, skin texture, skin luster, or skin roughness.

6. The composition according to any one of claims 1 to 5, wherein the bacteria is a gram-positive or gram-negative bacterium.

7. 7. The composition of claim 6, wherein the gram-positive bacterium is Weizmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, or Tetragenococcus halophilus.

8. 7. The composition of claim 6, wherein the gram-negative bacterium is Escherichia coli or Pantoea agglomerans.

9. The composition according to any one of claims 1 to 5, wherein the fungus is Saccharomyces cerevisiae or Aspergillus oryzae.

10. The composition according to any one of claims 1 to 5, which is a drug, a quasi-drug, a medical device, a cosmetic, or a food or drink.

11. A composition for promoting epithelial wound healing, comprising extracellular vesicles derived from bacteria and / or fungi.

12. A cell-stimulating composition comprising extracellular vesicles derived from bacteria and / or fungi.

13. The composition according to claim 12 , wherein the cell activation comprises promoting cell proliferation, promoting cell migration, or both.

14. A composition for improving epithelial barrier function, comprising extracellular vesicles derived from bacteria and / or fungi.

15. The composition according to any one of claims 11 to 14, wherein the bacteria is a gram-positive or gram-negative bacterium.

16. 16. The composition of claim 15, wherein the gram-positive bacterium is Weizmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, or Tetragenococcus halophilus.

17. 16. The composition of claim 15, wherein the gram-negative bacterium is Escherichia coli or Pantoea agglomerans.

18. The composition according to any one of claims 11 to 14, wherein the fungus is Saccharomyces cerevisiae or Aspergillus oryzae.

19. The composition according to any one of claims 11 to 14, which is a drug, a quasi-drug, a medical device, a cosmetic, or a food or drink.

20. A composition for external use comprising extracellular vesicles derived from bacteria and / or fungi.

21. The bacteria and / or fungi are selected from the group consisting of Weizmania coagulans, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecium, Tetragenococcus halophilus, Escherichia coli, Pantoea agglomerans, and the like.

21. The composition of claim 20, wherein the yeast strain is Saccharomyces agglomerans, Saccharomyces cerevisiae, or Aspergillus oryzae.

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