JPH0150382B2 - - Google Patents

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 technology is established to intentionally modify these protein resources into protein materials with functionality and nutritional properties suitable for structured foods, it will not only be possible to increase their utilization, but also to produce high-quality protein foods. can be made. One of the modification techniques is modification by enzyme modification, but at present, modification by hydrolase is the main method, 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 a crosslink between glutamine (abbreviated as Gln) and lysine (abbreviated as Lys) residues, which are abundant in food proteins. They discovered that it is possible to produce a gel-like substance and completed the present invention. That is, in the present invention, transglutaminase is added to 1 g of protein in a protein-containing solution with a protein concentration of 2% by weight or more.
This is a method for producing a gelled product, which is characterized by adding one or more units to a gel to form a gel. The protein used in the present invention is not limited by 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. In addition, animal proteins include milk protein, gelatin,
Collagen etc. can be illustrated. 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. 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.
If the concentration of the protein solution is less than 2% by weight,
It remains in solution or forms a precipitate and does not gel. Further, the pH of the protein-containing solution is preferably 6 to 9. One or more units of transglutaminase per 1 g of protein is added to this protein-containing solution to form a gel. This transglutaminase
Connellan et al.'s method [Journal of Biological
Chemistry, 246 (4), 1093 (1971)],
Prepared from guinea pig liver. That is, guinea pig liver is dispersed in a sucrose solution, centrifuged, the supernatant liquid is collected, and the crude transglucosidase is obtained by fractionating it with a diethylaminoethyl cellulose column. This is precipitated with 1% protamine sulfate and the precipitate is collected. Furthermore, after washing this precipitate with 0.2M Tris-acetate buffer, 0.05M ammonium sulfate-5mM Tris-HCl buffer (2mM ethylenediaminetetraacetic acid (hereinafter referred to as
Protamine was removed from the resulting extract using a carboxymethylcellulose column, and ammonium sulfate solution (containing 1M EDTA) was added to the oral fluid, followed by centrifugation to remove the precipitate. to recover. 10mM of precipitate
Tris-acetate buffer (1mM EPTA, 0.16M
(containing KCl) and centrifuged supernatant for 10 min.
% agarose (Bio Gel A-0.5M), and the resulting highly active fraction is concentrated by ultrafiltration to obtain purified transglutaminase. Other methods for preparing transglutaminase include the method of Clarke et al. [Archives of
Biochemistry and Biophysics, 79, 338 (1959)]
There is. That is, 600 ml of 0.25M sucrose solution is added to 300 g of guinea pig liver and homogenized. Centrifuge this to obtain a supernatant. Add sodium acetate to 0.01M, adjust pH to 5.0 with acetic acid, and centrifuge. Homogenize the resulting precipitate by adding 30 ml of 0.05M phosphate buffer (PH6.5). This suspension is centrifuged, the supernatant is dialyzed against 0.001M phosphate buffer (PH7.5), and this is used as a crude transglutaminase solution. In these methods, there is no problem even if the order of operations is changed or the amount added, concentration, PH value separation device, etc. are slightly changed. The protein concentration of transglutaminase obtained in this way was measured using the Lowry method.
Biological Chemistry, 193, 265 (1951)],
Enzyme activity was measured using the hydroxysamic acid method using N-carbobenzoxy-L-glutaminylglycine and hydroxyamine [Journal of Biological
Chemistry, 241(23), 5518 (1966)], the specific activity of the prepared enzyme solution is 6.0 to 13.0.
indicates a range of values. 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 -30°C and thawed for use. The transglutaminase thus obtained is added in an amount of 1 unit or more per 1 g of protein to form a gel. If the amount added is less than 1 unit, a highly viscous solution will result. Moreover, even if more than 2000 units are added, the effect does not change much. Transglutaminase adds Glu- to protein molecules.
It is known that Lye crosslinking occurs (JEFolk
and JSFinlayson “Advances in Protein
Chemistry”Vol.31 ed.by CBAnfinsen, JT
Edsall and FMRichards, Academic Press
Inc., New York, NY, 1977, pl) deduced from the following experimental data that the gel produced when transglutaminase was applied to a high protein solution was due to Glu-Lye crosslinking. Serves as a reaction site for transglutaminase
αsl casein with acetylated and succinylated Lys residues did not gel even when transglutaminase was applied. Since the reaction is carried out in the presence of dithiothreitol, which is an S-S ring agent, in the reaction solution, the gel is not mainly composed of S-S bonds. When the elastic modulus of normal gelatin gel obtained by heating and cooling and gelatin gel gelatinized with transglutaminase was measured, 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. In contrast, the gel produced by transglutaminase shows only a small amount of change even when the temperature changes, suggesting that the gel has strong covalent bonds. In fact, when both gels were exposed to temperatures above 40°C, the transglutaminase gel remained intact, but the regular gelatin gel melted. From the above, it is considered that the gel is produced by Glu-Lys crosslinking and not by SS crosslinking. The gelled product thus obtained gels in a relatively short time, that is, within 1 minute, and within 30 minutes at the most, and has gel physical properties equivalent to those of general gelled products. Further, the protein-containing solution used in the present invention is not limited to a simple mixture of protein and water, but may 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. as well 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 a material for immobilized enzymes. It can also be used as Example 1 Transglutaminase was prepared by the following method. Add about 2 drops of cold 0.25M sucrose solution to 800 g of guinea pig liver, homogenize at 20,000 rpm for 2 minutes,
Centrifugation (105,000 xg, 5°C, 1 hour) was performed to obtain a supernatant. This was mixed with 5mM Tris-HCl buffer (2mM
Equilibrated with MEDTA (PH7.5)
After adding and adsorbing to DEAE cellulose column,
Fractionation was performed using a gradient elution method in which the salt concentration of the above buffer was varied from 0M to 1.0M to obtain a fraction with high enzyme activity. Add 40 ml of 1% protamine sulfate while stirring gently, and centrifuge (14,600 x g, 15 minutes,
Collect the precipitate at 5℃), suspend it in 0.2M Tris-acetate buffer (PH6.0), homogenize and wash.
The precipitate was collected by centrifugation (2500 xg, 1 minute, 5°C). Transglutaminase was extracted from the precipitate by adding 5mM Tris-HCl buffer (containing 2mM EDTA, PH7.5) containing 0.05M ammonium sulfate and homogenizing. Repeat this three times,
The collected extract was added to a carboxymethyl cellulose column equilibrated with 5mM Tris-succinate buffer (containing 2mM EDTA, PH6.0) to remove protamine, and the filtrate was added with 1M EDTA (PH8.0) 2.4
After adding 47.4 g of ammonium sulfate and stirring well, the precipitate was collected by centrifugation (15,000 x g, 10 minutes, 5°C). This was mixed with 10mM Tris-acetate buffer (1mM
After dissolving in EDTA (containing 0.16M KCl, pH 6.0) and removing hardly soluble substances by centrifugation (27,000 x g, 30 minutes, 5°C), the supernatant was equilibrated with the same buffer.
Perform gel filtration with agarose (Bio Ge A-0.5M), collect the highly active fraction, and filter it for 10 to 20 minutes.
It was concentrated by ultrafiltration (UM-10, manufactured by Amicon) to a concentration of mg/ml to obtain a transglutaminase solution. This solution was stored frozen at -30°C or below, dissolved at appropriate times, and used (this solution was prepared by constantly operating at 5°C). Transglutaminase was allowed to act on the substrate proteins shown in Table 1 to obtain a gelled product.

【table】

[Table] Example 2 α _sl casein, Na-caseinate, soybean protein
11S globulin, water-extracted soy protein, 500mg each
0.1M Tris-HCl buffer (5mM CaCl ₂ , 20mM
Containing dithiothreitol (pH 7.6) was dissolved in 3.5 ml, 1.5 ml of soybean oil was added thereto, and homogenized at 20,000 rpm for 3 minutes to obtain an emulsion. When 0.09 units of transglutaminase was added to 1 mg of protein, a gelled product was immediately obtained. Example 3 2, 5, and 10% by weight solutions of α _sl casein, soybean protein 11S globulin, and soybean protein 7S globulin were prepared.
0.1M Tris-HCl buffer (5mM CaCl ₂ , 20mM
0.5 ml of M dithiothreitol (pH 7.6) was prepared, and transglutaminase was added to each at 37°C at a ratio of 0.1 unit per 1 mg of protein to determine whether gelation occurred. Obtained.

[Table] ○: Gelation △: Weak gel ×: Still in solution Example 4 A 5% by weight solution of α _sl casein and a 10% by weight solution of soybean protein 11S globulin were mixed in 0.1M Tris-HCl buffer (5mM CaCl ₂ , 200mM dithiothreitol (containing 200mM dithiothreitol, pH 7.6) was prepared, and to 0.8ml of these solutions, 5 x 10 ^-4 - 2.0 units of transglutaminase was added per 1mg of protein, and it was observed whether gelation occurred. The results shown in Table 3 were obtained.

[Table] Example 5 A Tris-HCl buffer solution containing 5mM CaCl ₂ and 20mM dithiothreitol with a pH of 7.0 to 9.0 was prepared, and used to prepare a 5% by weight α _sl casein solution.
0.8 ml of each 10% by weight soybean protein 11S globulin solution was prepared, 0.1 unit of transglutaminase was added to 1 mg of protein, and it was observed whether gelation occurred. The results are shown in Table 4.

[Table] ◎: Immediate gelation ○: It took some time for gelation ×: Example 6 as a solution Example 6 Pour 1 ml of the sample solution into a test piece preparation container with a diameter of 9.3 mm and a height of 15 mm, as shown below. gel to create a cylindrical gel, which was measured using a rheogram (Toyo Seiki Seisakusho Co., Ltd., CV-100) from 18 to 25.
The temperature was raised to ℃, and the storage elastic modulus at each temperature was measured. Gelatin cooling gel Add water to gelatin to make a 10% solution by weight, and after completely dissolving gelatin at 60℃ for 3 minutes,
Pour 1ml into a test piece making container and heat to 3℃.
The mixture was left to gel for 20 minutes, returned to room temperature, and measured. Gelatin TGase gel 0.1M to make a 10% solution by weight in gelatin
Add Tris-HCl solution (containing 5mM CaCl ₂ , 20mM dithioleitol, PH7.6) and incubate at 60°C for 30 minutes.
Dissolve the gelatin completely in minutes, pour it into a test piece preparation container, quickly add transglutaminase at a ratio of 0.1 unit to 1 mg of gelatin, and leave it at room temperature for 1 hour to gel.
It was measured. The results are shown in Figure 1. The storage modulus of gelatin cooled gels decreased significantly as the temperature increased, but in contrast, gelatin TGase gels were less affected by temperature changes. Example 7 1 ml of 0.1M Tris/ _HCl buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, pH 7.6) was prepared to give a concentration of 5% by weight for α sl casein and 10% by weight for soybean 11S globulin. , and add transglutaminase to this for 1 mg of protein.
A gel was obtained by adding 0.1 unit. This gel
After further maintaining the temperature at 100°C for 20 minutes, it was cooled to room temperature. Rheometer (Fudou Kogyo Co., Ltd., NRM-
2002J), the maximum load when a plunger (5Φ, ball type) was inserted was measured and used as the gel strength.
The results are shown in Table 5.

[Table] As can be seen from the table above, the gel strength increased with heat treatment in all cases.

[Brief explanation of drawings]

FIG. 1 shows the results of Example 6. In the figure, the horizontal axis is temperature (°C), the vertical axis is storage modulus (dyn/cm ² ),
The solid line represents the gelatin TGase gel of the present invention, and the dashed line represents the gelatin cooling gel.

[Claims]

1. An acid-resistant casein powder characterized by blending sodium caseinate or calcium caseinate with at least one type of cyclic phosphoric acid and at least one type of polysaccharide. 2. The powder according to item 1, wherein the cyclic phosphoric acid is one or more selected from the group consisting of sodium hexametaphosphate, sodium ultraphosphate, and phytic acid. 3 As polysaccharides, alginic acid, chondroitin, chondroitin sulfate, hyaluronic acid, tycuronic acid, colominic acid, λ-carrageenan, κ-carrageenan, caronine sulfate, polylipose phosphate, polyacetylgalactosamine phosphate, sodium alginate, carboxymethylcellulose, cellulose glycol 2. The powder according to item 1, which uses one or more selected from the group consisting of calcium acid, sodium cellulose glycolate, sodium starch glycolate, starch phosphate, and sodium polyacrylate.