JPH0799923A - Production of seasoning - Google Patents

Abstract

PURPOSE:To prepare a seasoning remarkably enriched in glutamic acid and improved in flavor and taste. CONSTITUTION:In a method for producing a seasoning by treating a protein raw material with a protease, the liquid culture product of Bacillus subtilis having an ability for producing gamma-GTP(gamma-glutamyltranspeptidase) and not giving the smell of fermented soybeans to the culture product is added to the protease- acting reactional system.

Description

Detailed Description of the Invention

[0001]

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 producing a seasoning having an improved flavor.

[0002]

2. Description of the Related Art γ-GTP (γ-glutamyl transpeptidase: γ-glutamyl transpept)
Idase) is an enzyme that converts glutamine, which has no taste, into glutamic acid, which has taste. Conventionally, when a proteolytic enzyme is allowed to act on a protein raw material such as soybean to produce a seasoning, in order to enhance glutamic acid, a microbial origin,
For example, Bacillus subtilis
The enzyme preparation of γ-GTP which has been fractionated and purified from the culture of Tilis) is used. However, in such a method using a γ-GTP preparation, the amount of glutamic acid produced and the flavor of the obtained seasoning are not always satisfactory.

[0003]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of eliminating the above-mentioned drawbacks and providing a seasoning in which glutamic acid is remarkably enhanced and which has an improved flavor.

[0004]

Therefore, the present inventors have
In order to achieve the above-mentioned object, as a result of intensive studies, a hydrolyzate of a protein raw material by an enzyme was identified as a liquid culture of Bacillus subtilis having a γ-GTP producing ability and the culture having no natto odor. Addition and action of the γ-GTP preparation fractionated and purified from the culture, compared with the case of adding and action, glutamic acid is significantly enhanced, the flavor is also significantly improved seasoning is obtained. Therefore, the present invention has been completed based on this finding.

That is, the present invention relates to a method for producing a seasoning by allowing a proteolytic enzyme to act on a protein raw material,
An improved flavor characterized by the addition of a liquid culture of Bacillus subtilis, which has the ability to produce γ-GTP and whose culture has substantially no natto odor, to a reaction system for the action of proteolytic enzymes. It relates to a method for producing seasonings.

The present invention will be described in detail below. In the present invention, a known method is adopted for the hydrolysis of the protein raw material with a proteolytic enzyme. That is, as the protein raw material, for example, soybean, defatted soybean (defatted soybean), dehulled soybean, soybean protein meal, wheat gluten, corn gluten and the like are used. These protein raw materials are usually subjected to denaturing treatment by, for example, steaming treatment, pressure-heated expansion treatment with saturated steam or heated steam, or heat-pressurized kneading expansion treatment with an extruder. The denatured protein raw material is added to the action reaction system of the proteolytic enzyme at a concentration of 0.1 to 30% to be hydrolyzed.

Any proteolytic enzyme may be used as long as it is used in foods. And, it may be a single enzyme or a complex enzyme. Examples of these include molds, bacteria, proteolytic enzymes of actinomycetes, and koji. Soy sauce koji or the like is the most preferable enzyme source because it contains a proteolytic enzyme and a protein raw material together, and is also a protein raw material.

[0008] Proteolytic enzymes are usually added in an amount of 1 to 20% based on the protein raw material (dry matter conversion). Hydrolysis by an enzyme is usually carried out under conditions of pH 3 to 12 and temperature of 30 to 70 ° C., preferably 40 to 60 ° C. for 10 to 300 hours, preferably 72 to 170 hours. In the hydrolysis step of the protein raw material, a carbohydrate raw material such as wheat, corn and starch, and a saccharifying enzyme such as amylase may be appropriately added and used by a conventional method. Furthermore, the hydrolysis is salt-free to 20
It may be carried out in the presence of up to about% salt.

Next, the Bacillus used in the present invention
A strain belonging to S. subtilis has the ability to produce γ-GTP,
Moreover, the liquid culture is a strain having no natto odor. Among them, those having a γ-GTP production ability of 2 units / ml or more are preferably used. As a typical strain thereof, for example, γ-G
Bacillus subtilis IAM1523 having a TP-producing ability of 2 units / ml or more, a mutant strain thereof, for example, no. 1349 etc. can be mentioned.

In the present invention, γ-of liquid culture
The GTP activity is determined by the method of Ogawa et al.
l and biological chemistry
y, 55, 2971, 1991). That is, γ-glutamyl-p-nitroanilide (γ-glutamyl-p-nitroanilide)
e) is a glutamyl group donor, glycylglycine (gl
ycylglycine) as its receptor,
The amount of enzyme that transfers 1 μmole of γ-glutamyl group to glycylglycine per minute was defined as 1 unit. In addition, for the selection of a strain of Bacillus subtilis whose culture has substantially no natto odor, the liquid culture was diluted 10 times with distilled water. As a result of the sensory test by the panelists of the above, it was decided that all the strains judged to be none were the relevant strains.

An example of the screening method of the strain used in the present invention is as follows. Agar nutrient medium (polypeptone 3%, yeast extract 1%, glucose 0.
1%, NaCl 0.5%, agar 1.5%, pH 7.0)
Plates are allowed to colonize any strain of Bacillus subtilis. On the other hand, a 0.7% agar plate (pH 8.0) containing 1.0 mM γ-glutamyl-p-nitroanilide, 50 mM glycylglycine, and 100 mM trishydroxyaminomethane-hydrochloric acid is prepared. This plate is placed on a plate medium on which the above strain has formed a colony. Then, by incubating at 30 to 40 ° C. for 10 to 60 minutes, γ-glutamyl-p-nitroanilide is decomposed and a yellow coloring band is formed around the colony. Colored bands of various sizes are formed around the colony depending on the degree of decomposition of the substance, that is, the degree of γ-GTP activity. Select a strain of Bacillus subtilis that is a colony with as large a chromogenic band as possible.

The above-mentioned arbitrary Bacillus subtilis strain may be a conserved strain or an unknown Bacillus subtilis strain obtained from nature. In addition, NTG (N-methyl-N'-nit) was added to cells of a strain having a natto odor.
It may be a mutant strain obtained by performing a known mutation treatment with ro-N-nitrosoguanidine), ultraviolet rays or the like.

Next, 1 to 5 ml of the white ears of the selected strain is a liquid medium (the agar nutrient medium without agar).
/ Inoculate a test tube (16 mm x 160 mm) and
100 ° C., 100-180 rpm for 15-100 hours,
Shake culture. For each of the liquid cultures thus obtained, the presence or absence of the natto odor is determined by the above method. A liquid culture having substantially no natto odor is further measured for γ-GTP activity, and a strain having γ-GTP producing ability is selected.

Next, a liquid culture of the strain of the present invention used in the present invention can be obtained by culturing in a conventional manner using a medium containing ordinary components, for example, as follows. 1) Medium: As a nitrogen source, usable inorganic or organic nitrogen compounds, such as ammonium sulfate, ammonium chloride, soybean powder (whole soybean or defatted soybean), yeast extract, peptone, meat extract, gelatin, Corn steep liquor, amino acid,
Soybean or wheat bran leachate is used. Examples of the carbon source include sugars such as glucose, maltose, sucrose, molasses, starch syrup, polysaccharide compounds such as various starches, various organic acids such as citric acid and malic acid, and inorganic salts such as manganese and phosphoric acid. ,sodium,
As salts of various ions such as potassium, magnesium and calcium, and as trace elements, vitamins such as biotin and thiamine are used. Then, these medium components are appropriately mixed. PH of the medium is 5-10
Adjusted with caustic potash or soda. Sterilization is performed under normal conditions, for example, 100 to 130 ° C. and 5 to 30 minutes.

2) Culture: A liquid culture method is used. At that time, it is preferable to perform aeration stirring and shaking culture. The aeration condition is 0.01 to 4 vvm, and the medium amount is 20 when shaking and stirring.
50 ~ 30 with ~ 200ml / 500ml Sakaguchi flask
It is 0 rpm. And the temperature is usually 10 to 100 ° C,
The temperature is preferably 30 to 45 ° C., and the time is 10 to 150 hours. The pH during culturing is 5-10. In this way, the γ-GTP activity is usually 2 units / ml or more,
In addition, a liquid culture having substantially no natto odor can be obtained.

The liquid culture used in the present invention is Bacillus
It may contain the bacterial cells of S. subtilis strains, or may be a culture supernatant obtained by removing the bacterial cells by centrifugation, filtration or the like. Further, they may be used as a dried product obtained by freeze-drying, spray-drying, low-temperature or warm-drying, or the like.

Next, the liquid culture produced as described above is added to the action reaction system of the proteolytic enzyme in the production of the seasoning. The enzyme may be added before, during, or after the action of the enzyme. When the liquid culture is added to the reaction system of the proteolytic enzyme after the action of the proteolytic enzyme, for example, 20 to 80 ° C., preferably 4 after the addition.
0 to 60 ° C., 5 minutes to 20 hours, preferably 10 minutes to 1
It is preferable to react for 0 hours. The amount of the liquid culture added to the reaction system of the action of the proteolytic enzyme is preferably 0.5 unit or more per g of the protein raw material dry matter in terms of γ-GTP activity. On the other hand, even if 30 units or more are added, the effect of the addition is hardly seen.

When the protein raw material is hydrolyzed, 0 to
A protein raw material and a carbohydrate raw material which have been subjected to a denaturation treatment in the presence of 18%, preferably 4 to 12% sodium chloride, a proteolytic enzyme such as koji, and the liquid culture of the present invention are charged and further cultured by a known method. The salt-tolerant lactic acid bacterium and the salt-tolerant yeast obtained as above are added as usual, and fermented by a known control method,
It is also possible to produce fermented seasonings. As the salt-tolerant lactic acid bacterium and the salt-tolerant yeast, those commonly used for brewing soy sauce miso may be used. For example, salt-tolerant lactic acid bacteria include Pediococcus halophilus IAM169
3, IAM1678, FERM-pNO. 1414 and the like are salt-tolerant yeasts such as Zygosaccharomyces rouxi.
i) ATCC13356, IFO0495, IFO05
05, Torulopsis
is ・ versatilis) IFO0652, IFO
01231 or the like can be used. Fermentation is 20 ~ 35 ℃,
It may be carried out for 3 to 60 days.

Although the seasoning of the present invention is produced as described above, it can be made into a solid seasoning such as powder and tablets by various known operations. By using the seasoning of the present invention, the seasoning-blended food of the present invention having an improved flavor can be produced. For example, 2-50%, preferably 10-40%, of the seasoning of the present invention may be added to grilled meat and grilled fish sauce, pond vinegar, various salad oils, dashi seasonings and the like.

[0020]

INDUSTRIAL APPLICABILITY In the production of a seasoning by hydrolyzing a protein raw material with a proteolytic enzyme, when the liquid culture of the strain of the present invention is used as described above, a γ-GTP preparation fractionated and purified from it is used. It is possible to produce a high-quality seasoning with a significantly improved flavor as compared with the case of using.

[0021]

EXAMPLES The present invention will be specifically described below based on examples. In the following examples, the sensory evaluation of seasonings was performed by a scoring method by 20 skilled panelists, and the comprehensive evaluation of flavors was scored according to the following criteria, and the average value was shown. Excellent, 5 points; Excellent, 4 points; Normal, 3 points
Points: Inferior, 2 points; Especially inferior, 1 point In addition, total nitrogen (TN) in seasonings is "Soy sauce test method"
(Published by The Japan Soy Sauce Research Institute) The method and glutamic acid (Glu) described above are Hitachi high-speed amino acid analyzer L
It was measured by -8500 type.

Example 1 Soy flour 2.0% (W / v), glucose 1% (w /
v), a medium containing 0.5% (w / v) salt (pH)
7.0) 10l was put into a 20l jar famenter and sterilized by autoclave at 120 ° C for 15 minutes. The Bacillus subtilis IAM1523 of the present invention was added to the medium.
The cell suspension of (the strain of the present invention) was inoculated at a rate of 10 ⁶ cells / ml, the temperature was 38 ° C., the aeration rate was 1 vvm, and the stirring number was 50.
The culture was performed at 0 rpm for 2 days. Further, the liquid culture has a natto odor and Bacillus subtilis IAM1212 (control strain 1) having a γ-GTP producing ability of 1.7 units, and natto odor and a γ-GTP producing ability of 3.0. Unit / ml Bacillus subtilis IAM1633 (control strain 2)
Was cultured in exactly the same manner as in IAM1523 above to obtain each liquid culture.

Next, 30% (w /
v) To each moromi containing 1700 ml of saline solution, 500 ml of a liquid culture of each strain (culture liquid containing cells) was added,
Furthermore, water was added so that the total amount of pumped water would be 2200 ml, and the usual method of soy sauce production was controlled, and fermentation for 6 months,
Aged. Furthermore, these moromi flavors were squeezed by a conventional method and fired to obtain a fermented seasoning. Table 1 shows the presence or absence of natto odor and the γ-GTP activity value of the obtained liquid culture,
Further, the TN and Glu analysis values and the sensory evaluation values of the obtained seasoning are shown.

Table 1 ─────────────────────────────────── Strain natto odor γ-GTP TN Glu Glu / TN Presence or absence of sensory evaluation value Activity (single (%) (%) / ml) ─────────────────────────────── ───── Inventive strain None 2.5 1.75 1.40 0.80 4.8 Control strain 1 Yes 1.7 1.70 1.20 0.71 2.1 Control strain 2 Yes 3.0 1.78 1.50 0.84 2.3 ───────────────────────────────────

From Table 1, the fermented seasoning obtained using the strain IAM1523 of the present invention is compared with the control strain,
It can be seen that the glutamic acid content is high and the sensory evaluation value is high.

Example 2 130 ml of water was added to 100 g of defatted soybean, and the mixture was kept at 150 ° C. for 2 minutes.
After steaming for minutes, the protease (Aspergillul)
bran malt 1 from s. oryzae ATCC 20386 strain
5 g of a precipitate obtained by adding 2 liters of cold ethanol to 1 liter of a koji extract obtained by extracting 4 kg of water with 4 g, and Bacillus subtilis IAM1523 strain were used to produce cells in the same manner as in Example 1. 100 ml of the liquid culture containing was added. Further, a supernatant liquid (bacterial body removing liquid culture) 27 obtained by removing the bacterial cells by centrifuging 300 ml of the bacterial cell-containing liquid culture
The same procedure as above was carried out except that 0 ml to 100 ml was taken and added instead of the cell-containing liquid culture. Salt was added to these so that the final concentration was 12%, and after stirring well, hydrolysis was carried out for 5 days while shaking at 45 ° C. This hydrolyzate was centrifuged to obtain a transparent seasoning (seasoning of the present invention). In addition, 100m of the liquid culture for removing the bacterial cells
chill 1 to 5 ° C, 200 ml cold ethanol at -20 ° C
Was added to form a γ-GTP-containing precipitate. The precipitate was collected by centrifugation and freeze-dried to obtain a purified ethanol fraction. A control seasoning was obtained in the same manner as described above, except that water was added to this and adjusted to 100 ml and used in place of the above liquid culture. Table 2 shows analytical values and the like of these seasonings.

Table 2 ──────────────────────────────────── Liquid culture γ-GTP TN Glu Glu / TN sensory evaluation value and enzyme activity (unit (%) (%) form / ml) ───────────────────────────── ──────── Cell-containing liquid culture 2.3 2.26 1.40 0.62 4.8 (invention) Cell-free liquid culture 2.1 2.28 1.40 0. 61 4.8 (Invention) Ethanol fractionated product 2.1 2.23 0.91 0.41 3.4 (Control) ──────────────────── ─────────────────

From Table 2, it can be seen that the seasoning of the present invention using each liquid culture was obtained by using the ethanol fraction purified product even though the γ-GTP activity of the liquid culture is equivalent to the ethanol fraction purified product. It can be seen that, compared with the obtained seasoning as a control, Glu is produced better and the sensory evaluation value is higher, showing a remarkable flavor improving effect.

[0029]

Example 3 Soy flour 2.0% (w / v), glucose 1
The IAM1523 strain was cultured in the same manner as in Example 1 using 20 l of a medium (pH 7.0) containing 50% (w / v) and 0.5% (w / v) sodium chloride to obtain a liquid culture. Next, 1500 g of soy sauce koji, 1700 ml of 30% (w / v) saline
The liquid culture was added to the moromi mash containing 0 ml and 30 m, respectively.
1, 200 ml, and powder obtained by freeze-drying 16 l of the liquid culture were dissolved in water to obtain γ-GTP activity of 30 and 4
What was adjusted so that it might be 0 unit was added, and water was added and adjusted so that the total pumping water might be 2000 ml, the same management as Example 1 was performed, and fermentation and aging were performed for 6 months.
In addition, to the lyophilized product of the liquid culture, the amount of saline solution added was reduced to a prescribed salt concentration, and water was added instead of the reduced salt water content. Further, the fermented seasoning was obtained by pressing and burning in the same manner as in Example 1. Third
The analytical values of the fermented seasoning are shown in the table.

Table 3 ──────────────────────────────────── Additive liquid γ-GTP TN Glu Glu / TN sensory evaluation value Culture amount Addition amount (single (%) (%) (ml) / protein raw material dry matter 1 g) ─────────────────────── ────────────── 0 0 1.78 1.42 0.80 3.3 120 0.5 1.76 1.49 0.85 4.6 600 2.4 1. 76 1.52 0.86 4.8 Lyophilized product 30.0 1.82 1.65 0.91 5.0 Lyophilized product 40.0 1.86 1.66 0.89 5.0 ──── ────────────────────────────────

From Table 3, the liquid culture of the present invention was prepared using γ-GT.
It has been found that the P activity is preferably 0.5 unit or more per 1 g of the protein raw material dry matter.

Example 4 1 liter of each fermented seasoning obtained in Example 1, 1 liter of mirin and water, and 300 g of sugar were mixed and stirred, and then heated and concentrated to about half to prepare a "broiled fish sauce". As a result of tasting with 20 skilled panelists using the sauce for teriyaki for the first time, all the panelists who made with Bacillus subtilis IAM1523 strain had other strains. It was evaluated to be preferable in comparison with.

Example 5 A liquid 500 obtained by extracting 750 ml of a fermented seasoning prepared by using a liquid culture for removing bacterial cells, 150 g of sugar, and 30 g of crushed bonito flakes in 600 ml of hot water in Example 2.
ml, 20 g of kelp, and 10 g of shiitake together were mixed with 100 ml of a liquid extracted with hot water to prepare a high-quality "dashi seasoning".

Front page continued (72) Inventor Hironaga Hashiba 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (1)

[Claims] 1. A liquid of Bacillus subtilis having a γ-GTP-producing ability and a culture thereof having substantially no natto odor, in a method for producing a seasoning by allowing a proteolytic enzyme to act on a protein raw material. A method for producing a seasoning having an improved flavor, which comprises adding a culture to a reaction system for the action of a protease.