JP3251199B2 - Method for producing cereal preparation with reduced allergen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a cereal preparation with reduced allergen.

[0002]

2. Description of the Related Art The ingestion of food, which is indispensable on a daily basis, sometimes causes an abnormal reaction in a living body. Among them, a pathological condition in which a substance in food is recognized as an antigen and presents symptoms such as asthma and eczema by a series of immunological mechanisms is called food allergy. Food allergic patients are estimated to be less than about 1% of the population, and especially in children, food allergies frequently occur as a cause of bronchial asthma, atopic dermatitis and the like. Food allergy, generally, milk, eggs, but such as soybean are said to be and the child to be the causative agent of food allergy is often,
In recent years, allergies due to grains such as rice and wheat have been increasing [Kazue Yamada et al., Pediatric Clinic, Vol.
The following (1985); Soichi Arai et al., Pediatric Internal Medicine, Vol. 22, No. 4
15 or less (1990)].

[0003] As a method of treating food allergy patients, in addition to symptomatic treatment with drugs, methods of restricting or not ingesting foods that cause allergies have been attempted.
Restricting food can hinder the maintenance and development of life. Therefore, a method of removing only allergic components and ingesting foods that do not impair other nutritional components has been studied.

In recent years, ingredients that cause food allergies,
That is, studies on allergens have been advanced, and in rice, it has been reported that globulin, a protein with a molecular weight of 16 kd, and many other protein molecules are allergens [Ryo Nakamura, Chemistry and Biology, Vol. 25, 739 et seq. (1987)] .

[0005] For proteins as cereal allergens, especially rice allergens, salt-soluble proteins have been mainly studied. Thus, for rice allergic patients, allergen-reducing rice salt thereof soluble protein was subjected to removal or reduction processing in the US have been given. However, the present inventor has found that there are patients who develop allergies even with treated rice in which the salt-soluble protein as a rice allergen has been removed or reduced, confirming the presence of a plurality of rice allergens other than salt-soluble protein, Furthermore, they identified their allergen proteins and confirmed that among these rice allergens, proteins with high homology exist not only in rice but also widely in cereals (particularly, corn and wheat), and were newly discovered. A method for producing a cereal preparation with reduced allergens has been found.

[0006]

Accordingly, an object of the present invention is to provide a novel method for producing a reduced allergen-reduced cereal preparation which can be given to more allergic patients than the conventional allergen-reduced cereal preparation. Is to do.
It is another object of the present invention to provide a novel method for producing a novel allergen-reduced rice preparation which can be given to more allergic patients than conventional allergen-reduced rice preparations.

[0007]

The problems SUMMARY OF THE INVENTION may, according to the invention, with respect to UDP-glucose starch cereal glucosyltransferase was deleted Gotoshi, and performing allergen-reducing treatment, allergen-reducing cereal preparations Can be achieved. In addition,
Akira, waxy varieties cereals, waxy tampa
Gene that expresses cereal-deficient cereal or waxy protein
Allergen reduction treatment for cereals that have been engineered
Allergen-reduced cereal preparation
It can also be achieved by a manufacturing method. The present invention also provides a salt-insoluble protein having a molecular weight of 60 kd, a molecular weight of 3
Salts insoluble proteins 3kd, and rice to which at least one protein is lacking or reduced chosen from salts insoluble protein or Ranaru group molecular weight 36 kd, and performing allergen-reducing treatment, allergen-reducing It can also be achieved by a method for producing rice preparations.

[0008] In the present specification, the molecular weight [unit: kilodalton (kd)] of a rice protein is determined by measuring a liquid sample containing a target protein by using sodium dodecyl sulfate (SDS) as a surfactant and a reducing agent (for example, A sample for electrophoresis obtained by treatment with mercaptoethanol) was subjected to SDS-polyacrylamide electrophoresis (hereinafter referred to as SD).
(S-PAGE). Detailed separation conditions will be described in detail in Examples.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
As used herein, the term “salt-insoluble protein having a molecular weight of 60 kd” refers to a salt-insoluble rice protein having a molecular weight of 6
The present inventors have found for the first time that the protein is a 0 kd protein, and that this protein is an allergen protein. A typical example is a waxy protein, a rice starch synthase [Okagaki et al., Pl.
ant Mol. Biol. (1992) Volume 19 Section 5
13 to 516]. Waxy protein (UDP
Glucose starch glucosyltransferase)
Consists of 532 amino acid residues. In the present specification, the “salt-insoluble protein having a molecular weight of 60 kd” does not need to have the activity as the waxy protein as long as it is a protein having allergenicity. Therefore, in the “salt-insoluble protein having a molecular weight of 60 kd”, in addition to the waxy protein itself, one or more amino acids are added, deleted or substituted in the amino acid sequence of the waxy protein, and Allergenic proteins are included.

[0010] UDP glucose starch glucosyltransferase is a kind of the above waxy protein, and is composed of various grains including rice (eg, wheat, corn, barley, rye, oats, millet, millet or millet). Widely distributed. In addition, UDP glucose starch glucosyltransferase has high homology in partial amino acid sequences in various grains including rice (eg, wheat, corn, barley, rye, oats, millet, millet or millet). In particular, rice has high homology with wheat and corn. In this specification, "UDP glucose starch glucosyltransferase" does not need to have enzymatic activity as long as it is a protein having allergenicity. Therefore, in the above-mentioned "UDP glucose starch glucosyltransferase", in addition to the UDP glucose starch glucosyl transferase itself, one or more amino acids are added, deleted or substituted in the amino acid sequence of UDP glucose starch glucosyl transferase, In addition, the above-mentioned allergenic proteins are included.

As used herein, the term "salt-insoluble protein having a molecular weight of 36 kd" refers to a salt-insoluble protein having a molecular weight of 36 kd among rice proteins, and is a novel protein discovered by the present inventors for the first time. The present inventors have also found that this novel protein is an allergen protein. N-terminal of this novel protein (positions 1 to 1)
The amino acid sequence at position 0) is as follows. Ser (Ala, Gly) -Lys-Gly-Ala
(Glu) -Gly-Met-Asn-Val-Val
-Phe 1-position "Ser (Ala, Gly)" is used as a 1-position amino acid serine (Ser) when determining the amino acid sequence.
Is detected in a major amount, and alanine (Ala) and glycine (Gly) are also simultaneously detected in small but non-negligible significant amounts, respectively. In addition, alanine (Ala) is also detected as amino acid at position 4 of “Ala (Glu)” at position 4, but glutamic acid (Gl
u) also means that it is simultaneously detected in a small but significant amount that cannot be ignored.

Therefore, in the above “salt-insoluble protein having a molecular weight of 36 kd”, the first and fourth positions are serine (Ser).
And proteins that are alanine (Ala),
Also included are proteins in which position 1 is replaced with alanine (Ala) or glycine (Gly) and / or position 4 is replaced with glutamic acid (Glu). Furthermore, in addition to the protein itself having the amino acid sequence at the N-terminus (positions 1 to 10), one or more amino acids in the entire amino acid sequence are added to the “salt-insoluble protein having a molecular weight of 36 kd”. Also included are proteins that have been deleted or substituted and have the allergenic properties described above.

[0013] In the present specification, the "salt-insoluble protein having a molecular weight of 33 kd" is a salt-insoluble protein having a molecular weight of 33 kd among rice proteins, and is a novel protein discovered by the present inventors for the first time. The present inventors have also found that this novel protein is an allergen protein. N-terminal of this novel protein (positions 1 to 1)
The amino acid sequence at position 5) is as follows. Ser (Ala, Gly) -Thr (Lys) -Gly
-Ala (Glu) -Gly-Met-Asn-Val
-Val-Phe-X-Gly (Thr, Ser) -A
la-Glu-Met

"Ser (Ala, Gly)" at the 1st position and "Ala (Glu)" at the 4th position have the same meaning as described above, and "Thr (Lys)" at the 2nd position also has a major amount of threonine. (Thr) and a significantly small amount of lysine (Lys) are detected.
(Thr, Ser) "also contains a major amount of glycine (G
ly) and significant small amounts of threonine (Thr) and serine (Ser). Also, 11
The "X" at the position means that the type of amino acid cannot be specified.

Therefore, in the “salt-insoluble protein having a molecular weight of 33 kd”, the first, second, fourth and twelfth positions include
Not only are proteins that are serine (Ser), threonine (Thr), alanine (Ala) and glycine (Gly), respectively, but also position 1 is substituted with alanine (Ala) or glycine (Gly) and / or position 2 is lysine. (L
ys) and / or glutamic acid at position 4 (G
lu) and / or at position 12 threonine (T
Proteins substituted with (hr) or (Ser) are also included. Furthermore, one or more amino acids in the entire amino acid sequence are added to the “salt-insoluble protein having a molecular weight of 33 kd” in addition to the protein itself having the N-terminal (positions 1 to 15) amino acid sequence. , Deleted or substituted, and the allergenic protein described above.

As used herein, the term "salt-insoluble protein having a molecular weight of 13 kd" is a salt-insoluble protein having a molecular weight of 13 kd among rice proteins, and the present inventors have first found that this protein is an allergen protein. . A representative example of its active form is prolamin, a rice seed storage protein [Yamagata et al., BioSci. Biotec
hnol. Biochem. (1992) Volume 56 Section 5
37]. Prolamin is expressed by a number of similar multigenes and consists of a population of proteins with slightly different amino acid sequences. In the present specification, the “salt-insoluble protein having a molecular weight of 13 kd” does not need to have the activity as the prolamin protein as long as it is a protein having allergenicity. Therefore,
The “salt-insoluble protein having a molecular weight of 13 kd” includes:
In addition to the prolamin protein group itself, a protein having one or more amino acids added, deleted or substituted in the amino acid sequence of the prolamin protein and having the above-mentioned allergenicity is included.

Hereinafter, the method of the present invention will be mainly described with respect to rice, but unless otherwise specified, it should be understood that the description relating to rice applies to other cereals. In the method of the present invention, cereals lacking or reducing UDP glucose starch glucosyltransferase are used as raw material cereals for the allergen reduction treatment. Further, as the raw rice for the allergen reduction treatment, rice having a low salt-insoluble protein (allergen) content, that is, a molecular weight of 60 kd,
Use is made of rice lacking or reduced in salt insoluble protein having a molecular weight of 33 kd and / or a molecular weight of 36 kd. As the cereal or rice having a low salt-insoluble protein content, a known cereal or rice can be used as it is, but it can also be produced by variety selection, creation of a mutant, or genetic engineering techniques. Grains having a low content of salt-insoluble protein having a molecular weight of 60 kd, or rice, include grains having a low waxy protein content, or rice, particularly waxy varieties,
For example, waxy rice, waxy wheat or waxy corn can be used. Most waxy varieties lack waxy proteins (Hirano Hirano, Seed Bioscience, Gakkai Shuppan Center, 1995 117-1.
22). Many waxy mutants have also been isolated by mutagen treatment, and mutants in which the protein is expressed in an inactive form, mutants lacking the protein itself, etc. have been obtained [Hirano et al., Proteins / Nucleic Acids]・ Enzyme (199
2) Vol. 37, pp. 1317-1325].

In genetic engineering techniques, it has been reported that good-tasting rice can be produced by suppressing the expression of this waxy protein by antisense technology and controlling the amylose content (for example,
JP-A-4-104791, JP-A-5-13981,
And JP-A-5-168482). For the suppression, as a promoter, a rice waxy promoter which is expressed in rice endosperm, a corn Ad
h1 promoter, cauliflower mosaic virus 35
An S promoter, a rice allergen promoter, or the like can be used. Also, as a terminator,
Nopaline synthase gene NOS terminator or cauliflower mosaic virus 35S RNA terminator can be mentioned.

Rice having a low content of salt-insoluble protein having a molecular weight of 13 kd includes rice having a low prolamin content,
For example, mutants with reduced prolamin or varieties with originally low prolamin content have been reported (Hikaru Sato et al., Seed Bioscience, Gakkai Shuppan Center, 1
995, pages 163-168). Prolamin expression can also be suppressed by the same antisense technology as described above.

For example, antisense waxy can be introduced into rice variety Koshihikari by the following method. A plasmid pRWX1 (JP-A-5-168482) having the antisense waxy gene prepared by Fujimura et al. And capable of reducing the amylose content in rice endosperm is prepared. In this plasmid, a part of the waxy gene is connected downstream of the rice allergen promoter in an antisense orientation, and further a terminator of the waxy gene is ligated. A large amount of plasmid pRWX1 DNA is prepared and introduced into a mature ripe embryo of rice cultivar Koshihikari by a particle gun method. After dehulling the mature seeds of rice variety Koshihikari, sterilization is performed for 30 minutes in a 3.5% calcium hypochlorite solution while degassing and stirring.
Then, after sufficiently washing with sterilized water, water is removed on sterilized filter paper, and 2,4-dichlorophenoxyacetic acid (2,4-
D) Containing 3 ppm, 0.4% gellan gum in N6 medium
Is placed on a callus induction medium (Japanese Patent Application Laid-Open No. 5-292555) and cultured at 25 ° C. in a dark place. Six days after the start of culture, only the enlarged embryo tissue is removed. The excised tissues are arranged on a 6 cm Petri dish containing a callus induction medium with the scutellum side up. The arrangement method is such that 10 to 20 pieces are arranged along a circumference having a radius of 1 cm to form a petri dish for shots.

As a particle gun device, for example,
A pneumatic Reebok model 260 can be used. Plasmid pRWX1 DNA and pUBA DN having a bialaphos resistance gene as a selection marker
A [Toki et al., PlantPhysiol. (199
2) Vol. 100, p. 1503] and 1: 1.
Add 16 μl of NA solution according to the method of Morikawa et al. (Plant Cell Engineering, 4,
43-48, 1992), coated with 1 μm gold particles, placed on a special projectile (bullet), and air-dried. 0.1mg gold particles per shot, 0.4μg DN
A, Two shots are shot on a shot petri dish under the conditions of a firing pressure pumping eight times and a distance between samples of 6 cm, and two genes are introduced. From the petri dish shot, the embryo tissue,
After replacing with a N6 medium containing 10 ppm of the selected drug bialaphos [Meiji Seika Co., Ltd.], culture at 25 ° C. in a dark place for 7 days, and then continue culture in the light place. After 4 to 8 weeks from the start of the culture, bialaphos-resistant callus by gene transfer is confirmed on the scutellum, the callus portion is cut out, and the culture is expanded and cultured in a new medium. From this callus, the method of Okii et al.
No. 92955) to regenerate the plant. It is confirmed by PCR that these transformants contain DNA derived from pRWX1. Furthermore, it is confirmed by Western blotting that the expression of waxy protein is suppressed in the obtained seeds. These seeds are sown to obtain a large amount of progeny seeds and can be used as raw rice for the allergen-reduced rice preparation according to the present invention.

By the same method as described above, antisense waxy can be introduced into rice varieties other than Koshihikari. Further, antisense prolamin can be introduced into various rice varieties. Further, the DNA sequence of a salt-insoluble protein having a molecular weight of 33 kd or a DNA sequence of a salt-insoluble protein having a molecular weight of 36 kd was analyzed, and its antisense DN was analyzed.
A can also be introduced.

The shape of the grain or rice used as a raw material for the allergen reduction treatment according to the present invention is not particularly limited. For example, rice grains after threshing and milling can be used as they are. Brown rice may be used. Either new rice or old rice (or new cereals or old cereals) can be used. In addition, those obtained by preparing (for example, pulverizing, puffing, or gelatinizing) those grains or rice can also be used.

The allergen reduction treatment in the method of the present invention can be performed by a known method. For example, a method of treating with a proteolytic enzyme, a method of contacting with a salt aqueous solution, a lower alcohol, an alkaline aqueous solution, and / or an acidic aqueous solution, or a method of fermenting with a solution in which saccharides and saccharide-decomposing microorganisms are dispersed, and further appropriately treating them Combined methods can be mentioned.

When a treatment with a proteolytic enzyme is carried out as the allergen reducing treatment, papain, collagenase, trasglutaminase or carboxypeptidase can be used as the proteolytic enzyme, and these enzymes are generally used for microorganisms or plants. It can be of any origin. The amount of the enzyme is preferably 0.01 to 5 parts by weight, more preferably 0.05 to 1 part by weight, based on 100 parts by weight of cereals or rice (for example, rice grains or rice pieces). If the amount of the enzyme is more than 5 parts by weight, proteins other than the allergen will be decomposed and bitter components will be produced, which is not preferable. On the other hand, when the amount of the enzyme is small, the decomposition of the allergen is not sufficient, which is not preferable. A required amount of enzyme can be dissolved or suspended in a buffer solution or the like, and cereals or rice (eg, rice grains or rice pieces) can be immersed in the solution. If necessary, a surfactant can be added, or the treatment can be performed under pressure or reduced pressure. The reaction conditions such as the reaction time and the reaction temperature can be appropriately determined in consideration of the optimum conditions of the enzyme activity and the throughput of cereals or rice. After completion of the enzymatic reaction, the enzyme is deactivated and the reaction is stopped. The method of deactivating is not particularly limited, and any known method (for example, heating) can be used.

In the case of treating cereal grains or cereal pieces or rice grains or rice pieces with an aqueous salt solution as the allergen reduction treatment, the type of salt used in the aqueous salt solution is not particularly limited. , Hydrochloride, sulfate, carbonate or phosphate) and alkali metal salts (for example, sodium salt or potassium salt). Particularly, sodium chloride, sodium carbonate, sodium polyphosphate or various phosphates are used. This is preferable because the efficiency of allergen reduction is high. The concentration of salt in the aqueous solution is 0.05M
~ 5M is preferred. If it exceeds 5M, the removal of allergen protein is not sufficient, which is not preferable. In order to prevent spoilage during and after the treatment with the salt solution, a small amount (10% to 20%) of alcohol (for example, ethanol) is used.
Can also be included in the aqueous salt solution. Further, a surfactant, a reducing agent, and the like can be included in the salt aqueous solution as needed. When performing the allergen reduction treatment, cereals or rice (for example, rice grains or rice pieces) are immersed in the salt aqueous solution. The immersion treatment may be either standing or stirring. Stirring may be performed by rotating the immersion vessel,
A stirring blade may be used. The temperature for the treatment is preferably 5 to 40 ° C., and the pH for the treatment is not particularly limited. The processing time is preferably from 10 minutes to 72 hours. After being immersed in an aqueous salt solution, desalting is performed. The desalting method is not particularly limited, and any known method (for example, exposure to running water) can be used.

As the allergen reduction treatment, the above treatment with an aqueous salt solution and the above treatment with a protease can be used in combination. In this case, the salt aqueous solution treatment can be performed before, during, or after the enzyme solution treatment. When the treatment with the salt solution is performed before or after the treatment with the enzyme solution, the respective treatments with the enzyme solution and the salt solution may be the same as those described above. When the treatment with the salt solution is performed during the treatment with the enzyme solution, the solution to be used is a solution in which the enzyme is further dissolved or suspended in the salt solution. That is, as the proteolytic enzyme to be used, generally, any enzyme of microbial or plant origin can be used. The amount of the enzyme is 0.01 to 5 with respect to 100 parts by weight of cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or flakes).
It is desirable that the amount be from 0.05 to 1 part by weight. If the amount of the enzyme is more than 5 parts by weight, proteins other than the allergen will be decomposed and bitter components will be produced, which is not preferable. On the other hand, when the amount of the enzyme is small, the decomposition of the allergen is not sufficient, which is not preferable. Examples of the kind of the salt in the aqueous salt solution used for the treatment include salts of inorganic acids (eg, hydrochloride, sulfate, carbonate or phosphate) and alkali metal salts (eg, sodium salt, potassium salt). Sodium carbonate, sodium polyphosphate or various phosphates are preferred because of their high allergen reduction efficiency. The concentration of the salt in the aqueous solution is 0.05 M to 5
M is preferred. If it exceeds 5M, it is not preferable because the removal of allergen protein becomes insufficient. A required amount of enzyme and salt are dissolved or suspended in a buffer solution or the like, and cereal (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice flakes) is immersed in the solution or suspension. If necessary, a surfactant, a small amount (10% to 20%) of alcohol (for example, ethanol), and / or a reducing agent may be added, or the pressure may be increased or decreased . Soaking treatment may be a stand may be stirred. Stirring may be performed by rotating the vessel in which the liquid is immersed, or by using a stirring blade. The reaction conditions such as the reaction time and the reaction temperature can be appropriately determined in consideration of the optimum conditions of the enzyme activity and the throughput of cereals or rice. Processing temperature is 5-4
0 ° C. is preferred, and the pH to be treated is not particularly limited. The processing time is preferably from 10 minutes to 72 hours. After treatment, wash with water to remove enzymes and salts.

As an allergen reduction treatment, when cereals (for example, cereal grains or flakes) or rice (for example, rice grains or flakes) are treated with an aqueous dispersion in which lactic acid bacteria are dispersed in an aqueous saccharide solution, the saccharide of the dispersion used is used. The concentration is adjusted to 2 to 10%, and the concentration of lactic acid bacteria is adjusted to 10 ⁶ to 10 ⁸ per 1 ml of the dispersion. As lactic acid bacteria, for example, Lactobac
Examples include those belonging to the genus illus or the genus Streptococcus. The sugar is not limited as long as the fungus to be used can assimilate, but monosaccharides, for example, glucose, fructose, maltose,
Or disaccharides, such as sucrose, are common.
Grains (for example, cereal grains or grain pieces) or rice (for example, rice grains or rice pieces) are immersed in the aqueous dispersion in which lactic acid bacteria are dispersed in an aqueous saccharide solution. The amount of the aqueous dispersion may be 2 to 20 times (v / w) the amount of cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or flakes). The immersion treatment may be performed in a stationary state, but it is more preferable that the immersion treatment is weakly stirred. The stirring can be carried out by rotating the vessel to be immersed or by using stirring blades. Reaction conditions such as reaction time and reaction temperature can be appropriately determined in consideration of the optimal conditions of lactic acid bacteria and the throughput of cereals or rice. The cereal (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice pieces) after the fermentation treatment is washed with water to remove lactic acid bacteria.

As the allergen reduction treatment, when treating cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice pieces) with a lactic acid bacteria dispersion in an aqueous saccharide solution,
The above treatment with a proteolytic enzyme and / or the above treatment with an aqueous salt solution can be used in combination. In that case, the treatment solution used is a solution in which the above-mentioned protease is dissolved or suspended in a lactic acid bacteria dispersion in an aqueous saccharide solution, and the types and concentrations of the saccharide, lactic acid bacteria, enzymes and / or salts are It may be the same as above. The protease is preferably an acidic protease having an optimum pH of 7.0 or less. That is, as the proteolytic enzyme to be used, generally, any enzyme of microbial or plant origin can be used. The amount of the enzyme is 0.01 to 5 parts by weight, particularly 0.1 to 100 parts by weight of cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or flakes).
The amount is desirably from 0.5 to 1 part by weight. If the amount of the enzyme is more than 5 parts by weight, proteins other than the allergen will be decomposed and bitter components will be produced, which is not preferable. On the other hand, if the amount of the enzyme is small, the decomposition of the allergen becomes insufficient, which is not preferable. Examples of the kind of the salt in the aqueous salt solution used for the treatment include salts of inorganic acids (for example, hydrochloride, sulfate, carbonate or phosphate) and alkali metal salts (for example, sodium salt or potassium salt). Sodium, sodium carbonate, sodium polyphosphate or various phosphates are preferable because of high allergen reduction efficiency. The concentration of the salt in the solution is preferably 0.05M to 5M. If it exceeds 5M, it is not preferable because the removal of allergen protein becomes insufficient. The required amount of sugar, lactic acid bacteria,
Enzymes and / or salts are dissolved or suspended in a buffer solution or the like, and grains (eg, cereal grains or grain pieces) or rice (eg, rice grains or rice pieces) are immersed in the solution or suspension. As needed,
A surfactant, a reducing agent, a small amount (10% to 20%) of alcohol (for example, ethanol) may be added, or the pressure may be increased or decreased. The immersion treatment may be either standing or stirring. Stirring may be performed by rotating the vessel in which the liquid is immersed, or by using a stirring blade. Reaction conditions such as reaction time and reaction temperature are appropriately determined in consideration of the optimal conditions of lactic acid bacteria, the optimal conditions of enzyme activity, and / or the amount of processed rice. Processing temperature is 5
-40 ° C is preferred, and the pH to be treated is not particularly limited. The processing time is preferably from 10 minutes to 72 hours. After the treatment, it is washed with water to remove lactic acid bacteria, sugars, enzymes and / or salts.

As the allergen reduction treatment, when cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice flakes) are treated with a lower alcohol, for example, ethanol, the ethanol is anhydrous [95% ( v / v)]
Ethanol or an aqueous ethanol solution can be used. The ethanol concentration of the aqueous ethanol solution is 5
It is desirably 0% or more, particularly 70% or more. If the ethanol concentration is less than 50%, the efficiency of the treatment and the removal operation decreases, which is not preferable. Hereinafter, anhydrous ethanol or an aqueous ethanol solution may be referred to as an ethanol treatment liquid. If necessary, a surfactant may be added to the ethanol treatment solution. Grains (eg, cereal grains or flakes) or rice (eg, rice grains or flakes) are immersed in the ethanol treatment liquid. The amount of the ethanol treatment liquid may be 2 to 20 times (v / w) the amount of cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice pieces). The immersion treatment may be allowed to stand, but stirring is more preferable. Stirring may be performed by rotating the vessel in which the liquid is immersed, or by using a stirring blade. The processing temperature is preferably 5 to 40C. The processing time is 1 minute to 72 hours, preferably 2 to 12 hours. After being immersed in the ethanol treatment liquid, the ethanol component is removed from the grains (for example, cereal grains or flakes) or rice (for example, rice grains or flakes). As a removing method, any known method (for example, a method of exposing to water) can be used.

In the case of treating cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice pieces) with an alkaline aqueous solution as the allergen reducing treatment, the alkaline aqueous solution may be a strong alkali salt having a pH of 7 to 10. Preferably, an aqueous solution adjusted to pH 8 to 10 can be used. It is not preferable that the pH of the aqueous solution used is less than 7, since the efficiency of the allergen reduction treatment and the operation of removing the alkali component decreases. The pH of the aqueous solution used is 10
If it exceeds, the protein fraction having rice characteristics such as glutelin is undesirably extracted and removed into the aqueous solution. Examples of the strong alkali salts include hydroxides such as sodium and potassium. In addition, you may add a surfactant to an alkaline aqueous solution as needed. Grains (for example, cereal grains or grain pieces) or rice (for example, rice grains or rice pieces) are immersed in the alkaline aqueous solution. The amount of the aqueous solution may be 2 to 20 times (v / w) the amount of cereals (for example, cereal grains or flakes) or rice (for example, rice grains or flakes). The immersion treatment may be allowed to stand,
It is more preferable to stir. Stirring may be performed by rotating the vessel in which the liquid is immersed, or by using a stirring blade. The processing temperature is preferably 5 to 40C. The processing time is 1 minute to 72 hours, preferably 2 to 12 hours. After immersion in an alkaline aqueous solution, the alkali content is removed. Any known method may be used for the removal, and examples thereof include a method of exposing to water or neutralizing with a dilute acid.

As the allergen reduction treatment, an alkaline aqueous solution may be used followed by an acid aqueous solution. In this case, the treatment with the alkaline aqueous solution may be the same as the above operation. An acid aqueous solution is an aqueous solution obtained by diluting an acid and having a pH of 5 or less.
The pH is not particularly limited as long as it is below,
It is an aqueous solution prepared below. The type of the acidic aqueous solution is not particularly limited, and an inorganic acid (eg, hydrochloric acid, phosphoric acid, or sulfuric acid)
Or an aqueous solution of an organic acid (such as acetic acid or citric acid). In addition, you may add a surfactant as needed. Grains (for example, cereal grains or cereal pieces) or rice (for example, rice grains or rice pieces) immersed in the alkaline aqueous solution are immersed in the acid aqueous solution. The amount of the aqueous solution may be 2 to 20 times (v / w) the amount of cereals (for example, cereal grains or cereal pieces) or rice (for example, rice grains or flakes). The immersion treatment may be allowed to stand, but stirring is more preferable. Stirring may be performed by rotating the vessel in which the liquid is immersed, or by using a stirring blade. The temperature for the treatment is preferably 5 to 40C. The processing time is 1 minute to 72 hours, preferably 2 to 12 hours. After immersion in an acidic aqueous solution, the acid content is removed. Any known method may be used as the removing method, and examples thereof include a method of exposing to water.

The allergen-reduced cereal preparation or the allergen-reduced rice preparation produced by the method of the present invention can be fed to allergic patients as they are, or the allergen-reduced cereal preparation can be used as a cereal-related food, or Brewing raw materials or brewed products can be mentioned, and as typical examples, wheat-related foods (eg, bakery products (eg, cakes, confectionery such as cookies, bread), udon, pasta, noodles, gyoza skins), Alternatively, corn-related foods [e.g., cereal, corn starch, tacos (dough made by kneading corn flour with water or oil or fat)], and further, from the allergen-reduced rice preparation, rice-related foods, such as , Rice crackers,
Mochi, udon or bread can also be prepared and given to allergy sufferers. The thus obtained allergen-reduced cereal preparation or cereal-related food, or allergen-reduced rice preparation or rice-related food can be taken by a wider variety of allergy patients while avoiding the onset of allergy.

[0034]

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.

[Experimental Example 1] [Allergenicity of salt-insoluble protein] (1) 1 kg of commercially available Koshihikari polished rice grains were immersed in 5 liters of a 0.1N sodium hydroxide aqueous solution, decompressed (20 mmHg) for 40 minutes, and then kept at a constant temperature of 40 ° C. Below,
Stir for 2 hours. Next, the rice grains were immersed in 5 liters of 0.1 molar hydrochloric acid aqueous solution for 2 hours, and the rice grains were washed in running water for 24 hours.
By drying, Koshihikari rice grains with reduced salt-soluble protein and reduced allergen were obtained. The obtained salt-soluble protein-removed allergen-reduced rice grains are pulverized, 1 g of the obtained pulverized rice is precisely weighed, and then a 10-fold volume extract (4 M urea aqueous solution is adjusted to pH 7.4 with 100 mM Tris buffer). The mixture was stirred at room temperature for 2 hours with a stirrer, centrifuged (10,000 rpm: 10 minutes), and the supernatant was collected. The supernatant was dialyzed against distilled water and then freeze-dried to obtain a urea extract (sample A) of salt-soluble protein-removed rice.

(2) The reaction of the sample A with an IgE antibody in the serum of a patient with rice allergy was measured by ELISA. That is, a phosphate buffer solution (hereinafter referred to as PB
Sample A was dissolved at 10 μg / ml in a solution in which urea was dissolved at 4 M in S), and 100 μl of the resulting solution was added to an ELISA plate Maxisorp (manufactured by Nunc Corporation). Coated at ° C. After washing the plate twice with PBS, 250 μl of 2% bovine serum albumin (hereinafter, BSA) was added,
Blocking was performed at 7 ° C. for 1 hour, followed by washing twice with PBS. Serum X of a patient diagnosed as having rice allergy or healthy adult serum (healthy human serum) was added to 0.2% BSA and 0.0%.
Dilute 10 times by volume with PBS containing 5% Tween 20, add 100 μl of each diluted solution to the plate,
The reaction was carried out at room temperature overnight. 0.05% Tween20
After washing the plate four times with PBS containing PBS (hereinafter, PBS-T), 100 μl of a 2,500-fold diluted solution of a sheep anti-human IgE antibody (manufactured by Vector) bound with biotin as a secondary antibody is added to the plate. In addition, the reaction was carried out at 37 ° C. for 2 hours. In addition, PBS-T containing 0.2% BSA was used for dilution of the antibody. After the reaction with the secondary antibody was completed, the plate was washed five times with PBS-T,
Avidin-conjugated peroxidase (manufactured by Boehringer) diluted to 0-fold volume was added to perform enzyme labeling. Again, PBS
The plate was washed 5 times with -T, and the substrate solution (0.034
100% of a 0.1% orthophenylenediamine solution to which% H ₂ O ₂ has been added. After accurately reacting at 37 ° C. for 30 minutes, color development was stopped by adding 40 μl of 4N—H ₂ SO ₄ and the absorbance (490 nm) was measured. All measurements were performed in duplicate, and the average was determined. The ELISA value in the case of using healthy human serum was 0.030, while the serum X of rice allergy patient was 0.267, and IgE antibody was detected in serum X of rice allergy patient.

(3) In order to detect the allergen molecules of the sample A, immunoblot was performed using patient serum X. That is, to the sample A corresponding to a protein amount of 2 mg, SDS was added to a final concentration of 1%, 2-mercaptoethanol was 1%, Tris-HCl was 10 mM, and glycerin was 20%, and each was added with distilled water. The whole was made up to 1 ml to prepare a sample for SDS-PAGE. The sample was heated at 100 ° C. for 2 minutes,
Bromophenol blue (BPB: B
romphenol blue) and centrifugation (10,000 × g; 10 minutes), followed by SDS-PA
Subjected to GE. As the SDS-PAGE gel, a mini gel system manufactured by Daiichi Pure Chemicals was used. SDS gradient mini gel having a polyacrylamide gel concentration of 10 to 20%,
Those having a migration distance of 8 cm and 12 wells were used. Electrophoresis was performed using a vertical cassette electrophoresis system (manufactured by Daiichi Kagaku) at 40 mA per gel at a constant current for 60 minutes. Running buffer is 0.025M-Tris,
PH 6.8 containing 0.19M glycine, 0.1% SDS
Of Tris-Glycine running buffer was used.

Each fractionated protein was transferred to a polyvinylidene fluoride membrane (hereinafter, PVDF membrane) using a semi-dry blotter. After blocking the transfer membrane with PBS-T containing 5% skim milk for 1 hour at 37 ° C., the transfer membrane was mounted on a mini blotter, and then washed with PBS-T containing 0.5% skim milk (hereinafter, PBS-TS). Patient serum X or healthy human serum diluted by a factor of 1 was injected into each lane and allowed to react at room temperature for 24 hours. 10 ml of 500-fold diluted biotin-conjugated sheep anti-human IgE antibody (0.5 mg / ml) (diluted with PBS-TS) as a secondary antibody
, Followed by avidin-conjugated peroxidase 500
10 ml of a U / ml diluent (diluted with PBS-TS) was added to perform enzyme labeling of the protein molecule reacted with the IgE antibody. The reacted allergen molecule was detected by adding a chromogenic substrate and staining. 0.05M for color development
Diaminobenzidine trihydrochloride / 0.01 M hydrogen peroxide was used. After reacting at 37 ° C. for 1 hour in the absence of light, the transfer film was washed with distilled water to stop the reaction. The reacted band and the intensity of the staining were measured using a densitometer [Ato Co., Ltd.
Manufactured by Densitograph Co., Ltd.]. The molecular weight of the reacted band was compared with a molecular weight marker (Bio-Rad: biotinylated marker) that was electrophoresed simultaneously. As a result, four bands reacting with patient serum X were detected, and their molecular weights were estimated to be 60 kd, 36 kd, 33 kd, and 13 kd from the molecular weight markers.

[0038]

[Experimental Example 2] [Purification of salt-insoluble protein remaining in rice treated with salt-soluble protein] Koshihikari 10 reduced in salt-soluble protein and allergen reduced obtained in Experimental Example 1 (1)
g was sufficiently crushed by a coffee mill. 6M urea-0.5% sodium lauryl sulfate solution 1 was added to the obtained rice powder.
After adding 50 ml and suspending well with an ultrasonic device,
Shake at room temperature for 2 hours. This extraction solution is 4000 rpm
And the resulting supernatant was put into a dialysis membrane and concentrated using Sephadex G-150 (manufactured by Pharmacia). The protein was separated from this concentrated solution by SDS-PAGE using a 5 to 20% gradient gel.
Stained with Coomassie Blue R250, 60kd, 36k
The four bands of d, 33 kd, and 13 kd were respectively cut out, put into a dialysis membrane, and the buffer for SDS-PAGE was 5 m each.
and electrophoretically extracted and recovered using a horizontal gel electrophoresis apparatus. This solution was put into a dialysis membrane again, and concentrated using Sephadex G-150 (manufactured by Pharmacia). Each sample was again subjected to SDS-PAGE using a 5 to 20% gradient gel to separate proteins. And it recovered similarly from the acrylamide gel. Thus, 60 kd, 36 kd, 33 kd,
And purified products of the 13 kd protein.

[0039]

[Experimental Example 3] [Identification of N-terminal amino acid sequence of salt-insoluble protein] 60 kd, 36 kd, 33 k purified in Experimental Example 2
d and 13 kd were subjected to SDS-PAGE on a 10-20% gradient gel to separate
Electroblotted onto DF membrane. This membrane was stained with Ponceau S dye, a portion of each protein band of interest was cut out, and the N-terminal amino acid sequence was determined using a protein sequencer (Perkin Elmer; model 492).

For the 60 kd protein, 17 amino acid residues from the N-terminus could be determined as follows. Ala-Thr-Gly-Ala-Gly-Met-A
sn-Val-Val-Phe-Val-Gly-Al
a-Glu-Met-Ala-Pro When the above amino acid sequence (see SEQ ID NO: 1 in the sequence listing) was searched against a protein database, a waxy protein [Okaga], a rice starch synthase, was found.
Ki et al., Plant Mol. Biol. (1992)
19, pp. 513-516].

With respect to the 36 kd protein, 10 amino acid residues from the N-terminus could be determined as follows. Ser (Ala, Gly) -Lys-Gly-Ala
(Glu) -Gly-Met-Asn-Val-Val
-Phe 1st place “Ser (Ala, Gly)” and 4th place “Al
“a (Glu)” has the same meaning as described above. Each amino acid sequence is shown as SEQ ID NO: 2 in the sequence listing.

With respect to the 33 kd protein, 15 amino acid residues from the N-terminus could be determined as follows. Ser (Ala, Gly) -Thr (Lys) -Gly
-Ala (Glu) -Gly-Met-Asn-Val
-Val-Phe-X-Gly (Thr, Ser) -A
la-Glu-Met 1st place “Ser (Ala, Gly)”, 2nd place “Thr
(Lys) "," Ala (Glu) "at position 4," X "at position 11 and" Gly (Thr, Ser) "at position 12
It has the same meaning as above. Each amino acid sequence is shown in SEQ ID NO: 3 in the sequence listing.

Each of the amino acid sequences of the 36 kd protein and the 33 kd protein showed high homology to the N-terminal amino acid sequence of the rice waxy protein.
Until now, a completely matching sequence has not been registered in the protein database, and is considered to be a novel protein.

For the 13 kd protein, 30 amino acid residues from the N-terminus could be determined as follows. Phe-Asp-Val-Leu-Gly-Gln-A
sn (Ser) -Ile (Tyr) -Arg-Gln-
Tyr-Gln-Val (Leu) -Gln-Ser-
Pro-Leu (Val) -Leu-Leu-Gln-
Gln-Gln-Val-Leu-Ser-Pro-T
In Asr (Ser) at position 7 of yr-Asn-Glu-Phe, asparagine (Asn) was detected as a major amino acid at position 7 in the determination of the amino acid sequence, but serine (Ser) was also small. It means that it was simultaneously detected in a significant but significant amount. Similarly, “Ile (Tyr)” in the 8th place is also
A major amount of isoleucine (Ile) and a significantly smaller amount of tyrosine (Tyr) were detected, and "Val (Le
u) ”, a major amount of valine (Val) and a significantly small amount of leucine (Leu) were detected, and further,“ Leu ”at position 17 was also detected.
(Val) "also means that a major amount of leucine (Leu) and a significantly smaller amount of valine (Val) were detected. These amino acid sequences are shown in SEQ ID NO: 4 in the sequence listing. As described above, also for the 13 kd protein, a plurality of amino acids were detected, and a mixture of a plurality of proteins, that is, polymorphism was shown.
3kd prolamin [Yamagata et al., BioSc
i. Biotechnol. Biochem. (199
2) Vol. 56, p. 537]. 13kd
A plurality of prolamins are registered as multiple genes having slightly different amino acid sequences.

[0045]

[Experimental example 4] [Immunoblot using anti-waxy serum] The method of Sano et al. [Theor. Appl. Genet. (198
4) Vol. 68, pp. 467-473], the waxy protein was partially purified from Koshihikari. The waxy protein and the purified 60 kd protein, 33 kd protein, and 36 kd protein prepared in Experimental Example 2 were fractionated by SDS-PAGE using a 10-20% gradient gel (Daiichi Kagaku). , P
Electroblotted onto VDF membrane. The membrane was reacted with rabbit anti-waxy serum, POD-labeled anti-rabbit IgG serum was used as a secondary antibody, and color was developed using OPD (ortho-phenylenediamine) as a substrate. The result is shown in FIG. In FIG. 1, lane 1 is a waxy protein partially purified from Koshihikari, lane 2 is a purified 60 kd protein prepared in Experimental Example 2, and lane 3 is a purified 33 kd protein prepared in Experimental Example 2. Lane 4 is the purified 36 kd protein prepared in Experimental Example 2 above, and
d, 66 kd, 45 kd, 31 kd and 22 kd are the positions of the molecular weight markers used. As is clear from FIG. 1, the 60 kd protein and the waxy protein produced color bands of the same size. The 33 kd and 36 kd proteins also reacted with the anti-waxy serum to produce a colored band. Therefore, the 60 kd protein was found to be a waxy protein. Furthermore, it was suggested that the 33 kd protein and the 36 kd protein are proteins related to the waxy protein, although the molecular weights are significantly different from the waxy protein.

[0046]

[Preparation Example 1] 1 kg of commercially available waxy rice grains were immersed in 5 liters of a 0.1N aqueous sodium hydroxide solution, and the pressure was reduced (20 minutes).
mmHg), and the mixture was stirred for 2 hours under a constant temperature condition of a water temperature of 40 ° C. Next, the rice was immersed in 5 liters of a 0.1 molar hydrochloric acid aqueous solution for 2 hours, washed with running water for 24 hours, and dried to obtain waxy rice grains reduced in salted water allergen.

[0047]

[Preparation Example 2] Instead of the commercially available waxy rice grains, except using commercially available waxy corn grain to obtain a brining allergen duck Chi corn grains by repeating the procedure described in Preparation Example 1.

[0048]

Preparation Example 3 1 kg of commercially available waxy rice was immersed in 5 liters of an enzyme solution prepared by dissolving 1 g of papain powder in PBS, depressurized (20 mmHg) for 40 minutes, and allowed to stand at a constant temperature of 40 ° C. for 16 hours. An enzymatic reaction was performed. After the reaction, the rice was washed in running water and freeze-dried to obtain allergen-reduced rice.

[0049]

[Preparation Example 4] in place of the commercial glutinous rice, except using commercially available waxy maize was obtained allergens reduction waxy maize Repeat the procedures described in Preparation Example 3.

[0050]

Comparative Preparation Example Rice was treated in the same manner as in Preparation Example 1 except that rice was changed to glutinous rice (Koshihikari) to obtain rice with reduced allergen by a conventional method.

[0051]

Experimental Example 5 The allergen-reduced rice obtained in Preparation Examples 1 and 3 and Comparative Preparation Example was pulverized, and 1 g of the obtained pulverized rice was precisely weighed.
After adding 0 mM Tris-HCl buffer; pH 7.4) and stirring with a stirrer for 2 hours, centrifugation (10,000 RP)
M; 10 minutes) to obtain a supernatant. The supernatant was dialyzed against distilled water and freeze-dried to obtain a urea extract. The sample obtained from Preparation Example 1 is referred to as Sample B, the sample obtained from Preparation Example 3 is referred to as Sample C, and the sample obtained from Comparative Preparation Example is referred to as Sample D. In the same manner as in the method described in Experimental Example 1 (2), Ig in serum X of rice allergic patients and serum in healthy subjects were added to these samples BD.
The reaction with the E antibody was measured by the ELISA method. As a result, the samples B and C for the serum X of the rice allergy patient
Of the sample D was 0.285, whereas the ELISA value of the sample was 0.18 and 0.15, respectively.
That is, if the result of sample D is 100, sample B becomes 63 and sample C becomes 53. Therefore, the allergen was sufficiently reduced. In addition, the ELISA values of healthy individuals were all less than 0.1.

[0052]

EXPERIMENTAL EXAMPLE 6 Samples B to D obtained in Experimental Example 5 were
The 60 kd protein fractionated by SDS-PAGE was detected by silver staining. That is, protein amount 2
mg of extract (samples B-D) at final concentrations of 1% SDS, 1% 2-mercaptoethanol, T
ris-HCl was added to 10 mM and glycerin to 20%, and the whole was made up to 1 ml with distilled water.
A sample for DS-PAGE was prepared. 100 ℃
For 2 minutes, and bromophenol blue (BPB: Bromophenol) to 0.05%.
blue), and centrifuged (10,000 × g;
Min), and then subjected to SDS-PAGE. 2-6
2. As the SDS-PAGE gel, a mini gel system manufactured by Daiichi Pure Chemicals was used. Polyacrylamide gel concentration 1
0-20% SDS gradient mini gel, migration distance 8
cm, 12 wells were used. Electrophoresis was performed using a vertical cassette electrophoresis system (manufactured by Daiichi Kagaku) at 40 mA per gel at a constant current for 60 minutes. As a running buffer, a Tris-glycine running buffer of pH 6.8 containing 0.025 M-Tris, 0.19 M glycine, and 0.1% SDS was used. The molecular weight was measured using SDS-PAGE molecular weight marker No. 1 (Daiichi Kagaku). The detection of the protein band separated by electrophoresis was performed by a silver staining method, and “Silver Staining Kit Wako 2” manufactured by Wako Pure Chemical Industries, Ltd. was used. A 60 kd molecule was detected from sample D, but not from samples B and C. Therefore, when allergen-reduced treated rice is manufactured using Koshihikari as a raw material, allergen molecules of 60 kd are detected. However, when mochi rice is used as a raw material, allergen molecules of 60 kd do not remain and allergens with a higher degree of reduction are obtained. It was found that reduced rice could be supplied.

[0053]

[Sequence list]

SEQ ID NO: 1 Sequence length: 17 Sequence type: amino acid Sequence type: Peptide sequence Ala Thr Gly Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro 1 5 10 15

SEQ ID NO: 2 Sequence length: 10 Sequence type: amino acid Sequence type: peptide Sequence characteristics Location: Other information: Xaa is Ser, Ala or Gly Location: 4 Other information: Xaa Is Ala or Glu

SEQ ID NO: 3 Sequence length: 15 Sequence type: amino acid Sequence type: peptide Sequence characteristics Location: 1 Other information: Xaa is Ser, Ala or Gly Location: 2 Other information: Xaa Is Thr or Lys Location: 4 Other information: Xaa is Ala or Glu Location: 11 Other information: Xaa is unknown Location: 12 Other information: Xaa is Gly, Thr or Ser sequence Xaa Xaa Gly Xaa Gly Met Asn Val Val Phe Xaa Xaa Ala Glu Met 1 5 10 15

SEQ ID NO: 4 Sequence length: 30 Sequence type: amino acid Sequence type: peptide Sequence characteristics Location: 7 Other information: Xaa is Asn or Ser Location: 8 Other information: Xaa is Ile Or Tyr Location: 13 Other information: Xaa is Val or Leu Location: 17 Other information: Xaa is Leu or Val Sequence Phe Asp Val Leu Gly Gln Xaa Xaa Arg Gln Tyr Gln Xaa Gln Ser 1 5 10 15 Pro Xaa Leu Leu Gln Gln Gln Val Leu Ser Pro Tyr Asn Glu Phe 20 25 30

[0058]

According to the method of the present invention, it is possible to provide a cereal preparation with reduced allergen and a rice preparation with reduced allergen that can be taken by a wider variety of cereal allergy patients and rice allergy patients.

[Brief description of the drawings]

FIG. 1 is a drawing showing the results of immunoblotting performed on purified waxy protein, purified 60 kd protein, purified 33 kd protein, and purified 36 kd protein using anti-waxy serum.

Continuing from the front page (72) Inventor Sohei Nojiri 8-4-1 Higashiogu, Arakawa-ku, Tokyo Inside the Allergen-Free Technology Research Institute, Inc. (72) Inventor Hiroyuki Anzai 8-4-1 Higashiogu, Arakawa-ku, Tokyo Inside the Allergen-Free Technology Research Institute, Inc. (72) Inventor, Keisuke Shimada 8-4-1 Higashi-Ogu, Arakawa-ku, Tokyo Inside the Laboratory Allergen-Free Technology Research Institute, Inc. (72) Kazuyuki Mogi, Hisashi Higashio-8, Arakawa-ku, Tokyo No.4-1, Allergen Free Technology Research Institute, Inc. (72) Inventor Kazuko Katsumata 8-4-1, Higashiogu, Arakawa-ku, Tokyo Inside Allergen Free Technology Research Institute, Inc. (72) Inventor Hiroshi Sugiyama Tokyo 8-4-1, Higashiogu, Arakawa-ku Inside Allergen-Free Technology Research Laboratories Co., Ltd. (72) Inventor Saburo Amano 8-4-1 Higashiogu, Arakawa-ku, Tokyo Formula company allergen-free technology within the Institute (56) Reference Patent flat 5-236889 (JP, A) JP flat 6-253758 (JP, A) (58 ) investigated the field (Int.Cl. ^7, DB name A23L 1/10 A23L 1/29-1/308 JICST file (JOIS) JAFIC file (JOIS)

Claims (7)

(57) [Claims] To 1. A cereal UDP-glucose starch glucosyltransferase was deleted Gotoshi, and performing allergen-reducing treatment, a manufacturing method of the allergen-reducing cereal preparations. (2) Waxy varieties cereal, waxy by mutagen treatment
Expression of a protein-deficient cereal or waxy protein
Allergen reduction for cereals with genetic control of cereals
Allergen-reduced cereals characterized by performing a keratinization treatment
Method of manufacturing the preparation. 3. The allergen reduction treatment comprises treating the cereal with a protease, bringing the cereal into contact with an aqueous salt solution, a lower alcohol, an alkaline aqueous solution, and / or an acidic aqueous solution, or dispersing saccharides and saccharide-degrading microorganisms. The method according to claim 1 or 2 , comprising fermenting with a solution. 4. The grain is wheat, claim 1-3 noise
A method according to any one of the preceding claims. 5. The grain is corn, claims 1 to
The method according to any one of claims 3 to 7 . 6. A salt-insoluble protein having a molecular weight of 60 kd,
A salt-insoluble protein having a molecular weight of 33 kd, and a molecular weight of 36
against the US at least one protein selected from salts insoluble protein or Ranaru group kd was deleted Gotoshi, and performing allergen-reducing treatment, a manufacturing method of the allergen-reducing rice preparations. 7. The allergen reduction treatment comprises treating the rice with a protease, contacting the rice with a salt aqueous solution, a lower alcohol, an alkaline aqueous solution, and / or an acidic aqueous solution, or dispersing saccharides and saccharide-degrading microorganisms. 7. The method of claim 6 , comprising fermenting with a solution.