JPH09172995A - Method for decreasing allergen of wheat flour and wheat flour having decreased allergen content - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a processed wheat food safe to a patient of cereal allergy by a sitologically allowable means by reacting a water-added wheat flour with a specific proteinase under neutral condition to decrease the allergen content of the wheat flour. SOLUTION: Wheat flour mixed with water or an aqueous solution of ethanol is reacted with a sitologically allowable proteinase having high collagenase-like activity under neutral condition. The enzyme preferably has a low amylase activity in addition to high collagenase-like activity and is most preferably bromelain. Preferably, 1 pt. of wheat flour is incorporated with 0.1-10 pts. of water or an aqueous solution of ethanol having a concentration of <=20% and reacted with 0.1-5wt.% (based on the wheat flour) of a proteinase. The present process gives a wheat having decreased allergen content and containing inactivated wheat allergen epidope, etc., having the amino acid sequence expressed by the formula.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for reducing allergen in wheat flour and wheat flour reduced in allergen by this method. More specifically, the present invention relates to a hypoallergenic wheat flour which is useful as a food material for cereal allergy patients, and a processed flour food made from this wheat flour.

[0002]

2. Description of the Related Art In recent years, the number of food allergy patients has been rapidly increasing worldwide, and allergy to rice, wheat, etc. has become a serious problem because those grains are the staple foods of people. . In order to solve this problem, the inventors of the present invention have developed hypoallergenic rice in 1990 (J. Food Sci., 55, p781, 1990; J. Fo).
od Sci., 55, p1105, 1990; Nutrition magazine, 44, p51, 1991;
Trends Food Sci., 4, 1993). The rice with reduced allergens was certified as a food for specified health use by the Ministry of Health and Welfare as the first functional food in 1993, and is now widely supplied to rice allergic patients.

On the other hand, although wheat flour has been used in large quantities as a raw material for bread, noodles, pasta, etc., which are staple foods of many people all over the world, up to now, it has been considered as a food science of this flour. Acceptable hypoallergenization has not been successful. In this regard, the inventors of the present invention isolated an antigenic peptide having the amino acid sequence of SEQ ID NO: 1 from a chymotrypsin hydrolyzate of gluten, which is used as an antigen by most wheat allergy patients (Biosci. Biotech. Bioch.
em., 59, p1596-1607, 1995), that the epitope (antigenic determinant) of this peptide is the amino acid sequence portion shown in SEQ ID NO: 2, and that the amino acid acid group essential for the antigenicity of this epitope is the N-terminal First glutamine residue (Gln)
And the 4th and 5th proline residues (Pro) (Proceedings of the 1995 Annual Meeting of the Japan Agricultural Chemistry). Then, the inventors of the present invention, for the purpose of inactivating the epitope of this wheat allergen, by treating with a collagenase that recognizes a proline residue, to lower the allergen flour, further bread and bread It has also been successfully processed into pasta (Bios
ci. Biotech. Biochem., 58, p388-390, 1994).

However, since this collagenase is an enzyme produced by the genus Clostridium, which is an anaerobic bacillus, it is not approved as a food additive, and the collagenase-treated hypoallergenized flour is a food product. It has a fundamental defect that it cannot be used as a raw material. This invention was made in view of the above circumstances, and a method capable of reducing the allergen of wheat flour by a method that is foodologically acceptable,
It is an object of the present invention to provide a low allergen-reduced wheat flour obtained thereby, and a wheat flour processed food using the low-allergenized wheat flour as a raw material.

[0005]

Means for Solving the Problems The present invention is to solve the above-mentioned problems by adding flour to which water or an aqueous ethanol solution has been added, which has a high collagenase-like activity and is food-processible. Provided is a method for reducing wheat allergen, which comprises reacting an enzyme under neutral conditions.

In this method for reducing the allergen of flour, the proteolytic enzyme has a high collagenase-like activity,
A preferred embodiment is an enzyme having low amylase activity. More specifically, bromelain is most preferred as the proteolytic enzyme used in the method of this invention due to its high collagenase-like activity and low amylase activity.
Further, in the above method, the amount is 0.05 to 100 times, preferably 0.1 to 1 times that of the flour.
0 times the amount of water or an aqueous ethanol solution having a concentration of 20% or less is 0.05 times to 100 times, preferably 0.1 times to 1 times.
Add 0 times amount, and 0.0% to flour
It is also a preferred embodiment to react 1% to 10% by weight, preferably 0.1% to 5% by weight of a proteolytic enzyme.

The method for reducing the allergen of wheat flour of the present invention can be applied to all wheat flour such as strong flour, semi-strong flour, medium strength flour, and weak flour. The present invention also relates to wheat flour which has been hypoallergenized by the above method, wherein the wheat allergen epitope having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 is inactivated. And a flour-processed food using this hypoallergenic flour as a raw material.
This processed flour food is any food made from wheat flour, such as bread, noodles, maimai or dumpling skin, pasta, pizza dough, and confectionery.

Further, the present invention also provides a method for producing a hypoallergenized bread, which comprises adding sodium hydrogen carbonate and citric acid to the above hypoallergenic wheat flour to generate carbon dioxide, and then baking the mixture. .

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The results of the test examples are shown below, and the embodiments of the present invention will be described in more detail. Test Example 1 This test was conducted in order to select a proteolytic enzyme which has a high collagenase-like activity and a low amylase activity and is food-acceptable. (1) Sample wheat flour : weak wheat flour (trade name: Cleopatra, manufactured by Showa Sangyo Co., Ltd.) was used. Enzymes : Protease A, Protease B, Protease S, Protease N, Pro Leather, Papain W-40
(Above, manufactured by Amano Pharmaceutical Co., Ltd.), bromelain (reagent name: bromelin, manufactured by Wako Pure Chemical Industries, Ltd.), and macrase (produced by Kaken Pharma Co., Ltd.) were used. (2) Test method Collagenase-like activity : Van Wa using FALGPA as a substrate
It was measured according to the method of rt (Anal. Biochem., 113, p356-365, 1981). Substrate concentration 0.05 mM; solvent 0.4 M
NaCl-10 mM CaCl ₂ -50 mM Tris-HCl buffer (pH 7.5); enzyme concentration was about 5 mg / ml, and FALGPA decomposition reaction was performed at 25 ° C. to give 324 nm.
The decrease in absorbance was measured for several minutes. The enzyme activity is [unit /
mg enzyme]. Here, 1 unit is the activity of hydrolyzing 1 μmol of FALGPA in 1 minute, and ΔA
= 0.0025 / min. Amylase activity : 1% gelatinized potato starch as a substrate,
Perform the enzyme reaction for 30 minutes at 37 ℃, and use Luchsinger and Cor
Nesky's method (Anal. Biochem., 4, p346-347, 1962)
The reducing group was quantified according to the method described in 1. The enzyme activity is [μm
ol glucose equivalent / min / mg enzyme]. (3) Test results The results of this test are as shown in Table 1. This Table 1
As is clear from the above, the collagenase-like activity of the tested proteolytic enzymes was higher in the order of bromelain> protease B> protease A = papain. The amylase activity of these four proteolytic enzymes was measured. As shown in Table 1, the amylase activities of proteases A and B were relatively high, and those of bromelain and papain were about 10 times lower. It became clear.

[0010]

[Table 1]

The fact that a proteolytic enzyme having a high collagenase-like activity is effective means that collagenase recognizes the proline residue of the wheat flour epitope (peptide of SEQ ID NO: 2) and inactivates it. It is clear from the conventional knowledge of the present inventors that it is possible to reduce allergens in Further, the fact that a protease having low amylase activity is effective means that when wheat starch is decomposed by amylase, the volume of wheat flour decreases,
This is because the sweetness is imparted and the processability is significantly impaired. In addition, since wheat allergic patients use the amylase inhibitor in wheat protein as an antigen, it is not possible to add amylase inhibitor to prevent the decomposition of wheat starch. It depends on what is possible.

From the above test results, therefore, it was confirmed that bromelain is most preferable as the proteolytic enzyme used in the method for reducing the allergen of flour of the present invention. Test Example 2 The pH dependence of the collagenase-like activity of bromelain was examined. (1) Sample The same FALGPA and Bromelain as in Test Example 1 were used. (2) Test method Collagenase-like activity was measured by the same method as in Test Example 1 under conditions of pH 5 to 10. (3) Test result The result of this test is as shown in FIG. The collagenase-like activity of bromelain is highest at pH 9, p
The activity was relatively high even in H5 to 8, but the activity decreased at pH 10. From this result, it was found that the antigenicity of wheat flour can be efficiently reduced by reacting bromelain under the condition of pH 9.

However, under such pH conditions,
Flavonoids in wheat flour are alkaline and turn yellow, which may cause the final product to be colored. The first requirement for low-allergen wheat flour is that it has no antigenicity, but at the same time, it is required that the quality of colors and the like is comparable to that of ordinary wheat flour. Therefore, in the method of the present invention, it was confirmed that it is necessary to set the conditions under which the protease can react with the flour under neutral conditions. Test Example 3 This test was carried out in order to search conditions under which bromelain could be reacted under neutral conditions to reduce the antigenicity of wheat flour. (1) Samples Products A and B whose manufacturing steps are shown in FIG. 2 were used. (2) Test method The product was tested for antigenicity by the following ELISA method. That is, the protein in the bromelain reaction product of wheat flour was extracted with a 4M urea-containing Tris-hydrochloric acid (UTH) buffer solution (pH 8.6) and centrifuged (1,300 × g:
5 minutes). The resulting supernatant has a protein concentration of about 1
It was diluted with UTH buffer to a concentration of 00 to 250 μg / ml. About 100 μl of the IgE-EL was used as an index for the binding property with the specific IgE antibody in the serum of wheat allergy patients (obtained from Urabune Hospital, Yokohama City University School of Medicine).
Antigenicity was measured by the ISA method (see FIG. 3). (3) Test results The results of this test are as shown in Table 2. As is clear from the results in Table 2, the flour (product B) obtained by adding the same amount of 10% ethanol and reacting bromelain at 37 ° C. for 8 hours had its antigenicity reduced to below the detection limit.

The product (Product A) obtained by adding 0.6 times the amount of water to the flour and reacting 1% by weight of bromelain at 37 ° C. has the antigenicity remaining in the reaction time of 1 to 4 hours. However, in the reaction product for 8 hours, the antigenicity was reduced to below the detection limit. From the above results, it was confirmed that by adding water or an aqueous ethanol solution to wheat flour, bromelain can be efficiently reacted even under neutral conditions to reduce the antigenicity of wheat flour.

[0015]

[Table 2]

Test Example 4 The following test was performed on the product A whose manufacturing process is shown in FIG. (1) Test method SDS-PAGE : Protein in 200 g of product A and untreated wheat flour was extracted with 1 ml of UTH buffer and centrifuged (1,300 xg: 5 minutes). An equal amount of Seprasol II (manufactured by Daiichi Pure Chemicals Co., Ltd.) was added to the obtained supernatant, and after boiling for 15 minutes, the method of Laemmli (Nature, 227, p680-68) was used.
5, 1970), electrophoresis was performed using a polyacrylamide gel (manufactured by Daiichi Pure Chemicals Co., Ltd.) with a concentration gradient of 10 to 20%.
Rabbit muscle phosphorylase b as a molecular weight marker
(MW, 97 kDa), bovine serum albumin (MW, 6
7 kDa), ovalbumin (MW, 43 kDa), bovine carbonate dehydratase (MW, 31 kDa), soybean trypsin inhibitor (MW, 22 kDa), chicken egg white lysozyme (MW, 14 kDa). After electrophoresis, Coomassie Brilliant Blue (R-250, Si
Staining was performed using gma). Resistography : 86 g of dry matter of freeze-dried product A was added to 74 ml of water and mixed, and a resist graph (manufactured by Brabender Instrument) was used at 30 ° C. for 63 r.
Stirred at pm for 20 minutes. An equivalent operation was performed on a mixture of untreated wheat flour (100 g) and water (60 ml). Farmography : Raw baker's yeast (manufactured by Oriental Yeast Co., Ltd.) was suspended in water at a concentration of 2 g / ml. This yeast suspension (0.345 ml) was mixed with a hypoallergenic flour batter (50 g) or flour dough (50 g) prepared by resistography, and a thermograph (A
R-1000, manufactured by Ato Co., Ltd.) was used to measure the amount of carbon dioxide generated and the amount retained on the dough at 24 ° C. for 90 minutes. (2) Test results As shown in FIG. 4, when SDS-PAGE was performed on the 4M urea extract of the product (product A) after 8 hours of bromelain reaction, most of the reaction products were from gel compared to untreated wheat flour. The molecular weight was reduced to the extent that it escaped. 20 kDa to 30
Since the component around kDa remained after the reaction, it is considered that this component is not antigenic.

Further, as shown in FIG. 5, the resist gram (A) of the allergen-reduced wheat flour (Product A) was flat as compared with that of the untreated wheat flour, showing that gluten was not formed. . However, since the allergen-reduced wheat flour has a carbon dioxide-retaining ability, as shown in the fermogram of FIG. 6, it was confirmed that wheat flour-processed foods such as bread can be produced using this flour. . Test Example 5 Bread was manufactured using the product A whose manufacturing process is shown in FIG. 2, and its characteristics were tested. (1) Sample Light wheat flour (1 kg) in 0.6 liter of water at 37 ° C.
Bromelain (10 g) was treated for 8 hours. Bromelain reaction mixture, salt 10g, glucose 10g, citric acid 1
3g, sorbitan monostearate (trade name: Emasol
S-10F, manufactured by Kao Co., Ltd.) 10 mg, and sodium hydrogencarbonate 20 g was further added and stirred. 30g of mixture
Each was quickly put in a 100 ml container or a muffin-type alumina cup, and baked in an oven at 180 ° C. for 20 minutes to produce bread.

[0018] In addition, bread made from wheat flour that has been reduced in allergen by collagenase treatment (Biosci. Biotech.
Biochem., 58, p388-390, 1994) was also produced. In addition,
No baker's yeast was used in the bread prepared in this test example. This is because the baker's yeast protein and the wheat allergen have similar amino acid sequences, and thus wheat allergic patients often show an allergic reaction to baker's yeast.
Therefore, bread was produced by generating carbon dioxide from sodium hydrogen carbonate and citric acid instead of baker's yeast. (2) Test method Specific volume : Loaf volume is determined by rapeseed substitution method (American Associa
tion of Cereal Chemists. in "Approved Methods of t
he AACC ", 8th Ed. The Association. St. Paul, Method
10-10B, 1983). The specific volume was expressed as loaf volume (ml) / loaf weight (g). Color evaluation : color parameters (L, a, b) at the top of the pan
The value) was measured using a color difference meter (SC-2-XCH, manufactured by Suga Test Instruments Co., Ltd.). (3) Test results The results of this test are shown in Table 3. This table 3
As is clear from the above, the wheat bread that was made hypoallergenic by the bromelain treatment had the same characteristics as those of the wheat bread that was made hypoallergenic by the collagenase treatment in both the specific volume and the color tone evaluation. Also, the commercially available English muffins had the same specific volume.

Further, FIG. 7 shows a photograph of the appearance (A) and cross section (B) of the produced bread. The addition of sorbitan monostearate resulted in a good texture of the bread inner layer. In addition, the addition of glucose caused Strecker decomposition and Maillard reaction, and the products produced very good bread color and aroma.

[0020]

[Table 3]

[0021]

Industrial Applicability As described in detail above, the method of the present invention enables a wheat flour to be made into a low allergen by a foodologically acceptable method, a hypoallergenized wheat flour obtained thereby, and this low flour. Provided is a processed food product of wheat flour which is made from allergenized wheat flour. As a result, it becomes possible to provide a safe processed food product of wheat flour for patients with wheat flour allergy.

[0022]

[Sequence list]

SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ser Gln Gln Gln Gln Pro Pro Phe Ser Gln Gln Gln Pro Pro Phe 1 5 10 15 Ser Gln Gln Gln Gln Pro Pro Phe Ser Gln Gln Gln Pro Pro Phe 20 25 30 SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide (Xaa represents any amino acid residue)

[Brief description of the drawings]

FIG. 1 is a histogram showing the pH dependence of collagenase-like activity of bromelain.

FIG. 2 is an example of a process for producing a hypoallergenic wheat flour by bromelain treatment.

FIG. 3 is a schematic diagram showing a method for measuring antigenicity by an ELISA method.

FIG. 4 is a diagram showing the results of SDS-PAGE of hypoallergenic wheat flour and untreated wheat flour by bromelain treatment.

FIG. 5 is a resist gram of hypoallergenic wheat flour (A) and untreated wheat flour (B) by bromelain treatment.

FIG. 6 is a fermogram of hypoallergenic wheat flour treated with bromelain.

[FIG. 7] Appearance (A) of bread prepared using wheat flour with reduced allergen by bromelain treatment, and its cross section (B).
FIG.

Claims (11)

[Claims] 1. A low allergen of wheat flour, which comprises reacting wheat flour to which water or an aqueous ethanol solution has been added with a proteolytic enzyme that has a high collagenase-like activity and is pharmaceutically acceptable under neutral conditions. Method. 2. The method for reducing wheat allergen according to claim 1, wherein the proteolytic enzyme is an enzyme having a low amylase activity. 3. The method for reducing allergen of flour according to claim 1, wherein the protease is bromelain. 4. The amount of wheat flour is 0.05 to 1 times that of wheat flour.
4. The method for reducing flour allergen according to claim 1, wherein the amount of water is 100 times. 5. The method for reducing allergen of wheat flour according to claim 1, wherein an ethanol aqueous solution having a concentration of 20% or less is added to wheat flour in an amount of 0.05 to 100 times. 6. From 0.01% to 1 of wheat flour
4. Reacting 0% by weight of proteolytic enzyme.
Method for reducing allergen in wheat flour. 7. A wheat allergen-reduced flour obtained by the method according to claim 1, wherein the wheat allergen epitope whose amino acid sequence is shown in SEQ ID NO: 2 is inactivated. Allergenized flour. 8. A wheat flour hypoallergenized by the method according to claim 1, wherein the wheat allergen epitope whose amino acid sequence is shown in SEQ ID NO: 3 is inactivated. Allergenized flour. 9. A wheat flour processed food using the hypoallergenic wheat flour according to claim 7 or 8 as a raw material. 10. A method for producing a low-allergenized bread, which comprises adding the sodium hydrogencarbonate and citric acid to the low-allergenized wheat flour of claim 7 or 8 to generate carbon dioxide, and then baking the mixture. 11. The sorbitan monostearate and glucose are added to the hypoallergenic wheat flour.
0 bread making method.