KR101439417B1 - Methods for Reducing Allergenicity of Rice by Heat Treatment - Google Patents

Abstract

The present invention relates to a method of reducing allergenicity by treating the grain with heat at 90-130 占 폚. The present invention provides a method for efficiently reducing allergenicity of cereals, especially rice, using heat treatment. According to the present invention, it is possible to reduce the allergic antigenicity of cereals, especially rice, to 10 ⁵ -fold, and the protein content can be maintained to be 50% or more of the pre-allergenic anti-allergy grain, . The present invention can greatly contribute to the prevention of allergic diseases caused by foods, especially rice, which are food allergies.

Description

Methods for Reducing Allergenicity of Rice by Heat Treatment [0002]

The present invention relates to a heat treatment method for reducing the allergic antigenicity of cereals, especially rice, and a heat-treated rice reduced in allergenicity.

Food allergies in industrialized countries in recent decades has shown a tendency to increase in incidence is about 3-6% from the children (Sanders DS. Mucosal integrity and barrier function in the pathogenesis of early lesions in Crohn's disease. J. Clin. Pathol. 58: 568572 (2005)). Food allergy is a type I hypersensitivity reaction that occurs in food entering the body through the digestive tract. In allergy patients, food allergens penetrate non-digested food (Gell PGH, Coombs RRA, eds., Oxford: England: Blackwell (1963); Tesaki S, et al. , an Active Compound against allergen absorption in Hypoallergenic Wheat Flour Produced by Enzymatic Modification Biosci Biotechnol Biochem 66:.... 1930-1935 (2002)), the intestinal absorption of these allergens can be called the first stage of a food allergy onset (Isobe N, Suzuki M, Oda M, Tanabe S. Enzyme-Modified Cheese Exerts Inhibitory Effects on Allergen Permeation in Rats Suffering from Indomethacin-Induced Intestinal Inflammation. Biosci . Biotechnol . Biochem . 72: 1740-1745 (2007)).

Egg, pork, peach, mackerel, chicken, milk, buckwheat and crab are known as food causative agents of food allergies. The symptoms of food allergies can be expected to be improved by thorough avoidance of causative foods, but in some cases symptoms may persist even if avoidance is difficult or avoided.

Rice is a representative grains that are consumed in stocks in Asia and gradually become health foods in the West. In Japan, using the rice was a staple of allergic asthma or severe atopic dermatitis associated with several reports of rice (Yamada K et al., Study of the role of cereal allergens in atopic dermatitis. J. Jpn. Pediatr., 91 : 888 (1975)). In the case of the Republic of Korea, there are few reports of allergic patients related to rice, but it is expected that there will be a considerable number of allergic patients related to rice in Japan.

Therefore, it is necessary to study how to reduce the allergenicity of cereals such as rice.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a technique capable of effectively reducing the allergic antigenicity of cereals, particularly rice, as a causative food of food allergy. As a result, the present inventors have found that when heat of 90-130 ° C is used, allergenicity is effectively reduced up to 10 ⁵ times, and the content of protein is maintained at 50% or more of the total amount of allergen- The present invention has been accomplished by confirming that it is possible to provide undiluted cereals.

It is therefore an object of the present invention to provide a method for reducing the allergic antigenicity of cereals through heat treatment.

Another object of the present invention is to provide an allergenic-reducing grain.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention relates to a method for reducing grain allergy antigenicity by treating the grain with heat at 90-130 占 폚.

The present inventors have sought to develop a technique capable of effectively reducing the allergic antigenicity of cereals, particularly rice, as a causative food of food allergy. As a result, the present inventors have found that when heat of 90-130 ° C is used, allergenicity is effectively reduced up to 10 ⁵ times, and the content of protein is maintained at 50% or more of the total amount of allergen- It was confirmed that it is possible to provide grain that is not deficient.

The present invention can be applied to various grains. For example, cereals to which the present invention may be applied include, but are not limited to, rice, buckwheat, wheat, barley, corn, sorghum, barley and oats. Preferably, the grain to which the present invention is applicable is rice.

The cereals to which the present invention is applied are not processed or processed before the enzyme treatment. According to a preferred embodiment of the present invention, the grain to which the present invention is applied is impregnated with water and subjected to an expansion process.

According to a preferred embodiment of the present invention, the heat of the present invention is 100-130 ° C at 1-2 atm, more preferably 110-130 ° C at 1-1.3 atm.

According to a preferred embodiment of the present invention, the treatment of the present invention reduces the content of 10-70 kDa protein in the cereal, more preferably decreases the protein content of 10-50 kDa, and even more preferably 10- More preferably a protein content of 10-30 kDa, even more preferably a protein content of 10-20 kDa, and most preferably a protein content of 12- Reduces the protein content of 14 kDa.

According to a preferred embodiment of the present invention, the protein of the present invention is thioredoxin or glutaredoxin, more preferably thioredoxin.

According to a preferred embodiment of the present invention, the treatment of the present invention maintains 50-90%, more preferably 60-90%, even more preferably 65-90%, of the protein content in the cereal, Most preferably 65-85%.

According to a preferred embodiment of the present invention, the treatment of the present invention is carried out for 1-150 minutes, more preferably at 90-110 < 0 > C for 40-150 minutes or at 110-130 & More preferably at 90-110 DEG C for 40-120 minutes or at 110-130 DEG C for 1-80 minutes and even more preferably at 90-110 DEG C for 40-100 minutes or at 110-130 DEG C For 1 to 70 minutes, most preferably 90-110 < 0 > C for 50-100 minutes or 110-130 < 0 > C for 5-70 minutes.

As evidenced in the following examples, it can be seen that by the treatment of the present invention, allergens in cereals, especially rice, can be reduced by up to 10 < ^{2 >} -10 < ⁵ > times to effectively reduce allergic antigenicity.

According to another aspect of the present invention, there is provided an allergen reduced grain having an allergic antigenicity reduced by 10 ² -10 ⁵ times as compared to a pre-reduced grain.

According to a preferred embodiment of the present invention, the allergen-reduced grain of the present invention is a grain having an allergic antigenicity reduced by 10 ³ -10 ⁵ times, and most preferably a grain reduced by 10 ⁴ -10 ⁵ times.

The allergenic reduction can be measured using any of the quantitative immunoassays in the art. For example, using an enzyme-linked immunosorbent assay (ELISA) method, the ELISA method preferably comprises: coating a rice allergen antigen on a solid phase; Dispensing a dilution solution of the primary antibody to the coated solid phase; Color development using a labeled secondary antibody; And an indirect ELISA comprising measuring the absorbance.

Since the allergy-reduced cereal of the present invention is produced according to the method of the present invention described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the cereal of the present invention has a protein content of 50-90%, more preferably 60-90%, even more preferably 65-90%, most preferably 65-90% 85%.

According to a preferred embodiment of the present invention, the grain of the present invention is rice.

According to a preferred embodiment of the present invention, the allergy-reduced grain of the present invention is reduced by the method of the present invention described above.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for effectively reducing the allergic antigenicity of cereals, particularly rice, by treating heat at a suitable temperature.

(b) According to the present invention, the allergic antigenicity of cereals, especially rice, can be reduced by up to 10 ⁵ times.

(c) According to the present invention, there is an advantage that the present invention can provide a cereal having no nutrient deficiency by maintaining a protein content of at least 50% with respect to the allergy-antigen-reduced cereal.

(d) The present invention can greatly contribute to the prevention of allergic diseases caused by food, especially rice, which is a food allergy.

Fig. 1 shows a process for producing rice protein (allergen).
Figure 2 shows the results of SDS-PAGE of rice protein fractions.
Fig. 3 shows the reactivity of the serum and the rice white extract of rice sensitized patients.
Figure 4 shows antibody titers of rabbit sera administered with rice protein. 1-1 and 1-2 are rabbit serum treated with 10-30 kDa rice protein, and 2-1 and 2-2 are rabbit serum treated with 30-∞ kDa rice protein.
FIG. 5 shows the results of SDS-PAGE of rice allergy antigen protein according to the heat treatment temperature.
FIG. 6 shows the results of an indirect competition ELISA test for the rice allergen antigen protein according to the heat treatment temperature.
FIG. 7 shows the result of SDS-PAGE on the rice allergy antigen protein according to the heat treatment (100 ° C) time.
FIG. 8 shows results of an indirect competition ELISA test for rice allergen antigen protein according to heat treatment (100 ° C) time.
FIG. 9 shows SDS-PAGE results of rice allergy antigen protein according to heat treatment (120 ° C) time.
FIG. 10 shows the result of an indirect competition ELISA test for rice allergy antigen protein according to heat treatment (120 ° C.).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Materials and methods

Rice protein extraction

Brown rice or white rice (Origin: Iksan, Jeollabuk - do, Korea Food Research Institute, Rice Center, Korea) was used for the extraction of rice protein. The sample was pulverized to 50 mesh or less using a crusher (CYCLOTEC, 1093 sample mill) and immersed in hexane for 24 hours to remove the fat and dried. 100 g of defatted brown rice and white rice were taken in 1 L PBS (pH 7.2, 140 mM NaCl, 2.5 mM KCl, 8 mM NaHPO ₄ .12H ₂ O and 1 mM KH ₂ PO ₄ ) solution for 48 hours. Proteins were extracted and centrifuged at 8,000 rpm for 30 minutes using a centrifuge (Sorvall RC 28S) to obtain supernatant. The supernatant was filtered through a filter paper (Toyo roshi kaisha, NO. 5C), and 5 fractions were obtained using a 1, 3, 10 kDa membrane of a filtration apparatus (Amicon, Milipo). At this time, fractions of 1 kDa or less containing PBS were discarded, and fractions of 1-∞ kDa, 1-3 kDa, 3-10 kDa, 10 kDa and 30 ∞ kDa or more were concentrated at 60 ° C. using a concentrator, (Fig. 1).

Rice protein fraction

(1) 1 - ∞ kDa fraction (white rice)

And filtered with a 1 kDa membrane using a filtration apparatus (Amicon, Millipore). At this time, to reduce the salt contained in PBS as much as possible, when 1 liter of PBS extract of white rice protein was left to be about 100 mL (based on one Amicon filtration device), 250 mL of distilled water was added and the mixture was filtered while being diluted. The filtrate was filtered at a rate of about 150 mL / day, diluted with 1 L of extract, and took about 4 days to filter. Approximately 250 mL of the extract remaining at the end was quantified using a 300 mL scalpel cylinder, and two 10% (30 mL) aliquots were taken. A total of 20% (60 mL) 1 kDa - ∞ fractions were collected and stored in a -20 ° C freezer and lyophilized.

(2) 1 - 3 kDa fraction (white rice)

The filtrate was further filtered with a 3 kDa membrane using a filtration apparatus (Amicon, Millipore) with a fraction of 1 kDa or more (240 mL). At this time, 100 mL of distilled water was added 2-3 times to maximally filter less than 3 kDa protein. Filtration was carried out at a rate of about 500 mL / day, and filtration took about one day. This fraction was concentrated to about 20 mL at 60 ° C. for 1 hour by a concentrator, stored in a -20 ° C. freezer, and freeze-dried.

(3) 3 - 10 kDa fraction (white rice)

The fractions of 1 - 3 kDa or more were filtered with a 10 kDa membrane using a filtration apparatus (Amicon, Milipo). At this time, 100 mL of distilled water was added 2-3 times to maximally filter less than 10 kDa protein. Filtration was carried out at a rate of about 150 mL / hr and filtration was terminated in 3-4 hours. This fraction was concentrated to about 20 mL at 60 ° C. for 1 hour by a concentrator, stored in a -20 ° C. freezer, and freeze-dried.

(4) 10 - 30 kDa fraction (white rice)

The fractions of 3 - 10 kDa or more were filtered with a 10 kDa membrane using a filtration apparatus (Amicon, Milipo). At this time, 100 mL of distilled water was added 2-3 times to maximally filter less than 10 kDa protein. Filtration was carried out at a rate of about 300 mL / hr and filtration was terminated in 1-2 hours. This fraction was concentrated to about 20 mL at 60 ° C. for 1 hour by a concentrator, stored in a -20 ° C. freezer, and freeze-dried. MS analysis of 10-30 kDa rice protein samples confirmed allergen proteins (gi 1398915, mass 17944), thioredoxin (gi 82407383, mass 14061) glutaredoxin (gi 485953, mass 11866) and the like.

(5) 30 - ∞ kDa fraction (white rice)

A fraction of 10 - 30 kDa was obtained, and the fraction remaining on the membrane was a protein having a molecular weight greater than 30 kDa. The fraction did not need to be concentrated because the concentration was high. It was observed that white precipitates were piled up, presumably because distilled water was added to the protein extracted with PBS. This fraction was also stored in a -20 ° C freezer and lyophilized.

Protein yield of rice

In the previous studies, the protein content of rice was known to be about 6.8%, and the protein extraction yield was 5.5%. The protein extracted from 90 g of white rice was fractionated into filtration membranes to obtain 2.97 g of 1-3 kDa, 0.36 g of 3-10 kDa, 0.07 g of 10-30 kDa and 0.35 g of 30-∞ kDa. The rabbit was injected with the 10-30 kDa rice protein and 30-∞ kDa rice protein to make an antibody.

White rice 10g Weight (g) yield(%) 1-∞ kDa (1) 0.56 5.6 1-∞ kDa (2) 0.54 5.4 Average 0.55 5.5

White rice 90g Weight (g) yield(%) 1-3 kDa 2.97 79.2 3-10 kDa 0.36 9.6 10-30 kDa 0.07 1.9 30-∞ kDa 0.35 9.3 Sum 3.75 100

protein gi mass( Da ) Thioredoxin 82407383 14061 Glutarezine 485953 11866

Rice protein fraction SDS - PAGE

SDS-PAGE was performed to determine the size of rice protein fractions. Gel was applied to 12% separating gel and 5% stacking gel. The sample was quantitated at 20 μg / μL and the sample buffer (1 M Tris-HCl: pH 6.8, 50% glycerol, 10 % SDS, 2-mercaptoethanol, 1% bromophenol blue) was added and heated at 100 ° C for 5 minutes before use. Electrophoresis was performed at 200 V for 30 minutes. After electrophoresis, the gel was stained with Coomassie staining solution (1.0 g Coomassie blue R-250, 450 ml methanol, 450 ml H ₂ O, 100 ml glacial acetic acid, 800 ml H ₂ O) Stained for 30 min and decolorized with coumadin staining solution (100 ml methanol, 100 ml glacial acetic acid, 800 ml H ₂ O). As a result, the whole protein showed many protein bands, but the band was most prominent in the range of 14-16 kDa. Protein bands did not appear in 1-3 and 3-10 fractions, suggesting that the low-molecular proteins had already passed through the gel. It was confirmed that protein bands appeared clearly in the 10-30 fraction (FIG. 2).

Ensure patient serum

Serum from rice allergy patients was purchased at Samsung Medical Center. Specific IgE antibodies were measured using ImmunoCAP (Phadia, Sweden) in order to obtain serum of patients sensitized to rice, and those with a serum level of 0.36 kU / L or more were considered positive. Allergic diseases were classified into atopic dermatitis, urticaria, asthma, allergic rhinitis, and allergic gastroenteritis, and related information such as clinical symptoms, family history, and dietary intake status of the patient were constructed in the database system. Blood was centrifuged and serum was stored at -70 ° C until analysis. The patient's serum used a storage method called VLS (vacuum like sealing) to compensate for problems such as storage location, data management of samples, and prevention of serum denaturation due to long-term storage. Among them, serum with high enough specific IgE for rice was selected and used for serum screening.

Specific IgE antibody test was performed as follows. First, CAP (capsule allergen product) with antigen was washed 8 times with washing solution, and this CAP was transferred to a container containing 50 μL of undiluted patient serum and allowed to react with serum for 30 minutes at room temperature. After completion of the reaction, the CAP was washed eight times, and 50 μL of a conjugate (enzyme-anti-IgE and β-galactosidase) was added thereto. The reaction was allowed to proceed at room temperature for 150 minutes, Fluorescence substrate and 0.01% 4-methylumbelliferyl-beta-D-galactoside) were added and reacted for 10 minutes. At the end of this procedure, 400 μL of blocking solution (sodium carbonate) was added thereto, and the reaction was allowed to proceed for 2 minutes. Then, the degree of color development was measured using FluoroCount 96. Absolute values were calculated using standard curves plotted against 6 concentrations of standard test solutions (0.35, 0.7, 3.5, 17.5, 50 and 100 kU / L) to obtain specific IgE concentrations. Serum was examined without dilution. The measurement limit was 0.35 U / mL and less than 100 U / mL, 0 U / mL for less than 0.35 U / mL and 101 U / mL for more than 100 U / mL Respectively.

Table 4 shows the test results of rice allergy patients tested at Samsung Medical Center. Serum was collected in 17 patients and 11 cases were positive in rice. Gender distribution was 8 males and 3 females, and the mean age was 3.36 ± 1.83 years. The mean of specific IgE for rice in serum was 16.38 ± 24.95 kU / L.

number Age (years) gender gun IgE  ( ku / L) Food specific antibodies ( ku / L) rice One 0 man 6530 89.8 2 3 Woman 1234 6.68 3 6 Woman 1476 5.11 4 0 man 480 7.78 5 0 man 3007 24.2 6 11 man 36170 6.03 7 0 man 926 9.93 8 15 man 5001 7.35 9 One man 3013 10.5 10 One Woman 2818 9.13 11 0 man 857 3.61

Identification of rice allergens using patient serum

The results of western blotting on rice protein using sera from 11 rice allergic patients are shown in FIG. The patient's individual responses were as follows: antigen of rice protein of about 50, 40, 20 and 16 kDa in patient 1 serum, 16 kDa and 20 kDa in patient 2 serum, 70 kDa and 30 kDa in patient 3 serum, 4 The patient's serum was 70, 50 and 14 kDa, the patient's serum was 20 kDa, the patient's serum was 14 kDa, the patient's serum was 14 kDa, the patient's serum was 20, 16 and 14 kDa, the patient's serum was 14 kDa, Serum was reacted with 14, kDa, 10, 50, 16, and 14 kDa, and 11 with 20 kDa rice protein antigens. Overall, seven proteins of approximately 70, 50, 40, 30, 20, 16 and 14 kDa reacted with sera from rice allergy patients.

In summary, the 14 kDa protein was found to react to 6 (55%) of the sera, the 20 kDa protein to 5 sera and the 16 kDa protein to 4 sera, Of allergens. In addition, the 70, 50 and 40 kDa antigens reacted with 2, 3 and 1 sera, respectively, and the 30 kDa antigen also reacted with 1 sera to judge them as minor allergens.

Heat treatment

For the heat treatment of rice protein, 5% rice protein was added to distilled water and seeded in a shaking waterbath (JSSB-30T, JSR, Australia) and an autoclave (AC-12, ℃, respectively.

Evaluation of allergenicity of rice protein ELISA  Experiment)

(1) Production of rabbit antibody (antiserum) using rice protein as allergen

3-3.5 Kg of rabbits (Central Lab animals, Korea) were purchased for antibody production and cultivated in a breeding ground for about one week. In the purified rabbits, the antibody to physiological saline was prepared by diluting the 10-kDa rice protein and 30-∞ kDa rice protein, which had been separated from rice, with 10 mg / mL of FCA in a ratio of 1: 1 One ml of rabbit was divided into two portions at the back, and injected first. Antigen injection was injected once every two weeks and FIA was used from the second. Blood collection was performed one week after the antigen injection. Partial blood collection was used until 1-3 weeks, and about 2 mL was taken from the venous blood outside the rabbit ear with a syringe. In the last 4th week, blood was drawn from the rabbit eyes and about 80 ml was taken. The collected blood was allowed to stand at room temperature for about 3 hours to solidify, and the blood cells adhering to the wall of the tube were smeared with a sterilizing rod. Serum was obtained by centrifugation at 12,000 RPM for 10 minutes. The serum was stored at -80 ° C with NaN ₃ (0.1%) as a preservative, and the activity of the antibody was confirmed by ELISA. The amount of blood that can be obtained from rabbits weighing 3.5-4 kg averaged 69 mL and the yield of serum was 47%. The blood sampling rate was the highest at 1-15 rabbits (85 mL), but the serum yield was the lowest at 43%.

number Blood sampling ( mL ) serum( mL ) yield(%) 1-1 85 36 42 1-2 53 24 45 2-1 74 38 51 2-2 57 30 52

(2) Indirect non-competition ELISA

In order to measure antibody titers in rabbit serum, indirect noncompetitive ELISA analysis

The protein extracted from rice was diluted to 2 μg / mL in a coating buffer (tris hydroxymethyl aminomethane 0.05M, pH 9.0). These were dispensed in 100 μL each into microplate wells and allowed to stand overnight at 4 ° C. This was washed three times with wash buffer (PBS with Tween 20, 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl and 0.05% Tween 20), and 100 μL of diluted rabbit serum was added to each well for 1 hour After the reaction, the cells were further washed three times, and 100 μL of a diluted HRP-labeled goat anti-rabbit IgG conjugate at 1 / 10,000 as a secondary antibody was added and reacted for 1 hour. After washing again 3 times, 100 μL of substrate solution (0.01% TMB in phosphate-citrate buffer, pH 5.0, 0.001% H ₂ O ₂ ) was added, and the reaction was stopped for 30 minutes and then 50 μL of 2 MH ₂ SO ₄ was added Absorbance was measured at 450 nm using a microplate reader (Emax, Molecular Devices). As a result, in the case of 1-1 and 1-2 rabbits injected with 10-30 kDa rice protein fraction, the antibody titer was almost 0.04 and 0.05 in the first antigen infusion, but the second antibody titer was 0.35 and 0.48 Absorbance. In the fourth test, antibody titers ranged from 0.79 to 0.90, and antibody titers from 1-2 rabbits were higher. For the 2-1 and 2-2 rabbits injected with 30-∞ kDa rice protein fraction, the 2-2 rabbits showed the same trend as the results of 1-1 and 1-2. Antibody titer was 0.04 at first injection, 0.35 at first injection and 0.69 at 4th injection. On the other hand, 2-1 rabbits rarely produced antibodies, presumably because the rabbits seemed to be a health problem, such as hemorrhaging during the experiment due to the rejection of the new environment. Therefore, 1-2 (RLP: rice low protein) and 2-2 (RHP: rice high protein) serum were used in rice allergy antigen test.

② In order to measure the degree of reduction of allergenicity of rice protein, indirect competition ELISA

The protein extracted from rice was diluted to 2 μg / mL in a coating buffer (tris hydroxymethyl aminomethane 0.05M, pH 9.0). These were dispensed in 100 μL each into microplate wells and allowed to stand overnight at 4 ° C. This was washed three times with wash buffer (phosphate buffered saline with PBST; 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20) and then diluted stepwise with rabbit serum diluted 1/500 One hydrolyzed rice protein hydrolyzate was mixed in equal amounts. 100 μL of the mixed solution was added to each well, followed by reaction for 1 hour to induce a competitive reaction. The resultant was further washed three times, and 100 μL of HRP-labeled goat anti-rabbit IgG conjugate as a secondary antibody was added and reacted for 1 hour. After washing again 3 times, 100 μL of substrate solution (0.01% TMB in phosphate-citrate buffer, pH 5.0, 0.001% H ₂ O ₂ ) was added, and the reaction was stopped for 30 minutes and then 50 μL of 2 MH ₂ SO ₄ was added Absorbance was measured at 450 nm using a microplate reader (Emax, Molecular Devices).

Experiment result

Allergic antigenicity according to heat treatment temperature of rice protein Abatement

The protein content of 5% rice protein solution was 7.94 μg / μL in average and showed no significant change with heating time (Table 6).

Protein quantification results of heat-treated rice protein  Heat treatment temperature (캜) Protein content (μg / μL) Control group 9.12 60 8.43 80 8.22 100 6.67 120 6.31

SDS electrophoresis was performed to measure the protein content of a specific size. As a result, the protein content of 12-14 kDa decreased with increasing heating temperature (FIG. 5). In the case of heating at 60 ° C and 80 ° C for 1 hour, the band of 12-14 kDa appeared, but the band disappeared at 100 ° C and 120 ° C.

In the indirect competition ELISA for measuring allergenic antigenicity reduction, allergic antigenicity was not decreased in the 60 ℃ and 80 ℃ heat treated samples where protein band of 12-14 kDa remained, but allergic antigen And the allergic antigenicity was reduced by 10000 times or more at the heat treatment at 120 ° C (FIG. 6).

Allergenicity of rice protein by heat treatment (100 ℃) Abatement  Experiment result

The protein content of 5% rice protein solution was 7.77 μg / μL on average, and there was no significant change with heating time (Table 7).

Protein quantification results of heat treatment (100 ℃) rice protein  Heat treatment time ( min ) Protein content (μg / μL) Control group 9.12 10 8.78 30 7.06 60 6.67 90 6.20

SDS electrophoresis was performed to measure the protein content of a specific size. As the heating time increased, the protein of 12-14 kDa tended to decrease (FIG. 7). The band was clearly visible at 10 minutes heating, but the protein band disappeared as the heating time increased from 30 to 90 minutes.

In the indirect competition ELISA for the reduction of allergenic antigenicity, the allergic antigen reduction effect was not observed in 10 minutes and 30 minutes heat treatment, And a reduction in allergenicity of about 1000 times or more was observed when heated for 90 minutes (FIG. 8).

Allergenicity of rice protein according to heat treatment time at 120 ℃ Abatement  Experiment result

The protein content of 5% rice protein solution was 7.77 μg / μL on average, and there was no significant change with heating time (Table 8).

Protein quantification results of rice protein treated with heat (120 ℃)  Heat treatment time ( min ) Protein content (μg / μL) Control group 9.12 10 7.40 20 7.10 40 7.06 60 6.31

SDS electrophoresis was performed to measure the protein content of a specific size. As the heating time increased, the protein of 12-14 kDa tended to decrease (FIG. 9). The band of 12-14 kDa disappeared when heated at 120 ℃ for 10-60 min.

Indirect competition ELISA was performed to measure the allergenic reduction of the allergen. As a result, it was observed that the antigenicity was reduced by 10,000 times or more in all the heat treatment for 10 to 60 minutes (FIG. 10).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (8)

A method of reducing rice allergic antigen by 10 ⁴ -10 ⁵ times by reducing the content of thioredoxin protein while maintaining the protein content in the rice at 65-85% by treating the rice with 120 ° C heat for 10-60 minutes. delete delete delete delete delete delete delete

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