JP2619933B2 - Method for producing high polymerization degree gelatin - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing highly polymerized gelatin. More specifically, the present invention relates to a method for producing a water-soluble highly polymerized gelatin by adding transglutaminase to a gelatin solution to cross-link polymerize the gelatin and terminating the cross-linking polymerization before the gelatin gels.

2. Description of the Related Art Gelatin is a water-soluble protein obtained by denaturing and solubilizing collagen, which is a major component of connective tissues such as animal bones and skin, and when the solution is cooled below the freezing point. Reversibly forms a gel. At present, industrial gelatin production methods include an acid method in which raw materials such as cow and pig skin or bone in which minerals have been eluted with hydrochloric acid are pretreated with an acid, and an alkaline method in which lime is used for pretreatment. Although present, these pretreatments are intended to break the naturally occurring crosslinks between collagen molecules or between their constituent chains, and to facilitate subsequent extraction with warm water. Therefore, the major components of the obtained gelatin are about
Monomer (α component), dimer (β component), trimer (γ component) of the constituent polypeptide chains of collagen with a molecular weight of 100,000 and their hydrolysis products The macromolecules leaving the cross-linking of the compound contain only trace amounts. Enzymatic gelatin obtained by directly decomposing the skin or the like of the raw material with an enzyme is the same as a component.

The characteristics of these gelatin components give various advantages to their viscosity, jelly strength, etc., for example, high viscosity or high jelly strength for the purpose of improving the texture of gel, etc.
When a high melting point was required, satisfactory results were not obtained. In addition, when it is used for industrial applications, its viscosity limit is a problem.

Conventionally, to improve the jelly strength, viscosity and melting point of gelatin for food applications, sodium cellulose sulfate,
It was necessary to add polysaccharides such as carrageenan, agar, sodium alginate, gums, thickeners such as oligosaccharides, and gelling agents. High polymers are also obtained by crosslinking gelatin itself, but most of them are due to aldehydes such as formalin and glutaraldehyde. It is intended to form a gelled product by cross-linking. Also, most of these crosslinking agents are not edible. Furthermore, when vegetable tannin is used as a cross-linking agent, if the gelatin concentration is low, the reactivity is low, and if the concentration is high, it is difficult to control the molecular weight because it cross-links the entire system.

On the other hand, transglutaminase is known to crosslink the γ-carboxyamide group of glutamine residue and the ε-amino group of lysine residue in protein molecules (FOLK,
JEand FINLAYSON, JS (1977) .Adv.Protein Chem., V
ol. 31, 1) and specific examples used for gelatin are not disclosed. Further, 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 industrially use this. The present inventors have found that in the case of gelatin, a crosslinking reaction does not occur effectively at such a low concentration range, but rather a hydrolysis reaction of a main chain pepride bond is preferred.

Further, as an example of transglutaminase acting in a high-concentration region of gelatin, Japanese Patent Application Laid-Open No. 58-149645 discloses Example 6 of "Method for producing gelled substance", which is a method for producing a gelled substance. The purpose is different from the "production method of highly polymerized gelatin" of the present invention. That is, this method aims at obtaining a gelatin gel by sufficiently cross-linking gelatin, and unlike a normal reversible gelatin gel, this gel does not form a sol by heating, and the dried product only swells, Does not dissolve in On the other hand, the highly polymerized gelatin according to the present invention performs this sol-gel conversion and is distinguished from the gelled product described in the above publication in that it has the basic properties of gelatin. Furthermore, the present inventors, in the case of gelatin, the solution is completely gelled by ε (γ-glutamyl) lysyl cross-linking and completes the cross-linking reaction until a gel is formed,
It has been found that when cooled to room temperature, gelation due to hydrogen bonds inherent in gelatin is prevented, and the gel becomes very brittle compared to the gel of raw gelatin. On the other hand, the gelatin of the present invention is in a solution state at a high temperature, and when gelatinized by cooling, the rubber elasticity of the gelatin, which mainly uses hydrogen bonding of the gelatin itself, exists and is enhanced.

Means for Solving the Problems It is an object of the present invention to improve the viscosity of a gelatin solution, the jelly strength of a gel when cooled and gelled, and the melting temperature by using an enzymatic cross-linking polymerization reaction to reduce the defects of ordinary gelatin. The point is to supplement. That is, a transglutaminase of 0.1 units or more, preferably 0.5 to 3 units per 1 g of gelatin is added to a gelatin solution having a concentration of 2 to 60% by weight, preferably 5 to 40% by weight, and a crosslinking reaction is performed. Deactivate the enzyme before it becomes a gel, or stop it by changing the reaction conditions to produce a lot of oligomers and high polymers of gelatin, have the desired viscosity in solution, and cool to gel. The purpose is to obtain a high polymerized gelatin which has a high jelly strength and a high melting temperature when formed.

Further, the present invention makes use of the characteristics of the enzyme reaction, by appropriately selecting the reaction pH, temperature, etc., and appropriately controlling the reaction rate, to obtain a high polymer of gelatin by a production method which can be industrially sufficiently controlled. The purpose is.

Another object of the present invention is to obtain a useful modified gelatin which can be widely used for food gelatin, industrial gelatin, photographic gelatin and the like.

A feature of the present invention is that a gelatin solution having a concentration of 2 to 60% by weight, preferably 5 to 40% by weight, is added with transglutaminase in an amount of 0.1 unit or more, preferably 0.5 to 3 units per 1 g of gelatin and subjected to crosslinking polymerization. Highly polymerized gelatin with improved physical modification that performs sol-gel conversion in the same way as ordinary gelatin by inactivating the enzyme before changing to a gelled product or changing the reaction conditions and stopping the reaction Is to manufacture.

The gel obtained by cooling the high-polymerized gelatin produced in this way has a larger elongation than the raw gelatin gel and a higher melting point than the raw gelatin gel in normal use, because the cross-linking due to hydrogen bonding of the gelatin itself is added to the cross-linking by the enzyme in addition to the cross-linking by the enzyme. Due to the high temperature, it can be used as high-viscosity gelatin and high-melting gelatin, and when used for food, it has a sticky texture that is difficult to melt in the mouth.

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

Furthermore, the gel obtained by cooling the product subjected to the deactivation operation by this manufacturing method does not undergo a secular change due to the continuation of the enzyme reaction or a water separation phenomenon accompanying the progress of the gelation reaction, and can maintain stability for a long time. It is possible.

According to the method for producing highly polymerized gelatin according to one aspect of the present invention, a method is used in which gelatin is previously polymerized and then dried to obtain highly polymerized gelatin, which is used in the same manner as ordinary gelatin. According to another aspect of the present invention, during the production process of a product containing gelatin, an enzyme can be added before molding to obtain a highly polymerized gelatin. In this case, since the solution used for molding is already completed after the reaction and is stable, it is compared with the method of mixing the unreacted solution with the enzyme in the above-mentioned "method for producing a gelled material" and molding to complete the reaction. It has the advantage that the handling operation is simplified.

The present invention can be applied to all commercially available gelatins (including unrefined gelatin "niwa"), and substantially differs between the types of raw materials used or gelatins produced by different production methods such as acidic gelatin and alkaline gelatin. There is no significant difference. The present inventors have found that it is preferable to use acidic gelatin, but it has been found that the cross-linking reaction proceeds to the same extent even in an alkaline gelatin having a low isoelectric point and a considerably small number of glutamine residues.

The transglutaminase used in the present invention is preferably a transglutaminase produced from actinomycetes `` Streptovertillium '', but the same applies to transglutaminase of another origin such as an enzyme extracted from guinea pig liver. Any material that exhibits a crosslinking reaction effect can be used.

It is preferable that the pH of the gelatin solution used in the present invention is 6-8, but even at pH 4-10, the reaction is possible if it takes a long time. Furthermore, this has a very great advantage that the reaction rate is reduced by removing the optimum pH of the enzyme reaction, the reaction is easily controlled, and the degree of the crosslinking reaction can be arbitrarily selected. The reaction temperature can be selected in the range from the melting temperature of the gel to the inactivation temperature of the enzyme, but is preferably selected in the range of 40-60 ° C. In a special case, after the enzyme is mixed into the gelatin solution, the mixture is immediately cooled to a temperature below the freezing point, and a crosslinking reaction is allowed to proceed for a predetermined time in a gel state. It is also possible to obtain polymerized gelatin.

The deactivation method used in the present invention includes deactivation by high-temperature heating, such as treatment at 75 ° C. or more at pH 7 for 10 minutes or more, or low p.
Denaturation of the enzyme with H, for example, treatment at 40 ° C. at pH 2 for 30 minutes or more. In addition, enzyme reaction inhibitors such as EDTA,
There are various deactivation methods by adding a deactivator. In addition to removing the pH from the optimum pH, the reaction can be stopped by stopping the reaction in a salt solution at a high temperature or by adding a low-molecular-weight reaction substrate such as an amino acid or a peptide. I can do it. Furthermore, when the enzyme concentration is low, the deactivation reaction also proceeds during the crosslinking reaction, and the enzyme may be actually deactivated during the reaction.

The production of the high polymer gelatin according to the present invention does not inhibit the enzymatic reaction in the gelatin solution, and includes polysaccharides, starch, dextrin, glycerin, gums, organic acids, sugars, inorganic salts, seasonings, coloring agents, spices, Paste, thickener, stabilizer,
Emulsifiers and other additives usually added in the use of gelatin can be blended.

Hereinafter, the present invention will be described in more detail based on examples and comparative examples, but this does not limit the present invention.

Example 1 Highly polymerized gelatin was produced as follows.

Water was added to 15 g of an acidic gelatin having a jelly strength of 244, a viscosity of 40, a melting point of 30 ° C. and an isoelectric point of 9.1 so as to have a concentration of 15% by weight, and after swelling, dissolved at 50 ° C. and adjusted to pH 7 with caustic soda. Transglutaminase Ajimoto's product; 12 mg of hydroxamic acid method (specific activity according to J. Biol. Chem., Vol. 241, 5518 (1966); 2.5 units / mg) suspended in a small amount of water and added After that, stirring was continued at 50 ° C. After 10 minutes, the solution was heated on a hot plate to rapidly raise the temperature to 80 ° C. and kept for 5 minutes to inactivate the enzyme. Thereafter, the solution was immediately immersed in a cold water bath, rapidly cooled to 60 ° C., poured into a stainless steel plate, and cooled in a refrigerator. The film-like gel having a thickness of 1 mm thus obtained was used as it was for measurement as a gel. The dried product was obtained by shredding this gel into a width of 3 mm, drying and crushing.

For comparison, a gelatin gel was produced in accordance with the "method for producing a gel" described in JP-A-58-149645, and the measurement was carried out together with untreated raw gelatin. The gelatin gel was produced in the same manner as described above, except that an enzymatic reaction was carried out until the gelatin solution gelled, and then cooled and cut into a desired shape.

The molecular weight distribution of the high polymer gelatin thus produced was measured by liquid chromatography and is shown in FIG.
(Column; Asahipak GS-620: mobile phase; M / 10 phosphate buffer: flow rate; 0.5 ml / m: temperature; 50 ° C .: sample; 0.5% * 20 μl: filtration: filter paper 5 μ: detection ultraviolet wavelength; 230 nm) In the gelatin produced by the method according to the example, the high-molecular-weight component more than the γ component is clearly increased as compared with the raw material gelatin, and a highly polymerized gelatin is formed.

The gel thus obtained was subjected to an original differential scanning calorimeter (Perkin Elmer DSC-7: sample gel; / 27 mg: heating rate; 5
FIG. 2 shows the data measured at (° C./m). Looking at the melting peaks of micelles in which the constituent polypeptide chains of gelatin form cross-linking points of the gels associated by hydrogen bonding, the gel obtained by the method according to this example shows a melting point higher than that of the raw gelatin. The temperature has a shoulder to a slightly high temperature, and the heat of fusion is increasing. This is a micelle due to hydrogen bonding caused by cooling, as in the case of raw gelatin, although the structure of the gel is slightly disordered,
The increase in enthalpy from 15.6 J / g to 27.7 J / g indicates that it is further stabilized by high polymerization. On the other hand, the gel according to the above-mentioned "method for producing a gelled substance" has a peak at the high temperature side and the melting temperature of the gel itself is high, but the enthalpy is only 9.4 J / g, and the transglutaminase at a high temperature is high. The cross-linking of covalent bonds by H. prevents the cross-linking by hydrogen bonding on cooling, indicating that the gel essentially differs from the gel according to the invention.

The physical properties of the gelatin obtained by the method according to this example are JIS
Jelly strength 250, viscosity 105, 10% gel melting temperature 48 ° C
Met. This is improved compared to the physical properties of the raw material gelatin.

Tensile test of gel obtained by cooling gelatin obtained by the method according to this example (Fudo rheometer: pulling speed; 6
cm / m) are shown in FIG. Compared to raw gelatin,
Clearly, the breaking strength and elongation are large. In addition, the gel obtained by the "method of producing a gelled material" has a small elongation, is brittle, and breaks immediately, indicating that the gel is formed by a small number of covalent bonds, not by general crosslinking by hydrogen bonds. .

Example 2 The same gelatin as in Example 1 was added with water to a concentration of 6% by weight based on 6 g of gelatin. After swelling, the mixture was dissolved at 40 ° C., and pH was adjusted with sodium hydroxide.
Adjusted to 7. Transglutaminase 4.8 was added to this aqueous solution.
After adding a suspension of mg in a small amount of water, stirring was continued at 40 ° C. Thirty minutes later, the solution was heated on a hot plate to rapidly raise the temperature to 80 ° C., and kept for 5 minutes to inactivate the enzyme. Then, the solution was immediately immersed in a cooling bath, rapidly cooled to 60 ° C., poured into a stainless steel plate, and cooled in a refrigerator. This gel was shredded, dried and then crushed to obtain a dried product.

The melting temperature of the 10% gel of the high-polymerized gelatin thus produced was 45 ° C. This is an improvement over the melting temperature of the raw gelatin. At this gelatin concentration and reaction temperature, a similar melting temperature was obtained even when the reaction time was 25-60 minutes.

Example 3 Water was added to 20 g of an alkali method gelatin having a jelly strength of 260, a viscosity of 40, a melting point of 31 ° C. and an isoelectric point of 5.0 so as to have a concentration of 20% by weight. When you measure this pH
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, the solution was heated on a hot plate to rapidly raise the temperature to 80 ° C. and kept for 5 minutes to inactivate the enzyme. Thereafter, the solution was immediately immersed in a cold water bath, rapidly cooled to 60 ° C., poured into a stainless steel plate, and cooled in a refrigerator. The thus obtained gel in the form of a film having a thickness of 1 mm was used as it was in the measurement. The dried product was obtained by shredding this gel into a width of 3 mm, drying and crushing.

The physical properties of gelatin obtained by the method according to this example were jelly strength 250, viscosity 110, and melting temperature of 10% gel at 60 ° C. This is improved compared to the physical properties of the raw material gelatin.

FIG. 4 shows the results of a tensile test of the gel obtained by cooling the gelatin obtained by the method according to the present embodiment. The breaking strength and elongation are clearly higher than those of the raw gelatin.
Further, the gel obtained by the above-mentioned "method for producing a gelled material" has a small elongation, is easily cut, and is brittle.

Example 4 The same 40 g of gelatin as in Example 3 was prepared by adding 16 g of glycerin and 77 g of water.
After swelling by pouring into a mixture of the above, the mixture was dissolved at 60 ° C. and adjusted to pH 7 with sodium hydroxide. After 16 mg of transglutaminase was suspended in a small amount of water and added 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. and kept for 5 minutes to inactivate the enzyme. Then immediately immerse the solution in a cold water bath and quench at 60 ° C,
Poured into stainless steel plate and cooled in refrigerator. The film-like gel having a thickness of 1 mm thus obtained was used as it was for measurement as a gel.

For comparison, a gel having the composition of the present example obtained by pouring it into a stainless steel plate without cooling at 80 ° C. without cooling and leaving the mixture at room temperature for 2 days was measured.

The melting temperature of the gel obtained by the method according to this example is 50 ° C.
Met. This is the melting temperature of the gel made from raw gelatin
Improved compared to 40 ° C. FIG. 5 shows the results of the tensile test of the gel. The elongation to the break point is clearly larger than that of the gel not treated with the enzyme. Also, the gel in which the enzyme was not inactivated has a small elongation and is brittle.

Example 5 Water was added to 75 g of an alkali method gelatin having a jelly strength of 185, a viscosity of 60, a melting point of 28 ° C. and an isoelectric point of 5.1 to a concentration of 15% by weight, and after swelling, was dissolved at 40 ° C. When this pH is measured, 6.1
Met. After transglutaminase (30 mg) was suspended in a small amount of water and added to the aqueous gelatin solution, stirring was continued at 40 ° C. Forty minutes later, when the solution began to thicken, hydrochloric acid was quickly injected to lower the pH to 4.2. Next, the solution was cooled to 30 ° C., adjusted to pH 2 with hydrochloric acid, and poured into a stainless steel plate.
Immediately gelled with cold water. After leaving the enzyme at room temperature for 1 hour to inactivate the enzyme, the gel was transferred to a refrigerator. The 3 mm thick film gel thus obtained was cut into 5 mm widths, placed in a net basket and immersed in running water for 3 hours. 40 swollen gel
When dissolved at ° C, the gelatin concentration was 5% by weight and the pH was 3.7. The mixture was poured into a stainless steel plate, gelled, and dried with cold air to obtain a gelatin polymer.

The gel obtained by cooling the gelatin obtained by the method according to this example had a melting temperature of 40 ° C. This is an improvement over the melting temperature of the raw gelatin gel at 28 ° C at pH 3.7.

Example 6 Water was added to 6 g of the same gelatin as in Example 5 to a concentration of 6% by weight, and after swelling, was dissolved at 40 ° C. After 4.8 mg of transglutaminase was suspended in a small amount of water and added to the above gelatin aqueous solution, stirring was continued at 40 ° C. After 70 minutes, where the solution thickened, 10 ml of acetic acid was quickly added to stop the reaction. The pH of this solution was measured to be 3.5.
Thereafter, the solution was poured into a stainless steel plate and cooled in a refrigerator.

The gel obtained by cooling the gelatin obtained by the method according to this example had a melting temperature of 33 ° C. This is an improvement over the melting temperature of the raw gelatin gel at 27 ° C at pH 3.2.

Effect of the Invention 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 high-polymerized gelatin of the present invention has a larger elongation to tension and a higher melting temperature than the raw gelatin gel in a normal use condition because the cross-linking by the gelatin itself is added to the cross-linking by the enzyme in addition to the cross-linking by the enzyme. Available as high viscosity gelatin and high melting point gelatin,
When used for edible use, it has a sticky texture that is hardly soluble in the mouth.

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

Furthermore, the gel obtained by cooling the high-polymerized gelatin subjected to the inactivation operation of the enzyme by this production method is free from a secular change due to the continuation of the enzymatic reaction and the water separation phenomenon accompanying the progress of the gelation reaction, and has a long time. It is possible to maintain stability over time.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a liquid chromatography chart showing the molecular weight distributions of highly polymerized gelatin and raw gelatin. FIG. 2 shows a gel of 15% by weight of each of the high-polymerized gelatin prepared in Example 1 and the starting gelatin.
4 is a chart showing the results of differential scanning calorimetry of gels of the same concentration obtained by “Method for producing gelled product” described in JP-A-149645. FIG. 3 is a stress-strain curve of a 15% by weight gel of the high-polymerized gelatin and the raw material gelatin produced in Example 1 and a gel of the same concentration obtained by the above-mentioned "Method for producing gelled product" in a tensile test. It is. FIG. 4 is a stress-strain curve of a tensile test of a gel of 20% by weight of each of the high-polymerized gelatin and the raw material gelatin produced in Example 3 and a gel of the same concentration obtained by the above-mentioned "Method for producing gelled product". It is. FIG. 5 shows the stress-strain curves of the high-polymerized gelatin prepared in Example 4 and the glycerin-containing gel of the raw material gelatin and the tensile test of the same composition obtained by the above-mentioned “Method for Producing Gelation”.

Claims (1)

(57) [Claims] (1) To a gelatin solution having a concentration of 2 to 60% by weight, 0.1 g or more of transglutaminase per 1 g of gelatin is added to carry out cross-linking polymerization, and then the enzyme is lost before the gelatin solution becomes a gel. A method for producing highly polymerized gelatin, which comprises activating or terminating the reaction.