JPH1175765A - Production of seasoning - Google Patents

Abstract

PROBLEM TO BE SOLVED: To specifically increase the content of an amino acid such as glutamic acid which is an UMAMI ingredient of an amino acid-based seasoning such as a soy sauce by adding a specified enzyme to a soybean protein that is a raw material. SOLUTION: When an amino acid-based seasoning (e.g. a soy sauce) using a soybean protein as a raw material is obtained, an X-prolylpeptidyl aminopeptidase (hereinafter referred to as DP TV) is added to afford the amino acid-based seasoning enriched with an UMAMI ingredient. The DP IV is obtained by taking out a Koji (a fungus) in, e.g. a Koji production process for producing a soy sauce Koji or a wheat bran Koji, adding a phosphoric acid buffer thereto, carrying out an extraction, filtering the resultant extract solution through a filter, preparing a crude enzyme liquid, then conducting a treatment such as salting out or chromatography and providing the refined DP IV. The DP IV may be added as an UMAMI ingredient enricher in any step of a mixing step for mixing soybeans after the denaturing treatment with wheat, the Koji production step and a charging step in the production of the soy sauce, but is preferably added in the charging step.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an amino acid seasoning using soybean protein as a raw material. More specifically, the present invention relates to a method for producing (brewing) soy sauce and an enhancer for umami components of soy sauce.

[0002]

2. Description of the Related Art In general, soy sauce is used as a vegetable material such as soybean and wheat in Aspergillus oryzae.
us oryzae), Aspergillus soya (As
soy sauce koji obtained by breeding koji molds such as C. pergillus sojae) into a saline solution, and fermenting and aging it. This is a traditional fermented seasoning in Japan.

[0003] In soy sauce brewing, besides koji mold, lactic acid bacteria (Pediococcus halophilus) are used.
halophilus), yeast (Zygosaccharomyces).
rouxii), Candida versatilis (Ca
Microorganisms such as ndida versatilis) are involved, and the action of these microorganisms produces soy sauce with excellent aroma and taste.

The taste of soy sauce is such that the five tastes of sweetness, umami, sourness, sourness and bitterness are exerted together in a complicated fermentation process. For example, soy sauce is produced by the decomposition of saccharides and proteins in the raw material by enzymes of Aspergillus, sweet components such as sugar, umami components such as amino acids and peptides, and sour components such as organic acids generated by lactic acid fermentation of lactic acid bacteria. It contains bitter ingredients such as bitter amino acids and peptides, which bring out the richness, and the salty taste of salt, and also contains flavor components such as alcohol and esters produced by alcoholic fermentation of yeast. It has been used as a seasoning.

[0005] In particular, the degradation of protein raw materials is caused by the action of various proteases of Aspergillus oryzae, which affects not only the yield of soy sauce but also the taste thereof. In soy sauce moromi (moromi), the decomposition of the protein raw material proceeds rapidly from the initial stage of the preparation. This is caused by the action of exopeptidase such as leucine aminopeptidase and acid carboxypeptidase on the polypeptide produced by endopeptidase in Aspergillus oryzae. , Amino acids and dipeptides to produce low-molecular peptides (Tatsuroro Tochikura, edited by "Science and Technology of Soy Sauce", pp. 174-176,
Japan Brewery Association (1988)).

However, acidic carboxypeptidase has an optimum pH as low as 3.7, and its action is considered to be extremely small at an initial pH of 5.5 to 6, during which amino acid production proceeds rapidly during soy sauce brewing. Leucine aminopeptidase is an exopeptidase that releases amino acids sequentially from the N-terminus of the substrate peptide chain. However, when a proline residue is present in the peptide, X- is one in which one amino acid is bonded to the imino group side of proline. Pro-Pept
The operation stops in the state of ide. Therefore, the amination rate of soy sauce reaching about 80% could not be explained by the action of leucine aminopeptidase or acidic carboxypeptidase.

[0007] The present inventor has found that X-Pro-Peptide acts on X-Pro-Peptide, which has never been reported in molds.
It has been found for the first time that a novel X-prolyl dipeptidyl aminopeptidase releasing Pro is produced by Aspergillus oryzae (Tachi et al., Ph.
ytochemistry, 31 (11), 3707-
3709 (1992)). X-prolyl dipeptidyl aminopeptidase is also known as dipeptidyl aminopeptidase IV (DP IV) (EC 3.4.4.15.5). DP IV produced by Aspergillus is a dimeric protein having a relative molecular weight of 280,000 and consisting of two subunits with a molecular weight of 145,000. This enzyme is similar to mammalian DP IV, especially pig kidney DP IV
The same relative molecular weight is shown. Hereinafter, in this specification,
X-prolyl dipeptidyl aminopeptidase produced by Aspergillus is simply referred to as DP IV.

[0008] DP IV acts on Gly-Pro-X binding and specifically produces Gly-Pro. The present inventors have considered that Gly-Pro is a peptide that is not degraded by other endopeptidases or exopeptidases, and found that the Gly-Pro content is correlated with the degradation of the raw protein in soy sauce brewing.
It has been clarified that the measurement of the ly-Pro content is one indicator of the aging of soy sauce (Tachi, J. Brew. S.
oc. Japan, 91 (2), 138-140 (19
96)).

However, no report has been made on how DP IV is involved in the taste of soy sauce, particularly umami, in brewing soy sauce.

[0010]

Generally, the rough index of taste of soy sauce is the total nitrogen content. Among them, it is known that the umami particularly depends greatly on the glutamic acid content. Therefore, in order to obtain soy sauce with excellent umami, it is necessary to increase the glutamic acid content of the total nitrogen such as amino acids and peptides generated in the soy sauce by hydrolysis of the raw material protein.

On the other hand, it has been proposed to brew soy sauce using only an enzyme agent without koji-making the raw material for brewing soy sauce (JP-A-49-81598 and JP-A-49-86).
594). This method is excellent in that it does not require large-scale facilities and long time for koji making, and strict temperature and humidity control for obtaining uniform koji is not required. The brewing method was not sufficiently satisfactory in the amount of ingredients.

[0012]

In order to improve the taste of soy sauce, the present inventor has conducted a small soy sauce preparation test and a DP test.
As a result of intensive studies on the role of IV for protein degradation and amino acid production, DP IV greatly contributes to the degradation of raw protein in soy sauce brewing and promotes the production of amino acids as already reported by the present inventors. Not only that, surprisingly, it has been found that the content of the umami components of soy sauce such as glutamic acid and aspartic acid is specifically increased, the umami of the soy sauce is improved, and the taste of the soy sauce is significantly affected. . The present invention has been made based on such findings.

That is, the present invention relates to a method for producing a seasoning, which comprises adding X-prolyl dipeptidyl aminopeptidase to a method for producing an amino acid seasoning using soybean protein as a raw material. Further, the present invention provides a step of mixing a protein material for brewing soy sauce and a carbohydrate material which have been subjected to a denaturation treatment, a koji making step of adding seed koji to the mixture, and a step of adding salt water to the obtained soy sauce koji (dekoji) A soy sauce production (brewing) method comprising a mashing step of fermenting and aging the mash by adding X-prolyl dipeptidyl aminopeptidase, preferably DP IV derived from Aspergillus oryzae. The above brewing method.

[0014]

DETAILED DESCRIPTION OF THE INVENTION As a raw material for brewing soy sauce, any conventionally used raw material can be used in the present invention. The two main ingredients for soy sauce brewing are protein ingredients and carbohydrate ingredients.

Soy is used as a protein material for soy sauce brewing. Examples of soybeans include defatted soybeans and whole soybeans. Gluten, a purified protein, or the like can be used as a protein material instead of soybean.

As the carbohydrate raw material for soy sauce brewing, wheat, preferably wheat is used. Among the components of wheat, carbohydrates, that is, starch is important, but also contains more than 10% of protein, and since the main raw material of soy sauce is protein, so-called hard wheat having a high protein content is preferably used. . It is also preferable to use koji barley obtained by milling wheat and then collecting components suitable for brewing soy sauce.
The carbohydrate raw material may include barley, bare barley, bran, perfumed, wheat gluten, barley, and the like, in addition to wheat. Also, various starches can be used as a carbohydrate raw material instead of wheat.

Hereinafter, the method for brewing soy sauce of the present invention will be described by exemplifying the brewing of concentrated soy sauce using soybean and wheat as protein raw materials and carbohydrate raw materials for soy sauce brewing, respectively (hereinafter simply referred to as protein raw materials and carbohydrate raw materials, respectively). Will be explained. In brewing soy sauce, a protein material and a carbohydrate material are first modified. As the denaturation treatment, any treatment method may be used as long as it is a modification treatment usually performed in soy sauce brewing to facilitate the growth of koji mold and the action of enzymes.

As the soybean denaturation treatment, NK treatment method,
Examples include a cooking method such as a continuous cooking method, an alcohol denaturation method, and a puffing method. As the denaturation treatment of protein raw materials such as gluten and purified protein, a steaming method, an alcohol denaturation method, or the like is used.

For the NK type treatment method, a rotary steaming apparatus is used industrially, and a normal rotary steaming can (NK can) has a vapor pressure of about 0.9 to 1.0 kg / cm ² . For 40 to 60 minutes. Recently, a continuous steaming method using a continuous steaming apparatus has been adopted, and according to this method, steaming at a higher pressure and for a shorter time has been put to practical use. A puffing treatment is also particularly preferred as a modification treatment of soybeans.
70 ° C., 4 to 7 kg / cm ² steam pressure for 2 minutes to
After contact for 15 seconds, the soybeans are swollen by rapidly releasing them into the atmosphere.

As a modification treatment of wheat, roasted mushrooms can be mentioned. Examples of the modification of various starches include a steaming method.
Roasting means roasting wheat with a wheat stirrer, which makes the starch alpha and facilitates the action of enzymes. The wheat that has been subjected to the roasting process is then broken.

The method for brewing soy sauce of the present invention is characterized in that
Except for adding PIV, a conventional method may be used. Hereinafter, the method for brewing soy sauce of the present invention will be described by taking as an example the brewing of concentrated soy sauce using soybeans and wheat as raw materials.

As described above, soybeans that have been subjected to the steaming treatment and wheat that has been subjected to the roasting and cracking treatment are mixed, and seed koji is added to the mixture for koji making. Koji making is a process in which koji mold is grown to produce proteolytic enzymes and saccharifying enzymes, and then saline is added to cause hydrolysis. The time required for koji making is typically about 45 hours (3 days koji) or about 72 hours (4 days koji). After the above-mentioned processing raw materials are put into an apparatus for koji making (filling), after a certain period of time according to a conventional method, stirring called maintenance (first maintenance, second maintenance) is performed to adjust the product temperature (raw material temperature). And make up for fresh air. Koji-making was traditionally performed using the koji lid method, but today, using an automatic koji-making device, labor saving, uniform temperature distribution, prevention of contamination by various bacteria, and achievement of enzyme titer in a short time are achieved. And so on.

As a koji mold used as a seed koji, a yellow koji mold is preferable. For example, Aspergillus oryzae (IAM2616, RIB128, RIB430,
RIB915), Aspergillus soya, Aspergillus tamari (IFO4359 etc.) and the like.
Of these, Aspergillus oryzae and Aspergillus soya are preferred. These koji molds are easily available and may be used as a single strain or strain, or a plurality of strains or strains may be used as a mixture. For example, commercial koji can be easily obtained as a mixture of a plurality of koji molds. Aspergillus oryzae used in the present invention is not limited to the above-described species or strains, and may be any of these variants, mutants, and mutants, as long as they are suitable for brewing the soy sauce of the present invention. it can.

The soy sauce koji (dekoji) thus prepared contains various enzymes such as protease and amylase, and these enzymes are important for fermentation and maturation in the moromi formed by the subsequent preparation. Has an effect. Among these enzymes, the DP IV according to the present invention is also included. In the brewing method of the present invention, DP IV derived from koji mold prepared separately in advance is further added to the soy sauce brewing raw material as described later. It is characterized by being added.

This soy sauce koji is then mixed with saline to make moromi. This is called preparation. The concentration of the salt solution to be added may be a conventional method, and the salt concentration of the moromi ripened by back calculation is 13%.
-20%, preferably about 17%. The amount of salt water (water pumping) may be in a conventional manner. Pumping water is expressed by the volume of saline used for a given raw material. For example, in the case of koji making using a total of 1 kl of soybean and wheat, the same amount (1 kl) of saline is added and charged. Is referred to as fetching 10 water, and usually 1 for deep soy sauce.
A saline solution of 0 to 12 water is used.

With this preparation, fermentation and ripening of moromi are performed. Fermentation and aging are carried out at 20 to 40 ° C, preferably 25 to 30 ° C, for 3 to 6 months, preferably for about 5 months. For example, if the temperature of moromi immediately after preparation is maintained at a low temperature of about 15 ° C., and then the room temperature is increased to about 28 ° C.,
Eventually, the product temperature will be about 28 ° C, and fermentation will be promoted. In this case, cool air is sent after about 5 months to raise the temperature of moromi to 15 ° C.
It is preferred to lower to. In the process of preparation, fermentation and ripening, the raw material components are decomposed by the enzyme in the koji in the initial stage after preparation of the moromi. Peptides and amino acids generated by this decomposition are used as nutrients, and thereafter, Pediococcus halophilus (Pediococcus halophilus) is used.
lactic acid bacteria, such as S. ophilus, and Zygosaccharomyces.
rouxii), soy sauce yeasts such as Candida versatilis
Candida yeast and the like begin to proliferate and exert an important effect on fermentation and maturation of moromi. 20-40 days after preparation,
Preferably, after 30 days, the yeast for soy sauce such as the above-mentioned T. saccharomyces luxii, and lactic acid bacteria such as Pediococcus halophilus may be added from the outside 7 to 20 days after the preparation.

The matured moromi is squeezed according to a conventional method to separate a liquid portion (freshly fried), and if necessary, various additives are added to the freshly fried and heated (burned) to a certain temperature, and finally heated. Soy sauce is obtained as a product.

In the present invention, in the above-mentioned method for brewing soy sauce, DP IV which has been separated from koji mold in advance is used.
Is added. Therefore, DP IV derived from Aspergillus oryzae according to the present invention can be used as a taste enhancer for soy sauce. Aspergillus includes the Aspergillus oryzae used as the seed koji, and Aspergillus oryzae RIB915 (Aspergill) having high DP IV activity.
us oryzae RIB915).

The one-island method (E. Ichishima,
"Methods in Enzymology", G.A.
E. FIG. Perlmann and L.A. Loland, 1st
9, p. 397 (1970)), by inoculating bran with Aspergillus oryzae RIB915, bran koji can be prepared. DP IV can be extracted from bran koji. DP IV can also be extracted from soy sauce koji, moromi or moromi filtrate.

The DP IV is obtained, for example, as follows. First, in the koji making process of soy sauce koji or bran koji,
The koji is taken out, a phosphate buffer solution is added thereto to perform extraction, and the mixture is filtered with filter paper to prepare a crude enzyme solution. Ammonium sulfate was added to the obtained crude enzyme solution to be saturated,
The precipitate precipitated by standing is filtered through celite to obtain a salted out ammonium sulfate. The obtained ammonium sulfate salted-out product is subjected to gel filtration, and if necessary, hydrophobic chromatography, regel filtration, ion exchange chromatography, sucrose density gradient isoelectric focusing, etc., to obtain purified DPIV.

The DP thus extracted and purified from the koji mold
IV, in the above-mentioned soy sauce brewing, as a flavor enhancer, a mixing step of soybean and wheat after the denaturation treatment, a koji making step,
It may be added in any of the preparation steps (including the fermentation / ripening step). Preferably, DP is used in the charging process.
Add IV.

In the addition of DP IV, the enzyme is preferably added in the form of an extract or powder together with saline. The addition enzyme DP IV in powder form is obtained by freeze-drying the extract. DP IV is added such that the DP IV activity is 0.01-0.3 nkat / g, and preferably 0.1-0.2 nkat / g.

The amount of the saline used at that time may be adjusted so that the amount of the saline after the addition of the saline in the preparation step becomes an amount in accordance with the usual method of soy sauce brewing. The salt concentration used is 13-20 in the matured moromi.
%, Preferably about 17%.

The DP IV activity is measured as follows. First, 0.45 mM glycyl-L-proline-p
-Nitroanilide (Gly-Pro-pNA) (pH
(7.5, dissolved in 20 mM phosphate buffer) 0.1 ml of the enzyme solution is added to 0.5 ml, and the reaction is carried out at 30 ° C. for 5 minutes. After adding 0.5 ml of 0.1 M HCl to stop the reaction, the absorbance of the released p-nitroaniline at 400 nm is measured, and 1 mol of p-nitroaniline is released per second at pH 7.5 and 30 ° C. Enzyme titer is 1 kata
As 1, the DPIV activity is determined.

[0035]

EXAMPLES The present invention will be described in more detail with reference to the following examples. However, the following examples are merely for illustrative purposes and do not limit the present invention in any way.

Example 1 (Preparation of umami ingredient enhancer DP IV) Using 6 kg of defatted soybeans and 6 kg of wheat, using "Ichishi" (manufactured by Bioc) as a seed koji, 4 kg at 30.degree.
Koji-making was performed for 8 hours to prepare soy sauce koji. In addition, the preparation of bran koji is performed using Aspergillus or
Using the yzae RIB915, the method of Ichijima was followed. Take 30 g of bran in a 1 liter Erlenmeyer flask,
After adding and mixing 21 ml of distilled water, the mixture was autoclaved to 12 ml.
Sterilized at 1 ° C for 20 minutes. A. From the storage slope oryza
e Two loops of RIB915 were inoculated and cultured at 30 ° C. for 72 hours to prepare bran koji.

Take soy sauce koji and bran koji, and add 10
A double volume of 20 mM phosphate buffer (pH 7.5) was added, and 4
C. for 3 hours. Filtration was performed in Step 2 to prepare a crude enzyme solution. DP IV in the koji making process of soy sauce koji
The activity increased rapidly from 18 hours to 30 hours of koji making, and reached 1.0 nkat / g koji in 48 hours. In addition, also in bran koji, DP IV activity was observed in the koji making process, and increased rapidly from 36 hours to 60 hours, and reached 1.0 nkat / g koji in 72 hours.

Ammonium sulfate was added to each of the above crude enzyme solutions so as to be 90% saturated, and left at 4 ° C. overnight.
The salted-out precipitate was subjected to celite filtration using a standard supercell to obtain an ammonium sulfate salted-out product. After Sephadex G-200 (Pharmacia) was buffered with 20 mM phosphate buffer (pH 7.5), the column (2 cm) was used.
x 95 cm). 1.7 g of ammonium sulfate precipitate
A solution dissolved in 3 ml of an mM phosphate buffer (pH 7.5) was added, and DP was added with a 20 mM phosphate buffer (pH 7.5).
IV was eluted. DP IV activity was 3 nkat / ml.

Example 2 (Effect of DP IV on Decomposition of Soy Protein in Soy Sauce Brewing) Preparation of bran koji and crude enzyme solution (hereinafter referred to as “Example 1”)
DP IV present enzyme solution) was prepared. 1.7 g of ammonium sulfate salting out product was added to 20 mM phosphate buffer (pH 7.5) 3
ml of the crude enzyme solution dissolved in a 20 mM phosphate buffer (p
H7.5) buffered with Sephacryl S-300 (Ph
armacia, 2 cm x 95 cm) and eluted with 20 mM phosphate buffer (pH 7.5).
The DP IV activity of the eluate was measured, and an enzyme solution from which DP IV had been removed (hereinafter referred to as a DP IV removal enzyme solution) was prepared.

On the other hand, an enzyme solution (hereinafter referred to as DP IV-added enzyme solution) was prepared by adding DP IV in an amount of 0.5 nkat to the above crude enzyme solution (enzyme solution containing DP IV). S
10 ml of distilled water was added to 300 mg of Oybean Flour (Type I, 52% protein, manufactured by SIGMA), heated at 121 ° C. for 10 minutes, and 10 ml of 1M phosphate buffer (pH 7) was added thereto. 2 m of DP IV removal enzyme solution or DP IV added enzyme solution
l and 1 ml of toluene at 60 ° C for 60 hours or 9 hours.
The enzyme reaction was performed for 0 hours. After completion of the reaction, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes, and the product filtered with filter paper (No. 2) was used as a soybean protein degradation product.

Ultra-filtration of the soybean protein hydrolyzate (USY
-1, molecular weight cut off 10,000, Advantech)
Thereafter, measurement was performed using an amino acid analysis system (LC-6A, OPA method, manufactured by Shimadzu Corporation). Column is Ship
ack ISC07 / S1504 using mobile phase A
Liquid: 0.2N sodium citrate solution (pH 3.
2), solution B: 0.6N sodium citrate + 0.6N
Boric acid solution (pH 10), solution C: 0.2N sodium hydroxide solution, 55 ° C., flow rate 0.3 ml / mi
n, Detection was performed under the analysis conditions of EX348 nm and Em450 nm. Table 1 shows the amino acid composition of the soy protein digest.
Shown in

[0042]

[Table 1]Table 1 Amino acidsComposition g (composition ratio%) Enzyme solution with DP IV removal Enzyme solution with DP IV DP IV added enzyme solution Asp 0.2 (1.2%) 2.3 (3.9%) 3.1 (4.9%) Thr 1.1 (6.6%) 4.4 (7.5%) 5.8 (9.2%) Ser 0.5 (3.0%) 2.6 (4.5%) 4.4 (7.0%) Glu 0.2 (1.2%) 6.8 (11. 6%) 7.8 (12.4%) Pro 0.1 (<1%) 2.9 (5.0%) 2.8 (4.5%) Gly 0.2 (1.2%) 1 2.4 (2.4%) 2.0 (3.2%) Ala 0.8 (4.8%) 3.3 (5.6%) 2.9 (4.6%) Val 1.0 ( 6.0%) 3.6 (6.1%) 2.8 (4.5%) Met 0.4 (2.4%) 1.4 (2.4%) 1.3 (2.1%) ) Ile 1.4 (8.4%) 4.0 (6.8%) 2.8 (4.5%) Leu 2.6 (15.6%) 6.4 (10) 3.9%) 4.3 (6.8%) Tyr 0.8 (4.8%) 2.6 (4.5%) 3.3 (5.2%) Phe 2.2 (13.2%) ) 4.0 (6.8%) 3.8 (6.0%) His 1.6 (9.6%) 2.8 (4.8%) 2.6 (4.1%) Lys 1. 3 (7.8%) 4.3 (7.3%) 6.5 (10.3%) Arg 2.3 (13.8%) 5.8 (9.9%) 6.7 (10.7%) Total 16.7 (100%) 58.6 (100%) 62.9 (100%) (Hydrolysis at 30 ° C. for 60 hours)

With regard to amino acids having taste,
In the enzyme solution containing DP IV, 6.8 mg of glutamic acid and 2.3 mg of aspartic acid were produced, and the amount of production increased about 30 times and 10 times that of the enzyme solution for removing DP IV, and the amount of proline was increased to 2.9 mg. . Further, in the DP IV-added enzyme solution, glutamic acid was 7.8 mg and aspartic acid was 3.1 mg.
The production of mg and 0.8 mg increased.

As for hydrophobic amino acids such as leucine and phenylalanine and basic amino acids such as histidine and arginine, their composition ratios (%)
However, in the enzyme solution with DP IV, the amount was reduced by about 50 to 70% as compared with the enzyme solution with DP IV removed, and further decreased in the enzyme solution with DP IV added.

As described above, DP IV specifically promotes the production of glutamic acid, aspartic acid, etc., which plays an important role in the taste (umami) of soy sauce, and enhances the taste of soy sauce. It turns out that it can be used for brewing.

[0046]

According to the soy sauce brewing method of the present invention, the content of amino acids such as glutamic acid and aspartic acid, which are umami components of soy sauce, can be specifically increased.
And DP IV can be used as a taste component enhancer of soy sauce.

Claims (5)

[Claims] 1. A method for producing an amino acid seasoning using soybean protein as a raw material, comprising adding X-prolyl dipeptidyl aminopeptidase. 2. The method according to claim 1, wherein the amino acid seasoning made from soy protein is soy sauce. 3. A step of mixing a denatured soy sauce brewing protein material and a carbohydrate material, a koji making step of adding seed koji to the mixture, and adding a saline solution to the obtained soy sauce koji to make moromi. A method for producing soy sauce comprising a preparation step of making, fermenting and aging the mixture, wherein X-prolyl dipeptidyl aminopeptidase is added. 4. The production method according to claim 1, wherein the X-prolyl dipeptidyl aminopeptidase is derived from Aspergillus oryzae. 5. A soy sauce flavor enhancer comprising X-prolyl dipeptidyl aminopeptidase as an active ingredient.