JPS58149645A - Preparation of gelatinized material - Google Patents

Abstract

PURPOSE:To obtain a gelatinized material, by adding transglutaminase to a protein-cotaining solution. CONSTITUTION:Both vegetable protein such as defatted soybean, etc. and animal protein such as milk protein, gelatin, collagen etc. can be used. When 1 unit transglutaminase based on 1g protein is added to a solution containing 2-15wt% protein, and, if necessary, a polysaccharide, seasoning, coloring matter, etc., the solution is gelatinized in a short time, to give a gelatinized material stable to heat. Transglutaminase forming crosslinking by covalent bonds between the residues of glutamin and lysine, extracted from the liver of a guinea pig is used as the transglutaminase.

Description

[Detailed description of the invention] The present invention relates to a novel method for producing a gelled product.

Among existing protein resources, the use of many of them is restricted due to reasons such as low biological value and poor functional properties. If a technology were established to intentionally modify these protein resources into functional and nutritious protein materials suitable for organized food, it would not only increase their utilization but also produce high-quality proteins. Can make food. Enzyme modification as one of the modification techniques
Although there are other types of modification, currently the main modification is based on hydrolase t, and there are few examples of the use of other enzymes.

The present inventors focused on transglutaminase, which is one of the acyltransferases, and formed crosslinks by opening glutamine (abbreviated as Gin) residues and lysine (abbreviated as Lys) residues, which have a high T content in food proteins. They discovered that it was possible to produce a gel-like substance, and completed the present invention.

That is, the present invention is a method for producing a gelled product, which is characterized by adding 1 unit or more of transglutaminase to 1 protein If to a protein-containing solution having a protein concentration of 2% by weight or more to form a gel.

The protein used in the present invention is not limited in its origin, and any protein such as vegetable protein or animal protein can be used. Examples of vegetable proteins include defatted oilseed products (defatted soybeans) and proteins separated from them. Furthermore, examples of animal proteins include milk protein, gelatin, and collagen.

A protein-containing solution containing 2% by weight or more of these proteins is prepared. The concentration of the protein-containing solution is desirably relatively high, usually 2% by weight or more, preferably 5% to 15% by weight.
That's fine. In this case, food additives such as starch, polysaccharides, seasonings, colorants, and spices can be added.

These amounts may be selected and added as appropriate within a range that does not inhibit subsequent gelation by transglutaminase.

When the concentration of the protein solution is less than 2 weights, it remains in a solution state or precipitates and does not gel. Further, the pH of the protein-containing solution is preferably 6 to 9.

Transglutaminase was added to this protein-containing solution.
1 unit or more is added to the gel to form a gel. This transglutaminase was prepared using the method of Connellan et al. (
Journal of Biological Chem
istry, 246(4), 1093 (1971
) Prepared from guinea pig liver according to ). That is, guinea pig liver is dispersed in a sucrose solution, centrifuged, the supernatant is collected, and this is fractionated using a diethylaminoethyl cellulose column to obtain crude transglucosidase.

This is precipitated with 1% protamine sulfate and the precipitate is collected. Further, this precipitate was washed with 0.2 M Tris-acetate buffer, and then washed with 0.05 M ammonium sulfate-5 mM Tris.
-Extraction using HCI buffer (containing 2mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA)), extracting the resulting extract using carpoxymethylcellulose to remove muteflotamine, and adding ammonium sulfate solution (IMED) to the oral fluid.
(contains TA) and centrifugation is performed to collect the precipitate. The precipitate was diluted with 10mM Trim-acetate buffer (1m
MEDTA.

The supernatant was dissolved in 10% agarose (Bio Get A-0) and centrifuged.
, 5M), and the resulting highly active fraction is concentrated by ultrafiltration to obtain purified transglutaminase.

Other methods for preparing transglutaminase include C1a
rke et al.'s method (Archives of Bioch
emistryand biophysics+ 79
+338 (1959)). That is, 30097 pieces of guinea pig liver, 600 pieces of 0.25 M sucrose solution.
- and homogenize.

Centrifuge this to obtain a supernatant.

Add sodium acetate to a concentration of 0.01 M, adjust the pH to 5, Or- with acetic acid, and centrifuge. 0.06 M in the resulting precipitate! Add 30 m of J acid buffer (pH 6,5) and homogenize.

This suspension is centrifuged, the supernatant is dialyzed against 0.001 M phosphate buffer (pH 7.5), and this is used as a crude transglutaminase solution.

In these methods, the order of operations may be changed or the amount added, concentration, pH value separation device, etc. may be slightly changed. The protein concentration of transglutaminase obtained in this way was measured using the p-z method (Journal of Biology).
cal Chemistry+ 193, 266
(1951)), the enzymatically active N-carbobenzoxy-
Hydroxamic acid method using glutaminylglycine and hydroxyamine (Journal of Biolog
ical Chemistry, 241 (23)
+5618 (1966)), the specific activities of the prepared enzyme solutions range from 6.0 to 13.0. Furthermore, when the molecular weight is measured by electrophoresis, the value is in the range of 80,000 to 90,000. This transglutaminase solution can be stored at a low temperature of about -3071' and thawed for use.

One or more units of transglutaminase thus obtained is added to each protein to form a gel. If the amount added is less than 1 unit, the solution becomes highly viscous, and even if more than 2000 units is added, the effect does not change much.

Glu-Lya is added to protein molecules by transglutaminase.
It is known that crosslinking occurs (J, E, Fo
lkAnfinsen+ J, T, Edsall
and F, M, Richards+Academ
ic Pre6Tn1. , New York, N.
It was inferred from the following experimental data that the gel generated when transglutaminase is applied to a protein solution is due to Glu-Lys crosslinking.

■ Lys serves as the reaction site for transglutaminase
Even when transglutaminase was applied to αs1 casein with acetylated and succinylated residues, no gelation occurred.

■ Because the reaction is carried out in the coexistence of dithiothreitol, a C15-S reducing agent, in the reaction solution, it is not a gel mainly composed of S-S bonds. ■ A normal gelatin gel obtained by heating and cooling. When we measured the elastic modulus of each gelatin gel gelated with gelatin and transglutaminase, we found that the elastic modulus of normal gelatin gel decreased significantly as the temperature increased. This is thought to be because the crosslinks that form the gel network structure are secondary bonds, rather than strong bonds such as covalent bonds, and these weak bonds break as the temperature rises. Compared to this gel, the amount of change in the transglutaminase gel was small even when the temperature changed, suggesting that it is a gel with strong covalent bonds. In fact, when both gels were exposed to 4 volts, the transglutaminase gel remained intact, but the normal gelatin gel melted.

As mentioned above, the gel is generated by Glu-Lys crosslinking, and it is thought that the gel is not due to S-s crosslinking.

The thus obtained gelled product gels in a relatively short time, that is, within 1 minute, and within 30 minutes at the most, and has gel properties equivalent to those of general gelled products.

Furthermore, the protein-containing solution used in the present invention is not limited to a simple mixture of protein and water, but may also be an emulsion containing a mixture of protein, water, and oil.

Furthermore, by heating this gelled product, a stronger gel can be made.

The gelled product of the present invention can be used as yogurt, jelly, etc. in the same way as conventional gelled foods, and can be produced without heating and is a heat-stable gel, so it can be used as a material for microcapsules or as an immobilized enzyme. It can also be used as a material.

Example 1 Transglutaminase was prepared by the following method. Approximately 2 ounces of a cold 0.26 M guinea pig liver solution at 800 fC was added, homogenized at 20,000 rpm for 2 minutes, and centrifuged (106,0 OOXf, 5 C for 11 hours) to obtain a supernatant.

Add this to 5mM*)jjsuψHCl buffer (2mM EDTA
After adding and adsorbing DEAE to a cellulose column equilibrated with pH 7.5), fractionation was performed using a gradient elution method in which the saline concentration of the above buffer was varied from 0M to 1.0M. I got both.

While stirring this slowly, add 40% of protamine monothiosulfate.
Add jI/ and centrifuge (14,600Xf.

Collect the precipitate (SC) for 15 minutes, suspend it in 0.2 M) Lis-acetate buffer (pH 6,0), homogenize, wash, and centrifuge (2,600Xf, 1 minute, 5t'). Then, the precipitate was collected.

From this precipitate, 5mM containing 0.05M ammonium sulfate)! A hydrochloric acid buffer solution (containing 2mM EDTA, pH 7.5) was added, homogenized, and transglutaminase was extracted. This was repeated three times, and the collected extract was added to a carboxymethyl cellulose column equilibrated with 5mM) lis-succinate buffer (containing 2mM EDTA, p) (6,0) to remove protamine, and the filtrate was IM EDTA (p)18.0) 2.4 wi
After adding t and 47.4 f of ammonium sulfate and stirring well, the precipitate was collected by centrifugation (!5,000Xf, 10 minutes, 5C).

This is 10mM)! JS Φ acetate buffer (1mM EDTA
Contains 0.16 M KCI, pH 6,0) and centrifugation (27,000xr, 30 minutes, SC)
After removing poorly soluble substances, the supernatant was equilibrated with the same buffer solution (1% agarose (Bio Gcl A-0,5
Gel filtration is performed with M), the highly active fraction is collected, and this is subjected to ultrafiltration (UM) to a concentration of 10 to 20 m
-10, manufactured by Amicon) to prepare a transglutaminase solution. This solution was stored frozen at -30C or below, dissolved and used at appropriate times (this solution was prepared by constantly operating at 5C).

Table 1r: Transglutaminase was allowed to act on the indicated substrate proteins to obtain gelled products.

Table 1 *I C, As Zittle et al.
, J, Diary Sci, +46 +1183(
+963) *2 V, H, Thanh et al, J,
Agric, FoodChem, +24(6),
+117 (1976) Example 2 α-casein, Na-caseinate, soybean protein 1 11S globulin, water-extracted soybean protein, 500mg each were mixed with 0.1M) Lis-HCl buffer (5mM CaC1,
, containing 20mM dithiothreitol, pH 7,6)3.
5d? Dissolve this and add 1.5 d of soybean oil to it for 200 min.
Homogenization was performed for 3 minutes at 0.00 rpm to obtain a homogenized emulsion.

To this, transglutaminase was added at 0.00% per 1 kg of protein.
When 09 units were added, gelatin was obtained immediately.

Example 3 A 2.5.10% by weight solution of α, l-casein, soybean protein 115 globulin, and soybean protein 7S glypurin was added to 0.1
M) Lis-HCl buffer (5mM CaC11,20
Transglutaminase was added to each protein at 37C for 0.5 d at pH 7.6) containing mM dithiothreitol.
It was added at a ratio of 0.1 unit to m91C to determine whether gelation occurred, and the results shown in Table 2 were obtained.

Table 2 0 gelatinization △: weak gel ×: remain as solution Example 4 5 weight ratio solution of αal casein and soybean protein I! A 10% by weight solution of S globulin was prepared with 0.1 M) Lis-HCl buffer (containing 5mM CaCIQ120mM dithiothreitol, pH 7.6), and these solutions were diluted with 0.1M (containing 5mM CaCIQ120mM dithiothreitol, pH 7.6).
For each 8-r pair, add transglutaminase to 1 m of protein.
When 5 x 10 to 2.0 units per gram were added and it was observed whether gelation occurred or not, the results shown in Table 3t were obtained.

Table 3 ■ = Immediate gelation. : Gelled for over 1 hour, but weak gel ×: Still in solution Example 5 A Tris-HCl buffer solution containing 11 g of 5 mM Ca and 20 mM dithiothreitol at pH 7.0 to pH 9.0 was prepared, and using 5% by weight α8. Casein solution and 10 wt% soy protein 115 globulin solution were each mixed with 0.8-
0.1 unit of transglutaminase was added to 11 g of protein, and it was observed whether gelation occurred. The results are shown in Table 4t.

Table 4 @: Immediately gelled ○: Slightly required time for gelation ×: Still in solution Example 6 Diameter 9.3tts, height 1.5d? Pour 1 m of the sample solution into a test piece preparation container, gel it as shown below to create a cylindrical gel, and apply it using a rheogram (Toyo Seiki Seisakusho ■, CV-100) with a 18 to 25 trowel.
The storage elastic modulus at each temperature was measured.

■ Add water to the gelatin to make a 10% gelatin cooling gel solution, completely dissolve the gelatin in 60211' for 3 minutes, and then add 1T.
111 was poured into a test piece preparation container and left to stand for 20 minutes in 3Cr-coated water to gel and return to room temperature for measurement.

■ Gelatin TGa@e Gel Add 0.1M Tris-HCl solution (contains 5mM CaCl, 20mM dithiothreitol, pH 7.6) to gelatin to make a 10% solution by weight, and completely dissolve gelatin at 60r for 3 minutes. Then, the mixture was poured into a test piece preparation container, and immediately transglutaminase was added at a ratio of 0.1 unit to 1 g of gelatin, and the gelatin was left to gel for 1 hour at room temperature, followed by measurement.

I will show you the results for the second time. The storage modulus of gelatin cooled gel decreases significantly as the temperature increases, but in comparison, gelatin TGase gel was less affected by temperature changes.

Example 7 α5, 5% by weight for casein and 10% by weight for soybean 115 globulin (0.1 M)
1 ml of lysate-hydrochloric acid buffer (containing 5mM CaCl, 20mM dithiothreitol, pH 7.6) was prepared, and 0.1 unit of transglutaminase was added to the protein IQE to obtain a gel. Add this gel to
After maintaining the temperature at 100 rpm for 20 minutes, it was cooled to room temperature.

The gel immediately after gelation and the heat-treated gel were measured using a rheometer (Fudou Kogyo ■, NRM-2002J).
The maximum load when a plunger (5Φ, pole type) was inserted was measured and defined as gel strength. The results are shown in Table 5.

Table 5 As can be seen from the above table, in both cases, the gel strength increased with heat treatment.

[Brief explanation of drawings]

FIG. 1 shows the results of Example 6. In the figure, the horizontal axis is temperature (r)
, the vertical axis is the storage modulus (dyn/d), the solid line shows the gelatin TGa@e gel of the present invention, and the broken line shows the gelatin cooling gel. Patent Applicant□ Ajinomoto Co., Inc. 11 N) Bottom C°C Procedural Amendment October 1980 et al. Kazuo Wakasugi, Commissioner of the Japan Patent Office 1, Indication of the Case, Patent Application No. 31978, 1982, 2, Title of Invention: Gelified Product Manufacturing method 3, relationship with the case of the person making the amendment Patent applicant address 5-8-7, Kyobashi-chome, Chuo-ku, Tokyo Contents of amendment (1) Specification - "protecnJ" on page 6, line 12
is corrected to rProteinJ. (2) Correct "r511M Tris" on page 9, line 7 of the specification to "5Il1M Tris." (3) rGcIJ on page 10, line 14 of the specification is replaced by rGe
Correct to lJ. (4) In Table 1 on page 11 of the specification, "Chemistry ■ and" in the milk protein column "Na-caseinate" is corrected to "Chemistry ■) and". (5) Correct rDacryJ to rDairyJ in the column of milk protein ■Na-caseinate in Table 1 on page 11 of the specification. (6) In Table 1 on page 11 of the specification, "weapon ■))" in the milk protein ■Na-caseinate column is corrected to "common benefit ■)." (7) "Flake" in the column for soybean protein 11S globulin in Table 1 on page 12 of the specification is corrected to "flake". (8) F Adjustment J in the 5th line from the bottom of page 16 of the specification is corrected to ``Preparation J.''
is corrected to "△nigelization". (10) rcal on page 17 of the specification, line 7 from
Correct 12 J to rCaC. (11) Correct “adjustment” in the 5th line from the bottom of No. 17 of the specification to rllllJ. (12) [Height 1.
Correct 5111114 to [height 15111].

Claims (1)

[Claims] Add transglutaminase to a protein-containing solution with a protein concentration of 2% by weight or more! A method for producing a gelled product, which comprises adding 1 unit or more to f to cause gelation.