Modifier for protein-containing compositions

Abstract

The purpose of the present invention is to provide a protein-containing composition in which the free amount of essential amino acids is increased. By using a protein deamidase and a protease derived from the genus Chryseobacterium, it is possible to improve the free amount of essential amino acids, foamability, foam stability, and / or emulsibility of a protein-containing composition.

Description

Technical Field

[0001] This invention relates to a processing technique for modifying compositions containing proteins. Background Technology

[0002] Protein is an essential nutrient that makes up the body. The efficiency of protein absorption is synonymous with the efficiency of amino acid digestion. In other words, efficient digestion is necessary for protein to be effectively introduced into the body.

[0003] The world population is projected to reach 9.7 billion by 2050, raising concerns that future protein demand will exceed supply (a protein crisis). This has led to a growing movement to replace animal protein with plant-based proteins, which have a lower environmental impact.

[0004] However, plant-based proteins are known to be less digestible than animal-based proteins (Non-Patent Literature 1), and the gap between food intake and nutrient absorption has also become a problem.

[0005] On the other hand, in the food industry, the modification of protein materials is receiving increasing attention for enhancing the added value of food. Such modifications include not only improving the digestibility of plant-based protein materials, but also enhancing the foaming properties, foam stability, and emulsifying properties of protein materials, including animal-based protein materials.

[0006] Existing technical documents Non-patent literature Non-Patent Literature 1: Eiyo to Shiro (Nutrition and Grains), Vol. 20, No. 4, pp. 259-266 Summary of the Invention

[0007] The technical problem that the invention aims to solve The object of this invention is to provide a technique for manufacturing modified protein-containing compositions. As mentioned above, plant proteins are less digestible than animal proteins; therefore, a first object of this invention is to provide a technique for manufacturing plant protein-containing compositions with increased levels of essential amino acids. A second object of this invention is to provide a technique for manufacturing protein-containing compositions with improved foaming properties, foam stability, or emulsifying properties.

[0008] Technical solutions for solving the problem The inventors conducted in-depth research and discovered that by treating plant-based protein materials with protein deamidase and protease derived from *Chlorella*, the free essential amino acid content in the resulting plant-based protein-containing food and beverage is increased. Furthermore, by treating the protein materials with protein deamidase and protease derived from *Chlorella*, the foaming properties, foam stability, or emulsifying properties of the resulting protein materials are improved. This invention was completed based on further repeated research following this discovery.

[0009] That is, the present invention provides an invention in the manner described below.

[0010] (A) A modifier for a protein-containing composition comprising a protein deamidase and a protease derived from the genus *Chlorella*, wherein the modification of the protein-containing composition is an improvement in the amount of essential amino acids in 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.

[0011] The use of a combination of protein deamidase and protease derived from Chlorella for modifying protein-containing compositions, wherein the modification of the protein-containing composition is an improvement in the amount of essential amino acids in the composition containing plant protein, the foaming properties of the composition containing protein, the foam stability of the composition containing protein, and / or the emulsifying properties of the composition containing protein.

[0012] The use of a combination of protein deamidase and protease derived from the genus *Chlorella* as a modifier for protein-containing compositions, wherein the modification of the protein-containing composition is an improvement in the amount of essential amino acids free in 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.

[0013] Item 1. An essential amino acid free enhancement agent for a composition containing plant protein, comprising a protein deamidase and a protease derived from the genus *Chlorella*.

[0014] Item 2. An essential amino acid free enhancement agent for the composition containing plant protein according to Item 1, wherein the protein deamidase is protein glutaminase.

[0015] Item 3. An essential amino acid free content enhancer for the composition containing plant protein according to items 1 and 2, wherein the plant protein is a protein selected from plants selected from the group consisting of legumes, cereals and seeds.

[0016] Item 4. A modifier for a protein-containing composition comprising a protein deamidase and a protease derived from the genus *Chlorella*, wherein the modification is an improvement in foaming properties, foam stability, and / or emulsifying properties.

[0017] (B) A method for modifying a protein-containing composition, comprising the step of acting a protein deamidase and a protease derived from Chlorella on the protein-containing composition, wherein the modification of the protein-containing composition is an improvement in the amount of essential amino acids in the plant protein-containing composition, the foaming property of the protein-containing composition, the foam stability of the protein-containing composition, and / or the emulsifying property of the protein-containing composition.

[0018] Item 5. A method for increasing the free essential amino acid content of a composition containing plant protein, comprising the step of acting a protein deamidase and a protease derived from Chlorella on the composition containing plant protein.

[0019] Item 6. A method for modifying a protein-containing composition, comprising the step of acting a protein deamidase and a protease derived from the genus *Chlorella* on the protein-containing composition, wherein the modification is an improvement in foaming properties, foam stability, and / or emulsifying properties.

[0020] (C) A method for manufacturing a processed protein-containing composition, comprising the step of acting a protein deamidase and a protease derived from Chlorella on the protein-containing composition, wherein the processed protein-containing composition is selected from any one of processed compositions containing plant-based proteins with increased free essential amino acids, and processed compositions containing proteins with improved foaming properties, foam stability, and / or emulsifying properties.

[0021] Item 7. A method for manufacturing a processed composition containing plant-based protein with increased free essential amino acids, comprising the step of acting a protein deamidase and a protease derived from Chlorella on the composition containing plant-based protein.

[0022] Item 8. A method for manufacturing a processed protein-containing composition with improved foaming properties, foam stability, and / or emulsifying properties, comprising the step of acting a protein deamidase and a protease derived from Chlorella on the protein-containing composition.

[0023] (D) A processed protein-containing composition obtained by a method comprising a step of acting a protein deamidase and a protease derived from the genus *Chlorella* on the protein-containing composition.

[0024] Item 9. A processed composition containing plant-based protein with increased free essential amino acids, obtained by a method comprising the steps of acting a protein deamidase and a protease derived from the genus *Chlorella* on the composition containing plant-based protein.

[0025] Item 10. A processed protein-containing composition with improved foaming properties, foam stability, and / or emulsifying properties, obtained by a method comprising the steps of acting a protein deamidase and a protease derived from Chlorella on the protein-containing composition.

[0026] Invention Effects According to the present invention, a technique is provided for manufacturing a composition containing plant-based protein with increased free essential amino acids, or for manufacturing a composition containing protein with improved foaming properties, foam stability, or emulsifying properties. Detailed Implementation

[0027] 1. Modifiers for protein-containing compositions The modifier for protein-containing compositions of the present invention, specifically the agent for increasing the free essential amino acid content of plant-based protein compositions (hereinafter also referred to as the "first modifier"), is characterized in that it contains a protein deamidase and a protease derived from *Chlorella*. Furthermore, the modifier for improving the foaming properties, foam stability, and / or emulsifying properties of protein-containing compositions of the present invention (hereinafter also referred to as the "second modifier") is also characterized in that it contains a protein deamidase and a protease derived from *Chlorella*. Hereinafter, the first modifier and the second modifier will be collectively referred to as "modifiers".

[0028] 1-1. Applications The first modifier of the present invention relates to an essential amino acid free content enhancer for compositions containing plant proteins, used to increase the free content of essential amino acids contained in compositions containing plant proteins.

[0029] Specifically, the first modifier of the present invention is used to increase the amount of essential amino acids in free amino acids compared to the amount of non-essential amino acids. The increase in the amount of free essential amino acids can be confirmed by an increase in the ratio of essential amino acids to the total amount of free amino acids compared to the following situations: cases where a combination of protein deamidase and proteases other than those derived from *Chlorella* is not used (i.e., cases where a composition containing plant proteins is treated under the same conditions without using a combination of protein deamidase and proteases other than those derived from *Chlorella*), and particularly cases where a combination of protein deamidase and proteases other than those derived from *Chlorella* is used (i.e., cases where a composition containing plant proteins is treated under the same conditions with a combination of protein deamidase and proteases other than those derived from *Chlorella*).

[0030] The second modifier of the present invention is used to improve the foaming properties, foam stability and / or emulsifying properties of protein-containing compositions.

[0031] The improvement in foaming properties can be confirmed by an increase in the total volume of the protein-containing composition foamed by homogenization treatment, including the foam, compared to the case where no combination of protein deamidase and proteases other than those derived from *Chlorella* is used (i.e., the protein-containing composition is treated under the same conditions without the use of protein deamidase and proteases other than those derived from *Chlorella*). The improvement in foam stability can be confirmed by suppressing the decrease in the total volume of the foamed protein-containing composition over time compared to the above case. The improvement in emulsifying properties can be confirmed by an increase in the turbidity of the emulsion composition prepared using the protein-containing composition and the oily base compared to the above case.

[0032] The composition containing plant protein, which is applicable as the first modifier of the present invention, the composition containing protein, which is applicable as the second modifier of the present invention, and the specific methods of using these modifiers are described in detail in "2. Modification method of composition containing protein".

[0033] 1-2. Protein deamidases Protein deamidase, together with proteases derived from the genus *Chlorella*, is an effective component in the modifier of the present invention. Specifically, protein deamidase is an effective component in the essential amino acid free content enhancer of the composition containing plant protein involved in the first modifier of the present invention, which helps to increase the free content of essential amino acids; and is an effective component in the second modifier of the present invention, which helps to improve foaming properties, foam stability, and / or emulsifying properties.

[0034] As a protein deamidase, there are no particular limitations on its type and source, as long as it is an enzyme that exhibits the function of decomposing the amide-containing side chain of a protein without accompanying peptide bond cleavage or protein cross-linking. Examples of protein deamidases include those disclosed in Japanese Patent Application Publication No. 2000-50887, Japanese Patent Application Publication No. 2001-218590, and International Patent Application Publication No. 2006 / 075772, which are derived from the genera *Chryseobacterium*, *Flavobacterium*, *Empedobacter*, *Sphingobacterium*, *Aureobacterium*, or *Myroides*. These protein deamidases can be used alone or in combination.

[0035] Examples of protein deamidases include protein glutaminase and protein asparaginase, and more broadly, protein arginine deiminase. Among these protein deamidases, protein glutaminase is preferably chosen from the viewpoint of further improving the free amount of essential amino acids, foaming properties, foam stability, and / or emulsifying properties.

[0036] Among these protein deamidases, from the viewpoint of further improving the free amount of essential amino acids, foaming properties, foam stability and / or emulsifying properties, protein deamidases derived from the genus *Chlorella* are more preferably cited, protein glutaminases derived from the genus *Chlorella* are more preferably cited, and protein glutaminases derived from the species *Chlorella utilis* are even more preferably cited.

[0037] Protein deamidases can be prepared from the culture medium of microorganisms that serve as the source of the aforementioned protein deamidases. Specific preparation methods include recovering protein deamidases from the culture medium or bacterial cells of these microorganisms. For example, when using protein deamidase-secreting microorganisms, the bacterial cells can be recovered from the culture medium beforehand by filtration, centrifugation, etc., and the enzyme can then be separated and / or purified. Alternatively, when using non-secreting protein deamidase microorganisms, the bacterial cells can be recovered from the culture medium beforehand, and the enzyme can be exposed by pressure treatment, ultrasonic treatment, etc., after breaking down the bacterial cells, and then separated and / or purified. Known protein separation and / or purification methods can be used without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatographic methods using ion exchange resins. The separated and / or purified enzyme can be powdered by freeze-drying, vacuum drying, or other drying methods. Furthermore, powdering can also be achieved using appropriate excipients and / or drying aids in these drying methods. In addition, the isolated and / or purified enzymes can be liquefied by adding appropriate additives and then filtration sterilization. Furthermore, commercially available products can also be used as protein deamidases.

[0038] 1-3. Proteases derived from the genus *Chlorella* The protease derived from *Chlorella* spp., together with protein deamidase, is an effective component in the modifier of the present invention. Specifically, the protease derived from *Chlorella* spp. is an effective component in the essential amino acid free content enhancer of the composition containing plant protein involved in the first modifier of the present invention, which helps to increase the free content of essential amino acids; and is an effective component in the second modifier of the present invention, which helps to improve foaming properties, foam stability, and / or emulsifying properties.

[0039] As a protease derived from the genus *Chlorella*, there are no particular limitations as long as it is an enzyme derived from the genus *Chlorella* that can hydrolyze the peptide bonds of proteins.

[0040] Specific examples of proteases derived from the genus *Chryseobacterium* include those 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* can be used alone or in combination. Among these *Chryseobacterium* proteases, from the viewpoint of further improving the free essential amino acid content, foaming properties, foam stability, and / or emulsifying properties, proteases derived from *Chryseobacterium proteolyticum* are preferably cited.

[0041] Proteases derived from the genus *Chlorella* can be prepared using known methods. For example, they can be readily prepared by culturing *Chlorella* bacteria and isolating the protease using known methods, or by using gene recombination technology.

[0042] 1-4. Other ingredients The modifier of the present invention can be any enzyme composition containing a protein deamidase and a protease derived from *Chlorella* spp., but other components besides the enzyme may also be included as acceptable additives and / or bases in the formulation of the enzyme, or may not contain additives and / or bases. Examples of such additives and bases include excipients, buffers, antioxidants, UV stabilizers, preservatives, pH adjusters, dispersants, emulsifiers, solubilizers, carriers, and solvents (water, etc.). These additives and bases may be used individually or in combination of two or more. Furthermore, the content of these additives and bases can be appropriately set according to the type of these components and / or the formulation form.

[0043] 1-5. Characteristics There are no particular limitations on the properties of the modifier for the protein-containing composition of the present invention, for example, dry formulations and liquid formulations in powder, fine granule, or particulate form can be mentioned.

[0044] 2. Modification method of protein-containing compositions As described above, the combination of protein deamidase and protease derived from *Chlorella* helps to increase the free essential amino acid content of compositions containing plant proteins, and also helps to improve the foaming properties, foam stability, and / or emulsifying properties of protein-containing compositions. Therefore, the present invention also provides: a method for increasing the free essential amino acid content of a composition containing plant proteins (hereinafter also referred to as the "first modification method"), comprising the step of acting a protein deamidase and a protease derived from *Chlorella* on a composition containing plant proteins; and a method for modifying a protein-containing composition (hereinafter also referred to as the "second modification method"), comprising the step of acting a protein deamidase and a protease derived from *Chlorella* on a composition containing proteins, wherein the modification is an improvement in foaming properties, foam stability, and / or emulsifying properties. Hereinafter, the first modification method and the second modification method are collectively referred to as the "modification method".

[0045] In the method for increasing the free essential amino acid content of a composition containing plant-based proteins according to the first modification method of the present invention, the terms "increasing the free essential amino acid content," "protein deamidase," and "protease derived from *Chlorella*" are as detailed in "1. Modifier for Protein-Containing Compositions" above. Similarly, in the second modification method of the present invention, the terms "increasing foaming properties, foam stability, and / or emulsifying properties," "protein deamidase," and "protease derived from *Chlorella*" are also as detailed in "1. Modifier for Protein-Containing Compositions" above.

[0046] 2-1. To enable protein deamidases and proteases derived from *Chlorella* to act on protein-containing compositions. process In the first modification method, in the step of acting a protein deamidase and a protease derived from *Chlorella* on the composition containing plant protein, a mixture of the composition containing plant protein and a protein deamidase and a protease derived from *Chlorella* can be prepared as needed, and the enzymatic reaction is performed. In the second modification method, in the step of acting a protein deamidase and a protease derived from *Chlorella* on the composition containing protein, a mixture of the composition containing protein and a protein deamidase and a protease derived from *Chlorella* can be prepared as needed, and the enzymatic reaction is performed. In the second modification method, the protein may include plant protein and animal protein.

[0047] 2-1-1. Compositions containing plant-based proteins The composition containing plant-based protein is an example of the enzyme treatment target in the first modification method and is suitable for the first modifying agent. Furthermore, the composition containing plant-based protein is an example of the enzyme treatment target in the second modification method and is suitable for the second modifying agent. The composition containing plant-based protein contains plant-based protein, and there are no particular limitations as long as it is in a form that can be absorbed by an organism.

[0048] Typically, the plant-based protein-containing composition used in this invention is a dietary composition containing plant-based protein and water. The properties of the plant-based protein-containing composition are not particularly limited; for example, examples include fluid states such as liquid, paste, and slurry (hereinafter, these fluid-state plant-based protein-containing compositions are collectively referred to as "liquids, etc.") and solid states. In the first modification method, the plant-based protein-containing composition can be either fluid or solid. In the second modification method, the plant-based protein-containing composition is fluid.

[0049] Specific examples of compositions containing plant-based proteins, as compositions prepared from unorganized protein materials, include: (i) liquids obtained by dispersing dried powder of plant-based protein materials (specifically, at least one of plant organs from the source plant of the plant protein, and materials whose protein content has been increased by removing at least a portion of components other than protein from the aforementioned plant organs. The same applies below.) in water; (ii) compositions in which plant-based protein materials are crushed and dispersed in water, and unorganized components such as the peel of the plant material are removed as needed by any means such as centrifugation, filtration, filter bags, or sieves. Liquids obtained by dissolving substances; (iii) liquids in which the protein content is increased by removing components other than plant proteins from the liquids in (i) or (ii) above; (iv) liquids obtained by mixing dried powder prepared from any of the liquids in (i) to (iii) above with water; and, preferably, when cereals are used as plant protein materials, (v) liquids obtained by further treating any of the liquids in (i) to (iv) above with amylase; as a composition prepared from texturized protein materials, (vi) texturized plant protein materials that swell with water can be cited as an example.

[0050] It should be noted that the textured plant protein material used in the preparation of the specific example described above (vi) is generally a food material known as a substitute for meat (meat-like material). A typical example of a textured plant protein material is a material obtained by extruding a mixture of raw materials containing plant protein and water using an extruder, drying or freezing it to create a meat-like texture. It should be noted that in this invention, the "meat" simulated by the textured plant protein material refers to the muscle of an edible animal; when referred to as "meat," its meaning includes not only the muscle of mammals and poultry but also the meat of fish and shellfish.

[0051] As a preferred example of a liquid composition containing plant-based proteins, so-called plant-based alternative milk (also known as plant milk) can be cited.

[0052] It should be noted that, in the following context, the "content of plant protein material" in a composition containing plant protein refers to the proportion of the dry weight of the components constituting the aforementioned plant organs in the composition when the composition is prepared using non-tissue plant protein material, and to the proportion of the dry weight of the tissue plant protein material when the composition is prepared using tissue plant protein material. For example, if the composition containing plant protein is a liquid or the like composed solely of components derived from plant organs and water, as in the specific examples (i) to (v) above, the "content of plant protein material" refers to the proportion of the dry weight of the liquid or the like. If the composition containing plant protein is a liquid or the like containing the specific examples (i) to (v) above and added components, the "content of plant protein material" refers to the proportion of the dry weight of the portion remaining after removing the added components from the liquid or the like. Furthermore, when the composition containing plant protein, as in the specific example described above (vi), is a swollen substance consisting only of tissueed plant protein material and water, the "content of plant protein material" refers to the proportion of the dry weight of the composition. When the composition containing plant protein is a swollen substance comprising the specific example described above (vi) and additives, the "content of plant protein material" refers to the proportion of the dry weight of the portion of the swollen substance after removing the additives.

[0053] The content of plant protein in plant-based protein materials is not particularly limited, but examples include 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 increasing the effect of increasing the free amount of essential amino acids, this 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 this content range is not particularly limited, but examples include 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 plant protein in plant-based protein materials include, for example, 5 to 90% by weight, preferably 10 to 85% by weight; when the plant protein is derived from legumes, for example, 5 to 90% by weight, preferably 30 to 85% by weight, more preferably 40 to 85% by weight, 45 to 85% by weight, or 50 to 80% by weight; when the plant protein is derived from cereals, 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.

[0054] Furthermore, the content of plant protein material in the composition containing plant protein is not particularly limited. For example, 0.05 to 80% by weight can be mentioned. When the composition containing plant protein is prepared from non-organized protein material, 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, more preferably 5 to 15% by weight, or 8 to 12% by weight can be mentioned.

[0055] 2-1-1-1. Plant-based protein Plant-based proteins can be derived from plants without particular restrictions. Examples include legumes such as soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupins, and kidney beans; cereals such as wheat, barley, oats, sorghum, rice, rye, buckwheat, barnyard millet, teff, quinoa, corn, and potatoes; seeds such as almonds, coconuts, peanuts, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, pine nuts, chia seeds, quinoa, amaranth seeds, canary seeds, and flax seeds; and natural proteins found in algae. Furthermore, the plant-based proteins in this invention can be any proteins derived from plants. Therefore, in addition to the aforementioned natural proteins, they can also be proteins obtained by chemically decomposing the aforementioned natural proteins using acids or alkalis, by enzymatically decomposing them using proteases, by chemically modified proteins using various reagents, or by artificially synthesizing peptides.

[0056] In this invention, the aforementioned plant-based proteins can be used alone or in combination of two or more. In the first modification method, from the viewpoint of further improving the free essential amino acid content, legume or cereal proteins are preferred, more preferably proteins from soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupins, kidney beans, and oats, and even more preferably proteins from soybeans and peas. In the second modification method, from the viewpoint of further improving emulsifying properties, cereal proteins are preferred, and more preferably proteins from oats.

[0057] The content of plant protein in the composition containing plant protein is not particularly limited, and for example, 0.01 to 50% by weight can be cited. In the first modification method, the content of plant protein in the composition containing plant protein can be, for example, 0.01 to 50% by weight. When the composition containing plant protein is prepared from non-organized protein material, for example, 0.01 to 50% by weight, 0.1 to 40% by weight, or 0.5 to 30% by weight can be cited, preferably 1 to 25% by weight, or 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 content of plant protein in the composition containing plant protein can 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.

[0058] 2-1-1-2. Other ingredients In a composition containing plant-based protein, any other ingredients may be included, or none may be included, besides the plant-based protein. Examples of other ingredients include components found in the plant from which the plant-based protein originates (sugars, lipids, etc.), as well as added ingredients (other food ingredients, food additives, etc.). Examples of food additives include thickeners, binders, flavorings, pH adjusters, buffers, colorings, and fragrances.

[0059] 2-1-2. Compositions containing animal protein Compositions containing animal protein are examples of enzyme-treated materials in the second modification method, and examples of suitable materials for the second modifier. There are no particular restrictions on the form in which the animal protein can be absorbed by an organism.

[0060] Typically, the animal protein-containing compositions used in this invention contain animal proteins in a fluid state, such as liquid, paste, or slurry. Specific examples of animal protein-containing compositions include liquids obtained by dispersing animal milk or dried animal milk in water, and liquids obtained by dispersing crudely purified or refined proteins from animal milk in water. It should be noted that, 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 components).

[0061] The content of animal protein material in the composition containing animal protein is not particularly limited, 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.

[0062] 2-1-2-1. Animal protein There are no particular restrictions on animal protein as long as its source is animal. Preferably, milk protein is an example; more preferably, whey and casein are examples; and even more preferably, whey is an example.

[0063] The content of animal protein in the composition containing animal protein is not particularly limited, for example, 0.03 to 30% by weight, preferably 0.08 to 20% by weight, or 0.8 to 8% by weight, preferably 1 to 6% by weight.

[0064] 2-1-2-2. Other ingredients In a composition containing animal protein, any other ingredients may be included, or none may be included, besides animal protein. Examples of other ingredients include components found in the animal protein source material (carbohydrates, lipids, etc.) and added ingredients (other food ingredients and food additives, etc.). Examples of food additives include thickeners, binders, seasonings, pH adjusters, buffers, colorants, and flavorings.

[0065] 2-1-3. Dosage of protein deamidase The amount of protein deamidase used is not particularly limited. In the first modification method, the amount used relative to 1g of plant protein material contained in the composition containing plant protein is, for example, 0.01U or more or 0.1U or more. From the viewpoint of further improving the effect of increasing the free amount of essential amino acids, 1U or more or 5U or more is preferred, more preferably 7.5U or more, further preferably 10U or more, and even more preferably 13U or more. The upper limit of the above-mentioned range of protein deamidase usage relative to 1g of plant protein material is not particularly limited. Examples include 400U or less, 300U or less, 200U or less, 150U or less, 100U or less, or 75U or less, preferably 60U or less or 50U or less, more preferably 40U or less or 30U or less, and further preferably 25U or less, 20U or less, or 17U or less.

[0066] In the second modification method, the amount of protein deamidase used, relative to the amount of plant protein material contained in the protein-containing composition, can be, for example, 0.01 U or more or 0.1 U or more. From the viewpoint of further improving foaming properties, foam stability, and / or emulsifying properties, it is preferably 1 U or more. There is no particular limitation on the upper limit of the above-mentioned range of protein deamidase usage relative to the amount of plant protein material, for example, 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.

[0067] In the second modification method, the amount of protein deamidase used, relative to the amount of animal protein material contained in the protein-containing composition, can be, for example, 0.1 U or more or 1 U or more. From the viewpoint of further improving the foaming properties, foam stability, and / or emulsifying properties, 3 U or more is preferred, 5 U or more is more preferred, and 7 U or more or 8 U or more is even more preferred. There is no particular limitation on the upper limit of the above-mentioned range of protein deamidase usage relative to 1 g of animal protein material; for example, 400 U or less, 200 U or less, 100 U or less, 50 U or less, or 30 U or less are examples, preferably 20 U or less, more preferably 15 U or less, and even more preferably 12 U or less.

[0068] Furthermore, in the first modification method, the amount of protein deamidase used relative to 1g of plant protein can be, for example, 0.01U or more, or 0.1U or more. From the viewpoint of further improving the effect of increasing the free amount of essential amino acids, 1U or more, or 5U or more, more preferably 7.5U or more, or 10U or more, even more preferably 13U or more, or 16U or more, and even more preferably 18U or more, 20U or more, or 22U or more are preferred. There is no particular limitation on the upper limit of the above-mentioned range of protein deamidase usage relative to 1g of plant protein. For example, 500U or less, 400U or less, 300U or less, 200U or less, 150U or less, 100U or less, or 80U or less are preferred, 70U or less, or 60U or less, more preferably 50U or less, or 45U or less, and even more preferably 40U or less, 35U or less, or 32U or less are preferred. In the first modification method, the specific range of the amount of protein deamidase used relative to 1g of plant protein can be, for example, 0.01-500U, 0.01-400U or 0.1-300U, preferably 1-200U, 5-150U or 5-100U, more preferably 7.5-80U or 10-70U, further preferably 13-60U, 16-50U, 18-45U, 20-45U, and even more preferably 22-40U, 22-35U or 22-32U.

[0069] In the second modification method, the amount of protein deamidase used relative to 1g of plant protein can be, for example, 0.01U or more or 0.1U or more. From the viewpoint of further improving foaming properties, foam stability, and / or emulsifying properties, 1U or more or 3U or more is preferred, 5U or more or 7U or more is more preferred, and 9U or more is even more preferred. There is no particular limitation on the upper limit of the above-mentioned range of protein deamidase usage relative to 1g of plant protein; for example, 100U or less, 50U or less, or 20U or less is preferred, and 15U or less is even more preferred. In the second modification method, the specific range of the amount of protein deamidase used relative to 1g of plant protein can be, for example, 0.01–100U, or 0.1–50U, 1–20U, 3–20U, more preferably 5–15U, or 7–15U, and even more preferably 9–15U.

[0070] In the second modification method, the amount of protein deamidase used relative to 1g of animal protein is, for example, 0.01U or more or 0.1U or more. From the viewpoint of further improving foaming properties, foam stability, and / or emulsifying properties, 1U or more or 3U or more is preferred, 5U or more or 7U or more is more preferred, and 9U or more is even more preferred. There is no particular limitation on the upper limit of the above-mentioned range of protein deamidase usage relative to 1g of animal protein; for example, 100U or less, 50U or less, or 20U or less is preferred, and 15U or less is even more preferred. In the second modification method, the specific range of the amount of protein deamidase used relative to 1g of animal protein is, for example, 0.01–100U, or 0.1–50U, 1–20U, 3–20U, more preferably 5–15U, or 7–15U, and even more preferably 9–15U.

[0071] It should be noted that regarding the activity of protein deamidases, using benzyloxycarbonyl-L-glutamylglycine (Z-Gln-Gly) as a substrate will release 1 [unit of enzyme] within 1 minute. μ The amount of enzyme in mol of ammonia is taken as 1 unit (1U).

[0072] 2-1-4. Amount of protease derived from Chlorella species used The amount of protease derived from *Chlorella* is not particularly limited. In the first modification method, the amount used relative to 1g of plant protein material contained in the composition containing plant protein is, for example, 0.05U or more. From the viewpoint of further improving the effect of increasing the free amount of essential amino acids, 0.1U or more or 1U or more is preferred, more preferably 5U or more or 7.5U or more, further preferably 10U or more or 12U or more, and even more preferably 15U or more or 18U or more is preferred. The upper limit of the above-mentioned range of the amount of protease derived from *Chlorella* relative to 1g of plant protein material is not particularly limited. Examples include 400U or less, 300U or less, 200U or less, 150U or less, 100U or less, or 90U or less, preferably 80U or less, 70U or less, or 60U or less, more preferably 50U or less or 40U or less, and further preferably 30U or less, 25U or less, or 22U or less.

[0073] In the second modification method, the amount of protease derived from *Chlorella* relative to the amount of plant protein material contained in the protein-containing composition can be, for example, 0.1 U or more or 1 U or more. From the viewpoint of further improving foaming properties, foam stability, and / or emulsifying properties, it is preferable to use 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. There is no particular limitation on the upper limit of the above-mentioned range of the amount of protease derived from *Chlorella* relative to the amount of plant protein material; for example, it can be 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.

[0074] In the second modification method, the amount of protease derived from *Chlorella* relative to the amount of animal protein material contained in the protein-containing composition can be, for example, 0.005 U or more. From the viewpoint of further improving foaming properties, foam stability, and / or emulsifying properties, the amounts can be 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 amounts of protease derived from *Chlorella* relative to the amount of animal protein material is not particularly limited; for example, amounts of 400 U or less, 200 U or less, or 100 U or less can be included.

[0075] Furthermore, in the first modification method, the amount of protease derived from *Chlorella* relative to 1g of plant protein used is, for example, 0.05U or more. From the viewpoint of further improving the effect of increasing the free amount of essential amino acids, 0.1U or more, or 1U or more, more preferably 5U or more, or 7.5U or more, even more preferably 10U or more, 12.5U or more, 15U or more, or 20U or more, and even more preferably 22U or more, or 24U or more are all preferred. The upper limit of the above-mentioned range of the amount of protease derived from *Chlorella* relative to 1g of plant protein is not particularly limited. For example, amounts of 1000U or less, 500U or less, 400U or less, 300U or less, 200U or less, 150U or less, 100U or less, or 90U or less are all preferred, 80U or less, 70U or less, or 60U or less are preferred, 55U or less, or 50U or less are more preferred, and 45U or less, or 40U or less are all preferred. In the first modification method, the specific range of the amount of protease derived from *Chlorella* relative to 1g of plant protein used is, for example, 0.05–1000U, 0.05–500U, or 0.05–400U, preferably 0.1–300U, 0.1–200U, or 1–150U, more preferably 5–100U or 7.5–90U, further preferably 10–80U, 12.5–70U, 15–60U, or 20–55U, and even more preferably 22–50U, 22–45U, or 24–40U.

[0076] In the second modification method, the amount of protease derived from *Chlorella* relative to the amount of plant protein contained in the protein-containing composition can be, for example, 1 U or more or 10 U or more. From the viewpoint of further improving foaming properties, foam stability, and / or emulsifying properties, it is preferable to use 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. There is no particular limitation on the upper limit of the above-mentioned range of amounts of protease derived from *Chlorella* relative to the amount of plant protein; for example, it can be 400 U or less, 300 U or less, 200 U or less, or 150 U or less. In the second modification method, the specific range of the amount of protease derived from Chlorella relative to 1g of plant protein can be, for example, 1-400U, 10-400U, preferably 15-400U or 20-400U, more preferably 40-400U, 50-400U, 60-400U, 70-400U, and even more preferably 80-400U, 90-400U, 90-300U, 90-200U or 90-150U.

[0077] In the second modification method, the amount of protease derived from *Chlorella* can be, for example, 0.1 U or more or 1 U or more, relative to the amount of animal protein contained in the protein-containing composition. From the viewpoint of further improving the foaming properties, foam stability and / or emulsifying properties, it is preferable to use 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 improving the foaming properties and / or emulsifying properties, it is even more preferable to use 30 U or more, 40 U or more or 50 U or more. From the viewpoint of further improving the emulsifying properties, it is even more preferable to use 60 U or more, 80 U or more, 90 U or more or 100 U or more. There is no particular limitation on the upper limit of the above-mentioned usage range of proteases derived from the genus Chlorella relative to 1g of animal protein. Examples include 400U or less, 300U or less, 200U or less, or 150U or less. From the viewpoint of improving foam stability, 100U or less, 60U or less, or 50U or less are preferred, and 30U or less are even more preferred. In the second modification method, the specific range of the amount of protease derived from Chlorella relative to 1g of animal protein can be, for example, 0.1–400U or 1–400U, preferably 7–400U, 10–400U, 10–200U, 10–150U, 13–100U, 17–60U, 20–50U, 20–30U, 13–400U, 17–400U, 20–400U, 30–400U, 40–400U, 50–400U, 60–400U, 80–400U, 90–400U, 90–300U, 90–200U, or 90–150U.

[0078] Regarding the ratio of protein deamidase to protease derived from Chlorella, it is determined based on the above-mentioned usage amounts for each enzyme. However, from the viewpoint of further improving the effect of increasing the free amount of essential amino acids, the following usage ratio can be preferred.

[0079] In the first modification method, the amount of protease derived from *Chlorella* relative to 1 U of protein deamidase used is preferably 0.005 U or more, more preferably 0.05 U or more or 0.1 U or more, further 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 above-mentioned range of the amount of protease derived from *Chlorella* relative to 1 U of protein deamidase used is, for example, 20 U or less. From the viewpoint of further improving the effect of increasing the free amount of essential amino acids, it is preferably 15 U or less, or 10 U or less, more preferably 8 U or less, or 6 U or less, further 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 protease derived from Chlorella relative to 1 U of protein deamidase used is preferably 0.005 to 20 U, more preferably 0.05 to 15 U or 0.1 to 10 U, further 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.

[0080] In the second modification method, the amount of protease derived from *Chlorella* relative to the amount of protein deamidase used per 1U is preferably 0.005U or more or 0.01U or more. From the viewpoint of further improving the foaming property, foam stability and / or emulsifying property, more preferably 0.05U or more, 0.1U or more or 0.3U or more, even more preferably 0.8U or more, 1U or more or 1.3U or more, even more preferably 1.8U or more or 2U or more. From the viewpoint of further improving the foaming property and / or emulsifying property, more preferably 2.5U or more, 4U or more or 5U or more. From the viewpoint of further improving the emulsifying property, particularly preferably 6U or more, 8U or more or 10U or more. As the upper limit of the above-mentioned usage range of proteases derived from the genus Chlorella relative to each 1U of protein deamidase, examples include 50U or less, 30U or less, 20U or less, or 15U or less. From the viewpoint of further improving foam stability, 10U or less is preferred, 6U or less is more preferred, or 5U or less is more preferred, and 3U or less is even more preferred. In the second modification method, the specific range of the amount of protease derived from Chlorella relative to 1 U of protein deamidase used can be exemplified as preferably 0.005–50 U, 0.01–50 U, more preferably 0.05–50 U, 0.1–50 U, 0.3–50 U, 0.8–50 U, 1–50 U, 1.3–50 U, 1.3–30 U, 1.3–20 U, 1.3–15 U, 1.3–10 U, 1.3–7 U, 1.3–5 U, 1.3–3 U, 1.8–50 U, 2–50 U, 2.5–50 U, 4–50 U, 5–50 U, 6–50 U, 8–50 U, 8–50 U, 8–30 U, 8–20 U, 8–15 U, or 8–10 U.

[0081] Regarding the activity of proteases, it is determined using casein as a substrate via the Folin method. That is, using casein as a substrate, the enzyme reaction is carried out using conventional methods at a pH set according to the optimal pH of the protease being tested, yielding an activity equivalent to 1 [unit of protein] within 1 minute. μ The increase in enzyme amount of the chromogenic substance in Folin solution containing g of tyrosine is taken as 1 unit (1U) to determine the activity of the protease.

[0082] 2-1-5. Reaction Operation and Processing Conditions The methods for preparing the plant-based protein mixture in the first modification method and the protein mixture in the second modification method are not particularly limited. Furthermore, the treatment conditions (temperature, time, pH, etc.) for these mixtures are not particularly limited as long as the effects of the present invention are achieved. The order in which the protein deamidase and the protease derived from *Chlorella* act are not particularly limited; various enzymes can act in any order, or both enzymes can act simultaneously; it is preferable that both enzymes act simultaneously.

[0083] The processing temperature for the plant-based protein mixture in the first modification method and the protein mixture in the second modification method can be, for example, 4 to 80°C, preferably 8 to 70°C. The lower limit of this temperature range can be 15°C, 30°C, or 45°C, and the upper limit can be 65°C, 60°C, or 55°C. Furthermore, the processing time in the first modification method is not particularly limited, for example, 0.1 to 72 hours, preferably 12 to 36 hours. The processing time in the second modification method is also not particularly limited, for example, 0.1 to 12 hours, preferably 0.5 to 6 hours, more preferably 1 to 3 hours. The processing pH (at 25°C) for the plant-based protein mixture can be, for example, 2 to 9, preferably 3 to 8, more preferably 5 to 7.6, further preferably 6 to 7.4, and even more preferably 6.5 to 7.2.

[0084] These treatment conditions are appropriately selected based on the optimal temperature and pH of the enzyme used and / or the desired effect of the invention (improvement in the amount of essential amino acids released, foaming properties, foam stability, and / or emulsification). Furthermore, the optimal treatment conditions can be determined through preliminary experiments.

[0085] 2-2. Other processes The modification method of the present invention may include steps other than the step of acting on the aforementioned protein deamidase and protease derived from *Chlorella*, or it may not include any other steps. Examples of other steps include the step of preparing the composition containing plant protein used in the first modification method or the composition containing protein (containing plant protein or containing animal protein) used in the second modification method, an enzyme inactivation step, a cooling step, a filtration step, etc. These other steps may be performed individually or in combination of two or more steps.

[0086] The process for preparing a composition containing plant or animal protein can be carried out by any method for preparing a composition containing plant or animal protein.

[0087] For example, when using plant-based milk substitutes (plant milk) as a composition containing plant protein, it can be prepared using 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 a composition containing plant or animal protein, any food additives such as seasonings, pH adjusters, buffers, colorants, and flavorings can be added, and / or any treatment other than treatment using protein deamidases and proteases derived from *Chlorella* (e.g., fermentation treatment such as lactic acid fermentation) can be performed, or these operations can be omitted.

[0088] 3. Method for manufacturing a processed protein-containing composition The present invention also provides the following manufacturing methods: a method for manufacturing a processed composition containing plant-based protein with increased free essential amino acids (hereinafter also referred to as the "first manufacturing method"), comprising the step of acting a protein deamidase and a protease derived from *Chlorella* on the composition containing plant-based protein; and a method for manufacturing a processed composition containing protein with improved foaming properties, foam stability, and / or emulsifying properties (hereinafter also referred to as the "second manufacturing method"), comprising the step of acting a protein deamidase and a protease derived from *Chlorella* on the composition containing protein. Hereinafter, the first manufacturing method and the second manufacturing method will be collectively referred to as "manufacturing methods".

[0089] In the first manufacturing method of the present invention, the terms "increasing the free amount of essential amino acids," "protein deamidase," and "protease derived from *Chlorella*" are detailed as described in "1. Modifier for a protein-containing composition" above; the term "step of acting protein deamidase and protease derived from *Chlorella* on a composition containing plant protein" is detailed as described in "2. Modification method for a protein-containing composition" above. In the second manufacturing method of the present invention, the terms "increasing foaming properties," "increasing foam stability," "increasing emulsifying properties," "protein deamidase," and "protease derived from *Chlorella*" are detailed as described in "1. Modifier for a protein-containing composition" above; the term "step of acting protein deamidase and protease derived from *Chlorella* on a composition containing protein" is detailed as described in "2. Modification method for a protein-containing composition" above.

[0090] The manufacturing method of the present invention may include steps other than the step of acting on a protein deamidase and a protease derived from *Chlorella*, or it may not include other steps. Among the "other steps," in addition to the steps detailed in "2. Method for modifying a protein-containing composition" above, any cooking steps can be performed to prepare the final food and beverage form of the processed plant protein-containing composition to be manufactured.

[0091] As an example of a cooking process, for instance, if the composition containing plant protein used in the first manufacturing method is in a textured state, the composition, after being treated with protein deamidase and a protease derived from *Chlorella*, can be shaped into a form suitable for the desired form, and then heated and cooked as needed, thereby obtaining a processed composition containing plant protein (food). Regarding the heating and cooking method, those skilled in the art can appropriately determine it according to the type of food. Specifically, examples of heating and cooking methods include boiling, roasting (roasting, toasting, baking, grilling, broiling), steaming, frying, etc. These heating and cooking methods can be used individually or in combination.

[0092] 4. Processed protein-containing compositions The present invention also provides the following compositions: a processed composition containing plant-based protein with increased free essential amino acids (hereinafter referred to as "the first processed protein-containing composition"), obtained by a method comprising the step of acting a protein deamidase and a protease derived from *Chlorella* on the composition containing plant-based protein; and a processed protein-containing composition with improved foaming properties, foam stability, and / or emulsifying properties (hereinafter referred to as "the second processed protein-containing composition"), obtained by a method comprising the step of acting a protein deamidase and a protease derived from *Chlorella* on the composition containing protein. Hereinafter, the first processed protein-containing composition and the second processed protein-containing composition are collectively referred to as "processed protein-containing compositions".

[0093] The first processed protein-containing composition of the present invention is a composition processed using protein deamidase and a protease derived from *Chlorella* to increase the free amount of essential amino acids. The second processed protein-containing composition of the present invention is a composition processed using protein deamidase and a protease derived from *Chlorella* to improve foaming properties, foam stability, and / or emulsifying properties.

[0094] In the first processed protein-containing composition of the present invention, the terms "increase in the amount of free essential amino acids," "protein deamidase," and "protease derived from *Chlorobacterium*" are detailed in "1. Modifier for protein-containing compositions" above. The terms "method comprising the step of acting a protein deamidase and a protease derived from *Chlorobacterium* on a composition containing plant-based proteins" are detailed in "2. Modification method for protein-containing compositions" and "3. Method for manufacturing a processed protein-containing composition" above. In the second processed protein-containing composition of the present invention, the terms "increase in foaming properties," "increase in foam stability," "increase in emulsifying properties," "protein deamidase," and "protease derived from *Chlorobacterium*" are detailed in "1. Modifier for protein-containing compositions" above. The terms "step of acting a protein deamidase and a protease derived from *Chlorobacterium* on a composition containing proteins" are detailed in "2. Modification method for protein-containing compositions" and "3. Method for manufacturing a processed protein-containing composition" above.

[0095] The specific form of the protein-containing composition processed according to the present invention can be selected from any food or beverage form.

[0096] When the plant-based protein-containing composition used as the material for the first processed protein-containing composition of the present invention is in a textured form, this form can refer to processed foods of livestock, poultry, and / or surimi. That is, as the first processed protein-containing composition of the present invention, meat-like processed foods (referring to foods that mimic processed foods of livestock, poultry, and / or surimi) can be cited. More preferably, as the first processed protein-containing composition of the present invention, meat-like and / or poultry-like processed foods (referring to foods that imitate processed foods of livestock and / or poultry) can be cited. Such meat-like and / or poultry processed foods are simply foods that are cooked by shaping and heating meat containing livestock and / or poultry. Specific examples include hamburger patties, meatballs, patties, meat strips, fried patties, and snacks.

[0097] When the plant-based protein composition used as the material for the protein-containing composition of the first processing of the present invention is in a non-organized state, specific forms of the protein-containing composition of the first processing of the present invention may include plant-based milk substitutes, yogurt substitutes, cheese substitutes, ice cream substitutes, etc., and preferably plant-based milk substitutes and yogurt substitutes.

[0098] Specific forms of protein-containing compositions that are processed in the second way according to the present invention include plant-based milk substitutes, cream substitutes, ice cream substitutes, and animal dairy products (animal milk, cream, ice cream).

[0099] Example The present invention will be specifically described below with examples, but the present invention is not to be construed as being limited to the following examples.

[0100] [A. A composition containing protein] Use the protein materials shown in Table 1.

[0101] [Table 1]

[0102] [B-1. Protein deamidase] As a protein deamidase, a protein glutaminase derived from Chryseobacterium proteolyticum (manufactured by Amano Enzyme Co., Ltd.) is used. This protein deamidase will also be referred to as "PG" below.

[0103] The activity of protein deamidases was determined by the following method.

[0104] Add 0.1 mL of the sample solution containing protein deamidase to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, incubate at 37°C for 10 minutes, then add 1 mL of 0.4 M TCA solution to stop the reaction. As a control group, add 1 mL of 0.4 M TCA solution to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, then add 0.1 mL of the sample solution containing protein deamidase, and incubate at 37°C for 10 minutes.

[0105] For the solution obtained above, the amount of ammonia generated in the reaction solution was determined using Ammonia-test Wako (Fujifilm and Koichi Chemicals Co., Ltd.). A calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) was prepared using an ammonia standard solution (ammonium chloride), and the ammonia concentration in the reaction solution was determined using this standard curve.

[0106] 1 will be generated within 1 minute μ The enzyme activity of protein deamidase is calculated using the following mathematical formula, where mol of ammonia is taken as 1 unit (1U). In the formula, the reaction solution volume is 2.1, the enzyme solution volume is 0.1, and Df is the dilution factor of the enzyme solution. Additionally, 17.03 is the molecular weight of ammonia.

[0107] [Number 1] Protein deamidase activity (U / mL) = Ammonia concentration in reaction solution (mg / L) × (1 / 17.03) × (reaction solution volume / enzyme solution volume) × (1 / 10) × Df [B-2. Protease derived from the genus *Chlorella*] As a protease derived from the Chryseobacterium genus, a protease derived from Chryseobacterium proteolyticum is used.

[0108] The protease activity value was determined by the following method.

[0109] Heat 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) at 37°C for 10 minutes, then add 1 mL of the sample solution containing the protease and immediately vortex to mix. After incubating the liquid at 37°C for 10 minutes, add 5 mL of trichloroacetic acid solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid) and vortex to mix. Incubate again at 37°C for 30 minutes and then filter. Discard the initial 3 mL of filtrate, measure 2 mL of the subsequent filtrate, add 5 mL of 0.55 mol / L sodium carbonate solution and 1 mL of Folin-Ciocalteu solution (1→3), vortex thoroughly, and incubate at 37°C for 30 minutes. For this liquid (enzyme reaction solution), using water as a control, measure the absorbance (AT) at 660 nm.

[0110] In addition, 1 mL of the sample solution containing protease was measured, and 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 to mix. Then, 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was added, and the mixture was shaken to mix immediately. The mixture was then placed at 37°C for 30 minutes. Otherwise, the same procedure as the enzyme reaction solution was performed to obtain the liquid (blank group), and its absorbance AB was measured.

[0111] Take 1 mL, 2 mL, 3 mL, and 4 mL of 1 mg / mL tyrosine standard stock solution (0.2 mol / L hydrochloric acid), and add 0.2 mol / L hydrochloric acid test solution to each to prepare 100 mL. Take 2 mL of each liquid, add 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1→3), shake to mix immediately, and let stand at 37°C for 30 minutes. For these liquids, take 2 mL of 0.2 mol / L hydrochloric acid test solution and repeat the above operation to obtain liquids, which are used as controls. Measure the absorbance A1, A2, A3, and A4 at a wavelength of 660 nm. Plot the absorbance A1, A2, A3, and A4 as the vertical axis and the amount of tyrosine in 2 mL of each liquid as the horizontal axis. μ Using g) as the horizontal axis, construct a standard curve and calculate the amount of tyrosine corresponding to an absorbance difference of 1. μ g).

[0112] It will bring the equivalent of 1 tyrosine within 1 minute. μ The increase in enzyme amount of chromogenic substance in g of Folin solution is taken as 1 unit (1U).

[0113] [Number 2] Protease activity (U / g, U / mL) = (AT-AB)×F×11 / 2×1 / 10×1 / M AT: Absorbance of the enzyme reaction solution AB: Absorbance of the blank group F: The amount of tyrosine when the absorbance difference is 1 as determined by the tyrosine standard curve. μ g) 11 / 2: Conversion factor when converting to the total liquid volume after the reaction stops. 1 / 10: Conversion factor when converting to reaction time per minute. M: The amount of sample (g or mL) in 1 mL of the sample solution. [B-3. Proteases derived from other bacteria] As proteases, proteases derived from Bacillus licheniformis and proteases derived from Bacillus amyloliquefaciens are used.

[0114] The proteases were determined using the method described under [B-2. Proteases derived from the genus *Chlorella*].

[0115] [C. Manufacturing of processed protein-containing compositions] [C-1] First composition containing protein The plant protein material shown in [A. Protein-containing Composition] above was mixed with water to prepare a liquid plant protein-containing composition (pH approximately 7) containing 10% by weight of the plant protein material. The enzyme specified in Table 2 was added to the plant protein-containing composition and incubated at 50°C for 24 hours. The enzyme was then inactivated by treating in a boiling water bath for 10 minutes. After the enzyme inactivation process, the mixture was cooled to room temperature to obtain the processed plant protein-containing composition.

[0116] [C-2] Second composition containing protein The plant protein material shown in [A. Protein-containing Composition] above was suspended in deionized water (final concentration 10% by weight), boiled until completely gelatinized, and then 10 U / g-starch α-amylase was added. The mixture was incubated at 60°C for 30 minutes to prepare a liquid composition containing plant protein (pH approximately 7). The enzyme specified in Table 3 was added to the composition containing plant protein, and the mixture was incubated at 50°C for 2 hours. Then, the enzyme was inactivated by treating at 100°C for 5 minutes. After the enzyme inactivation process, the mixture was cooled to room temperature to obtain the processed composition containing plant protein (processed oat milk).

[0117] Additionally, the animal protein material shown in [A. Protein-Containing Composition] above was suspended in deionized water (final concentration 5% by weight) to prepare a liquid composition containing animal protein (pH approximately 7). The prescribed 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. Then, the enzyme was inactivated by treating at 100°C for 5 minutes. After the enzyme inactivation process, the mixture was cooled to room temperature to obtain the processed composition containing animal protein.

[0118] [D. Amino acid analysis] The processed composition containing plant protein obtained in [C-1] above was centrifuged (15000×g, 10 minutes) to obtain a supernatant. The obtained supernatant was then subjected to 0.45... μ The sample was filtered through a filter to obtain the amino acid analysis sample. For the amino acid analysis sample, the free amino acid content was analyzed using an amino acid analysis apparatus (using an Agilent 1260 Infinity II LC system) according to the apparatus's operating procedures. Specifically, first, the free essential amino acids (essential AAs: histidine, threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, lysine) and non-essential amino acids (non-essential AAs: tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, arginine) were determined. The percentage (%) of essential amino acids in the total free amino acid content of the amino acid analysis sample was calculated.

[0119] [E. Foaming properties] The 50 mL of the processed protein-containing liquid composition obtained in [C-2] above was homogenized at 18000 rpm for 30 minutes and immediately transferred to a 100 mL graduated cylinder. The total volume VF0 of the composition, including foam, was measured, and the "foaming property" was calculated using the following formula.

[0120] [Number 3] Foaming property (%) = 100 × (VF0 - 50) / 50 [F. Foam Stability] Similar to the [E. Foaming] test described above, 50 mL of the processed protein-containing liquid composition obtained in [C-2] was homogenized at 18000 rpm for 30 minutes, immediately transferred to a 100 mL graduated cylinder, and the volume VF0, including the foam, was measured. The volume VF30, representing the total volume of the composition after standing for 30 minutes, was measured, and the "foam stability" was calculated using the following formula.

[0121] [Number 4] Foam stability (%) = 100 × (VF30 / VF0) [G. Emulsifying properties] The 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 10000 rpm for 2 minutes to prepare an emulsified composition. 50 mL of the freshly prepared emulsified composition was then taken... μ Add L to 5 mL of 0.1% SDS solution. Measure its turbidity (A0) at absorbance of 500 nm and calculate "emulsification" using the following formula.

[0122] [Number 5] Emulsifying properties (m) 2 / g) = (2×2.303×A0) / (0.25×protein weight) [Experimental Example 1] Using the plant-based protein materials (soybean protein material or pea protein material) shown in Table 1 of [A. Protein-containing Composition] above, a processed plant-based protein composition was prepared by the method of "[C-1] First Protein-containing Composition" in [C. Manufacturing of Processed Protein-containing Composition] above. Furthermore, the amount of PG added was 30 U and the amount of protease added was 39 U per 1g of soybean protein, and the amount of PG added was 18.8 U and the amount of protease added was 24.4 U per 1g of pea protein.

[0123] For the processed compositions containing plant-based proteins, the experiments in [D. Amino Acid Analysis] were performed to calculate the percentage (%) of essential amino acids in the total amount of free amino acids. The results are shown in Table 2.

[0124] [Table 2]

[0125] As shown in Table 2, it can be confirmed that by treating plant protein materials with PG and proteases derived from the genus *Chlorella*, the amount of free essential amino acids in the processed compositions containing plant proteins increases compared to treatment with proteases derived from other bacteria.

[0126] [Experimental Example 2] A processed protein-containing composition was prepared using the plant-based protein material (oat protein material) or animal-based protein material (whey protein material) shown in Table 1 of [A. Protein-containing Composition] above, by the method of "[C-2] Second Protein-containing Composition" in [C. Manufacturing of Processed Protein-containing Composition] above.

[0127] For the processed protein-containing compositions, the above-described tests for [E. foaming properties], [F. foam stability], and [G. emulsifying properties] were performed. The results are shown in Tables 3 and 4.

[0128] [Table 3]

[0129] [Table 4]

[0130] As shown in Tables 3 and 4, it can be confirmed that treating the protein material with PG and proteases derived from *Chlorella* improves the foaming properties, foam stability, and emulsifying properties of the processed protein-containing compositions. It should be noted that, similar to Table 2, the free essential amino acid content is also increased in the processed protein-containing compositions in Table 3.

Claims

1. A composition containing plant protein essential amino acid free amount improver, characterized in that, A protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

2. The essential amino acid free amount enhancer for a composition containing plant protein according to claim 1, wherein, The protein deamidating enzyme is a protein glutaminase.

3. The essential amino acid free amount enhancer for a composition containing plant protein according to claim 1, wherein, The vegetable protein is a protein of a plant selected from the group consisting of legumes, cereals, and seeds.

4. A modifier for a composition containing a protein, characterized by, A protein deamidating enzyme and a protease derived from the genus Chryseobacterium, wherein the modification is an improvement in foaming property, foam stability, and / or emulsifying property.

5. A method for increasing the free amount of essential amino acids in a composition containing vegetable proteins, characterized by, A process comprising subjecting a composition containing a vegetable protein to a protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

6. A method for modifying a composition containing a protein, characterized by, A process comprising subjecting a composition containing a protein to a protein deamidating enzyme and a protease derived from the genus Chryseobacterium, wherein the modification is an improvement in foaming property, foam stability, and / or emulsifying property.

7. A method for producing a processed plant protein-containing composition with an increased free amount of essential amino acids, characterized by, A process comprising subjecting a composition containing a vegetable protein to a protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

8. A method for producing a processed protein-containing composition having improved foamability, foam stability, and / or emulsifiability, characterized by, A process comprising subjecting a composition containing a protein to a protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

9. A processed plant protein-containing composition with increased free essential amino acids, characterized by, A product obtained by a process comprising subjecting a composition containing a vegetable protein to a protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

10. A processed protein-containing composition having improved foamability, foam stability and / or emulsifiability, characterized in that, A product obtained by a process comprising subjecting a composition containing a protein to a protein deamidating enzyme and a protease derived from the genus Chryseobacterium.

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