Low-molecular-weight protein hydrolysate for plant-based meat substitute using defatted hemp seed meal having effects of preventing, improving and treating muscle atrophy or sarcopenia and its manufacturing method

Abstract

The present invention relates to a low molecular weight protein hydrolysate for plant-based meat substitute using defatted hemp seed meal, which has the effects of preventing, improving, and treating muscle atrophy or sarcopenia, and a method for producing the same.

Description

Technology Field

[0001] delete

[0002] The present invention relates to a low molecular weight protein hydrolysate for plant-based meat substitute using defatted hemp seed meal, which has the effects of preventing, improving, and treating muscle atrophy or sarcopenia, and a method for producing the same. Background Technology

[0003] Muscle atrophy is a condition resulting from the weakening of muscles and refers to the loss of muscle tissue. The most common cause is a lack of exercise, but it is known that reduced physical activity due to injury or disease, nutritional deficiencies, and genetic factors can also cause muscle atrophy. Additionally, a decrease in protein synthesis in the body due to aging can lead to muscle atrophy, and nutritional deficiencies caused by dieting are also a cause.

[0004] Furthermore, muscle loss and atrophy are closely correlated. Looking at the current state of the treatment industry for age-related sarcopenia and muscle atrophy, the decline in physical function and muscle strength among the elderly—caused by spinal diseases, cancer, aging, or internal and surgical issues—is a major problem in the aging society. Preventing the decline in muscle function due to aging—that is, stopping muscle atrophy and maintaining contractile function and muscle elasticity—is a crucial factor for healthy physical activity and independent daily living in old age.

[0005] Therefore, in accordance with the global trend of an aging society, efforts are needed to maintain the musculoskeletal function of the elderly to increase their quality of life and healthy life expectancy, and research and the development of related products are required to improve muscle strength and mitigate muscle atrophy.

[0006] As one of the proactive measures to address various muscle loss issues, there is a demand for more fundamental muscle health management methods through the development of muscle health improvement technologies. Furthermore, the development of functional materials to prevent muscle function decline and recover from muscle atrophy can serve as a countermeasure for preventing and improving diseases related to muscle strength loss caused by an aging society.

[0007] Meanwhile, regarding technologies developed for the improvement of muscle atrophy, the development of products utilizing natural extracts such as Schisandra chinensis extract, red sea cucumber enzyme hydrolysate, Angelica gigas extract, Cnidium officinale extract, Paeonia lactiflora extract, and Eucommia ulmoides extract is underway, but the effectiveness is currently insufficient.

[0008] Therefore, there is a need to develop new materials that improve muscle strength or muscle atrophy, which are highly effective in improving muscle atrophy without causing other side effects. Prior art literature

[0010] Korean Patent Registration No. 10-2403795 (May 25, 2022) Korean Patent Publication No. 10-2022-0134101 (October 5, 2022) The problem to be solved

[0011] The objective of the present invention is to provide a method for producing a low molecular weight protein hydrolysate for plant-based meat substitute using defatted hemp seed meal, which exhibits excellent improvement effects on muscle atrophy and sarcopenia, and plant-based meat substitute containing the low molecular weight protein hydrolysate. means of solving the problem

[0013] The objective of the present invention is achieved by providing a method for producing a low-molecular-weight protein hydrolysate for plant-based meat substitutes using defatted hemp seed meal, characterized by comprising: an acidity adjustment step of mixing distilled water with defatted hemp seed meal and adjusting the pH to 10; a supernatant separation step of separating the supernatant after centrifuging the mixture with the acidity adjusted through the acidity adjustment step; a protein extraction step of extracting protein by adjusting the pH of the supernatant separated through the supernatant separation step to 5; an ultrasonic heat treatment step of mixing distilled water with the protein extracted through the protein extraction step and heat-treating it after irradiating it with ultrasound; and a hydrolysis step of mixing a hydrolytic enzyme with the protein extract that has undergone ultrasonic irradiation and heat treatment through the ultrasonic heat treatment step and reacting it to reduce the molecular weight.

[0014] In addition, the objective of the present invention can also be achieved by providing a method for producing a low-molecular-weight protein hydrolysate for plant-based meat substitutes using hemp seed defatted meal, characterized by comprising: an acidity adjustment step of mixing distilled water and sodium hydroxide with hemp seed defatted meal to adjust the pH to 10; a supernatant separation step of separating the supernatant after centrifuging the mixture whose acidity has been adjusted through the acidity adjustment step; a protein extraction step of extracting protein by adjusting the pH of the supernatant separated through the supernatant separation step to 5; an ultrasonic heat treatment step of mixing distilled water with the protein extracted through the protein extraction step and heat-treating it after irradiating it with ultrasound; and a hydrolysis step of mixing a hydrolytic enzyme with the protein extract that has undergone ultrasonic irradiation and heat treatment through the ultrasonic heat treatment step and reacting it to reduce the molecular weight.

[0015] In addition, the objective of the present invention can also be achieved by providing a method for producing a low-molecular-weight protein hydrolysate for plant-based meat substitutes using hemp seed defatted meal, characterized by comprising: an acidity adjustment step of mixing distilled water and sodium hydroxide with hemp seed defatted meal to adjust the pH to 10; a supernatant separation step of separating the supernatant after centrifuging the mixture with the acidity adjusted through the acidity adjustment step; a protein extraction step of mixing hydrochloric acid with the supernatant separated through the supernatant separation step to adjust the pH to 5 and extracting protein; an ultrasonic heat treatment step of mixing distilled water with the protein extracted through the protein extraction step and heat-treating it after irradiating it with ultrasound; and a hydrolysis step of mixing a hydrolytic enzyme with the protein extract that has undergone ultrasonic irradiation and heat treatment through the ultrasonic heat treatment step and reacting it to reduce the molecular weight.

[0016] According to a preferred feature of the present invention, the ultrasound is irradiated for 10 to 20 minutes with an intensity of 250 to 350 W.

[0017] According to a more preferred feature of the present invention, the heat treatment is carried out at a temperature of 90 to 110°C for 10 to 20 minutes.

[0018] According to a more preferred feature of the present invention, the hydrolysis step is performed by sequentially treating the protein extract with hydrolytic enzymes of different components.

[0019] According to a more preferred feature of the present invention, the hydrolytic enzyme is composed of an alkalase or a flavozyme.

[0020] According to a more preferred feature of the present invention, the hydrolysis step comprises mixing 3 to 5 parts by weight of alkalase with 100 parts by weight of the protein extract and treating at a temperature of 45 to 55°C for 55 to 65 minutes, and then further mixing 3 to 5 parts by weight of flavozyme and treating at a temperature of 45 to 55°C for 55 to 65 minutes.

[0022] In addition, the objective of the present invention can also be achieved by providing a plant-based meat substitute containing a low molecular weight protein hydrolysate produced by the above-described manufacturing method as an active ingredient.

[0023] According to a preferred feature of the present invention, the plant-based meat substitute comprises a low molecular weight protein hydrolysate, gluten, and a flavor enhancer. Effects of the invention

[0025] The method for preparing a low molecular weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal according to the present invention and the plant-based meat substitute containing the low molecular weight protein hydrolysate exhibit excellent improvement effects on muscle atrophy and sarcopenia, and provide a low molecular weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal and plant-based meat substitute containing the low molecular weight protein hydrolysate.

[0026] In addition, a method for producing an optimal low-molecular-weight protein hydrolysate from defatted hemp seed meal that has preventive, improving, and therapeutic effects on muscle atrophy or sarcopenia has been established. When the low-molecular-weight protein hydrolysate from defatted hemp seed meal produced by the method of the present invention was administered to mice with induced muscle atrophy, it was confirmed that muscle strength and motor skills were improved, weight loss due to muscle weakness was improved, and grip strength enhancement and muscle strengthening effects were excellent. Therefore, the low-molecular-weight protein hydrolysate from defatted hemp seed meal produced by the method of the present invention has the effect of being usefully utilized in the manufacture of functional foods and pharmaceuticals for muscle strengthening along with the improvement of muscle atrophy or sarcopenia. Brief explanation of the drawing

[0028] FIG. 1 is a flowchart illustrating a method for preparing a low-molecular-weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal according to the present invention. FIG. 2 shows the results of HLPC size exclusion chromatography, which analyzes the degree of hydrolysis according to treatment conditions for a protein extract of defatted hemp seed meal in one embodiment of the present invention (HPI: hemp seed protein extract, CON: group of hemp seed protein extracts treated identically without enzyme addition, AF: enzyme-treated group of hemp seed protein extract (alcalase and flavozyme), HEAT: enzyme-treated group of hemp seed protein extract after heat treatment, SONIC: enzyme-treated group of hemp seed protein extract after ultrasonic treatment, HEAT+SONIC: enzyme-treated group of hemp seed protein extract after heat treatment and ultrasonic treatment). Figure 3 is a graph showing the content of low molecular weight (3 kDa or less) peptides and high molecular weight (more than 3 kDa) peptides contained in a protein hydrolysate obtained according to treatment conditions for a protein extract of hemp seed defatted meal in one embodiment of the present invention, expressed as the area % of an HPLC analysis graph. Figure 4 shows the results of analyzing the content of free amino acids contained in the protein hydrolysate obtained under different treatment conditions for the protein extract of hemp seed defatted meal in one embodiment of the present invention using TNBS analysis. Figure 5 shows SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining images of protein hydrolysates obtained under different treatment conditions for a protein extract of hemp seed defatted meal in one embodiment of the present invention. Figure 6 is a schematic diagram of the process for a mouse animal experiment to confirm the effect of the low molecular weight protein hydrolysate of hemp seed defatted meal according to the present invention on improving muscle atrophy in one embodiment of the present invention. Figure 7 shows the results of measuring changes in body weight and muscle strength over time for each mouse in the normal group (NC), muscle atrophy induction group (DEX), hemp seed defatted meal protein extract administration group (HPI), low dose administration group of hemp seed defatted meal protein hydrolysate (250 mg / kg administration group: HPH-L), and high dose administration group of hemp seed defatted meal protein hydrolysate (500 mg / kg administration group: HPH-H). Figure 8 shows the results of measuring autonomous movement for each mouse in the normal group (NC), the muscle atrophy-induced group (DEX), and the high-dose administration group of the hemp seed defatted meal protein hydrolysate of the present invention (500 mg / kg administration group: HPH-H). Figure 9 shows the muscle photographs and muscle weight measurements for each mouse in the normal group (NC), muscle atrophy induction group (DEX), hemp seed defatted meal protein extract administration group (HPI), low dose administration group of hemp seed defatted meal protein hydrolysate (250 mg / kg administration group: HPH-L), and high dose administration group of hemp seed defatted meal protein hydrolysate (500 mg / kg administration group: HPH-H). Figures 10 and 11 show the results of microscopic observation of muscle fibers and measurements of the cross-sectional area of ​​muscle fibers in the calf muscles (Figure 9) and thigh muscles (Figure 10) after isolating the calf muscles (Figure 9) and thigh muscles (Figure 10) from each mouse of the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), low dose administration group of hemp seed defatted meal protein hydrolysate (250 mg / kg administration group: HPH-L), and high dose administration group of hemp seed defatted meal protein hydrolysate (500 mg / kg administration group: HPH-H). Figure 12 shows the results of analyzing the expression levels of proteins involved in muscle breakdown (MuRF1, MAFbx) and muscle synthesis (Myf5, MyoD, MyHC) by Western blot after obtaining protein lysates from each mouse in the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), low dose administration group of hemp seed defatted meal protein hydrolysate (250 mg / kg administration group: HPH-L) and high dose administration group of hemp seed defatted meal protein hydrolysate (500 mg / kg administration group: HPH-H) after isolating calf muscles. Figure 13 shows the results of analyzing the expression levels of proteins involved in muscle breakdown (MuRF1, MAFbx) and muscle synthesis (Myf5, MyoD, MyHC) by Western blot after obtaining protein lysates from thigh muscles isolated from mice of the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), low dose administration group of hemp seed defatted meal protein hydrolysate (250 mg / kg administration group: HPH-L) and high dose administration group of hemp seed defatted meal protein hydrolysate (500 mg / kg administration group: HPH-H). Figure 14 shows the results of isolating calf muscles from mice of the normal group (NC), muscle atrophy induction group (DEX), hemp seed defatted meal protein extract administration group (HPI), low dose administration group of hemp seed defatted meal protein hydrolysate (250 mg / kg administration group: HPH-L), and high dose administration group of hemp seed defatted meal protein hydrolysate (500 mg / kg administration group: HPH-H), obtaining protein lysates, and analyzing the expression levels of apoptosis-related proteins through Western blot. Specific details for implementing the invention

[0029] Hereinafter, preferred embodiments of the present invention and the physical properties of each component are described in detail. This description is intended to be sufficient for a person skilled in the art to easily practice the invention, and does not imply that the technical scope and concept of the present invention are limited thereby.

[0031] The method for producing a low-molecular-weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal according to the present invention comprises: an acidity adjustment step (S101) in which distilled water is mixed with hemp seed defatted meal and the pH is adjusted to 10; a supernatant separation step (S103) in which the supernatant is separated after centrifuging the mixture in which the acidity is adjusted through the acidity adjustment step (S101); a protein extraction step (S105) in which the pH of the supernatant separated through the supernatant separation step (S103) is adjusted to 5 to extract protein; an ultrasonic heat treatment step (S107) in which distilled water is mixed with the protein extracted through the protein extraction step (S105) and heat treatment is performed after irradiating with ultrasound; and a hydrolysis step (S109) in which a hydrolytic enzyme is mixed with the protein extract in which ultrasound irradiation and heat treatment have been performed through the ultrasonic heat treatment step (S107) and reacted to produce a low-molecular-weight product.

[0033] The above acidity adjustment step (S101) is a step of mixing distilled water with hemp seed defatted meal and adjusting the pH to 10, and it is preferable that the process consists of preparing a mixture by mixing 1500 to 2500 parts by weight of distilled water with 100 parts by weight of hemp seed defatted meal, and then mixing sodium hydroxide into the mixture to adjust the pH of the mixture to 10.

[0034] The hemp seeds mentioned above are seeds of hemp (cannabis) and are considered one of the superfoods because they contain a large amount of nutrients such as protein and amino acids. They are a low-calorie, protein-rich food that contains more unsaturated fatty acids, such as omega-3 and omega-6, than mackerel, and also contain various essential amino acids.

[0035] Therefore, as research on the useful biological activities of hemp seeds is reported, interest in manufacturing functional materials using them is increasing, but there have been no reports of materials that can prevent, improve, or treat muscle atrophy or sarcopenia using defatted hemp seed meal.

[0036] At this time, the defatted meal refers to the residue of seeds remaining after oil extraction, and in the present invention, defatted meal of hemp seeds was used.

[0037] As described above, the mixture with a pH adjusted to 10 increases the amount of protein eluted from the hemp seed defatted meal contained in the mixture, and at this time, it is preferable to use sodium hydroxide with a 2-normal concentration.

[0039] The supernatant separation step (S103) is a step of separating the supernatant after centrifuging the mixture whose acidity has been adjusted through the acidity adjustment step (S101). It consists of a process of separating the supernatant of the mixture from which foreign substances have been removed by centrifuging the mixture, whose pH has been adjusted to 10 through the acidity adjustment step (S101), at a speed of 2500 to 3500 rpm.

[0041] The protein extraction step (S105) above is a step of extracting protein by adjusting the pH of the supernatant separated through the supernatant separation step (S103) to 5, and it is preferable that the process is carried out by mixing hydrochloric acid into the supernatant separated through the supernatant separation step (S103) to adjust the pH of the supernatant to 5.

[0042] As described above, when the pH of the supernatant is adjusted to 5, the supernatant becomes acidic, and the protein components contained in the supernatant precipitate, making it easy to recover.

[0043] At this time, it is preferable to use hydrochloric acid that has a 2-normal concentration.

[0045] The above ultrasonic heat treatment step (S107) is a step of mixing distilled water with the protein extracted through the above protein extraction step (S105) and heat-treating it after irradiating it with ultrasound. It is preferable that the process comprises mixing 1500 to 2500 parts by weight of distilled water with 100 parts by weight of the protein extracted through the above protein extraction step (S105), irradiating it with ultrasound of an intensity of 250 to 350 W for 10 to 20 minutes, and then heat-treating the mixture irradiated with ultrasound at a temperature of 90 to 110°C for 10 to 20 minutes.

[0046] If the above ultrasonic and heat treatment processes are performed outside the range of conditions described above, problems arise in which hydrolytic activity is reduced or proteins are denatured, resulting in decreased physiological activity.

[0048] The above hydrolysis step (S109) is a step of mixing and reacting a hydrolytic enzyme with the protein extract, which has undergone ultrasonic irradiation and heat treatment through the above ultrasonic heat treatment step (S107), to reduce its molecular weight. It consists of a process of sequentially treating the protein extract, which has undergone ultrasonic irradiation and heat treatment through the above ultrasonic heat treatment step (S107), with hydrolytic enzymes of different components.

[0049] At this time, it is preferable that the above hydrolytic enzyme consists of an alkalase or a flavourzyme.

[0050] In addition, the hydrolysis step (S109) is more preferably composed of a process in which 3 to 5 parts by weight of alkalase are mixed with 100 parts by weight of a protein extract that has undergone ultrasonic irradiation and heat treatment through the ultrasonic heat treatment step (S107), treated at a temperature of 45 to 55°C for 55 to 65 minutes, and then 3 to 5 parts by weight of flavozyme are additionally mixed and treated at a temperature of 45 to 55°C for 55 to 65 minutes.

[0051] In addition, after the hydrolysis step (S109) performed by the above process is completed, it is preferable to proceed with a process of heat-treating the mixture hydrolyzed through the hydrolysis step (S109) at a temperature of 85 to 95°C for 10 to 20 minutes to inactivate hydrolytic enzymes such as alkalase and flavozyme.

[0052] When the hydrolysis step (S109) is carried out through the above process, the production of a low molecular weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal according to the present invention is completed, and the low molecular weight protein hydrolysate produced through the above process contains a large amount of low molecular weight peptides of 3 kDa or less and also contains a large amount of free amino acids.

[0054] By undergoing the hydrolysis step carried out by the above process, the production of a low molecular weight protein hydrolysate for plant-based meat substitute using the hemp seed defatted meal according to the present invention is completed, and a plant-based meat substitute containing the low molecular weight protein hydrolysate produced by the above manufacturing method as an active ingredient can be provided.

[0055] At this time, the plant-based meat substitute may comprise a low molecular weight protein hydrolysate, gluten, and a flavor enhancer, and may be manufactured by a compression molding process of a mixture formed by mixing 0.5 to 1.5 parts by weight of gluten and 0.5 to 1.5 parts by weight of a flavor enhancer with 100 parts by weight of the low molecular weight protein hydrolysate.

[0056] At this time, the flavor enhancer is not particularly limited and any ingredient capable of imparting an aroma and taste similar to meat may be used, but it is preferable to use one composed of water, coconut oil, and xanthan gum.

[0058] In addition, the present invention confirmed the activity of improving, preventing, or treating muscle atrophy or sarcopenia of the low molecular weight protein hydrolysate produced through the above process.

[0059] To this end, when a low molecular weight protein hydrolysate prepared through the present invention was administered to mice that had induced muscle atrophy or sarcopenia by administering dexamethasone, it was found that the body weight lost due to muscle atrophy or sarcopenia was recovered, grip strength was improved, and reduced exercise volume was improved, and it was found that there was an increase in muscle mass and a muscle strengthening effect.

[0060] In addition, in another embodiment of the present invention, it was confirmed that the symptoms of muscle fiber reduction due to muscle atrophy or sarcopenia were improved by the administration of the low molecular weight protein hydrolysate of the hemp seed defatted meal of the present invention, and that the reduction of muscle fibers was effectively inhibited. Furthermore, this effect was found to be more effective in the group treated with the low molecular weight protein hydrolysate of the hemp seed defatted meal obtained by hydrolysis compared to the group treated with the protein extract extracted from the hemp seed defatted meal (before hydrolysis treatment; HPI).

[0061] In addition, in another embodiment of the present invention, the low molecular weight protein hydrolysate of the hemp seed defatted meal of the present invention was analyzed to affect the expression of proteins involved in muscle breakdown and muscle synthesis.

[0062] As a result, in the group treated with the low molecular weight protein hydrolysate of the hemp seed defatted meal of the present invention, the protein expression levels of MuRF1 and MAFbx, which are ubiquitin ligase (Ubiquitin ligase E3) transferases that induce muscle atrophy, were found to be decreased, while the expression of Myf5, MyoD, and MyHC, which are involved in muscle differentiation and synthesis, was found to be increased.

[0063] Therefore, through the above results, the inventors found that the low-molecular-weight protein hydrolysate produced through the present invention inhibits the expression of proteins that induce muscle atrophy and simultaneously promotes the expression of proteins that induce muscle differentiation and muscle synthesis, thereby enabling the prevention, improvement, and treatment of muscle diseases such as muscle atrophy or sarcopenia.

[0064] Furthermore, the inventors analyzed whether the low-molecular-weight protein hydrolysate produced through the present invention also affects the expression of proteins related to apoptosis in muscle tissue.

[0065] As a result, in the muscle cells of the group with induced muscle atrophy, the expression levels of Bax, known to induce apoptosis, and cleaved caspase-3, which occurs when apoptosis occurs, were found to be significantly increased compared to the normal group, whereas in the group treated with the low molecular weight protein hydrolysate prepared through the present invention, the expression levels of Bax and cleaved caspase-3 were found to be suppressed.

[0066] At the same time, in the group in which muscle atrophy was induced, the expression of Bcl-2, which is known to have a function of inhibiting apoptosis, was found to be reduced compared to the normal group, whereas in the group treated with the low molecular weight protein hydrolysate produced through the present invention, the expression of Bcl-2 was found to be increased.

[0067] These results indicate that the hemp seed defatted meal low molecular weight protein hydrolysate of the present invention can effectively inhibit the death of muscle cells caused by muscle atrophy or sarcopenia by regulating the expression of proteins associated with apoptosis in muscle.

[0068] Based on the above results, the inventors found that a composition containing a low-molecular-weight protein hydrolysate produced through the present invention can be used as a material for improving, preventing, or treating muscle atrophy or sarcopenia.

[0069] Therefore, the present invention can provide a health functional food composition for improving muscle atrophy or sarcopenia or strengthening muscles, comprising a low molecular weight protein hydrolysate of hemp seed defatted meal prepared by the method of the present invention as an active ingredient.

[0070] The health functional food composition for improving muscle atrophy or sarcopenia or strengthening muscles according to the present invention may contain, in addition to containing low molecular weight protein hydrolysate of defatted hemp seed meal as an active ingredient, various flavoring agents or natural carbohydrates as additional ingredients, as in conventional food compositions.

[0071] Examples of the natural carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, such as conventional sugars like dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The flavoring agents described above may advantageously include natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.).

[0072] The health functional food composition of the present invention can be formulated 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, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0073] In addition to the active ingredients of the present invention, the above food composition may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents 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. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0074] In addition, the present invention provides a health functional food for improving muscle atrophy or sarcopenia or strengthening muscles, comprising a low molecular weight protein hydrolysate of hemp seed defatted meal produced by the method of the present invention as an active ingredient.

[0075] 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 improving muscle atrophy or sarcopenia or strengthening muscles.

[0076] In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body as defined in Article 6727 of the Health Functional Foods Act, and means consuming it for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0077] The health functional food of the present invention may include conventional food additives, and unless otherwise specified, suitability as a food additive is determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the food additive code approved by the Korea Food and Drug Administration.

[0078] Items listed in the above food additives code include, for example, chemically synthesized products such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0079] For example, a health functional food in tablet form may be produced by granulating a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives using a conventional method, and then adding a lubricant or the like and compression molding, or by directly compression molding the mixture. In addition, the health functional food in tablet form may contain a binder or the like as needed.

[0080] Among the capsule-type health functional foods, hard capsules can be manufactured by filling a conventional hard capsule with a mixture in which the active ingredient of the present invention is mixed with additives such as excipients, and soft capsules can be manufactured by filling a capsule base such as gelatin with a mixture in which the active ingredient of the present invention is mixed with additives such as excipients. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0081] In addition, the health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., using a previously known method, and if necessary, it can be coated with sucrose or other coating agents, or the surface can be coated with a substance such as starch or talc.

[0082] In addition, a granular health functional food can be manufactured into a granular form by a previously known method of mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc., and may contain flavoring agents, stimulants, etc. as needed.

[0083] The above health functional foods may be beverages, meat, chocolate, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0084] In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of muscle atrophy or sarcopenia comprising a low molecular weight protein hydrolysate of hemp seed defatted meal prepared by the method of the present invention as an active ingredient.

[0085] The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

[0086] The above pharmaceutical composition may preferably be formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

[0087] The formulation form of the above pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops, or injectable liquids. For example, for formulation into the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, trackercanth, or sodium oleate; sodium stearate; magnesium stearate; sodium benzoate; sodium acetate; sodium chloride; etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, which are appropriate in the art.

[0088] The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to treatment, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the strength of administration, and the judgment of the prescriber. The determination of the dosage based on these factors is within the level of a person skilled in the art, and generally, the dosage is in the range of 0.01 mg / kg / day to approximately 2,000 mg / kg / day.

[0089] A more preferred dosage is 1 mg / kg / day to 500 mg / kg / day. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the invention in any way.

[0090] The pharmaceutical composition of the present invention may be administered to mammals, such as rats, livestock, and humans, by various routes. Any mode of administration may be anticipated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebrovascular injection.

[0092] Hereinafter, a method for producing a low molecular weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal according to the present invention and the physical properties of the low molecular weight protein hydrolysate for plant-based meat substitute produced through the method will be explained with reference to examples.

[0094] <Example 1>

[0095] Defatted hemp seed meal purchased from the market was mixed with distilled water at a weight ratio of 1:20 (defatted meal:distilled water), and then 2N NaOH was added to adjust the pH to 10 to elute the protein. Subsequently, the precipitated impurities were removed by centrifugation, and only the supernatant was collected. Then, 2N HCl was added and the pH was adjusted to the isoelectric point of 5 to leach the protein extract, followed by separation. 100 parts by weight of the obtained protein extract were mixed with 2,000 parts by weight of distilled water and subjected to 300W ultrasound for 15 minutes, followed by heat treatment at 100°C for 15 minutes. Subsequently, 4 parts by weight of alkalase were mixed with 100 parts by weight of the heat-treated protein extract and subjected to enzymatic treatment at 50°C for 60 minutes. Afterward, 4 parts by weight of flavozyme were additionally mixed and subjected to enzymatic treatment at 50°C for 60 minutes, followed by heating at 90°C for 15 minutes to prepare a low molecular weight protein hydrolysate for plant-based meat substitutes using defatted hemp seed meal. (HEAT+SONIC)

[0097] <Comparative Example 1>

[0098] A low molecular weight protein hydrolysate for plant-based meat substitute was prepared using defatted hemp seed meal, in the same manner as in Example 1 above, but without ultrasonic irradiation or heat treatment. (AF)

[0100] <Comparative Example 2>

[0101] A low molecular weight protein hydrolysate for plant-based meat substitute was prepared using defatted hemp seed meal, in the same manner as in Example 1 above, but without ultrasonic irradiation. (HEAT)

[0103] <Comparative Example 3>

[0104] A low molecular weight protein hydrolysate for plant-based meat substitute was prepared using defatted hemp seed meal, in the same manner as in Example 1 above, but without performing a heat treatment process. (SONIC)

[0106] HPLC SIZE EXCLUSION analysis of low molecular weight protein hydrolysates for plant-based meat substitutes using hemp seed defatted meal prepared through Example 1 and Comparative Examples 1 to 3 was performed and is shown in Figure 2 below.

[0107] {However, for the HPLC size exclusion analysis of low molecular weight protein hydrolysates, a Shodex SUGAR KS-804 column (8.0 mm × 300 mm, 7 μm) was used for sample analysis in an HPLC (High Performance Liquid Chromatography, Waters, USA), and detection was performed using a Refractive Index Detector (RI) with a sample injection volume of 20 μL. 100% distilled water was used as the mobile phase, and analysis was performed under the conditions shown in Table 1 below.}

[0108]

[0109]

[0110] As shown in Figure 1 below, the groups treated with the enzymes alcalase and flavourzyme showed an increase in the peak corresponding to low molecular weight peptides compared to the group not treated with the enzymes, and it can be confirmed that the peak corresponding to low molecular weight peptides of 3 kDa or less increased significantly.

[0112] In addition, based on the HPLC analysis results of low molecular weight protein hydrolysates for plant-based meat substitutes using hemp seed defatted meal prepared through Example 1 and Comparative Examples 1 to 3 above, the area % values ​​were converted into a graph to analyze the trends of low molecular weight peptides of 3 kDa or less and high molecular weight peptides of more than 3 kDa for the hydrolysates of each experimental group, and are shown in Figure 3 below.

[0113] As shown in Figure 3 below, in the experimental groups treated with alcalase and flavourzyme, the proportion of low molecular weight peptides increased, while the proportion of high molecular weight peptides decreased. In particular, in the group that underwent pretreatment with ultrasound and heat treatment before enzyme treatment, the content of low molecular weight peptides of 3 kDa or less was found to be the highest, while the content of high molecular weight peptides exceeding 3 kDa was found to be the lowest.

[0114] These results indicate that when producing protein hydrolysates from hemp seed defatted meal, if pretreatment with ultrasound and heat is performed before enzyme treatment, the hydrolysis efficiency of the enzyme is increased, allowing for the highest content of low molecular weight peptides of 3 kDa or less to be obtained.

[0116] In addition, TNBS (2,4,6-trinitrobenzene sulfonic acid) analysis was performed to measure the amino acid content of the low molecular weight protein hydrolysate for plant-based meat substitutes using the hemp seed defatted meal prepared through Example 1 and Comparative Examples 1 to 3 above, and the results are shown in Figure 4 below.

[0117] {However, TNBS analysis can analyze the content of free amino acids contained in the sample by quantitatively measuring free amino groups. Specifically, for TNBS analysis, the sample and standard reagent solutions were dissolved in 0.1M sodium bicarbonate at pH 8.5 at concentrations of 20–200 µg / ml. 250 µl of 0.01% (w / v) TNBSA solution was mixed with 500 µl of each sample and standard reagent solution. The reaction was then carried out in a 37°C incubator for 2 hours. Subsequently, 250 µl of 10% SDS and 125 µl of 1N HCl were added to each reaction mixture to stop the reaction. Free amino acids were analyzed by measuring absorbance at a wavelength of 335 nm, and the absorbance values ​​of the samples were plotted on a leucine standard curve to be expressed as values.}

[0118] As shown in Figure 4 below, compared to the group that was not treated with enzymes, the group that underwent hydrolysis with enzyme treatment showed an increase in the content of free amino acids and an increase in the degree of hydrolysis, and it can be confirmed that the degree of hydrolysis increased further in the group that underwent pretreatment with ultrasound and heat treatment.

[0120] In addition, the degree of hydrolysis of the low molecular weight protein hydrolysate for plant-based meat substitutes using the hemp seed defatted meal prepared through Example 1 and Comparative Examples 1 to 3 was analyzed via DS-PAGE electrophoresis. The measured results are shown in Figure 5 below.

[0121] {However, DS-PAGE electrophoresis analysis was performed using a 15% gel. Each sample was diluted to a concentration of 10 mg / ml, 20 µl was loaded onto the gel, and electrophoresis was performed at 85 V for approximately 2 hours. Subsequently, the analysis was conducted under conditions where the samples were stained with Coomassie Brilliant Blue reagent and then destained using 30% methanol containing 10% acetic acid.}

[0122] As shown in Figure 5 below, a dark protein band was observed in the group of hemp seed protein extract (HPI) that was not treated with enzymes, whereas almost no protein bands were observed in the groups that underwent hydrolysis through enzyme treatment. These results indicate that all proteins contained in the protein extract of defatted hemp seed meal were effectively hydrolyzed into low-molecular-weight peptides by enzyme treatment.

[0124] In addition, the protective effect against muscle atrophy of the low molecular weight protein hydrolysate for plant-based meat substitute using the hemp seed defatted meal prepared through Example 1 above was analyzed and is shown in Figures 7 to 9 below.

[0125] However, the protective effect against muscle atrophy was assessed through the analysis of body weight and grip strength, the analysis of the effect of inhibiting the reduction of exercise volume, and the analysis of the effect of inhibiting muscle loss, and mice were used as experimental animals.

[0126] At this time, male 7-week-old C57BL / 6N mice were purchased from Coatec Co., Ltd. (Pyeongtaek, Korea) and used as experimental animals, and were reared under controlled temperature (23±2℃), humidity (50±5%), and a 12-hour light-dark cycle. After a 1-week adaptation period, all experimental animals were randomized into 1) a normal control group, 2) a dexamethasone administration group, 3) a dexamethasone + protein extract of defatted hemp seed meal (500 mg / kg) administration group, 4) a dexamethasone + protein hydrolysate of defatted hemp seed meal (250 mg / kg) administration group, and 5) a protein hydrolysate of defatted hemp seed meal (500 mg / kg) administration group. During the experimental period (28 days), the protein extract of the defatted hemp seed meal or the protein hydrolysate of the defatted hemp seed meal was administered orally daily, and each control group was fed an equal amount of saline. To induce muscle atrophy in mice, dexamethasone dissolved in saline was administered intraperitoneally daily for 2 weeks at a concentration of 10 mg / kg body weight, 2 weeks after the start of oral administration, while the control group was treated with only saline in the same manner. During the experimental period, all experimental animals were allowed to freely consume a normal diet and drinking water. Blood was collected by cardiac blood sampling after anesthesia via injection of Avertin (2,2,2-tribromoethanol; Sigma-Aldrich Co.), and a total of four leg muscles—quadriceps, tibialis anterior, gastrocnemius, and splenic muscles—were excised from each mouse and stored at -40°C. A schematic diagram of the animal experiment described above is shown in Fig. 6.

[0127] In addition, body weight and grip strength analysis were performed using a method that measures body weight and analyzes grip strength on the aforementioned mouse experimental group. These analyses were measured on days 0, 7, 14, 21, 23, 25, and 28, respectively, following a one-week adaptation period. Grip strength was measured in kgf units, and a stainless steel mesh plate was attached to the measuring device. After confirming that the experimental animal grasped the mesh plate of the experimental tool with all its legs, the grip strength was measured by pulling the tail at a constant speed until the grip was released. Three measurements were obtained for each animal, and the average value was calculated.

[0128] In addition, the analysis of the above-mentioned effect of inhibiting the decrease in activity was performed by measuring voluntary wheel running. This was conducted on the 21st day of adaptation to the mouse animal model, and the number of times the mouse ran freely on the wheel for 24 hours was measured.

[0129] In addition, muscle weights were measured and muscles were observed for each of the aforementioned mouse experimental groups. Muscle photography and weight measurement were performed simultaneously with dissection after the experimental period (28 days). Photography was conducted by fixing the camera to maintain a constant distance between the muscles, and the weight measurements were adjusted to body weight.

[0130] As shown in Figure 7 below, the analysis of body weight and grip strength revealed that the body weight loss of mice due to muscle atrophy was most effectively suppressed in the group administered with the low molecular weight protein hydrolysate of the hemp seed defatted meal prepared in the present invention (HPH-L) (6a). In the analysis of grip strength, the group administered with the low molecular weight protein hydrolysate of the hemp seed defatted meal of the present invention was also found to be superior, and the group administered with the low molecular weight protein hydrolysate at a concentration of 500 mg / kg showed a superior grip strength effect compared to the normal control group.

[0131] In addition, as shown in Figure 8 below, the analysis of the effect of inhibiting the decrease in activity level showed that in mice with muscle atrophy induced by administering dexamethasone, the activity level was lower compared to the normal group, whereas in the group administered the low molecular weight protein hydrolysate of the hemp seed defatted meal of the present invention (500 mg / kg administration group: HPH-H), it was confirmed that the symptoms of decreased activity level observed in the muscle atrophy mice were improved.

[0132] In addition, as shown in Figure 9 below, the results of an investigation into four muscles isolated from each mouse—the quadriceps femoris, tibialis anterior, gastrocnemius, and splenic muscles—showed that compared to the normal group, the group with sarcopenia induced by dexamethasone administration showed muscle loss in all four muscles. Meanwhile, in the case of the group administered the low molecular weight protein hydrolysate of the hemp seed defatted meal of the present invention (HPH-L; 250 mg / kg and HPH-H; 500 mg / kg), muscle loss was improved, and muscle mass was restored to a level similar to normal.

[0134] In addition, the inhibitory effect on muscle fiber reduction of a low molecular weight protein hydrolysate for plant-based meat substitute using the hemp seed defatted meal prepared through Example 1 above was analyzed and is shown in Figures 10 to 11 below.

[0135] {However, to investigate the inhibitory effect on muscle fiber reduction, calf and thigh muscles were isolated from the aforementioned mouse experimental group. The calf and thigh muscles were refrigerated in 10% formalin immediately after dissection for tissue fixation. For tissue staining, each muscle was thinly sliced ​​to a thickness of 4–5 µm and stained with hematoxylin for 2 minutes followed by eosin for 1 minute. Subsequently, images were taken using an optical microscope (Leica Microsystems, Wetzlar, Germany) for histological analysis, and the cross-sectional area (CSA) of the muscle fibers was quantified using Image j software (64-bit Java 1.8.0_172).}

[0136] As shown in Figures 10 to 11 below, compared to the normal group, the decrease in muscle fibers in the calf and thigh muscles of mice with induced muscle atrophy was significantly increased, whereas in the group administered the low molecular weight protein hydrolysate obtained from the hemp seed defatted meal of the present invention, the decrease in muscle fibers was effectively inhibited. In particular, it was confirmed that the group treated with the hydrolyzed low molecular weight protein hydrolysate more effectively inhibited the decrease in muscle fibers in both the calf and thigh compared to the group treated with the protein extract from the hemp seed defatted meal (before hydrolysis treatment; HPI), and the decrease in muscle fibers was inhibited in a concentration-dependent manner with the low molecular weight protein hydrolysate treatment.

[0137] Therefore, it can be seen that the low molecular weight protein hydrolysate for plant-based meat substitute using the hemp seed defatted meal prepared through Example 1 of the present invention can effectively improve, prevent, or treat muscle atrophy or muscle loss.

[0139] In addition, the effects of a low molecular weight protein hydrolysate for plant-based meat substitute using the hemp seed defatted meal prepared in Example 1 above on muscle degradation and muscle synthesis-related protein expression were analyzed and are shown in Figures 12 to 13 below.

[0140] However, the effects on the expression of proteins related to muscle degradation and muscle synthesis were analyzed using Western blot. RiPA buffer was added to calf and thigh muscles (samples stored at -40°C or below) collected from each of the mouse experimental groups, homogenized, and centrifuged to obtain the supernatant. The concentration of proteins contained in the supernatant was quantified using the BCA protein assay method. Subsequently, electrophoresis was performed on 8% or 12% SDS-PAGE, and the proteins were transferred to 0.45 μm polyvinylidene difluoride membranes. The membranes were then blocked with a 3% BSA and 0.1% Tween-20 solution at room temperature for 1 hour, followed by the addition of a primary antibody and overnight reaction at 4°C. After washing with TBS-T, a horseradish peroxidase secondary antibody was added and reacted for 1 hour. Subsequently, the expressed protein levels were confirmed using the Fusion Solo 6S EDGE imaging system (Vilber, Marne-la-Vallee, France) and quantified using Fusion-capt software (Vilber).

[0141] The primary antibodies used at this time were Myf5 (ab125078; Abcam, Cambridge, UK), MyoD (MA1-41017; Invitrogen, USA), MHC (sc-376157; Santa Cruz Biotechnology, USA), MuRF1 (ab183094; Abcam, Cambridge, UK), and MAFbx (ab168372; Abcam, Cambridge, UK).

[0142] As shown in Figure 12 below, in the case of calf muscles, the protein expression levels of MuRF1 and MAFbx, ubiquitin ligase E3 transferases that induce muscle atrophy, were found to be reduced in a concentration-dependent manner in the group treated with hemp seed protein hydrolysate (HPH), whereas the expression of Myf5, MyoD, and MyHC, which are involved in muscle differentiation and synthesis, was found to be increased.

[0143] In addition, as shown in Figure 13 below, in the case of thigh muscles, similar to the results for calf muscles, the protein expression levels of muscle atrophy regulatory factors were found to be decreased in the group treated with hemp seed protein hydrolysate (HPH), whereas the expression levels of proteins involved in muscle differentiation and synthesis were found to be increased.

[0144] In addition, the above results were confirmed to be significantly increased in the group treated with hemp seed protein hydrolysate (HPH) compared to the group treated with hemp seed defatted meal protein extract (HPI), which was the sample before hemp seed protein hydrolysate.

[0146] In addition, the effect of the low molecular weight protein hydrolysate for plant-based meat substitute using the hemp seed defatted meal prepared through Example 1 above on the expression of apoptosis-related proteins was analyzed, and furthermore, the effect of the low molecular weight protein hydrolysate obtained from the hemp seed defatted meal of the present invention on the expression of apoptosis-related proteins was analyzed.

[0147] At this time, Western blots were performed on the calf muscle samples of the mouse experimental group obtained by the same method as above, except that Bax (2772, Cell Signaling, USA), Bcl-2 (ab692; Abcam, Cambridge, UK), and Cleaved Caspase-3 (Asp175, Cell Signaling, USA) were used as primary antibodies, respectively, and the expression levels of Bax, Bcl-2, and Cleaved Caspase-3 proteins were analyzed.

[0148] As shown in Figure 14 below, in the case of the muscle atrophy-inducing group (DEX), the expression of caspase-3, a cell death-inducing factor, was found to be significantly increased compared to the normal group (NC), whereas in the groups treated with the hemp seed protein hydrolysate of the present invention (HPH-L, HPH-H), the expression of caspase-3 was found to be decreased, and the expression of Bax, a cell death-inducing factor, was found to be effectively suppressed, while the expression of Bcl-2, known as a factor that inhibits cell death, was found to be increased. Meanwhile, in the group treated with the hemp seed defatted meal protein extract, which is the sample prior to hemp seed protein hydrolysate (HPI), no significant effect similar to that observed in the group treated with the hemp seed protein hydrolysate was confirmed.

[0149] These results mean that the low molecular weight protein hydrolysate obtained from the defatted hemp seed meal according to the present invention can effectively inhibit apoptosis occurring in the area where muscle atrophy or sarcopenia has developed, thereby maintaining muscle mass and improving muscle strength.

[0151] Accordingly, the method for preparing a low molecular weight protein hydrolysate for plant-based meat substitute using hemp seed defatted meal according to the present invention and the plant-based meat substitute containing the low molecular weight protein hydrolysate provide a low molecular weight protein hydrolysate for plant-based meat substitute that exhibits excellent improvement effects on muscle atrophy and sarcopenia, and a plant-based meat substitute containing the low molecular weight protein hydrolysate. Explanation of the symbols

[0153] S101 ; pH control stage S103 ; Supernatant separation step S105 ; Protein extraction step S107 ; Ultrasonic heat treatment step S109 ; Hydrolysis step

Claims

Claim 1 Plant-based alternative meat using defatted hemp seed meal, characterized by comprising a low molecular weight protein hydrolysate produced by including the following manufacturing method and having effects for preventing and improving muscle atrophy or sarcopenia: an acidity adjustment step of mixing distilled water with defatted hemp seed meal and adjusting the pH to 10; a supernatant separation step of separating the supernatant after centrifuging the mixture whose acidity has been adjusted through the acidity adjustment step; a protein extraction step of extracting protein by adjusting the pH of the supernatant separated through the supernatant separation step to 5; and an ultrasonic heat treatment step of mixing distilled water with the protein extracted through the protein extraction step, irradiating with ultrasound, and then heat-treating. The method comprises a hydrolysis step in which a hydrolytic enzyme is mixed and reacted with a protein extract, which has undergone ultrasonic irradiation and heat treatment through the ultrasonic heat treatment step, to reduce the molecular weight; wherein the ultrasonic is irradiated for 10 to 20 minutes at an intensity of 250 to 350 W, the heat treatment is carried out for 10 to 20 minutes at a temperature of 90 to 110°C, and the hydrolysis step comprises a process of mixing 1,500 to 2,000 parts by weight of distilled water and 3 to 5 parts by weight of alkalase with 100 parts by weight of the protein extract and treating at a temperature of 45 to 55°C for 55 to 65 minutes, and then additionally mixing 3 to 5 parts by weight of flavozyme and treating at a temperature of 45 to 55°C for 55 to 65 minutes. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete

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