KR102663146B1 - Composition for preventing, improving or treating sarcopenia comprising a mealworm larvae protein or its hydrolysate - Google Patents

Abstract

A pharmaceutical composition for preventing, improving or treating muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient; food composition; Health functional food; and feed composition. The brown mealworm larval protein of the present invention or its hydrolyzate can effectively inhibit the expression of Myostatin, and the composition containing it as an active ingredient reduces muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. It can be usefully used in the pharmaceutical, food, and feed industries as a composition for preventing, improving, or treating various muscle diseases caused by .

Description

Composition for preventing, improving or treating sarcopenia comprising a mealworm larvae protein or its hydrolysate as an active ingredient {Composition for preventing, improving or treating sarcopenia comprising a mealworm larvae protein or its hydrolysate}

A pharmaceutical composition for preventing, improving or treating muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient; food composition; Health functional food; and feed composition.

Korea entered an aging society in 2000 with the elderly population accounting for 7.2% of the total population, and is expected to enter a super-aging society (more than 20%) in 2050 (2013 statistics on the elderly, Statistics Korea). A person's muscle mass decreases with age (approximately 10-15% between the ages of 50 and 70, and more than 30% between the ages of 70 and 80), and muscle strength and function weaken accordingly, which is called sarcopenia. It is said that Senile sarcopenia is a major cause that limits the independent living of the elderly by causing activity and walking difficulties. In addition, sarcopenia lowers the basal metabolic rate, increases insulin resistance, promotes the development of type 2 diabetes, and increases the risk of high blood pressure and cardiovascular disease by 3-5 times.

The concept of sarcopenia began in 1989 when Irwin Rosenberg introduced the term 'sarcopenia', which, with its origins in Greek, is a combination of the words "sarx" meaning muscle and "penia" meaning decreased. Sarcopenia is associated with aging and refers to a decrease in muscle strength due to a decrease in muscle mass. Here, “muscle” refers to skeletal muscle and has nothing to do with smooth muscle. In other words, sarcopenia refers to a loss of skeletal muscle mass mainly distributed in the extremities, cachexia, a state of significant muscle loss that occurs in the terminal stages of malignant tumors, and muscle wasting due to acute diseases such as the flu. It is distinct from wasting, or primary muscle disease, and should be viewed purely as a result of gradual skeletal muscle decline associated with aging.

Currently, there are no drugs approved for the treatment of sarcopenia, and drug repositioning technology that applies myostatin inhibitors or existing FDA-approved treatments for other diseases to sarcopenia is being developed.

Myostatin is a polypeptide growth factor belonging to the TGF-β superfamily. TGF-β has a large number of isoforms, which are known to be involved in cell proliferation, apoptosis, differentiation, and bone formation and maintenance (Massague & Chen, 2000). Myostatin belongs to growth differentiation factor (GDF) number 8, is involved in tissue growth and development, and acts by activating the Smad signaling system. In addition, it has been reported to affect bone formation and regeneration by inhibiting the cell cycle and proliferation of progenitor cells by the p21 gene. Myostatin is mainly produced in skeletal muscle cells and causes muscle loss and strength reduction in an autocrine manner. It inhibits the expression of IGF-1 or Follistatin, which are involved in muscle hypertrophy, and thereby inhibits the expression of IGF-1 and Follistatin, which are proteins in myoblasts. It is known to inhibit synthesis and cell proliferation.

Conventionally, antibodies to inhibit only the function of myostatin have been manufactured to treat sarcopenia, but side effects have been reported, and research on anti-myostatin materials using natural products is also actively underway.

Against this background, the present inventors have made extensive research efforts to develop an agent that can effectively treat sarcopenia, and as a result, brown mealworm larvae protein and its hydrolyzate effectively inhibit the expression of myostatin, preventing or preventing sarcopenia. The present invention was completed by confirming that treatment is possible.

Korean Patent Publication No. 10-2019-0088393 Korean Patent Publication No. 10-2020-0009543

Therefore, the purpose of the present invention is to provide a pharmaceutical composition that can effectively prevent or treat muscle diseases.

Another object of the present invention is to provide a food composition that can effectively prevent or improve muscle disease.

Another object of the present invention is to provide a health functional food that can effectively prevent or improve muscle diseases.

Another object of the present invention is to provide a feed additive composition that can effectively prevent or improve muscle disease.

In order to achieve the purpose of the present invention as described above,

The present invention provides a food composition for preventing or improving muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient.

In one embodiment of the present invention, the brown mealworm larvae protein is prepared by: a) pulverizing dried brown mealworm larvae; b) adding ethanol to the ground product to degrease it; c) adding sodium hydroxide to the defatted brown mealworm larvae and mixing them, followed by centrifugation to obtain a precipitate; and d) desalting the obtained precipitate and then freeze-drying it.

In one embodiment of the present invention, the hydrolyzate can be prepared by hydrolyzing brown mealworm larval protein by treating it with alcalase, flavorzyme, or a mixture thereof.

In one embodiment of the present invention, the brown mealworm larval protein or its hydrolyzate can inhibit Myostatin expression.

In one embodiment of the present invention, the muscle disease may be a muscle disease caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration.

In one embodiment of the present invention, the muscle disease is atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spine syndrome, and amyotrophic lateral cord syndrome. It may be selected from the group consisting of sclerosis (amyotrophic lateral sclerosis), Charcot-Marie-Tooth disease, and sarcopenia.

In addition, the present invention provides a health functional food for preventing or improving muscle disease containing brown mealworm larval protein or its hydrolyzate as an active ingredient.

In one embodiment of the present invention, the health functional food may be selected from the group consisting of beverages, meat, confectionery, noodles, rice cakes, bread, gum, candy, ice cream, and alcoholic beverages.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient.

In addition, the present invention provides a feed additive composition for preventing or improving muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient.

The brown mealworm larval protein of the present invention or its hydrolyzate can effectively inhibit the expression of Myostatin, and the composition containing it as an active ingredient reduces muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. It can be usefully used in the pharmaceutical, food, and feed industries as a composition for preventing, improving, or treating various muscle diseases caused by .

Figure 1 shows the manufacturing process diagram of brown mealworm larval protein (MPI) and its hydrolyzate (MPH) prepared through Examples 1 and 2 according to the present invention.
Figure 2 evaluates the cytotoxicity of brown mealworm larval protein (MPI) and its hydrolysates (MPHAF, AF-LT, AF-TT, AF-MT) of the present invention.
Figure 3 shows the protein hydrolyzate of brown mealworm larvae (MPHAF, MPHFA, MPHME, MPHA, MPHF) of the present invention prepared under different hydrolysis conditions, treated with C2C12 cells at a concentration of 0.01 mg/mL, and then treated with myostatin (Myostatin). ) This is the result of measuring the relative mRNA expression level.
Figure 4 shows the results of measuring the relative mRNA expression level of Myostatin after treating C2C12 cells with hydrolysates of each size (AF-LT, AF-TT, AF-MT) of MPHAF at a concentration of 0.01 mg/mL. .
Figure 5 shows the relative Myostatin promoter luciferdase activity measured after treating C2C12 cells with brown mealworm larval protein (MPI) and its hydrolyzate (MPH) of the present invention at a concentration of 0.01 mg/mL.
Figure 6 shows the expression of inflammatory cytokines (IL) after treating macrophages inflamed with LPS with the brown mealworm larval protein hydrolyzate (MPHAF, MPHFA, MPHME, MPHA, MPHF) of the present invention prepared under different hydrolysis conditions. This is the result of evaluating the degree of inhibition of -6, TNF-a, IL-1b) expression.
Figure 7 shows the degree of inhibition of inflammatory cytokine (IL-6, TNF-a, IL-1b) expression after treating macrophages inflamed with LPS with size-specific hydrolysates of MPHAF (AFLT, AFTT, AFMT). It is a result.
Figure 8 shows the available amino group concentration of brown mealworm larval protein hydrolyzate (MPHAF, MPHFA, MPHME, MPHA, MPHF) of the present invention prepared under different hydrolysis conditions.
Figure 9 shows the protein concentration of brown mealworm larval protein hydrolyzate (MPHAF, MPHFA, MPHME, MPHA, MPHF) of the present invention prepared under different hydrolysis conditions.
Figure 10 shows the measurement of protein concentration of hydrolysates (AF-LT, AF-TT, AF-MT) by size of MPHAF.
Figure 11 shows the SDS-PAGE pattern of the brown mealworm larval protein hydrolyzate of the present invention prepared under different hydrolysis conditions.
Figure 12 shows the results of evaluating the antioxidant activity of brown mealworm larval protein hydrolysates (MPHAF, MPHFA, MPHME, MPHA, MPHF) of the present invention prepared under different hydrolysis conditions.
Figure 13 shows the results of evaluating the antioxidant activity of hydrolysates (AF-LT, AF-TT, AF-MT) by size of MPHAF.

The present invention relates to a pharmaceutical composition for preventing or treating muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient.

The term “prevention” used in the present invention refers to all actions that suppress or delay the onset of muscle disease by administering the composition to an individual.

The term “treatment” used in the present invention refers to any action in which the symptoms of a muscle disease are improved or beneficially changed by the composition.

The term “brown mealworm larval protein” used in the present invention refers to protein extracted from brown mealworm larvae.

The brown mealworm larvae protein is prepared by: a) pulverizing dried brown mealworm larvae; b) adding ethanol to the ground product to degrease it; c) adding sodium hydroxide to the defatted brown mealworm larvae and mixing them, followed by centrifugation to obtain a precipitate; and d) desalting the obtained precipitate and then freeze-drying it.

The term “hydrolyzate of brown mealworm larval protein” used in the present invention refers to a substance prepared by treating brown mealworm larval protein with a hydrolytic enzyme and then hydrolyzing it.

The hydrolyzate can be prepared by hydrolyzing brown mealworm larval protein by treating it with alcalase, flavorzyme, or a mixture thereof as a hydrolytic enzyme. Alternatively, the hydrolyzate can be prepared by hydrolyzing brown mealworm larval protein by sequentially treating it with alcalase and flavorzyme or by treating it in the reverse order.

In one embodiment of the present invention, the hydrolyzate is treated with 0.5% (w/v) alcalase for 12 hours using brown mealworm larval protein as a substrate, and then treated with 0.5% (w/v) flavorzyme for 12 hours. It may be a hydrolyzate obtained through processing.

In another embodiment of the present invention, the hydrolyzate was treated with 0.5% (w/v) alcalase for 12 hours using brown mealworm larval protein as a substrate, and then treated with 0.5% (w/v) flavorzyme for 12 hours. The hydrolyzate obtained through treatment may have a molecular weight of 10 KDa or more.

The brown mealworm larval protein of the present invention or its hydrolyzate can exhibit the effect of preventing or treating muscle disease by inhibiting the expression of Myostatin.

The term “Myostatin (MSTN)” used in the present invention is a protein that regulates muscle growth, belongs to the transforming growth factor-β (TGF-β) family, and is a growth and differentiation factor (GDF). -8).

The term “muscle disease” used in the present invention refers to a disease caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration.

The term “muscle” used in the present invention comprehensively refers to tendons, muscles, and tendons, and “muscle function” refers to the ability to exert force by muscle contraction, and the muscle has the maximum contractile force to overcome resistance. These include muscular strength, which is the ability to exert force, muscular endurance, which is the ability to express how long or how many times a muscle can repeat contraction and relaxation with a given weight, and quickness, which is the ability to exert strong force in a short period of time. This “muscle function” is proportional to muscle mass, and “improving muscle function” means improving muscle function for the better.

In one embodiment of the present invention, the muscle disease is atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spine syndrome, and amyotrophic lateral sclerosis. (Lou Gehrig's disease, amyotrophic lateral sclerosis), rigid spine syndrome (rigid spinsesyndrome), Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease), and sarcopenia (sarcopenia). It is not limited to this. In addition, the muscle wasting or degeneration occurs due to inherited factors, acquired factors, aging, etc., and muscle wasting is characterized by gradual loss of muscle mass and weakening and degeneration of muscles, especially skeletal muscles, voluntary muscles, and cardiac muscles.

In one embodiment of the present invention, the brown mealworm larval protein or its hydrolyzate contained in the composition is not particularly limited, but is specifically 0.001% to 99% by weight, more specifically 0.01% by weight, based on the total weight of the composition. It may include weight% to 50% by weight. Meanwhile, the brown mealworm larval protein of the present invention or its hydrolyzate may be included in the composition at a concentration of 0.0001 to 1000 μg/ml.

The composition of the present invention is a pharmaceutical composition containing brown mealworm larval protein or a hydrolyzate thereof as an active ingredient, and can be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to these active ingredients, and the auxiliaries include Excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, or flavoring agents can be used.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the composition, one or more buffering agents (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch (corn starch, wheat starch, rice starch, potato). starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl. -It is prepared by mixing cellulose or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.

Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, or preservatives. You can. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, or suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be applied externally to the skin; Injections administered intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterineally, intrathecally, or intracerebrovascularly; Transdermal administration; Alternatively, it can be formulated in the form of a nasal inhalant according to methods known in the art.

The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations are included. In the above, transdermal administration means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

For inhalation administration, the compounds used according to the invention may be packaged in pressurized packs or using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a text generally known in all pharmaceutical chemistry.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. That is, the total effective amount of the pharmaceutical composition of the present invention can be administered to the patient as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. You can. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response regulators.

The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing brown mealworm larval protein or its hydrolyzate as an active ingredient. When the pharmaceutical composition for preventing and treating muscle disease of the present invention is used as an external skin agent, fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, and foaming agents are added. ), fragrance, surfactant, water, ionic emulsifier, non-ionic emulsifier, filler, metal ion sequestrant, chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic activator, lipophilic activator. Or it may contain adjuvants commonly used in the field of dermatology, such as lipid vesicles or any other ingredients commonly used in topical skin preparations. Additionally, the ingredients may be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for preventing and treating muscle diseases of the present invention is provided as an external skin preparation, it is not limited thereto, but may be in the form of an ointment, patch, gel, cream, or spray.

Additionally, the present invention relates to the use of a composition containing brown mealworm larval protein or a hydrolyzate thereof as an active ingredient for the production of a medicine for preventing or treating muscle diseases. The composition of the present invention containing the above-described brown mealworm larval protein or its hydrolyzate as an active ingredient can be used for the production of a medicine for preventing or treating muscle diseases.

Additionally, the present invention relates to a method for preventing or treating muscle disease, comprising administering a therapeutically effective amount of brown mealworm larval protein or a hydrolyzate thereof to a mammal.

The term “mammal” used in the present invention refers to a mammal that is the subject of treatment, observation, or experiment, and preferably refers to a human.

As used herein, the term “therapeutically effective amount” refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as considered by a researcher, veterinarian, physician, or other clinician. This includes amounts that lead to alleviation of the symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by a person skilled in the art, and can be determined based on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, and general health condition. , gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs can be adjusted according to various factors. In the treatment method of the present invention, in the case of adults, the brown mealworm larval protein of the present invention or its hydrolyzate can be administered at a dose of 0.0001 mg/kg to 1000 mg/kg once or several times a day. The capacity is not particularly limited.

In the treatment method of the present invention, the pharmaceutical composition of the present invention can be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. there is.

In the present invention, the pharmaceutical composition can be used by formulating or using in combination brown mealworm larval protein or a hydrolyzate thereof and a conventionally known muscle disease treatment agent.

Additionally, the present invention provides a food composition containing brown mealworm larval protein or a hydrolyzate thereof as an active ingredient.

In addition to containing brown mealworm larval protein or its hydrolyzate as an active ingredient, the food composition may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same way as the pharmaceutical composition above and 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, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the active ingredient (brown mealworm larval protein or hydrolyzate thereof), various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants, and thickening agents (cheese, chocolate, etc.) ), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The active ingredient (brown mealworm larval protein or hydrolyzate thereof) of the present invention is a material derived from natural products and has almost no side effects like chemicals, so it can be safely used even when taken for a long period of time for the purpose of providing functionality to improve muscle diseases. there is.

That is, the food composition of the present invention can be usefully used as a functional food composition for preventing or improving muscle diseases.

In addition, the present invention provides a health functional food for preventing or improving muscle disease containing brown mealworm larval protein or its hydrolyzate as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving muscle diseases.

In the present invention, “health functional food” refers to food manufactured and processed using raw materials or ingredients with functional properties useful to the human body in accordance with Act No. 6727 on Health Functional Food, and nutrients for the structure and function of the human body. It means ingestion for the purpose of controlling health or obtaining useful health effects such as physiological effects.

The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards.

Items listed in the “Food Additive Code” include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high-quality pigment, and guar gum; Mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations are included.

For example, a health functional food in the form of tablets is prepared by granulating a mixture of the active ingredient of the present invention (brown mealworm larval protein or hydrolyzate thereof) with excipients, binders, disintegrants and other additives in a conventional manner, It can be compression molded by adding a lubricant, etc., or the mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary.

Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention (brown mealworm larval protein or hydrolyzate thereof) with additives such as excipients, and soft capsules can be A mixture of the active ingredient of the present invention (brown mealworm larval protein or hydrolyzate thereof) with additives such as excipients can be prepared by filling a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

The health functional food in the form of a ring can be prepared by molding a mixture of the active ingredient of the present invention (brown mealworm larval protein or its hydrolyzate) and excipients, binders, disintegrants, etc. by a known method, and can be prepared as needed. Depending on the surface, it can be peeled with white sugar or another peeling agent, or the surface can be coated with substances such as starch or talc.

The health functional food in the form of granules can be prepared in granular form by mixing the active ingredient of the present invention (brown mealworm larval protein or hydrolyzate thereof) with excipients, binders, disintegrants, etc., using a known method. Depending on the product, it may contain flavoring agents, flavoring agents, etc.

As confirmed in the following examples, the health functional food containing the brown mealworm larva protein or its hydrolyzate as an active ingredient of the present invention has an excellent effect of suppressing the expression of Myostatin, reducing atony, Muscular atrophy, muscular dystrophy, myasthenia, cachexia, rigid spine syndrome, amyotrophic lateral sclerosis (Lou Gehrig's disease), Charcot-Marie disease -It is effective in preventing or improving muscle diseases such as tooth disease and sarcopenia.

The health functional foods may include beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

Additionally, the present invention provides a cosmetic composition for preventing or improving muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient. The cosmetic composition is not particularly limited, but can be used externally on the skin or taken orally.

The cosmetic composition of the present invention contains brown mealworm larval protein or its hydrolyzate as an active ingredient and is used as a basic cosmetic composition (face wash, pack, body wash such as lotion, cream, essence, cleansing foam, and cleansing water) along with dermatologically acceptable excipients. It can be manufactured in the form of oil), color cosmetic compositions (foundation, lipstick, mascara, makeup base), hair product compositions (shampoo, rinse, hair conditioner, hair gel), and soap.

The excipients are not limited thereto, but may include, for example, skin emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners, and solvents. In addition, it may additionally contain fragrances, pigments, disinfectants, antioxidants, preservatives, and moisturizers, and may include thickeners, inorganic salts, synthetic polymer materials, etc. for the purpose of improving physical properties. For example, when preparing a face wash and soap using the cosmetic composition of the present invention, it can be easily produced by adding the brown mealworm larva protein or its hydrolyzate to a regular face wash and soap base. When producing cream, it can be produced by adding brown mealworm larval protein or its hydrolyzate to a general oil-in-water (O/W) cream base. Here, synthetic or natural materials such as proteins, minerals, vitamins, etc. can be added for the purpose of improving physical properties, such as fragrances, chelating agents, pigments, antioxidants, preservatives, etc.

In addition, the present invention provides a feed additive composition for preventing or improving muscle disease comprising brown mealworm larval protein or a hydrolyzate thereof as an active ingredient.

Specifically, the brown mealworm larval protein of the present invention or its hydrolyzate shows an overall muscle strength increase effect due to an increase in bone muscle strength or an improvement in muscle function, so it can be included in a feed additive as a growth promoter for animals or livestock.

The term "feed additive" used in the present invention refers to a substance added to feed for various effects such as supplementing nutrients and preventing weight loss, improving digestibility of fiber in feed, improving meat quality, preventing reproductive disorders and improving conception rate, and preventing high temperature stress in the summer. It refers to substances. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and includes mineral preparations such as sodium bicarbonate (sodium bicarbonate), bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, vitamin preparations such as kerotene, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complex, protective amino acid preparations such as methionine and lysic acid, protective fatty acid preparations such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast culture Water, live bacteria such as mold fermentation products, yeast, etc. may be additionally included.

The feed additive composition according to the present invention is not particularly limited and can be applied to any individual for the purpose of increasing overall muscle strength and promoting growth by increasing skeletal muscle mass, improving muscle function, or improving bone density. The subjects include animals, such as non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, such as cynomolgous monkeys and Refers to mammals, including chimpanzees. In another embodiment, the subject is a livestock animal (e.g., horse, cow, pig, etc.) or a pet animal (e.g., dog or cat).

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Example 1>

Protein extraction from brown mealworm larvae

In this experiment, protein from brown mealworm larvae was extracted. The extracted brown mealworm larvae protein (Mealworm Protein Isolate) was abbreviated as ‘MPI’.

<1-1> Raw material for brown mealworm larvae

Brown mealworm larvae were purchased in dried form from Edible Bug Co., Seoul, Korea, ground, prepared into powder form using a 1.4 mm sieve, and stored refrigerated at 4°C before use.

<1-2> Degreasing brown mealworm larvae

The efficiency of protein extraction is increased through the degreasing process of brown mealworm larvae. That is, 99.5% ethanol was added to brown mealworm larvae at a ratio of 1:5, and then extracted in a shaking bath (VS-1205SW1, Vision Scientific Co., Ltd, Daejeon, Korea) at 40°C for 60 minutes. After repeating this process twice, the ethanol was volatilized and dried for 12 hours.

<1-3> Brown mealworm larvae protein extraction process

0.25M NaOH was added to the defatted brown mealworm larvae at a ratio of 1:15, and then mixed at 40°C for 60 minutes using a hot plate & magnetic stirrer (Vision Science Co., Korea). Afterwards, centrifugation was performed at 4200 rpm at 4°C for 15 minutes using a centrifuge (VS-24SMTi, Vision Science Co., Ltd, Korea), and the supernatant was collected and the pH was adjusted to 4.5. Afterwards, the precipitate was obtained by centrifugation at 4200 rpm for 15 minutes at 4°C.

<1-4> Sediment dialysis and freeze-drying

The obtained precipitate was placed in a dialysis bag (12KDa MWCO; Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and desalted for 12 hours. Afterwards, it was freeze-dried for 36 hours to obtain the final brown mealworm larval protein (Mealworm Protein Isolate, MPI) of the present invention.

<Example 2>

Brown mealworm larvae protein hydrolyzate

In this experiment, hydrolyzate of brown mealworm larval protein was prepared using proteolytic enzyme. Mealworm Protein Hydrolysate was abbreviated as ‘MPH’.

<2-1> Raw material brown mealworm larvae protein

Brown mealworm larval protein (MPI) prepared through <Example 1> was used as a raw material.

<2-2> Setting conditions for protein hydrolysis of brown mealworm larvae

In order to establish the optimal manufacturing conditions for brown mealworm larval protein hydrolyzate, the conditions of substrate concentration, enzyme concentration, pH, reaction temperature, and reaction time were set as follows with reference to previous research. Substrate concentration (1%), enzyme concentration (alcalase, flavorzyme, alcalase+flavorzyme 1%), pH (8), reaction temperature (55℃), reaction time (12 hours or 24 hours)

<2-3> Brown mealworm larval protein autoenzyme inactivation

Protein powder was dispersed in a pH 8 buffer according to the set substrate concentration conditions, and the autologous enzyme was inactivated in a water bath at 85°C for 20 minutes.

<2-4> Creation of pH environment for protein hydrolysis in brown mealworm larvae

The protein solution with the autologous enzyme inactivated was transferred to another water bath set to a reaction temperature appropriate for each condition, cooled, and adjusted to the set pH using 1N NaOH.

<2-5> Obtaining hydrolyzate of protein from brown mealworm larvae

The enzyme (Alcalase (Novozymes A/S.Co., Ltd., Bagsvaerd, Denmark), flavorzyme) concentration for each substrate is treated to 1% (w/v) and pH is maintained at 8. Hydrolysis was performed. Enzyme treatment methods and reaction times are shown in detail in Table 1 below. Afterwards, it was centrifuged and only the supernatant was taken to obtain a hydrolyzate, which was then freeze-dried for 36 hours to obtain the final hydrolyzate of the brown mealworm larval protein of the present invention.

- MPHA: 1% (w/v) hydrolyzate from alcalase treatment for 24 hours

- MPHF: Hydrolyzate from 1% (w/v) flavorzyme treatment for 24 hours

- MPHME: Hydrolyzate from complex enzyme (0.5% (w/v) alcalase & 0.5% (w/v) flavorzyme) treatment for 24 hours

- MPHAF: Hydrolyzate treated with 0.5% (w/v) alcalase for 12 hours and then 0.5% (w/v) flavorzyme for 12 hours.

- MPHFA: Hydrolyzate from 0.5% (w/v) flavorzyme treatment for 12 hours followed by 0.5% (w/v) alcalase treatment for 12 hours

Enzyme treatment method and reaction time of protein hydrolyzate from brown mealworm larvae Sample name Enzyme treatment conditions (% of substrate (w/v), enzyme type, reaction time) MPHA (1%(w/v), alcalase, 24h) MPHF (1%(w/v), flavorzyme, 24h) MPHME (0.5%(w/v) alcalase + 0.5%(w/v) flavourzyme, 24h) MPHAF (0.5%(w/v), alcalase, 12h) → (0.5%(w/v), flavourzyme, 12h) MPHFA (0.5%(w/v), flavourzyme, 12h) → (0.5%(w/v), alcalase, 12h)

<2-6> Obtaining hydrolyzate according to size of MPHAF

The hydrolyzate obtained under MPHAF conditions was centrifuged at Rcf 4200 for 25 minutes using Pierce™ Protein Concentrator PES, 10K MWCO (Thermo Scientific™, MA, USA). The unseparated layer yielded ‘AF-MT’, a hydrolyzate with a molecular weight greater than 10 kDa. The separated layer was centrifuged again using Pierce ™ Protein Concentrator PES, 3K MWCO (Thermo Scientific ™, MA, USA) to separate 'AF-TT' hydrolyzate with molecular weight greater than 3 kDa and less than 10 kDa and 'AF' hydrolyzate with molecular weight less than 3 kDa. -LT' was obtained.

- AF-LT: Hydrolyzate with a molecular weight of 3 kDa or less in MPHAF

- AF-TT: Hydrolyzate with a molecular weight of 3 to 10 kDa in MPHAF

- AF-MT: Hydrolyzate with a molecular weight of 10 kDa or more in MPHAF

<Experimental Example 1>

Effect on myostatin expression

<1-1> Cytotoxicity evaluation (MTT Assay)

Before measuring the expression level of myostatin, an MTT experiment was performed to measure the toxicity of the sample.

After culturing C2C12 mouse normal cells, a myoblast cell line, in DEME medium for 24 hours, the medium was removed from the medium in which the cells were grown, washed with PBS, and trypsin was injected. When cells are separated from the Petri dish, 10% FBS reagent is added, centrifuged to settle the cells, and the supernatant is discarded. Afterwards, 10 ul of tryphan blue reagent was injected into the cell counting slide and the number of cells was counted using an Automated Cell Counting device. After confirming the total number of cells, the cells and medium sufficient for culture were mixed, injected into a well plate, and cultured in an incubator at 37°C for more than 24 hours. The medium of C2C12 cells was removed and replaced with 1 mL of new medium. . Samples (WPI, MPI, MPHAF, AF-LT, AF-TT, AF-MT) were diluted step by step using distilled water to concentrations of 1 mg/mL, 0.1 mg/mL, and 0.01 mg/mL, then added to the medium. It was placed in an incubator at ℃. After 24 hours, C2C12 cells cultured at 37°C were taken out, the cell medium was removed, and 10 ul of MTT reagent was injected per well. After reacting for 2 hours in an incubator at 37°C, the medium and MTT reagent were removed from the well, 50 ul of DMSO was injected, and the number of living cells was counted using a Microplate Spectrophotometer (Epoch, BioTek).

As a result, as shown in Figure 2 and Table 2, all samples (MPI, MPHAF, AF-LT, AF-TT, AF-MT) except for 10 mg/ml WPI showed higher cell survival rates than the control group at each concentration. indicated.

Cytotoxicity evaluation of protein hydrolyzate from brown mealworm larvae Sample Treatment concentration average Standard Deviation WPI con 0.898 0.035 1mg/ml 0.787 0.282 0.1mg/ml 1.026 0.114 0.01mg/ml 0.95 0.089 MPI con 0.898 0.035 1mg/ml 1.142 0.048 0.1mg/ml 1.167 0.027 0.01mg/ml 1.168 0.021 MPHAF con 0.898 0.035 1mg/ml 1.239 0.134 0.1mg/ml 1.068 0.122 0.01mg/ml 1.218 0.143 AF-LT con 0.898 0.035 1mg/ml 1.32 0.115 0.1mg/ml 1.331 0.116 0.01mg/ml 1.123 0.088 AF-TT con 0.898 0.035 1mg/ml 1.172 0.087 0.1mg/ml 1.14 0.103 0.01mg/ml 1.031 0.29 AF-MT con 0.898 0.035 1mg/ml 1.263 0.026 0.1mg/ml 0.994 0.267 0.01mg/ml 1.166 0.099

<1-2> Measurement of relative mRNA expression level of myostatin

The level of myostatin expression was measured to confirm the effectiveness of preventing and improving sarcopenia according to the hydrolysis conditions of brown mealworm larval protein and the size of the hydrolyzate. As a positive control, WPI whey protein isolate powder purchased from PUREUNBIN Co., Yeongcheon-si, Gyeongsangbuk-do, Korea was used.

After confirming the concentration of the sample without cytotoxicity through Experimental Example <1-1>, C2C12 cells were cultured in an incubator at 37°C for more than 24 hours. The medium of C2C12 cells cultured for more than 24 hours was removed, and 1 mL of medium was injected. Afterwards, 1 ul of sample (MPI, MPH) with a concentration of 0.01 mg/mL dissolved in distilled water was injected into the well (PBS was injected into the control group), mixed, and placed in an incubator at 37°C to react for 24 hours. Afterwards, the medium was removed and washed twice in total with PBS. After washing, 200 ul of TRIzol was injected into the well, left at room temperature for 5 minutes, and then 40 ul of chloroform was added and mixed. After centrifugation at 13,200 rpm and 4°C for 15 minutes, 120 ul of isopropanol was mixed with the supernatant and left at room temperature for 10 minutes. After 10 minutes, the supernatant was removed by centrifugation at 13,200 rpm and 4°C for 15 minutes, and then 100 ul of cold 70% ethanol was injected, followed by centrifugation at 13,200 rpm and 4°C for 2 minutes. At this time, the 70% cold ethanol injection process was repeated twice. After removing 70% ethanol and injecting DEPC (Diethyl pyrocarbonate), the concentration of RNA was measured using a Microplate Spectrophotometer (Epoch, BioTek). After measuring the RNA concentration of the sample, the sample and DEPC-water were additionally injected accordingly, and 10ul of TOPreal™ qPCR 2X PreMIX (SYBR Green with high ROX) was injected. The mixed samples were transferred to Zipperstrip Strip PCR Tubes and DNA was amplified using a Thermal Cycler (Bio-Rad) device. After completion of amplification, it was transferred to a microcentrifuge tube and 2 ul of sample, 5 ul of 2X mastermix, 2.5 ul of pure water, and 0.5 ul of primer were injected into the PCR plate. Afterwards, mGAPDH and mMSTN were each injected into one sample. Finally, it was sealed and the amplified DNA was detected using a Real-Time PCR (Applied Biosystems, Quantstudio3) device.

As a result, as shown in Figure 3 and Table 3, compared to MPI (brown mealworm larval protein), MPH (hydrolyzate of brown mealworm larval protein) according to hydrolysis conditions showed lower myostatin expression and was effective in improving muscle loss. It was confirmed that it exists. Among them, MPHAF showed the lowest myostatin expression level.

Afterwards, as a result of confirming the myostatin expression level of the fractions of MPHAF, which showed the lowest myostatin expression level, by molecular size, as shown in Figure 4 and Table 4, in the hydrolyzate (AF-MT) of 10 kDa or more, It showed the lowest level of myostatin expression, which was lower than that of WPI, the positive control group, confirming that it had the best effect on improving muscle loss.

Relative myostatin expression level (MSTN/GAPDH) according to myoblast (C2C12) treatment of brown mealworm larval protein hydrolyzate according to hydrolysis conditions. Sample average Standard Deviation CON One 0.223 WPI 0.299 0.04 MPI 0.661 0.036 MPHAF 0.369 0.093 MPHFA 0.54 0.041 MPHME 0.397 0.082 MPHA 0.434 0.091 MPHF 0.597 0.04

Relative myostatin expression level (MSTN/GAPDH) according to treatment of myogenic cells (C2C12) of hydrolyzate fractionated by MPHAF molecular size. Sample average Standard Deviation CON One 0.16 WPI 0.299 0.189 MPI 0.638 0.094 MPHAF 0.389 0.238 AF-LT 0.594 0.429 AF-TT 0.452 0.219 AF-MT 0.118 0.096

<1-3> Measurement of relative myostatin promoter activity

A luciferdase and beta-galactosidase dual luminescence-based genetic assay was used to measure the myostatin promoter activity of MPI and MPH. Luciferdase activity analysis was first performed, and then beta-galactosidase quantitative analysis was performed to increase the accuracy of gene expression analysis.

In detail, the MSTN (Myostatin) promoter (-534 to +132, 667 bp) was inserted into the pGL4.15 vector using XhoI and BglII. 24 hours after injecting 4×10 ⁵ cells into 12 wells, the cells were washed with PBS and 900 μl of Opti-MEM media was added. Afterwards, pGL4.15-MSTN promoter (1 μg, firfly luc) and pRL-TK (200ng, renilla luc) were simultaneously mixed with Lipofectamine 2000 (2 μl, Invitrogen) in 100 μl of Opti-MEM media, and 100 μl was added to the cells. Add them one by one for co-transfection. At this time, pGL4.15 empty vector was used as a negative control. After 4 hours, the medium was changed, and after 12 hours, luciferase activity (luminescence) was measured using the Dual-Luciferase® Reporter Assay System (Promega) and a luminometer (EnSpire Mμltimode Plate Reader, PerkinElmer). pRL-TK was used as a loading control.

As a result, as shown in Figure 5, MPI showed a higher myostatin promoter activity than the control group, and MPH showed a lower myostatin promoter activity than the control group, indicating that MPH was an inhibitor of myostatin, a substance that interferes with muscle synthesis. By lowering activity, it was judged that the effect on sarcopenia would be greater than MPI.

For reference, MPH shown in Figure 5 used a hydrolyzate of brown mealworm larval protein prepared by treating the substrate with 1% alkalase for 12 hours (conditions except treatment time are the same as MPHA).

<Experimental Example 2>

Measurement of inhibition of inflammatory cell expression (anti-inflammatory effect)

To determine the inhibition rate of inflammatory cell expression in MPH samples according to MPI and hydrolysis conditions and size, LPS was used in macrophages to observe the expression levels of IL-6, TNF-a, and IL-1b, which are pro-inflammatory cytokines in the blood. WPI (Whey protein isolate) was used as a positive control. A mixture of LPS (Lipopolysaccharide) and distilled water was used as a negative control. That is, LPS was used to induce inflammation, and samples were processed at different concentrations to observe the expression levels of IL-6, IL-1b, and TNF-a, which are inflammation-inducing cytokines in the blood. For reference, pro-inflammatory cytokines in the blood promote the breakdown of myofibrillar proteins, thereby reducing protein synthesis and, as a result, directly causing muscle wasting.

Prior to measuring the expression level of inflammatory cytokines, Bradford assay was performed to measure the protein concentration of each sample. Briefly, 100 μl of 10-fold serially diluted sample was mixed with 5 ml of analysis reagent, and the absorbance was measured at 595 nm. For the calibration curve, absorbance was measured by diluting 1 mg/mL γ-globulin standard solution to a concentration of 10-100 μl/100 μl and injecting the analysis reagent in the same manner as the sample.

As a result, as shown in Figures 6 and 7, when macrophages were treated with LPS, an inflammatory factor, the expression levels of inflammatory cytokines IL-6, TNF-a, and IL-1b were found to increase, while It was confirmed that inflammatory cytokines were suppressed in a concentration-dependent manner in all experimental groups in which each sample was treated with LPS. These results showed that all samples reduced the expression of inflammatory cytokines. In detail, all experimental groups showed superior IL-1b inhibitory activity compared to the positive control group (WPI), and in particular, the experimental group treated with MPHME showed the best TNF-a inhibitory effect compared to the positive control group (WPI). Meanwhile, all experimental groups except those treated with MPHA or MPHAF showed superior IL-6 inhibition effect compared to the positive control group (WPI).

As a result of summarizing the above, it was confirmed that the experimental group treated with MPHME as a hydrolyzate showed excellent TNFa, IL-1b, and IL-6 inhibitory activities, and had the best anti-inflammatory effect.

<Experimental Example 3>

Degree of hydrolysis, analysis of constituent amino acids and measurement of molecular weight of protein hydrolyzate from brown mealworm larvae

<3-1> TNBS assay

In this experiment, in order to measure the degree of hydrolysis of hydrolyzates prepared under different hydrolysis conditions (see Example <2-5> above), TNBS assay was used to measure the degree of hydrolysis of each protein hydrolyzate as an indicator. The concentration of available amino groups was measured. For reference, when a protein is hydrolyzed, the available amino groups increase in proportion to the degree of hydrolysis, so this can be an indicator of the peptide content.

In detail, the degree of hydrolysis of MPH prepared under each condition was measured using TNBS (2,4,6-Trinitrobenzene Sμlfonic Acid) solution. A standard solution was prepared at a concentration of 2-20 μg/ml using 0.1M sodium bicarbonate (pH 8.5) solution (Reaction buffer, RB) and L-Leucine. Afterwards, TNBS solution (working reagent, WR) was prepared at a concentration of 0.01% (v/v) using RB. 250 μl of WR was added to 500 μl of each sample and standard solution and reacted at 37°C for 2 hours. Afterwards, 250 μl of 10% SDS and 125 μl of 1N HCl were added to stop the reaction, and the absorbance was measured at 335 nm.

As a result, as shown in Figure 8 and Table 5, MPHF (1%, flavourzyme, 24h) showed the lowest available amino group concentration (31.283 mg/g) among the hydrolysates according to hydrolysis conditions. On the other hand, MPHAF ((0.5%, alcalase, 12h) → (0.5%, flavourzyme, 12h)) showed the highest available amino group concentration (50.323 mg/g), indicating that enzyme treatment under MPHAF conditions was used when hydrolyzing mealworms. It was confirmed that the degree of hydrolysis was the highest.

Available amino group concentration (mg/g) of protein hydrolyzate from brown mealworm larvae according to hydrolysis conditions Sample Available amino group concentration (mg/g) Standard Deviation WPI 15.008 0.139 MPI 12.139 0.276 MPHAF 50.323 0.643 MPHFA 44.412 2.771 MPHME 47.912 0.96 MPHA 46.556 0.547 MPHF 31.283 2.355

<3-2> BCA assay

In order to measure the degree of hydrolysis of hydrolyzates prepared under different hydrolysis conditions (see Example <2-5> above), the amount of change in protein structure was measured using the BCA method.

In detail, the BCA reagent was mixed at A:B = 50:1 to prepare a working reagent (WR) and a BSA standard solution at each concentration (0-2000 μg/ml). 2 ml of WR was dispensed into 0.1 ml of the standard solution and 0.1 ml of the sample and reacted at 37°C for 30 minutes. After the reaction was cooled for 10 minutes, the absorbance was measured at 562 nm.

As a result, as shown in Figure 9 and Table 6, MPI (56.882 mg/g) had a higher protein content than MPH (MPHF: 19.84 mg/g, MPHFA: 14.02 mg/g, MPHME: 12.43 mg/g, MPHA: 11.88 mg/g, MPHAF: 10.52 mg/g), MPHAF showed the lowest protein concentration among MPHs, indicating that the hydrolysis conditions for MPHAF resulted in the most hydrolysis.

Afterwards, as a result of checking the protein content of the fractions of MPHAF with the highest degree of hydrolysis according to molecular size, as shown in Figure 10 and Table 7, WPI (182.19 mg/g) had the highest protein content, and MPI (145.69 mg/g) g), MPHAF (123.66 mg/g), and fractions (AF-MT: 113.97 mg/g, AF-TT: 90.19 mg/g, AF-LT: 20.45 mg/g).

Protein concentration of brown mealworm larval protein hydrolyzate according to hydrolysis conditions (mg/g) sample BCA concentration (mg/g) MPI 56.882 MPHAF 10.518 MPHFA 14.018 MPHME 12.427 MPHA 11.882 MPHF 19.836

Protein concentration of hydrolyzate fractionated by molecular size of MPHAF (mg/g) Sample average Standard Deviation WPI 182.19 2.107 MPI 145.69 1.157 MPHAF 123.66 2.258 AF-LT 20.45 1.637 AF-TT 90.19 2.332 AF-MT 113.97 3.388

<3-3> Constituent amino acid analysis

In this experiment, in order to confirm the effect of the constituent amino acids on sarcopenia, MPHAF, which has the highest hydrolysis degree in hydrolysis by enzyme treatment, was used to measure the amino acid composition and content of fractions fractionated by molecular size using HPLC amino acid analysis experiments. did.

First, 0.05 g of the sample was placed in a decomposition tube, 2 ml of 6N HCl was added, and acid hydrolysis was performed in a vacuum at 105°C for 24 hours. Afterwards, the filtrate was diluted 100 times in distilled water and filtered through a 0.45μm nylon syringe filter and used as an analysis sample. The conditions of the amino acid analysis device are shown in Table 8 below. After unit correction for the experimental results, the content of 17 amino acids was measured, and the results are shown in detail in Table 9.

Conditions for amino acid analysis equipment Measurement Condition Instrument YL9100 HPLC System, Young in Chromass Co. Anyang, Korea Column HPLC Column, 3.9 mm×150mm Column temp. 37℃ Mobile phase A: 10% Waters accq-tag Eluent A concentrate B: 60% ACN Wave length Ex: 250mm, Em: 395mm

Amino acids composed of hydrolyzate fractionated by molecular size of MPHAF WPI MPI AF-LT AF-TT AF-MT amino acid average standard
Deviation average standard
Deviation average standard
Deviation average standard
Deviation average standard
Deviation histidine 1.693 0.682 2.385 0.605 0.754 0.1 1.003 0.179 1.234 0.048 isoleucine 3.908 0.364 4.086 0.112 1.343 0.033 1.852 0.052 1.871 0.062 leucine 9.125 0.31 8.35 0.178 3 0.08 3.841 0.053 3.832 0.076 Lysine 8.04 1.093 5.505 0.343 2.471 0.045 3.004 0.175 2.952 0.059 methionine 2.57 0.087 0.787 0.045 1.253 0.024 0.804 0.016 0.712 0.07 Phenylalanine 2.455 0.065 4.136 0.177 1.354 0.026 1.886 0.061 1.937 0.08 tryptophan N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. threonine 6.528 0.546 3.653 0.185 1.02 0.067 1.483 0.05 1.555 0.105 Valine 3.843 0.087 3.645 0.113 1.047 0.02 2.292 0.041 2.285 0.048 Total essential amino acids 38.161 1.812 32.547 0.773 12.243 0.234 16.166 0.142 16.378 0.345 alanine 4.504 0.059 7.194 0.278 3.012 0.153 3.48 0.153 3.362 0.102 arginine 1.26 0.096 4.63 0.608 2.226 0.135 2.288 0.074 2.334 0.082 aspartic acid 10.452 0.805 10.786 0.087 1.523 0.169 4.649 0.233 4.847 0.387 cysteine 0.177 0.001 0.607 0.032 0.255 0.007 0.311 0.004 0.299 0.009 glutamic acid 16.425 1.371 12.459 0.839 2.743 0.052 5.827 0.107 5.889 0.25 glycine 1.673 0.353 7.559 0.544 2.715 0.025 3.348 0.018 3.324 0.113 Proline 5.886 0.153 5.949 0.242 1.592 0.046 2.478 0.076 2.456 0.064 serine 5.483 0.369 6.059 0.246 1.644 0.022 2.648 0.034 2.587 0.102 tyrosine 2.493 0.009 8.536 0.445 3.59 0.105 4.375 0.062 4.208 0.121 total amino acids 86.514 3.864 96.325 1.81 31.543 3.88 45.57 2.014 45.684 2.252 hydrophobic amino acids 33.963 1.275 41.706 2.263 15.316 0.539 19.981 0.552 19.779 0.586 hydrophilic amino acids 41.558 3.084 42.1 1.019 10.775 0.349 19.294 0.275 19.385 0.843 BCAA 16.875 0.758 16.081 0.245 5.391 0.133 7.986 0.133 7.989 0.192

<3-4> SDS-PAGE

The amount of low-molecular-weight peptides produced by decomposition of hydrolysates prepared under different hydrolysis conditions (see Example <2-5>) was measured using SDS-PAGE.

In detail, in order to prepare the protein content of the sample to 50 μg/10 μl, 0.057 g of each sample was mixed in 5 ml of distilled water, referring to the BCA experiment results. After mixing 10 μl of the sample with 10 μl of 2x laemmli buffer, 5 μl of mercaptoethanol was injected and heated at 95°C for 5 minutes. Afterwards, the pretreated sample was injected into a 15% SDS gel and electrophoresed at 80V-120V. The gel after electrophoresis was stained using Coomassie brilliant blue solution, and destained using Coomassie brilliant blue R-250 destaining solution containing acetic acid and methanol. Afterwards, the polyacrylamide gel was scanned and imaged.

As a result, as shown in Figure 11, when looking at the patterns, it was confirmed that high molecular weight peptides were present in MPHF among the hydrolysates, while the largest amount of low molecular weight peptides of 6 kDa or less were produced in MPHAF.

<Experimental Example 4>

Antioxidant activity measurement

In this experiment, the ABTS radical scavenging activity test was used to determine the effect of hydrolyzate produced by enzyme treatment on oxidative stress, which can affect sarcopenia.

In detail, to form radicals in the reagent, 7.4mM ABTS and potassium persulfate were mixed in a 1:1 ratio and stored in a cool, dark place for 24 hours. Afterwards, the ABTS reagent was diluted with distilled water so that an absorbance of 0.7 ± 0.02 was measured at a wavelength of 734 nm using a spectrophotometer. Each sample was diluted in distilled water to a concentration of 500ug/ml, mixed with ABTS reagent at a ratio of 1:1, stored in a cool and dark place for 10 minutes, and then measured for absorbance at 760nm. At this time, the blank sample was a sample extraction solution and distilled water, and the control sample was a mixture of ABTS solution and distilled water in a ratio of 1:1. The ABTS radical scavenging rate (%) was calculated using the absorbance measurement results.

As a result, as shown in Figure 12 and Table 10, compared to MPI, MPH showed a higher ABTS radical scavenging ability under each hydrolysis condition, confirming that it has a high antioxidant ability. Among the hydrolysates, MPHAF (93.55%) was found to have the highest antioxidant capacity.

Afterwards, as a result of confirming the ABTS radical scavenging ability of the fractions of MPHAF by molecular size, it was confirmed that the fraction of 10 kDa or more (AF-MT 97.24%) had the highest antioxidant ability, as shown in Figure 13 and Table 11.

ABTS radical scavenging rate (%) of protein hydrolyzate from brown mealworm larvae according to hydrolysis conditions Sample average Standard Deviation WPI 49.508 0.835 MPI 30.4 0.783 MPHAF 93.546 1.132 MPHFA 83.73 1.513 MPHME 84.983 0.673 MPHA 80.162 0.095 MPHF 34.443 0.72

ABTS radical scavenging rate (%) of hydrolyzate fractionated by MPHAF molecular size Sample average Standard Deviation WPI 49.508 0.835 MPI 30.4 0.783 MPHAF 93.546 1.132 AF-LT 51.887 0.287 AF-TT 65.779 1.282 AF-MT 97.241 0.989

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

WPI: Whey protein isolate
MPI: Mealworm Protein Isolate
MPHA: hydrolyzate from 1% alcalase treatment for 24 hours.
MPHF: Hydrolyzate from 1% flavorzyme treatment for 24 hours
MPHME: Hydrolyzate from complex enzyme (0.5% (w/v) alcalase & 0.5% (w/v) flavorzyme) treatment for 24 hours.
MPHAF: Hydrolyzate after treatment with 0.5% (w/v) Alcalase for 12 hours and then with 0.5% (w/v) Flavorzyme for 12 hours.
MPHFA: Hydrolyzate from 0.5% (w/v) flavorzyme treatment for 12 hours followed by 0.5% (w/v) alcalase treatment for 12 hours.
AF-LT: Hydrolyzate with a molecular weight of 3 KDa or less in MPHAF
AF-TT: Hydrolyzate with a molecular weight of 3 to 10 KDa from MPHAF
AF-MT: Hydrolyzate with a molecular weight of 10 KDa or more in MPHAF

Claims (10)

A food composition for preventing or improving muscle disease,
The food composition contains hydrolyzate of brown mealworm larval protein as an active ingredient,
The hydrolyzate was obtained by treating brown mealworm larval protein with 0.5% (w/v) alcalase for 12 hours and then treating it with 0.5% (w/v) flavorzyme for 12 hours. The hydrolyzate had a molecular weight of 10 KDa or more. A food composition for preventing or improving muscle disease, characterized in that it is a hydrolyzate having a large size. According to paragraph 1,
The brown mealworm larvae protein is prepared by: a) pulverizing dried brown mealworm larvae; b) adding ethanol to the ground product to degrease it; c) adding sodium hydroxide to the defatted brown mealworm larvae and mixing them, followed by centrifugation to obtain a precipitate; and d) a food composition characterized in that it is manufactured through a process comprising the step of desalting the obtained precipitate and then freeze-drying it. delete According to paragraph 1,
A food composition characterized in that the hydrolyzate of the brown mealworm larval protein inhibits the expression of Myostatin. According to any one of paragraphs 1, 2, and 4,
A food composition characterized in that the muscle disease is a muscle disease caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. According to clause 5,
The muscle diseases include atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spine syndrome, and amyotrophic lateral sclerosis (Lou Gehrig's disease). , Charcot-Marie-Tooth disease, and sarcopenia. A food composition characterized in that it is selected from the group consisting of. As a health functional food for preventing or improving muscle disease,
The health functional food contains hydrolyzate of brown mealworm larval protein as an active ingredient,
The hydrolyzate was obtained by treating brown mealworm larval protein with 0.5% (w/v) alcalase for 12 hours and then treating it with 0.5% (w/v) flavorzyme for 12 hours. The hydrolyzate had a molecular weight of 10 KDa or more. A health functional food for preventing or improving muscle disease, characterized in that it is a hydrolyzate with a large size. In clause 7,
The health functional food is a health functional food selected from the group consisting of beverages, meat, confectionery, noodles, rice cakes, bread, gum, candy, ice cream, and alcoholic beverages. A pharmaceutical composition for preventing or treating muscle disease,
The pharmaceutical composition contains hydrolyzate of brown mealworm larval protein as an active ingredient,
The hydrolyzate was obtained by treating brown mealworm larval protein with 0.5% (w/v) alcalase for 12 hours and then treating it with 0.5% (w/v) flavorzyme for 12 hours. The hydrolyzate had a molecular weight of 10 KDa or more. A pharmaceutical composition for preventing or treating muscle disease, characterized in that it is a hydrolyzate having a large size. A feed additive composition for preventing or improving muscle disease,
The feed additive composition contains hydrolyzate of brown mealworm larval protein as an active ingredient,
The hydrolyzate was obtained by treating brown mealworm larval protein with 0.5% (w/v) alcalase for 12 hours and then treating it with 0.5% (w/v) flavorzyme for 12 hours. The hydrolyzate had a molecular weight of 10 KDa or more. A feed additive composition for preventing or improving muscle disease, characterized in that it is a hydrolyzate having a large size.