Liquid preparations containing transglutaminase

Abstract

To provide a liquid preparation that shows improved stability of transglutaminase.SOLUTION: A liquid preparation is provided containing transglutaminase and one or more kinds selected from the group consisting of glycine, proline, serine, glutamate, aspartate and organic acid salts, wherein a total content of the liquid preparation having the of one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts is 2 wt.% or more or 5 wt.% or more, and wherein the liquid preparation shows improved stability of transglutaminase and is easily utilized in the field of food.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a liquid formulation containing transglutaminase, and more particularly to a liquid formulation having improved stability of transglutaminase. [Background technology]

[0002] Transglutaminase is an enzyme that catalyzes protein cross-linking by condensing the amino group of glutamine residues in proteins with primary amines and transferring the substituent on the amine to the glutamine residue, and is widely used in meat modification and processing, etc. From the perspective of enzyme stability, preparations containing transglutaminase are provided as solid preparations such as powders and granules. However, when transglutaminase is allowed to act on a substrate, it is often dissolved in an appropriate solvent and used as a liquid formulation. Conventional solid formulations require dissolution in a solvent before use, which makes them inconvenient and also poses the risk of microbial contamination when dissolved in a solvent.

[0003] Disclosed liquid preparations containing transglutaminase include a preparation in which transglutaminase is contained in a polyol-water suspension containing 25% to 100% by weight of polyol and having a pH value in the range of 4.4 to 5.1 (Patent Document 1), and a preparation in which transglutaminase is dissolved together with a water activity adjuster, a redox potential controller, a preservative, and a pH adjuster (Patent Document 2). However, the formulation described in Patent Document 1 requires that the pH of the polyol-water suspension be controlled within a very narrow range of 4.4 to 5.1. Moreover, the formulation described in Patent Document 2 requires the incorporation of a considerable number of stabilizers to stabilize the transglutaminase, which may impose limitations on the use of the enzyme formulation.

[0004] On the other hand, it has been disclosed that sugars or sugar alcohols or amino acids are used as stabilizers in freeze-dried preparations of transglutaminase (Patent Documents 3 and 4). However, the techniques described in Patent Documents 3 and 4 relate to freeze-dried preparations of transglutaminase, which is blood coagulation factor XIII, and are not intended to stabilize liquid preparations of transglutaminase used in the food industry.

[0005] Therefore, there is a demand for a liquid formulation of transglutaminase that is easy to use in the food industry and highly stable. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2014-532421 [Patent Document 2] Chinese Patent No. 105462950 [Patent Document 3] Patent No. 3530300 [Patent Document 4] Patent No. 6244079 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, an object of the present invention is to provide a liquid preparation containing transglutaminase, which has improved stability of transglutaminase and is easy to use in the food industry. [Means for solving the problem]

[0008] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that the stability of transglutaminase in a liquid formulation can be improved by adding one or more elements selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts to the liquid formulation at a total content of 2% by weight or more or 5% by weight or more, thereby completing the present invention.

[0009] That is, the present invention relates to the following. [1] A liquid preparation containing transglutaminase and one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts, wherein the total content of the one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts is 5% by weight or more. [2] The liquid formulation described in [1], wherein the transglutaminase is derived from a microorganism. [3] The liquid formulation according to [1] or [2], wherein the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts and organic acid salts is 10% by weight or more. [4] The liquid preparation according to any one of [1] to [3], wherein the organic acid salt is one or more selected from the group consisting of acetate, citrate, and gluconate. [5] The liquid formulation according to any one of [1] to [4], wherein the glutamate, aspartate and organic acid salt are each a sodium salt. [6] The liquid formulation according to any one of [1] to [5], wherein the liquid formulation has a pH of 4 to 7. [7] A liquid preparation containing transglutaminase and one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts, wherein the total content of the one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts is 2% by weight or more. [8] A method for producing a liquid preparation, comprising adding transglutaminase to a solvent together with one or more species selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts, and dissolving the same, wherein the total content of the one or more species selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts is 5% by weight or more. [9] The method according to [8], wherein the transglutaminase is derived from a microorganism.

[10] The method according to [8] or [9], wherein the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts and organic acid salts is 10% by weight or more.

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

[10] , wherein the organic acid salt is one or more selected from the group consisting of acetate, citrate, and gluconate.

[12] The method according to any one of [8] to

[11] , wherein the glutamic acid salt, the aspartic acid salt and the organic acid salt are each a sodium salt.

[13] The method for producing a liquid formulation according to any one of [8] to

[12] , which comprises adjusting the pH of the liquid formulation to 4 to 7.

[14] A method for producing a liquid preparation, comprising adding transglutaminase to a solvent together with one or more species selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts, and dissolving the same, wherein the total content of the one or more species selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts is 2% by weight or more.

[15] A method for stabilizing transglutaminase in a liquid formulation, comprising adding one or more species selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts to the liquid formulation containing transglutaminase so that the total content of these species is 5% by weight or more.

[16] The stabilization method according to

[15] , wherein the transglutaminase is derived from a microorganism.

[17] The stabilization method according to

[15] or

[16] , wherein the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts is 10% by weight or more.

[18] The stabilization method according to any one of

[15] to

[17] , wherein the organic acid salt is one or more selected from the group consisting of acetate, citrate, and gluconate.

[19] The stabilization method according to any one of

[15] to

[18] , wherein the glutamic acid salt, the aspartic acid salt and the organic acid salt are each a sodium salt.

[20] The stabilization method according to any one of

[15] to

[19] , which comprises adjusting the pH of the liquid formulation to 4 to 7.

[21] A method for stabilizing transglutaminase in a liquid formulation, comprising adding one or more species selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts to the liquid formulation containing transglutaminase so that the total content of these species is 2% by weight or more.

[22] A food to which the liquid formulation according to any one of [1] to [6] has been added.

[23] A food to which the liquid formulation described in [7] has been added. [Effects of the Invention]

[0010] According to the present invention, a liquid formulation of transglutaminase having improved stability of transglutaminase can be provided. The liquid preparation of the present invention does not require dissolving transglutaminase in a solvent when used, making it highly convenient and reducing the risk of microbial contamination, etc. Furthermore, it is suitable for use in the food industry. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the residual rate of transglutaminase activity for each liquid preparation of Examples 1 to 5 and Comparative Example 1 in Test Example 1. [Figure 2] FIG. 1 shows the residual rate of transglutaminase activity for each liquid preparation of Examples 1-1 to 1-3 and Comparative Example 2 in Test Example 2(1). [Figure 3] FIG. 1 shows the residual rate of transglutaminase activity for each liquid preparation of Examples 2-1 to 2-4 and Comparative Example 3 in Test Example 2(2). [Figure 4] FIG. 1 shows the residual rate of transglutaminase activity for each liquid preparation of Examples 3-1 to 3-3 and Comparative Example 4 in Test Example 2(3). [Figure 5] FIG. 1 shows the residual rate of transglutaminase activity for each liquid preparation of Examples 4-1 to 4-4 and Comparative Example 5 in Test Example 2(4). [Figure 6] FIG. 1 shows the relationship between the pH of a liquid preparation and transglutaminase activity for a glycine-added group and a glycine-free group in Test Example 3. [Figure 7] FIG. 1 is a graph showing the residual rate of transglutaminase activity for each liquid preparation of Example 6 and Comparative Examples 6-1 and 6-2 in Test Example 4. [Figure 8] FIG. 1 is a graph showing the residual rate of transglutaminase activity for each liquid preparation of Example 7 and Comparative Examples 7-1 and 7-2 in Test Example 4. [Figure 9] FIG. 1 shows the residual rate of transglutaminase activity for each liquid preparation of Example 8 and Comparative Examples 8-1 and 8-2 in Test Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a liquid formulation containing transglutaminase and having improved stability of the transglutaminase (hereinafter also referred to as "the formulation of the present invention" in this specification).

[0013] The preparation of the present invention contains transglutaminase and one or more members selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts at a concentration of 5% by weight or more. In another aspect, the formulation of the present invention contains transglutaminase together with one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts at a concentration of 2% by weight or more.

[0014] Transglutaminase (protein-glutamine γ-glutamyltransferase) is a transferase that catalyzes the reaction of condensing the amino group of glutamine residues in proteins with a primary amine, transferring the substituent on the amine to the glutamine residue, and producing 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. Therefore, transglutaminase is preferably used in the modification and processing of meat such as fish meat and livestock meat.

[0015] As the transglutaminase, calcium-independent transglutaminase obtained from a microorganism is preferably used. Examples of calcium-independent transglutaminases derived from microorganisms include transglutaminases 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" provided by Ajinomoto Co., Inc. and others can also be used. The content of transglutaminase in the preparation of the present invention is preferably 1 U (unit) to 1,000 U, more preferably 10 U to 350 U, per 1 g of the preparation of the present invention. The enzymatic activity of transglutaminase can be measured and calculated, for example, by the hydroxamate method. Specifically, a reaction is carried out using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates, and the hydroxamic acid produced in the reaction is allowed to form an iron complex 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, thereby calculating the enzymatic activity. Herein, 1 U is defined as the amount of enzyme that produces 1 μmol of hydroxamic acid per minute at 37°C and pH 6.0 (see JP-A-64-027471).

[0016] In the formulation of the present invention, glycine contained together with transglutaminase is 2-aminoacetic acid, which has the simplest structure among the amino acids that constitute proteins and is classified as a non-polar side chain amino acid. Proline is pyrrolidine-2-carboxylic acid and is a cyclic amino acid. Serine is 2-amino-3-hydroxypropionic acid, a hydroxyamino acid that is classified as a polar, uncharged side chain amino acid. In the formulation of the present invention, glycine, proline and serine are each contained in a free form. Glutamate is a salt of 2-aminopentanedioic acid, which is classified as an acidic polar side chain amino acid. Aspartate is a salt of 2-aminobutanedioic acid, a class of acidic polar side chain amino acids. Proline, serine, glutamate and aspartate may be used in any of the L-, D- and DL-forms, but are preferably in the L- and DL-forms, and more preferably in the L-form.

[0017] In the preparation of the present invention, the glutamate and aspartate specifically include salts with inorganic bases, organic bases, inorganic acids, organic acids, and amino acids.

[0018] Examples of salts with inorganic bases include salts with alkali metals such as lithium, sodium and potassium, salts with alkaline earth metals such as magnesium and calcium, and ammonium salts. Examples of salts with organic bases include salts with alkanolamines such as monoethanolamine, diethanolamine and triethanolamine, and salts with heterocyclic amines such as morpholine and piperidine. Examples of salts with inorganic acids include salts with hydrohalic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, etc. Examples of salts with organic acids include salts with monocarboxylic acids such as formic acid, acetic acid, and propanoic acid; salts with saturated dicarboxylic acids such as oxalic acid, malonic acid, malic acid, and succinic acid; salts with unsaturated dicarboxylic acids such as maleic acid and fumaric acid; salts with tricarboxylic acids such as citric acid; and salts with keto acids such as α-ketoglutaric acid. Examples of salts with amino acids include salts with aliphatic amino acids such as glycine and alanine; salts with aromatic amino acids such as phenylalanine; salts with basic amino acids such as lysine; and salts with amino acids that form lactams such as pyroglutamic acid.

[0019] From the viewpoint of solubility in the formulation of the present invention and the effect of stabilizing transglutaminase, alkali metal salts such as sodium salts are preferably used as glutamate and aspartate. In the preparations of the present invention, not only normal salts but also acid salts (hydrogen salts) of the glutamic acid salts and aspartic acid salts can be suitably used, and hydrates thereof can also be used. When hydrates of glutamate and aspartate are used as glutamate and aspartate, the contents of glutamate and aspartate in the preparation of the present invention are expressed as the contents converted into anhydrous forms.

[0020] In the present invention, the above-mentioned glycine, proline, serine, glutamate, and aspartate may be extracted and purified from naturally occurring animals and plants, or may be obtained by chemical synthesis, fermentation, enzymatic methods, genetic recombination, or the like. Alternatively, commercially available products provided by various companies may be used.

[0021] In the preparation of the present invention, the organic acid salt contained together with transglutaminase is an acid salt of an organic compound, and can be used without any particular limitation as long as it can be dissolved in the liquid preparation, is edible, and is usable in food. Examples of suitable organic acid salts include salts of saturated monocarboxylic acids such as formic acid, acetic acid, and propanoic acid; salts of unsaturated monocarboxylic acids such as sorbic acid; salts of hydroxymonocarboxylic acids such as glycolic acid, lactic acid, and glyceric acid; salts of saturated dicarboxylic acids such as oxalic acid, malonic acid, and succinic acid; salts of unsaturated dicarboxylic acids such as maleic acid and fumaric acid; salts of malic acid, alcoholic acid, and the like. Salts of hydroxydicarboxylic acids such as tartaric acid; salts of hydroxytricarboxylic acids such as citric acid and isocitric acid; salts of keto acids such as pyruvic acid, oxaloacetic acid and α-ketoglutaric acid; salts of aldonic acids having about 5 to 6 carbon atoms such as gluconic acid, galactonic acid and mannonic acid; salts of aldaric acids having about 5 to 6 carbon atoms such as glucaric acid, galactaric acid and mannaric acid; and salts of uronic acids having about 5 to 6 carbon atoms such as fructuronic acid, glucuronic acid, galacturonic acid and mannuronic acid; and the like are preferably used, with acetates, citrates and gluconates being more preferably used. Acetate, citrate, and the like are also preferred in that they function as buffers, as will be described later.

[0022] Examples of the salts of the organic acids include salts with alkali metals such as lithium, sodium, and potassium; salts with alkaline earth metals such as magnesium and calcium; and salts with inorganic bases such as ammonium salts; and salts with organic bases such as salts with alkanolamines such as monoethanolamine, diethanolamine, and triethanolamine; and salts with heterocyclic amines such as morpholine and piperidine. Alkali metal salts are preferably used, and sodium salts are more preferably used. In the preparation of the present invention, not only normal salts but also acid salts (hydrogen salts) can be suitably used as the organic acid salts, and hydrates thereof can also be used. When a hydrate is used as the organic acid salt, the content of the organic acid salt in the preparation of the present invention is expressed as the content converted into the anhydrate.

[0023] In the present invention, the organic acid salt may be any of those extracted and purified from naturally occurring animals and plants, or those obtained by chemical synthesis, fermentation, enzymatic methods, genetic recombination methods, etc., or commercially available products provided by various companies may be used.

[0024] In the preparation of the present invention, one or more compounds can be selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts and organic acid salts. The formulation of the present invention contains one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts in a total content of 5% by weight or more, preferably 10% by weight or more. In another embodiment of the present invention, the formulation of the present invention contains one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts in a total content of 2% by weight or more. On the other hand, the total content of one or more amino acids selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts in the formulation of the present invention is usually 20% by weight or less. If the total content of the amino acids exceeds 20% by weight, the stabilizing effect on transglutaminase plateaus, and the effect commensurate with the content of the amino acids cannot be expected, which is uneconomical.

[0025] The formulation of the present invention is a liquid formulation in which transglutaminase and one or more members selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts are dissolved in a solvent. Here, the term "liquid preparation" refers to a preparation that is in the form of a solution at room temperature, and includes preparations that are in the form of a viscous liquid. The term "room temperature" refers to the room temperature defined in the General Rules of the Japanese Pharmacopoeia, 17th Edition, i.e., 1°C to 30°C. As a solvent for dissolving transglutaminase and the above amino acids, etc., water suitable for food production such as purified water, deionized water, tap water, etc. is preferably used. Furthermore, as described below, buffer solutions such as acetate buffer and phosphate buffer can also be used as the solvent.

[0026] In the present invention, the pH of the formulation of the present invention is preferably 4-7, more preferably 4-6, and even more preferably 5-6, from the viewpoint of the stability of transglutaminase. The pH of the preparation of the present invention is measured at 20°C by the conventional glass electrode method. The pH of the formulation of the present invention can be adjusted using a pH adjuster or buffer. Any pH adjuster or buffer can be used without particular limitation, as long as it is capable of adjusting the pH of the solution containing transglutaminase and the amino acids, etc., to the desired range and is edible. Examples of such pH adjusters or buffers include hydrochloric acid, citric acid, sodium citrate, succinic acid, acetic acid, sodium acetate, potassium hydroxide, sodium hydroxide, sodium bicarbonate, sodium carbonate, lactic acid, sodium lactate, phosphoric acid, trisodium phosphate, sodium hydrogen phosphate, and sodium dihydrogen phosphate. Of these, citric acid, sodium citrate, acetic acid, sodium acetate, phosphoric acid, sodium hydrogen phosphate, sodium dihydrogen phosphate, etc. are preferably used. The formulation of the present invention can also be prepared using, as a solvent, a citrate buffer, acetate buffer, phosphate buffer, or the like, whose pH has been adjusted to 4 to 7. The concentration of the various buffering agents in such buffers is preferably about 0.01 M to 1 M.

[0027] Furthermore, the formulation of the present invention may contain food additives such as thickening stabilizers (sodium alginate, xanthan gum, sodium carboxymethylcellulose, etc.), preservatives (sodium benzoate, sodium edetate, potassium sorbate, etc.), antioxidants (ascorbic acid, erythorbic acid, etc.), etc., within the scope that does not impair the characteristics of the present invention.

[0028] The formulation of the present invention can be produced by adding transglutaminase and one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts to a solvent such as water, together with a pH adjuster or buffer, and other food additives as needed, and then mixing and dissolving them. The formulation of the present invention can also be produced by adding transglutaminase and one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts, together with other food additives as necessary, to a buffer solution whose pH has been adjusted to preferably 4 to 7, mixing, and dissolving.

[0029] In the preparation of the present invention, the stability of transglutaminase is improved, and it can be stored for a long period of time under normal storage conditions (for example, refrigerated storage). Therefore, there is no need to dissolve it in a solvent when using it, which is highly convenient, and the risk of microbial contamination, etc. occurring during the process of dissolving it in a solvent is reduced.

[0030] The present invention also provides a method for producing a stable liquid formulation containing transglutaminase (hereinafter also referred to as the "production method of the present invention" in this specification).

[0031] The production method of the present invention comprises adding transglutaminase to a solvent and dissolving it together with one or more members selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts. In the manufacturing method of the present invention, the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts in the liquid preparation is 5% by weight or more, and preferably 10% by weight or more. In another embodiment of the manufacturing method of the present invention, the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts in the liquid formulation is 2% by weight or more. On the other hand, if the total content of the above amino acids etc. exceeds 20% by weight, the stabilizing effect on transglutaminase plateaus, and therefore the total content of one or more selected from the group consisting of glycine, proline, serine, glutamate, aspartate and organic acid salts in a liquid formulation is usually 20% by weight or less. The transglutaminase, glycine, proline, serine, glutamic acid salt, aspartic acid salt and organic acid salt, as well as the solvent for dissolving them, are as described above for the formulation of the present invention.

[0032] In the production method of the present invention, the pH of the liquid formulation is controlled preferably to 4 to 7, more preferably to 4 to 6, and even more preferably to 5 to 6, by using a pH adjuster or buffer, or by using a buffer solution as a solvent. The pH adjuster or buffer and buffer solution, and the method for measuring pH are as described above for the formulation of the present invention.

[0033] In the production method of the present invention, food additives other than pH adjusters or buffers, such as thickening stabilizers, preservatives, antioxidants, etc., can also be added within the scope of not impairing the characteristics of the present invention. The same applies to such other food additives as described above.

[0034] The manufacturing method of the present invention stabilizes transglutaminase, allowing for long-term storage under normal storage conditions (e.g., refrigeration), and provides a highly convenient liquid formulation that does not require dissolution in a solvent when used.

[0035] Furthermore, the present invention provides a method for stabilizing transglutaminase in a liquid formulation (hereinafter also referred to as "the stabilization method of the present invention" in this specification).

[0036] The stabilization method of the present invention involves adding one or more species selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts to a liquid formulation containing transglutaminase so that the total content of these species is 5% by weight or more. In the stabilization method of the present invention, the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts is preferably 10% by weight or more. In another embodiment of the stabilization method of the present invention, the total content of one or more selected from the group consisting of glycine, proline, serine, glutamic acid salts, aspartic acid salts, and organic acid salts is 2% by weight or more. In addition, if the total content of the above amino acids, etc. exceeds 20% by weight, the stabilizing effect on transglutaminase plateaus, so the total content of one or more amino acids selected from the group consisting of glycine, proline, serine, glutamate, aspartate, and organic acid salts in a liquid preparation is usually 20% by weight or less. The transglutaminase, glycine, proline, serine, glutamic acid salts, aspartic acid salts and organic acid salts, as well as the solvent used in the liquid preparation, are as described above for the preparation of the present invention.

[0037] In the stabilization method of the present invention, the pH of a liquid formulation containing transglutaminase is controlled to preferably 4 to 7, more preferably 4 to 6, and even more preferably 5 to 6, by using a pH adjuster or buffer, or by using a buffer as a solvent. The pH adjuster or buffer and buffer solution, and the method for measuring pH are as described above for the formulation of the present invention.

[0038] In the stabilization method of the present invention, food additives other than pH adjusters or buffers, such as thickening stabilizers, preservatives, antioxidants, etc., can also be added within limits that do not impair the characteristics of the present invention. Such other food additives are also as described above for the formulation of the present invention.

[0039] The stabilization method of the present invention stabilizes transglutaminase in a liquid formulation, allowing it to be stored for long periods under normal storage conditions (e.g., refrigerated storage), and provides a highly convenient liquid formulation that does not require dissolution in a solvent when used.

[0040] Furthermore, the present invention provides a food product containing the formulation of the present invention (hereinafter also referred to as "the food product of the present invention" in this specification). The foods of the present invention preferably include processed meat foods (for example, processed salted meat products such as ham and bacon; meat paste products such as sausages, hamburger steaks, meatballs, shumai, gyoza, meat buns, meatballs, and minced meat cutlets), processed seafood foods (for example, fish sausages, kamaboko, chikuwa, satsumaage, hampen, fish balls, shrimp dumplings, etc.), cooked rice foods (for example, cooked white rice, red rice, pilaf, seasoned rice, rice porridge, risotto, rice balls, sushi, boxed lunches, rice noodles, etc.), noodles (for example, udon, pasta, Japanese soba noodles, Chinese noodles, yakisoba noodles, instant noodles that have been fried or dried, gyoza and shumai wrappers, etc.), grain flour (for example, wheat flour, barley flour, corn flour, buckwheat flour, rye flour, oat flour, millet flour, Examples of tofu include, but are not limited to, tofu products (e.g., pea flour, soy flour, etc.) and processed foods thereof, dairy products (e.g., yogurt, cheese, cream, butter, butter oil, cheese, concentrated whey, ice cream, concentrated milk, condensed milk, cream powder, whey powder, buttermilk powder, milk powder, fermented milk, lactic acid bacteria drinks, dairy drinks, etc.), tofu (e.g., silken tofu, firm tofu, soft tofu, filled tofu, etc.) and processed products thereof (e.g., tofu namaage, silken namaage, baked tofu, fried tofu (thick fried tofu, thin fried tofu, sushi fried tofu, etc.); tofu paste products made from soy milk, tofu dough, isolated soy protein, etc. (e.g., tofu tofu, chikuwa, tofu kamaboko, etc.); koyadofu; soy milk and tofu desserts (e.g., soy milk pudding, soy milk jelly, soy milk yogurt, etc.)).

[0041] The amount of the formulation of the present invention added to the food of the present invention can be appropriately determined depending on the form of the food of the present invention, the types of raw materials contained in the food, the processing method, etc., but it is preferably added so that the transglutaminase titer per 1 g of food is 0.00001 U to 1000 U, more preferably 0.0001 U to 100 U, and even more preferably 0.001 U to 10 U.

[0042] The food of the present invention can be produced by adding general food additives to food materials such as meat and the preparation of the present invention, as needed, according to general food production methods.

[0043] According to the present invention, stabilized transglutaminase acts on food materials such as meat, making it possible to provide foods with improved texture. [Example]

[0044] The present invention will be described in more detail below with reference to examples.

[0045] [Examples 1 to 5, Comparative Example 1] Liquid preparation containing transglutaminase 17.6 wt% of microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) was dissolved in 72.4 wt% of 0.05 M phosphate buffer (pH = 6.0) together with 10 wt% each of glycine, proline, serine, sodium glutamate, and sodium aspartate to prepare the liquid formulations of Examples 1 to 5. On the other hand, the same amount of the above transglutaminase was dissolved in 72.4 wt % of 0.05 M phosphate buffer (pH=6.0) together with 10 wt % of lysine hydrochloride to prepare a liquid formulation of Comparative Example 1.

[0046] [Test Example 1] Evaluation of the stabilizing effect of transglutaminase The stability of transglutaminase in each of the liquid preparations of Examples 1 to 5 and Comparative Example 1 was evaluated by an accelerated test under high-temperature storage as follows. Each of the liquid preparations of Examples 1 to 5 and Comparative Example 1 was stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid preparation was measured by the hydroxamate method described above. A liquid preparation in which only transglutaminase was dissolved in phosphate buffer was used as a control and treated in the same manner. The stability of transglutaminase in each liquid formulation is shown in Table 1 and Figure 1, where the residual transglutaminase activity when stored at 44°C is expressed as a percentage of the transglutaminase activity when stored at 4°C.

[0047] [Table 1]

[0048] As shown in Table 1 and FIG. 1, the residual activity of transglutaminase in the control was 62%. In contrast, the liquid preparations of Examples 1 to 5 of the present invention showed a high residual transglutaminase activity rate of around 90%. On the other hand, in the liquid preparation of Comparative Example 1, the residual activity of transglutaminase was 66%, which was similar to that of the control.

[0049] The above results of Test Example 1 confirmed that glycine, proline, serine, sodium glutamate, and sodium aspartate each have a stabilizing effect on transglutaminase in the liquid preparation. On the other hand, lysine hydrochloride, which is a salt of an amino acid other than those mentioned above, did not show any stabilizing effect on transglutaminase.

[0050] [Test Example 2] Examination of the effect of amino acid or amino acid salt content on transglutaminase stabilization As described below, the effect of the content of amino acids or amino acid salts in the liquid formulation on the stability of transglutaminase was evaluated by an accelerated test under high-temperature storage.

[0051] (1) Glycine content Glycine was added to and dissolved in 0.05 M phosphate buffer (pH = 6.0) containing 17.6 wt% microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) and 0.1 wt% sodium benzoate at 5 wt%, 10 wt%, and 20 wt%, and the total amount was adjusted to 100 wt% with 0.05 M phosphate buffer (pH = 6.0) to prepare liquid formulations designated Examples 1-1, 1-2, and 1-3, respectively. In addition, a liquid preparation prepared in the same manner but with a glycine content of 1% by weight was used as Comparative Example 2. The liquid preparations of Examples 1-1 to 1-3 and Comparative Example 2 were stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid preparation was measured by the hydroxamate method described above. A liquid preparation prepared in the same manner without the addition of glycine served as a control and was treated in the same manner. The transglutaminase activity when each liquid preparation was stored at 44°C is shown in Figure 2 as the residual rate (%) of transglutaminase activity when stored at 4°C.

[0052] As shown in Figure 2, the liquid preparations of Examples 1-1 to 1-3 containing glycine at concentrations of 5 wt%, 10 wt%, and 20 wt% each showed a higher residual transglutaminase activity than the control. On the other hand, in the liquid preparation of Comparative Example 2, which contained 1 wt % glycine, the residual activity of transglutaminase was similar to that of the control.

[0053] (2) Proline content Proline was added to and dissolved in 0.05 M phosphate buffer (pH = 6.0) containing 17.6 wt% microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) and 0.1 wt% sodium benzoate at concentrations of 5 wt%, 10 wt%, 20 wt%, and 30 wt%, and the total amount was adjusted to 100 wt% with 0.05 M phosphate buffer (pH = 6.0) to prepare liquid formulations designated as Examples 2-1, 2-2, 2-3, and 2-4, respectively. In addition, a liquid preparation prepared in the same manner but with a proline content of 1 wt % was used as Comparative Example 3. The liquid formulations of Examples 2-1 to 2-4 and Comparative Example 3 were stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid formulation was measured by the hydroxamate method described above. A liquid formulation prepared in the same manner but without the addition of proline served as a control and was treated in the same manner. The transglutaminase activity when each liquid preparation was stored at 44°C is shown in Figure 3 as a residual rate (%) relative to the transglutaminase activity when stored at 4°C.

[0054] 3, the liquid formulations of Examples 2-1 to 2-4, which contained proline at concentrations of 5 wt%, 10 wt%, 20 wt%, and 30 wt%, exhibited higher residual transglutaminase activity than the control. The residual transglutaminase activity in the liquid formulation of Example 2-4, which contained 30 wt%, was not significantly different from the residual activity in the liquid formulation of Example 2-3, which contained 20 wt%, suggesting that the stabilizing effect on transglutaminase activity plateaus when the proline content exceeds 20 wt%. On the other hand, in the liquid preparation of Comparative Example 3, which contained 1 wt % proline, the residual activity of transglutaminase was similar to that of the control.

[0055] (3) Serine content Serine was added to and dissolved in 0.05 M phosphate buffer (pH = 6.0) containing 17.6 wt% microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) and 0.1 wt% sodium benzoate at 5 wt%, 10 wt%, and 20 wt%, and the total amount was adjusted to 100 wt% with 0.05 M phosphate buffer (pH = 6.0) to prepare liquid formulations designated as Examples 3-1, 3-2, and 3-3, respectively. In addition, a liquid preparation prepared in the same manner but with a serine content of 1 wt % was used as Comparative Example 4. The liquid formulations of Examples 3-1 to 3-3 and Comparative Example 4 were stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid formulation was measured by the hydroxamate method described above. A liquid formulation prepared in the same manner without adding serine served as a control and was treated in the same manner. The transglutaminase activity when each liquid preparation was stored at 44°C is shown in Figure 4 as the residual rate (%) of transglutaminase activity when stored at 4°C.

[0056] As shown in Figure 4, the liquid preparations of Examples 3-1 to 3-3 containing serine at concentrations of 5 wt%, 10 wt%, and 20 wt%, respectively, showed a higher residual transglutaminase activity than the control. On the other hand, in the liquid preparation of Comparative Example 4, which contained 1 wt % serine, the residual activity of transglutaminase was similar to that of the control.

[0057] (4) Monosodium glutamate content To 0.05 M phosphate buffer (pH = 6.0) containing 17.6 wt% microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) and 0.1 wt% sodium benzoate, sodium glutamate was added and dissolved at 5 wt%, 10 wt%, 20 wt%, and 30 wt%, and the total amount was adjusted to 100 wt% with 0.05 M phosphate buffer (pH = 6.0) to prepare liquid formulations designated as Examples 4-1, 4-2, 4-3, and 4-4, respectively. In addition, a liquid preparation prepared in the same manner but with a sodium glutamate content of 1 wt % was used as Comparative Example 5. The liquid preparations of Examples 4-1 to 4-4 and Comparative Example 5 were stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid preparation was measured by the hydroxamate method described above. A liquid preparation prepared in the same manner but without the addition of sodium glutamate served as a control and was treated in the same manner. The transglutaminase activity when each liquid preparation was stored at 44°C is shown in Figure 5 as the residual rate (%) of transglutaminase activity when stored at 4°C.

[0058] 5, the liquid formulations of Examples 4-1 to 4-4, which contained sodium glutamate at concentrations of 5 wt%, 10 wt%, 20 wt%, and 30 wt%, exhibited higher residual transglutaminase activity than the control. The residual transglutaminase activity in the liquid formulation of Example 4-4, which contained 30 wt% sodium glutamate, was not significantly different from the residual activity in the liquid formulation of Example 4-3, which contained 20 wt% sodium glutamate, suggesting that the stabilizing effect on transglutaminase activity plateaus when the sodium glutamate content exceeds 20 wt%. On the other hand, in the liquid preparation of Comparative Example 5, in which the content of sodium glutamate was 1% by weight, the residual activity of transglutaminase was similar to that of the control.

[0059] The results of Test Example 2 above showed that a stabilizing effect on transglutaminase was observed when the content of each of glycine, proline, serine, and sodium glutamate in the liquid formulation was 5% by weight or more, and that an even better stabilizing effect was obtained when the content of each of glycine, proline, serine, and sodium glutamate was 10% by weight or more. It was also suggested that the stabilizing effect on transglutaminase plateaued when the contents of glycine, proline, serine, and sodium glutamate exceeded 20% by weight.

[0060] [Test Example 3] Examination of the effect of pH of liquid formulation on the stabilization of transglutaminase As described below, the effect of the pH of the liquid formulation on the stabilization of transglutaminase was evaluated by an accelerated test using high-temperature storage. For the glycine-added group, a sample containing microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) and 10 wt% glycine was prepared according to the liquid formulation shown in Table 2. In addition, in each sample of the glycine-added group, glycine was replaced with each buffer solution to prepare each sample of the glycine-free group. The samples from the glycine-added and glycine-free groups were stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid preparation was measured by the hydroxamate method described above. The results are shown in Figure 6.

[0061] [Table 2]

[0062] As shown in FIG. 6, in the liquid preparations with a pH of 4 to 6, the samples with glycine added had higher transglutaminase activity than the samples without glycine added. Furthermore, in the samples containing glycine, when the pH of the liquid preparation was 5 to 6, it was found that the transglutaminase activity was particularly high.

[0063] The above results of Test Example 3 suggest that when the formulation of the present invention has a pH of 4 to 6, a good transglutaminase stabilizing effect is observed, and when the liquid formulation has a pH of 5 to 6, transglutaminase activity is maintained at a higher level.

[0064] [Examples 6 to 8, Comparative Examples 6-1 to 8-2] Liquid preparations containing transglutaminase 17.6 wt% of microbial transglutaminase (1,000 U / g, "Activa TG", Ajinomoto Co., Inc.) was dissolved in 72.4 wt% of 0.05 M phosphate buffer (pH = 6.0) together with 10 wt% each of sodium acetate, sodium gluconate, and sodium citrate to prepare the liquid formulations of Examples 6 to 8. Liquid preparations of Comparative Examples 6-1 to 8-2 were prepared in the same manner as in the above Examples, except that the contents of sodium acetate, sodium gluconate, and sodium citrate were set to 1% by weight and 0.1% by weight, respectively.

[0065] [Test Example 4] Evaluation of the stabilizing effect of transglutaminase The stability of transglutaminase in each of the liquid preparations of Examples 6 to 8 and Comparative Examples 6-1 to 8-2 was evaluated by an accelerated test under high-temperature storage as follows. Each of the liquid preparations of Examples 6 to 8 and Comparative Examples 6-1 to 8-2 was stored at 4°C and 44°C for 24 hours, and the transglutaminase activity in each liquid preparation was measured by the hydroxamate method described above. A liquid preparation in which only transglutaminase was dissolved in phosphate buffer was used as a control and treated in the same manner. The stability of transglutaminase in each liquid preparation is shown in Figures 7 to 9 as the residual rate (%) of transglutaminase activity when stored at 44°C relative to transglutaminase activity when stored at 4°C.

[0066] As shown in Figures 7 to 9, the residual activity of transglutaminase was clearly improved in each of the liquid formulations of Examples 6 to 8, which contained 10% by weight of sodium acetate, sodium gluconate, and sodium citrate, compared to the control. However, in the liquid formulations of Comparative Examples 6-1, 7-1 and 8-1, in which the sodium acetate, sodium gluconate and sodium citrate contents were each 1 wt%, and Comparative Examples 6-2, 7-2 and 8-2, in which the sodium acetate, sodium gluconate and sodium citrate contents were each 0.1 wt%, the residual transglutaminase activity was similar to that of the control.

[0067] The above results of Test Example 4 showed that transglutaminase was stabilized by organic acid salts such as sodium acetate, sodium gluconate, and sodium citrate, and that transglutaminase was well stabilized when the content of the organic acid salts in the liquid formulation was 10% by weight. [Industrial Applicability]

[0068] As described above in detail, the present invention can provide a liquid formulation of transglutaminase with improved stability of transglutaminase. The liquid preparation of the present invention does not require dissolving transglutaminase in a solvent when used, making it highly convenient and reducing the risk of microbial contamination, etc. Furthermore, it is suitable for use in the food industry. Furthermore, according to the present invention, it is possible to provide a food containing a food material such as meat that has been treated with transglutaminase and has improved stability by adding the liquid preparation of the present invention described above.

[0069] This application is based on patent application No. 2018-057185 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A liquid preparation containing transglutaminase and one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine, and having improved stability of transglutaminase, wherein the transglutaminase is a calcium-independent transglutaminase derived from a microorganism, and the total content of one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine is 5% by weight or more (excluding those containing cross-linkable proteins or polypeptides).

2. 2. The liquid formulation according to claim 1, wherein the total content of one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine is 10% by weight or more.

3. 3. The liquid formulation according to claim 1 or 2, wherein the alkali metal salts of aspartic acid, acetic acid, and gluconic acid are each sodium salts.

4. The liquid formulation according to any one of claims 1 to 3, wherein the liquid formulation has a pH of 4 to 7.

5. A liquid preparation containing transglutaminase and one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine, and having improved stability of transglutaminase, wherein the transglutaminase is a calcium-independent transglutaminase derived from a microorganism, and the total content of one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine is 2% by weight or more (excluding those containing cross-linkable proteins or polypeptides).

6. A method for producing a liquid preparation in which transglutaminase is stabilized, comprising adding transglutaminase to a solvent together with one or more members selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine, and dissolving the same, wherein the transglutaminase is a calcium-independent transglutaminase derived from a microorganism, and the total content of one or more members selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine is 5% by weight or more.

7. 7. The method according to claim 6, wherein the total content of one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine is 10% by weight or more.

8. 8. The method according to claim 6 or 7, wherein the alkali metal salts of aspartic acid, acetic acid, and gluconic acid are each a sodium salt.

9. The method according to any one of claims 6 to 8, comprising adjusting the pH of the liquid formulation to 4 to 7.

10. A method for producing a liquid preparation in which transglutaminase is stabilized, comprising adding transglutaminase to a solvent together with one or more members selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine, and dissolving the same, wherein the transglutaminase is a calcium-independent transglutaminase derived from a microorganism, and the total content of one or more members selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine is 2% by weight or more.

11. A method for stabilizing transglutaminase in a liquid preparation, comprising adding one or more members selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine to the liquid preparation containing transglutaminase so that the total content of these members is 5% by weight or more, wherein the transglutaminase is a calcium-independent transglutaminase derived from a microorganism.

12. The stabilization method according to claim 11, wherein one or more selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine are added so that the total content is 10% by weight or more.

13. The method for stabilization according to claim 11 or 12, wherein the alkali metal salt of aspartic acid, the alkali metal salt of acetic acid, and the alkali metal salt of gluconic acid are each a sodium salt.

14. The method for stabilization according to any one of claims 11 to 13, comprising adjusting the pH of the liquid formulation to 4 to 7.

15. A method for stabilizing transglutaminase in a liquid preparation, comprising adding one or more members selected from the group consisting of alkali metal salts of aspartic acid, alkali metal salts of acetic acid, alkali metal salts of gluconic acid, proline, and serine to the liquid preparation containing transglutaminase so that the total content of these members is 2% by weight or more, wherein the transglutaminase is a calcium-independent transglutaminase derived from a microorganism.

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