CN110055295A - The method that phosphorylation-enzymatic isolation method prepares low irritability wheat gliadin - Google Patents
The method that phosphorylation-enzymatic isolation method prepares low irritability wheat gliadin Download PDFInfo
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- CN110055295A CN110055295A CN201910358125.4A CN201910358125A CN110055295A CN 110055295 A CN110055295 A CN 110055295A CN 201910358125 A CN201910358125 A CN 201910358125A CN 110055295 A CN110055295 A CN 110055295A
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Classifications
-
- 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
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- 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
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- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
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- Nutrition Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses the methods that phosphorylation-enzymatic isolation method prepares low irritability wheat gliadin, belong to wheat processing technical field.The present invention is the anaphylaxis by combining chemistry with enzymolysis processing method reduction wheat gliadin, pass through the measurement of the structure to treated alcohol soluble protein, and compare the sensitization of treated wheat gliadin and untreated wheat gliadin, it was found that there was no significant difference compared with the allergy parameter of unsensitized mouse for the sensitization of treated wheat gliadin, illustrate that phosphorylation of the invention-enzymolysis processing mode effectively reduces the sensitization of wheat gliadin.
Description
Technical field
The invention belongs to wheat processing technical fields, are related to phosphorylation-enzymatic isolation method and prepare low irritability wheat gliadin
Method.
Background technique
Wheat is one of three grande culture object of the world, is the important food composition of the mankind.Many staple foods are wheat products, such as
Bread, noodles, steamed bun and other baking goods.Although wheat has important role, Triticum in daily life
In the Typical allergic food of the World Food Programme (FAO) report.The albumen that can be caused allergic reaction in wheat containing sensitization,
Even if taking in a small amount of wheat flour, it is also possible to be had a huge impact to the health of highly sensitive patient.From January 1 in 2006
Day rises, and 8 kinds of anaphylactogens such as wheat are included in label range in american legislation.This means that there are many people to wheat and wheat products
Allergy.In in the past few decades, the same with others allergy food, Wheat Dood Allergy phenomenon occurrence frequency is higher and higher.The whole world
There is nearly 1% population to Wheat Dood Allergy, the children of 0.4-1.3%, which eat wheat or wheat products, allergic reaction, occurs
The a little higher than adult of probability 0.2-0.9%.Although the anaphylactic phenomenon of wheat takes place frequently, currently the only reply is small
The most effective way of wheat allergy is to carry out reasonable working process to it.
According to deliquescent difference, wheat gluten can be divided into water-soluble albumin, the globulin of salt dissolubility and not
Dissolubility albumen.Wherein, insoluble protein is known as prolamin, is made of alcohol soluble protein and glutenin, alcohol soluble protein and wheat
Glutelin collectively constitutes Gluten.Alcohol soluble protein accounts for the 40% of wheat total protein, molecular weight ranges 28-55kDa.According to biochemistry
Feature and hereditary capacity, alcohol soluble protein can be divided into four kinds of hypotypes such as α, β, ω, γ again.The content of α alcohol soluble protein, accounts for about wheat
The 15-30% of total protein.
Different types of wheat gluten can cause different allergic symptoms.Caused allergy after common intake wheat gluten
Phenomenon has chylous diarrhea, asthma and rhinitis etc..When doing Allergic skin test, the alcohol soluble protein in wheat gluten is because being rich in Wheat Dood Allergy
Former IgE combination epitope, the important indicator for becoming detection, diagnosing Wheat Dood Allergy.Meanwhile if by specific processing method, drop
Content or structure low or that change the alcohol soluble protein in wheat, removal or reduction anaphylactogen, can achieve reduces Wheat Dood Allergy
Effect.
Epitope is the special chemical group for being present in antigenic surface, determining antigentic specificity, the property of epitope
Matter, number and steric configuration determine the specificity of antigen.The structure of epitope is divided into two class of comformational epitope and linear epitope.It is low
Duplicate Gln-Gln-Gln-Pro-Pro is the epitope that IgE is combined in the glutenin subunit of molecular weight.Alcohol soluble protein structure packet
A duplicate amino acid sequence, referred to as central field are included, this central field Pro-rich and glutamine.
The second level and tertiary structure of albumen are the key that immune system identification epitopes.Allergen protein in food is processed
Cheng Zhong may be along with the change of Protein Epitopes when structure changes, this also will affect in body immune system
A possibility that cell recognition alcohol soluble protein epitope.This provides possibility for anaphylactogen is reduced or removed by working process.
In recent years, in the production process of wheat products, a variety of different working process modes be applied to wheat flour or
The pretreatment of Gluten.The common processing method applied to Gluten is divided into physics, chemistry and three kinds of enzyme hydrolysis method.Using compared with
More physical methods includes ultrasound, damp and hot water-bath and microwave treatment etc..Chemical modification method includes phosphorylation, acetylation and deacylation
Amine etc..In enzymolysis processing, there are alkali protease, papain and pancreatin using more enzyme.Using stronger single
Physical treatment mode is affected to the rheological equationm of state of flour or Gluten, is unfavorable for the processing in later period and the formation of product.And
The physical treatment mode of moderate strength is then smaller on the influence of the structure of wheat gluten, for example, using the physical treatment of moderate strength
Mode handles wheat flour or Gluten, although remain the working properties of the good dough of wheat flour or Gluten,
The physical treatment of moderate strength can only second level to albumen and tertiary structure bring small variation, and this small variation is simultaneously
The sensitization of allergen protein cannot be effectively reduced.And application of the chemical treatment method in terms of food is also fewer and fewer, firstly, plus
The additive of high dose is not esthetically acceptable to the consumers increasingly in work treatment process, although in addition, chemical treatments can larger journey
Degree influences the structure of wheat gluten, but about hypersensitive change situation of the alcohol soluble protein after chemical treatment there are still dispute,
This is related with the difference for the treatment of conditions, for example, the difference of deamidation treatment conditions will cause hypersensitive reduction of alcohol soluble protein
Or it increases.Currently, still concentrating on enzyme modification about the hypersensitive most efficient method for reducing or eliminating albumen in food
On, but, the application of these researchs still has some difficulties, such as alcohol soluble protein allergen protein has special resistance to digestive ferment and makees
With, it is not easy to be rapidly digested by an enzyme in a body hydrolysis, is not easy to make its complete hydrolysis using single enzyme or individual method of enzymatically treating, it is therefore, normal to need
It wants a variety of enzyme collaborations to digest wheat gliadin, causes enzymatic isolation method that there is higher cost and flour product may be destroyed
The shortcomings that middle others active constituent.
Summary of the invention
[technical problem]
For the existing method for solving the problems, such as food allergy, there are costly, effect is uncertain, not easy to operate, it is difficult to
The disadvantages of standardization is mass produced, and is easy to produce serious bad flavor.
[technical solution]
The present invention provides a kind of methods for preparing low irritability wheat gliadin.The method is first to use tripolyphosphate
Sodium first carries out phosphatizing treatment to Gluten, then is hydrolyzed with alkali protease, then extracts wheat gliadin.It is low
Sensitization wheat gliadin refers to and never passes through the wheat gliadin extracted in the Gluten that removing anaphylactogen is handled
It compares, into after animal body, the allergy indication decline of animal body or the wheat gliadin of disappearance.
It is described with sodium tripolyphosphate first to Gluten phosphatizing treatment, be to mix Gluten with phosphate buffer, so
After add sodium tripolyphosphate, sodium tripolyphosphate reacts with Gluten.Further, the solid-liquid of Gluten and phosphate buffer
Than for 1g:(10~12.5) mL, the pH of phosphate buffer is 8.5~9.5.Further, the additive amount of sodium tripolyphosphate and paddy
The mass ratio of protein powder is 1:4.5~1:5.5, and sodium tripolyphosphate adds in batches.Further, the additive amount of sodium tripolyphosphate
Mass ratio with Gluten is 1:4.5~1:5.5, and sodium tripolyphosphate is divided into 3 equal portions and reaction system is added, opens respectively in reaction
Begin, reaction starts rear 10~15min and reaction starts addition when rear 25~30min；Reaction temperature is 25 DEG C, and the reaction time is
1h is stirred and is controlled the pH of reaction system in 8.5~9.5 ranges.
Described is hydrolyzed with alkali protease, is addition under the conditions of 60 DEG C~65 DEG C, 9.0~9.5 pH
Alkali protease carries out enzymatic hydrolysis reaction.Further, the additive amount of the alkali protease of every gram of Gluten is 180U~200U.
Further, the time of the enzymatic treatment can be 1 hour, and entire reaction process is stirred continuously, and be protected with 2M NaOH
The pH for holding reaction system is 9.0~9.5.
In one embodiment of the invention, the buffer of phosphatizing treatment is the phosphate buffer of pH9.5 or 9.0,
The solid-to-liquid ratio of Gluten and phosphate buffer is 1:10.
In one embodiment of the invention, the mass ratio of the additive amount of the sodium tripolyphosphate and Gluten is 1:
5。
In one embodiment of the invention, the temperature of phosphatizing treatment is 25 DEG C, and the processing time is 1h.
In one embodiment of the invention, when hydrolysis by novo is handled, the pH of reaction system is 9.5.
In one embodiment of the invention, when hydrolysis by novo is handled, the additive amount of alkali protease are as follows: every
200U alkali protease is added in gram Gluten.
In one embodiment of the invention, the termination method of enzyme hydrolysis processing is to increase the temperature of system
To 85 DEG C, 15min is handled, alkali protease is inactivated.
It is described untreated identical with the extracting mode of gliadin in treated Gluten, untreated and processing
Gluten in gliadin extracting method are as follows: the pH of treated Gluten system is adjusted to neutrality, and is freezed dry
Dry is powdery.Gluten is carried out with anhydrous ether to stay overnight ungrease treatment, after being repeated twice, after the anhydrous ether that volatilizees, is added
65% ethyl alcohol, 1h is extracted at 25 DEG C, and the solid-to-liquid ratio of extraction is 1:10.The supernatant 5000g of extraction is centrifuged 15min, by 40 DEG C,
90mbar rotary evaporation removes the ethyl alcohol in supernatant, obtains gliadin after freeze-drying.
The present invention also provides a kind of methods of the anaphylactogen in removing Gluten, are first with sodium tripolyphosphate first to Gluten
Phosphatizing treatment is carried out, then is hydrolyzed with alkali protease.
It is described with sodium tripolyphosphate first to Gluten phosphatizing treatment, be to mix Gluten with phosphate buffer, so
After add sodium tripolyphosphate, sodium tripolyphosphate reacts with Gluten.Further, the solid-liquid of Gluten and phosphate buffer
Than for 1g:(10~12.5) mL, the pH of phosphate buffer is 8.5~9.5.Further, the additive amount of sodium tripolyphosphate and paddy
The mass ratio of protein powder is 1:4.5~1:5.5, and sodium tripolyphosphate adds in batches.Further, the additive amount of sodium tripolyphosphate
Mass ratio with Gluten is 1:4.5~1:5.5, and sodium tripolyphosphate is divided into 3 equal portions and reaction system is added, opens respectively in reaction
Begin, reaction starts rear 10~15min and reaction starts addition when rear 25~30min；Reaction temperature is 25 DEG C, and the reaction time is
1h is stirred and is controlled the pH of reaction system in 8.5~9.5 ranges.
Described is hydrolyzed with alkali protease, is addition under the conditions of 60 DEG C~65 DEG C, 9.0~9.5 pH
Alkali protease carries out enzymatic hydrolysis reaction.Further, the additive amount of the alkali protease of every gram of Gluten is 180U~200U.
Further, the time of the enzymatic treatment can be 1 hour, and entire reaction process is stirred continuously, and be used in combination
It is 9.0~9.5 that 2M NaOH, which keeps the pH of reaction system,.
In one embodiment of the invention, the buffer of phosphatizing treatment is the phosphate buffer of pH9.5 or 9.0,
The solid-to-liquid ratio of Gluten and phosphate buffer is 1:10.
In one embodiment of the invention, the mass ratio of the additive amount of the sodium tripolyphosphate and Gluten is 1:
5。
In one embodiment of the invention, the temperature of phosphatizing treatment is 25 DEG C, and the processing time is 1h.
In one embodiment of the invention, when hydrolysis by novo is handled, the pH of reaction system is 9.5.
In one embodiment of the invention, when hydrolysis by novo is handled, the additive amount of alkali protease are as follows: every
200U alkali protease is added in gram Gluten.
In one embodiment of the invention, the termination method of enzyme hydrolysis processing is to increase the temperature of system
To 85 DEG C, 15min is handled, alkali protease is inactivated.
[beneficial effect]
(1) present invention process step is simple, the time of processing is short, the temperature of processing is low, avoids physical treatment mode
Disadvantage more than time length, energy consumption.
(2) by chemical treatments in conjunction with enzyme hydrolysis, the improvement of the rheological equationm of state of dough is not only improved, can also effectively be dropped
The anaphylaxis of low gliadin reduces the dosage of chemical reagent.The second level of wheat gliadin after phosphatizing treatment and
Tertiary structure has conspicuousness variation, using enzymatic hydrolysis will be not easy in wheat gliadin it is being digested, there is antigen determination section
The segment hydrolysis of position, reduces its molecular weight, antigenicity reduces, to reduce the sensitization of wheat gliadin.
(3) present invention employs the additive amounts of the sodium tripolyphosphate of median dose, and reaction system is added portionwise, and avoid
The variation of biggish pH occurs for reaction system.Lower treatment temperature also reduces the consumption of the energy.
(4) sensitization of the gliadin of combined processing mode is lower than the gliadin that single mode is handled.This hair
The gliadin of bright preparation is more easy to digest, safety, no bad flavor, without bitter taste.
Detailed description of the invention
It is described in detail by referring to the following drawings to made by treated wheat gliadin, feature of the invention, mesh
And advantage will become more apparent upon.
Pass through the structure change of phosphorylation-enzymolysis processing wheat gliadin in Fig. 1 embodiment 1.(A) secondary structure
Content；(B) intrinsic fluorescence spectroscopy；(C) surface hydrophobic；(D) free sulfhydryl groups content.Raw is the untreated molten egg of wheat alcohol
White, Phos-Alcalase is the wheat gliadin of phosphorylated-enzymolysis processing.
SDS- in Fig. 2 embodiment 1 by phosphorylation-enzymolysis processing wheat gliadin after in vitro digestion
PAGE map.(A): peptic digest 10min；(B): peptic digest 120min；(C): intestinal digestion 10min；Swimming lane 1 is untreated wheat
Alcohol soluble protein, swimming lane 2 are phosphorylation-enzymolysis processing wheat gliadin.
The serum of the allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Fig. 3 A embodiment 1
The variation of IgE concentration.
The serum of the allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Fig. 3 B embodiment 1
The variation of Histamine concentrations.
In Fig. 4 embodiment 1 the relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization because
Son and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Fig. 5 embodiment 2；(A): IgE
The variation of concentration；(B): the variation of serum Histamine concentrations.
In Fig. 6 embodiment 2 the relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization because
Son and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Fig. 7 embodiment 3；(A): IgE
The variation of concentration；(B): the variation of serum Histamine concentrations.
In Fig. 8 embodiment 3 the relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization because
Son and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Fig. 9 embodiment 4；(A): IgE
The variation of concentration；(B): the variation of serum Histamine concentrations.
The relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 10 embodiment 4
The factor and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 11 comparative example 1；(A):
The variation of IgE concentration；(B): the variation of serum Histamine concentrations.
The relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 12 comparative example 1
The factor and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 13 comparative example 2；(A):
The variation of IgE concentration；(B): the variation of serum Histamine concentrations.
The relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 14 comparative example 2
The factor and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 15 comparative example 3；(A):
The variation of IgE concentration；(B): the variation of serum Histamine concentrations.
The relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 16 comparative example 3
The factor and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
The allergy characterization of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 17 comparative example 4；(A):
The variation of IgE concentration；(B): the variation of serum Histamine concentrations.
The relevant cell of allergy of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization in Figure 18 comparative example 4
The factor and transcription factor expression are horizontal.(A): IFN-γ；(B): IL-4；(C): IL-5.
Specific embodiment
Related detecting method:
Secondary structure measurement: the secondary structure of Fourier's infrared method measurement wheat gliadin is used.32 times scanning and
4cm-1Under the conditions of record sample background scanning optical spectrum.Potassium bromide grinding drying after with sample ground and mixed, after being pressed into chip,
It is measured under record condition identical with background.Each sample does parallel three times.
Intrinsic fluorescence spectroscopy: the fluorescence spectrum of Fluorescence Spectrometer measurement wheat gliadin is used.The concentration of sample is
0.5mg/mL, excitation wavelength 280nm, launch wavelength range are 300-500nm.Scanning speed is 1200nm/min, when reaction
Between be 0.5s, voltage 500V, excitation and transmitting seam be 5nm.
Surface hydrophobic measurement: using the surface hydrophobic of ANS determination of color wheat gliadin.Wheat gluten is dissolved in
The phosphate buffer of pH 7.0, protein concentration range are 0.05-0.5mg/mL, and the 8.0mM of 20 μ L is added in every 4mL sample solution
ANS, the excitation wavelength of measurement is 390nm, launch wavelength 470nm.Fluorescence intensity makees slope of a curve table with protein concentration
Levy the surface hydrophobic of wheat gliadin.
The measurement of free sulfhydryl groups: Tris-glycine-EDTA solution of the wheat gliadin in 5mL of 3mg is weighed
30min, every 10min oscillation is primary, Ellman ' the s reagent of 40 μ L of addition, stores 30min at 25 DEG C after covering masking foil.
Measure the light absorption value at 412nm.
In-vitro simulated untreated and phosphorylation-enzymolysis processing wheat gliadin pipe intestinal digesting.Weigh the wheat of 8mg
Alcohol soluble protein mixes with the simulation peptic digest liquid (SGF) of 3.86mL in centrifuge tube, the 0.3M CaCl of 1 μ L is then added2With
The water of 139 μ L.0.16mL porcine pepsin is added after preheating in 37 DEG C of water-baths in each centrifuge tube, is placed in 37 DEG C of water-baths and reacts
2h.100 μ L sample digestions are taken to do SDS-PAGE analysis in 10min and 120min respectively.After digesting 2h, with 30 μ L NaHCO3
Terminate reaction.It takes the peptic digest sample of 2mL to mix with the simulation intestinal digestion liquid (SIF) of 1.1mL, after 37 DEG C of preheatings, 0.5mL is added
Pancreatic juice, be placed in 37 DEG C of digestion 4h.Sample digestion is taken to be SDS- in peptic digest 10min, 120min and intestinal digestion 10min respectively
PAGE analysis.
The measurement of associated allergic factor level in serum: the concentration of IgE in serum, histamine, IL-4, IL-5 are all made of
ELISA kit measurement.According to the concentration for illustrating to measure each index of each kit.
RNA is extracted and real-time quantitative fluorescence PCR: being extracted from C2C12 cell and tissue sample always using Trizol reagent
RNA, and RNA concentration is measured with Nanodrop.The first chain is synthesized using Prime Script RT system (Takara)
cDNA.Real-time quantitative PCR carries out in ABI STEP-ONE7900 RT-PCR system.Each sample does parallel three times.
The present invention carries out sensitization processing to BALB/c mouse by the gliadin in intraperitoneal injection wheat.
Embodiment 1
100g Gluten is weighed in the beaker for the phosphate buffer for being 9.5 equipped with 1000mL pH, 1000mL buffer is pre-
25 DEG C of water-baths preheating 30min are placed in, guarantee that entire reaction system be reaction temperature is 25 DEG C, and with the pH of pH measurement reaction system
Value, monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.Weigh 20g trimerization
Sodium phosphate is divided into three equal parts and is slowly added in reaction system, according to the numerical value change of pH meter, the NaOH of 2M is constantly added dropwise, makes pH
Stablize 9.0~9.5, reacts 1h.
It places the beaker in 65 DEG C of water-baths, the pH for adjusting reaction system is 9.0~9.5, and the basic protein of 20000U is added
Enzyme is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, reaction system is placed in 85 DEG C of water-baths
15min carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, and pellet frozen is dried.
The wheat gliadin in Gluten after extraction process: claim paddy of the 50g after above-mentioned phosphorylation-enzymolysis processing
Protein powder, be added 500mL 65% ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotates
It is freeze-dried after evaporating ethanol, obtains phosphorylation-enzymolysis processing wheat gliadin.Similarly, never pass through phosphorus
It is extracted in acidification-enzymolysis processing Gluten and obtains wheat gliadin.
Wheat gliadin after not phosphorylated-enzymolysis processing and phosphorylated-enzymolysis processing is dissolved in 0.01mol/
The PBS of L, pH 7.4, concentration 1mg/mL.Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitization after mixing in equal volume
Mouse.Intraperitoneal injection for the first time is first day, and the injection dosage of every mouse is 0.1mL, respectively again at the 7th day and the 14th day
Sensitization, total sensitization is three times.Every group of mouse has 5, and control group (CON) injects isometric PBS, in addition two groups inject respectively without
Phosphorylation-enzymolysis processing wheat gliadin (NT) and phosphorylation-enzymolysis processing wheat gliadin (Phos-
Alcalase).28th day, the index and spleen index of mouse is measured, relevant cell factor in IgE, Histamine concentrations and spleen in serum
MRNA expression.
As shown in Figure 1, the secondary structure of the wheat gliadin after (A) phosphorylation-enzymolysis processing has apparent change,
Middle alpha-helix, beta sheet and β-corner have apparent change.(B) fluorescence intensity has apparent reduction, and the position at top goes out
Existing Red Shift Phenomena, illustrates that the tertiary structure of wheat gliadin changes, and the micro-loop of tryptophan therein and tyrosine
Border also changes.(C) surface hydrophobic of phosphorylation-enzymolysis processing wheat gliadin has significant decrease, illustrates albumen
The position of the hydrophobic grouping on surface significantly changes, and also reflects the variation of the tertiary structure of albumen.(D) phosphorylation-enzymatic hydrolysis
The free SH content of the wheat gliadin of processing has significant increase, illustrates the fracture of the chemical bond of albumen, further illustrates
Significant change occurs for the three-level and quaternary structure of albumen.
As shown in Fig. 2, the in vitro digestion of phosphorylation-enzymolysis processing wheat gliadin the experimental results showed that, phosphorus
Acidification-enzymolysis processing wheat gliadin is easier to be digested, and is almost digested after enteron aisle 10min.
As shown in figure 3, compared with the control group, use is phosphorylated-mouse of the wheat gliadin sensitization of enzymolysis processing
The equal conspicuousness of every allergy index increase, and (A) serum of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization
IgE concentration；(B) serum Histamine concentrations have a conspicuousness reduction than untreated wheat gliadin, and with unsensitized mouse phase
Than there was no significant difference for the allergy feature of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization.
As shown in figure 4, compared with the control group, use is phosphorylated-mouse of the wheat gliadin sensitization of enzymolysis processing
The equal conspicuousness of allergy relevant cell factor increase, and the mistake of the mouse of phosphorylation-enzymolysis processing wheat gliadin sensitization
Quick relevant cell factor has conspicuousness reduction than untreated wheat gliadin.
Embodiment 2
100g Gluten is weighed in the beaker for the phosphate buffer for being 8.5~9.5 equipped with 1000mL pH, 1000mL is slow
Fliud flushing is preset in 25 DEG C of water-baths preheating 30min, guarantees that entire reaction system be reaction temperature is 25 DEG C, and measure reactant with pH
The pH value of system monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.It weighs
17.8g sodium tripolyphosphate is divided into three equal parts and is slowly added in reaction system, according to the numerical value change of pH meter, is constantly added dropwise 2M's
NaOH stablizes pH 8.5~9.5, reacts 1h.
It places the beaker in 65 DEG C of water-baths, the pH for adjusting reaction system is 9.0~9.5, and the basic protein of 20000U is added
Enzyme is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, reaction system is placed in 85 DEG C of water-baths
15min carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, and pellet frozen is dried.
The wheat gliadin in Gluten after extraction process: claim paddy of the 50g after above-mentioned phosphorylation-enzymolysis processing
Protein powder, be added 500mL 65% ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotates
It is freeze-dried after evaporating ethanol, obtains phosphorylation-enzymolysis processing wheat gliadin.Similarly, never pass through phosphorus
It is extracted in acidification-enzymolysis processing Gluten and obtains wheat gliadin.
Wheat gliadin after not phosphorylated-enzymolysis processing and phosphorylated-enzymolysis processing is dissolved in 0.01mol/
The PBS of L, pH 7.4, concentration 1mg/mL.Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitization after mixing in equal volume
Mouse.Intraperitoneal injection for the first time is first day, and the injection dosage of every mouse is 0.1mL, respectively again at the 7th day and the 14th day
Sensitization, total sensitization is three times.Every group of mouse has 5, and control group (CON) injects isometric PBS, in addition injects and does not locate respectively for two groups
The alcohol soluble protein (NT) and phosphorylation-enzymolysis processing alcohol soluble protein (Phos-Alcalase) of reason.28th day, measure mouse
Index and spleen index, in serum in IgE, Histamine concentrations and spleen relevant cell factor mRNA expression.As a result such as Fig. 5, figure
6。
Embodiment 3
100g Gluten is weighed in the beaker for the phosphate buffer for being 8.5~9.5 equipped with 1000mL pH, 1000mL is slow
Fliud flushing is preset in 25 DEG C of water-baths preheating 30min, guarantees that entire reaction system be reaction temperature is 25 DEG C, and measure reactant with pH
The pH value of system monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.It weighs
17.8g sodium tripolyphosphate is divided into three equal parts and is slowly added in reaction system, according to the numerical value change of pH meter, is constantly added dropwise 2M's
NaOH stablizes pH 8.5~9.5, reacts 1h.
It places the beaker in 65 DEG C of water-baths, the pH for adjusting reaction system is 9.0~9.5, and the basic protein of 18000U is added
Enzyme is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, reaction system is placed in 85 DEG C of water-baths
15min carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, and pellet frozen is dried.
The wheat gliadin in Gluten after extraction process: claim paddy of the 50g after above-mentioned phosphorylation-enzymolysis processing
Protein powder, be added 500mL 65% ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotates
It is freeze-dried after evaporating ethanol, obtains phosphorylation-enzymolysis processing wheat gliadin.Similarly, never pass through phosphorus
It is extracted in acidification-enzymolysis processing Gluten and obtains wheat gliadin.
Wheat gliadin after not phosphorylated-enzymolysis processing and phosphorylated-enzymolysis processing is dissolved in 0.01mol/
The PBS of L, pH 7.4, concentration 1mg/mL.Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitization after mixing in equal volume
Mouse.Intraperitoneal injection for the first time is first day, and the injection dosage of every mouse is 0.1mL, respectively again at the 7th day and the 14th day
Sensitization, total sensitization is three times.Every group of mouse has 5, and control group (CON) injects isometric PBS, in addition injects and does not locate respectively for two groups
The alcohol soluble protein (NT) and phosphorylation-enzymolysis processing alcohol soluble protein (Phos-Alcalase) of reason.28th day, measure mouse
Index and spleen index, in serum in IgE, Histamine concentrations and spleen relevant cell factor mRNA expression.As a result such as Fig. 7, figure
8。
Embodiment 4
100g Gluten is weighed in the beaker for the phosphate buffer for being 8.5~9.5 equipped with 1250mL pH, 1000mL is slow
Fliud flushing is preset in 25 DEG C of water-baths preheating 30min, guarantees that entire reaction system be reaction temperature is 25 DEG C, and measure reactant with pH
The pH value of system monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.It weighs
14.5g sodium tripolyphosphate is divided into three equal parts and is slowly added in reaction system, according to the numerical value change of pH meter, is constantly added dropwise 2M's
NaOH stablizes pH 8.5~9.5, reacts 1h.
It places the beaker in 65 DEG C of water-baths, the pH for adjusting reaction system is 9.0~9.5, and the basic protein of 18000U is added
Enzyme is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, reaction system is placed in 85 DEG C of water-baths
15min carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, and pellet frozen is dried.
The wheat gliadin in Gluten after extraction process: claim paddy of the 50g after above-mentioned phosphorylation-enzymolysis processing
Protein powder, be added 500mL 65% ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotates
It is freeze-dried after evaporating ethanol, obtains phosphorylation-enzymolysis processing wheat gliadin.Similarly, never pass through phosphorus
It is extracted in acidification-enzymolysis processing Gluten and obtains wheat gliadin.
Wheat gliadin after not phosphorylated-enzymolysis processing and phosphorylated-enzymolysis processing is dissolved in 0.01mol/
The PBS of L, pH 7.4, concentration 1mg/mL.Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitization after mixing in equal volume
Mouse.Intraperitoneal injection for the first time is first day, and the injection dosage of every mouse is 0.1mL, respectively again at the 7th day and the 14th day
Sensitization, total sensitization is three times.Every group of mouse has 5, and control group (CON) injects isometric PBS, in addition injects and does not locate respectively for two groups
The alcohol soluble protein (NT) and phosphorylation-enzymolysis processing alcohol soluble protein (Phos-Alcalase) of reason.28th day, measure mouse
Index and spleen index, in serum in IgE, Histamine concentrations and spleen relevant cell factor mRNA expression.As a result such as Fig. 9, figure
10。
Comparative example 1
100g Gluten is weighed in the beaker for the phosphate buffer for being 8.5~9.5 equipped with 2000mL pH, 1000mL is slow
Fliud flushing is preset in 37 DEG C of water-baths preheating 30min, guarantees that entire reaction system be reaction temperature is 37 DEG C, and measure reactant with pH
The pH value of system monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.It weighs
20g sodium tripolyphosphate is divided into three equal parts and is slowly added in reaction system, according to the numerical value change of pH meter, is constantly added dropwise 2M's
NaOH stablizes pH 8.5~9.5, reacts 2h.
It places the beaker in 65 DEG C of water-baths, the pH for adjusting reaction system is 9.0~9.5, and the basic protein of 10000U is added
Enzyme is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, reaction system is placed in 85 DEG C of water-baths
15min carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, and pellet frozen is dried.
The wheat gliadin in Gluten after extraction process: claim paddy of the 50g after above-mentioned phosphorylation-enzymolysis processing
Protein powder, be added 500mL 65% ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotates
It is freeze-dried after evaporating ethanol, obtains phosphorylation-enzymolysis processing wheat gliadin.Similarly, never pass through phosphorus
It is extracted in acidification-enzymolysis processing Gluten and obtains wheat gliadin.
Wheat gliadin after not phosphorylated-enzymolysis processing and phosphorylated-enzymolysis processing is dissolved in 0.01mol/
The PBS of L, pH 7.4, concentration 1mg/mL.Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitization after mixing in equal volume
Mouse.Intraperitoneal injection for the first time is first day, and the injection dosage of every mouse is 0.1mL, respectively again at the 7th day and the 14th day
Sensitization, total sensitization is three times.Every group of mouse has 5, and control group (CON) injects isometric PBS, in addition injects and does not locate respectively for two groups
The alcohol soluble protein (NT) and phosphorylation-enzymolysis processing alcohol soluble protein (Phos-Alcalase) of reason.28th day, measure mouse
Index and spleen index, in serum in IgE, Histamine concentrations and spleen relevant cell factor mRNA expression.As a result such as Figure 11, figure
12。
Comparative example 2
100g Gluten is weighed in the beaker for the phosphate buffer for being 8.5~9.5 equipped with 1000mL pH, 1000mL is slow
Fliud flushing is preset in 25 DEG C of water-baths preheating 30min, guarantees that entire reaction system be reaction temperature is 25 DEG C, and measure reactant with pH
The pH value of system monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.It weighs
20g sodium tripolyphosphate is divided into three equal parts and is slowly added in reaction system, according to the numerical value change of pH meter, is constantly added dropwise 2M's
NaOH stablizes pH 8.5~9.5, reacts 30min.
It places the beaker in 65 DEG C of water-baths, the pH for adjusting reaction system is 9.0~9.5, and the basic protein of 20000U is added
Enzyme is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, reaction system is placed in 85 DEG C of water-baths
15min carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, and pellet frozen is dried.
The wheat gliadin in Gluten after extraction process: claim paddy of the 50g after above-mentioned phosphorylation-enzymolysis processing
Protein powder, be added 500mL 65% ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotates
It is freeze-dried after evaporating ethanol, obtains phosphorylation-enzymolysis processing wheat gliadin.Similarly, never pass through phosphorus
It is extracted in acidification-enzymolysis processing Gluten and obtains wheat gliadin.
Wheat gliadin after not phosphorylated-enzymolysis processing and phosphorylated-enzymolysis processing is dissolved in 0.01mol/
The PBS of L, pH 7.4, concentration 1mg/mL.Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitization after mixing in equal volume
Mouse.Intraperitoneal injection for the first time is first day, and the injection dosage of every mouse is 0.1mL, respectively again at the 7th day and the 14th day
Sensitization, total sensitization is three times.Every group of mouse has 5, and control group (CON) injects isometric PBS, in addition injects and does not locate respectively for two groups
The alcohol soluble protein (NT) and phosphorylation-enzymolysis processing alcohol soluble protein (Phos-Alcalase) of reason.28th day, measure mouse
Index and spleen index, in serum in IgE, Histamine concentrations and spleen relevant cell factor mRNA expression.As a result such as Figure 13, figure
14。
Comparative example 3 is individually handled using sodium tripolyphosphate
100g Gluten is weighed in the beaker for the phosphate buffer for being 9.5 equipped with 1000mL pH, 1000mL buffer is pre-
25 DEG C of water-baths preheating 30min are placed in, guarantee that entire reaction system be reaction temperature is 25 DEG C, and with pH measurement reaction system
PH value monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.Weigh 20g tri-
Polyphosphate sodium is divided into quarter and is slowly added in reaction system, according to the numerical value change of pH meter, the NaOH of 2M is constantly added dropwise, makes
PH stablizes 9.5, reacts 1h.
The wheat gliadin in Gluten after extraction process: claiming 50g treated Gluten, and 500mL 65% is added
Ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotary evaporation freezes after removing ethyl alcohol
It is dry, obtain wheat gliadin.Similarly, wheat gliadin is extracted from untreated Gluten.
By untreated and treated, wheat gliadin is dissolved in 0.01mol/L, the PBS of pH 7.4, concentration 1mg/mL.
Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitized mice after mixing in equal volume.Intraperitoneal injection for the first time is first
It, the injection dosage of every mouse is 0.1mL, and the sensitization again at the 7th day and the 14th day, is total to sensitization three times respectively.Every group of mouse
There are 5, control group (CON) injects isometric PBS, in addition injects untreated alcohol soluble protein (NT) and phosphoric acid respectively for two groups
Change-enzymolysis processing alcohol soluble protein (Phos-Alcalase).28th day, the index and spleen index of mouse is measured, IgE, histamine in serum
The mRNA expression of relevant cell factor in concentration and spleen.
The result shows that:
Individual phosphatizing treatment to the structure of wheat gliadin there is no the change of conspicuousness, phosphatizing treatment it is small
There was no significant difference for the mouse of the allergy feature of the mouse of gliadin sensitization and untreated wheat gliadin sensitization.Knot
Fruit such as Figure 15, Figure 16.
Comparative example 4 is individually handled using alkali protease
100g Gluten is weighed in the beaker for the phosphate buffer for being 9.0~9.5 equipped with 1000mL pH, 1000mL is slow
Fliud flushing is preset in 65 DEG C of water-baths preheating 30min, guarantees that entire reaction system be reaction temperature is 65 DEG C, and measure reactant with pH
The pH value of system monitors the variation of pH.Under the revolving speed of blender 100rpm, it is dispersed in Gluten in buffer solution system.It is added
The alkali protease of 20000U is maintained at 9.0~9.5 with the pH that the NaOH of 2M adjusts reaction system, after digesting 1h, by reactant
System is placed in 15min in 85 DEG C of water-baths, carries out destroy the enzyme treatment.Then 4000g is centrifuged 15min, abandons supernatant, at pellet frozen is dry
Reason.
The wheat gliadin in Gluten after extraction process: claiming 50g treated Gluten, and 500mL 65% is added
Ethyl alcohol and 25 DEG C at extract 1h.5000g is centrifuged 15min, takes supernatant, 40 DEG C, 90mbar rotary evaporation freezes after removing ethyl alcohol
It is dry, obtain wheat gliadin.Similarly, wheat gliadin is extracted from untreated Gluten.
By untreated and treated, wheat gliadin is dissolved in 0.01mol/L, the PBS of pH 7.4, concentration 1mg/mL.
Again with the aluminum hydroxide adjuvant of 40mg/mL by 1:1 sensitized mice after mixing in equal volume.Intraperitoneal injection for the first time is first
It, the injection dosage of every mouse is 0.1mL, and the sensitization again at the 7th day and the 14th day, is total to sensitization three times respectively.Every group of mouse
There are 5, control group (CON) injects isometric PBS, in addition injects untreated alcohol soluble protein (NT) and phosphoric acid respectively for two groups
Change-enzymolysis processing alcohol soluble protein (Phos-Alcalase).28th day, the index and spleen index of mouse is measured, IgE, histamine in serum
The mRNA expression of relevant cell factor in concentration and spleen.
The result shows that:
Individual phosphatizing treatment to the structure of wheat gliadin there is no the change of conspicuousness, phosphatizing treatment it is small
There was no significant difference for the mouse of the allergy feature of the mouse of gliadin sensitization and untreated wheat gliadin sensitization.Knot
Fruit such as Figure 17, Figure 18.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for preparing low irritability wheat gliadin, which is characterized in that first with sodium tripolyphosphate first to Gluten
Phosphatizing treatment is carried out, then is hydrolyzed with alkali protease, wheat gliadin is then extracted.
2. a kind of method for preparing low irritability wheat gliadin according to claim 1, which is characterized in that described
With sodium tripolyphosphate first to Gluten phosphatizing treatment, it is to mix Gluten with phosphate buffer, then adds tripolyphosphate
Sodium, sodium tripolyphosphate react with Gluten.
3. a kind of method for preparing low irritability wheat gliadin according to claim 2, which is characterized in that Gluten
Solid-to-liquid ratio with phosphate buffer is 1g:(10~12.5) mL, the pH of phosphate buffer is 8.5~9.5.
4. a kind of method for preparing low irritability wheat gliadin according to claim 2 or 3, which is characterized in that three
The additive amount of polyphosphate sodium and the mass ratio of Gluten are 1:4.5~1:5.5, and sodium tripolyphosphate adds in batches.
5. a kind of method for preparing low irritability wheat gliadin according to claim 4, which is characterized in that trimerization phosphorus
The additive amount of sour sodium and the mass ratio of Gluten are 1:4.5~1:5.5, and sodium tripolyphosphate is divided into 3 equal portions and reaction system is added, point
Do not start in reaction, react start after 10~15min and reaction start after 25~30min when addition；Reaction temperature is 25 DEG C,
Reaction time is 1h, stirs and controls the pH of reaction system in 8.5~9.5 ranges.
6. a kind of any method for preparing low irritability wheat gliadin, feature exist according to claim 1~5
In, it is described to be hydrolyzed with alkali protease, it is to add alkaline egg under the conditions of 60 DEG C~65 DEG C, 9.0~9.5 pH
White enzyme carries out enzymatic hydrolysis reaction.
7. a kind of method for preparing low irritability wheat gliadin according to claim 6, which is characterized in that every gram of paddy
The additive amount of the alkali protease of protein powder is 180U~200U.
8. a kind of method of the anaphylactogen in removing Gluten, which is characterized in that first first carried out to Gluten with sodium tripolyphosphate
Phosphatizing treatment, then be hydrolyzed with alkali protease.
9. the method for the anaphylactogen in a kind of removing Gluten according to claim 8, which is characterized in that described with three
Polyphosphate sodium is to mix Gluten with phosphate buffer first to Gluten phosphatizing treatment, then adds sodium tripolyphosphate, three
Polyphosphate sodium reacts with Gluten；The solid-to-liquid ratio of Gluten and phosphate buffer is 1g:(10~12.5) mL, phosphoric acid buffer
The pH of liquid is 8.5~9.5；The additive amount of sodium tripolyphosphate and the mass ratio of Gluten are 1:4.5~1:5.5, sodium tripolyphosphate
It adds in batches；Described is hydrolyzed with alkali protease, is added under the conditions of 60 DEG C~65 DEG C, 9.0~9.5 pH
Alkali protease is added to carry out enzymatic hydrolysis reaction；The additive amount of the alkali protease of every gram of Gluten is 180U~200U.
10. any the method is prepared according to claim 1~8 low irritability wheat gliadin or wanted by right
Seek 9 the methods treated Gluten, and the low irritability that any the method is prepared according to claim 1~8
Wheat gliadin or treated that Gluten is preparing the application in food by claim 9 the method.
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| PCT/CN2020/087101 WO2020221180A1 (en) | 2019-04-30 | 2020-04-27 | Method for preparing hypoallergenic wheat gliadin by means of phosphorylation-enzymatic method |
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|---|---|---|---|---|
| WO2020221180A1 (en) * | 2019-04-30 | 2020-11-05 | 江南大学 | Method for preparing hypoallergenic wheat gliadin by means of phosphorylation-enzymatic method |
| CN112220005A (en) * | 2020-09-28 | 2021-01-15 | 集美大学 | Hypoallergenic scallop meat porridge and preparation method thereof |
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Non-Patent Citations (4)
| Title |
|---|
| YING LI等: "The potential of papain and alcalase enzymes and process optimizations to reduce allergenic gliadins in wheat flour", 《FOOD CHEM》 * |
| 孟良玉等: "小麦致敏蛋白的研究进展", 《食品与发酵工艺》 * |
| 朱永胜: "STP磷酸化处理对小麦面筋蛋白酶解特性的影响", 《河南工业大学学报自然科学版》 * |
| 毛炜翔等: "小麦过敏研究进展", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020221180A1 (en) * | 2019-04-30 | 2020-11-05 | 江南大学 | Method for preparing hypoallergenic wheat gliadin by means of phosphorylation-enzymatic method |
| CN112220005A (en) * | 2020-09-28 | 2021-01-15 | 集美大学 | Hypoallergenic scallop meat porridge and preparation method thereof |
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