Method for producing texturized vegetable protein with enhanced functionality adding hempseed residue

Abstract

The present invention relates to a method for producing a plant tissue protein with added hemp seed meal, characterized by comprising the steps of: introducing a plant tissue protein material containing hemp seed meal powder into an extrusion molding machine and then extruding it to produce an extruded product; and drying the extruded product produced; the plant tissue protein with added hemp seed meal produced by the above method; and a processed food produced using the plant tissue protein with added hemp seed meal.

Description

Technology Field

[0001] The present invention relates to a method for producing plant tissue protein with added hemp seed meal, characterized by comprising the steps of: mixing plant tissue protein materials of isolated soy protein, wheat gluten, corn starch, modified starch, maltodextrin, and hemp seed meal powder; introducing the mixed plant tissue protein materials into an extrusion molding machine and then extruding to produce an extruded product; and drying the extruded product produced, and to a plant tissue protein with added hemp seed meal produced by the said method. Background Technology

[0002] Hempseed (Hempseed, Seed of Cannabis sativa Hemp (L.) refers to the seeds of the cannabis plant, an annual herb belonging to the Cannabis genus, in an edible state after the hallucinogenic component THC (Tetrahydrocanabinol) found in the shell has been removed. Its origin is Central Asia, and it is currently cultivated in various regions around the world. Hemp seeds have a nutty yet soft texture, exhibiting a taste similar to other nuts. They are nutritionally excellent due to their high content of protein, essential amino acids, and minerals, and because of their high fat content (25–35%), they are also used as edible oil by pressing. Furthermore, hemp seed oil is rich in polyunsaturated fatty acids such as alpha-linolenic acid (ALA) and gamma-linolenic acid (GLA), and is known to be effective in reducing cholesterol, alleviating vascular inflammation, and treating skin diseases and atopic dermatitis. In traditional Korean medicine, it is called Majain and is used for intractable constipation, diabetes, various pain disorders, menstrual irregularities, skin diseases, and dysentery; recently, its inhibitory effect on neuroinflammation and neuroprotective activity have become well known.

[0003] Plant-based meat is a food product manufactured using plant-based ingredients to mimic the taste of meat, rather than animal-based materials like meat; it is also referred to as plant-based meat substitute. Examples of plant-based meat include wheat meat, made using gluten found in wheat flour; rice meat, made using rice; and soy meat, made by mixing soy protein and gluten. Recently, there has been a growing trend of vegetarians adopting a plant-based diet due to concerns such as global warming and animal welfare, leading to the emergence of the alternative meat market as a new consumer trend.

[0004] To make soybean protein have a meat texture, methods such as the spinning process, thermoplastic extrusion, and steam texturization have been used. The extrusion process is a representative method that enables mass production and commercialization and can form a texture similar to meat. The extrusion process refers to a process in which vegetable protein is textured and broken down, rearranged, and coagulated through the combined action of pressure, heat, and mechanical shear force using an extruder called an extruder to produce TVP (TVP).

[0005] Texturized Vegetable Protein (TVP) is manufactured using soy protein as the main ingredient through an extrusion process. Defatted soy flour or concentrated soy protein is thoroughly mixed with water and then extruded under high pressure while heated in an extruder; this causes the soy protein molecules to coagulate with an directional orientation, resulting in a texture that has a chewing sensation similar to meat. The texture and taste of the final TVP product are significantly influenced by the types and ratios of the mixed ingredients, as well as the moisture content, heating temperature, and heating time. Currently, commercially used TVP is low-moisture TVP in pellet form, which is manufactured into a lump shape by adding a binder after hydration and is used in food products.

[0006] When TVPs are used as raw materials for plant-based meat, they must possess texture, flavor, and appearance similar to meat. Furthermore, their texture must not be destroyed even during the rehydration that inevitably occurs during cooking, and they must also possess functional properties such as water retention and fat absorption similar to meat. However, due to the low cohesion between TVPs, the form of the alternative meat is unstable, and it is currently difficult to achieve a texture similar to actual meat.

[0007] Regarding conventional patented technologies related to hemp seeds, Korean Registered Patent No. 2583021 discloses a method for manufacturing a hemp milk beverage using hemp seeds, and Korean Published Patent No. 2022-0143526 discloses red bean porridge using hemp seeds, but there is no method for manufacturing plant tissue protein with added hemp seed meal according to the present invention. The problem to be solved

[0008] The present invention was devised in response to the above-mentioned needs, and the objective of the present invention is to provide a method for producing plant tissue protein with added hemp seed meal, which has a texture similar to meat tissue and has optimized binding power, cohesive power, and physical properties, and excellent quality and palatability, and a method for producing an alternative meat product using the plant tissue protein with added hemp seed meal, in the process of producing plant tissue protein using hemp seeds, which are a functional material. means of solving the problem

[0009] To solve the above problem, the present invention provides a method for producing plant tissue protein with added hemp seed meal, characterized by comprising the steps of: (1) preparing hemp seed meal powder by grinding hemp seeds from which oil has been separated by roasting and pressing hemp seeds; (2) mixing isolated soy protein, wheat gluten, corn starch, modified starch, and maltodextrin with the plant tissue protein material of the hemp seed meal powder prepared in step (1); (3) preparing an extruder by feeding the mixed plant tissue protein material from step (2) into an extrusion molding machine and then extruding it; and (4) drying the extruder prepared in step (3).

[0010] In addition, the present invention provides a plant tissue protein with added hemp seed meal prepared by the above method.

[0011] In addition, the present invention provides an alternative meat product manufactured using plant tissue protein with added hemp seed meal. Effects of the invention

[0012] The hemp seeds used in the production of the plant tissue protein of the present invention are nutritionally superior due to their high content of protein, essential amino acids, and minerals, and are a functional material known to be effective in anti-inflammation, memory enhancement, blood circulation, and anticancer effects. It has been confirmed that they have antioxidant, anti-inflammatory, whitening, and muscle strengthening effects, so the plant tissue protein using the hemp seeds of the present invention can also be expected to have the functional effects of hemp seeds.

[0013] In addition, it is possible to provide high-quality plant tissue protein that has a texture, water retention, and puffing ability similar to meat, while minimizing the destruction of texture even during rehydration, which is an essential process during cooking.

[0014] In addition, the plant tissue protein of the present invention is expected to help prevent cardiovascular diseases and lifestyle diseases because it has a high protein content but relatively low levels of fat, saturated fatty acids, and cholesterol compared to animal proteins.

[0015] In addition, when manufacturing alternative meat products using the plant tissue protein of the present invention, excellent palatability is exhibited in terms of taste, texture, and flavor, which has the advantage of being more preferred by consumers. Brief explanation of the drawing

[0016] Figure 1 is a graph comparing the NO (Nitric oxide) production inhibitory ability of the hemp seed meal extract of the present invention according to concentration. CON: untreated group, LPS: 1 μg / mL treated group, EGCG: LPS+EGCG treated group, 50: LPS+hemp seed meal extract 50 μg / mL treated group, 100: LPS+hemp seed meal extract 100 μg / mL treated group, 200: LPS+hemp seed meal extract 200 μg / mL treated group. Figure 2 is a graph comparing the melanin production inhibitory ability of the hemp seed meal extract of the present invention according to concentration. CON: untreated group, α-MSH: α-MSH (Melanin stimulating hormone) 200 nM treated group, Arbutin: α-MSH + arbutin 100 µg / mL treated group, 50: α-MSH + hemp seed meal extract 50 µg / mL treated group, 100: α-MSH + hemp seed meal extract 100 µg / mL treated group, 200: α-MSH + hemp seed meal extract 200 µg / mL treated group. Figure 3 is a graph comparing the diameter of myotube cells according to the concentration of the hemp seed meal extract of the present invention. CON: untreated group, DEX: dexamethasone 100 μM treated group, 50: DEX+hemp seed meal extract 50 μg / mL treated group, 100: DEX+hemp seed meal extract 100 μg / mL treated group, 200: DEX+hemp seed meal extract 200 μg / mL treated group. Figure 4 is a photograph comparing the appearance of plant tissue protein extruded at different screw speeds before and after drying. Figure 5 is a photograph comparing the appearance of plant tissue protein before and after drying, extruded at different injection port temperatures. Figure 6 is a photograph comparing the appearance of plant tissue protein extruded with different water supply rates before and after drying. Figure 7 is a photograph comparing the appearance of plant tissue proteins extruded with different mixing ratios before and after drying. Figure 8 is a photograph comparing the appearance of plant tissue protein extruded with different mixing ratios of hemp seeds before and after drying. Specific details for implementing the invention

[0017] To achieve the objective of the present invention, the present invention

[0018] (1) A step of producing hemp seed meal powder by grinding hemp seeds from which oil has been separated by roasting and pressing the hemp seeds;

[0019] (2) A step of mixing the plant tissue protein material of isolated soy protein, wheat gluten, corn starch, modified starch and maltodextrin with the hemp seed meal powder prepared in step (1);

[0020] (3) A step of preparing an extruded product by feeding the mixed plant tissue protein material from step (2) above into an extrusion molding machine and then extruding it; and

[0021] (4) A method for producing plant tissue protein with added hemp seed meal is provided, characterized by including a step of drying the extruded product produced in step (3) above.

[0022] In the method for producing plant tissue protein with added hemp seed meal according to the present invention, the hemp seed meal powder of step (1) can preferably be produced by roasting hemp seeds at 160 to 170°C for 10 to 14 minutes, then pressing to separate the oil, and then grinding the hemp seeds. More preferably, it can be produced by roasting hemp seeds at 165°C for 12 minutes, then pressing to separate the oil, and then grinding the hemp seeds. Preparing the hemp seed meal powder by pre-treating under the above conditions allowed for the removal of oil, which reduced the greasy and fishy taste, while exhibiting a nutty flavor. Additionally, when added in an appropriate amount during the production of plant tissue protein, puffing occurs well and the physical properties are excellent, allowing for production in a state suitable for plant tissue protein production. However, if the production conditions fall outside the above range, there is a problem in that puffing does not occur well during the production of plant tissue protein and the physical properties are not suitable.

[0023] In addition, in the method for producing plant tissue protein with added hemp seed meal according to the present invention, step (2) may preferably mix plant tissue protein materials based on the total weight of the plant tissue protein materials, comprising 43-47% by weight of isolated soy protein, 38-42% by weight of wheat gluten, 2-6% by weight of corn starch, 1-5% by weight of modified starch, 1-5% by weight of maltodextrin, and 3-7% by weight of hemp seed meal powder; more preferably, based on the total weight of the plant tissue protein materials, may mix plant tissue protein materials comprising 45% by weight of isolated soy protein, 40% by weight of wheat gluten, 4% by weight of corn starch, 3% by weight of modified starch, 3% by weight of maltodextrin, and 5% by weight of hemp seed meal powder. By mixing plant tissue protein materials with the above-mentioned types of materials and mixing ratios, it was possible to produce a plant tissue protein with excellent physical properties such as binding strength, cohesiveness, gumminess, chewiness, and elasticity, and with the materials blending well to produce a plant tissue protein with excellent flavor and taste.

[0024] In addition, in the method for producing plant tissue protein with added hemp seed meal according to the present invention, the extruder of step (3) can preferably be produced by extruding the plant tissue protein material after feeding it into an extrusion molding machine controlled to a screw speed of 1500~1600 rpm, a sample feed rate of 180~220 g / min, a water supply rate of 150~200 cc / min, and an extrusion port temperature of 175~190℃, and more preferably by extruding the plant tissue protein material after feeding it into an extrusion molding machine controlled to a screw speed of 1500~1600 rpm, a sample feed rate of 200 g / min, a water supply rate of 170 cc / min, and an extrusion port temperature of 175~190℃. Extruding under the above conditions allowed for the production of a high-quality plant tissue protein that has water retention capacity, puffing power, and physical properties similar to meat, while minimizing the destruction of texture upon rehydration.

[0025] In addition, in the method for producing plant tissue protein with added hemp seed meal according to the present invention, the drying in step (4) can preferably be performed by drying the extruded product at 50 to 70°C for 10 to 14 hours, and more preferably by drying the extruded product at 60°C for 12 hours. By drying under the above conditions, a texture similar to meat is formed so that it does not crack easily, and it can be dried while exhibiting a porous structure, binding well, and minimizing deformation.

[0026] The method for producing plant tissue protein with added hemp seed meal according to the present invention, more specifically, is as follows:

[0027] (1) A step of preparing hemp seed meal powder by roasting hemp seeds at 160~170℃ for 10~14 minutes, then pressing to separate the oil, and grinding the hemp seeds;

[0028] (2) A step of mixing plant tissue protein materials based on the total weight of the plant tissue protein materials, comprising 43-47% by weight of isolated soy protein, 38-42% by weight of wheat gluten, 2-6% by weight of corn starch, 1-5% by weight of modified starch, and 1-5% by weight of maltodextrin, and 3-7% by weight of the hemp seed meal powder prepared in step (1);

[0029] (3) A step of preparing an extruded product by feeding the mixed plant tissue protein material from step (2) into an extrusion molding machine controlled to a screw speed of 1500~1600 rpm, a sample feed rate of 180~220 g / min, a water feed rate of 150~200 cc / min, and an extrusion port temperature of 175~190℃, and then extruding it; and

[0030] (4) The method may include a step of drying the extruded product prepared in step (3) at 50 to 70°C for 10 to 14 hours, and

[0031] More specifically,

[0032] (1) A step of preparing hemp seed meal powder by roasting hemp seeds at 165°C for 12 minutes, pressing to separate oil, and then grinding the hemp seeds;

[0033] (2) A step of mixing plant tissue protein materials based on the total weight of the plant tissue protein materials, comprising 45% by weight of isolated soy protein, 40% by weight of wheat gluten, 4% by weight of corn starch, 3% by weight of modified starch, and 3% by weight of maltodextrin, and 5% by weight of the hemp seed meal powder prepared in step (1);

[0034] (3) A step of preparing an extruded product by feeding the mixed plant tissue protein material from step (2) into an extrusion molding machine controlled to a screw speed of 1500~1600 rpm, a sample feed rate of 200 g / min, a water feed rate of 170 cc / min, and an extrusion port temperature of 175~190℃, and then extruding it; and

[0035] (4) The step of drying the extruded product prepared in step (3) at 60°C for 12 hours may be included.

[0036] The present invention also provides a plant tissue protein with added hemp seed meal prepared by the above method.

[0037] The present invention also provides an alternative meat product prepared using the plant tissue protein with added hemp seed meal. The alternative meat product may preferably be an alternative meat sausage, an alternative meat ham, an alternative meat bacon, an alternative meat tteokgalbi, an alternative meat patty, or an alternative meat meatball, but is not limited thereto.

[0039] The present invention will be described in detail below with reference to manufacturing examples and embodiments. However, the following manufacturing examples and embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the following manufacturing examples and embodiments.

[0041] Preparation Example 1. Texturized Vegetable Protein from Hemp Seed Meal

[0042] (1) Hemp seeds were roasted at 165°C for 12 minutes, then pressed to separate the oil, and the hemp seeds were ground to produce hemp seed meal powder.

[0043] (2) Based on the total weight of the TVP material, 45% by weight of isolated soy protein (ISP), 40% by weight of wheat gluten, 4% by weight of corn starch, 3% by weight of modified starch and 3% by weight of maltodextrin and 5% by weight of the hemp seed meal powder prepared in step (1) above were mixed.

[0044] (3) An extruded product was produced by extrusion molding using an extrusion molding machine with a round die having a diameter of 1 cm, with the screw speed set to 1500~1600 rpm, the sample feed rate to 200 g / min, the water feed rate to 150 cc / min, the barrel temperatures to 90℃-180~195℃-180~190℃, and the injection port temperature to 175~190℃.

[0045] (4) The extruded product prepared in step (3) above was dried at 60°C for 12 hours.

[0047] Preparation Example 2. Hemp seed meat substitute sausage

[0048] A sausage mixture was prepared by mixing the hemp seed meal plant tissue protein of Preparation Example 1 with sausage ingredients. The prepared sausage mixture was filled into a sausage casing and heat-treated.

[0050] Experimental method

[0051] 1. Extraction method and extraction yield of hemp seed meal

[0052] The sample was mixed with 70% ethanol solvent at a ratio of 10 times the sample weight and extracted by stirring at 180 rpm for 3 hours at room temperature. After extraction, centrifugation was performed at 12,000 rpm and 4°C for 10 minutes, and the supernatant was collected, filtered using a 0.45 µm filter, and used in the experiment.

[0053] To measure the yield of solubilized components after extraction, 1 mL of each extract was placed in an aluminum dish, and the 70% ethanol extract was dried at 60°C for 3 hours, 80°C for 1 hour, and 90°C for 30 minutes to measure the content. The solid content was calculated as a percentage using the difference in weight before drying, and the extraction yield was expressed as a percentage by dividing the weight of the sample used by the total amount of solubilized components.

[0055] 2. Analysis of Functional Components and Evaluation of Antioxidant Activity of Hemp Seed Meal

[0056] 2.1. Analysis of Total Polyphenol Content

[0057] 100 µl of 2N Folin-Ciocalteau's phenol reagent solution was added to 100 µl of a sample of a specific concentration diluted with an extraction solvent and reacted at room temperature for 3 minutes, after which 1 mL of 0.7 M Na2CO3 solution was added and reacted at room temperature for 1 hour. Absorbance was measured at 750 nm using a microplate reader, and the content was calculated using a standard curve with tannic acid.

[0059] 2.2. ABTS Radical Scavenging Activity

[0060] For the measurement of ABTS radical scavenging activity, 7 mM ABTS and 2.45 mM potassium persulfate were mixed and left at room temperature for 24 hours in the dark to generate ABTS free radicals. The solution was then diluted with distilled water using a microplate reader until the absorbance at 734 nm reached 0.7–0.9. 100 µl of the diluted ABTS solution was added to 50 µl of the sample and incubated in the dark for 5 minutes to measure the absorbance at 734 nm. The antioxidant activity of ABTS was calculated as the percentage decrease in absorbance between the group with the added sample solution and the group without, and the IC50, which is the concentration of the sample required to reduce ABTS antioxidant activity by 50%, was used. 50 It was expressed as a value.

[0062] 2.3. DPPH Radical Scavenging Activity

[0063] For the measurement of DPPH radical scavenging activity, 0.2 mM DPPH (2,2-Diphenyl-1-picryl hydrazyl) was diluted with 99% ethanol using a microplate reader after shielding from light, until the absorbance at 517 nm was 0.5–0.7. 50 µl of the diluted DPPH solution was added to 100 µl of the sample and reacted in the dark for 10 minutes. The absorbance was measured at 517 nm, and the activity was calculated as the percentage decrease in absorbance between the added and unadded groups. The IC50, which is the concentration required to scavenge 50% of DPPH free radicals, was determined. 50 It was expressed as a value.

[0065] 3. Evaluation of Anti-inflammatory Efficacy

[0066] The anti-inflammatory efficacy was evaluated by quantifying the ability to inhibit NO production after treating mouse-derived macrophages, specifically RAW264.7 cells, with the inflammatory mediator LPS (Lipopolysaccharide).

[0068] 3.1. RAW264.7 Cell Line Culture

[0069] RAW264.7 cells were purchased from the ATCC (American Type Culture Collection) and cultured at 1×10⁶ using DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal Bovine Serum) and 1% Penicillin / Streptomycin. 6 They were cultured at a concentration of cells / mL for 24 hours at 37℃ and 5% CO2 conditions.

[0071] 3.2. Measurement of NO (Nitric Oxide) Production Inhibition Ability

[0072] NO concentration was measured using Griess reagent. RAW264.7 cells were placed in a 96-well plate at a ratio of 1 × 10⁶ 6 Cells were inoculated into wells and cultured for 24 hours, after which the medium was replaced with serum-free DMEM and the sample was treated simultaneously. After 1 hour, a final dose of 1 µg / mL of LPS was added, followed by incubation for 24 hours. After incubation, an equal volume of Griess reagent was reacted with the supernatant for 10 minutes, and the absorbance was measured at 540 nm using a spectrophotometer and expressed as a percentage relative to the LPS-alone treatment group.

[0074] 3.3. Measurement of Cytotoxicity (MTT assay)

[0075] Cytotoxicity was assessed using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RAW264.7 cells were deposited in 96-well plates at a ratio of 1 × 10⁶ 5Cells were inoculated into wells and cultured for 24 hours, after which the medium was changed to serum-free DMEM. Simultaneously, the sample was added, followed by 24 hours of incubation. After incubation, 10 µl of 5 mg / mL MTT solution was dispensed into each well, and a reduction reaction was induced for 3 hours. The supernatant was removed, and 100 µl of DMSO (Dimethyl sulfoxide) solution was added to completely dissolve the purple formazan crystals. The degree of color development was measured by absorbance at 570 nm using a spectrophotometer and expressed as a percentage relative to the untreated group.

[0077] 4. Evaluation of Skin Whitening Efficacy

[0078] Skin whitening efficacy was evaluated by quantifying the ability to inhibit melanin production after inducing melanin production in mouse melanoma cells (B16F10 cells) by treating them with melanin-stimulating hormone (α-MSH).

[0080] 4.1. Culture of B16F10 Cell Line

[0081] B16F10 cells were purchased from ATCC (American Type Culture Collection) and cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal Bovine Serum) and 1% Penicillin / Streptomycin at 37°C and 5% CO2.

[0083] 4.2. Measurement of Melanin Biosynthesis Inhibitory Ability

[0084] 0.5 × 10⁶ B16F10 cells in 12 wells 5Cells were inoculated to a cell / well ratio and cultured for 24 hours. After 24 hours, fresh medium and samples were added, along with 200 nM α-MSH (Melanin-stimulating hormone) and arbutin (100 µg / mL) as a positive control, and cultured for 48 hours. Subsequently, the cells were washed twice with PBS, and Lysis buffer (1% Triton X-100, 0.2 mM PMSF in 0.1 M Sodium phosphate buffer) was added. The mixture was then incubated at -70°C for 1 hour. After incubation at room temperature for approximately 30 minutes, the cells were harvested and centrifuged at 4°C at 12,000 rpm for 15 minutes. The supernatant was used to measure tyrosinase activity, while the precipitate was used to measure melanin content. The cell precipitate was dissolved by adding 200 µl of Lysis buffer (10% Dimethyl sulfoxide (DMSO) in 1N NaOH) at 80°C for 1 hour, and the absorbance was measured at 405 nm. The amount of melanin was determined using a standard straight line established by synthetic melanin.

[0086] 4.3. Measurement of Cytotoxicity (MTT assay)

[0087] Cytotoxicity was assessed using the MTT assay. B16F10 cells were deposited in 96-well plates at a density of 0.5 × 10⁶ 5 Cells were inoculated into wells and cultured for 24 hours, after which the medium was replaced with fresh DMEM. Simultaneously, the sample was added, followed by 24 hours of incubation. After incubation, 10 µl of 5 mg / mL MTT solution was dispensed into each well, and the reduction reaction was induced for 3 hours. The supernatant was removed, and 100 µl of DMSO solution was added to completely dissolve the purple formazan crystals. The degree of color development was measured by absorbance at 570 nm using a spectrophotometer and expressed as a percentage relative to the untreated group.

[0089] 5. Evaluation of efficacy in improving muscle atrophy

[0090] The efficacy of muscle strengthening was evaluated by inducing muscle differentiation and muscle atrophy inhibitory activity in C2C12 cells with DEX (Dexamethasone) and measuring the length and area of ​​muscle fibers using Jenner-Giemsa staining.

[0092] 5.1. C2C12 Cell Line Culture

[0093] C2C12 cells were obtained from the ATCC (American Type Culture Collection) and used. The cells were cultured in DMEM containing 10% FBS and 1% P / S as the culture medium at 37°C and 5% CO2.

[0095] 5.2. Differentiation of Myofibrillar Cells

[0096] For C2C12 myoblast cell culture, growth medium containing 90% DMEM (Dulbecco's Modified Eagle Medium), 10% FBS (Fetal Bovine Serum), and 1% antibiotic-antimycotic was used, and the cells were cultured at 37°C and 5% CO2. To prevent overcrowding caused by cell proliferation, a cell density of approximately 50–60% was maintained, and subculture was performed every 48 hours to maintain an optimal number. To differentiate C2C12 myoblasts into myotubes, a differentiation medium containing 90% DMEM, 2% horse serum, and 1% antibiotic-antimycotic was used. The samples were applied to the cells every two days for 6 days, and myotube formation was observed. In addition, to induce muscle atrophy, 100 μM of DEX (Dexamethasone) was applied to cells on day 6 of differentiation, and the samples were treated at the same concentration as before to observe morphological changes in myotube cells after 48 hours. To confirm the changes in cells occurring during the proliferation and differentiation process, observations were made using an optical microscope (Le ica Biosystems, Hesse, Germany), and the diameter of the myotube cells was measured using Image J software (National Institute of Health, Version v1.32j).

[0098] 5.3. Measurement of Cytotoxicity (MTT assay)

[0099] 1×10⁶ C2C12 myoblast cells in a 96-well plate 5Cells were seeded at a cell / well level and cultured for 24 hours, after which the cell surface was washed with PBS solution. Subsequently, equal amounts of medium and the sample dissolved in PBS were added to each well at different concentrations and cultured for 24 hours. After culture, 1 µg / mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, and the area was shielded from light and reacted at 37°C for 1 hour. After removing all the supernatant, DMSO was added to dissolve the generated formazan, and then the plate was placed on a microplate reader (SpectraMax ® Cell viability was calculated by measuring absorbance at 570 nm using ABS.

[0101] 6. Measurement of physical properties of plant tissue protein with added functional materials

[0102] The texture of the hydrated plant tissue protein was measured 10 times using a physical property measuring instrument (SUNRHEO-METER, CR3000 EX-L, Japan) and calculated as the average value. A No. 1 probe (diameter 2 cm) was used, and under measurement conditions of a support movement speed of 100 mm / min and a maximum stress of 20 kg, measurements were taken before and after drying using 1.5–2.0 cm samples. After drying, the samples were rehydrated in water for 12 hours, then the water was removed for 15 minutes, and hardness, elasticity, and cohesiveness were measured.

[0104] Example 1. Evaluation of Functional Components and Efficacy of Hemp Seed Meal

[0105] 1) Functional ingredients and antioxidant activity

[0106] The extraction yield of the 70% ethanol extract of hemp seed meal was confirmed to be 8.48%, and the total polyphenol content was 8.85 mg / g. In addition, antioxidant activity was determined by the IC50, which is the sample concentration required to reduce ABTS and DPPH by 50%. 50 The values ​​were expressed as values ​​and measured at concentrations of 4,709.37 and 8,730.62 μg / mL, respectively.

[0107] Total Polyphenol Content and Antioxidant Activity of Hemp Seed Meal Extract item Analysis results Extraction yield (%) 8.48% Total polyphenol content (mg / g) 8.85±0.74 ㎎ / g * IC 50 (㎍ / mL) ABTS 4,709.37 µg / mL DPPH 8,730.62 µg / mL

[0109] 2) Anti-inflammatory effects

[0110] Before measuring the NO production inhibitory ability of hemp seed pear extract on RAW264.7 cells, a cytotoxicity test using an MTT assay was conducted to determine a concentration that does not exhibit toxicity within the cells, and the experiment was carried out at a concentration that did not exhibit toxicity.

[0111] To confirm the anti-inflammatory efficacy, RAW264.7 macrophages were treated with the extract and stimulated with LPS to measure the inhibitory effect on NO production. The results showed that, with the LPS-treated group considered as 100%, the hemp seed meal extract reduced NO production in a concentration-dependent manner, and at a concentration of 200 μg / mL, it inhibited NO production to a level similar to that of the positive control (EGCG-treated group) (Fig. 1).

[0113] 3) Skin whitening effect

[0114] Before measuring the whitening efficacy of hemp seed meal extract using B16F10 cells, cytotoxicity was first measured to identify the concentration that did not cause toxicity to the cells, and the experiment was conducted at the concentration that did not show toxicity. The melanin content of the hemp seed extract was measured using B16F10 cells. Melanin production was induced by treatment with α-MSH, and the experiment was conducted with hemp seed extract at concentrations of 50, 100, and 200 μg / mL. As a result, it was confirmed that melanin biosynthesis was inhibited in a concentration-dependent manner when the α-MSH treatment group was considered as 100%, and at a concentration of 200 μg / mL, melanin production was inhibited to a level similar to that of the positive control (arbutin treatment group) (Fig. 2).

[0116] 4) Evaluation of efficacy in improving muscle atrophy

[0117] To determine the effect of hemp seed meal extract on the differentiation process of myotubes using C2C12 myoblasts, cytotoxicity was first evaluated to confirm that the concentrations did not show toxicity to cells in all sample treatment groups compared to the control group that was not treated with the sample.

[0118] Muscle atrophy refers to the exponential decrease in skeletal muscle mass over time due to a reduction in the number of myofibrils. To evaluate the protective activity against muscle atrophy along with the C2C12 cell differentiation-promoting efficacy of hemp seed meal extract, the diameter of differentiated C2C12 myotubes was quantified by measuring the diameter after treating the samples at different concentrations with DEX (Dexamethasone 100 μM). As a result, a loss of myotubes was confirmed in the DEX-treated group compared to the control group. It was confirmed that this was improved at a concentration of 200 μg / mL of hemp seed meal extract when treated with DEX and the samples together (Fig. 3).

[0120] Example 2. Evaluation of quality characteristics of plant tissue protein according to screw speed

[0121] The production of TVP (ISP, mixed with wheat gluten and corn starch) was carried out at screw speeds (1000, 1200, 1300, 1400, 1500 rpm), and a commercially available TVP product was used as a control. As a result of the experiment, it was confirmed that as the screw speed increased, the pressure increased, the expansion rate of the extruded TVP increased, the water retention capacity was high, and the characteristic texture of TVP was formed. Therefore, a speed of 1500 rpm or higher was finally selected (Fig. 4, Table 2, Table 3).

[0122] Measurement of physical properties of plant tissue protein before drying Screw speed (rpm) Diameter (cm) Physical properties Before drying After drying Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 1.98 - 1000 0.94 0.72 Below measurable specifications 1200 1.04 0.92 771,400 92.84 62.56 3,249 3,065 -0.43 1300 1.08 1.03 789,780 83.16 56.70 2,737 2,149 -0.86 1400 1.30 1.08 1,515,600 82.25 51.15 4,815 5,339 -0.80 1500 1.36 1.23 1,699,800 87.18 65.44 6,838 6,150 -1.29

[0124] Measurement of physical properties of rehydrated plant tissue proteins Screw speed (rpm) Conservative power (%) Physical properties Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 74.7 22,562 86.44 79.75 56.04 45.14 -0.27 1000 66.6 Below measurable specifications 1200 63.8 65,770 77.54 54.66 111.65 73.33 -1.00 1300 63.5 62,863 83.63 47.48 160.23 134.08 -2.00 1400 60.3 113,666 96.38 56.43 264.35 252.50 -2.60 1500 64.6 106,083 90.57 63.28 562.41 631.15 -2.28

[0126] Example 3. Evaluation of quality characteristics of plant tissue protein according to temperature conditions

[0127] The production of plant tissue proteins (ISP, a mixture of wheat gluten and corn starch) was carried out according to the barrel's extrusion temperature. As a result of using a commercially available plant tissue protein product as a control, it was confirmed that all values, except for cohesiveness, were higher than those of the control. Measurement of the puffing rate confirmed that the puffing rate increased as the temperature increased. Conversely, it was confirmed that the water-holding capacity decreased as the temperature increased. Comparison of plant tissue proteins according to temperature revealed that while the puffing rate of TVP increased with increasing temperature, high hardness and low water-holding capacity were observed due to a lack of porous structure, so it was selected to be conducted within 190℃ (Fig. 5, Table 4, Table 5).

[0128] Measurement of physical properties of plant tissue protein before drying Temperature (°C) Diameter (cm) Physical properties Before drying After drying Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 1.98 - 150~160 0.94 0.72 1,267,340 83.81 51.82 5,433 3,919 -0.50 160~175 1.04 0.92 1,977,200 80.22 66.70 8,125 6,406 -1.50 175~185 1.08 1.03 1,881,000 82.85 63.08 7,951 6,563 -1.57 185~195 1.30 1.08 2,671,000 83.60 68.24 5,883 5,517 -1.17

[0130] Measurement of physical properties of rehydrated plant tissue proteins Temperature (°C) Conservative power (%) Physical properties Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 74.7 22,562 86.44 79.75 56.04 45.14 -0.27 150~160 63.7 104,123 115.87 25.53 49.99 60.01 -0.80 160~175 63.7 281,500 89.73 55.42 536.84 450.50 -2.00 175~185 61.3 162,330 83.94 43.59 455.11 262.14 -2.17 185~195 60.1 187,660 90.77 59.28 400.02 410.84 -1.57

[0132] Example 4. Evaluation of quality characteristics of plant tissue protein according to moisture inflow

[0133] After using an extrusion port with a diameter of 1 cm, the production of plant tissue protein (ISP, a mixture of wheat gluten and corn starch) was carried out according to moisture inflow rates (100, 150, 200, 250, and 300 cc / min), and a commercially available plant tissue protein product was used as a control. As a result, at 150 cc / min or less, the plant tissue protein did not expand, and at 200 cc / min or more, the plant tissue protein was not compressed during extrusion due to the high moisture content. Finally, the plant tissue protein was ideally compressed and expanded at a moisture content of 150–200 cc / min (Fig. 6, Table 6, Table 7).

[0134] Measurement of physical properties of plant tissue protein before drying Water inflow (cc / min) Diameter (cm) Physical properties Before drying After drying Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 1.98 - 100 0.94 0.72 1,875,800 80.17 61.22 6,903 5,590 -0.86 150 1.04 0.92 1,637,800 83.97 60.20 6,564 5,187 -1.17 200 1.08 1.03 393,650 95.25 62.73 2,067 1,914 -0.40 250 1.30 1.08 Below measurable specifications 300 1.36 1.23 Below measurable specifications

[0136] Measurement of physical properties of rehydrated plant tissue proteins Water inflow (cc / min) Conservative power (%) Physical properties Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 74.7 22,562 86.44 79.75 56.04 45.14 -0.27 100 64.1 146,800 86.91 65.10 574.26 526.72 -2.43 150 62.0 266,155 87.65 71.27 817.84 807.10 -0.72 200 66.0 115,110 89.08 50.43 193.38 164.13 -1.00 250 62.1 Below measurable specifications 300 66.2 Below measurable specifications

[0138] Example 5. Evaluation of quality characteristics of plant tissue protein according to mixing ratio

[0139] Quality characteristics were evaluated using plant tissue proteins prepared with different types of ingredients and mixing ratios. Samples were divided into undried and rehydrated forms, and commercially available plant tissue protein products were used as the control group.

[0140] As a result of measuring physical properties, it was confirmed that the hardness of most samples decreased when measured after drying following soaking, due to the high porous structure. As a result of measuring diameter, when modified starch was added, the plant tissue protein expanded, but proper binding did not occur, such as tearing in the middle. The highest diameter was observed when modified starch, maltodextrin, and corn starch were added together (Fig. 7, Table 8, Table 9).

[0141] Measurement of physical properties of plant tissue protein before drying item Diameter (cm) Physical properties Before drying After drying Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 1.98 - * TVP 1 1.30 1.08 2,671,000 83.60 68.24 5,883 5,517 -1.17 TVP 2 1.63 1.58 1,193,250 79.31 62.46 4,330 3,409 -1.62 TVP 3 1.63 1.50 1,085,105 82.51 64.83 4,223 3,312 -0.89 TVP 4 1.17 1.13 2,125,714 81.67 65.93 9,085 7,293 -1.87 TVP 5 1.41 1.33 749,280 85.85 67.36 3,834 3,273 -0.29 TVP 6 1.29 1.24 1,237,120 79.46 63.80 4,963 3,826 -0.71 TVP 7 1.70 1.63 664,685 81.03 65.24 2,755 2,202 -0.67

[0142] * TVP 1(ISP:Wheat Gluten:Corn Starch = 50:40:10), TVP 2(ISP:Wheat Gluten:Modified Starch = 50:40:10), TVP 3(ISP:Wheat Gluten:Modified Starch:Corn Starch = 50:40:5:5), TVP 4(ISP:Wheat Gluten:Maltodextrin = 50:40:10), TVP 5(ISP:Wheat Gluten:Maltodextrin:Corn Starch = 50:40:5:5), TVP 6(ISP:Wheat Gluten:Modified Starch:Maltodextrin = 50:40:5:5), TVP 7(ISP:Wheat Gluten:Corn Starch:Modified Starch:Maltodextrin = 50:40:4:3:3)

[0143] Measurement of physical properties of rehydrated plant tissue proteins item Conservative power (%) Physical properties Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 71.3 33,761 84.41 80.15 84.42 70.45 -0.66 *TVP 1 60.1 187,660 90.77 59.28 400.02 410.84 -1.57 TVP 2 70.3 79,104 83.00 57.26 300.14 249.90 -1.33 TVP 3 69.4 66,347 94.00 82.38 331.55 310.15 -0.17 TVP 4 63.1 64,396 88.25 70.94 282.66 262.12 0.00 TVP 5 68.0 115,670 89.69 68.52 445.15 344.60 -0.18 TVP 6 58.1 165,812 89.56 76.38 803.47 736.14 0.00 TVP 7 68.3 67,496 88.23 73.54 323.55 286.22 -0.30

[0144] *TVP 1(ISP:Wheat Gluten:Corn Starch = 50:40:10), TVP 2(ISP:Wheat Gluten:Modified Starch = 50:40:10), TVP 3(ISP:Wheat Gluten:Modified Starch:Corn Starch = 50:40:5:5), TVP 4(ISP:Wheat Gluten:Maltodextrin = 50:40:10), TVP 5(ISP:Wheat Gluten:Maltodextrin:Corn Starch = 50:40:5:5), TVP 6(ISP:Wheat Gluten:Modified Starch:Maltodextrin = 50:40:5:5), TVP 7(ISP:Wheat Gluten:Corn Starch:Modified Starch:Maltodextrin = 50:40:4:3:3)

[0146] Example 6. Evaluation of quality characteristics of plant tissue protein according to the ratio of hemp seed meal powder

[0147] Plant tissue protein was prepared according to hemp seed meal ratios (5%, 10%, 25%) using an extrusion nozzle with a diameter of 1 cm, and a commercially available plant tissue protein product was used as a control for comparison. As a result, the swelling rate decreased as the hemp seed meal ratio increased, and no swelling occurred in the 25% addition group (Fig. 8). In addition, when quality characteristics were measured and compared, the hardness was found to be low in the 25% addition group because no swelling occurred, while the diameter and water-holding capacity were high in the 5% addition group, so it was decided to use 5% addition.

[0148] Measurement of physical properties of plant tissue protein before drying item Diameter (cm) Physical properties Before drying After drying Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 1.98 - *Control group 1.7 1.63 664,685 81.03 65.24 2,755 2,202 -0.67 TVP 1 1.56 1.42 1,227,375 82.40 63.75 2,010.93 2,542.93 -0.80 TVP 2 1.14 1.14 1,243,561 80.52 34.28 1,042.23 1,745.23 -0.63 TVP 3 0.85 0.74 1,256,125 79.70 27.43 506.27 722.23 -0.50

[0149] * Control group (ISP:Wheat gluten:Corn starch:Modified starch:Maltodextrin = 50:40:4:3:3), TVP 1 (ISP:Wheat gluten:Hemp seed meal:Corn starch:Modified starch:Maltodextrin = 45:40:5:4:3:3), TVP 2 (ISP:Wheat gluten:Hemp seed meal:Corn starch:Modified starch:Maltodextrin = 40:40:10:4:3:3), TVP 3 (ISP:Wheat gluten:Hemp seed meal:Corn starch:Modified starch:Maltodextrin = 25:40:25:4:3:3)

[0151] Measurement of physical properties of rehydrated plant tissue proteins item Conservative power (%) Diameter (cm) Physical properties Hardness (gf / ㎠) Springiness(%) Cohesiveness (%) Gumminess(gf) Brittleness(gf) Adhesiveness(gf) Control 72.1 2.18 43,520 85.24 84.06 95.63 76.80 -0.38 *Control group 68.3 1.82 67,496 88.23 73.54 323.55 286.22 -0.30 TVP 1 67.3 1.74 249,020 89.33 73.12 490.66 484.67 0.00 TVP 2 62.3 1.40 156,750 87.07 76.42 490.44 455.08 -0.29 TVP 3 53.6 0.88 64,210 88.66 63.85 164.11 146.82 -0.43

[0152] * Control group (ISP:Wheat gluten:Corn starch:Modified starch:Maltodextrin = 50:40:4:3:3), TVP 1 (ISP:Wheat gluten:Hemp seed meal:Corn starch:Modified starch:Maltodextrin = 45:40:5:4:3:3), TVP 2 (ISP:Wheat gluten:Hemp seed meal:Corn starch:Modified starch:Maltodextrin = 40:40:10:4:3:3), TVP 3 (ISP:Wheat gluten:Hemp seed meal:Corn starch:Modified starch:Maltodextrin = 25:40:25:4:3:3)

[0154] Example 7. Sensory characteristics of meat substitute sausages according to the type of plant tissue protein

[0155] Meat substitute sausages were prepared using the method of Preparation Example 2 with different types of plant tissue proteins, and their sensory characteristics were compared. For the sensory characteristics, 30 sensory evaluators were asked to select the meat substitute sausage they preferred regarding taste, smell, and texture. As a result, the meat substitute sausage made with plant tissue protein containing hemp seed meal received more selections (Table 12).

[0156] Sensory characteristics of plant-based sausages Types of plant tissue proteins taste smell Texture ISP:Wheat Gluten:Corn Starch:Modified Starch:Maltodextrin = 50:40:4:3:3 4 people 12 people 7 people ISP:Wheat Gluten:Hemp Seed Meal:Corn Starch:Modified Starch:Maltodextrin = 45:40:5:4:3:3 26 people 18 people 23 people

Claims

Claim 1 (1) a step of preparing hemp seed meal powder by grinding hemp seeds from which oil has been separated by roasting and pressing the hemp seeds; (2) a step of mixing plant tissue protein materials based on the total weight of the plant tissue protein materials, comprising 43-47% by weight of isolated soy protein, 38-42% by weight of wheat gluten, 2-6% by weight of corn starch, 1-5% by weight of modified starch, and 1-5% by weight of maltodextrin, and 3-7% by weight of the hemp seed meal powder prepared in step (1); (3) a step of preparing an extruder by feeding the mixed plant tissue protein materials from step (2) into an extrusion molding machine and then extruding the mixture; and (4) a step of drying the extruder prepared in step (3), characterized by comprising the steps of preparing a plant tissue protein with added hemp seed meal. Claim 2 delete Claim 3 A method for producing plant tissue protein with added hemp seed meal according to claim 1, characterized in that the extruder of step (3) is fed into an extrusion molding machine controlled to a screw speed of 1500~1600 rpm, a sample feed rate of 180~220 g / min, a water feed rate of 150~200 cc / min, and an injection port temperature of 175~190℃, and then extruded. Claim 4 In claim 1, (1) a step of preparing hemp seed meal powder by roasting hemp seeds at 160~170℃ for 10~14 minutes, pressing to separate oil, and then grinding the hemp seeds; (2) a step of mixing a plant tissue protein material comprising, based on the total weight of the plant tissue protein material, 43~47 wt% isolated soy protein, 38~42 wt% wheat gluten, 2~6 wt% corn starch, 1~5 wt% modified starch, and 1~5 wt% maltodextrin, and 3~7 wt% of the hemp seed meal powder prepared in step (1); (3) a step of mixing the plant tissue protein material from step (2) in an extrusion molding machine controlled to a screw speed of 1500~1600 rpm, a sample feed rate of 180~220 g / min, a water supply rate of 150~200 cc / min, and an extrusion port temperature of 175~190℃ A method for producing plant tissue protein with added hemp seed meal, characterized by comprising the steps of: (4) producing an extruded product by extrusion molding after input; and (4) drying the extruded product produced in step (3) at 50 to 70°C for 10 to 14 hours. Claim 5 Plant tissue protein with added hemp seed meal, prepared by any one of the methods of paragraphs 1, 3, and 4. Claim 6 An alternative meat product manufactured using plant tissue protein with added hemp seed meal according to Paragraph 5.

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