JP3302872B2 - Allergen-reducing method for flour and low-allergen flour - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for reducing the allergen of wheat flour and to a wheat flour reduced in allergen by the method. More specifically, the present invention relates to a low-allergen wheat flour useful as a food material for cereal allergy patients, and a processed flour food made from the flour.

[0002]

2. Description of the Related Art In recent years, the number of patients suffering from food allergies has been increasing rapidly worldwide. Among them, allergies to rice, wheat, etc. have become a serious problem since those grains are the staple food of people. . The inventors of the present invention have developed low-allergen rice in 1990 to solve this problem (J. Food Sci., 55, p781, 1990; J. Fo).
od Sci., 55, p1105, 1990; Nutrition Journal, 44, p51, 1991;
Trends Food Sci., 4, 1993). This allergen-reduced rice 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 allergy patients.

[0003] On the other hand, although flour is used in large quantities as a raw material for bread, noodles, pasta, and the like, which is the staple food of many people around the world, to date, the flour of this flour has not been used in foods. Acceptable hypoallergenization has not been successful. In this regard, the inventors of the present invention have isolated an antigenic peptide having the amino acid sequence of SEQ ID NO: 1 from gluten chymotrypsin hydrolyzate, which is used as an antigen by the majority of wheat allergic 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 the amino acid acid group essential for antigenicity of this epitope is N-terminal. First glutamine residue (Gln)
And the fourth and fifth proline residues (Pro) (Abstracts of the 1995 Annual Meeting of the Japanese Society of Agricultural Chemistry). Then, the inventors of the present invention aimed to inactivate the epitope of the wheat allergen, by treating with a collagenase recognizing a proline residue, the wheat flour was reduced to an allergen, and the flour was further reduced to bread and bread. Processing into pasta has also been successful (Bios
ci. Biotech. Biochem., 58, p388-390, 1994).

However, since this collagenase is an enzyme produced by the anaerobic bacterium Clostridium sp., It has not been approved as a food additive, and collagenase-treated allergen-reduced flour is not used in foods. It had a fundamental disadvantage that it could not be used as a raw material. The present invention has been made in view of the circumstances as described above, and a method capable of reducing the allergen of flour by a food-acceptable method,
An object of the present invention is to provide a low-allergen wheat flour obtained therefrom and a processed flour food product using the low-allergen wheat flour as a raw material.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems by providing a wheat flour to which water or an ethanol aqueous solution is added, having a high collagenase-like activity and a food-acceptable proteolysis. Provided is a method for reducing allergen of flour, which comprises reacting an enzyme under neutral conditions.

[0006] In this method for reducing the allergen of wheat flour, the proteolytic enzyme has, in addition to high collagenase-like activity,
In a preferred embodiment, the enzyme has low amylase activity. More specifically, bromelain is most preferred as a proteolytic enzyme for use in the method of the present 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 the amount of flour.
0 times the amount of water or 20% or less aqueous ethanol solution is 0.05 to 100 times, preferably 0.1 to 1 time.
0 times the amount, and 0.0
In a preferred embodiment, the protease is reacted with 1% to 10% by weight, preferably 0.1% to 5% by weight of a protease.

The method for reducing allergen of wheat flour according to the present invention can be applied to all kinds of flour such as strong flour, semi-strong flour, medium flour and soft flour. The present invention also provides a wheat flour reduced in allergen by the above method, wherein the wheat allergen epitope having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 is inactivated. And a processed flour food using the reduced allergen wheat flour as a raw material.
This processed flour food is, for example, any food made from flour such as bread, noodles, shumai or gyoza skin, pasta, pizza dough, and confectionery.

Further, the present invention also provides a method for producing a low-allergen bread, characterized in that sodium bicarbonate and citric acid are added to the above-mentioned low-allergen wheat flour to generate carbon dioxide and then baked. .

[0009]

Hereinafter, the results of test examples will be shown, and the embodiments of the present invention will be described in more detail. Test Example 1 This test was performed in order to select a proteolytic enzyme having a high collagenase-like activity and a low amylase activity, and being pharmaceutically acceptable. (1) Sample flour : Soft flour (trade name: Cleopatra, manufactured by Showa Sangyo KK) was used. Enzymes : Protease A, Protease B, Protease S, Protease N, Proleather, Papain W-40
(Above, manufactured by Amano Pharmaceutical Co., Ltd.), bromelain (reagent name: bromelin, manufactured by Wako Pure Chemical Industries, Ltd.), and Maclase (manufactured by Kaken Pharma Co., Ltd.). (2) Test method Collagenase-like activity : Van Wa using FALGPA as substrate
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): The enzyme concentration was about 5 mg / ml, and the FALGPA degradation reaction was carried out at 25 ° C. to 324 nm.
Was measured for a few minutes. Enzyme activity is [unit /
mg enzyme]. Here, 1 unit is the activity of hydrolyzing 1 μmol of FALGPA in one minute, and ΔA
= 0.0025 / min. Amylase activity : Substrate is 1% gelatinized potato starch,
Enzyme reaction was performed at 37 ° C for 30 minutes.
Nesky's method (Anal. Biochem., 4, p346-347, 1962)
The reducing group was quantified according to the method described in (1). Enzyme activity was [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 can be seen, the collagenase-like activity of the proteolytic enzymes tested was higher in the order of bromelain> protease B> protease A = papain. When the amylase activities of these four kinds of proteases were measured, as shown in Table 1, the amylase activities of proteases A and B were relatively high, and those of bromelain and papain were as low as about one tenth. It became clear.

[0010]

[Table 1]

[0011] The fact that a protease having a high collagenase-like activity is effective means that collagenase recognizes and inactivates the proline residue of the wheat flour epitope (the peptide of SEQ ID NO: 2), and the It is clear from the inventors' conventional knowledge that can be reduced to allergen. In addition, the fact that a protease having low amylase activity is effective means that when wheat starch is degraded by amylase, the volume of flour decreases,
In addition, because the sweetness is imparted and the processability is significantly impaired, and in addition, since wheat allergy patients use the amylase inhibitor in wheat protein as an antigen, it is not possible to add an amylase inhibitor to prevent the degradation of wheat starch. Depends on what is possible.

Accordingly, the above test results confirmed that bromelain is particularly preferred as the protease used in the method for reducing wheat flour allergen 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 under the conditions of pH 5 to 10 by the same method as in Test Example 1. (3) Test results The results of this test are as shown in FIG. The collagenase-like activity of bromelain is highest at pH 9, with p
H5 to 8 were relatively high, but at pH 10, the activity was reduced. From this result, it was found that the antigenicity of flour can be efficiently reduced by reacting bromelain under the condition of pH 9.

However, under such pH conditions,
Since the flavonoids in the flour are alkaline and turn yellow, the final product may be colored. The first requirement for a low-allergen wheat flour is that it has no antigenicity, but at the same time it is required that the quality such as color is comparable to that of ordinary wheat flour. Therefore, it was confirmed that in the method of the present invention, it was necessary to set conditions under which the protease could react with flour under neutral conditions. Test Example 3 This test was conducted in order to allow bromelain to react under neutral conditions and to search for conditions under which the antigenicity of flour could be reduced. (1) Samples Products A and B whose production steps were shown in FIG. 2 were used. (2) Test method The antigenicity of the product was tested by the following ELISA method. That is, the protein in the bromelain reaction product of wheat flour was extracted with a 4 M urea-containing Tris-HCl (UTH) buffer (pH 8.6) and centrifuged (1,300 × g:
5 minutes). The obtained supernatant is used when the protein concentration is about 1
It diluted with UTH buffer so that it might be set to 00-250 microgram / ml. The IgE-EL of 100 μl was determined using the binding to a specific IgE antibody in wheat allergy patient serum (obtained from Urafune Hospital, Yokohama City University School of Medicine) as an index.
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 evident from the results in Table 2, the wheat 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.

A product (product A) obtained by adding 0.6 times the amount of water to flour and reacting 1% by weight of bromelain at 37 ° C. shows that the antigenicity remains in a reaction time of 1 to 4 hours. However, in the 8-hour reaction, the antigenicity was reduced below the detection limit. From the above results, it was confirmed that by adding water or an aqueous ethanol solution to wheat flour, even under neutral conditions, bromelain could efficiently react and reduce the antigenicity of wheat flour.

[0015]

[Table 2]

Test Example 4 The following test was conducted on the product A whose production process was shown in FIG. (1) Test method SDS-PAGE : 200 g of product A and protein in untreated flour were extracted with 1 ml of UTH buffer and centrifuged (1,300 × g: 5 minutes). An equal volume of Seprasol II (manufactured by Daiichi Kagaku) is added to the obtained supernatant, and the mixture is boiled for 15 minutes, followed by the method of Laemmli (Nature, 227, p680-68).
5, 1970) using a polyacrylamide gel (manufactured by Daiichi Kagaku) having 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 carbonic acid dehydratase (MW, 31 kDa), soybean trypsin inhibitor (MW, 22 kDa), and chicken egg white lysozyme (MW, 14 kDa). After electrophoresis, Coomassie Brilliant Blue (R-250, Si
gma). Resist Photography: added and mixed water 74ml to 86g in terms of dry matter of the product A lyophilized, 30 ° C. using a resist graph (Brabender Instrument Co.), 63r
Stirred at pm for 20 minutes. The same operation was performed for a mixture of untreated 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 low-allergen wheat flour batter (50 g) or wheat flour dough (50 g) prepared by resistography, and then added to a thermograph (A).
(R-1000, manufactured by ATTO Co., Ltd.) at 24 ° C. for 90 minutes to measure the amount of carbon dioxide generated and the amount retained in the dough. (2) Test results As shown in FIG. 4, when the 4M urea extract of the product (product A) after 8 hours of bromelain reaction was subjected to SDS-PAGE, the reaction products were mostly from the gel compared to untreated flour. The molecular weight was reduced enough to escape. 20kDa-30
Since a component around kDa remained even after the reaction, this component is considered to have no antigenicity.

As shown in FIG. 5, the resistgram (A) of the allergen-reduced wheat flour (product A) was flatter than that of the untreated wheat flour, indicating that no gluten was formed. . However, as shown in the thermogram of FIG. 6, since the allergen-reduced flour has a carbon dioxide retention ability, it was confirmed that it can be used to produce processed flour foods such as bread using the same. . Test Example 5 Bread was manufactured using the product A whose manufacturing process was shown in FIG. 2, and its characteristics were tested. (1) Sample Weak flour (1 kg) in 0.6 liter of water at 37 ° C.
It was treated with bromelain (10 g) for 8 hours. In the bromelain reaction solution, 10 g of sodium chloride, 10 g of glucose, 1 citric acid
3g, sorbitan monostearate (trade name: Emasol
(S-10F, manufactured by Kao Corporation) 10 mg, and sodium hydrogencarbonate 20 g was further added and stirred. 30g of mixture
Each was quickly put into a 100 ml container or a muffin-type alumina cup and baked in an oven at 180 ° C. for 20 minutes to produce bread.

Further, bread made from wheat flour reduced in allergen by collagenase treatment is used as a raw material (Biosci. Biotech.
Biochem., 58, p388-390, 1994). In addition,
No baker's yeast was used in the bread prepared in this test example. This is because the protein of baker's yeast and wheat allergen have similar amino acid sequences, and therefore, wheat allergy patients often show an allergic reaction to baker's yeast.
For this reason, bread was produced by generating carbon dioxide from sodium bicarbonate and citric acid instead of baker's yeast. (2) Test method Specific volume : Loaf volume was changed to 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 Tone Rating : Color parameters (L, a, b) on top of bread
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 as shown in Table 3. This Table 3
As is clear from the above, the wheat bread reduced in allergen by the bromelain treatment had the same characteristics as the wheat bread reduced in allergen by the collagenase treatment in both specific volume and color tone evaluation. In addition, the commercial English muffin had the same specific volume.

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

[0020]

[Table 3]

[0021]

As described in detail above, the method of the present invention can reduce the allergen of flour by a food-acceptable method, the allergen-reduced wheat flour obtained thereby, Provided is a processed flour food made from allergenized flour. Thereby, it becomes possible to provide safe flour processed foods to flour allergic patients.

[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 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 the collagenase-like activity of bromelain.

FIG. 2 is an example of a process for producing allergen-reduced flour by bromelain treatment.

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

FIG. 4 shows the results of SDS-PAGE of allergen-reduced wheat flour and untreated wheat flour by bromelain treatment.

FIG. 5 is a resistgram of the allergen-reduced wheat flour (A) and the untreated wheat flour (B) obtained by bromelain treatment.

FIG. 6 is a thermogram of reduced allergen wheat flour by bromelain treatment.

FIG. 7 shows the appearance (A) of a bread prepared using low-allergen wheat flour by bromelain treatment and its cross section (B).
FIG.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-6-253758 (JP, A) JP-A-2-167040 (JP, A) Biosci. Biotech. Biochem. , November 23, 1994, Vol. 58, No. 11, pp. 2061-2065 Journal of the Japanese Society of Agricultural Chemistry, July 5, 1995, Vol. 69, special issue, p. 165 (58) Field surveyed (Int. Cl. ⁷ , DB name) A23L 1/10-1/105 A21D 2/00-17/00 JICST file (JOIS)

Claims (9)

(57) [Claims] 1. A method for reducing allergen of wheat flour, which comprises reacting bromelain with a flour to which water or an aqueous ethanol solution has been added under neutral conditions. 2. The flour is 0.05 to 1 times the flour.
2. The method for reducing wheat flour allergen according to claim 1 , wherein 00 times the amount of water is added. 3. The method for reducing wheat flour allergen according to claim 1 , wherein an aqueous ethanol solution having a concentration of 20% or less is added to the flour in an amount of 0.05 to 100 times. 4. 0.01% to 1% of flour
2. The method according to claim 1 , wherein 0% by weight of bromelain is reacted. 5. A flour that hypoallergenic by a method according to claims 1 to 4, low, characterized in that flour allergen epitope shows the amino acid sequence in SEQ ID NO: 2 is inactivated Allergenized flour. 6. A flour that hypoallergenic by a method according to claims 1 to 4, low, characterized in that flour allergen epitope shows the amino acid sequence in SEQ ID NO: 3 has been inactivated Allergenized flour. 7. A processed flour food made from the allergen-reduced wheat flour according to claim 5 or 6 . 8. The hypoallergenic wheat flour of claim 5 or 6, wherein the production method of hypoallergenic bread, characterized in that the addition of sodium bicarbonate and citric acid is calcined mixture was allowed to generate carbon dioxide. 9. Sorbitan / low-allergen wheat flour
9. The method according to claim 8 , wherein monostearate and glucose are added.