JPH0787968A - Promotion of transglutaminase reaction - Google Patents

Abstract

PURPOSE:To effectively obtain a protein-containing material having improved properties and palatability, etc., by treating protein of a substrate with transglutaminase after adding monosodium L-glutamate to promote protein- bridging reaction. CONSTITUTION:After adding 1.0-30g of monosodium L-glutamate, the substrate consisting of 1.0g of protein (e.g. alpha S1-casein) is subjected to enzyme reaction with 0.01-2000 U of transglutaminase at 5 deg.C for 12hr. This process promotes the protein-bridging reaction by transglutaminase and effectively performs the improvements of heat resistance and water retention of gelatinized protein and palatability, e.g. elasticity of protein-containing material. Further, an enzyme which is produced by a microorganism, e.g. an actinomyces: Streptoverticillium griseocarneum IFO 12776 or extracted from an animal organ, e.g. the liver of a guinea pig or the heart of a fish is preferably used as the transglutaminase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for promoting the reaction of transglutaminase to efficiently improve the quality of a protein-containing material. According to the present invention, it is expected that the enzymatic reaction time will be shortened and the deterioration of the quality of the protein-containing material and the proliferation of microorganisms that occur during the enzymatic reaction will be reduced. Examples of the quality improvement of the protein-containing material include acceleration of gelation of protein, improvement of heat resistance and water retention of gelled product of protein, improvement of texture such as elasticity of protein-containing material.

[0002]

BACKGROUND ART Transglutaminase is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain. Parachloromercury benzoic acid, N-ethylmaleimide, monoiodoacetic acid and the like are known as substances that inhibit the reaction, but no substance that promotes the reaction is known.

[0003]

The object of the present invention is to promote the protein cross-linking reaction of transglutaminase.

[0004]

The transglutaminase used in the present invention (hereinafter referred to as the present enzyme) includes the actinomycete Streptoverticillium griseocarneum (Str).
eptoverticillium griseoca
rneum) IFO 12776, Streptoverticillium cinnamonium sub-SP cinnamonium (Streptoverticillium cinn)
aoneum sub sp. cinnamoneu
m) IFO 12852, Streptoverticillium
Mobara Ensu (Streptoverticilliu)
m mobaraense IFO 13819, Streptomyces
sp. ) No. 83 and Streptomyces lavendulae
e) No. There are the present enzyme produced by microorganisms such as 466 and the present enzyme extracted from animals such as liver of guinea pig and heart of fish. The microorganism-produced enzyme is calcium-independent, whereas the animal-derived enzyme is calcium-dependent.

The present enzyme has the following physicochemical properties of 1 to 11. 1. Action Crosslinks the γ-carboxamide group of glutamine residues and the ε-amino group of lysine residues in protein molecules.

2. Optimum pH The optimum pH of this enzyme derived from actinomycetes is 6 at the time of reaction at 37 ° C. for 10 minutes using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates.
Approximately 7 and the optimum pH of this enzyme of animal origin under the same conditions
Is around 6.

3. Optimum temperature The optimum temperature of this enzyme derived from actinomycetes is 4 when reacting with benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates at pH 6 and 10 minutes.
The temperature is around 5 to 55 ° C, and under the same conditions, the enzyme of animal origin is 5
It is around 0 to 55 ° C.

4. pH stability This enzyme derived from actinomycetes retains almost 100% activity when kept at 37 ° C for 10 minutes at pH 5-9, and this enzyme derived from animal was kept at 37 ° C for 10 minutes at pH 6-7.5. Sometimes almost 100% activity remains.

5. Temperature stability The enzyme derived from actinomycetes retains 74% activity when kept at pH 7 at 50 ° C for 10 minutes, and the enzyme derived from animal retains 40% activity under the same conditions.

6. Substrate specificity This enzyme does not react when the substrates are benzyloxycarbonylasparaginylglycine, benzyloxycarbonylglutamine and glycylglutaminylglycine. However, the reactivity is highest when the substrate is benzyloxycarbonylglutaminylglycine.

7. Effect of Inhibitor The activity of this enzyme is inhibited by parachloromercury benzoic acid, N-ethylmaleimide and monoiodoacetic acid.

8. Effect of metal ion The activity of this enzyme is inhibited by copper ion and zinc ion.

9. Isoelectric point The present enzyme derived from actinomycetes has a pI of 9 to 10, and the present enzyme derived from an animal has a pI of 4.5 (by ampholine isoelectric focusing).

10. Molecular weight The present enzyme derived from actinomycetes is around 40,000, and the enzyme derived from animals is around 90,000 (by SDS disc electrophoresis).

11. Enzyme activity measurement method Reagent A (0.2 M Tris-HCl buffer (pH 6.0), 0.1 M hydroxylamine,
Consisting of 0.01M reduced glutathione and 0.03M benzyloxycarbonyl-L-glutaminylglycine)
Add 0.5 ml and mix and react at 37 ° C. for 10 minutes. Then 0.5 ml of Reagent B (consisting of 3N hydrochloric acid, 12% trichloroacetic acid and 5% ferric chloride (dissolved in 0.1N hydrochloric acid)) was added to stop the reaction to form an iron complex, and then 525 nm Measure the absorbance at. As a control, the absorbance of the same reaction was performed using a solution in which the enzyme was inactivated by heating in advance, and the absorbance was measured to determine the difference in the absorbance with the enzyme solution. Separately, a calibration curve is prepared using L-glutamic acid γ-monohydroxamic acid instead of the present enzyme solution. The enzyme unit that produces 1 μmol of hydroxamic acid per minute is defined as 1 unit (1 U), and the desired unit of the enzyme solution is calculated.

The protein as a substrate has a lysine residue and a glutamine residue, and is not limited by its origin and properties as long as it is acted on by this enzyme, it is not limited to plant protein or animal protein. Any microbial protein, algal protein, etc. can be used. In addition, proteins other than those mentioned above, which are partially cleaved with protease, synthetic peptides, and various chemically modified proteins having a glutamine residue or a lysine residue are also substrates for this enzyme.

Examples of the protein-containing material are seafood, beef (beef, pork, lamb, etc.) chicken, soybean, wheat, rice, milk,
Gelatin, nuts, etc. and their processed products such as fish meat, fish paste, ham / sausage, soy milk, soybean tissue protein, vegetable protein powder, gluten, noodles, cakes, etc.
There are biscuits, cheese, ice cream and yogurt.

L-glutamic acid monosodium salt (hereinafter referred to as MSG) is an umami component of kelp. MSG
Decomposes gluten or Micrococcus glutamicus
It is obtained by fermentation.

M or a protein-containing material may be added to M under the following conditions:
SG is added and the enzyme is allowed to act. However, the mixing order of these three is not specific and can be appropriately performed as necessary.

When the protein-containing material can be kneaded with a liquid or marine product such as milk, and additives such as noodles can be kneaded during its production, the enzyme and MSG may be directly added to the protein-containing material. . It is preferable that MSG and this enzyme are added in this order or simultaneously. When the protein-containing material is a solid such as meat or rice, it is difficult to add it directly, so the protein-containing material is dipped in an aqueous solution containing MSG and an aqueous solution containing the present enzyme for a certain period of time.
As for the dipping method, it is preferable to carry out MSG and the present enzyme in this order or at the same time as in the above-mentioned addition method. The working conditions are as follows: MSG 0.1 to 30 g is added to protein 1.0 g, and the enzyme 0.001 to 2000 U is added to 0 to 60 g.
It is operated at a temperature of 5 ° C and a pH of 5 to 10. MSG, the action amount of the enzyme, the reaction time, the reaction temperature, the pH, etc. are adjusted according to the desired quality improvement level based on the above-mentioned physicochemical properties.

[0021]

[Action] When the enzyme acts on the protein serving as the substrate, M
Addition of SG promotes the crosslinking reaction between the γ-carboxamide group of the glutamine residue and the ε-amino group of the lysine residue in the protein molecule. Therefore, the effect of accelerating the crosslinking reaction by adding MSG was investigated in the following manner.

As a substrate protein, 1.0% αS1-
Using 10 ml of an aqueous casein solution, 0.5 g of MSG was added at the same time as 1.0 U of the present enzyme to carry out an enzyme reaction at 5 ° C. for 12 hours, and then the change in the molecular weight of αS1-casein in the reaction solution was measured by gel filtration. . In addition, the molecular weight of αS1-casein was measured by gel filtration in the same manner by using the one without addition of the present enzyme and MSG as a control solution and the one without addition of MSG as a comparison solution. The results of gel filtration of the reaction solution are shown in FIG. 2, the results of the control solution are shown in FIG. 3, and the results of the comparative solution are shown in FIG. The measurement conditions of gel filtration at this time are as follows. 1. Column: TSKgel G300SWXL 7.5 x 6
00 mm 2. Eluent: 0.1M phosphate buffer (containing 0.3M sodium sulfate) 3. Flow rate: 0.5 ml / min. 4. Temperature: room temperature 5. Detection: UV280nm

[Figure 1]

[Fig. 2]

FIG. 3 shows that the molecular weight of 298,803 (3
4.46%), 36,274 (59.19%) of αS1
-When the enzyme acts on casein, the molecular weight becomes 265,5.
83 (74.53%) was polymerized, and when MSG was added to act this enzyme, the molecular weight was 469,970 (87.9).
It can be confirmed that the polymer is further polymerized to 5%).
The concentration of free MSG remaining after the acid was added to the reaction solution to precipitate the protein was 0.1 g / 10 ml, which was the same as the concentration before the reaction. That is, it is considered that MSG is not introduced into αS1-casein in the reaction solution, and the action of MSG promotes the cross-linking reaction of this enzyme.

[0023]

[Example] (Example 1) 100 m of 10% sodium caseinate aqueous solution
5 g of MSG was added at the same time as 10 U of this enzyme, and allowed to act at 5 ° C. for 12 hours. The viscosity of this reaction solution was measured using a B-type viscometer.

(Comparative Example 1) In Example 1, an aqueous solution without addition of MSG and the present enzyme is Comparative Example 1-1, an aqueous solution without addition of the present enzyme is Comparative Example 1-2, and MSG is not added. This aqueous solution was used as Comparative Example 1-3. The viscosity of each aqueous solution was measured in the same manner as in Example 1. Example 1
Table 1 shows the measurement results of Comparative Example 1-1, Comparative Example 1-2, and Comparative Example 1-3.

[0025]

[Table 1]

(Example 2) 100 g of raw peeled shrimp (Brown variety from India) was immersed in 70 g of a 5% MSG aqueous solution, refrigerated and allowed to stand for 3 hours, and then 10 U of this enzyme was added to this aqueous solution.
Was added, and the mixture was refrigerated and left overnight. A retort pouch was filled with 20 g of shrimp treated with the enzyme and 100 ml of water, sealed, and then retort-heated at 121 ° C. for 20 minutes to obtain a retort-heated shrimp (hereinafter referred to as retort shrimp).
A sensory evaluation was performed on the retort shrimp produced in the above manner.

(Comparative Example 2) A retort shrimp produced by the same procedure as in Example 2 except that MSG and the present enzyme were not added, and Comparative Example 2-1 was obtained without adding the present enzyme. A retort shrimp was produced by the same procedure except for Comparative Example 2-2, and MSG was not added, and a retort shrimp was produced by the same procedure as Comparative Example 2-3. Sensory evaluation was performed on the produced retort shrimp in the same manner as in Example 2. Table 2 shows the evaluation results of Example 2, Comparative Example 2-1, Comparative Example 2-2, and Comparative Example 2-3.
Summarized in.

[0028]

[Table 2]

[0029]

[Effect] According to the present invention, the protein cross-linking reaction by transglutaminase can be promoted, and the quality of the protein-containing material can be improved in a shorter time than when only transglutaminase is allowed to act.

[0030]

[Brief description of drawings]

FIG. 1 shows the distribution of the molecular weight of 1.0% αS1-casein reacted with this enzyme and MSG by gel filtration.

FIG. 2 shows the molecular weight distribution of 1.0% αS1-casein by gel filtration.

[Fig. 3] 1.0% α reacted with this enzyme by gel filtration
The molecular weight distribution of S1-casein is shown.

Claims (3)

[Claims] 1. A method for accelerating a transglutaminase reaction, which comprises adding L-glutamic acid monosodium salt when acting transglutaminase on a protein serving as a substrate. 2. A transglutaminase, wherein L-glutamic acid monosodium salt is added when a transglutaminase is allowed to act on a material containing a substrate protein in an amount of 1.0% by weight or more (hereinafter referred to as a protein-containing material). To accelerate the reaction of. 3. L-glutamic acid monosodium salt 1.0 to 30 g is added to protein 1.0 g, and transglutaminase 0.01 to 2000 U is allowed to act. A method for accelerating the transglutaminase reaction described.