Modifier for protein-containing composition

Abstract

The purpose of the present invention is to provide a protein-containing composition having an improved essential amino acid release amount. By using a protein deamidase and a protease derived from the genus Chryseobacterium, it is possible to improve the essential amino acid release amount, foamability, foam stability, and / or emulsifiability of a protein-containing composition.

Description

Modifiers for protein-containing compositions

[0001] The present invention relates to processing techniques for modifying protein-containing compositions.

[0002] Protein is one of the important nutrients that make up the body. The efficiency of protein intake is synonymous with the efficiency of digestion into amino acids. In other words, efficient digestion is essential for effective absorption of protein into the body.

[0003] The world population is expected to reach 9.7 billion by 2050, and there are concerns that future protein demand will exceed supply (a protein crisis). In this context, there is an accelerating trend to replace animal protein with plant-based protein, which has a lower environmental impact.

[0004] However, it is known that plant proteins are less digestible than animal proteins (Non-Patent Document 1), and the gap between food intake and nutrient absorption is also a problem.

[0005] Meanwhile, in the food industry, modification of protein materials is attracting increasing attention as a way to increase the added value of foods. Such modifications include not only improving the digestibility of vegetable protein materials, but also improving the properties of protein materials, including animal protein materials, such as foaming ability, foam stability, and emulsifying ability.

[0006] Nutrition and Food, Vol. 20, No. 4, pp. 259-266

[0007] The present invention aims to provide a technology for producing a modified protein-containing composition. As described above, vegetable proteins are less digestible than animal proteins. Therefore, a first object of the present invention is to provide a technology for producing a vegetable protein-containing composition with an improved amount of released essential amino acids. A second object of the present invention is to provide a technology for producing a protein-containing composition with improved foaming properties, foam stability, or emulsifying properties.

[0008] As a result of extensive research, the present inventors have found that treating a vegetable protein material with protein deamidase and a protease derived from the genus Chryseobacterium improves the amount of released essential amino acids in the resulting vegetable protein-containing food or drink, and that treating a protein material with protein deamidase and a protease derived from the genus Chryseobacterium improves the foaming properties, foam stability, or emulsifying properties of the resulting protein material. The present invention was completed based on this finding and through further research.

[0009] That is, the present invention provides the following aspects.

[0010] (A) A modifying agent for a protein-containing composition, comprising protein deamidase and a protease derived from the genus Chryseobacterium, wherein the modification of the protein-containing composition is to improve the amount of essential amino acids released from a plant-based protein-containing composition, the foaming properties of the protein-containing composition, the foam stability of the protein-containing composition, and / or the emulsifying properties of the protein-containing composition. Use of a combination of protein deamidase and a protease derived from the genus Chryseobacterium for modifying a protein-containing composition, wherein the modification of the protein-containing composition is to improve the amount of essential amino acids released from a plant-based protein-containing composition, the foaming properties of the protein-containing composition, the foam stability of the protein-containing composition, and / or the emulsifying properties of the protein-containing composition. Item 1. An agent for improving the amount of free essential amino acids in a vegetable protein-containing composition, comprising protein deamidase and a protease derived from the genus Chryseobacterium. Item 2. The agent for improving the amount of free essential amino acids in a vegetable protein-containing composition according to Item 1, wherein the protein deamidase is protein glutaminase. Item 3. The agent for improving the amount of free essential amino acids in a vegetable protein-containing composition according to Item 1 or 2, wherein the vegetable protein is a protein from a plant selected from the group consisting of pulses, cereals, and nuts and seeds. Item 4. A modifying agent for a protein-containing composition, comprising a protein deamidase and a protease derived from the genus Chryseobacterium, wherein the modification is an improvement in foaming ability, foam stability, and / or emulsifying ability.

[0011] (B) A method for modifying a protein-containing composition, comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on the protein-containing composition, wherein the modification of the protein-containing composition is to improve the amount of free essential amino acids of the plant protein-containing composition, the foaming properties of the protein-containing composition, the foam stability of the protein-containing composition, and / or the emulsifying properties of the protein-containing composition. Item 5. A method for improving the amount of free essential amino acids of a plant protein-containing composition, comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on the plant protein-containing composition. Item 6. A method for modifying a protein-containing composition, comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on the protein-containing composition, wherein the modification is to improve the foaming properties, foam stability, and / or emulsifying properties.

[0012] (C) A method for producing a processed protein-containing composition, comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a protein-containing composition, wherein the processed protein-containing composition is either a processed plant protein-containing composition with an improved amount of essential amino acid release, or a processed protein-containing composition with improved foaming properties, foam stability, and / or emulsifying properties. Item 7. A method for producing a processed plant protein-containing composition with an improved amount of essential amino acid release, comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a plant protein-containing composition. Item 8. A method for producing a processed protein-containing composition with improved foaming properties, foam stability, and / or emulsifying properties, comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a protein-containing composition.

[0013] (D) A processed protein-containing composition obtained by a method comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a protein-containing composition. Item 9. A processed plant protein-containing composition with an improved amount of essential amino acid release, obtained by a method comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a plant protein-containing composition. Item 10. A processed protein-containing composition with improved foamability, foam stability, and / or emulsifying properties, obtained by a method comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a protein-containing composition.

[0014] According to the present invention, a technique for producing a vegetable protein-containing composition having an improved amount of free essential amino acids, or a protein-containing composition having improved foaming properties, foam stability, or emulsifying properties is provided.

[0015] 1. Protein-Containing Composition Modifiers Among the protein-containing composition modifiers of the present invention, an agent for improving the amount of essential amino acid release in a vegetable protein-containing composition (hereinafter also referred to as a "first modifier") is characterized by containing protein deamidase and a protease derived from the genus Chryseobacterium. Furthermore, among the protein-containing composition modifiers of the present invention, a modifier used to improve the foaming properties, foam stability, and / or emulsifying properties of the protein-containing composition (hereinafter also referred to as a "second modifier") is also characterized by containing protein deamidase and a protease derived from the genus Chryseobacterium. Hereinafter, the first and second modifiers are collectively referred to as "modifiers."

[0016] 1-1. Uses The agent for improving the free amount of essential amino acids in a vegetable protein-containing composition, which is the first modifying agent of the present invention, is used to improve the free amount of essential amino acids contained in a vegetable protein-containing composition.

[0017] Specifically, the first modifier of the present invention is used for increasing the amount of essential amino acids more than the amount of non-essential amino acids among the released amino acids. The improvement in the amount of released essential amino acids can be confirmed by an increase in the ratio of the amount of essential amino acids to the total amount of free amino acids compared to when a combination of protein deamidase and a protease other than that derived from the genus Chryseobacterium is not used (i.e., when a vegetable protein-containing composition is treated under the same conditions except for not using the combination of protein deamidase and a protease other than that derived from the genus Chryseobacterium), particularly when a protein deamidase and a protease other than that derived from the genus Chryseobacterium are used (i.e., when a vegetable protein-containing composition is treated under the same conditions except for using protein deamidase and a protease other than that derived from the genus Chryseobacterium).

[0018] The second modifier of the present invention is used to improve the foaming properties, foam stability, and / or emulsifying properties of a protein-containing composition.

[0019] Improved foamability can be confirmed by an increase in the total volume, including the foam, of a protein-containing composition foamed by homogenization compared to when the combination of protein deamidase and a protease other than that derived from the genus Chryseobacterium is not used (i.e., when the protein-containing composition is treated under the same conditions except for not using the combination of protein deamidase and a protease other than that derived from the genus Chryseobacterium); improved foam stability can be confirmed by a suppressed degree of decrease in the total volume of the foamed protein-containing composition over time compared to the above case; and improved emulsifiability can be confirmed by an increase in the turbidity of an emulsion composition prepared using the protein-containing composition and an oily base compared to the above case.

[0020] The vegetable protein-containing composition to which the first modifying agent of the present invention is applied, the protein-containing composition to which the second modifying agent of the present invention is applied, and specific methods for using these modifying agents are described in detail in "2. Method for modifying a protein-containing composition."

[0021] 1-2. Protein deamidase Protein deamidase, together with the protease derived from the genus Chryseobacterium, is an active ingredient in the modifying agent of the present invention. That is, protein deamidase is an active ingredient that contributes to improving the amount of released essential amino acids in the agent for improving the amount of released essential amino acids in a plant protein-containing composition according to the first modifying agent of the present invention, and is an active ingredient that contributes to improving the foaming property, foam stability, and / or emulsifying property in the second modifying agent of the present invention.

[0022] The protein deamidase is not particularly limited in type, origin, etc., as long as it is an enzyme that exhibits the action of degrading an amide group-containing side chain of a protein without cleaving peptide bonds or crosslinking of proteins. Examples of protein deamidases include protein deamidases derived from the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides, which are disclosed in JP 2000-50887 A, JP 2001-218590 A, and WO 2006 / 075772. These protein deamidating enzymes may be used singly or in combination of two or more kinds.

[0023] Examples of protein deamidating enzymes include protein glutaminase and protein asparaginase, and in a broad sense, also include protein arginine deiminase. Among these protein deamidating enzymes, protein glutaminase is preferred from the viewpoint of further increasing the amount of essential amino acid release, foaming properties, foam stability, and / or emulsifying properties.

[0024] Among these protein deamidating enzymes, from the viewpoint of further increasing the amount of essential amino acid release, foaming properties, foam stability, and / or emulsifying properties, more preferred are protein deamidating enzymes derived from the genus Chryseobacterium, even more preferred are protein glutaminases derived from the genus Chryseobacterium, and even more preferred are protein glutaminases derived from the species Chryseobacterium proteolyticum.

[0025] Protein deamidase can be prepared from a culture solution of a microorganism from which the above-mentioned protein deamidase is derived. Specific preparation methods include methods of recovering protein deamidase from the culture solution or cells of the above-mentioned microorganism. For example, when a protein deamidase-secreting microorganism is used, the cells can be recovered from the culture solution in advance by filtration, centrifugation, or the like, as necessary, and the enzyme can then be separated and / or purified. Furthermore, when a protein deamidase-nonsecreting microorganism is used, the cells can be recovered from the culture solution in advance by pressure treatment, ultrasonic treatment, or the like, as necessary, and the enzyme can then be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method, without any particular limitation, and examples thereof include centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins, etc. The separated and / or purified enzyme can be powdered by a drying method such as freeze-drying or vacuum drying, and can also be powdered using an appropriate excipient and / or drying aid in the drying method. The isolated and / or purified enzyme can also be liquefied by adding an appropriate additive and sterilizing by filtration. Alternatively, commercially available protein deamidating enzymes can be used.

[0026] 1-3. Chryseobacterium-Derived Protease The Chryseobacterium-derived protease is an active ingredient in the modifying agent of the present invention, together with protein deamidase. That is, the Chryseobacterium-derived protease is an active ingredient that contributes to improving the amount of essential amino acid release in the agent for improving the amount of essential amino acid release in a plant protein-containing composition according to the first modifying agent of the present invention, and is an active ingredient that contributes to improving the foaming property, foam stability, and / or emulsifying property in the second modifying agent of the present invention.

[0027] The protease derived from the genus Chryseobacterium is not particularly limited as long as it is an enzyme derived from the genus Chryseobacterium that hydrolyzes peptide bonds in proteins.

[0028] Specific examples of proteases derived from the genus Chryseobacterium include proteases derived from Chryseobacterium nematophagum, Chryseobacterium cucumeris, Chryseobacterium lactis, Chryseobacterium rhizoplane, Chryseobacterium joostei, Chryseobacterium shigense, Chryseobacterium proteolyticum, Chryseobacterium gleum, and Chryseobacterium soil. These proteases derived from the genus Chryseobacterium may be used singly or in combination of two or more. Among these proteases derived from the genus Chryseobacterium, proteases derived from the species Chryseobacterium proteolyticum are preferred from the viewpoint of further enhancing the effects of improving the amount of essential amino acid release, foaming properties, foam stability, and / or emulsifying properties.

[0029] The protease derived from the genus Chryseobacterium can be prepared by known methods. For example, it can be easily prepared by culturing bacteria of the genus Chryseobacterium and isolating the protease using known means, or by a method using genetic recombination technology.

[0030] 1-4. Other Components The modifying agent of the present invention may be an enzyme composition containing protein deamidase and a protease derived from the genus Chryseobacterium. The modifying agent may or may not contain additives and / or bases acceptable for formulation of enzyme preparations as components other than the enzyme. Examples of such additives and bases include excipients, buffers, antioxidants, UV protection agents, preservatives, antiseptics, pH adjusters, dispersants, emulsifiers, solubilizers, carriers, and solvents (e.g., water). These additives and bases may be used alone or in combination of two or more. The content of these additives and bases may be appropriately determined depending on the type of components and / or the formulation form.

[0031] 1-5. Properties The properties of the modifier for the protein-containing composition of the present invention are not particularly limited, and examples include dry preparations in the form of powder, fine particles, or granules, and liquid preparations.

[0032] 2. Method for Modifying a Protein-Containing Composition As described above, the combination of protein deamidase and a protease derived from the genus Chryseobacterium contributes to improving the amount of essential amino acids released from a plant protein-containing composition, and also contributes to improving the foaming properties, foam stability, and / or emulsifying properties of the protein-containing composition. Therefore, the present invention further provides a method for improving the amount of essential amino acids released from a plant protein-containing composition, comprising the step of treating a plant protein-containing composition with protein deamidase and a protease derived from the genus Chryseobacterium (hereinafter also referred to as the "first modification method"), and a method for modifying a protein-containing composition, comprising the step of treating a protein deamidase and a protease derived from the genus Chryseobacterium, wherein the modification is to improve the foaming properties, foam stability, and / or emulsifying properties (hereinafter also referred to as the "second modification method"). Hereinafter, the first and second modification methods are collectively referred to as the "modification methods."

[0033] In the method for improving the amount of free essential amino acids in a vegetable protein-containing composition according to the first modification method of the present invention, the "improvement of the amount of free essential amino acids," the "protein deamidase," and the "protease derived from the genus Chryseobacterium" are as described in detail above in "1. Modifiers for protein-containing compositions." In addition, in the second modification method of the present invention, the "improvement of foaming property, foam stability, and / or emulsifying property," the "protein deamidase," and the "protease derived from the genus Chryseobacterium" are as described in detail above in "1. Modifiers for protein-containing compositions."

[0034] 2-1. Step of allowing protein deamidase and Chryseobacterium genus-derived protease to act on a protein-containing composition In the first modification method, in the step of allowing protein deamidase and Chryseobacterium genus-derived protease to act on a vegetable protein-containing composition, a vegetable protein mixture containing the vegetable protein-containing composition, protein deamidase, and Chryseobacterium genus-derived protease is appropriately prepared, and a treatment is carried out to allow the enzymatic reaction to proceed. In the second modification method, in the step of allowing protein deamidase and Chryseobacterium genus-derived protease to act on a protein-containing composition, a protein mixture containing the protein-containing composition, protein deamidase, and Chryseobacterium genus-derived protease is appropriately prepared, and a treatment is carried out to allow the enzymatic reaction to proceed. In the second modification method, the protein includes vegetable protein and animal protein.

[0035] 2-1-1. Vegetable protein-containing composition The vegetable protein-containing composition is an example of a target for enzyme treatment in the first modification method and an example of a target for application of the first modifying agent. The vegetable protein-containing composition is also an example of a target for enzyme treatment in the second modification method and an example of a target for application of the second modifying agent. There are no particular limitations on the vegetable protein-containing composition, as long as it contains vegetable protein and is in a form that can be ingested by a living organism.

[0036] Typically, the vegetable protein-containing composition used in the present invention is a composition for consumption or drinking, containing vegetable protein and water. The vegetable protein-containing composition is not particularly limited in its form, and may be, for example, a fluid form such as liquid, slurry, or paste (hereinafter, these fluid vegetable protein-containing compositions are collectively referred to as "liquid, etc."), or a solid form. In the first modification method, the vegetable protein-containing composition may be fluid or solid. In the second modification method, the vegetable protein-containing composition is fluid.

[0037] Specific examples of vegetable protein-containing compositions prepared from unstructured protein materials include (i) liquids obtained by dispersing a dry powder of a vegetable protein material (specifically, at least one of a plant organ of a plant from which the vegetable protein is derived and a material obtained by increasing the protein content by removing at least a portion of components other than the protein from the plant organ; the same applies hereinafter) in water; and (ii) compositions prepared by crushing and dispersing a vegetable protein material in water, and optionally removing insoluble matter derived from the skin of the plant material by any means such as centrifugation, filtration, a filter bag, or a sieve. (iii) a liquid obtained by removing components other than vegetable protein from the liquid (i) or (ii) above to increase the protein content; (iv) a liquid obtained by mixing water with a dried powder prepared from any of the liquids (i) to (iii) above; and, preferably when cereals are used as the vegetable protein material, (v) a liquid obtained by further treating any of the liquids (i) to (iv) above with amylase. An example of a liquid prepared from a textured protein material is (vi) a textured vegetable protein material swollen with water.

[0038] The textured vegetable protein material used in preparing the specific example (vi) above is a food material generally known as a meat substitute (imitation meat). A typical example of a textured vegetable protein material is a material obtained by extruding a raw material mixture containing vegetable protein and water using an extruder or the like, and then drying or freezing it to form a meat-like texture. In the present invention, the "meat" that the textured vegetable protein material imitates refers to the muscle of an animal that is used for food, and the term "meat" is used to encompass not only the muscle of mammals and birds, but also the flesh of seafood.

[0039] A preferred example of a liquid vegetable protein-containing composition is so-called vegetable milk substitute (also called vegetable milk).

[0040] In the following, the "content of plant protein material" in a plant protein-containing composition refers to the proportion of the dry weight of the components constituting the plant organ contained in the plant protein-containing composition in the case of a plant protein-containing composition prepared using a non-textured plant protein material, and refers to the proportion of the dry weight of the structured plant protein material in the case of a plant protein-containing composition prepared using a textured plant protein material. For example, when the plant protein-containing composition is a liquid or the like composed only of components derived from a plant organ and water, as in the above specific examples (i) to (v), the "content of plant protein material" refers to the proportion of the dry weight of the liquid or the like. When the plant protein-containing composition is a liquid or the like containing one of the above specific examples (i) to (v) and an additional ingredient, the "content of plant protein material" refers to the proportion of the dry weight of the liquid or the like excluding the additional ingredient. Furthermore, when the vegetable protein-containing composition is a swollen product composed only of a textured vegetable protein material and water, as in the specific example (vi) above, the "content of vegetable protein material" refers to the percentage of the dry weight of the composition. When the vegetable protein-containing composition is a swollen product containing the specific example (vi) above and an additional ingredient, the "content of vegetable protein material" refers to the percentage of the dry weight of the swollen product excluding the additional ingredient.

[0041] The vegetable protein content of the vegetable protein material is not particularly limited, but may be, for example, 5% by weight or more, 10% by weight or more, 13% by weight or more, 15% by weight or more, 20% by weight or more, 25% by weight or more, or 30% by weight or more. From the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, the content is preferably 35% by weight or more, more preferably 40% by weight or more, and even more preferably 45% by weight or more. The upper limit of the content range is not particularly limited, but may be, for example, 90% by weight or less, preferably 85% by weight or less, 80% by weight or less, more preferably 70% by weight or less, even more preferably 60% by weight or less, and even more preferably 55% by weight or less. Specific ranges for the content of the vegetable protein contained in the vegetable protein material include, for example, 5 to 90% by weight, and preferably 10 to 85% by weight; when the vegetable protein is derived from pulses, the content is, for example, 5 to 90% by weight, preferably 30 to 85% by weight, and more preferably 40 to 85% by weight, 45 to 85% by weight, or 50 to 80% by weight; when the vegetable protein is derived from grasses, the content is, for example, 5 to 90% by weight, preferably 10 to 85% by weight, 10 to 60% by weight, 10 to 40% by weight, 10 to 20% by weight, or 13 to 20% by weight.

[0042] The content of the vegetable protein material in the vegetable protein-containing composition is not particularly limited, but may be, for example, 0.05 to 80% by weight. When the vegetable protein-containing composition is prepared from a non-structured protein material, the content may be, for example, 0.05 to 80% by weight, 0.1 to 60% by weight, or 1 to 40% by weight, preferably 2 to 30% by weight or 3 to 20% by weight, and more preferably 5 to 15% by weight or 8 to 12% by weight.

[0043] 2-1-1-1. Vegetable Proteins Vegetable proteins are not particularly limited as long as they are derived from plants, and examples include pulses such as soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupin beans, and kidney beans; cereals such as wheat, barley, oats, sorghum, rice, rye, buckwheat, barnyard millet, foxtail millet, teff, quinoa, corn, and potatoes; nuts and seeds such as almonds, coconuts, peanuts, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, pine nuts, hemp seeds (industrial hemp), chia seeds, chia seeds, amaranth, canary seeds, and linseed; and natural proteins contained in algae. Furthermore, the plant protein of the present invention may be any protein derived from a plant, and may be, in addition to the above-mentioned natural proteins, proteins obtained by chemical partial hydrolysis of the above-mentioned natural proteins using acid, alkali, etc., proteins obtained by enzymatic partial hydrolysis using proteases, etc., proteins chemically modified with various reagents, or proteins obtained by artificial peptide synthesis.

[0044] In the present invention, the above-mentioned vegetable proteins may be used alone or in combination of two or more. In the first modification method, from the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, preferred are proteins of pulses or cereals, more preferred are proteins of soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupin beans, kidney beans, and oats, and even more preferred are proteins of soybeans and peas. In the second modification method, from the viewpoint of further enhancing emulsifiability, preferred are proteins of cereals, more preferred are proteins of oats.

[0045] The vegetable protein content in the vegetable protein-containing composition is not particularly limited, but may be, for example, 0.01 to 50% by weight. In the first modification method, the vegetable protein content in the vegetable protein-containing composition may be, for example, 0.01 to 50% by weight. When the vegetable protein-containing composition is prepared from a non-structured protein material, the vegetable protein content may be, for example, 0.01 to 50% by weight, 0.1 to 40% by weight, or 0.5 to 30% by weight, preferably 1 to 25% by weight, 2 to 20% by weight, more preferably 2.5 to 15% by weight, or 3 to 10% by weight. In the second modification method, the vegetable protein content in the vegetable protein-containing composition may be, for example, 0.01 to 50% by weight, preferably 0.1 to 40% by weight, more preferably 0.5 to 30% by weight, even more preferably 1 to 25% by weight, 1 to 20% by weight, 1 to 15% by weight, 1 to 10% by weight, or 1 to 5% by weight.

[0046] 2-1-1-2. Other Components The vegetable protein-containing composition may or may not contain any other components in addition to the vegetable protein. Examples of other components include components contained in the vegetable protein's derived plant (carbohydrates, lipids, etc.) and additional components (other food ingredients, food additives, etc.). Examples of food additives include thickeners, binders, seasonings, pH adjusters, buffers, colorants, and flavorings.

[0047] 2-1-2. Animal protein-containing composition The animal protein-containing composition is an example of a target for enzyme treatment in the second modification method and an example of a target for application of the second modifying agent. There are no particular restrictions on the animal protein-containing composition, as long as it is in a form that can be ingested by a living body.

[0048] Typically, the animal protein-containing composition used in the present invention contains animal protein and has fluidity such as liquid, slurry, or paste. Specific examples of animal protein-containing compositions include animal milk, liquids obtained by dispersing dried animal milk in water, and liquids obtained by dispersing crudely purified or purified protein from animal milk in water. Hereinafter, the "content of animal protein material" in the animal protein-containing composition refers to the proportion of the dry weight of the animal protein-containing composition (excluding the weight of added ingredients).

[0049] The content of the animal protein material in the animal protein-containing composition is not particularly limited, but may be, for example, 0.05 to 40% by weight, preferably 0.1 to 30% by weight, or 1 to 10% by weight, preferably 2 to 7% by weight.

[0050] 2-1-2-1. Animal Protein The animal protein is not particularly limited as long as it is derived from an animal, but milk protein is preferred, whey and casein are more preferred, and whey is even more preferred.

[0051] The content of animal protein in the animal protein-containing composition is not particularly limited, but may be, for example, 0.03 to 30 wt %, preferably 0.08 to 20 wt %, or 0.8 to 8 wt %, preferably 1 to 6 wt %.

[0052] 2-1-2-2. Other Components In addition to the animal protein, the animal protein-containing composition may or may not contain any other components. Examples of other components include components contained in the animal protein's derived material (carbohydrates, lipids, etc.) and added components (other food ingredients, food additives, etc.). Examples of food additives include thickeners, binders, seasonings, pH adjusters, buffers, colorants, and flavorings.

[0053] 2-1-3. Amount of Protein Deamidase Used The amount of protein deamidase used is not particularly limited, and in the first modification method, the amount used per gram of vegetable protein material contained in the vegetable protein-containing composition may be, for example, 0.01 U or more, or 0.1 U or more. From the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, the amount is preferably 1 U or more, or 5 U or more, more preferably 7.5 U or more, even more preferably 10 U or more, and even more preferably 13 U or more. The upper limit of the range of the amount of protein deamidase used per gram of vegetable protein material is not particularly limited, and examples thereof include 400 U or less, 300 U or less, 200 U or less, 150 U or less, 100 U or less, or 75 U or less, preferably 60 U or less, or 50 U or less, more preferably 40 U or less, or 30 U or less, and even more preferably 25 U or less, 20 U or less, or 17 U or less.

[0054] In the second modification method, the amount of protein deamidase used per 1 g of vegetable protein material contained in the protein-containing composition is, for example, 0.01 U or more, or 0.1 U or more. From the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying ability, the amount is preferably 1 U or more. The upper limit of the range of the amount of protein deamidase used per 1 g of vegetable protein material is not particularly limited, and examples include 100 U or less, 50 U or less, or 20 U or less, preferably 10 U or less, more preferably 5 U or less, and even more preferably 3 U or less. In the second modification method, the amount of protein deamidase used per 1 g of animal protein material contained in the protein-containing composition is, for example, 0.1 U or more, or 1 U or more. From the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying ability, the amount is preferably 3 U or more, more preferably 5 U or more, and even more preferably 7 U or more or 8 U or more. The upper limit of the range of the amount of protein deamidase used per 1 g of the animal protein material is not particularly limited, and examples thereof include 400 U or less, 200 U or less, 100 U or less, 50 U or less, or 30 U or less, preferably 20 U or less, more preferably 15 U or less, and even more preferably 12 U or less.

[0055] In the first modification method, the amount of protein deamidase used per 1 g of vegetable protein is, for example, 0.01 U or more or 0.1 U or more, and from the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, the amount is preferably 1 U or more or 5 U or more, more preferably 7.5 U or more or 10 U or more, even more preferably 13 U or more or 16 U or more, and even more preferably 18 U or more, 20 U or more, or 22 U or more. The upper limit of the range of the amount of protein deamidase used per 1 g of vegetable protein is not particularly limited, and examples thereof include 500 U or less, 400 U or less, 300 U or less, 200 U or less, 150 U or less, 100 U or less, or 80 U or less, preferably 70 U or less or 60 U or less, more preferably 50 U or less or 45 U or less, and even more preferably 40 U or less, 35 U or less, or 32 U or less. In the first modification method, a specific range of the amount of protein deamidase used per 1 g of vegetable protein is, for example, 0.01 to 500 U, 0.01 to 400 U, or 0.1 to 300 U, preferably 1 to 200 U, 5 to 150 U, or 5 to 100 U, more preferably 7.5 to 80 U or 10 to 70 U, even more preferably 13 to 60 U, 16 to 50 U, 18 to 45 U, or 20 to 45 U, and even more preferably 22 to 40 U, ​​22 to 35 U, or 22 to 32 U.

[0056] In the second modification method, the amount of protein deamidase used per 1 g of vegetable protein is, for example, 0.01 U or more, or 0.1 U or more. From the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying properties, the amount is preferably 1 U or more or 3 U or more, more preferably 5 U or more or 7 U or more, and even more preferably 9 U or more. The upper limit of the range of the amount of protein deamidase used per 1 g of vegetable protein is not particularly limited, and examples include 100 U or less, 50 U or less, or 20 U or less, and preferably 15 U or less. In the second modification method, specific ranges of the amount of protein deamidase used per 1 g of vegetable protein are, for example, 0.01 to 100 U, or 0.1 to 50 U, 1 to 20 U, 3 to 20 U, more preferably 5 to 15 U, or 7 to 15 U, and even more preferably 9 to 15 U. In the second modification method, the amount of protein deamidase used per 1 g of animal protein is, for example, 0.01 U or more, or 0.1 U or more. From the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying ability, the amount is preferably 1 U or more or 3 U or more, more preferably 5 U or more or 7 U or more, and even more preferably 9 U or more. The upper limit of the range of the amount of protein deamidase used per 1 g of animal protein is not particularly limited, and examples include 100 U or less, 50 U or less, or 20 U or less, and preferably 15 U or less. In the second modification method, specific ranges of the amount of protein deamidase used per 1 g of animal protein are, for example, 0.01 to 100 U, or 0.1 to 50 U, 1 to 20 U, 3 to 20 U, more preferably 5 to 15 U, or 7 to 15 U, and even more preferably 9 to 15 U.

[0057] Regarding the activity of protein deamidase, one unit (1 U) is defined as the amount of enzyme that liberates 1 μmol of ammonia per minute using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as a substrate.

[0058] 2-1-4. Amount of Chryseobacterium-derived protease used The amount of Chryseobacterium-derived protease used is not particularly limited, but in the first modification method, the amount used per 1 g of plant protein material contained in the plant protein-containing composition is, for example, 0.05 U or more, and from the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, the amount is preferably 0.1 U or more, or 1 U or more, more preferably 5 U or more, or 7.5 U or more, even more preferably 10 U or more, or 12 U or more, and even more preferably 15 U or more, or 18 U or more. The upper limit of the range of the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein material is not particularly limited, but may be, for example, 400 U or less, 300 U or less, 200 U or less, 150 U or less, 100 U or less, or 90 U or less, preferably 80 U or less, 70 U or less, or 60 U or less, more preferably 50 U or less, or 40 U or less, and even more preferably 30 U or less, 25 U or less, or 22 U or less.

[0059] In the second modification method, the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein material contained in the protein-containing composition is, for example, 0.1 U or more or 1 U or more, and from the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying properties, is preferably 2 U or more or 2.5 U or more, more preferably 5 U or more, 5.5 U or more, 6 U or more, 7 U or more, or 8 U or more, and even more preferably 10 U or more or 12 U or more. The upper limit of the above-mentioned range of the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein material is not particularly limited, and examples include 400 U or less, 200 U or less, 100 U or less, 50 U or less, 30 U or less, or 20 U or less. In the second modification method, the amount of Chryseobacterium-derived protease used per 1 g of animal protein material contained in the protein-containing composition is, for example, 0.005 U or more, and from the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying properties, is preferably 0.05 U or more, 0.5 U or more, or 1 U or more, more preferably 5 U or more or 10 U or more, even more preferably 15 U or more or 20 U or more, even more preferably 35 U or more, 40 U or more, or 50 U or more, and even more preferably 70 U or more, 80 U or more, or 85 U or more. The upper limit of the above-mentioned range of the amount of Chryseobacterium-derived protease used per 1 g of animal protein material is not particularly limited, but examples include 400 U or less, 200 U or less, or 100 U or less.

[0060] In the first modification method, the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein is, for example, 0.05 U or more, and from the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, it is preferably 0.1 U or more, or 1 U or more, more preferably 5 U or more, or 7.5 U or more, even more preferably 10 U or more, 12.5 U or more, 15 U or more, or 20 U or more, and even more preferably 22 U or more, or 24 U or more. The upper limit of the range of the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein is not particularly limited, but examples include 1000 U or less, 500 U or less, 400 U or less, 300 U or less, 200 U or less, 150 U or less, 100 U or less, or 90 U or less, preferably 80 U or less, 70 U or less, or 60 U or less, more preferably 55 U or less, or 50 U or less, and even more preferably 45 U or less, or 40 U or less. In the first modification method, specific ranges of the amount of Chryseobacterium-derived protease used per 1 g of plant protein include, for example, 0.05 to 1000 U, 0.05 to 500 U, or 0.05 to 400 U, preferably 0.1 to 300 U, 0.1 to 200 U, or 1 to 150 U, more preferably 5 to 100 U or 7.5 to 90 U, even more preferably 10 to 80 U, 12.5 to 70 U, 15 to 60 U, or 20 to 55 U, and even more preferably 22 to 50 U, 22 to 45 U, or 24 to 40 U.

[0061] In the second modification method, the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein contained in the protein-containing composition is, for example, 1 U or more or 10 U or more, and from the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying properties, is preferably 15 U or more or 20 U or more, more preferably 40 U or more, 50 U or more, 60 U or more, 70 U or more, and even more preferably 80 U or more, 90 U or more, or 100 U or more. The upper limit of the range of the amount of Chryseobacterium-derived protease used per 1 g of vegetable protein is not particularly limited, but examples include 400 U or less, 300 U or less, 200 U or less, or 150 U or less. In the second modification method, specific ranges of the amount of Chryseobacterium-derived protease used per 1 g of plant protein include, for example, 1 to 400 U, 10 to 400 U, preferably 15 to 400 U or 20 to 400 U, more preferably 40 to 400 U, 50 to 400 U, 60 to 400 U, 70 to 400 U, and even more preferably 80 to 400 U, 90 to 400 U, 90 to 300 U, 90 to 200 U, or 90 to 150 U. In the second modification method, the amount of Chryseobacterium-derived protease used is, for example, 0.1 U or more or 1 U or more per gram of animal protein contained in the protein-containing composition. From the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying properties, the amount is preferably 7 U or more, 10 U or more, or 13 U or more, more preferably 17 U or more or 20 U or more. From the viewpoint of further enhancing the effects of improving foamability and / or emulsifying properties, the amount is even more preferably 30 U or more, 40 U or more, or 50 U or more. From the viewpoint of further enhancing the effects of improving emulsifying properties, the amount is even more preferably 60 U or more, 80 U or more, 90 U or more, or 100 U or more. The upper limit of the above-mentioned range of usage amount of Chryseobacterium-derived protease per 1 g of animal protein is not particularly limited, but may be, for example, 400 U or less, 300 U or less, 200 U or less, or 150 U or less. From the viewpoint of improving foam stability, the amount is preferably 100 U or less, 60 U or less, or 50 U or less, and more preferably 30 U or less.In the second modification method, specific ranges of the amount of Chryseobacterium-derived protease used per 1 g of animal protein include, for example, 0.1 to 400 U, or 1 to 400 U, preferably 7 to 400 U, 10 to 400 U, 10 to 200 U, 10 to 150 U, 13 to 100 U, 17 to 60 U, 20 to 50 U, 20 to 30 U, 13 to 400 U, 17 to 400 U, 20 to 400 U, 30 to 400 U, 40 to 400 U, 50 to 400 U, 60 to 400 U, 80 to 400 U, 90 to 400 U, 90 to 300 U, 90 to 200 U, or 90 to 150 U.

[0062] The ratio of protein deamidase to the protease derived from the genus Chryseobacterium to be used is determined based on the above-mentioned amounts of each enzyme used. From the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, the following ratios are preferred.

[0063] In the first modification method, the amount of the protease derived from the genus Chryseobacterium used per 1 U of protein deamidase is preferably 0.005 U or more, more preferably 0.05 U or more, or 0.1 U or more, even more preferably 0.25 U or more, 0.5 U or more, or 0.7 U or more, and even more preferably 0.8 U or more, 1 U or more, 1.1 U or more, or 1.2 U or more. The upper limit of the range of the amount of the protease derived from the genus Chryseobacterium used per 1 U of protein deamidase is, for example, 20 U or less, and from the viewpoint of further enhancing the effect of improving the amount of released essential amino acids, the amount is preferably 15 U or less, or 10 U or less, more preferably 8 U or less, or 6 U or less, even more preferably 5 U or less, or 4 U or less, and even more preferably 3 U or less, 2 U or less, or 1.5 U or less. In the first modification method, the specific range of the amount of the protease derived from the genus Chryseobacterium to be used per 1 U of protein deamidase is preferably 0.005 to 20 U, more preferably 0.05 to 15 U, or 0.1 to 10 U, even more preferably 0.25 to 8 U, 0.5 to 6 U, or 0.7 to 5 U, and even more preferably 0.8 to 4 U, 1 to 3 U, 1.1 to 2 U, or 1.2 to 1.5 U.

[0064] In the second modification method, the amount of protease derived from the genus Chryseobacterium used per 1 U of protein deamidase is preferably 0.005 U or more or 0.01 U or more. From the viewpoint of further enhancing the effects of improving foamability, foam stability, and / or emulsifying properties, the amount is more preferably 0.05 U or more, 0.1 U or more, or 0.3 U or more, even more preferably 0.8 U or more, 1 U or more, or 1.3 U or more, and even more preferably 1.8 U or more or 2 U or more. From the viewpoint of further enhancing the effects of improving foamability and / or emulsifying properties, the amount is even more preferably 2.5 U or more, 4 U or more, or 5 U or more. From the viewpoint of further enhancing the effects of improving emulsifying properties, the amount is particularly preferably 6 U or more, 8 U or more, or 10 U or more. The upper limit of the range of the amount of protease derived from the genus Chryseobacterium used per 1 U of protein deamidase is, for example, 50 U or less, 30 U or less, 20 U or less, or 15 U or less, and from the viewpoint of further improving foam stability, is preferably 10 U or less, more preferably 6 U or less, or 5 U or less, and even more preferably 3 U or less. In the second modification method, specific ranges of the amount of protease derived from the genus Chryseobacterium used per 1 U of protein deamidase include preferably 0.005 to 50 U, 0.01 to 50 U, more preferably 0.05 to 50 U, 0.1 to 50 U, 0.3 to 50 U, 0.8 to 50 U, 1 to 50 U, 1.3 to 50 U, 1.3 to 30 U, 1.3 to 20 U, 1.3 to 15 U, 1.3 to 10 U, 1.3 to 7 U, 1.3 to 5 U, 1.3 to 3 U, 1.8 to 50 U, 2 to 50 U, 2.5 to 50 U, 4 to 50 U, 5 to 50 U, 6 to 50 U, 8 to 50 U, 8 to 50 U, 8 to 30 U, 8 to 20 U, 8 to 15 U, or 8 to 10 U.

[0065] Protease activity is measured by the Folin method using casein as a substrate. That is, protease activity is measured by carrying out an enzymatic reaction using casein as a substrate at a pH determined by a standard method according to the optimum pH of the protease to be measured, and one unit (1 U) is defined as the amount of enzyme that causes an increase in the Folin test solution color substance equivalent to 1 μg of tyrosine per minute.

[0066] 2-1-5. Reaction Procedure and Treatment Conditions There are no particular limitations on the methods for preparing the vegetable protein mixture in the first modification method and the protein mixture in the second modification method. Furthermore, there are no particular limitations on the treatment conditions (temperature, time, pH, etc.) for these mixtures, as long as the effects of the present invention are obtained. There are no particular limitations on the order in which the protein deamidase and the Chryseobacterium protease are allowed to act, and the enzymes may be allowed to act sequentially in any order, or both enzymes may be allowed to act simultaneously, but it is preferable that both enzymes be allowed to act simultaneously.

[0067] The treatment temperatures for the vegetable protein mixture in the first modification method and the protein mixture in the second modification method are, for example, 4 to 80°C, preferably 8 to 70°C. The lower limit of the temperature range may be 15°C, 30°C, or 45°C, and the upper limit of the temperature range may be 65°C, 60°C, or 55°C. The treatment time in the first modification method is not particularly limited, but may be, for example, 0.1 to 72 hours, preferably 12 to 36 hours. The treatment time in the second modification method is also not particularly limited, but may be, for example, 0.1 to 12 hours, preferably 0.5 to 6 hours, and more preferably 1 to 3 hours. The treatment pH (25°C) of the vegetable protein mixture is, for example, 2 to 9, preferably 3 to 8, more preferably 5 to 7.6, even more preferably 6 to 7.4, and even more preferably 6.5 to 7.2.

[0068] These treatment conditions are appropriately selected depending on the optimum temperature and pH of the enzyme used, and / or the desired effect of the present invention (improvement of the amount of essential amino acid release, foaming properties, foam stability, and / or emulsifying properties). The optimum treatment conditions can be determined through preliminary experiments.

[0069] 2-2. Other Steps The modification method of the present invention may or may not include other steps in addition to the step of reacting the above-mentioned protein deamidase and Chryseobacterium-derived protease. Examples of such other steps include a step of preparing the vegetable protein-containing composition used in the first modification method or the protein-containing composition (vegetable protein-containing composition or animal protein-containing composition) used in the second modification method, an enzyme inactivation step, a cooling step, a filtration step, etc. These other steps may be performed singly or in combination of two or more steps.

[0070] The step of preparing a plant or animal protein-containing composition can be carried out by any method for preparing a plant or animal protein-containing composition.

[0071] For example, when a plant-based milk substitute (plant-based milk) is used as the plant protein-containing composition, it can be prepared by any plant-based milk substitute preparation method depending on the type of plant from which the plant protein is derived. In the process of preparing the plant or animal protein-containing composition, any food additive such as a seasoning, pH adjuster, buffer, colorant, or flavoring may be further blended, and / or any treatment other than the treatment with protein deamidase and Chryseobacterium-derived protease (for example, fermentation treatment such as lactic acid fermentation) may or may not be performed.

[0072] 3. Method for Producing a Processed Protein-Containing Composition The present invention also provides a method for producing a processed plant protein-containing composition having an improved amount of released essential amino acids, which comprises the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a plant protein-containing composition (hereinafter also referred to as the "first production method"), and a method for producing a processed protein-containing composition having improved foaming properties, foam stability, and / or emulsifying properties, which comprises the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a protein-containing composition (hereinafter also referred to as the "second production method"). Hereinafter, the first and second production methods are collectively referred to as the "production methods."

[0073] In the first production method of the present invention, the "improvement of the amount of released essential amino acids," "protein deamidase," and "protease derived from the genus Chryseobacterium" are as described in detail above in "1. Modifier for protein-containing composition," and the "step of allowing protein deamidase and protease derived from the genus Chryseobacterium to act on a vegetable protein-containing composition" is as described in detail above in "2. Method for modifying a protein-containing composition." In the second production method of the present invention, the "improvement of foaming property," "improvement of foam stability," "improvement of emulsifying property," "protein deamidase," and "protease derived from the genus Chryseobacterium" are as described in detail above in "1. Modifier for protein-containing composition," and the "step of allowing protein deamidase and protease derived from the genus Chryseobacterium to act on a protein-containing composition" is as described in detail above in "2. Method for modifying a protein-containing composition."

[0074] The production method of the present invention may or may not include other steps in addition to the step of reacting protein deamidase and Chryseobacterium-derived protease. In the "other steps", in addition to those detailed in "2. Method for modifying a protein-containing composition" above, any cooking step can be performed to prepare the processed plant protein-containing composition to be produced in the final form of a food or drink.

[0075] As an example of the cooking step, for example, when a textured vegetable protein-containing composition is used in the first production method, the composition after treatment with protein deamidase and Chryseobacterium protease can be molded into a desired shape and cooked as needed to obtain a processed vegetable protein-containing composition (food). The cooking method can be appropriately determined by those skilled in the art depending on the type of food. Specific cooking methods include boiling, baking (roasting, toasting, baking, grilling, broiling), steaming, frying, etc. These cooking methods may be used alone or in combination.

[0076] 4. Processed Protein-Containing Composition The present invention also provides a processed plant protein-containing composition with an improved amount of essential amino acid release, obtained by a method comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a plant protein-containing composition (hereinafter referred to as the "first processed protein-containing composition"), and a processed protein-containing composition with improved foaming properties, foam stability, and / or emulsifying properties, obtained by a method comprising the step of allowing protein deamidase and a protease derived from the genus Chryseobacterium to act on a protein-containing composition (hereinafter referred to as the "second processed protein-containing composition"). Hereinafter, the first processed protein-containing composition and the second processed protein-containing composition are also collectively referred to as the "processed protein-containing compositions."

[0077] The first processed protein-containing composition of the present invention is a composition that has been processed by protein deamidase and a protease derived from the genus Chryseobacterium to improve the amount of essential amino acid release. The second processed protein-containing composition of the present invention is a composition that has been processed by protein deamidase and a protease derived from the genus Chryseobacterium to improve foaming property, foam stability, and / or emulsifying property.

[0078] In the first processed protein-containing composition of the present invention, the "improvement of the amount of released essential amino acids," the "protein deamidase," and the "Chryseobacterium-derived protease" are as described in detail above in "1. Modifier for protein-containing composition," and the "method comprising the step of acting protein deamidase and Chryseobacterium-derived protease on a plant protein-containing composition" is as described in detail above in "2. Method for modifying protein-containing composition" and "3. Method for producing processed protein-containing composition." In the second processed protein-containing composition of the present invention, the "improvement of foaming property," the "improvement of foam stability," the "improvement of emulsifying property," the "protein deamidase," and the "Chryseobacterium-derived protease" are as described in detail above in "1. Modifier for protein-containing composition," and the "step of acting protein deamidase and Chryseobacterium-derived protease on a protein-containing composition" is as described in detail above in "2. Method for modifying protein-containing composition" and "3. Method for producing processed protein-containing composition."

[0079] The specific form of the processed protein-containing composition of the present invention can be selected from any food or drink form.

[0080] When a textured vegetable protein-containing composition is used as an ingredient of the first processed protein-containing composition of the present invention, the form can conform to that of meat, poultry, and / or fish paste processed foods. That is, the first processed protein-containing composition of the present invention includes meat-like processed foods (foods that imitate meat, poultry, and / or fish paste processed foods). More preferably, the first processed protein-containing composition of the present invention includes meat and / or poultry-like processed foods (foods that imitate meat and / or poultry processed foods). Such meat and / or poultry processed foods may be any food that is prepared by shaping and heating meat from meat and / or poultry, and specific examples thereof include hamburgers, meatballs, patties, meatloaf, minced meat cutlets, dim sum, etc.

[0081] When a non-structured vegetable protein-containing composition is used as the raw material for the first processed protein-containing composition of the present invention, specific forms of the first processed protein-containing composition of the present invention include vegetable milk substitutes, yogurt substitutes, cheese substitutes, ice cream substitutes, etc., and preferably vegetable milk substitutes and yogurt substitutes.

[0082] Specific forms of the second processed protein-containing composition of the present invention include plant-based milk substitutes, cream substitutes, ice cream substitutes, and animal-based dairy products (animal milk, cream, ice cream).

[0083] The present invention will be specifically described below with reference to examples, but the present invention should not be construed as being limited to the following examples.

[0084] A. Protein-Containing Compositions The protein materials shown in Table 1 were used.

[0085] Protein deamidase] Protein glutaminase derived from Chryseobacterium proteolyticum (manufactured by Amano Enzyme Inc.) was used as the protein deamidase. Hereinafter, this protein deamidase will also be referred to as "PG."

[0086] Protein deamidase activity was measured by the following method. 0.1 mL of a sample solution containing protein deamidase was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture was allowed to stand at 37°C for 10 minutes. Then, 1 mL of 0.4 M TCA solution was added to stop the reaction. As a blank, 1 mL of 0.4 M TCA solution was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and 0.1 mL of the sample solution containing protein deamidase was further added, followed by standing at 37°C for 10 minutes.

[0087] The amount of ammonia generated in the reaction solution was measured using an Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) The ammonia concentration in the reaction solution was determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0088] The activity of protein deamidase was calculated using the following formula, where 1 unit (1 U) is the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the volume of the reaction solution is 2.1, the volume of the enzyme solution is 0.1, Df is the dilution factor of the enzyme solution, and 17.03 is the molecular weight of ammonia.

[0089]

[0090] [B-2. Chryseobacterium-Derived Protease] Chryseobacterium proteolyticum-derived protease was used as the Chryseobacterium-derived protease. Protease activity was measured by the following method. 5 mL of a 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was heated at 37°C for 10 minutes, and then 1 mL of a sample solution containing protease was added and immediately shaken. This solution was left at 37°C for 10 minutes, and then 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid) was added and shaken, and the mixture was again left at 37°C for 30 minutes and filtered. The first 3 mL of filtrate was discarded, and the next 2 mL of filtrate was measured, to which 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added, shaken well, and allowed to stand for 30 minutes at 37° C. The absorbance AT of this solution (enzyme reaction solution) at a wavelength of 660 nm was measured, using water as a control.

[0091] Separately, 1 mL of a sample solution containing protease was measured, 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid) was added, and the mixture was shaken. 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was then added, the mixture was immediately shaken, and the mixture was allowed to stand at 37°C for 30 minutes. This mixture (blank) was treated in the same manner as the enzyme reaction solution described above, except that the absorbance AB was measured.

[0092] 1 mL, 2 mL, 3 mL, and 4 mL of a 1 mg / mL tyrosine standard stock solution (0.2 mol / L hydrochloric acid) were measured, and 0.2 mol / L hydrochloric acid test solution was added to each to make a 100 mL solution. 2 mL of each solution was measured, and 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added. The mixture was immediately shaken and left at 37°C for 30 minutes. For each of these solutions, 2 mL of 0.2 mol / L hydrochloric acid test solution was measured and the resulting solution was used as a control. Absorbances A1, A2, A3, and A4 at a wavelength of 660 nm were measured. A calibration curve was created, plotting absorbances A1, A2, A3, and A4 on the vertical axis and the amount of tyrosine (μg) in 2 mL of each solution on the horizontal axis, and the amount of tyrosine (μg) per 2 mL of each solution was calculated. The amount of enzyme that causes an increase in the colored substance of Folin's test solution equivalent to 1 μg of tyrosine per minute was defined as 1 unit (1 U).

[0093]

[0094] [B-3. Proteases derived from other bacteria] Proteases derived from Bacillus licheniformis and Bacillus amyloliquefaciens were used as proteases. The proteases were measured by the method described in [B-2. Proteases derived from Chryseobacterium genus].

[0095] [C. Production of processed protein-containing composition] [C-1] First protein-containing composition The vegetable protein material shown in [A. Protein-containing composition] above was mixed with water to prepare a liquid vegetable protein-containing composition (pH approximately 7) containing 10% by weight of vegetable protein material. A predetermined amount of enzyme shown in Table 2 was added to the vegetable protein-containing composition, and the mixture was incubated at 50°C for 24 hours. The mixture was treated in a boiling water bath for 10 minutes to carry out an enzyme inactivation step. After the enzyme inactivation step, the mixture was cooled to room temperature to obtain a processed vegetable protein-containing composition.

[0096] [C-2] Second Protein-Containing Composition The vegetable protein material shown in [A. Protein-Containing Composition] above was suspended in deionized water (final concentration 10 wt%) and boiled until completely gelatinized. Then, 10 U / g-starch of α-amylase was added, and the mixture was incubated at 60°C for 30 minutes to prepare a liquid vegetable protein-containing composition (pH approximately 7). The predetermined amount of enzyme shown in Table 3 was added to the vegetable protein-containing composition, and the mixture was incubated at 50°C for 2 hours. The mixture was then treated at 100°C for 5 minutes to inactivate the enzyme. After the enzyme inactivation step, the mixture was cooled to room temperature to obtain a processed vegetable protein-containing composition (processed oat milk). The animal protein material shown in [A. Protein-Containing Composition] above was suspended in deionized water (final concentration 5 wt%) to prepare a liquid animal protein-containing composition (pH approximately 7). The predetermined amount of enzyme shown in Table 4 was added to the animal protein-containing composition, and the mixture was incubated at 50°C for 2 hours. Thereafter, the enzyme was inactivated by treating at 100° C. for 5 minutes. After the enzyme inactivation step, the mixture was cooled to room temperature to obtain a processed animal protein-containing composition.

[0097] [D. Amino Acid Analysis] The processed vegetable protein-containing composition obtained in [C-1] above was centrifuged (15,000 × g, 10 minutes) to obtain a centrifugal supernatant. The obtained centrifugal supernatant was filtered through a 0.45 μm filter to obtain a sample for amino acid analysis. The sample for amino acid analysis was analyzed for free amino acid content using an amino acid analyzer (amino acid analysis using an Agilent 1260 Infinity II LC system) according to the instrument's protocol. Specifically, first, the free essential amino acids (essential AA: histidine, threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, lysine) and non-essential amino acids (non-essential AA: tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, arginine) were measured. The ratio (%) of essential amino acids to the total amount of free amino acids in the sample for amino acid analysis was calculated.

[0098] [E. Foaming Property] 50 mL of the processed protein-containing liquid composition obtained in [C-2] above was homogenized at 18,000 rpm for 30 minutes and immediately transferred to a 100 mL measuring cylinder. The total volume of the composition including the foam (VF0) was measured, and the "foaming property" was calculated using the following formula.

[0099] [F. Foam stability] As in the test of [E. Foamability] above, 50 mL of the processed protein-containing liquid composition obtained in [C-2] above was homogenized at 18,000 rpm for 30 minutes and immediately transferred to a 100 mL measuring cylinder, and the total volume of the composition including the foam, VF0, was measured. After standing for 30 minutes, the total volume of the composition, VF30, was measured, and the "foam stability" was calculated using the following formula.

[0100] [G. Emulsifying Property] 30 mL of the processed protein-containing liquid composition obtained in [C-2] above was mixed with 10 mL of canola oil and homogenized at 10,000 rpm for 2 minutes to prepare an emulsion composition. 50 μL of the emulsion composition immediately after preparation was taken and added to 5 mL of 0.1 wt % SDS solution. The turbidity (A0) was measured at an absorbance of 500 nm, and the "emulsifying property" was calculated using the following formula.

[0101] [Test Example 1] A processed vegetable protein-containing composition was prepared using the vegetable protein material (soybean protein material or pea protein material) shown in Table 1 of [A. Protein-containing composition] above, according to the method of "[C-1] First protein-containing composition" of [C. Production of processed protein-containing composition] above. The amounts of PG added per gram of soybean protein were 30 U, the amounts of protease added were 39 U, and the amounts of PG added per gram of pea protein were 18.8 U, and the amounts of protease added were 24.4 U.

[0102] The processed vegetable protein-containing composition was subjected to the test described above in [D. Amino acid analysis], and the ratio (%) of essential amino acids to the total amount of free amino acids was calculated. The results are shown in Table 2.

[0103]

[0104] As shown in Table 2, it was confirmed that by treating a vegetable protein material with PG and a protease derived from the genus Chryseobacterium, the amount of essential amino acids released from the processed vegetable protein-containing composition was increased compared to treatment with proteases derived from other bacteria.

[0105] [Test Example 2] Using the vegetable protein material (oat protein material) or animal protein material (whey protein material) shown in Table 1 of [A. Protein-containing composition] above, a processed protein-containing composition was prepared by the method of "[C-2] Second protein-containing composition" in [C. Production of processed protein-containing composition] above.

[0106] The processed protein-containing composition was subjected to the tests for [E. Foaming ability], [F. Foam stability], and [G. Emulsifying ability] described above. The results are shown in Tables 3 and 4.

[0107]

[0108]

[0109] As shown in Tables 3 and 4, it was confirmed that the foaming properties, foam stability, and emulsifying properties of the processed protein-containing composition were improved by treating the protein material with PG and Chryseobacterium protease. As with Table 2, the amount of free essential amino acids was also increased for the processed protein-containing compositions in Table 3.

Claims

1. An agent for enhancing the release amount of essential amino acids from a vegetable protein-containing composition, comprising protein deamidase and a protease derived from the genus Chryseobacterium.

2. The agent for enhancing the release amount of essential amino acids in a vegetable protein-containing composition according to claim 1, wherein the protein deamidase is protein glutaminase.

3. An agent for enhancing the release amount of essential amino acids from a vegetable protein-containing composition according to claim 1, wherein the vegetable protein is a protein from a plant selected from the group consisting of pulses, cereals, and nuts and seeds.

4. A modifier for a protein-containing composition, comprising protein deamidase and a protease derived from the genus Chryseobacterium, wherein the modification is to improve foaming property, foam stability, and / or emulsifying property.

5. A method for increasing the amount of free essential amino acids in a vegetable protein-containing composition, comprising the step of allowing protein deamidating enzyme and a protease derived from the genus Chryseobacterium to act on the vegetable protein-containing composition.

6. A method for modifying a protein-containing composition, comprising the step of reacting a protein deamidase and a protease derived from the genus Chryseobacterium with the protein-containing composition, wherein the modification is to improve foaming properties, foam stability, and / or emulsifying properties.

7. A method for producing a processed vegetable protein-containing composition having an improved amount of free essential amino acids, comprising the step of reacting a vegetable protein-containing composition with a protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

8. A method for producing a processed protein-containing composition having improved foaming properties, foam stability, and / or emulsifying properties, comprising a step of reacting a protein-containing composition with protein deamidase and a protease derived from the genus Chryseobacterium.

9. A processed vegetable protein-containing composition having an improved amount of released essential amino acids, obtained by a method comprising the step of acting a protein deamidating enzyme and a protease derived from the genus Chryseobacterium on a vegetable protein-containing composition.

10. A processed protein-containing composition having improved foaming properties, foam stability, and / or emulsifying properties, obtained by a method including a step of acting a protein deamidase and a protease derived from the genus Chryseobacterium on a protein-containing composition.

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