JP2005198582A - Method for making wheat flour low allergenic, low allergenic wheat flour obtained by the method and wheat flour processed food - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique to beneficially decrease allergens contained in wheat flour, especially an allergen (protein of allergy cause) of food-dependent exercise-induced anaphylaxis contained in wheat flour, especially a method for beneficially decreasing IgE-bound epitope of allergen contained in wheat flour, to obtain wheat flour made low allergenic by the method, and to provide wheat flour processed food having the low allergic wheat flour as the raw material. <P>SOLUTION: The method for decreasing the allergen contained in wheat flour comprises bring wheat flour into contact with an extract of a yeast or malt in the presence of a water-based medium to dissolve the wheat allergen in the wheat flour. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for reducing allergens of wheat flour, the allergenized wheat flour obtained thereby, and processed foods using such flour, and in particular, development of food-dependent exercise-induced anaphylaxis among food allergies. The present invention relates to a technique that can advantageously prevent or suppress.

  Food-dependent exercise-induced anaphylaxis is a disease that develops anaphylaxis symptoms due to the addition of exercise load after the intake of certain food. Specifically, as symptom, for example, appearance of wheal or erythema is observed, and sometimes blood pressure is lowered and consciousness is lost.

  In recent years, the number of reports of food-dependent exercise-induced anaphylaxis has been increasing in Japan, where various foods such as wheat, shrimp, squid, crabs and nuts have been reported to be the cause. (Non-Patent Document 1). As a basic treatment for such food-dependent exercise-induced anaphylaxis, the causative food removal therapy is currently effective. However, because there are many processed foods that use such causative foods as raw materials on the market, foods containing allergens are obliged to be clearly stated in the raw material labeling, but all of them should be avoided. It has become very difficult. For this reason, development of foods with reduced allergens has been underway.

  In addition, wheat is the most frequently reported as a causative food for food-dependent exercise-induced anaphylaxis. In wheat-dependent exercise-induced anaphylaxis, it is known that the amount of wheat consumed correlates with the induction of anaphylactic symptoms. That is, if the wheat is allergen-reduced and the allergen-reduced wheat is used, the induction of the patient's symptoms can be prevented or suppressed.

  On the other hand, even healthy individuals are often sensitized to allergens, but in this case as well, the amount of antigen ingested is greatly affected. It is desirable to ingest, and such a reduced allergen wheat product is also useful as an allergy-preventing food.

  By the way, as a technique for reducing the allergen of wheat, various techniques have been proposed so far. For example, Patent Document 1 discloses proteolytic degradation that has high collagenase-like activity and is pharmaceutically acceptable. A technique for reducing the allergen of flour by reacting an enzyme (specifically, bromelain) with the flour has been clarified. Patent Document 2 discloses that a wheat protein is dispersed in water using an organic acid, a mineral acid is added, and 30% to 90% of glutamine and aspartic acid residues contained in the wheat protein are deamidated. Thus, a technique for obtaining low allergenized wheat has been disclosed. Furthermore, in Patent Document 3, after mixing wheat flour and carbohydrase to hydrolyze the carbohydrate allergen contained in the wheat flour, the protease was mixed to decompose the protein allergen, thereby reducing the allergen. A method for producing wheat flour has been clarified, and it is described that actinase and alkaline protease are suitable as proteases. Patent Documents 4 to 6 disclose techniques for producing processed flour foods such as sweets and noodles using reduced allergen flour.

  However, the flour allergen-reduced by the method as described above is intended to inactivate the wheat allergen epitope (QQQPP) shown in SEQ ID NO: 7, and such flour is used. Even if processed foods are manufactured, it is difficult to effectively prevent or suppress the development of wheat allergy, especially food-dependent exercise-induced anaphylaxis. In addition, if only a single enzyme is used, there is a risk of developing allergies to that enzyme.

JP-A-9-172959 JP 2000-69915 A Japanese Patent Laid-Open No. 2001-258889 JP 2000-342203 A JP 2000-342204 A JP 2001-275553 A Satoshi Harada and two others, “Study by Aggregation of Food-Dependent Exercise-Induced Anaphylaxis (FDEIA) Reported in Japan”, Allergy, 49 (11), p. 1066-1073. Masanobu Morita, 5 others, “Fast ω-gliadin is a major allergen in wheat-dependent exercise-induced anaphylaxis”, Journal of Dermat Journal of Dermatological Science, 2003 (33), p. 99-104.

  Here, the present invention has been made in the background of such circumstances, and the problem to be solved is an allergen contained in wheat, particularly an allergen of food-dependent exercise-induced anaphylaxis contained in flour ( It is to provide a technique for decomposing and advantageously reducing allergen-causing proteins. Specifically, the present invention provides a method for advantageously reducing the IgE-binding epitopes of allergens contained in wheat flour, flour allergen-reduced by such a method, and processed flour foods using such low-allergen flour as a raw material. There is to do.

  And as a result of intensive studies to solve such problems, the present inventors have found that ω-gliadin contained in wheat is a major causative antigen of wheat-dependent exercise-induced anaphylaxis ( Non-patent document 2). Furthermore, as a result of repeated research, the amino acid sequences (QQIPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQLPQQQ, QQYPQQQ) represented by SEQ ID NO: 1 to SEQ ID NO: 6 in the ω-gliadin are respectively wheat-dependent exercises. It was found to be the major IgE binding epitope responsible for causing allergic reactions of induced anaphylaxis. They have found that yeast and koji mold extracts are effective in degrading such epitopes.

  Accordingly, the present invention has been completed based on such findings, and the first aspect thereof is to bring yeast or koji extract into contact with wheat flour in the presence of an aqueous medium. Thus, there is a method for reducing the allergen of wheat flour, which comprises decomposing the wheat allergen in the flour.

  Moreover, in the second aspect of the method for reducing allergens of wheat flour according to the present invention, examples of the wheat allergen include allergy-causing proteins of food-dependent exercise-induced anaphylaxis.

  Furthermore, in the third aspect of the method for reducing allergens of wheat flour according to the present invention, the wheat allergen has any one of the epitopes of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6.

  In addition, in the 4th aspect of this invention, the said aqueous medium is made to exist in the ratio of 10-5000 weight part with respect to 1000 weight part of the said flour.

  Moreover, in the fifth aspect of the method for reducing allergen of flour according to the present invention, the yeast in a crushed state is added at a ratio of 10 to 1000 parts by weight with respect to 1000 parts by weight of the flour. A configuration in which an extract of yeast is brought into contact with the flour will be employed.

  Furthermore, in the preferable sixth aspect of the present invention, the contact of the koji extract with the wheat flour is performed by mixing koji in a ratio of 10 to 1000 parts by weight with respect to 1000 parts by weight of water. On the other hand, a configuration is employed in which the extract is added at a ratio of 10 to 5000 parts by weight with respect to 1000 parts by weight of the flour.

  By the way, the present invention is also intended for low allergen wheat, and the embodiment of low allergen wheat flour characterized in that it has been reduced to allergen by the method for reducing allergen of wheat flour as described above. And

  Furthermore, the present invention is also intended for processed flour foods, and is obtained by using the reduced allergen flour obtained by the method for reducing allergens of wheat flour as described above. This is the first aspect.

  In the second aspect of the processed flour food according to the present invention, bread, cake, cookies, biscuits, crackers, noodles, or tempura can be exemplified as the processed flour food.

  And according to the first aspect described above in the method for reducing allergen of wheat flour according to the present invention, the extract of yeast or koji, which are microorganisms, is brought into contact with flour in the presence of an aqueous medium. The enzyme contained in such an extract effectively decomposes allergens in wheat flour, so that low allergen is advantageously achieved. Moreover, in the wheat flour obtained by such a method, the allergen is inactivated.

  In addition, yeast and koji extracts (mixtures of various enzymes) that have been widely used in the field of brewing and fermented foods have been adopted to react with allergens in wheat flour and inactivate these allergens. Therefore, unlike the case where a single purified enzyme is used, the risk of developing an allergy due to the enzyme is low, food safety can be secured to a very high level, and the cost can be reduced. .

  Further, in the second embodiment of the method for reducing allergens of wheat flour according to the present invention, the allergy of flour includes, in particular, food-dependent exercise-induced anaphylaxis, and yeast and koji extracts are proteins that cause such allergies. Is advantageously reduced.

  Furthermore, in the third aspect of the method for reducing allergens of wheat flour according to the present invention, the flour allergen includes a protein or peptide fragment containing any of the epitopes of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6. Thus, yeast and koji extracts effectively degrade such epitopes.

  In addition, according to the fourth aspect of the method for reducing allergen of flour according to the present invention, the aqueous medium is present in a ratio of 10 to 5000 parts by weight with respect to 1000 parts by weight of the flour. In other words, since the amount of the aqueous medium is 1 to 500% of the amount of flour, it is processed as it is without drying the reduced allergen flour, bread, noodles, sweets, tempura, etc. It is possible to produce various processed foods.

  Further, according to the fifth and sixth aspects of the method for reducing allergens of wheat flour according to the present invention, if a predetermined amount of yeast or koji extract is brought into contact with flour in the presence of an aqueous medium, a simpler operation is possible. Thus, it is possible to easily obtain a low allergen flour that is safe as a food or food additive.

  Moreover, in the allergen-reduced wheat flour according to the present invention, among allergens of wheat, in particular, the allergen (IgE-binding epitope) of food-dependent exercise-induced anaphylaxis is effectively degraded, and the occurrence of such allergies is caused. This is advantageously prevented or suppressed.

  Furthermore, in the processed wheat food according to the present invention, since the allergenized flour as described above is used as a raw material, the development of wheat allergy, particularly food-dependent exercise-induced anaphylaxis, is advantageously prevented or suppressed. It is done. Accordingly, processed flour foods such as bread, cakes, cookies, biscuits, crackers, noodles, and tempura according to the present invention are also effective as an allergy prevention food.

  By the way, in the present invention, the allergen is a causative substance that causes an allergic reaction when orally ingesting wheat itself or foods containing wheat, and is particularly dependent on food (wheat) among causative substances contained in flour. The amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6 (QQIPQQQ, QQFPQQQ, QQSPEQQ, which may induce a reaction by binding to IgE antibody in ω-gliadin, which is a major causative antigen of sexual exercise-induced anaphylaxis, QQSPQQQ, QQLPQQQ, or QQYPQQQ) is a protein or peptide fragment.

  And by reducing the main IgE-binding epitopes of allergens contained in wheat flour as described above, the onset of allergy is prevented or suppressed. In the present invention, wheat allergens having such epitopes are In the presence of an aqueous medium, it has a great feature in that it is decomposed using an extract of yeast or koji.

  Thus, in the present invention, yeast and koji extracts that have been widely used in the field of brewing and fermented foods, specifically, a mixture of various enzymes contained in yeast and koji are used. Therefore, wheat flour treated with such an extract is excellent in safety to the human body and can be sufficiently tolerated as a food. In addition, since yeast and straw are not special microorganisms, the production cost of low allergenized flour can be kept low. Moreover, since the extract is a mixture of various enzymes and is not a purified single enzyme, the risk that the enzyme itself becomes an allergen and develops an allergy is extremely low. That is, since the wheat allergen is decomposed by a plurality of enzymes, the amount of each enzyme can be made small, and the risk of being sensitized by the enzyme becomes extremely low.

  The yeast employed in the present invention is not particularly limited as long as it is acceptable as a food. For example, the genus Saccharomyces (for example, used for brewing alcohol, producing alcohol, or bread making) is used. Saccharomyces) yeast, beer yeast, sake yeast, shochu yeast, wine yeast such as wine, baker's yeast, soy sauce yeast, miso yeast, pickled yeast, etc., and at least one of these yeasts The extract obtained from is advantageously used.

  On the other hand, the koji employed in the present invention is a product obtained by breeding mold on starch such as rice, wheat, soybean, bran or the like, as is well known. Examples of such koji include sake lees, miso, soy sauce koji, mirin koji, shochu, amazake koji, and the like, and an extract extracted from at least one of these koji is advantageously used. It will be.

  By the way, in order to obtain an extract from yeast or straw as described above, any conventionally known method can be employed. For example, in the case of yeast, since various enzymes are present in the cell, the yeast can be crushed using a known crusher such as a ball mill, or the cell wall can be crushed by sonication to extract the cell contents. Is used, and the yeast in which the cell wall has been crushed can be used as it is as an extract. In addition, when crushing yeast mechanically, in addition to the method of crushing solid yeast as it is, aqueous systems such as distilled water, purified water, tap water, electrolyzed water (acidic water, alkaline water), milk, skimmed milk powder, etc. For example, a method of suspending about 10 to 5000 parts by weight of yeast with respect to 1000 parts by weight of the medium and then performing a crushing operation as described above to take out various enzymes in cells can be employed. Then, the solid or powdery extract thus obtained and the extract in which the cell contents are dissolved are used. In addition, when using the extract, it is desirable to remove scum consisting of the cell walls of yeast by performing centrifugation, filtration, or the like.

  On the other hand, in rice bran, mold grown on starch such as rice and wheat produces various enzymes outside the cells, so it is possible to use rice bran as it is. In order to avoid such inconveniences, since wastes such as wheat are mixed in wheat flour, it can be used in aqueous media such as distilled water, purified water, tap water, electrolyzed water (acidic water, alkaline water), milk, and skimmed milk powder. It is desirable to extract various enzymes and use such an extract. Such an extract is, for example, mixed with 10 to 1000 parts by weight of soot that is 1 to 100% of its weight with respect to 1000 parts by weight of an aqueous medium, at 4 to 45 ° C. for 1 to 24 hours, After performing the extraction treatment, it can be obtained by carrying out centrifugation, filtration, etc., and in the extract obtained in this way, the enzyme produced by Aspergillus oryzae is dissolved. .

  Thus, yeast and koji extracts obtained by the above-described methods include not only proteolytic enzymes (proteases) that degrade proteins or peptide fragments having the amino acid sequences described above, but also starch degrading enzymes and lipids. Various degrading enzymes such as degrading enzymes are also included. Then, the enzyme in such an extract decomposes the epitope of the amino acid sequence found by the present inventors, thereby advantageously preventing or suppressing the onset of wheat allergy, particularly wheat-dependent exercise anaphylaxis. To get.

  As described above, the allergens of wheat flour are reduced by using the yeast or koji extract. In the present invention, the presence of a predetermined amount of an aqueous medium is required to efficiently decompose the wheat allergen. Below, the extract and flour are brought into contact. At this time, the amount of the aqueous medium is not particularly limited, but is 10 to 5000 parts by weight corresponding to 1 to 500% of the weight with respect to 1000 parts by weight of flour subjected to the low allergen treatment. It is desirable that an aqueous medium is present. This is because if the amount of the aqueous medium is too small, the decomposition of the wheat allergen by the extract may not be performed well. In addition, if the amount of the aqueous medium is too large, it becomes impossible to use the wheat flour subjected to the allergen-reducing treatment as it is as a raw material for food processing. In other words, if the amount of the aqueous medium is large, the allergen-treated wheat flour is contained in a large amount of the aqueous medium, which requires a drying step and takes time and increases the manufacturing cost of the processed food. Because it will be. In the present invention, the aqueous medium is not limited to water itself such as distilled water, purified water, and tap water, but may be water, for example, electrolyzed water (or electrolyzed water). It is also possible to use known aqueous solutions such as acidic water, alkaline water), milk, skim milk powder, and ethanol water.

  In addition, as long as the wheat allergen treatment is performed, as long as it contains wheat allergen, it is not limited at all by the type of production area, strong flour, medium flour, weak flour, etc. Thus, the allergen-reducing treatment according to the present invention can be performed. In addition, the wheat flour may be processed, such as raw, dried, frozen, heated, or kneaded, such as wheat gluten. In addition, when the aqueous medium is previously contained in the flour, it is needless to say that the amount of the aqueous medium including the aqueous medium is desirably adjusted so that the amount of the aqueous medium is as described above. By the way.

  More specifically, in order to reduce the allergen of wheat flour using yeast extract, first, flour and yeast extract are mixed in the presence of an aqueous medium, and such a mixture or liquid mixture is mixed. The wheat allergen may be allowed to react with the enzyme contained in the extract, preferably at a temperature in the range of 5 to 60 ° C., more preferably 18 to 50 ° C., and preferably 1 to 24 hours. At this time, if the temperature is too low, the activity of the enzyme contained in the extract is reduced, making it difficult to efficiently decompose the wheat allergen epitope. On the other hand, if the temperature is too high, the enzyme There is a risk of heat deactivation. In addition, if the reaction time is too short, it is difficult to sufficiently decompose the wheat allergen epitope, while if the reaction time is too long, many proteins in the flour are decomposed, for example, when producing bread, There is a possibility that the function required for flour cannot be performed, such as bread does not swell and becomes hard bread.

  In addition, the amount of yeast extract used is not particularly limited, but in an amount of 1 to 100% of the weight of the flour, in other words, 10 to 1000 per 1000 parts by weight of the flour. An amount that is a ratio of parts by weight is preferably employed. However, if the amount of the extract to be used is too small, the target wheat allergen may not be efficiently decomposed. This is because it is not only economical, but also a decrease in the flavor of the flour and a decrease in physical properties (hardness stress, cohesiveness, adhesion, gum stress).

  Also, in the case of reducing the allergen of wheat flour using the koji extract, first, the wheat flour and koji extract are mixed in the presence of an aqueous medium, and such a mixture or mixture is preferably The wheat allergen may be allowed to react with the enzyme contained in the extract at a temperature in the range of 5 to 60 ° C., more preferably 18 to 50 ° C., and preferably 1 to 24 hours. At this time, if the temperature is too low, the activity of the enzyme contained in the extract is reduced, making it difficult to efficiently decompose the wheat allergen epitope. On the other hand, if the temperature is too high, the enzyme There is a risk of heat deactivation. In addition, if the reaction time is too short, it is difficult to sufficiently decompose the wheat allergen epitope, while if the reaction time is too long, many proteins in the flour are decomposed, for example, when producing bread, There is a possibility that the function required for flour cannot be performed, such as bread does not swell and becomes hard bread.

  Further, when the extract as described above is employed as the koji extract, the amount of the extract added is preferably 10 to 5000 parts by weight with respect to 1000 parts by weight of the flour, that is, It is desirable to add so that it may become 1 to 500% of the amount of flour. This is because if the amount of the extract is too small, the amount of the enzyme will decrease and the target wheat allergen may not be efficiently decomposed. In addition to being uneconomical, there is a risk that the odor of rice cake will adhere to the flour, the flavor of the flour may be reduced, and physical properties (hardness stress, cohesiveness, adhesion, gum stress) may be reduced. Because there is.

  As described above, in the present invention, low allergen can be advantageously realized by a simple operation in which wheat flour and a specific microorganism extract are brought into contact with each other in the presence of an aqueous medium. Yes, in this reduced allergen flour (low allergen flour), wheat allergens in wheat, especially allergen epitopes of food-dependent exercise-induced anaphylaxis (amino acids shown in SEQ ID NOs: 1 to 6) Array) can be effectively decomposed. In the present invention, since extracts of safe microorganisms (yeast, koji) widely used in the fields of brewing and fermented foods are employed, the safety as food or food additives is extremely high. It can be secured.

  By the way, the allergen-reduced wheat flour obtained as described above is used as a raw material for food as it is or after being dried, and is used as a bread, cake, cookie, biscuit, cracker, noodles, or tempura. Etc. will be manufactured according to a conventional method.

  In addition, when producing a flour processed food using low allergenized flour, a low allergenized flour is obtained separately, and in the same manner as the conventional method for producing the desired flour processed food, It is also possible to produce a wheat flour processed food while allergenizing. For example, when manufacturing bread, the following method can be employed. That is, to the wheat flour (strong flour), the above-mentioned yeast and koji extracts are added together with sugar, salt, yeast, butter, milk, water, and other moisture, which are raw materials for bread production. To ask. And if primary fermentation, secondary fermentation, etc. are performed at normal fermentation temperature, the decomposition | disassembly of the wheat allergen by the extract of yeast or koji will be performed with fermentation of bread dough. If the bread dough subjected to the final fermentation is baked at a high temperature, bread made of reduced allergen flour is produced. Thus, if the allergen reduction of wheat flour and the production of processed wheat food can be performed simultaneously, the production time of the allergen-reduced food can be advantageously shortened.

  The exemplary embodiments of the present invention have been described in detail above, but these are merely examples, and the present invention is not limited in any way by specific descriptions according to such embodiments. It should be understood that this is not to be interpreted.

  Hereinafter, some examples of the present invention will be shown and the present invention will be more specifically clarified, but the present invention is not limited by the description of such examples. It goes without saying. In addition to the following examples, the present invention includes various changes and modifications based on the knowledge of those skilled in the art without departing from the spirit of the present invention, in addition to the above specific description. It should be understood that improvements can be made.

<Test Example 1 (Epitope degradation activity measurement test)>
First, a quenching fluorescent substrate represented by the following general formula (I) having a 2- (N-methylamino) benzoyl (Nma) group and a 2,4-dinitrophenyl (Dnp) group is referred to as “analytical biotechnology”. According to the synthesis method described in Chemistry (Analytical Biochemistry), 212, p. 58-64 (1993), a peptide chain is extended and constructed sequentially by a solidification method using an ordinary automatic synthesizer, and hydrogen fluoride. It was synthesized by separating from the resin by treatment and deprotection, and separating the target product by reverse phase HPLC. Then, the quenching fluorescent substrate was dissolved in DMSO to prepare a substrate solution having a concentration of 1 mM. The quenching fluorescent substrate represented by the following general formula (I) has an average molecular weight of 1936.5, and has a peptide bond in the amino acid sequence represented by any one of SEQ ID NOs: 1, 2, 4, and 5. When cut, it has the property of emitting fluorescence (excitation wavelength 340 nm, measurement wavelength 440 nm).
Ac-rrK (Nma) QQXPQQQK (Dnp) r-NH ₂ (I)
[In the formula, X = I / F / S / L (31: 6: 32: 31)]

On the other hand, 1 × 10 ⁹ cultured yeasts were placed in a 50 mM phosphate buffer (pH 6.8) and crushed with beads at a temperature of 4 ° C. Thereafter, the suspension was centrifuged at a rotational speed of 13000 rpm for 15 minutes, and a supernatant was obtained to prepare a yeast extract. Further, 6.8 g of rice bran was put in 100 ml of tap water, extracted at room temperature for 3 hours, allowed to stand, and then the supernatant was taken to prepare a koji extract.

The substrate solution prepared above was diluted 100-fold with 100 mM phosphate buffer (pH 7.0). Next, 200 μl of the diluted substrate solution was injected into each well of a 96-well plate, equilibrated at 37 ° C. for 3 minutes, and the fluorescence intensity (initial blank) was measured. Thereafter, 5 μl of each extract obtained above is added and allowed to react under a temperature condition of 37 ° C., and the fluorescence intensity is measured immediately after the addition of the extract, after 10 minutes, 20 minutes and 30 minutes. The results obtained are shown in FIG. For comparison, 5 μl of buffer solution was added to the substrate solution instead of each extract, and the fluorescence intensity was measured in the same manner. The results are shown in FIG. The fluorescence intensity was measured at an excitation wavelength (λ _ex ): 355 nm and a measurement wavelength (λ _em ): 460 nm.

  As is clear from the results of FIG. 1, when the yeast extract or koji extract was added, the fluorescence intensity increased with the passage of time. Depending on the enzyme contained in the yeast extract or koji extract, It was observed that any or all of the wheat allergen epitopes shown in SEQ ID NOs: 1, 2, 4, 5 were cleaved or degraded.

<Test example 2 (allergen measurement of wheat flour)>
(A) Extract-free additive-treated product 1 ml of 100 mM phosphate buffer (pH 7.4) is added to 0.5 g of wheat flour (Nisshin Flour Camellia Powerful Flour) and suspended, and maintained at 37 ° C. for 20 hours. did. This was designated as an extract-free product.

(B) Koji extract processed product 0.5 g of wheat flour (Nisshin Flour Camellia strong flour), 0.5 ml of Koji extract with a protein concentration of 0.33 mg / ml, and 100 mM phosphate buffer (pH 7.4) 0.5 ml of the mixture was suspended, and reacted at 37 ° C. for 20 hours to obtain a koji extract processed product. The koji extract was prepared in the same manner as in <Test Example 1> above.

(C) Yeast extract processed product 0.5 g of wheat flour (Nisshin Flour made Camellia strong flour), 0.5 ml of yeast extract with a protein concentration of 5.22 mg / ml, and 100 mM phosphate buffer (pH 7.4) And 0.5 ml of the suspension was added and suspended at 37 ° C. for 20 hours to obtain a yeast extract treated product. The yeast extract was prepared in the same manner as in <Test Example 1> Test Example 1 above.

  Then, using various processed products as described above, whether or not the IgE-binding epitope sequences in the yeast and koji extract-treated fractions were reduced was examined by a dot blot method. That is, the extract non-added processed product, the koji extract processed product, and the yeast extract processed product obtained above were spotted on a PVDF membrane (Millipore) so that the amount of flour was 2 μg, Blocking was overnight with TBST buffer (50 mM Tris-buffered saline, 1% Tween 20, pH 7.4) containing 5% skim milk.

  On the other hand, a rabbit was immunized with the synthetic QQSPEQQQFPQQQIPQQQ peptide shown in SEQ ID NO: 8 to obtain serum. And it confirmed that the IgG fraction which refine | purified this serum by affinity column reacts with all of the peptide which has an amino acid sequence shown by sequence number 1,2,3.

  Then, the PVDF membrane was transferred into a solution obtained by diluting the IgG fraction of the rabbit serum 5000 times using TBST buffer containing 5% skim milk, and allowed to react at room temperature for 1 hour. Next, the PVDF membrane was washed 3 times with TBST buffer, and then reacted for 1 hour in a peroxidase (HRP) -labeled anti-rabbit IgG antibody (BIOSOURCE) solution diluted 30000 times with TBST buffer. Then, the amount of bound HRP-labeled anti-rabbit IgG antibody was detected by autoradiography using ECL Plus Western Blotting Detection Regents (Amersham Bioscience). In addition, the intensity of the spot is calculated by the image analysis software Gel-Pro Analyzer (Media Cybernetic), and the antibody reaction rate in the non-extracted product is determined as 100% from the obtained spot intensity value. The rates were calculated and the results obtained are shown in Table 1 below.

  As is clear from the results of Table 1, in the yeast extract processed product and the koji extract processed product, the intensity of the spot is reduced, and at least one epitope sequence of SEQ ID NOs: 1 to 3 is present. It can be seen that it has been decomposed. Moreover, it can be predicted from the value of the antibody reaction rate that the yeast extract processed product and the koji extract processed product can advantageously suppress the occurrence of allergic reaction.

<Test Example 3 (Bread dough and bread allergen measurement and patient serum IgE reactivity)>
First, the following three types of liquids were prepared.
(A) Tap water (b) Straw extract (supernatant)
100 ml of water and 6.8 g of water were mixed and incubated at room temperature for 3 hours with stirring every 30 minutes. And the supernatant of this liquid was taken out as a koji extract.
(C) Yeast extract (supernatant)
To 1000 g of water, 200 g of commercially available yeast was added and stirred and mixed. Then, using a ball mill, the yeast cells are crushed over about 1 hour and 10 minutes, and the resulting crushed liquid is centrifuged at 3000 rpm for 10 minutes, and the supernatant is extracted from the yeast extract. As taken out.

  Then, for 100 g of wheat flour (Yokoyama Milling Ezo deer strong powder), add 50 ml of any of the three liquids prepared above, baker's yeast 6 g and 15 g of salt, which are ordinary bread ingredients, I kneaded the dough. Then, the proofer (electric fermenter) was set at a temperature of 37 ° C. and a humidity of 80%, and primary fermentation: 3 hours 10 minutes and secondary fermentation: 2 hours 20 minutes. In addition, the temperature of bread dough was measured at the time of this fermentation, and the obtained temperature was shown in following Table 2. In addition, after secondary fermentation, bread was baked at baking temperatures of 210 ° C. (upper heat) and 200 ° C. (lower heat) for 11 minutes. All the finished breads were fully swelled as bread.

  In addition, by using a part of bread dough (hereinafter abbreviated as “dough”) after secondary fermentation and part of baked bread (hereinafter abbreviated as “bread”), the following allergen measurement and patient serum IgE reactivity It was confirmed.

-Allergen measurement-
1. For the dough, 1 g of the dough was added to 5 ml of a mixture of 100 mM phosphate buffer (pH 7.4): glycerol = 1: 1 and suspended using a glass homogenizer. On the other hand, for bread, 1 g of bread was added to 7.5 ml of a mixture of 100 mM phosphate buffer (pH 7.4): glycerol = 1: 1 and suspended using a glass homogenizer.
2. Each such suspension was centrifuged at a rotational speed of 12000 rpm for 10 minutes.
3. Then, 10 μl of the supernatant was diluted 100 times with a mixture of 100 mM phosphate buffer (pH 7.4): glycerol = 1: 1.
4). 50 μl of the above diluted solution 3 was spotted on a PVDF membrane (Millipore) using a dot blotter.
5). Then, blocking was performed overnight at 4 ° C. with a TBST buffer solution (50 mM Tris-buffered saline, 1% Tween 20, pH 7.4) containing 5% skim milk.
6). On the other hand, rabbits were immunized with the synthetic peptide represented by SEQ ID NO: 8 to obtain serum. And it confirmed that the IgG fraction which refine | purified this serum by affinity column reacts with all of the peptide which has an amino acid sequence shown by sequence number 1,2,3. The IgG fraction of the rabbit serum was diluted 5000 times with a TBST buffer containing 5% skim milk, and the PVDF membrane was transferred into the solution and reacted at room temperature for 1 hour. 7). Next, the PVDF membrane was washed three times with TBST buffer, and then reacted for 1 hour in a peroxidase (HRP) -labeled anti-rabbit IgG antibody (BIOSOURCE) solution diluted 30000 times with TBST buffer.
8). Then, the amount of bound HRP-labeled anti-rabbit IgG antibody was detected by autoradiography using ECL Plus Western Blotting Detection Regents (Amersham Bioscience). In addition, the intensity of the spot was calculated with the image analysis software Gel-Pro Analyzer (Media Cybernetic), and from the value of the obtained spot intensity, the allergen content when using tap water as the liquid was assumed to be 100%. The allergen content was calculated, and the results obtained are shown in Table 3 below.

  As is clear from the results in Table 3, the dough and bread made of flour brought into contact with the extract (extract) of koji and yeast have a reduced allergen content and cause allergy. It can be seen that the resulting epitope sequence is reduced. It can also be seen that bread has less allergen than dough.

-Reactivity of patient serum IgE-
1-5. The same operations as 1 to 5 in the above “allergen measurement” were performed.
6). Serum prepared by collecting blood from three wheat-dependent exercise-induced anaphylaxis patients (A to C) was diluted 10-fold with TBST buffer containing 5% skim milk, and the PVDF membrane was transferred into the diluted solution. The reaction was allowed to proceed overnight at 4 ° C.
7). Next, the PVDF membrane was washed three times with TBST buffer, and then reacted for 1 hour in a peroxidase (HRP) -labeled anti-human IgE antibody (BIOSOURCE) solution diluted 10,000 times with TBST buffer.
8). The amount of bound HRP-labeled anti-human IgE antibody was detected by autoradiography using ECL Plus Western Blotting Detection Regents (Amersham Biosciences). Further, the intensity of the spot is calculated by the image analysis software Gel-Pro Analyzer (Media Cybernetic), and the IgE reactivity when using tap water as the liquid is calculated as 100% from the obtained spot intensity value. IgE reactivity was calculated, and the results obtained are shown in Table 4 below.

  As is clear from the results of Table 4, in the bread dough and bread made of wheat flour contacted with the koji or yeast extract (extract), the IgE reactivity of the patient's serum is also reduced. It can be seen that low allergenization can be advantageously realized.

  As is apparent from the above description, in the allergen-reduced flour produced according to the method for reducing allergen of wheat flour according to the present invention, a predetermined allergen is advantageously decomposed. Therefore, foods using such flour are not only safe to eat for patients who have an allergic reaction to wheat, but also preventive foods that do not suffer from wheat allergies for healthy people. As can be provided.

In an Example, it is a graph which shows the relationship between reaction time and fluorescence intensity.

Claims (9)

  A method for reducing allergens of wheat flour, comprising decomposing wheat allergens in the flour by bringing the extract of yeast or koji into contact with the flour in the presence of an aqueous medium.   The method for reducing allergens of wheat flour according to claim 1, wherein the wheat allergen is an allergy-causing protein of food-dependent exercise-induced anaphylaxis.   The method for reducing allergen of wheat flour according to claim 1 or 2, wherein the wheat allergen has any one of the epitopes of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6.   The method for reducing allergen of wheat flour according to any one of claims 1 to 3, wherein the aqueous medium is present in a ratio of 10 to 5000 parts by weight with respect to 1000 parts by weight of the wheat flour.   The yeast extract is brought into contact with the flour by adding 10 to 1000 parts by weight of the crushed yeast with respect to 1000 parts by weight of the flour. The method for reducing allergens of wheat flour according to any one of the above.   The contact of the koji extract with the wheat flour prepares the koji extract by mixing koji in a ratio of 10 to 1000 parts by weight with respect to 1000 parts by weight of water, while the extract is used as the wheat flour. The method for reducing allergen of wheat flour according to any one of claims 1 to 4, wherein the method is carried out by adding at a ratio of 10 to 5000 parts by weight per 1000 parts by weight of.   The allergen-reduced wheat flour which has been reduced to allergen by the method for reducing allergen of wheat flour according to any one of claims 1 to 6.   Processed wheat flour manufactured using the allergen-reduced wheat according to claim 7. The flour processed food according to claim 8, which is bread, cake, cookies, biscuits, crackers, noodles, or tempura.

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