JP7559336B2 - Methods for stabilizing enzymes in liquid formulations - Google Patents

Description

The present invention relates to a method for stabilizing an enzyme in a liquid preparation, and more specifically, to a method for stabilizing an enzyme in a liquid preparation containing transglutaminase and a method for storing the liquid preparation.

In the food industry, enzyme preparations such as transglutaminase used for modifying and processing fish and livestock meat are usually provided as solid preparations in powder or granule form from the viewpoint of stability. For stabilization of transglutaminase powder, it is known to use a package in which an outer bag with characteristic gas barrier properties inside and a stabilizer such as an oxygen absorber, dehydrating agent, nitrogen generating agent, or carbon dioxide generating agent are contained and the outer bag is sealed (Patent Document 1), or to stabilize transglutaminase using stabilizers, oxygen absorbers, or packaging materials (Patent Document 2).

However, when transglutaminase or the like is allowed to act on a substrate, it is often dissolved in an appropriate solvent and used as a liquid preparation. Conventional solid preparations require dissolution in a solvent before use, which is inconvenient and poses the risk of microbial contamination when dissolved in a solvent.

On the other hand, it is known that enzymes such as transglutaminase are prone to a decrease in enzymatic activity in a liquid state. To address such a decrease in activity, for example, liquid preparations containing transglutaminase include a preparation in which transglutaminase is contained in a polyol-water suspension having a pH value within a specific narrow range and which contains polyol (Patent Document 3), a preparation in which transglutaminase is dissolved together with a water activity regulator, an oxidation-reduction potential controller, a preservative, and a pH adjuster (Patent Document 4), and a liquid preparation in which the stability of transglutaminase is improved by adding specific amounts of glycine, proline, serine, glutamate, aspartate, and organic acid salts (Patent Document 5). However, no liquid preparation is known that can be easily prepared and can be stored for long periods under conditions other than refrigeration.

Patent No. 3422137 Patent No. 3281368 Special table 2014-532421 publication Chinese Patent No. 105462950 WO2019/182123

The present invention aims to provide a method for stabilizing the enzyme activity in a liquid preparation containing an enzyme such as transglutaminase even at room temperature or higher, and a storage method for maintaining the enzyme activity in the liquid preparation for a long period of time.

As a result of extensive research aimed at solving the above problems, the inventors discovered that by storing a liquid formulation of transglutaminase in a container made of a packaging material with a specific oxygen permeability, the enzymes such as transglutaminase in the liquid formulation can remain at high levels even at room temperature or above, and thus completed the present invention.

That is, the present invention relates to the following.
[1] (1) a step of preparing a liquid formulation by dissolving an enzyme in a solvent; and (2) a step of storing the liquid formulation in a packaging container made of a packaging material having an oxygen transmission rate of 0.1 cc or less per day at 23° C., 50% RH, and 1 atmosphere when the container has a volume of 100 cc.
13. A method for stabilizing an enzyme in a liquid formulation comprising:
[2] The method according to [1], wherein the enzyme is transglutaminase.
[3] The method according to [1] or [2], wherein 1 U to 1,000 U of transglutaminase is dissolved per 1 g of the liquid preparation.
[4] The method according to any one of [1] to [3], comprising a step of adding one or more carboxylates selected from the group consisting of glutamate, aspartate, acetate, citrate, and gluconate in an amount of 5 wt % to 25 wt % based on the total weight of the liquid formulation.
[5] The method according to [4], wherein the carboxylate is one or more selected from the group consisting of sodium glutamate and sodium gluconate.
[6] The method according to any one of [1] to [5], comprising adjusting the pH of the liquid formulation to 4 to 7.
[7] The method according to any one of [1] to [6], wherein the packaging material comprises a material selected from the group consisting of polyethylene terephthalate, polyethylene terephthalate having a gas barrier film, and aluminum.
[8] The method according to any one of [1] to [7], wherein the packaging material further comprises an oxygen absorbing layer.
[9] The method according to any one of [1] to [8], wherein the storage temperature is 1° C. or higher and 45° C. or lower.
[10] The method according to any one of [1] to [8], wherein the remaining activity of the enzyme after storage at 34° C. for one month is 75% or more.
[11] (1) a step of preparing a liquid formulation by dissolving an enzyme in a solvent; and (2) a step of storing the liquid formulation in a packaging container made of a packaging material having an oxygen transmission rate of 0.1 cc or less per day at 23°C, 50% RH, and 1 atmosphere when the container has a volume of 100 cc.
A method for storing a liquid formulation comprising the steps of:
[12] The method according to [11], wherein the enzyme is transglutaminase.
[13] The method according to [11] or [12], wherein 1 U to 1,000 U of transglutaminase is dissolved per 1 g of the liquid preparation.
[14] The method according to any one of [11] to [13], comprising a step of adding one or more carboxylates selected from the group consisting of glutamate, aspartate, acetate, citrate, and gluconate in an amount of 5 wt % to 25 wt % based on the total weight of the liquid formulation.
[15] The method according to [14], wherein the carboxylate is one or more selected from the group consisting of sodium glutamate and sodium gluconate.
[16] The method according to any one of [11] to [15], comprising adjusting the pH of the liquid formulation to 4 to 7.
[17] The method according to any one of [11] to [16], wherein the packaging material comprises a material selected from the group consisting of polyethylene terephthalate, polyethylene terephthalate having a gas barrier film, and aluminum.
[18] The method according to any one of [11] to [17], wherein the packaging material further comprises an oxygen absorbing layer.
[19] The method according to any one of [11] to [18], wherein the storage temperature is 1° C. or higher and 45° C. or lower.
[20] The method according to any one of [11] to [18], wherein the remaining activity of the enzyme after storage at 34° C. for one month is 75% or more.

According to the present invention, it is possible to provide a liquid preparation in which enzyme activity is maintained for a long period of time even at room temperature or higher.
According to the present invention, the liquid preparation can be stored without refrigeration equipment, and therefore can be conveniently stored and more widely distributed.

FIG. 1 is a graph showing the effect of differences in container materials on the residual rate of transglutaminase activity in liquid preparations in Test Example 1. FIG. 13 is a graph showing the effect of the oxygen scavenging function of the container and the addition of monosodium glutamate (MSG) on the residual rate of transglutaminase activity of a liquid preparation stored for 1, 2, and 3 months in Test Example 2. FIG. 13 is a graph showing a comparison of the effect of adding MSG or sodium gluconate on the residual enzyme activity rate when an aluminum container with a deoxidizing function and an aluminum container without such function are used in Test Example 2.

The present invention provides a method for stabilizing an enzyme in a liquid formulation (hereinafter also referred to as the "stabilization method of the present invention"), which comprises the steps of dissolving an enzyme in a solvent to prepare a liquid formulation, and storing the liquid formulation in a packaging container made of a packaging material having a specific oxygen permeability.

1. A step of dissolving an enzyme in a solvent to prepare a liquid preparation The enzyme is not particularly limited, but examples thereof include transglutaminase, 4-α-glucanotransferase, α-amylase, α-glucosyltransferase, β-amylase, glucoamylase, α-glucosidase, maltotetrahydrolase, glucose oxidase, etc., among which transglutaminase is preferred. As the transglutaminase, a calcium-independent one obtained from a microorganism is preferably used.
Examples of calcium-independent transglutaminase derived from microorganisms include transglutaminase produced by actinomycetes belonging to the genus Streptomyces, and 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., etc., can also be used.

The content of transglutaminase in the liquid preparation of the present invention is preferably 1 U (unit) to 1,000 U per gram of the liquid preparation of the present invention, and more preferably 10 U to 350 U.

The enzyme 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 calculated from a calibration curve, whereby the enzyme activity can be calculated. In this specification, the amount of enzyme that produces 1 μmol of hydroxamic acid per minute at 37° C. and pH = 6.0 is defined as 1 U (see JP-A-64-027471).

From the viewpoint of further increasing the stability of the enzyme, it is preferable to add a carboxylate to the liquid preparation.
The carboxylates include glutamate, aspartate, acetate, citrate and gluconate, and they can be used alone or in combination of two or more kinds.

Specific examples of salts of carboxylic acids include salts with inorganic bases, organic bases, inorganic acids, organic acids, and salts with amino acids.

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, and the like.
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.

From the viewpoint of solubility in the liquid preparation of the present invention and the effect of stabilizing transglutaminase, an alkali metal salt such as a sodium salt is preferably used as the carboxylate.
In the liquid preparation of the present invention, not only normal salts but also acid salts (hydrogen salts) can be suitably used as the above-mentioned carboxylates, and hydrates thereof can also be used.
When these hydrates are used as the carboxylates, the content of each carboxylate in the preparation of the present invention is expressed as the content converted into the anhydrate.

In the present invention, the carboxylate may be one extracted and purified from naturally occurring animals and plants, or one obtained by chemical synthesis, fermentation, enzymatic or recombinant methods, or it may be one commercially available from various companies.

Glutamate and aspartate can be used in the L-, D-, or DL- configuration, but the L- and DL- configurations are preferred, and the L-configuration is even more preferred.

In the present invention, the carboxylates dissolved together with the enzyme are contained in an amount of usually 5% by weight or more, preferably 10% by weight or more, in terms of total carboxylate content relative to the total weight of the liquid preparation, from the viewpoint of enzyme stability.
On the other hand, the total content of carboxylates in the liquid preparation of the present invention is usually 25% by weight or less, and preferably 20% by weight or less, because if the total content of carboxylates exceeds 25% by weight, the stabilizing effect of transglutaminase plateaus and the effect commensurate with the content of carboxylates cannot be expected, which is not economical.

In the present invention, the liquid preparation is a liquid preparation in which an enzyme is dissolved in a solvent.
Here, the term "liquid preparation" refers to a preparation in the form of a solution at room temperature, and also includes a preparation in the form of a viscous liquid.
The term "room temperature" refers to room temperature as defined in the General Rules of the Japanese Pharmacopoeia, 17th Edition, i.e., 1°C to 30°C.
For example, examples of solvents for dissolving enzymes such as transglutaminase and carboxylates include water suitable for food production such as purified water, deionized water, and tap water, and buffer solutions such as acetate buffer, phosphate buffer, etc. Among these, acetate buffer and phosphate buffer are preferred, and phosphate buffer is more preferred.

The temperature of the solvent in which the transglutaminase and the carboxylate are dissolved is usually 0 to 70°C, preferably 4 to 50°C.
The time required for dissolution is appropriately set, but is usually 0.1 to 1440 minutes.

The method of the present invention may include a step of adjusting the pH of the liquid preparation. From the viewpoint of the stability of an enzyme, for example, transglutaminase, the pH is usually adjusted to 4 to 7, preferably 4 to 6, and more preferably 5 to 6.
In the present invention, the pH of the liquid preparation is measured at 20° C. by a conventional glass electrode method.

The pH can be adjusted using a pH adjuster or a buffer. As the pH adjuster or buffer, any pH adjuster or buffer can be used without particular limitation as long as it can adjust the pH of the solution containing an enzyme such as transglutaminase and a carboxylate to a 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 step of adjusting the pH in the method of the present invention may be carried out simultaneously with or after the dissolving step. Alternatively, the pH of the solvent such as citrate buffer, acetate buffer, phosphate buffer, etc. may be adjusted to 4 to 7 before dissolving. The concentration of various buffering agents in such buffers in the liquid preparation is preferably about 0.01 M to 1 M.

The method of the present invention may include a bubbling step, which can remove oxygen and suppress a decrease in enzyme activity.
Bubbling can be carried out by a conventional method, for example, by contacting an inert gas with a liquid preparation stored in a tank or the like to prevent a decrease in enzyme activity due to oxygen and maintain the activity.
Examples of inert gases include nitrogen and argon, with nitrogen being preferred since it is tasteless, odorless and has low solubility in water and therefore does not affect the product.

Furthermore, the method of the present invention may include a step of adding food additives such as thickening stabilizers (sodium alginate, xanthan gum, sodium carboxymethylcellulose, etc.), preservatives (sodium benzoate, sodium edetate, potassium sorbate, etc.), and antioxidants (ascorbic acid, erythorbic acid, etc.) to the liquid formulation and a mixing step, within the scope of the present invention.

2. Step of storing the liquid formulation in a packaging container made of a packaging material having low oxygen permeability By storing the liquid formulation obtained by the above steps in a packaging container made of a packaging material having low oxygen permeability, enzymes such as transglutaminase in the liquid formulation are stabilized, and the liquid formulation can be stored for a long period of time.

In the present invention, "oxygen transmission amount" is an index of how much oxygen permeates a container or the material of the container (film, etc.), and "low oxygen transmission amount" means that when the volume of the container is 100 cc, the oxygen transmission amount per day at 1 atmosphere is 0.1 cc or less, as measured from outside the container at 23°C and 50% RH using a device such as a MOCON oxygen transmission rate measuring device (OX-TRAN 2/61) filled with nitrogen in an empty container. From the viewpoint of stabilizing the enzyme, 0.05 cc or less is preferable, 0.01 cc or less is more preferable, and 0 cc is particularly preferable.
Examples of packaging materials with low oxygen permeability in the present invention (hereinafter sometimes abbreviated as packaging materials in the present invention) include, from the viewpoint of stabilizing the enzyme, polyethylene terephthalate (PET), polyethylene terephthalate having a gas barrier film, aluminum, nylon, nylon having a gas barrier film, ethylene-vinyl alcohol copolymer (EVOH), polyvinyl alcohol (PVA), etc. Among these, polyethylene terephthalate having a gas barrier film and aluminum are preferred.
Examples of the gas barrier film include diamond-like carbon (DLC) film, SiC, SiO, SiOC, SiN, SiON, SiONC, Si-containing diamond carbon, and other Si-containing films, and alumina films, but DLC films are preferred from the viewpoints of gas barrier properties and chemical stability. The gas barrier film may also be a laminate of multiple films of different compositions.
Examples of the DLC film include an amorphous carbon film, a hydrogenated amorphous carbon film, a tetrahedral amorphous carbon film, and a hydrogenated tetrahedral amorphous carbon film.

The packaging material in the present invention may be any of the above materials alone or a laminated packaging material in which a plurality of different materials are layered.
For example, the packaging material in the present invention is composed of an aluminum-containing layer which is an oxygen barrier layer. The aluminum-containing layer is a layer containing a metal foil such as aluminum or a metal vapor deposition film, and preferably has high oxygen barrier properties.

In addition to the oxygen barrier layer, the packaging material may also be composed of an oxygen absorbing layer, a nylon layer, a sealant layer, etc. It may also have an outer layer on the outside.

The oxygen absorbing layer is a layer that contains an oxygen absorbent and has an oxygen scavenging function. Any conventionally known oxygen absorbent can be used, but from the standpoint of hygiene and oxygen absorption capacity, those that use iron powder as the main component and an alkali metal halide such as sodium chloride or calcium chloride or an alkaline earth metal halide as an oxidation promoter are particularly suitable. The oxygen absorbing layer can be made by kneading the oxygen absorbent with polyolefin or the like.

The sealant layer, also known as the heat seal layer, is made of a material such as polyolefin, and is preferably colored white.

When polyolefin is used as the material for each of the above layers, it is preferable that such polyolefin is polypropylene or a polypropylene copolymer with a propylene content of 70% or more.

In such packaging materials, there are no particular limitations on the outer layer that may be present on the outside, and materials such as polyethylene terephthalate (PET) can be used.

The thickness of the packaging material in the present invention can usually be about 200 μm or less.
For example, the thickness of the nylon layer can usually be set to 3 to 40 μm, taking into consideration the overall thickness of the packaging material.

Specific examples of packaging materials in the present invention include PET materials such as laminated packaging materials in which PET is coated with a DLC film, laminated packaging materials in which the above-mentioned materials are further subjected to special shrink treatment, laminated packaging materials in which a nylon layer is provided between the oxygen barrier layer and the oxygen absorbing layer, such as PET/aluminum (AL) foil/nylon (NY) layer/oxygen absorbing layer/sealant layer, and aluminum laminated packaging materials such as PET/AL foil/NY layer/intermediate layer/oxygen absorbing layer/sealant layer in which an intermediate layer such as polyolefin is provided between the NY layer and the oxygen absorbing layer.

The packaging container made of the packaging material in the present invention is a packaging container characterized in that at least a part of it is made of the above-mentioned laminated packaging material, and the shape is not particularly limited as long as the enzyme is stabilized, but examples of the shape include a bag, pack-in-box, bottle, and molded product.

The method for producing a packaging container made of the packaging material of the present invention is not particularly limited, and can be any conventional method as appropriate.

In the present invention, the liquid preparation can be stored in the above-mentioned packaging container by a conventional method. In order to remove oxygen during storage, an inert gas such as nitrogen may be simultaneously sealed in the head space in the container (the space above the beverage).
After the liquid preparation has been stored, it is desirable to seal the packaging container, and the sealing method can be a conventional method.

In the present invention, the storage temperature is usually 1°C or higher, preferably 4°C or higher, from the viewpoint of stabilizing the enzyme in the liquid preparation, and from the same viewpoint, the upper limit is 45°C or lower, preferably 35°C or lower, and more preferably 24°C or lower.

The storage period is not particularly limited as long as the enzyme activity in the liquid preparation is maintained, but is usually within 3 years, preferably within 2 years, and more preferably within 1 year.

In the present invention, "stabilization" refers to a state in which an enzyme maintains high activity during storage.
For example, in a transglutaminase-containing liquid preparation, the residual activity of transglutaminase after storage for one month at 34° C. is usually 75% or more, preferably 80% or more, more preferably 85% or more, and particularly preferably 90% or more. The upper limit is usually 100%.
The residual activity of transglutaminase can be measured by the hydroxamate method as described above.

The present invention further provides a method for storing a liquid formulation (hereinafter also referred to as the "storage method of the present invention"), comprising the steps of: preparing a liquid formulation by dissolving an enzyme in a solvent; and storing the liquid formulation in a packaging container made of a packaging material having an oxygen transmission rate of 0.1 cc or less per day at 23°C, 50% RH, and 1 atmospheric pressure when the container has a volume of 100 cc.

In the preservation method of the present invention, each step, various definitions and preferred ranges are as described above.
In the present invention, the storage temperature is usually 1°C or higher, preferably 4°C or higher, from the viewpoint of stabilizing the enzyme in the liquid formulation, and the upper limit is 45°C or lower, preferably 35°C or lower, and more preferably 24°C or lower, from the same viewpoint.

In the preservation method of the present invention, the preservation period is not particularly limited as long as the enzyme activity is maintained, but is usually within 2 years, preferably within 1 year.

The preservation method of the present invention may also include a step of adding food additives other than pH adjusters or buffers, such as thickening stabilizers, preservatives, bactericides, antioxidants, etc., in accordance with the above, and other steps, to the extent that the characteristics of the present invention are not impaired.

The present invention will be described in more detail below with reference to the following examples.

Test Example 1: Examination of packaging materials A microbial transglutaminase (1,000 U/g, "Activa TG", Ajinomoto Co., Inc.) was dissolved in 0.05 M phosphate buffer (pH 6.0) as shown in Table 1 to prepare a liquid TG sample.
100 g of liquid TG sample was sealed in a polyethylene (PE) container (about 150 cc), a barrier polyethylene terephthalate (barrier PET) container (about 100 cc), and an aluminum container (about 250 cc), and stored at 4° C., 24° C., 34° C., and 44° C. (accelerated storage test at high temperatures). The barrier PET container is a container whose surface is coated with a diamond-like carbon (DLC) material.
The oxygen transmission rate of each container material was measured using a MOCON oxygen transmission rate measuring device (OX-TRAN 2/61). At 23°C, 50% RH, and 1 atm, the oxygen transmission rate of each material when the container volume is 100 cc is 0.174 cc per day for PE, 0.006 cc for barrier PET, and 0 (oxygen does not transmit) for aluminum containers in a state without cracks. At 44°C, 78% RH, the oxygen transmission rate of PE is 0.434 cc per day, 0.014 cc for barrier PET, and 0 (oxygen does not transmit) for aluminum containers in a state without cracks.
As a control, a liquid TG sample was placed in a 1.5 ml microtube and stored at -80°C.
After one month, the samples were collected, and the transglutaminase activity of each sample was measured by the hydroxamate method, and the residual rate relative to the enzyme activity of the sample stored at -80°C was calculated.

図１に示すように、バリアＰＥＴ容器及びアルミ容器で保存したトランスグルタミナーゼの活性残存率はＰＥ容器に比べて高いことが認められた。

As shown in FIG. 1, it was found that the residual activity of transglutaminase stored in the barrier PET container and aluminum container was higher than that in the PE container.

[Test Example 2] Evaluation of the stabilizing effect of transglutaminase Microbial transglutaminase was dissolved in 0.05 M phosphate buffer (pH 6.0) as shown in Table 2, and 10% MSG was added to the monosodium glutamate (MSG)-added group, and 10% sodium gluconate was added to the sodium gluconate-added group.
100 g of liquid TG sample was sealed in an aluminum container with oxygen scavenging function (250 cc) and an aluminum container without oxygen scavenging function (250 cc), and stored at 4° C., 24° C., 34° C., and 44° C. (accelerated storage test at high temperatures). The aluminum container with oxygen scavenging function is a container made of a laminated packaging material consisting of an aluminum foil layer and an oxygen absorbing layer.
As a control, a liquid TG sample was placed in a 1.5 ml microtube and stored at -80°C.
After 1 month, 2 months, and 3 months, samples were collected, the transglutaminase activity of each sample was measured by the hydroxamate method, and the residual rate relative to the enzyme activity of the sample stored at -80°C was calculated.

As shown in FIG. 2, no significant difference was observed in the residual activity of transglutaminase when stored at 4° C., but when stored at 24° C., the residual activity was higher in the group without MSG in the group in the container with oxygen scavenging function.
When stored at 34°C, almost no transglutaminase remained in the MSG-free group stored in a container without oxygen scavenging function.
On the other hand, when stored in a container with an oxygen scavenging function, the residual rate decreased over time, whereas in the MSG-added group, the enzyme activity was maintained at a high level, especially when stored in a container with an oxygen scavenging function.

A comparison between MSG and sodium gluconate is shown in Figure 3. Note that a test on the sodium gluconate-added group stored at 34°C for 3 months has not been conducted and is therefore not shown.
It was confirmed that the addition of sodium glutamate and sodium gluconate exhibited a significant stabilizing effect on transglutaminase when stored at 34°C, and that the stabilizing effect was further enhanced when stored in an aluminum container with an oxygen scavenging function.In addition, it was confirmed that the addition of sodium gluconate exhibited an even greater stabilizing effect when stored in an aluminum container with an oxygen scavenging function when stored at 44°C.

The present invention provides a liquid formulation that can be stored and distributed at or above room temperature.

Claims (14)

(1) a step of dissolving transglutaminase in a solvent to prepare a liquid formulation; and (2) a step of storing the liquid formulation in a packaging container made of a packaging material having an oxygen transmission rate of 0.1 cc or less per day at 23° C., 50% RH, and 1 atmosphere when the container has a volume of 100 cc.
1. A method for stabilizing transglutaminase in a liquid formulation comprising:
(1) is a step of preparing a liquid formulation by dissolving 1 U to 1,000 U of transglutaminase per 1 g of the liquid formulation and adding 5 wt % to 25 wt % of glutamate or gluconate based on the total weight of the liquid formulation;
(2) The packaging material is a laminated packaging material in which PET is coated with a diamond-like carbon (DLC) film , a laminated packaging material having PET/aluminum foil/nylon layer/oxygen absorbing layer/sealant layer, or an aluminum laminated packaging material having PET/aluminum foil/nylon layer/intermediate layer/oxygen absorbing layer/sealant layer ,
A method in which the residual activity of transglutaminase is 85% or more after storage at 34°C for one month. The method according to claim 1, in which 10 U to 350 U of transglutaminase is dissolved per 1 g of the liquid preparation. The method according to claim 1, comprising the step of adding 10% to 20% by weight of glutamate or gluconate based on the total weight of the liquid formulation. The method of claim 3, wherein the glutamate or gluconate is sodium gluconate. The method according to any one of claims 1 to 4, comprising a step of adjusting the pH of the liquid formulation to 4 to 7. The method according to any one of claims 1 to 5, wherein the storage temperature is 1°C or higher and 45°C or lower. The method according to any one of claims 1 to 6, wherein the residual activity of transglutaminase after storage at 34°C for one month is 90% or more. (1) a step of dissolving transglutaminase in a solvent to prepare a liquid formulation; and (2) a step of storing the liquid formulation in a packaging container made of a packaging material having an oxygen transmission rate of 0.1 cc or less per day at 23° C., 50% RH, and 1 atmosphere when the container has a volume of 100 cc.
A method for preserving a liquid formulation, comprising:
(1) is a step of preparing a liquid formulation by dissolving 1 U to 1,000 U of transglutaminase per 1 g of the liquid formulation and adding 5 wt % to 25 wt % of glutamate or gluconate based on the total weight of the liquid formulation;
(2) The packaging material is a laminated packaging material in which PET is coated with a diamond-like carbon (DLC) film , a laminated packaging material having PET/aluminum foil/nylon layer/oxygen absorbing layer/sealant layer, or an aluminum laminated packaging material having PET/aluminum foil/nylon layer/intermediate layer/oxygen absorbing layer/sealant layer ,
A method in which the residual activity of transglutaminase is 85% or more after storage at 34°C for one month. The method according to claim 8, in which 10 U to 350 U of transglutaminase is dissolved per 1 g of the liquid preparation. The method according to claim 8 or 9, comprising the step of adding 10% to 20% by weight of glutamate or gluconate based on the total weight of the liquid formulation. The method of claim 10, wherein the glutamate or gluconate is sodium gluconate. The method according to any one of claims 8 to 11, comprising adjusting the pH of the liquid formulation to 4 to 7. The method according to any one of claims 8 to 12, wherein the storage temperature is 1°C or higher and 45°C or lower. The method according to any one of claims 8 to 13, wherein the residual activity of transglutaminase after storage at 34°C for one month is 90% or more.