JP2000069915A - Allergen-reduced wheat protein and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To readily produce an allergen-reduced wheat protein according to a simple method for treatment without causing remarkable lowering of molecular weight. SOLUTION: A wheat protein is dispersed in water with an organic acid and a mineral acid is then added to deamidate 30-90% of glutamic acid residues and asparagine residues contained in the wheat protein. The resultant substance is subsequently precipitated under weak acidic conditions (pH 4-5) to produce an allergen-reduced wheat protein. The produced allergen-reduced wheat protein has usefulness as an allergen-reduced wheat protein material or a protein enhancer for processing a low-allergic food and can be utilized for various foods.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a wheat protein with reduced allergen and a method for producing the same.

[0002]

2. Description of the Related Art It has been said that intense atopic dermatitis is related to food allergens of rice and wheat, and it has recently been found that the tendency is increasing. As a result of the investigation, it is said that there are many severe cases of atopic dermatitis patients who are positive for food allergens, especially those who are allergic to wheat.

[0003]

In view of the above circumstances, the present invention provides a wheat protein in which the wheat allergen is reduced and which can be consumed by a wheat allergic patient, and a method for producing a wheat protein having a reduced allergen. The purpose is to provide.

[0004]

Means for Solving the Problems The present inventors have developed a glutamine residue and an asparagine residue which are contained in wheat protein allergens (low-molecular-weight glutenin, α-gliadin, γ-gliadin, etc.) in a large amount, and particularly low-molecular-weight glutenin. A glutamine residue in the QQQPP (glutamine, glutamine, glutamine, proline, proline) sequence portion, which is assumed to be one of the epitopes, is deamidated to a glutamic acid residue, thereby achieving low allergenization of wheat protein. As a result of intensive research, the present invention has been completed.

[0005] That is, the present invention relates to a method for preparing a glutamine residue and an asparagine residue contained in wheat protein in an amount of 30% to 90%.
% Of allergen-reduced wheat protein.
The present invention also provides a method of dispersing wheat protein in water using an organic acid, adding a mineral acid, and deamidating 30% to 90% of glutamine residues and asparagine residues contained in the wheat protein. This is a method for producing a low-allergen wheat protein, which is characterized in that it is neutralized to a weak acid and precipitated.

[0006] As a raw material of the allergen-reduced wheat protein of the present invention, wheat gluten is usually used. The process for producing the allergen-reduced wheat protein of the present invention using wheat gluten will be described below.

[First step] Wheat gluten is uniformly dispersed in an organic acid solution which does not substantially decompose wheat gluten, for example, in an organic acid solution selected from lactic acid, acetic acid and citric acid, preferably in a lactic acid solution. Disperse. Organic acid is 0.01 ~
0.1N (prescribed), preferably 0.02 to 0.06N,
More preferably, those having 0.02 to 0.04 N are used. The wheat gluten is uniformly dispersed while being added little by little to 2 to 10 times, preferably 5 times, the amount of the organic acid. Means for uniformly dispersing wheat gluten in the organic acid solution include, for example, means for stirring with a homomixer.
At that time, neither heating nor cooling is required, and the heating can be performed at room temperature. In addition, reducing agents such as sulfites and ascorbic acid may be used in combination for dispersion.

[Second step] Next, a mineral acid is added to the dispersion in which the wheat gluten is dispersed, and deamidation is advanced. While warming and stirring the dispersion, 0.5-5 times, preferably 1-4 times, the total amount of the expected glutamine and asparagine residues is added little by little to the mineral acid.
Maintain at 0 to 100 ° C. for about 30 minutes to 6 hours. Mineral acids include sulfuric acid, nitric acid, preferably hydrochloric acid. Mineral acids are usually added as dilute aqueous solutions. The deamidation rate can be adjusted by changing conditions such as the type and amount of the mineral acid used, the heating temperature and the heating time. The amount of glutamine residues and the amount of asparagine residues are predicted by the amino acid composition of wheat gluten.

[Third step] After cooling, the reaction solution is weakly acidic (p
The product precipitated as H4-5) is removed by filtration or centrifugation and dried. To make the reaction solution weakly acidic, an alkali solution (eg, sodium hydroxide solution, potassium hydroxide solution, sodium carbonate, etc.) is used. The allergen-reduced wheat protein of the present invention has an isoelectric point of p
H4-5, and in this step, the reaction solution is neutralized to near the isoelectric point. The following methods can be used to measure the isoelectric point of the allergen-reduced wheat protein of the present invention. First, 0.1N hydrochloric acid was added dropwise to 50 ml of a 1 w / v% allergen-reduced wheat protein aqueous solution with stirring, and p
The change in H is measured to obtain a titration curve. The point where the slope of the obtained titration curve becomes the gentlest is estimated as the isoelectric point.

[Fourth Step] A part of the product is dispersed in water, and the acidity is determined by titration. The product is, if necessary, mixed with a calculated amount of sodium carbonate and ground to form a soluble fine powder. Alternatively, the aqueous solution of the product is neutralized to pH 7 with sodium hydroxide or sodium carbonate, and spray-dried with a spray drier.

[0011] The wheat protein obtained through the above steps is
As is clear from the results of the experimental examples described later, allergen is reduced by deamidation of the amide group contained in the wheat protein. The deamidation rate is at least 10% or more, usually 30% or more, preferably 50% or more.
% Or more.

[0012] Even if all of the amide groups contained in wheat protein are not deamidated, the purpose of reducing allergens can be achieved by deamidating the glutamine residue constituting the QQQPP sequence. On the other hand, when the deamidation ratio exceeds 90%, the molecular weight of the decomposed product is advanced, and the high molecular weight fraction decreases. By highly deamidating wheat protein in this way, the properties of the protein are weakened, and the properties such as gel-forming ability may be far from wheat gluten. Therefore, when a low-allergen wheat protein having a large molecular weight is required, the deamidation ratio is preferably 90% or less, and preferably less than 80%.

The allergen-reduced wheat protein of the present invention is neutral and alkaline, exhibits high water solubility, and has excellent emulsification stability, dispersibility and surface tension lowering ability. Further, the isoelectric point is pH 4-5, the color tone is pale yellow in powder, and when dissolved, it becomes a thin emulsion-like solution. Since the product of the present invention has little odor and no bitterness, it can be applied to foods. Because of its high water solubility and excellent emulsion stability, it is particularly useful as an emulsifying / dispersing agent (among others, an emulsifying / dispersing agent for food). Therefore, processed meat products such as ham, sausage and hamburger, processed fish products such as kamaboko and bamboo rings, bread, cake,
It has an effect of improving the quality of baked confectionery such as cookies, biscuits and crackers, emulsified foods such as dressings, noodles, batters, and various other foods, and is useful as a highly safe food material.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto. In addition, the calculation method of the deamidation rate in an Example is based on the following method.

[Method for calculating deamidation rate] The amount of ammonia in the reaction solution produced by deamidation by acid hydrolysis was quantified according to the Conway microdiffusion method. The calculation is as follows.

Deamidation ratio (%) = (Ammonia content in reaction solution / Ammonia content in excessively decomposed solution ^* ) × 100

^* 200g of raw gluten (protein content 25%)
Was dispersed in 400 ml of 0.025N lactic acid, and 35 w / w%
Amount of ammonia in the reaction mixture after adding 100 g of hydrochloric acid and heating and stirring at 100 ° C for 6 hours

Example 1 1.0 m of a 0.78 w / v% sodium bisulfite solution
200 g of raw wheat gluten (protein content: 25%) is added little by little to 400 ml of 0.025N lactic acid to which l has been added, and uniformly dispersed by a homomixer. 25 g of concentrated hydrochloric acid (35 w / w%) was added to this dispersion under stirring, and
For 3 hours to allow the deamidation to proceed. After standing to cool, pH
The product is neutralized to around 4 and the product precipitated near the isoelectric point is removed by centrifugation. The whole amount is dispersed in about three times the amount of water, and an aqueous solution neutralized and dissolved to pH 6.5 with caustic soda is spray-dried with a spray drier to produce the desired product. Gain 4
2 g, yield 84%, deamidation rate 30.0%, water solubility,
It was a pale yellow fine powder.

Example 2 1.0 m of a 0.78 w / v% sodium bisulfite solution
200 g of raw wheat gluten (protein content: 25%) is added little by little to 400 ml of 0.025N lactic acid to which l has been added, and uniformly dispersed by a homomixer. 25 g of concentrated hydrochloric acid (35 w / w%) was added to this dispersion under stirring,
For 1 hour to allow the deamidation to proceed. After standing to cool, pH
The product is neutralized to around 4 and the product precipitated near the isoelectric point is removed by filtration and dried. The acidity is determined by titration,
The mixture was pulverized with a calculated amount of anhydrous sodium carbonate required for neutralization to obtain a water-soluble target substance. 43 g, yield about 80
%, Deamidation ratio 50.0%, water-soluble, pale yellow fine powder.

Example 3 1.0 m of a 0.78 w / v% sodium bisulfite solution
200 g of raw wheat gluten (protein content: 25%) is added little by little to 400 ml of 0.025N lactic acid to which l has been added, and uniformly dispersed by a homomixer. 50 g of concentrated hydrochloric acid (35 w / w%) was added to this dispersion under stirring, and
For 4 hours to allow the deamidation to proceed. After standing to cool, pH
The product is neutralized to around 4 and the product precipitated near the isoelectric point is removed by filtration and dried. The acidity is determined by titration,
The mixture was pulverized with a calculated amount of anhydrous sodium carbonate required for neutralization to obtain a water-soluble target substance. 36 g, yield about 65
%, A deamidation rate of 90.0%, and a water-soluble, pale yellow fine powder. Table 1 shows the results of measuring the amino acid composition of this product.
Shown in

[0021]

[Table 1]

Table 2 shows the reaction conditions of the above Examples 1 to 3 and the results obtained by fractionating the obtained allergen-reduced wheat protein by filtration. The amount of hydrochloric acid in Table 2 represents the number of g of hydrochloric acid per 100 g of raw gluten (protein content 25%). A molecular weight of about 40,000 daltons or more was defined as a high molecular fraction, and a molecular weight of 5,000 to 10,000 daltons was defined as a low molecular fraction.

[Molecular weight measurement] 5% w / v of Examples 1-3
After passing the aqueous solution through a filter having a pore size of 0.45 μm, the molecular weight was measured by high performance liquid chromatography. As a standard substance, γ-globulin (156,00
0), ovalbumin (45,000), lysozyme (17,500), angiotensin (1,269),
A calibration curve was created using enkephalin (555).

Chromatographic conditions Column: Shim-pack Diol-150 (7.
9φ × 250) (manufactured by Shimadzu Corporation) Pump: LC-6A (manufactured by Shimadzu Corporation) Detector: SPD-6AV 280 nm (manufactured by Shimadzu Corporation) Mobile phase: 10 mM phosphate buffer (pH 7) + 0.2 M sodium sulfate Temperature: room temperature

[0025]

[Table 2]

Test Example: Test Method for Allergenicity to Wheat Allergy The samples of Examples 1 to 3 and untreated wheat gluten as a control were used to compare their allergen-reducing effects. RAST (Radioallergosorbent) for serological diagnosis of allergens
test), the RAS representing the amount of IgE to wheat
T value was measured, and RA of 0.35 or more with respect to wheat protein was measured.
The sera of subjects who showed an ST value response were selected and tested.
Serum from a healthy subject with a RAST value of 0.34 or less was used as a control. Note that R representing the total IgE amount of the test subject and the healthy subject
The IST (Radioimmunosorbent test) value is also shown in Table 3.

[Allergy Test] The samples of Examples 1 to 3 and untreated wheat gluten were dissolved in the sample treatment solutions of Test Methods 1 and 2 so as to be 4 mg / ml.
The sample treatment solution of Test Method 1 is a 2 w / v% SDS (sodium dodecyl sulfate) solution, and the sample treatment solution of Test Method 2 is 0.1 M Tris-HCl (pH 7.0) containing 8 M urea.
4) liquid, and both sample processing liquids are 1 v as a reducing agent.
/ V% mercaptoethanol. This was dropped on a nitrocellulose membrane, reacted with the serum of a subject and a healthy person, further reacted with a goat anti-human IgE-alkaline phosphatase conjugate, and quantified using a densitometer and image analysis software. Table 3 shows test results
Shown in

[0028]

[Table 3]

In the table, ND indicates that no detection was possible, and NDn indicates that no measurement was performed.

When the reaction was measured for the serum of a wheat RAST-positive person, as shown in Table 3, all the subjects showed a clear reaction with untreated wheat gluten, but 30%
By deamidation, subjects A, C, H and L became less responsive, and after 50% deamidation, subjects D, E, F, G and J showed less responsiveness. After 90% deamidation, all subjects showed little response. As a result, it was confirmed that the deamidation of wheat protein reduced allergen. It is understood that this effect is due to the fact that the glutamine, particularly the QQQPP sequence, which is one of the epitopes, contained in the allergen in the wheat protein is reduced by the deamidation treatment and the allergen is reduced.

[0031]

According to the method for producing a low-allergen wheat protein of the present invention, the molecular weight does not significantly decrease,
The low-allergenized wheat protein can be easily deamidated by a simple processing method. The allergen-reduced wheat protein of the present invention is a low-allergen wheat protein material,
Alternatively, it is highly useful as a protein enhancer for processing hypoallergenic foods and can be used for various foods.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toru Kishimoto Kyoto University Uji City Gokasho (no address) Inside Kyoto University Food Science Institute (72) Inventor Keiko Ichise Gokasho Uji City Kyoto Prefecture (no address) Inside the Food Science Research Institute, Kyoto University Prefectural University of Medicine Pediatrics (72) Inventor Futari Sugiura 7-16 Kasugacho, Takatsuki-shi, Osaka Glyco Nutrition Food Co., Ltd. (72) Inventor Atsushi Tsuda 7-16 Kasugacho, Takatsuki-shi, Osaka Glico Nutrition Food Co., Ltd. Inside

Claims (2)

[Claims] An allergen-reduced wheat protein obtained by deamidating 30% to 90% of glutamine residues and asparagine residues contained in wheat protein. 2. Wheat protein is dispersed in water using an organic acid, and mineral acid is added to deamidate 30% to 90% of glutamine residues and asparagine residues contained in the wheat protein. A method for producing a hypoallergenic wheat protein, comprising precipitating under acidic conditions.