KR102080719B1 - Composition for preventing, treating sarcopenia or improving muscle strength or increasing muscle mass containing a fermented soybean - Google Patents

Abstract

The composition according to one aspect includes a fermented medicinal soybean fermented with Korean infants and young lactic acid bacteria, and can prevent or treat muscle diseases by promoting muscle differentiation and inhibiting degradation of muscle proteins.

Description

Composition for preventing, treating sarcopenia or improving muscle strength or increasing muscle mass containing a fermented soybean}

The present invention relates to a composition for preventing, treating or enhancing muscle diseases containing medicinal soybean fermented products.

The elderly population is on the rise in recent years, and the UN estimates that by 2050 there will be over two billion people over 60 years old. Muscle loss and muscle weakness are one of the major body changes caused by aging, and it is known to progress gradually from 40 years old. Skeletal muscles are the largest organs, reaching 50% of the total body weight, and sarcopenia has a major impact on quality of life, including degrading physical capacity and increasing the risk of falls and fractures. It is expected to be active.

However, there is no effective alternative to muscle reduction. Hormonal therapy is typically used to treat muscle loss, but when it is not very effective and side effects are being reported, new alternatives are needed. Meanwhile, although many studies have demonstrated that protein intake and muscle production are inseparable, 50% of the population over 65 years of age consume protein below the Korean nutrition standard (2015 National Nutrition Statistics). Soybeans contain high-quality protein and are also more economical than other foods. It is also a favorite food among consumers and contains many health functionalities.Phytochemicals such as isoflavones as well as soy protein are used to treat chronic diseases due to antioxidant, anti-inflammatory and other physiological activities. It has been reported that the risk is lowered. Among them, black beans have been used as a herb since ancient times, and some studies have shown that black beans have higher antioxidant effects than yellow beans. Unlike soybeans, black soybeans also contain anthocyanins and proanthocyanidins in their shells in addition to isoflavones.

When consuming protein as a muscle ingredient, not only whether or not it meets the daily recommended amount, but also the degree of absorption of the protein in the body, the US Food and Drug Administration (FDA) recently said that the soy protein produced by fermentation has the highest digestive absorption rate. It was announced. Fermentation not only facilitates digestive absorption, but also produces many metabolites, doubling the health functionality. Thus, fermented soy protein or soy anthocyanin, procyanidins, isoflavones, etc. are expected to be more effective for muscle production and muscle differentiation.

One aspect is the prevention of muscle disease containing a fermented medicinal bean as an active ingredient, inoculated and fermented by Bifidobacterium animalis subsp. Lactis. LDTM 8102 (KCTC13392BP) strain to the medicinal beans or It provides a therapeutic pharmaceutical composition.

Another aspect is the prevention of muscle diseases containing a fermented medicinal soybean as an active ingredient, inoculated and fermented with a strain of Bifidobacterium animalis subsp. Lactis. LDTM 8102 (KCTC13392BP) in the soybean. It is to provide a food composition for improvement.

Another aspect is to promote muscle differentiation, muscle regeneration, containing the fermented medicinal bean as an active ingredient, inoculated and fermented with Bifidobacterium animalis subsp.Lactis LDTM 8102 (KCTC13392BP) strains in the medicinal beans Or to provide a composition for muscle strengthening.

Another aspect is to provide a method for preparing a medicinal soybean fermentation comprising inoculating and fermenting a Bifidobacterium animalis subsp . Lactis LDTM 8102 (KCTC13392BP) strain to the medicinal beans.

One aspect is the prevention of muscle disease containing a fermented medicinal bean as an active ingredient, inoculated and fermented with a strain of Bifidobacterium animalis subsp. Lactis. LDTM 8102 (KCTC13392BP) to the soybean Provided is a therapeutic pharmaceutical composition. Another aspect is a muscle disease comprising administering to a subject a medicinal bean fermentation product, inoculated and fermented with a strain of Bifidobacterium animalis subsp. Lactis. LDTM 8102 (KCTC13392BP) to the subject. Provides a way to treat it.

In the present specification, the term “yak bean” refers to small black beans whose shell is black and smaller in size than normal black beans. There are rats, small blue beans, medicinal beans, dawon beans, socheong No. 2, and much smaller than ordinary black beans, but The flavone family of useful ingredients, genistein and daidzein, are much higher than other black beans.

As used herein, the term “ Bifidobacterium animalis subsp. Lactis. LDTM 8102 (KCTC13392BP) strain” is isolated from feces of Korean infants and is a beta-glucosidase (β- It can be effectively used for bioconversion of biomass natural products including rice, barley, milk, soybeans, ginseng, algae, woody system, etc. through microbial metabolism.

As used herein, the term "fermented product" refers to the modification or transformation of raw materials by physical and chemical methods using lactic acid bacteria, and in nature, the desired result can be changed in a matter of days or months that can be changed after tens of thousands of years. It means the decomposition process that can be obtained. Specifically, the fermentation product is a fermentation product produced by mixing the medicinal beans into the lactic acid bacteria culture medium and inoculating and fermenting the lactic acid bacteria, wherein the lactic acid bacteria is Bifidobacterium animalis subsp. Lactis. LDTM 8102 (KCTC13392BP) strain. In addition, the fermented product may not only mean a product fermented by the lactic acid bacteria, but may also include a fermentation extract obtained by filtering the fermentation filtrate or fermented product after fermenting the medicinal beans with the lactic acid bacteria.

The medicinal beans may be pretreated before fermentation. Specifically, the weak beans may be pretreated by performing roasting or grinding. In one embodiment, when the pretreatment is roasting, the roasting may be performed for 5 to 30 minutes at a temperature condition of 100 to 300 ℃. Specifically, the roasting is 100 to 300 ℃, 100 to 250 ℃, 100 to 200 ℃, 100 to 150 ℃, 150 to 300 ℃, 150 to 250 ℃, 150 to 200 ℃, 200 to 300 ℃, 160 to 300 ℃ Or it may be carried out under the conditions of 160 to 250 ℃. In addition, the roasting may be performed for 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, 5 to 15 minutes, 5 to 10 minutes, 7 to 30 minutes, 7 to 20 minutes, or 10 to 30 minutes. have. At this time, when the roasting temperature is less than the above range, the flavor of the medicinal beans is not maintained, there is a problem that the efficacy of preventing or treating muscle diseases is lowered, and when the roasting temperature is exceeded, the bitter taste of the medicinal beans becomes stronger, consumer preference There is a decreasing problem. In addition, when the roasting time is less than the above range, there is a problem that the stability of the taste is not maintained when applied as a food composition, if the above range, there is a problem that the weak soybeans are carbonized.

The extraction may be extracted from the fermented medicinal beans with water, C1 to C4 lower alcohol or a mixture thereof. For example, the lower alcohol used to extract the weak soybean may be ethanol or methanol. After extracting the weak soybeans during extraction may be to extract water, alcohol or a mixture thereof by adding 2 to 20 times the weight of the weak beans. For example, the extract may be added by adding 3 to 10 times. Extraction temperature is 30 ℃ to 100 ℃, 60 ℃ to 100 ℃. It may be 30 ℃ to 90 ℃, 30 ℃ to 80 ℃, 60 ℃ to 90 ℃, or 60 ℃ to 80 ℃. Extraction time may be 1 hour to 10 hours, 2 hours to 8 hours, 2 to 5 hours, 3 to 7 hours or 4 to 6 hours. As the extraction method, both cold needle, ultrasonic extraction or reflux cooling extraction method can be used. Extraction times may be one to five times, two to four times, three to five times repeated extraction.

In one embodiment, the dose of the fermentation may be included at a concentration of 1 to 30 μg / mL relative to the total composition. The dose of the fermentation product is, for example, 1 to 30 µg / mL, 1 to 25 µg / mL, 1 to 20 µg / mL, 3 to 30 µg / mL, 5 to 25 µg / mL, 5 to 20 µg / mL Or, it may be included in a concentration of 10 to 20 μg / mL. At this time, if the fermented soybean is less than the weight part, the muscle differentiation is lowered and the decomposition of muscle protein is promoted, there is a problem that difficult to exert the prevention or treatment effect of muscle disease, if the soybean fermentation exceeds the weight part There may be problems such as toxicity in the body.

The composition can be used for the prevention or treatment of muscle diseases. The muscle diseases include, for example, atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis (Lou Gehrig's disease, amyotrophic lateral sclerosis, rigid spinse syndrome, Charcot-Marie-Tooth disease, or sarcopenia. In one embodiment, the sarcopenia may be senile sarcopenia. Specifically, the fermented product may be used for the prevention or treatment of senile muscular dystrophy by increasing the expression of early and late muscle differentiation factors. In addition, the fermentation may be used for the prevention or treatment of muscle diseases by reducing the expression of muscle protein differentiation factor.

Pharmaceutical compositions for the prevention or treatment of muscle diseases according to one embodiment are oral formulations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. It can be formulated and used in the form of and may include suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions for formulation.

The carrier or excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicide, cellulose, methyl cellulose, undetermined. And various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

In the case of formulation, it may be prepared by using diluents or excipients such as fillers, weights, binders, wetting agents, disintegrating agents, and surfactants which are commonly used.

Solid preparations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like with fermented medicinal beans. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for preventing or treating muscle diseases according to one embodiment depends on the condition, body weight, extent of disease, drug form, route of administration and duration of the patient, but may be appropriately selected by those skilled in the art. However, for the desired effect, it may be administered at 0.0001 to 2,000 mg / kg, preferably at 0.001 to 2,000 mg / kg. Administration may be administered once a day or may be divided several times. However, the scope of the present invention is not limited by the above dosage.

The pharmaceutical composition for preventing or treating muscle diseases according to one embodiment may be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Another aspect is to promote muscle differentiation, muscle regeneration, containing the fermented medicinal bean as an active ingredient, inoculated and fermented with Bifidobacterium animalis subsp.Lactis LDTM 8102 (KCTC13392BP) strains in the medicinal beans Or it provides a composition for muscle strengthening. Specific contents of the composition are as described above. In one embodiment, the composition may be to increase muscle mass or promote muscle production. In such muscle differentiation promoting, muscle regeneration or muscle strengthening applications, muscle growth may occur by increasing fiber size and / or by increasing fiber count. In addition, the increase in muscle mass improves the growth of muscles, especially among the body components, which can increase muscle mass through physical exercise and endurance, and increase muscle mass by administering a substance having an effect of increasing muscles in the body. . Muscle regeneration may also be the formation of new muscle fibers from myoblasts.

Another aspect is the prevention of muscle disease containing the fermented medicinal bean as an active ingredient, inoculated and fermented with Bifidobacterium animalis subsp.Lactis. LDTM 8102 (KCTC13392BP) strains. Or it provides a food composition for improvement. Specific contents of the composition are as described above.

In the food composition for preventing or improving muscle diseases according to one embodiment, when using the fermented medicinal beans as an additive of the food composition, it may be added as it is or used with other foods or food ingredients, according to a conventional method It can be used properly. The mixed amount of the active ingredient can be appropriately determined depending on the purpose of use, such as prevention, health or treatment.

Formulations of food compositions may be in the form of powders, granules, pills, tablets, capsules, as well as in the form of general foods or beverages.

There is no restriction | limiting in particular in the kind of said food, The foodstuff which can add the said substance is a dairy product containing meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, etc. , Various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., may include all of the food in the usual sense.

In general, the fermented medicinal soybeans may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the raw material. However, in the case of long-term intake for health and hygiene or for health control, the amount may be below the above range, and the present invention has no problem in terms of safety in terms of using fractions from natural products. The above amount can also be used.

The beverage in the food composition according to one embodiment may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in a conventional beverage. The natural carbohydrates described above may be monosaccharides such as glucose, fructose, disaccharides such as maltose, sucrose and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, erythritol and the like. As the sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, a food composition for preventing or improving muscle diseases according to one embodiment includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, and pH adjusting agents. And stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks. In addition, the composition for improving sleep of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The ratio of such additives is not limited, but is generally selected from the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the food composition.

Another aspect provides a method for preparing a medicinal soybean fermentation comprising inoculating and fermenting a Bifidobacterium animalis subsp . Lactis LDTM 8102 (KCTC13392BP) strain to the medicinal beans. In one embodiment, the method may include roasting the soybeans at 100 to 300 ° C. for 5 to 30 minutes before the step of fermentation. Details of the roasting temperature and time are as described above. The prepared soybean fermented product may further include extracting with water, C1 to C4 lower alcohol, or a mixture thereof to prepare an extract. Details of the step of preparing the extract is as described above.

As described above, the composition according to the present invention, fermented with infants, specifically Bifidobacterium animalis subsp.Lactis LDTM 8102 (KCTC13392BP) strain derived from infants of Koreans Compared to the fermented soybeans, which did not undergo the fermentation step, it was confirmed that it promotes muscle differentiation in muscle cells and inhibits degradation of muscle proteins. Therefore, the composition containing the fermentation may be used for the prevention or treatment of muscle diseases by promoting muscle differentiation of the human body and inhibiting muscle reduction.

The composition according to one aspect may be used for the prevention or treatment of muscle diseases by promoting muscle differentiation in muscle cells and inhibiting the degradation of muscle proteins. In addition, muscle differentiation, muscle regeneration, muscle mass gain through the muscle strengthening effect can be used to promote muscle production or improve muscle function. In addition, the composition contains a weak soybean is harmless to the human body does not cause adverse effects on normal cells has the advantage that there is no harm to the human body for long-term use.

Figure 1a is a photograph confirming the expression of the early muscle differentiation factor in the mouse muscle cells treated in Example 1 and Comparative Examples 1 and 2.
1B is a graph quantifying the expression level of muscle differentiation initial factors expressed in mouse muscle cells treated with Example 1 and Comparative Examples 1-2.
Figure 2a is a photograph confirming the expression of late muscle differentiation factor in the mouse muscle cells treated in Example 1 and Comparative Examples 1-2.
Figure 2b is a graph quantifying the expression level of late muscle differentiation factor expressed in mouse muscle cells treated in Example 1 and Comparative Examples 1-2.
Figure 3a is a photograph confirming the muscle differentiation promoting efficacy in the mouse muscle cells treated in Example 1 and Comparative Examples 1 to 2 through IF staining.
Figure 3b is a graph quantifying the number of nuclei in the root canal cells compared to the total number of nuclei in the mouse muscle cells treated in Example 1 and Comparative Examples 1 and 2 (fusion index).
Figure 4a is a photograph confirming the expression of muscle protein degradation factors in the mouse muscle cells treated in Example 1 and Comparative Examples 1-2.
Figure 4b is a graph quantifying the expression of MuRF-1 in mouse muscle cells treated with Example 1 and Comparative Examples 1 and 2.
Figure 4c is a graph quantifying the expression of Atrogin-1 in mouse muscle cells treated with Example 1 and Comparative Examples 1-2.
5A is a photograph confirming myhc expression in mouse muscle cells treated with Example 1 by Western blot.
5B is a graph quantifying myhc expression in mouse muscle cells treated with Example 1. FIG.
Figure 6a is a photograph confirming myhc expression in mouse muscle cells treated in Example 2 by Western blot.
6B is a graph quantifying myhc expression in mouse muscle cells treated with Example 2. FIG.
Figure 7a is a photograph confirming myhc expression by Western blot after the treatment of Example 1 to the muscle cells of the mice that did not induce aging.
Figure 7b is a graph quantifying myhc expression after treatment of Example 1 to the muscle cells of mice not induced aging.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Preparation of Medicinal Bean Fermentation

A medicinal soybean fermented product was prepared using Bifidobacterium animalis subsp. Lactis. LDTM 8102 (Accession No .: KCTC13392BP) isolated from feces of Korean infants. Specifically, Bifidobacterium animalis subsp. Lactis. LDTM 8102 (Accession No .: KCTC13392BP), which is mixed with glycerol and frozen at −80 ° C., is inoculated onto MRSC medium at 37 ° C. , The pre-culture process was performed for 20 to 24 hours in anaerobic conditions. Thereafter, 50 g / L of weak soybean powder was inoculated with the above strain culture medium, which was subjected to a pre-culture process to increase activity, and then cultured under anaerobic conditions maintained at 37 ° C. The samples collected during the fermentation were lyophilized by heat treatment, and the samples were added with 0, 5, 10, 30, 50, and 70% fermented alcohol (vegetable alcohol) at a rate of 10 ml per 0.5 g, respectively. After bathing for 2 hours at < RTI ID = 0.0 > Then, the mixture was allowed to stand at room temperature for 1 hour, cooled, and centrifuged at 5000 rpm for 10 minutes to separate the supernatant. The separated supernatant was filtered using a 0.2 μm syrine filter, and all the spirits were removed in a 40 ° C. vacuum condenser, and then lyophilized to powder. Thereafter, the lyophilized powder was dissolved in 50% DMSO and used as a fermented soybean.

Example 2. Preparation of Fermented Medicinal Beans

After inoculation of the strain culture medium with increased activity by performing a pre-culture process to 50-100 g / L medicinal soybean powder roasted at 160 to 230 ° C. for 15 to 25 minutes, shaking culture was performed under anaerobic conditions maintained at 37 ° C. Except that, the fermented medicinal beans were prepared in the same manner as in Example 1.

[Comparative Example]

Comparative Example 1. Preparation of Fermented Medicinal Soybeans

A medicinal soybean fermented product was prepared in the same manner as in Example 1, except that fermented medicinal soybeans using Lactobacillus rhamnosus GG (LGG) lactic acid bacteria.

Comparative Example 2. Preparation of Fermented Medicinal Soybeans

Bifidobacterium animalis subsp . Lactis ( Bifidobacterium animalis subsp . Lactis) A fermented medicinal soybean was prepared in the same manner as in Example 1, except that fermented medicinal soybean using KCTC 5854 lactic acid bacteria.

Experimental Example

Confirmation of Root Reduction Efficacy of Medicinal Soybean Fermentation

In order to confirm the sensitizing effect of reducing senile muscles of Example 1 and Comparative Examples 1 and 2, Western blot analysis was performed on the expression levels of myoD and myhc, which are biomarkers related to muscle differentiation. Specifically, after seeding mouse muscle cells C2C12 in a 6 cm dish, DMEM (Dulbecco) added with 10% Fetal Bovine Serum and 1% Antibiotic-antimycotic -modified Eagle medium) was incubated for 72 hours in a 37 ℃, 10% CO ₂ incubator (Forma Scientific Co., Marjetta, OH, USA). Subsequently, while incubating the medium with DMEM (Dulbecco-modified Eagle medium) containing 2% Horse Serum and 1% Antibiotic-antimycotic, inducing aging with TGF-beta, The fermented products of Example 1 and Comparative Examples 1 and 2 were treated, respectively, to determine whether the medicinal soybean fermented product recovered the aged muscle cells due to TGF-beta. Since different biomarkers have different expression timings, the initial differentiation factor myoD expression level was identified 24 hours after the fermentation of the soybean fermented cells, and the late differentiation factor myhc expression level was 96 hours later using RIPA buffer. Scraped off Then, the protein was quantified using protein assay reagent kits (Bio-Rad Laboratories, Hercules, CA, USA) after centrifugation at 13000g for 10 minutes to extract pure protein. The extracted protein was electrophoresed using SDS polyacrylamide gel and then transferred to nitrocellulose blotting membrane (GE Healthcare Life Science, Amersham, UK). Thereafter, the membrane was blocked with 5% skim milk for 1 hour and then treated with primary antibodies of myoD and myhc diluted 1: 1000 for 16 hours in a 4 ° C. refrigerator. After washing the primary antibody three times for 10 minutes with TBS-T buffer, the secondary antibody diluted 1: 5000 concentration in 5% skim milk was treated for 1 hour. The secondary antibody was then washed 5 times for 8 min with TBS-T buffer. Thereafter, the degree of luminescence of the myogenic biomarker protein was confirmed in the dark room using an ECL detection kit, and a protein band was developed on the film using a develping buffer. The expression level of the protein was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Figure 1a is a photograph confirming the expression of the early muscle differentiation factor in the mouse muscle cells treated in Example 1 and Comparative Examples 1 and 2.

1B is a graph quantifying the expression level of muscle differentiation initial factors expressed in mouse muscle cells treated with Example 1 and Comparative Examples 1-2.

Figure 2a is a photograph confirming the expression of late muscle differentiation factor in the mouse muscle cells treated in Example 1 and Comparative Examples 1-2.

Figure 2b is a graph quantifying the expression level of late muscle differentiation factor expressed in mouse muscle cells treated in Example 1 and Comparative Examples 1-2.

As shown in FIGS. 1A and 1B, it was confirmed that the expression level of myoD, which is an early differentiation factor, was higher in the mouse muscle cells treated with Example 1 than in the mouse muscle cells treated with Comparative Examples 1-2. 2A and 2B, it was confirmed that the expression level of myhc, which is a late muscle differentiation factor, was higher in the mouse muscle cells treated with Example 1 than in the mouse muscle cells treated with Comparative Examples 1-2. . That is, the weak soybean fermentation product according to one aspect promotes muscle differentiation not only in the early stages of muscle differentiation but also in the late stage, and may be used for the prevention or treatment of senile myotropia.

Confirmation of the effect of promoting the differentiation of medicinal soybeans

In order to confirm the effect of promoting muscle differentiation of Example 1 and Comparative Examples 1 and 2, immunofluorescence staining was performed. Specifically, after seeding mouse muscle cells C2C12 on 8 well chamber slides (Thermo Scientific), 10% Fetal Bovine Serum and 1% Antibiotic-antimycotic Incubated for 72 hours in a 37 ° C., 10% CO ₂ incubator (Forma Scientific Co., Marjetta, OH, USA) using the added Dulbecco-modified Eagle medium (DMEM). Subsequently, while incubating the medium with DMEM (Dulbecco-modified Eagle medium) containing 2% Horse Serum and 1% Antibiotic-antimycotic, inducing aging with TGF-beta, The fermented products of Example 1 and Comparative Examples 1 and 2, respectively, were treated to determine whether the medicinal soybean fermented product recovered the aged muscle cells due to TGF-beta. For 96 hours in medium with DMEM (Dulbecco-modified Eagle medium) with 2% Horse Serum and 1% Antibiotic-antimycotic containing TGF-beta and medicinal soybean ferment. After incubation, the medium was suctioned, washed with PBS, and fixed for 4 minutes with 4% formaldehyde. Afterwards, the cells were penetrated with 0.2% Triton X 100 for 10 minutes to allow dyes to enter the cells. After washing with PBS, it was blocked with 20% normal goat serum for 1 hour. The myhc primary antibody was then treated and stored in a 4 ° C. refrigerator for 16 hours. Subsequently, the secondary antibody was treated, the nuclei were stained with DAPI, and myhc proteins and nuclei in the root canal cells were identified using a confocal microscope. According to the following Equation 1, the differentiation of mouse myoblasts into myotubes was confirmed.

[Equation 1]

Fusion Index = number of nuclei in myotubes / total nucleus of muscle cells

Figure 3a is a photograph confirming the muscle differentiation promoting efficacy in the mouse muscle cells treated in Example 1 and Comparative Examples 1 to 2 through IF staining.

Figure 3b is a graph quantifying the number of nuclei in the root canal cells compared to the total number of nuclei in the mouse muscle cells treated in Example 1 and Comparative Examples 1 and 2 (fusion index).

As shown in Figure 3a, the expression of myhc was reduced in Comparative Examples 1 to 2 induced aging with TGF-beta compared to the control group did not induce aging with TGF-beta. On the other hand, when Example 1 was treated it was confirmed that the expression of myhc increased. In addition, the increase in the number of nuclei in the root canal cells was found to increase muscle cell differentiation. . In addition, as shown in Figure 3b, it was confirmed that the number of nuclei in the root canal cells compared to the total number of nuclei in the mouse muscle cells treated with Example 1 compared to the mouse muscle cells treated with Comparative Examples 1-2. That is, the composition according to one aspect may promote differentiation of muscle cells by promoting muscle canal cell formation.

Expression of Muscle Protein Degradation Related Factors in Medicinal Soybean Fermentation

The increase in muscle protease complex and ubiquitin enzyme is closely related to myopathy, and it was confirmed by Western blot whether the expression of muscle protein degradation-related factors of Example 1 and Comparative Examples 1 and 2 was expressed. Specifically, the mouse myocytes were differentiated for 96 hours to form a myofibril bundle, and then treated with fermentations of Example 1 and Comparative Examples 1 and 2 while inducing aging with TGFb-beta. After 24 hours, proteins were scraped off using RIPA buffer and centrifuged at 13000 x g for 10 minutes to extract pure protein. Subsequently, the protein was assayed using protein assay reagent kits (Bio-Rad Laboratories, Hercules, CA, USA), and the extracted protein was electrophoresed using SDS polyacrylamide gel, followed by nitrocellulose blotting membrane (GE Healthcare Life Science). , Amersham, UK). After blocking the membrane with 5% skim milk for 1 hour, muscle proteinase complex and ubiquitin enzymes Murf-1 and Atrogin-1 were diluted 1: 1000 as the primary antibody and treated for 16 hours in a 4 ° C. refrigerator. After washing the primary antibody three times for 10 minutes with TBS-T buffer, the secondary antibody diluted 1: 5000 in 5% skim milk was treated for 1 hour. Then, the secondary antibody was washed by repeating 5 times for 8 minutes with TBS-T buffer. Then, the degree of luminescence of the muscle proteinase complex and ubiquitin enzyme was confirmed in the dark room using the ECL detection kit, and the protein band was developed on the film using the develping buffer. The expression level of the protein was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Figure 4a is a photograph confirming the expression of muscle protein degradation factors in the mouse muscle cells treated in Example 1 and Comparative Examples 1-2.

Figure 4b is a graph quantifying the expression of MuRF-1 in mouse muscle cells treated with Example 1 and Comparative Examples 1 and 2.

Figure 4c is a graph quantifying the expression of Atrogin-1 in mouse muscle cells treated with Example 1 and Comparative Examples 1-2.

As shown in Figures 4a to 4c, the expression levels of muscle protein degrading factors (MURF-1, Atrogin-1) in the mouse muscle cells treated with Example 1 compared to the mouse muscle cells treated with Comparative Examples 1 and 2 It was confirmed that the low. That is, the weak soybean fermentation product according to one aspect may be used for preventing or treating myotropia by inhibiting the degradation of muscle protein.

Expression of Muscle Protein Degradation Related Factors after Pretreatment

In order to confirm the recovery effect on the aged muscle cells of the pretreated medicinal soybean fermentation, the amount of myhc expression in Example 1 without pretreatment and Example 2 with pretreatment was analyzed by Western blot. Specifically, after seeding mouse muscle cells C2C12 in a 6 cm dish, DMEM (Dulbecco) added with 10% Fetal Bovine Serum and 1% Antibiotic-antimycotic -modified Eagle medium) was incubated for 72 hours in a 37 ℃, 10% CO ₂ incubator (Forma Scientific Co., Marjetta, OH, USA). Subsequently, while incubating the medium with DMEM (Dulbecco-modified Eagle medium) containing 2% Horse Serum and 1% Antibiotic-antimycotic, inducing aging with TGF-beta, Example 1 and Example 2 were each treated with (capacity). After 96 hours, the protein was scraped off using RIPA buffer, centrifuged at 13000g for 10 minutes to extract pure protein, and the protein was quantified using protein assay reagent kits (Bio-Rad Laboratories, Hercules, CA, USA).

Figure 5a is a photograph confirming myhc expression in mouse muscle cells treated in Example 1 by Western blot, Figure 5b is a graph quantifying the expression of the myhc.

Figure 6a is a photograph confirming myhc expression in mouse muscle cells treated with Example 2 by Western blot, Figure 6b is a graph quantifying the expression of the myhc.

As shown in Figure 5 and Figure 6, it can be seen that the expression of myhc increased in Example 2 subjected to pretreatment.

Muscle Enhancer and Muscle Differentiation Effect of Medicinal Soybean Fermentation

In order to confirm the effect of muscle enhancement and muscle differentiation of Example 1, the expression level of myhc (myosin heavy chain), which is a late muscle differentiation biomarker, was measured by Western blot analysis. Specifically, after seeding mouse muscle cells C2C12 in a 6 cm dish, DMEM (Dulbecco) added with 10% Fetal Bovine Serum and 1% Antibiotic-antimycotic -modified Eagle medium) was incubated for 72 hours in a 37 ℃, 10% CO ₂ incubator (Forma Scientific Co., Marjetta, OH, USA). Thereafter, the medium was changed to DMEM (Dulbecco-modified Eagle medium) to which 2% horse serum (Horse Serum) and 1% antibiotic-antimycotic were added, and the fermentation product of Example 1 was used. Was treated once every 24 hours for a total of 96 hours to determine whether the fermented soybeans promote differentiation of muscle cells. After 96 hours, the protein was scraped off using RIPA buffer, centrifuged at 13000g for 10 minutes to extract pure protein, and protein quantitated using protein assay reagent kits (Bio-Rad Laboratories, Hercules, CA, USA). . The extracted protein was electrophoresed using SDS polyacrylamide gel and then transferred to nitrocellulose blotting membrane (GE Healthcare Life Science, Amersham, UK). Thereafter, the membrane was blocked with 5% skim milk for 1 hour and then treated with a primary antibody of myhc diluted 1: 1000 for 16 hours in a 4 ° C. refrigerator. After washing the primary antibody three times for 10 minutes with TBS-T buffer, the secondary antibody diluted 1: 5000 concentration in 5% skim milk was treated for 1 hour. The secondary antibody was then washed 5 times for 8 min with TBS-T buffer. Thereafter, the degree of luminescence of the myogenic biomarker protein was confirmed in the dark room using an ECL detection kit, and a protein band was developed on the film using a develping buffer. The expression level of the protein was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Figure 7a is a photograph confirming myhc expression by Western blot after the treatment of Example 1 to the muscle cells of the mice that did not induce aging.

Figure 7b is a graph quantifying myhc expression after treatment of Example 1 to the muscle cells of mice not induced aging.

As shown in FIG. 7, when the mouse muscle cells were treated with Example 1, myhc expression was significantly increased compared to the control group, so that muscle differentiation was promoted.

Claims (13)

Food for the prevention or improvement of muscle diseases containing fermented medicinal soybeans as an active ingredient, inoculated and fermented with Bifidobacterium animalis subsp.Lactis.LDTM 8102 (KCTC13392BP) strain The composition of claim 1, wherein the muscle disease is a muscle disease due to muscle function decline, muscle loss, muscle wasting or muscle degeneration. The food composition of claim 1, wherein the fermentation product comprises fermentation filtrate or fermentation extract. delete The method of claim 1, wherein the muscle disease is atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis (Lou Gehrig's disease) , amyotrophic lateral sclerosis, rigid spinse syndrome, Charcot-Marie-Tooth disease and sarcopenia. The food composition of claim 4, wherein the sarcopenia is senile muscular dystrophy. The food composition of claim 1 which promotes muscle differentiation. The food composition of claim 1 which inhibits the degradation of muscle protein. The food composition of claim 1, wherein the medicinal beans are pretreated. The food composition of claim 8, wherein the pretreatment is processed by performing roasting or grinding. Pharmaceutical for the prevention or treatment of muscle diseases containing fermented medicinal soybean as an active ingredient, inoculated and fermented with Bifidobacterium animalis subsp.Lactis LDTM 8102 (KCTC13392BP) strain The composition, wherein the muscle disease is a muscle disease due to muscle function decline, muscle loss, muscle wasting or muscle degeneration. Promotes muscle differentiation, muscle regeneration or muscle strengthening containing fermented medicinal soybeans as an active ingredient, inoculated with Bifidobacterium animalis subsp.Lactis LDTM 8102 (KCTC13392BP) strains. Composition. Bifidobacterium animalis subsp . Lactis LDTM 8102 (KCTC13392BP) inoculated and fermented to the soybeans. The method according to claim 12, wherein before the step of fermentation, comprising about roasting the soybeans at 100 to 300 ℃ for 5 to 30 minutes.

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