Agents and compositions for promoting myoblast differentiation into myotube, agents and compositions for muscle enhancement, agents and compositions for improving muscle capability, agents and compositions for improving muscle endurance, agents and compositions for improving basal metabolism, methods for producing decomposed whey protein and peptide-containing compositions

Abstract

To provide agents and compositions for promoting myoblast differentiation into myotube with excellent safety and capability, agents and compositions for muscle enhancement with excellent safety and capability, agents and compositions for improving muscle capability with excellent safety and capability, agents and compositions for improving endurance with excellent safety and capability, agents and compositions for improving basal metabolism with excellent safety and capability, methods for producing decomposed whey protein containing a peptide having an amino acid sequence DISL (SEQ ID NO:1) and a peptide having an amino acid sequence DISLL (SEQ ID NO:2) which are safe and useful for promoting myoblast differentiation into myotube, enhancing muscle, improving muscle capability, improving endurance, and improving basal metabolism, and compositions containing a peptide having an amino acid sequence DISL (SEQ ID NO:1) and a peptide having an amino acid sequence DISLL (SEQ ID NO:2) being safe and useful for myoblast differentiation promotion into myotube, muscle enhancement, muscle capability improvement, endurance improvement, and basal metabolism improvement.SOLUTION: Disclosed is an agent for promoting differentiation of myoblast into myotube, comprising at least any one of a peptide having an amino acid sequence DISL (SEQ ID NO:1) and a peptide having an amino acid sequence DISLL (SEQ ID NO:2).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for promoting myotube differentiation of myoblasts and a composition for promoting myotube differentiation of myoblasts, a muscle-building agent and a composition for muscle-building, a muscle performance improver and a composition for improving muscle performance, an endurance improver and a composition for improving endurance, a basal metabolic rate improver and a composition for improving basal metabolic rate, a method for producing a whey protein hydrolysate, and a peptide-containing composition. [Background technology]

[0002] Muscles are formed by the differentiation of muscle cells. In muscle cell differentiation, myoblasts, which are precursor cells, proliferate to a certain number of cells, migrate to the pre-muscle region, and fuse with each other to become differentiated multinucleated myotubes. After that, genes that express muscle-specific traits such as muscle contractile proteins and specific enzymes are expressed in the myotubes, and the muscle contractile apparatus is constructed. Since strengthening muscles and enhancing their functions is important for maintaining and improving motor function and maintaining physical health, various muscle strengthening means have been studied. For example, a muscle differentiation inducer containing a fermented extract of rice bran and / or rice germ as an active ingredient (see, for example, Patent Document 1) and a myotube cell differentiation promoter containing a component derived from sugarcane or bagasse as an active ingredient (see, for example, Patent Document 2) have been proposed.

[0003] PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is known as a factor that controls energy metabolism in the body. PGC-1α was identified as a transcriptional coactivator that activates transcription by the nuclear receptor PPARγ in brown adipose tissue. It has since been revealed that PGC-1α interacts with not only PPARγ but also many nuclear receptors and various transcription factors, and controls the expression of target genes as a transcriptional coactivator. PGC-1α is abundantly expressed in the heart, skeletal muscle, liver, kidney, brown adipose tissue, brain, etc., and is increased by cold stimulation, nutritional state, exercise, etc. It is known that its expression is significantly increased in skeletal muscle during exercise (see, for example, Non-Patent Document 1). Based on this control effect on energy production, the functions of PGC-1α are known to be involved in muscle hypertrophy (see, for example, Non-Patent Document 2) and improved endurance (see, for example, Non-Patent Document 3).

[0004] Incidentally, there have been several reports on the enzymatic degradation of proteins using enzymes originating from Bacillus licheniformis to produce functional peptides (see, for example, Patent Documents 3 and 4). However, there have been no reports on the enzymatic degradation of whey protein using enzymes originating from Bacillus licheniformis to produce both a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). Furthermore, there have been no reports that the peptide consisting of the amino acid sequence DISL (sequence number 1) and the peptide consisting of the amino acid sequence DISLL (sequence number 2) have the functionality of promoting myotube differentiation of myoblasts, strengthening muscles, improving muscle performance, improving endurance, and improving basal metabolism. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2017-193497 A [Patent Document 2] JP 2020-169126 A [Patent Document 3] Special Publication No. 2022-550029 [Patent Document 4] Special Publication No. 2011-530274 [Non-patent literature]

[0006] [Non-Patent Document 1] Nomura K et al., Anti-Aging Medicine, 11, 3, 36-41, 2015. [Non-Patent Document 2] JL Ruas et al., Cell, 151, 6, 1319-1331, 2012. [Non-Patent Document 3] Handschin C et al., Nature, 454, 7203, 463-469, 2008. Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, although muscle differentiation inducers and myotube cell differentiation promoters have been proposed, there is a strong demand for the development of new materials and manufacturing methods thereof that are highly safe and useful for promoting myotube differentiation of myoblasts, building muscle, improving muscle performance, improving endurance, and improving basal metabolism.

[0008] The present invention aims to solve the above-mentioned problems in the prior art and to achieve the following objectives: That is, the present invention aims to provide a myoblast myotube differentiation promoter and a composition for promoting myoblast myotube differentiation, which are highly safe and have an excellent effect of promoting myoblast myotube differentiation. Another object of the present invention is to provide a muscle-building agent and a muscle-building composition which are highly safe and have an excellent muscle-building effect. Another object of the present invention is to provide a muscle performance improving agent and a muscle performance improving composition which are highly safe and have an excellent muscle performance improving effect. Another object of the present invention is to provide an endurance enhancing agent and composition for improving endurance which are highly safe and have an excellent endurance improving effect. Another object of the present invention is to provide a basal metabolism enhancer and a composition for enhancing basal metabolism which are highly safe and have an excellent basal metabolism enhancing effect. Another object of the present invention is to provide a method for producing whey protein hydrolysates containing a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), which are highly safe and useful for promoting myotube differentiation of myoblasts, building muscle, improving muscle performance, improving endurance, and improving basal metabolism. Another object of the present invention is to provide a peptide-containing composition comprising a peptide having the amino acid sequence DISL (SEQ ID NO: 1) and a peptide having the amino acid sequence DISLL (SEQ ID NO: 2), which are highly safe and useful for promoting myotube differentiation of myoblasts, building muscle, improving muscle performance, improving endurance, and improving basal metabolism. [Means for solving the problem]

[0009] As a result of intensive research conducted by the present inventors to solve the above problems, they have discovered that by hydrolyzing whey protein using an enzyme originating from Bacillus licheniformis, whey protein hydrolysates containing a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) are produced, and that these peptides are useful for promoting myotube differentiation of myoblasts, building muscle, improving muscle performance, improving endurance, and improving basal metabolism.

[0010] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> The agent for promoting myotube differentiation of myoblasts is characterized by comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). <2> The above <1> The myoblast myotube differentiation promoter according to claim 1, The composition is for promoting myotube differentiation of myoblasts, and is characterized by being ingested by oral, nasal, or enteral administration. <3> The muscle-building agent is characterized by containing at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). <4> The above <3> The muscle-enhancing agent according to claim 1, The composition for muscle building is characterized by being ingested by oral, nasal, or enteral administration. <5> The muscle performance enhancer comprises at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). <6> The above <5> The muscle performance enhancer according to claim 1, The composition for improving muscle performance is characterized in that it is ingested by oral administration, nasal administration, or enteral administration. <7> The endurance-improving agent comprises at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). <8> The above <7> The endurance enhancer according to claim 1, The composition for improving endurance is characterized in that it is ingested by oral administration, nasal administration, or enteral administration. <9> The basal metabolism enhancer is characterized by comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). <10> The above <9> The basal metabolic rate enhancer according to claim 1, The composition for improving basal metabolism is characterized by being ingested by oral administration, nasal administration, or enteral administration. <11> A method for producing a whey protein hydrolysate comprising a peptide having the amino acid sequence DISL (SEQ ID NO: 1) and a peptide having the amino acid sequence DISLL (SEQ ID NO: 2), comprising: It involves breaking down whey proteins using an enzyme derived from Bacillus licheniformis, The method is characterized in that the pH of the whey protein solution containing the whey protein is adjusted to 7.6 to 12.0, and a reaction caused by the enzyme originating from Bacillus licheniformis is initiated. <12> Preparing the whey protein solution having a whey protein content of 5 to 30% by mass; Adding the enzyme derived from Bacillus licheniformis in an amount of 1 to 10% by mass based on the solid content of the whey protein solution; The reaction with the enzyme originating from Bacillus licheniformis is carried out at 35 to 60° C. for 1 to 6 hours. <11> This is a method according to the present invention. <13> A peptide-containing composition comprising a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), The peptide-containing composition is characterized in that the content of the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) in the peptide-containing composition is 0.003% by mass or more, and the content of the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) in the peptide-containing composition is 0.050% by mass or more. Effect of the Invention

[0011] The myotube differentiation promoter and composition for promoting myoblast myotube differentiation of the present invention can solve the above-mentioned problems in the past and achieve the above-mentioned objective, and can provide a myotube differentiation promoter and composition for promoting myoblast myotube differentiation of myoblasts that are highly safe and have excellent myotube differentiation promoting effect. The muscle-building agent and muscle-building composition of the present invention can solve the above-mentioned conventional problems and achieve the above-mentioned object, and can provide a muscle-building agent and muscle-building composition that are highly safe and have an excellent muscle-building effect. The muscle performance enhancer and muscle performance improving composition of the present invention can solve the above-mentioned conventional problems and achieve the above-mentioned object, and can provide a muscle performance enhancer and muscle performance improving composition that are highly safe and have an excellent muscle performance improving effect. The endurance improving agent and endurance improving composition of the present invention can solve the above-mentioned problems in the conventional art and achieve the above-mentioned objective, and can provide an endurance improving agent and endurance improving composition that are highly safe and have an excellent endurance improving effect. The basal metabolic rate enhancer and composition for improving basal metabolic rate of the present invention can solve the above-mentioned problems in the past and achieve the above-mentioned objective, and can provide a basal metabolic rate enhancer and composition for improving basal metabolic rate that are highly safe and have an excellent basal metabolic rate enhancing effect. The method for producing whey protein hydrolysates of the present invention can solve the above-mentioned problems in the prior art and achieve the above-mentioned object, and can provide a method for producing whey protein hydrolysates containing a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), which are highly safe and useful for promoting myotube differentiation of myoblasts, building muscle, improving muscle performance, improving endurance, and improving basal metabolism. The peptide-containing composition of the present invention can solve the above-mentioned problems in the past and achieve the above-mentioned object, and can provide a peptide-containing composition comprising a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), which is highly safe and useful for promoting myotube differentiation of myoblasts, building muscle, improving muscle performance, improving endurance, and improving basal metabolism. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the results of calculating the resting energy expenditure in Test Example 3. [Diagram 2] FIG. 2 is a graph showing the results of investigating the correlation between resting energy expenditure and muscle mass after 12 weeks of intake in Test Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] (Agent for promoting myotube differentiation of myoblasts, muscle strengthening agent, muscle performance improver, endurance improver, basal metabolism improver) The agent for promoting myoblast myotube differentiation of the present invention contains at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), and further contains other components as necessary. As used herein, promoting the myotube differentiation of myoblasts refers to increasing the myotube differentiation rate of myoblasts compared to before the use of the agent for promoting myotube differentiation of myoblasts.

[0014] The muscle-building agent of the present invention contains at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), and further contains other components as necessary. In the present specification, "building muscle" refers to promoting muscle synthesis compared to before the use of the muscle-building agent. The muscle-building agent is preferably based on at least one of the effects of promoting myotube differentiation of myoblasts and promoting expression of the myogenin gene in myoblasts, but is not limited to these effects.

[0015] The muscle performance improver of the present invention contains at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), and further contains other components as necessary. In this specification, improving muscle performance means improving the ability of resistance exercise (strength training, weight training) etc., compared to before the use of the muscle performance enhancer. The muscle performance enhancer is preferably based on the effect of promoting the expression of PGC-1α gene in myoblasts, but is not limited to this effect.

[0016] The endurance-enhancing agent of the present invention contains at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), and further contains other components as necessary. In the present specification, improving endurance means improving muscle endurance compared to before using the endurance improver. The endurance improver is preferably based on the action of promoting the expression of PGC-1α gene in myoblasts, but is not limited to this action.

[0017] The basal metabolism enhancer of the present invention contains at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), and further contains other components as necessary. In this specification, improving basal metabolism (hereinafter, sometimes referred to as "promoting basal metabolism") means increasing the amount of energy consumed by basal metabolism compared to before the use of the basal metabolism enhancer. Whether or not the amount of energy consumed by basal metabolism has increased can be confirmed, for example, by examining the amount of energy consumed at rest.

[0018] <Peptide> The myoblast myotube differentiation promoter, muscle-building agent, muscle performance improver, endurance improver, or basal metabolism improver contains at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) (hereinafter, these may be collectively referred to as "peptide"). Either one of the peptides may be used, or both may be used in combination.

[0019] It was not previously known that the peptide has the effects of promoting myotube differentiation of myoblasts, strengthening muscle, improving muscle performance, improving endurance, and improving basal metabolism, and is useful as an agent for promoting myotube differentiation of myoblasts, strengthening muscle, improving muscle performance, improving endurance, or improving basal metabolism, and this is a new discovery by the present inventors.

[0020] The peptides also include salts thereof. The salt is not particularly limited and can be appropriately selected depending on the purpose. Examples of the salt include sodium salt, potassium salt, and hydrochloride salt.

[0021] The peptides used may be those produced by general chemical synthesis methods such as solid phase and liquid phase methods, or those produced by the method for producing whey protein hydrolysates of the present invention described below.

[0022] The content of the peptide in the myoblast myotube differentiation promoter, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolic improver is not particularly limited and can be selected appropriately depending on the purpose. The myoblast myotube differentiation promoter, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolic rate improver may consist of only the peptide, or may be a formulation of the peptide in any dosage form.

[0023] <Other ingredients> The other components are not particularly limited as long as they do not impair the effects of the present invention and can be appropriately selected depending on the purpose, and examples thereof include proteins, peptides other than the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), free amino acids or salts thereof, carriers, excipients, fillers, preservatives, stabilizers, binders, pH adjusters, buffers, thickeners, gelling agents, preservatives, antioxidants, etc. These may be used alone or in combination of two or more.

[0024] The content of the other components in the myoblast myotube differentiation promoter, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolic rate improver is not particularly limited and can be selected appropriately depending on the purpose.

[0025] <Application> The uses of the myoblast myotube differentiation promoter, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolic rate improver are not particularly limited and can be appropriately selected depending on the purpose, and include a wide range of uses such as pharmaceuticals, quasi-drugs, and food and beverages.

[0026] The myoblast myotube differentiation promoter is highly safe and has an excellent effect of promoting myotube differentiation of myoblasts, and therefore can be suitably used, for example, as an active ingredient of a composition for promoting myotube differentiation of myoblasts, which will be described later.

[0027] The muscle-building agent is highly safe and has an excellent muscle-building effect, and therefore can be suitably used, for example, as an active ingredient of a muscle-building composition described below.

[0028] The muscle performance enhancer is highly safe and has an excellent muscle performance enhancing effect, and therefore can be suitably used, for example, as an active ingredient of a muscle performance enhancing composition described below.

[0029] The endurance enhancing agent is highly safe and has an excellent endurance enhancing effect, and therefore can be suitably used, for example, as an active ingredient of a composition for improving endurance, which will be described later.

[0030] The basal metabolism enhancer is highly safe and has an excellent basal metabolism enhancing effect, and therefore can be suitably used, for example, as an active ingredient of a basal metabolism enhancing composition described below.

[0031] The myoblast myotube differentiation promoter, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolic rate improver are preferably applied to humans, but may also be applied to animals other than humans.

[0032] The agent for promoting myotube differentiation of myoblasts, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolism improver may be administered orally or parenterally, and preferably may be administered orally, nasally, or enterally.

[0033] The dosage form of the myoblast myotube differentiation promoter, muscle-strengthening agent, muscle performance improver, endurance improver, or basal metabolism improver is not particularly limited, and a known dosage form can be appropriately selected depending on the purpose. Examples of the dosage form include oral preparations such as tablets, pills, hard capsules, soft capsules, microcapsules, powders, granules, and liquids; and parenteral preparations such as injections, suppositories, and liniments. The method for producing the various dosage forms of the myoblast myotube differentiation promoter, the muscle-building agent, the muscle performance improver, the endurance improver, or the basal metabolic rate improver is not particularly limited, and any known method can be appropriately selected.

[0034] The method, amount, area of ​​use, period of use, interval of use, etc. of the myoblast myotube differentiation promoter, muscle strengthening agent, muscle performance improver, endurance improver, or basal metabolic improver are not particularly limited and can be appropriately selected taking into consideration the age and health condition of the subject, etc.

[0035] The amount of use (sometimes referred to as dosage or intake amount) of the myoblast myotube differentiation promoter, the muscle-strengthening agent, the muscle performance improver, the endurance improver, or the basal metabolic improver is not particularly limited and can be appropriately selected depending on the purpose, and examples of the total amount of the peptide include 75 to 75,000 μg / day, 75 to 7,500 μg / day, etc.

[0036] The myoblast myotube differentiation promoter, muscle-building agent, muscle performance improver, endurance improver, or basal metabolic improver are highly safe and can be used on a daily basis, and can therefore be used continuously, for example, at least once a day.

[0037] In addition, the myoblast myotube differentiation promoter can be used as a reagent for research into the mechanism of action of promoting myoblast myotube differentiation, the muscle-enhancing agent can be used as a reagent for research into the mechanism of action of the muscle-enhancing action, the muscle performance improver can be used as a reagent for research into the mechanism of action of the muscle performance improvement action, the endurance improver can be used as a reagent for research into the mechanism of action of the endurance improvement action, and the basal metabolic rate improver can be used as a reagent for research into the mechanism of action of the basal metabolic rate improvement action.

[0038] (Composition for promoting myotube differentiation of myoblasts, composition for muscle building, composition for improving muscle performance, composition for improving endurance, composition for improving basal metabolism) The composition for promoting myotube differentiation of myoblasts of the present invention is ingested by oral administration, nasal administration, or enteral administration, and contains the myotube differentiation promoter of the present invention and further contains other components as necessary. The muscle-building composition of the present invention is to be ingested by oral administration, nasal administration, or enteral administration, and contains the muscle-building agent of the present invention and further contains other ingredients as necessary. The composition for improving muscle performance of the present invention is to be ingested by oral, nasal or enteral administration, and contains the muscle performance improver of the present invention and further contains other ingredients as necessary. The endurance-enhancing composition of the present invention is to be ingested by oral, nasal or enteral administration, and contains the endurance-enhancing agent of the present invention and further contains other ingredients as necessary. The composition for improving basal metabolism of the present invention is to be ingested by oral administration, nasal administration, or enteral administration, and contains the basal metabolism enhancer of the present invention, and further contains other ingredients as necessary.

[0039] <Agent for promoting myoblast myotube differentiation, muscle building agent, muscle performance improver, endurance improver, basal metabolism improver> The agent for promoting myotube differentiation of myoblasts is the agent for promoting myotube differentiation of myoblasts of the present invention described above. The content of the myoblast myotube differentiation promoter in the composition for promoting myoblast myotube differentiation is not particularly limited and can be appropriately selected depending on the form of the composition for promoting myoblast myotube differentiation, etc. For example, the total amount of the peptide used may be the same as the amount of the myoblast myotube differentiation promoter used above. The composition for promoting myoblast myotube differentiation may consist only of the myoblast myotube differentiation promoter.

[0040] The muscle-building agent is the muscle-building agent of the present invention described above. The content of the muscle-building agent in the muscle-building composition is not particularly limited and can be appropriately selected depending on the form of the muscle-building composition, and for example, the amount used is set to be the same as the amount of the muscle-building agent used as the total amount of the peptide. The muscle-building composition may consist of only the muscle-building agent.

[0041] The muscle performance improver is the muscle performance improver of the present invention described above. The content of the muscle performance enhancer in the muscle performance improving composition is not particularly limited and can be appropriately selected depending on the form of the muscle performance improving composition, for example, the total amount of the peptides used can be the same as the amount of the muscle performance enhancer used above. The muscle performance improving composition may consist only of the muscle performance enhancer.

[0042] The endurance improver is the endurance improver of the present invention described above. The content of the endurance enhancer in the endurance improving composition is not particularly limited and can be appropriately selected depending on the form of the endurance improving composition, and can be, for example, an amount used that is the same as the amount of the endurance enhancer used as the total amount of the peptides. The endurance improving composition may consist of only the endurance enhancer.

[0043] The basal metabolic rate enhancer is the basal metabolic rate enhancer of the present invention described above. The content of the basal metabolism enhancer in the composition for improving basal metabolism is not particularly limited and can be appropriately selected depending on the form of the composition for improving basal metabolism, and for example, the total amount of the peptide used can be the same as the amount of the basal metabolism enhancer used above. The composition for improving basal metabolism may consist of only the basal metabolism enhancer.

[0044] <Other ingredients> The other components in the composition for promoting myotube differentiation of myoblasts, the composition for muscle building, the composition for improving muscle performance, the composition for improving endurance, or the composition for improving basal metabolism are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include the same components as those in the above-mentioned agent for promoting myotube differentiation of myoblasts, the muscle building agent, the muscle performance improver, the endurance improver, or the basal metabolism improver. These may be used alone or in combination of two or more.

[0045] The content of the other components in the composition for promoting myotube differentiation of myoblasts, the composition for muscle building, the composition for improving muscle performance, the composition for improving endurance, or the composition for improving basal metabolism is not particularly limited and can be selected appropriately depending on the purpose.

[0046] <Aspects> The composition is not particularly limited as long as it is ingested orally, nasally, or enterally, and can be appropriately selected according to the purpose, and examples thereof include the oral preparations and food and drink described above. Here, food and drink include medicines, quasi-drugs, foods (including beverages, the same applies below), supplements, etc. that are ingested orally, nasally, or enterally. The food and supplement are sometimes called functional foods. The food and supplement also include foods for specified health uses, foods with nutritional functions, and foods with functional claims.

[0047] The composition for promoting myotube differentiation of myoblasts, the composition for muscle building, the composition for improving muscle performance, the composition for improving endurance, or the composition for improving basal metabolism is preferably applied to humans, but may also be applied to animals other than humans. When applied to animals other than humans, the composition can be used, for example, as feed.

[0048] The type of the food and drink is not particularly limited and can be appropriately selected depending on the purpose. Examples include soft drinks (carbonated drinks, non-alcoholic drinks, juices, coffee drinks, tea drinks, mineral water, sports drinks, etc.), milk drinks, soy milk drinks, fermented milk, lactic acid bacteria drinks, cocoa, alcoholic drinks (beer, sparkling wine, other brewed alcohol, liqueur, sake, amazake, wine, shochu, etc.), drinks prepared from instant powders, other drinks; bread, cereals, confectioneries (biscuits, cookies, chocolate, etc.), processed foods (dried bonito flakes, salted fish, kusaya, etc.), pickles (pickled vegetables, kimchi, pickles), fermented foods (natto, cheese, yogurt, etc.), seasonings (soy sauce, miso, vinegar, etc.), liquid foods, etc.

[0049] The method for producing the composition for promoting myotube differentiation of myoblasts, the composition for muscle building, the composition for improving muscle performance, the composition for improving endurance, or the composition for improving basal metabolism is not particularly limited, and can be appropriately selected depending on the aspect of the composition for promoting myotube differentiation of myoblasts, the composition for muscle building, the composition for improving muscle performance, the composition for improving endurance, or the composition for improving basal metabolism.

[0050] The amount, site of use, period of use, interval of use, etc. of the composition for promoting myotube differentiation of myoblasts, the composition for muscle strengthening, the composition for improving muscle performance, the composition for improving endurance, or the composition for improving basal metabolism are not particularly limited and can be appropriately selected depending on the purpose. For example, the amount of use can be the same as the amount of the above-mentioned agent for promoting myotube differentiation of myoblasts, the muscle strengthening agent, the muscle performance improving agent, the endurance improving agent, or the basal metabolism improving agent.

[0051] The composition for promoting myotube differentiation of myoblasts, the composition for muscle building, the composition for improving muscle performance, the composition for improving endurance, and the composition for improving basal metabolism are highly safe and can be used on a daily basis.

[0052] As described above, the myotube differentiation promoter of myoblasts and the composition for promoting myotube differentiation of myoblasts of the present invention have an excellent effect of promoting myotube differentiation of myoblasts. Therefore, the present invention also relates to a method for promoting myotube differentiation of myoblasts, which comprises administering to a subject at least one of the agent for promoting myotube differentiation of myoblasts and the composition for promoting myotube differentiation of myoblasts.

[0053] As described above, the muscle-building agent and muscle-building composition of the present invention have an excellent muscle-building effect. Therefore, the present invention also relates to a method for building muscle, which comprises administering to a subject at least one of the muscle-building agent and the muscle-building composition.

[0054] As described above, the muscle performance enhancing agent and composition for improving muscle performance of the present invention have an excellent muscle performance improving effect. Therefore, the present invention also relates to a method for improving muscle performance, which comprises administering to a subject at least one of the muscle performance enhancer and the muscle performance enhancing composition.

[0055] As described above, the endurance enhancing agent and composition for improving endurance of the present invention have an excellent endurance improving effect. Therefore, the present invention also relates to a method for improving endurance, which comprises administering to a subject at least one of the endurance-improving agent and the composition for improving endurance.

[0056] As described above, the basal metabolism enhancer and the composition for enhancing basal metabolism of the present invention have an excellent basal metabolism enhancing effect. Therefore, the present invention also relates to a method for improving basal metabolism, which comprises administering to a subject at least one of the basal metabolism enhancer and the basal metabolism improving composition.

[0057] (Method for producing whey protein hydrolysate) The method for producing a whey protein hydrolysate of the present invention is a method for producing a whey protein hydrolysate containing a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2), and includes at least an enzyme treatment step and may further include other steps as necessary. The method for producing a whey protein hydrolysate of the present invention produces a whey protein hydrolysate containing both a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

[0058] <Enzyme treatment process> The enzyme treatment step is a step of decomposing whey protein using an enzyme derived from Bacillus licheniformis.

[0059] <<Whey Protein>> The whey protein is a water-soluble material obtained by concentrating, separating, fractionating, and the like, whey protein in animal milk, including cow's milk, and contains α-lactalbumin, β-lactoglobulin, and the like. Specifically, for example, whey protein concentrate and whey protein isolate can be used. Examples of commercially available products include "Wheyco W80 (manufactured by Wheyco)" and "PROGEL800 (manufactured by FrieslandCampina DMV)".

[0060] The whey protein used may be one appropriately prepared by a known method, or a commercially available product may be used.

[0061] <<Enzymes originating from Bacillus licheniformis>> The enzyme originating from Bacillus licheniformis is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include proteases, etc. The enzyme originating from Bacillus licheniformis may be used alone or in combination of two or more kinds.

[0062] The enzyme originating from Bacillus licheniformis may be appropriately prepared or a commercially available product. Specific examples of the commercially available products include proteases such as Orientase 22BF (HBI Corporation), Bioprase SP-20FG (Nagase ChemteX Corporation), and Protin SD-AY10 (Amano Enzyme Inc.).

[0063] The method for preparing the enzyme originating from Bacillus licheniformis is not particularly limited and can be appropriately selected depending on the purpose. For example, Bacillus licheniformis is cultured, and the resulting culture is subjected to a cell disruption treatment, such as ultrasonic treatment or pressurization, as necessary. Then, unnecessary cell walls and the like are removed by appropriately combining filtration, centrifugation, ultrafiltration, salting out, dialysis, chromatography, and the like, and the contents that become the enzyme are recovered. The culture conditions for Bacillus licheniformis (temperature, aeration and stirring conditions, pH of the medium, culture time, etc.) are not particularly limited and can be appropriately selected depending on the species and characteristics of the microorganism, the culture scale, etc.

[0064] <<Enzyme treatment>> The method of the enzyme treatment is not particularly limited and can be appropriately selected depending on the purpose. For example, there is a method in which the enzyme derived from Bacillus licheniformis is added to a whey protein solution obtained by dissolving the whey protein in a solvent such as water, and the reaction is allowed to proceed.

[0065] The content of the whey protein in the whey protein solution used in the enzyme treatment is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 5 to 30% by mass, more preferably 10 to 20% by mass.

[0066] The amount of the enzyme originating from Bacillus licheniformis used in the enzyme treatment is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 1 to 10 mass % relative to the solid content of the whey protein solution, more preferably 2 to 8 mass %, and particularly preferably 3 to 6 mass %.

[0067] The pH of the whey protein solution at the start of the enzyme reaction is not particularly limited as long as it is 7.6 to 12.0 and can be appropriately selected depending on the purpose. However, from the viewpoints of large peptide production amount and excellent flavor, the pH is preferably 8.0 to 11.0, and more preferably 8.5 to 10.0.

[0068] The temperature for the enzyme reaction is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 35 to 60°C, more preferably 45 to 55°C, from the viewpoints of enzyme activity and thermal stability.

[0069] The time for the enzyme reaction is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 1 to 6 hours, and more preferably 2 to 4 hours.

[0070] The method for confirming whether or not the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) have been produced by the enzyme treatment is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include liquid chromatography mass spectrometry (LC / MS).

[0071] <Other processes> The other steps are not particularly limited as long as they do not impair the effects of the present invention and can be appropriately selected depending on the purpose. For example, there can be mentioned a step of increasing the temperature of the enzyme treatment liquid after the enzyme treatment and maintaining it for a certain period of time to inactivate the enzyme.

[0072] (Peptide-Containing Composition) The peptide-containing composition of the present invention contains at least a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), and further contains other components as necessary.

[0073] <Peptide> The peptide consisting of the amino acid sequence DISL (sequence number 1) and the peptide consisting of the amino acid sequence DISLL (sequence number 2) are the same as the peptides in the above-mentioned myoblast myotube differentiation promoter, muscle-enhancing agent, muscle performance improver, endurance improver, or basal metabolic improver.

[0074] The content of the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) in the peptide-containing composition is not particularly limited as long as it is 0.003% by mass or more and can be appropriately selected depending on the purpose, but is preferably 0.01% by mass or more, more preferably 0.03% by mass or more, and particularly preferably 0.1% by mass or more.

[0075] The content of the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) in the peptide-containing composition is not particularly limited as long as it is 0.050% by mass or more and can be appropriately selected depending on the purpose, but is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, and particularly preferably 1.0% by mass or more.

[0076] <Other ingredients> The other components in the peptide-containing composition are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose, and examples thereof include the same as the other components in the myoblast myotube differentiation promoter, muscle strengthener, muscle performance improver, endurance improver, or basal metabolism improver described above. These may be used alone or in combination of two or more.

[0077] The contents of other components in the peptide-containing composition are not particularly limited and can be appropriately selected depending on the purpose.

[0078] The method for producing the peptide-containing composition is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include the above-mentioned method for producing whey protein hydrolysate of the present invention. The peptide-containing composition can be prepared by concentrating the above-mentioned peptide-containing composition to obtain a peptide-containing composition containing a desired amount of peptide.

[0079] The method for concentrating the peptide is not particularly limited, and a general technique can be appropriately selected. For example, the peptide can be concentrated by appropriately combining techniques such as chromatography (e.g., gel filtration, ion exchange chromatography, affinity chromatography, silica gel chromatography, HPLC, etc.); crystallization; salting out; organic solvent precipitation; and ultrafiltration.

[0080] The use of the peptide-containing composition is not particularly limited and can be appropriately selected depending on the purpose. EXAMPLES

[0081] The present invention will be specifically described below by way of examples, but the present invention is not limited to these examples in any way.

[0082] Example 1 10 g of whey protein was added to 90 g of water and dispersed while heating at 50 to 60° C. Furthermore, a sodium hydroxide solution was added to adjust the pH to 9, thereby preparing a whey protein solution. To the whey protein solution, 0.3 g (3.0% by mass based on the solid content of the whey protein solution) of protease originating from Bacillus licheniformis (Orientase 22BF, HIV Inc.) was added, and the enzyme reaction was carried out while maintaining the temperature at 60°C. After 3 hours, the temperature was raised to 90°C and maintained for 30 minutes to inactivate the protease. The pH of the resulting enzyme reaction solution was adjusted to 7 with sodium hydroxide to obtain an enzymatically decomposed whey protein solution. The obtained enzymatically hydrolyzed whey protein solution was diluted with 50% acetonitrile, and liquid chromatography mass spectrometry (LC / MS) was used to confirm whether or not a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2) were produced. The content of each peptide in the enzymatically hydrolyzed whey protein solution (based on solids) was approximately 0.04 to 0.07 mass% for the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1), and approximately 0.06 to 0.08 mass% for the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

[0083] Example 2 10 g of whey protein was added to 90 g of water and dispersed while heating at 50 to 60° C. Furthermore, a sodium hydroxide solution was added to adjust the pH to 9, thereby preparing a whey protein solution. 0.44 g (4.4% by mass based on the solid content of the whey protein solution) of protease derived from Bacillus licheniformis (Biophrase SP-20FG, Nagase Chemtex Corporation) was added to the whey protein solution, and the enzyme reaction was carried out while maintaining the temperature at 50°C. After 3 hours, the temperature was raised to 90°C and maintained for 30 minutes to inactivate the protease. The pH of the resulting enzyme reaction solution was adjusted to 7 with sodium hydroxide to obtain an enzymatically hydrolyzed whey protein solution. The obtained enzymatically decomposed whey protein solution was diluted with 50% acetonitrile, and the presence or absence of a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2) was confirmed using LC / MS. The content of each peptide in the enzymatically hydrolyzed whey protein solution (based on solids) was approximately 0.02 mass% for the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1), and approximately 0.18 mass% for the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

[0084] Example 3 10 g of whey protein was added to 90 g of water and dispersed while heating at 50 to 60° C. Furthermore, a sodium hydroxide solution was added to adjust the pH to 9, thereby preparing a whey protein solution. 0.55 g (5.5% by mass based on the solid content of the whey protein solution) of protease derived from Bacillus licheniformis (Protin SD-AY10, Amano Enzyme Inc.) was added to the whey protein solution, and the enzyme reaction was carried out while maintaining the temperature at 50°C. After 3 hours, the temperature was raised to 90°C and maintained for 30 minutes to inactivate the protease. The pH of the resulting enzyme reaction solution was adjusted to 7 with sodium hydroxide to obtain an enzymatically hydrolyzed whey protein solution. The obtained enzymatically decomposed whey protein solution was diluted with 50% acetonitrile, and the presence or absence of a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2) was confirmed using LC / MS. The content of each peptide in the enzymatically hydrolyzed whey protein solution (based on solids) was approximately 0.02 mass% for the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1), and approximately 0.25 mass% for the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

[0085] Comparative Example 1 10 g of whey protein was added to 90 g of water and dispersed while heating at 50 to 60° C. Furthermore, a sodium hydroxide solution was added to adjust the pH to 8, thereby preparing a whey protein solution. 0.3 g (3.0% by mass based on the solid content of the whey protein solution) of a protease not derived from Bacillus licheniformis (Samoase, Amano Enzyme Co., Ltd.) was added to the whey protein solution, and the enzyme reaction was carried out while maintaining the temperature at 65° C. After 3 hours, the temperature was raised to 90° C. and maintained for 30 minutes to inactivate the protease. The resulting enzyme reaction solution was adjusted to pH 7 with sodium hydroxide to obtain an enzymatically hydrolyzed whey protein solution. The obtained enzymatically hydrolyzed whey protein solution was diluted with 50% acetonitrile, and LC / MS was used to confirm whether or not a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2) were produced, but the production of neither peptide was confirmed.

[0086] Comparative Example 2 10 g of whey protein was added to 90 g of water and dispersed while heating at 50 to 60° C. Furthermore, a sodium hydroxide solution was added to adjust the pH to 9, thereby preparing a whey protein solution. 0.3 g (3.0% by mass based on the solid content of the whey protein solution) of a protease not derived from Bacillus licheniformis (Biophrase OP, Nagase Chemtex Corporation) was added to the whey protein solution, and the enzyme reaction was carried out while maintaining the temperature at 65°C. After 3 hours, the temperature was raised to 90°C and maintained for 30 minutes to inactivate the protease. The pH of the resulting enzyme reaction solution was adjusted to 7 with sodium hydroxide to obtain an enzymatically decomposed whey protein solution. The obtained enzymatically decomposed whey protein solution was diluted with 50% acetonitrile, and the presence or absence of a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2) was confirmed using LC / MS. The production of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) was not confirmed, and the content of the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) in the enzymatically hydrolyzed whey protein solution (based on solids) was approximately 0.12% by mass.

[0087] The results of Examples 1 to 3 and Comparative Examples 1 and 2 are shown in Tables 1 and 2 below. Example 1 was carried out twice, with the results of the first run being shown in Table 1 and the results of the second run being shown in Table 2.

[0088] [Table 1]

[0089] [Table 2]

[0090] From the above results, it was confirmed that an enzymatically decomposed whey protein solution containing both the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) can be obtained by enzymatically decomposing whey protein using an enzyme originating from Bacillus licheniformis. On the other hand, when an enzyme not originating from Bacillus licheniformis was used, an enzymatically decomposed whey protein solution containing both the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) was not obtained.

[0091] [material] In the following Test Examples 1 and 2, the materials shown below were used. (cell) Normal human myoblasts (Lonza, Lot. No.: 21TL076030, 21 years old, male, Caucasian) (Culture medium) Growth medium composition: SkGM-2 Composition of differentiation medium: DMEM-F12 medium supplemented with 2% horse serum (Test reagents, etc.) SkGM-2 (Lonza, CC-3245) · DMEM-F12 (Gibco, 11330-032) Horse serum (HS, Thermo Fisher Scientific, 16050-122) · DPBS(-)(Sigma, D8537-500ML) 0.05% Trypsin-EDTA solution (Gibco, 25300-054) 75cm 2 Flask (Corning, 430641U) 96-well black plate (Perkin Elmer, 6055300) 4% paraformaldehyde in phosphate buffer (Nacalai tesque, 09154-14) Bovine serum albumin (BSA, Sigma-Aldrich, A9647) · TritonX-100 (ICN Biomedicals, 807426) · Hoechst33342, trihydrochloride, trihydrate(Thermo Fisher Scientific, H1399) · Myosin 4 Monoclonal Antibody (MF20) (anti-MHC antibody, Thermo Fisher Scientific, 14-6503-37) · F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 555 (Thermo Fisher Scientific, A-21425) NucleoSpin® RNA Plus (Takara Bio, U0984B) · Multi-purpose refrigerated centrifuge (TOMY, MDX-310) CO2 incubator (Thermo Fisher Scientific, 370) Phase contrast microscope (Olympus, IX73) Confocal quantitative image cytometer (Yokogawa Electric, CellVoyager CQ1) NanoDrop TMOne Ultra Trace UV-Visible Spectrophotometer (Thermo Fisher Scientific) · Agilent 2100 Bioanalyzer(Agilent Technologies)

[0092] [Cell preculture] The cells used in the following Test Examples 1 and 2 were pre-cultured as follows. Normal human myoblasts were cultured at 75 cm 2 The flask was placed in a 75cm 2 The flask was placed in a CO2 incubator (5% CO2, 37°C, humidified) and normal human myoblasts were cultured. The medium was changed every 1-2 days, and the cells were harvested when they reached 60% confluence and used in the test. The cells were passaged as follows. After washing the cells with DPBS(-), the cells were detached using a 0.05% trypsin-EDTA solution, and trypsin was neutralized by adding SkGM-2. The cell suspension was then collected in a 15 mL centrifuge tube and centrifuged (room temperature, 180 x g, 5 minutes). The supernatant was removed, and fresh growth medium was added to stir the cells, and the cell number was counted using a cell counting board. The cells were suspended to the desired cell density using SkGM-2, and seeded into a 96-well black plate to be used in the test.

[0093] (Test Example 1: Myotube cell differentiation promotion test) The cells were seeded in a 96-well black plate at a density of 7,500 cells / 100 μL / well and cultured in a CO2 incubator (5% CO2, 37°C, humidified). The next day, the medium was replaced with differentiation medium (DMEM-F12 medium supplemented with 2% HS) supplemented with each test substance (see Table 3) and solvent control, or growth medium (SkGM-2) supplemented with solvent control (N=3). After 48 hours, the following MHC staining was performed. After incubation, the medium was removed, 4%-paraformaldehyde phosphate buffer (4℃) was added, and the cells were fixed by incubating at 4℃ for 15 minutes. Next, the wells were washed three times with DPBS(-), and then blocking / membrane permeabilization solution (3%BSA / 0.3% Triton-X 100 / DPBS(-)) was added and incubated at room temperature for 30 minutes. Next, the solution was replaced with a primary antibody solution (prepared by adding 1 / 300 volume of anti-MHC antibody to 3% BSA / DPBS(-)) and incubated overnight at 4℃. After washing three times with 3% BSA / DPBS(-), the solution was replaced with a secondary antibody solution (prepared by adding 1 / 500 volume of secondary antibody and 1 / 1000 volume of Hoechst33342 to 3% BSA / DPBS(-)) and incubated at room temperature for 2 hours. After washing three times with DPBS(-), DPBS(-) was added and images were taken using a confocal quantitative image cytometer (Yokogawa Electric, CellVoyager CQ1). After acquiring images, nuclei and MHC-positive nuclei were detected and counted, and the myotube differentiation rate (fusion index (%): number of MHC-positive nuclei / total number of nuclei x 100) was calculated to quantify the myotube differentiation rate. Measurements were performed with N=3, and the mean ± standard error (SE) was calculated. A significant difference test between the two groups was performed using the Student's t-test (significance level p<0.05). The results of the myotube differentiation rate (fusion index) measurement are shown in Table 3 below.

[0094] [Table 3] In Table 3, "#" indicates that there was a significant difference (p<0.05) between "negative control, water, proliferation medium" and "$" indicates that there was a significant difference (p<0.05) between "negative control, water, differentiation medium". The test substance "whey protein" used was the same as the whey protein used in Example 1, and the "enzyme-treated whey protein product" used was the "enzyme-hydrolyzed whey protein solution" obtained in Example 1.

[0095] As shown in Table 3, when a whey protein enzyme-treated product containing a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1), a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), or a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) (sometimes referred to as "whey protein hydrolysate containing a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2)") was added, the myotube differentiation rate (fusion index) was significantly increased compared to when the test substance was not added.

[0096] (Test Example 2: DNA chip test) The cells were seeded in a 96-well black plate at a density of 7,500 cells / 100 μL / well and cultured in a CO2 incubator (5% CO2, 37°C, humidified). The next day, the differentiation medium was replaced with a differentiation medium (DMEM-F12 medium supplemented with 2% HS) supplemented with each test substance (see Table 4) and a solvent control (N=1). After 24 hours, the cells were harvested and RNA was extracted using NucleoSpin® RNA Plus. The quality was confirmed using NanoDrop and Bioanalyzer, and samples with A260 / A280 of 1.5 or more and RIN (RNA Integrity Number) of 7.0 or more were used for testing. DNA chips were run using the SurePrint G3 Human GE Microarray 8 x 60K Ver3.0, and after normalizing the data, gene expression analysis was performed for the negative control and samples at each time point. The results of representative gene expression related to muscle differentiation are shown in Table 4.

[0097] [Table 4] In Table 4, "nd" stands for not detected. The test substance "whey protein" used was the same as the whey protein used in Example 1, and the "enzyme-treated whey protein product" used was the "enzyme-hydrolyzed whey protein solution" obtained in Example 1.

[0098] As shown in Table 4, a comparison of gene variations between individuals showed that the addition of a peptide consisting of the amino acid sequence DISL (sequence number 1) or a peptide consisting of the amino acid sequence DISLL (sequence number 2) increased the expression of the myogenin gene, a gene that induces myotube differentiation, compared to the negative control, and also increased the expression of the PGC-1α gene, which is involved in sugar uptake and increased mitochondrial function. In addition, when an enzyme-treated whey protein product containing a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2) (sometimes referred to as "whey protein hydrolysate containing a peptide consisting of the amino acid sequence DISL (sequence number 1) and a peptide consisting of the amino acid sequence DISLL (sequence number 2)") was added, although the fold change of the myogenin gene was negative, the value was higher than that of whey protein, and it was therefore confirmed to have a certain degree of effect.

[0099] Muscles are formed by the differentiation of muscle cells, in which precursor cells, myoblasts, proliferate to a certain number of cells, migrate to the pre-muscle region, and fuse with each other to become differentiated multinucleated myotubes, which then become muscle fibers, thereby becoming muscles. In a myotube differentiation promotion test using human myoblasts (Test Example 1), it was confirmed that the addition of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) or a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) promoted myotube differentiation compared to the negative control. In addition, as shown in Test Example 2, the expression of the myogenin gene, which is important for inducing differentiation of muscle cells, was increased by 1.8 times with the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and by 1.6 times with the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), suggesting that these peptides promote the induction of expression and thereby promote differentiation into myotubes, i.e., promote muscle synthesis and exert a muscle-building effect. Furthermore, a comparison of gene expression levels in Test Example 2 showed that the PGC-1α gene expression level in myoblasts to which a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) or a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) was added was increased by 2.0 times for the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and 1.7 times for the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) compared to the gene expression level in the negative control. Since it has been reported that PGC-1α improves mitochondrial function and the expression of GLUT4, which is important for sugar uptake (see Non-Patent Document 1 mentioned above), it was suggested that these peptides would improve muscle performance and endurance.

[0100] (Test Example 3: Human Test) The effect of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) or a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) on basal metabolism was tested as follows.

[0101] The subjects were healthy adults aged 20 years or older and under 65 years (7 men, 11 women; age 47.6±8.0 years) who consumed the test beverages listed below once a day for 12 weeks. <Test beverage> A powdered beverage containing 10 g of the whey protein enzymatic hydrolyzate was suspended in water to prepare a test beverage. The amount of the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) in the 10 g of the whey protein enzymatic hydrolyzate was 1 mg for the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and 24.3 mg for the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

[0102] Resting energy expenditure (kcal / min) was measured before and 12 weeks after ingestion of the test beverage using an Aeromonitor AE-310S (Minato Medical Science Co., Ltd.). Measurement of resting energy expenditure began after fasting for 12 hours or more. The Aeromonitor AE-310S is a device that measures carbon dioxide output (VCO2) and oxygen intake (VO2) and automatically calculates resting energy expenditure (kcal / min).

[0103] In addition, muscle mass was measured using a body composition analyzer (InBody3.2, Biospace Corporation) before and 12 weeks after ingestion of the test beverage.

[0104] The average resting energy expenditure (kcal / min) before and after 12 weeks of test drink intake is shown in Figure 1. As shown in Figure 1, the average resting energy expenditure (kcal / min) was 5.12 (kcal / min) before intake and 5.45 (kcal / min) after 12 weeks of intake, and was significantly higher after 12 weeks of intake compared to before intake (paired t-test: p<0.01). Therefore, it was confirmed that the present invention improves basal metabolism.

[0105] Figure 2 shows the results of investigating the correlation between resting energy expenditure (kcal / min) (vertical axis) and muscle mass (kg) (horizontal axis) after 12 weeks of intake. As shown in Figure 2, a significant positive correlation was confirmed between resting energy expenditure (kcal / min) and muscle mass (kg) after 12 weeks of intake (Pierson's correlation coefficient r = 0.75, t = 4.60, p = 0.00).

[0106] From the results of the above-mentioned Test Examples 1 and 2, it has been confirmed that muscle differentiation is promoted according to the present invention. Therefore, it is suggested that muscle mass is increased by continuously taking at least one of the peptides consisting of the amino acid sequence DISL (SEQ ID NO: 1) and the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2). In addition, based on the results of Test Examples 1 to 3, it is presumed that the intake of the peptides promotes muscle differentiation, and as a result, the increase in muscle mass leads to an increase in resting energy consumption, i.e., an improvement in basal metabolism.

Claims

1. An agent for promoting myotube differentiation of myoblasts, comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

2. The myoblast myotube differentiation promoter according to claim 1, A composition for promoting myotube differentiation of myoblasts, which is administered orally, nasally, or enterally.

3. A muscle-building agent comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

4. The muscle-enhancing agent according to claim 3, A composition for building muscle mass, which is ingested by oral, nasal, or enteral administration.

5. A muscle performance improving agent comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

6. The muscle performance enhancer according to claim 5, A composition for improving muscle performance, which is ingested by oral, nasal or enteral administration.

7. An endurance improving agent comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

8. The endurance enhancer according to claim 7, A composition for improving endurance, which is ingested by oral, nasal or enteral administration.

9. A basal metabolic rate enhancer comprising at least one of a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2).

10. The basal metabolic rate enhancer according to claim 9, A composition for improving basal metabolism, which is ingested by oral, nasal or enteral administration.

11. A method for producing a whey protein hydrolysate comprising a peptide having the amino acid sequence DISL (SEQ ID NO: 1) and a peptide having the amino acid sequence DISLL (SEQ ID NO: 2), comprising: The method comprises degrading whey protein using an enzyme derived from Bacillus licheniformis, The method comprises adjusting the pH of the whey protein solution containing the whey protein to 7.6 to 12.0 to initiate a reaction with the enzyme originating from Bacillus licheniformis.

12. Preparing the whey protein solution having a whey protein content of 5 to 30% by mass; Adding the enzyme derived from Bacillus licheniformis in an amount of 1 to 10% by mass based on the solid content of the whey protein solution; The method according to claim 11, comprising carrying out the reaction with the enzyme originating from Bacillus licheniformis at 35 to 60° C. for 1 to 6 hours.

13. A peptide-containing composition comprising a peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) and a peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2), A peptide-containing composition, characterized in that the content of the peptide consisting of the amino acid sequence DISL (SEQ ID NO: 1) in the peptide-containing composition is 0.003% by mass or more, and the content of the peptide consisting of the amino acid sequence DISLL (SEQ ID NO: 2) in the peptide-containing composition is 0.050% by mass or more.

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