CN110055295B - Method for preparing hypoallergenic wheat alcohol soluble protein by phosphorylation-enzymolysis method - Google Patents
Method for preparing hypoallergenic wheat alcohol soluble protein by phosphorylation-enzymolysis method Download PDFInfo
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- 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
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- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
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Abstract
The invention discloses a method for preparing hypoallergenic wheat alcohol soluble protein by a phosphorylation-enzymolysis method, and belongs to the technical field of wheat processing. The method reduces the allergenicity of the wheat gliadin by combining a chemical and enzymolysis treatment method, finds that the allergenicity of the treated wheat gliadin has no significant difference compared with the allergenicity parameters of an unsensitized mouse by measuring the structure of the treated gliadin and comparing the allergenicity of the treated wheat gliadin and the unsensitized wheat gliadin, and proves that the phosphorylation-enzymolysis treatment method effectively reduces the allergenicity of the wheat gliadin.
Description
Technical Field
The invention belongs to the technical field of wheat processing, and relates to a method for preparing hypoallergenic wheat alcohol soluble protein by a phosphorylation-enzymolysis method.
Background
Wheat is one of three major crops in the world and is an important food composition for human beings. Many staple foods are wheat products such as bread, noodles, steamed bread and other baked products. Despite its important role in daily life, wheat belongs to a common allergic food reported by the world Food and Agriculture Organization (FAO). Wheat contains allergenic proteins that cause allergic reactions, and even a small intake of wheat flour can have a significant impact on the health of highly sensitive patients. From 1/2006, 8 allergens such as wheat are listed in the label range in U.S. legislation. This means that many people are allergic to wheat and wheat products. In the past decades, wheat allergy has been occurring more and more frequently, as in other allergic foods. Nearly 1% of the population in the world is allergic to wheat, and 0.4-1.3% of children eat wheat or wheat products and have allergic reaction, and the probability of occurrence is slightly higher than that of adults 0.2-0.9%. Although wheat causes frequent allergic phenomena, the only currently most effective way to deal with wheat allergy is to process it rationally.
Wheat proteins can be classified into water-soluble albumin, salt-soluble globulin and insoluble protein according to solubility. Wherein the insoluble protein is called prolamin, and is composed of prolamin and glutenin, and the prolamin and glutenin together constitute glutelin powder. The alcohol soluble protein accounts for 40% of total wheat protein, and has a molecular weight of 28-55 kDa. According to biochemical characteristics and genetic characteristics, prolamins can be divided into four subtypes of alpha, beta, omega, gamma and the like. The content of alpha alcohol soluble protein is about 15-30% of total wheat protein.
Different types of wheat proteins cause different allergic symptoms. Common allergic phenomena caused by wheat protein ingestion include celiac disease, asthma, rhinitis and the like. When the allergen is detected, the alcohol soluble protein in the wheat protein becomes an important index for detecting and diagnosing the wheat allergy because the alcohol soluble protein is rich in the wheat allergen IgE binding epitope. Meanwhile, if the content or the structure of alcohol soluble protein in the wheat is reduced or changed and the allergen is removed or reduced through a specific processing mode, the effect of reducing the wheat allergy can be achieved.
An epitope is a specific chemical group present on the surface of an antigen that determines the specificity of the antigen, the nature, number and spatial configuration of which determines the specificity of the antigen. The structure of antigenic epitopes is divided into conformational epitopes and linear epitopes. The repeating Gln-Gln-Gln-Pro-Pro in the low molecular weight glutenin subunit is an epitope for IgE binding. Prolamin structures comprise a repetitive sequence of amino acids, called the central domain, which is rich in proline and glutamine.
The secondary and tertiary structure of proteins is critical for the recognition of epitopes by the immune system. The allergic protein in the food may be accompanied by the change of the epitope of the protein when the structure of the allergic protein is changed in the processing process, which also affects the possibility that the cell in the immune system of the body recognizes the epitope of the prolamin. This provides the possibility of reducing or removing allergens by processing.
In recent years, various processing methods have been applied to pretreatment of wheat flour or wheat gluten in the production of wheat products. Common processing methods applied to wheat gluten are divided into three methods, namely physical, chemical and enzymatic hydrolysis. The physical methods which are applied more frequently comprise ultrasound, wet hot water bath, microwave treatment and the like. Chemical modification methods include phosphorylation, acetylation, deamidation, and the like. In the enzymolysis treatment, alkaline protease, papain and pancreatin are used as more enzymes. The use of a stronger single physical treatment means has a greater impact on the rheological properties of the flour or gluten, which is detrimental to the later processing and product formation. While the medium-intensity physical treatment has little influence on the structure of the wheat protein, for example, the medium-intensity physical treatment is used for treating the wheat flour or the wheat gluten, and although the good dough processing property of the wheat flour or the wheat gluten is kept, the medium-intensity physical treatment only can slightly change the secondary and tertiary structures of the protein, and the slight change can not effectively reduce the sensitization of the allergenic protein. While the use of chemical treatment methods in food products is becoming less and less, firstly, high doses of additives are becoming less and less acceptable to consumers during processing, and furthermore, although chemical treatment methods can greatly influence the structure of wheat proteins, there is still controversy about the change in the allergenicity of chemically treated prolamines, which is related to the differences in the treatment conditions, for example, the differences in the deamidation treatment conditions can cause a decrease or increase in the allergenicity of prolamines. Currently, the most effective methods for reducing or eliminating the allergy of protein in food are focused on enzymatic modification, but there are some difficulties in the application of these researches, such as that the allergic protein such as prolamin has special resistance to digestive enzymes, is not easily digested and hydrolyzed by enzymes, and is not easily hydrolyzed completely by using a single enzyme or a single enzyme treatment method, so that multiple enzymes are often required to carry out enzymolysis on wheat prolamin in a synergistic manner, which causes the defects that the enzymolysis method has high cost and may damage other active ingredients in the flour product.
Disclosure of Invention
[ problem ] to
Aiming at the defects of high cost, uncertain effect, difficult operation, difficult standardized large-scale production, easy generation of serious bad flavor and the like of the existing method for solving the problem of food allergy.
[ solution ]
The invention provides a method for preparing low-allergenicity wheat alcohol soluble protein. The method comprises the steps of firstly carrying out phosphorylation treatment on wheat gluten by using sodium tripolyphosphate, then carrying out hydrolysis treatment by using alkaline protease, and then extracting gliadin. Hypoallergenic gliadin refers to gliadin that has a reduced or eliminated allergic indication in animals when compared to gliadin extracted from gluten that has not been treated to remove allergens.
The method is characterized in that the sodium tripolyphosphate is used for carrying out phosphorylation treatment on the wheat gluten, the wheat gluten is mixed with a phosphate buffer solution, then the sodium tripolyphosphate is added, and the sodium tripolyphosphate reacts with the wheat gluten. Further, the solid-to-liquid ratio of the wheat gluten to the phosphate buffer solution is 1 g: (10-12.5) mL, and the pH value of the phosphate buffer solution is 8.5-9.5. Further, the mass ratio of the addition amount of the sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding the sodium tripolyphosphate in batches. Furthermore, the mass ratio of the addition amount of the sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding 3 equal parts of sodium tripolyphosphate into the reaction system, and adding the sodium tripolyphosphate into the reaction system at the beginning of the reaction, 10-15 min after the beginning of the reaction and 25-30 min after the beginning of the reaction respectively; the reaction temperature is 25 ℃, the reaction time is 1h, and the pH of the reaction system is stirred and controlled within the range of 8.5-9.5.
The alkaline protease is used for hydrolysis treatment, and the alkaline protease is added for enzymatic hydrolysis reaction at the temperature of 60-65 ℃ and the pH value of 9.0-9.5. Furthermore, the addition amount of the alkaline protease per gram of the wheat gluten is 180-200U. Furthermore, the enzymatic treatment time can be 1 hour, the whole reaction process is continuously stirred, and 2M NaOH is used for keeping the pH value of the reaction system to be 9.0-9.5.
In one embodiment of the present invention, the buffer solution for phosphorylation is phosphate buffer solution with ph9.5 or 9.0, and the solid-to-liquid ratio of wheat gluten to the phosphate buffer solution is 1: 10.
in one embodiment of the present invention, the mass ratio of the addition amount of the sodium tripolyphosphate to the vital wheat gluten is 1: 5.
in one embodiment of the present invention, the temperature of the phosphorylation treatment is 25 ℃ and the treatment time is 1 hour.
In one embodiment of the present invention, the pH of the reaction system in the alkaline protease hydrolysis treatment is 9.5.
In one embodiment of the present invention, the alkaline protease is added in the hydrolysis treatment with the alkaline protease in an amount of: 200U of alkaline protease is added into every gram of wheat gluten.
In one embodiment of the present invention, the enzymatic hydrolysis treatment is terminated by raising the temperature of the system to 85 ℃ for 15min to inactivate the alkaline protease.
The extraction method of gliadin in the untreated and treated wheat gluten is the same, and comprises the following steps: the pH of the treated gluten system was adjusted to neutral and freeze dried to a powder form. Carrying out overnight degreasing treatment on wheat gluten by using anhydrous ether, repeating twice, volatilizing the anhydrous ether, adding 65% ethanol, extracting for 1h at 25 ℃, wherein the solid-to-liquid ratio of extraction is 1: 10. centrifuging the extracted supernatant at 5000g for 15min, removing ethanol from the supernatant by rotary evaporation at 40 deg.C and 90mbar, and freeze drying to obtain gliadin.
The invention also provides a method for removing the allergen in the vital gluten, which comprises the steps of firstly carrying out phosphorylation treatment on the vital gluten by using sodium tripolyphosphate and then carrying out hydrolysis treatment by using alkaline protease.
The method is characterized in that the sodium tripolyphosphate is used for carrying out phosphorylation treatment on the wheat gluten, the wheat gluten is mixed with a phosphate buffer solution, then the sodium tripolyphosphate is added, and the sodium tripolyphosphate reacts with the wheat gluten. Further, the solid-to-liquid ratio of the wheat gluten to the phosphate buffer solution is 1 g: (10-12.5) mL, and the pH value of the phosphate buffer solution is 8.5-9.5. Further, the mass ratio of the addition amount of the sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding the sodium tripolyphosphate in batches. Furthermore, the mass ratio of the addition amount of the sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding 3 equal parts of sodium tripolyphosphate into the reaction system, and adding the sodium tripolyphosphate into the reaction system at the beginning of the reaction, 10-15 min after the beginning of the reaction and 25-30 min after the beginning of the reaction respectively; the reaction temperature is 25 ℃, the reaction time is 1h, and the pH of the reaction system is stirred and controlled within the range of 8.5-9.5.
The alkaline protease is used for hydrolysis treatment, and the alkaline protease is added for enzymatic hydrolysis reaction at the temperature of 60-65 ℃ and the pH value of 9.0-9.5. Furthermore, the addition amount of the alkaline protease per gram of the wheat gluten is 180-200U.
Furthermore, the enzymatic treatment time can be 1 hour, the whole reaction process is continuously stirred, and 2M NaOH is used for keeping the pH value of the reaction system to be 9.0-9.5.
In one embodiment of the present invention, the buffer solution for phosphorylation is phosphate buffer solution with ph9.5 or 9.0, and the solid-to-liquid ratio of wheat gluten to the phosphate buffer solution is 1: 10.
in one embodiment of the present invention, the mass ratio of the addition amount of the sodium tripolyphosphate to the vital wheat gluten is 1: 5.
in one embodiment of the present invention, the temperature of the phosphorylation treatment is 25 ℃ and the treatment time is 1 hour.
In one embodiment of the present invention, the pH of the reaction system in the alkaline protease hydrolysis treatment is 9.5.
In one embodiment of the present invention, the alkaline protease is added in the hydrolysis treatment with the alkaline protease in an amount of: 200U of alkaline protease is added into every gram of wheat gluten.
In one embodiment of the present invention, the enzymatic hydrolysis treatment is terminated by raising the temperature of the system to 85 ℃ for 15min to inactivate the alkaline protease.
[ advantageous effects ]
(1) The method has the advantages of simple process steps, short treatment time and low treatment temperature, and avoids the defects of long time and high energy consumption of a physical treatment mode.
(2) The combination of the chemical treatment mode and the enzymatic hydrolysis is beneficial to the improvement of the rheological property of the dough, and can also effectively reduce the anaphylaxis of gliadin and reduce the dosage of chemical reagents. The secondary and tertiary structures of the phosphorylated wheat alcohol soluble protein have significant changes, and the fragments which are difficult to be enzymolyzed and have antigenic determinant parts in the wheat alcohol soluble protein are hydrolyzed by using enzymatic hydrolysis, so that the molecular weight of the fragments is reduced, the antigenicity of the fragments is reduced, and the allergenicity of the wheat alcohol soluble protein is reduced.
(3) The invention adopts the addition amount of the sodium tripolyphosphate with medium dosage and adds the sodium tripolyphosphate into the reaction system in batches, thereby avoiding the reaction system from generating larger pH change. Lower processing temperatures also reduce energy consumption.
(4) The allergenicity of gliadin in the composite treatment mode is lower than that of gliadin treated in the single mode. The gliadin prepared by the invention is easier to digest, safe, free of bad flavor and bitter.
Drawings
The features, objects and advantages of the present invention will become more apparent from the detailed description of the treated gliadin protein with reference to the following drawings.
Figure 1 structural changes in phospho-enzymatically treated gliadin in example 1. (A) The content of secondary structure; (B) endogenous fluorescence spectroscopy; (C) surface hydrophobicity; (D) free thiol content. Raw is unprocessed gliadin of wheat, and Phos-Alcalase is phosphorylation-enzymolysis processed gliadin of wheat.
FIG. 2 SDS-PAGE patterns of phosphorylated-enzymatically treated gliadin in example 1 after simulated digestion in vitro. (A) The method comprises the following steps Stomach digestion for 10 min; (B) the method comprises the following steps Digesting in stomach for 120 min; (C) the method comprises the following steps Intestinal digestion for 10 min; lane 1 is untreated gliadin, lane 2 is phospho-enzymatically treated gliadin.
Figure 3A change in serum IgE concentration for allergy characterization of phospho-enzymatically treated gliadin-sensitized mice in example 1.
Figure 3B change in serum histamine concentration for allergy characterization of phospho-enzymatically treated gliadin-sensitized mice in example 1.
FIG. 4 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice of example 1. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
FIG. 5 allergy characterization of phospho-enzymatically treated gliadin-sensitized mice in example 2; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
FIG. 6 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice of example 2. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
FIG. 7 allergy characterization of phospho-enzymatically treated gliadin-sensitized mice in example 3; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
FIG. 8 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice in example 3. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
FIG. 9 allergy characterization of phospho-enzymatically treated gliadin-sensitized mice of example 4; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
FIG. 10 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice of example 4. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
FIG. 11 allergy characterization of phosphorylated-enzymatically treated gliadin sensitized mice in comparative example 1; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
FIG. 12 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice of comparative example 1. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
Figure 13 allergy characterization of phosphorylated-enzymatically treated gliadin-sensitized mice in comparative example 2; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
Figure 14 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice in comparative example 2. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
Figure 15 allergy characterization of phosphorylated-enzymatically treated gliadin-sensitized mice in comparative example 3; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
Figure 16 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice in comparative example 3. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
Figure 17 allergy characterization of phosphorylated-enzymatically treated gliadin-sensitized mice in comparative example 4; (A) the method comprises the following steps Changes in IgE concentration; (B) the method comprises the following steps Change in serum histamine concentration.
Figure 18 expression levels of allergy-associated cytokines and transcription factors in phospho-enzymatically treated gliadin-sensitized mice in comparative example 4. (A) The method comprises the following steps IFN-gamma; (B) the method comprises the following steps IL-4; (C) the method comprises the following steps IL-5.
Detailed Description
The related detection method comprises the following steps:
and (3) secondary structure determination: the secondary structure of wheat gliadin was determined using fourier infrared method. At 32 scans and 4cm-1Scanning spectra of the background of the sample were recorded under the conditions. And grinding and drying the potassium bromide, grinding and mixing the potassium bromide with the sample, pressing the mixture into a wafer, and measuring the wafer under the same recording condition with the background. Triplicate runs were made for each sample.
Endogenous fluorescence spectroscopy: the fluorescence spectrum of wheat gliadin was determined using a fluorescence spectrometer. The concentration of the sample is 0.5mg/mL, the excitation wavelength is 280nm, and the emission wavelength range is 300-500 nm. The scanning speed is 1200nm/min, the reaction time is 0.5s, the voltage is 500V, and the excitation and emission gap is 5 nm.
Surface hydrophobicity measurement: and (3) measuring the surface hydrophobicity of the wheat gliadin by adopting an ANS color rendering method. Wheat protein was dissolved in phosphate buffer pH 7.0 at a protein concentration range of 0.05-0.5mg/mL, 20. mu.L of 8.0mM ANS was added per 4mL of sample solution, and the measured excitation wavelength was 390nm and the emission wavelength was 470 nm. The slope of the curve of the fluorescence intensity along with the protein concentration represents the surface hydrophobicity of the wheat gliadin.
Determination of free thiol groups: weighing 3mg of gliadin in 5mL of Tris-glycine-EDTA solution for 30min, oscillating once every 10min, adding 40 μ L of Ellman's reagent, covering with tinfoil paper, and storing at 25 deg.C for 30 min. The absorbance at 412nm was measured.
In vitro simulation of gastrointestinal digestion of untreated and phospho-enzymatically treated gliadins of wheat. Weighing 8mg of gliadin in a centrifuge tube, mixing with 3.86mL of simulated gastric digestive juice (SGF), and adding 1. mu.L of 0.3M CaCl2And 139. mu.L of water. After each centrifuge tube was preheated in a 37 ℃ water bath, 0.16mL of porcine pepsin was added and placed in a 37 ℃ water bath for reaction for 2 h. 100 μ L of the digested sample was taken at 10min and 120min for SDS-PAGE analysis. After digestion for 2h, 30. mu.L NaHCO was used3The reaction was terminated. 2mL of the gastric digestion sample was mixed with 1.1mL of simulated intestinal digestion (SIF), preheated at 37 ℃, added with 0.5mL of pancreatic juice, and digested at 37 ℃ for 4 h. SDS-PAGE analysis was performed on samples digested in the stomach for 10min, 120min and digested in the intestine for 10 min.
Determination of the levels of relevant allergic factors in serum: the concentrations of IgE, histamine, IL-4 and IL-5 in serum were determined by ELISA kit. The concentration of each index was measured according to the instructions of each kit.
RNA extraction and real-time quantitative fluorescent PCR: total RNA was extracted from C2C12 cell and tissue samples using Trizol reagent and RNA concentration was determined using Nanodrop. First strand cDNA was synthesized using Prime Script RT System (Takara). Real-time quantitative PCR was performed on the ABI STEP-ONE7900 RT-PCR system. Triplicate runs were made for each sample.
According to the invention, the sensitization treatment is carried out on the BALB/c mouse by injecting gliadin in wheat into the abdominal cavity.
Example 1
100g of wheat gluten is weighed in a beaker filled with 1000mL of phosphate buffer solution with the pH value of 9.5, 1000mL of buffer solution is pre-placed in a water bath at 25 ℃ for preheating for 30min, the reaction temperature of the whole reaction system is ensured to be 25 ℃, the pH value of the reaction system is measured by using the pH value, and the change of the pH value is monitored. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 20g of sodium tripolyphosphate, dividing into three equal parts, slowly adding into a reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter to stabilize the pH at 9.0-9.5, and reacting for 1 h.
Placing the beaker in a water bath at 65 ℃, adjusting the pH of the reaction system to 9.0-9.5, adding 20000U alkaline protease, adjusting the pH of the reaction system to 9.0-9.5 by using 2M NaOH, performing enzymolysis for 1h, placing the reaction system in a water bath at 85 ℃ for 15min, and performing enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: weighing 50g of the wheat gluten subjected to phosphorylation-enzymolysis treatment, adding 500mL of 65% ethanol, and extracting at 25 ℃ for 1 h. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain phosphorylation-enzymolysis treated gliadin. Similarly, gliadin was extracted from gluten which had not been subjected to the phospho-enzymatic treatment.
Dissolving gliadin which is not subjected to phosphorylation-enzymolysis treatment and is subjected to phosphorylation-enzymolysis treatment in PBS (phosphate buffer solution) with the concentration of 1mg/mL and the pH value of 0.01mol/L and pH value of 7.4. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. There were 5 mice per group, a control group (CON) injected with PBS of equal volume, and the other two groups injected with gliadin (NT) without and with Phos-Alcalase, respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice.
As shown in fig. 1, (a) secondary structure of the phospho-enzymatically treated gliadin is significantly altered, with significant changes in α -helix, β -sheet and β -turn. (B) The fluorescence intensity is obviously reduced, and the position of the highest peak has a red shift phenomenon, which indicates that the tertiary structure of the wheat gliadin is changed, and the microenvironment of tryptophan and tyrosine in the wheat gliadin is also changed. (C) The surface hydrophobicity of the phosphorylated-enzymatically treated wheat gliadin is obviously reduced, which shows that the position of hydrophobic groups on the surface of the protein is obviously changed, and the change of the tertiary structure of the protein is also reflected. (D) The free SH content of the wheat gliadin treated by phosphorylation and enzymolysis is obviously increased, which shows that the chemical bond of the protein is broken, and further shows that the tertiary structure and the quaternary structure of the protein are obviously changed.
As shown in fig. 2, the results of the in vitro simulated digestion experiments of the phosphorylated-enzymatically treated gliadin show that the phosphorylated-enzymatically treated gliadin is more easily digested and is almost completely digested after 10min in the intestinal tract.
As shown in fig. 3, each allergy index of mice sensitized with gliadin that was not subjected to phosphorylation-enzymolysis was significantly increased compared to the control group, and (a) serum IgE concentration of mice sensitized with gliadin that was subjected to phosphorylation-enzymolysis; (B) serum histamine concentrations were significantly reduced compared to untreated gliadin, and there was no significant difference in the allergic characteristics of phosphorylated-enzymatically treated gliadin sensitized mice compared to non-sensitized mice.
As shown in fig. 4, the mice sensitized with the wheat gliadin that was not subjected to the phosphorylation-enzymolysis treatment all had a significant increase in the allergy-related cytokines compared to the control group, while the mice sensitized with the phosphorylation-enzymolysis treatment wheat gliadin had a significant decrease in the allergy-related cytokines compared to the untreated wheat gliadin.
Example 2
Weighing 100g of wheat gluten in a beaker filled with 1000mL of phosphate buffer solution with the pH of 8.5-9.5, pre-placing 1000mL of buffer solution in a 25 ℃ water bath for preheating for 30min, ensuring that the reaction temperature of the whole reaction system is 25 ℃, measuring the pH value of the reaction system by using the pH value, and monitoring the change of the pH value. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 17.8g of sodium tripolyphosphate, dividing into three equal parts, slowly adding into a reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter to stabilize the pH value at 8.5-9.5, and reacting for 1 h.
Placing the beaker in a water bath at 65 ℃, adjusting the pH of the reaction system to 9.0-9.5, adding 20000U alkaline protease, adjusting the pH of the reaction system to 9.0-9.5 by using 2M NaOH, performing enzymolysis for 1h, placing the reaction system in a water bath at 85 ℃ for 15min, and performing enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: weighing 50g of the wheat gluten subjected to phosphorylation-enzymolysis treatment, adding 500mL of 65% ethanol, and extracting at 25 ℃ for 1 h. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain phosphorylation-enzymolysis treated gliadin. Similarly, gliadin was extracted from gluten which had not been subjected to the phospho-enzymatic treatment.
Dissolving gliadin which is not subjected to phosphorylation-enzymolysis treatment and is subjected to phosphorylation-enzymolysis treatment in PBS (phosphate buffer solution) with the concentration of 1mg/mL and the pH value of 0.01mol/L and pH value of 7.4. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice. The results are shown in FIG. 5 and FIG. 6.
Example 3
Weighing 100g of wheat gluten in a beaker filled with 1000mL of phosphate buffer solution with the pH of 8.5-9.5, pre-placing 1000mL of buffer solution in a 25 ℃ water bath for preheating for 30min, ensuring that the reaction temperature of the whole reaction system is 25 ℃, measuring the pH value of the reaction system by using the pH value, and monitoring the change of the pH value. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 17.8g of sodium tripolyphosphate, dividing into three equal parts, slowly adding into a reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter to stabilize the pH value at 8.5-9.5, and reacting for 1 h.
Placing the beaker in a water bath at 65 ℃, adjusting the pH of the reaction system to 9.0-9.5, adding 18000U of alkaline protease, adjusting the pH of the reaction system to 9.0-9.5 by using 2M NaOH, performing enzymolysis for 1h, placing the reaction system in a water bath at 85 ℃ for 15min, and performing enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: weighing 50g of the wheat gluten subjected to phosphorylation-enzymolysis treatment, adding 500mL of 65% ethanol, and extracting at 25 ℃ for 1 h. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain phosphorylation-enzymolysis treated gliadin. Similarly, gliadin was extracted from gluten which had not been subjected to the phospho-enzymatic treatment.
Dissolving gliadin which is not subjected to phosphorylation-enzymolysis treatment and is subjected to phosphorylation-enzymolysis treatment in PBS (phosphate buffer solution) with the concentration of 1mg/mL and the pH value of 0.01mol/L and pH value of 7.4. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice. The results are shown in FIG. 7 and FIG. 8.
Example 4
Weighing 100g of wheat gluten into a beaker filled with 1250mL of phosphate buffer solution with the pH of 8.5-9.5, pre-placing 1000mL of buffer solution into a water bath at 25 ℃ for preheating for 30min, ensuring that the reaction temperature of the whole reaction system is 25 ℃, measuring the pH value of the reaction system by using the pH value, and monitoring the change of the pH value. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 14.5g of sodium tripolyphosphate, dividing into three equal parts, slowly adding into a reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter, stabilizing the pH value at 8.5-9.5, and reacting for 1 h.
Placing the beaker in a water bath at 65 ℃, adjusting the pH of the reaction system to 9.0-9.5, adding 18000U of alkaline protease, adjusting the pH of the reaction system to 9.0-9.5 by using 2M NaOH, performing enzymolysis for 1h, placing the reaction system in a water bath at 85 ℃ for 15min, and performing enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: weighing 50g of the wheat gluten subjected to phosphorylation-enzymolysis treatment, adding 500mL of 65% ethanol, and extracting at 25 ℃ for 1 h. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain phosphorylation-enzymolysis treated gliadin. Similarly, gliadin was extracted from gluten which had not been subjected to the phospho-enzymatic treatment.
Dissolving gliadin which is not subjected to phosphorylation-enzymolysis treatment and is subjected to phosphorylation-enzymolysis treatment in PBS (phosphate buffer solution) with the concentration of 1mg/mL and the pH value of 0.01mol/L and pH value of 7.4. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice. The results are shown in FIGS. 9 and 10.
Comparative example 1
Weighing 100g of wheat gluten in a beaker filled with 2000mL of phosphate buffer solution with the pH of 8.5-9.5, pre-placing 1000mL of buffer solution in a 37 ℃ water bath for preheating for 30min, ensuring that the reaction temperature of the whole reaction system is 37 ℃, measuring the pH value of the reaction system by using the pH value, and monitoring the change of the pH value. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 20g of sodium tripolyphosphate, dividing into three equal parts, slowly adding into a reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter to stabilize the pH value at 8.5-9.5, and reacting for 2 h.
Placing the beaker in a water bath at 65 ℃, adjusting the pH of the reaction system to 9.0-9.5, adding 10000U of alkaline protease, adjusting the pH of the reaction system to 9.0-9.5 by using 2M NaOH, performing enzymolysis for 1h, placing the reaction system in a water bath at 85 ℃ for 15min, and performing enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: weighing 50g of the wheat gluten subjected to phosphorylation-enzymolysis treatment, adding 500mL of 65% ethanol, and extracting at 25 ℃ for 1 h. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain phosphorylation-enzymolysis treated gliadin. Similarly, gliadin was extracted from gluten which had not been subjected to the phospho-enzymatic treatment.
Dissolving gliadin which is not subjected to phosphorylation-enzymolysis treatment and is subjected to phosphorylation-enzymolysis treatment in PBS (phosphate buffer solution) with the concentration of 1mg/mL and the pH value of 0.01mol/L and pH value of 7.4. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice. The results are shown in FIGS. 11 and 12.
Comparative example 2
Weighing 100g of wheat gluten in a beaker filled with 1000mL of phosphate buffer solution with the pH of 8.5-9.5, pre-placing 1000mL of buffer solution in a 25 ℃ water bath for preheating for 30min, ensuring that the reaction temperature of the whole reaction system is 25 ℃, measuring the pH value of the reaction system by using the pH value, and monitoring the change of the pH value. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 20g of sodium tripolyphosphate, dividing into three equal parts, slowly adding into a reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter to stabilize the pH value at 8.5-9.5, and reacting for 30 min.
Placing the beaker in a water bath at 65 ℃, adjusting the pH of the reaction system to 9.0-9.5, adding 20000U alkaline protease, adjusting the pH of the reaction system to 9.0-9.5 by using 2M NaOH, performing enzymolysis for 1h, placing the reaction system in a water bath at 85 ℃ for 15min, and performing enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: weighing 50g of the wheat gluten subjected to phosphorylation-enzymolysis treatment, adding 500mL of 65% ethanol, and extracting at 25 ℃ for 1 h. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain phosphorylation-enzymolysis treated gliadin. Similarly, gliadin was extracted from gluten which had not been subjected to the phospho-enzymatic treatment.
Dissolving gliadin which is not subjected to phosphorylation-enzymolysis treatment and is subjected to phosphorylation-enzymolysis treatment in PBS (phosphate buffer solution) with the concentration of 1mg/mL and the pH value of 0.01mol/L and pH value of 7.4. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice. The results are shown in FIG. 13 and FIG. 14.
Comparative example 3 treatment with sodium tripolyphosphate alone
100g of wheat gluten is weighed in a beaker filled with 1000mL of phosphate buffer solution with the pH value of 9.5, 1000mL of buffer solution is pre-placed in a water bath at the temperature of 25 ℃ for preheating for 30min, the reaction temperature of the whole reaction system is ensured to be 25 ℃, the pH value of the reaction system is measured by using the pH value, and the change of the pH value is monitored. The gluten was dispersed in the buffer system at 100rpm of the stirrer. Weighing 20g of sodium tripolyphosphate, dividing into four equal parts, slowly adding into the reaction system, continuously dropwise adding 2M NaOH according to the numerical value change of a pH meter to stabilize the pH value at 9.5, and reacting for 1 h.
Extracting the gliadin in the treated wheat gluten: 50g of the treated wheat gluten was weighed out and extracted with 500mL of 65% ethanol at 25 ℃ for 1 hour. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain gliadin. Similarly, gliadin is extracted from untreated gluten.
Untreated and treated gliadin were dissolved in 0.01mol/L PBS, pH 7.4, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice.
The results show that:
the structure of the wheat gliadin is not obviously changed by the single phosphorylation treatment, and the allergic characteristics of the mice sensitized by the phosphorylation treatment wheat gliadin are not obviously different from those of the mice sensitized by the untreated wheat gliadin. The results are shown in FIG. 15 and FIG. 16.
Comparative example 4 treatment with alkaline protease alone
Weighing 100g of wheat gluten in a beaker filled with 1000mL of phosphate buffer solution with the pH value of 9.0-9.5, pre-placing 1000mL of buffer solution in a 65 ℃ water bath for preheating for 30min, ensuring that the reaction temperature of the whole reaction system is 65 ℃, measuring the pH value of the reaction system by using the pH value, and monitoring the change of the pH value. The gluten was dispersed in the buffer system at 100rpm of the stirrer. 20000U of alkaline protease is added, 2M NaOH is used for adjusting the pH value of the reaction system to be 9.0-9.5, after 1 hour of enzymolysis, the reaction system is placed in a water bath at 85 ℃ for 15min for enzyme deactivation treatment. Then, the mixture was centrifuged at 4000g for 15min, the supernatant was discarded, and the precipitate was lyophilized.
Extracting the gliadin in the treated wheat gluten: 50g of the treated wheat gluten was weighed out and extracted with 500mL of 65% ethanol at 25 ℃ for 1 hour. Centrifuging at 5000g for 15min, collecting supernatant, rotary evaporating at 40 deg.C and 90mbar to remove ethanol, and freeze drying to obtain gliadin. Similarly, gliadin is extracted from untreated gluten.
Untreated and treated gliadin were dissolved in 0.01mol/L PBS, pH 7.4, at a concentration of 1 mg/mL. Then mixed with 40mg/mL aluminum hydroxide adjuvant according to the proportion of 1: 1, sensitizing the mouse after equal volume and uniform mixing. The first intraperitoneal injection was the first day, the injection dose per mouse was 0.1mL, and sensitization was performed again on day 7 and day 14, respectively, for a total of three times. Mice were injected with 5 mice per group, a control group (CON) with an equal volume of PBS, and two groups were injected with untreated prolamin (NT) and phospho-enzymatically treated prolamin (Phos-Alcalase), respectively. On day 28, spleen indices, serum IgE, histamine concentrations, and mRNA expression levels of relevant cytokines in the spleen were determined for the mice.
The results show that:
the structure of the wheat gliadin is not obviously changed by the single phosphorylation treatment, and the allergic characteristics of the mice sensitized by the phosphorylation treatment wheat gliadin are not obviously different from those of the mice sensitized by the untreated wheat gliadin. The results are shown in FIGS. 17 and 18.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. A method for preparing low-sensitization gliadin is characterized in that sodium tripolyphosphate is used for carrying out phosphorylation treatment on wheat gluten, alkaline protease is used for carrying out hydrolysis treatment, and then gliadin is extracted;
the mass ratio of the addition amount of the sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding the sodium tripolyphosphate in batches;
the alkaline protease is used for hydrolysis treatment, the alkaline protease is added for enzymatic hydrolysis reaction at the temperature of 60-65 ℃ and under the condition of pH of 9.0-9.5, and the addition amount of the alkaline protease per gram of wheat gluten is 180-200U.
2. The method for preparing hypoallergenic wheat gliadin according to claim 1, wherein the phosphorylation of the gluten with sodium tripolyphosphate is carried out by mixing the gluten with a phosphate buffer, adding sodium tripolyphosphate, and reacting the sodium tripolyphosphate with the gluten.
3. The method of claim 2, wherein the hypoallergenic wheat gliadin protein is present in the sample; the solid-liquid ratio of the wheat gluten to the phosphate buffer solution is 1 g: (10-12.5) mL, and the pH value of the phosphate buffer solution is 8.5-9.5.
4. The method for preparing hypoallergenic wheat gliadin according to claim 3, wherein the mass ratio of the addition amount of sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding 3 equal parts of sodium tripolyphosphate into the reaction system, and adding the sodium tripolyphosphate into the reaction system at the beginning of the reaction, 10-15 min after the beginning of the reaction and 25-30 min after the beginning of the reaction respectively; the reaction temperature is 25 ℃, the reaction time is 1h, and the pH of the reaction system is stirred and controlled within the range of 8.5-9.5.
5. A method for removing allergen in vital gluten is characterized in that firstly sodium tripolyphosphate is used for carrying out phosphorylation treatment on the vital gluten, and then alkaline protease is used for carrying out hydrolysis treatment, wherein the phosphorylation treatment on the vital gluten by the sodium tripolyphosphate is carried out by mixing the vital gluten with a phosphoric acid buffer solution, then the sodium tripolyphosphate is added, and the sodium tripolyphosphate reacts with the vital gluten; the solid-liquid ratio of the wheat gluten to the phosphate buffer solution is 1 g: (10-12.5) mL, wherein the pH value of the phosphate buffer solution is 8.5-9.5; the mass ratio of the addition amount of the sodium tripolyphosphate to the wheat gluten is 1: 4.5-1: 5.5, adding the sodium tripolyphosphate in batches; the alkaline protease is used for hydrolysis treatment, and the alkaline protease is added for enzymatic hydrolysis reaction at the temperature of 60-65 ℃ and the pH value of 9.0-9.5; the addition amount of the alkaline protease per gram of the wheat gluten is 180-200U.
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| 小麦致敏蛋白的研究进展;孟良玉等;《食品与发酵工艺》;20120430;第38卷(第4期);第148页第2.1节-149页第3节 * |
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