JPH0286743A - Production of gelatin having high polymerization degree - Google Patents

Abstract

PURPOSE:To obtain the subject gelatin having high jelly strength and high melting point by crosslinking and polymerizing a gelatin solution having a specific concentration with a transglutaminase and terminating the reaction before the gelatin of the gelatin. CONSTITUTION:A gelatin solution having a concentration of 2-60wt.% is produced by adding water to gelatin and the gelatin is crosslinked and polymerized by the addition of >=0.1 unit of transglutaminase (based on 1g of gelatin) originated from an actinomycete (e.g., Streptoverticillium). The crosslinking reaction is terminated before the gelatin of the gelatin by inactivating the enzyme or changing the reaction condition to obtain the objective gelatin suitable for a gelatin for food, gelatin for industrial use, gelatin for photographic use, etc.

Description

[Detailed description of the invention] Head of the Joto Department ■ Branch The present invention relates to a method for producing highly polymerized gelatin.

More specifically, the present invention relates to a method for producing water-soluble highly polymerized gelatin by adding transglutaminase to a gelatin solution, crosslinking polymerizing gelatin, and stopping the crosslinking polymerization before gelatin gelatinizes.

and ゛ Begelatin is a water-soluble protein obtained by hydrolyzing collagen, which is a major component of connective tissue fibers such as animal bone and skin, and its solution reversibly forms a gel when cooled below the freezing point. Current industrial gelatin manufacturing methods include an acid method in which raw materials such as cow or pig skin or bones from which the acetic acid has been eluted with hydrochloric acid are pretreated with acid, and a pretreatment with lime solution. Although alkaline methods exist, the purpose of these pretreatments is to cleave naturally occurring crosslinks between collagen molecules or its constituent chains to facilitate subsequent extraction with hot water. Therefore, the main component of the gelatin obtained is about 1
Most of the monomers (α component), dimers (β component), and dimers (T component) of the constituent polypeptide chains of collagen, which have a molecular weight of 0,000,000, and their hydrolysis products, are natural There are only trace amounts of macromolecules that remain cross-linked. In addition, enzymatic gelatin, which is obtained by directly decomposing raw materials such as skin with enzymes, has similar ingredients.

The characteristics of these gelatin components give various advantages in terms of viscosity, jelly strength, etc., but for example, high viscosity, high jelly strength, high melting point, etc. are required for the purpose of improving gel quality. In some cases, satisfactory results were not obtained. Furthermore, when it was intended for industrial use, its viscosity limit was a problem.

Conventionally, to improve the jelly strength, viscosity and melting point of gelatin for food use, polysaccharides such as sodium cellulose sulfate, carrageenan, agar and sodium alginate, thickeners such as gums and oligosaccharides, and gelling agents have been added. I needed to. In addition, gelatin itself has been crosslinked to obtain high polymers, but most of these are based on aldehydes such as formalin and glutaraldehyde, which are difficult to control due to their high reactivity, and the formation of high polymers. The aim is to form a gelled product through cross-linking rather than through cross-linking. Also, most of these crosslinkers are not edible. Furthermore, when using vegetable tannin as a crosslinking agent, if the gelatin concentration is low, the reactivity is low, and if the concentration is high, the entire system will be crosslinked, making it difficult to control the molecular weight.

On the other hand, transglutaminase takes the γ carboxamide group of glutamine residues and the ε of gin residues in protein molecules.
It is known to crosslink amino groups (FOLK,
J, E, and FINLAYSON, J, S, (19
77).

Adv, Protein Chem,, vol, 31+
1). Specific examples of use in gelatin are not disclosed. Furthermore, in the method described in this document, the protein concentration used is low, at most 1% by weight, and no attempt has been made to utilize it industrially. The present inventors have found that in the case of gelatin, the crosslinking reaction does not occur effectively in such a low concentration range, but rather the hydrolysis reaction of the peptide bonds in the main chain takes precedence.

Furthermore, as an example of using transglutaminase in a high concentration region of gelatin, there is Example 6 of ``Method for producing a gelled product'' in Japanese Patent Application Laid-Open No. 58149645.
This is a method for producing a gelled product, and its purpose is different from the "method for producing highly polymerized gelatin" of the present invention. In other words, the purpose of this method is to sufficiently crosslink gelatin to obtain a gelatin gel, and unlike ordinary reversible gelatin gels, this gel does not turn into a sol when heated; it only swells when dried, and does not absorb water. does not dissolve in On the other hand, the highly polymerized gelatin according to the present invention undergoes this sol-gel conversion and is distinguished from the gelatin of the above publication in that it has the basic properties of gelatin. Furthermore, in the case of gelatin, the present inventors found that when the solution is completely gelled by ε (T-glutamyl) lysyl cross-linking and the cross-linking reaction is completed to form a gelled product, when the gelatin is cooled to room temperature, It was discovered that gelation due to its own hydrogen bonds was hindered, resulting in a gel that was much more brittle than that of the raw material gelatin. On the other hand, the gelatin of the present invention is in a solution state at high temperatures, and when it is cooled to gel, it mainly uses hydrogen bonds in the gelatin body, so the rubber elasticity of gelatin is present and enhanced.

The purpose of the present invention is to improve the viscosity of a gelatin solution, the jelly strength and melting temperature of gelatin when cooled and gelated, and to overcome the defects of ordinary gelatin by using an enzymatic cross-linking polymerization reaction. That is, in a gelatin solution with a concentration of 2-60% by weight, preferably 5-40% by weight, 0.0.
After adding 1 unit or more of transglutaminase, preferably 0.5 to 3 units, and causing a crosslinking reaction, the enzyme is deactivated before it becomes a gelled product, or the reaction conditions are changed to stop the reaction. Generates a large amount of gelatin oligomers and high polymers and has the desired viscosity in solution state.
The aim is also to obtain highly polymerized gelatin that has high jelly strength and high melting temperature when cooled and gelatinized.

Furthermore, the present invention makes use of the characteristics of enzymatic reactions to appropriately select the reaction pH, temperature, etc., and appropriately control the reaction rate, thereby making it possible to obtain a highly polymerized product of gelatin using a production method that is industrially fully controllable. The purpose is

A further object of the present invention is to obtain a useful modified gelatin that can be widely used as food gelatin, industrial gelatin, photographic gelatin, and the like.

A feature of the invention is that the gelatin solution has a concentration of 2-60% by weight, preferably 5-40% by weight, and 70% by weight for gelatin IB.
By adding 1 unit or more, preferably 0.5-3 units of transglutaminase and crosslinking polymerization, the enzyme is deactivated before it becomes a gelled product, or the reaction conditions are changed to stop the reaction. The objective is to produce highly polymerized gelatin with improved physical properties that undergoes sol-gel conversion in the same way as ordinary gelatin.

The gel obtained by cooling the highly polymerized gelatin produced in this way is crosslinked by enzymes and hydrogen bonds in the gelatin body, so under normal usage conditions it has a greater elongation against tension than the raw gelatin gel and melts. Because of its high temperature, it can be used as high-viscosity gelatin and high-melting-point gelatin, and when used for food, it is difficult to melt during the day and exhibits a sticky texture.

In this production method, by changing the temperature of the solution during the crosslinking reaction, it is possible to set the melting temperature of the gel when the resulting highly polymerized gelatin is cooled to a desired temperature.

Furthermore, the gel obtained by cooling the product that has been deactivated using this production method does not change over time due to the continuation of the enzymatic reaction or undergo syneresis due to the progress of the gelation reaction, and can remain stable for a long period of time. It is possible.

According to a method for producing highly polymerized gelatin according to one embodiment of the present invention, gelatin is polymerized in advance and then dried to obtain highly polymerized gelatin, which is used in the same manner as ordinary gelatin. According to another aspect of the invention, enzymes can be added during the manufacturing process of gelatin-containing products prior to shaping to obtain highly polymerized gelatin. In this case, the solution used for molding has already completed the reaction and is stable, so compared to the above-mentioned method of "Method J for producing gelled product, in which unreacted solution is mixed with enzyme and molded to complete the reaction." It has the advantage of simplifying handling operations.

The present invention is applicable to all commercially available gelatins; for example, there is no substantial difference between acid-processed gelatin and alkaline-processed gelatin. The present inventors have found that although it is preferable to use acidic gelatin, the crosslinking reaction proceeds to the same extent even with alkaline gelatin, which has a low isoelectric point and contains considerably less glutamine residues.

The transglutaminase used in the present invention is preferably one produced from the actinomycete "Streptovertsillium," but transglutaminase of a different origin, such as an enzyme extracted from guinea pig liver, may also be used. Any material that exhibits a crosslinking reaction effect can be used.

Although it is preferable that the pH of the gelatin solution used in the present invention is 6-8, the reaction can be carried out even at pl+4-10 if it takes a long time. Furthermore, this has the great advantage of slowing down the reaction rate by removing the optimum pH for the enzymatic reaction, making it easier to control the reaction, and allowing the degree of crosslinking reaction to be arbitrarily selected. The reaction temperature can be selected within the range from above the melting temperature of the gel to below the inactivation temperature of the enzyme, but is preferably selected within the range of 40-60'C. In even more special cases, after mixing the enzyme with the gelatin solution, it is immediately cooled to below the freezing point, the crosslinking reaction is allowed to proceed for a predetermined period of time in the gelatin state, and then the enzyme is deactivated. It is also possible to obtain polymerized gelatin.

Inactivation methods used in the present invention include inactivation by high temperature heating, such as p117 treatment at 75°C or higher for 10 minutes or more, and enzyme denaturation by low pn, such as p112 treatment at 40°C for 30 minutes or more. There is. In addition, E
[There are various methods of deactivation, including those by adding enzyme reaction inhibitors such as lTA, and deactivators. For stopping the reaction, p
In addition to removing l+ from the optimum pH, the reaction can be stopped using a high-temperature salt solution, or by adding a low-molecular reaction substrate such as an amino acid peptide, which can be appropriately selected depending on the purpose. Furthermore, when the enzyme concentration is low, a deactivation reaction also proceeds simultaneously during the crosslinking reaction, and there are cases where the enzyme is actually completely deactivated during the reaction.

The production of high polymer gelatin according to the present invention includes polysaccharides, starch, dextrin, glycerin, gums, organic acids, saccharides, inorganic salts, seasonings, colorants, spices, and other ingredients in the gelatin solution within a range that does not inhibit the enzymatic reaction. Thickeners, thickeners, stabilizers, emulsifiers, and other additives that are usually added when using gelatin can be added.

EXAMPLES Hereinafter, the present invention will be explained in more detail based on Examples and Comparative Examples, but these are not intended to limit the present invention.

Disaster relief ■1 Highly polymerized gelatin was produced as follows.

Jelly strength: 244, viscosity: 40, melting point: 30°C, isoelectric point: 9.
Water was added to 15 g of acid gelatin No. 1 so as to give a concentration of 15 times overheating, and after swelling, the mixture was dissolved at 50°C, and the pH was adjusted to 7 with caustic soda. Transglutaminase (manufactured by Amano Pharmaceutical; hydroxamic acid method (J, Biol, Chem,
+Vo1.241.5518 (1966)) Specific activity: 2.5 units/mg) After adding 12 mg of the suspension in a small amount of water, stirring was continued at 50°C. After 10 minutes, heat the solution rapidly to 80°C on hot plate-L.
The temperature was raised to 100% and maintained for 5 minutes to inactivate the enzyme. Thereafter, the solution was immediately immersed in a cold water bath to be rapidly cooled to 60°C, poured onto a stainless steel plate, and cooled in a refrigerator. The thus obtained film-like gel having a thickness of 1111 was used as it was for measurement. A dried product was obtained by cutting this gel into pieces with a width of 3IIIfi, drying them, and then pulverizing them.

For comparison, a gelatin gel was produced according to the "Method for producing gelatin" described in JP-A-58-149645, and was measured together with untreated raw material gelatin. The gelatin gel was produced in the same manner as above except that the gelatin solution was subjected to an enzymatic reaction until gelatinized, then cooled and shredded into desired shapes.

The molecular weight distribution of the highly polymerized gelatin thus produced was measured by liquid chromatography and is shown in FIG. (
Column; Asahipak G5-620: Mobile phase; M/10 phosphate buffer; flow rate: 0.5ml/m
temperature; 50°C: sample; 0.5% umbrella 20ttl: n filter; filtration 5μ: detection ultraviolet wavelength; The high molecular weight components above are increasing,
Highly polymerized gelatin is formed.

In addition, the gel thus obtained was measured using a differential scanning calorimeter (PerkinElmer DSC-7: sample gel; 27 mg:
The data measured at a heating rate of 5° C./m) are shown in FIG. Looking at the melting peak of micelles, which form cross-linking points in the gel where the constituent polypeptide chains of gelatin are associated with each other through hydrogen bonds, the gel obtained by the method of this example has a lower melting point than the gel of the raw material gelatin. Although the temperature has fallen slightly, it remains high and the heat of fusion is increasing. Although this structure is slightly disordered, the crosslinking points of the gel are micelles due to hydrogen bonds generated by cooling, as in the case of raw material gelatin, and the enthalpy increases from 1!5.6 J/g to 27.7 J/g. The increase indicates further stabilization due to high polymerization. On the other hand, the gel produced according to the above-mentioned "method for producing a gelled product" has a tail on the high temperature side, and although the melting temperature of the gel itself is high, the enthalpy is 9.4 J/gL or nothing (transformer at high temperature). Covalent cross-linking by glutaminase precludes cross-linking by hydrogen bonds upon cooling, indicating that this gel is essentially different from the gel according to the invention.

The physical properties of gelatin obtained by the method according to this example are JIS
The jelly strength is 250. The melting temperature of the viscosity 105.10% gel was 48°C. This is improved compared to the physical properties of raw material gelatin.

A tensile test (Fudau rheometer: tensile speed: 6
cn+/m) results are shown in FIG.

Compared to the raw material gelatin, the breaking strength and elongation are clearly higher. In addition, the gel obtained by the ``method for producing gelled products'' has low elongation, becomes brittle, and breaks quickly, indicating that the gel is not entirely crosslinked by hydrogen bonds but is formed by a small number of covalent bonds. .

Water was added to 6 g of the same gelatin as in Example 1 to give a concentration of 6% by weight, and after swelling, it was dissolved at 40"C and the pH was adjusted to 7 with caustic soda. Transglutaminase 4.8B was added to this aqueous solution. After adding the suspension in a small amount of water, stirring was continued at 40°C. After 30 minutes, the solution was heated on a hot plate to rapidly raise the temperature to 80°C and kept for 5 minutes. The enzyme was inactivated. Thereafter, the solution was immediately immersed in a cooling bath to be rapidly cooled to 60"C, poured onto a stainless steel plate, and cooled in a refrigerator. This gel was cut into pieces, dried, and then ground to obtain a dry product.

The melting temperature of the 10% gel of highly polymerized gelatin thus produced was 45°C. This is improved compared to the melting temperature of raw material gelatin. At this gelatin concentration and reaction temperature, similar melting temperatures were obtained even with reaction times of 25-60 minutes.

1 base Uzery strength 260, viscosity 40, melting point 31°C 1 isoelectric point 5
．． Water was added to 20 g of alkaline method gelatin to give a concentration of 20% by weight, and after swelling, the gelatin was dissolved at 50°C.

When this pH was measured, it was 5.2. After adding 24 mg of transglutaminase suspended in a small amount of water to this aqueous gelatin solution, stirring was continued at 50°C.

After 15 minutes, heat the solution on a hot plate to rapidly
The temperature was raised to 0°C and kept for 5 minutes to inactivate the enzyme.

Thereafter, the solution was immediately immersed in a cold water bath and cooled to 60°C, poured onto a stainless steel plate, and cooled in a refrigerator. The thus obtained film-like gel having a thickness of lll111 was used as it was for measurement.

A dried product was obtained by cutting this gel into pieces with a width of 3M, drying them, and then crushing them.

The physical properties of the gelatin obtained by the method according to this example are as follows: jelly strength: 250%, viscosity: 110.10%, gel melting temperature: 60°
It was C. This is improved compared to the physical properties of raw material gelatin.

FIG. 4 shows the results of a tensile test of the cooled gelatin obtained by the method of this example.

Compared to the raw material gelatin, the breaking strength and elongation are clearly higher. Further, the gel obtained by the above-mentioned "method for producing gelled product" has low elongation, easily breaks, and is brittle.

Example 1 40 g of the same gelatin as in Roe Example 3 was added to a mixture of 16 g of glycerin and 77 g of water to swell it, then melted at 60°C and adjusted to pH 7 with caustic soda. After adding 1 g of transglutaminase 16II suspended in a small amount of water to this aqueous solution, stirring was continued at 60"C. After 10 minutes, the solution was heated on a hot plate to rapidly raise the temperature to 80 °C. The solution was kept for 5 minutes to inactivate the enzyme.Then, the solution was immediately immersed in a cold water bath to rapidly cool it to 60'C, poured into a stainless steel plate, and cooled in the refrigerator. The film-like gel was used as it was for measurement.

For comparison, a gel containing the formulation of this example was obtained by pouring it directly onto a stainless steel plate and cooling it without inactivating it at 80° C., and the gel was left at room temperature for 2 days and then measured.

The melting temperature of the gel obtained by the method according to this example is 50''
It was C. This is improved compared to the melting temperature of gel made from raw material gelatin, which is 40°C. Further, the results of the gel tensile test are shown in FIG. The elongation to the breaking point is clearly greater than that of the gel not treated with enzymes. In addition, the gel that did not inactivate the enzyme had less elongation and was brittle.

Penetrating jelly strength 185, viscosity 60, melting point 28°C1 isoelectric point 5
．． Water was added to 75 g of alkaline gelatin No. 1 to give a concentration of 15% by weight, and after swelling, the gelatin was dissolved at 40°C. This p
When H was measured, it was 6.1. After suspending 30 mg of transglutaminase in a small amount of water and adding it to the aqueous gelatin solution, stirring was continued at 40°C. After 40 minutes, when the solution started to thicken, hydrochloric acid was quickly injected to lower the pH to 4.2. The solution was then cooled to 30'C and treated with hydrochloric acid at p.
After raising the temperature to H2, it was poured into a stainless steel plate and immediately gelled with cold water. After inactivating the enzyme by leaving it at room temperature for 1 hour, the gel was transferred to a refrigerator. 3 obtained in this way
A film-like gel having a thickness of ffi11 was cut into pieces of 5-mm width, placed in a fine basket, and immersed in running water for 3 hours. When the swollen gel is dissolved at 40°C, the gelatin concentration is 5% by weight, ρ■
It was 3.7. The mixture was poured into a stainless steel plate, gelled, and dried with cold air to obtain a gelatin polymer.

The melting temperature of the gel obtained by cooling the gelatin obtained by the method of Hatago Honjitsu was 40°C. This is improved compared to the melting temperature of raw material gelatin gel at pH 3.7 of 28°C.

Actual U Water was added to 6 g of the same gelatin as in Example 5 to give a concentration of 6% by weight, and after swelling, the gelatin was dissolved at 40°C. After 4.8 mg of transglutaminase was suspended in a small amount of water and added to the gelatin aqueous solution, stirring was continued at 40°C. 7
After 0 minutes, when the solution started to thicken, acetic acid 101 was quickly added to stop the reaction. When the pH of this solution was measured, it was PH3.5. Thereafter, the solution was poured onto a stainless steel plate and cooled in a refrigerator.

The melting temperature of the gel obtained by cooling the gelatin obtained by the method of this example was 33°C. This is improved compared to the melting temperature of raw gelatin gel at ρ■3.2 of 27°C.

Main points 1. Effects The highly polymerized gelatin produced by the method of the present invention is soluble in water and can be used under the same conditions as ordinary gelatin. The gel obtained by cooling the highly polymerized gelatin of the present invention has a higher elongation under tension and a higher melting temperature than the raw gelatin gel under normal usage conditions because crosslinking by enzymes and hydrogen bonding of the gelatin body is added. It can be used as high viscosity gelatin, high melting point gelatin, and when used for food, it is difficult to dissolve during the day and has a sticky texture.

In the production method of the present invention, by changing the temperature of the solution during the crosslinking reaction, it is possible to set the melting temperature of the gel when the obtained highly polymerized gelatin is cooled to a desired temperature.

Furthermore, the gel obtained by cooling highly polymerized gelatin that has been subjected to enzyme deactivation using this production method does not undergo any deterioration over time due to the continuation of the enzymatic reaction or the syneresis phenomenon associated with the progress of the gelation reaction, and can be used for long periods of time. It is possible to maintain stability over a period of time.

[Brief explanation of the drawing]

FIG. 1 is a liquid chromatography chart showing the molecular weight distribution of highly polymerized gelatin and raw material gelatin. Figure 2 shows a gel containing 15% by weight of each of the highly polymerized gelatin and raw material gelatin produced in Example 1 and JP-A No. 5
"Method for producing gelled product" described in Publication No. 8-149645
It is a chart showing the results of differential scanning calorimetry of the obtained gels of the same concentration. Figure 3 shows stress-strain curves of tensile tests of 15% by weight gels of the highly polymerized gelatin and raw material gelatin produced in Example 1, and gels of the same concentration obtained by the above-mentioned "Method for producing gelled products". It is. Figure 4 shows stress-strain curves of tensile tests of 20% by weight gels of the highly polymerized gelatin and raw material gelatin produced in Example 3, and gels of the same concentration obtained by the above-mentioned "Method for producing gelled products". It is. FIG. 5 shows the glycerin-blended gel of the highly polymerized gelatin produced in Example 4 and the raw material gelatin, and the "
2 is a stress-strain curve of a tensile test of the same composition obtained by the method for producing a gelled product. Patent author Nippi Gelatin T Gyo Co., Ltd. Ajinomoto Co., Inc. (4 others)

Claims (1)

[Claims] 1 g of gelatin in a gelatin solution with a concentration of 2-60% by weight
Highly polymerized gelatin characterized by adding 0.1 unit or more of transglutaminase to cross-link polymerization, and then deactivating the enzyme or stopping the reaction before the gelatin solution becomes a gelatinized product. manufacturing method.