Nucleus format abnormality improver

Abstract

To provide a nucleus format abnormality improver, progerin accumulation inhibitor for improving various symptoms and diseases in various tissues in a living body, especially on the skin of a living body, improving various symptoms such as reduction of horny cell function caused by nucleus format abnormality or progerin accumulation, and an external preparation for preventing / improving for preventing and improving, reduction of horny cell layer function caused by nucleus format abnormality and / or progerin accumulation.SOLUTION: The invention uses a fermentation product which is obtained by fermentation using lactic acid bacteria, with soybean milk as a matrix. The fermentation product obtained by fermentation using the lactic acid bateria with soybean milk as the matrix, has high effects for improving various symptoms such as reduction of horny cell layer function caused by nucleus format abnormality and progerin accumulation, in addition, a nucleus format abnormality improver or the like is used continuously or consecutively for repeating improvement of the nucleus format abnormality and suppression of progerin accumulation, thereby preventing the nucleus format abnormality and progerin accumulation, and by the nucleus format abnormality improver, it is possible to improve nucleus format abnormality in various tissues in a living body such as the skin of the living body, suppress the progeriin accumulation, for preventing various symptoms such as reduction of the horny cell layer function.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for improving nuclear morphological abnormalities, an agent for inhibiting progerin accumulation, and an external skin preparation for preventing and improving stratum corneum function decline caused by nuclear morphological abnormalities and / or progerin accumulation, which contain a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria. [Background technology]

[0002] The skin is located at the boundary between the body and the outside world, and has a skin barrier function to prevent the intrusion of substances from the outside and the evaporation of water from inside the skin, and plays an important role in maintaining the body. The skin is an epithelium with a thickness of 100 to 200 μm, consisting of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum, which is the outermost layer of the skin, functions as a physical and chemical barrier between the body and the external environment, and also has the function of regulating the texture and appearance of the skin. Therefore, maintaining a healthy stratum corneum is essential for maintaining a healthy body and skin. The stratum corneum is composed of keratinocytes, which are formed by the terminal differentiation of epidermal cells in the stratum basale, and the intercellular lipids that surround them. The stratum corneum function is determined by the important roles played by components in keratinocytes, such as keratin fibers with SS bonds that are generated during terminal differentiation, filaggrin, natural moisturizing factors, and the cornified envelope. In addition, intercellular adhesion and intercellular lipids such as ceramide also play important roles. It is important that these are produced and matured normally, and that the stratum corneum is not only formed normally, but that its functions are properly exerted and maintained (Non-Patent Document 1).

[0003] The nuclear lamina is a complex and highly organized protein meshwork that covers the nucleoplasmic surface of the nuclear inner membrane, and is a backing structure that mechanically supports and maintains the nuclear morphology. In humans, the nuclear morphology is generally maintained as a sphere except for some cells, so that the nuclear morphology is observed as a near perfect circle in a two-dimensional microscope image. The nuclear lamina is mainly composed of intermediate filament proteins A-type and B-type lamins, and not only maintains the nuclear morphology, but also has various functions necessary for life support, such as acting as a scaffold for proteins that function around the nuclear membrane, anchoring chromatin and transcription factors inside the nucleus to the nuclear periphery, compartmentalizing the genome required for DNA transcription, replication, repair, etc., interacting with the cytoskeleton outside the nucleus, and participating in signal transduction and material transport between the nucleus and the cytoplasm. For this reason, it is known that abnormalities in nuclear morphology are mainly accompanied by abnormalities in the proteins that make up the nuclear lamina, and cause problems specific to multicellular organisms, such as tissue-specific genetic diseases and aging (Non-Patent Document 2).

[0004] A well-known example of a disease associated with the above-mentioned nuclear morphological abnormality is Hutchinson-Gilford Progeria Syndrome (HGPS), which is caused by a congenital genetic abnormality. It has been revealed that in HGPS patients, abnormal processing of the A-type lamin gene causes the production of an abnormal protein called progerin, which leads to abnormal nuclear morphology and accelerated aging (Non-Patent Document 3). The main symptoms of this disease include poor growth in height and weight, as well as noticeable premature aging changes such as skin aging, hypercholesterolemia, accelerated arteriosclerosis, diabetes, osteoporosis, senile cataracts, graying of hair, and hair loss as the symptoms progress. In particular, in the skin, although major differentiation marker proteins such as keratin 1, keratin 10, loricrin, and filaggrin in the epidermis are normally expressed and localized in the basal layer, spinous layer, and granular layer, insufficient maintenance of stratum corneum hydration and defective formation of the intercellular lipid matrix occur in the stratum corneum, which is the uppermost layer of the epidermis (Non-Patent Document 4), and it has been shown that, for example, the skin surface is rough and shiny (Non-Patent Document 5). This suggests that in order for the stratum corneum to function normally, including its function of regulating the texture and appearance of the skin, it is important not only to form a normal stratum corneum by promoting differentiation in the epidermis, but also to properly exert and maintain the functions of the formed stratum corneum, and that nuclear morphological abnormalities are deeply involved not only in the manifestation of various aging phenomena but also in skin changes such as a decrease in stratum corneum function in the epidermis. Progerin accumulation and nuclear morphological abnormalities similar to those observed in HGPS have also been confirmed in healthy individuals. For example, their frequency is higher in elderly people than in newborns, suggesting that nuclear morphological abnormalities are related to intrinsic aging that accompanies aging (Non-Patent Document 6). Anti-aging components have been discovered by a screening method that uses the abnormal state of nuclear morphology in cultured cells as an indicator (Patent Document 1). However, research into the biological reactions through which nuclear morphological abnormalities promote and manifest aging phenomena and skin changes has only just begun, and knowledge about the detailed mechanisms is currently limited.

[0005] Fermented soy milk (lactic acid bacteria fermented soy milk liquid), obtained by fermenting soy milk, a milky liquid extracted from soybean seeds, with lactic acid bacteria, was known to have a whitening effect on B16 cells, an antioxidant effect, etc. (Patent Document 2), but its effect on the nuclear morphology and progerin accumulation of human skin cells, and its effect on the stratum corneum functions related to these, had not been investigated at all. Furthermore, the relationship between the whitening and antioxidant effects of B16 cells and the nuclear morphology and progerin accumulation of human skin cells and the stratum corneum functions related to these was also unknown. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2013-257347 A [Patent Document 2] Japanese Patent Application Publication No. 3-127713 [Non-patent literature]

[0007] [Non-Patent Document 1] Drug Delivery System,22(4),424-432,2007 [Non-Patent Document 2] Biomedicines, 2020, Jul;8(7):188 [Non-Patent Document 3] Aging Res Rev., 2017, Jan;33:18-29 [Non-Patent Document 4] Aging Cell, 2014, Apr;13(2):292-302 [Non-Patent Document 5] Case Reports in Dentistry, Volume 2013, Article ID 631378, 4pages [Non-Patent Document 6] Cells, 2020, Mar;9(3):718 [Non-Patent Document 7] Cosmetics & Toiletries, 2013 Oct;Trifluoroacetyl-Tripeptide-2 to Target Senescence for Anti-aging Benefits Summary of the Invention [Problem to be solved by the invention]

[0008] The objective of the present invention is to provide a nuclear morphological abnormality improving agent or progerin accumulation inhibitor that has the effect of improving various diseases and symptoms in various tissues in the body, particularly various symptoms in the skin such as nuclear morphological abnormalities and decreased stratum corneum function caused by progerin accumulation, and an external skin preparation for preventing and improving nuclear morphological abnormalities and / or decreased stratum corneum function caused by progerin accumulation. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to achieve the above object, and as a result have achieved the above object by using a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria. That is, the present invention includes the following preferred embodiments. [1] An agent for improving nuclear morphological abnormalities, comprising a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria. [2] A progerin accumulation inhibitor, characterized in that it contains a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria. [3] A topical skin preparation for preventing and improving stratum corneum dysfunction caused by nuclear morphological abnormalities and / or progerin accumulation, characterized in that it contains a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria. [4] Use of a skin topical composition containing a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria for the prevention and improvement of stratum corneum dysfunction caused by nuclear morphological abnormalities and / or progerin accumulation (excluding use in treating humans). Effect of the Invention

[0010] The fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria is highly effective in improving various symptoms such as nuclear morphological abnormality and stratum corneum dysfunction caused by progerin accumulation. In addition, the nuclear morphological abnormality improving agent, progerin accumulation inhibitor, and skin external preparation for preventing and improving stratum corneum dysfunction caused by nuclear morphological abnormality and progerin accumulation of the present invention can be used continuously or successively to repeatedly improve nuclear morphological abnormality and inhibit progerin accumulation, thereby preventing nuclear morphological abnormality and progerin accumulation, and as a result, improving nuclear morphological abnormality and inhibiting progerin accumulation in various tissues in the body including the skin, and thereby preventing various symptoms such as stratum corneum dysfunction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below.

[0012] In the present invention, the soymilk to be fermented by lactic acid bacteria is not particularly limited as long as it is a milky liquid extracted from soybean seeds, and can be obtained, for example, by soaking soybean seeds in water, grinding them, boiling them down, and then separating the solid and liquid by filtration, centrifugation, etc. Soymilk is defined in detail as a beverage in the JAS standard established by the Minister of Agriculture, Forestry and Fisheries specifying the types (items) of agricultural and forestry products, the methods for handling agricultural and forestry products, the methods for testing, etc., but the soymilk in the present invention does not have to fall under this standard or definition as long as it meets the contents described below.

[0013] The soybean seeds used for preparing soymilk are not particularly limited as long as they are seeds of soybean (Glycine max (L.) Merr.) belonging to the genus Glycine in the family Fabaceae. For example, seeds of yellow soybeans, green soybeans, black soybeans, red soybeans, brown soybeans, and white soybeans, which are broadly classified according to the color of the seeds, and seeds of large, medium, small, and very small soybeans, which are broadly classified according to the size of the seeds, can be used. However, for reasons of industrial production, work, and the like, it is acceptable for parts other than the seeds (e.g., awns, flag leaves, and culms) to be included to a certain extent during the preparation process. The seeds may be used raw, or may be dried or semi-dried in advance. The shape of the seeds is not particularly limited, and the seeds may be used as they are when harvested, or may be used after being chopped or crushed into fine particles.

[0014] The method for preparing soy milk is roughly divided into three steps: a soaking step, a grinding step, and a heating step, but each step is not necessarily essential, and the order of each step is not limited. If it does not interfere with the use of the obtained soy milk, some steps may be omitted, and other pretreatment steps may be added before, after, or between each step. For example, a method of soaking soybean seeds in a solvent such as water and wet grinding them, a method of steaming soybean seeds, adding a solvent such as water, and wet grinding them, a method of soaking soybean seeds in hot water and wet grinding them, a method of dry grinding soybean seeds, adding a solvent such as water, and boiling them, a method of squeezing and extracting them from raw soybean seeds, and the like can be used.

[0015] In the soaking step of soybean seeds to obtain soymilk, a method of soaking soybean seeds in a solvent at 20 to 30°C for 4 hours or more, preferably 16 hours, can be used. When soybean seeds are added to a high-temperature solvent at 90°C or higher, such as hot water, the soaking step can be performed without setting a specific soaking time. As the soaking solvent, water can be preferably used, but is not particularly limited. As long as it does not interfere with the subsequent grinding step, an aqueous solution of an inorganic salt (sodium chloride, potassium chloride, magnesium chloride, ammonium carbonate, etc.), or a buffer solution (phosphate buffer, acetate buffer, Tris-HCl buffer, carbonate buffer, borate buffer, etc.), an aqueous solution of an inorganic acid (hydrochloric acid, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), an aqueous solution of an organic acid (acetic acid, citric acid, lactic acid, succinic acid, ascorbic acid, fumaric acid, malic acid, etc.), an aqueous solution of a surfactant (saponin, lecithin, etc.), a low-alcohol solution, or a mixture thereof can be used. Coals (methanol, ethanol, propanol, butanol, etc.), ketones (acetone, ethyl methyl ketone, etc.), hydrocarbons (propane, butane, hexane, cyclohexane, etc.), ethers (diethyl ether, etc.), glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), halogenated hydrocarbons (dichloromethane, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc.), acetates (ethyl acetate, methyl acetate, etc.), or mixtures thereof can be used. The mixing ratio (weight ratio) of soybean seeds to the solvent is generally 1:1 to 1:10, preferably 1:2 to 1:5, calculated based on the dry weight of the seeds. The soaked solvent may be removed, or may be used as it is as the solvent for the next step without being removed.

[0016] In the grinding process of soybean seeds to obtain soy milk, a method of dry grinding before adding a solvent and soaking, or a method of wet grinding after adding a solvent and soaking can be used. A grinding method using shear force, shear stress, friction force, etc. by grinding media, a grinding method using shear stress or friction force between abrasives, a grinding method by colliding high-pressure fluids, a grinding method by passing through a narrow gap, etc. can be used.

[0017] In the heating step of soybean seeds to obtain soymilk, a method of heating at 60 to 70° C. for 3 hours or more, or at 70 to 90° C. for 1 hour or more, preferably at 90 to 120° C. for 1 minute or more can be used. As long as components derived from proteins and the like contained in the soybean seeds are eluted, a method of soaking soybean seeds in a solvent and crushing them, followed by boiling, a method of steaming soybean seeds before crushing, or a method of adding a high-temperature solvent such as hot water to the soybean seeds can be used.

[0018] After preparation, soy milk may be used for fermentation as it is, or may be subjected to solvent substitution or purification treatment according to a conventional method, or may be used after being diluted or concentrated to an appropriate concentration if necessary. In some cases, the liquid phase after solid-liquid separation may be solidified according to a conventional method such as spray drying or freeze drying, and further pulverized to powder form if necessary before use.

[0019] The soy milk prepared by the above method is preferably sterilized as necessary before being subjected to the fermentation step to remove unwanted bacteria that may hinder fermentation. In this case, the method of removing unwanted bacteria may be a method of washing and sterilizing the soybean seeds to be used in advance with sterilizing ethanol or the like, and then extracting the soy milk with sterilized water to obtain the soy milk, or a method of heat sterilization after obtaining the soy milk from the soybean seeds. As the heat sterilization method, an autoclave sterilization method in which heating is performed at 105 to 121°C for 10 to 20 minutes, or an intermittent sterilization method in which the soy milk is held at 80 to 90°C for 60 to 120 minutes and is repeated once a day for 2 to 3 days, is generally used. However, this sterilization step is not essential. If it does not hinder the next fermentation step, this sterilization step may be omitted.

[0020] The lactic acid bacteria used for fermentation in the present invention is not particularly limited, but from the viewpoint of versatility, it is preferable to use Lactobacillus delbrueckii. It is preferable to pre-culture in a basic medium before subjecting it to the fermentation process. At this stage, the culture may be performed in a conventional manner as long as the lactic acid bacteria can grow. The composition of the basic medium for lactic acid bacteria is sufficient if it contains a minimum carbon source, nitrogen source, and phosphorus source, but it is preferable to use a medium developed as a medium that shows good growth of lactic acid bacteria as a whole, such as MRS medium (MRS medium composition: peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween80 1g, K2HPO4 2g, sodium acetate 5g, diammonium citrate 2g, MgSO4·7H2O 0.2g, MnSO4·nH2O 0.05g, purified water 1L). Of course, medium compositions other than those mentioned above can also be applied to the present invention, so long as they are substances that lactic acid bacteria can assimilate and grow in. Furthermore, the basal medium may contain various additives, such as aqueous solutions of inorganic salts (sodium chloride, potassium chloride, magnesium chloride, ammonium carbonate, etc.), or buffer solutions (phosphate buffer, acetate buffer, Tris-HCl buffer, carbonate buffer, borate buffer, etc.), aqueous solutions of inorganic acids (hydrochloric acid, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), aqueous solutions of organic acids (acetic acid, citric acid, lactic acid, succinic acid, ascorbic acid, fumaric acid, malic acid, etc.), aqueous solutions of surfactants (saponin, lecithin, etc.), lower alcohols (methanol, ethanol, propanol, butanol, etc.), ketones (acetone, ethyl methyl ketone, etc.), Hydrocarbons (propane, butane, hexane, cyclohexane, etc.), ethers (diethyl ether, etc.), glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), halogenated hydrocarbons (dichloromethane, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc.), acetates (ethyl acetate, methyl acetate, etc.), sugars (glucose, fructose, sucrose, maltose, oligosaccharides, etc.) or mixtures thereof can be added, but when added, it is preferable to add them within a range that does not affect the growth of lactic acid bacteria. Furthermore, in the subsequent fermentation step, the above various components may be contained in the solvent at a concentration within a range that does not affect the fermentation of lactic acid bacteria.

[0021] The fermented product of the present invention is a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria. The fermented product as referred to in the present invention includes the entire culture obtained after fermentation, a part of the culture, or a product thereof that has been further processed by extraction, concentration, purification, or the like. Specifically, the term "fermentation culture liquid" includes a fermentation culture liquid which is a mixture of a solid phase derived from the substrate, bacteria, etc. present in the fermentation system after a fermentation process and a liquid phase derived from the basal medium, solvent, etc. present in the fermentation system after the fermentation process (in the present application, the mixture after fermentation is referred to as the fermentation culture liquid); the liquid phase of the fermentation culture liquid obtained by filtering the fermentation culture liquid, etc.; a liquid obtained by diluting or concentrating the liquid phase of the fermentation culture liquid; a dried product obtained by evaporating the solvent of the liquid phase of the fermentation culture liquid; an extract obtained by further extracting from this dried product; a liquid obtained by diluting or concentrating this extract; the solid phase of the fermentation culture liquid which is a residue from filtering the fermentation culture liquid, an extract obtained by further extracting from the solid phase of the fermentation culture liquid; a liquid obtained by diluting or concentrating this extract; a dried product obtained by evaporating the solvent of this extract liquid; an extract obtained by further extracting from this dried product; a liquid obtained by diluting or concentrating this extract liquid; or a crude or purified product thereof.

[0022] The fermentation process for obtaining the fermented soy milk is not particularly limited, but for example, the soy milk prepared by the methods of

[0014] to

[0017] can be inoculated with lactic acid bacteria and fermented. The amount of lactic acid bacteria to be inoculated is not particularly limited, but it is preferably 10 5 ~10 10The range of cells / g. The fermentation conditions are 5°C to 50°C, preferably 25 to 40°C, and fermentation can be performed for 1 to 15 days, preferably 3 to 10 days. If necessary, the basal medium described in

[0020] may be added in advance in a conventional manner and fermented. Although stationary fermentation is sufficient, shaking culture or aeration culture can be performed to shorten the fermentation time. When the above fermentation process is completed, it is preferable to sterilize the fermentation culture liquid that has been fermented under the above conditions for 15 to 120 minutes at 80 to 120°C for about 15 to 120 minutes in order to stop the fermentation. However, this sterilization process is not essential. If it does not interfere with the use of the obtained fermented product, this sterilization process may be omitted. The fermentation culture liquid that has been fermented may be used as it is as a fermented product, or the liquid phase may be separated by solid-liquid separation means such as filtration or centrifugation to obtain a fermented product. In some cases, the solid phase after solid-liquid separation of the fermentation culture liquid may be used as the fermented product as is, or this solid phase may be further extracted with a solvent similar to the solvent that can be used in the soaking step in preparing soymilk as described in

[0015] , and the liquid phase may be separated and used as the fermented product as is, or, generally and preferably, by solid-liquid separation means such as filtration or centrifugation.

[0023] After preparation, the fermented soymilk may be used as it is, or may be subjected to purification treatment, disruption treatment by ultrasonication, or the like according to a conventional method, or may be diluted or concentrated to an appropriate concentration if necessary. In some cases, the liquid phase after solid-liquid separation may be solidified according to a conventional method such as spray drying or freeze drying, and further pulverized to powder form if necessary before use. In addition, when the fermented soy milk is used as each agent of the present invention, it is sufficient that the fermented soy milk is contained as an active ingredient of each agent, and the fermented soy milk may be used as it is, or may be mixed with a general base. The final form may be any form such as liquid, emulsion, gel, solid, powder, granule, etc., and optional ingredients used in external skin preparations may be appropriately mixed as needed within a range that does not impair the effects. Examples of the optional ingredients include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, preservatives, fragrances, various medicinal ingredients, pH adjusters, neutralizers, etc. Examples of the final form include, for example, skin external preparations, basic cosmetics such as milky lotion, cream, lotion, essence, gel, pack, and facial cleanser, makeup cosmetics such as lipstick, makeup base, foundation, and makeup pressed powder, cleansing cosmetics such as facial cleanser, body shampoo, and soap, and further bath additives and sunscreen cosmetics, but are not limited to these. The amount of fermented soy milk in each agent may be appropriately adjusted depending on the desired effect, but is preferably in the range of 0.0001 to 10% by mass, and optimally 0.001 to 1% by mass, calculated as evaporation residue.

[0024] Nuclear morphological abnormalities are a phenomenon in which the nuclear membrane and nuclear lamina structures that cover the nucleus, an organelle that constitutes the cells of eukaryotes, and form a spherical nucleus, become distorted. Nuclear morphological abnormalities can be evaluated, for example, by observing stained nuclei under a microscope and comparing the average nuclear circularity of two cell groups of the same origin. If the nuclear circularity is lower than that of a reference cell group, it can be considered that nuclear morphological abnormalities have occurred. Conversely, if the nuclear circularity is higher than that of the reference cell group, it can be considered that the nuclear morphological abnormality has been improved.

[0025] Inhibition of progerin accumulation means suppressing the accumulation of progerin in cells. Progerin accumulation in cells can be suppressed by suppressing the expression of progerin mRNA or the production of progerin protein, or by removing or degrading the produced progerin protein from cells. EXAMPLES

[0026] Examples of the effect test of the fermented soy milk of the present invention are shown below. In addition, examples of application of the fermented soy milk to external skin preparations are described, but the present invention is not limited to the examples described here.

[0027] <Preparation of lactic acid bacteria to be inoculated into the extract> The lactic acid bacteria used was Lactobacillus delbrueckii. One loopful of the lactic acid bacteria strain was inoculated into 30 mL of MRS medium and cultured at 30°C for 3 days to obtain a lactic acid bacteria culture solution. (MRS medium composition: 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 1 g Tween 80, 2 g K2HPO4, 5 g sodium acetate, 2 g ammonium dihydrogen citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·nH2O, 1 L of purified water)

[0028] <Preparation of soy milk> 100 mL of purified water was added to 20 g of pre-dried soybean (variety: Fukuyutaka) seeds, and the seeds were soaked at room temperature for 16 hours, then crushed in a mixer to obtain a soybean seed slurry. The slurry was boiled at 90°C for 10 minutes, and the solids were removed by filtration to obtain soy milk. <Preparation of fermented product> 5 mL of the lactic acid bacteria culture solution was added to the obtained soy milk and thoroughly stirred. The soy milk inoculated with the lactic acid bacteria was subjected to static culture at 30°C for 3 days, and then sterilized at 105°C for 15 minutes. After sterilization, the solid matter was removed by filtration to obtain the fermented soy milk. Purified water was used as a negative control, and trifluoroacetyl tripeptide-2, a known progerin accumulation inhibitor that suppresses the accumulation of progerin, a substance that causes nuclear morphological abnormalities, was used as a comparative control (Non-Patent Document 7).

[0029] <Cell culture> Human epidermal keratinocytes were dispersed in EpiLife medium, with 60 μM calcium (Thermo Fisher Scientific) prepared by adding Human Keratinocyte Growth Supplement Kit (HKGS Kit, Thermo Fisher Scientific) according to the standard method, and then plated at 5.0 × 10 4 The cells were seeded at cells / well and cultured for 24 hours at 5% CO2 and 37°C. After that, the cultured cells were divided into a nuclear morphological abnormality treatment group and an untreated group and manipulated. <Nuclear morphological abnormality treatment area> For the cells treated with nuclear morphological abnormalities, Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific), Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific), and antisense oligonucleotide (ASO: 5'-AGUGACCGTGACACUGGA-3') were mixed in the usual manner to prepare an ASO transfection solution. The ASO transfection solution was added to the medium and cultured at 5% CO2 and 37°C for 24 hours. The cell culture medium containing the ASO transfection solution was removed, and fresh EpiLife medium was added. Then, soybean milk fermentation product was added to the medium to a final concentration of 0.01%, or trifluoroacetyl tripeptide-2 was added to a final concentration of 0.05%. As a negative control, purified water was added to the medium instead of each sample, and cultured at 5% CO2 and 37°C for 2 days. <Untreated area> For untreated cells, a control solution was prepared by mixing purified water instead of ASO with Lipofectamine 2000 Transfection Reagent and Opti-MEM Reduced Serum Medium. The control solution was added to the medium and cultured at 37°C with 5% CO2 for 24 hours. The cell culture medium containing the control solution was removed and fresh EpiLife medium was added. As a negative control, purified water was added to the medium instead of each sample and cultured at 37°C with 5% CO2 for 2 days.

[0030] <Quantitative determination of progerin expression level> For the cells in each treatment group, mRNA was extracted using RNeasy Mini Kit (QIAGEN) according to a standard protocol. Then, reverse transcription was performed using PrimeScript RT Reagent Kit (Takara Bio) according to a standard protocol to synthesize cDNA. Using the obtained cDNA as a template, the expression levels of Progerin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; used as a housekeeping gene) were measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) according to a standard protocol using the following primers and enzymes. The primers used were the sense primer for Progerin (5'-ACTGCAGCAGCTCGGGG-3'), the antisense primer for Progerin (5'-TCTGGGGGCTCTGGGC-3'), the sense primer for GAPDH (5'-CCACATCGC TCAGACACCAT-3'), and the antisense primer for GAPDH (5'-TGACCAGGC GCCCAATA-3'). Power SYBR Green Master Mix (Applied Biosystems) was used for PCR reactions, and gene expression was analyzed by the comparative Ct method. In other words, the change in gene expression level due to the addition of the test substance was calculated as a relative amount to the Progerin Ct value in the nuclear morphological abnormality treatment / purified water addition group, which was corrected by the GAPDH Ct value, and the significant difference from the value in Comparative Example 2 was tested by the Tukey-Kramer multiple comparison test.

[0031] <Nuclear morphology abnormality measurement> For the cells in each treatment group, the cell culture medium was removed, the cells were washed with PBS(-), and then PBS(-) containing 4% paraformaldehyde was added and fixed at 20°C for 15 minutes. The treatment solution was removed, the cells were washed with PBS(-), and then PBS(-) containing DAPI at 1 μg / mL was added, and nuclear staining was performed at 20°C for 5 minutes. The treatment solution was removed, PBS(-) was added, and a fluorescent image was obtained with a fluorescent microscope (Keyence). From the obtained fluorescent images, the nuclear circularity was calculated by image analysis using the Analyze particles function of the image analysis software ImageJ, and the significant difference from the value of Comparative Example 5 was examined by Tukey-Kramer's multiple comparison test.

[0032] [Table 1]

[0033] Table 1 shows the results of the inhibitory effect of fermented soy milk on the accumulation of progerin in human epidermal keratinocytes. When the Progerin expression ratios of Comparative Example 1, which was an untreated sample in which purified water was added to human epidermal keratinocytes as a negative control, and Comparative Example 2, which was treated with nuclear morphology abnormality, were compared, with the Progerin expression ratio of Comparative Example 2 being taken as 1, the Progerin expression ratio of Comparative Example 1 was 0.094, which was a significant difference, and it was confirmed that nuclear morphology abnormality was indeed induced in epidermal keratinocytes by the nuclear morphology abnormality treatment. The Progerin expression ratio of Comparative Example 3, which was added with 0.05% trifluoroacetyl tripeptide-2 used as a positive control, was 0.583, and it was confirmed that the Progerin expression level was significantly lower than that of Comparative Example 2, which was added with purified water as a negative control. On the other hand, the Progerin expression ratio of Example 1, which was added with 0.01% fermented soy milk, was 0.169, and it was confirmed that the Progerin expression level was significantly and remarkably lower than that of Comparative Example 2, which was added with purified water as a negative control. Therefore, it was shown that the fermented soy milk can exert a significant progerin expression inhibitory effect at a lower concentration in human epidermal keratinocytes than known progerin accumulation inhibitors that suppress the accumulation of progerin, a substance that causes nuclear morphological abnormalities, and furthermore, can exert a progerin accumulation inhibitory effect by inhibiting progerin expression. From the above, it was suggested that the fermented soy milk has a significantly high progerin expression inhibitory effect, and furthermore, a progerin accumulation inhibitory effect by inhibiting progerin expression.

[0034] [Table 2]

[0035] Table 2 shows the results of the effect of fermented soy milk in improving nuclear morphological abnormalities in epidermal keratinocytes. The nuclear circularity of Comparative Example 4, which was untreated by adding purified water as a negative control to epidermal keratinocytes, was 0.843, while the nuclear circularity of Comparative Example 5, which was treated with nuclear morphology abnormality, was 0.820, and a significant difference was confirmed, confirming that nuclear morphology abnormality was indeed induced in epidermal keratinocytes by nuclear morphology abnormality treatment. The nuclear circularity of Comparative Example 6, which was added with 0.05% trifluoroacetyl tripeptide-2 used as a positive control, was 0.835, and no significant difference was confirmed with Comparative Example 5, which was added with purified water as a negative control. On the other hand, the nuclear circularity of Example 2, which was added with 0.01% fermented soy milk, was 0.842, and nuclear morphology abnormality was significantly improved compared to Comparative Example 5, which was added with purified water as a negative control. Therefore, it was shown that the fermented soy milk can exert a nuclear morphology abnormality suppressing effect on human epidermal keratinocytes even at a concentration lower than the concentration at which a known progerin accumulation inhibitor that suppresses the accumulation of progerin, a causative substance of nuclear morphology abnormality, does not necessarily exert an effect to a degree that is recognized as substantial usefulness.

[0036] Next, a formulation example of a skin external preparation containing the fermented soy milk of the present invention is shown, but the present invention is not limited thereto. In the following, the numerical value written after the component name indicates the blend amount, and each example is expressed as a total of 100 mass %. All parts mean parts by mass, and all % means mass %. The fermented soy milk shown in each formulation example below was prepared using soy milk extracted by crushing dried soybean seeds with a homogenizer, adding hot water at 100 ° C., and heating for 5 minutes according to the method described in

[0028] , and the blend amount is shown as mass % converted to evaporation residue. In each formulation example, the effect of the present application was confirmed.

[0037] <Prescription Examples> (Formulation Example 1) Cosmetic cream (mass%) a) Beeswax: 2.0 b) Stearyl alcohol: 5.0 c) Stearic acid 8.0 d) Squalane: 10.0 e) Self-emulsifying glyceryl monostearate: 3.0 f) Polyoxyethylene cetyl ether (20E.O.) 1.0 g) Fermented soy milk: 1.0 h) 1,3-Butylene glycol 5.0 i) Potassium hydroxide 0.3 j) Preservatives and antioxidants: appropriate amount k) Purified water...remainder Total: 100 Manufacturing method Dissolve a) through f) by heating and keep at 80°C. Dissolve h) through k) by heating and keep at 80°C, add to a) through f) and emulsify, then cool to 40°C with stirring. Then add g) and stir to dissolve uniformly.

[0038] (Formulation Example 2) Emulsion (% by mass) a) Beeswax 0.5 b) Vaseline: 2.0 c) Squalane: 8.0 d) Sorbitan sesquioleate: 0.8 e) Polyoxyethylene oleyl ether (20E.O.) 1.2 f) Fermented soy milk: 0.001 g) 1,3-Butylene glycol: 7.0 h) Carboxyvinyl polymer: 0.2 i) Potassium hydroxide 0.1 j) Purified water...remainder k) Preservatives and antioxidants: appropriate amount l) Ethanol: 7.0 Total: 100 Manufacturing method Dissolve a) through e) by heating and keep at 80°C. Dissolve g) through k) by heating and keep at 80°C, add to a) through e) and emulsify, then cool to 50°C with stirring. Add f) and l) at 50°C, and stir and cool to 40°C.

[0039] (Formulation Example 3) Lotion (% by mass) a) Fermented soy milk: 0.01 b) Glycerin: 5.0 c) Polyoxyethylene sorbitan monolaurate (20E.O.) 1.0 d) Ethanol: 6.0 e) Fragrance...appropriate amount f) Preservatives and antioxidants: appropriate amount g) Purified water...remainder Total: 100 Manufacturing method Mix a) through g) and dissolve uniformly.

[0040] (Formulation Example 4) Facial cleanser (mass%) a) Stearic acid: 12.0 b) Myristic acid: 14.0 c) Lauric acid 5.0 d) Jojoba oil: 3.0 e) Glycerin: 10.0 f) Sorbitol: 15.0 g) 1,3-Butylene glycol: 10.0 h) POE(20) glycerol monostearate 2.0 i) Potassium hydroxide 5.0 j) Water...Remainder k) Chelating agent: appropriate amount l)Fragrance...appropriate amount m) Fermented soy milk ··· 0.05 Total: 100 Manufacturing method Heat a) to h) to dissolve and keep at 70℃. Dissolve i) in j) and add to a) to h) to saponify. Then add k) and l) and cool with stirring. Add m) at 50℃ and stir and cool to 40℃.

[0041] (Formulation Example 5) Essence (mass%) a) Fermented soy milk: 10.0 b) Carboxyvinyl polymer: 0.05 c) L-arginine: appropriate amount d) Glycerin: 5.0 e) Polyoxyethylene sorbitan monolaurate (20E.O.) 1.0 f) Ethanol: 6.0 g)Fragrance...appropriate amount h) Preservatives and antioxidants: appropriate amount i) Purified water...remainder Total: 100 Manufacturing method Disperse b) in a portion of i), then add c) to adjust the pH to 6.5. Then mix a) to i) and dissolve uniformly.

[0042] (Formulation Example 6) Cosmetic gel (mass%) a) Fermented soy milk: 0.1 b) Carboxyvinyl polymer: 0.5 c) Sodium hydroxide 0.05 d) Methyl parahydroxybenzoate 0.1 e) bisethoxydiglycol cyclohexane-1,4-dicarboxylate 0.5 f) Polyglyceryl-10 (eicosanedioic acid / tetradecanedioic acid) 0.5 g) PEG / PPG / Polybutylene Glycol-8 / 5 / 3 Glycerin··0.5 h) Polyoxyethylene (60E.O.) hydrogenated castor oil ··· 0.1 i)Fragrance...appropriate amount j) Preservatives and antioxidants: appropriate amount k) Purified water...remainder Total: 100 Manufacturing method Disperse b) in a portion of k), then add c). Then mix a) through k) together and dissolve uniformly.

[0043] (Formulation Example 7) Lipstick (mass%) a) Fermented soy milk: 0.0001 b) Candelilla wax: 9.0 c) Solid Paraffin: 8.0 d) Beeswax: 5.0 e) Carnauba wax: 5.0 f) Ranilin: 11.0 g) Castor oil... balance h) Cetyl 2-ethylhexanoate 0.5 i) Isopropyl myristate ester: 10.0 j) Titanium dioxide: 5.0 k) Red No. 201 ··· 0.6 l) Red No. 202...1.0 m) Red No. 223...0.2 n)Fragrance...appropriate amount o) Preservatives and antioxidants: appropriate amount Total: 100 Manufacturing method Disperse j) to m) in a part of g) and process with a roller. Then mix a) to o), heat and melt, and disperse uniformly. After dispersion, pour into a mold and rapidly cool to form a stick shape.

[0044] (Formulation Example 8) Powder foundation (mass%) a) Fermented soy milk: 0.0001 b) Squalane: 10.0 c) Sorbitan sesquioleate: 3.5 d) Preservatives and antioxidants: appropriate amount e) Titanium dioxide: 13.0 f) Sericite: 25.0 g) Talc... remainder h) Red iron oxide···0.8 i) Yellow iron oxide 2.5 j) Black iron oxide 0.1 Total: 100 Manufacturing method Mix e) to j) and crush them in a crusher. Then add the mixed and dissolved a) to d) and mix them with stirring. Crush them again and press them into a metal plate container.

[0045] (Formulation Example 9) Bath additives (mass%) a) Fermented soy milk: 0.5 b) bisethoxydiglycol cyclohexane-1,4-dicarboxylate 0.2 c) Polyglyceryl-10 (eicosanedioic acid / tetradecanedioic acid) 0.2 d) PEG / PPG / Polybutylene glycol-8 / 5 / 3 Glycerin 0.2 e) Sodium bicarbonate 50.0 f) Sodium sulfate... balance g)Fragrance...appropriate amount h) Preservatives and antioxidants: appropriate amount Total: 100 Manufacturing method Mix a) to h) uniformly. [Industrial Applicability]

[0046] As described above in detail, it was confirmed that the fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria has a remarkable effect of improving nuclear morphological abnormalities and suppressing progerin accumulation. It is known that nuclear morphological abnormalities and progerin accumulation are deeply involved not only in the expression of various aging phenomena such as skin aging, hypercholesterolemia, accelerated arteriosclerosis, diabetes, osteoporosis, senile cataracts, gray hair, hair loss, and other premature aging changes, and aging changes due to endogenous programmed aging associated with aging, but also in skin changes such as reduced stratum corneum function in the epidermis. Therefore, by applying the nuclear morphological abnormality improving agent, progerin accumulation inhibitor, or external skin preparation for preventing and improving reduced stratum corneum function caused by nuclear morphological abnormalities and / or progerin accumulation of the present invention, various symptoms such as reduced stratum corneum function caused by nuclear morphological abnormalities and progerin accumulation are expected to be improved. Therefore, the fermented product can be effectively used to prevent and improve reduced stratum corneum function caused by nuclear morphological abnormalities and progerin accumulation.

Claims

1. An agent for improving nuclear morphological abnormalities, comprising a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria.

2. A progerin accumulation inhibitor comprising a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria.

3. An external skin preparation for preventing and improving stratum corneum dysfunction caused by nuclear morphological abnormalities and / or progerin accumulation, characterized in that it contains a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria.

4. Use of a skin topical composition containing a fermented product obtained by fermenting soy milk as a substrate with lactic acid bacteria for the prevention and improvement of reduced stratum corneum function caused by nuclear morphological abnormalities and / or progerin accumulation (excluding use in treating humans).

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