WO2020221180A1 - 磷酸化-酶解法制备低致敏性小麦醇溶蛋白的方法 - Google Patents

磷酸化-酶解法制备低致敏性小麦醇溶蛋白的方法 Download PDF

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WO2020221180A1
WO2020221180A1 PCT/CN2020/087101 CN2020087101W WO2020221180A1 WO 2020221180 A1 WO2020221180 A1 WO 2020221180A1 CN 2020087101 W CN2020087101 W CN 2020087101W WO 2020221180 A1 WO2020221180 A1 WO 2020221180A1
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gluten
wheat
gliadin
sodium tripolyphosphate
wheat gliadin
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French (fr)
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王立
薛腊梅
李言
钱海峰
孙娟
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
    • A23J1/125—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the technical field of wheat processing and relates to a method for preparing hypoallergenic wheat gliadin by a phosphorylation-enzymatic hydrolysis method.
  • Wheat is one of the three major crops in the world and an important food component for humans. Many staple foods are wheat products, such as bread, noodles, steamed bread and other baked products. Although wheat plays an important role in daily life, wheat is a common allergic food reported by the World Food and Agriculture Organization (FAO). Wheat contains allergenic proteins that can cause allergic reactions. Even a small amount of wheat flour may have a great impact on the health of highly sensitive patients. Since January 1, 2006, eight allergens including wheat have been included in the label range in the US legislation. This means that many people are allergic to wheat and wheat products. In the past few decades, like other allergic foods, the frequency of wheat allergy has increased.
  • FEO World Food and Agriculture Organization
  • wheat protein can be divided into water-soluble albumin, salt-soluble globulin and insoluble protein.
  • the insoluble protein is called prolamin, which is composed of prolamin and glutenin, and prolamin and glutenin together form gluten.
  • Gliadin accounts for 40% of the total protein in wheat, and its molecular weight ranges from 28-55kDa.
  • gliadin can be divided into four subtypes: ⁇ , ⁇ , ⁇ , and ⁇ . The content of alpha gliadin accounts for about 15-30% of the total wheat protein.
  • Different types of wheat protein can cause different allergy symptoms.
  • Common allergies caused by the intake of wheat protein include celiac disease, asthma and rhinitis.
  • the gliadin in wheat protein is rich in wheat allergen IgE binding epitopes, and becomes an important indicator for detecting and diagnosing wheat allergy.
  • specific processing methods are used to reduce or change the content or structure of gliadin in wheat, and to remove or reduce allergens, the effect of reducing wheat allergies can be achieved.
  • An epitope is a special chemical group that exists on the surface of an antigen and determines the specificity of the antigen. The nature, number and spatial configuration of the antigen epitope determine the specificity of the antigen.
  • the structure of epitopes is divided into conformational epitopes and linear epitopes.
  • the repeated Gln-Gln-Gln-Pro-Pro in the low molecular weight glutenin subunit is the epitope bound by IgE.
  • the gliadin structure includes a repetitive amino acid sequence called the central domain, which is rich in proline and glutamine.
  • the secondary and tertiary structure of the protein is the key to the immune system to recognize epitopes.
  • proteins when their structure changes, they may be accompanied by changes in protein epitopes, which will also affect the possibility of cells in the body's immune system to recognize gliadin epitopes. This provides the possibility to reduce or remove allergens through processing.
  • the medium-strength physical treatment is used to treat wheat flour or gluten, although the good dough processing properties of wheat flour or gluten are retained.
  • moderate-intensity physical treatment can only bring about small changes to the secondary and tertiary structure of the protein, and this small change cannot effectively reduce the allergenicity of allergic proteins.
  • the application of chemical treatment methods in food is also becoming less and less.
  • high-dose additives during processing are less and less accepted by consumers.
  • chemical treatment methods can greatly affect the structure of wheat protein, There is still controversy about the changes in the allergenicity of gliadin after chemical treatment, which is related to the difference in treatment conditions. For example, the difference in deamidation treatment conditions will cause the allergy of gliadin to decrease or increase.
  • the present invention provides a method for preparing hypoallergenic wheat gliadin.
  • the method is to first phosphorylate the gluten with sodium tripolyphosphate, then hydrolyze the gluten with alkaline protease, and then extract the wheat gliadin.
  • Hypoallergenic wheat gliadin refers to wheat whose allergic signs decrease or disappear after entering the animal body compared with wheat gliadin extracted from gluten that has not undergone allergen removal treatment Gliadin.
  • the method can adopt the following steps:
  • alkaline protease Under the conditions of 60°C ⁇ 65°C and pH 9.0 ⁇ 9.5, add alkaline protease to carry out the enzymatic hydrolysis reaction, the addition amount of alkaline protease is 180U ⁇ 200U/g gluten;
  • step (2) The pH of the gluten system after the enzymatic hydrolysis in step (2) is adjusted to neutral, dried and defatted, and the wheat prolamin is extracted with ethanol.
  • the gluten In the first phosphorylation treatment of gluten with sodium tripolyphosphate, the gluten is mixed with a phosphate buffer, and then sodium tripolyphosphate is added, and the sodium tripolyphosphate reacts with the gluten. Furthermore, the solid-liquid ratio of gluten to phosphate buffer is 1 g: (10-12.5) mL, and the pH of the phosphate buffer is 8.5-9.5. Further, the mass ratio of the addition amount of sodium tripolyphosphate to the gluten powder is 1:4.5-1:5.5, and the sodium tripolyphosphate is added in batches. Furthermore, the addition amount of sodium tripolyphosphate and the mass ratio of gluten are 1:4.5 ⁇ 1:5.5.
  • Sodium tripolyphosphate is added into the reaction system in 3 equal parts, respectively at the start of the reaction and 10 ⁇ 10 after the start of the reaction. 15min and 25-30min after the start of the reaction; the reaction temperature is 25°C, the reaction time is 1h, stirring and controlling the pH of the reaction system within the range of 8.5-9.5.
  • the hydrolysis treatment with alkaline protease is to add alkaline protease to carry out enzymatic hydrolysis under the conditions of 60°C-65°C and pH 9.0-9.5. Further, the addition amount of alkaline protease per gram of gluten powder is 180U-200U. Furthermore, the time of the enzymatic treatment can be 1 hour, the whole reaction process is continuously stirred, and 2M NaOH is used to maintain the pH of the reaction system at 9.0-9.5.
  • the buffer for phosphorylation is a phosphate buffer with pH 9.5 or 9.0, and the solid-liquid ratio of gluten to phosphate buffer is 1:10.
  • the mass ratio of the added amount of sodium tripolyphosphate to the gluten is 1:5.
  • the temperature of the phosphorylation treatment is 25° C., and the treatment time is 1 h.
  • the pH of the reaction system is 9.5.
  • the addition amount of the alkaline protease is: 200 U alkaline protease is added per gram of gluten.
  • the method for terminating the enzymatic hydrolysis treatment is to raise the temperature of the system to 85° C. and treat it for 15 minutes to inactivate the alkaline protease.
  • the said extracting wheat gliadin is: adjusting the pH of the processed gluten system to neutral, and freeze-drying to powder.
  • the gluten was degreasing treatment with anhydrous ether overnight. After repeating twice, after evaporating the anhydrous ether, 65% ethanol was added, and extraction was performed at 25°C for 1 hour. The solid-liquid ratio of the extraction was 1:10.
  • the extracted supernatant was centrifuged at 5000 g for 15 minutes, and the ethanol in the supernatant was removed by rotary evaporation at 40° C. and 90 mbar, and gliadin was obtained after freeze-drying.
  • the method of extracting gliadin in the gluten without phosphorylation-enzymatic hydrolysis treatment and after treatment is the same.
  • the present invention also provides a method for removing allergens from gluten powder, which is to first phosphorylate the gluten powder with sodium tripolyphosphate, and then perform hydrolysis treatment with alkaline protease.
  • the gluten In the first phosphorylation treatment of gluten with sodium tripolyphosphate, the gluten is mixed with a phosphate buffer, and then sodium tripolyphosphate is added, and the sodium tripolyphosphate reacts with the gluten. Furthermore, the solid-liquid ratio of gluten to phosphate buffer is 1 g: (10-12.5) mL, and the pH of the phosphate buffer is 8.5-9.5. Further, the mass ratio of the addition amount of sodium tripolyphosphate to the gluten powder is 1:4.5-1:5.5, and the sodium tripolyphosphate is added in batches. Furthermore, the addition amount of sodium tripolyphosphate and the mass ratio of gluten are 1:4.5 ⁇ 1:5.5.
  • Sodium tripolyphosphate is added into the reaction system in 3 equal parts, respectively at the start of the reaction and 10 ⁇ 10 after the start of the reaction. 15min and 25-30min after the start of the reaction; the reaction temperature is 25°C, the reaction time is 1h, stirring and controlling the pH of the reaction system within the range of 8.5-9.5.
  • the hydrolysis treatment with alkaline protease is to add alkaline protease to carry out enzymatic hydrolysis under the conditions of 60°C-65°C and pH 9.0-9.5. Further, the addition amount of alkaline protease per gram of gluten powder is 180U-200U. Furthermore, the time of the enzymatic treatment can be 1 hour, the whole reaction process is continuously stirred, and 2M NaOH is used to maintain the pH of the reaction system at 9.0-9.5.
  • the buffer for phosphorylation is a phosphate buffer with pH 9.5 or 9.0, and the solid-liquid ratio of gluten to phosphate buffer is 1:10.
  • the mass ratio of the added amount of sodium tripolyphosphate to the gluten is 1:5.
  • the temperature of the phosphorylation treatment is 25° C., and the treatment time is 1 h.
  • the pH of the reaction system is 9.5.
  • the addition amount of the alkaline protease is: 200 U alkaline protease is added per gram of gluten.
  • the method for terminating the enzymatic hydrolysis treatment is to raise the temperature of the system to 85° C. and treat it for 15 minutes to inactivate the alkaline protease.
  • the present invention has simple process steps, short processing time, and low processing temperature, which avoids the disadvantages of long time and high energy consumption of physical processing methods.
  • the present invention uses a medium-dose addition amount of sodium tripolyphosphate and adds it to the reaction system in batches to avoid a large pH change in the reaction system.
  • the lower processing temperature also reduces energy consumption.
  • the sensitization of gliadin in the compound treatment is lower than that in the single treatment.
  • the gliadin prepared by the invention is easier to digest, safe, and has no bad flavor and no bitter taste.
  • Figure 1 Structural changes of wheat gliadin after phosphorylation-enzymatic hydrolysis in Example 1; (A) content of secondary structure; (B) endogenous fluorescence spectrum; (C) surface hydrophobicity; (D) Free sulfhydryl content.
  • Raw is untreated wheat gliadin, Phos-Alcalase is wheat gliadin treated with phosphorylation and enzymolysis.
  • FIG. 2 The SDS-PAGE profile of the wheat prolamin treated by phosphorylation-enzymatic hydrolysis in Example 1 after simulated digestion in vitro; (A): gastric digestion for 10 min; (B): gastric digestion for 120 min; (C): intestine Digestion for 10 minutes; Lane 1 is untreated wheat gliadin, Lane 2 is phosphorylation-enzymatic hydrolysis treatment of wheat gliadin.
  • Figure 4 The expression levels of allergy-related cytokines and transcription factors in the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Example 1; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 5 Allergy characteristics of the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Example 2; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Fig. 6 The expression levels of allergy-related cytokines and transcription factors in the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Example 2; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 7 Allergy characteristics of the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Example 3; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Fig. 8 The expression levels of allergy-related cytokines and transcription factors in the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Example 3; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 9 Allergy characteristics of mice sensitized with wheat prolamin treated with phosphorylation-enzymatic hydrolysis in Example 4; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Fig. 10 The expression levels of allergy-related cytokines and transcription factors in the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Example 4; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 11 Allergy characteristics of mice sensitized with wheat prolamin treated with phosphorylation-enzymatic hydrolysis in Comparative Example 1; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Figure 12 The expression levels of allergy-related cytokines and transcription factors in the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 1; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 13 Allergy characteristics of phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 2; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Figure 14 The expression levels of allergy-related cytokines and transcription factors in the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 2; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 15 Allergy characteristics of the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 3; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Figure 16 The expression levels of allergy-related cytokines and transcription factors in phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 3; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Figure 17 Allergy characteristics of the phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 4; (A): changes in IgE concentration; (B): changes in serum histamine concentration.
  • Figure 18 The expression levels of allergy-related cytokines and transcription factors in phosphorylated-enzymatically treated wheat prolamin-sensitized mice in Comparative Example 4; (A): IFN- ⁇ ; (B): IL-4; (C): IL-5.
  • Endogenous fluorescence spectrum Use a fluorescence spectrometer to measure the fluorescence spectrum of wheat gliadin.
  • the concentration of the sample is 0.5 mg/mL
  • the excitation wavelength is 280 nm
  • the emission wavelength range is 300-500 nm.
  • the scanning speed is 1200nm/min
  • the reaction time is 0.5s
  • the voltage is 500V
  • the excitation and emission slits are 5nm.
  • Determination of free sulfhydryl groups Weigh 3 mg of wheat prolamin in 5 mL of Tris-glycine-EDTA solution for 30 min, shake once every 10 min, add 40 ⁇ L of Ellman’s reagent, cover with tin foil and store at 25°C for 30 min. Measure the absorbance at 412nm.
  • the concentrations of IgE, histamine, IL-4, and IL-5 in the serum were all determined with an ELISA kit. Determine the concentration of each indicator according to the instructions of each kit.
  • RNA extraction and real-time quantitative fluorescent PCR Use Trizol reagent to extract total RNA from C2C12 cells and tissue samples, and use Nanodrop to measure RNA concentration.
  • the Prime Script RT system (Takara) was used to synthesize the first strand cDNA.
  • Real-time quantitative PCR is performed on the ABI STEP-ONE7900 RT-PCR system. Each sample is made three times in parallel.
  • the present invention sensitizes BAL B/c mice by intraperitoneal injection of gliadin in wheat.
  • Extraction of wheat prolamin in gluten after treatment weigh 50g of gluten after phosphorylation-enzymatic hydrolysis treatment, add 500mL-65% ethanol and extract for 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, 40°C, 90mbar rotary evaporation to remove ethanol and freeze-dry to obtain phosphorylated-enzymatically treated wheat gliadin. Similarly, wheat gliadin is extracted from gluten that has not undergone phosphorylation-enzymatic hydrolysis treatment.
  • the wheat prolamin that has not been treated by phosphorylation-enzymatic hydrolysis and after phosphorylation-enzymatic hydrolysis is dissolved in 0.01 mol/L, pH 7.4 PBS, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There are 5 mice in each group.
  • the control group (CON) was injected with an equal volume of PBS, and the other two groups were injected with wheat gliadin (NT) without phosphorylation-enzymatic hydrolysis and wheat alcohol with phosphorylation-enzymatic hydrolysis. Phos-Alcalase.
  • mice's spleen index, serum IgE and histamine concentrations, and mRNA expression levels of related cytokines in the spleen were measured.
  • mice sensitized with wheat gliadin without phosphorylation-enzymatic hydrolysis treatment had significantly increased allergic indicators, while phosphorylation-enzymatic hydrolysis treated wheat
  • A Serum IgE concentration of gliadin-sensitized mice
  • B Serum histamine concentration was significantly lower than that of untreated wheat gliadin, and compared with non-sensitized mice, phosphorylation- There was no significant difference in the allergic characteristics of the mice sensitized with wheat prolamin treated by enzymatic hydrolysis.
  • the allergy-related cytokines of the mice sensitized with wheat gliadin without phosphorylation-enzymatic hydrolysis treatment were significantly increased, while the phosphorylation-enzymatic hydrolysis treatment wheat
  • the allergy-related cytokines of gliadin-sensitized mice were significantly lower than that of untreated wheat gliadin.
  • Extraction of wheat prolamin in gluten after treatment weigh 50g of gluten after phosphorylation-enzymatic hydrolysis treatment, add 500mL-65% ethanol and extract for 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, rotate and evaporate at 40°C, 90mbar to remove ethanol and freeze-dry to obtain phosphorylated-enzymatically treated wheat prolamin. Similarly, wheat gliadin is extracted from gluten that has not undergone phosphorylation-enzymatic hydrolysis treatment.
  • the wheat prolamin that has not been treated by phosphorylation-enzymatic hydrolysis and after phosphorylation-enzymatic hydrolysis is dissolved in 0.01 mol/L, pH 7.4 PBS, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group (CON) was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin
  • Extraction of wheat prolamin in gluten after treatment weigh 50g of gluten after phosphorylation-enzymatic hydrolysis treatment, add 500mL-65% ethanol and extract for 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, 40°C, 90mbar rotary evaporation to remove ethanol and freeze-dry to obtain phosphorylated-enzymatically treated wheat gliadin. Similarly, wheat gliadin is extracted from gluten that has not undergone phosphorylation-enzymatic hydrolysis treatment.
  • the wheat prolamin that has not been treated by phosphorylation-enzymatic hydrolysis and after phosphorylation-enzymatic hydrolysis is dissolved in 0.01 mol/L, pH 7.4 PBS, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group (CON) was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin
  • Extraction of wheat prolamin in gluten after treatment weigh 50g of gluten after phosphorylation-enzymatic hydrolysis treatment, add 500mL-65% ethanol and extract for 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, 40°C, 90mbar rotary evaporation to remove ethanol and freeze-dry to obtain phosphorylated-enzymatically treated wheat gliadin. Similarly, wheat gliadin is extracted from gluten that has not undergone phosphorylation-enzymatic hydrolysis treatment.
  • the wheat prolamin that has not been treated by phosphorylation-enzymatic hydrolysis and after phosphorylation-enzymatic hydrolysis is dissolved in 0.01 mol/L, pH 7.4 PBS, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group CON was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin
  • Extraction of wheat prolamin in gluten after treatment weigh 50g of gluten after phosphorylation-enzymatic hydrolysis treatment, add 500mL-65% ethanol and extract for 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, rotate and evaporate at 40°C, 90mbar to remove ethanol and freeze-dry to obtain phosphorylated-enzymatically treated wheat prolamin. Similarly, wheat gliadin is extracted from gluten that has not undergone phosphorylation-enzymatic hydrolysis treatment.
  • the wheat prolamin that has not been treated by phosphorylation-enzymatic hydrolysis and after phosphorylation-enzymatic hydrolysis is dissolved in 0.01 mol/L, pH 7.4 PBS, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group CON was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin
  • Extraction of wheat prolamin in gluten after treatment weigh 50g of gluten after phosphorylation-enzymatic hydrolysis treatment, add 500mL-65% ethanol and extract for 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, 40°C, 90mbar rotary evaporation to remove ethanol and freeze-dry to obtain phosphorylated-enzymatically treated wheat gliadin. Similarly, wheat gliadin is extracted from gluten that has not undergone phosphorylation-enzymatic hydrolysis treatment.
  • the wheat prolamin that has not been treated by phosphorylation-enzymatic hydrolysis and after phosphorylation-enzymatic hydrolysis is dissolved in 0.01 mol/L, pH 7.4 PBS, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group (CON) was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin
  • Extract the wheat gliadin in the processed gluten weigh 50g of the processed gluten, add 500mL 65% ethanol and extract 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, 40°C, 90mbar rotary evaporation to remove the ethanol and freeze-dry to obtain wheat gliadin. Similarly, wheat gliadin is extracted from untreated gluten.
  • the untreated and treated wheat gliadin was dissolved in 0.01 mol/L, pH 7.4 PBS, and the concentration was 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group (CON) was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin
  • Extract the wheat gliadin in the processed gluten weigh 50g of the processed gluten, add 500mL 65% ethanol and extract 1h at 25°C. Centrifuge at 5000g for 15min, take the supernatant, 40°C, 90mbar rotary evaporation to remove the ethanol and freeze-dry to obtain wheat gliadin. Similarly, wheat gliadin is extracted from untreated gluten.
  • the untreated and treated wheat gliadin was dissolved in 0.01 mol/L, pH 7.4 PBS, and the concentration was 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant in a 1:1 equal volume to sensitize mice.
  • the first intraperitoneal injection was the first day, and the injection dose of each mouse was 0.1 mL.
  • the mice were sensitized again on the 7th day and the 14th day, for a total of three sensitization. There were 5 mice in each group.
  • the control group (CON) was injected with an equal volume of PBS, and the other two groups were injected with untreated prolamin (NT) and phosphorylated-enzymatically treated prolamin (Phos-Alcalase).
  • NT untreated prolamin
  • Phos-Alcalase phosphorylated-enzymatically treated prolamin

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Abstract

公开了磷酸化-酶解法制备低致敏性小麦醇溶蛋白的方法，属于小麦加工技术领域。通过结合化学与酶解处理方法降低小麦醇溶蛋白的过敏性，处理后的小麦醇溶蛋白的致敏性与未致敏的小鼠的过敏参数相比无显著性差异，本发明的磷酸化-酶解处理方式有效降低了小麦醇溶蛋白的致敏性。

Description

磷酸化-酶解法制备低致敏性小麦醇溶蛋白的方法 技术领域
本发明属于小麦加工技术领域，涉及磷酸化-酶解法制备低致敏性小麦醇溶蛋白的方法。
背景技术
小麦是世界三大农作物之一，是人类重要的食物组成。很多主食均是小麦制品，如面包、面条、馒头以及其他烘焙制品。尽管小麦在日常生活中有着重要的角色，但是小麦属于世界粮农组织(FAO)报告的常见过敏食物。小麦中含有致敏性的能引起过敏反应的蛋白，即使摄入少量的小麦粉，也可能会对高度敏感病人的健康产生很大的影响。自2006年1月1日起，美国立法中小麦等8种过敏原列入标签范围。这意味着有许多人对小麦以及小麦制品过敏。在过去的几十年里，同其他的过敏食品一样，小麦过敏现象发生频率越来越高。全球有将近1％的人口对小麦过敏，0.4-1.3％的儿童食用小麦或小麦制品有过敏反应，其发生的概率稍高于成年人的0.2-0.9％。虽然，小麦导致过敏的现象频发，但是，目前唯一应对小麦过敏的最有效的方式是对其进行合理的加工处理。
根据溶解性的不同，可以将小麦蛋白分为水溶性的清蛋白、盐溶性的球蛋白和不溶性蛋白。其中，不溶性蛋白称为醇溶谷蛋白，由醇溶蛋白和麦谷蛋白组成，醇溶蛋白和麦谷蛋白共同组成谷朊粉。醇溶蛋白占小麦总蛋白的40％，分子量范围为28-55kDa。根据生化特点和遗传特性，醇溶蛋白又可以分为α、β、ω、γ等四种亚型。α醇溶蛋白的含量，约占小麦总蛋白的15-30％。
不同类型的小麦蛋白会引起不同的过敏症状。常见的摄入小麦蛋白后引起的过敏现象有乳糜泻、哮喘和鼻炎等。在做过敏原检测时，小麦蛋白中的醇溶蛋白因富含小麦过敏原IgE结合表位，成为检测、诊断小麦过敏的重要指标。同时，如果通过特定的加工方式，降低或改变小麦中的醇溶蛋白的含量或结构，去除或降低过敏原，可以达到降低小麦过敏的效果。
抗原表位是存在于抗原表面的、决定抗原特异性的特殊化学基团，抗原表位的性质、数目和空间构型决定抗原的特异性。抗原表位的结构分为构象表位和线性表位两类。低分子量的麦谷蛋白亚基中重复的Gln-Gln-Gln-Pro-Pro是IgE结合的表位。醇溶蛋白结构包括一个重复的氨基酸序列，称为中心域，这个中心域富含脯氨酸和谷氨酰胺。
蛋白的二级和三级结构是免疫系统识别表位的关键。食品中的过敏蛋白在加工过程中，其结构发生改变时，可能会伴随着蛋白抗原表位的改变，这也会影响机体免疫系统中细胞识别醇溶蛋白表位的可能性。这为通过加工处理降低或去除过敏原提供了可能性。
近年来，在小麦制品的生产过程中，各种不同的加工处理方式被应用于小麦粉或谷朊粉的预处理。常见的应用于谷朊粉的加工方法分为物理、化学和酶水解法三种。应用较多的物理方法包括超声、湿热水浴和微波处理等。化学改性方法包括磷酸化、乙酰化和脱酰胺等。在酶解处理中，应用较多的酶有碱性蛋白酶、木瓜蛋白酶和胰酶。使用较强的单一的物理处理方式对面粉或谷朊粉的流变性质影响较大，不利于后期的加工和产品的形成。而中等强度的物理处理方式则对小麦蛋白的结构影响较小，例如，采用中等强度的物理处理方式来处理小麦粉或谷朊粉，虽然保留了小麦粉或谷朊粉的良好的面团的加工性质，但是，中等强度的物理处理只能对蛋白的二级和三级结构带来微小的变化，而这种微小的变化并不能有效降低过敏蛋白的致敏性。而化学处理方法在食品方面的应用也越来越少，首先，加工处理过程中高剂量的添加剂越来越不被消费者接受，另外，化学处理方式虽然能较大程度影响小麦蛋白的结构，但关于化学处理后的醇溶蛋白的过敏性的改变情况仍存在争议，这与处理条件的不同有关，例如，脱酰胺处理条件的不同会造成醇溶蛋白的过敏性的降低或升高。目前，关于降低或消除食品中蛋白的过敏性的最有效的方法还是集中在酶法改性上，但，这些研究的应用尚存在一些困难，如醇溶蛋白等过敏蛋白对消化酶具特殊的抗拒作用，不易被酶消化水解，采用单一的酶或单独的酶处理方法不易使其完全水解，因此，常需要多种酶协同对小麦醇溶蛋白进行酶解，造成酶解法具有成本较高并可能会破坏面粉制品中其他的活性成分的缺点。
发明内容
[技术问题]
针对现有的解决食品过敏性问题的方法存在费用高、效果不确定、不易操作，难以规范化大规模生产，容易产生严重不良风味等缺点。
[技术方案]
本发明提供了一种制备低致敏性小麦醇溶蛋白的方法。所述方法是先用三聚磷酸钠先对谷朊粉进行磷酸化处理，再用碱性蛋白酶进行水解处理，然后提取小麦醇溶蛋白。低致敏性小麦醇溶蛋白，是指与从没有经过脱除过敏原处理的谷朊粉中提取的小麦醇溶蛋白相比，进入动物体后，动物体的过敏指征下降或消失的小麦醇溶蛋白。
所述方法可以采用如下步骤：
(1)用三聚磷酸钠先对谷朊粉进行磷酸化处理
将谷朊粉与磷酸缓冲液混合，谷朊粉与磷酸缓冲液的固液比为1g：(10～12.5)mL，磷酸缓冲液的pH为8.5～9.5，然后添加三聚磷酸钠，三聚磷酸钠与谷朊粉发生反应；
(2)用碱性蛋白酶进行水解处理
在60℃～65℃、pH 9.0～9.5条件下，添加碱性蛋白酶进行酶促水解反应，碱性蛋白酶的添加量为180U～200U/g谷朊粉；
(3)提取小麦醇溶蛋白
将步骤(2)酶解处理后的谷朊粉体系的pH调节为中性，并干燥、脱脂，用乙醇提取出小麦醇溶蛋白。
所述的用三聚磷酸钠先对谷朊粉磷酸化处理，是将谷朊粉与磷酸缓冲液混合，然后添加三聚磷酸钠，三聚磷酸钠与谷朊粉发生反应。进一步地，谷朊粉与磷酸缓冲液的固液比为1g：(10～12.5)mL，磷酸缓冲液的pH为8.5～9.5。进一步地，三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分批次添加。更进一步地，三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分为3等份加入反应体系，分别在反应开始、反应开始后10～15min以及反应开始后25～30min时加入；反应温度为25℃，反应时间为1h，搅拌并控制反应体系的pH在8.5～9.5范围内。
所述的用碱性蛋白酶进行水解处理，是在60℃～65℃、pH 9.0～9.5条件下，添加碱性蛋白酶进行酶水解反应。进一步地，每克谷朊粉的碱性蛋白酶的添加量为180U～200U。更进一步地，所述的酶法处理的时间可以是1小时，整个反应过程不断搅拌，并用2M NaOH保持反应体系的pH为9.0～9.5。
在本发明的一种实施方式中，磷酸化处理的缓冲液为pH9.5或9.0的磷酸缓冲液，谷朊粉与磷酸缓冲液的固液比为1：10。
在本发明的一种实施方式中，所述的三聚磷酸钠的添加量与谷朊粉的质量比为1：5。
在本发明的一种实施方式中，磷酸化处理的温度是25℃，处理时间为1h。
在本发明的一种实施方式中，碱性蛋白酶水解处理时，反应体系的pH为9.5。
在本发明的一种实施方式中，碱性蛋白酶水解处理时，碱性蛋白酶的添加量为：每克谷朊粉中添加200U碱性蛋白酶。
在本发明的一种实施方式中，所述的酶水解处理的终止办法是将体系的温度升高至85℃，处理15min，使碱性蛋白酶失活。
所述的提取小麦醇溶蛋白为：将处理后的谷朊粉体系的pH调节为中性，并冷冻干燥为粉状。用无水乙醚对谷朊粉进行过夜脱脂处理，重复两次后，挥发无水乙醚后，加入65％乙醇，25℃下提取1h，提取的固液比为1：10。提取的上清液5000g离心15min，通过40℃，90mbar旋转蒸发除去上清液中的乙醇，冷冻干燥后得到麦醇溶蛋白。未经磷酸化-酶解处理和处理后的谷朊粉中的麦醇溶蛋白的提取方式相同。
本发明还提供一种脱除谷朊粉中的过敏原的方法，是先用三聚磷酸钠先对谷朊粉进行磷酸化处理，再用碱性蛋白酶进行水解处理。
所述的用三聚磷酸钠先对谷朊粉磷酸化处理，是将谷朊粉与磷酸缓冲液混合，然后添加三聚磷酸钠，三聚磷酸钠与谷朊粉发生反应。进一步地，谷朊粉与磷酸缓冲液的固液比为1g：(10～12.5)mL，磷酸缓冲液的pH为8.5～9.5。进一步地，三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分批次添加。更进一步地，三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分为3等份加入反应体系，分别在反应开始、反应开始后10～15min以及反应开始后25～30min时加入；反应温度为25℃，反应时间为1h，搅拌并控制反应体系的pH在8.5～9.5范围内。
所述的用碱性蛋白酶进行水解处理，是在60℃～65℃、pH 9.0～9.5条件下，添加碱性蛋白酶进行酶水解反应。进一步地，每克谷朊粉的碱性蛋白酶的添加量为180U～200U。更进一步地，所述的酶法处理的时间可以是1小时，整个反应过程不断搅拌，并用2M NaOH保持反应体系的pH为9.0～9.5。
在本发明的一种实施方式中，磷酸化处理的缓冲液为pH9.5或9.0的磷酸缓冲液，谷朊粉与磷酸缓冲液的固液比为1：10。
在本发明的一种实施方式中，所述的三聚磷酸钠的添加量与谷朊粉的质量比为1：5。
在本发明的一种实施方式中，磷酸化处理的温度是25℃，处理时间为1h。
在本发明的一种实施方式中，碱性蛋白酶水解处理时，反应体系的pH为9.5。
在本发明的一种实施方式中，碱性蛋白酶水解处理时，碱性蛋白酶的添加量为：每克谷朊粉中添加200U碱性蛋白酶。
在本发明的一种实施方式中，所述的酶水解处理的终止办法是将体系的温度升高至85℃，处理15min，使碱性蛋白酶失活。
[有益效果]
(1)本发明工艺步骤简单、处理的时间短、处理的温度低，避免了物理处理方式的时间长、能耗多的缺点。
(2)将化学处理方式与酶水解结合，既有利于面团的流变性质的改善，也可有效降低麦醇溶蛋白的过敏性，降低了化学试剂的用量。磷酸化处理后的小麦醇溶蛋白的二级和三级结构有显著性变化，利用酶法水解将小麦醇溶蛋白中不易被酶解的、具有抗原决定部位的片段水解，降低其分子量，其抗原性降低，从而降低小麦醇溶蛋白的致敏性。
(3)本发明采用了中等剂量的三聚磷酸钠的添加量，并分批加入反应体系，避免了反应 体系发生较大的pH的变化。较低的处理温度也减少能源的消耗。
(4)复合处理方式的麦醇溶蛋白的致敏性低于单一方式处理的麦醇溶蛋白。本发明制备的麦醇溶蛋白更易消化，安全，无不良风味、无苦味。
附图说明
通过参照以下附图对处理后的小麦醇溶蛋白所作的详细描述，本发明的特征、目的和优点将会变得更明显。
图1实施例1中经过磷酸化-酶解处理的小麦醇溶蛋白的结构变化；(A)二级结构的含量；(B)内源性荧光光谱；(C)表面疏水性；(D)游离巯基含量。Raw为未处理的小麦醇溶蛋白，Phos-Alcalase为经磷酸化-酶解处理的小麦醇溶蛋白。
图2实施例1中经过磷酸化-酶解处理的小麦醇溶蛋白经体外模拟消化后的SDS-PAGE图谱；(A)：胃消化10min；(B)：胃消化120min；(C)：肠消化10min；泳道1为未处理的小麦醇溶蛋白，泳道2为磷酸化-酶解处理的小麦醇溶蛋白。
图3实施例1中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：血清IgE浓度的变化；(B)：血清组胺浓度的变化。
图4实施例1中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图5实施例2中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图6实施例2中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图7实施例3中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图8实施例3中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图9实施例4中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图10实施例4中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图11对比例1中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图12对比例1中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图13对比例2中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图14对比例2中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图15对比例3中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图16对比例3中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
图17对比例4中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏表征；(A)：IgE浓度的变化；(B)：血清组胺浓度的变化。
图18对比例4中磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关的细胞因子和转录因子表达水平；(A)：IFN-γ；(B)：IL-4；(C)：IL-5。
具体实施方式
相关检测方法：
二级结构测定：使用傅里叶红外法测定小麦醇溶蛋白的二级结构。在32次扫描和4cm -1条件下记录样品的背景的扫描光谱。溴化钾研磨烘干后与样品研磨混合，压成晶片后，在与背景相同的记录条件下测定。每个样品做三次平行。
内源性荧光光谱：使用荧光光谱仪测定小麦醇溶蛋白的荧光光谱。样品的浓度为0.5mg/mL，激发波长为280nm，发射波长范围为300-500nm。扫描速度为1200nm/min，反应时间为0.5s，电压为500V，激发和发射缝为5nm。
表面疏水性测定：采用ANS显色法测定小麦醇溶蛋白的表面疏水性。小麦蛋白溶于pH 7.0的磷酸缓冲液，蛋白浓度范围为0.05-0.5mg/mL，每4mL样品溶液中加入20μL的8.0mM的ANS，测定的激发波长为390nm，发射波长为470nm。荧光强度随蛋白浓度作曲线的斜率表征小麦醇溶蛋白的表面疏水性。
游离巯基的测定：称取3mg的小麦醇溶蛋白于5mL的Tris-glycine-EDTA溶液30min，每10min振荡一次，加入40μL的Ellman’s reagent，覆盖锡箔纸后25℃下存放30min。测定412nm处的吸光值。
体外模拟未处理和磷酸化-酶解处理的小麦醇溶蛋白胃肠道消化。称取8mg的小麦醇溶蛋 白于离心管中，与3.86mL的模拟胃消化液(SGF)混合，然后加入1μL的0.3M CaCl 2和139μL的水。每个离心管在37℃水浴中预热后加入0.16mL猪胃蛋白酶，置于37℃水浴中反应2h。分别在10min和120min时取100μL消化样品做SDS-PAGE分析。消化2h后，用30μL NaHCO 3终止反应。取2mL的胃消化样品与1.1mL的模拟肠消化液(SIF)混合，37℃预热后，加入0.5mL的胰液，置于37℃消化4h。分别在胃消化10min、120min和肠消化10min取消化样品做SDS-PAGE分析。
血清中相关过敏因子水平的测定：血清中IgE，组胺，IL-4，IL-5的浓度均采用ELISA试剂盒测定。按照各试剂盒的说明测定各指标的浓度。
RNA提取和实时定量荧光PCR：使用Trizol试剂从C2C12细胞和组织样品中提取总RNA，并用Nanodrop对RNA浓度进行测定。使用Prime Script RT系统(Takara)合成第一链cDNA。实时定量PCR在ABI STEP-ONE7900 RT-PCR系统上进行。每个样品做三次平行。
本发明通过腹腔注射小麦中的麦醇溶蛋白对BAL B/c小鼠进行致敏处理。
实施例1
称取100g谷朊粉于装有1000mLpH为9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于25℃水浴预热30min，保证整个反应体系为反应温度为25℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取20g三聚磷酸钠，分为三等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在9.0～9.5，反应1h。
将烧杯置于65℃水浴中，调节反应体系的pH为9.0～9.5，加入20000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g经过上述磷酸化-酶解处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋转蒸发除去乙醇后冷冻干燥，得到磷酸化-酶解处理的小麦醇溶蛋白。类似地，从没有经过磷酸化-酶解处理的谷朊粉中提取得到小麦醇溶蛋白。
将未经磷酸化-酶解处理和经磷酸化-酶解处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注 射未经磷酸化-酶解处理的小麦醇溶蛋白(NT)和磷酸化-酶解处理的小麦醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。
如图1所示，(A)磷酸化-酶解处理后的小麦醇溶蛋白的二级结构有明显的改变，其中α-螺旋，β-折叠和β-转角有明显的改变。(B)荧光强度有明显的降低，且最高峰的位置出现红移现象，说明小麦醇溶蛋白的三级结构发生改变，并且其中的色氨酸和酪氨酸的微环境也发生变化。(C)磷酸化-酶解处理的小麦醇溶蛋白的表面疏水性有显著降低，说明蛋白表面的疏水基团的位置发生显著改变，也反映出蛋白的三级结构的变化。(D)磷酸化-酶解处理的小麦醇溶蛋白的游离SH含量有显著的增加，说明蛋白的化学键的断裂，进一步说明了蛋白的三级和四级结构发生显著的变化。
如图2所示，磷酸化-酶解处理的小麦醇溶蛋白的体外模拟消化实验结果表明，磷酸化-酶解处理的小麦醇溶蛋白更容易被消化，在肠道10min后几乎完全被消化。
如图3所示，与对照组相比，用未经磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的各项过敏指标均显著性增加，而磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的(A)血清IgE浓度；(B)血清组胺浓度比未处理的小麦醇溶蛋白有显著性降低，且与未致敏的小鼠相比，磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏特征无显著性差异。
如图4所示，与对照组相比，用未经磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关细胞因子均显著性增加，而磷酸化-酶解处理的小麦醇溶蛋白致敏的小鼠的过敏相关细胞因子比未处理的小麦醇溶蛋白有显著性降低。
实施例2
称取100g谷朊粉于装有1000mLpH为8.5～9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于25℃水浴预热30min，保证整个反应体系为反应温度为25℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取17.8g三聚磷酸钠，分为三等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在8.5～9.5，反应1h。
将烧杯置于65℃水浴中，调节反应体系的pH为9.0～9.5，加入20000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g经过上述磷酸化-酶解处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋 转蒸发除去乙醇后冷冻干燥，得到磷酸化-酶解处理的小麦醇溶蛋白。类似地，从没有经过磷酸化-酶解处理的谷朊粉中提取得到小麦醇溶蛋白。
将未经磷酸化-酶解处理和经磷酸化-酶解处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。结果如图5，图6。
实施例3
称取100g谷朊粉于装有1000mLpH为8.5～9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于25℃水浴预热30min，保证整个反应体系为反应温度为25℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取17.8g三聚磷酸钠，分为三等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在8.5～9.5，反应1h。
将烧杯置于65℃水浴中，调节反应体系的pH为9.0～9.5，加入18000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g经过上述磷酸化-酶解处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋转蒸发除去乙醇后冷冻干燥，得到磷酸化-酶解处理的小麦醇溶蛋白。类似地，从没有经过磷酸化-酶解处理的谷朊粉中提取得到小麦醇溶蛋白。
将未经磷酸化-酶解处理和经磷酸化-酶解处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。结果如图7，图8。
实施例4
称取100g谷朊粉于装有1250mLpH为8.5～9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于25℃水浴预热30min，保证整个反应体系为反应温度为25℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取14.5g三聚磷酸钠，分为三等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在8.5～9.5，反应1h。
将烧杯置于65℃水浴中，调节反应体系的pH为9.0～9.5，加入18000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g经过上述磷酸化-酶解处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋转蒸发除去乙醇后冷冻干燥，得到磷酸化-酶解处理的小麦醇溶蛋白。类似地，从没有经过磷酸化-酶解处理的谷朊粉中提取得到小麦醇溶蛋白。
将未经磷酸化-酶解处理和经磷酸化-酶解处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。结果如图9，图10。
对比例1
称取100g谷朊粉于装有2000mL pH为8.5～9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于37℃水浴预热30min，保证整个反应体系为反应温度为37℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取20g三聚磷酸钠，分为三等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在8.5～9.5，反应2h。
将烧杯置于65℃水浴中，调节反应体系的pH为9.0～9.5，加入10000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g经过上述磷酸化-酶解处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋 转蒸发除去乙醇后冷冻干燥，得到磷酸化-酶解处理的小麦醇溶蛋白。类似地，从没有经过磷酸化-酶解处理的谷朊粉中提取得到小麦醇溶蛋白。
将未经磷酸化-酶解处理和经磷酸化-酶解处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。结果如图11，图12。
对比例2
称取100g谷朊粉于装有1000mLpH为8.5～9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于25℃水浴预热30min，保证整个反应体系为反应温度为25℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取20g三聚磷酸钠，分为三等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在8.5～9.5，反应30min。
将烧杯置于65℃水浴中，调节反应体系的pH为9.0～9.5，加入20000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g经过上述磷酸化-酶解处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋转蒸发除去乙醇后冷冻干燥，得到磷酸化-酶解处理的小麦醇溶蛋白。类似地，从没有经过磷酸化-酶解处理的谷朊粉中提取得到小麦醇溶蛋白。
将未经磷酸化-酶解处理和经磷酸化-酶解处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。结果如图13，图14。
对比例3单独采用三聚磷酸钠进行处理
称取100g谷朊粉于装有1000mLpH为9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于25℃水浴预热30min,，保证整个反应体系为反应温度为25℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。称取20g三聚磷酸钠，分为四等份缓慢加入反应体系中，根据pH计的数值变化，不断滴加2M的NaOH，使pH稳定在9.5，反应1h。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋转蒸发除去乙醇后冷冻干燥，得小麦醇溶蛋白。类似地，从未经处理的谷朊粉中提取小麦醇溶蛋白。
将未处理和处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天，每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。
结果表明：
单独的磷酸化处理对小麦醇溶蛋白的结构并没有显著性的改变，磷酸化处理的小麦醇溶蛋白致敏的小鼠的过敏特征与未处理的小麦醇溶蛋白致敏的小鼠无显著性差异。结果如图15，图16。
对比例4单独采用碱性蛋白酶进行处理
称取100g谷朊粉于装有1000mLpH为9.0～9.5的磷酸缓冲液的烧杯中，1000mL缓冲液预置于65℃水浴预热30min，保证整个反应体系为反应温度为65℃，并用pH测定反应体系的pH值，监测pH的变化。在搅拌器100rpm的转速下，使谷朊粉分散在缓冲液体系中。加入20000U的碱性蛋白酶，用2M的NaOH调节反应体系的pH保持在9.0～9.5，酶解1h后，将反应体系置于85℃水浴中15min，进行灭酶处理。接着4000g离心15min，弃上清，沉淀冷冻干燥处理。
提取处理后的谷朊粉中的小麦醇溶蛋白：称50g处理后的谷朊粉，加入500mL 65％的乙醇与25℃下提取1h。5000g离心15min，取上清，40℃，90mbar旋转蒸发除去乙醇后冷冻干燥，得小麦醇溶蛋白。类似地，从未经处理的谷朊粉中提取小麦醇溶蛋白。
将未处理和处理后的小麦醇溶蛋白溶于0.01mol/L，pH 7.4的PBS，浓度为1mg/mL。再与40mg/mL的氢氧化铝佐剂按1：1等体积混合均匀后致敏小鼠。第一次腹腔注射为第一天， 每只小鼠的注射剂量为0.1mL，分别在第7天和第14天再次致敏，共致敏三次。每组小鼠有5只，对照组(CON)注射等体积的PBS，另外两组分别注射未处理的醇溶蛋白(NT)和磷酸化-酶解处理的醇溶蛋白(Phos-Alcalase)。第28天，测定小鼠的脾脏指数，血清中IgE、组胺浓度以及脾脏中相关细胞因子的mRNA表达水平。
结果表明：
单独的磷酸化处理对小麦醇溶蛋白的结构并没有显著性的改变，磷酸化处理的小麦醇溶蛋白致敏的小鼠的过敏特征与未处理的小麦醇溶蛋白致敏的小鼠无显著性差异。结果如图17，图18。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (8)

  1. 一种制备低致敏性小麦醇溶蛋白的方法，其特征在于，包括以下步骤：
    (1)用三聚磷酸钠先对谷朊粉进行磷酸化处理
    将谷朊粉与磷酸缓冲液混合，谷朊粉与磷酸缓冲液的固液比为1g：(10～12.5)mL，磷酸缓冲液的pH为8.5～9.5，然后添加三聚磷酸钠，三聚磷酸钠与谷朊粉发生反应；
    (2)用碱性蛋白酶进行水解处理
    在60℃～65℃、pH 9.0～9.5条件下，添加碱性蛋白酶进行酶促水解反应，碱性蛋白酶的添加量为180U～200U/g谷朊粉；
    (3)提取小麦醇溶蛋白
    将步骤(2)酶解处理后的谷朊粉体系的pH调节为中性，并干燥、脱脂，用乙醇提取出小麦醇溶蛋白。
  2. 根据权利要求1所述的一种制备低致敏性小麦醇溶蛋白的方法，其特征在于，步骤(1)中三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分批次添加。
  3. 根据权利要求2所述的一种制备低致敏性小麦醇溶蛋白的方法，其特征在于，三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分为3等份加入反应体系，分别在反应开始、反应开始后10～15min以及反应开始后25～30min时加入；反应温度为25℃，反应总时间为1h，搅拌并控制反应体系的pH在8.5～9.5范围内。
  4. 根据权利要求1所述的一种制备低致敏性小麦醇溶蛋白的方法，其特征在于，步骤(2)中酶促水解反应的时间是1小时，整个反应过程中不断搅拌，并用2M NaOH保持反应体系的pH为9.0～9.5。
  5. 根据权利要求1所述的一种制备低致敏性小麦醇溶蛋白的方法，其特征在于，步骤(3)所述提取小麦醇溶蛋白，是将步骤(2)酶解处理后的谷朊粉体系的pH调节为中性，并冷冻干燥为粉状，用无水乙醚对谷朊粉进行脱脂处理，重复两次后，挥发无水乙醚后，加入乙醇提取麦醇溶蛋白，除去提取液中的乙醇，并进行冷冻干燥，得到小麦醇溶蛋白。
  6. 一种脱除谷朊粉中的过敏原的方法，其特征在于，先用三聚磷酸钠先对谷朊粉进行磷酸化处理，再用碱性蛋白酶进行水解处理。
  7. 根据权利要求6所述的一种脱除谷朊粉中的过敏原的方法，其特征在于，所述的用三聚磷酸钠先对谷朊粉磷酸化处理，是将谷朊粉与磷酸缓冲液混合，然后添加三聚磷酸钠，三聚磷酸钠与谷朊粉发生反应；谷朊粉与磷酸缓冲液的固液比为1g：(10～12.5)mL，磷酸缓冲液的pH为8.5～9.5；三聚磷酸钠的添加量与谷朊粉的质量比为1：4.5～1：5.5，三聚磷酸钠分批次添加；所述的用碱性蛋白酶进行水解处理，是在60℃～65℃、pH 9.0～9.5条件下，添加碱性蛋白酶进行酶水解反应；每克谷朊粉的碱性蛋白酶的添加量为180U～200U。
  8. 根据权利要求1～5任一所述方法制备得到的低致敏性小麦醇溶蛋白或经过权利要求7所述方法处理后的谷朊粉，以及根据权利要求1～5任一所述方法制备得到的低致敏性小麦醇溶蛋白或经过权利要求7所述方法处理后的谷朊粉在制备食品中的应用。
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