Exosome production promoter and composition containing the exosome production promoter

Abstract

To provide an exosome production promoter that is highly capable of promoting the production of exosomes in vivo, while ensuring high safety, and also to provide an exosome production promoter-containing composition that contains the exosome production promoter.SOLUTION: An exosome production promoter contains a fermented product yielded by fermenting a composition containing soybean extract, defatted rice bran, and yeast extract with Bacillus subtilis. Preferably, the Bacillus subtilis is Bacillus subtilis var natto. Also preferably, the solid content of the yeast extract in the composition is 0.1 mass% or more and 1.0 mass% or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an exosome production promoter and a composition containing the exosome production promoter. [Background technology]

[0002] It is known that membrane vesicles (extracellular vesicles) secreted by cells exist in a variety of particles, differing in their generation mechanism, size, and constituent molecules.

[0003] In particular, exosomes derived from endosomes have recently been recognized as beauty ingredients that are incorporated into cosmetics and other products.

[0004] When exosomes are taken up by fibroblasts, they exert various cosmetic effects, such as promoting cell proliferation and wound healing.

[0005] Cosmetic compositions for skin regeneration containing exosomes, particularly stem cell-derived exosomes, which are expected to have such beneficial effects on the skin, have been proposed (see, for example, Patent Document 1).

[0006] However, isolating and purifying exosomes themselves is technically difficult, and the amount that can be produced at one time is extremely small, making them very expensive.Furthermore, there are safety concerns regarding compositions containing purified exosomes derived from stem cells.

[0007] To solve these problems, efforts are underway to develop exosome production promoters that can promote exosome production in vivo without administering exosomes themselves; however, no such promoters have been found that can sufficiently promote exosome production. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 2018-184446 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide an exosome production promoter that has a high ability to promote exosome production in the body and is highly safe, and to provide an exosome production promoter-containing composition that contains the exosome production promoter. [Means for solving the problem]

[0010] These objects can be achieved by the present invention as set forth in (1) to (6) below. (1) A fermented product obtained by fermenting a composition containing soybean extract, defatted rice bran, and yeast extract with Bacillus subtilis. death, The proportion of the solid content of the fermented product in the total solid content is 0.1% by mass or more and 2.0% by mass or less. An exosome production promoter characterized by:

[0011] (2) The exosome production promoter according to (1) above, wherein the Bacillus subtilis is Bacillus subtilis natto.

[0012] (3) The exosome production promoter according to (1) or (2) above, wherein the content of the solid content of the yeast extract in the composition is 0.1% by mass or more and 1.0% by mass or less.

[0013] (4) A composition containing an exosome production promoter, characterized by containing the exosome production promoter according to any one of (1) to (3) above.

[0014] (5) The composition containing an exosome production promoter according to (4), wherein the composition is liquid.

[0015] (6) The composition containing an exosome production promoter according to (4) or (5) above, wherein the composition is an external preparation. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide an exosome production promoter that has a high ability to promote exosome production in the body and is highly safe, and to provide an exosome production promoter-containing composition that contains the exosome production promoter. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the relative expression levels of CD63 in each example and each comparative example relative to the control. [Figure 2] FIG. 2 is a diagram showing the relative expression levels of CD9 in each Example and Comparative Example relative to the control. DETAILED DESCRIPTION OF THE INVENTION

[0018] Preferred embodiments of the present invention will be described in detail below. [1] Exosome production promoter First, the exosome production-promoting agent of the present invention will be described.

[0019] The exosome production promoter of the present invention is characterized by containing a fermented product obtained by fermenting a composition containing soybean extract, defatted rice bran, and yeast extract with Bacillus subtilis.

[0020] This makes it possible to provide an exosome production promoter that has high exosome production-promoting ability in the body and is highly safe.

[0021] [1-1] Fermented products The exosome production promoter of the present invention contains at least the fermented product, i.e., a fermented product obtained by fermenting a composition containing soybean extract, defatted rice bran, and yeast extract with Bacillus subtilis.

[0022] [1-1-1] Composition to be subjected to fermentation The composition to be fermented by Bacillus subtilis, i.e., a composition containing soybean extract, defatted rice bran, and yeast extract, will be described below.

[0023] [1-1-1-1] Soybean extract The composition subjected to fermentation by Bacillus subtilis comprises a soy extract.

[0024] The soybean extract is an extract of soybeans. More specifically, the soybean extract may be, for example, an extract obtained by extracting soybeans with a solvent (water, hot water, an organic solvent such as alcohol (ethanol, etc.), or a water-organic solvent mixture).

[0025] There are no particular limitations on the extraction method or conditions for obtaining soybean extract. For example, the soybeans used as the raw material for extraction may be uncrushed or crushed. Furthermore, as long as the quality of the extract can be maintained, pretreatment such as impurity removal may be performed. In addition to water or hot water, the extraction solvent may also include alcohols such as methanol and ethanol, esters such as ethyl acetate, organic solvents such as n-hexane, and mixtures of these organic solvents with water or hot water. Preferred extraction solvents are water or hot water, alcohols (e.g., methanol and ethanol), and mixtures of water or hot water with alcohol.

[0026] The temperature of the hot water is, for example, 40°C or higher and 100°C or lower, preferably 50°C or higher and 80°C or lower, and more preferably 50°C or higher and 70°C or lower.

[0027] The pH of the extraction solvent during extraction is, for example, 3 or more and 7 or less, preferably 4 or more and 6 or less, and more preferably 4 or more and 5 or less. This allows the stability of the various components contained in the extraction raw material to be maintained.

[0028] The extraction temperature is not particularly limited, but room temperature or heated extraction is preferred. In the case of heated extraction, the heating temperature is, for example, 40°C to 100°C, preferably 50°C to 80°C, and more preferably 50°C to 70°C. This allows for efficient extraction.

[0029] The soybean extract preferably has a Brix value of 10.0 or more and 20.0 or less, more preferably 12.0 or more and 18.0 or less, and even more preferably 13.0 or more and 17.0 or less.

[0030] This allows the solid content of soybean extract in the composition to be suitably high, and eliminates the need for excessive concentration or other treatments during preparation of the soybean extract, which is advantageous from the standpoints of productivity, cost, etc. Furthermore, it is possible to more effectively prevent unintended deterioration, denaturation, etc. of the constituent components during concentration, etc. The Brix value is a measure of the solid content concentration in the soybean extract.

[0031] The soybean extract preferably contains raffinose and stachyose, which are oligosaccharides specific to soybeans. This promotes the growth of the bacteria and further improves the production of fermentation metabolites. Hereinafter, raffinose and stachyose will be collectively referred to as "soybean oligosaccharides."

[0032] When the soybean extract contains soybean oligosaccharides, the content of soybean oligosaccharides in the composition is preferably 0.5% by mass or more and 10.0% by mass or less, and more preferably 1.5% by mass or more and 4.0% by mass or less.

[0033] This shortens the lag period of the bacteria, and as a result, the fermentation process can be completed in a shorter time.

[0034] The solid content of the soybean extract in the composition is preferably 10% by mass or more and 25% by mass or less, and more preferably 13% by mass or more and 20% by mass or less. This significantly improves the production of fermentation metabolites and reduces costs.

[0035] [1-1-1-2]Defatted rice bran The composition subjected to fermentation by Bacillus subtilis comprises defatted rice bran. Rice bran normally contains about 20% by mass of oil, and defatted rice bran is obtained by subjecting rice bran to a degreasing process.

[0036] The use of such defatted rice bran not only serves as a source of high-quality nitrogen and vitamins that promote bacterial growth, but also eliminates the growth-inhibiting effect of oxidized oil on bacteria.

[0037] The defatted rice bran preferably has an oil content of 15% by mass or less, more preferably 13% by mass or less. This makes the above-mentioned effects more pronounced.

[0038] The rice variety used as the raw material for defatted rice bran is not particularly limited, and examples include Koshihikari, Hinohikari, Yumehikari, Hoshinoyume, Hitomebore, Akitakomachi, Yukihikari, Nanatsuboshi, and Hakucho, as well as pigmented rice such as black rice (purple black rice) such as Kitanomurasaki, Meosakuramurasaki, Asamurasaki, and Okunomurasaki, and red rice such as Beniroman, Beni-goromo, Yuyakemochi, and Benisomemochi.

[0039] The content of defatted rice bran in the composition is preferably 0.003% by mass or more and 0.080% by mass or less, more preferably 0.005% by mass or more and 0.070% by mass or less, and even more preferably 0.010% by mass or more and 0.050% by mass or less.

[0040] This shortens the lag period of the bacteria, and as a result, the fermentation process can be completed in a shorter time.

[0041] When the soybean extract contains soybean oligosaccharides, when the content of soybean oligosaccharides in the composition is XP [% by mass] and the content of defatted rice bran in the composition is XB [% by mass], it is preferable that the relationship between XP and XB is 30≦XP / XB≦400, and it is more preferable that the relationship between XP and XB is 130≦XP / XB≦200.

[0042] This shortens the lag period of the bacteria, thereby shortening the fermentation process and also increasing the production of fermentation metabolites.

[0043] [1-1-1-3] Yeast extract The composition subjected to fermentation by Bacillus subtilis comprises yeast extract.

[0044] Yeast extract is an extract of yeast. For example, yeast extract can be obtained by extracting cultured yeast through autolysis.

[0045] Autolysis of yeast can be carried out by known methods. For example, yeast recovered after completion of culture is suspended in water or a buffer solution to obtain a yeast suspension, which is used as an autolysis liquid. By keeping this autolysis liquid at 30°C or higher and 55°C or lower, the yeast can be decomposed by enzymes originally present in the yeast cells and extracted.

[0046] Autolysis may be carried out under controlled or uncontrolled conditions, with the pH of the yeast suspension preferably being in the range of 4 to 7.

[0047] Examples of the yeast include bacteria of the genus Saccharomyces, Shizosaccharomyces, Pichia, Candida, Kluyveromyces, Williopsis, Debaryomyces, Galactomyces, Torulaspora, Rhodotorula, Yarrowia, and Zygosaccharomyces.

[0048] Among these, Candida tropicalis, Candida lypolitica, Candida utilis, Candida sake, and Saccharomyces cerevisiae are preferred, and Saccharomyces cerevisiae and Candida utilis are more preferred.

[0049] The yeast may be a natural yeast (yeast whose genes have not been artificially modified) or a mutant strain.

[0050] The culture format is not particularly limited and can be appropriately determined depending on, for example, the culture scale, the intended use of the obtained culture, and the like.

[0051] The solid content of the yeast extract in the composition is preferably 0.1% by mass or more and 1.0% by mass or less, more preferably 0.2% by mass or more and 0.9% by mass or less, and even more preferably 0.3% by mass or more and 0.8% by mass or less.

[0052] This allows for more optimal supply of trace elements such as vitamins, minerals, and amino acids that promote bacterial growth, thereby further increasing the amount of metabolic products produced.

[0053] The content of amino nitrogen derived from the yeast extract in the composition is preferably 0.005% by mass or more and 0.070% by mass or less, more preferably 0.010% by mass or more and 0.063% by mass or less, and even more preferably 0.015% by mass or more and 0.021% by mass or less. This makes it possible to more suitably increase the amount of metabolites produced.

[0054] The amino nitrogen content can be determined by the Pope-Stevens cuprate method.

[0055] [1-1-1-4] Other ingredients The composition to be fermented by Bacillus subtilis may contain ingredients other than those mentioned above. Examples of such ingredients include corn steep liquor, blackstrap molasses, wheat bran, soybean pulp, casein peptone, meat peptone, soy peptone, casamino acids, vitamins, glycerin fatty acid esters, sucrose fatty acid esters, silicone resins, etc.

[0056] [1-1-2] Bacillus subtilis The Bacillus subtilis used for fermenting the composition will now be described.

[0057] Bacillus subtilis is a representative type of aerobic spore-forming bacteria.

[0058] The Bacillus subtilis is not particularly limited, but for example, Bacillus natto can be suitably used. This makes it possible to more suitably increase the amount of metabolites produced.

[0059] The Bacillus subtilis is preferably Bacillus subtilis natto, which is easily available and inexpensive. Commercially available common Bacillus subtilis can be used as the Bacillus subtilis. A mutant strain of Bacillus subtilis may also be used.

[0060] [1-1-3] Fermentation conditions The fermented product is obtained by fermenting a composition containing soybean extract, defatted rice bran, and yeast extract with Bacillus subtilis. The fermented product is usually obtained by inoculating Bacillus subtilis into a medium containing soybean extract, defatted rice bran, and yeast extract, and culturing the mixture under appropriate conditions. The fermentation culture conditions for obtaining the fermented product are not particularly limited. The fermentation culture is usually performed with aeration and stirring.

[0061] The medium is not particularly limited as long as it allows the Bacillus subtilis to grow in. The medium is usually a liquid medium, but may also be a solid medium.

[0062] The pH of the medium (particularly the pH during fermentation) is preferably 5.5 or more and 8.5 or less, more preferably 6.0 or more and 8.0 or less, and even more preferably 6.5 or more and 7.5 or less. This allows the stability of the various components contained in the extraction raw material to be maintained in an optimal state.

[0063] The culture temperature is preferably 15°C to 50°C, more preferably 20°C to 45°C, even more preferably 30°C to 45°C, and most preferably 35°C to 43°C.

[0064] The culture time is preferably 24 hours or more and 60 hours or less, more preferably 27 hours or more and 54 hours or less, and even more preferably 30 hours or more and 48 hours or less.

[0065] Furthermore, various nutrients such as sugars, and acids, alkalis, etc. for adjusting the pH may be added to the medium. Furthermore, the fermentation may be mixed fermentation or continuous fermentation.

[0066] [1-1-4] Other conditions The form of the fermented product is not particularly limited. For example, the fermented product may be a fermentation broth obtained by fermentation culture or a liquid obtained by filtering the fermented broth. Furthermore, the fermented product may be the fermented broth that has been sterilized or pH adjusted as necessary, or that has been subjected to post-treatments such as deodorization, decolorization, and purification using an ion exchange resin, an activated carbon column, or a dialysis membrane. Furthermore, the fermented product may be a concentrated liquid or paste-like product obtained by concentrating the fermented broth. Alternatively, the fermented product may be a solid product such as a powder obtained by removing liquid components from the fermented broth by a known method such as freeze-drying.

[0067] The content of the fermented product in the exosome production promoter of the present invention is not particularly limited, but the proportion of the solid content of the fermented product in the total solid content constituting the exosome production promoter is preferably 0.1% by mass or more and 2.0% by mass or less, and more preferably 0.5% by mass or more and 1.2% by mass or less.

[0068] This allows the exosome production promoter to have a higher ability to promote exosome production.

[0069] [1-2] Other ingredients The exosome production-promoting agent of the present invention may contain at least the fermented product, but may also contain components other than the fermented product. Hereinafter, such components will be referred to as "other components" in this section.

[0070] Examples of other components include components other than the fermented product that have the function of promoting exosome production. When such components are contained together with the fermented product, for example, they act synergistically, thereby improving the exosome production promoting ability of the exosome production promoter as a whole.

[0071] The content of other components in the exosome production-promoting agent of the present invention is not particularly limited, but is preferably 5% by mass or less, and more preferably 1% by mass or less.

[0072] [2] Composition containing an exosome production promoter Next, the composition containing the exosome production promoter of the present invention will be described.

[0073] The composition containing an exosome production enhancer of the present invention is characterized by containing the exosome production enhancer described above. In other words, the composition containing an exosome production enhancer of the present invention further contains, in addition to the exosome production enhancer described above, a component that does not promote exosome production.

[0074] This makes it possible to provide an exosome production promoter-containing composition containing an exosome production promoter that is highly capable of promoting exosome production in vivo and highly safe. In particular, it is possible to obtain the effects of components other than the exosome production promoter while maintaining the ability to promote exosome production in vivo. For example, by including a component with a cosmetic effect other than the ability to promote exosome production, a synergistic effect with the exosome production promoter can be obtained, thereby making the cosmetic effect of the exosome production promoter-containing composition as a whole particularly excellent. Furthermore, the ease of handling of the exosome production promoter-containing composition as a whole is improved.

[0075] [2-1] Exosome production promoter The composition containing the exosome production promoter of the present invention contains the exosome production promoter of the present invention.

[0076] The solid content of the fermented product in the composition containing the exosome production promoter of the present invention is preferably 0.01% by mass or more, and more preferably 0.03% by mass or more.

[0077] This allows the exosome production promoter to have a higher ability to promote exosome production.

[0078] In particular, when the composition containing an exosome production promoter of the present invention is liquid, the content of the solid matter of the fermented product in the composition containing an exosome production promoter is preferably 0.01% by mass or more and 1.0% by mass or less, and more preferably 0.03% by mass or more and 0.4% by mass or less.

[0079] This improves the ease of handling of the exosome production promoter-containing composition, for example, improving the ease of applying the exosome production promoter-containing composition to the skin and spraying it using a sprayer, while also increasing the exosome production-promoting ability of the exosome production promoter.

[0080] [2-2] Liquid components The composition containing the exosome production promoter of the present invention may further contain, as a component other than the exosome production promoter of the present invention, for example, a liquid component that dissolves or disperses at least a portion of the components of the exosome production promoter of the present invention.

[0081] Examples of such liquid components include water, monohydric alcohols such as ethanol, and polyhydric alcohols such as 1,3-butylene glycol, 1,3-propanediol, glycerin, and ethylhexylglycerin. In particular, the polyhydric alcohol components can function as a solvent and dispersion medium, as well as a moisturizing component. Furthermore, by including these polyhydric alcohol components and mixing them with the powder composition during use, the moisturizing effect described above can be more pronounced due to the synergistic effect with the effect of retinol, which promotes the turnover of epidermal cells in the skin.

[0082] When the composition containing an exosome production promoter of the present invention is a liquid composition, the content of the liquid components (including liquid components derived from the exosome production promoter (such as liquid components derived from the fermentation product)) in the composition containing an exosome production promoter is preferably 60.0% by mass or more and 99.0% by mass or less, more preferably 70.0% by mass or more and 98.0% by mass or less, and even more preferably 80.0% by mass or more and 97.0% by mass or less.

[0083] This makes it possible to sufficiently enhance the exosome production-promoting ability of the exosome production-promoting agent-containing composition as a whole, while improving the ease of handling of the exosome production-promoting agent-containing composition, and thereby improving, for example, the ease of applying the exosome production-promoting agent-containing composition to the skin and the ease of spraying it using a sprayer.

[0084] The liquid component preferably contains at least water, and the content of water (including water derived from the exosome production promoter (such as water derived from the fermentation product)) in the composition containing the exosome production promoter is preferably 50.0% by mass or more and 93.0% by mass or less, more preferably 60.0% by mass or more and 90.0% by mass or less, and even more preferably 65.0% by mass or more and 87.0% by mass or less.

[0085] This makes it possible to sufficiently enhance the exosome production-promoting ability of the exosome production-promoting agent-containing composition as a whole, while improving the ease of handling of the exosome production-promoting agent-containing composition, and thereby improving, for example, the ease of applying the exosome production-promoting agent-containing composition to the skin and the ease of spraying it using a sprayer.

[0086] [2-3] Other ingredients The composition containing an exosome production enhancer of the present invention may contain at least the exosome production enhancer, but may also contain components other than the exosome production enhancer. Hereinafter, such components will be referred to as "other components" in this section.

[0087] Examples of other ingredients include excipients, cosmetic ingredients other than exosome production promoters, plant extracts, vitamins, vitamin-like substances, minerals, oils and fats, thickeners, gelling agents, surfactants, pH adjusters, antioxidants, colorants, stabilizers such as lactose, solubilizers such as glutamic acid and aspartic acid, preservatives, pH adjusters, humectants, emulsifiers, dispersants, emulsions, solutions, suspensions, elixirs, fragrances, flavorings, other active ingredients (medicinal ingredients), and the like.

[0088] Examples of thickeners include xanthan gum. Examples of pH adjusters include citric acid and sodium citrate.

[0089] Examples of preservatives include phenoxyethanol. An example of the surfactant is hydrogenated castor oil (PEG-4).

[0090] However, the content of other components in the composition containing the exosome production promoter is preferably 5% by mass or less, and more preferably 1% by mass or less.

[0091] [2-4] Other conditions The composition containing an exosome production-promoting agent of the present invention may be in any state, for example, liquid, solid, semi-solid, etc. Furthermore, the composition containing an exosome production-promoting agent of the present invention may be supported on a substrate. In this case, the substrate may be in any form, for example, cotton-like or sheet-like.

[0092] In particular, the composition containing the exosome production promoter is preferably in a liquid form. This further improves the ease of handling of the exosome production promoter, and can further improve, for example, the ease of applying the exosome production promoter to the skin and the ease of spraying using a sprayer.

[0093] The composition containing an exosome production promoter may be, for example, an oral agent, but is preferably an external agent.

[0094] This makes it possible to more suitably promote collagen production from fibroblasts and more suitably enhance the barrier function of epidermal keratinocytes.

[0095] The exosome production promoter-containing composition of the present invention can be used in contact with the skin (including nails) or mucous membranes of animals (including humans), including pharmaceuticals; quasi-drugs; skin care products such as lotions, creams, emulsions, serums, whitening agents, and facial cleansers (including facial cleansers); lip products such as lip balm and lipstick; cosmetics such as makeup bases, blushers, and foundation creams; nail care products such as polish, polish remover, strengtheners, lengtheners, hardeners, cuticle removers, and softeners; hair removal products such as shaving creams and lotions, depilatories, and after-shave skin conditioners; hair care products such as hair treatments and shampoos; and other products such as soaps, body soaps, hand soaps, anti-aging agents, antiperspirants, sunscreens, suntan accelerators, perfumes, cooling sprays, and bath additives. Among these, the exosome production promoter-containing composition of the present invention is preferably selected from the group consisting of lotions, creams, emulsions, and serums.

[0096] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these. [Example]

[0097] Next, specific examples of the present invention will be described. However, the present invention is not limited to the descriptions in these examples.

[0098] [3] Manufacturing of exosome production promoters Example 1 First, soybean extract, defatted rice bran, and yeast extract were prepared.

[0099] The soybean extract used was a concentrate of soybean broth, which had a Brix value of 15.0 and contained soybean oligosaccharides.

[0100] The defatted rice bran used was obtained by subjecting rice bran to a defatting treatment and had an oil content of 13% by mass.

[0101] The yeast extract used was powdered MEAT P1G manufactured by Asahi Group Foods Co., Ltd. The proportion of amino nitrogen in the yeast extract was 6.0±1.0% by mass.

[0102] The soybean extract, defatted rice bran, and yeast extract were mixed in a predetermined ratio, sterilized at 121°C for 21 minutes, and then cooled to 45°C. The composition thus obtained had a soybean extract solids content of 17.5% by mass, a soybean oligosaccharide content of 2.7% by mass, a defatted rice bran content of 0.016% by mass, and a yeast extract solids content of 0.58% by mass.

[0103] To this was added Bacillus natto cultured under specified conditions, i.e., Bacillus natto cultured for 28 hours at 37°C in a medium prepared in the same manner as above, in which 100 parts by mass of soybean extract was added to 0.5 parts by mass of the same yeast extract as above. Fermentation was carried out at 37°C for 40 hours, and then a purification process was carried out to obtain a liquid fermented product. The solid content of the obtained fermented product was 1.0% by mass.

[0104] Example 2 An exosome production promoter was produced in the same manner as in Example 1, except that Bacillus subtilis A (NBRC3134) was used instead of Bacillus natto.

[0105] Example 3 An exosome production promoter was produced in the same manner as in Example 1, except that Bacillus subtilis B (NBRC13719) was used instead of Bacillus natto.

[0106] Example 4 An exosome production promoter was produced in the same manner as in Example 1, except that the amounts of yeast extract and soybean extract used were changed to prepare a composition so that the content of yeast extract in the composition used for fermentation was 0.1% by mass.

[0107] Example 5 An exosome production promoter was produced in the same manner as in Example 1, except that the amounts of yeast extract and soybean extract used were changed to prepare a composition so that the content of yeast extract in the composition used for fermentation was 1.0% by mass.

[0108] (Comparative Example 1) An exosome production promoter was produced in the same manner as in Example 1, except that Bacillus subtilis natto was not used.

[0109] (Comparative Example 2) An exosome production promoter was produced in the same manner as in Example 1, except that yeast extract was not used.

[0110] (Comparative Example 3) An exosome production promoter was produced in the same manner as in Example 1 except that red bran was used instead of defatted rice bran.

[0111] Comparative Example 4 An exosome production promoter was produced in the same manner as in Example 1, except that lactic acid bacteria (NBRC3070: Lactobacillus plantarum) were used instead of Bacillus natto.

[0112] (Comparative Example 5) An exosome production promoter was produced in the same manner as in Example 1, except that yeast (NBRC0555: Saccharomyces cerevisiae) was used instead of Bacillus subtilis natto.

[0113] [4] Evaluation The exosome production promoter obtained as described above was evaluated as follows.

[0114] [4-1] Evaluation of the exosome production promoting effect The cells used were human adipose-derived mesenchymal stem cells (HMSC-AD, manufactured by Lifeline Cell Technology), and the culture medium used was StemLife BM basal medium supplemented with StemLife MSC LifeFactors proliferation additive set (manufactured by Kurabo).

[0115] The thawing and implantation of frozen cells was performed according to the manual. After counting the number of cells from the thawed cell suspension on a clean bench, the cells were transplanted at the recommended density of 2,500 cells / cm. 2 The cells were seeded into a T75 cell culture flask (BM Instruments) so that the cell coverage was 100%. After culturing for 24 hours at 37°C under 5% CO2, the medium was changed. If the cell coverage was below 40%, the medium was changed every other day. After the cell coverage exceeded 40%, the medium was changed daily. When the cell coverage reached 70%, the culture was terminated and the cells were collected by trypsinization.

[0116] The collected cells were seeded at 100,000 cells / well in a 6-well plate (BM Instruments) and cultured at 37°C under 5% CO2 until the cells reached subconfluence. The culture supernatant was then aspirated, and 2 mL of the liquid exosome production promoter prepared in each Example and Comparative Example was diluted 1:10 with sterile water and added to each well. The cells were then cultured for an additional 48 hours. After incubation, the culture supernatant was collected from each well, and the amounts of exosome-specific markers CD63 and CD9 in the culture supernatant were measured using the ELISA method described below. The cells were washed with PBS(-) and lysed in RIPA Buffer (Fujifilm WAKO Pure Chemicals) to prepare cell lysates. Total protein content of the cell lysate samples was quantified using a BCA Protein Assay kit (Pierce). The culture medium without sample was used as a control. CD9 and CD63 are markers specific to exosomes, and measuring them makes it possible to quantify exosomes.

[0117] [4-1-1] Evaluation by promoting CD63 production Measurements were performed using a commercially available CD63 / CD63 Exosome ELISA kit, Human (Cosmo Bio) for quantifying human exosomes. Specifically, 100 μL of the culture supernatant and CD63 standard bead solution collected as described above were added to each well of an ELISA plate and incubated at room temperature for 2 hours. After the antigen-antibody reaction, the reaction solution in each well was completely removed and the plate was washed three times with 300 μL of wash buffer. 100 μL of diluted HRP-labeled anti-CD63 antibody was added to each well and incubated at room temperature for 2 hours. After the reaction, the plate was washed three times with 300 μL of wash buffer. 100 μL of color-developing substrate solution was added to each well and incubated at room temperature for 20 minutes in the dark, followed by the addition of 50 μL of stop solution. Measurements were performed using a microplate reader (TakaraBio) at 450 nm, and the amount of CD63 was calculated using the standard calibration curve.

[0118] In addition, the amount of protein in the cell lysate stored as described above was measured using a separate transparent plate (Thermo Fisher Scientific) with a BCA Protein Assay kit (Pierce).

[0119] To eliminate the influence of cell number, the CD63 expression level (ng / mg protein) was calculated as a ratio to total protein, and the relative value to the control was calculated. These results are shown in Figure 1. Figure 1 shows that excellent results were obtained in each of the above examples, whereas unsatisfactory results were obtained in each of the comparative examples.

[0120] [4-1-2] Evaluation by promoting CD9 production Measurements were performed using a commercially available CD9 / CD9 Exosome ELISA kit, Human (Cosmo Bio) for quantifying human exosomes. Specifically, 100 μL of the culture supernatant and CD9 standard bead solution collected as described above were added to each well of an ELISA plate and incubated at room temperature for 2 hours. After the antigen-antibody reaction, the reaction solution in each well was completely removed and the plate was washed three times with 300 μL of wash buffer. 100 μL of diluted HRP-labeled anti-CD9 antibody was added to each well and incubated at room temperature for 2 hours. After the reaction, the plate was washed three times with 300 μL of wash buffer. 100 μL of color-developing substrate solution was added to each well and incubated at room temperature for 20 minutes in the dark, followed by the addition of 50 μL of stop solution. Measurements were performed using a microplate reader (TakaraBio) at a wavelength of 450 nm, and the amount of CD9 was calculated using the standard calibration curve.

[0121] In addition, the amount of protein in the cell lysate stored as described above was measured using a separate transparent plate (Thermo Fisher Scientific) with a BCA Protein Assay kit (Pierce).

[0122] To eliminate the influence of cell number, the CD9 expression level (ng / mg protein) was calculated as a ratio to total protein, and the relative value to the control was calculated. These results are shown in Figure 2. Figure 2 shows that excellent results were obtained in each of the above examples, whereas unsatisfactory results were obtained in each of the comparative examples.

[0123] [4-2] Usability of the composition containing the exosome production promoter First, a lotion was prepared as an exosome production enhancer-containing composition using the exosome production enhancer of each of the examples and comparative examples. More specifically, the lotion was prepared as an exosome production enhancer-containing composition by mixing 10.0 parts by mass of the exosome production enhancer, 5.0 parts by mass of 1,3-butylene glycol, 3.0 parts by mass of 1,3-propanediol, and 82.0 parts by mass of purified water.

[0124] Five female subjects used each of the lotions obtained as described above, and answered a questionnaire about their skin condition before and after use. More specifically, they applied each lotion to the left cheek twice a day, morning and night, for 14 consecutive days, and compared their skin condition 12 hours after the end of the test with their skin condition before the test. Then, each subject calculated a score for each of the following items: skin dryness, skin firmness, and makeup application, based on the following evaluation criteria.

[0125] 2: I felt much better after the exam. 1: I felt better after the exam. 0: No change before and after the test. -1: It got worse after the exam. -2: It got much worse after the exam.

[0126] For each of the lotions according to the Examples and Comparative Examples, the total score for each subject was calculated for skin dryness, skin firmness, and makeup application, and the average of the total scores for the five subjects was calculated. The higher the average total score, the better the feel in use. The results of [4-2] above are summarized in Table 1.

[0127] [Table 1]

[0128] It can be seen from Table 1 that excellent results were obtained in each of the examples, whereas unsatisfactory results were obtained in each of the comparative examples.

Claims

1. The present invention contains a fermented product obtained by fermenting a composition containing soybean extract, defatted rice bran, and yeast extract with Bacillus subtilis, The exosome production promoter, characterized in that the proportion of the solid content of the fermentation product in the total solid content is 0.1% by mass or more and 2.0% by mass or less.

2. The exosome production promoter according to claim 1, wherein the Bacillus subtilis is Bacillus subtilis natto.

3. The exosome production promoter according to claim 1, wherein the content of the solid content of the yeast extract in the composition is 0.1% by mass or more and 1.0% by mass or less.

4. A composition containing an exosome production promoter, comprising the exosome production promoter according to any one of claims 1 to 3.

5. The exosome production-promoting agent-containing composition according to claim 4, wherein the exosome production-promoting agent-containing composition is liquid.

6. The composition containing an exosome production promoter according to claim 4, wherein the composition is an external preparation.

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