Method for improving softening of plant protein-containing product

Abstract

The purpose of the present application is to provide a technique to improve softening of a plant protein-containing product by addition of phytase during the production of the product. The present application discloses, as an embodiment, a method for producing a plant protein-containing product, the method comprising: (1) a step for adding a phytic acid-degrading enzyme to a production raw material; and (2) a step for adding, to the production raw material, an enzyme that contributes to the formation of a protein crosslinking structure.

Description

Method for improving the softening of vegetable protein-containing products

[0001] In one embodiment, the present invention provides a method for producing a vegetable protein-containing product, which includes the steps of (1) adding a phytate-degrading enzyme to a production raw material, and (2) adding an enzyme that contributes to the formation of a cross-linked structure of a protein to the production raw material, and is useful, for example, in the food industry.

[0002] Animal protein materials such as livestock products and marine products have traditionally been popular ingredients in the food industry. However, due to the recent rise in environmental awareness and health consciousness, the market for plant-based products ("plant protein-containing products") manufactured using plant protein materials such as beans and grains as alternatives to animal protein is expanding. While the importance of plant protein materials is increasing, plant proteins contain phytic acid, which binds with minerals (iron, calcium, magnesium, etc.) to become insoluble and inhibit absorption by the body of the consumer. Therefore, it is desirable to remove phytic acid. For this reason, one method of removing phytic acid is to decompose phytic acid using phytase (a representative phytate-degrading enzyme), which is widely used in the production process of plant protein-containing products (see, for example, Patent Documents 1 and 2).

[0003] JP-A No. 2023-500467 WO 2003 / 043478

[0004] Under these circumstances, the influence of phytase addition on the physical properties of products has not been widely recognized among those skilled in the art. However, the present inventors have, through their investigations, found for the first time that the addition of phytase has an adverse effect on the physical properties of vegetable protein-containing products, i.e., that the hardness of the products decreases and the products become "softened." The inventors then faced a new problem: in order to provide vegetable protein-containing products with an improved texture, it is necessary to overcome this adverse effect on the physical properties, i.e., "softening of the products." The present invention aims to provide a new technology that can solve the previously unrecognized problem of product softening caused by the addition of phytase, while maintaining the previously desired effect of improving mineral absorbability achieved by the addition of phytase.

[0005] As a result of extensive research, the present inventors have found that the above-mentioned problems can be solved by adding an enzyme that contributes to the formation of cross-linked structures in proteins in addition to the addition of a phytate-degrading enzyme during the production of a vegetable protein-containing product, and have thus completed the present invention. The present invention will be described below by specifically listing embodiments for solving the problems. However, the present invention is not limited to these embodiments.

[0006] [1] A method for producing a vegetable protein-containing product, comprising: (1) adding a phytate-degrading enzyme to a production raw material; and (2) adding an enzyme that contributes to the formation of a cross-linked structure of a protein to the production raw material. [2] The production method according to [1] above, wherein the phytate-degrading enzyme is phytase. [3] The production method according to [1] or [2] above, wherein the enzyme that contributes to the formation of a cross-linked structure of a protein is one or more selected from enzymes that act directly on proteins to form cross-linked structures and enzymes that act indirectly on proteins to form cross-linked structures. [4] The production method according to [3] above, wherein the enzyme that contributes to the formation of a cross-linked structure of a protein is one or more selected from transglutaminase, glucose oxidase, and ascorbic acid oxidase. [5] The production method according to any of [1] to [4] above, wherein the production raw material further contains an animal protein. [6] The manufacturing method according to any one of [1] to [5] above, wherein the amount of the phytate-degrading enzyme added is in the range of 0.01 to 100,000 U / g activity relative to the weight of the vegetable protein material in the raw materials. [7] The manufacturing method according to any one of [1] to [6] above, wherein the amount of the enzyme contributing to the formation of a cross-linked structure of protein added is in the range of 0.0001 to 250 U / g activity relative to the total weight of the vegetable protein material and the animal protein material in the raw materials (hereinafter also referred to as "total protein material weight") when the enzyme acts directly on the protein to form a cross-linked structure. [8] The manufacturing method according to any one of [1] to [6] above, wherein the amount of the enzyme contributing to the formation of a cross-linked structure of protein added is in the range of 0.001 to 10,000 U enzymatic activity relative to 1 g of the substrate glucose when the enzyme acts indirectly on the protein by acting on the glucose substrate contained in the raw materials to form a cross-linked structure. [9] The production method according to any one of the above [1] to [6], wherein the amount of the enzyme added that contributes to the formation of a crosslinked structure of a protein is in the range of 0.001 to 10,000 U of enzyme activity per 1 g of the substrate L-ascorbic acids, when the enzyme acts on L-ascorbic acids, which are substrates contained in the production raw materials, to indirectly act on the protein to form a crosslinked structure.

[10] The method according to any one of [1] to [9] above, wherein the vegetable protein-containing product is a food or a food ingredient.

[11] The method according to any one of [1] to

[10] above, wherein the vegetable protein-containing product is semi-solid or solid.

[0007]

[12] A method for improving softening of a plant protein-containing product caused by the addition of a phytate-degrading enzyme to raw materials for production, the method comprising adding an enzyme that contributes to the formation of cross-linked structures of proteins to the raw materials for production of the plant protein-containing product.

[13] The method for improving the softening of a plant protein-containing product described above in

[12] , wherein the phytate-degrading enzyme is phytase.

[14] The method for improving the softening of a plant protein-containing product described above in

[12] or

[13] , wherein the enzyme that contributes to the formation of cross-linked structures of proteins is one or more selected from enzymes that act directly on proteins to form cross-linked structures and enzymes that act indirectly on proteins to form cross-linked structures.

[15] The method for improving the softening of a plant protein-containing product described above in

[14] , wherein the enzyme that contributes to the formation of cross-linked structures of proteins is one or more selected from transglutaminase, glucose oxidase, and ascorbic acid oxidase.

[16] The production method described in any of

[12] to

[15] , wherein the raw materials for production further contain animal protein.

[17] The method for improving food according to any one of

[12] to

[16] above, wherein the amount of phytate-degrading enzyme added is in the range of 0.01 to 100,000 U / g activity relative to the weight of the vegetable protein material in the raw materials.

[18] The method for improving food according to any one of

[12] to

[17] above, wherein the amount of enzyme contributing to the formation of a cross-linked structure of protein added is in the range of 0.0001 to 250 U / g activity relative to the total weight of the vegetable protein material and the animal protein material in the raw materials ("total protein material weight"), when the enzyme acts directly on the protein to form a cross-linked structure.

[19] The method for improving food according to any one of

[12] to

[17] above, wherein the amount of enzyme contributing to the formation of a cross-linked structure of protein added is in the range of 0.001 to 10,000 U enzymatic activity relative to 1 g of the substrate glucose, when the enzyme acts indirectly on the protein by acting on the glucose substrate contained in the raw materials to form a cross-linked structure.

[20] The method for improving according to any one of

[12] to

[17] above, wherein the amount of the enzyme added that contributes to the formation of a crosslinked structure of a protein is in the range of 0.001 to 10,000 U of enzyme activity per 1 g of the substrate L-ascorbic acids, when the enzyme forms a crosslinked structure by acting indirectly on the protein through acting on L-ascorbic acids, which are substrates contained in the production raw materials.

[21] The method for improving according to any one of

[12] to

[20] above, wherein the vegetable protein-containing product is a food product or a food ingredient.

[22] The method for improving according to any one of

[12] to

[21] above, wherein the vegetable protein-containing product is in a semi-solid or solid form.

[0008]

[23] An agent for improving the softening of vegetable protein-containing products caused by adding a phytate-degrading enzyme to the production raw materials, the agent containing an enzyme that contributes to the formation of a cross-linked structure of proteins.

[0009] According to one embodiment of the present invention, there is provided a method for producing a vegetable protein-containing product, which includes the steps of (1) adding a phytate-degrading enzyme to a production raw material, and (2) adding an enzyme that contributes to the formation of a cross-linked structure of a protein to the production raw material.

[0010] Fig. 1 shows a schematic diagram of a measurement sample in "Hardness Measurement" in the Examples described later. Fig. 2 shows a schematic diagram of a measurement sample during measurement in "Hardness Measurement" in the Examples described later. Fig. 3 shows the measurement results in "Hardness Measurement" in the Examples described later.

[0011] The present invention will be described below with reference to its embodiments, but the present invention is not limited thereto. Those skilled in the art may modify the following embodiments in various aspects without departing from the spirit of the present invention, and such modifications are also within the scope of the present invention.

[0012] One embodiment disclosed by the present invention is "a method for producing a vegetable protein-containing product, comprising: (1) a step of adding a phytate-degrading enzyme to a production raw material; and (2) a step of adding an enzyme that contributes to the formation of a cross-linked structure of a protein to the production raw material" (Embodiment A).

[0013] In conventional methods, the addition of phytase, a typical phytate-degrading enzyme, decomposes phytic acid contained in vegetable proteins, and the resulting phytic acid binds with minerals (iron, calcium, magnesium, etc.) to become insoluble, thereby preventing a decrease in bioavailability. However, as detailed in the Examples below, a "problem associated with the addition of phytase" has been identified: the vegetable protein-containing product produced with the addition of phytase softens and loses hardness. Product hardness significantly affects the texture upon consumption, and improvement of this problem is an urgent issue. Embodiment A relates to a method for producing a vegetable protein-containing product in which the above-mentioned problem associated with the addition of phytase-degrading enzymes, such as phytase, is improved by adding an enzyme that contributes to the formation of cross-linked structures in the vegetable protein during the production process, thereby restoring the hardness of the product. This embodiment is described in detail below.

[0014] (Enzymes) The "phytate-degrading enzyme" is not particularly limited as long as it can catalyze the hydrolysis of phosphate esters of phytic acid. For example, phytase is an example. If commercially available, the commercially available product can be purchased and used. The "enzyme that contributes to the formation of a crosslinked structure of proteins" is not particularly limited as long as it is an enzyme that can crosslink proteins. The crosslinking may take any of the following forms: 1) an enzyme acts directly on a protein to form a crosslinked structure (direct crosslinking), or 2) an enzyme acts on another substrate to produce a chemical substance, which then acts on the protein to form a crosslinked structure (indirect crosslinking). For example, a representative enzyme for direct crosslinking is transglutaminase. Representative enzymes for indirect crosslinking include glucose oxidase and ascorbic acid oxidase. When glucose oxidase or ascorbic acid oxidase is used, glucose or L-ascorbic acid, respectively, is used as a substrate to generate hydrogen peroxide, which then acts on the protein to form a crosslinked structure. Note that a person skilled in the art can select an appropriate "enzyme that contributes to the formation of a cross-linked structure of a protein" by taking into consideration various factors such as reactivity with the target protein, cost, difficulty in obtaining approval and licensing, ease of procurement, etc. If the enzyme is commercially available, it can be purchased and used. One type of "enzyme that contributes to the formation of a cross-linked structure of a protein" may be used, or two or more types may be used in combination.

[0015] The order of the step of adding the phytate-degrading enzyme to the raw materials for production and the step of adding the enzyme that contributes to the formation of cross-linked protein structures to the raw materials for production is not particularly limited, and the steps of adding both enzymes can be performed in any of the following modes: 1) adding the phytate-degrading enzyme and then the enzyme that contributes to the formation of cross-linked protein structures, 2) adding the phytate-degrading enzyme and then the enzyme that contributes to the formation of cross-linked protein structures, or 3) adding the phytate-degrading enzyme and the enzyme that contributes to the formation of cross-linked protein structures simultaneously. Those skilled in the art can carry out this embodiment by appropriately selecting the order of addition depending on the type of the target vegetable protein-containing product and the raw materials for its production.

[0016] (Production Raw Materials) "Production raw materials" refer to materials other than phytate-degrading enzymes and enzymes that contribute to the formation of protein cross-linking structures used in the production of a vegetable protein-containing product. These raw materials can be appropriately selected depending on the desired form of the "vegetable protein-containing product." Examples of such raw materials include vegetable protein materials (e.g., legumes such as soybeans, peas, and broad beans; grains such as rice, oats, and wheat; and nuts such as almonds, cashews, and walnuts). While the raw materials themselves can be used as production raw materials, vegetable proteins extracted and purified from the raw materials (e.g., powdered soy protein, granular soy protein, pea protein, and broad bean protein) can also be used as vegetable protein materials. If any of these are commercially available, they can be purchased and used. In addition to the above-mentioned vegetable protein materials, the production raw materials can further contain water, edible oils and fats, seasonings, binders, and other common food additives depending on the desired form of the vegetable protein-containing product, as long as the characteristics of this embodiment are not impaired.

[0017] The edible oils and fats that can be used are not particularly limited as long as they are edible, and examples thereof include vegetable oils and fats such as salad oil, corn oil, soybean oil, sesame oil, rapeseed oil, rice oil, safflower oil, cottonseed oil, sunflower oil, perilla oil, olive oil, palm oil, peanut oil, almond oil, coconut oil, avocado oil, cacao butter, peanut butter, palm oil, and shortening; and animal oils and fats such as fish oil, beef tallow, lard, and milk fat.

[0018] The seasoning may be any food or food additive used to flavor dishes, without any particular limitation. Examples of the seasoning include salt, sugars (e.g., glucose, fructose, sucrose, lactose, dextrin, etc.), starches (e.g., wheat starch, rice starch, potato starch, corn starch (cornstarch), waxy cornstarch, tapioca starch, etc.), soy protein, soy flour, vegetable powders (e.g., carrot, onion, celery, Chinese cabbage, cabbage, etc.) and extracts thereof, seaweed powders (e.g., kelp, wakame, etc.) and extracts thereof, mushroom powders (e.g., matsutake, shiitake, shimeji, etc.) and extracts thereof, soy sauce, mirin, sake, yeast extract, umami seasonings (e.g., amino acid seasonings, nucleic acid seasonings, etc.), onion powder, garlic powder, black pepper, sweeteners, acidulants, spices, etc. Examples of binders include commonly used ones such as breadcrumbs.

[0019] Other common food additives include emulsifiers (glycerin fatty acid esters, sucrose fatty acid esters, saponin, lecithin, etc.), manufacturing agents (phosphates, silicon dioxide, etc.), preservatives (benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, ε-polylysine, etc.), antioxidants (ascorbic acid, erythorbic acid, catechin, etc.), fungicides (thiabenzoxol, fludioxonil, etc.), colorings (annatto pigment, turmeric pigment, gardenia pigment, etc.), flavorings (synthetic flavorings such as ethyl acetoacetate and anisaldehyde, natural flavorings such as squid, shrimp, crab), gum arabic, tamarind seed gum, etc. One or more of the above-mentioned edible oils and fats, seasonings, binders, and other common food additives can be added as needed, and the amount added can be determined according to the usual amount.

[0020] In this embodiment, animal protein materials may be included in the raw materials as long as the characteristics of the product are not impaired. In this case, a hybrid product containing both plant and animal proteins can be obtained. Examples of such animal protein materials include animal-derived materials such as beef, pork, chicken, eggs, and milk, as well as seafood-derived materials such as white meat from salmon and sea bream, and red meat from tuna and bonito. As described above, this embodiment encompasses both 1) cases in which the raw materials contain only plant protein materials and 2) cases in which the raw materials contain both plant and animal protein materials. Hereinafter, both cases will be collectively referred to as "total protein materials." The total weight of both will also be referred to as "total protein material weight."

[0021] (Amount of Enzyme Added) (1) Phytate-Degrading Enzyme Phytate-degrading enzyme is preferably added to raw materials in an amount appropriate for decomposing phytic acid contained in vegetable proteins and preventing the phytic acid from binding with minerals (iron, calcium, magnesium, etc.) and becoming insoluble. This amount can be determined appropriately by those skilled in the art, taking into account the raw materials, etc. The enzymatic activity of a phytate-degrading enzyme can be determined appropriately by those skilled in the art according to the definition of 1 unit (1 U) of the enzyme used. For example, in the case of phytase, as used herein, phosphate is produced by the action of phytase on sodium phytate as a substrate at 37°C and pH 5.5, and the absorbance of the resulting phosphate-molybdic acid complex produced by reacting the resulting phosphate with ammonium molybdate is measured at a wavelength of 380 nm, and the amount of phosphate is determined using a calibration curve. The amount of enzyme required to produce 1 μmol of phosphate from sodium phytate per minute is defined as 1 unit (1 U). From the viewpoint of phytate-degrading enzyme activity, the phytate-degrading enzyme is added so that the activity is, for example, in the range of 0.001 to 100,000 U / g relative to the weight of the vegetable protein material in the production raw material, more preferably in the range of 0.01 to 10,000 U / g, and even more preferably in the range of 0.1 to 1,000 U / g.

[0022] (2) Enzymes Contributing to the Formation of Cross-Linked Protein Structures The amount of enzymes added that contribute to the formation of cross-linked protein structures can be determined appropriately depending on the type of vegetable protein-containing product to be produced, the hardness of the desired product, and other factors. As described above, cross-linking can be achieved in either of the following ways: 1) an enzyme acts directly on a protein to form a cross-linked structure (direct cross-linking); or 2) an enzyme acts on another substrate to produce a chemical substance that acts on the protein to form a cross-linked structure (indirect cross-linking). For example, a representative enzyme for direct cross-linking is transglutaminase. Representative enzymes for indirect cross-linking include glucose oxidase and ascorbic acid oxidase. When glucose oxidase or ascorbic acid oxidase is used, glucose or L-ascorbic acid, respectively, is used as a substrate to generate hydrogen peroxide, which then acts on the protein to form a cross-linked structure. The amount of enzyme to be added to the production raw materials can be determined appropriately by one skilled in the art, taking into consideration the type of enzyme used (cross-linking mode) and the production raw materials (e.g., the total type of protein material and the type of substrate). The amounts of enzymes to be added will be explained below using transglutaminase (an enzyme for direct cross-linking), glucose oxidase, and ascorbic acid oxidase (enzymes for indirect cross-linking) as specific examples. Those skilled in the art will be able to determine the appropriate amounts of enzymes to be added by referring to these specific examples.

[0023] 1) When transglutaminase is used In this specification, the enzymatic activity of transglutaminase is measured by reacting a reaction system containing benzyloxycarbonyl-L-glutamylglycine and hydroxylamine as substrates in a Tris buffer solution at 37°C and pH 6.0 with transglutaminase, forming an iron complex with the hydroxamic acid produced in the presence of trichloroacetic acid, measuring the absorbance at 525 nm, and determining the amount of hydroxamic acid using a calibration curve. One unit (1 U) of the enzyme is defined as the amount of enzyme required to produce 1 μmol of hydroxamic acid per minute (see Japanese Patent Laid-Open No. 64-27471). From the viewpoint of transglutaminase activity, transglutaminase is preferably added so as to achieve an activity in the range of, for example, 0.0001 to 250 U / g, more preferably 0.001 to 100 U / g, and even more preferably 0.01 to 50 U / g, relative to the weight of the total protein material in the production raw material.

[0024] 2) When Glucose Oxidase is Used In this specification, the enzymatic activity of glucose oxidase is measured by generating hydrogen peroxide by the action of glucose oxidase on glucose as a substrate in the presence of oxygen at 30°C and pH 5.6, and then measuring the absorbance at 500 nm of the quinoneimine dye generated by the action of peroxidase on the generated hydrogen peroxide in the presence of aminoantipyrine and phenol. The amount of enzyme required to oxidize 1 μmol of glucose per minute is defined as 1 unit (1 U). In this embodiment, when glucose oxidase is used as the enzyme that contributes to the formation of a crosslinked structure of a protein, it is necessary to add glucose, which serves as a substrate, to the production raw material to which the enzyme is added. However, this does not apply if the production raw material itself already contains glucose. The amount of glucose may be 0.00001 to 99 wt %, preferably 0.0001 to 95 wt %, more preferably 0.001 to 90 wt %, and even more preferably 0.01 to 80 wt %, based on the total weight of the raw materials. Even if the raw materials themselves contain glucose, glucose can be added externally to adjust the amount to the above range. In this embodiment, when glucose oxidase is used as the enzyme that contributes to the formation of a crosslinked structure of the protein, the glucose oxidase is added so that the enzyme activity per 1 g of the substrate (glucose) is, for example, 0.001 to 10,000 U, preferably 0.01 to 5,000 U, more preferably 0.1 to 3,000 U, and even more preferably 1 to 1,000 U.

[0025] 3) When Ascorbic Acid Oxidase is Used In this specification, the enzymatic activity of ascorbic acid oxidase is measured by reacting ascorbic acid with L-ascorbic acid as a substrate in the presence of oxygen at 30°C and pH 5.6, and quantifying the amount of L-ascorbic acid lost by measuring the absorbance at 245 nm. The amount of enzyme required to oxidize 1 μmol of L-ascorbic acid per minute is defined as 1 unit (1 U). In this embodiment, when ascorbic acid oxidase is used as the enzyme contributing to the formation of a crosslinked protein structure, it is necessary to add an L-ascorbic acid substrate to the production raw material to which the enzyme is added. However, this does not apply when the production raw material itself already contains an L-ascorbic acid (for example, a material containing a large amount of ascorbic acid (e.g., acerola powder, etc.)). Here, L-ascorbic acids refer to ascorbic acid, ascorbic acid salts, or compounds with a modified ascorbic acid skeleton. Examples include salts with alkali metals or alkaline earth metals (e.g., sodium ascorbate, calcium ascorbate, etc.), provitamins such as ascorbic acid 2-glucoside, and ascorbic acid esters (e.g., ascorbic acid palmitate, ascorbic acid stearate, etc.). Among these, ascorbic acids and sodium ascorbate are preferred. The amount of L-ascorbic acids may be 0.00001 to 99 wt %, preferably 0.0001 to 95 wt %, more preferably 0.001 to 90 wt %, and even more preferably 0.01 to 80 wt %, based on the total weight of the raw materials. Even when the raw materials themselves contain L-ascorbic acids, the amount can be adjusted by adding externally the L-ascorbic acids to achieve the above-mentioned amount. Furthermore, in this embodiment, when ascorbic acid oxidase is used, the amount of ascorbic acid oxidase added is such that the enzyme activity is in the range of, for example, 0.001 to 10,000 U, preferably 0.01 to 5,000 U, more preferably 0.1 to 3,000 U, and even more preferably 1 to 1,000 U per 1 g of the substrate (L-ascorbic acids) of the enzyme.

[0026] (Production of a Vegetable Protein-Containing Product) In this embodiment, step (1) of adding a phytate-degrading enzyme to the raw materials and step (2) of adding an enzyme that contributes to the formation of a cross-linked protein structure to the raw materials can be performed by methods commonly used in the art. Each enzyme is added to the raw materials, mixed, and subjected to the enzymatic reaction step (3). The order of mixing the raw materials and the enzymes is not particularly limited. For example, all or part of the raw materials may be premixed. Those skilled in the art can determine the appropriate mixing order. The enzymatic reaction step (3) can be performed by methods commonly used in the art, for example, by heating the mixture at 0 to 70°C for 0 minutes to 72 hours. The enzymatic reaction may be performed with appropriate stirring. After the enzymatic reaction is complete, an enzyme deactivation step may be included, if necessary. The enzymatic reaction step (3) may be performed separately for step (1) of adding the phytate-degrading enzyme and step (2) of adding the enzyme that contributes to the formation of a cross-linked protein structure. In the manufacturing method of this embodiment, the enzyme-treated manufacturing raw materials prepared in steps (1) to (3) can be further subjected to a heating step, a cooling step, and / or a freezing step, etc., as necessary, to produce the final vegetable protein-containing product.

[0027] As the heating means, conventional cooking means such as steaming, baking, frying, etc. can be employed, and the heating can be carried out using cooking equipment such as a steamer, oven, convection oven, steam convection oven, fryer, etc. The time and temperature of the heating step are not particularly limited and can be appropriately determined by a person skilled in the art depending on the type of desired vegetable protein-containing product, but in one embodiment, heating can be carried out at 50°C to 150°C, preferably 70°C to 120°C, in another embodiment at 150°C to 250°C, preferably 150°C to 220°C, for usually 1 minute to 60 minutes, preferably 5 minutes to 30 minutes, and in another preferred embodiment, 1 minute to 20 minutes, preferably 2 minutes to 15 minutes.

[0028] As the cooling means, ordinary cooling means employed in food production can be used, such as cooling at room temperature, cooling with air, cooling with running water, etc. As the freezing means, ordinary freezing means employed in food production can be used, such as freezing in a freezer at about -20°C to -40°C, quick freezing, etc. From the viewpoint of maintaining the quality of the plant protein-containing product during the storage period until it is provided to the consumer, quick freezing is preferred.

[0029] (Plant Protein-Containing Product) The plant protein-containing product produced in this embodiment may be consumed as a food product itself, or may be used as a food ingredient and then further cooked or otherwise processed to prepare a final food product. The form of the plant protein-containing product produced in this embodiment is not particularly limited, but is preferably semi-solid or solid. Specific examples include plant-based meat products (e.g., hamburger steak), plant-based dairy products (e.g., cheese, yogurt, butter, etc.), plant-based fish products, plant-based egg products, textured plant proteins, protein bars, etc.

[0030] Another embodiment disclosed by the present invention is "(B) a method for improving the softening of a vegetable protein-containing product caused by the addition of a phytate-degrading enzyme to the raw materials for production, comprising adding an enzyme that contributes to the formation of a cross-linked structure of protein to the raw materials for production of the vegetable protein-containing product" (Embodiment B). Embodiment B relates to a method for improving the softening of a vegetable protein-containing product caused by the addition of a phytate-degrading enzyme to the raw materials for production, by adding an enzyme that contributes to the formation of a cross-linked structure of protein in the process of producing the vegetable protein-containing product.

[0031] This embodiment is carried out by adding an enzyme that contributes to the formation of cross-linked structures of proteins in a process for producing a vegetable protein-containing product, which includes a step of adding a phytate-degrading enzyme to raw materials for production. The "enzyme that contributes to the formation of cross-linked structures of proteins" and the "phytate-degrading enzyme" used in this embodiment, as well as the manner of use (e.g., the amount added, the raw materials to which the enzyme is added, the vegetable protein-containing product and its production method, etc.), can be referenced from the explanation in the above embodiment A.

[0032] Another embodiment disclosed by the present invention is "an agent for improving the softening of vegetable protein-containing products caused by adding a phytate-degrading enzyme to raw materials for production, the agent containing an enzyme that contributes to the formation of a cross-linked structure of proteins (Embodiment C)."

[0033] This embodiment relates to the use of an enzyme that contributes to the formation of cross-linked protein structures to improve the softening of vegetable protein-containing products caused by the addition of a phytate-degrading enzyme to raw materials during the production process. More specifically, in this embodiment, the enzyme that contributes to the formation of cross-linked protein structures is provided in the form of an improving agent used for the above-mentioned application. The improving agent may be the enzyme itself that contributes to the formation of cross-linked protein structures, or it may be in the form of a composition containing the enzyme that contributes to the formation of cross-linked protein structures. The form of the composition is not particularly limited, and examples thereof include solids (including powders, granules, etc.), liquids (including slurries, etc.), gels, pastes, etc. The composition may further contain a base commonly used in the food flavoring field. When the composition is in the form of a liquid, examples of the base include water, ethanol, glycerin, propylene glycol, various animal and vegetable oils, etc. When the composition is in a solid form, examples of bases include starch, dextrin, cyclodextrin, various sugars such as sucrose and glucose, proteins, peptides, salt, solid fats, silicon dioxide, and mixtures thereof, as well as yeast cells and various powdered extracts. The composition may further contain, for example, excipients, pH adjusters, antioxidants, thickening stabilizers, sweeteners (e.g., sugars), acidulants, spices, colorants, etc., as long as the purpose of this embodiment is not impaired. The composition may be produced by methods conventional in the art. The composition may be subjected to, for example, concentration, drying, decolorization, etc., either alone or in combination.

[0034] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto. Those skilled in the art may modify the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also included within the scope of the present invention.

[0035] Example 1 (1) Preparation of Gel Bars The ingredients were mixed using a hand blender (Bamix, manufactured by CHERRY TERRACE Inc.) according to the formulation shown in Table 1 below. The resulting mixture was filled into a vinyl casing film (Krehalon Film, manufactured by Kureha Corporation) with a diameter of 48 mm and a length of 300 mm, and then heated at 40°C for 30 minutes (enzyme reaction step), subsequently heated at 90°C for 30 minutes (enzyme inactivation step), and cooled in a refrigerator (5°C) overnight to obtain gel bars. The compositions of the prepared gel bars are shown in Tables 1-1 and 1-2 below. The gel bar in Test Group 1 is a "control group" to which neither phytase nor transglutaminase preparation (TG preparation) was added. The gel rods in test plots 2 to 5 are "phytase / TG preparation added group 1," which is based on the addition of phytase (0.1% by weight) to the gel rods in test plot 1. More specifically, the gel rods in test plot 2 are "phytase added group 1," which is the gel rods in test plot 1 to which only phytase (0.1% by weight) has been added. The gel rods in test plots 3 to 5 are "phytase + TG preparation added groups 1(1) to (3)," which are the gel rods in test plot 2 to which a predetermined amount of TG preparation has been added. The gel rods in Test Plots 6 to 9 were "Phytase / TG Preparation Added Group 2," which was based on the gel rod in Test Plot 1 to which phytase (0.2 wt%) had been added. More specifically, the gel rod in Test Plot 6 was "Phytase Added Group 2," in which only phytase (0.2 wt%) had been added to the gel rod in Test Plot 1. The gel rods in Test Plots 7 to 9 were "Phytase + TG Preparation Added Groups 2(1) to (3)," in which a predetermined amount of TG preparation had been added to the gel rod in Test Plot 6. The "transglutaminase preparation (TG preparation)" was a preparation composed of 10% transglutaminase and 90% dextrin.

[0036]

[0037]

[0038] Table 2 below shows the enzymatic activity of the added phytase and transglutaminase.

[0039]

[0040] (2) Measurement of Hardness The hardness of the gel rods obtained in (1) above was measured using a texture analyzer (TA-XT plus, Stable Micro Systems). The gel rods were removed from the casing and cut into 30 mm wide pieces to obtain cylindrical gel rods for measurement. A schematic diagram is shown in Figure 1. The samples, which had been refrigerated (5°C) until immediately before measurement, were placed on the sample stage with the cut surfaces facing up and down. A spherical probe with a diameter of 5 mm attached to the texture analyzer was penetrated through the center of the top surface of the sample at a compression rate of 2 mm / sec, and the force (breaking strength (g)) at which the probe broke the gel rod was measured. Figure 2 shows a schematic diagram of the measurement method. The measured breaking strengths are shown in Tables 3-1 and 3-2 below. A summary of the measurement results is shown in Figure 3.

[0041]

[0042]

[0043] The test results in Tables 3-1 and 3-2 above show that although the groups to which phytase alone was added (Test Plots 2 and 6) showed a significant decrease in breaking strength compared to the control group (Test Plot 1), the groups to which transglutaminase was also added (Test Plots 3 to 5 and Test Plots 7 to 9) showed a sufficient increase in breaking strength. These results demonstrate that the addition of transglutaminase (an enzyme that contributes to the formation of cross-linked structures in proteins) improves the hardness of vegetable protein products softened by the addition of phytase. The degree of improvement can be adjusted as necessary by, for example, adjusting the amount of the enzyme that contributes to the formation of cross-linked structures in proteins added or the enzyme reaction time.

[0044] Example 2: Application to plant-based cheese (1) Preparation of plant-based cheese All raw materials were placed in a heated mixer (Thermomix™21, manufactured by Vorwerk) according to the blending ratios shown in Tables 4-1 and 4-2 below, and mixed for 5 minutes at 50°C and speed 2.5. The temperature was set to 90°C, and mixed for 8 minutes at speed 2.5. The mixture was poured into molds and refrigerated at 5°C for 12 hours to prepare various plant-based cheeses. Test plots 1 to 5 vary in the amount of "phytase" and the amount of "transglutaminase preparation (TG preparation)."

[0045]

[0046]

[0047] Table 5 below shows the enzyme activity of the added phytase as well as the transglutaminase.

[0048]

[0049] (2) Measurement of Hardness The hardness of the plant-based cheese obtained in (1) above was measured using a texture analyzer (TA-XTplus, Stable Micro Systems). The plant-based cheese was removed from the mold and cut into a 30 mm x 15 mm x 15 mm rectangular parallelepiped to serve as a measurement sample. The sample, which had been refrigerated (5°C) until immediately before measurement, was placed on the sample stage, and a spherical probe with a diameter of 5 mm attached to the texture analyzer was penetrated through the center of the top surface of the sample at a compression speed of 1 mm / sec. The force (breaking strength (g)) required for the probe to break the plant-based cheese was measured. The measured breaking strengths are shown in Table 6 below.

[0050]

[0051] A sensory evaluation of hardness was also conducted according to the following criteria. The evaluation was conducted by a panel of three experts. The results are shown in Table 7. 3: Hardness equivalent to the control 2: Slightly softer than the control 1: Much softer than the control

[0052]

[0053] The test results showed that although the addition of phytase alone reduced breaking strength compared to the control (Tests 2 and 4), the addition of transglutaminase significantly improved breaking strength (Tests 3 and 5). These results demonstrate that the addition of transglutaminase improves the hardness of plant-based cheese softened with phytase.

[0054] Example 3: Application to plant-based hamburger steak (1) Preparation of plant-based hamburger steak Plant-based hamburger steaks were prepared according to the blending ratios shown in Table 8 below. First, granular soy protein was reconstituted with water. The reconstituted powdered soy protein and all other ingredients (powdered soy protein, methylcellulose, shortening, seasonings, enzymes) were mixed at low speed in a mixer for 120 seconds. The mixture was taken by hand and formed into patties of 80 g each. The formed patties were grilled at 180°C for 2.5 minutes on each side to obtain plant-based hamburger steaks.

[0055]

[0056] Table 9 below shows the enzyme activity of the added phytase as well as the transglutaminase.

[0057]

[0058] (2) Measurement of Hardness The hardness of the obtained plant-based hamburger was measured using a texture analyzer (TA-XTplus, manufactured by Stable Micro Systems). The plant-based hamburger was cut into a 20 mm x 20 mm x 15 mm rectangular parallelepiped to serve as a measurement sample. The sample, which had been refrigerated (5°C) until immediately before measurement, was placed on the sample stage, and a cylindrical probe with a diameter of 15 mm attached to the texture analyzer was penetrated through the center of the top surface of the sample at a compression speed of 2 mm / sec. The force (breaking strength (g)) required for the probe to break the plant-based hamburger was measured. The measured breaking strengths are shown in Table 10 below.

[0059]

[0060] (Sensory Evaluation) A sensory evaluation of hardness was carried out by a panel of three experts according to the following criteria: 3: Hardness equivalent to that of the control 2: Slightly softer than the control 1: Much softer than the control

[0061] The test results showed that although the addition of phytase alone reduced breaking strength compared to the control (Test Group 2), the addition of transglutaminase significantly improved breaking strength (Test Group 3). These results demonstrate that the addition of transglutaminase improves the hardness of plant-based cheese softened with phytase.

[0062] In one embodiment, the present invention provides a method for producing a plant protein-containing product, which includes (1) adding a phytate-degrading enzyme to a production raw material, and (2) adding an enzyme that contributes to the formation of a cross-linked structure of a protein to the production raw material, and is useful, for example, in the field of food. This application is based on Japanese Patent Application No. 2024-060252 (filing date: April 3, 2024), the contents of which are incorporated in full herein.

Claims

1. A method for producing a vegetable protein-containing product, comprising: (1) adding a phytate-degrading enzyme to the raw materials; and (2) adding an enzyme that contributes to the formation of a cross-linked structure of protein to the raw materials.

2. The method according to claim 1, wherein the phytate-degrading enzyme is phytase.

3. The manufacturing method according to claim 1, wherein the enzyme that contributes to the formation of a cross-linked structure of a protein is one or more selected from enzymes that act directly on proteins to form cross-linked structures and enzymes that act indirectly on proteins to form cross-linked structures.

4. The method according to claim 3, wherein the enzyme that contributes to the formation of a cross-linked structure of the protein is one or more selected from the group consisting of transglutaminase, glucose oxidase, and ascorbic acid oxidase.

5. The method of claim 1, wherein the raw materials further contain animal protein.

6. The method according to any one of claims 1 to 5, wherein the amount of phytate-degrading enzyme added is in the range of 0.01 to 100,000 U / g activity relative to the weight of the vegetable protein material in the raw material.

7. A production method according to any one of claims 1 to 5, wherein the amount of enzyme added that contributes to the formation of a cross-linked structure of protein is in the range of 0.0001 to 250 U / g activity relative to the total weight of the plant protein material and animal protein material in the production raw materials (hereinafter also referred to as "total protein material weight"), when the enzyme acts directly on the protein to form the cross-linked structure.

8. A production method according to any one of claims 1 to 5, wherein the amount of enzyme added that contributes to the formation of a crosslinked structure of a protein is in the range of 0.001 to 10,000 U of enzyme activity per 1 g of glucose substrate, when the enzyme acts on glucose, a substrate contained in the production raw material, to indirectly act on the protein to form a crosslinked structure.

9. A production method according to any one of claims 1 to 5, wherein the amount of enzyme added that contributes to the formation of a crosslinked structure of a protein is in the range of 0.001 to 10,000 U of enzyme activity per 1 g of L-ascorbic acids as substrate, when the enzyme acts on L-ascorbic acids, which are substrates contained in the production raw materials, to indirectly act on the protein to form a crosslinked structure.

10. The method according to any one of claims 1 to 5, wherein the plant protein-containing product is a food product or a food ingredient.

11. The method according to any one of claims 1 to 5, wherein the vegetable protein-containing product is in a semi-solid or solid form.

12. A method for improving the softening of a vegetable protein-containing product caused by adding a phytate-degrading enzyme to the raw materials for producing the vegetable protein-containing product, the method comprising adding an enzyme that contributes to the formation of a cross-linked structure of protein to the raw materials for producing the vegetable protein-containing product.

13. The improved method according to claim 12, wherein the phytate-degrading enzyme is phytase.

14. The improvement method according to claim 12, wherein the enzyme that contributes to the formation of a cross-linked structure of a protein is one or more selected from enzymes that act directly on proteins to form cross-linked structures and enzymes that act indirectly on proteins to form cross-linked structures.

15. The improved method according to claim 14, wherein the enzyme that contributes to the formation of a cross-linked structure of the protein is one or more selected from the group consisting of transglutaminase, glucose oxidase, and ascorbic acid oxidase.

16. The method of claim 12, wherein the raw materials further contain animal protein.

17. The method for improving food quality according to any one of claims 12 to 16, wherein the amount of phytate-degrading enzyme added is in the range of 0.01 to 100,000 U / g activity relative to the weight of the vegetable protein material in the raw materials for production.

18. The method for improving a protein according to any one of claims 12 to 16, wherein the amount of enzyme added that contributes to the formation of a cross-linked structure of protein is in the range of 0.0001 to 250 U / g activity relative to the total weight of the plant protein material and the animal protein material in the raw materials for production (hereinafter also referred to as "total protein weight"), when the enzyme acts directly on the protein to form the cross-linked structure.

19. The improved method according to any one of claims 12 to 16, wherein the amount of enzyme added that contributes to the formation of a cross-linked structure of a protein is in the range of 0.001 to 10,000 U of enzyme activity per 1 g of glucose substrate, when the enzyme acts on glucose, a substrate contained in the raw material for production, to indirectly act on the protein to form a cross-linked structure.

20. The method according to any one of claims 12 to 16, wherein the amount of enzyme added that contributes to the formation of a cross-linked structure of a protein is in the range of 0.001 to 10,000 U of enzyme activity per 1 g of L-ascorbic acid, the substrate, when the enzyme acts on L-ascorbic acid, which is a substrate contained in the raw material for production, to indirectly act on the protein to form a cross-linked structure.

21. The method for improving vegetable protein according to any one of claims 12 to 16, wherein the vegetable protein-containing product is a food product or a food ingredient.

22. The method for improving vegetable protein according to any one of claims 12 to 16, wherein the vegetable protein-containing product is in a semi-solid or solid form.

23. An agent for improving the softening of vegetable protein-containing products, which is produced by adding phytate-degrading enzyme to the raw materials used for production, and which contains an enzyme that contributes to the formation of cross-linked structures in proteins.

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