JPH05236889A - Allergen-reduced rice preparation, method for producing the same, and processed food containing the same - Google Patents

Abstract

(57) [Summary] [Object] To provide an allergen-reduced rice preparation, a method for producing the same, and a processed food containing the same. [Structure] Rice with a low glutelin and / or prolamin content is treated with an aqueous salt solution to obtain a molecular weight of 12,000 to 30,0
The 00, 30,000-40,000 and 50,000-60,000 proteins were substantially removed. [Effects] Allergen proteins are efficiently removed,
Moreover, an allergen-reduced rice preparation can be provided without impairing the original quality of rice.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an allergen-reduced rice preparation, a method for producing the same, and a processed food containing the same.
In particular, the present invention relates to allergen-reduced rice that can be used as food for patients who develop an allergic reaction to rice.

[0002]

2. Description of the Related Art In recent years, the number of food allergy patients has been rapidly increasing. This is due to the large amount of protein intake that accompanies westernization of eating habits, as well as other factors (for example, exhaust gas air pollution,
Or, changes in lifestyle such as Western-style houses with high airtightness and carpet are complicatedly overlapped, and various substances existing in the living environment are transformed into allergens.

A rapid increase in allergies to rice is one example. This type of food allergy has become common not only in children but also in adults, and it causes great psychological distress not only to the physical and mental distress of the individual but also to the parents and the whole family. Attempts have been made to treat food allergy patients by restricting or not allowing food intake. However, limiting food can interfere with life support and development. Therefore, it is one of the preferred methods to remove foods that cause allergies and to eat foods that do not damage other nutritional components.

As this kind of method, for example, Japanese Patent Laid-Open No. 2-16
As described in Japanese Patent No. 7040, a proteolytic enzyme is allowed to act to remove proteins involved in allergies contained in rice, and the solubility of 10% trichloroacetic acid in salt is 80%. A method of hydrolyzing until the above and removing the soluble component is known. However, this method has not yet been able to sufficiently remove the allergen, and 20 to 30% of rice allergic patients have not been improved.

As an antigenic component (allergen) specific to a patient who is allergic to rice, electrophoresis (S
Molecular weight when analyzed by DS-PAGE 12,000-30,0
00, especially proteins with a molecular weight of around 16,000 have been identified, but other antigenic components (allergens) have not been clarified and could not be identified. Naturally, any component can be selectively removed or reduced. It was not clear what should be done or what would be appropriate. Hereinafter, in the present specification, the molecular weight of a protein is S
It shall be calculated from the result of DS-PAGE.

[0006]

Under these circumstances, the present inventors have made a detailed analysis of rice and found that the above-mentioned molecular weight of 12,000 to 30,000 was found for rice allergic patients who are ineffective with conventional low allergen rice. , Especially molecular weight 1
Besides protein around 6,000, molecular weight 30,000-40,000
Especially 33,000 to 35,000 proteins and molecular weight 50,000 to 6
It was found that the fraction of 0,000 proteins, especially those with a molecular weight of 52,000-57,000, is an important protein involved in allergy. Furthermore, in the method using salt water as described above, the molecular weight of 3
Protein fraction of 0,000-40,000 and molecular weight 50,000-60,
It was also found that the 000 protein fraction could not be removed satisfactorily.

The present inventors have made diligent efforts to solve such problems and provide as many rice allergy patients as possible, and as a result, salt extraction using rice with a low glutelin and / or prolamin content has led to excellent results. The inventors have found that allergen-reduced rice can be produced, and completed the present invention. That is, an object of the present invention is to provide an allergen-reduced rice preparation that is extremely effective as food for patients who develop an allergic reaction to rice.

[0008]

Accordingly, the present invention is directed to a molecular weight of 12,000 to 30,000, 30,000 to 40,000 and 50,000 to 60,0 contained in rice having a low glutelin and / or prolamin content.
00 protein substantially removed. Further, the present invention is characterized in that rice having a low glutelin and / or prolamin content is treated with an aqueous salt solution.
Molecular weight of rice with low glutelin and / or prolamin content 12,000-30,000, 30,000-40,000 and 50,000-
It also relates to a method of making a rice preparation from which 60,000 proteins have been substantially removed. Furthermore, the present invention relates to glutelin and / or
Or the molecular weight contained in rice with low prolamin content 12,000-
It also relates to a processed rice food product obtained using a rice preparation from which 30,000, 30,000 to 40,000 and 50,000 to 60,000 proteins have been substantially removed.

The allergen-reduced rice preparation of the present invention comprises a protein having a molecular weight of 12,000 to 30,000, especially a molecular weight of about 16,000, a molecular weight of 30,000 to 40,000, and particularly a molecular weight of 33,000 to 3
5,000 proteins, and proteins having a molecular weight of 50,000 to 60,000, especially 52,000 to 57,000, have been removed or reduced.

The rice preparation of the present invention can be prepared by subjecting rice having a low glutelin and / or prolamin content to an allergen-reducing treatment. Here, the allergen reduction treatment is a protein extraction operation using an aqueous salt solution. In the allergen-reducing treatment, rice having a low glutelin and / or prolamin content is stirred in a salt aqueous solution with the product temperature at the time of extraction above the freezing point to 15 ° C. or less, and a protein extraction treatment with a salt aqueous solution is performed. The allergen protein can be efficiently removed and separated in an extremely short time and with a small number of extractions. The product temperature is the temperature of the salt aqueous solution in which rice is dispersed.
When rice, an aqueous salt solution, water, or an enzyme or a surfactant used in the present invention is used, each or all of them may be cooled in advance to a freezing point of the respective substance or higher to 15 ° C. or lower before use. More preferably, and after each or all of the above substances are mixed, they may be cooled to a temperature above the freezing point and below 15 ° C. Furthermore, by adding a proteolytic enzyme to the above salt aqueous solution, protein elution and removal are promoted, and when a surfactant is added to this, the proteolytic enzyme permeates rice and its action is promoted. preferable.

The low glutelin rice with a low glutelin content used in the present invention has a glutelin content of 5 in the protein.
-50%, preferably 5-35%, more preferably 10
~ 20%, low prolamin rice with a low prolamin content has a prolamin content in the protein of 0.5 to 5%, preferably 0.5 to 3%. Used in the state of rice flour.
The rice that produces low glutelin rice, which is the raw material of the present invention, is a variety of NM67 improved strains produced by the National Institute for Agrobiological Resources, Ministry of Agriculture and Fisheries, and the rice that produces low prolamin rice is 8 made in the laboratory
There are 7KG20-179 varieties of rice.

In the method of the present invention, a surfactant and / or a proteolytic enzyme can be optionally used, and as the proteolytic enzyme, those described in JP-A No. 2-167040 can be used. As the surfactant, it is particularly preferable to use polyglycerin ester or lysophospholipid (for example, lysolecithin). Preferable proteolytic enzymes used for promoting elution of the protein to be removed include, for example, papain, bromelain, trypsin, pepsin, pancreatin, actinase and α-chymotrypsin. The amount of proteolytic enzyme used is 0.05 to 5 parts by weight with respect to 100 parts by weight of the salt aqueous solution, and the amount of surfactant used is 100 parts by weight of the salt aqueous solution.
About 0.02 to 5 parts by weight is preferable. Further, prior to the enzymatic reaction, the enzymatic reaction is promoted by subjecting the salt aqueous solution containing rice to a pressure reduction or a pressure treatment. Decompression degree is 10
It is suitable that the pressure is -50 mmHg and the pressure is 2-10 kg / cm ² .

In the allergen-reduced rice of the present invention, an aqueous salt solution is added to rice with a low glutelin content and the product temperature is above the freezing point to 1
It is obtained by stirring the mixture at 5 ° C or lower and then eluting the eluted protein fraction. When the product temperature is below the freezing point, the amount of allergen protein extracted once decreases and the extraction efficiency decreases. If the product temperature exceeds 15 ° C, the extraction efficiency will decrease as in the case below the freezing point. On the other hand, stationary separation,
Even in solid-liquid separation operations such as centrifugation, filtration separation, and membrane separation, the efficiency is inferior in terms of time and yield as compared with the case of treating at 15 ° C or lower. In the present invention, after the extraction treatment, the preparation can be desalted or without desalting.

In the allergen-reduced rice preparation of the present invention, 1 g of the powder was added with 10 ml of 1M NaCl to give 30
It is extremely effective if the protein concentration in the supernatant becomes 500 μg / ml or less, preferably 200 μg / ml or less, and further 100 μg / ml or less when stirred at room temperature for a minute.

As the salt solution which is the extractant solution of the present invention, saline is most suitable, but various other phosphates, carbonates, sodium salts, potassium salts, calcium salts, sulfates or hydrochlorides, For example, an aqueous solution of potassium chloride, sodium sulfate, sodium polyphosphate, sodium bicarbonate, sodium carbonate or the like can be used. A suitable salt water concentration is 0.05 to 3 molar concentration, preferably 0.5 to 1.5 molar concentration.

Specifically, one of the preferred embodiments of the method of the present invention will be described.
A saline solution having a concentration of M to 3 M and having a freezing temperature or higher is added, and the temperature of the product is adjusted to the freezing temperature or higher to 15 ° C. or lower and stirred for 2 minutes to 48 hours. Stir for 24 hours, for powder, 2 minutes to 8 hours, preferably 5 minutes to 3 hours, and have a molecular weight of 10,000 to 3
0,000 Protein fraction with molecular weight of around 16,000, molecular weight 30,000-40,000 Protein fraction with molecular weight of around 33,000 and molecular weight 50,000-60,000 with molecular weight of 52,0
The protein fraction of 00 to 57,000 is eluted and then separated by a method such as standing or centrifugation to obtain the allergen-reduced rice preparation of the present invention. This operation is repeated several times if necessary. Usually, about 1 to 3 times is enough. Further, it is naturally possible to carry out this operation not in a batch system but in a continuous system. It is also possible to elute with a salt aqueous solution and then wash with water to remove salt or more strictly remove water-eluting allergen. The target protein fraction is sufficiently eluted,
Whether or not an allergen-reduced rice preparation has been obtained can be determined by confirming the presence of the protein fraction to be removed by electrophoresis, high performance liquid chromatography, or the like. For more precise measurement, an immunological analysis method such as an enzyme immunoassay method or a radioimmunoassay method may be used.

The thus-prepared allergen-reduced rice preparation of the present invention may be used as it is, or may be dried and powdered or granulated depending on the intended use. The drying method may be any method as long as it is a method used for drying food. For example, spraying, vacuum, hot air, freezing, sun, electromagnetic waves, or a drying method combining these may be used. For powdering, for example, roll type, pestle type,
A shock method and the like can be mentioned.

The low allergenized rice preparation obtained by the present invention may be used as it is after being cooked, or may be used as a rice cracker such as hail, rice cracker, rice cake, rice noodles, rye, etc. It can also be used for the processing applications, and a delicious cooked product equivalent to ordinary rice can be obtained. Further, it can be used together as a part of other food ingredients for allergic patients, and can be used for producing macaroni, spaghetti, udon, buckwheat, bread, cookies, confectionery, etc. containing hypoallergenic rice, for example. Of course, it can be used not only for humans but also for animal feed such as pet food. Therefore, its use and usage are not particularly limited.

Next, an inspection method for confirming the effect of the present invention will be described in the following experimental examples. Experimental Example 1 Grinded product 1 of low glutelin rice (glutelin content 20%; rice obtained from rice of NM67 improved low glutelin line) 1
76 ml Tris citrate buffer solution (pH 7.4) containing 10 ml of 1M-NaCl was added to g, and the mixture was stirred for 14 hours at 4 ° C., then centrifuged for 20 minutes (10000 × G), and the supernatant (extract) Separated. The extract was dialyzed against 20 mM phosphate buffer (pH 7.4) containing 0.15 mM NaCl using a dialysis tube having a pore size molecular weight of 3500 cut at 4 ° C. for 24 hours. The dialyzed extract was freeze-dried to obtain a salt extract (hereinafter referred to as extract A). The rice of the NM67 improved low glutelin line used in this experimental example was produced by the following method. That is, seeds of Japanese clam (rice variety) were treated with ethyleneimine to induce mutation, and the obtained seeds were cultivated to obtain rice. The rice thus obtained was subjected to protein analysis to obtain a target mutant variety having a low glutelin content. Low-prolamin rice 87KG20-179 is
It is obtained in the same manner by performing γ-ray irradiation instead of the treatment with ethyleneimine.

The rice residue after extraction is 1M NaCl for 14 hours.
20 ml of 76 mM Tris citrate buffer (pH 7.4) containing
It was stirred at and washed with water for another 2 hours. At this time, the protein concentration of the supernatant was 14 μg / ml (total 10 ml). After freeze-drying the rice residue, 10 ml of a urea extractant (76 mM Tris citrate buffer containing 7 M urea and 20 mM 2-mercaptoethanol) was added to 0.8 g of the freeze-dried product, and the mixture was stirred at room temperature for 10 minutes. Centrifuge for 20 minutes (10000 x G)
The supernatant (urea extract) was separated with and purified in the same manner as the salt extract (hereinafter referred to as extract B).

Next, the extract A and the extract B corresponding to a protein amount of 2 mg were respectively added with SDS at a final concentration of 1% and 2-
Mercaptoethanol 1%, Tris-hydrochloric acid buffer solution 1 mM and glycerin 20% were added to each, and distilled water was added to 1 ml to prepare an electrophoresis (SDS-PAGE) sample. Both samples were heat-treated at 100 ° C. for 2 minutes, and BPB (bromophenol blue) was added so as to be 0.05%. 5 μl of the sample was added to 10 to 20% gradient SDS-polyacrylamide gel and 70 at 40 mA.
The extract A and the extract B were fractionated by electrophoresis for minutes. Next, the fractionated components of the extract A and the extract B were electrophoretically transferred onto an Immobilon P membrane manufactured by Millipore at 80 mA constant current for 1 hour. After blocking the transfer membrane with 5% skim milk, the serum of a rice allergy patient and the control adult serum without rice allergy were reacted at room temperature for 14 hours. After washing the membrane, biotin-conjugated anti-human I
A 500-fold dilution of gE antibody (manufactured by Tago) and avidin conjugated with peroxidase (1000-fold dilution) were each added to 2
The reaction was carried out at 37 ° C for a time, and the IgE antibody bound to the membrane was enzyme-labeled.

On the other hand, DAB (3,3-diamino-benzidine tetrah
25 ml of 50 mM Tris-HCl buffer (pH
7.6) Dissolve in 100 ml, hydrogen peroxide (30%) 50
μl was added to prepare a color developing solution. This color-developing solution was added to the transfer membrane to detect IgE antibody. As is clear from Tables 1 and 2 showing the results, patient serum (serum No. 1 to N
o. 7) strongly reacted with the extract A, particularly with the protein component fractionated to have a molecular weight of 16,000 (Table 1). On the other hand, patient serum hardly reacted with extract B,
No difference from normal human serum (serum No. 8 to No. 10) was observed, and the fraction component specifically recognized by patient serum was extract B.
Was not observed (Table 2).

Experimental Example 2 To 1 g of the low-glutelin rice crushed product used in Example 1, 10 ml of the urea extractant described in Experimental Example 1 was added, and
Extract and purify rice protein in the same manner as
Extract C was obtained. Extract B and Extract C of Experimental Example 1
1 g of each of the above was dissolved in 10 ml of a urea extractant, and 100 μl of the solution was added to 900 μl of a 0.5 M saline solution containing 50% glycerin, and the mixture was stirred at 4 ° C. for 14 hours. After centrifugation (10,000 × G) for 20 minutes, supernatants were obtained and designated as Extract B and Extract C, respectively.

A rice allergic patient was placed in a prone position, and one arm was sterilized with alcohol cotton and naturally dried, and then the extract B, the extract C and the control (50% glycerin, 0.5M NaCl solution) were used as the antigen solution. It was added drop by drop. A sterilized needle was pierced into the skin in an oblique direction through the dropped antigen solution, and 20 minutes later, the presence or absence of wheal and redness around it was determined. Judgment was made by Sheldon JM, et al. (A
manual of clinical Allergy 159 WBSaunders Compan
y Philadelphia and London, 1967). That is, if it is the same as the control, it is negative (-), if it is difficult to judge whether redness is recognized (±), redness is recognized, but if the diameter is 21 mm or less (+), there is redness of 21 mm or more. If there was no wheal (++), and if both redness and wheal were observed, it was (+++). The results are shown in Table 3. As is clear from Table 3, in Extract B, no protein serving as an allergen was found, and in Extract C, it was found. That is, it is shown that rice treated with salt water does not have a protein that becomes an allergen.

Experimental Example 3 A salt water extract was treated in the same manner as in Experimental Example 1 except that a powder of commercially available polished rice was used in place of the low glutelin rice, and the resulting salt water extract was extracted with an extract E and extracted with urea from the salt water extraction residue. The liquid was designated as Extract F. As is clear from Tables 4 and 5 showing the results, the patient sera (serum No. 1 to No. 7) strongly reacted with the extract E, and especially with the protein component fractionated to have a molecular weight of 30,000 or less. It reacted strongly (Table 4). Furthermore, patient serum (serum N
o. 1-No. 7) hardly reacted with the protein component fractionated into extract F having a molecular weight of 30,000 or less, particularly the protein component fractionated into a molecular weight of 16,000,
It reacts with 30,000 to 40,000 or 50,000 to 70,000 fractionated proteins, and the difference from normal human serum is recognized on the high molecular side, and the fraction component specifically recognized by patient serum is also found in extract F. (Table 5).

Experimental Example 4 Urea extractant 10 described in Experimental Example 1 was added to 1 g of commercially available ground rice.
ml was added, and rice protein was extracted and purified in the same manner as in Experimental Example 1 to obtain an extract G. Extract F and extract G
Was determined in the same manner as in Experimental Example 2. The results are shown in Table 6.

[0027]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 2 kg of low glutelin rice (rice obtained from rice of NM67 improved low glutelin line used in Example 1 above; glutelin content 20%) was placed in a 10 liter container and 1 MN
Add 7 kg of aCl solution, add 1.5 g of decaglycerin monooleate (trade name: MO750) and 25 g of proteolytic enzyme (protease N amano), and then add 10 ° C.
Stir at the product temperature of 12 hours, and after stirring, centrifuge (8,000r
pm) and the precipitate was obtained. This operation was similarly repeated twice. Next, 8 liters of water was added to the obtained precipitate and the mixture was stirred for 2 hours, and the supernatant was removed. The precipitate obtained by repeating this again was dried with a hot air dryer to obtain 1.9 kg of the low allergen rice of the present invention. 19 people who tested this hypoallergenic rice negatively on the skin reaction of rice allergic patients,
There was one positive person. However, negative means (-) and positive means (++) or more. When 6 rice allergic patients were ingested in an amount of 25 g per day for 1 week, no onset due to ingestion was observed.

Example 2 2 kg of low-prolamin rice (rice obtained from rice of line 87KG20-179: prolamin content 3%) was placed in a 10-liter container, 7 kg of 1M NaCl solution was added, and 1.0 g of lysolecithin was further added. The mixture was stirred at a product temperature of 13 ° C for 24 hours. The subsequent operation was the same as in Example 1. When this low allergen rice was examined by an intradermal reaction in rice allergic patients, 16 were negative and 1 was positive. However, negative means (-) and positive means (++) or more. When 5 rice allergic patients were ingested in an amount of 25 g per day for 1 week, no onset due to ingestion was observed.

Example 3 5 kg of the low-glutelin rice used in Example 1 was placed in a 30 liter container and 0.5M CaCl ₂ and 0.5M at a product temperature of 30 ° C.
After 20 liters of a mixed solution of Na ₂ SO ₄ and 30 g of proteolytic enzyme (pronase) were added and allowed to stand for 30 minutes, the salt water was cooled to 7 ° C. and stirred for 3 hours. After stirring, the mixture was sieved to separate from an aqueous salt solution. Then water was poured over the sieve until the salt was gone. The obtained rice was placed in a rotary dryer and dried with warm air at 45 ° C. to obtain 4.5 kg of the low allergen rice of the present invention. This hypoallergenic rice was tested in the skin reaction of rice allergic patients, 18 negative, 0 positive
It was a name. However, negative (-) and positive (++)
The above is said. In addition, 20 people per day for 7 rice allergy patients
When ingested in an amount of g for 11 days, no onset due to ingestion was observed.

Comparative Example 1 1.85 kg of treated rice was obtained by treating in the same manner as in Example 1 except that 2 kg of commercially available polished rice (Sasanishi) was used. When this treated rice was examined by an intradermal reaction of rice allergic patients, 14 were negative and 6 were positive.

Processed Food Production Example A rice cracker was produced from the rice obtained in Example 1 and Comparative Example 1 by a conventional method. That is, rice was steamed, kneaded, molded, frozen, dried, then baked, soy sauce was applied, and dried to obtain a rice cracker. Table 7 shows the results of comparison of the texture, baking color and flavor of the rice crackers thus obtained. That is, the rice cracker produced from the rice obtained in Example 1 was superior.

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, an allergen-reduced rice preparation can be provided in which allergen proteins are efficiently removed and the original quality of rice is not impaired. It brings benefits.

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

[Table 3]

[0036]

[Table 4]

[0037]

[Table 5]

[0038]

[Table 6]

[0039]

[Table 7]

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kazufumi Tsubaki 7-35, Higashiohisa, Arakawa-ku, Tokyo Within Asahi Denka Co., Ltd. (72) Inventor Takashi Suzuki 7-35, Higashiohisa, Arakawa-ku, Tokyo Asahi Denka Co., Ltd.

Claims (3)

[Claims] 1. A molecular weight of 12,000 to 30,000, 30,000 to 40,0 contained in rice having a low glutelin and / or prolamin content.
A rice preparation substantially free of 00 and 50,000-60,000 proteins. 2. The rice having a low glutelin and / or prolamin content is treated with an aqueous salt solution, and the rice having a low glutelin and / or prolamin content has a molecular weight of 12,000 to 30,000, 30,000 to 40,000 and 50,000 to 60,000.
A method for producing a rice preparation from which the protein of 1. has been substantially removed. 3. A molecular weight of 12,000 to 30,000, 30,000 to 40,0 contained in rice having a low glutelin and / or prolamin content.
A processed rice food product obtained using a rice preparation substantially free of 00 and 50,000-60,000 proteins.