Modifier for animal protein

Abstract

The purpose of the present invention is to provide a technology for modifying animal protein.　A modified animal protein-containing dry composition obtained by a method including an enzyme treatment step for treating an animal protein-containing composition with a protein deamidase to obtain an enzyme-treated composition and a drying step for removing water from the enzyme-treated composition can improve a prescribed characteristic in the presence of water. Further, a modified muscle protein-containing food obtained by a method including an enzyme treatment step for treating a food material containing muscle protein with a protein deamidase has increased stress.

Description

Modifier for animal proteins

[0001] The present invention relates to a processing technique for modifying animal proteins.

[0002] Protein is one of the important nutrients that constitute the body and is ingested from food and drink products. Animal protein is superior to plant protein in terms of digestibility, amino acid score, and other aspects, and can be called an excellent protein source in this regard.

[0003] Among the functions generally possessed by food and drink materials, physical functional properties such as emulsifying property, foaming property, water retention, and stress bring added value to quality, palatability and / or processability. Chemical modification and enzymatic modification have been proposed as techniques for changing these functions. In particular, unlike chemical treatment, enzymatic modification is easily accepted by consumers, and thus has been studied and put into practical use in the food and drink industry.

[0004] Animal proteins also have various functional properties such as gel-forming property, emulsifying property, water retention, and foaming property, and various modifications have been proposed through enzymatic modification. For example, transglutaminase, laccase, and tyrosinase, which are protein cross-linking enzymes, are known as enzymes that modify at least any of the above functional properties (Non-Patent Document 1). In particular, transglutaminase is an Activa transglutaminase preparation for meat processing (R) and has been commercialized as TG-H. This commercially available preparation acts on meat proteins to improve breaking strength, thereby providing chewy processed meat products.

[0005] Research Report of Grants-in-Aid for Scientific Research, June 14, 2023, Grant Number 20K05913

[0006] In the food and drink industry, modification of protein materials has attracted increasing attention in terms of increasing the added value of food and drink products. Regarding the modification of animal proteins, there is a demand for new options for functional modification techniques, or the creation of modification techniques for imparting further properties.

[0007] Therefore, an object of the present invention is to provide a technique for modifying animal proteins.

[0008] Generally, the functional properties of protein materials are exhibited in the presence of water. On the other hand, it is desirable for protein materials to be distributed in a dried state before being processed into food and beverages by food and beverage manufacturers in the presence of water, or before being prepared for use in food and beverages by consumers in the presence of water. However, once animal proteins are dried, their original functional properties are often not obtained even when resuspended in water. Therefore, the first object of the present invention is to provide a dried animal protein that has been treated to impart modified properties and that can exhibit those modified properties in the presence of water.

[0009] Furthermore, as mentioned above, the protein cross-linking enzyme transglutaminase can improve chewiness by acting on muscle proteins. On the other hand, there are few options for enzyme preparations for meat processing that can improve the chewiness of foods such as meat containing muscle proteins. Therefore, the second objective of the present invention is to provide a processing technology that can increase the stress of foods containing muscle proteins.

[0010] In other words, the object of the present invention is to provide a dried product that is suitable for the first object described above, or to provide a processing technology that is suitable for the second object described above.

[0011] As a result of diligent research by the inventors to solve the above first objective, it was found that a dried product of an animal protein-containing composition treated with a protein deamidation enzyme can exhibit predetermined modification properties (improved properties selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment) in the presence of water.

[0012] Furthermore, as a result of diligent research by the inventors to solve the second objective mentioned above, it was discovered that treating muscle protein with a protein deamidation enzyme increases stress.

[0013] This invention was completed by further investigation based on these findings. Specifically, this invention provides the invention in the following embodiments.

[0014] [A] Item A-1. A method for modifying a dry composition containing animal protein in the presence of water, comprising an enzyme treatment step of treating the animal protein-containing composition with a protein deamidation enzyme to obtain an enzyme-treated composition, and a drying step of removing water from the enzyme-treated composition, wherein the properties are selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment. Item A-2. The method for modifying according to Item A-1, wherein the animal protein is a protein from an animal selected from the group consisting of mammals, birds, reptiles, amphibians, fish, mollusks, and crustaceans. Item A-3. A modifier comprising a protein deamidation enzyme for improving the properties of a dry composition containing animal protein in the presence of water, wherein the properties are selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment. Item A-4. A modified animal protein-containing dry composition obtained by a method comprising: an enzyme treatment step of treating an animal protein-containing composition with a protein deamide enzyme to obtain an enzyme-treated composition; and a drying step of removing water from the enzyme-treated composition, wherein the dry composition, when mixed with water, is intended to obtain properties selected from the group consisting of foaming ability, foam stability, emulsification, emulsification stability, aggregation inhibition, and protein solubility after heat treatment.

[0015] [B] Item B-1. A method for increasing the stress of a food containing muscle protein, comprising an enzymatic treatment step of treating a food material containing muscle protein with a protein deamidation enzyme. Item B-2. The method according to item B-1, wherein the muscle protein is beef muscle protein. Item B-3. The method according to item B-1 or B-2, wherein the food material containing muscle protein is not cooked. Item B-4. The method according to any one of items B-1 to B-3, wherein the food material containing muscle protein is unrefined meat. Item B-5. A stress increasing agent for a food containing muscle protein, comprising a protein deamidation enzyme. Item B-6. A modified muscle protein-containing food obtained by a method comprising an enzymatic treatment step of treating a food material containing muscle protein with a protein deamidation enzyme.

[0016] According to the present invention, a technology for modifying animal proteins is provided.

[0017] Specifically, according to the first aspect of the present invention, a dried animal protein that has been treated to impart modification properties is provided, which can exhibit said modification properties (improved properties selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment) in the presence of water.

[0018] Furthermore, according to the second aspect of the present invention, a processing technique is also provided that can increase the stress of food products containing muscle protein.

[0019] The images show (A) the appearance of test solutions (without heat treatment) obtained by mixing a PG-treated casein-containing dry composition (PG-casein) and an untreated casein-containing dry composition (Non-casein) with water, and (B) the appearance of a test solution (with heat treatment) obtained by boiling the test solution (without heat treatment).

[0020] [A] 1. Modification Method The modification method of the present invention is a method for improving the properties of a dried composition containing animal protein in the presence of water, and comprises a predetermined enzyme treatment step and a drying step. The properties are selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment (hereinafter also referred to as "predetermined properties"). A modified animal protein-containing dried composition is obtained by the modification method of the present invention.

[0021] [A] 1-1. Enzyme treatment process: In the enzyme treatment process, the animal protein-containing composition is treated with a protein deamidation enzyme to obtain an enzyme-treated composition.

[0022] [A] 1-1-1. Compositions containing animal protein There are no particular restrictions on compositions containing animal protein, as long as they are ingestible by living organisms. Typically, the compositions containing animal protein used in the present invention contain animal protein and water, and have fluid properties such as liquid, slurry, and paste (hereinafter referred to as "liquid, etc.").

[0023] Specific examples of animal protein-containing compositions include: (i) liquids obtained by dispersing a dried powder of an animal protein material (specifically, tissue (muscle tissue, etc.), body fluid (milk, etc.), or egg from the animal from which the protein originates, and at least one of the materials obtained by removing at least a portion of non-protein components from the said tissue, body fluid, or egg to increase the concentration of the protein; the same applies hereinafter) in water; (ii) liquids obtained by crushing and dispersing an animal protein material in water and removing insoluble matter as necessary by any means such as centrifugation, filtration, filter bag, or sieve; (iii) liquids obtained by increasing the concentration of the protein from the liquids in (i) or (ii) above by removing non-protein components; and (iv) liquids obtained by mixing a dried powder prepared from any of the liquids in (i) to (iii) above with water.

[0024] In the following, "content of animal protein material" in an animal protein-containing composition refers to the proportion of the dry weight of the animal protein-containing composition (excluding the weight of added components).

[0025] The content of animal protein material in the animal protein-containing composition is not particularly limited, but examples include 0.05 to 40% by weight, preferably 0.1 to 30% by weight, or 1 to 20% by weight, preferably 5 to 15% by weight.

[0026] There are no particular restrictions on the animals from which the animal protein is derived. For example, animals include mammals, birds, reptiles, amphibians, fish, mollusks, and crustaceans. Examples of mammals include pigs, cattle, sheep, deer, horses, kangaroos, and rabbits. Examples of birds include chickens, quail, turkeys, and ducks. Examples of reptiles include crocodiles and snakes. Examples of amphibians include frogs. Examples of fish include sardines, tuna, salmon, and cod. Examples of mollusks include octopuses and squid. Examples of crustaceans include crabs and shrimp. In this invention, the animal protein may be derived from one type of animal, or a combination of protein derived from two or more types of animals may be used.

[0027] Specific examples of animal proteins include muscle protein, milk protein (whey, casein, etc.), and egg white (albumin, etc.).

[0028] The content of animal protein in the animal protein material (dry weight) is not particularly limited, but for example, it can be 0.05 to 100% by weight, preferably 0.5 to 100% by weight, more preferably 5 to 100% by weight, even more preferably 30 to 100% by weight, and even more preferably 50 to 100% by weight.

[0029] The content of animal protein in the animal protein-containing composition is not particularly limited, but examples include 0.05 to 30% by weight, preferably 0.5 to 25% by weight, or 1 to 20% by weight, more preferably 5 to 15% by weight.

[0030] Compositions containing animal protein may or may not contain other components besides animal protein. Examples of other components include components found in the source material of the animal protein (such as carbohydrates and / or lipids), and additives (such as other ingredients, seasonings, and / or food additives). Examples of food additives include one or more types of thickeners, binders, pH adjusters, buffers, colorants, and flavorings.

[0031] [A] 1-1-2. Protein deamidases The type and origin of protein deamidases are not particularly limited, as long as they are enzymes that degrade the amide group-containing side chains of proteins without cleaving peptide bonds or crosslinking proteins. Examples of protein deamide enzymes include those disclosed in Japanese Patent Publication No. 2000-50887, Japanese Patent Publication No. 2001-218590, and International Publication No. 2006 / 075772, which are derived from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides. These protein deamide enzymes may be used individually or in combination.

[0032] Examples of protein deamide enzymes include protein glutaminase and protein asparaginase, and in a broader sense, protein arginine deiminase can also be included. Among these protein deamide enzymes, protein glutaminase is preferred from the viewpoint of further enhancing the predetermined properties of the modified animal protein-containing dried composition in the presence of water.

[0033] Among these protein deamide enzymes, from the viewpoint of further enhancing the predetermined properties of the modified animal protein-containing dried composition in the presence of water, protein deamide enzymes derived from the genus Chryseobacterium are more preferable, protein glutaminases derived from the genus Chryseobacterium are even more preferable, and protein glutaminases derived from the species Chryseobacterium proteoricum are even more preferable.

[0034] Protein deamide enzymes can be prepared from the culture medium of the microorganism from which the above-mentioned protein deamide enzymes originate. Specific preparation methods include recovering the protein deamide enzyme from the culture medium or cells of the above-mentioned microorganisms. For example, when using a protein deamide enzyme-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using a protein deamide enzyme-non-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the cells can be crushed by pressurization, sonication, etc., to expose the enzyme, and then the enzyme can be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration. Commercially available protein deamidation enzymes can also be used.

[0035] The amount of protein deamidase used is not particularly limited, but as the amount used per gram of animal protein in the animal protein-containing composition, for example, it may be 0.01 U or more, or 0.05 U or more. From the viewpoint of further enhancing the predetermined properties of the modified animal protein-containing dried composition in the presence of water, it is preferably 0.1 U or more, more preferably 1 U or more, even more preferably 5 U or more, or 10 U or more, and even more preferably 15 U or more, or 18 U or more. The upper limit of the above range of the amount of protein deamidase used per gram of animal protein is not particularly limited, but for example, it may be 200 U or less, 100 U or less, 50 U or less, or 30 U or less, preferably 25 U or less, or 23 U or less. Specific ranges for the amount of protein deamidase used per gram of animal protein in the protein-containing composition include 0.01 to 200 U, 0.05 to 100 U, 0.1 to 50 U, 1 to 50 U, 5 to 30 U, 10 to 30 U, 15 to 25 U, or 18 to 23 U.

[0036] For the protein deamidation enzyme activity, one unit (1 U) is defined as the amount of enzyme that releases 1 μmol of ammonia per minute using benzyloxycarbonyl-L-glutaminilglycine (Z-Gln-Gly) as the substrate.

[0037] In the enzyme treatment step, it is not necessary to use other enzymes other than protein deamidase, or other enzymes may be used in combination as long as they do not impair the effects of the present invention. Examples of other enzymes that may or may not be used in combination include one or more of the following: protein-cutting enzymes (proteases, peptidases, etc.), protein-crosslinking enzymes (laccases, transglutaminases, etc.), lipid-degrading enzymes (lipases, etc.), starch-degrading enzymes (amylases, etc.). In preferred embodiments of the present invention, it is preferable not to use other enzymes other than protein deamidase.

[0038] [A] 1-1-3. Reaction Procedure and Enzyme Treatment Conditions The reaction procedure in the enzyme treatment step is not particularly limited, and typically a protein mixture containing an animal protein composition and a protein deamidase together with water is subjected to conditions that allow the enzymatic reaction to proceed.

[0039] The conditions for enzymatic treatment of the protein mixture (temperature, time, pH, etc.) are not particularly limited as long as the effects of the present invention are obtained.

[0040] Examples of processing temperatures include 0 to 80°C, 4 to 80°C, or 8 to 70°C, preferably 15 to 65°C, 30 to 65°C, more preferably 40 to 65°C, and even more preferably 45 to 55°C. The processing time is not particularly limited, but examples include 0.1 to 24 hours, preferably 6 to 20 hours, and even more preferably 12 to 18 hours. Examples of processing pH (at 25°C) include 4 to 9, preferably 5 to 8, more preferably 6 to 7.6, and even more preferably 6.5 to 7.4.

[0041] These processing conditions are appropriately selected according to the optimal temperature, optimal pH, and / or the desired effect of the present invention (the effect of improving the predetermined properties of an animal protein-containing dry composition in the presence of water) of the enzyme used. The optimal processing conditions can be determined through preliminary experiments.

[0042] [A] 1-2. Drying process In the drying process, water is removed from the enzyme-treated composition. Known drying methods can be used to remove water, such as freeze-drying and vacuum drying. Freeze-drying is preferred from the viewpoint of further enhancing the predetermined properties of the modified animal protein-containing dried composition in the presence of water.

[0043] [A] 1-3. Other steps The modification method of the present invention may or may not include other steps other than the enzyme treatment step and the drying step described above. Examples of such other steps include a step of preparing an animal protein-containing composition to be subjected to the enzyme treatment step, an enzyme deactivation step, a cooling step, a filtration step, etc. If other steps are included, one of these other steps may be performed alone, or two or more steps may be performed in combination.

[0044] The step of preparing the animal protein-containing composition can be performed by any method. For example, the animal protein-containing composition can be prepared based on the descriptions of (i) to (iv) in the specific examples of the animal protein-containing composition described in the above "[A] 1-1-1. Animal Protein-Containing Composition".

[0045] In the step of preparing the animal protein-containing composition, any optional food additives such as seasonings, pH adjusters, buffers, coloring agents, and flavorings may be optionally added, and / or any treatment other than enzymatic treatment (for example, fermentation treatment such as lactic acid fermentation) may or may not be performed.

[0046] The enzyme deactivation step, cooling step, and / or filtration step can be performed between the enzymatic treatment step and the drying step. The conditions for the enzyme deactivation step may be appropriately determined according to the thermal properties of the enzyme used. For example, the temperature is 80 to 100°C, preferably 90 to 100°C, more preferably 95 to 100°C, and the treatment time is, for example, 5 to 15 minutes, preferably 8 to 12 minutes.

[0047] [A] 1-4. Modified Animal Protein-Containing Dry Composition The modified animal protein-containing dry composition obtained by the modification method of the present invention has improved predetermined properties in the coexistence with water, as compared with an animal protein-containing dry composition obtained without performing the above-described predetermined enzymatic treatment step. Therefore, the modified animal protein-containing dry composition can be mixed with water and used to obtain properties selected from the group consisting of foamability, foam stability, emulsifying property, emulsion stability, aggregation inhibition property, and protein solubility after heat treatment.

[0048] Examples of products obtained by mixing the modified animal protein-containing dry composition with water include foods and drinks or food and drink materials that utilize the predetermined properties in the final form or the cooking process form. Examples of these foods and drinks or food and drink materials include processed egg products (chawanmushi, mayonnaise, meringue, etc.), bread or confectionery (pancakes, cookies, etc.), dairy products (milk, cheese, yogurt, non-whipped cream, whipped cream, ice cream, butter, etc.), and aqueous extracts of animal protein materials.

[0049] [A] 2. Modifier The modifier of the present invention comprises a protein deamidation enzyme and is intended to improve predetermined properties of a dry composition containing animal protein in the presence of water.

[0050] [A] 2-1. Applications The modifier of the present invention is used to improve a predetermined property, namely, a property selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment.

[0051] The improvement in foaming ability can be confirmed by the fact that the total volume of foam in the liquid composition, obtained by mixing a dry composition containing modified animal protein, obtained by the method described in "[A] 1. Modification Method" above using the modifier of the present invention, with water and homogenizing it, is increased compared to the case where a protein deamidation enzyme is not used (i.e., when a dry composition containing animal protein obtained under the same conditions except that a protein deamidation enzyme is not used), is increased. The improvement in foam stability can be confirmed by the fact that the degree of decrease over time of the total volume of the foamed liquid composition is suppressed compared to the above case. The improvement in emulsification ability can be confirmed by the fact that the turbidity of the emulsion composition, prepared by mixing the dry composition containing modified animal protein with an oily base and water and emulsifying it, is increased compared to the above case. The improvement in emulsion stability can be confirmed by the fact that the degree of decrease over time of the turbidity of the emulsion composition is suppressed compared to the above case. The improvement in aggregation inhibition can be confirmed by the fact that the amount of filtrate obtained by filtering a liquid composition prepared by mixing a dry composition containing modified animal protein with water through filter paper is increased compared to the above case. The improvement in protein solubility after heat treatment can be confirmed by the fact that the amount of dissolved protein obtained by spectrophotometric measurement is increased compared to the above case when a liquid composition containing a dry composition containing modified animal protein and water is subjected to the heat denaturation temperature of animal protein that has not been treated with protein deamidase enzymes.

[0052] [A] 2-2. Active Ingredients The details of the protein deamidase, which is the active ingredient in the modifier of the present invention, are as described in "[A] 1. Modification Method" above.

[0053] The modifier of the present invention may contain only a protein deamidase as an active ingredient and may not contain other components (e.g., other enzymes), or it may contain other components (e.g., other enzymes) as long as it does not impair the effects of the present invention. Among such other components, whether present or not, examples of other enzymes include one or more of the following: protein-cutting enzymes (proteases, peptidases, etc.), protein-crosslinking enzymes (laccases, transglutaminases, etc.), lipid-degrading enzymes (lipases, etc.), starch-degrading enzymes (amylases, etc.). In preferred embodiments of the present invention, the active ingredient preferably does not contain other enzymes other than a protein deamidase, and more preferably does not contain other components other than a protein deamidase.

[0054] [A] 2-3. Other Components The modifier of the present invention may be an enzyme composition containing a protein deamidation enzyme, but it may or may not contain other components other than the enzyme, such as additives and / or bases that are acceptable in the formulation of the enzyme preparation. Examples of such additives and bases, whether present or absent, include excipients, buffers, antioxidants, UV inhibitors, preservatives, pH adjusters, dispersants, emulsifiers, solubilizers, carriers, solvents (water, etc.), etc. When using additives and bases, one of these additives and bases may be used alone, or two or more may be used in combination. Furthermore, the content of these additives and bases may be appropriately set according to the type of component and / or the formulation form, etc.

[0055] [A] 2-4. Properties The properties of the modifier of the present invention are not particularly limited, but examples include powdered, finely granulated, granular dried formulations and liquid formulations.

[0056] [A] 2-5. For other specific methods of using the modifier of the present invention, please refer to the above section "[A] 1. Modification Method".

[0057] [B] 1. Stress Increase Method The stress increase method of the present invention is a method for increasing the stress of a food product containing muscle protein (specifically, a cooked product of a food product containing muscle protein), and includes a predetermined enzyme treatment step. By the stress increase method of the present invention, a modified muscle protein-containing food product with increased stress can be obtained. Note that the term "food product" includes not only the final form intended for consumption, but also forms that are intended for secondary processing.

[0058] [B] 1-1. Enzyme treatment process In the enzyme treatment process, food material containing muscle protein is treated with a protein deamidation enzyme. This yields a modified muscle protein-containing food product.

[0059] [B] 1-1-1. Food materials containing muscle protein There are no particular restrictions on food materials containing muscle protein, as long as they are ingestible by living organisms. Examples of muscle protein include myofibrillar proteins and meat matrix proteins. Examples of myofibrillar proteins include myosin, actin, actomyosin, tropomyosin, troponin, connectin, etc. Examples of meat matrix proteins include collagen, elastin, etc. Food materials containing muscle protein may contain one type of muscle protein alone or two or more types in combination.

[0060] Preferred food ingredients containing muscle protein include the muscles of mammals, birds, reptiles, amphibians, fish, mollusks, and crustaceans. Examples of mammals include pigs, cattle, sheep, deer, horses, kangaroos, and rabbits. Examples of birds include chickens, quail, turkeys, and ducks. Examples of reptiles include crocodiles and snakes. Examples of amphibians include frogs. Examples of fish include sardines, tuna, salmon, and cod. Examples of mollusks include octopuses and squid. Examples of crustaceans include crabs and shrimp.

[0061] Food materials containing muscle protein can take the following forms: non-minced (for example, unrefined meat and refined meat; refined meat here refers to processed meat made by binding together scraps of meat that are not minced with a binder, also called compressed meat; non-refined meat refers to muscle tissue that has not been processed with a binder (may be in the form of meat pieces, sliced ​​meat, chunks of meat, etc.); and minced. Non-minced materials include grilled or boiled meat (steak, grilled meat, yakiniku, roast beef, stewed meat, curry meat, etc.); grilled or boiled fish, mollusks, or crustaceans, or uncooked muscle tissue used as ingredients for these. Examples of minced ingredients include molded products of minced muscle tissue, such as processed minced meat products of livestock such as sausages, meatballs, hamburgers, meatballs, meatloaf, and patties, or uncooked meat varieties thereof; and processed fish products such as kamaboko, hanpen, chikuwa, tsumire, fish ham, fish sausage, fish meatballs, and tuna nuggets, or uncooked surimi products thereof.

[0062] If the food material containing muscle protein is not minced, it may undergo pre-treatment other than heating. Such pre-treatments include physical treatments such as cutting, removing tendons, pounding, and piercing.

[0063] In a preferred embodiment, the method for increasing the stress of a food containing muscle protein according to the present invention can suppress cooking losses due to heat cooking while increasing the stress. From this viewpoint, preferred examples of food materials containing muscle protein include uncooked food materials, specifically the uncooked muscle tissue, the uncooked meat, and the uncooked surimi. From a similar viewpoint, preferred examples of food materials containing muscle protein include unbound meat, which does not use a binding agent (binder) that serves as a means of suppressing cooking losses.

[0064] Food ingredients containing muscle protein may also contain components (such as lipids and / or carbohydrates) that were typically present in the source material from which the animal protein originated, in addition to animal protein itself.

[0065] Furthermore, food ingredients containing muscle protein may or may not contain additives (other ingredients, seasonings, and / or food additives, etc.). Examples of food additives include one or more types of thickeners, binders, pH adjusters, buffers, colorants, and flavorings.

[0066] [B] 1-1-2. Protein deamidases The type and origin of protein deamidases are not particularly limited, as long as they are enzymes that degrade the amide group-containing side chains of proteins without cleaving peptide bonds or crosslinking proteins. Examples of protein deamide enzymes include those disclosed in Japanese Patent Publication No. 2000-50887, Japanese Patent Publication No. 2001-218590, and International Publication No. 2006 / 075772, which are derived from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides. These protein deamide enzymes may be used individually or in combination.

[0067] Examples of protein deamide enzymes include protein glutaminase and protein asparaginase, and in a broader sense, protein arginine deiminase can also be included. Among these protein deamide enzymes, protein glutaminase is preferred from the viewpoint of further increasing the stress on foods containing muscle protein.

[0068] Among these protein deamide enzymes, from the viewpoint of further increasing the stress on foods containing muscle protein, a more preferable protein deamide enzyme is derived from the genus Chryseobacterium, even more preferably a protein glutaminase derived from the genus Chryseobacterium, and even more preferably a protein glutaminase derived from the species Chryseobacterium proteoricum.

[0069] Further details regarding protein deamidases are as described above in "[A] 1-1-2. Protein Deamidases".

[0070] The amount of protein deamidase used is not particularly limited, but as an amount per gram of food material containing muscle protein, for example, it may be 0.01 U or more, or 0.05 U or more. From the viewpoint of further increasing the stress of the food containing muscle protein, it is preferably 0.1 U or more, more preferably 1 U or more, even more preferably 5 U or more, or 10 U or more, and even more preferably 15 U or more, or 18 U or more. The upper limit of the above range of the amount of protein deamidase used per gram of food material containing muscle protein is not particularly limited, but for example, it may be 200 U or less, 100 U or less, 50 U or less, or 30 U or less, preferably 25 U or less, or 23 U or less. Specific ranges for the amount of protein deamidase used per gram of food material containing muscle protein include 0.01 to 200 U, 0.05 to 100 U, 0.1 to 50 U, 1 to 50 U, 5 to 30 U, 10 to 30 U, 15 to 25 U, or 18 to 23 U.

[0071] In the enzyme treatment step, it is not necessary to use other enzymes other than protein deamidase, or other enzymes may be used in combination as long as they do not impair the effects of the present invention. Examples of other enzymes that may or may not be used in combination include one or more of the following: protein-cutting enzymes (proteases, peptidases, etc.), protein-crosslinking enzymes (laccases, transglutaminases, etc.), lipid-degrading enzymes (lipases, etc.), starch-degrading enzymes (amylases, etc.). In preferred embodiments of the present invention, it is preferable not to use other enzymes other than protein deamidase.

[0072] [B] 1-1-3. Reaction Operation and Enzyme Treatment Conditions The reaction operation in the enzyme treatment step is not particularly limited. Typically, in the enzyme treatment step, the food material containing muscle protein can be brought into contact with a protein deamidase in the presence of water and subjected to conditions that allow the enzymatic reaction to proceed. More specifically, the food material containing muscle protein can be immersed in a solution containing a protein deamidase and water; the food material containing muscle protein can be injected with a solution containing a protein deamidase and water; and / or the food material containing muscle protein (containing water) can be coated with a dry enzyme preparation containing a protein deamidase and subjected to conditions that allow the enzymatic reaction to proceed.

[0073] The conditions (temperature, time, pH, etc.) for the enzymatic treatment of food materials containing muscle protein are not particularly limited as long as the effects of the present invention are obtained.

[0074] Examples of processing temperatures include 0 to 80°C, 4 to 70°C, or 8 to 60°C, preferably 15 to 55°C, 20 to 55°C, more preferably 30 to 55°C, and even more preferably 35 to 45°C. The processing time is not particularly limited, but examples include 0.1 to 24 hours, preferably 1 to 12 hours, and more preferably 3 to 6 hours. Examples of processing pH (at 25°C) include 4 to 9, preferably 5 to 8, more preferably 6 to 7.6, and even more preferably 6.5 to 7.4.

[0075] These processing conditions are appropriately selected according to the optimal temperature, optimal pH, and / or the desired effect of the present invention (increasing the stress of food containing muscle protein) of the enzyme used. The optimal processing conditions can be determined through preliminary experiments.

[0076] [B] 1-2. Other Steps The stress-increasing method of the present invention may or may not include other steps besides the enzyme treatment step described above. Examples of such other steps include a step of preparing food materials containing muscle protein, an enzyme deactivation step, a cooling step, a heating and cooking step, etc. If other steps are included, one of these other steps may be performed alone, or two or more steps may be performed in combination.

[0077] In the process of preparing food materials containing muscle protein, molding, pre-processing, seasoning, and / or cooking should be carried out based on the specific examples of food materials described in "[B] 1-1-1. Food Materials Containing Muscle Protein" above. Preferably, cooking is not performed in the process of preparing food materials containing muscle protein.

[0078] The enzyme deactivation, cooling, and heating steps can be performed after the enzyme treatment step. The conditions for the enzyme deactivation step can be appropriately determined according to the thermal characteristics of the enzyme used, but examples include 80 to 100°C, preferably 90 to 100°C, more preferably 95 to 100°C, for 5 to 15 minutes, preferably 8 to 12 minutes.

[0079] In the cooking process, the necessary heating method can be used depending on the specific form of the food material containing muscle protein. For example, roasting (roasting, toasting, baking, grilling, broiling), boiling (stewing, bain-marie), steaming, frying, etc., can be used.

[0080] [B] 1-3. Modified Muscle Protein-Containing Food The modified muscle protein-containing food obtained by the stress-increasing method of the present invention has increased stress compared to the muscle protein-containing food obtained without the predetermined enzyme treatment step described above. Normally, increased stress leads to increased cooking losses. However, in a preferred embodiment of the present invention, the modified muscle protein-containing food has increased stress, yet the cooking losses (specifically, cooking losses due to heating) are suppressed compared to the muscle protein-containing food obtained without the predetermined enzyme treatment step described above. More specifically, the decrease in weight after heating (%) relative to the weight before heating (100%), i.e., cooking loss (%), is suppressed to, for example, 0.99 times or less, specifically, for example, 0.5 to 0.99 times, 0.7 to 0.98 times, 0.8 to 0.97 times, 0.9 to 0.96 times, or 0.93 to 0.95 times, compared to the cooking loss (%) in the muscle protein-containing food obtained without the predetermined enzyme treatment step described above. For this reason, preferred examples of modified muscle protein-containing foods include those that have undergone the above-mentioned heat cooking process in their preparation, or those that have not undergone the above-mentioned heat cooking process in their preparation, but are used for the purpose of being subjected to the said heat cooking process.

[0081] [B] 2. Stress increasing agent The stress increasing agent of the present invention contains a protein deamidation enzyme and is intended to increase the stress of food containing muscle protein (specifically, cooked food containing muscle protein).

[0082] [B] 2-1. Applications The stress increasing agent of the present invention is used to increase the stress of food containing muscle protein after cooking. The increase in stress can be confirmed by the fact that the stress of the modified muscle protein-containing food (after cooking) obtained by the method described in "[B] 1. Method for Increasing Stress" above using the stress increasing agent of the present invention is increased compared to the case where a protein deamidation enzyme is not used (i.e., the muscle protein-containing food (after cooking) obtained under the same conditions except that a protein deamidation enzyme is not used).

[0083] [B] 2-2. Active Ingredients The details of the protein deamidase, which is the active ingredient in the stress increasing agent of the present invention, are as described in "[B] 1. Method for Increasing Stress" above.

[0084] The stress increasing agent of the present invention contains only a protein deamidase as an active ingredient and does not have to contain other components (e.g., other enzymes), or it may contain other components (e.g., other enzymes) as long as it does not impair the effects of the present invention. Among such other components, whether or not they are included, examples of other enzymes include one or more of the following: protein cleaving enzymes (proteases, peptidases, etc.), protein cross-linking enzymes (laccases, transglutaminases, etc.), lipid-degrading enzymes (lipases, etc.), starch-degrading enzymes (amylases, etc.). In preferred embodiments of the present invention, the active ingredient preferably does not contain other enzymes other than the protein deamidase, and more preferably does not contain other components other than the protein deamidase.

[0085] [B] 2-3. Other Components The stress increasing agent of the present invention may be an enzyme composition containing a protein deamidation enzyme, but it may or may not contain other components other than the enzyme, such as additives and / or bases that are acceptable in the formulation of the enzyme preparation. Examples of such additives and bases, whether present or absent, include excipients, buffers, antioxidants, UV inhibitors, preservatives, pH adjusters, dispersants, emulsifiers, solubilizers, carriers, solvents (water, etc.), etc. When using additives and bases, one of these additives and bases may be used alone, or two or more may be used in combination. Furthermore, the content of these additives and bases may be appropriately set according to the type of component and / or the formulation form, etc.

[0086] [B] 2-4. Properties The properties of the stress increasing agent of the present invention are not particularly limited, but examples include powdered, finely granulated, granular dried formulations and liquid formulations.

[0087] [B] 2-5. For other specific methods of using the stress increasing agent of the present invention, please refer to the above section "[B] 1. Method of increasing stress".

[0088] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0089] [Animal Protein-Containing Compositions] The animal protein materials (in a dried state) or food materials containing muscle protein shown in Table 1 were used. Pork powder, beef powder, and chicken powder are dried powders of pork, beef, and chicken, respectively.

[0090] [Protein Deamide Enzyme] As the protein deamide enzyme, we used protein glutaminase derived from Chryseobacterium proteolyticum (manufactured by Amano Enzyme Co., Ltd.). Hereafter, this protein deamide enzyme will also be referred to as "PG".

[0091] The protein deamidase activity was measured by the following method: 0.1 mL of 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 left to stand at 37°C for 10 minutes. The reaction was then stopped by adding 1 mL of 0.4 M TCA solution. 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 then 0.1 mL of sample solution containing protein deamidase was added. The mixture was left to stand at 37°C for 10 minutes.

[0092] The amount of ammonia produced in the reaction solution was measured using the Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) for the solution obtained as described above. 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).

[0093] The activity of the protein deamidase was calculated using the following formula, with one unit (1 U) defined as the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the reaction volume is 2.1, the enzyme solution volume is 0.1, and Df is the dilution ratio of the enzyme solution. Also, 17.03 is the molecular weight of ammonia.

[0094]

[0095] [[A] Modification Method] [[A]-1. Preparation of Modified Animal Protein-Containing Dry Composition] Protein glutaminase was added to an aqueous suspension containing 10% by weight of animal protein material to a concentration of 20 U / g-protein, and the mixture was reacted overnight at 50°C. After the reaction, the mixture was boiled at 100°C for 10 minutes. It was then cooled to room temperature and freeze-dried. This yielded a modified animal protein-containing dry composition (with PG treatment). For comparison, an animal protein-containing dry composition (without PG treatment) was obtained by the same procedure except that protein glutaminase was not added.

[0096] [A]-2. Evaluation of various properties] A modified animal protein-containing dry composition (with PG treatment) or a comparative animal protein-containing dry composition (without PG treatment) was mixed with distilled water to a concentration of 5% by weight (for evaluation of foaming ability, foam stability, emulsification ability, emulsification stability, and aggregation inhibition) or 0.45 mg / mL (for evaluation of protein solubility after heat treatment), and the following properties were confirmed for the resulting liquid composition (hereinafter simply referred to as "liquid composition").

[0097] [Foaming Properties] A 50 mL liquid composition was homogenized at 18,000 rpm for 30 minutes and immediately transferred to a 100 mL graduated cylinder. The total volume VF0 of the liquid composition, including the foam, was measured, and the "foaming properties" were calculated using the following formula. The results are shown in Table 2.

[0098]

[0099] As shown in Table 2, the dried composition containing animal protein (treated with PG) showed an improvement in foaming ability in the presence of water. In particular, this improvement was remarkably significant for milk casein.

[0100] [Foam Stability] Similar to the [Foaming Properties] test described above, 50 mL of the liquid composition was homogenized at 18,000 rpm for 30 minutes, and immediately transferred to a 100 mL graduated cylinder. The total volume VF0 of the liquid composition, including the foam, was measured. After standing for 30 minutes, the total volume VF30 of the liquid composition was measured, and the "foam stability" was calculated using the following formula. The results are shown in Table 3.

[0101]

[0102] As shown in Table 3, the dried composition containing animal protein (with PG treatment) showed an improvement in foam stability in the presence of water.

[0103] [Emulsifying properties] An emulsion composition was prepared by mixing 30 mL of the liquid composition with 10 mL of canola oil and homogenizing it at 10,000 rpm for 2 minutes. 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 500 nm absorbance, and the "emulsifying properties" were calculated using the following formula. The results are shown in Table 4.

[0104]

[0105] As shown in Table 4, the dried composition containing animal protein (treated with PG) showed an improvement in emulsification properties in the presence of water. In particular, this improvement was remarkably significant for porcine protein.

[0106] [Emulsification Stability] Similar to the [Emulsification] test described above, an emulsion composition was prepared by mixing 30 mL of the liquid composition with 10 mL of canola oil and homogenizing it at 10,000 rpm for 2 minutes. 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 500 nm absorbance. Furthermore, 50 μL of the emulsion composition, after being left for 10 minutes, was taken and added to 5 mL of 0.1 wt% SDS solution, and the turbidity (A10) was measured at 500 nm absorbance. The "emulsification stability" was calculated using the following formula. The results are shown in Table 5.

[0107]

[0108] As shown in Table 5, the dried composition containing animal protein (treated with PG) showed an improvement in emulsification stability in the presence of water. In particular, this improvement was remarkably significant for casein.

[0109] [Agglutination Inhibition] A 20 mL liquid composition was filtered through filter paper (Qualitative filter paper #1, manufactured by Whatman). After 30 minutes, the volume (mL) of the filtered filtrate (aqueous extract of animal protein material) was measured and used as an indicator of aggregation inhibition. The results are shown in Table 6.

[0110]

[0111] As shown in Table 6, the dried composition containing animal protein (with PG treatment) showed an improved effect in inhibiting aggregation in the presence of water.

[0112] [Protein solubility after heat treatment] After preparing the liquid composition, it was stirred at room temperature for 5 minutes to prepare the test solution (without heat treatment). Furthermore, the test solution (without heat treatment) was boiled for 10 minutes to prepare the test solution (with heat treatment).

[0113] Figure 1 shows photographs of the appearance of the test solution (without heat treatment) and the test solution (with heat treatment).

[0114] The amount of soluble protein in the test solution (without heat treatment) and the test solution (with heat treatment) was quantified using the following BCA method. <Protein content test method - BCA method (bicinchonic acid method)> Pierce BCA Protein Assay Kits (manufactured by Pierce) were used. 50 mL of BCA Protein Assay Reagent (Reagent A Pierce) and 1 mL of BCA Protein Assay Reagent (Reagent B Pierce) were placed in a 50 mL stoppered Erlenmeyer flask and mixed to prepare the BCA reagent. Four milliliters of BCA reagent were measured into a test tube (15 x 150 mm), left at 37°C for exactly 10 minutes, and then 0.2 mL of either the unheated test solution or the heat-treated test solution was added and immediately mixed. This solution was left at 37°C for exactly 30 minutes and then cooled with running water. The absorbance (AT) at a wavelength of 562 nm was measured using water as a control. Separately, four milliliters of BCA reagent were left at 37°C for exactly 10 minutes, and then 0.2 mL of water was added and mixed. The absorbance (AB) was measured using the same procedure. A calibration curve showing the relationship between albumin concentration and absorbance (562 nm) was also created. The protein concentration (mg / g or mg / mL) in the sample solution was calculated from the difference in absorbance (AT - AB) between the calibration curve created with albumin and the sample solution.

[0115] As shown in Figure 1(A), the casein-containing dry composition that was not treated with PG (non-casein) did not solubilize when mixed with water, and the actual protein concentration measured was below the detection limit. In contrast, the casein-containing dry composition that was treated with PG (PG-casein) solubilized when mixed with water, and the actual protein concentration measured was 0.41 mg / mL.

[0116] Furthermore, as shown in Figure 1(B), the casein-containing dry composition that was not treated with PG (Non-casein) remained unsoluble even after boiling after mixing with water, and the actual protein concentration measured was below the detection limit. In contrast, the casein-containing dry composition that was treated with PG (PG-casein) remained dissolved even after boiling after mixing with water, and the actual protein concentration measured was 0.40 mg / mL, indicating that it was hardly affected by boiling.

[0117] [B. Method for increasing stress] [B.-1. Preparation of modified muscle protein-containing food] Beef tenderloin was moderately pierced with a fork, wrapped in plastic wrap, and lightly pounded with a rolling pin to make the thickness uniform (10 mm). A small amount of salt was rubbed onto both sides of the beef. 20 g of beef tenderloin and 5 mL of distilled water were placed in a resealable plastic bag, and protein glutaminase was mixed in at a ratio of 20 U per 1 g of beef. The mixture was incubated at 40°C for 4 hours. This yielded a modified muscle protein-containing food (with PG treatment). For comparison, the same process was carried out except that protein glutaminase was not added to obtain a muscle protein-containing food (without PG treatment).

[0118] [B]-2. Measurement of stress and cooking loss after heating. Modified muscle protein-containing food (with PG treatment) and a comparative muscle protein-containing food (without PG treatment) were each grilled over medium heat for 2 minutes on each side. After cooling to room temperature, a rheometer (manufactured by Sun Science Co., Ltd.) was used to measure the stress required for the plunger to be pushed in to 50% of the sample thickness using a 20 mm diameter plunger. The weight before and after grilling was also measured, and the decrease in weight after grilling (weight before grilling - weight after grilling) (%), with the weight before grilling set as 100%, was obtained as the cooking loss (%). The results are shown in Table 7.

[0119]

[0120] As shown in Table 7, the modified muscle protein-containing food (with PG treatment) showed an increase in stress after cooking, reaching 1.6 times that of the untreated food. Furthermore, although cooking losses would normally increase with increased stress, cooking losses were unexpectedly suppressed.

Claims

1. A method for modifying a dried composition containing animal protein in the presence of water, comprising: an enzyme treatment step of treating the animal protein-containing composition with a protein deamide enzyme to obtain an enzyme-treated composition; and a drying step of removing water from the enzyme-treated composition, wherein the properties are selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment.

2. The modification method according to claim 1, wherein the animal protein is a protein from an animal selected from the group consisting of mammals, birds, reptiles, amphibians, fish, mollusks, and crustaceans.

3. A modifier comprising a protein deamidase for improving the properties of a dry composition containing animal protein in the presence of water, wherein the properties are selected from the group consisting of foaming ability, foam stability, emulsification ability, emulsification stability, aggregation inhibition, and protein solubility after heat treatment.

4. A modified animal protein-containing dry composition obtained by a method comprising: an enzyme treatment step of treating an animal protein-containing composition with a protein deamide enzyme to obtain an enzyme-treated composition; and a drying step of removing water from the enzyme-treated composition, wherein the dry composition, when mixed with water, is intended to obtain properties selected from the group consisting of foaming ability, foam stability, emulsification, emulsification stability, aggregation inhibition, and protein solubility after heat treatment.

5. A method for increasing the stress of a food containing muscle protein, comprising an enzymatic treatment step of treating the food material containing muscle protein with a protein deamidation enzyme.

6. The method according to claim 5, wherein the muscle protein is beef muscle protein.

7. The method according to claim 5 or 6, wherein the food material containing muscle protein is not cooked by heat.

8. The method according to claim 5 or 6, wherein the food material containing muscle protein is unrefined meat.

9. A stress enhancer for muscle protein-containing foods, containing a protein deamidation enzyme.

10. A modified muscle protein-containing food obtained by a method including an enzymatic treatment step of treating a food material containing muscle protein with a protein deamidation enzyme.

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