Enzyme preparation for cheese analog

Abstract

The present invention provides: an enzyme preparation for a cheese analog, the preparation containing (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase, and improving both the meltability and extensibility when the cheese analog is heated and the shredding suitability when cold; and a method for producing a cheese analog in which these properties have been improved.

Description

Enzyme preparation for cheese analogues

[0001] The present invention relates to an enzyme preparation for cheese analogs that improves both the properties of cheese analogs when heated and their shredding suitability when cooled, a method for producing cheese analogs that have both of these properties, and a method for increasing the hardness of cheese analogs that have the properties when heated.

[0002] In recent years, growing awareness of animal welfare and health has led to an expansion of the market for plant-based proteins, i.e., dairy-free cheeses and cheese analogs. A known method for producing dairy-free cheese substitutes involves using dairy-free plant-based milk and enzymes such as proteases, lipases, and transglutaminases, as well as microorganisms (Patent Document 1). Meanwhile, cheese analogs, also known as analog cheeses or imitation cheeses, are foods that contain added starch, and in which part or all of the fat and protein of cheese is replaced with plant-derived ingredients, without fermentation or aging processes, and are processed to have an appearance and texture similar to that of cheese. However, in addition to appearance and texture at room temperature, good meltability and / or extensibility upon heating are also desired from the perspectives of cooking suitability and texture. To address this issue, it has been previously known to treat cheese analogs with proteases and α-glucosidases to impart meltability and / or extensibility upon heating similar to that of cheese (Patent Document 2). It is also known to allow transglutaminase to act on cheese analogues to impart a smooth texture (Patent Document 3).

[0003] Demand is also increasing for cheese analogs such as shredded cheese and sliced ​​cheese, which are widely used for both home and commercial use, such as in pizzas, salads, and as toppings or ingredients in snack products.

[0004] Special table 2016-502868 publication WO2023 / 033188 publication WO2023 / 033187 publication

[0005] The present invention addresses the problem that, when a cheese analogue with improved meltability and / or spreadability upon heating is produced by allowing a carbohydrate-modifying enzyme such as α-glucosidase (also abbreviated as AG) and a protease to act on a cheese analogue containing starch and protein, the hardness of the cheese analogue decreases upon cooling (during production), resulting in a decrease in so-called shredding suitability during the shredding step and a decrease in manufacturability. Therefore, the present invention aims to provide a technology that improves both the properties upon heating and the shredding suitability upon cooling.

[0006] The present inventors have conducted extensive research to solve the above problems and have found that by treating a cheese analog with a protease, a carbohydrate-modifying enzyme such as AG or glucoamylase (also abbreviated as GA), and further with transglutaminase (also abbreviated as TG), it is possible to produce a cheese analog that maintains its properties when heated while improving its hardness when cooled, thereby improving its shredding suitability. Based on this finding, the present inventors have conducted further extensive research and have completed the present invention.

[0007] That is, the present invention provides the following: [1] An enzyme preparation for a cheese analogue, comprising (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase. [2] The preparation according to [1], wherein (B) is α-glucosidase, glucoamylase, or a mixture thereof. [2-1] The preparation according to [1], wherein (B) is glucoamylase. [3] The preparation according to [1], [2], or [2-1], wherein the cheese analogue comprises a protein, a starch, and an oil. [4] The preparation according to any of [1] to [3], wherein the cheese analogue is a shredded cheese analogue. [5] The preparation according to [4], wherein the shredded cheese analogue is a shredded cheese analogue. [6] The preparation according to any of [1] to [5], wherein 1 g of the enzyme preparation contains 100 to 1,000,000 U of (A), 0.1 to 100,000 U of (B), and 0.1 to 200 U of (C). [6-1] The preparation according to [6], which contains 0.1 to 100 U of (B) α-glucosidase per 1 g of the enzyme preparation. [6-2] The preparation according to [6], which contains 0.1 to 100,000 U of (B) glucoamylase per 1 g of the enzyme preparation. [7] The preparation according to any one of [6] to [6-2], which is used so as to be added in an amount of 0.1 to 50% by weight based on the total weight of the cheese analog. [8] (1) A method for producing a cheese analog, which comprises a step of allowing a mixture containing a protein, starch, and an oil to react with (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase. [9] The production method according to [8], wherein (B) is α-glucosidase, glucoamylase, or a mixture thereof. [9-1] The production method according to [8], wherein (B) is glucoamylase.

[10] The production method according to [8], [9], or [9-1], wherein the protein is a plant-derived protein.

[11] The method of any one of [8] to

[10] , further comprising (2) a step of heating the mixture obtained in step (1), (3) a step of cooling the heated mixture, and (4) a step of cutting the cooled mixture.

[12] The method of

[11] , wherein the cheese analog is a shredded cheese analog.

[13] The method of

[12] , wherein the shredded cheese analog is a shredded cheese analog.

[14] The manufacturing method according to any one of [8] to

[13] , wherein step (1) involves the action of 0.001 to 100,000 U of (A), 0.0001 to 10,000 U of (B), and 0.0001 to 1,000 U of (C) per 1 g of the mixture. [14-1] The manufacturing method according to any one of [8] to

[13] , wherein step (1) involves the action of 0.001 to 100,000 U of (A), 0.0001 to 100,000 U of (B), and 0.0001 to 1,000 U of (C) per 1 g of the mixture. [14-2] The manufacturing method according to [14-1], wherein step (1) involves the action of 0.0001 to 10,000 U of (B) α-glucosidase per 1 g of the mixture. [14-3] The method according to [14-1], wherein the step (1) involves the application of 0.001 to 100,000 U of (B) glucoamylase per 1 g of the mixture.

[15] A method for increasing the hardness of a cheese analog, comprising the step of applying (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase to a mixture containing protein, starch, and fat.

[0008] According to the present invention, the cheese exhibits appropriate melting properties and extensibility when heated, but maintains appropriate hardness when shredded, allowing shredded or sliced ​​cheese analogs to be produced without loss.

[0009] Figure 1 shows a flowchart for producing cheese analogs. Figure 2 shows a method for calculating the meltability and shredability of cheese analogs. Figure 3 shows photographs of the meltability and shredability of cheese analogs when heated in Test Example 1. Figure 4 shows photographs of the meltability and shredability of cheese analogs when heated in Test Example 2. Figure 5 shows photographs of the meltability and shredability of cheese analogs when heated in Test Example 3. Figure 6 shows photographs of the meltability and shredability of cheese analogs when heated in Test Example 4.

[0010] 1. Enzyme Preparation The present invention relates to an enzyme preparation for cheese analogues (also referred to as the preparation of the present invention) which comprises (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase.

[0011] (A) Protease The protease used in the present invention is an enzyme that catalyzes the hydrolysis of peptide bonds in proteins. Any protease with substrate specificity and reaction characteristics that has the activity and can degrade proteins can be used in the present invention. Furthermore, the protease's origin is not particularly limited, and it can be derived from any source, including plants (e.g., papaya), mammals, fish, and microorganisms (e.g., Aspergillus, Bacillus, and Rhizopus), and recombinant enzymes may also be used. In the present invention, the activity unit of an endoprotease is defined as the amount of enzyme that, using casein as a substrate, causes an increase in the Folin test solution color substance equivalent to 1 μg of tyrosine per minute. In the present invention, the activity unit of an exoprotease is defined as the activity of producing 1 μmol of p-nitroaniline per minute using L-leucyl-p-nitroanilide as a substrate (1 unit (1 U)). In the present invention, proteases include endo / exoproteases, endoproteases, exoproteases, and combinations thereof.

[0012] In the formulation of the present invention, the protease is preferably selected from the group consisting of (1) an endo- / exo-protease, (2) a combination of an endo- and exo-protease, (3) an exo-protease, and (4) an endo-protease; more preferably selected from the group consisting of (1) an endo- / exo-protease, (2) a combination of an endo- and exo-protease, and (4) an endo-protease; and even more preferably selected from the group consisting of (1) an endo- / exo-protease, and (2) a combination of an endo- and exo-protease.

[0013] The endo- / exo-protease used in the present invention is an enzyme that hydrolyzes peptide bonds within proteins and peptide bonds at the terminals of proteins to produce several peptides or amino acids. The endo- / exo-protease used in the present invention may be a commercially available product, such as Proteax (manufactured by Amano Enzyme Inc.; derived from Aspergillus oryzae), Peptidase R (manufactured by Amano Enzyme Inc.; derived from Rhizopus oryzae), Denateam AP (manufactured by Nagase ChemteX Corporation; derived from Aspergillus oryzae), or food-grade purified papain (manufactured by Nagase ChemteX Corporation; derived from papaya latex).

[0014] The endoprotease used in the present invention is an enzyme that hydrolyzes peptide bonds within proteins to produce several peptides. The endoprotease used in the present invention may be a commercially available product, and examples thereof include Protin SD-NY10 (manufactured by Amano Enzyme Inc.; derived from Bacillus amyloliquefaciens), Protin SD-AY10 (manufactured by Amano Enzyme Inc.; derived from Bacillus licheniformis), Denapsin 2P (manufactured by Nagase ChemteX Corporation; derived from Aspergillus niger), and Bioprase SP-20FG (manufactured by Nagase ChemteX Corporation; derived from Bacillus licheniformis).

[0015] The exoprotease used in the present invention is an enzyme that hydrolyzes peptide bonds at the amino or carboxyl terminal of a protein to release amino acids. The exoprotease used in the present invention may be a commercially available product. Examples of the exoprotease used in the present invention include aminopeptidase (purified product). For example, Denateam LEP 10P (manufactured by Nagase ChemteX Corporation) is included.

[0016] (B) Carbohydrate-Modifying Enzymes The carbohydrate-modifying enzymes used in the present invention are classified into enzymes with carbohydrate-degrading activity, enzymes with glycosyltransferase activity, and enzymes that oxidize sugars. However, the carbohydrate-modifying enzymes used in the present invention are not particularly limited as long as they do not inhibit the effects of the present invention. Among these, enzymes with carbohydrate-degrading activity (also called carbohydrate-degrading enzymes), which hydrolyze glycosidic bonds in carbohydrates, are preferred. Examples of carbohydrate-modifying enzymes used in the present invention include carbohydrate-degrading enzymes such as α-amylase, β-amylase, invertase, maltotriohydrolase, pullulanase, amyloglucosidase, α-glucosidase, β-glucosidase, isoamylase, glucoamylase, pectinase, cellulase, and hemicellulase. Among these, α-glucosidase (EC 3.2.1.20) and glucoamylase, which are exo-enzymes that hydrolyze α-1,4 glycosidic bonds in glucose units to produce α-glucose, are preferred. The origin of the carbohydrate-modifying enzyme is not particularly limited and may be any of plants, animals, and microorganisms. Specific examples include those derived from filamentous fungi, preferably from the genus Aspergillus, and more preferably from Aspergillus niger. Recombinant enzymes produced by genetic engineering techniques may also be used. These carbohydrate-modifying enzymes may be used alone or in combination, and may be produced by conventional methods or commercially available products. One unit (U) of α-glucosidase is defined as the amount of enzyme that produces 1 μg of glucose using α-methyl-D-glucoside as a substrate at pH 5.0 and 40°C in 60 minutes (reference: Japan Food Additives Association, 4th Edition, Voluntary Standards for Existing Food Additives, "Transglucosidase Activity Measurement Method"). One unit (U) of glucoamylase is also defined as the activity that produces a reducing power equivalent to 10 mg of glucose from soluble starch at pH 5.0 and 40°C in 30 minutes.

[0017] (C) Transglutaminase Transglutaminase (protein-glutamine γ-glutamyltransferase) is a transferase that catalyzes the reaction of condensing the amino group of a glutamine residue in a protein with a primary amine, transferring a substituent on the amine to the glutamine residue, and generating ammonia. Typically, the amino group of a lysine residue in a protein is used as the primary amine, and transglutaminase acts as a cross-linking enzyme.

[0018] As the transglutaminase, calcium-independent transglutaminase obtained from a microorganism is preferably used. Examples of calcium-independent transglutaminase derived from a microorganism include transglutaminase produced by actinomycetes belonging to the genus Streptomyces, which can be obtained according to the method described in Japanese Patent No. 2572716, but commercially available products such as "Activa TG-K" and "Activa TG-S" available from Ajinomoto Co., Inc. and others can also be used. The enzymatic activity of transglutaminase can be measured and calculated, for example, by the hydroxamate method. That is, a reaction is carried out using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates, and an iron complex of the hydroxamic acid produced in the reaction is formed in the presence of trichloroacetic acid. The absorbance at 525 nm is then measured, and the amount of hydroxamic acid produced is determined from a calibration curve, whereby the enzymatic activity can be calculated. In this specification, the amount of enzyme that produces 1 μmol of hydroxamic acid in 1 minute at 37° C. and pH 6.0 is defined as 1 U (see Japanese Patent Application Laid-Open No. 64-027471).

[0019] The cheese analogue of the present invention is a cheese substitute food that resembles cheese and is produced by reacting (A) to (C) with a mixture containing protein, carbohydrates such as starch, and fats and oils. Cheese analogues include those that use milk-derived ingredients such as milk protein and cheese for flavor, and plant-based (plant-derived) cheese analogues that do not use animal protein. The cheese analogue of the present invention is preferably a plant-based cheese analogue.

[0020] The "protein" used in the present invention refers to a protein typically used in food applications, and includes non-animal proteins such as plant-derived proteins, microbial-derived proteins, and fungal-derived proteins. Examples of proteins used in the present invention include plant-derived proteins such as almond protein, soybean protein, pea protein, chickpea protein, broad bean protein, lentil protein, oat protein, chia seed protein, rapeseed protein, and floating weed protein; microbial-derived proteins; and fungal-derived proteins. Of these, plant-derived proteins are preferred, with almond protein, soybean protein, pea protein, broad bean protein, and lentil protein being preferred, and pea protein, broad bean protein, and lentil protein being more preferred. One type of protein may be used, or two or more types may be used in combination.

[0021] In the cheese analog of the present invention, the protein content is usually 0.1% by weight or more, preferably 0.2% by weight or more, more preferably 0.5% by weight or more, even more preferably 1% by weight or more, even more preferably 2% by weight or more, 3% by weight or more, 4% by weight or more, or 5% by weight or more, particularly preferably 8% by weight or more or 12% by weight or more, relative to the total weight of the cheese analog, and the upper limit is usually 50% by weight or less, preferably 40% by weight or less, more preferably 30% by weight or less, even more preferably 20% by weight or less, and particularly preferably 16% by weight or less. Specifically, in the cheese analog of the present invention, the protein content is usually 0.1 to 50% by weight, preferably 0.2 to 40% by weight, more preferably 0.5 to 30% by weight, even more preferably 1 to 20% by weight, even more preferably 2 to 20% by weight, 3 to 20% by weight, 4 to 20% by weight, or 5 to 20% by weight, particularly preferably 8 to 16% by weight or 12 to 16% by weight, relative to the total weight of the cheese analog. In the cheese analogue of the present invention, if the protein content is less than 0.1% by weight relative to the cheese analogue, the cheese analogue will tend to be lacking in nutritional value and will be more susceptible to oil separation during production, while if the protein content exceeds 50% by weight, the cheese analogue will tend to have a powdery texture and be less tasty and will be more susceptible to gelling during production.

[0022] The term "starch" as used herein refers to raw starch or modified starch derived from plants that is commonly used in food applications. Examples of starch in the present invention include rice starch, sago starch, tapioca starch, waxy corn starch (waxy cornstarch), regular corn starch, potato starch, wheat starch, dry-heat-treated starches derived from these plants, and chemically-treated starches derived from these plants, such as hydroxypropylated phosphate cross-linked starch, acetylated adipate cross-linked starch, acetylated phosphate cross-linked starch, acetylated oxidized starch, sodium octenyl succinate starch, acetate starch, oxidized starch, hydroxypropyl starch, phosphate monoesterified phosphate cross-linked starch, phosphorylated starch, and phosphate cross-linked starch, with waxy corn starch (waxy cornstarch) and tapioca starch being preferred. The starch may be used alone or in combination of two or more kinds.

[0023] In the cheese analog of the present invention, the starch content is typically 5% by weight or more, preferably 7.5% by weight or more, more preferably 10% by weight or more, even more preferably 12.5% ​​by weight or more, and particularly preferably 18% by weight or more, based on the total weight of the cheese analog, with the upper limit typically being 70% by weight or less, preferably 60% by weight or less, more preferably 50% by weight or less, even more preferably 40% by weight or less, and particularly preferably 22% by weight or less. Specifically, in the cheese analog of the present invention, the starch content is typically 5 to 70% by weight, preferably 7.5 to 60% by weight, more preferably 10 to 50% by weight, even more preferably 12.5 to 40% by weight, and particularly preferably 18 to 22% by weight, based on the total weight of the cheese analog. By ensuring that the starch content in the cheese analog of the present invention falls within the above range, the effect of imparting a smooth texture and shape retention similar to that of dairy cheese can be obtained. In the cheese analogue of the present invention, if the starch content is less than 5% by weight, based on the total weight of the cheese analogue, the cheese analogue will tend to lack shape retention, be excessively soft, and be unpalatable, while if it exceeds 70% by weight, the cheese analogue will tend to have a powdery texture and be unpalatable, and the viscosity of the mixture during production will tend to increase excessively, making mixing difficult.

[0024] The "oils and fats" used in the present invention refer to oils and fats that are commonly used in food applications. Examples of the oils and fats used in the present invention include vegetable oils and fats such as coconut oil, palm oil, rapeseed oil, soybean oil, corn oil, safflower oil, and cacao oil; and animal oils and fats such as beef tallow, lard, and chicken fat. Vegetable oils and fats are preferred, and coconut oil is more preferred. The oils and fats may be used alone or in combination of two or more.

[0025] In the cheese analog of the present invention, the fat / oil content is usually 2.5% by weight or more, preferably 5% by weight or more, more preferably 7.5% by weight or more, even more preferably 10% by weight or more, and particularly preferably 18% by weight or more, relative to the total weight of the cheese analog, and the upper limit is usually 70% by weight or less, preferably 60% by weight or less, more preferably 50% by weight or less, even more preferably 40% by weight or less, and particularly preferably 22% by weight or less. More specifically, the fat / oil content of the cheese analog of the present invention is usually 2.5 to 70% by weight, preferably 5 to 60% by weight, more preferably 7.5 to 50% by weight, even more preferably 10 to 40% by weight, and particularly preferably 18 to 22% by weight, relative to the total weight of the cheese analog. In the cheese analogue of the present invention, if the fat content is less than 2.5% by weight based on the total weight of the cheese analogue, the cheese analogue will tend to have a powdery texture and be unpalatable, and will tend to lack meltability after heating. If the fat content exceeds 70% by weight, the cheese analogue will tend to lack shape retention, be excessively soft, and be unpalatable, and will tend to experience oil separation during production.

[0026] In the cheese analogue of the present invention, the weight ratio of starch to protein (starch:protein) is usually 1:0.001-100, preferably 1:0.005-80, more preferably 1:0.01-50, and even more preferably 1:0.025-40.

[0027] In the cheese analogue of the present invention, the weight ratio of starch, protein, and fat (starch:protein:fat) is usually 1:0.001-100:0.001-100, preferably 1:0.005-80:0.005-80, more preferably 1:0.01-50:0.01-50, and even more preferably 1:0.025-40:0.025-40.

[0028] The cheese analog of the present invention may contain additives commonly used in the food industry in addition to the above-mentioned components. Examples of additives include flavorings (e.g., cheddar flavoring (powder, liquid), parmesan flavoring (powder, liquid), camembert flavoring (powder, liquid), cream cheese flavoring (powder, liquid)), seasonings (e.g., salt, yeast extract, lactic acid), coloring agents, excipients (dextrin, lactose), various amino acids, thickening polysaccharides (e.g., gum arabic, xanthan gum, tamarind seed gum, guar gum, locust bean gum, carrageenan, agar), proteases, carbohydrate-modifying enzymes, and enzymes other than TG. The amount of additive used is, for example, 1 to 30% by weight based on the cheese analog.

[0029] The cheese analog of the present invention contains the above ingredients, and is treated with the above enzymes to produce a block of cheese analog through the process described below. However, sliced ​​or finely shredded cheese is preferred. Such cheese is also called cut cheese, sheared cheese, shredded cheese, or cut cheese. The enzyme preparation of the present invention is preferably applied to shredded cheese such as sliced ​​cheese or shredded cheese.

[0030] The enzyme preparation for cheese analogs of the present invention improves the quality, such as texture and appearance, of cheese analogs, or enhances their manufacturability. Texture refers to improved meltability and / or spreadability upon heating, resulting in a pleasant sensation, such as a firm texture or a pleasant feel on the tongue when placed in the mouth. Appearance refers to an appetizing shape suitable for cooking. Manufacturability refers to whether a cheese analog is suitable for production with the desired quality, and when cutting, it refers to the absence of sticking to a shredding machine and the suppression of adhesion between shredded cheese pieces. In the present invention, the "heating" in "meltability and / or spreadability upon heating" refers to heating to melt and / or spread the cheese analog during cooking or food processing using the cheese analog as an ingredient. The heating temperature is, for example, 70 to 200°C, and the heating time is, for example, 1 to 20 minutes. "During heating" refers to the period immediately after heating is completed (e.g., within 20 minutes). In the present invention, "meltability" refers to the property of a cheese analog to liquefy, melt, and spread. In the present invention, "extensibility" refers to the property of a cheese analogue to stretch like strings. In the present invention, "improved meltability upon heating" refers to the fact that the meltability upon heating of a cheese analogue produced with the addition of an enzyme of the present invention is improved compared to the meltability upon heating of a cheese analogue produced without the addition of an enzyme. In the present invention, "improved extensibility upon heating" refers to the fact that the extensibility upon heating of a cheese analogue produced with the addition of an enzyme of the present invention is improved compared to the extensibility upon heating of a cheese analogue produced without the addition of an enzyme. In this specification, the effect of improving "meltability and / or extensibility upon heating" can be evaluated, for example, by the method described in the Examples below or a method similar thereto.

[0031] The enzyme preparation of the present invention is not particularly limited in its form as long as it contains (A) to (C). For example, the preparation of the present invention may contain (A) to (C) together, or may be in the form of a kit in which (A) to (C) are prepared separately and then combined before use. When (A) to (C) are contained together, the activity of each enzyme contained per gram of the enzyme preparation of the present invention is as follows: (A): endo- or endo- / exo-protease, usually 100 to 1,000,000 U, preferably 2,000 to 100,000 U, more preferably 10,000 to 50,000 U; (B): carbohydrate-modifying enzyme, usually 0.1 to 100,000 U, preferably 1 to 20,000 U, more preferably 5 to 5,000 U; α-glucosidase, usually 0.1 to 100 U, preferably 1 to 50 U, more preferably 5 to 40 U; glucoamylase, usually 0.1 to 100,000 U, preferably 1 to 20,000 U, more preferably 10 to 5,000 U; and (C): usually 0.1 to 200 U, preferably 1 to 100 U, more preferably 10 to 50 U.

[0032] More specifically, when (A) to (C) are contained together in the enzyme preparation of the present invention, the ratio of each enzyme to the total amount of enzymes contained in the enzyme preparation of the present invention is as follows: (A): endo- or endo- / exo-protease, usually 1 to 40 parts by weight, preferably 5 to 20 parts by weight, (B): α-glucosidase, usually 1 to 40 parts by weight, preferably 5 to 30 parts by weight, glucoamylase, usually 0.001 to 5 parts by weight, preferably 0.1 to 1 part by weight, and (C): usually 0.1 to 5 parts by weight, preferably 1 to 5 parts by weight, more preferably 1 to 3 parts by weight. The weight ratio of enzyme to additives other than enzymes in the enzyme preparation of the present invention can be, for example, 5 to 10:95 to 90.

[0033] In the case of a kit format in which the enzymes are prepared separately and then combined before use, the amount of enzyme contained in each preparation can be adjusted appropriately to achieve the above activity.

[0034] The formulation of the present invention may be added at any step in the production process of a cheese analog, as described below. For example, the formulation of the present invention is typically added in an amount of 0.1 to 50 wt %, preferably 0.5 to 10 wt %, and more preferably 1 to 5 wt %, based on the total weight of the cheese analog. When only a very small amount of enzyme is required, the formulation of the present invention may be prepared into an enzyme solution of a measurable concentration, and the solution may be diluted before addition.

[0035] In addition to the above enzymes, the formulation of the present invention may further contain other food additives such as excipients such as dextrin, starch, modified starch, reduced maltose, seasonings such as meat extract, proteins such as vegetable protein, gluten, egg white, gelatin, casein, protein hydrolysates, partial protein hydrolysates, emulsifiers, chelating agents such as citrates and polymerized phosphates, reducing agents such as glutathione and cysteine, alginic acid, kansui (alkaline water), oils and fats, colorants, acidulants, flavorings, etc.

[0036] The formulation of the present invention may be in the form of a liquid, paste, granules, or powder.

[0037] 2. Method for Producing Cheese Analogues The present invention also includes a method for producing a cheese analogue, which comprises the step of (1) allowing a mixture containing a protein, starch, and fats and oils to react with (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase (hereinafter sometimes abbreviated as "the production method of the present invention").

[0038] In the production method of the present invention, the step (1) of allowing the enzymes (A) to (C) to act on the mixture is not particularly limited as long as the enzymes can be brought into contact with or coexist with the mixture. For example, the enzymes may be brought into direct contact with the mixture, or an enzyme water may be prepared from the enzymes and water and then brought into contact with the mixture. Furthermore, the enzymes (A) to (C) may be brought into contact with the mixture simultaneously or at different times.

[0039] The reacting step also includes a step of mixing the mixture with an enzyme and heating to a temperature suitable for the enzyme reaction. The heating step may be a step of heating with stirring. More specifically, the reacting step is a step of heating and mixing (emulsifying) fats and oils, starch, protein, and optional additives (e.g., seasonings, various amino acids, excipients, flavorings, coloring agents, thickening polysaccharides), and enzymes (A) to (C) with stirring, and allowing the enzymes (A) to (C) to react with the fats and oils, starch, and protein in the mixture (enzyme reaction step). The heating temperature is typically 30 to 90°C, preferably 40 to 80°C, and more preferably 50 to 70°C. The heating time is typically 0.1 to 60 minutes, preferably 1 to 30 minutes, and more preferably 3 to 10 minutes. The pH during contact with each enzyme is not particularly limited, but is typically 4 to 10.

[0040] In step (1) of the production method of the present invention, the amount of each enzyme added per 1 g of the mixture (cheese analogue) is as follows: (A): endo-type: usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, more preferably 0.1 to 1,000 U; exo-type: usually 0.0001 to 10,000 U, preferably 0.001 to 1,000 U, more preferably 0.01 to 100 U; The endo / exo protease is usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, more preferably 0.1 to 1,000 U. When an endo / exo protease is used, the ratio of the endo protease activity to the exo protease activity in the endo / exo protease (endo protease activity:exo protease activity) is usually 1 U: 0.000000001 to 10,000,000 U, preferably 1 U: 0.0000001 to 100,000 U, more preferably 1 U: 0.00001 to 1,000 U (the same applies below); (B): usually 0.0001 to 100,000 U, preferably 0.001 to 10,000 U, more preferably 0.01 to 1,000 U; and (C): Usually 0.0001 to 1000 U, preferably 0.001 to 100 U, more preferably 0.01 to 10 U.

[0041] More specifically, in step (1) of the production method of the present invention, the amount of each enzyme added per 1 g of the mixture (cheese analogue) is as follows: (A): endo-type: usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, further preferably 0.1 to 5,000 U, and more preferably 0.1 to 1,000 U; exo-type: usually 0.0001 to 10,000 U, preferably 0.001 to 1,000 U, and more preferably 0.01 to 100 U; The endo / exo protease is usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, further preferably 0.1 to 5,000 U, and even more preferably 0.1 to 1,000 U. When an endo / exo protease is used, the ratio of endo protease activity to exo protease activity in the endo / exo protease (endo protease activity:exo protease activity) is usually 1 U:0.000000001 to 10,000,000 U, preferably 1 U:0.0000001 to 100,000 U, and more preferably 1 U:0.00001 to 1,000 U (the same applies hereinafter). (B): In the case of α-glucosidase, usually 0.0001 to 10,000 U, preferably 0.001 to 1,000 U, more preferably 0.01 to 100 U; in the case of glucoamylase, usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, more preferably 0.1 to 1,000 U; and (C): usually 0.0001 to 1,000 U, preferably 0.001 to 100 U, more preferably 0.01 to 10 U.

[0042] More specifically, in step (1) of the production method of the present invention, the amount of each enzyme added per gram of protein in the mixture is as follows: (A): endo type, usually 0.01 to 1,000,000 U, preferably 0.1 to 100,000 U, more preferably 1 to 10,000 U, exo type, usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, more preferably 0.1 to 1,000 U, endo type / exo type, usually 0.01 to 1,000,000 U, preferably 0.1 to 100,000 U, more preferably 1 to 10,000 U, (B): usually 0.001 to 1,000,000 U, preferably 0.01 to 100,000 U, more preferably 0.1 to 10,000 U, and (C): Usually 0.001 to 10,000 U, preferably 0.01 to 1,000 U, more preferably 0.1 to 100 U.

[0043] More specifically, in step (1) of the production method of the present invention, the amount of each enzyme added per gram of protein in the mixture is as follows: (A): endo-type, usually 0.01 to 1,000,000 U, preferably 0.1 to 100,000 U, more preferably 1 to 50,000 U, more preferably 1 to 10,000 U; exo-type, usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, more preferably 0.1 to 1,000 U; endo-type / exo-type, usually 0.01 to 1,000,000 U, preferably 0.1 to 100,000 U, more preferably 1 to 50,000 U, more preferably 1 to 10,000 U; (B): α-glucosidase, usually 0.001 to 100,000 U, preferably 0.01 to 10,000 U, more preferably 0.1 to 1,000 U; In the case of glucoamylase, it is usually 0.01 to 1,000,000 U, preferably 0.1 to 100,000 U, more preferably 1 to 10,000 U; (C): it is usually 0.001 to 10,000 U, preferably 0.01 to 1,000 U, more preferably 0.1 to 100 U.

[0044] Similarly, in step (1) of the production method of the present invention, the amount of each enzyme added per gram of starch in the mixture is as follows: (A): endo type, usually 0.005 to 500,000 U, preferably 0.05 to 50,000 U, more preferably 0.5 to 5,000 U, exo type, usually 0.0005 to 50,000 U, preferably 0.005 to 5,000 U, more preferably 0.05 to 500 U, endo type / exo type, usually 0.005 to 500,000 U, preferably 0.05 to 50,000 U, more preferably 0.5 to 5,000 U, (B): usually 0.0005 to 500,000 U, preferably 0.005 to 50,000 U, more preferably 0.05 to 5,000 U, and (C): Usually 0.0005 to 5000 U, preferably 0.005 to 500 U, more preferably 0.05 to 50 U.

[0045] More specifically, in step (1) of the production method of the present invention, the amount of each enzyme added per gram of starch in the mixture is as follows: (A): endo type, usually 0.005 to 500,000 U, preferably 0.05 to 50,000 U, more preferably 0.5 to 25,000 U, and more preferably 0.5 to 5,000 U; exo type, usually 0.0005 to 50,000 U, preferably 0.005 to 5,000 U, and more preferably 0.05 to 500 U; endo type / exo type, usually 0.005 to 500,000 U, preferably 0.005 to 50,000 U, more preferably 0.5 to 25,000 U, and more preferably 0.05 to 5,000 U; (B): In the case of α-glucosidase, usually 0.0005 to 50,000 U, preferably 0.005 to 5,000 U, more preferably 0.05 to 500 U; in the case of glucoamylase, usually 0.005 to 500,000 U, preferably 0.05 to 50,000 U, more preferably 0.5 to 5,000 U; and (C): usually 0.0005 to 5,000 U, preferably 0.005 to 500 U, more preferably 0.05 to 50 U.

[0046] In the production method of the present invention, when (A) an endoprotease and (B) are used, the ratio of the amounts of the endoprotease and (B) added (endoprotease:(B) α-glucosidase) is, for example, 1 U: 0.000000001 to 10,000,000 U, preferably 1 U: 0.0000001 to 100,000 U, and more preferably 1 U: 0.00001 to 1,000 U.

[0047] In the production method of the present invention, when (A) an endoprotease and (B) a glucoamylase are used, the ratio of the amounts of the endoprotease and (B) added (endoprotease:(B)) is, for example, 1 U: 0.00000001 to 100,000,000 U, preferably 1 U: 0.000001 to 1,000,000 U, and more preferably 1 U: 0.0001 to 10,000 U.

[0048] When (A) an exoprotease and (B) an α-glucosidase are used in the production method of the present invention, the ratio of the amounts of exoprotease and (B) added (exoprotease:(B)) is usually 1 U:0.00000001 to 100,000,000 U, preferably 1 U:0.000001 to 1,000,000 U, and more preferably 1 U:0.0001 to 10,000 U.

[0049] When an exoprotease (A) and a glucoamylase (B) are used in the production method of the present invention, the ratio of the amounts of exoprotease and (B) added (exoprotease:(B)) is usually 1 U:0.000000001 to 10,000,000 U, preferably 1 U:0.0000001 to 100,000 U, and more preferably 1 U:0.00001 to 1,000 U.

[0050] In the production method of the present invention, when (A) endo / exo protease and (B) α-glucosidase are used, the ratio of the amounts of endo / exo protease and (B) added (endo / exo protease:(B)) is, for example, 1 U:0.000000001 to 10,000,000 U, preferably 1 U:0.0000001 to 100,000 U, and more preferably 1 U:0.00001 to 1,000 U.

[0051] In the production method of the present invention, when (A) endo / exo protease and (B) glucoamylase are used, the ratio of the amounts of endo / exo protease to (B) added (endo / exo protease:(B)) is, for example, 1 U:0.00000001 to 100,000,000 U, preferably 1 U:0.000001 to 1,000,000 U, and more preferably 1 U:0.0001 to 10,000 U.

[0052] In the production method of the present invention, when an endoprotease (A) and (C) are used, the ratio of the amounts of the endoprotease and (C) added (endoprotease:(C)) is, for example, 1 U:0.000000001 to 1,000,000 U, preferably 1 U:0.0000001 to 10,000 U, and more preferably 1 U:0.00001 to 100 U.

[0053] When an exoprotease (A) and (C) are used in the production method of the present invention, the ratio of the amounts of the exoprotease and (C) added (exoprotease:(C)) is, for example, 1 U:0.00000001 to 10,000,000 U, preferably 1 U:0.000001 to 100,000 U, and more preferably 1 U:0.0001 to 1,000 U.

[0054] In the production method of the present invention, when (A) endo / exo protease and (C) are used, the ratio of the amounts of (A) endo / exo protease to (C) added ((A) endo / exo protease:(C)) is, for example, 1 U:0.000000001 to 1,000,000 U, preferably 1 U:0.0000001 to 10,000 U, and more preferably 1 U:0.00001 to 100 U.

[0055] When (B) α-glucosidase and (C) are used in the production method of the present invention, the ratio of the amounts of (B) and (C) added ((B):(C)) is usually 1 U: 0.00000001 to 10,000,000 U, preferably 1 U: 0.000001 to 100,000 U, and more preferably 1 U: 0.0001 to 1,000 U.

[0056] When (B) glucoamylase and (C) are used in the production method of the present invention, the ratio of the amounts of (B) and (C) added ((B):(C)) is usually 1 U:0.000000001 to 1,000,000 U, preferably 1 U:0.0000001 to 10,000 U, and more preferably 1 U:0.00001 to 100 U.

[0057] The production method of the present invention is characterized by including a step of allowing the enzymes (A) to (C) to act on the above mixture, which is a cheese analog raw material, but other steps, such as the following conventional steps, can be applied: (2) a step of heating the mixture obtained in step (1), (3) a step of cooling the heated mixture, and (4) a step of cutting the cooled mixture. In addition to the above, the production method may also include, as appropriate, a step of adding other seasoning liquids or additives, a step of mixing with seasoning liquids, a step of stirring them, etc.

[0058] For example, the mixing step and the stirring step can be carried out by a method known in the food production field, and examples thereof include mixing using a mixer used in the production of cheeses, such as a food processor, a cooker-type emulsifier, a kettle-type emulsifier, a vertical high-speed shear emulsifier, or a scraped-surface heat exchanger.

[0059] In the production method of the present invention, step (2) is more specifically a step of further heating the mixture obtained in step (1) with stirring at a temperature at which the starch gelatinizes to obtain a mixture containing gelatinized starch, followed by a step of sterilizing the mixture. The heating temperature in step (2) is not particularly limited as long as it can inactivate the enzyme and further sterilize the mixture, but is typically 50 to 120°C, preferably 60 to 120°C, and more preferably 70 to 120°C. The heating time is typically 0.1 to 60 minutes, preferably 1 to 60 minutes, and more preferably 5 to 60 minutes.

[0060] In the production method of the present invention, the step (3) is, more specifically, a step of pouring the mixture obtained in the step (2) into a mold and cooling it to obtain a cheese analog. The cooling method in the step (3) is not particularly limited, but the heated product obtained in the step (2) is usually cooled to -20 to 20°C, preferably -10 to 10°C, and more preferably 0 to 10°C. The heated product obtained in the step (2) may be filled into a desired container and cooled.

[0061] In the production method of the present invention, step (4) more specifically includes a step of cutting the cheese analog obtained in step (3) into blocks and a step of cutting it into sliced ​​cheese or shredded cheese. For example, this step can be used to produce shredded cheese by shredding it to a predetermined size. The specific shred size can be adjusted as appropriate and is not particularly limited, but examples of the size include 4.5 to 10 mm x 30 mm, and for thin slices, 1.0 to 2.0 mm x 60 mm.

[0062] In the production method of the present invention, the pH of the mixture containing fats and oils, starch, and proteins to be reacted with (A) to (C) is, for example, pH 3 to 6.

[0063] In the production method of the present invention, the mixture containing fats and oils, starch, and proteins to be reacted with (A) to (C) contains water. The amount of water used is, for example, 5 to 80% by weight, preferably 15 to 70% by weight, more preferably 25 to 60% by weight, and even more preferably 35 to 50% by weight, based on the weight of the cheese analogue.

[0064] In the production method of the present invention, the cheese analog may contain additives commonly used in the food industry in addition to the above-mentioned components. Examples of additives include flavorings (e.g., cheddar flavoring (powder, liquid), parmesan flavoring (powder, liquid), camembert flavoring (powder, liquid), cream cheese flavoring (powder, liquid)), seasonings (e.g., salt, yeast extract, lactic acid), coloring agents, excipients (dextrin, lactose), various amino acids, thickening polysaccharides (e.g., gum arabic, xanthan gum, tamarind seed gum, guar gum, locust bean gum, carrageenan, agar), proteases, carbohydrate-modifying enzymes, and enzymes other than TG. The amount of additive used is, for example, 1 to 30% by weight based on the cheese analog.

[0065] In the production method of the present invention, the definitions and preferred ranges of (A) to (C) and the cheese analogue are the same as those described in the formulation of the present invention.

[0066] The cheese analog produced by the production method of the present invention can be used as a food product as is or in combination with other ingredients or general foods. In this specification, the term "food" broadly encompasses anything that can be orally ingested (excluding pharmaceuticals), and includes not only so-called "foods" but also beverages, health supplements, health functional foods (e.g., foods for specified health uses, foods with functional claims, foods with nutrient functions), supplements, etc. Foods using the cheese analog produced by the production method of the present invention include foods that are eaten after heating, such as pizza, gratin, doria, and lasagna.

[0067] The present invention also includes a method for increasing the hardness of a cheese analog, comprising the step of (1) treating a mixture containing protein, starch, and fat with (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase (hereinafter referred to as the "hardness-increasing method of the present invention"). The method for increasing hardness of a cheese analog includes the step of treating a mixture containing fat, starch, and protein with (A) a protease and (B) a carbohydrate-modifying enzyme, which results in a hardness at room temperature that is lower than the hardness suitable for cutting. Therefore, the method further treats the mixture with (C) a transglutaminase to increase the hardness and improve the production efficiency of cutting, etc. "Increasing the hardness" means increasing or adjusting the hardness to a level suitable for cutting, and "hardness suitable for cutting" refers to the normal hardness of cheese that is known as hard cheese and is used after shredding. A hardness suitable for cutting is, for example, a shred rate of 90% or more.

[0068] In the method for increasing hardness of the present invention, the definitions and preferred ranges of (A) to (C) and the cheese analogue are the same as those described in the production method of the present invention.

[0069] The present invention will be described in more detail below based on examples and test examples, but the present invention is not limited to these.

[0070] [Test Example 1] Prototype Procedure: The raw materials were weighed according to the blending ratios shown in Table 1 and emulsified using a heating mixer (Thermomix TM21, manufactured by Vorwek) at 70°C for 5 minutes with stirring (enzyme reaction step) according to the flow chart in Figure 1. The mixture was then heated at 90°C for 8 minutes with stirring (enzyme inactivation step). The resulting mixture was filled into a mold and cooled in a refrigerator (5°C) for 48 hours to obtain a cheese analog. The cheese was also cut into circles to examine the properties of the cheese analog.

[0071]

[0072] AG: α-glucosidase, manufactured by Amano Enzyme Inc., 120 U / g. The amount of α-glucosidase in the cheese analogs of experimental plots 2A to 4A was converted to 0.36 U of enzyme activity per 1 g of cheese analog. PRO: Proteax (trade name), manufactured by Amano Enzyme Inc., 218,000 U / g (endo- and exo-proteases), 1,400 U / g (exo- and endo-proteases). The amounts of protease in the cheese analogs of experimental plots 2A to 4A were converted to 436 U of endo- and 2.8 U of exo-proteases per 1 g of cheese analog. TG: Transglutaminase, 1300 U / g. The amount of transglutaminase in the cheese analog of experimental plot 3A was converted to 0.13 U of enzyme activity per 1 g of cheese analog.

[0073] Measurement Method (Evaluation of Meltability of Cheese Analogues Upon Heating) The meltability of cheese analogues upon heating was evaluated using the method shown in Figure 2. Cheese analogues (weight: 2 g, size: diameter 2 cm, thickness: 2 mm) were cut out, placed on a circular sheet, and heated in an oven at 180°C for 10 minutes. After heating, the distance that the cheese analogues had melted and spread was measured at four points using the circular sheet of cheese analogue, and the average value was calculated. Meltability was evaluated from the calculated values ​​according to the following criteria.

[0074] ◎: 3.51mm~ ○: 2.76~3.50mm △: 2.01~2.75mm ×: ~2.00mm

[0075] (Evaluation of Shredding Suitability) Using the method shown in Figure 2, a cheese analog (weight: 70 g, size: width 5 cm, length 3 cm, height 3 cm) was shredded at a laboratory temperature of 15°C using a shredder (Professional SaladShooter Electric Slicer, manufactured by Presto, 15 shredding holes x 2 rows). The shred rate was calculated from the amount of cheese analog remaining in the shredder and the amount of shredded cheese analog. The shredding suitability was evaluated from the calculated shredding rate according to the following criteria: ◎: 96.1% - ○: 93.1% - 96.0% △: 90.1% - 93.0% ×: up to 90.0%

[0076] Effect of Addition of TG Samples shown in Table 1 were prepared and evaluated based on the above criteria using the method shown in Figure 2. The results are shown in Table 2 and Figure 3.

[0077]

[0078] When the cheese analogue is not added with enzymes, it does not melt or spread much when heated, but the addition of AG and PRO increases the melting and spreading (improved meltability). However, the cheese analogue with AG and PRO added had a shred rate that was reduced by 5% or more compared to the cheese analogue without enzymes, and a large amount of cheese analogue remained in the shredder, which is likely to lead to reduced productivity. It was confirmed that the addition of TG improved the shred rate while maintaining meltability.

[0079] [Test Example 2] Study on TG concentration The samples in Table 3 were prepared in the same manner as in Test Example 1 and evaluated using the method in Figure 2 based on the criteria of Test Example 1. The results are shown in Table 4 and Figure 4.

[0080]

[0081]

[0082] It was found that the shred rate improved by adding 0.01% or more of TG, and that the shred rate was significantly improved by adding 0.03% or more. It was also found that even when 0.05% of TG was added, the melting property was higher than that of cheese analogs to which no enzyme was added. [Test Example 3] Examination of GA Concentration The samples in Table 5 were prepared in the same manner as in Test Example 1, except that AG was replaced with glucoamylase (GA), and evaluated according to the criteria of Test Example 1 using the method of Figure 2. The results are shown in Table 6 and Figure 5.

[0083]

[0084] GA: 250,000 U / g glucoamylase for sake brewing, "Amano" SD (trade name), manufactured by Amano Enzyme Inc. The amount of glucoamylase in the cheese analogues of test plots 1C to 4C was 2.5 U in terms of enzyme activity per 1 g of cheese analogue.

[0085]

[0086] It was found that even when GA and PRO were used to improve meltability, adding 0.01% or more of TG improved the shred rate. [Test Example 4] Study of lentil protein Except for changing the protein to lentil protein, the samples in Table 7 were prepared in the same manner as in Test Example 1, and evaluated according to the criteria of Test Example 1 using the method in Figure 2. The results are shown in Table 8 and Figure 6.

[0087]

[0088] GA: Amano SD (trade name) glucoamylase for sake brewing, manufactured by Amano Enzyme Inc., 250,000 U / g. The amount of glucoamylase in the cheese analogs of test plots 2D and 3D was equivalent to 2.5 U of enzyme activity per 1 g of cheese analog. PRO: Proteax (trade name), manufactured by Amano Enzyme Inc., 218,000 U / g (endo-activity), 1,400 U / g (exo-activity) (endo- / exo-protease). The amount of protease in the cheese analogs of test plots 2D and 3D was equivalent to 2,180 U of endo-activity and 14 U of exo-activity per 1 g of cheese analog. TG: Transglutaminase, 1,300 U / g. The amount of transglutaminase in the cheese analog of test plot 3D was equivalent to 0.13 U of enzyme activity per 1 g of cheese analog.

[0089]

[0090] Even cheese analogs using lentil protein did not melt or spread much when heated without the addition of enzymes (1D), but the addition of GA and PRO increased the melting spread from 2 mm to 5 mm (2D) (improved meltability). Furthermore, by adding TG (3D), it was confirmed that even when lentil protein was used, the shredding rate increased dramatically from 50.7% to 94.0% while maintaining meltability.

[0091] (Ingredients used) Waxy cornstarch: Waxy cornstarch MD (Nihon Shokuhin Kagaku) ​​Tapioca starch: NOVATION3300 (Ingredient) Gum arabic: Superstab AA (Nexira) Tamarind seed gum: Glyloid 2A (Sumitomo Pharma) Coconut oil: Organic premium coconut oil Cocowell (Cocowell) Pea protein: Nutralys F85M (Rocket) Fava bean protein: VITESSENCE Prista P360 (Ingredient) Salt: Nakuru M (Naikai Salt Industry) Lactic acid: Lactic Acid Powder (Mezzoni Foods) PRO: Protease: Proteax (Amano Enzyme) AG (α-Glucosidase): α-Glucosidase "Amano", Amano Enzyme Inc. GA: Glucoamylase for sake brewing "Amano" SD, Amano Enzyme Inc. Lentil protein: Organic lentil protein (GUZEN)

[0092] According to the present invention, by allowing (A) a protease, (B) a carbohydrate-modifying enzyme, and (C) a transglutaminase to act on a mixture containing fats and oils, starch, and protein, it is possible to efficiently produce cheese analogs such as shredded cheese and sliced ​​cheese that have improved meltability and / or spreadability when heated.

[0093] This application is based on patent application No. 2023-194015 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. An enzyme preparation for a cheese analogue comprising (A) a protease, (B) a carbohydrate-modifying enzyme and (C) a transglutaminase.

2. The formulation of claim 1, wherein (B) is α-glucosidase, glucoamylase or a mixture thereof.

3. The formulation of claim 1, wherein (B) is a glucoamylase.

4. The formulation of claim 1, wherein the cheese analog comprises protein, starch and fat.

5. The formulation of claim 1, wherein the cheese analog is a shredded cheese analog.

6. The formulation of claim 5, wherein the shredded cheese analog is a shredded cheese analog.

7. The preparation according to claim 1, which contains 100 to 1,000,000 U of (A), 0.1 to 100,000 U of (B), and 0.1 to 200 U of (C) per gram of the enzyme preparation.

8. The formulation according to claim 7, which is used in an amount of 0.1 to 50% by weight based on the total weight of the cheese analogue.

9. (1) A method for producing a cheese analogue, comprising the step of reacting a mixture containing protein, starch and fats with (A) a protease, (B) a carbohydrate-modifying enzyme and (C) a transglutaminase.

10. The process of claim 9, wherein (B) is α-glucosidase, glucoamylase or a mixture thereof.

11. The method of claim 9, wherein (B) is a glucoamylase.

12. The method according to claim 9, wherein the protein is a plant-derived protein.

13. The method of claim 9, further comprising the steps of: (2) heating the mixture obtained in step (1); (3) cooling the heated mixture; and (4) cutting the cooled mixture.

14. The method of claim 13, wherein the cheese analog is a shredded cheese analog.

15. The method of claim 14, wherein the shredded cheese analog is a shredded cheese analog.

16. The method according to claim 9, wherein step (1) is a step of reacting 0.001 to 100,000 U of (A), 0.0001 to 100,000 U of (B), and 0.0001 to 1,000 U of (C) per 1 g of the mixture.

17. A method for increasing the hardness of a cheese analog, comprising the step of reacting a mixture containing protein, starch and fat with (A) a protease, (B) a carbohydrate-modifying enzyme and (C) a transglutaminase.

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