Exosome secretion-promoting composition, mitochondria-activating composition, longevity-associated gene-activating composition, foods and beverages, and pharmaceuticals

Abstract

The purpose of the present invention is to provide an exosome secretion-promoting composition for promoting exosome secretion from intestinal epithelial cells.　The exosome secretion-promoting composition for promoting exosome secretion from intestinal epithelial cells contains lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, a culture supernatant of the lactic acid bacteria, and / or an extract from the lactic acid bacteria as active ingredients.

Description

Compositions for promoting exosome secretion, compositions for activating mitochondria, compositions for activating longevity genes, foods and beverages, pharmaceuticals

[0001] The present invention relates to a composition for promoting exosome secretion, a composition for activating mitochondria, a composition for activating longevity genes, a food or beverage for promoting exosome secretion, and a pharmaceutical for promoting exosome secretion. More specifically, the present invention relates to a composition that promotes the secretion of exosomes having cosmetic and anti-aging effects, as well as a food or beverage and a pharmaceutical containing the said composition.

[0002] Exosomes are a type of extracellular vesicle secreted by most cell types. They are particles approximately 30-150 nm in size, covered by the same lipid bilayer membrane as cells. They contain nucleic acids such as mRNA and miRNA, as well as proteins such as enzymes. Exosomes secreted from cells have recently attracted attention for their role in intercellular interactions.

[0003] Exosomes secreted by mesenchymal stem cells derived from bone marrow cells and adipocytes are known to contain genes, proteins, and growth factors related to cell differentiation, proliferation, and regeneration, and expectations for the clinical application of exosomes are rising. For example, exosomes secreted by mesenchymal stem cells are known to show therapeutic effects against various diseases, and compositions for the treatment of kidney disease (Patent Document 1) and cosmetic compositions for skin whitening, wrinkle improvement, or skin regeneration (Patent Document 2) using exosomes as an active ingredient have been developed. Furthermore, it is known that promoting exosome secretion can lead to the suppression of skin cell aging (Patent Document 3) and the prevention of Alzheimer's disease (Patent Document 4).

[0004] Japanese Patent Publication No. 2023-501510, Japanese Patent Publication No. 2018-184446, Japanese Patent Publication No. 2021-187787, Japanese Patent Publication No. 2021-83415

[0005] Thus, exosomes are expected to act on various organs such as muscles, skin, and the brain (nerve cells), exerting cosmetic and anti-aging effects. Therefore, compositions that promote exosome secretion are thought to exhibit excellent cosmetic and anti-aging effects.

[0006] However, compositions that can promote exosome secretion have not yet been sufficiently investigated.

[0007] As a result of diligent research to solve the above problems, the inventors of this invention discovered that exosomes obtained using specific cells and lactic acid bacteria act on cells such as muscle cells and activate longevity genes, thus completing the present invention.

[0008] In other words, the present invention provides inventions relating to the following embodiments: [Claim 1] An exosome secretion promoting composition for promoting exosome secretion from intestinal epithelial cells, comprising as an active ingredient a lactic acid bacterium belonging to the genus Fructobacillus, a culture of the lactic acid bacterium, a culture supernatant of the lactic acid bacterium, and / or an extract of the lactic acid bacterium. [Claim 2] The exosome secretion promoting composition according to Claim 1, wherein the lactic acid bacterium is Fructobacillus fructosus. [Claim 3] The exosome secretion promoting composition according to Claim 1 or 2, wherein the lactic acid bacterium is Fructobacillus fructosus OS-1010. [Item 4] An exosome secretion-promoting composition according to any one of Items 1 to 3, wherein exosomes secreted from intestinal epithelial cells act on muscle cells to activate mitochondria, TFAM genes, or longevity genes. [Item 5] An exosome secretion-promoting composition according to any one of Items 1 to 4, wherein exosomes secreted from intestinal epithelial cells act on skin cells to activate mitochondria or longevity genes. [Item 6] An exosome secretion-promoting composition according to any one of Items 1 to 5, wherein exosomes secreted from intestinal epithelial cells act on skin cells to activate hyaluronic acid synthase genes, collagen synthase genes, MFAP-4 genes, elastin genes, or NAMPT genes. [Item 7] An exosome secretion-promoting composition according to any one of Items 1 to 6, wherein exosomes secreted from intestinal epithelial cells act on skin cells to suppress elastin-degrading enzyme genes or hyaluronic acid-degrading enzyme genes. [Claim 8] The exosome secretion promoting composition according to any one of Claims 5 to 7, wherein the skin cells are fibroblasts or epidermal cells. [Claim 9] The exosome secretion promoting composition according to any one of Claims 1 to 8, wherein exosomes secreted from intestinal epithelial cells act on nerve cells to activate mitochondria. [Claim 10] The mitochondria activating composition containing exosomes secreted from intestinal epithelial cells using the exosome secretion promoting composition according to any one of Claims 1 to 9.[Item 11] A composition for activating longevity genes, containing exosomes secreted from intestinal epithelial cells using the exosome secretion-promoting composition described in any one of Items 1 to 3. [Item 12] A food or beverage for promoting exosome secretion, containing the exosome secretion-promoting composition described in any one of Items 1 to 3. [Item 13] A pharmaceutical product for promoting exosome secretion, containing the exosome secretion-promoting composition described in any one of Items 1 to 3.

[0009] According to the present invention, it is possible to provide a novel type of exosome secretion-promoting composition for promoting exosome secretion from intestinal epithelial cells.

[0010] The exosome secretion-promoting composition of the present invention (hereinafter sometimes referred to as "the composition of the present invention") has the effect of causing intestinal epithelial cells to secrete exosomes that have mitochondrial activating effects and longevity gene (e.g., Sirt1 gene) activating effects.

[0011] Exosomes secreted from intestinal epithelial cells using the composition of the present invention have the effect of activating TFAM genes, longevity genes, and mitochondria in muscle cells. As a result, it may produce anti-aging effects such as suppression of muscle atrophy, suppression of muscle strength decline, and suppression of muscle cell aging. Specifically, it can be expected to suppress muscle strength decline and muscle mass reduction associated with aging and immobility.

[0012] Furthermore, exosomes secreted from intestinal epithelial cells using the composition of the present invention have the effect of activating mitochondria, longevity genes, hyaluronic acid synthase genes, collagen synthase genes, microfibril-binding protein 4 (MFAP-4) genes, elastin genes, and / or NAMPT genes in skin cells. In addition, exosomes secreted from intestinal epithelial cells using the composition of the present invention have the effect of suppressing elastin-degrading enzyme (Elastase) genes and / or hyaluronic acid-degrading enzyme genes. As a result, it is possible to achieve beauty-related effects such as suppression of skin cell aging, promotion of hyaluronic acid production, and promotion of elastin production. Specifically, anti-aging of the skin, skin regeneration, improvement of skin dullness, improvement of skin firmness, improvement of skin wrinkles, and improvement of pore visibility can be expected.

[0013] Furthermore, exosomes secreted from intestinal epithelial cells using the exosome secretion-promoting composition of the present invention have the effect of activating longevity genes and / or mitochondria in nerve cells. As a result, it may have the effect of suppressing the aging of nerve cells. Specifically, it can be expected to suppress or improve the decline of cognitive function (especially memory).

[0014] According to the present invention, lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria are provided as active ingredients for an exosome secretion promoting composition for promoting exosome secretion by intestinal epithelial cells.

[0015] More specifically, for example, lactic acid bacteria belonging to Fructobacillus fructosus, such as Fructobacillus fructosus strain NBRC-3516 and Fructobacillus fructosus strain OS-1010 (accession number: NITE BP-03818), cultures of the lactic acid bacteria, culture supernatants of the lactic acid bacteria, and / or extracts of the lactic acid bacteria are provided as active ingredients in an exosome secretion promoting composition for promoting exosome secretion by intestinal epithelial cells.

[0016] This figure shows the number of mitochondria in muscle cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the area of ​​mitochondria in muscle cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the membrane potential of mitochondria in muscle cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the Sirt1 gene expression level in muscle cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the TFAM gene expression level in muscle cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the number of mitochondria in skin cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the area of ​​mitochondria in skin cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the membrane potential of mitochondria in skin cells to which exosomes obtained in Test Example 2 were added, as a relative value with the control set to 1. This figure shows the Sirt1 gene expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the Sirt3 gene expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the hyaluronic acid synthase gene (HAS2) expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the type I collagen synthase gene (COL1A1) expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the microfibril-binding protein 4 (MFAP-4) gene expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the elastin gene (ELN) expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the NAMPT gene expression level in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1.This figure shows the expression level of the hyaluronic acid-degrading enzyme gene (HYAL1) in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the expression level of the elastin-degrading enzyme (Elastase) gene in skin cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the number of mitochondria in nerve cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the mitochondrial area in nerve cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1. This figure shows the mitochondrial membrane potential in nerve cells treated with exosomes obtained in Test Example 2, as a relative value with the control set to 1.

[0017] The exosome secretion-promoting composition for promoting exosome secretion from intestinal epithelial cells of the present invention (hereinafter referred to as "the exosome secretion-promoting composition of the present invention") contains as active ingredients a lactic acid bacterium belonging to the genus Fructobacillus, a culture of the lactic acid bacterium, the culture supernatant of the lactic acid bacterium, and / or an extract of the lactic acid bacterium. The exosome secretion-promoting composition will be described in detail below. Unless otherwise specified, terms used herein shall be interpreted in the sense commonly used in the art.

[0018] [1. Active Ingredients] The exosome secretion promoting composition of the present invention contains lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria as active ingredients.

[0019] [1-1. Lactic acid bacteria belonging to the genus Fructobacillus] Examples of lactic acid bacteria belonging to the genus Fructobacillus (hereinafter also referred to as "specified lactic acid bacteria") include Fructobacillus durionis, Fructobacillus tropaeoil, Fructobacillus fructosus, Fructobacillus americanaquae, and Fructobacillus apis. Fructobacillus apis), Fructobacillus broussonetiae, Fructobacillus cardui, Fructobacillus evanidus, Fructobacillus ficulneus, Fructobacillus papyriferae, Fructobacillus papyrifericola Examples include *Papyrifericola*, *Fructobacillus parabroussonetiae*, and *Fructobacillus pseudophyculneus*.

[0020] More specific examples of lactic acid bacteria belonging to the genus Fructobacillus include Fructobacillus fructosus, and among these, Fructobacillus fructosus strain NBRC3516 and Fructobacillus fructosus strain OS-1010 (NITE BP-03818) are mentioned, with Fructobacillus fructosus strain OS-1010 (NITE BP-03818) being particularly preferred.

[0021] [1-2. Form of the Active Ingredient] The form of the active ingredient in the present invention is at least one of the above-mentioned specified lactic acid bacteria, the culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and the extract of the lactic acid bacteria. The active ingredient may be used alone in any one of these forms, or in combination of multiple forms.

[0022] [1-2-1. Preparation of Lactic Acid Bacteria] When the active ingredient is in the form of lactic acid bacteria, the active ingredient includes the cellular components of the lactic acid bacteria and the products that are or were originally present in the lactic acid bacteria. The lactic acid bacteria may be either live or dead. In the case of dead bacteria, the cellular components may be crushed. In the case of crushed bacteria, some of the cellular components may be removed. Furthermore, the lactic acid bacteria may be in the form of a cellular powder dried by means of freeze-drying, shelf-drying, spray-drying, etc.

[0023] Lactic acid bacteria can be obtained by a manufacturing method that includes a step of culturing one or more of the above-mentioned specified lactic acid bacteria in a culture medium (sometimes referred to as the "culturing step"), and a step of separating and recovering the bacterial cells (sometimes referred to as the "separation and recovery step"). In the separation and recovery step, the separation and recovery method includes, for example, solid-liquid separation and bacterial cell washing. The manufacturing method may also include a step of drying the lactic acid bacteria cells (sometimes referred to as the "drying step"). Furthermore, the manufacturing method may also include a step of crushing the lactic acid bacteria (sometimes referred to as the "bacterial cell crushing step").

[0024] The culture medium used in the culture process includes a medium used for expansion culture (pre-culture medium) and a medium used for production culture (main culture medium). The main culture medium can be prepared by adding additives to the medium used as the pre-culture medium. The medium is preferably a liquid medium, but it may also be an agar medium. The medium contains a carbon source, and in addition to the carbon source, it also contains a nitrogen source, minerals, etc.

[0025] Carbon sources include carbohydrates and carbohydrate materials. Carbohydrates include sugars (monosaccharides, disaccharides, oligosaccharides), polysaccharides, and sugar alcohols. Specific examples of carbohydrates include lactose, sucrose, glucose, starch, xylitol, and dextrose. Carbohydrate materials can be any organic composition containing carbohydrates, such as milk and its processed products (skim milk powder, whey, milk powder, condensed milk, etc.), soy milk and its processed products (soy milk hydrolysate, etc.), grains, fruits, and vegetables. Milk can be derived from any mammal such as cows, goats, sheep, buffalo, camels, llamas, donkeys, yaks, horses, and reindeer. Carbohydrates may be isolated or contained in carbohydrate materials. For example, fructose (carbohydrate) may be used in the form contained in fruits (carbohydrate materials). Among these carbon sources, glucose is preferred. These carbon sources may be used individually or in combination of multiple types.

[0026] The concentration of the carbon source in the culture medium is not particularly limited and can be set appropriately depending on the type of culture medium and culture method, but for example, 0.5 to 4 w / w%, preferably 1 to 3 w / w%, and more preferably 1.5 to 2.5 w / w% can be cited.

[0027] Any inorganic or organic nitrogen source can be used as the nitrogen source. Examples include yeast extract (such as brewer's yeast), meat extract, proteins such as casein; protein hydrolysates such as peptone (such as protease peptone), peptides, and nitrogen-containing salts such as ammonium salts (such as ammonium citrate) and nitrates. These nitrogen sources may be used individually or in combination.

[0028] The concentration of the nitrogen source in the culture medium is not particularly limited and can be set appropriately according to the type of medium and culture method, but for proteins, for example, 0.3 to 4 w / w%, preferably 0.5 to 3 w / w%, and more preferably 1 to 2 w / w%; for peptides, for example, 0.1 to 2 w / w%, preferably 0.3 to 1.8 w / w%, and more preferably 0.5 to 1.5 w / w%; and for nitrogen-containing salts, for example, 0.03 to 1.5 w / w%, preferably 0.05 to 1 w / w%, and more preferably 0.1 to 0.5 w / w%.

[0029] Examples of minerals include manganese (e.g., manganese sulfate and other manganese salts), zinc, iron, sodium (e.g., sodium acetate and other sodium salts), potassium (e.g., dipotassium bisulfate, dipotassium hydrogen phosphate and other potassium salts), magnesium (e.g., magnesium sulfate and other magnesium salts), calcium, phosphorus (e.g., dipotassium hydrogen phosphate and other phosphates), sulfur (e.g., manganese sulfate, potassium bisulfate, magnesium sulfate and other sulfates), and trace elements. Among these minerals, manganese, sodium, magnesium, and potassium are preferred. These minerals may be used individually or in combination.

[0030] The concentration of minerals in the culture medium is not particularly limited and can be set appropriately according to the type of culture medium and culture method, but for example, in the case of manganese salts, examples include 0.001 to 0.01 w / w%, preferably 0.003 to 0.008 w / w%; for sodium salts, examples include 0.05 to 1.5 w / w%, preferably 0.1 to 1 w / w%; for magnesium salts, examples include 0.001 to 0.02 w / w%, preferably 0.005 to 0.015 w / w%; for potassium salts, examples include 0.05 to 1 w / w%, preferably 0.1 to 0.5 w / w%; for phosphates, examples include 0.05 to 1 w / w%, preferably 0.1 to 0.5 w / w%; and for sulfates, examples include 0.001 to 0.04 w / w%, preferably 0.005 to 0.02 w / w%.

[0031] In addition to the components mentioned above, the culture medium may also contain other components such as vitamins (e.g., B vitamins), surfactants (e.g., nonionic surfactants (e.g., Tween), anionic surfactants (e.g., SDS), antibacterial agents (e.g., triclosan), and antibiotics (e.g., monesin). These other components may be used individually or in combination. Among these other components, surfactants are preferred, and nonionic surfactants are more preferred.

[0032] The concentrations of other components in the culture medium are not particularly limited and can be set appropriately depending on the type of other components, the type of culture medium, the culture method, etc. However, if a surfactant is included, the concentration of the surfactant can be, for example, 0.01 to 0.5 w / w%, preferably 0.05 to 0.3 w / w%.

[0033] The culture conditions are not particularly limited, as long as they are conditions under which lactic acid bacteria belonging to the genus Fructobacillus can grow.

[0034] The culture temperature can be any temperature that is optimal for the lactic acid bacteria belonging to the genus Fructobacillus to be cultured, for example, 26 to 40°C, preferably 27 to 38°C, more preferably 28 to 36°C, and even more preferably 29 to 34°C. The culture time can be set appropriately according to the type of lactic acid bacteria belonging to the genus Fructobacillus to be cultured, for example, 4 to 48 hours, preferably 8 to 36 hours, and more preferably 12 to 24 hours.

[0035] Regarding the operations during cultivation, it is not necessary to stir the culture medium during cultivation. Furthermore, as an example of a specific cultivation procedure, the above-described cultivation can be performed as a production culture (main culture) for a certain period of time, and before that, an expansion culture (pre-culturing) can be performed in a small amount of medium (for example, 1 / 6 to 1 / 4 of the main culture medium by volume). The culture conditions for pre-culturing can be set appropriately according to the type of lactic acid bacteria belonging to the genus Fructobacillus, and the above-described conditions can be adopted. Furthermore, in the main culture, the culture obtained in the pre-culturing can be inoculated into the main culture medium such that the OD660 is, for example, 0.01 to 0.04, preferably 0.01 to 0.03.

[0036] In the separation and recovery step, solid-liquid separation of the culture solution can be carried out by filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dewatering, pressure dewatering, belt press, centrifugal concentration and dewatering, multi-disc dewatering, etc., and the obtained bacterial cells can be washed.

[0037] In the drying step, the bacterial cells can be dried using drying methods such as freeze drying, shelf drying, spray drying, etc.

[0038] In the cell disruption step, the cells are disrupted by an arbitrary method (for example, a disruption method by ultrasonic waves). The cells may be dried. In the cell disruption step, a part of the cell components may be removed from the disrupted cells.

[0039] [1-2-2. Cultures of lactic acid bacteria] When the active ingredient is in the form of a culture of lactic acid bacteria, the active ingredient includes the culture medium components containing the products of the lactic acid bacteria together with the lactic acid bacteria. The culture of lactic acid bacteria may be a culture produced using one of the above-mentioned predetermined lactic acid bacteria alone, or a culture produced using a combination of multiple species, or a mixture of a culture produced using one species alone and a culture produced using another species alone.

[0040] A part of the culture medium components used for culturing the lactic acid bacteria may be removed from the culture medium components contained in the culture. Also, the lactic acid bacteria contained in the culture may be either live bacteria or dead bacteria. In the case of dead bacteria, the cells may be disrupted. Also, when the cells are disrupted, a part of the cell components may be removed.

[0041] The culture of lactic acid bacteria can be obtained by a production method including the above-mentioned culturing step. The production method may include the above-mentioned separation and recovery step, drying step, and / or cell disruption step in addition to the culturing step. The details of each step are as described in the above "1-2-1. Preparation of lactic acid bacteria".

[0042] [1-2-3. Culture supernatant of lactic acid bacteria] When the active ingredient is in the form of the culture supernatant of lactic acid bacteria, the active ingredient contains the medium components containing the products of the lactic acid bacteria. The culture supernatant of lactic acid bacteria may be a culture supernatant produced by using one of the above-mentioned predetermined lactic acid bacteria alone, or a culture supernatant produced by using a combination of multiple species, or a mixture of a culture supernatant produced by using one species alone and a culture supernatant produced by using another species alone.

[0043] The medium components contained in the culture supernatant may have a part of the medium components used for the culture of the lactic acid bacteria removed.

[0044] The culture supernatant of lactic acid bacteria can be obtained by a production method including the above-mentioned culture step and separation and recovery step. In addition to the culture step and the separation and recovery step, the production method may include the above-mentioned drying step and / or cell disruption step. The details of each step are as described in the above "1-2-1. Preparation of lactic acid bacteria".

[0045] [1-2-4. Extract of lactic acid bacteria] When the active ingredient is in the form of an extract of lactic acid bacteria, the active ingredient is a multi-component composition obtained by subjecting the above-mentioned lactic acid bacteria or the culture of lactic acid bacteria to an extraction treatment. Examples of the extraction solvent used for the preparation of the extract include water; monohydric lower alcohols having 1 to 4 carbon atoms such as ethanol and isopropanol; polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, and glycerin; and polar solvents such as mixtures thereof. Further, the temperature condition during extraction is preferably room temperature (for example, 5 to 35°C, preferably 20 to 30°C). Specific methods for preparing the extract include, for example, a method of preparing a suspension containing the above-mentioned lactic acid bacteria or the culture of lactic acid bacteria or their pulverized products in an extraction solvent and obtaining an extract (extract solution) by solid-liquid separation of the suspension, or a method of obtaining a powder (extract powder) by drying the extract as necessary.

[0046] [1-3 Content of active ingredient] The content of the active ingredient contained in the composition for promoting exosome secretion of the present invention is not particularly limited and can be appropriately determined according to the effect to be imparted.

[0047] The amount of the active ingredient contained in the exosome secretion promoting composition of the present invention may be, for example, 0.00001 to 50% by weight, preferably 0.0001 to 5% by weight, based on the dry weight of lactic acid bacteria belonging to the genus Fructobacillus. The above dry weight equivalent refers to the dry weight of the active ingredient if the active ingredient is lactic acid bacteria or a culture of lactic acid bacteria; if the active ingredient is a culture supernatant of lactic acid bacteria, it refers to the dry weight of the lactic acid bacteria used to obtain the culture supernatant; and if the active ingredient is an extract, it refers to the dry weight of the lactic acid bacteria used to obtain the extract.

[0048] [2. Other Components] In addition to the above-mentioned active ingredients, the exosome secretion-promoting composition of the present invention may or may not contain other pharmacological components as needed. Examples of such pharmacological components include anti-inflammatory agents, antioxidants, bactericides, cooling agents, vitamins, mucopolysaccharides, and the like.

[0049] Furthermore, the exosome secretion-promoting composition of the present invention may or may not contain bases and / or additives as necessary to achieve a desired formulation. Such bases and / or additives are not particularly limited to the extent that they are pharmaceutically acceptable, but examples include aqueous bases such as water and monohydric lower alcohols having 1 to 4 carbon atoms (ethanol, isopropanol, etc.); oily bases such as naturally derived oils (vegetable oils, animal oils, processed oils thereof), mineral oils, ester oils, fatty acid alkyl esters, fatty acids, fatty acid esters, and higher alcohols; surfactants; polyhydric alcohols (glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, etc.); and additives such as cooling agents, preservatives, fragrances, colorants, viscosity modifiers, pH adjusters, wetting agents, stabilizers, antioxidants, UV absorbers, chelating agents, adhesives, buffers, solubilizers, and preservatives.

[0050] [3. Formulation and Product Classification] The formulation of the exosome secretion-promoting composition of the present invention is not particularly limited and may be liquid, semi-solid (cream, gel, ointment, paste), solid (granules, fine granules, powder, tablet, capsule), etc. Furthermore, the exosome secretion-promoting composition of the present invention may be a non-emulsified formulation such as an aqueous formulation or an oily formulation, or an emulsified formulation such as an oil-in-water emulsion or a water-in-oil emulsion.

[0051] Furthermore, product classifications of the exosome secretion-promoting composition of the present invention include food and beverages and pharmaceuticals. Specifically, the food and beverages of the present invention contain the exosome secretion-promoting composition. Also, the pharmaceuticals of the present invention contain the exosome secretion-promoting composition.

[0052] While not particularly limited to food and beverages, examples include fermented milk (such as drinkable yogurt), lactic acid bacteria beverages, milk beverages (such as coffee milk and fruit milk), tea-based beverages (such as green tea, black tea, and oolong tea), fruit and vegetable-based beverages (beverages containing fruit juices such as orange, apple, and grape, and vegetable juices such as tomato and carrot), alcoholic beverages (such as beer, sparkling wine, and wine), carbonated beverages, soft drinks, and water-based beverages; and processed foods such as fermented milk (such as set-type yogurt and soft yogurt), confectionery, instant foods, and seasonings.

[0053] Furthermore, functional foods can also be cited as food and beverages. Functional foods refer to foods that have a certain function on the living body, and examples include health functional foods such as Foods for Specified Health Uses (including conditionally designated FOSHU [Foods for Specified Health Uses]) and nutrient function foods, foods with functional claims, foods for special dietary uses, nutritional supplements, health supplements, supplements (for example, in various dosage forms such as tablets, coated tablets, sugar-coated tablets, capsules and liquids), and beauty foods (for example, diet foods). Moreover, functional foods may also be foods for special dietary uses such as foods for the sick, powdered milk for pregnant and lactating women, infant formula, foods for the elderly, and foods for nursing care.

[0054] Pharmaceuticals include oral preparations, as well as topical preparations (including quasi-drugs) that include skin preparations and mucosal preparations. Dosage forms of oral preparations include tablets, coated tablets, sugar-coated tablets, capsules, and liquids, while dosage forms of topical preparations include liquids (including lotions, sprays, aerosols, and emulsions), foams, ointments, creams, gels, and patches.

[0055] Among these product classifications, food and beverages are preferred from the viewpoint of further enhancing the effects of the present invention.

[0056] [4. Uses] The exosome secretion promoting composition of the present invention is used for purposes such as inhibiting muscle atrophy, inhibiting muscle weakness, inhibiting muscle cell aging, anti-aging of the skin, skin regeneration, improving skin dullness, improving skin firmness, improving skin wrinkles, improving the appearance of pores, maintaining and improving cognitive function, and maintaining and improving memory.

[0057] [5. Dosage] When the exosome secretion-promoting composition of the present invention is administered orally, the amount of the active ingredient is, for example, 0.01 to 200 mg / kg / day, preferably 0.1 to 20 mg / kg / day, and can be administered orally 1 to 5 times a day.

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0059] [Test Example 1: Preparation of Lactic Acid Bacteria Powder] Fructobacillus fructosus OS-1010 strain was inoculated into 30 ml of MRS medium (pre-culture medium) manufactured by Difco, and cultured statically at 30°C for 24 hours. The resulting culture solution was inoculated into 1 L of MRS medium (main culture medium) so that the OD660 was 0.02, and cultured with stirring at 30°C for 12 hours at pH 6.0-7.0. The resulting culture solution was subjected to centrifugation to recover the bacterial cells. The recovered bacterial cells were washed with 1 L of 0.85 w / w% KCl aqueous solution. The washed bacterial cells were subjected to centrifugation again to recover the bacterial cells. After sterilization of the recovered bacterial cells, freeze-drying was performed to obtain lactic acid bacteria powder.

[0060] [Test Example 2: Adjustment of Caco-2 cell secreted exosomes etc. following lactic acid bacteria treatment] Caco-2 cells 1 × 10６ Seeds were sown in 100 mm dishes to achieve a cells / dish ratio, and then incubated in DMEM medium containing 10% FBS at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under the specified conditions. After culturing, a suspension of lactic acid bacteria powder suspended in PBS was prepared, and the suspension was added to the culture medium to achieve a concentration of 100 μg / mL of lactic acid bacteria powder. After culturing, the cell culture medium was collected in a tube, and cells were separated from the culture supernatant by centrifugation at 300 G for 5 minutes. Next, cell fragments were separated by centrifugation at 1200 G for 20 minutes, and extracellular vesicles larger than exosomes were separated by further centrifugation at 10000 G for 30 minutes.

[0061] The culture supernatant was concentrated 10-fold by ultrafiltration, and then exosomes were purified using the MagCapture Exosome Isolation PS Kit ver. 2 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the prescribed procedure. The purified exosomes were quantified using the MicroBCA Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.) as protein equivalents. These are referred to as "lactic acid bacteria-treated exosomes."

[0062] Next, in the preparation of the lactic acid bacteria-treated exosomes described above, exosomes were obtained in the same manner as before, except that the same amount of PBS used was added to the culture medium after culturing Caco-2 cells, without adding the FBS suspension of lactic acid bacteria powder. In the following experiments, the samples using these exosomes were used as the control.

[0063] [Effects on muscle cells] (1-1. Culture of muscle cells) Mouse skeletal muscle cells, C2C12 cells, were used as a skeletal muscle cell model. 2 × 10 ５ Seeds were sown in a 6-well plate to achieve a cells / well ratio, and then incubated in DMEM medium containing 10% FBS at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under the specified conditions. After culturing, the culture medium was replaced with DMEM medium containing 2% horse serum and 5% CO2. ２Differentiation induction was performed by culturing under specific conditions. The culture medium was changed every two days, and on the 10th day after differentiation induction, lactic acid bacteria-treated exosomes obtained in Test Example 2 were added to a final concentration of 180 ng / ml, and the culture was continued for another 24 hours.

[0064] (1-2. Confirmation of effects on mitochondria) The cells obtained in 1-1 were stained using the following method, and the number, area, and membrane potential activity of mitochondria were quantified. The cells were stained with 250 nM MitoTracker Red CMXRos (Thermo Fisher Scientific Co., Ltd.) at 37°C for 30 minutes, and then stained with 200 nM MitoTracker Green FM (Thermo Fisher Scientific Co., Ltd.) at 37°C for 30 minutes. Finally, the cells were stained with Hoechst 33342 (Dojin Chemical Laboratories Co., Ltd.) at 37°C for 30 minutes. The stained cells were analyzed using IN Cell Analyzer 2200 (Cytiva Corporation) to quantitatively analyze the number of mitochondria, mitochondrial area, and mitochondrial membrane potential. The results of the control obtained by performing the same procedure except for the change in exosomes were set to 1, and the relative values ​​of each were calculated. The results are shown in Figures 1 to 3.

[0065] As shown in Figures 1 to 3, the number of mitochondria, mitochondrial area, and mitochondrial membrane potential of muscle cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added were all improved compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have a function of activating mitochondria, as they improve the number of mitochondria, mitochondrial area, and mitochondrial membrane potential of muscle cells.

[0066] (1-3. Confirmation of the effect on gene expression levels) RNA was prepared from the cells obtained in 1-1 using the High Pure RNA Isolation Kit (manufactured by Roche Diagnostics K.K.) according to the prescribed procedure. RT-qPCR was performed using the GoTaq 1-Step RT-PCR System (manufactured by Promega Corporation) according to the prescribed procedure. The gene expression levels were relativized using the β-actin gene expression level as an internal standard for each gene expression level, and the gene expression levels were calculated. The control result was set to 1, and the relative values ​​were determined. The results are shown in Figures 4 and 5.

[0067] As shown in Figure 4, the Sirt1 gene expression level in muscle cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was higher than that of the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving Sirt1 gene expression.

[0068] As shown in Figure 5, the expression levels of TFAM genes, which are transcription factors responsible for mitochondrial DNA transcription and replication, and mitochondrial metabolic regulation, were improved in muscle cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added, compared to the control group. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving TFAM gene expression levels.

[0069] [Effects on skin cells] (2-1. Culture of skin cells) Immortalized Human Skin Fibroblast (Appled Biological Materials, Richmond, BC, Canada), a type of skin cell, was used. 3 × 10 ４ Seeds were sown in 96-well plates to achieve a cells / well ratio, and then incubated in DMEM medium containing 10% FBS at 37°C and 5% CO2. ２ The cells were cultured for 24 hours under the specified conditions. After culturing, the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added to a final concentration of 450 ng / ml, and the cells were cultured for another 24 hours.

[0070] (2-2. Confirmation of effects on mitochondria) The number of mitochondria, mitochondrial area, and mitochondrial membrane potential of dermal fibroblasts obtained in 2-1 were quantitatively analyzed using the same method as described in 1-2. The control results were set to 1 for calculations, and the relative values ​​for each were determined. The results are shown in Figures 6 to 8.

[0071] As shown in Figures 6 to 8, the number of mitochondria, mitochondrial area, and mitochondrial membrane potential of dermal fibroblasts to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added were all improved compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of activating mitochondria, as they improve the number of mitochondria, mitochondrial area, and mitochondrial membrane potential of skin cells.

[0072] (2-3. Confirmation of the effect on gene expression levels) RNA was recovered from the cells obtained in 2-1 using the same method as described in 1-3, and the expression levels of various genes were measured. The gene expression levels of each gene were relativized using the gene expression level of β-actin as an internal standard, and the gene expression levels were calculated. The control result was set to 1, and the relative values ​​of each gene expression level were determined. The results are shown in Figures 9 to 17.

[0073] As shown in Figure 9, the Silt1 gene expression level in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was higher than that of the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the Silt1 gene expression level in skin cells.

[0074] As shown in Figure 10, the Silt3 gene expression level in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was higher than that of the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the Silt3 gene expression level in skin cells.

[0075] As shown in Figure 11, the expression level of hyaluronic acid synthase gene (HAS2) in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was higher than that of the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the expression level of hyaluronic acid synthase gene (HAS2) in skin cells.

[0076] As shown in Figure 12, the expression level of type I collagen synthase gene (COL1A1) in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was improved compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the expression level of type I collagen synthase gene (COL1A1) in skin cells.

[0077] As shown in Figure 13, the expression level of the MFAP-4 gene, which is essential for elastin formation in skin cells, was improved when lactic acid bacteria-treated exosomes obtained in Test Example 2 were added compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the expression level of the MFAP-4 gene in skin cells.

[0078] As shown in Figure 14, the expression level of the elastin gene (ELN), which is essential for elastin production in skin cells, was improved when lactic acid bacteria-treated exosomes obtained in Test Example 2 were added compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the expression level of the elastin gene (ELN) in skin cells.

[0079] As shown in Figure 15, the expression level of the NAMPT gene, an NMN synthase, in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was improved compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of improving the expression level of the NAMPT gene in skin cells.

[0080] As shown in Fig. 16, the expression level of the hyaluronidase gene (HYAL1) in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was lower than that of the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of reducing the expression level of the hyaluronidase gene (HYAL1) in skin cells.

[0081] As shown in Fig. 17, the expression level of the elastase gene in skin cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added was lower than that of the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have the function of reducing the expression level of the elastase gene in skin cells.

[0082] As can be understood from the results of Figs. 9 to 17, the lactic acid bacteria-treated exosomes obtained in Test Example 2 enhance the synthesis of proteins related to skin elasticity and firmness and further suppress the degradation of those proteins. Therefore, it can be said that they have the effect of improving or maintaining skin elasticity and firmness.

[0083] [Effect on nerve cells] (3-1. Cultivation of nerve cells) SH-SY5Y cells, which are human neuroblastoma cells, were used as human nerve model cells. They were seeded in a 96-well black plate at 6×10 ３ cells / well and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO ２ for 24 hours under the conditions. After cultivation, the lactic acid bacteria-treated exosomes recovered in Test Example 2 were added so that the final concentration was 900 ng / ml, and they were further cultured for 24 hours.

[0084] (3-2. Confirmation of the effect on mitochondria) In the same manner as the method shown in 1-2, the number, area, and mitochondrial membrane potential of the mitochondria of the nerve cells obtained in 3-1 were quantitatively analyzed. The results of the control obtained by performing the same operations except for changing the exosomes were taken as 1 for calculation, and the respective relative values were obtained. The results are shown in Figs. 18 to 20.

[0085] As shown in Figures 18 to 20, the number of mitochondria, mitochondrial area, and mitochondrial membrane potential of nerve cells to which the lactic acid bacteria-treated exosomes obtained in Test Example 2 were added were all improved compared to the control. From this, it can be said that the lactic acid bacteria-treated exosomes obtained in Test Example 2 have a function of activating mitochondria, as they improve the number of mitochondria, mitochondrial area, and mitochondrial membrane potential of nerve cells.

Claims

1. An exosome secretion promoting composition for promoting exosome secretion from intestinal epithelial cells, comprising as an active ingredient lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria.

2. The exosome secretion promoting composition according to claim 1, wherein the lactic acid bacterium is Fructobacillus fructosus.

3. The exosome secretion promoting composition according to claim 1 or 2, wherein the lactic acid bacterium is Fructobacillus fructosus OS-1010.

4. The exosome secretion promoting composition according to claim 1 or 2, wherein exosomes secreted from intestinal epithelial cells act on muscle cells to activate mitochondria, TFAM genes, or longevity genes.

5. The exosome secretion promoting composition according to claim 1 or 2, wherein exosomes secreted from intestinal epithelial cells act on skin cells to activate mitochondria or longevity genes.

6. The exosome secretion promoting composition according to claim 1 or 2, wherein exosomes secreted from intestinal epithelial cells act on skin cells to activate hyaluronic acid synthase genes, collagen synthase genes, MFAP-4 genes, elastin genes, or NAMPT genes.

7. The exosome secretion promoting composition according to claim 1 or 2, wherein exosomes secreted from intestinal epithelial cells act on skin cells to suppress elastin-degrading enzyme genes or hyaluronic acid-degrading enzyme genes.

8. The exosome secretion promoting composition according to claim 5, wherein the skin cells are fibroblasts or epidermal cells.

9. The exosome secretion promoting composition according to claim 1 or 2, wherein exosomes secreted from intestinal epithelial cells act on nerve cells to activate mitochondria.

10. A composition for activating mitochondria, comprising exosomes secreted from intestinal epithelial cells using the exosome secretion-promoting composition according to claim 1 or 2.

11. A composition for activating longevity genes, comprising exosomes secreted from intestinal epithelial cells using the exosome secretion-promoting composition according to claim 1 or 2.

12. Food or beverage for promoting exosome secretion containing the exosome secretion-promoting composition according to claim 1 or 2.

13. A pharmaceutical product for promoting exosome secretion containing the exosome secretion-promoting composition according to claim 1 or 2.

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