Method for producing processed proteins

Abstract

This invention provides a method for producing processed proteins that enhance the cross-linking effect of proteins. [Solution] A method for producing a processed protein is provided, comprising a crosslinking step of reacting a protein with laccase and transglutaminase, wherein the protein is soy protein, tofu, or fermented soy milk.

Description

Technical Field

[0001] The present invention relates to a method for producing a processed protein. More specifically, the present invention relates to a processing technology for enhancing the cross-linking effect of proteins.

Background Art

[0002] Proteins are subjected to cross-linking treatment for a predetermined purpose. For example, in the purpose of improving existing processed foods and creating processed foods having new taste characteristics, etc., proteins as food materials are subjected to cross-linking treatment.

[0003] As a means used for cross-linking proteins, transglutaminase is known. Transglutaminase cross-links between the side-chain carbamoyl group of the glutamine residue of a protein and the side-chain amino group of a lysine residue by an isopeptide bond (Non-Patent Document 1). For example, Patent Documents 1 and 2 describe that the strength of tofu is enhanced by using transglutaminase in the production of tofu.

[0004] Further, Patent Document 3 describes cross-linking proteins with laccase, and further describes that laccase cross-links proteins by forming a Schiff base between the ε-amino group of lysine and another amino group.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] As mentioned above, methods utilizing specific enzymes are known for crosslinking proteins, but in order to meet the growing need for modifications in food products, there is a desire for technologies that further enhance the crosslinking effect.

[0008] Therefore, the present invention aims to provide a processing technology that enhances the crosslinking effect of proteins. [Means for solving the problem]

[0009] The inventors have discovered that combining laccase and transglutaminase significantly enhances the crosslinking effect of proteins. Considering that the crosslinking effect obtained by laccase alone is generally poor even when varying the amount used, and that both laccase and transglutaminase use the same lysine side-chain amino acid as a substrate, the significant enhancement of the protein crosslinking effect through this combination of enzymes was unexpected. This unexpected effect from the combination was so pronounced that even when laccase was used in such large quantities that the protein crosslinking effect was nearly saturated, further addition of transglutaminase dramatically increased the crosslinking effect. Based on these findings, the present invention was completed through further investigation. Specifically, the present invention provides the invention in the following embodiments.

[0010] Item 1. A method for producing a modified protein, comprising a crosslinking step of reacting a protein with laccase and transglutaminase. Item 2. The method for producing the processed protein according to Item 1, wherein the protein is soy protein. Item 3. A method for producing a processed protein according to item 1 or 2, wherein the processed protein is tofu. Item 4. A method for producing a processed protein according to item 1 or 2, wherein the processed protein is fermented soy milk. Item 5. A method for producing a processed protein according to any one of Items 1 to 4, wherein 0.02 U or more of the laccase is used per gram of the protein. Item 6. A method for producing a processed protein according to any one of items 1 to 5, wherein 30 U or more of the laccase is used per 1 g of the protein. Item 7. A method for producing a processed protein according to any one of items 1 to 5, wherein 0.02 U or more of the transglutaminase is used per gram of the protein. Item 8. A method for producing a processed protein according to any one of items 1 to 7, wherein 0.0004 U or more of the transglutaminase is used per 1 U of the laccase. Item 9. A method for producing a processed protein according to any one of items 1 to 8, wherein the laccase is derived from Trametes hirsuta. [Effects of the Invention]

[0011] According to the present invention, a processing technology is provided that enhances the crosslinking effect of proteins. [Brief explanation of the drawing]

[0012] [Figure 1] This shows the relationship between the presence or absence of laccase treatment and the concentration of laccase in tofu production, and the strength of the tofu, as obtained from preliminary test examples. [Figure 2] The relationship between the presence or absence of laccase treatment and the presence or absence and concentration of transglutaminase treatment in tofu production, as obtained in Test Example 1, and the strength of the tofu is shown. [Modes for carrying out the invention]

[0013] The present invention relates to a method for producing a processed protein, characterized by including a crosslinking step in which the protein is treated with laccase and transglutaminase. The method for producing the processed protein will be described in detail below.

[0014] The source of the protein used in the present invention is not particularly limited, and materials containing protein can be used without particular limitation. Examples of materials containing protein include materials used in various industrial fields such as food materials, medical materials, and industrial materials. Specific examples of proteins include plant proteins and animal proteins. Examples of plant proteins include legume proteins such as soybean protein and kudzu bean protein; cereal proteins such as wheat protein, rye protein, oat protein, and corn protein. Examples of animal proteins include fish protein, livestock meat protein, egg protein, milk protein, tendon protein (such as gelatin and collagen).

[0015] These proteins may be used alone or in combination of multiple types. Among these proteins, from the viewpoint of further enhancing the cross-linking effect of the protein, plant proteins are preferably used, more preferably legume proteins are used, and particularly preferably soybean protein is used.

[0016] As a specific form of these proteins, materials containing protein prepared in an appropriate form (materials containing protein) are used. Specifically, the material containing protein only needs to be in a form in which the protein and the enzyme can efficiently contact. Preferably, in the case of animal proteins, examples include ground meat of animal food ingredients (such as fish and livestock meat), minced meat; egg liquids such as whole liquid eggs, egg white liquids, and egg yolk liquids; animal milks such as cow's milk and goat's milk. In the case of plant proteins, examples include plant milks, typically, juice (milk) squeezed from water-absorbed products of food ingredients (such as legumes like soybeans and kudzu beans; cereals such as wheat, rye, oats, and corn), as well as bean powders and cereal powders. For tendon proteins (such as gelatin and collagen), purified proteins can be used as materials containing protein.

[0017] Materials containing these proteins may be used alone or in combination of multiple types. Among the materials containing these proteins, from the perspective of further enhancing the cross-linking effect of the proteins, plant milk is preferably used, and soy milk is more preferably used.

[0018] The protein content in plant milk is not particularly limited. For example, it may be 3 g / 100 mL or more, preferably 3.8 g / 100 mL or more, more preferably 4.2 g / 100 mL or more, still more preferably 4.8 g / 100 mL or more, and particularly preferably 5 g / 100 mL or more. The upper limit of the protein content range in plant milk is not particularly limited. For example, it may be 8 g / 100 mL or less. The solid content of the plant milk material (for example, the soybean solid content in the case of soy milk) is not particularly limited. For example, it may be 6% by weight or more, preferably 8% by weight or more, more preferably 9% by weight or more, still more preferably 9.5% by weight or more, and particularly preferably 10% by weight or more. The upper limit of the solid content range of the plant milk material is not particularly limited. For example, it may be 18% by weight or less or 16% by weight or less.

[0019] In addition, as long as the effects of the present invention are not impaired, the material containing the protein may contain quality improvers such as emulsifiers (sucrose fatty acid esters, phospholipids, monoglycerin fatty acid esters, organic acid monoglycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyglycerin fatty acid esters, propylene glycol fatty acid esters, etc.), pH adjusters (sodium carbonate, sodium hydrogen carbonate, calcium carbonate, etc.), coloring agents, fragrances, seasonings (salt, sugar (sucrose), etc.) according to the form of the target processed protein.

[0020] The laccase used in this invention is an enzyme (EC 1.10.3.2) having phenol oxidase activity. Specific examples of laccase include those derived from microorganisms such as fungi and bacteria, and more specifically, laccases derived from genera such as Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Pycnoporus, Pyricularia, Trametes, Rhizoctonia, Rigidoporus, Coprinus, Psatyrella, Myceliophtera, Schtalidium, Polyporus, Phlebia, and Coriolus.

[0021] These laccases may be used individually or in combination. Among these laccases, those derived from the genus Trametes are preferred, and those derived from Trametes hirsuta are particularly preferred, from the viewpoint of further enhancing the protein cross-linking effect.

[0022] There are no particular limitations on the amount of laccase used, but for example, an amount of 0.02 U or more per gram of protein is used. From the viewpoint of further enhancing the crosslinking effect of proteins, the amount of laccase used per gram of protein is preferably 0.2 U or more, more preferably 2 U or more, even more preferably 10 U or more, even more preferably 30 U or more, and even more preferably 45 U or more. There are no particular limitations on the upper limit of the range of laccase used, but for example, an amount of 500 U or less, 200 U or less, 100 U or less, 80 U or less, 60 U or less, or 55 U or less per gram of protein is used.

[0023] Regarding laccase activity, one unit of enzyme is defined as the amount of enzyme that increases the absorbance at 405 nm by 1.0 OD per minute when 0.1 ml of enzyme solution is added to 3 ml of a 1.0 mg / ml solution of the substrate 2,2'-Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) at 25°C.

[0024] The transglutaminase used in this invention is an enzyme (EC 2.3.2.13) having transglutaminase activity. Transglutaminases include both calcium-dependent transglutaminases that require calcium for activity expression and calcium-independent transglutaminases that do not require calcium for activity expression. Specific examples of transglutaminases include those derived from microorganisms, mammals, fish, etc. More specifically, these include microorganisms belonging to genera such as Streptomyces, Bacillus, and Geobacillus; mammals such as guinea pigs (liver), cattle (blood), and pigs (blood); and fish such as salmon, sea bream, and cod.

[0025] These transglutaminases may be used individually or in combination of multiple types. Among calcium-dependent and calcium-independent transglutaminases, calcium-independent transglutaminases are preferred from the viewpoint of further enhancing the protein crosslinking effect. Furthermore, among the above transglutaminases, microbial transglutaminases are preferred from the viewpoint of further enhancing the protein crosslinking effect, more preferably transglutaminases derived from the genus Streptomyces, and particularly preferably transglutaminases derived from Streptomyces mobaraensis.

[0026] The amount of transglutaminase used is not particularly limited, but examples of amounts used per gram of protein include 0.001U or more, 0.01U or more, or 0.02U or more. From the viewpoint of further enhancing the crosslinking effect of proteins, the amount of transglutaminase used per gram of protein is preferably 0.2U or more, more preferably 0.5U or more, even more preferably 1U or more, even more preferably 1.5U or more, and even more preferably 2U or more, 5U or more, 10U or more, or 15U or more. The upper limit of the range of transglutaminase used is not particularly limited, but examples of amounts used per gram of protein include 200U or less, 100U or less, 50U or less, 30U or less, 25U or less, or 20U or less.

[0027] Regarding the activity of transglutaminase, one unit is defined as the amount of enzyme that produces 1 μmol of hydroxamic acid per minute when benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine are reacted at 37°C.

[0028] The ratio of laccase to transglutaminase used is determined by the amount of each enzyme used, but from the viewpoint of further enhancing the protein crosslinking effect, preferably the amount of transglutaminase used per 1U of laccase is 0.00001U or more, 0.0001U or more, or 0.0004U or more, more preferably 0.004U or more, even more preferably 0.01U or more, even more preferably 0.02U or more, even more preferably 0.03U or more, particularly preferably 0.04U or more, 0.1U or more, 0.2U or more, or 0.3U or more. There is no particular upper limit to the range of transglutaminase used per 1U of laccase, but examples include 10U or less, 5U or less, 2U or less, 1U or less, 0.6U or less, 0.5U or less, or 0.4U or less.

[0029] In the crosslinking process, a protein composition containing a protein-containing material, laccase, and transglutaminase is prepared by mixing the protein-containing material with laccase and transglutaminase, typically by adding laccase and transglutaminase to the protein-containing material, and the protein composition is maintained under heating conditions to allow the crosslinking of proteins by laccase and transglutaminase to proceed.

[0030] The temperature during the preparation of the protein composition of the protein-containing material is not particularly limited, and the material may be in an unheated state or a heated state, but it is preferably in a heated state. The temperature when the material is heated is not particularly limited, but preferably it is a temperature that promotes crosslinking, as described later.

[0031] Furthermore, in the crosslinking process, in addition to crosslinking the protein, processing according to the desired form of the processed protein may be carried out simultaneously. For example, if the form of the processed protein is tofu, a coagulation reaction may be carried out simultaneously, and if it is a fermented product such as fermented soy milk (such as yogurt made from soy milk), fermentation may be carried out simultaneously.

[0032] Therefore, the protein composition subjected to the crosslinking process may include, in addition to the protein-containing material, laccase, and transglutaminase, other materials necessary to form the desired processed protein. Examples of other materials include coagulants used to form tofu, and fermentation bacteria (such as yogurt starter cultures) used to form fermented products such as fermented soy milk (such as yogurt such as soy milk yogurt).

[0033] The coagulant is not particularly limited, and any coagulant commonly used in tofu production can be used. Specifically, examples of coagulants include salt coagulants and acid coagulants. Examples of salt coagulants include magnesium chloride, magnesium sulfate, calcium sulfate, calcium chloride, etc., and examples of acid coagulants include glucono delta-lactone, etc.

[0034] These coagulants may be used individually or in combination of multiple types. Among these, salt coagulants are preferred, and magnesium chloride is more preferred, from the viewpoint of further enhancing the crosslinking effect of proteins.

[0035] The amount of coagulant used is not particularly limited, but examples include 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, or 0.5% by weight or more. From the viewpoint of further enhancing the crosslinking effect of proteins, the amount of coagulant used is preferably 0.75% by weight or more, more preferably 1% by weight or more, even more preferably 1.5% by weight or more, and even more preferably 2% by weight or more. The upper limit of the range of coagulant used is not particularly limited, but examples include 4% by weight or less, or 3% by weight or less.

[0036] The fermentation bacteria used, such as yogurt starter cultures, are not particularly limited and can be appropriately determined considering the optimal temperatures for laccase and transglutaminase. For example, yogurt starter cultures can include thermophilic species with an optimal growth temperature of around 37°C to 45°C, and mesophilic species with an optimal growth temperature of 20°C to 30°C, but either type of species can be used. In other words, even if the cross-linking process is carried out under heating conditions, not only thermophilic species but also mesophilic species can be used. Examples of thermophilic species include those belonging to the genera Streptococcus and Enterococcus. Examples of mesophilic species include those belonging to the genera Lactococcus (preferably Lactococcus lactis subsp. cremoris), Lactobacillus, and Acetobacter (preferably Acetobacter orientalis).

[0037] These yogurt starter cultures may be used individually or in combination of two or more. Among these yogurt starter cultures, species belonging to the genera Lactococcus, Lactobacillus, and Acetobacter are preferred, more preferably Lactococcus lactis subsp. cremoris and Acetobacter orientalis, and even more preferably a combination of these yogurt starter cultures.

[0038] The temperature at which the protein composition is supplied and crosslinking is promoted can be appropriately determined based on the optimal temperatures of laccase and transglutaminase, and the form of the target processed protein, etc., but for example, 35 to 70°C, preferably 38 to 60°C, and more preferably 40 to 57°C is mentioned. More specifically, if the form of the target processed protein is tofu, the temperature for promoting crosslinking can be appropriately determined based on the optimal temperatures of laccase and transglutaminase, etc., but for example, 50 to 70°C, preferably 52 to 60°C, and more preferably 54 to 57°C is mentioned. Also, if the form of the target processed protein is a fermented product such as fermented soy milk (such as yogurt such as soy milk yogurt), the temperature for promoting crosslinking can be appropriately determined based on the optimal temperatures of laccase and transglutaminase and the optimal temperature for the growth of fermentation bacteria (such as yogurt bacteria), etc., but for example, 35 to 50°C, preferably 38 to 46°C, and more preferably 40 to 44°C is mentioned.

[0039] The time required for crosslinking is not particularly limited and depends on the processes that should be carried out simultaneously in the crosslinking process (for example, coagulation in the case of tofu, fermentation in the case of fermented products such as fermented soy milk, etc.), the scale of the protein composition, etc., but for example, it is 30 minutes or more, preferably 1 hour or more. There is no particular upper limit to the time range for crosslinking, but for example, it is 30 hours or less, 24 hours or less, 12 hours or less, 8 hours or less, or 4 hours or less.

[0040] After the cross-linking reaction is complete, any treatment suitable for the morphology of the processed protein can be performed as appropriate. Examples of such treatments include boiling, baking (roasting, toasting, baking, grilling, broiling), steaming, and deep-frying. These treatments may be used individually or in combination.

[0041] The specific form of the processed protein (i.e., cross-linked protein) obtained by the manufacturing method of the present invention is determined according to the type of protein, the type of material containing the protein, and the cross-linking effect to be obtained, but examples include tofu such as firm tofu, silken tofu, and packaged tofu; fermented products such as fermented soy milk and fermented animal milk (yogurt such as soy milk yogurt and animal milk yogurt, cheese, etc.); other soy processed products such as soy meat, fried tofu, and tofu hamburgers; fish paste products such as kamaboko, chikuwa, hanpen, fish sausage, and tsumire; processed meat products such as hamburgers, sausages, meatballs, and minced cutlets; processed egg products such as rolled omelets, egg fillings, scrambled eggs, shredded omelets, omelet sheets, and long eggs; processed grain products such as noodles, confectionery, and bread; viscoelastic candies such as jelly, gummies, and chewing candies; and food adhesives.

[0042] Among these processed proteins, tofu, fermented soy milk (preferably soy milk yogurt), and other soy products are preferred from the viewpoint of enjoying an even more enhanced crosslinking effect, tofu and other soy products are more preferred, and tofu is even more preferred.

[0043] The crosslinking effect that the processed protein should obtain by the manufacturing method of the present invention is not particularly limited as long as it is a property that changes due to protein crosslinking, and examples include improved organizational properties such as increased compressive strength, increased viscoelasticity, and improved gel-forming ability. [Examples]

[0044] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0045] (1) Method for measuring enzyme activity (1-1) Method for measuring laccase activity In the following test examples, the enzyme activity of laccase was measured using 2,2'-Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS, Boehringer Mannheim) as the substrate by the method described below.

[0046] ABTS was dissolved at a concentration of 1.0 mg / ml in 25 mM citrate buffer (pH 3.2) to prepare the substrate solution. 3.0 ml of this substrate solution was placed in a cuvette, preheated to 25°C, and then 0.1 ml of enzyme solution was added and stirred. The mixture was incubated at 25°C, and the absorbance at 405 nm was measured after 1 minute and 3 minutes. Under these conditions, the amount of enzyme that increases the absorbance at 405 nm by 1.0 OD per minute was defined as 1 unit (U).

[0047] (1-2) Method for measuring transglutaminase activity In the following test examples, the enzyme activity of transglutaminase was measured using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates, and the following substrate solutions and colorimetric solutions were used in the method described below.

[0048] (substrate solution) The solution was prepared by dissolving 2.42 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 0.70 g of hydroxyammonium hydrochloride, 0.31 g of reduced glutathione, and 1.01 g of Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) in distilled water to a total volume of 100 mL (pH 6.0). (Color-developing solution) It was prepared by mixing 30 mL of 3M hydrochloric acid solution, 30 mL of 12% by weight trichloroacetic acid solution, and 30 mL of 5% by weight iron(III) chloride solution.

[0049] The enzyme was diluted to an appropriate concentration with 200 mM Tris-HCl (pH 6.0) to prepare a sample solution. 10 μL of substrate solution was added to 10 μL of sample solution and mixed, then reacted at 37°C for 10 minutes. 100 μL of color development solution was added to stop the reaction and form an Fe complex, after which the absorbance at 525 nm was measured. As a control, the absorbance of a sample solution that had been pre-inactivated by heat was measured and the absorbance difference between it and the uninactivated sample solution was determined. Separately, a calibration curve was created using L-glutamic acid-γ-monohydroxamic acid instead of the sample solution, and the amount of hydroxamic acid produced was determined from the absorbance difference. Enzyme activity that produces 1 μmol of hydroxamic acid per minute was defined as 1 unit (U).

[0050] (2) Material Soy milk: Organic soy milk manufactured by Meilaku Group Co., Ltd., protein concentration 5g / 100mL, soy solids 10% by weight Laccase: Laccase derived from Trametes hirsuta Transglutaminase: Transglutaminase derived from Streptomyces mobaraensis

[0051] [Example of a preliminary examination] Tofu was prepared by heating 20 mL of soy milk at 55°C for 5 minutes, adding 30 U, 60 U, or 120 U of laccase aqueous solution per gram of protein and mixing for 5 seconds, or by adding 0.6 mL of 10% magnesium chloride aqueous solution (final concentration 1.0% by weight) without adding laccase aqueous solution and mixing for 5 seconds, then heating the soy milk composition at 55°C for 1 hour to prepare tofu, and finally cooling the resulting tofu to 4°C.

[0052] The strength (Firmness (N); specifically, compressive strength) of the obtained tofu was measured using a rheometer (COMPAC-100II, Sun Science Co., Ltd.). The measurement conditions were: Mode: 20, Adapter: No. 13, Repeat: 1, Indentation distance: 5 mm. The results are shown in Figure 1.

[0053] As shown in Figure 1, the strength of tofu prepared using 30U of laccase was 1.3 times that of tofu prepared with only a coagulant (22N), specifically an increase of only 7N, indicating a slight increase in tofu strength due to laccase. On the other hand, increasing the amount of laccase further did not significantly increase the strength of the tofu, suggesting that the laccase substrate was almost completely depleted.

[0054] [Test Example 1] Tofu was prepared by heating 20 mL of soy milk at 55°C for 5 minutes, adding laccase aqueous solution in the amount shown in Table 1 per gram of protein and mixing for 5 seconds, adding transglutaminase aqueous solution in the amount shown in Table 1 per gram of protein and mixing for 5 seconds, and adding 0.6 mL of 10% by weight magnesium chloride aqueous solution (final concentration 1.0% by weight) and mixing for 5 seconds to prepare a soy milk composition. Tofu was prepared by heating the soy milk composition at 55°C for 1 hour (crosslinking process with coagulation), and the obtained tofu was cooled to 4°C (Comparative Examples 1-1 to 1-6, Examples 1-1 to 1-4).

[0055] [Table 1]

[0056] The strength (Firmness (N); specifically, compressive strength) of the obtained tofu was measured using a rheometer (COMPAC-100II, Sun Science Co., Ltd.). The measurement conditions were: Mode: 20, Adapter: No. 13, Repeat: 1, Indentation distance: 5 mm. The results are shown in Figure 2.

[0057] As shown in Figure 2, the strength of the tofu from Comparative Examples 1-2 to 1-5, which were prepared using transglutaminase treatment in combination with a coagulant, was increased compared to the tofu from Comparative Example 1-1, which was prepared using only a coagulant. Specifically, compared to the strength of the tofu from Comparative Example 1-1 (20N), the strength increased 3.0 times (specifically, an increase of 39N) when the amount of transglutaminase added per gram of soy protein was 0.02U (Comparative Example 1-2), 5.4 times (specifically, an increase of 88N) when it was 0.2U (Comparative Example 1-3), 8 times (specifically, an increase of 140N) when it was 2U (Comparative Example 1-4), and 8.3 times (specifically, an increase of 145N) when it was 20U (Comparative Example 1-4).

[0058] On the other hand, in the preparation of Comparative Examples 1-6, which were prepared using only laccase treatment in combination with a coagulant, the strength was only increased to a mere 1.4 times (specifically, an increase of 8N) compared to the strength of the tofu of Comparative Example 1-1 (20N), despite using 50U of laccase per gram of soy protein, similar to the results shown in the preliminary test example above.

[0059] In contrast, as shown in the preliminary test example above, when 50 U of laccase was used per gram of soy protein, it was thought that the substrate of laccase (especially the amino group of the lysine side chain) was almost completely depleted. However, in the tofu prepared using a combination of laccase and transglutaminase, which has the same substrate (amino group of the lysine side chain), the strength was increased 4.2 times compared to the strength of the tofu of Comparative Example 1-1 (20 N) when 0.02 U of transglutaminase per gram of soy protein was used (Example 1-1). (Specifically, an increase of 64N) When combined with 0.2U of transglutaminase (Example 1-2), the strength was increased 7.3 times (specifically, an increase of 126N) When combined with 2U of transglutaminase (Example 1-4), the strength was increased 10 times (specifically, an increase of 183N) When combined with 20U of transglutaminase (Example 1-5), the strength was increased 10 times (specifically, an increase of 180N) A superior strength-enhancing effect was observed that exceeded the additive effect of the strength-enhancing effects of laccase and transglutaminase individually.

[0060] Furthermore, as is clear from the comparison between Comparative Examples 1-2 and 1-3 and Comparative Example 1-6, transglutaminase has been shown to have a higher strength-enhancing effect than laccase. In particular, when the amount of transglutaminase added per gram of soy protein exceeds 2U (Comparative Examples 1-4 and 1-5), almost no strength-enhancing effect is observed, suggesting that the substrate of transglutaminase is depleted. In contrast, in Examples 1-1 to 1-4, where transglutaminase was used in combination with laccase, an average strength enhancement of 37N was confirmed compared to Comparative Examples 1-2 to 1-5, where transglutaminase was used alone. Comparing Comparative Examples 1-4 and 1-5 with Examples 1-3 and 1-4, an average strength enhancement of 39N was confirmed in Examples 1-3 and 1-4 compared to Comparative Examples 1-4 and 1-5, where transglutaminase was used alone. Considering that the strength enhancement in Comparative Examples 1-6, which used laccase alone, was only 8N, it can be said from this perspective that the tofu of Examples 1-1 to 1-4 exhibited a superior strength enhancement effect that exceeded the additive effect of the strength enhancement effects.

[0061] Furthermore, since laccase and transglutaminase share a common substrate (amino groups in the side chains of lysine residues), it is reasonable to predict that if laccase and transglutaminase coexist, the reaction efficiency of transglutaminase will decrease as a portion of its substrate is lost due to the reaction by laccase. In addition, although laccase has substrates different from those of transglutaminase (specifically tyrosine residues), the protein molecules targeted by the enzymatic reaction are common to both laccase and transglutaminase. Therefore, the protein molecules cross-linked by laccase's tyrosine residues will experience steric hindrance, reducing the opportunity for contact between lysine residues and transglutaminase, which is also reasonable to predict as a decrease in the reaction efficiency of transglutaminase. Considering these points, it is reasonable to predict that when laccase and transglutaminase coexist, the potential of each enzyme for enhancing soy protein cannot be fully realized, and therefore, even an additive effect cannot be expected, let alone a synergistic effect. Therefore, it was unexpected that the combined use of laccase and transglutaminase, as shown in Examples 1-1 to 1-4, could synergistically enhance the strength of the tofu.

[0062] [Test Example 2] Sucrose was added to 20 mL of soy milk to a final concentration of 5% by weight, sterilized by autoclaving, and cooled and kept warm at 42°C. While kept warm at 42°C, 50 U of laccase per 1 g of soy protein was added and mixed for 5 seconds, 20 U of transglutaminase per 1 g of soy protein was added and mixed for 5 seconds, yogurt starter cultures (Lactococcus lactis subsp. Cremoris FC and Acetobacter orientalis FA) were added to a total final concentration of 0.2% by weight and mixed for 5 seconds, incubated at 42°C for 24 hours (cross-linking process involving fermentation), cooled to 4°C, and soy milk yogurt (Example 2) was prepared. Furthermore, soy milk yogurt was prepared in the same manner except that neither laccase nor transglutaminase was used (Comparative Example 2-1), soy milk yogurt was prepared in the same manner using only laccase among laccase and transglutaminase (Comparative Example 2-2), and soy milk yogurt was prepared in the same manner using only transglutaminase among laccase and transglutaminase (Comparative Example 2-3).

[0063] The firmness (N) of the obtained soy milk yogurt was measured in the same manner as in Test Example 1.

[0064] As a result, compared to the strength of the soy milk yogurt of Comparative Example 2-1, which was not treated with either laccase or transglutaminase, the strength of the soy milk yogurt of Comparative Example 2-2, which was treated only with laccase, was increased by 1.25 times, the strength of the soy milk yogurt of Comparative Example 2-3, which was treated only with transglutaminase, was increased by 1.33 times, while the strength of the soy milk yogurt of Example 2, which was treated with both laccase and transglutaminase, was increased by 1.63 times. In other words, the soy milk yogurt of Example 2 showed a superior strength-enhancing effect that exceeded the additive effect of the strength-enhancing effects of laccase and transglutaminase individually (an effect of 1.58 times enhancement).

Claims

1. A method for producing a modified protein, comprising a crosslinking step in which a protein is treated with laccase and transglutaminase.

2. The method for producing a processed protein according to claim 1, wherein the protein is soy protein.

3. The method for producing a processed protein according to claim 1 or 2, wherein the processed protein is tofu.

4. The method for producing a processed protein according to claim 1 or 2, wherein the processed protein is fermented soy milk.

5. A method for producing a processed protein according to any one of claims 1 to 4, wherein 0.02 U or more of the laccase is used per 1 g of the protein.

6. A method for producing a processed protein according to any one of claims 1 to 5, wherein 30 U or more of the laccase is used per 1 g of the protein.

7. A method for producing a processed protein according to any one of claims 1 to 6, wherein 0.02 U or more of the transglutaminase is used per gram of the protein.

8. A method for producing a processed protein according to any one of claims 1 to 7, wherein 0.0004 U or more of the transglutaminase is used per 1 U of the laccase.

9. A method for producing a processed protein according to any one of claims 1 to 8, wherein the laccase is derived from Trametes hirsuta.

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