JPH07236449A - Method for fractionating allergen in soybean milk - Google Patents

Abstract

PURPOSE:To readily fractionate a main allergen hardly being influenced by temperature conditions in centrifugation by adding a reducing agent such as a sulfurous acid compound to soybean milk and then centrifuging the resultant mixture. CONSTITUTION:This method for fractionating an allergen comprises adding a sulfurous acid compound, a glutathione compound and/or a cysteine compound to soybean milk and then centrifuging the resultant mixture. Thereby, a main allergen (GM30) specifically migrates into an upper layer and the amount thereof migrated is increased to readily carry out the fractionation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating allergens from soymilk for treating allergies, diagnosing allergies, etc. More specifically, it relates to the production of soymilk with reduced allergens and allergens in soymilk. The present invention relates to a method for separating allergens in soymilk, which is suitable for collecting soybean milk.

[0002]

2. Description of the Related Art Food allergy refers to a phenomenon in which an abnormal hypersensitivity reaction is caused when a specific food is eaten, resulting in a pathological condition, generally an egg, It is often caused by milk, soybean, rice, wheat, buckwheat, etc. Symptoms of allergy include rhinitis, eczema, diarrhea,
There are urticaria, etc., and these treatment methods include drug-based methods and finding foods that cause allergies, and removing the causative foods from the patient's diet until the risk of recurrence disappears, so-called elimination I have food therapy. And since the allergen component is in a specific protein fraction, the protein is decomposed by acting a proteolytic enzyme on the allergen-containing food, or the allergen is removed, or by a substitute food produced using a raw material without allergen, Subjected to elimination food therapy.

However, in the method of degrading a protein, the functional properties of the protein such as gelation property are lost.
It has the drawback of limited use. On the other hand, the major allergen of soybean (GM30) is soybean 34-kDa oil body-assoici
It has been confirmed to be ated protein (34 kilodalton oil body associated protein), and it is known that by centrifuging soymilk, this major allergen (GM30) is transferred to the oil body pad formed in the upper layer. Has been.

Therefore, it is an object of the present invention to provide a method for specifically separating a major allergen (GM30) in soymilk and easily separating the allergen in soymilk without degrading proteins. . It is also an object of the present invention to provide a method for producing soymilk with reduced allergen, which is capable of surely removing a causative agent of allergic symptoms from soymilk without impairing the taste and function of soymilk itself. . A further object of the present invention is to provide a method for collecting an allergen that can be used as a drug or a reagent for allergy treatment and allergy diagnosis.

[0005]

[Means for Solving the Problems] The inventors of the present invention focused on the fact that the major allergen (GM30) migrates to the upper layer upon centrifugation, and examined the method for separating the allergen in soymilk, We filed a patent application to find out that it is possible to effectively sort by conditions (Japanese Patent Application No. 6-2
8277). Then, after further examination, by adding at least one reducing agent, for example, a sulfite compound, a glutathione compound or a cysteine compound to soymilk and centrifuging the mixture, the temperature conditions during centrifugation are almost affected. However, it was found that the major allergen (GM30) specifically migrated to the upper layer, the amount of migration increased, and it could be easily separated.

By further separating the upper layer after centrifugation, soy milk with reduced allergen can be obtained,
The present invention has been completed based on the finding that the main allergen (GM30) can be efficiently collected by extraction from the upper layer using an alkaline aqueous solution. That is, the present invention has achieved the above object based on the above findings, and provides a method for separating allergens in soymilk, which comprises adding a reducing agent to soymilk and then centrifuging.

The method for separating allergens from soymilk according to the present invention, the method for producing soymilk with reduced allergens and the method for collecting allergens based on the method will be specifically described below. Soybean main allergen (GM30) is soybean 34-kD
Since it is an oil body-associated protein, it is necessary that the soybean milk contains oil and fat. Therefore, the soybean milk of the present invention is made from whole soybean or dehulled soybean. The soymilk can be prepared from the raw material by soaking the raw material in water, grinding the raw material, and separating the okara from the raw material without heating the obtained raw material.

It is also possible to utilize isolated soybean protein. In this case, soybean oil is added to the isolated soybean protein in an amount equal to or more than the amount of soybean oil contained in ordinary soymilk. Can be prepared. The unheated soy milk thus prepared is centrifuged, and a reducing agent is added to the soy milk at that time. As the reducing agent to be added, if there is no problem in food hygiene, a reducing agent known per se can be used. For example, a sulfite compound, a glutathione compound or a cysteine compound can be mentioned as a preferable one.

The sulfite compound is one which produces sulfite ions in an aqueous system, and examples thereof include alkali metal (sulfite, bisulfite, pyrosulfite, metabisulfite, potassium or sodium) salts of sulfite, and other water-soluble salts. Can be mentioned. The addition amount depends on the protein content in soymilk, but is 0.5% by weight or more, and preferably 1.0% by weight or more based on the soybean milk. The glutathione compound or cysteine compound is glutathione, cysteine, or their hydrochlorides, and the addition amount is 5 mM or more, preferably 10 m with respect to soymilk.
It is M or more.

The soy milk added with the reducing agent is then centrifuged. Centrifugation is more effective as the centrifugal acceleration increases, and centrifugation is performed at a centrifugal acceleration of 16,000 (xg) or more, particularly preferably a centrifugal acceleration of 25,000 to 500,000 (xg).
Centrifugation is preferably performed at a low temperature, for example, at a low temperature of 5 ° C. or lower, and by doing so, the enzyme action in soymilk during centrifugation can be suppressed. The centrifugation time is preferably 5 minutes to 60 minutes.

By centrifuging under such conditions, the major allergen (GM30) in soymilk can be transferred to the upper layer and separated. Then, the upper layer is separated to obtain soymilk with reduced allergen. On the other hand, the allergen can be efficiently collected from the separated upper layer by the method described below. In the case of centrifugation at a temperature of 10 ° C. or higher, antioxidants such as tocopherol and sodium ascorbate are added or lipoxygenase deficiency is added to suppress the production of soybean odor due to the action of lipoxygenase during the centrifugation operation. Lost soybeans can also be used. It is also possible to inactivate the enzyme by heating soymilk and centrifuge it, but in the heated soymilk, the major allergen (G
M30) upper layer transition is not recognized.

From the upper layer (oil body pad) separated by centrifugation as described above, the allergen can be easily obtained by extracting with an alkaline aqueous solution. For example, to the separated upper layer, an alkaline aqueous solution of about 0.1 M such as sodium carbonate is added in an amount of 10 to 50 times, and the mixture is uniformly dispersed by a homogenizer, and then stirred at a low temperature for 1 hour. Then, centrifuge at a centrifugal acceleration of 50,000 (× g) or more to obtain a transparent water-soluble section (sodium carbonate aqueous solution section) in the lower layer. Since the major allergen (GM30) is dissolved in this section, it is dialyzed with cellophane tube or the like, concentrated with an ultrafiltration membrane or the like, and freeze-dried to obtain the major allergen (GM30).

Since the soymilk obtained in the present invention does not lose the functional characteristics of protein and the allergen is reduced, if a soymilk beverage or tofu is produced using this soymilk as a raw material, a soymilk beverage with reduced allergen or It becomes tofu and can be suitably used for allergen patients. Further, the main allergen (GM30) obtained in the present invention is purified as necessary, and for hyposensitization therapy, which is attracting attention as a root allergy treatment method.
Alternatively, it can be effectively used as a diagnostic reagent for allergy diagnosis.

Experimental conditions will be described below, and the conditions of centrifugation in the method for separating allergens from soymilk according to the present invention will be described. Experimental Example [Adding amount of reducing agent and its evaluation] Whole soybeans were immersed in tap water for 16 hours, drained, and then ground while adding 4 times the amount of water. The obtained Kure was filtered to separate soymilk and okara. Cysteine was added in an amount of 0.1 to 100 mM to 5 ml of this soy milk, and 28,800 (xg was used using an ultracentrifuge (L60 type, SW65 rotor) manufactured by Beckman.
) (20,000 rpm) and soy milk at 5 ° C., and the upper layer of each was sampled and evaluated by the following method. Evaluation method: The upper layer obtained by centrifugation was used as an extraction buffer [sodium dodecyl sulfate (SDS) 1%, 2-mercaptoethanol 10 mM, Tris-HCl buffer 50 mM].
Then, fill up to 5 ml. This sample was appropriately diluted with the above extraction buffer, added to SDS 1%, 2-mercaptoethanol 1%, Tris-HCl buffer 1 mM, glycerin 20%, and subjected to electrophoresis (SDS-PAG).
E) Prepare the sample. Each sample was heat-treated at 95 ° C. for 4 minutes, and BPB (bromophenol blue) was added so as to be 0.05%. 5 μl of the sample is added to a 12% SDS-polyacrylamide gel and subjected to electrophoresis at 100 V for 90 minutes for fractionation. Next, the fractionated components were electrophoretically transferred onto an Immobilon P membrane manufactured by Millipore at a constant current of 80 mA for 1 hour. After blocking the transfer membrane with 5% skim milk, mouse monoclonal IgG antibody specific to soybean allergen (GM30) was reacted for 30 minutes at room temperature, the membrane was washed, and then detected by ABC method. That is, the biotin-labeled anti-mouse IgG antibody was reacted for 30 minutes at 37 ° C., and the biotin-labeled peroxidase-conjugated avidin was reacted for 30 minutes to label the membrane-bound IgG with peroxidase.

Diaminobenzidine tetrahydrochloride (DAB) is added to this to detect the allergen (GM30). The density of the (GM30) band of each sample was measured with a densitometer (GS670 manufactured by Bio-Rad), and expressed as 100 for the control (soy milk without centrifugation). The results are shown in Table 1.

[0016]

[Table 1]

It can be seen from Table 1 that the major allergen (GM30) is remarkably transferred to the upper layer by centrifugation of soy milk containing 5 mM or more of cysteine.

[0018]

EXAMPLES Hereinafter, the method for separating allergens from soymilk according to the present invention will be specifically described with reference to Examples. The present invention is not limited to the following examples. (Example 1) Whole soybeans were soaked in tap water for 16 hours, allowed to swell sufficiently, and then ground with 5 times amount of water to form gou,
This soybean milk was filtered to obtain soy milk. This soy milk has a protein concentration of 4%
Prepared by adding 10 mM of cysteine to the mixture, stirring, and then centrifuging at 118,000 (× g) (40,000 rpm) for 20 minutes at a temperature of 5 ° C (centrifuge, Hitachi Koki C
D40Y type continuous ultracentrifuge for Hitachi production), the upper layer was separated. The upper layer is subjected to the allergen collection method described in Example 3 below.

When the soybean milk with the upper layer separated was evaluated by the method described in the experimental example, the major allergen (GM30) was 15 when the major allergen (GM30) of the soybean milk not centrifuged was 100. The soymilk was flavored and then sterilized at 95 ° C. for 5 minutes to obtain drinking soymilk.

(Example 2) Whole soybeans were immersed in tap water for 16 hours, sufficiently swelled, and then ground with 5 times amount of water to form a go, which was filtered to obtain soy milk. Sodium milk was added with 1.5% of sodium bisulfite, stirred sufficiently, and then centrifuged under the same conditions as in Example 1 to separate the upper layer. When the soybean milk with the upper layer separated was evaluated by the method described in the experimental example, the main allergen (GM30) was 15, when the main allergen (GM30) of the soybean milk that was not centrifuged was 100.

The soybean milk was adjusted to pH 4.5 with 6N hydrochloric acid to precipitate the protein isoelectrically, and then treated with a continuous centrifuge (screw decanter) to obtain a precipitate. Water was added to this precipitate to a protein concentration of 5%, neutralized with caustic soda, heated at a temperature of 95 ° C. for 5 minutes, and cooled to give a final glucose concentration of 0.3%. After adding an aqueous solution of delta lactone and filling the container, a temperature of 95
Tofu was obtained by heating and coagulating in hot water of ℃ for 60 minutes. This tofu had the same texture as usual.

(Embodiment 3) For 100 g of the upper layer (oil body pad) separated in Embodiment 1, 1000 ml of 0.1
M sodium carbonate was added, the oil body pad was uniformly dispersed with a homogenizer, and the mixture was stirred at low temperature (temperature 5 ° C.) for 1 hour. Next, Beckman ultracentrifuge (L6
Type 0, Type 35 rotor) 80,000 (× g) (35,
(000r.pm) at a temperature of 5 ° C. for one day (15 hours) for centrifugation to obtain a transparent aqueous sodium carbonate fraction in the lower layer. This classification is 100 with an ultrafiltration device (molecular cutoff of 1000).
The solution was concentrated to ml, and then dialyzed by a cellophane tube by a conventional method. The dialyzed product obtained was freeze-dried to obtain 2.1 g of a dry powder. The dried powder was electrophoresed on a 12% SDS-polyacrylamide gel and the major allergen (GM30)
The purity was measured and found to be 85%.

[0023]

INDUSTRIAL APPLICABILITY As described above, according to the method for fractionating allergens in soymilk according to the present invention, the major allergen (GM30) in soymilk can be specifically separated and easily fractionated without degrading proteins. In addition, in the method for producing soymilk with reduced allergens according to the present invention, substances that cause the onset of allergic symptoms can be reliably removed from soymilk without impairing the taste and function of soymilk itself. Furthermore, by collecting the allergen according to the present invention, the collected allergen can be purified if necessary and used as a drug or a reagent for allergy treatment, allergy diagnosis and the like.

Claims (4)

[Claims] 1. A method for separating allergens from soy milk, which comprises adding a reducing agent to soy milk and then centrifuging. 2. A method for separating allergens in soymilk, which comprises adding at least one selected from a sulfite compound, a glutathione compound or a cysteine compound as the reducing agent according to claim 1, and then centrifuging. 3. A soymilk having reduced allergen, which is produced by removing the upper layer after the centrifugation from the soymilk by the method for separating allergen in soymilk according to claim 1 or 2. Manufacturing method. 4. The method for separating allergen in soymilk according to claim 1 or 2, wherein the upper layer after the centrifugation is collected from the soymilk, and an alkaline aqueous solution is added to the collected layer to extract the allergen in the soymilk. A method for collecting an allergen, which comprises: