JP3395397B2 - Seasoning manufacturing method - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a seasoning, and more particularly to a method for converting glutamic acid, which determines a desirable taste of a seasoning, into a protein material. The present invention relates to a method for producing a seasoning or a seasoning material having a high glutamic acid content, which is separated more effectively and at a high concentration. [0002] A method for producing a soy sauce or a soy sauce-like seasoning or a raw material of the seasoning by allowing an enzyme active substance containing a protein hydrolase to act on a protein raw material or a protein raw material containing a peptide. Many reports have already been made on (for example, Japanese Patent Publication No. 57-18859).
No., JP-B-63-94974). In addition, when a protein hydrolyzing enzyme is allowed to act on a protein raw material, glutamine in free form which does not contribute to taste is produced at a considerably high concentration despite having glutamic acid as a constituent part thereof. Attempts have been made to increase the concentration of glutamic acid in the hydrolyzate by allowing glutamine to act on this free form of glutamine, and some of these methods have already been put into practical use (for example, Japanese Patent Publication No. 17828, and JP-B-49-15789. [0004] Furthermore, free glutamine is unstable in a hydrolyzate, easily cyclizes (pyrolyzes), and changes into pyroglutamic acid where glutaminase or the like hardly acts. It is an obstacle in improving the concentration of glutamic acid in the medium. [0005] In summary, the conventional method in which a protein hydrolase is allowed to act on a protein raw material and a simple hydrolysis reaction is performed is limited as a method for effectively releasing bound glutamic acid present in the protein raw material. There is no other way. [0006] In the present invention, the bound glutamic acid present in the protein raw material is effectively converted into free glutamic acid in the hydrolyzate by the action of protein hydrolase. An object of the present invention is to obtain a seasoning or a seasoning raw material containing glutamic acid at a high concentration while keeping the released glutamic acid stable without changing it. [0007] In order to solve the above-mentioned problems and objects, the present inventors reexamined the related art in detail in detail, and furthermore, from the new viewpoint, variously. As a result of the study, prior to causing the protein hydrolase to act on the protein raw material, the ability to preferentially catalyze the deamidation reaction over the cross-linking reaction as compared with conventionally known transglutaminase was examined. When the transglutaminase active substance is allowed to act on the protein raw material and then the protein hydrolase active substance is acted on, the free form of glutamic acid is very effectively released or released into the hydrolyzate. Glutamic acid can be stably preserved in the hydrolyzate without any change, so seasonings or seasonings containing glutamic acid in high concentration It has been found that raw materials can be manufactured. The present inventors have completed the present invention based on this finding. That is, the present invention is to act on a protein material a transglutaminase active substance having the ability to preferentially catalyze a deamidation reaction over a crosslinking reaction,
This is a method for producing a seasoning, which comprises reacting a protein hydrolase active substance. The protein raw material in the present invention includes various plant protein raw materials, animal protein raw materials or microbial protein raw materials, partial hydrolysates thereof, peptides derived from these various protein raw materials, and their derivatives. A mixture of raw materials is used. Examples of vegetable protein raw materials include whole soybeans, soybean meal, soybean flakes, soybean crushed cake, isolated soybean protein, flour, isolated wheat gluten, and corn gluten. [0011] Animal protein raw materials include bird meat, seafood meat, processed products thereof, fish meal, fish solvable, casein, isolated whey protein, cottage cheese, and the like.
And a partially hydrolyzed silk thread. [0012] Microbial protein raw materials include yeast cells,
The yeast extract, the amino acid fermentation cells, and the extract are exemplified. As a transglutaminase active substance having a transglutaminase activity having an ability to catalyze a deamidation reaction preferentially over a crosslinking reaction, a deamidation reaction is preferentially performed over a crosslinking reaction. Guinea pig liver transglutaminase preparation having catalytic ability, microbial transglutaminase preparation having ability to catalyze deamidation reaction preferentially over crosslinking reaction, deamidation reaction over crosslinking reaction A fish transglutaminase preparation having the ability to catalyze preferentially, a culture of a modified bacterium in which the ability to produce these enzymes has been modified by introducing a plasmid that expresses it in a microbial cell such as Escherichia coli; A processed product of a bacterium culture is exemplified. A transglutaminase having an ability to catalyze a deamidation reaction preferentially over this crosslinking reaction is
It has a protein structure and an amino acid sequence that are related to a modified protein (mutein) with respect to conventionally known transglutaminase. Therefore, transglutaminase having the ability to preferentially catalyze the deamidation reaction over the cross-linking reaction can be obtained by a method similar to a known biochemical or genetic engineering method for preparing a modified protein. Can be prepared. However, the transglutaminase used in the method of the present invention, that is, the transglutaminase having the ability to catalyze the deamidation reaction preferentially over the crosslinking reaction, is used as described above. The present invention is not limited to the modified protein of glutaminase, and any transglutaminase prepared by any preparation method can be used as long as the transglutaminase has such ability. is there. The proteolytic enzyme active substances which act on the protein material after the transglutaminase active substance is acted on include various proteinase preparations, protease preparations, peptidase preparations, and the like. A mixture containing the proteolytic enzyme, and its source and raw material are not particularly limited. Moreover, endo-type protease, exo-type protease, or a mixture thereof may be used. Further, the proteolytic enzyme active substance does not need to be a purified sample, and may be, for example, rice koji, barley koji, or koji mold liquid medium culture. In order to cause a transglutaminase active substance having the ability to catalyze a deamidation reaction preferentially over a cross-linking reaction to act on a protein raw material, the protein raw material and the transglutaminase are used in an aqueous medium. A method of contacting the active substance is employed. The activity range of this enzyme is 5 to 55 ° C., pH
Since it is 4 to 10, the contact condition is also selected from this range. Also, the contacting time is suitably 0.1 to 24 hours. Since the activity of the protease is often impaired by the coexistence of the protease, it is not advisable to carry out the next step for causing the active substance of the protease to act simultaneously. In order to cause the protein hydrolyzing enzyme active substance to act on the protein raw material after the transglutaminase active substance having the ability to catalyze the deamidation reaction preferentially over the cross-linking reaction, the aqueous In the medium, a method of contacting the protein raw material and the protein hydrolase active substance after the transglutaminase active substance is allowed to act on the medium is employed. The conditions for the contact in this case are selected from the activity range of the selected protease. Also, the contact time is suitably 0.5 to 12 hours. In this step, even if a transglutaminase active substance coexists, the activity of the protein hydrolase is not affected, so that the process proceeds from the previous step to this step without performing any special intermediate treatment. You can do it. The protein hydrolyzate after the action of the protein hydrolase active substance is used as it is, or after necessary processing steps such as solid-liquid separation, concentration and drying, as a seasoning or its raw material. . Transglutamine used in the present invention
, A transglutaminase having the ability to preferentially catalyze the deamidation reaction over the cross-linking reaction, is an enzyme newly discovered by the present inventors, and its preparation method and specific activity are described below. Specific examples thereof will be described in Reference Examples 1 and 2 described below. The preparation method and specific activity of this transglutaminase are already published as described at the end of each Reference Example. Hereinafter, the content of the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. Example 1 Transglutaminase TGM1, TGM1 used in this example as a transglutaminase having the ability to preferentially catalyze a deamidation reaction over a crosslinking reaction
GM2 or TGM3 is prepared by the method of Reference Example 1 described later, and its specific activity is as described in Reference Example 2. Transglutaminase TGM1, TGM
2 or TGM3, 0.1 g each, 5 g each of soy protein powder or wheat protein powder newly separated from defatted soy flour or flour by alkali extraction isoelectric precipitation
And 1 L of a phosphate buffer of pH 7 under weak stirring at 37 ° C. for 2.0 hours. Then the pH was adjusted to 7
After re-adjustment, 0.1 g each of exo-type peptidase standard “Protease M” (trade name: product of Amano Pharmaceutical Co., Ltd.) was added, and the mixture was weakly stirred at 37 ° C. for 2.0 hours. The contact was continued for a further period. In this case, no solid was present in any of the hydrolysis samples, and it was confirmed that the protein powder was completely dissolved. On the other hand, a sample to which 0.1 mL of N hydrochloric acid was added without adding the above-mentioned transglutaminase (Control 1) and a sample to which the above-mentioned transglutaminase was not added (Control 2) 0.1% of a transglutaminase TG standard prepared from a culture of Escherichia coli into which the plasmid pKTG1 for expressing guinea pig liver transglutaminase was introduced.
The same treatment was performed for the sample to which g was added (Control 3). The transglutaminase T used in Control 3 was used.
The G standard has the usual transglutaminase activity that preferentially catalyzes the crosslinking reaction. For the total of 12 kinds of hydrolyzed samples described above, the total amount of glutamic acid (GH) detected by ordinary amino acid analysis (liquid chromatography) using an ion exchange column was determined, and the microkerdale method was used. The ratio of the amount of glutamic acid to the total amount of nitrogen (GH / TN) was calculated by quantifying the total amount of nitrogen (TN). The results are summarized in Table 1. [Table 1] A modified transglutaminase TGM1,
In the sample using TGM2 or TGM3, the ratio of the amount of glutamic acid to the total amount of nitrogen (GH / TN) was higher than that of the control 1, control 2 and control 3 samples, even when the protein material was soy protein or wheat protein. Equal or higher numbers were observed. This fact is due to the contact with the transglutaminase TGM1, TGM2 or TGM3 which has the ability to catalyze the deamidation reaction preferentially over the crosslinking reaction.
This indicates that glutamic acid is effectively released from the protein raw material and is preserved in the hydrolyzed solution. The sample treated with TGM1, TGM2 or TGM3 was not treated with acid unlike the sample of Control 1, so that monochloropropanol and dichloropropanol which are considered to have an adverse effect on the human body were used. There is an advantage that an organochlorine compound such as toluene does not by-produce at all. Reference Example 1 == Example of Preparation of Modified Transglutaminase TGM1, TGM2 or TGM3 = Polymerase Chain Reaction (PCR) Using Plasmid pKTG1 for Escherichia coli Expression of Guinea Pig Liver Transglutaminase as a Template Site-directed mutagenesis method (a reaction for rapidly and easily amplifying only specific sites of a DNA strand using a primer in which specific sites have been mutated; "Recombinant DNA Experiment Notes", edited by Kazuki Onodera, pp. 112-115) Published by Hyundai Kogyosha), three types of modified transglutaminase TG
Plasmid pK expressing M1, TGM2 or TGM3
TGM1, pKTGM2 or pKTGM3 were constructed.
The modified bacteria obtained by introducing these plasmids into Escherichia coli DH5α were cultured, and TGM1, TGM2 or TGM3 was prepared from the cultured cells. Also, they were expressed at the same level as in the case of Escherichia coli into which pKTG1 was introduced. Escherichia coli, Echerichia coli AJ13 transformed with plasmid pKTGM1 expressing the modified transglutaminase TGM1
No. 037 has been deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology, and its deposit number is FERM P-1.
4513. Further, a modified transglutaminase TGM
, Escherichia coli A transformed with plasmid pKTGM2 expressing
J13038 has been deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology, and its deposit number is FERM.
P-14514. Further, a modified transglutaminase TGM
, Escherichia coli A transformed with plasmid pKTGM3 expressing
J13039 has been deposited with the National Institute of Advanced Industrial Science and Technology, Ministry of International Trade and Industry, and its deposit number is FERM.
P-14515. In addition, Escherichia coli and Echerichia coli AJ123 transformed with guinea pig liver transglutaminase expression plasmid pKTG1.
No. 70 is deposited at the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry, and its deposit number is FERM P-10.
008. Further, (1) a product obtained by culturing Escherichia coli having the plasmid pKTG1 for expressing guinea pig liver transglutaminase in Escherichia coli; and (2) the modified transglutaminase TGM1, TGM2 and TGM3.
And a product obtained by culturing Escherichia coli having plasmids pKTGM1, pKTGM2 and pKTGM3, respectively, and (3) as a control, a plasmid vector carrying no guinea pig liver transglutaminase-expressing vector-pKK233- Extract of a culture of E. coli harboring E. coli and (4) transglutaminase G. extracted from guinea pig liver. P. L. Was electrophoresed on SDS polyacrylamide gel,
With the exception of (3), it was found that almost all the other samples had almost the same molecular weight. In (3), the presence of a protein corresponding to guinea pig liver transglutaminase protein could not be recognized under the electrophoresis conditions. FIG. 1 is a schematic diagram showing the results of electrophoresis on SDS polyacrylamide gel. The modified sites of TGM1, TGM2 or TGM3 with respect to the unmodified transglutaminase TG are as follows. TGM1: Two aspartic acid residues at residues 231 and 232 in the central negative charge region were converted to asparagine residues. TGM2: C-terminal negative charge region, 445, 448, 4
Five glutamic acid residues at residues 49, 450 and 452 were converted to glutamine residues. TGM3: shares TGM1 and TGM2 modification sites.
In addition, DN of unmodified guinea pig liver transglutaminase was used.
The A sequence and the amino acid sequence are described in JP-A-1-300.
No. 889, for example. Reference Example 2 = Confirmation of specific activity of modified transglutaminase TGM1, TGM2 or TGM3 = (1) Suppression of crosslinking reaction: Modified transglutaminase TGM1, TGM2 or TGM
In the crosslinking reaction represented by _monodansyl cadaverine, which is a fluorescent primary amine, to acetylated α _s1- casein, GM3 has an enzymatic affinity K for this protein substrate.
While m was the same as the unmodified transglutaminase TG, the maximum reaction rate Vmax was reduced by 2.5 times or more, indicating that suppression of the crosslinking reaction had occurred. It should be noted that the enzymatic affinity K for the protein substrate
m and the maximum reaction rate Vmax are defined by the following equations. Km = (Vmax / v-1) [S] Vmax = (Km + [S]) v / [S] where v is the production rate of the product and [S] is the substrate concentration. Table 2 shows the measured values for the enzymatic affinity Km with the protein substrate and the maximum reaction rate Vmax. [Table 2] Further, after the reaction starts, the ratio of α _s1- casein monomer present in the reaction system measured in 30 minute units increases rapidly with time in the unmodified transglutaminase TG reaction system. However, in the reaction system of the modified transglutaminase TGM1, TGM2 or TGM3, there was almost no change, and it was confirmed that the reaction was maintained and suppressed to 90 to 95% of the initial value (see FIG. 2). (2) No change in the deamidation reaction: The deamidation of the modified transglutaminase TGM1, TGM2 or TGM3 is carried out by the unmodified transglutaminase TG.
Were almost at the same level as those of No. 1 and no substantial difference was observed between the two (see FIG. 3). (3) Modified transglutaminase TGM
1. Enzymological behavior of TGM2 or TGM3: As described above, TGM1, TGM2 or TGM3 is deamidated while inhibition of the cross-linking reaction is occurring as compared to unmodified transglutaminase TG Since there is no change in the reaction,
When a protein hydrolyzing enzyme is allowed to act on a protein substrate on which TGM1, TGM2 or TGM3 has acted, bound glutamic acid constituting the protein substrate is easily and without change, in other words, effectively released. And is released into the hydrolyzate. The matters described in Reference Examples 1 and 2 have already been published as follows. "Koji Ikura et al." Site-Specific Mutations in the Negatively Charged Region Conserved in Transglutaminase Molecules "Announced at the 66th Annual Meeting of the Biochemical Society of Japan: Abstracts, 733 pages, Abstracts Date, August 1993 Month 2
5, oral presentation, October 2, 1993] [0048] As described above, the present invention hydrolyzes bound glutamic acid present in a protein raw material as free glutamic acid. Since glutamic acid can be effectively released into the product and the released glutamic acid can be stably maintained without change, there is an effect that a seasoning or a seasoning material containing glutamic acid at a high concentration can be produced.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a product obtained by culturing Escherichia coli having a plasmid pKTG1 for expressing guinea pig liver transglutaminase in E. coli, and a modified transglutaminase TGM
Plasmid pKT expressing 1, TGM2 and TGM3
Each product obtained by culturing E. coli having GM1, pKTGM2 and pKTGM3, respectively, and an extract of a culture of E. coli having plasmid pKK233-2 which does not carry a guinea pig liver transglutaminase gene as a control; And transglutaminase G. extracted from guinea pig liver. P. L. FIG. 4 is a schematic diagram showing the results of electrophoresis on SDS polyacrylamide gel for the present invention. In the figure, the vertical axis is the molecular weight (KDa)
, And the horizontal axis represents the above product or G.P. P. L. Is shown. FIG. 2. Guinea pig liver transglutaminase TG;
The modified transglutaminase TGM1, TGM2,
FIG. 3 is a diagram showing the activity of a crosslinking reaction with TGM3. In FIG. 2, the vertical axis indicates the _αs1- casein monomer ratio (%) after the crosslinking reaction, and the horizontal axis indicates the reaction time (minutes). A black circle indicates TG, a white triangle indicates TGM1, a white circle indicates TGM2, and a black triangle indicates TGM3. FIG. 3. Guinea pig liver transglutaminase TG;
The modified transglutaminase TGM1, TGM2,
It is a diagram showing the activity of deamidation reaction with TGM3. In FIG. 3, the vertical axis shows the amount of ammonia (mM) generated in the deamidation reaction, and the horizontal axis shows the reaction time (minute). Also, a black circle represents TG, a white triangle represents TGM1, a white circle represents TGM2,
Black triangles indicate TGM3, respectively.

Continuation of front page (56) References JP-A-48-82068 (JP, A) JP-A-4-126039 (JP, A) JP-A-63-36797 (JP, A) JP-A-7-23740 (JP) JP-A-7-75569 (JP, A) JP-A-6-225775 (JP, A) JP-B-47-37542 (JP, B1) JP-B-49-15789 (JP, B1) JP-B-A1 48-43637 (JP, B1) (58) Fields investigated (Int. Cl. ⁷ , DB name) A23L 1/228 A23L 1/23

Claims (1)

(57) [Claims] [Claim 1] A protein raw material is allowed to react with a transglutaminase active substance having an ability to preferentially catalyze a deamidation reaction over a cross-linking reaction. A method for producing a seasoning, which comprises reacting a degrading enzyme active substance.