阅读说明：本技术 一种制备低敏性乳清蛋白肽的方法 (Method for preparing hypoallergenic whey protein peptide ) 是由 涂宗财 陈海奇 胡月明 王辉 黎勇强 夏余鸿 杨一帆 于 2021-08-09 设计创作，主要内容包括：本发明公开了一种制备低敏性乳清蛋白肽的方法,明属于食品加工技术领域,涉及一种利用糖基化反应协同蛋白酶酶解复合改性制备低敏性乳清蛋白肽的技术。该方法具体为：配制乳清蛋白-低聚果糖溶液,冷冻干燥后制得固样,于过热蒸汽条件下进行糖基化反应,用胃蛋白酶进行第一步酶解,再用胰蛋白酶进行第二步酶解,利用μm级别滤膜滤出蛋白质分子,最后冷冻干燥处理制得低敏性乳清蛋白肽。通过本发明加工技术制备得到的低敏性乳清蛋白肽,能够有效降低其致敏性,并且通过硒等营养物质的添加增加了其附加值,同时有效抑制其糖基化反应末期产物的生成。(The invention discloses a method for preparing hypoallergenic whey protein peptide, belongs to the technical field of food processing, and relates to a technology for preparing hypoallergenic whey protein peptide by utilizing glycosylation reaction in cooperation with protease enzymolysis composite modification. The method specifically comprises the following steps: preparing a whey protein-fructo-oligosaccharide solution, freeze-drying to obtain a solid sample, carrying out glycosylation reaction under the condition of superheated steam, carrying out first-step enzymolysis by using pepsin, carrying out second-step enzymolysis by using trypsin, filtering out protein molecules by using a filter membrane of a mu m level, and finally carrying out freeze-drying treatment to obtain the hypoallergenic whey protein peptide. The hypoallergenic whey protein peptide prepared by the processing technology can effectively reduce the allergenicity of the hypoallergenic whey protein peptide, increases the added value of the hypoallergenic whey protein peptide by adding nutrient substances such as selenium and the like, and effectively inhibits the generation of the final product of the glycosylation reaction.)

1. A method for preparing hypoallergenic whey protein peptides, characterized in that said method comprises the following steps:

(1) preparation of whey protein solution

Firstly, preparing food-grade baking soda with solute mass fraction of 1%, mixing the food-grade baking soda with whey protein, and then adding ferric chloride, selenious acid and L-ascorbic acid into the mixture to prepare whey protein solution: (ii) a

(2) Glycosylation treatment

Dissolving the whey protein solution prepared in the step (1) and fructo-oligosaccharide with deionized water, mixing uniformly, freezing and drying to prepare a solid sample, and reacting for 2-3 minutes under the condition of superheated steam at 140 ℃ to obtain a sample after high-temperature glycosylation;

(3) enzymolysis

A. Adjusting the pH value of the sample in the step (2) to 2.0 by using a hydrochloric acid solution, adding pepsin according to the ratio of 1: 50-100 (w/w) of enzyme to the sample, and performing shake enzymolysis for 1-2 hours at 37 ℃ to obtain a sample A;

B. adjusting the pH value of the sample A to 7.0 by using a sodium hydroxide solution, adding trypsin according to the ratio of enzyme to the sample A1: 25-50 (w/w), and performing shake enzymolysis for 1-2 hours at 37 ℃ to obtain a sample B;

C. placing the sample B in a boiling water bath, terminating the reaction after 7-10 minutes, and cooling to room temperature;

(4) and (3) filtering: filtering the enzyme-inactivated product on a filter membrane with the level of mum to separate out protein molecules;

(5) freezing and concentrating: freezing and concentrating the filtrate in a refrigerator at-80 deg.C for 12 hr;

(6) and (3) freeze drying: and (4) freeze-drying the sample subjected to freeze concentration in the step (5) to obtain the hypoallergenic whey protein peptide.

2. The method for preparing hypoallergenic whey protein peptides according to claim 1, characterized in that: in the step (1), the ratio of the raw materials is whey protein, food-grade baking soda, ferric chloride, selenious acid and L-ascorbic acid is 10: 0.1-0.3: 0.01-0.03: 0.1-0.15: 0.3-0.45 according to the mass parts of solutes.

3. The method for preparing hypoallergenic whey protein peptides according to claim 1, characterized in that: and (3) dissolving the whey protein solution and the fructo-oligosaccharide in deionized water according to the mass ratio of 1: 1 of the solute in the step (2) and uniformly mixing.

Technical Field

The invention belongs to the technical field of food processing, and particularly relates to a method for preparing hyposensitive whey protein peptide.

Background

Cow milk has the advantages of rich nutrition, easy absorption and digestion, and the like, but is one of common foods causing allergy, and the allergic symptoms induced by cow milk mainly include skin reactions, respiratory symptoms, intestinal symptoms and systemic reactions.

Cow milk allergy is a non-specific reaction and is generally caused by allergens such as casein, beta-lactoglobulin and alpha-lactalbumin in cow milk. The allergic reaction caused by cow milk is generally induced by the mediation of IgE, and how to reduce the allergenicity of protein in cow milk is the key point for preparing hypoallergenic protein peptide.

The glycosylation reaction is the most common reaction in food thermal processing, belongs to the early stage of Maillard reaction, and means that condensation occurs between reducing sugar and protein containing free amino groups, glycosylation can affect the linear epitope of the protein, and further reduce the sensitization of the protein. Enzymatic hydrolysis is a method of limited hydrolysis of proteins using proteases, which is effective in reducing the sensitization of proteins. For example, CN 109897879B discloses a preparation method of hypoallergenic ovalbumin peptide, which utilizes enzymatic modification to effectively reduce allergenicity; however, the raw material and glycosylation reaction of the method have poor inhibition efficiency on the whey protein allergen, and the glycosylation reaction process of the method is carried out towards the end stage, so that a glycosylation reaction end-stage product is generated, and the product is combined with various tissue cells of a human body and destroys the tissue cells, thereby causing harm to the human body.

Disclosure of Invention

Aiming at the defects and problems in the prior art, the invention aims to provide a method for preparing hyposensitive whey protein peptide, which reduces the sensitization of whey protein by utilizing glycosylation reaction of high-temperature over-steam and combining with a protein enzymolysis technology, thereby obtaining the hyposensitive whey protein peptide.

The invention is realized by the following technical scheme:

the technical process adopted by the invention is as follows: firstly, preparing a whey protein solution, freeze-drying to prepare a solid sample, carrying out glycosylation reaction under the condition of superheated steam, carrying out first-step enzymolysis by using pepsin, carrying out second-step enzymolysis by using trypsin, filtering out protein molecules by using a filter membrane of a mu m level, and finally carrying out freeze-drying treatment to prepare the hypo-sensitive whey protein peptide.

The method comprises the following specific steps

1. Preparation of whey protein solution: firstly, preparing food-grade baking soda with solute mass fraction of 1%, mixing the food-grade baking soda with whey protein, and then adding ferric chloride, selenious acid and L-ascorbic acid into the mixture, wherein the specific proportions according to the solute mass fraction are as follows: whey protein, food grade baking soda, ferric chloride, selenious acid and L-ascorbic acid are 10: 0.1-0.3: 0.01-0.03: 0.1-0.15: 0.3-0.45;

2. and (3) glycosylation treatment: dissolving and uniformly mixing the whey protein solution and the fructo-oligosaccharide with deionized water according to the mass ratio of 1: 1 of the solute, wherein the mass of the deionized water is 5 times of that of the total solute; uniformly mixing, freezing and drying to prepare a solid sample, and reacting for 2-3 minutes under the condition of superheated steam at 140 ℃ to obtain a glycosylated sample;

3. enzymolysis

(1) Adjusting the pH value of the sample to 2.0 by using 1.0mol/L hydrochloric acid solution, adding pepsin according to the ratio of 1: 50-100 (w/w) of enzyme to the sample, and performing shake enzymolysis for 1-2 hours at 37 ℃ to obtain a sample A;

(2) adjusting the pH value of the sample A to 7.0 by using 1.0mol/L sodium hydroxide solution, adding trypsin according to the ratio of enzyme to the sample A1: 25-50 (w/w), and performing shake enzymolysis for 1-2 hours at 37 ℃ to obtain a sample B;

(3) placing the sample B in a boiling water bath, terminating the reaction after 7-10 minutes, and cooling to room temperature;

4. and (3) filtering: filtering the enzyme-inactivated product on a filter membrane with the level of mum to separate out protein molecules;

5. freezing and concentrating: freezing and concentrating the filtrate in a refrigerator at-80 deg.C for 12 hr;

6. and (3) freeze drying: and (4) freeze-drying the frozen and concentrated sample to obtain the hypoallergenic whey protein peptide.

According to the invention, fructo-oligosaccharide and beta-lactoglobulin are subjected to glycosylation reaction to reduce allergenicity; the sensitization epitope of the beta-lactoglobulin mostly contains lysine, and glycosylation mainly comprises the reaction of lysine in protein and reducing sugar, so that the glycosylation reaction can reduce the reactivity of the beta-lactoglobulin and IgE, and further reduce the sensitization.

The selection of raw materials, the parameter proportion and the superheated steam condition of the invention have the functions of regulating and controlling the glycosylation reaction, inhibiting the glycosylation reaction from going to the final stage and effectively inhibiting the generation of the final product of the glycosylation reaction.

The invention not only prepares the whey protein peptide, but also adds selenium which is an essential trace element and has the functions of preventing cardiovascular diseases, improving immunity and the like.

Therefore, by the technical process, the hypoallergenic whey protein peptide can be prepared, and the beneficial effects are as follows: can prepare a whey protein peptide, can effectively reduce the allergenicity thereof; the added value of the selenium is increased by adding nutrient substances such as selenium and the like; the generation of the final product of the glycosylation reaction is inhibited through the raw material parameters and the reaction regulation.

Drawings

Fig. 1 is a graph comparing the antigenicity of whey protein peptides prepared in each example and comparative example.

Detailed Description

The invention is further described below with reference to the accompanying drawings and specific embodiments.

Example one

1. Preparation of whey protein solution: firstly, preparing food-grade baking soda with solute mass fraction of 1%, firstly mixing the food-grade baking soda and whey protein according to the ratio of whey protein, food-grade baking soda, ferric chloride, selenious acid and L-ascorbic acid (10: 0.1: 0.01: 0.1: 0.3) in the solute mass fraction, and then adding ferric chloride, selenious acid and L-ascorbic acid into the mixture to obtain whey protein solution.

2. And (3) glycosylation treatment: dissolving and uniformly mixing the whey protein solution and the fructo-oligosaccharide with deionized water according to the mass ratio of 1: 1 of the solute, wherein the mass of the deionized water is 5 times of that of the total solute; after being uniformly mixed, the mixture is frozen and dried to prepare a solid sample, and the solid sample reacts for 2 minutes under the condition of superheated steam at the temperature of 140 ℃ to obtain a glycosylated sample.

3. Enzymolysis

(1) Adjusting the pH value of the sample to 2.0 by using 1.0mol/L hydrochloric acid, adding pepsin according to the ratio of 1: 50(w/w) of enzyme to the sample, and carrying out shake enzymolysis for 1 hour at the temperature of 37 ℃ to obtain a sample A;

(2) the pH of the sample A was adjusted to 7.0 with 1.0mol/L sodium hydroxide solution, trypsin was added at a ratio of 1: 25(w/w) of enzyme to the sample, and the mixture was subjected to shake enzymolysis at 37 ℃ for 1 hour to obtain a sample B.

(3) The reaction was terminated by placing sample B in a boiling water bath for 7 minutes and cooling to room temperature.

4. And (3) filtering: the enzyme-inactivated product is filtered on a filter membrane with the level of mum to separate out protein molecules.

5. Freezing and concentrating: the filtrate was concentrated by freezing in a refrigerator at-80 ℃ for 12 hours.

6. And (3) freeze drying: and freeze-drying the concentrated sample to obtain the hypoallergenic whey protein peptide.

Example two

1. Preparation of whey protein solution: firstly, preparing food-grade baking soda with solute mass fraction of 1%, firstly mixing the food-grade baking soda and whey protein according to the ratio of whey protein, food-grade baking soda, ferric chloride, selenious acid and L-ascorbic acid (10: 0.2: 0.02: 0.1: 0.3) in the solute mass fraction, and then adding ferric chloride, selenious acid and L-ascorbic acid into the mixture to obtain whey protein solution.

2. And (3) glycosylation treatment: dissolving and uniformly mixing the whey protein solution and the fructo-oligosaccharide with deionized water according to the mass ratio of 1: 1 of the solute, wherein the mass of the deionized water is 5 times of that of the total solute; after being uniformly mixed, the mixture is frozen and dried to prepare a solid sample, and the solid sample reacts for 2 minutes under the condition of superheated steam at the temperature of 140 ℃ to obtain a glycosylated sample.

3. Enzymolysis

(1) Adjusting the pH value of the sample to 2.0 by using 1.0mol/L hydrochloric acid, adding pepsin according to the ratio of 1: 50(w/w) of enzyme to the sample, and carrying out shake enzymolysis for 2 hours at the temperature of 37 ℃ to obtain a sample A;

(2) the pH of the sample A was adjusted to 7.0 with 1.0mol/L sodium hydroxide solution, trypsin was added at a ratio of 1: 25(w/w) of enzyme to the sample, and the mixture was subjected to shake enzymolysis at 37 ℃ for 2 hours to obtain a sample B.

(3) The reaction was terminated by placing sample B in a boiling water bath for 8 minutes and cooling to room temperature.

4. And (3) filtering: the enzyme-inactivated product is filtered on a filter membrane with the level of mum to separate out protein molecules.

5. Freezing and concentrating: the filtrate was concentrated by freezing in a refrigerator at-80 ℃ for 12 hours.

6. And (3) freeze drying: and freeze-drying the concentrated sample to obtain the hypoallergenic whey protein peptide.

EXAMPLE III

1. Preparation of whey protein solution: firstly, preparing food-grade baking soda with solute mass fraction of 1%, firstly mixing the food-grade baking soda and whey protein according to the ratio of whey protein, food-grade baking soda, ferric chloride, selenious acid and L-ascorbic acid (10: 0.2: 0.02: 0.15: 0.45) in the solute mass fraction, and then adding ferric chloride, selenious acid and L-ascorbic acid into the mixture to obtain whey protein solution.

2. And (3) glycosylation treatment: dissolving and uniformly mixing the whey protein solution and the fructo-oligosaccharide with deionized water according to the mass ratio of 1: 1 of the solute, wherein the mass of the deionized water is 5 times of that of the total solute; after being uniformly mixed, the mixture is frozen and dried to prepare a solid sample, and the solid sample reacts for 3 minutes under the condition of superheated steam at the temperature of 140 ℃ to obtain a glycosylated sample.

3. Enzymolysis

(1) Adjusting the pH value of the sample to 2.0 by using 1.0mol/L hydrochloric acid, adding pepsin according to the ratio of 1: 100(w/w) of enzyme to the sample, and performing shake enzymolysis for 1 hour at the temperature of 37 ℃ to obtain a sample A;

(2) the pH of the sample A was adjusted to 7.0 with 1.0mol/L sodium hydroxide solution, trypsin was added at a ratio of 1: 50(w/w) of enzyme to the sample, and the mixture was subjected to shake enzymolysis at 37 ℃ for 1 hour to obtain a sample B.

(3) The reaction was terminated by placing sample B in a boiling water bath for 9 minutes and cooling to room temperature.

4. And (3) filtering: the enzyme-inactivated product is filtered on a filter membrane with the level of mum to separate out protein molecules.

5. Freezing and concentrating: the filtrate was concentrated by freezing in a refrigerator at-80 ℃ for 12 hours.

6. And (3) freeze drying: and freeze-drying the concentrated sample to obtain the hypoallergenic whey protein peptide.

Example four

1. Preparation of whey protein solution: firstly, preparing food-grade baking soda with solute mass fraction of 1%, firstly mixing the food-grade baking soda and whey protein according to the ratio of whey protein, food-grade baking soda, ferric chloride, selenious acid and L-ascorbic acid (10: 0.3: 0.03: 0.15: 0.45) in the solute mass fraction, and then adding ferric chloride, selenious acid and L-ascorbic acid into the mixture to obtain whey protein solution.

2. And (3) glycosylation treatment: dissolving and uniformly mixing the whey protein solution and the fructo-oligosaccharide with deionized water according to the mass ratio of 1: 1 of the solute, wherein the mass of the deionized water is 5 times of that of the total solute; after being uniformly mixed, the mixture is frozen and dried to prepare a solid sample, and the solid sample reacts for 3 minutes under the condition of superheated steam at the temperature of 140 ℃ to obtain a glycosylated sample.

3. Enzymolysis

(1) Adjusting the pH value of the sample to 2.0 by using 1.0mol/L hydrochloric acid, adding pepsin according to the ratio of 1: 100(w/w) of enzyme to the sample, and carrying out shake enzymolysis for 2 hours at the temperature of 37 ℃ to obtain a sample A;

(2) the pH of the sample A was adjusted to 7.0 with 1.0mol/L sodium hydroxide solution, trypsin was added at a ratio of 1: 50(w/w) of enzyme to the sample, and the mixture was subjected to shake enzymolysis at 37 ℃ for 2 hours to obtain a sample B.

(3) The reaction was terminated by placing sample B in a boiling water bath for 10 minutes and cooling to room temperature.

4. And (3) filtering: the enzyme-inactivated product is filtered on a filter membrane with the level of mum to separate out protein molecules.

5. Freezing and concentrating: the filtrate was concentrated by freezing in a refrigerator at-80 ℃ for 12 hours.

6. And (3) freeze drying: and freeze-drying the concentrated sample to obtain the hypoallergenic whey protein peptide.

Comparative example 1

The procedure of the preparation of whey protein peptides in example two was followed except that only whey protein and food grade baking soda were added in step 1 and the ratio of whey protein to food grade baking soda was 10: 0.2 and the other processing steps were unchanged to obtain protein peptides.

Comparative example No. two

The procedure of the preparation of the whey protein peptide of example two was followed except that only whey protein and ferric chloride were added in step 1 and the ratio of whey protein to ferric chloride was 10: 0.02 and the protein peptide was obtained unchanged from the other processing steps.

Comparative example No. three

The procedure of example two was followed except that only whey protein, selenious acid and L-ascorbic acid were added in step 1, and whey protein, selenious acid and L-ascorbic acid were 10: 0.1: 0.3, and the other processing steps were unchanged to obtain the protein peptides.

Comparative example No. four

The procedure of the preparation of whey protein peptides in example two was followed, except that only whey protein was added in step 1 and the protein peptides were obtained unchanged by the other processing steps.

The whey protein peptides prepared in examples one to four and comparative examples one to four were subjected to antigenicity analysis by enzyme-linked immunosorbent assay, the concentration of the whey protein peptide was adjusted to 5. mu.g/mL, and the whey protein peptide (100. mu.L/well) was added to the microplate and allowed to stand overnight at 4 ℃; wash the microplate three times (200 μ L/well) with PBST, pat dry; BSA was added for blocking (250. mu.L/well), left at 37 ℃ for 2 hours, washed and then patted dry; adding rabbit anti-OVA serum (50 mu L/hole) into the ELISA plate, standing at 37 ℃ for 1 hour, washing the plate, and then drying by patting; adding goat anti-rabbit secondary antibody (200 mu L/hole, HRP enzyme label) into the enzyme label plate, standing for 1 hour at 37 ℃, washing the plate and then drying by patting; adding TMB for color development; adding sulfuric acid to terminate the reaction; the absorbance was measured and recorded at 490nm and the results are shown in FIG. 1.

The antigenicity of the protein peptide was shown by measuring the absorbance at 490nm in each case, and low antigenicity indicated low sensitization of the protein peptide.

The protein peptide is finally obtained from the whey protein-fructo-oligosaccharide solution through the steps of glycosylation, enzymolysis, filtration, freeze-drying and the like, as can be seen from figure 1, the food-grade baking soda, ferric chloride and nano-selenium are added in the preparation of the solution to inhibit the antigenicity of the product, but the inhibition effect is more obvious when the three work synergistically, and the prepared whey protein peptide has lower allergenicity.

The foregoing merely represents preferred embodiments of the invention, which are described in some detail and detail, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.