JP3417461B2 - Soy protein hydrolyzate, method for producing the same, and foods using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a soybean protein degradation product obtained by degrading β-conglycinin and Gly m Bd30K, which are major allergens of soybean protein, and a food product using the same.

[0002]

2. Description of the Related Art In recent years, an increasing number of so-called food allergic patients have an abnormal hypersensitivity reaction such as rhinitis, eczema, diarrhea and urticaria when eating a specific food, and sometimes become a serious morbid state. These treatment methods include symptomatic treatment with drugs and so-called elimination food therapy for removing causative substances that become allergens from the patient's diet. Among food allergies, eggs, milk, since specific proteins contained in soybeans are recognized as the three major allergens, it is common to introduce elimination food therapy, but among the above allergens, soy protein is Included in traditional Japanese foods such as tofu, frozen tofu, and yuba, and a wide range of foods containing this as a raw material, as well as the function of soybean proteins, such as emulsifying property, gel forming property, water retention property, oil retention property, and foaming property. In recent years, the number of foods that use soybean proteins that utilize such functionality has rapidly increased, making elimination diet therapy difficult.

By the way, soybean allergen protein is Gly m Bd 30K as a soybean protein component having high reactivity with IgE antibody obtained from a patient with atopic disease to soybean.
It has been reported that the α subunit and β subunit of β-conglycinin were identified (J. Nutr. Sci. Vitaminol., 555-565 (1991)).

Among these soybean allergen proteins, an attempt has been made to reduce the allergen by separating or degrading a protein called Glym Bd 30K, which has the highest frequency of onset in allergic patients, from soybean. For example, as an allergen removal from soy milk, JP 7-236444 JP, JP 7-236449, JP 8-308506, Gly m Bd 30K has a high affinity with the lipid component in soy milk. , A method utilizing transfer to supernatant under high centrifugal acceleration is disclosed.
Further, as described in JP-A-7-236427 and JP-A-9-37720, 7S of the main storage protein at a specific pH and ionic strength is used.
A method utilizing the difference in solubility between the 11S fraction and Gly mBd 30K is disclosed.

As an attempt to decompose Gly m Bd 30K with an enzyme, JP-A-6-253758, JP-A-7-203890,
JP-A-7-236439, JP-A-8-56600, JP-A-9
There is known a method in which a protease is allowed to act on soybean or soybean protein that has been heat-denatured as described in Japanese Patent No. 23822.

However, in the above method, a high centrifugal acceleration is required to separate Gly m Bd 30K and a high ionic strength is required to separate the 7S and 11S fractions from Gly m Bd 30K. Since a salt process is required, there are industrial difficulties, and β-conglycinin, which has a high frequency of onset in allergic patients, remains next to Gly m Bd 30K. Also, in the method of degrading Gly m Bd 30K with an enzyme, a protease acts on soybeans and soybean proteins that have been heat-denatured, so that not only Gly m Bd 30K but also the major storage protein
Both the 7S and 11S fractions were decomposed, and the physical properties of soybean protein suitable for foods, such as gel forming property, were almost lost, and there was a drawback that the use was limited.

[0007]

In view of the above circumstances, the present invention provides a low allergen soybean protein hydrolyzate in which β-conglycinin and Gly m Bd 30K, which are major allergens of soybean protein, are decomposed, and a food using the same. To aim. In view of the above circumstances, the present invention aims at a soybean protein decomposition product in which major allergens of soybean protein are uniformly decomposed and non-allergens are hardly decomposed, and a food using the same.

[0008]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that β-conglycinin and Gly in soybean protein can be obtained by allowing a protease to act on soybean protein under specific conditions. The present invention has been completed based on the finding that m Bd 30 K can be selectively decomposed and a target soybean protein hydrolyzate can be obtained.

That is, in the present invention, the ratio of the amount of glycinin to the amount of β-conglycinin in soybean protein is 5 or more, and the ratio of Gly m Bd 30K amount to the amount of crude protein in soybean protein is 0.5.
It is a soy protein hydrolyzate having a content of not more than%. Β- in soy protein
The ratio of the amount of glycinin to the amount of conglycinin is 5 or more and the amount of Gly m Bd 30K to the amount of crude protein in soybean protein is 0.
At 5% or less, 0.22M trichloroacetic acid soluble nitrogen rate is 5 to 30%
The soybean protein degradation product is preferred. The present invention also provides a soybean protein hydrolyzate-containing food containing the soybean protein hydrolyzate as part or all of the protein raw material. The present invention also provides
A method for producing a soybean protein hydrolyzate, which comprises degrading low-denatured soybean protein with a protease derived from Bacillus subtilis (natto) above 50 ° C and below 90 ° C. Low-denaturation defatted soybean NS
I (nitrogen solubility coefficient) is preferably 60 or more. It is preferable to decompose so that the TCA% of the reaction solution after decomposition becomes 5 to 30%. It is preferable to decompose at pH = 5 to 10 for 5 minutes to 2 hours.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The soybean protein degradation product of the present invention comprises β-conglycinin and Gly which are major allergens of soybean protein.
m Bd 30K is decomposed and reduced.

In general, the major storage proteins in soybean, glycinin and β-conglycinin, for example, in the case of general soybeans produced in the United States, the component ratio is usually glycinin / β-conglycinin ratio = 1.3 to 1.5. Processed products also maintain their component ratio.

The soybean protein hydrolyzate of the present invention has a ratio of 5 or more because β-conglycinin is selectively decomposed.

The amount of Gly m Bd 30K is usually about 2% with respect to the amount of crude protein of soybean protein (Kjeldahl nitrogen amount x 6.25), but Gly m Bd 30K is selectively present in the soybean protein hydrolyzate of the present invention. Since it has been decomposed into
% Or less, preferably 0.2% or less.

A method for investigating the change in the content of each component in soybean protein can be evaluated by separating soybean protein into each component by SDS-electrophoresis. In the case of β-conglycinin and glycinin, SDS-electrophoresis gel was stained with Coomassie blue, and the density of bands of β-conglycinin, glycinin and other proteins was measured with a densitometer, and β-conglycinin and glycinin for the entire area of the area were measured. The glycinin / β-conglycinin ratio can be determined from the area area ratio of.

Further, in the case of Gly m Bd 30K, Gly m Bd was similarly extracted from a gel obtained by separating soybean protein by SDS-electrophoresis.
EC using 30K protein transferred to PVDF (polyvinylidin difluoride) membrane and Gly m Bd 30K monoclonal antibody
A method of detecting by L method (manufactured by Amersham) (sensing fluorescence emission by peroxidase with an X-ray film) can be measured with high sensitivity (see the method of JP-A-7-236427). And the content is the purified Gly m Bd 30K protein (Gly m Bd 30K protein is purified by T. Ogawa et.al., Biosci.
Biotech.Biochem., 57, 1030 (1993)), and the density of each of the crude proteins detected by SDS-electrophoresis and the density detected by the above method was measured using a densitometer. When a calibration curve is prepared in Fig. 3, the band area area of the Gly m Bd 30K protein is proportional to the crude protein mass of the Gly m Bd 30K protein, and the Gly m Bd 30K protein amount can be calculated. Then, the soybean protein with a known crude protein amount was detected by the above method, and Gl
The amount of Gly m Bd 30K protein can be calculated from the calibration curve by examining the band area area of ym Bd 30K protein.
The amount of ym Bd 30K (hereinafter referred to as Gly m Bd 30K%) is required.

The soybean protein hydrolyzate of the present invention contains 0.22M.
Trichloroacetic acid soluble nitrogen rate (TCA% below) is 5 ~
Degradation of 30%, preferably 7 to 20%.

Next, the present invention is a soybean protein degradation product-containing food containing the above-mentioned soybean protein degradation product as a part or all of the protein raw material.

The soybean protein hydrolyzate of the present invention contains β-conglycinin and Gly m Bd 30 which are major allergens of soybean protein.
Since K is decomposed and glycinin, which is not an allergen, is hardly decomposed, it can be widely used for general foods in which soybean protein has been conventionally used.

Examples of soy protein-containing foods include tofu,
In addition to fried foods, cancerous foods, etc., meat products such as ham, sausages, hamburgers, meatballs, fish paste products such as kamaboko, fried kamaboko and bamboo rings, and prepared foods, protein drinks, baby milk powder and other beverages are exemplified. Foods using these soybean protein decomposition products, depending on the amount of soybean protein contained, 10 soybean allergens contained in foods using soybean protein other than the soybean protein decomposition product of the present invention. Since it is reduced to less than 10%, it is suitable for elimination diet therapy for soybean food allergy patients.

An example of the method for producing the soybean protein hydrolyzate of the present invention will be described below. First, by degrading low-denatured soybean protein with a specific temperature range and with a specific protease, glycinin, which is a main component of soybean protein, is hardly decomposed, and β-conglycinin and G, which are the main allergens of soybean protein, are decomposed.
There is a method of selectively decomposing only ly m Bd 30K.

In this method, the reaction temperature is important and the temperature is 50 ° C.
It is suitable to carry out the enzyme reaction at a temperature above 90 ° C and below 90 ° C, preferably 60 to 80 ° C. Below this temperature, the degree of decomposition of β-conglycinin, Gly m Bd 30K is low, and in denatured soybean protein above this or excessively heated, β-conglycinin, Gly m Bd 30K as well as glycinin are decomposed, and soybean protein is decomposed. Β-conglycinin and Gly m Bd 30K, which are major allergens of soybean, cannot be selectively decomposed, and the functionality originally possessed by soybean proteins, such as gel-forming property, is almost lost. Therefore, as the soybean protein used, soybean, full-fat soybean milk mainly composed of soybean protein, defatted soybean milk, concentrated soybean protein, soybean protein isolate, etc., but the processing treatment without protein denaturation or limited to low denaturation The processed soybean product is preferable, and it is not limited to a variety, a production area, or the like. Generally, defatted soybean obtained by subjecting soybean to low-temperature extraction treatment with N-hexane as an extraction solvent is suitable as a raw material, and in particular, a low-denaturation defatted soybean having an NSI (nitrogen solubility coefficient) of 60 or more, preferably 80 or more. Skim soy milk extracted from water with water, concentrated soy protein, and isolated soy protein are preferably used.

The protease to be used must be an enzyme or enzyme agent having a proteolytic activity at a temperature higher than 50 ° C. and lower than 90 ° C., preferably 60 to 80 ° C., and is derived from Bacillus subtilis (natto). Protease derived from Bacillus subtilis such as subtilisin which is a protease, protin (trade name; manufactured by Daiwa Kasei Co., Ltd.) which is a commercially available protease, and proleather (trade name; manufactured by Amano Pharmaceutical Co., Ltd.) is preferably used. Proteases derived from these Bacillus subtilis (natto) can selectively decompose β-conglycinin and Gly m Bd 30K, which are the major allergens of soybean protein, at the above reaction temperature.

The enzyme reaction step is carried out during the soybean protein production step. For example, when producing isolated soy protein,
A suspension of water-neutralized acid-precipitated curd obtained by isoelectric precipitation of defatted soymilk extracted with water from low-denaturation defatted soybeans is adjusted to more than 50 ° C and less than 90 ° C, preferably 60 to 80 ° C. Protease is added and the degradation reaction is carried out. After the completion of the reaction, the reaction solution is sterilized (also serves to inactivate the enzyme) and dried if necessary. Further, the reaction solution can be fractionated at around pH 6 which is the isoelectric point of glycinin, and the precipitated fraction can be collected, hydrated, neutralized and sterilized, and dried if necessary. The amount of protease added, the reaction pH, and the reaction time depend on β-conglycinin and Gly m B
Although it may be determined from the degree of decomposition of d 30K, the TCA% of the reaction solution after decomposition is 5 to 30%, preferably about 7 to 20%. Generally, the amount of protease added is 0.001 to 1% with respect to the solid content of the soybean protein suspension, preferably
Optimum pH of enzyme agent that uses reaction pH in the range of 0.01 to 0.5%
Or stable pH, usually pH = 5-10, preferably pH = 6-9
Then, the reaction may be carried out for 5 minutes to 2 hours, preferably 10 minutes to 1 hour, and continuous reaction can also be carried out by passing the solution through a column on which protease is immobilized.

[0024]

EXAMPLES Hereinafter, the embodiments of the present invention will be specifically described with reference to Examples, but the technical scope of the present invention is not limited by these.

Production Example 1 To 10 kg of low-denaturation defatted soybean (nitrogen solubility coefficient; NSI> 80) defatted with N-hexane was added 10 times the amount of water, and the mixture was adjusted to 1 at room temperature and pH 7.
After time extraction, centrifugation was performed to obtain 95 kg of defatted soy milk. Hydrochloric acid was added to this to adjust the pH to 4.5, and the mixture was centrifuged to remove whey to obtain 10 kg of acid precipitation curd. Add water to the acid precipitation card,
After adjusting the pH to 7 with 20% NaOH, the suspension was adjusted to 70 ° C. and 0.2% protein (trade name;
(Manufactured by Daiwa Kasei Co., Ltd.) was added and the enzyme reaction was carried out for 30 minutes. The reaction solution was sterilized by heating at 140 ° C. for 15 seconds, and the solution was spray-dried to obtain 3.5 kg of a soybean protein decomposition product. As a control, soybean protein was obtained by a method excluding the enzymatic reaction performed by adding protin in the above production process. Example 1 10 micrograms of the soybean protein decomposition product obtained in the above Production Example 1
The gel was stained with Coomassie blue by SDS-electrophoresis, and the density of each band of β-conglycinin and glycinin was quantified with a densitometer to determine the glycinin / β-conglycinin ratio. Similarly, 2 micrograms of soybean protein degradation product
DS-electrophoresed gel was transferred to PVDF membrane and Gly m Bd 30K
Detection of Gly m Bd 30K using monoclonal antibody
It was quantified. Glycinin / β-conglycinin ratio = 12.7, G
LymBd 30K% = 0.1%, TCA% = 13%, and only β-conglycinin and GlymBd 30K were decreased. Example 2 Tofu was prepared by mixing 8 parts of the soybean protein hydrolyzate obtained in the above Production Example 1, 91.7 parts of water and 0.3 part of gluconodeltalactone and heating at 95 ° C. for 30 minutes. Production Example 1 The texture was comparable to that of tofu prepared in the same manner with the control soybean protein. Example 3 6 parts of the soybean protein hydrolyzate obtained in the above Production Example 1, fish meat surimi 30
Part, soybean oil 29 parts, flavoring agent 5 parts, and water 30 parts were mixed with a food cutter, packed in a casing tube, and heated at 90 ° C. for 30 minutes to prepare sausage. Production Example 1 The texture was comparable to that of a sausage prepared in the same manner with the control soybean protein. Comparative Example 1 and Comparative Example 2 When the reaction temperature for enzymatic decomposition was 37 ° C. in the manufacturing process of the above Production Example 1 (Comparative Example 1), or in the manufacturing process of the above Production Example 1, water was added to the acid-precipitated curd to obtain pH 7 Soybean protein was prepared by subjecting the suspensions prepared in step 1 to heat treatment at 100 ° C. for 10 minutes prior to the enzymatic reaction, and subsequently performing the enzymatic reaction in the same manner as in Production Example 1 (Comparative Example 2). When analyzed in the same manner as in Example 1, the soybean protein of Comparative Example 1 had a glycinin / β-conglycinin ratio = 1.3 and Gly m Bd 30K% =
1.8%, TCA% = 6%, and in the soybean protein of Comparative Example 2, glycinin / β-conglycinin ratio = 2.6, Gly m Bd 30K%
= 0.4%, TCA% = 35%. Comparative Example 3 Using the soybean protein decomposition product obtained in Comparative Example 2, tofu was prepared in the same manner as in Example 2, but it was brittle and could not be eaten. Comparative Example 4 A sausage was prepared in the same manner as in Example 3 using the soybean protein degradation product obtained in Comparative Example 2, but was significantly brittle as compared to the sausage prepared in the same manner as in the Production Example 1 control soy protein. It was a thing. Comparative Example 5 A soybean hydrolyzate was prepared in the same manner as in Production Example 1 except that the protease used for enzymatic degradation in the production process of Production Example 1 was changed to 0.05% of papain (manufactured by Nippon Biocon Co., Ltd.). When analyzed in the same manner as in Example 1, the soybean protein of Comparative Example 5 had a glycinin / β-conglycinin ratio of 1
3.1, Gly m Bd 30K% = 0.6%, TCA% = 13% Gly m
The decrease in Bd 30K was slightly small.

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, a soybean protein decomposition product obtained by decomposing β-conglycinin and Gly m Bd 30K, which are major allergens of soybean protein, and a food using the same can be supplied, and it can be applied to soybean food allergy patients. It is now possible to use it as a protein source and food suitable for elimination diet therapy.

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Claims (7)

(57) [Claims] 1. A soybean hydrolyzate in which the ratio of the amount of glycinin to the amount of β-conglycinin in soybean protein is 5 or more and the ratio of the amount of Gly m Bd 30K to the amount of crude protein in soybean protein is 0.5% or less. 2. The ratio of 0.22M trichloroacetic acid-soluble nitrogen in which the ratio of the amount of glycinin to the amount of β-conglycinin in soybean protein is 5 or more and the amount of Gly m Bd30K is 0.5% or less relative to the amount of crude protein in soybean protein. Is a soy protein hydrolyzate containing 5 to 30%. 3. A soybean protein hydrolyzate-containing food containing the soybean protein hydrolyzate according to claim 1 or 2 as a part or all of a protein raw material. 4. A process for producing soybean protein hydrolyzate, characterized by degradation with a protease derived from a low-denatured soybean protein to 90 ° C. and less than Bacillus subtilis exceed 50 ° C.. 5. The NSI (nitrogen solubility coefficient) of low-denaturation defatted soybean is 6
The production method according to claim 4, which is 0 or more. 6. The method according to claim 4, wherein the reaction solution after decomposition is decomposed so that the TCA% becomes 5 to 30%. 7. The method of decomposing at pH = 5 to 10 for 5 minutes to 2 hours.
6. The method according to any one of to 6.