KR102772699B1 - Method for preparing high concentration branched chain amino acid with improvement activity of muscle function - Google Patents

Abstract

The present invention relates to a muscle protection composition comprising, as an active ingredient, a soybean fermentation product by Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP). As a result, compared to the conventionally known protease enzyme treatment, the branched chain amino acid content is significantly increased, thereby improving the muscle protection, muscle disease prevention or treatment effect, and improving palatability, so that it can be utilized as a health functional food.

Description

Method for preparing high concentration branched chain amino acid with improvement activity of muscle function

The present invention relates to a muscle protection composition comprising a fermented soybean product as an effective ingredient and a method for producing the same, and more particularly, to a muscle protection composition comprising a fermented soybean product with an increased branched-chain amino acid (BCAA) content as an effective ingredient and a method for producing the same.

The protein material market is on a steady increase worldwide and is currently estimated to be worth over 20 trillion won. Protein materials are applied to various industries including the food industry for various purposes, and the protein materials mainly used are classified into whey protein, egg yolk protein, gelatin, single-cell protein, soy protein, fish protein, gluten, and other plant-based proteins and are used for a wide range of purposes. In particular, plant-based proteins are expanding their use and marketability due to the wellness trend. In particular, the protein supplement products mainly enjoyed by people who exercise are forming a large market due to the wellness trend and the bodybuilder craze. In the United States, the sports nutrition market is about 20 trillion won and is growing by more than 10% annually. Among them, the protein supplement product market is one of the markets with the largest growth, at 5 trillion won.

Recently, due to the increased interest in protein intake, the limited product market of protein supplements has been expanding to include various protein materials in general foods to increase protein content. In the past, animal protein materials were mainly used, but recently, with the increase in consumer interest in the wellness trend and the increase in caution regarding animal protein sources such as mad cow disease, foot-and-mouth disease, and avian influenza, interest in plant-based protein materials with a relatively safe image is increasing. The material that is receiving particular attention in this interest is soy protein. Currently, the soy protein market forms the largest market among plant-based protein markets. However, due to the nature of soybeans as raw materials, there are concerns about allergies and the constantly occurring GMO issue, so many consumers are increasingly interested in new plant-based protein materials that can alleviate these concerns.

Branched-chain amino acids have three main characteristics. First, they provide direct energy (J. Nutr. 136: 269S-273S, 2006). Most amino acids are transported to the liver and metabolized, but branched-chain amino acids have the characteristic of bypassing the liver and being taken directly from peripheral tissues. BCAA is oxidized in peripheral tissues to produce CO2 and energy. Second, they provide a substrate for gluconeogenesis (J. Nutr. 136: 264S-268S, 2006). Gluconeogenesis is a reaction that directly synthesizes glucose in the body, and this reaction occurs in the liver, and the glucose produced is released into the blood and used in various places. At this time, branched-chain amino acids decomposed in muscles provide resources for glucose synthesis in the liver, namely alanine and pyruvate, which are precursors of glucose synthesis. Third, they inhibit protein decomposition and promote synthesis in muscles. : In cases such as sepsis, the concentration of amino acids in the circulating blood is very high because the use of glucose within cells is abnormal, so the body catabolizes muscles, the main storage site for body proteins, to supply energy, and in the process of obtaining energy directly from branched-chain amino acids, other amino acids are also released from the muscles into the blood. In this case, when branched-chain amino acids are injected into the bloodstream, muscle protein decomposition is suppressed and protein synthesis is promoted.

Research is being conducted on the production of hydrolysates containing a large amount of branched-chain amino acids from vegetable proteins, and technology using corn gluten hydrolysates has also been studied. Corn is widely consumed as a crop, but it is one of the representative GMO crops, so consumers are concerned and wary. In addition, research is being conducted on a method for producing rice protein hydrolysates containing a high amount of branched-chain amino acids using rice protein, a natural vegetable protein, as a raw material.

Korean Patent Publication No. 2020-0124083

The purpose of the present invention is to provide a muscle protection food composition that uses plant-based protein materials such as soybeans to increase the content of branched-chain amino acids, thereby minimizing side effects on the human body, while protecting muscle proteins, promoting muscle protein synthesis, and inhibiting muscle protein decomposition, and also improving palatability, so that it can be utilized as a health functional food.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle diseases, which can effectively treat various muscle diseases such as muscular dystrophy and sarcopenia, by increasing the content of branched-chain amino acids while using plant-based protein materials such as soybeans, thereby protecting muscle proteins, promoting muscle protein synthesis, and enhancing the efficacy of inhibiting muscle protein decomposition while minimizing side effects on the human body.

According to one aspect of the present invention,

A food composition for muscle protection is provided, which contains, as an active ingredient, a fermented soybean product fermented by Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

The above soybean fermentation product may be a soybean fermentation product obtained by fermenting soybean enzymatic hydrolysate treated with any one enzyme selected from serine protease, neutral protease, cysteine protease, and leucine protease, by the Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

The above soybean enzymatic hydrolysate may be treated by leucine protease.

The above beans may be at least one selected from soybeans, peas, fava beans, small beans, peas, broad beans, black beans, lima beans, and Egyptian beans.

The above fermented soybean product may have a branched chain amino acid content of 50 to 120 mg/g based on the supernatant of a hot water extract of 0.1 g of soybean/10 ml of water.

The above muscle protection food composition may be for inhibiting the synthesis of myostatin, a muscle protein decomposition factor.

The above muscle protection food composition may be for promoting the synthesis of p-mTOR, a muscle protein synthesis factor.

According to another aspect of the present invention,

A pharmaceutical composition for preventing or treating muscle disease is provided, which contains, as an active ingredient, a fermented soybean product fermented by Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

The above soybean fermentation product may be a soybean fermentation product obtained by fermenting soybean enzymatic hydrolysate treated with any one enzyme selected from serine protease, neutral protease, cysteine protease, and leucine protease, by the Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

The above fermented soybean product may have a branched chain amino acid content of 50 to 120 mg/g based on the supernatant of a hot water extract of 0.1 g of soybean/10 ml of water.

The pharmaceutical composition for preventing or treating the above muscle disease may be for inhibiting the synthesis of myostatin, a muscle protein decomposition factor.

The pharmaceutical composition for preventing or treating the above muscle disease may be for promoting the synthesis of p-mTOR, a muscle protein synthesis factor.

The above muscle disease may be any one selected from muscular atrophy, sarcopenia, atony, muscular dystrophy, myasthenia gravis, and amyotrophic lateral sclerosis.

According to another aspect of the present invention,

A method for producing a muscle protection composition is provided, comprising the step of producing a fermented soybean product by fermenting soybean powder with Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

Before the above step, a step of enzymatically decomposing the soybean powder with any one enzyme selected from serine protease, neutral protease, cysteine protease, and leucine protease may be additionally performed.

The muscle protection food composition of the present invention has the effect of protecting muscle proteins, promoting muscle protein synthesis, and inhibiting muscle protein decomposition while minimizing side effects on the human body by increasing the content of branched-chain amino acids while using plant-based protein materials such as soybeans, and also improving palatability, so that it can be utilized as a health functional food.

The pharmaceutical composition for preventing or treating muscle diseases of the present invention uses plant-based protein materials such as soybeans, thereby increasing the content of branched-chain amino acids, thereby minimizing side effects on the human body while protecting muscle proteins, promoting muscle protein synthesis, and enhancing the efficacy of inhibiting muscle protein decomposition, thereby effectively treating various muscle diseases such as muscular dystrophy and sarcopenia.

Figure 1 shows the results of branched-chain amino acid (BCAA) content analysis according to Experimental Example 1.
Figure 2 shows the amino acid analysis results of fermented soybean according to Experimental Example 2.
Figure 3 shows the results of SDS-PAGE analysis according to Experimental Example 3.
Figure 4 is a SEM image according to Experimental Example 3.
Figure 5 shows the FTIR analysis results according to Experimental Example 3.
Figure 6 shows the results of taste change analysis according to Experimental Example 4.
Figure 7 shows the results of cytotoxicity analysis according to Experimental Example 5.
Figure 8 shows the results of cell viability analysis according to Experimental Example 5.
Figure 9 shows the results of Western blotting analysis according to Experimental Example 5.

The present invention can be modified in various ways and has various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a specific description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The pharmaceutical composition for preventing or treating muscle disease of the present invention contains, as an active ingredient, a fermented soybean product fermented by Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

Preferably, the soybean fermentation product may be a soybean fermentation product obtained by fermenting a soybean enzymatic hydrolyzate treated with any one enzyme selected from serine protease, neutral protease, cysteine protease, and leucine protease, by the Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

The above muscle disease may be any one selected from muscular atrophy, sarcopenia, atony, muscular dystrophy, myasthenia gravis, and amyotrophic lateral sclerosis.

The above beans may be at least one selected from soybeans, peas, fava beans, small beans, peas, broad beans, black beans, lima beans, and Egyptian beans, and soybeans are preferably used.

The above-mentioned fermented soybean product may have a branched chain amino acid content of 50 to 120 mg/g, more preferably 80 to 120 mg/g, based on a supernatant of a hot water extract of 0.1 g of soybean/10 ml of water.

The above pharmaceutical composition for preventing or treating muscle disease can be used to inhibit the synthesis of myostatin, a muscle protein decomposition factor, and to promote the synthesis of p-mTOR, a muscle protein synthesis factor.

The above soybean fermentation product may be an extract of soybean fermentation product.

The above extract may be extracted using an extraction solvent that is water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof.

The term "extract" used herein also includes fractions obtained by further fractionating an extract. That is, the extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. In addition, fractions obtained by passing the extract or fraction through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), and other additionally performed purification methods are also included in the extract of the present invention.

The term "comprising as an active ingredient" used herein means comprising a sufficient amount to achieve the efficacy or activity of the soybean fermentation. In one specific embodiment of the present invention, the soybean fermentation is contained in the composition of the present invention in an amount of, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg or more, still more preferably 100 mg/kg or more, still more preferably 250 mg/kg or more, and most preferably 0.1 g/kg or more. Since the soybean fermentation is a natural product and does not cause side effects to the human body even when administered in excessive amounts, a person skilled in the art can select and implement the quantitative upper limit included in the composition of the present invention within an appropriate range.

The pharmaceutical composition of the present invention can be prepared using, in addition to the above-mentioned effective ingredient, a pharmaceutically suitable and physiologically acceptable excipient, and the excipient may include an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a glidant, or a flavoring agent.

The above pharmaceutical composition may be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the effective ingredients described above for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an orally acceptable, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture, if desired or necessary. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweetener, acacia, tracheacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

In the composition formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used. Additionally, antioxidants, buffers, bacteriostatic agents, and other conventional additives can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition as an injectable formulation such as an aqueous solution, suspension, or emulsion, or as a pill, capsule, granule, or tablet.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and oral administration is preferred.

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and thereby. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersant or stabilizer.

In addition, the present invention provides a food composition for muscle protection containing, as an effective ingredient, a fermented soybean product fermented by Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).

Specific details of the muscle protection food composition of the present invention are the same as those of the pharmaceutical composition for preventing or treating muscle disease described above, so specific details will be referred to that part.

The food composition according to the present invention can be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, bread, ramen, other noodles, gum, ice cream, vitamin complexes, health supplements, etc.

The food composition of the present invention may contain not only the fermented soybean as an effective ingredient, but also components commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates described above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured into a drink or beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to the fermented soybean of the present invention.

The present invention provides a health functional food comprising a food composition containing the fermented soybean as an effective ingredient. A health functional food means a food prepared by adding a fermented soybean to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing the same in the form of encapsulation, powder, suspension, etc., and which has a specific health effect when consumed. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of the fermented soybean added to such a health functional food varies depending on the type of the target health functional food and cannot be uniformly specified. However, it may be added within a range that does not damage the original taste of the food, and is usually in the range of 0.01 to 50 wt%, preferably 0.1 to 20 wt%, with respect to the target food. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100 wt%, preferably 0.5 to 80 wt%. In one specific example, the health functional food of the present invention may be in the form of pills, tablets, capsules or beverages.

Hereinafter, a method for manufacturing the muscle protection composition of the present invention will be described.

First, soybean powder is enzymatically hydrolyzed with one enzyme selected from serine protease, neutral protease, cysteine protease, and leucine protease. At this time, it is more preferable to use leucine protease.

Next, the enzymatic hydrolyzate of soybean powder by the commercial enzyme is fermented with Bacillus amyloliquefaciens NY-PSH 124-1 strain to produce a fermented soybean product.

In some cases, the step of enzymatic treatment with the protease described above may be omitted, and a fermented soybean product may be produced by treatment alone with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Hereinafter, preferred examples are presented to help understand the present invention, but the following examples are only illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and technical idea of the present invention, and it is natural that such changes and modifications fall within the scope of the appended patent claims.

[Example]

Example 1: Treatment with commercial enzyme Alcalase (AL) & B. A. NY-PSH 124-1 strain treatment

Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP) was cultured in Tryptic soy broth (TSB, soybean powder, 2%, sucrose 0.25%, sodium chloride, 0.5%, casein peptone 1.7%, disodium phosphate, pH adjusted to 7) at 37°C and 100 rpm for 24 h, centrifuged at 8,000 ×g at 4°C for 30 min, and the supernatant was lyophilized to prepare.

First, commercial enzyme Alcalase (AL) (derived from Bacillus Licheniformis, serine protease, 2.4 U/g) was added at 0.1 wt% based on the raw soybean powder to soybean powder (1 g/10 ml) mixed with water, and enzyme treatment was completed by treating at 37°C for 3 hours and then inactivating at 100°C for 10 minutes.

Next, 5 wt% of Bacillus amyloliquefaciens NY-PSH 124-1 strain was added to the enzyme-treated soybean powder, and the reaction was performed at 37°C and 100 rpm for 20 hours, followed by inactivation by treatment at 100°C for 10 minutes to complete fermentation, thereby obtaining a soybean fermented product treated with a commercial enzyme/strain complex.

Example 2: Treatment with commercial enzyme Neutrase (NU) & B. A. NY-PSH 124-1 strain treatment

A soybean fermentation product was produced under the same conditions as in Example 1, except that the commercial enzyme Neutrase (NU) (derived from Bacillus amyloliquefaciens , Neutral protease, 0.8 U/g) was used instead of the commercial enzyme Alcalase (AL).

Example 3: Treatment with commercial enzyme ProteaseMAX (Max) & B. A. NY-PSH 124-1 strain treatment

A soybean fermentation product was produced under the same conditions as in Example 1, except that the commercial enzyme ProteaseMAX (Max) (derived from Bacillus subtillus , Neutral Protease 2.4 U/g) was used instead of the commercial enzyme Alcalase (AL).

Example 4: Treatment with commercial enzyme Collupulin MG (Col) & B. A. NY-PSH 124-1 strain treatment

A soybean fermentation product was produced under the same conditions as in Example 1, except that the commercial enzyme Collupulin MG (Col) (derived from Papaya, Cysteine Protease 4.6 U/g) was used instead of the commercial enzyme Alcalase (AL).

Example 5: Treatment with commercial enzyme Protamex (Pro) & treatment with B. A. NY-PSH 124-1 strain

A soybean fermentation product was produced under the same conditions as in Example 1, except that the commercial enzyme Protamex (Pro) (derived from Aspergillus oryzae , Leucine protease 700 U/g) was used instead of the commercial enzyme Alcalase (AL).

Example 6: Treatment with commercial enzyme Protana Prime (Pri) & treatment with B. A. NY-PSH 124-1 strain

A soybean fermentation product was produced under the same conditions as in Example 1, except that the commercial enzyme Protana Prime (Pri) (derived from Aspergillus oryzae , Leucine protease 500 U/g) was used instead of the commercial enzyme Alcalase (AL).

Example 7: Treatment with commercial enzyme Flavourzyme (Fla) & treatment with B. A. NY-PSH 124-1 strain

A soybean fermentation product was produced under the same conditions as in Example 1, except that the commercial enzyme Flavourzyme (Fla) (derived from Aspergillus oryzae , Leucine protease 500 U/g) was used instead of the commercial enzyme Alcalase (AL).

Example 8: Treatment of B. A. NY-PSH 124-1 strain alone

A soybean fermentation product was produced under the same conditions as in Example 1, except that no commercial enzyme treatment was performed and only Bacillus amyloliquefaciens NY-PSH 124-1 strain was used.

Comparative Example 1: Treatment with commercial enzyme Alcalase (AL) alone

A soybean enzyme hydrolysate was prepared under the same conditions as in Example 1, except that treatment with the commercial enzyme Alcalase (AL) alone was performed without performing treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Comparative Example 2: Treatment with commercial enzyme Neutrase (NU) alone

A soybean enzyme hydrolysate was prepared under the same conditions as in Example 1, except that treatment with the commercial enzyme Neutrase (NU) alone was performed without performing treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Comparative Example 3: Treatment with commercial enzyme ProteaseMAX (Max) alone

A soybean enzyme hydrolysate was prepared under the same conditions as in Example 1, except that treatment with the commercial enzyme ProteaseMAX (Max) alone was performed without performing treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Comparative Example 4: Treatment with commercial enzyme Collupulin MG (Col) alone

A soybean enzyme hydrolysate was prepared under the same conditions as in Example 1, except that treatment with the commercial enzyme Collupulin MG (Col) alone was performed without performing treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Comparative Example 5: Treatment with commercial enzyme Protamex (Pro) alone

A soybean enzyme hydrolysate was prepared under the same conditions as in Example 1, except that treatment with the commercial enzyme Protamex (Pro) alone was performed without performing treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Comparative Example 6: Commercial Enzyme Protana Prime (Pri) Treatment Alone

A soybean enzyme hydrolysate was prepared under the same conditions as in Example 1, except that treatment with the commercial enzyme Protana Prime (Pri) alone was performed without treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

Comparative Example 7: Treatment with commercial enzyme Flavourzyme (Fla) alone

A soybean enzyme hydrolysate was produced under the same conditions as in Example 1, except that treatment with the commercial enzyme Flavourzyme (Fla) alone was performed without treatment with the Bacillus amyloliquefaciens NY-PSH 124-1 strain.

[Experimental example]

Experimental Example 1: Analysis of branched-chain amino acid (BCAA) content

To investigate the BCAA content, TLC (TLC Silica gel 60 F254, Merck, 200 x 200 x 0.25 mm) was used. 0.1 g of the sample was dissolved in 10 ml of distilled water, left at 100℃ for 10 minutes, and the supernatant separated by centrifugation (10,000 rpm, 4℃, 10 minutes) was used for the experiment. The developing solvent used was nitromethane: 1-propanol: DW (2:5:1.5, v:v:v), and color development was performed using 0.2% ninhydrin color developing reagent at 105℃ for 6 minutes. Using this analytical method, the branched-chain amino acid (BCAA) content (mg/g DW) of the fermented soybeans manufactured according to Examples 1 to 7 and Comparative Examples 1 to 7 was measured, and the results are summarized in Fig. 1 and Table 1 below.

division AL NU Max Col Pro Pri Fla BCAA (mg/g DW) Commercial enzymes
(Comparative Example 1-7) 29.80a 29.12a 29.35a 6.86c 20.43b 26.42a 23.08a Commercial enzymes +
BA NY124
(Example 1-7) 81.07d 86.25cd 94.12b 59.93e 102.11a 91.87bc 103.89a Growth rate (%) 272.1c 296.1c 320.7c 873.8a 499.8b 347.7c 450.2c

According to this, when commercial enzyme treatment was additionally performed prior to fermentation of soybean powder with Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP), the BCAA production increase rate was measured to be significantly higher. In particular, in Example 7, where the Bacillus amyloliquefaciens NY-PSH 124-1 strain was combinedly treated with the commercial enzyme Flavourzyme (Fla), the BCAA production rate was measured to be the highest, and the BCAA content was found to increase by about 4.5 times compared to Comparative Example 7 where the commercial enzyme Flavourzyme (Fla) was treated alone.

Experimental Example 2: Amino Acid Analysis of Soybean Fermentation

HPLC analysis was performed to determine the amino acid composition of soybean fermentation. Agilent 1216 infinity LC (Agilent Technologies, Santa Clara, CA, USA) was used, and the column was AdvanceBio AAA (Agilent Technologies, 4.6 x 100 mm, 27 ㎛). The detection wavelength was 338 nm, the solvent flow rate was 0.8 ㎖/min, and the column temperature was fixed at 40℃. The mobile phase was 10 mM Na ₂ HPO ₄ , 10 mM Na ₂ B ₄ O ₇ buffer (pH 8.2) as solvent A, and ACN-MeOH-DW (45:45:10, v:v:v) as solvent B, which gave a gradient. The solvent gradient of the mobile phase started with A: 98%, B: 2%, then became A: 43%, B: 57% at 13.4 min, A: 0%, B: 100% from 13.5 to 15.7 min, and A: 98%, B: 2% from 15.8 to 18 min. Agilent's Amino Acid Standard (1 nmol/㎕ in 0.1 M HCl) was used as a standard, and the standard curve was prepared by diluting it to 0.025, 0.05, 0.1, 0.25, and 0.5 mM with distilled water, and quantification was performed by creating a calibration curve.

The amino acid analysis results of the soybean fermented products of Examples 7 and 8 analyzed according to the above method are summarized in Fig. 2 and Table 2 below.

(mg/mL) Unprocessed Example 8 Example 7 aspartic acid 0.00 1.78 15.55 glutamic acid 0.00 7.47 46.40 serine 0.00 0.00 13.48 histidine 0.65 3.95 8.43 glycine 0.43 2.57 10.50 thereonine 0.00 6.05 18.62 arginine 0.00 0.00 0.00 Alanine 3.61 7.17 22.25 tyrosine 0.00 3.57 9.33 cysteine 0.00 0.00 0.00 methionine 0.00 5.23 6.82 phenylalanine 1.06 11.08 21.95 valine 1.57 16.54 32.27 isoleucine 1.32 10.73 23.45 leucine 2.81 31.77 50.22 Lysine 2.64 19.67 2.11 proline 0.00 23.66 31.08 BCAA (mg/g DW) 5.70 + 0.00 59.04 + 7.04 105.94 + 1.13 TAA (mg/g DW) 14.08 + 0.00 151.25 + 5.05 312.47 + 3.20

Accordingly, in Example 8, where Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP) was treated alone, the branched-chain amino acid (BCAA) content was measured to be 59.04 mg and the total amino acid (TAA) content was 151.2 mg, which is a 10-fold increase in total amino acids (TAA) and branched-chain amino acids (BCAA) compared to the untreated case. In Example 7, the total amino acids (TAA) and branched-chain amino acids (BCAA) were found to increase by about 2-fold compared to Example 5.

Experimental Example 3: Characteristics of fermented soybean powder

(1) SDS-PAGE analysis

The protein content was quantitatively measured at 560 nm using a BCA (Bio-Rad) protein kit with the supernatant of the sample using BSA (bovine serum albumin) protein as a standard with a spectrophotometer. For the size analysis of the protein sample, the protein quantified with the BCA kit was adjusted to 10–50 mg/㎖, and 5X SDS-loading buffer (0.5 M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 10% b-mercaptoethanol) was added. The proteins were separated by size on a discontinuous SDS-PAGE gel (12–14% resolving 4% stacking), and visualized by staining with commassie R-Blue staining. The photographs (a) and SDS-PAGE analysis results (b) of the untreated soybean powder group and the fermented soybeans of Examples 7 and 8 are shown in Fig. 3. Accordingly, when examining the degree of protein decomposition, only proteins of 35 KDa in size remained in the fermented soybean sample of Example 8 compared to the untreated group, and in the case of the fermented soybean sample of Example 7, it was confirmed that the proteins were decomposed and existed in small sizes at the peptide level.

(2) SEM analysis

The morphology of the soybean enzyme-treated powder particles was observed at 2000x magnification using a scanning electron microscope (ZEMINI SEM electron microscope (FEI Co., The Netherlands), and the SEM images of the untreated group and the samples of Examples 7 and 8 are shown in Fig. 4. According to this, compared to the soybean powder of the untreated group, it can be confirmed that the structure of the soybean powder of Examples 7 and 8 was collapsed, many pores were formed in the tissue, and the tissue swelled like a balloon, making it easy to dissolve.

(3) FTIR analysis

In order to measure the change in the chemical characteristics of the soybean fermentation, FTIR analysis (Fourier transform infra-red spectroscopy, Perkin Elmer Spectrum 400) was performed and the results are shown in Fig. 5. According to this, the sample of Example 7 treated with the enzyme/strain complex consists of three peak ranges in the FTIR spectrum: 1) 3500~2900 cm ^-1 , 2) 1700~1300 cm ^-1 , and 3) 1200~900 cm ^-1 . According to the results of previous studies, for each range, 1) means OH parasitism and mainly indicates water or ethanol characteristics; 2) mainly indicates the presence, increase, or production of phenol compounds and represents the main characteristics of flavonoids or phenols; and 3) indicates the expansion or increase of carbonyl groups, and an increase in the curve height indicates an increase in C=O bonds. According to this, the peak patterns for the untreated group and the sample of Example 8 are similar, and the peak of Example 7 showed strong peaks in all three sections, showing an increase in the creation of aromatic rings through the presence of OH groups and the combination of phenol compounds.

Experimental Example 4: Analysis of taste changes in fermented soybean products

An electronic tongue (Astree2, Alpha MOS, Toulouse, France) was used to analyze the taste changes in fermented soybean products. After filtering (Whatman No. 6, Whatman International Ltd, Kent, UK), 25 ml of the diluted sample was placed in a glass container and analyzed using an automatic sample analyzer. The electronic tongue used a module with seven sensors (Sensor array # 5, Alpha MOS, Toulouse, France). Among the seven sensors, SPS (803-0155) and GPS (803-0140) sensors were used as standards for calibration, and SRS (sour, astringent, bitter; 803-0135), STS (salty, spicy, metallic; 803-0145), UMS (umami, salty, astringent; 803-0150), SWS (sweet, sour; 803-0160), BRS (bitter, astringent; 803-0165, Alpha MOS, Toulouse, France) sensors were used. Rather than measuring individual chemical components, the seven sensors sensed the overall taste and converted each sensor sensitivity into a taste score in the range of 0-12. For each sensor, the mean (m) and standard deviation (∂) of all data were calculated, and X'=|(X-m)|/∂ was calculated based on the mean (X) of the repeated data sensor values for each sample. This value was used to represent the relative score of taste. The measurement of the samples was repeated five times, and the sensor was rinsed after the analysis of a single sample. The software provided by Alpha MOS (Alpha soft 14.1 version, Alpha MOS, Toulouse, France) was used for statistical processing.

Accordingly, the taste measurement results for the untreated group, BCAA, Example 7, and Example 8 are shown in Fig. 6 and Table 3 below.

Experimental group acidity,
Astringent sweetness Salty Savory Correction factor acerbity Correction factor AHS PKS CTS NMS CPS ANS SCS water 9.1 3.8 5.9 8.7 2.9 4 3 BCAA 8.2 3.7 3.5 8.6 3.8 3.4 3.8 Unprocessed 4.1 5.4 4.3 4.4 7.3 5.6 8.3 Example 8 4.8 8.1 7.1 4.1 8 7.5 7.0 Example 7 4.9 8.2 8.6 5.3 6.9 8.5 6.8

Accordingly, the soybean fermented products of Examples 7 and 8 showed minimal changes in sourness and astringency compared to the untreated products, but greatly improved sweetness and savory tastes, and increased saltiness and bitterness. These results can be seen as changes due to the increase in BCAA.

Experimental Example 5: Analysis of muscle cell protection effect

Mouse-derived muscle cells C2C12 were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS), and passaged when the cell growth rate was approximately 80%. For muscle cell differentiation, horse serum (HS) was used instead of FBS, and the cells were treated for a total of 5 days.

(1) Cytotoxicity evaluation

To determine cytotoxicity in muscle cells, an MTT assay was performed. C2C12 cells were seeded in a 96-well plate at a concentration of 1 × 10 ⁶ cells/㎖ and treated with various concentrations of BCAA, untreated soybean powder, and the fermented soybean of Example 7 for 1 hour, then H ₂ O ₂ was additionally treated and cultured for 24 hours. MTT (2 mg/㎖) was treated for 4 hours, and DMSO was added after all the culture medium was removed. The cytotoxicity results were measured for absorbance at 540 nm and calculated as a % compared to the untreated group, and the results are shown in Fig. 7. According to this, BCAA confirmed intracellular safety up to 10 ㎍/㎖ and untreated soybean powder 200 ㎍/㎖, and thereafter, experiments were performed at a concentration of 10 ㎍/㎖ for BCAA, 100 ㎍/㎖ for untreated soybean powder and fermented soybean.

(2) Measurement of protein expression

The muscle protein protection ability was analyzed by inducing muscle cell necrosis through _H2O2 treatment using C2C12 (Mice skeletal muscle) cell line. C2C12 cells were washed once with PBS solution, lysed on ice for 10 minutes _with lysis buffer (RIPA buffer with protease and phosphatase inhibitor cocktails), and the dissolved proteins were collected in a microtube and centrifuged (13,000 rpm, 4℃, 20 min) to collect the supernatant. The collected supernatant was quantified for protein using the Bradford method, and 20 μg of protein was separated by electrophoresis on a 10% SDS-PAGE gel. The SDS-PAGE gel was transferred to a nitrocellulose membrane and blocked with 5% skimmed milk to remove nonspecific proteins. Primary antibodies were used to bind Myostatin (1:1,000 #PA5-47034 Invitrogen), B-actin (1:5,000 sc-47778 santa cruz), mTOR (1:1,000, #2972 Cell Signaling Technology), antip-mTOR (1:1,000, #2448 Cell Signaling Technology), and then reacted with secondary antibodies containing horseradish peroxidase. The target protein was identified using an ECL kit. The target protein expression quantification was calculated after correction for the β-actin value.

The cell viability measurement results for the H ₂ O ₂ treatment group (positive control group), BCAA treatment group, single strain treatment group of Example 8, and enzyme/strain complex treatment group of Example 7 for C2C12 cells are shown in Fig. 8. According to this, the cell viability of the H ₂ O ₂ treatment group decreased to about 60% compared to the untreated group, and it was confirmed that the cell viability was restored to about 85% by the positive control group, BCAA protein. The soybean fermentation treatment group of Example 7 of the present invention showed that muscle cells were protected from 60% to 75%.

Additionally, the results of Western blotting analysis according to the above method are shown in Figure 9.

According to this, myostatin protein, which is related to muscle protein decomposition, significantly decreased in the BCAA treatment group compared to the H ₂ O ₂ treatment group. In addition, the soybean fermentation treatment groups of Examples 7 and 8 showed a significant decrease in myostatin expression compared to the BCAA treatment group and the H ₂ O ₂ treatment group. In particular, it was confirmed that the soybean fermentation treatment group of Example 7 had a remarkable effect in inhibiting muscle protein decomposition, as it was reduced by up to 50% compared to the H ₂ O ₂ treatment group.

In addition, the p-mTOR expression level was measured to determine the degree of increase in muscle protein synthesis. According to this, it was observed that p-mTOR/mTOR increased by about 160% in the BCAA treatment group compared to the H ₂ O ₂ treatment group, and p-mTOR/mTOR of all experimental samples significantly increased compared to the H ₂ O ₂ treatment group, and in particular, the soybean fermentation treatment group of Example 7 showed a high expression level similar to the BCAA treatment group, confirming that it exhibited an excellent effect in promoting muscle protein synthesis.

Below, a formulation example of a composition containing the bile extract of the present invention is described, but the present invention is not intended to be limited thereto but is merely intended to be described specifically.

Preparation Example 1. Preparation of a mountain product

500 mg of fermented soybean of Example 7

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled into a sealed bag to produce a powdered preparation.

Preparation Example 2. Preparation of tablets

300 mg of fermented soybean of Example 7

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by pressing them according to the usual method for manufacturing tablets.

Preparation Example 3. Preparation of capsules

200 mg of fermented soybean of Example 7

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above ingredients are mixed according to the conventional capsule manufacturing method and filled into a gelatin capsule to manufacture a capsule.

Preparation Example 4. Preparation of injection

600 mg of fermented soybean of Example 7

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

It is manufactured with the above ingredient content per ampoule according to the manufacturing method of a conventional injection.

Preparation Example 5. Preparation of liquid preparation

7.5 g of fermented soybean of Example 7

10 g of isoflavone

Mannitol 5 g

Appropriate amount of purified water

According to the usual method for manufacturing a liquid, each ingredient is dissolved in purified water, an appropriate amount of lemon flavor is added, the above ingredients are mixed, purified water is added, and the total amount is adjusted to 100 g. The liquid is then filled into a brown bottle and sterilized.

Preparation Example 6. Preparation of granules

1,900 mg of fermented soybean of Example 7

Vitamin mixture appropriate amount

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate monobasic 15 mg

55 mg of dibasic calcium phosphate

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a preferred example of mixing ingredients relatively suitable for granules, but the mixing ratio may be arbitrarily modified, and granules may be manufactured by mixing the above ingredients according to a conventional method for manufacturing granules, and then used to manufacture a health functional food composition according to a conventional method.

Preparation Example 7. Manufacturing of functional beverages

1,900 mg of fermented soybean of Example 7

1,000 mg of citric acid

100 g of oligosaccharide

2 g of plum concentrate

1 g of taurine

Add purified water to make a total of 900 mL

The above ingredients are mixed according to a conventional health beverage manufacturing method, stirred and heated at 85°C for about 1 hour, the resulting solution is filtered, placed in a sterilized 2 L container, sealed and sterilized, then refrigerated and used to manufacture the functional beverage composition of the present invention.

The above composition ratio is a preferred example of mixing ingredients suitable for relatively preferred beverages, but the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demand country, and intended use.

Above, the embodiments of the present invention have been described, but those skilled in the art will be able to modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., within the scope that does not depart from the spirit of the present invention described in the claims, and this will also be considered to be included within the scope of the rights of the present invention.

Korea Research Institute of Bioscience and Biotechnology KCTC14294BP 20200903

Claims (16)

A food composition for muscle protection comprising, as an active ingredient, a fermented soybean product obtained by fermenting a soybean enzymatic hydrolyzate enzymatically treated with leucine protease derived from Aspergillus oryzae and then with Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP). delete delete In the first paragraph,
A food composition for muscle protection, characterized in that the beans are at least one selected from soybeans, peas, fava beans, small beans, peas, fava beans, black beans, lima beans, and Egyptian beans. In the first paragraph,
A muscle protection food composition characterized in that the above soybean fermentation product has a branched chain amino acid content of 50 to 120 mg/g based on a hot water extract supernatant of 0.1 g of soybean/10 ml of water. In the first paragraph,
The above muscle protection food composition is a muscle protection food composition characterized in that it inhibits muscle protein decomposition by inhibiting the synthesis of myostatin, a muscle protein decomposition factor. In the first paragraph,
The above muscle protection food composition is a muscle protection food composition characterized in that it promotes muscle protein synthesis by promoting the synthesis of p-mTOR, a muscle protein synthesis factor. A pharmaceutical composition for preventing or treating muscle disease, comprising as an active ingredient a fermented soybean product, wherein the fermented soybean product is processed by an enzyme-processed soybean hydrolyzate derived from Aspergillus oryzae with a leucine protease, and the fermented soybean product is fermented by Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP).
A pharmaceutical composition for preventing or treating muscle disease, characterized in that the muscle disease is any one selected from muscular atrophy, sarcopenia, and muscular dystrophy. delete delete In Article 8,
A pharmaceutical composition for preventing or treating muscle disease, characterized in that the branched chain amino acid content of the soybean fermentation product is 50 to 120 mg/g based on the supernatant of the hot water extract of 0.1 g of soybean/10 ml of water. In Article 8,
The above pharmaceutical composition for preventing or treating muscle disease is a pharmaceutical composition for preventing or treating muscle disease, characterized in that it inhibits muscle protein decomposition by inhibiting the synthesis of myostatin, a muscle protein decomposition factor. In Article 8,
The above pharmaceutical composition for preventing or treating muscle disease is a pharmaceutical composition for preventing or treating muscle disease, characterized in that it promotes muscle protein synthesis by promoting the synthesis of p-mTOR, a muscle protein synthesis factor. delete A step of producing a soybean enzymatic hydrolyzate by enzymatically treating soybean powder with leucine protease derived from Aspergillus oryzae;
A method for producing a muscle protection composition, comprising: a step of fermenting the soybean enzyme hydrolyzate with Bacillus amyloliquefaciens NY-PSH 124-1 strain (Accession No. KCTC 14294BP) to produce a soybean fermentation product. delete