CN113981028A

CN113981028A - Method for producing wheat oligopeptide by multi-enzyme synergistic method

Info

Publication number: CN113981028A
Application number: CN202111328593.0A
Authority: CN
Inventors: 黄继红; 侯银臣; 廖爱美; 魏兆军; 董得平; 王晓曦; 杨玉岭; 潘龙; 臧立华; 杜丽平; 冯军伟
Original assignee: Henan University of Technology
Current assignee: Henan University of Technology
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-01-28

Abstract

The invention relates to a method for producing wheat oligopeptide by a multi-enzyme synergistic method. The method comprises the following steps: mixing pulp, adding a certain amount of pure water for adjusting pH into a mixing tank; receiving emulsion in a blending section, and performing enzymolysis for two times; the secondary enzymolysis liquid passes through an ultrafiltration membrane to remove macromolecular protein; filtering the ultrafiltrate with nanofiltration membrane to remove small molecular salt and oligopeptide substance lower than 100 Da; spray drying the product to finally obtain the water-soluble oligopeptide with the water content of 7 percent, and the obtained product has high purity and high yield.

Description

Method for producing wheat oligopeptide by multi-enzyme synergistic method

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for extracting oligopeptide from wheat by utilizing multiple enzymes.

Background

The wheat oligopeptide is a micromolecular polypeptide substance obtained by extracting protein from a natural food, namely wheat protein powder, and then carrying out directional enzyme digestion and a specific small peptide separation technology. The oligopeptide can inhibit cholesterol increase. The wheat oligopeptide can promote insulin secretion, and the functional substance is oligomethionine, and can be used for regulating blood sugar of human and improving diabetes symptoms. The wheat peptide can block the action of angiotensin enzyme, thus has the effect of lowering blood pressure. One of the characteristics of the wheat oligopeptide is that the wheat oligopeptide contains homoglutamine, can effectively regulate nerves and can be used as a special nutrient substance in the case of intestinal dysfunction. Has ACE inhibiting, immunoregulating, antioxidant and other bioactivity, and can stimulate lymphocyte proliferation, enhance macrophage phagocytic function, raise body's resistance to infection of outer pathogen, lower body's morbidity, etc. The wheat oligopeptide is a wheat proteolysis product, can inhibit the activity of angiotensin converting enzyme, and can prevent angiotensinogen from being converted into angiotensin II which can increase blood pressure, thereby physiologically reducing blood pressure and having no effect on normal blood pressure.

Disclosure of Invention

The invention aims to provide a method for producing wheat oligopeptide by a multi-enzyme synergistic method, and in order to achieve the aim, the following technical scheme is adopted in the application:

a method for producing wheat oligopeptide by a multi-enzyme synergistic method comprises the following steps: size mixing, secondary enzymolysis, ultrafiltration, nanofiltration and drying.

The step of size mixing comprises the steps of adding pure water for adjusting the pH value into a mixing tank, directly feeding wet gluten separated in the production process into the mixing tank, stirring while feeding, and pumping into an enzymolysis tank after emulsion is formed.

The secondary enzymolysis step comprises the steps of receiving emulsion in the size mixing step, and carrying out primary enzymolysis: adding the compounded No. 1 enzyme liquid while stirring, and hydrolyzing at a certain pH value and temperature; and (3) carrying out second enzymolysis: adding No. 2 compound enzyme liquid while stirring, and hydrolyzing at certain pH value and temperature.

The enzyme in the No. 1 enzyme solution is selected from Alcalase protease, neutral protease AS1398, animal and plant proteolytic enzyme, neutral protease Nutrase and pepsin.

The enzyme in the No. 1 enzyme solution is selected from Alcalase protease.

The pH value of the first enzymolysis is 7-9, the temperature is 40-60 ℃, and the hydrolysis time is 2-6 hours.

The enzyme in the No. 2 enzyme solution is selected from one or more of Alcalase protease, neutral protease AS1398, animal and plant proteolytic enzyme, neutral protease Nutrase, pepsin, papain and flavourzyme.

The pH value of the second enzymolysis is 6-7, the temperature is 40-50 ℃, and the hydrolysis time is 3-5 hours.

Drawings

FIG. 1 is a standard graph of tyrosine concentration versus absorbance;

FIG. 2 is a standard curve graph of glycine solution concentration versus absorbance;

FIG. 3 is a graph of Degree of Hydrolysis (DH) versus time;

FIG. 4(a) is a graph showing the relationship between pH and alkali consumption;

FIG. 4(b) is a graph showing the relationship between the enzyme concentration and the alkali consumption;

FIG. 4(c) is a graph showing the relationship between time and alkali consumption;

FIG. 4(d) is a graph showing the relationship between temperature and alkali consumption;

FIG. 5 is an electropherogram.

Examples

The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention.

The material and the method are as follows:

wheat gluten starch production in starch factories

Alkaline serine protease Alcalaes protease Denmark Novicin (China) Inc

Kaihin Hainin, a Kingchao industries Ltd

Shunhua bioengineering, Inc. of pepsin

Neutral protease AS1398 Stannless enzyme preparation plant

Neutral protease Nutrase Denmark Nuweixin (China)

Animal and plant proteolytic enzyme Guangxi Nanning Longbo bioengineering Co., Ltd

Peking Biochemical products Co., Ltd of papain

Flavor protease Jibao (Qingdao) Biotech Co., Ltd

Hydrochloric acid, sodium hydroxide, lysine and the like are all of analytical pure grades on the market.

Beijing Rayleigh Analyzer Co, UV ultraviolet Spectrophotometer

Hitachi 835-50 amino acid automatic analyzer Tongji university electromechanical plant

Shanghai laboratory instruments Co., Ltd of 101-2A digital display type electrothermal blowing drying oven

JJ-1 electric Agitator, Changzhou Guohua appliances Co., Ltd

Precision PH meter Shanghai Lei magnetic instrument factory

Constant temperature water bath kettle Yao city oriental electrical instrument factory

High speed centrifuge Beijing centrifuge plant

Constant temperature drying oven Shanghai Neisster Instrument plant

Electronic balance Shanghai Mei Teller-Tollido instrument factory

Shanghai fiber inspection Instrument Co Ltd of digestion furnace

The preparation method comprises the following steps:

wheat gluten is used as a raw material, the hydrolysis degree of 6 proteases on the gluten is compared, and alkaline protease is selected as an enzyme for the first step of enzymolysis, so that the optimal hydrolysis condition and dosage are determined; and further carrying out enzymolysis by adopting papain and flavourzyme. And centrifuging, microfiltering, ultrafiltering and spray drying the product to obtain an oligopeptide mixture.

Six different proteases were selected for hydrolysis. Preparing 5% solution from wheat gluten, adding alkaline protease, neutral protease, pepsin and animal and plant protein hydrolase into 100mL of 2000u/g protein, drawing up appropriate temperature and pH value of each enzyme according to data, determining hydrolysis degree and protein conversion rate after hydrolyzing for 5h, comparing hydrolysis capacity of each enzyme, determining content of free amino acid in hydrolysate, and finally determining the optimal protease by integrating the indexes.

Carrying out first enzymolysis: according to the preparation experiment and related data, carrying out L on four factors of hydrolysis reaction temperature, pH value, enzyme dosage and substrate concentration₉(3⁴) Orthogonal experiments, see table 1, determine the optimal hydrolysis conditions based on the degree of hydrolysis.

TABLE 1 orthogonal test factor horizon

The hydrolysis was carried out under the selected hydrolysis conditions as shown in the above table, and different hydrolysis times were selected, and the optimum hydrolysis time was determined using the degree of hydrolysis (ninhydrin method) as an index.

In order to prepare high F value oligopeptide with special amino acid composition, namely product with low content of aromatic amino acid and high content of branched chain amino acid, the second enzymolysis needs protease to cut off the aromatic amino acid at the tail end of the peptide chain, so that the aromatic amino acid is free, and the complete dearomatization is hoped. The protease releasing aromatic amino acid is preferably actin and secondly papain, but the actin is expensive and not suitable for industrial production, so that papain and flavourzyme are selected for enzymolysis from the practical production point of view, and the flavourzyme is added, so that the bitter taste of the peptide can be removed, and the hydrolysis degree of the peptide is increased. The main factors influencing the enzymolysis effect are as follows: pH (A), enzyme dosage [ E ]/[ S ]% (B), time (C) and temperature (D). The optimum process conditions are determined by adopting an orthogonal test method and taking the alkali consumption as an examination index, and the factor level is shown in table 2. After the reaction is finished, the pH is adjusted to 2.8, the enzymolysis liquid is heated to 90 ℃, and the temperature is preserved for 10min for enzyme deactivation. Then rapidly cooling to room temperature, and centrifuging the hydrolysate for 15min (4000 r/min).

TABLE 2 orthogonal experimental design for complex enzyme hydrolysis process

The determination of the hydrolysis degree adopts an indantrione method and a pH-stat method; the determination of the amino nitrogen adopts a formaldehyde potentiometric titration method; the branched chain amino acid has a maximum absorption peak at 220nm, and an ultraviolet spectrophotometer is used for detection; the aromatic amino acid has a maximum absorption peak at 280nm, and an ultraviolet spectrophotometer is used for detection; amino acid components are automatically analyzed by an amino acid analyzer; the molecular weight distribution of the oligopeptide mixture with the high F value adopts a sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (SDS-APGE) electrophoresis method; the enzyme activity is measured by adopting a Folin-phenol reagent method.

The protein conversion was determined by the following method:

according to the method introduced by Mannheim, 10mL of protein hydrolysate obtained after hydrolysis and centrifugation is taken, 10mL of 10% trichloroacetic acid (TCA) is added, the mixture is uniformly mixed and then stands for 30min, a centrifuge is used for centrifuging for 10min at 4000r/min, 5mL of centrifuged supernatant is taken for digestion, and the volume of the digested solution is determined by a 100mL volumetric flask. And finally, determining the protein content by using a Kjeldahl method. Protein conversion was calculated.

Calculating the formula:

x-conversion of protein;

dissolving the P-enzymolysis solution in 10 percent of TCA;

P₀dissolving 10% TCA in the non-enzymatic hydrolysate;

S₀-total nitrogen content of substrate protein.

The relationship between tyrosine concentration and absorbance is shown in table 3:

TABLE 3 relationship of tyrosine concentration to Absorbance value

The absorbance is used as ordinate, the tyrosine concentration is used as abscissa, the standard curve is drawn as shown in figure 1, the relationship between A and C is: C-100.03A-1.0006 (R)²＝0.9988)。

The results of the measurement of the absorbance value and the calculation of the enzyme activity when the hydrolysis time t is 10min are shown in Table 4.

TABLE 4 Activity of various proteases

Determination of conditions for the first enzymatic hydrolysis:

the standard curve of the degree of hydrolysis is plotted in table 5 and fig. 2.

TABLE 5 Glycine solution concentration vs. absorbance

The absorbance was plotted on the ordinate and the glycine concentration on the abscissa, to obtain a standard curve, as shown in FIG. 2.

Since glycine has a molecular weight of 75.07, it can be converted into-NH₂μ mol number of radicals:

C＝0.3162A+7.73×l0^-5(R²＝0.9956)

measuring the light absorption value of the hydrolysate to calculate-NH₂μ mol of radicals.

Screening for optimal proteases: six different enzymes were selected and compared for the degree of hydrolysis of gluten, and the results are shown in table 6. The amino acids in the hydrolysate were analyzed, the free amino acid content of several proteases hydrolyzing vital gluten is shown in Table 7, and the two indexes are combined to determine the best enzyme.

TABLE 6 degree of hydrolysis of wheat gluten by different proteases

As can be seen from table 6, the ability of several enzymes to hydrolyze vital gluten is, in order: alcalase, alkaline protease (made in China), neutral protease AS1398, animal and plant proteolytic enzyme, neutral protease Nuchase and pepsin.

The alkaline protease Alcalase catalyzes the hydrolysis of gluten to a greater degree because gluten has a greater solubility in alkaline solutions so that the enzyme and protein can be sufficiently contacted and hydrolyzed. In addition, Alcalase, a serine protease, is a non-specific protein endopeptidase that acts mainly on peptide bonds containing tyrosine, phenylalanine, and tryptophan, and cleaves the peptide chain from within the peptide chain to form two peptides, which better decompose natural proteins and peptides. The degree of hydrolysis of gluten by pepsin is relatively small, and pepsin can rapidly hydrolyze a peptide chain formed by phenylalanine residues, but gluten prolamin is difficult to disperse in an acidic aqueous solution, so that pepsin cannot sufficiently exert a catalytic effect.

TABLE 7 ratio of free BCAA and AAA in Total free amino acids

Furthermore, as can be seen from table 7, there are large differences in the free amino acids produced by the various enzymatic hydrolysis of gluten, and since the present experiment is intended to obtain a peptide solution with a high branched chain amino acid content and a low aromatic amino acid content, the branched chain amino acids should be retained as much as possible during hydrolysis so as to be present in the form of peptide bonds, and the aromatic amino acids should be released most. Among these enzymes, Alcalase acts on wheat gluten to produce relatively few free branched amino acids and relatively many free aromatic amino acids. In view of the yield of the hydrolysate and the economic efficiency, Alcalase protease was selected as the enzyme for the test in this test, in combination with the above test results.

Determination of optimal hydrolysis conditions: the technological conditions of the protease hydrolysis of the protein not only have great influence on the hydrolysis effect of the protein, but also have influence on the content of peptides in the final protein hydrolysate, so that the reasonable determination of the hydrolysis conditions of the protein has important significance. Jens Ader-Missen states that for any proteolytic reaction, when the enzyme and substrate are determined, and the extent of an enzymatic reaction is determined, the following hydrolysis parameters, pH, temperature (T), ratio of enzyme concentration to substrate concentration (E/S), and substrate concentration (S), are determined. Based on the results of the single-factor experiment and the related data, a four-factor three-level orthogonal test was performed to evaluate the hydrolysis effect by the degree of hydrolysis. The test results are shown in Table 8.

TABLE 8L₉(3⁴) Alcalase enzymatic hydrolysis vital wheat gluten orthogonal test result

From table 8 and it can be seen that the magnitude order of the effect of each factor on the degree of hydrolysis is: pH > temperature > enzyme to substrate concentration ratio > substrate concentration. So the optimal combination of hydrolysis conditions was finally determined as A1B1C2D1 and, considering the overall efficiency, the pH was determined to be 8.0, the temperature 55 ℃, the enzyme to substrate concentration ratio was 0.3% o, the substrate concentration was 7.5%. Empirical tests have shown that a degree of hydrolysis of 12.62% is obtained under these conditions.

Selection of hydrolysis time: the effect of different times on the hydrolysis effect of wheat gluten was studied and the results are shown in fig. 3. As can be seen from fig. 3, the degree of hydrolysis is continuously increased as the hydrolysis time is prolonged. The increasing range of the hydrolysis degree in the first hour is larger, when the hydrolysis time reaches about 4 hours, the increasing trend of the hydrolysis degree is slower, and the preferred hydrolysis time is selected to be 4 hours comprehensively. This is because soluble proteins and insoluble proteins are contained in the substrate protein, and upon hydrolysis of the soluble proteins, sensitive peptide bonds are rapidly cleaved at the initial stage, and insensitive peptide bonds are cleaved at the later stage; in the enzymatic hydrolysis of insoluble substrate proteins, enzymes are adsorbed on the surface of insoluble proteins, first hydrolyzing polymeric peptide chains loosely bound to the insoluble proteins, and then slowly hydrolyzing peptide bonds in the compact protein center. Furthermore, the concentration of soluble peptides in the reaction mixture increases, which in turn inhibits the rate of hydrolysis. Thus, initially, a large number of peptide bonds are hydrolysed, the rate of hydrolysis is rapid, and over time the rate of enzymatic hydrolysis decreases and an equilibrium state is reached.

Determination of optimal conditions for the second enzymatic hydrolysis

As can be seen from the extreme difference analysis of the orthogonal test in Table 9, the influence degree of each factor on the combined enzymolysis effect of papain and flavourzyme is A>B>C>D, i.e. pH value>Enzyme dosage>Time>Temperature, the optimal combination of the conditions of the final determination of the enzymatic hydrolysis is: a. the₂B₃C₃D₂。

TABLE 9 orthogonal test results and analysis of Complex enzyme hydrolysis of wheat gluten

FIG. 4 visually reflects the influence of pH, enzyme dosage, [ E ]/[ S ] on alkali consumption, and the optimal conditions of the process, namely pH 6.5, enzyme dosage [ E ]/[ S ] of 0.3 ‰, time 5h, temperature 45 ℃, and alkali consumption of 1.6l ml, are obtained.

Influence of pH on alkali consumption: the pH value is one of the main factors influencing the enzymatic reaction. The enzyme molecule is a special protein molecule, which has one or several active sites, which consist of binding sites and catalytic sites. The binding site functions to bind directly to the substrate, while the catalytic site catalyzes a specific reaction of the substrate. Groups in the active site are usually located at distant positions in the primary structure of the protein, and may be located on different peptide chains, but they must be close together in a certain relative position in the steric structure of the enzyme to form the active center (active site). The active site of the enzyme is therefore only present when the enzyme protein is held in a spatial configuration and exerts its catalytic action. The groups of the binding site and the catalytic site are sensitive to pH changes of the reaction system, and the dissociation state of the groups changes along with the pH changes, and the changes affect the special conformation of the enzyme molecules. In addition, Alaclase hydrolysate as a substrate in the system also shows different dissociation states along with the change of pH value. Each enzyme-catalyzed reaction system has an optimum pH value, so that the pH value directly influences the combination and catalysis of the enzyme and the substrate and is one of the main factors of the enzymolysis reaction.

As can be seen from FIG. 4(a), the enzymolysis effect is best and the alkali consumption is greatest when the pH is 6.5, which is the optimum pH for the papain and the flavourzyme complex enzyme to act on the Alaclase hydrolysate. When the pH value deviates from the pH value, the spatial conformation of the enzyme is changed, and the enzyme activity is reduced.

Influence of enzyme dosage on alkali consumption: at a certain substrate concentration, the conversion of substrate depends on the enzyme concentration. Therefore, the enzyme concentration greatly affects the enzymatic action, and as can be seen from FIG. 4(b), the larger the enzyme concentration, the larger the alkali consumption. The reason is that the more enzyme molecules, the more probability of contact with the peptide chain, and the more aromatic amino acids are released in a given period of time, the better the enzymolysis effect.

Influence of time on alkali consumption: as can be seen from FIG. 4(c), as the enzymolysis reaction proceeds, more enzyme cutting sites are exposed, so that the alkali consumption of the complex enzyme during the enzymolysis increases with the increase of time, and when the hydrolysis time is 5 hours, the alkali consumption is the largest, and under the condition of the time, the enzymolysis effect is the best.

Influence of temperature on alkali consumption: the reaction catalyzed by each enzyme has an optimum temperature, and the reaction speed of the enzyme is the fastest. The influence of temperature on the enzymatic reaction includes two aspects: on one hand, when the temperature is increased, the enzymolysis reaction speed is also increased, which is the same as that of the common chemical reaction; on the other hand, the enzyme is gradually denatured with increasing temperature, i.e., the rate of enzymatic reaction is decreased by decreasing the amount of active enzyme. The optimum reaction temperature is a temperature at which the enzymatic reaction rate reaches a maximum in a specific reaction system, and is a result of the balance of these two processes, and below the optimum temperature, the former effect is dominant, and above the optimum temperature, the latter effect is dominant, and thus the enzymatic activity rapidly decreases and the reaction rate rapidly decreases. The optimal reaction temperature is not the characteristic physical constant of the enzyme, and the optimal reaction temperature exists only in the enzymolysis reaction in a certain reaction system within a certain action time. As can be seen from FIG. 4(d), 45 ℃ is the optimum temperature for the enzymatic hydrolysis, and the enzymatic hydrolysis is most effective and the alkali consumption is the greatest at this temperature.

Amino acid composition analysis of high F-number oligopeptides: the amino acid composition and the content of the high F-number oligopeptide solution were measured by an automatic amino acid analyzer, and the results are shown in Table 10.

TABLE 10 composition of amino acids of high F-number oligopeptide mixture

And (3) calculating the yield of the F value and the high F value oligopeptide according to the following formula according to the mol number of the amino acids in the table:

note: apart from protein, gluten powder contains a small amount of non-protein components, which are ignored in this test.

The molecular weight of the high F-number oligopeptide is less than 1000Da as determined by SDS-PAGE electrophoresis, as shown in FIG. 5.

Six kinds of protease are screened. The results show that Alcalase is the preferred enzyme for the hydrolysis of wheat gluten.

Using four-factor three-level using L₉(3⁴) The optimal conditions for hydrolyzing the wheat gluten by the Alcalase protease are determined by orthogonal experimental design as follows: the pH value is 8.0, the temperature is 55 ℃, the concentration ratio of enzyme to substrate is 0.3 per mill, and the concentration of substrate is 5 percent; the study on the relationship between the hydrolysis time and the degree of hydrolysis showed that the preferred hydrolysis time was 4 hours.

By using L₉(3⁴) The optimal hydrolysis conditions of papain and flavourzyme complex enzyme are determined by orthogonal experimental design: pH 6.5, enzyme dosage [ E]/[S]0.3 per mill, 5 hours of time, 45 ℃ of temperature and 1.61ml of alkali consumption.

The prepared oligopeptide with the high F value is light yellow and tasteless, and has the molecular weight of less than 1000 Da. The F value is 45.2, and the product yield is 42.3%.

Pilot scale production:

(1) size mixing: adding a certain amount of pH-adjusted pure water into a blending tank, directly feeding wet gluten (dry basis weight 40Kg) separated in the production process into the blending tank, stirring while feeding to obtain emulsion, and feeding into a 500L enzymolysis tank.

(2) Receiving emulsion in a blending section, and performing enzymolysis for two times, wherein the enzymolysis for the first time is as follows: adding the compounded No. 1 enzyme liquid (the dry basis weight of the alkaline protease is 0.12Kg) while stirring; controlling the pH value to be 8.0, the temperature to be 55 ℃ and the hydrolysis time to be 4 h;

(3) and (3) carrying out second enzymolysis: adding No. 2 compound enzyme solution (papain and flavourzyme, dry basis weight 0.12Kg) while stirring; the pH value is controlled to be 6.5, the temperature is controlled to be 45 ℃, and the time is controlled to be 5 h.

(4) And (4) filtering the secondary enzymolysis solution with an ultrafiltration membrane to remove macromolecular proteins.

(5) The ultrafiltrate is filtered by a nanofiltration membrane to remove small molecular salt and oligopeptide substances lower than 100 Da.

(6) The product is sprayed and dried, and 16.92Kg of water-soluble oligopeptide mixture product with the water content of 7 percent is finally obtained.

TABLE 11 enlarged production data sheet

The pilot product is off-white and powdery, has no bad smell, and other indexes are detected by Q/HNFT05-2013 standard, and all indexes meet the standard requirements.

Claims

1. A method for producing wheat oligopeptide by a multi-enzyme synergistic method is characterized by comprising the following steps: size mixing, secondary enzymolysis, ultrafiltration, nanofiltration and drying.

2. The method for producing wheat oligopeptide according to claim 1 by multi-enzyme synergistic method, wherein the method comprises the following steps: the step of size mixing comprises the steps of adding pure water for adjusting the pH value into a mixing tank, directly feeding wet gluten separated in the production process into the mixing tank, stirring while feeding, and pumping into an enzymolysis tank after emulsion is formed.

3. The method for producing wheat oligopeptide according to claim 1 by multi-enzyme synergistic method, wherein the method comprises the following steps: the secondary enzymolysis step comprises the steps of receiving emulsion in the size mixing step, and carrying out primary enzymolysis: adding the compounded No. 1 enzyme liquid while stirring, and hydrolyzing at a certain pH value and temperature; and (3) carrying out second enzymolysis: adding No. 2 compound enzyme liquid while stirring, and hydrolyzing at certain pH value and temperature.

4. The method for producing wheat oligopeptide according to claim 3 by using multi-enzyme synergistic method, wherein the method comprises the following steps: the enzyme in the No. 1 enzyme solution is selected from Alcalase protease, neutral protease AS1398, animal and plant proteolytic enzyme, neutral protease Nutrase and pepsin.

5. The method for producing wheat oligopeptide according to claim 3 by using multi-enzyme synergistic method, wherein the method comprises the following steps: the enzyme in the No. 1 enzyme solution is selected from Alcalase protease.

6. The method for producing wheat oligopeptide according to claim 3 by using multi-enzyme synergistic method, wherein the method comprises the following steps: the pH value of the first enzymolysis is 7-9, the temperature is 40-60 ℃, and the hydrolysis time is 2-6 hours.

7. The method for producing wheat oligopeptide according to claim 3 by using multi-enzyme synergistic method, wherein the method comprises the following steps: the enzyme in the No. 2 enzyme solution is selected from one or more of Alcalase protease, neutral protease AS1398, animal and plant proteolytic enzyme, neutral protease Nutrase, pepsin, papain and flavourzyme.

8. The method for producing wheat oligopeptide according to claim 3 by using multi-enzyme synergistic method, wherein the method comprises the following steps: the pH value of the second enzymolysis is 6-7, the temperature is 40-50 ℃, and the hydrolysis time is 3-5 hours.