KR20230036871A - Manufacturing method of tofu for preventing or improving muscle disease using mealworm larva protein - Google Patents

Abstract

The present invention relates to a manufacturing method of tofu for preventing or alleviating muscle diseases containing brown mealworm larvae protein, brown mealworm larvae protein isolate, or hydrolysate of the brown mealworm larvae protein isolate as an active ingredient. According to the manufacturing method of the present invention, by adjusting pH of a mixture of soybean flour, brown mealworm larvae protein, and purified water to 9.0, reacting for a certain period of time, and then separating soy milk, the brown mealworm larvae protein is well mixed (integrated) within the tofu during coagulation to improve quality. In addition, as the brown mealworm larvae protein is included as an active ingredient along with the soybean flour and a heating step is additionally performed at a gel formation temperature of the brown mealworm larvae protein separately from a heating step at a gel formation temperature of the soybean, gel formation in soy milk can be effectively induced. In addition, by using glucono-delta-lactone and transglutaminase together as a coagulant, coagulation of the soy milk, which has a very small protein molecular weight (hydrolysate of the brown mealworm larvae protein isolate) and does not coagulate, can be effectively induced.

Description

Manufacturing method of tofu for preventing or improving muscle disease using mealworm larva protein}

The present invention relates to a method for producing tofu for preventing or improving muscle diseases, comprising brown mealworm larval protein, an extract of brown mealworm larval protein, or a hydrolyzate of brown mealworm larval protein extract as an active ingredient.

Sarcopenia is a syndrome in which muscle (Sarx) loss (penia), that is, the overall amount and strength of skeletal muscle is gradually lost, reducing the quality of life and causing limitations in physical activity. Year disease code (ICD-10 code) was assigned. Muscle loss causes significant impairment in the performance of normal daily life functions, and also affects the increase in chronic diseases. In addition, sarcopenia due to aging is a disease that one out of five elderly people in Korea suffer from.

Muscle atrophy is caused by malnutrition or long-term muscle inactivity, and occurs when the balance between normal protein synthesis and degradation is disrupted and protein is decomposed.

As the world population continues to increase, a new food source is needed, and edible insects are attracting attention as the most suitable protein source, and accordingly, the demand for high-protein, high-nutritional food is increasing.

In 2013, the Food and Agriculture Organization (FAO) announced a plan to promote edible insects as a future food resource and selected them as one of the policies to solve the upcoming food shortage problem. The Ministry of Drug Safety recognized brown mealworm larvae as a new food ingredient.

Brown mealworm larvae, which are edible insects, contain a sufficient amount of energy as a food resource, high protein, unsaturated fatty acids, and micronutrients (copper, iron, magnesium, manganese, biotin, pantothenic acid, etc.) As it is systematically established, it is highly valued as a highly nutritious food resource to solve future food shortages.

Bioactive peptides obtained through protein hydrolysis are generally defined as small molecular weight peptides that have physiological activity, and are usually composed of 3 to 20 amino acids, and the activity of the peptide is Varies. In addition, it can be easily absorbed into the living body due to its small size, and has the advantage of having various functional properties.

On the other hand, soybean (soybean) is highly valued as a high-quality vegetable protein material, and is a nutritionally excellent food resource, such as a high proportion of unsaturated fatty acids and dietary fiber. In addition, since it contains a large amount of phytochemicals such as isoflavones, phenols, and saponins, its physiological function is also attracting attention.

Soybean processed foods are divided into fermented and non-fermented foods, and tofu, a representative non-fermented food, has the advantage of being high in essential amino acids and calcium, and rich in micronutrients such as B vitamins. In addition, tofu is a food familiar to the elderly, ranking third among the foods that are consumed by the elderly over 60 in Korea. In addition, tofu has a soft texture and high digestibility, so it is preferred by the elderly with weak teeth and digestion.

However, compared to animal protein, vegetable protein has lower calorie intake, cholesterol, and saturated fat intake, but has limitations such as lower content of branched amino acids (BCAA) essential for protein synthesis and lower body absorption. In addition, the content of essential amino acids is lower than that of animal protein, so mixing and ingesting the two protein sources can help with balanced amino acid intake and efficient protein intake.

However, the preventive effect of muscle atrophy of processed foods studied so far is insignificant. In particular, in terms of the effect of physiologically active peptides, the improvement of the antioxidant function of milk protein hydrolysates in the body, the prevention of oxidation reactions in processed foods, and the new antibacterial properties of egg white hydrolysates Although there are studies on peptides, antihypertensive, antithrombotic, and immunomodulatory functions, studies on the production of protein hydrolysates effective for sarcopenia and the development of products using them are insufficient.

Under this background, the present inventors prepared tofu containing brown mealworm larvae protein, extract of brown mealworm larvae protein, or hydrolyzate of brown mealworm larva protein extract as an active ingredient, and the tofu has excellent antioxidant and anti-inflammatory activity. And it was confirmed that it can effectively improve muscle atrophy. In addition, in the case of the tofu of the present invention, it can be usefully used as a customized food for the elderly or patients with masticatory or swallowing disorders who have difficulty in eating because of its excellent gastric digestibility and low gel strength, in addition to the effect of improving muscle atrophy. The present invention was completed by confirming that there is.

Korean Patent Publication No. 10-2021-0064915 Korean Patent Publication No. 10-2021-0012963

Therefore, an object of the present invention is to provide a tofu manufacturing method that can effectively prevent or improve muscle diseases using brown mealworm larval protein.

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

The present invention is a) mixing brown mealworm larval protein, brown mealworm larval protein extract, or brown mealworm larval protein extract hydrolyzate in a weight ratio of 1: 1 with soy flour and adding distilled water thereto to mix; b) adjusting the pH of the mixture to 9.0, mixing for 30 to 60 minutes, and then separating okara and soybean milk; c) adjusting the pH of the separated soybean milk to 7.0 and then firstly heating it at 70°C; d) secondarily heating the firstly heated soymilk at 95° C.; e) cooling the secondarily heated soybean milk to 50° C. and then adding and mixing a coagulant; f) first reacting the mixture at 50°C; And g) providing a tofu manufacturing method for preventing or improving muscle diseases, comprising the step of performing a secondary reaction at 85 ° C. and then cooling the mixture subjected to the primary reaction at room temperature.

In one embodiment of the present invention, the extract of the brown mealworm larvae protein is obtained by: i) crushing the dried brown mealworm larvae; ii) degreasing by adding ethanol to the pulverized material; iii) adding sodium hydroxide to the degreased brown mealworm larvae and mixing them, followed by centrifugation to obtain a precipitate; and iv) desalting the obtained precipitate and then freeze-drying it.

In one embodiment of the present invention, the hydrolyzate of the brown mealworm larva protein extract can be prepared by sequentially treating the brown mealworm larva protein extract with alcalase and flavorzyme for hydrolysis.

In one embodiment of the present invention, proteins in soybean and brown mealworm larvae can be extracted through step b).

In one embodiment of the present invention, a gel of protein of the brown mealworm larvae can be formed through step c).

In one embodiment of the present invention, the coagulant in step e) may be gluconodeltalactone and transglutaminase.

In one embodiment of the present invention, the head may suppress the expression of a gene related to muscle loss.

In one embodiment of the present invention, the gene associated with muscle loss may be selected from the group consisting of Myostatin, MuRF 1 and Atrogin-1.

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 muscular disease is atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, rigid spine syndrome, amyotrophic lateral It may be selected from the group consisting of sclerosis (Lou Gehrig's disease, amyotrophic lateral sclerosis), Charcot-Marie-Tooth disease and sarcopenia.

The tofu manufacturing method of the present invention adjusts the pH of the mixture of soybean meal, brown mealworm larvae protein and purified water to 9.0, reacts for a certain period of time, and then separates the soybean milk so that the brown mealworm larvae protein is well mixed (integrated) in the tofu when coagulated, resulting in quality has an enhancing effect. In addition, in the present invention, as the brown mealworm larva protein is included as an active ingredient along with soybean meal, a heating step at the gel formation temperature of the brown mealworm larva protein is additionally performed separately from the heating step at the gel formation temperature of the soybean milk, so that the soybean milk Gel formation can be effectively induced. In addition, in the present invention, by using gluconodeltalactone and transglutaminase together as coagulants, it is possible to effectively induce coagulation of soymilk that does not coagulate due to the very small molecular weight of protein (hydrolyzate of brown mealworm larvae protein extract). .

On the other hand, the tofu prepared by the method of the present invention has excellent antioxidant and anti-inflammatory activity and effectively improves muscle atrophy by containing brown mealworm larvae protein, extract of brown mealworm larvae protein, or hydrolyzate of brown mealworm larva protein extract as an active ingredient. can make it In particular, the head of the present invention has an excellent effect of suppressing the expression of Myostatin, MuRF 1 and Atrogin-1, which are genes related to muscle loss, so that it is effective in reducing muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. It can be usefully used as a functional food to prevent or improve various muscle diseases caused by In addition, in the case of the tofu of the present invention, in addition to the muscle atrophy improvement effect, it has excellent gastrointestinal digestibility and low gel strength, so it can be usefully used as a customized food for the elderly or patients with masticatory or swallowing disorders who have difficulty in eating food. can

1 shows a manufacturing process diagram of brown mealworm larval protein extract (MPI) and hydrolyzate (MPH) thereof according to the present invention.
Figure 2a shows the storage modulus (G') and loss modulus (G”) of the brown mealworm larvae protein extract (MPI) according to the present invention, and 2b is the loss coefficient (tan δ) of the brown mealworm larva protein extract (MPI) is shown.
Figure 3 shows a manufacturing process diagram of soymilk and tofu according to the present invention.
Figure 4 is the result of performing SDS-PAGE to track changes in protein subunits of soymilk and tofu according to the present invention (M: protein marker, 1: soymilk made with 100% soybean flour, 2: 50% soybean flour and 50% soybean flour) Soymilk made from brown mealworm larvae powder, 3: Soymilk made from 50% soy flour and 50% protein extract from brown mealworm larvae, 4: Soymilk made from 50% soybean flour and 50% hydrolyzate of protein extract from brown mealworm larvae, 5: Tofu made with 100% soybean flour, 6: Tofu made with 50% soybean flour and 50% brown mealworm larvae powder, 7: Tofu made with 50% soybean flour and 50% brown mealworm larvae protein extract, 8: Tofu made with 50% soybean flour and brown mealworm larvae protein extract Tofu prepared with 50% hydrolyzate of mealworm larvae protein extract).
Figure 5 shows the change in pH of tofu according to the storage period according to the present invention (S: tofu made of 100% soy flour, SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder, SMPI: soy flour 50% and Tofu made with 50% brown mealworm larvae protein extract (MPI), SMPH: tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
Figure 6 shows the in vitro ileum digestibility of tofu according to the present invention (S: tofu made of 100% soybean flour, SM: tofu made of 50% soybean flour and 50% brown mealworm larvae powder, SMPI: Tofu made with 50% soy flour and 50% brown mealworm larva protein extract (MPI), SMPH: tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
7 shows the gel strength of tofu according to the present invention (S: tofu made of 100% soybean flour, SM: tofu made of 50% soybean flour and 50% brown mealworm larvae powder, SMPI: soybean flour 50% and brown mealworm larvae powder) Tofu made with 50% larval protein extract (MPI), SMPH: tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH)).
8a and 8b respectively show the storage modulus (G') and loss modulus (G”) of the tofu according to the present invention (S: tofu made of 100% soybean flour, SM: 50% soybean flour and 50 brown mealworm larvae powder) % tofu, SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI), SMPH: tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract ).
9 shows the loss factor (tan δ) of tofu according to the present invention (S: tofu made of 100% soy flour, SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder, SMPI: soy flour 50 % and tofu prepared with 50% brown mealworm larvae protein extract (MPI), SMPH: tofu prepared with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
10 shows the storage modulus (G′) and loss modulus (G“) of tofu according to frequency (S: tofu made of 100% soybean flour, SM: made of 50% soybean flour and 50% brown mealworm larvae powder). Tofu, SMPI: Tofu prepared with 50% soy flour and 50% protein extract (MPI), SMPH: Tofu prepared with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
11 is a measurement of the chemical structure of tofu according to the present invention using FTIR spectrophotometry (S: tofu made of 100% soy flour, SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder, SMPI : Tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI), SMPH: Tofu made with 50% soybean flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract, (1): 3274 cm ^-1 , (2): 2916 cm ^-1 , (3): 2849 cm ^-1 , (4): 1742 cm ^-1 , (5): 1628 cm ^-1 , (6): 1541 cm ^-1 , (7 ) 1039 cm ^-1 ).
12 is a SEM image showing the microstructure of the surface of tofu according to the present invention (S: tofu made of 100% soybean flour, SM: tofu made of 50% soybean flour and 50% brown mealworm larvae powder, SMPI: soybean flour 50% and tofu made with 50% brown mealworm larvae protein extract (MPI), SMPH: tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
13 shows the total phenol and total flavonoid content of tofu according to the present invention (S: tofu made with 100% soy flour, SM: tofu made with 50% soy flour and 50% brown mealworm larvae powder, SMPI: soy flour 50 % and tofu prepared with 50% brown mealworm larvae protein extract (MPI), SMPH: tofu prepared with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
14 shows the ABTS+ and DPPH radical scavenging activity of tofu according to the present invention (S: tofu made with 100% soy flour, SM: tofu made with 50% soy flour and 50% brown mealworm larvae powder, SMPI: soy flour 50 % and tofu prepared with 50% brown mealworm larvae protein extract (MPI), SMPH: tofu prepared with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
15 is a measurement of cell viability according to the concentration (0 to 2000 μg/ml) treatment of tofu extract according to the present invention (S: tofu made of 100% soybean flour, SM: 50% soybean flour and brown mealworm larvae powder) Tofu made with 50% SMPI: Tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI), SMPH: Made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract. tofu).
Figure 16 shows the inhibitory effect of LPS-induced proinflammatory cytokines (TNF-α, IL-1b, IL-6) according to the treatment of tofu extract (200 μg / mL) according to the present invention (S: 100 % Tofu made with soy flour, SM: Tofu made with 50% soy flour and 50% brown mealworm larvae powder, SMPI: Tofu made with 50% soy flour and brown mealworm larvae protein extract (MPI) 50%, SMPH: Soy flour 50% and tofu prepared with 50% hydrolyzate (MPH) of brown mealworm larvae protein extract).
Figure 17 confirms the weight change of mice according to tofu intake of the present invention in a mouse model of muscular atrophy induced by dexamethasone (DMSO con: DMSO-treated normal diet mouse group, DEXA con: dexamethasone-treated normal diet mouse group, DEXA -S: dexamethasone treated S diet mouse group, DEXA-SM: dexamethasone treated SM diet mouse group, DEXA-SMPI: dexamethasone treated SMPI diet mouse group, DEXA-SMPH: dexamethasone treated SMPH diet mouse group).
Figure 18 confirms the grip strength of mice according to tofu intake of the present invention in a mouse model of muscular atrophy induced by dexamethasone (DMSO con: DMSO-treated normal diet mouse group, DEXA con: dexamethasone-treated normal diet mouse group, DEXA- S: dexamethasone treated S diet mouse group, DEXA-SM: dexamethasone treated SM diet mouse group, DEXA-SMPI: dexamethasone treated SMPI diet mouse group, DEXA-SMPH: dexamethasone treated SMPH diet mouse group).
Figure 19 confirms the muscle fiber area of mouse muscle fibers according to tofu intake of the present invention in a mouse model of muscular atrophy induced by dexamethasone (DMSO con: DMSO-treated normal diet mouse group, DEXA con: dexamethasone-treated normal diet mouse group, DEXA-S: dexamethasone treated S diet mouse group, DEXA-SM: dexamethasone treated SM diet mouse group, DEXA-SMPI: dexamethasone treated SMPI diet mouse group, DEXA-SMPH: dexamethasone treated SMPH diet mouse group).
20 is a measurement of the expression levels of genes (Myostatin, MuRF 1, Atrogin-1) related to muscle reduction according to tofu intake of the present invention in a mouse model of muscular atrophy induced by dexamethasone (DMSO con: DMSO-treated normal diet mice group, DEXA con: dexamethasone-treated mouse group on normal diet, DEXA-S: dexamethasone-treated mouse group on S diet, DEXA-SM: dexamethasone-treated mouse group on SM diet, DEXA-SMPI: dexamethasone-treated mouse group on SMPI diet, DEXA -SMPH: dexamethasone treated SMPH diet mice group).
Figure 21 is a measurement of the expression levels of muscle synthesis-related genes (MyHC1, MyHC2A, MyHC2X, MyHC2B) according to tofu intake of the present invention in a mouse model of muscular atrophy induced by dexamethasone (DMSO con: DMSO-treated normal diet mouse group , DEXA con: dexamethasone-treated mouse group on normal diet, DEXA-S: dexamethasone-treated mouse group on S diet, DEXA-SM: dexamethasone-treated mouse group on SM diet, DEXA-SMPI: dexamethasone-treated mouse group on SMPI diet, DEXA- SMPH: dexamethasone treated SMPH diet mice group).
Figure 22 confirms the grip strength of mice according to the diet of the tofu (SMPH) of the present invention and the hydrolyzate (MPH) of protein extract of brown gourd larvae in a mouse model of muscular atrophy induced by dexamethasone (SMPH + Dexa: SMPH diet treated with dexamethasone mouse group, MPH+Dexa: dexamethasone treated MPH diet mouse group).
23 is a measurement of the expression levels of genes related to muscle reduction according to the diet of the tofu (SMPH) of the present invention and the hydrolyzate (MPH) of brown mealworm larval protein extract in a mouse model of muscular atrophy induced by dexamethasone (SMPH+Dexa: dexamethasone-treated SMPH diet mouse group, MPH+Dexa: dexamethasone-treated MPH diet mouse group).

The present invention is a) mixing brown mealworm larval protein, brown mealworm larval protein extract, or brown mealworm larval protein extract hydrolyzate in a weight ratio of 1: 1 with soy flour and adding distilled water thereto to mix; b) adjusting the pH of the mixture to 9.0, mixing for 30 to 60 minutes, and then separating okara and soybean milk; c) adjusting the pH of the separated soybean milk to 7.0 and then firstly heating it at 70°C; d) secondarily heating the firstly heated soymilk at 95° C.; e) cooling the secondarily heated soybean milk to 50° C. and then adding and mixing a coagulant; f) first reacting the mixture at 50°C; And g) relates to a tofu manufacturing method for preventing or improving muscle disease, comprising the step of cooling the mixture at room temperature after the secondary reaction at 85 ° C.

The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of muscle disease when a subject consumes the tofu.

The term "improvement" used in the present invention refers to any action that improves or beneficially changes the symptoms of muscle disease when a subject consumes the tofu.

The term “brown mealworm larvae protein” used in the present invention is a protein derived from brown mealworm larvae, and means brown mealworm larvae powder.

As used in the present invention, the term “extract of brown mealworm larvae protein” refers to an extract extracted by adding a solvent to brown mealworm larvae.

The term "hydrolyzate of brown mealworm larvae protein extract" used in the present invention refers to a material prepared by treating the protein extract of brown mealworm larvae with a hydrolase and then hydrolyzing it.

In one embodiment of the present invention, the extract of the protein of the brown mealworm larvae is obtained by: i) grinding the dry matter of the brown mealworm larvae; ii) degreasing by adding ethanol to the pulverized material; iii) adding sodium hydroxide to the degreased brown mealworm larvae and mixing them, followed by centrifugation to obtain a precipitate; and iv) desalting the obtained precipitate and then freeze-drying it.

In another embodiment of the present invention, the hydrolyzate of the brown mealworm larva protein extract can be prepared by hydrolyzing the extract of brown mealworm larvae protein by sequentially treating the extract with alcalase and flavozyme.

In the tofu manufacturing method of the present invention, step a) is a step of preparing a mixed solution in which soybean flour and brown mealworm larvae protein are mixed. This is a step of mixing the hydrolyzate in a weight ratio of 1:1 and then adding distilled water thereto.

In the tofu manufacturing method of the present invention, step b) is a step of extracting proteins from the mixed solution and separating the soymilk containing these proteins. Specifically, after adjusting the pH of the mixed solution obtained through step a) to 9.0, It is a step of mixing for 30 to 60 minutes and then separating it into okara and soybean milk using a filter cloth.

Since the brown mealworm larvae protein has the highest solubility at pH 9, by adjusting the pH of the mixed solution obtained in step a) to 9.0, the brown mealworm larvae protein can be dissolved in the solution to become soymilk.

In the case of manufacturing tofu using conventional insect powder, soymilk (soybean milk) is prepared first and then insect powder is added to the tofu, so that when the soybean milk is coagulated, the powder does not crack in the tofu and the insect powder does not mix well. There is a problem that is not.

In the present invention, after adjusting the pH of the mixture of soy flour, brown mealworm larvae protein and purified water to 9.0 through steps a) and b), reacting for a certain period of time, and then separating the soymilk, the brown mealworm larva protein is well mixed in tofu when coagulated. (Convergence) can lead to the effect of improving quality.

In the tofu manufacturing method of the present invention, step c) is the step of first heating the soybean milk. Specifically, after adjusting the pH of the soybean milk separated through step b) to 7.0, the first step is performed at 69° C. to 70° C. It is a step of heating and maintaining for 5 to 15 minutes.

In the present invention, in the preparation of tofu containing brown mealworm larvae protein, the temperature setting for gel formation of brown mealworm larva protein was preceded, and as a result, the storage modulus (G') and loss modulus (G”) of It was confirmed that the gel formation temperature (gel point) was reached by forming the junction.

Therefore, in the tofu manufacturing method of the present invention, a heating step at the gel formation temperature of the brown mealworm larvae protein is additionally included, separately from the heating step at the soybean gel formation temperature.

In the tofu manufacturing method of the present invention, step d) is a step of secondary heating the soybean milk. It is a step to keep for a minute.

Through step d) of the present invention, a soybean gel can be formed.

In the tofu manufacturing method of the present invention, step e) is a step of cooling the heated soybean milk and then adding and mixing a coagulant. After cooling, gluconodeltalactone and transglutaminase as coagulants are added and mixed.

In the present invention, by using gluconodeltalactone and transglutaminase together as coagulants, it is possible to effectively induce coagulation of soymilk that does not coagulate due to a very small protein molecular weight (hydrolyzate of brown mealworm larvae protein extract).

In one embodiment of the present invention, the gluconodeltalactone may be added at a concentration of 2% (w / v) of soymilk, and transglutaminase (Tgase) may be added to 5U / g protein. can

In the tofu manufacturing method of the present invention, step f) is a step of first reacting a mixture obtained by adding a coagulant to soybean milk at 50 ° C. Specifically, the mixture obtained through step e) is reacted at 50 ° C for 4 hours. By doing so, transglutaminase, a coagulant, can be activated to cause a protein cross-linking reaction.

In the tofu manufacturing method of the present invention, step g) is a step of cooling the first reaction mixture at room temperature after the second reaction at 85 ° C. In detail, the first reaction mixture obtained through step f) is cooled at 85 ° C. for 30 minutes to inactivate transglutaminase, activate gluconodeltalactone, another coagulant, and finally cool at room temperature to prepare uncompressed tofu.

The tofu prepared by sequentially performing the steps a) to g) of the present invention prevents muscle diseases by suppressing the expression of genes related to muscle loss such as Myostatin, MuRF 1 and Atrogin-1, or may have an ameliorating effect.

The term "myostatin" used in the present invention is a protein that regulates muscle growth and belongs to the TGF-β (transforming growth factor-β) family and is a growth and differentiation factor-8 (GDF-8). )am.

The term “MuRF 1” used in the present invention is also known as E3 ubiquitin-protein ligase TRIM63, and is an abbreviation for Muscle RING Finger 1 as a protein marker related to muscular atrophy.

The term "Atrogin-1" used in the present invention is an E3 ubiquitin ligase as a component of the UPP (ubiquitin proteasome pathway) that causes muscle atrophy along with MuRF 1, and is induced in the early process of muscle atrophy to reduce muscle mass. It is known to increase in advance.

As used herein, the term "muscle disease" refers to a disease caused by muscle deterioration, 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 "muscular function" means the ability to exert force by contraction of muscles, and the contractile force to overcome the resistance of the muscles to the maximum. It includes muscular strength, which is the ability to exert force, muscular endurance, which is the ability to indicate how long or how many times a muscle can contract and relax repeatedly with a given weight, and instantaneous power, which is the ability to exert strong force in a short time. This “muscle function” is proportional to muscle mass, and “improvement of muscle function” means to improve muscle function better.

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

The present inventors prepared tofu by sequentially performing the steps a) to g), and as a result, the tofu has excellent antioxidant and anti-inflammatory activities, and can effectively improve muscle atrophy by reducing the expression of muscle loss genes. confirmed that there is

In detail, tofu prepared by sequentially performing steps a) to g) of the present invention had high total phenolic and total flavonoid contents, and excellent ABTS+ and DPPH radical scavenging activities (see FIGS. 13 and 14). In addition, the tofu prepared by sequentially performing the steps a) to g) of the present invention effectively inhibited the expression of pro-inflammatory cytokines (IL-6, TNF-a, IL-1b) induced by LPS (Fig. see 16). For reference, pro-inflammatory cytokines can reduce protein synthesis by promoting the degradation of myofibrillar proteins, resulting in direct muscle wasting. Thus, when the expression of pro-inflammatory cytokines can be effectively inhibited, muscle degradation can be prevented.

In addition, the head prepared by sequentially proceeding with the steps a) to g) of the present invention increases the grip strength of the mouse when fed to the dexamethasone-induced muscular atrophy mouse model (see FIG. 18), and related to muscle reduction The mRNA expression of the genes Myostatin, MuRF 1, and Atrogin-1 was effectively reduced (see FIG. 20). As described above, myostatin inhibits muscle development, and MuRF 1 and Atrogin-1 correspond to components of the ubiquitin enzyme complex that causes muscle atrophy. shrinkage can be prevented.

Therefore, the tofu prepared by sequentially performing the steps a) to g) of the present invention can effectively prevent or improve muscle-related diseases such as muscle loss and muscle atrophy.

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

<Example>

1. Materials and Methods

<1-1> Material preparation

Soybean (Glycine max, L.) powder was purchased from Yeongwol Nonghyup (Yeongwol-gun, South Korea). Brown mealworm larvae were purchased as dried products from Edible Bug Co., Seoul, Korea, and were ground and prepared in powder form using a 1.4 mm sieve. Glucono-δ-lactone (GDL) was purchased from Daejeong Chemical and Gold (Siheung, Korea). Microbial transglutaminase (Biobong TG-WM; 100 U/g activity) powder was purchased from Kinry Food Ingredients Co., Ltd. (Shanghai, China).

<1-2> Preparation of Mealworm Protein Isolate (MPI)

After adding 99.5% ethanol to brown mealworm larvae at a ratio of 1:5 (w/v), they are degreased through a process of extraction in a shaking bath (VS-1205SW1, Vision Scientific Co., Ltd, Daejeon, Korea) for 60 minutes at 40°C. did After repeating this process twice, ethanol was volatilized and dried for 12 hours. After adding 0.25M NaOH to the defatted brown mealworm larvae at a ratio of 1:15 (w/v), they were mixed using a hot plate & magnetic stirrer (Vision Science Co., Korea) at 40 ° C. for 60 minutes. Then, centrifugation was performed for 15 minutes at 4200 rpm at 4° C. using a centrifuge (VS-24SMTi, Vision Science Co., Ltd, Korea), and the supernatant was collected and the pH was adjusted to 4.5. Subsequently, a precipitate was obtained by centrifugation at 4° C. at 4200 rpm for 15 minutes. The obtained precipitate was desalted for 12 hours in a dialysis bag (12KDa MWCO; Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Then, it was lyophilized for 36 hours to obtain the protein extract of the brown mealworm larvae of the present invention (hereinafter, simply abbreviated as 'MPI'). The manufacturing process of brown mealworm larvae protein extract (MPI) is shown in detail in FIG.

<1-3> Manufacture of mealworm larvae protein hydrolysate (MPH) of brown mealworm larvae protein extract

First, the conditions of substrate concentration, enzyme concentration, pH, reaction temperature, and reaction time were set as follows with reference to previous studies in order to establish the optimal manufacturing conditions for the hydrolyzate of the protein extract of brown mealworm larvae. Substrate concentration (1% (w / v)), enzyme / substrate concentration (alcalase & flavorzyme 1% (w / v)), pH (8), reaction temperature (55 ℃), reaction time (each enzyme 12 hours per star, 24 hours total).

1% (w/v) MPI was dispersed in a pH 8.0 buffer solution (Samcheon Chemical Co., Ltd.; Pyeongtaek, Korea), heated at 85°C for 20 minutes, and cooled to 55°C. The dispersion pH was adjusted to 8.0 with 0.1N NaOH and 0.1N HCl. Then, 5 mg of Alcalase (Novozymes, Bagsvaerd, Denmark) per g of the dispersion was added and stirred for 12 hours (20L stirring water bath; SWB-20L03; Cleaver Scientific Ltd.; Warwickshire, UK). Then, 5 mg of flavozyme (Novozymes, Bagsvaerd, Denmark) was added and hydrolysis was performed while maintaining pH 8 at 55° C. for 12 hours. Thereafter, the hydrolyzed dispersion was heated at 100° C. for 10 minutes to inactivate the enzyme, centrifuged at 4° C. at 4200 rpm for 10 minutes to obtain a supernatant, and then freeze-dried the supernatant using a vacuum freeze dryer. Thus, a hydrolyzate (hereinafter simply abbreviated as 'MPH') of the brown mealworm larvae protein extract of the present invention was prepared. Thus prepared MPH was stored at -20 ℃. The manufacturing process of the hydrolyzate (MPH) of the brown mealworm larvae protein extract is shown in detail in FIG.

<1-4> MPI gel formation temperature point derivation

An MPI solution was prepared using distilled water at a concentration of 15% (w/v) and then mixed for 30 minutes. Thereafter, the pH was adjusted to 7 using a 0.1M NaOH, 0.1M HCl solution, and a temperature sweep was measured under the following temperature ramp conditions using a rotational rheometer (AR1500ex, TA Instruments, USA).

Condition: Heating from 20℃ to 90℃ (1℃/min) -> 5 minutes at 90℃

For reference, in preparing the tofu containing the brown mealworm larvae of the present invention, the temperature setting for gel formation of the brown mealworm larva protein must be preceded. The tofu of the present invention was prepared by applying the gel formation temperature of the MPI thus derived.

<1-5> Tofu manufacturing

As raw materials for tofu production, soybean flour (Soy), brown mealworm larvae powder (M), brown mealworm larvae protein extract (MPI), and brown mealworm larvae protein extract hydrolyzate (MPH) were refrigerated and stored at 4 ° C before use. Soybean flour (Soy) was mixed with mealworm larvae powder (M), MPI, and MPH at a ratio of 1:1 (w/w) and mixed with distilled water at a ratio of 1:6 and mixed for 30 minutes. After adjusting the pH of the mixed solution to 9.0 with 0.1N NaOH, the mixture was mixed for 30 minutes to extract proteins in soybean and brown mealworm larvae. Thereafter, okara and soymilk were separated using a filter cloth. The separated soybean milk was adjusted to pH 7.0 with 0.1N HCl, heated to 70℃, the gel formation temperature of brown mealworm larvae protein, and maintained for 10 minutes, then reheated to 95℃, the gel formation temperature of soybean, and maintained for 10 minutes. did Thereafter, the soy milk was cooled to 50 ° C., glucono-delta-lactone (GDL) was added at 2% (w / v) of the soy milk, and transglutaminase (Transglutaminase, Tgase) was added to form 5U Tgase/g protein soy milk and mixed for 2 minutes. The mixture was reacted at 50°C for 4 hours (Tgase reaction temperature) and then at 85°C for 30 minutes (Tgase inactivation and GDL activation temperature). After all the reactions were completed, resting at room temperature for 30 minutes to obtain uncompressed tofu. The tofu manufacturing process of the present invention is shown in detail in FIG. 3, and in Table 1 below, the preparation of tofu with brown larvae produced through this experiment is summarized.

Tofu containing brown mealworm larvae of the present invention division Tofu Sample Name Ingredients control group S soy 100% experimental group SM 50% soy + 50% mealworm larvae (M) SMPI soy 50% + mealworm protein isolate (MPI) 50% SMPH 50% soy + 50% mealworm protein hydrolysate (MPH)

<1-6> Analysis of general components

The content of general ingredients for each tofu produced was analyzed according to the AOAC method. Crude moisture content was investigated by heating each tofu sample in an oven at 105 °C overnight, and the dried product was used for general component analysis. Ash content was determined by incinerating each tofu sample in a muffle furnace at 600°C for 5 hours. The crude fat content was measured according to the Soxhlet method using an auto extractor (HSOX-6; Hanil Co., Seoul, Korea), and the crude protein content was measured using an auto digestor (HDG-P, Hanil Co.) and a distillation device ( HKD-P, Hanil Co.) was estimated according to the Kjeldahl method with a factor of 6.25N. Carbohydrate content was quantified by excluding crude water content, crude ash content, crude protein and crude fat content from 100%.

<1-7> Measurement of constituent amino acid content

Freeze-dried tofu samples were used to measure amino acid content. That is, 0.05 g of lyophilized tofu powder was placed in a vacuum hydrolysis tube (Thermo Fisher Scientific), 2 mL of 6N HCl was added, and then heated in a dry oven for 24 hours (105° C.). After 24 hours, filtration and 100-fold dilution were analyzed by high-performance liquid chromatography (HPLC; YL 9100, YL instrument; Anyang, Korea) (see Table 2). Crystals were monitored by fluorescence detection at 37° C. using a 250 nm excitation wavelength (Ex) and a 395 nm emission wavelength (Em). Mobile phases A and B used for elution were 10% (v/v) eluent A buffer (Waters) and 60% (v/v) acetonitrile (HPLC grade; Burdick &Jackson; Muskegon, MI, USA), respectively. The amino acid concentration of each tofu sample was determined by calibration with a standard amino acid (Waters).

Gradient Profiles for High-Performance Liquid Chromatography (HPLC) Separations for Amino Acid Composition Analysis Time Flow rate %A %B Curve Initial One 100 0 6 0.5 One 98 2 6 15 One 93 7 6 19 One 90 10 6 32 One 67 33 6 33 One 67 33 6 34 One 0 100 6 37 One 0 100 6 38 One 100 0 6 50 One 10 0 6

<1-8> Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was used to analyze proteins present in soymilk and tofu with some modifications according to the method described by Kao, Su, and Lee (2003) (Effect of calcium sulfate concentration in soymilk on the microstructure of firm tofu and the protein constitutions in tofu whey.Journal of Agricultural and Food Chemistry, 51(21), 6211-6216.). Briefly, 50 mg of lyophilized tofu sample was dispersed in 1 mL of Tris-glycine buffer (86 mM Tris-90 mM glycine-4 mM Na2EDTA, pH 8.0) and placed in a sonicating water bath (JAC Ultrasonic 2010P; Jinwoo Co., Ltd.) for protein extraction. Engineering; Gyeonggi-do) for 1 hour. The extract was then centrifuged at 12,000×g and 4° C. for 20 minutes. The protein concentration of the supernatant was estimated using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Continuous buffer system (0.025 M Tris-HCl [Sigma-Aldrich Chemical Co.; St. Louis, MO, USA], 0.192 M glycine [Daejung], and 0.1% w/v SDS [Bio-Rad Laboratories Inc.; Hercules, California, USA], pH 8.3) and electrophoresis was performed using 4-20% precast gel (Luminano; Seoul, Korea). A protein extract (containing 35 μg protein) was prepared under reducing conditions. Protein molecular weight was estimated using a 6-170 kDa marker (GenDEPOT; Barker, TX, USA). Electrophoresis was performed at 125V for 80 minutes. Separated protein bands were stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories Inc.) overnight and then destained with a destaining solution containing 50% (v/v) methanol and 10% (v/v) acetic acid. did

<1-9> pH change during storage period

During the storage period, organic acids and acetic acids are produced in tofu due to decay and decomposition by microorganisms. This lowers the pH of the tofu steeping liquid, so the degree of pH reduction within the same period is related to the shelf life of the tofu. In this experiment, 2 g of tofu was immersed in 20 mL of distilled water, stored at 4° C. for 18 days, and the pH of the immersion solution was measured using a pH meter (720 p Istek Co.; Seoul, Korea).

<1-10> In vitro ( In vitro ) ileum digestibility

The digestibility of tofu was measured by the in vitro ileal digestibility method described by Recharla et al. (Recharla, N., Kim, D., Ramani, S., Song, M., Park, J. , Balasubramanian, B., ... Park, S. (2019).Dietary multi-enzyme complex improves in vitro nutrient digestibility and hind gut microbial fermentation of pigs.PLoS One, 14(5), e0217459.). Specifically, after mixing 25mL sodium phosphate buffer (0.1M, pH 6.0) and 10mL 0.2M HCl solution with 1 g of sample dry matter (DM), the pH of the mixed solution was adjusted to pH 2 (pepsin activity pH). Thereafter, a pepsin solution (10 mg/mL, Sigma-Aldrich Chemical Co.) and a chloramphenicol solution (0.5 g/100 mL with 99.5% ethanol, Sigma-Aldrich Chemical Co.) were added and reacted at 39° C. for 6 hours. Then, sodium phosphate buffer (0.2M, pH 6.8) and 0.6M NaOH solution were added, the pH of the mixture was adjusted to 6.8 (pancreatin active pH), and 1mL porcine pancreatin solution (50mg/mL, Sigma-Aldrich Chemical Co.) was added to the flask. After reacting at 39° C. for 18 hours, a 20% sulfosalicylic acid solution was added and reacted at room temperature for 30 minutes. Then, the sample was filtered through a crucible glass filter (FN1200-2G; Corning Life Science Co.; Oneonta, NY, USA) containing 500 mg Celite. Each test flask was washed with 1% sulfosalicylic acid solution, 95% ethanol and 99.5% acetone, and the residue was poured into a crucible. Finally, the crucible was heated at 100 ° C. overnight and allowed to cool to weigh the residue, and the digestibility was calculated using the following formula.

<1-11> Measurement of changes in gel strength and flow characteristics

The gel strength of tofu was measured using a texture analyzer (COMPAC-100II, Sun Sci. Co., Ltd.; Tokyo, Japan). Tofu was prepared in a cylindrical stainless steel cup (diameter 38 mm, height 68 mm). The gel strength measurement conditions were as follows; adapter no. 25 (dia: 20 mm), distance 7 mm, table speed 60 mm/min, replicate. Three replicate tests were performed on each sample at room temperature. A temperature sweep test was conducted to measure the flow characteristics of tofu over time. After adding two types of coagulants (Tgase, GDL) to soymilk, the storage modulus (G') and loss modulus (G”) were measured using a rotational rheometer (Anton Paar MCR 302 rheometer). The loss factor, tan δ, is expressed as a ratio (G”/G’). The temperature was maintained at 50 ° C for 4 hours, heated to 85 ° C at 5 ° C / min, and maintained for 30 minutes. Thereafter, cooling was performed at a rate of 5 °C/min to 25 °C. In addition, the modulus of elasticity according to the frequency (1-100 Hz) at a constant strain (0.5% strain) was observed through a frequency sweep test of the prepared tofu.

<1-12> Fourier transform infrared spectroscopy

Infrared spectra of freeze-dried tofu samples were obtained using FTIR spectroscopy (Cary 630; Agilent Inc.; Santa Clara, CA, USA). A sample was placed on the cleaned crystal and the DialPath was rotated to bring it into contact with the sample. Spectra were obtained from accumulations of 40 scans/min at 400-4000 cm ⁻¹ . Data were analyzed using Resolution Pro Software (Agilent Technologies, version 5.2.0 (CD 846)) and MicroLab PC software (Agilent Technologies).

<1-13> Scanning electron microscope (SEM)

The microstructure of the surface of tofu specimens was evaluated using a field emission scanning electron microscope (Quanta 650 FEG; Eindhoven, The Netherlands). Lyophilized tofu samples were cut into cubes less than 2 mm in size and coated with platinum (208 HR; Cressington, England). The microstructure was observed at 100×, 500×, 1000×, and 5000× at an accelerating voltage of 10 kV.

<1-14> Evaluation of antioxidant activity

[Measurement of total phenol content and flavonoid content]

Total phenolic content (TPC) of tofu samples was measured using the Folin-Ciocalteau reagent (Dixit, Bhatnagar, Kumar, Rani, Manjaya, & Bhatnagar, 2010). In detail, each 1 mg/mL sample was extracted using 70% (v/v) ethanol at room temperature and Whatman No. Filtered through 4 filter paper. Then, 30 μL sample extract was added to 120 μL distilled water, 30 μL Folin's reagent and 70 μL 10% sodium carbonate solution. After incubating the homogenate at 20° C. for 2 hours, absorbance at 725 nm was monitored using a microplate reader (Multiskan, Thermo Fisher Scientific). Total phenol content (TPC) was expressed as gallic acid equivalents (GAE) per gram of lyophilized sample.

Total flavonoid content (TFC) was measured by slightly modifying the method described by Kim, Choi, Yu, Kim, Lee, and Lee (2012). Specifically, a 0.2 g sample was dispersed in 80% (v/v) ethanol and stirred. The dispersion was prepared in Whatman No. Filtered through 4 filters. Then, 0.5mL sample extract was mixed with 5mL 90% (v/v) diethylene glycol and 0.5mL 1N NaOH. The mixture was vortexed and held at ambient temperature (25° C.) for 60 minutes. Then, absorbance was measured at 420 nm using a UV/visible spectrophotometer (Ultrospec 2100 pro.; Amersham Biosciences Co.; Piscataway, NJ, USA). Total flavonoid content (TFC) was expressed as mg naringin mg equivalents/g sample by preparing a standard calibration curve using naringin (Sigma-Aldrich Chemical Co.).

[DPPH and ABTS+ radical scavenging activity]

Antioxidant activity was measured through ABTS+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. ABTS + radical scavenging activity was 70% ( v/v) After mixing 20 uL of sample extract extracted with ethanol and 180 uL of ABTS+ solution (Sigma-Aldrich Chemical Co.), it was left in the dark for 10 minutes, and the absorbance was measured at 734 nm. DPPH radical scavenging activity was 70% After mixing 200 uL of sample extract extracted with (v/v) ethanol and 50 uL of DPPH solution (0.005 g/100mL 99.5 (v/v) ethanol, Sigma-Aldrich Chemical Co.), leave it in the dark for 30 minutes. The absorbance was measured at 520 nm, and the radical scavenging ability was calculated by the following formula.

Here, Abs _sample represents the absorbance of the test sample, Abs _blank represents the absorbance of the blank, and Abs _control represents the absorbance of the control group. Results were expressed as the sample concentration required for 50% inhibition (IC ₅₀ ). Ascorbic acid (Sigma-Aldrich Chemical Co.) was used as a positive control.

<1-15> Evaluation of anti-inflammatory activity

[3-(4,5-dimethylazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay]

Freeze-dried tofu samples were extracted using distilled water (10 mg/mL) at room temperature for 48 hours according to the method described by Hong, Yang, Kim, Eom, Lew, and Kang (2012) (Hong, J.- W., Yang, G.-E., Kim, YB, Eom, SH, Lew, J.-H., & Kang, H. (2012) Anti-inflammatory activity of cinnamon water extract in vivo and in vitro LPS -induced models. BMC Complementary and Alternative Medicine, 12(1), 1-8.). Whatman no. After filtering with 4 filter paper, the extract was used for analysis. Cell viability was measured using the MTT assay. Specifically, C2C12 cells (5 x 10 ⁴ cells/well) were seeded in a 96-well plate and incubated at 37°C for 24 hours. Different concentrations of the extract were then added to each well along with the culture medium. After culturing at 37° C. for 12 hours, the culture medium was removed. Fresh culture medium (Hyclone; Logan, UT, USA) containing 0.5 mg/mL MTT reagent (Sigma-Aldrich Chemical Co.) was added and reacted at 37°C for 4 hours. The medium was then removed and 50 μL dimethyl sulfoxide (DMSO) was added to solubilize the purple formazan crystals. Absorbance was measured at 540 nm using a microplate reader (SPL; Seoul, Korea).

[In vitro ( In vitro ) LPS treatment and enzyme-linked immunosorbent assay (ELISA)]

C2C12 cells were maintained in Dulbecco's modified Eagle medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Hyclone). After inoculating 3×10 ⁵ cells/well in a 96-well plate, inflammation was induced by reacting with 200ng/mL LPS for 30 hours. The inflamed cells were cultured for 18 hours with 200 μg/mL sample extract and the levels of TNF-α, IL-1β, and IL-6 in the cell culture medium were measured using an ELISA kit (TNF-α, IL-1β, and IL-6 mouse ELISA kits; Komabiotech; Seoul, Korea) were used for evaluation.

<1-16> Inhibitory effect of inflammatory cell expression in dexamethasone-induced muscular atrophy mouse model

[Mouse model of muscular dystrophy induced by dexamethasone]

8-week-old male mice (C57BL, ~25 g) were reared at room temperature and under 12/12 hour light/dark cycle conditions, and muscle atrophy was induced by injecting 30 uL of dexamethasone (DEXA) three times a week. As for the diet, Purina mouse diet chow (ND) and ND were supplied with a diet in which 15% was replaced with freeze-dried tofu samples, and were freely consumed with drinking water ( ad libitum ). Mice were randomly divided into six groups as shown in the table below.

Dexamethasone treatment and dietary composition by mouse group group Dexamethasone treatment or not diet DMSO con X ND DEXA-con O ND DEXA-S O ND (85%) + S (15%) DEXA-SM O ND (85%) + SM (15%) DEXA-SMPI O ND (85%) + SMPI (15%) DEXA-SMPH O ND (85%) + SMPH (15%)

[Weight and grip strength test]

The body weight of the mice was expressed by measuring the difference between the first day and the last day (the day of sacrifice). The mouse's grip strength (unit: N), which can be an indicator of muscle mass, was measured on the last day of sacrifice using a Bioseb grip strength meter (BIO-GS3; BIOScience and Experimental Biology; Largo, FL, USA), which was divided by body weight. Calculated as N/g body weight.

[Histological staining and cross-sectional area measurement]

The calf muscles (calf muslce) of the sacrificed mice were fixed with a 10% formalin solution and then cut off by embedding using paraffin. It was transferred to a glass slide, stained with hematoxylin and eosin (H&E, Sigma-Aldrich Chemical Co.), and observed through a microscope. Images were analyzed using Image J software (version 1.43, National Institute of Health; Bethesda, MD, USA). The average value was calculated by measuring the area of 10 muscle fibers.

[Real-time polymerase chain reaction (PCR)]

RT-qPCR was performed as previously described (Oh et al., 2018; ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome. Metabolism , 85, 286-297.). In detail, total RNA was isolated from calf muscle or C2C12 cells using RNAiso Plus reagent (Takara; Shiga, Japan), and the amount of RNA was measured with a spectrophotometer (SPL). Then, cDNA was synthesized using the PrimScriptTM RT Reagent Kit with gDNA Eraser (Takara), and then mixed with specific primers, SYBR Premix Ex TaqTM II, ROX Plus. The relative expression of muscle degradation related genes (myostatin, MuRF 1, Atrogin-1) and muscle synthesis related genes (MyCH isofomers) was quantified relative to the expression level of GAPDH, a housekeeping gene (internal control), using the 2 ^-ΔΔ Ct method. calculated through

<1-17> Measurement of expression levels of genes related to grip strength and muscle loss according to SMPH and MPH diets in dexamethasone-induced muscular atrophy mouse models

[Mouse model of muscular dystrophy induced by dexamethasone]

8-week-old male mice (C57BL, ~25 g) were reared at room temperature and under 12/12 hour light/dark cycle conditions, and muscle atrophy was induced by injecting 30 uL of dexamethasone (DEXA) three times a week. As for the diet, Purina mouse diet chow (ND) and ND were supplied with a diet in which 15% was replaced with freeze-dried tofu samples, and were freely consumed with drinking water ( ad libitum ). Mice were randomly divided into 2 groups as shown in the table below.

Dexamethasone treatment and dietary composition by mouse group group Dexamethasone treatment or not diet SMPH+Dexa O ND (85%) + SMPH (15%) MPH+Dexa O ND (85%) + MPH (15%)

[Grip strength test]

The grip strength of the mouse group (SMPH+Dexa) fed a diet supplemented with 15% of SMPH tofu powder and the mouse group (MPH+Dexa) fed a diet supplemented with 15% MPH powder was measured using a Bioseb grip strength meter (BIO-GS3; The mouse was measured immediately before sacrifice using BIOScience and Experimental Biology; Largo, FL, USA). Grip strength was expressed as the value (N/g body weight) divided by the force (N) right before the grip was released when the mouse holding the grid was pulled.

[Real-time polymerase chain reaction (PCR)]

RT-qPCR was performed as previously described (Oh et al., 2018; ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome. Metabolism , 85, 286-297.). In detail, total RNA was isolated from the calf muscles of mice of the SMPH+Dexa and MPH+Dexa groups using RNAiso Plus reagent (Takara; Shiga, Japan), and the amount of RNA was measured with a spectrophotometer (SPL). Then, cDNA was synthesized using the PrimScriptTM RT Reagent Kit with gDNA Eraser (Takara), and then mixed with specific primers, SYBR Premix Ex TaqTM II, ROX Plus. The relative expression of muscle degradation related genes (myostatin, MuRF 1, Atrogin-1) was calculated by relative quantification with the expression level of GAPDH, a housekeeping gene (internal control), using the 2 ^-ΔΔ Ct method.

<1-18> Statistical analysis

All analyzes except SDS-PAGE, rheological measurements, FTIR spectral analysis and SEM were performed in triplicate. Data were analyzed using the SPSS statistical package (SPSS 24.0; IBM; Chicago, IL, USA). Significant differences between data were tested using one-way analysis of variance (ANOVA) at p<0.05, and post hoc comparisons were performed using Duncan's multiple range test. Comparisons between the two groups were performed using an independent 2-sample t-test.

2. Results

<2-1> MPI gel formation temperature setting

A temperature sweep test was performed to measure the gel formation temperature of MPI. The junction between the storage modulus and the loss modulus becomes the gel point, and the loss factor, tan δ, is expressed as a ratio (G”/G'), so the point at which the value of tan δ becomes less than 1 This is also the gel formation temperature. As a result, as shown in FIG. 2, in MPI, G' and G" intersect at around 69.8 ° C (see FIG. 2a), and tan δ is less than 1, so the gel point can be confirmed (see FIG. 2b).

<2-2> General Ingredient Content of Tofu

The approximate composition of tofu is shown in detail in Table 5 below. SM had higher water (85.34%) and crude fat (0.85%) contents than S, but lower crude protein (4.22%), ash (0.55%), and carbohydrate (9.04%) contents. SM showed the highest crude fat content because mealworm larvae contained a large amount of crude fat (about 33% of dry weight). SMPI showed the highest crude protein (10.99%) content. The crude protein (6.63%) content of SMPH was lower than that of SMPI, and the crude ash (3.10%) content of SMPH was high, which is thought to be due to the addition of acid or base to adjust pH in the hydrolysis process. These results suggest that replacement of 50% soybean flour with mealworm larvae, MPI and MPH during tofu manufacturing has a significant effect on major constituents, especially protein and lipid content (p<0.05).

Content of general ingredients in tofu Samples Moisture (%) Protein (%) Lipid (%) Ash (%) Carbohydrates (%) S 82.44± ^0.11b 6.53± ^0.19b 0.44± ^0.01c 0.85± ^0.05b 9.74±0.24 ^a SM 85.34± ^0.61a 4.22± ^0.02c 0.85±0.05 ^a 0.55± ^0.03c 9.04±0.66 ^a SMPI 82.43± ^0.12b 10.99± ^0.44a 0.24±0.01 ^d 0.83± ^0.01b 5.51± ^0.54c SMPH 82.92± ^0.15b 6.63± ^0.06b 0.76± ^0.02b 3.10±0.15 ^a 6.64± ^0.22b S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.

<2-3> Component Amino Acid Content

17 amino acids were detected in tofu (see Table 6). There was no significant difference in essential amino acid, branched chain amino acid, and total amino acid contents of S and SM (p>0.05). However, the total amino acid contents of SMPI (63.82 g/100 g sample) and SMPH (46.97 g/100 g sample) were found to be quite high. In addition, the essential amino acid content of SMPI (24.28 g/100 g sample) and SMPH (18.22 g/100 g sample) was significantly higher than that of S (10.52 g/100 g sample). In addition, the branched chain amino acid content of SMPI (9.92 g/100 g sample) and SMPH (7.81 g/100 g sample) was higher than that of S (4.14 g/100 g sample). These results indicate that the amino acid content of tofu can be improved by replacing soy flour with MPI or MPH when preparing tofu. The methionine and cysteine content of vegetable proteins, such as soybean protein, is generally lower than that of animal proteins, such as mealworm larvae proteins, which are considered high quality. In fact, S had the lowest content of sulfur amino acids such as methionine and cysteine compared to SM, SMPI, and SMPH. Also, insects (particularly mealworm larvae) are high in branched-chain amino acids, which helps prevent muscle wasting or breakdown. This suggests that tofu, which is rich in branched-chain amino acids such as leucine, isoleucine, and valine, will be helpful for the elderly with muscle loss.

Constituent amino acid content of tofu Samples S SM SMPI SMPH ASP 4.54± ^1.47b 4.37± ^0.26b 7.86±2.23 ^a 5.93± ^0.40ab SER 2.02± ^0.54b 2.05± ^0.12b 3.93±0.74 ^a 2.84± ^0.25b GLU 4.67± ^2.77b 5.92± ^0.27ab 9.60± ^2.21b 6.66± ^1.45ab GLY 1.54± ^0.34c 1.51± ^0.06c 3.70±0.66 ^a 2.57± ^0.30b HIS 0.78± ^0.18c 0.88± ^0.08c 1.91±0.27 ^a 1.31± ^0.16b ARG 0.80± ^0.07b 0.80± ^0.45b 1.81±0.30 ^a 1.40±0.15 ^a THR 0.61± ^0.05b 0.61± ^0.34b 1.37±0.23 ^a 1.06± ^0.11a ALA 1.48± ^0.49b 1.58± ^0.10b 3.79± ^0.97a 2.80±0.28 ^a PRO 1.65± ^0.41b 2.21± ^0.13b 3.29± ^0.66a 2.36± ^0.22b CYS 0.92± ^0.12c 1.10± ^0.44bc 2.07±0.34 ^a 1.62± ^0.11ab TYR 1.13± ^0.16c 1.60± ^0.09c 5.26±0.44 ^a 3.79± ^0.41b VAL 1.31± ^0.30b 1.56± ^0.10b 3.71±0.83 ^a 2.97± ^0.26a MET 0.28± ^0.06c 0.32± ^0.02c 0.73± ^0.12a 0.53± ^0.05b LYS 3.38± ^1.26b 3.32± ^0.18b 5.72± ^1.61a 4.13± ^0.24ab ILE 0.68± ^0.03b 0.74± ^0.17b 1.36± ^0.29a 1.13± ^0.11a LEU 2.14± ^0.47c 2.20± ^0.19c 4.85±1.04 ^a 3.71± ^0.33b PHE 1.33± ^0.22c 1.39± ^0.05c 2.86±0.24 ^a 2.15± ^0.24b Hydrophilic 10.42±2.36 ^c 11.50± ^1.19c 24.28±4.80 ^a 18.22± ^1.77b Hydrophobic 18.86± ^5.64b 20.64± ^1.18b 39.53 ± 8.34 ^a 28.74± ^3.18b Total EAAs ¹⁾ 10.52±1.97 ^c 11.01± ^0.63c 22.50± ^4.61a 17.00± ^1.49b Total BCAAs ²⁾ 4.14± ^0.61b 4.50± ^0.32b 9.92±2.16 ^a 7.81±0.70 ^a Total amino acids 31.78± ^7.42b 31.45± ^1.81b 63.82± ^13.14a 46.97± ^{4.94b 1)} essential amino acids
²⁾ branched chain amino acids
S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.
According to ^ad Duncan's multiple range test (p<0.05), there is a significant difference between different initials in each treatment group

<2-4> SDS-PAGE

SDS-PAGE was performed to track changes in protein subunits in soymilk and tofu.

As a result, as shown in FIG. 4, two major protein fractions, 7S globulin (β-coglycinin) and 11S globulin (glycinin), which play an important role in gelation and protein network formation, were detected in soymilk (lanes 1-4). After heating and coagulating the soymilk, the intensity of the bands representing 7S and 11S, except for SMPH, rapidly decreased. This suggests that large proteins aggregate and AB subunits are formed by SS bridges between acidic and basic polypeptides. However, the aggregation strength of SMPH was relatively low due to its low molecular weight detected at the bottom of lane 8.

<2-5> pH change during storage period

The pH change after storage for 18 days is shown in FIG. 5 . It was found that the pH of tofu continuously decreased with the lapse of storage period (4.42~4.45 on day 0 and 3.81~4.20 on day 18). The decrease in pH due to storage may be due to an increase in organic acids such as lactic acid and acetic acid produced by microbial decomposition. The pH of S and SMPI changed from 4.45 on day 0 to 4.01 and 3.94 on day 18, respectively. SM showed the greatest drop in pH from day 0 to day 18 (4.42-3.81), whereas the pH value of SMPH showed the least decrease from day 0 to day 18 (4.43-4.20).

<2-6> In vitro ( In vitro ) ileum digestibility

The digestibility of tofu is shown in FIG. 6 . In vitro ileal digestibility of all samples showed a significant difference (p<0.05). S showed the lowest digestibility (58.35%) due to anti-nutritional components including phytate, trypsin inhibitor, tannins or protein bound starch. SM showed a higher digestibility than S (64.61%) because the digestibility of animal protein (mealworm larvae) was higher than that of plant protein (soybean). SMPI was found to be more digestible than SM because the chitin level in SM is higher than that in SMPI and chitin reduces digestibility in vitro. In particular, the SMPH sample showed a remarkably superior digestibility compared to other samples.

<2-7> Gel strength and rheological properties

The gel strength of the tofu is shown in detail in FIG. 7 . The gel strength of SM (135.0 g/cm 2 ) was determined to be significantly lower than that of S (504.3 g/cm ² ). The gelation properties of thermally induced gels are affected by protein hydrophobicity and lipids can inhibit protein gelation due to the interference of protein-protein-polymer interactions. It can also be explained by a decrease in the thiol group content due to lipid oxidation. On the other hand, SMPI (927.9 g/cm ² ) showed the highest gel strength because it had the highest protein concentration. A high protein concentration is related to the degree of exposure of hydrophobic regions and sulfhydryl groups during heat denaturation, which in turn promotes soymilk coagulation. Conversely, SMPH (32.0 g/cm ² ) exhibited the lowest gel strength because the degree of gel formation was low due to MPH's inherent properties such as low molecular weight. Rheological changes during the tofu manufacturing process can be a factor in determining the quality of tofu. As shown in FIGS. 8 and 9 , the time- and temperature-dependent variation patterns of the storage modulus (G′), loss modulus (G″), and loss modulus (tan δ) were similar. After heating at 50 °C, the viscoelasticity (G' and G") of all samples gradually improved with a cross-linked network formed by TGase. The sharp increase in G' in the initial heating stage suggests the generation of a gel network and formation of a gel. Compared to S, in SMPI, G' and G" increased more rapidly. It also increases slowly in SM and SMPH, indicating an order of gel formation initiation rates. During heating from 50 °C to 85 °C, the viscoelasticity decreased due to non-covalent bond breakage at high temperatures. In addition, G' and G" increased significantly in all samples, suggesting gel network strengthening in the cooling step. Tan δ decreased in all samples during the first few minutes of heating, indicating a firm and elastic protein network. Tan δ of The smaller the value, the closer it is to the solid property, and the larger the value, the closer it is to the liquid property. Therefore, it can be seen that the value of Tan δ, which is G”/G’ of the manufactured tofu, is SMPH > SM > S > SMPI, and SMPI is the most solid close, and SMPH was confirmed to be the closest to liquid.

In addition, the elastic modulus according to the frequency (1-100 Hz) at a constant strain (0.5% strain) was observed through a frequency sweep test of the prepared tofu (see FIG. 10). A frequency sweep provides information about the viscous and elastic profiles of a sample under constant strain. As a result, as shown in FIG. 10, it was found that G' and G" are SMPI > S > SM > SMPH. As G′ and G″ according to frequency are larger, they are closer to solid properties, and smaller ones, they are closer to liquid properties. It can be seen that the above results are consistent with the trend in FIG.

<2-8> FTIR Spectra

The chemical structure of the tofu samples was determined using FTIR spectrophotometry, and the results are shown in detail in FIG. 11 . The wide band between 3500 and 3200 cm ^-1 corresponds to the hydroxyl group (-OH) group, and the band around 3274 cm ^-1 (peak No. 1) may be due to the hydroxyl group (-OH) stretching vibration found in all heads. The peaks at 2916 cm ^-1 and 2849 cm ^-1 (peaks 2 and 3) correspond to aldehyde CH stretching vibrations, which are present in cellular components such as proteins, carbohydrates and lipids, such as methyl groups (-CH3) and methylene groups (- CH2-). The peak (peak 4) at 1742 cm ^-1 corresponds to the ester carbonyl functional group. Peaks 2, 3 and 4 are present in S and SM samples due to triglyceride content, whereas peaks 2, 3 and 4 are not present in SMPI and SMPH due to low lipid content due to fat removal. The peaks at 1628 cm ^-1 and 1541 cm ^-1 (peaks 5 and 6) correspond mainly to amide I, which is attributed to C=O bond vibrational bending, which results in several folded component bands representing various protein fragment secondary structures. and amide II is attributed to NH bending, respectively. Amide I band spectrum consists of α-helices (1650-1660 cm ^-1 ), β-sheets (1618-1640 cm ^-1 and 1670-1690 cm ^-1 ), β-turns (1660-1670 cm ^-1 ) and random coils. (1690-1700 cm ^-1 ); The α-helix and β-turn content decreased after enzymatic hydrolysis. Moreover, the amide II band, which is mainly a mixture of in-plane NH bending and CN stretching, also consistently decreased with enzymatic degradation of the peptide backbone. Therefore, SMPH was found to have the lowest peak 5 and 6 intensities. The peak at 1039 cm ^-1 (peak 7) indicates that the complex was formed by the electrostatic interaction between the carboxyl group (-COO-) of most proteins and the amine group (-NH3+) of chitosan, also in the soybean protein-chitosan interaction. hydrogen bonding occurs. This suggests that chitosan extracted from the shells of mealworm larvae of SM affects the peak intensity at 1039 cm ^-1 , so SM exhibits the highest intensity at 1039 cm ^-1 due to chitosan vibration.

<2-9> Microstructure of tofu

In order to confirm the structural characteristics of the tofu, surface images at magnifications of 500-5000 were visualized using SEM, and the results are shown in detail in FIG. 12 . S was found to have a much denser and more uniform structure than the other samples. Compared to S, SM was found to have honey-comb-shaped pores due to dispersed fat globules. On the other hand, SMPI exhibited large cavities and rough structures due to protein aggregation, and SMPH showed dense and homogeneous small pores due to the hydrolyzate being evenly dispersed in the protein aggregates.

<2-10> Total phenol content and total flavonoid content

Total phenol content and total flavonoid content of SM, SMPI and SMPH were measured differently depending on the mealworm larval protein type used.

As a result, as shown in FIG. 13 and Tables 7-8, compared to S (8.27 mg GAE / g), SM, SMPI, and SMPH showed significantly higher total phenol content due to the high phenol content of mealworm larvae (12.83 -22.66 mg GAE/g). SMPH showed the highest total phenol content (22.66 mg GAE/g) because it releases bound polyphenols in a free state through hydrolysis. On the other hand, S had the highest total flavonoid content (0.72 mg NE/g), and SMPI and SMPH had the lowest total flavonoid content (0.40 and 0.47 mg NE/g, respectively). This may be because soybeans are rich in isoflavones, including genistein and daidzein. On the other hand, SMPH was investigated to have a higher total flavonoid content than SMPI.

Total phenol content (unit: concentration (mg GAE/g)) sample average Standard Deviation S 8.27 0.48 SM 13.77 1.11 SMPI 12.83 0.40 SMPH 22.66 2.59 S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.

Total flavonoid content (unit: naringin mg equivalents/g) sample average Standard Deviation S 0.72 0.05 SM 0.58 0.08 SMPI 0.40 0.03 SMPH 0.47 0.03 S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.

<2-11> ABTS+ and DPPH free radical scavenging activity

Antioxidant activity of tofu was determined according to ABTS+ and DPPH free radical scavenging activity compared to positive control (ascorbic acid). The IC ₅₀ values for ascorbic acid are 0.02 μg/ml and 0.03 μg/ml, respectively.

As a result, as shown in FIG. 14 and Tables 9-10, the IC ₅₀ values for ABTS+ free radical scavenging activities of S, SM, SMPI, and SMPH were 7.00, 5.00, 2.31, and 1.56 μg/mL, respectively. Also, the DPPH free radical scavenging activity showed a similar trend to that of ABTS+. SMPH had the lowest IC ₅₀ values in both ABTS+ and DPPH activities (p<0.05). This may be because the reactive groups of bioactive peptides are exposed through proteolysis, increasing antioxidant activity. The antioxidant capacity was related to the total phenolic content and appeared to follow the same trend (see Figure 13). This is because phenolic compounds are secondary metabolites with antioxidant effects.

ABTS+ radical scavenging activity (unit: IC _50, μg/mL) sample average Standard Deviation S 7.00 0.15 SM 5.00 0.10 SMPI 2.31 0.10 SMPH 1.56 0.02 vitC 0.02 0.00 S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.

DPPH radical scavenging activity (Unit: IC _50, μg/mL) sample average Standard Deviation S 0.31 0.02 SM 0.41 0.07 SMPI 0.13 0.01 SMPH 0.11 0.01 vitC 0.03 0.00 S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.

<2-12> Cell viability

To confirm the cytotoxicity of tofu, C2C12 cells were treated with tofu extract at a concentration of 0 to 2000 μg/ml for 24 hours, and then MTT assay was performed.

As a result, as shown in FIG. 15, no significant difference in survival rate was found in the concentration range of 0-200 μg/mL. Accordingly, an inflammatory cytokine expression inhibition experiment was conducted at a concentration of 200 μg/mL. On the other hand, in the concentration range of 1000-2000 μg/mL, the SMPH-treated group had the highest cell viability, but there was no significant difference from other treatments (p>0.05).

<2-13> LPS-induced apoptosis inhibitory effect

In order to determine the inhibition rate of inflammatory cell expression of the sample, the expression levels of IL-6, TNF-a, and IL-1b, which are inflammatory cytokines in the blood, were observed using lipopolysaccharide (LPS) in C2C12 cells.

For reference, the production of pro-inflammatory cytokines such as TNF-α, IL-1b, and IL-6 is essential to determine the initial inflammation that can develop into systemic inflammatory response syndrome. In addition, LPS is a potent pro-inflammatory activator of macrophages that function as innate immune cells both in vivo and in vitro. Inflammation-inducing cytokines in the blood promote the degradation of myofibrillar proteins to reduce protein synthesis, resulting in direct muscle wasting.

As a result, as shown in FIG. 16 and Table 11, the expression of pro-inflammatory cytokines was markedly increased in cells upon LPS treatment, and the increased expression of pro-inflammatory cytokines was markedly reduced when tofu extract was treated together. I was able to confirm. In particular, treatment with SM, SMPI, and SMPH significantly reduced the expression of pro-inflammatory cytokines compared to S. These results suggest that SM, SMPI, and SMPH have higher inhibitory ability against LPS-induced TNF-α compared to S.

Expression level of pro-inflammatory cytokines TNFa IL-1b IL-6 Con 1±0.17 1±0.07 1±0.29 S 0.67±0.05 1.18±0.29 0.67±0.05 SM 0.7±0.33 1.21±0.33 1.24±0.49 SMPI 0.79±0.16 1.43±0.33 1.22±0.18 SMPH 0.86±0.33 1.25±0.35 0.93±0.2 LPS 14.01±1.6 8.83±1.63 5.18±2.03 LPS+S 3.81±0.96 4.84±0.85 3.59±1.7 LPS+SM 1.77±0.73 1.72±0.46 0.8±0.18 LPS+SMPI 1.51±0.26 1.79±0.23 0.84±0.07 LPS+SMPH 0.54±0.26 0.73±0.14 0.43±0.11 S: tofu made with 100% soy flour;
SM: tofu made of 50% soy flour and 50% brown mealworm larvae powder;
SMPI: tofu made with 50% soy flour and 50% brown mealworm larvae protein extract (MPI);
SMPH: Tofu made with 50% soy flour and 50% hydrolyzate (MPH) of brown mealworm larvae protein extract.

<2-14> Weight change

In a mouse model of muscular atrophy induced by dexamethasone (DEXA), changes in body weight of mice according to tofu consumption were measured.

For reference, when dexamethasone, a synthetic glucocorticoid, is administered, muscle protein is broken down and weight is reduced. A number of studies have shown that mice treated with dexamethasone experience weight and muscle loss. Dexamethasone stimulates the production of reactive oxygen species (ROS) in vivo and in vitro, and ROS is one of the factors that may play an essential role in inducing muscle loss due to oxidative damage of muscle by aging-related overproduction of ROS.

As a result, as shown in FIG. 17, it was found that the body weight of the mice was significantly lowered after treatment with dexamethasone, except for the DEXA-SM (-0.07 kg) and DEXA-SMPH (-0.39 kg) groups. It was confirmed that there was no significant difference in weight change between the SM and SMPH intake groups and the DMSO control group even though they were treated with dexamethasone. Through the above results, it was found that intake of SM and SMPH alleviated muscle atrophy caused by dexamethasone in mice.

<2-15> Grip Strength and Proximal Cross-section Area

In a mouse model of dexamethasone (DEXA)-induced muscular atrophy, grip strength of the forelimbs was compared to determine whether tofu intake improved muscle function.

As a result, as shown in FIG. 18, the DEXA-administered group, except for the DEXA-SMPH (0.044 N/g body weight) group, showed a significant decrease in gripping force, and among them, the DEXA-SMPI group had the lowest (0.030 N/g body weight) group. g body weight).

In addition, as shown in FIG. 19, the muscle fiber root area was significantly lower in the DEXA-treated group (1562.70 μm ² ) compared to the DMSO control group (1562.70 μm ² ), and in the SM-treated group (1378.33 μm ² ) among the treated groups. The reduction in near-section area was the lowest, followed by S (1283.80 μm ² ), SMPH (1259.83 μm ² ), and SMPI (1169.37 μm ² ). These results suggest that SM and SMPH intake alleviate dexamethasone-induced muscle atrophy.

<2-16> Effects on gene expression related to muscle reduction and synthesis

To investigate the mechanism of muscle degradation, the expression levels of genes related to muscle loss (Myostatin, MuRF 1, Atrogin-1) and genes related to muscle synthesis (MyHC1, MyHC2A, MyHC2X, MyHC2B) were measured using RT (real time)-PCR. was measured.

As a result, as shown in FIG. 20 and Table 12, when dexamethasone was treated, the mRNA expression of Myostatin, MuRF 1, and Atrogin-1, which are genes related to muscle loss, increased markedly, but the expression of the above genes was significant according to tofu intake (Except for the expression level of Atrogin-1 in SM).

In addition, as shown in FIG. 21 and Table 13, when dexamethasone was treated, the expression levels of MyHC2A, MyHC2X, and MyHC2B mRNAs, which are genes related to muscle synthesis, were significantly reduced (except for MyHC1). However, there was no significant difference in the expression level between the tofu consumption groups.

Through these results, it was confirmed that intake of tofu had no significant effect on muscle synthesis, but intake of SM and SMPH among brown meal replacement tofu had the effect of suppressing muscle atrophy by lowering the mRNA expression level of muscle loss genes. .

Gene expression level related to muscle loss according to tofu intake (unit: arbitrary units) gene group DMSO con DEXA-con DEXA-S DEXA-SM DEXA-SMPI DEXA-SMPH Myostatin 1.00 5.57 3.18 1.05 2.22 1.31 MuRF1 1.00 4.23 3.96 1.91 5.48 1.43 Atrogin1 1.00 3.15 2.71 1.55 1.11 0.96

Gene expression level related to muscle synthesis according to tofu intake (unit: arbitrary unit) gene group DMSO con DEXA-con DEXA-S DEXA-SM DEXA-SMPI DEXA-SMPH MyHC1 1.00 0.74 0.19 0.29 0.50 0.53 MyHC2A 1.00 0.44 0.71 0.42 0.80 0.53 MyHC2X 1.00 0.22 0.71 0.36 0.69 0.69 MyHC2B 1.00 0.43 0.55 0.40 0.58 0.79

<2-17> Expression levels of genes related to grip strength and muscle loss according to SMPH and MPH diets

In a mouse model of muscular atrophy induced by dexamethasone, the expression levels of genes related to grip strength and muscle loss according to SMPH and MPH diets were compared.

As a result, as shown in FIG. 22 and Table 14, the grip strength was 0.044 ± 0.0022 N / g body weight in the case of the SMPH + Dexa group and 0.030 ± 0.0011 N / g body weight in the case of the MPH + Dexa group, and proceeded with the SMPH diet One mouse showed relatively high grip strength.

In addition, as shown in Figure 23 and Table 15, when comparing the expression levels of Myostatin, MuRF-1, and Atrogin-1, which are genes related to muscle loss, the SMPH + Dexa group was significantly superior to the MPH + Dexa group in all factors. showed a very low number.

Through the above results, it was confirmed that muscle atrophy can be effectively improved when ingested in the form of tofu by adding it to tofu rather than simply ingesting mealworm larvae protein hydrolyzate (MPH).

Grip strength according to SMPH and MPH diets (unit: N/g body weight) group grip strength SMPH+Dexa 0.044±0.0022 MPH+Dexa 0.030±0.0011

Gene expression levels associated with muscle loss following SMPH and MPH diets (unit: arbitrary units) gene group SMPH+Dexa MPH+Dexa Myostatin 1.43 17.18 MuRF1 0.96 16.97 Atrogin1 1.31 4.09

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can 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 limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (10)

a) mixing brown mealworm larvae protein, brown mealworm larval protein extract, or brown mealworm larval protein extract hydrolyzate in a weight ratio of 1:1 with soybean flour and adding distilled water thereto to mix;
b) adjusting the pH of the mixture to 9.0, mixing for 30 to 60 minutes, and then separating okara and soybean milk;
c) adjusting the pH of the separated soybean milk to 7.0 and then firstly heating it at 70°C;
d) secondarily heating the firstly heated soymilk at 95° C.;
e) cooling the secondarily heated soybean milk to 50° C. and then adding and mixing a coagulant;
f) first reacting the mixture at 50°C; and
g) A method for producing tofu for preventing or improving muscle diseases, comprising the step of cooling the mixture at room temperature after the second reaction at 85 ° C. According to claim 1,
The extract of the brown mealworm larvae protein is obtained by: i) crushing the dried brown mealworm larvae; ii) degreasing by adding ethanol to the pulverized material; iii) adding sodium hydroxide to the degreased brown mealworm larvae and mixing them, followed by centrifugation to obtain a precipitate; And iv) characterized in that produced through a process comprising the step of desalting the obtained precipitate and then freeze-drying, tofu manufacturing method for preventing or improving muscle diseases. According to claim 1,
The hydrolyzate of the protein extract of the brown mealworm larvae is prepared by sequentially treating the extract of the protein of the brown mealworm larvae with alcalase and flavorzyme to hydrolyze the tofu manufacturing method for preventing or improving muscle diseases. According to claim 1,
Tofu manufacturing method for preventing or improving muscle disease, characterized by extracting protein in soybean and brown mealworm larvae through step b). According to claim 1,
A method for producing tofu for preventing or improving muscle diseases, characterized in that through step c) forming a gel of protein of brown gourd larvae. According to claim 1,
In step e), the coagulant is gluconodeltalactone and transglutaminase, characterized in that, tofu manufacturing method for preventing or improving muscle disease. According to claim 1,
The tofu is a tofu manufacturing method for preventing or improving muscle diseases, characterized in that for suppressing the expression of genes related to muscle loss. According to claim 7,
The gene related to muscle loss is characterized in that selected from the group consisting of Myostatin, MuRF 1 and Atrogin-1, tofu manufacturing method for preventing or improving muscle diseases. According to any one of claims 1 to 8,
The muscle disease is a tofu manufacturing method for preventing or improving muscle disease, characterized in that it is a muscle disease due to muscle decline, muscle loss, muscle atrophy, muscle wasting or muscle degeneration. According to claim 9,
The muscle disease is atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, rigid spine syndrome, amyotrophic lateral sclerosis (Lou Gehrig's disease, amyotrophic lateral sclerosis) , Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease), characterized in that selected from the group consisting of sarcopenia, tofu manufacturing method for preventing or improving muscle disease.

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