KR102644928B1 - Manufacturing method of tofu for preventing or improving muscle disease using mealworm larva protein - Google Patents

Abstract

The present invention relates to a method of producing tofu for preventing or improving muscle disease comprising brown mealworm larval protein, brown mealworm larval protein extract, or hydrolyzate of brown mealworm larval protein extract as an active ingredient. The tofu manufacturing method of the present invention adjusts the pH of the mixture of soybean flour, brown mealworm larval protein, and purified water to 9.0, reacts for a certain period of time, and then separates the soy milk, so that the brown mealworm larval protein is well mixed (integrated) into the tofu when coagulated, thereby improving the quality. This has an improving effect. In addition, in the present invention, since brown mealworm larval protein is included as an active ingredient in addition to soybean flour, 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 soybeans, thereby producing soymilk. can effectively lead to gel formation. In addition, in the present invention, by using gluconodelta lactone and transglutaminase together as coagulants, the coagulation of soymilk, which does not coagulate, can be effectively induced because the molecular weight of the protein is very small (hydrolyzate of protein extract of brown mealworm larvae). .

Description

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

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

Sarcopenia is a syndrome in which the loss (penia) of muscles (Sarx), that is, the overall amount and strength of skeletal muscles is gradually lost, which reduces the quality of life and limits physical activity. In the United States, sarcopenia is diagnosed in 2016. A disease code (ICD-10 code) was assigned. Muscle loss causes significant impairment in performing normal daily life functions and also contributes to the increase in chronic diseases. In addition, sarcopenia due to aging is a disease that affects one in five elderly people in Korea.

Muscle atrophy is caused by nutritional deficiency or lack of use of muscles for a long period of time. It occurs when the balance between normal protein synthesis and breakdown is disrupted and protein is broken down.

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

In 2013, the Food and Agriculture Organization of the International Organization (FAO) announced a plan to revitalize edible insects as a future food resource, citing it as one of the policies to solve the upcoming food shortage problem. In line with this, in Korea, in 2014, edible insects were introduced. The Ministry of Drug Safety recognized brown mealworm larvae as a new food ingredient.

Brown mealworm larvae, which are edible insects, contain sufficient energy as a food resource and a large amount of high protein, unsaturated fatty acids, and micronutrients (copper, iron, magnesium, manganese, biotin, pantothenic acid, etc.), and there is a system for raising and supplying them in large quantities. Because it is systematically constructed, it has high value as a highly nutritious food resource to solve the food shortage predicted in the future.

Bioactive peptides obtained through hydrolysis of proteins are generally defined as peptides with low molecular weight and physiological activity. They are usually composed of 3 to 20 amino acids, and the activity of the peptide varies depending on the composition or sequence of the amino acids. Varies. In addition, it has the advantage of being small in size, easily absorbed into the body, and having various functional properties.

Meanwhile, soybeans have high value as a high-quality vegetable protein material and are a nutritionally excellent food resource due to their high unsaturated fatty acid ratio and dietary fiber. In addition, it contains a large amount of phytochemicals such as isoflavones, phenols, and saponins, so its physiological functions are also receiving 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 vitamin B. In addition, tofu is a food familiar to the elderly, ranking third among the foods consumed by those aged 60 or older in Korea. In addition, tofu has a soft texture and is highly digestible, so it is preferred by elderly people with weak teeth and digestion.

However, while vegetable protein has lower calorie intake, cholesterol, and saturated fat intake than animal protein, it has limitations such as low content of branched amino acids (BCAA), which are essential for protein synthesis, and low absorption in the body. In addition, the content of essential amino acids is lower than that of animal protein, so consuming a mixture of the two protein sources can help achieve balanced amino acid intake and efficient protein intake.

However, the effect of processed foods studied so far on preventing muscle atrophy is minimal, and in particular, the effects of bioactive peptides include improving the antioxidant function of milk protein hydrolyzate in the body, preventing oxidation reactions in processed foods, and developing new antibacterial properties of egg white hydrolyzate. Although there are studies on peptides, antihypertensive, antithrombotic, and immunomodulatory functions, there is insufficient research in terms of manufacturing protein hydrolysates that are effective in sarcopenia and developing products using them.

Under this background, the present inventor prepared tofu containing brown mealworm larval protein, an extract of brown mealworm larva protein, or a hydrolyzate of a brown mealworm larval protein extract as an active ingredient, and the tofu had excellent antioxidant and anti-inflammatory activities. It was confirmed that muscle atrophy can be effectively improved. In addition, in the case of the tofu of the present invention, in addition to the effect of improving muscle atrophy, it has excellent gastrointestinal digestibility and low gel strength, so it can be useful as a customized food for elderly people who have difficulty eating food or patients with mastication or swallowing disorders. The present invention was completed by confirming that it exists.

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

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

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

The present invention relates to the following steps: a) mixing brown mealworm larval protein, extract of brown mealworm larval protein, or hydrolyzate of brown mealworm larval protein extract with soybean flour at a weight ratio of 1:1 and adding distilled water to the mixture; b) adjusting the pH of the mixed solution to 9.0, mixing for 30 to 60 minutes, and then separating into okara and soymilk; c) adjusting the pH of the separated soymilk to 7.0 and then first heating it at 70°C; d) second heating the first heated soymilk liquid at 95°C; e) Cooling the second heated soy milk liquid to 50°C and then adding and mixing a coagulant; f) subjecting the mixture to a primary reaction at 50°C; and g) subjecting the primary reaction mixture to a secondary reaction at 85°C and then cooling it to room temperature. It provides a method of producing tofu for preventing or improving muscle disease.

In one embodiment of the present invention, the extract of brown mealworm larvae protein is prepared by: i) pulverizing dried brown mealworm larvae; ii) adding ethanol to the ground product to degrease it; ⅲ) adding sodium hydroxide to the defatted brown mealworm larvae, mixing them and centrifuging them 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 protein extract of brown mealworm larvae can be prepared by sequentially treating the extract of brown mealworm larvae protein with alcalase and flavorzyme to hydrolyze it.

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 from 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 tofu can suppress the expression of genes related to muscle loss.

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

The tofu manufacturing method of the present invention adjusts the pH of the mixture of soybean flour, brown mealworm larval protein, and purified water to 9.0, reacts for a certain period of time, and then separates the soy milk, so that the brown mealworm larval protein is well mixed (integrated) into the tofu when coagulated, thereby improving the quality. This has an improving effect. In addition, in the present invention, since brown mealworm larvae protein is included as an active ingredient in addition to soybean flour, a heating step at the gel formation temperature of the brown mealworm larvae protein is additionally performed separately from the heating step at the gel formation temperature of the soybeans. It can effectively lead to gel formation. In addition, in the present invention, by using gluconodelta lactone and transglutaminase together as coagulants, the coagulation of soymilk, which does not coagulate, can be effectively induced because the molecular weight of the protein is very small (hydrolyzate of protein extract of brown mealworm larvae). .

On the other hand, tofu produced by the method of the present invention contains brown mealworm larval protein, brown mealworm larval protein extract, or hydrolyzate of brown mealworm larva protein extract as an active ingredient, so it has excellent antioxidant and anti-inflammatory activity and effectively improves muscle atrophy. You can do it. In particular, the tofu 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, and can prevent muscle function decline, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. It can be useful as a functional food to prevent or improve various muscle diseases caused by muscle tissue. In addition, in the case of the tofu of the present invention, in addition to the effect of improving muscle atrophy, it has excellent gastrointestinal digestibility and low gel strength, so it can be useful as a customized food for elderly people who have difficulty eating food or patients with mastication or swallowing disorders. You can.

Figure 1 shows a manufacturing process diagram of brown mealworm larval protein extract (MPI) and its hydrolyzate (MPH) according to the present invention.
Figure 2a shows the storage modulus (G') and loss modulus (G") of the brown mealworm larval protein extract (MPI) according to the present invention, and 2b shows the loss coefficient (tan δ) of the brown mealworm larval protein extract (MPI). It represents.
Figure 3 shows a manufacturing process diagram of soymilk and tofu according to the present invention.
Figure 4 shows the results of 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 larva powder, 3: Soymilk made with 50% soybean flour and 50% brown mealworm larva protein extract, 4: Soymilk made with 50% soybean flour and 50% hydrolyzate of brown mealworm larva protein extract, 5: Tofu made with 100% soybean flour, 6: Tofu made with 50% soybean flour and 50% brown mealworm larva powder, 7: Tofu made with 50% soybean flour and 50% brown mealworm larva protein extract, 8: 50% soybean flour and brown. Tofu made with 50% hydrolyzate of mealworm larva protein extract).
Figure 5 shows the pH change by storage period of tofu according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: 50% soybean flour and Tofu made with 50% brown mealworm larval protein extract (MPI), SMPH: tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larva protein extract (MPH)).
Figure 6 shows the in vitro ileal digestibility of tofu according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI), SMPH: tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larval protein extract (MPH)).
Figure 7 shows the gel strength of tofu according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: 50% soybean flour 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 larval protein extract (MPH)).
Figures 8a and 8b show the storage modulus (G') and loss modulus (G") of tofu according to the present invention, respectively (S: tofu made with 100% soybean flour, SM: 50% soybean flour and brown mealworm larva powder 50% Tofu made with %, SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI), SMPH: Tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larval protein extract (MPH). ).
Figure 9 shows the loss coefficient (tan δ) of tofu according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: soybean flour 50% % and tofu made with 50% brown mealworm larval protein extract (MPI), SMPH: tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larva protein extract (MPH)).
Figure 10 shows the storage modulus (G') and loss modulus (G") of tofu according to frequency (S: tofu made with 100% soybean flour, SM: made with 50% soybean flour and 50% brown mealworm larva powder) Tofu, SMPI: Tofu prepared from 50% soybean flour and 50% brown mealworm larval protein extract (MPI), SMPH: Tofu prepared from 50% soybean flour and 50% hydrolyzate of brown mealworm larval protein extract (MPH)).
Figure 11 shows the chemical structure of tofu according to the present invention measured using FTIR spectrophotometry (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI : Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI), SMPH: Tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larva protein extract (MPH), (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 ).
Figure 12 is an SEM image showing the microstructure of the tofu surface according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: 50% soybean flour and tofu prepared with 50% brown mealworm larval protein extract (MPI), SMPH: tofu prepared with 50% soybean flour and 50% hydrolyzate of brown mealworm larval protein extract (MPH)).
Figure 13 shows the total phenol and total flavonoid content of tofu according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: soybean flour 50% % and tofu made with 50% brown mealworm larval protein extract (MPI), SMPH: tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larva protein extract (MPH)).
Figure 14 shows the ABTS+ and DPPH radical scavenging activity of tofu according to the present invention (S: tofu made with 100% soybean flour, SM: tofu made with 50% soybean flour and 50% brown mealworm larva powder, SMPI: soybean flour 50% % and tofu made with 50% brown mealworm larval protein extract (MPI), SMPH: tofu made with 50% soybean flour and 50% hydrolyzate of brown mealworm larva protein extract (MPH)).
Figure 15 is a measurement of cell survival rate according to treatment at different concentrations (0 ~ 2000 μg/ml) of tofu extract according to the present invention (S: tofu made with 100% soybean flour, SM: 50% soybean flour and brown mealworm larva powder Tofu made with 50% soybean flour, SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI), SMPH: made with 50% soybean flour and 50% hydrolyzate of brown mealworm larval protein extract (MPH). tofu).
Figure 16 shows the inhibitory effect of LPS-induced pro-inflammatory cytokines (TNF-α, IL-1b, IL-6) according to treatment with tofu extract (200 μg/mL) according to the present invention (S: 100 % Tofu made from soybean flour, SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder, SMPI: Tofu made from 50% soybean flour and 50% brown mealworm larva protein extract (MPI), SMPH: 50% soybean flour. and tofu prepared with 50% hydrolyzate (MPH) of protein extract of brown mealworm larvae).
Figure 17 confirms the change in body weight of mice according to ingestion of tofu of the present invention in the dexamethasone-induced muscular atrophy mouse model (DMSO con: DMSO-treated regular diet mouse group, DEXA con: dexamethasone-treated regular 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 shows the grip strength of mice according to ingestion of tofu of the present invention in a mouse model of muscle atrophy induced by dexamethasone (DMSO con: DMSO-treated regular diet mouse group, DEXA con: dexamethasone-treated regular 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 shows the periapical cross-sectional area of mouse muscle fibers according to ingestion of tofu of the present invention in the dexamethasone-induced muscular atrophy mouse model (DMSO con: DMSO-treated regular diet mouse group, DEXA con: dexamethasone-treated regular 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 20 shows the measurement of the expression levels of genes (Myostatin, MuRF 1, Atrogin-1) related to muscle loss following ingestion of tofu of the present invention in a dexamethasone-induced muscular atrophy mouse model (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 21 shows the measurement of the expression levels of genes related to muscle synthesis (MyHC1, MyHC2A, MyHC2X, MyHC2B) according to the tofu intake of the present invention in the dexamethasone-induced muscular atrophy mouse model (DMSO con: DMSO-treated regular 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 22 shows the grip strength of mice according to the tofu (SMPH) and hydrolyzate of brown mealworm larval protein extract (MPH) diet of the present invention in a dexamethasone-induced muscular atrophy mouse model (SMPH+Dexa: dexamethasone-treated SMPH diet Mouse group, MPH+Dexa: dexamethasone-treated MPH diet mouse group).
Figure 23 is a measurement of the expression level of genes related to muscle loss according to the tofu (SMPH) and hydrolyzate (MPH) diet of the protein extract of brown mealworm larvae of the present invention in a dexamethasone-induced muscular atrophy mouse model (SMPH+Dexa: Dexamethasone-treated SMPH diet mouse group, MPH+Dexa: dexamethasone-treated MPH diet mouse group).

The present invention relates to the following steps: a) mixing brown mealworm larval protein, extract of brown mealworm larval protein, or hydrolyzate of brown mealworm larval protein extract with soybean flour at a weight ratio of 1:1 and adding distilled water to the mixture; b) adjusting the pH of the mixed solution to 9.0, mixing for 30 to 60 minutes, and then separating into okara and soymilk; c) adjusting the pH of the separated soymilk to 7.0 and then first heating it at 70°C; d) second heating the first heated soymilk liquid at 95°C; e) Cooling the second heated soymilk liquid to 50°C and then adding and mixing a coagulant; f) subjecting the mixture to a primary reaction at 50°C; and g) subjecting the primary reaction mixture to a secondary reaction at 85°C and then cooling it to room temperature. It relates to a method of producing tofu for preventing or improving muscle disease.

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

The term “improvement” used in the present invention refers to any action in which the symptoms of a muscle disease are improved or beneficially changed when an individual consumes the tofu.

The term “brown mealworm larval protein” used in the present invention refers to protein derived from brown mealworm larvae and brown mealworm larva powder.

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

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

In one embodiment of the present invention, the extract of brown mealworm larvae protein is prepared by: i) pulverizing dried brown mealworm larvae; ii) adding ethanol to the ground product to degrease it; ⅲ) adding sodium hydroxide to the defatted brown mealworm larvae, mixing them and centrifuging them 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 protein extract of brown mealworm larvae can be prepared by sequentially treating the extract of brown mealworm larvae protein with alcalase and flavorzyme to hydrolyze it.

In the tofu manufacturing method of the present invention, step a) is a step of preparing a mixed solution of soybean flour and brown mealworm larval protein. Specifically, soybean flour is mixed with brown mealworm larval protein, brown mealworm larva protein extract, or brown mealworm larval protein extract. This is the step of mixing the hydrolyzate at a weight ratio of 1:1 and then adding and mixing distilled water.

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

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

Conventionally, when tofu is manufactured using insect powder, soymilk (soymilk liquid) is first made and then insect powder is added to it. When the soymilk coagulates, the powder inside the tofu crackles and the insect powder does not mix well. There is a problem that is not there.

In the present invention, the pH of the mixture of soybean flour, brown mealworm larvae protein, and purified water is adjusted to 9.0 through steps a) and b) above, then reacted for a certain period of time, and then the soymilk is separated, so that the brown mealworm larvae protein is well mixed in the tofu when coagulated. (By harmonizing), the effect of improving quality can be derived.

In the tofu manufacturing method of the present invention, step c) is a step of first heating the soy milk. Specifically, the pH of the soy milk separated through step b) is adjusted to 7.0, and then the first heat is heated at 69°C to 70°C. This is the step of heating and maintaining for 5 to 15 minutes.

In the present invention, in manufacturing tofu containing brown mealworm larval protein, temperature setting for gel formation of brown mealworm larval protein was preceded, and as a result, the storage modulus (G') and loss modulus (G") were changed around 69.8°C. It was confirmed that the gel formation temperature (gel point) was reached by forming a point contact.

Accordingly, the tofu manufacturing method of the present invention additionally includes a heating step at the gel formation temperature of brown mealworm larval protein, separately from the heating step at the gel formation temperature of soybeans.

In the tofu manufacturing method of the present invention, step d) is a step of secondary heating the soy milk liquid. Specifically, the primary heated soy milk liquid obtained through step c) is secondarily heated at 95° C. for 5 to 15 minutes. This is a stage that is maintained for a minute.

A soybean gel can be formed through step d) of the present invention.

In the tofu manufacturing method of the present invention, step e) is a step of cooling the heated soymilk and then adding and mixing a coagulant. Specifically, the second heated soymilk obtained through step d) is heated to 50°C. After cooling, glucono delta lactone and transglutaminase are added and mixed as coagulants.

In the present invention, by using gluconodelta lactone and transglutaminase together as coagulants, it is possible to effectively coagulate soymilk that does not coagulate because the molecular weight of the protein is very small (hydrolyzate of brown mealworm larval protein extract).

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

In the tofu manufacturing method of the present invention, step f) is the first step of reacting a mixture of soy milk with a coagulant 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 subjecting the first reaction mixture to a second reaction at 85°C and then cooling it at room temperature. Specifically, the first reaction mixture obtained through step f) above is reacted at 85°C. This is the step of producing non-pressed tofu by reacting for 30 minutes to inactivate transglutaminase, activating gluconodeltalactone, another coagulant, and finally cooling at room temperature.

Tofu prepared by sequentially proceeding with steps a) to g) of the present invention prevents muscle disease by suppressing the expression of genes related to muscle loss such as Myostatin, MuRF 1, and Atrogin-1, or It can have an improving effect.

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

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

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

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

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

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

The present inventor produced tofu by sequentially performing the steps a) to g) above, and as a result, the tofu not only has excellent antioxidant and anti-inflammatory activities, but can also effectively improve muscle atrophy by reducing the expression of muscle loss genes. It was confirmed that it exists.

In detail, tofu prepared by sequentially proceeding with steps a) to g) of the present invention had high total phenol and total flavonoid contents and excellent ABTS+ and DPPH radical scavenging activities (see FIGS. 13 and 14). In addition, the tofu prepared by sequentially proceeding with steps a) to g) of the present invention effectively suppressed the expression of pro-inflammatory cytokines (IL-6, TNF-a, IL-1b) induced by LPS (Figure 16). For reference, pro-inflammatory cytokines promote the breakdown of myofibrillar proteins, thereby reducing protein synthesis and, as a result, can directly cause muscle wasting. Accordingly, if the expression of pro-inflammatory cytokines can be effectively suppressed, muscle breakdown can be prevented.

In addition, the tofu prepared by sequentially proceeding with steps a) to g) of the present invention increases the grip strength of the mouse when fed to a mouse model of muscular atrophy induced by dexamethasone (see FIG. 18), and has It effectively reduced the mRNA expression of the genes Myostatin, MuRF 1, and Atrogin-1 (see Figure 20). As seen above, myostatin inhibits muscle development, and MuRF 1 and Atrogin-1 are components of the ubiquitin enzyme complex that causes muscle atrophy. If the expression of these genes can be reduced, muscle It can prevent atrophy.

Therefore, tofu prepared by sequentially proceeding with 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 for illustrating 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 Agricultural Cooperative (Yeongwol-gun, Korea). Brown mealworm larvae were purchased in dried form from Edible Bug Co. (Seoul, Korea), ground, and prepared in powder form using a 1.4 mm sieve. Glucono-δ-lactone (GDL) was purchased from Daejeong Chemical (Siheung, Korea). Microbial transglutaminase (Biobong TG-WM; 100 U/g activity) powder was purchased from Kinry Food Ingredients Co., Ltd. (Shanghai, China).

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

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

<1-3> Preparation of hydrolyzate of brown mealworm larvae protein extract (Mealworm larvae protein hydrolysate, MPH)

First, in order to establish the optimal production conditions for the hydrolyzate of brown mealworm larval protein extract, the conditions of substrate concentration, enzyme concentration, pH, reaction temperature, and reaction time were set as follows with reference to previous research. Substrate concentration (1% (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 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 using 0.1N NaOH and 0.1N HCl. Subsequently, 5 mg of Alcalase (Novozymes, Bagsvaerd, Denmark) per g of dispersion was added and stirred for 12 hours (20 L stirring water bath; SWB-20L03; Cleaver Scientific Ltd.; Warwickshire, UK). Then, 5 mg of flavorzyme (Novozymes, Bagsvaerd, Denmark) was added and hydrolyzed while maintaining pH 8 at 55°C for 12 hours. Afterwards, the hydrolyzed dispersion was heated at 100°C for 10 minutes to inactivate the enzyme, centrifuged at 4200rpm for 10 minutes at 4°C to obtain a supernatant, and the supernatant was freeze-dried using a vacuum freeze dryer. Thus, a hydrolyzate (hereinafter simply abbreviated as 'MPH') of the protein extract of brown mealworm larvae of the present invention was prepared. MPH prepared in this way was stored at -20°C. The manufacturing process of hydrolyzate (MPH) of brown mealworm larvae protein extract is shown in detail in Figure 1.

<1-4> Derivation of gel formation temperature point of MPI

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

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

For reference, when producing tofu containing brown mealworm larvae of the present invention, temperature setting for gel formation of brown mealworm larva protein must be set in advance. The tofu of the present invention was manufactured by applying the gel formation temperature of MPI derived in this way.

<1-5> Tofu production

As raw materials for tofu production, soybean flour (Soy), brown mealworm larva powder (M), brown mealworm larva protein extract (MPI), and hydrolyzate of brown mealworm larva protein extract (MPH) were used and stored refrigerated at 4°C. Mealworm larvae powder (M), MPI, and MPH were mixed in soy flour (Soy) at a 1:1 ratio (w/w), and mixed with distilled water at a 1:6 ratio and mixed for 30 minutes. The pH of the mixed solution was adjusted to 9.0 with 0.1N NaOH and mixed for 30 minutes to extract proteins in soybean and brown mealworm larvae. Afterwards, the okara and soy milk were separated using a filter cloth. The pH of the separated soymilk was adjusted to 7.0 with 0.1N HCl, heated to 70°C, the gel formation temperature of brown mealworm larval protein, and held for 10 minutes, then reheated to 95°C, the gel formation temperature of soybeans, and held for 10 minutes. did. Afterwards, 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 make 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 reactions were completed, the tofu was left at room temperature for 30 minutes to obtain unpressed tofu. The tofu production process of the present invention is shown in detail in Figure 3, and Table 1 below summarizes the production of brown mealworm larva tofu produced through this experiment.

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

<1-6> General ingredient analysis

The general ingredient content of each manufactured tofu was analyzed according to the AOAC method. The crude water 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 at 600°C for 5 hours in a muffle furnace. 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 ( It was estimated according to the Kjeldahl method with a factor of 6.25N using HKD-P, Hanil Co.). Carbohydrate content was quantified at 100% excluding crude oil, ash, crude protein, and crude fat content.

<1-7> Measurement of amino acid content

Freeze-dried tofu samples were used to measure amino acid content. That is, 0.05 g of freeze-dried 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, it was filtered and diluted 100 times and 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 250 nm excitation wavelength (Ex) and 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 measured by correcting with standard amino acids (Waters).

Gradient profile for high-performance liquid chromatography (HPLC) separation 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 according to the method described by Kao, Su, and Lee (2003) with some modifications (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 (86mM Tris-90mM glycine-4mM Na2EDTA, pH 8.0) and placed in an ultrasonic bath (JAC Ultrasonic 2010P; Jinwoo Co., Ltd.) for protein extraction. Engineering; Gyeonggi-do) and sonicated for 1 hour. The extract was then centrifuged at 12,000 × g and 4°C for 20 min. Protein concentration of the supernatant was estimated using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). A 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), electrophoresis was performed using 4-20% precast gel (Luminano; Seoul, Korea). Protein extracts (containing 35 μg protein) were prepared under reducing conditions. Protein molecular weight was estimated using the 6-170 kDa marker (GenDEPOT; Barker, TX, USA). Electrophoresis was performed at 125V for 80 minutes. The isolated 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 acid are produced in tofu due to decay and decomposition by microorganisms. This lowers the pH of the tofu soaking liquid, and the degree of pH decrease 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 and 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 ) Ileal 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.). In detail, 1 g of sample dry matter (DM) was mixed with 25 mL sodium phosphate buffer (0.1 M, pH 6.0) and 10 mL 0.2 M HCl solution, and then the pH of the mixture was adjusted to pH 2 (pepsin activity pH). Afterwards, pepsin solution (10 mg/mL, Sigma-Aldrich Chemical Co.) and 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. Afterwards, 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 activity pH), and then 1mL porcine pancreatin solution (50mg/mL, Sigma-Aldrich Chemical) was added. Co.) was added to the flask. After reacting at 39°C for 18 hours, 20% sulfosalicylic acid solution was added and reacted at room temperature for 30 minutes. Thereafter, the sample was filtered through a crucible-type 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 left to cool to weigh the residue, and the digestion rate was calculated using the following equation.

<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 repeated tests were performed on each sample at room temperature. A temperature sweep test was conducted to measure the flow characteristics of tofu by manufacturing time. After adding two types of coagulants (Tgase, GDL) to the soy milk, 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, then heated to 85°C at 5°C/min and maintained for 30 minutes. Afterwards, it was cooled to 25°C at a rate of 5°C/min. In addition, the elastic modulus according to frequency (1-100 Hz) at a constant strain rate (0.5% strain) was observed through a frequency sweep test of the manufactured 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). The sample was placed on the cleaned crystal and the DialPath was rotated to make contact with the sample. Spectra were obtained from an accumulation 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 head target surface was evaluated using a field emission scanning electron microscope (Quanta 650 FEG; Eindhoven, The Netherlands). Freeze-dried tofu samples were cut into cubes less than 2 mm and coated with platinum (208 HR; Cressington, England). The microstructure was observed at 100×, 500×, 1000×, and 5000× at an acceleration voltage of 10 kV.

<1-14> Antioxidant activity evaluation

[Measurement of total phenol content and flavonoid content]

The 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 purified using Whatman No. 4 Filtered through 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 the homogenate was incubated at 20°C for 2 hours, the absorbance at 725 nm was monitored using a microplate reader (Multiskan, Thermo Fisher Scientific). Total phenolic 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). In detail, 0.2 g sample was dispersed in 80% (v/v) ethanol and stirred. The dispersion was treated with Whatman No. Filtered through 4 filters. Then, 0.5 mL sample extract was mixed with 5 mL 90% (v/v) diethylene glycol and 0.5 mL 1N NaOH. The mixture was vortexed and kept at ambient temperature (25°C) for 60 minutes. The absorbance was then 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 creating a standard calibration curve using naringin (Sigma-Aldrich Chemical Co.).

[DPPH and ABTS+ radical scavenging ability]

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) 20 uL of sample extract extracted with ethanol and 180 uL of ABTS+ solution (Sigma-Aldrich Chemical Co.) were mixed, left in the dark for 10 minutes, and absorbance was measured at 734 nm. DPPH radical scavenging ability was 70%. (v/v) 200 uL of sample extract extracted with ethanol and 50 uL of DPPH solution (0.005 g/100mL 99.5 (v/v) ethanol, Sigma-Aldrich Chemical Co.) were mixed and left in the dark for 30 minutes. Absorbance was measured at 520 nm, and radical scavenging ability was calculated using the formula below.

Here, Abs _sample represents the absorbance of the experimental 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) analysis]

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. 4 After filtering with filter paper, the extract was used for analysis. Cell viability was measured using MTT assay. In detail, 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 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. Then, the medium was 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 LPS treatment and enzyme-linked immunosorbent assay (ELISA)]

C2C12 cells were maintained in Dulbecco's modified Eagle's 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. Inflammatory cells were cultured with 200 μg/mL sample extract for 18 hours, 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). It was evaluated using ELISA kits; Komabiotech; Seoul, Korea).

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

[Dexamethasone-induced muscular atrophy mouse model]

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

Dexamethasone treatment and dietary composition for each mouse group group Dexamethasone treatment or not diet DMSO con X N.D. DEXA con O N.D. 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 test]

The body weight of the mouse was expressed by measuring the difference between the first and 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), divided by body weight. Calculated as N/g body weight.

[Histological staining and cross-sectional area measurements]

The calf muscle of the sacrificed mouse was fixed with a 10% formalin solution, embedded in paraffin, and sectioned. This 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 area of 10 muscle fibers was measured and the average value was calculated.

[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 spectrophotometrically (SPL). Then, cDNA was synthesized using the PrimScriptTM RT reagent kit containing gDNA Eraser (Takara) and then mixed with specific primers, SYBR Premix Ex TaqTM II and ROX Plus. The relative expression of muscle breakdown-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. It was calculated through

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

[Dexamethasone-induced muscular atrophy mouse model]

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

Dexamethasone treatment and dietary composition for each 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 with a diet supplemented with 15% of MPH powder were measured using a Bioseb grip strength meter (BIO-GS3; BIOScience and Experimental Biology; Largo, FL, USA) was used to measure mice before they were sacrificed. Grip strength was expressed as the force (N) just before losing the grip when pulling the mouse holding the grid divided by the body weight (N/g body weight).

[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 in the SMPH+Dexa and MPH+Dexa groups using RNAiso Plus reagent (Takara; Shiga, Japan), and the amount of RNA was measured spectrophotometrically (SPL). Then, cDNA was synthesized using the PrimScriptTM RT reagent kit containing gDNA Eraser (Takara) and then mixed with specific primers, SYBR Premix Ex TaqTM II and ROX Plus. The relative expression of muscle breakdown-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 two groups were performed using an independent two-sample t-test.

2. Results

<2-1> Setting the gel formation temperature of MPI

A temperature sweep test was performed to measure the gel formation temperature of MPI. The point of contact between the storage modulus and the loss modulus becomes the gel formation temperature (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 Figure 2, in MPI, G' and G" intersect around 69.8°C (see Figure 2a), and tan δ becomes less than 1, so the gel point was confirmed (see Figure 2b).

<2-2> General ingredient content of tofu

The approximate composition of tofu is shown in detail in Table 5 below. Compared to S, SM had higher moisture (85.34%) and crude fat (0.85%) content, but lower crude protein (4.22%), ash (0.55%), and carbohydrate (9.04%) content. SM showed the highest crude fat content because mealworm larvae contain 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 ash content (3.10%) of SMPH was higher, which is believed to be due to the addition of acid or base to adjust pH during the hydrolysis process. These results suggest that 50% soybean meal replacement with mealworm larvae, MPI and MPH during tofu manufacturing had a significant effect on the main composition, especially protein and lipid content (p<0.05).

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

<2-3> Component amino acid content

Seventeen amino acids were detected in tofu (see Table 6). There was no significant difference in the essential amino acid, branched chain amino acid, and total amino acid contents of S and SM (p>0.05). However, the total amino acid content of SMPI (63.82 g/100 g sample) and SMPH (46.97 g/100 g sample) was found to be significantly high. Additionally, 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). Additionally, 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 replacing soybean flour with MPI or MPH when manufacturing tofu can improve the amino acid content of tofu. The methionine and cysteine content of plant-based proteins, such as soy protein, is generally lower than that of animal-based proteins, such as mealworm larval protein, 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. Additionally, insects (especially mealworm larvae) are high in branched chain amino acids, which helps prevent muscle wasting and breakdown. This suggests that tofu, which is rich in branched-chain amino acids such as leucine, isoleucine, and valine, may be helpful for elderly people with muscle loss.

Amino acid content of tofu Samples S SM SMPI SMPH ASP 4.54±1.47 ^b 4.37± ^0.26b 7.86± ^2.23a 5.93± ^0.40ab SER 2.02± ^0.54b 2.05± ^0.12b 3.93± ^0.74a 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± ^0.66a 2.57± ^0.30b HIS 0.78± ^0.18c 0.88± ^0.08c 1.91± ^0.27a 1.31± ^0.16b ARG 0.80± ^0.07b 0.80± ^0.45b 1.81± ^0.30a 1.40± ^0.15a THR 0.61± ^0.05b 0.61± ^0.34b 1.37± ^0.23a 1.06± ^0.11a ALA 1.48± ^0.49b 1.58± ^0.10b 3.79± ^0.97a 2.80± ^0.28a PRO 1.65± ^0.41b 2.21± ^0.13b 3.29± ^0.66a 2.36± ^0.22b CYS 0.92± ^0.12c 1.10±0.44 ^b.c. 2.07± ^0.34a 1.62± ^0.11ab T.Y.R. 1.13± ^0.16c 1.60± ^0.09c 5.26± ^0.44a 3.79± ^0.41b VAL 1.31± ^0.30b 1.56± ^0.10b 3.71± ^0.83a 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± ^1.04a 3.71± ^0.33b PHE 1.33± ^0.22c 1.39± ^0.05c 2.86± ^0.24a 2.15± ^0.24b Hydrophilic 10.42± ^2.36c 11.50± ^1.19c 24.28± ^4.80a 18.22±1.77 ^b Hydrophobic 18.86± ^5.64b 20.64±1.18 ^b 39.53± ^8.34a 28.74± ^3.18b Total EAAs ¹⁾ 10.52± ^1.97c 11.01± ^0.63c 22.50± ^4.61a 17.00±1.49 ^b Total BCAAs ²⁾ 4.14± ^0.61b 4.50± ^0.32b 9.92± ^2.16a 7.81± ^0.70a 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 from 100% soybean flour;
SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder;
SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI);
SMPH: Tofu made from 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH).
According to ^ad Duncan's multiple range test (p<0.05), there is a significant difference between different initials in each treatment section.

<2-4> SDS-PAGE

SDS-PAGE was performed to track changes in protein subunits of soy milk and tofu.

As a result, as shown in Figure 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 soy milk (lanes 1-4). After heating and coagulating soy milk, the intensity of the bands representing 7S and 11S decreased rapidly, except for SMPH. 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 18 days of storage is shown in Figure 5. The pH of tofu was found to continuously decrease as the storage period elapsed (4.42~4.45 on day 0, 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 decrease in pH from day 0 to day 18 (4.42-3.81), while SMPH showed the least decrease in pH value from day 0 to day 18 (4.43-4.20).

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

The digestibility of tofu is shown in Figure 6. The in vitro ileal digestibility of all samples showed significant differences (p<0.05). S showed the lowest digestibility (58.35%) due to anti-nutritional components including phytate, trypsin inhibitor, tannin, or protein-bound starch. SM was found to have a higher digestibility than S (64.61%) because the digestibility of animal protein (mealworm larvae) is higher than that of plant protein (soybeans). SMPI appeared to be more digestible than SM because the chitin level in SM was higher than that in SMPI and chitin reduced in vitro digestibility. In particular, SMPH samples showed significantly better digestibility than other samples.

<2-7> Gel strength and rheological properties

The gel strength of tofu is shown in detail in Figure 7. The gel strength of SM (135.0 g/cm 2 ) was measured to be significantly lower than that of S (504.3 g/cm ² ). The gelation properties of heat-induced gels are affected by protein hydrophobicity, and lipids can inhibit protein gelation due to interference with protein-protein polymer interactions. This can also be explained by a decrease in thiol group content due to lipid oxidation. Meanwhile, SMPI (927.9g/cm ² ) showed the highest gel strength because it had the highest protein concentration. High protein concentration is related to the degree of exposure of hydrophobic regions and sulfhydryl groups during heat denaturation, which ultimately promotes soymilk coagulation. On the contrary, SMPH (32.0 g/cm ² ) showed the lowest gel strength because the degree of gel formation was low due to MPH's unique characteristics 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 Figures 8 and 9, the time- and temperature-dependent variation patterns of 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 the cross-linked network formed by TGase. The rapid increase in G' in the initial heating stage suggests the generation of a gel network and gel formation. Compared to S, in SMPI, G' and G" increased faster. It also increases slowly in SM and SMPH, indicating the order of gel formation onset rates. During heating from 50°C to 85°C, the viscoelasticity decreased due to non-covalent bond destruction at high temperature. Additionally, G' and G" increased significantly in all samples, suggesting gel network strengthening during the cooling step. Tan δ decreased in all samples during the first few minutes of heating, indicating a rigid and elastic protein network. Tan δ of The smaller the value, the closer it is to solid properties, and the larger it is, the closer it is to liquid properties. Therefore, it was found that the value of Tan δ, which is G”/G’ of the manufactured tofu, was SMPH > SM > S > SMPI, with SMPI being the closest to solid. It was close, and SMPH was confirmed to be closest to liquid.

In addition, the elastic modulus according to frequency (1-100 Hz) at a constant strain rate (0.5% strain) was observed through a frequency sweep test of the manufactured tofu (see Figure 10). A frequency sweep provides information about the viscous and elastic profile of a sample under constant strain. As a result, as shown in Figure 10, it was found that G' and G" were SMPI > S > SM > SMPH. The larger G' and G" according to frequency, the closer to solid characteristics, and the smaller, closer to liquid characteristics. It can be seen that the above results are consistent with the trend in FIG. 9.

<2-8> FTIR spectra

The chemical structure of the tofu sample was determined using FTIR spectrophotometry, and the results are shown in detail in Figure 11. The broad band between 3500 and 3200 cm ^-1 corresponds to the hydroxyl (-OH) group, and the band around 3274 cm ^-1 (peak No. 1) may be due to the hydroxyl (-OH) stretching vibration found in all tofu. The peaks at 2916 cm ^-1 and 2849 cm ^-1 (peaks 2 and 3) correspond to aldehyde CH stretching vibrations, which are associated with methyl groups (-CH3) and methylene groups (-CH3) present in cellular components such as proteins, carbohydrates and lipids. It can be attributed to CH2-). The peak at 1742 cm ^-1 (peak 4) 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 absent 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 to amide I, mainly due to C=O bond vibrational bending, resulting in several folded component bands representing various protein fragment secondary structures. and amide II, respectively, are due to NH bending. Amide I band spectra are α-helix (1650-1660 cm ^-1 ), β-sheet (1618-1640 cm ^-1 and 1670-1690 cm ^-1 ), β-turn (1660-1670 cm ^-1 ) and random coil. (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 decreased consistently with enzymatic digestion of the peptide backbone. Therefore, SMPH appeared to have the lowest peak 5 and 6 intensities. The peak at 1039 cm ^-1 (peak 7) indicates that a complex was formed by electrostatic interaction between the carboxyl group (-COO-) of soybean protein and the amine group (-NH3+) of chitosan, and also in the soy protein-chitosan interaction. Hydrogen bonding occurs. This suggests that chitosan extracted from the shell of mealworm larvae affects the peak intensity at 1039 cm ^-1 of SM, so SM shows the highest intensity at 1039 cm ^-1 due to chitosan vibration.

<2-9> Microstructure of tofu

To confirm the structural characteristics of the head, surface images at 500-5000 times magnification were visualized using SEM, and the results are shown in detail in Figure 12. S appeared to have a much more dense and uniform structure than other samples. SM was found to have honeycomb-shaped pores because the fat globules were more dispersed compared to S. On the other hand, SMPI showed large cavities and a rough structure due to protein aggregation, while SMPH showed the characteristics of having dense and homogeneous small cavities due to the hydrolyzate being evenly dispersed in the protein aggregates.

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

The total phenolic content and total flavonoid content of SM, SMPI and SMPH were determined differently depending on the type of mealworm larval protein used.

As a result, as shown in Figure 13 and Table 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 had the highest total phenol content (22.66 mg GAE/g) because it released bound polyphenols in a free state through hydrolysis. On the other hand, S had the highest total flavonoid content among the samples (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. Meanwhile, SMPH was found 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 from 100% soybean flour;
SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder;
SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI);
SMPH: Tofu made from 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH).

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 from 100% soybean flour;
SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder;
SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI);
SMPH: Tofu made from 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH).

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

The antioxidant capacity of tofu was determined based on ABTS+ and DPPH free radical scavenging abilities compared to the 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 Figure 14 and Tables 9-10, the IC ₅₀ values for ABTS+ free radical scavenging activity of S, SM, SMPI, and SMPH were measured to be 7.00, 5.00, 2.31, and 1.56 μg/mL, respectively. Additionally, DPPH free radical scavenging activity showed a similar trend to ABTS+. SMPH had the lowest IC ₅₀ value for both ABTS+ and DPPH activities (p<0.05). This may be because the reactive groups of bioactive peptides are exposed through protein hydrolysis, thereby increasing antioxidant activity. Antioxidant capacity was found to be related to total phenol content and 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 from 100% soybean flour;
SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder;
SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI);
SMPH: Tofu made from 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH).

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 from 100% soybean flour;
SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder;
SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI);
SMPH: Tofu made from 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH).

<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 Figure 15, there was no significant difference in survival rate in the concentration range of 0-200 μg/mL. Accordingly, an experiment to inhibit inflammatory cytokine expression was conducted at a concentration of 200 μg/mL. Meanwhile, in the concentration range of 1000-2000 μg/mL, the SMPH treatment group had the highest cell survival rate, but there was no significant difference from other treatments (p>0.05).

<2-13> LPS-induced apoptosis inhibitory effect

To determine the inhibition rate of inflammatory cell expression in the sample, lipopolysaccharide (LPS) was used in C2C12 cells to observe the expression level of IL-6, TNF-a, and IL-1b, which are pro-inflammatory cytokines in the blood.

Of note, the production of pro-inflammatory cytokines such as TNF-α, IL-1b, and IL-6 is essential for determining the initial inflammation that can develop into systemic inflammatory response syndrome. Additionally, LPS is a potent pro-inflammatory activator of macrophages, which function as innate immune cells in vivo and in vitro. Inflammatory cytokines in the blood promote the breakdown of myofibrillar proteins, thereby reducing protein synthesis and ultimately causing direct muscle wasting.

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

Pro-inflammatory cytokine expression level 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 from 100% soybean flour;
SM: Tofu made from 50% soybean flour and 50% brown mealworm larva powder;
SMPI: Tofu made with 50% soybean flour and 50% brown mealworm larval protein extract (MPI);
SMPH: Tofu made from 50% soybean flour and 50% hydrolyzate of brown mealworm larvae protein extract (MPH).

<2-14> Weight change

In a mouse model of muscular atrophy induced by dexamethasone (DEXA), the change in body weight of mice according to tofu intake was measured.

For reference, when dexamethasone, a synthetic glucocorticoid, is administered, muscle proteins are broken down and weight loss occurs. Many 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 the induction of muscle loss due to oxidative damage to muscle caused by age-related ROS overproduction.

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

<2-15> Grip strength and root cross-sectional area

Forelimb grip strength was compared to determine whether tofu consumption improves muscle function in a mouse model of dexamethasone (DEXA)-induced muscular atrophy.

As a result, as shown in Figure 18, the DEXA-administered group, excluding the DEXA-SMPH (0.044 N/g body weight) group, showed a significant decrease in grip strength, and among them, the DEXA-SMPI group had the lowest (0.030 N/g body weight). g body weight).

Additionally, as shown in Figure 19, the apical cross-sectional area of muscle fibers was found to be significantly lower in the DEXA-treated group (1562.70 μm ² ) compared to the DMSO control group (1562.70 μm ² ), and among the treatment groups, it was found to be significantly lower in the SM-treated group (1378.33 μm ² ). The decrease in root cross-sectional area was the lowest, and the decrease in root cross-sectional area was found to be lower in that order: S (1283.80 μm ² ), SMPH (1259.83 μm ² ), and SMPI (1169.37 μm ² ). These results suggest that consumption of SM and SMPH alleviates dexamethasone-induced muscle atrophy.

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

To investigate the mechanism of muscle breakdown, 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. It was measured.

As a result, as shown in Figure 20 and Table 12, when treated with dexamethasone, the mRNA expression of Myostatin, MuRF 1, and Atrogin-1, which are genes related to muscle loss, was significantly increased, but the expression of these genes was significant depending on tofu intake. It was found to decrease significantly (except for the expression level of Atrogin-1 in SM).

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

Through these results, it was confirmed that consumption of tofu had no significant effect on muscle synthesis, but consumption of SM and SMPH, a substitute for brown mealworm tofu, had the effect of suppressing muscle atrophy by lowering the mRNA expression level of muscle loss genes. .

Expression level of genes related to muscle loss following tofu consumption (unit: arbitrary unit) 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

Expression level of genes 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 level of genes related to grip strength and muscle loss according to SMPH and MPH diets

The expression levels of genes related to grip strength and muscle loss according to SMPH and MPH diets were compared in a dexamethasone-induced muscular atrophy mouse model.

As a result, as shown in Figure 22 and Table 14, the grip strength was 0.044±0.0022 N/g body weight in the SMPH+Dexa group and 0.030±0.0011 N/g body weight in the MPH+Dexa group, resulting in 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 more significant in all factors than the MPH+Dexa group. It showed a very low value.

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

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

Claims (10)

a) mixing soybean meal with brown mealworm larval protein, extract of brown mealworm larval protein, or hydrolyzate of brown mealworm larval protein extract at a weight ratio of 1:1 and adding distilled water to the mixture;
b) adjusting the pH of the mixed solution to 9.0, mixing for 30 to 60 minutes, and then separating into okara and soymilk;
c) adjusting the pH of the separated soymilk to 7.0 and then first heating it at 70°C;
d) second heating the first heated soymilk liquid at 95°C;
e) Cooling the second heated soy milk liquid to 50°C and then adding and mixing a coagulant;
f) subjecting the mixture to a primary reaction at 50°C; and
g) A method of producing tofu for preventing or improving muscle disease, comprising the step of subjecting the first reaction mixture to a second reaction at 85°C and then cooling it at room temperature. According to paragraph 1,
The extract of the protein of brown mealworm larvae includes the steps of i) pulverizing dried brown mealworm larvae; ii) adding ethanol to the ground product to degrease it; ⅲ) adding sodium hydroxide to the defatted brown mealworm larvae, mixing them and centrifuging them to obtain a precipitate; and iv) a method of producing tofu for preventing or improving muscle disease, characterized in that it is manufactured through a process comprising desalting the obtained precipitate and then freeze-drying it. According to paragraph 1,
The hydrolyzate of the protein extract of brown mealworm larvae is a method of producing tofu for preventing or improving muscle disease, characterized in that it is produced by sequentially treating the extract of brown mealworm larval protein with alcalase and flavorzyme to hydrolyze it. According to paragraph 1,
A tofu manufacturing method for preventing or improving muscle disease, characterized in that the protein in soybeans and brown mealworm larvae is extracted through step b). According to paragraph 1,
A method of producing tofu for preventing or improving muscle disease, characterized in that a gel of brown mealworm larval protein is formed through step c). According to paragraph 1,
A method of producing tofu for preventing or improving muscle disease, wherein the coagulant in step e) is gluconodelta lactone and transglutaminase. According to paragraph 1,
A method of producing tofu for preventing or improving muscle disease, characterized in that the tofu suppresses the expression of genes related to muscle loss. In clause 7,
A method of producing tofu for preventing or improving muscle disease, wherein the gene related to muscle loss is selected from the group consisting of Myostatin, MuRF 1, and Atrogin-1. According to any one of claims 1 to 8,
A method of producing tofu for preventing or improving muscle disease, characterized in that the muscle disease is a muscle disease caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. According to clause 9,
The muscle diseases include atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spine syndrome, and amyotrophic lateral sclerosis (Lou Gehrig's disease). , Charcot-Marie-Tooth disease, and sarcopenia.