JP3055729B2 - Allergen-reduced rice preparation, method for producing the same, and processed food containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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 have an allergic reaction to rice.

[0002]

2. Description of the Related Art In recent years, food allergy sufferers are increasing rapidly. This is due to the high protein intake associated with westernized diets, as well as other factors such as exhaust air pollution,
Or, changes in lifestyle such as Western style houses with carpets and airtightness are complicatedly overlapped, and various substances existing in the living environment are transformed into allergens.

[0003] A sudden increase in allergy to rice is one example. Such food allergies are more common in adults as well as children, and cause great mental and physical distress to parents and the whole family, as well as to the physical and mental distress of the individual. As a treatment method for a food allergy patient, a method of restricting or not allowing food to be taken has been attempted. However, restricting food can hinder the maintenance and development of life. Therefore, it is one of the preferable methods to remove only allergic components and to ingest foods that do not damage other nutritional components.

[0004] 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 acted on to remove allergic proteins contained in rice, and the solubility of 10% trichloroacetic acid to salt-soluble protein in rice is 80%. A method is known in which hydrolysis is performed until the above is reached to remove soluble components. However, this method has not yet sufficiently eliminated allergens, and 20-30% of rice allergic patients have not been improved.

As an antigen component (allergen) specific to a patient who is allergic to rice, electrophoresis (S
DS-PAGE) when analyzed by molecular weight 12,000-30,0
00, especially proteins with a molecular weight of around 16,000 have been identified, but other antigen components (allergens) are not clear and could not be identified, so of course, any components can be selectively removed or reduced. It was not clear what to do and 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 analyzed rice in detail, and found that the above-mentioned molecular weight of 12,000 to 30,000 for patients with rice allergy ineffective with conventional low allergen rice. , Especially molecular weight 1
Around 6,000 proteins, molecular weight 30,000-40,000
Especially 33,000-35,000 protein and molecular weight 50,000-6
It was found that the fraction of 0,000 proteins, especially those having a molecular weight of 52,000 to 57,000, is an important protein involved in allergy. Furthermore, in the method using salt water as described above, even when a surfactant or an enzyme is used in combination, the molecular weight is 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 sufficiently removed.

The present inventors have made intensive efforts to solve such problems and to provide as many rice allergic patients as possible, and as a result, excellent rice is extracted by using salty water with low glutelin and / or prolamin content. They found that allergen-reduced rice could be produced, and completed the present invention. That is, an object of the present invention is to provide an allergen-reduced rice preparation which is extremely effective as food for patients who have an allergic reaction to rice.

[0008]

Accordingly, the present invention provides a method for producing rice having a low glutelin and / or prolamin content of 12,000 to 30,000, 30,000 to 40,000 and 50,000 to 60,000.
Glu from which allergen proteins have been substantially removed.
It relates to a preparation of rice with a low content of tellin and / or prolamin . Further, the present invention is characterized in that a rice having a low glutelin and / or prolamin content is treated with an aqueous salt solution, and a molecular weight of 12,000 to 30,000, 30,000 to 40,000 and 50,000 contained in rice having a low glutelin and / or prolamin content. 0
It also relates to a method of producing a rice preparation from which 00-60,000 proteins have been substantially removed. Further, the present invention relates to a rice having a low molecular weight of 12,0 and a low glutelin and / or prolamin content.
Array of 00～30,000,30,000～40,000 and 50,000～60,000
Glutelin substantially free of rugen protein; and
And / or a processed rice food obtained using a low-prolamin rice preparation.

[0009] The allergen-reduced rice preparation of the present invention is a protein having a molecular weight of 12,000 to 30,000, especially a protein having a molecular weight of about 16,000, a molecular weight of 30,000 to 40,000, 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.

The rice preparation of the present invention can be prepared by subjecting rice having low glutelin and / or prolamin content to an allergen reduction treatment. Here, the allergen reduction treatment is a protein extraction operation using an aqueous salt solution. In the allergen reduction treatment, by performing a protein extraction treatment with a salt aqueous solution in which rice with a low glutelin and / or prolamin content is stirred in a salt aqueous solution with the product temperature at the time of extraction being higher than the freezing point to 15 ° C or lower. In a very short time and with a small number of extractions, allergen proteins can be efficiently removed and separated. The product temperature is the temperature of the aqueous salt solution in which the rice is dispersed.
Rice used in the present invention, aqueous solution of salt, water, and when using enzymes and surfactants, including them, each or all of them should be cooled to the freezing point of the respective substance or higher and 15 ° C or lower before use. More preferably, after each or all of the above substances are blended, the mixture may be cooled to a temperature from the freezing point to 15 ° C. Further, the addition of a protease to the salt aqueous solution promotes the elution and removal of the protein, and when a surfactant is added to the solution, the protease permeates the rice and promotes its action. preferable.

[0011] The low glutelin rice having a low glutelin content used in the present invention has a glutelin content of 5% in protein.
~ 50%, preferably 5-35%, more preferably 10%
Low prolamin rice having a prolamin content of 0.5% to 5%, preferably 0.5% to 3%, has a prolamin content of 0.5% to 5%, and is a refined or unpolished grain. Used in the state of rice flour.
Rice that produces low glutelin rice as a raw material of the present invention includes a variant rice of the NM67 improved line produced by the National Institute for Agrobiological Resources, and the rice that produces low prolamin rice includes 8 made in the laboratory
There are 7KG20-179 varieties of rice.

In the method of the present invention, a surfactant and / or a protease may be optionally used. As the protease, those described in JP-A-2-167040 can be used. As the surfactant, use of a polyglycerin ester or lysophospholipid (for example, lysolecithin) is particularly preferable. Preferred proteolytic enzymes used to promote the elution and removal of the protein to be removed include, for example, papain, bromelain, trypsin, pepsin, pancreatin, actinase, α-chymotrypsin and the like. The amount of the protease used is 0.05 to 5 parts by weight with respect to 100 parts by weight of the aqueous salt solution, and the amount of the surfactant used is 100 parts by weight of the aqueous salt 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 pressure treatment. Decompression degree is 10
５０50 mmHg, and the degree of pressurization is suitably ² to 10 kg / cm ² .

[0013] The allergen-reduced rice of the present invention is prepared by adding an aqueous solution of salt to low glutelin content rice, and keeping the product temperature above the freezing point to 1
It is obtained by stirring at a temperature of 5 ° C. or lower and removing the eluted protein fraction. When the product temperature falls below the freezing point, the amount of allergen protein extracted at one time decreases, and the extraction efficiency decreases. When the product temperature exceeds 15 ° C., the extraction efficiency decreases as in the case where the temperature is below the freezing point. Meanwhile, stationary separation,
In solid-liquid separation operations such as centrifugation, filtration separation, membrane separation, etc., the efficiency is inferior in terms of time and yield as compared with the case where the treatment is carried out at 15 ° C. or lower. In the present invention, after the extraction treatment, a preparation can be obtained without desalting or desalting.

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

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

Specifically, one preferred embodiment of the method of the present invention is as follows.
A salt solution having a concentration of M to 3M or higher and a freezing temperature or higher is added, and the temperature of the product is set to the freezing temperature or higher and 15 ° C or lower, and the mixture is stirred for 2 minutes to 48 hours. Time to 24 hours, if the powder, 2 minutes to 8 hours, preferably 5 minutes to 3 hours, stirring, molecular weight 10,000 to 3
0,000 especially the protein fraction with a molecular weight of around 16,000, the molecular weight of 30,000 to 40,000, especially the protein fraction with a molecular weight of around 33,000 and the molecular weight of 50,000 to 60,000, especially the 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 sufficient. In addition, it is naturally possible to carry out this operation not in a batch system but in a continuous system. After elution with an aqueous salt solution, the salt may be removed by washing with water or the water-eluting allergen may be more strictly removed. The protein fraction of interest 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-obtained allergen-reduced rice preparation of the present invention is used as it is or is dried, powdered or granulated, depending on the use. The drying method may be any method that is used for drying foods. For example, spraying, vacuum, hot air, freezing, solar radiation, electromagnetic waves, or a drying method combining these can be used. Powdering is also possible, for example, roll type, mortar type,
A method such as an impact method may be used.

The allergen-reduced rice preparation obtained according to the present invention can be used as it is as cooked rice or used as a raw material for rice crackers, rice crackers such as rice crackers, rice cakes, rice noodles, gyuhi, or sake or vinegar as well as ordinary rice. It can also be used for processing applications, and a delicious cooked product equivalent to ordinary rice can be obtained. Furthermore, it can be used as a part of other food ingredients for allergy sufferers, and can be used for the production of macaroni, spaghetti, udon, buckwheat, bread, cookies, confectionery and the like containing low allergen rice. 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 with reference to the following experimental examples. Experimental Example 1 Pulverized product of low glutelin rice (20% glutelin content; rice obtained from NM67 improved low glutelin strain rice)
A 76 mM tris citrate buffer (pH 7.4) containing 10 ml of 1M-NaCl was added to the g, and the mixture was stirred for 14 hours at 4 ° C., centrifuged (10000 × G) for 20 minutes, and the supernatant (extract) Was isolated. The extract was dialyzed against a 20 mM phosphate buffer (pH 7.4) containing 0.15 mM NaCl at 4 ° C. for 24 hours using a dialysis tube having a cut-off molecular weight of 3,500. The dialyzed extract was freeze-dried to obtain a salt extract (hereinafter referred to as extract A). The rice of the above-mentioned NM67 improved low glutelin strain used in this experimental example was produced by the following method. That is, the seeds of Japanese Masari (rice varieties) 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. In addition, low prolamin rice 87KG20-179,
It is similarly obtained by performing gamma 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
And further washed with water for 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, followed by stirring at room temperature for 10 minutes. Centrifugation for 20 minutes (10000 x G)
The supernatant (urea extract) was separated 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 the protein amount of 2 mg were SDS 1% and 2-
Electrocaptophoresis (SDS-PAGE) samples were prepared by adding 1% of mercaptoethanol, 1 mM of Tris-HCl buffer, and 20% of glycerin to make 1 ml with distilled water. Both samples were heat-treated at 100 ° C. for 2 minutes, and BPB (bromophenol blue) was added to a concentration of 0.05%. 5 μl of the sample was added to a 10-20% gradient SDS-polyacrylamide gel, and 70 μg at 40 mA.
Extract A and extract B were fractionated by electrophoresis for 1 minute. Next, the fraction components of Extract A and Extract B were electrophoretically transferred to Immobilon P membrane manufactured by Millipore at a constant current of 80 mA for 1 hour. After blocking the transfer membrane with 5% skim milk, the sera of rice allergic patients and the control adult serum without rice allergy were reacted for 14 hours at room temperature. After washing the membrane, biotin-conjugated anti-human I
A 500-fold diluted solution of the gE antibody (manufactured by Tago) and avidin bound to peroxidase (a 1000-fold diluted solution) were each used for 2 times.
The reaction was carried out at 37 ° C. for a time, and the IgE antibody bound to the membrane was labeled with an enzyme.

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

Experimental Example 2 To 1 g of the crushed low glutelin rice used in Experimental Example 1, 10 ml of the urea extractant described in Experimental Example 1 was added, and rice protein was extracted and purified in the same manner as in Experimental Example 1. Was performed to obtain an extract C. 1 g of each of Extract B and Extract C of Experimental Example 1 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, followed by stirring at 4 ° C for 14 hours. did. After centrifugation (10,000 × G) for 20 minutes, the supernatant was obtained and used as extract B and extract C, respectively.

After the rice allergy patient is placed in a prone position, the entire arm is disinfected with alcohol cotton and air-dried. Then, extract B, extract C and a control (50% glycerin, 0.5M NaCl solution) are used as antigen solutions. It was added drop by drop. The sterilized needle was pierced into the skin in an oblique direction through the dropped antigen solution, and after 20 minutes, the presence or absence of wheal and redness around it was determined. The judgment was made by the method of Sheldon JM, et al. (A
manual of clinical Allergy 159 WBSaunders Compan
y Philadelphia and London, 1967). That is, negative (-) when the same as the control, redness is difficult when it is difficult to determine whether redness is observed (±), but redness is 21% or less (+) when the diameter is 21 mm or less. When there was no wheal, the score was (++), and when both redness and wheal were observed, the score was (++). Table 3 shows the results. As is clear from Table 3, extract B did not show any protein as an allergen and extract C did. In other words, this indicates that the rice that has been treated with salt water does not have any protein as an allergen.

EXPERIMENTAL EXAMPLE 3 The same procedure as in Experimental Example 1 was carried out except that commercially available milled rice flour was used in place of low glutelin rice, and the resulting brine extract was extracted with extract E, and urea extraction of the brine extract residue was performed. The liquid was used as extract F. As is clear from Tables 4 and 5, which show the results, the patient sera (serum Nos. 1 to 7) reacted strongly with the extract E, and in particular, the protein components fractionated to a molecular weight of 30,000 or less. It reacted strongly (Table 4). Furthermore, patient serum (serum N
o. 1 to No. 7) did not substantially react with the protein component fractionated to a molecular weight of 30,000 or less, especially the protein component fractionated to a molecular weight of 16,000 of the extract F,
Reacted with 30,000-40,000 or 50,000-70,000 fractionated proteins, a difference from healthy human serum was observed on the high molecular side, and a fraction component specifically recognized by patient serum was also found in extract F (Table 5).

Experimental Example 4 The urea extractant 10 described in Experimental Example 1 was added to 1 g of milled commercial polished rice.
Then, 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 used in the same manner as in Experimental Example 2. Table 6 shows the results.

[0027]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1 Low glutelin rice; put (the NM67 improved low glutelin rice obtained from rice strains used in experiment example 1 20% glutelin content) 2 kg to the container 10 liter, 1M
After adding 7 kg of NaCl solution, adding 1.5 g of decaglycerin monooleate (trade name: MO750) and 25 g of proteolytic enzyme (protease N Amano),
The mixture was stirred for 12 hours at a product temperature of 10 ° C., and after centrifugation, a centrifuge (8,
000 rpm) to obtain a precipitate. This operation was 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. This was repeated again, and the resulting precipitate was dried with a hot-air drier to obtain 1.9 kg of the low-allergen rice of the present invention. Examination of the hypoallergenic rice by the intradermal reaction of a rice allergic patient revealed 19 negative and 1 positive. However, negative means (-) and positive means (++) or more. In addition, when 6 rice allergy patients were ingested at an amount of 25 g / day for 1 week, no onset due to the 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 a 1M NaCl solution was added, and 1.0 g of lysolecithin was further added. The mixture was stirred at a temperature of 13 ° C. for 24 hours. Subsequent operations were the same as in Example 1. Examination of this hypoallergenic rice by the intradermal reaction of a rice allergic patient revealed 16 negative and 1 positive. However, negative means (-) and positive means (++) or more. In addition, when five rice allergy patients were ingested at an amount of 25 g per day for one week, no onset due to the ingestion was observed.

Example 3 5 kg of low glutelin rice used in Example 1 was placed in a 30 liter container, and 0.5 M CaCl ₂ and 0.5 M at a product temperature of 30 ° C.
After adding 20 liters of a mixed solution of Na ₂ SO ₄ and 30 g of proteolytic enzyme (pronase), and allowed to stand for 30 minutes, the temperature of the salt water was cooled to 7 ° C., followed by stirring for 3 hours. After stirring, the mixture was separated from the aqueous salt solution by sieving. Then, water was poured on the sieve until there was no salt. 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. When this hypoallergenic rice was examined by an intradermal reaction of rice allergic patients, 18 negative and 0 positive
Was a name. However, negative means (-) and positive means (++)
It says above. In addition, 20 rice a day
When g was taken for 11 days, no onset due to the ingestion was observed.

Comparative Example 1 The procedure of Example 1 was repeated, except that 2 kg of commercially available polished rice (Sasanishiki) was used to obtain 1.85 kg of treated rice. When the treated rice was examined by an intradermal reaction of a rice allergy patient, 14 were negative and 6 were positive.

Example of Processed Food Production From the rice obtained in Example 1 and Comparative Example 1, rice crackers were produced by a conventional method. That is, the rice was steamed, kneaded, molded, frozen, dried, subsequently baked, further soy sauced, and dried to obtain a rice cracker. Table 7 shows the results of comparing the texture, baked color and flavor of the rice crackers thus obtained. That is, the rice crackers produced from the rice obtained in Example 1 were more excellent.

[0032]

According to the present invention, an allergen protein can be efficiently removed and a rice preparation with reduced allergen can be provided without impairing the original quality of rice. It brings benefits.

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

[Table 3]

[0036]

[Table 4]

[0037]

[Table 5]

[0038]

[Table 6]

[0039]

[Table 7]

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Kazufumi Tsubaki 7-35-35 Higashiogu, Arakawa-ku, Tokyo Asahi Denka Kogyo Co., Ltd. (72) Inventor Takashi Suzuki 7-35-35 Higashiogu, Arakawa-ku, Tokyo Asahi In Denka Kogyo Co., Ltd. (56) References JP-A-5-103617 (JP, A) Agricultural and Biological Chemistry (1991) Vol. 55, No. 2, p. 509-513 (58) Field surveyed (Int. Cl. ⁷ , DB name) A23L 1/10 A23L 1/015 BIOSIS (DIALOG)

Claims (3)

(57) [Claims] 1. A rice having a low glutelin and / or prolamin content, having a molecular weight of 12,000 to 30,000, 30,000 to 40,0.
The glutelin and / or prolamin content has been substantially removed from 00 and 50,000-60,000 allergen proteins .
Low rice preparation. 2. The method of claim 1, wherein the rice having a low glutelin and / or prolamin content is treated with an aqueous salt solution, wherein 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 has been substantially removed. 3. A rice having a low glutelin and / or prolamin content having a molecular weight of 12,000 to 30,000, 30,000 to 40,0.
Low glutelin and / or prolamin content with substantially allergen proteins of 00 and 50,000-60,000 removed
A processed rice food obtained using a rice preparation.