Method for preparing hypoallergenic whey protein through combination of enzymolysis and glycosylation

Abstract

The invention belongs to the technical field of dairy products and food biology, and particularly relates to a method for preparing hypoallergenic whey protein through combination of an enzyme method and glycosylation and application of the hypoallergenic whey protein. The method specifically comprises the following steps: carrying out enzymolysis on whey protein isolate by using alkaline protease to prepare a whey protein hydrolysate, adding allose, uniformly mixing, carrying out glycosylation reaction under a freeze-drying condition, and finally, carrying out ultrafiltration and dialysis to remove redundant sugar, so as to prepare a sample, namely the low-sensitization whey protein. According to the whey protein prepared by the method, the sensitization is effectively inhibited, the low sensitization after digestion is ensured, and the problem that the sensitization is increased in the digestion process is avoided.

Description

Technical Field

[0001] The invention belongs to the field of dairy products and food biotechnology, and specifically relates to a method for preparing low-allergenic whey protein by enzymatic method combined with glycosylation and application thereof. Background Art

[0002] Cow's milk is rich in nutrients and bioactive substances and is an important source of high-quality protein for humans. The main proteins contained in cow's milk protein are casein and whey protein, of which casein accounts for about 80% of cow's milk protein, whey protein accounts for about 20% of the total cow's milk protein, and there are a small amount of other proteins. Among them, whey protein is the main raw material for the production of milk powder. It is rich in nutrients and provides high-quality protein and micronutrients in a form that is easy for humans to absorb. It is an important part of a diversified diet, but some people will have allergic reactions when eating dairy products, especially infants and young children will cause more severe allergic reactions than adults when eating dairy products. This may be due to the immature digestive system and low immunity of infants and young children. For people who are allergic to dairy products, completely refusing to eat dairy products seems to be the best choice, but the nutrition it provides is indispensable to people, especially for infants and young children. The infant milk powder they eat contains sufficient essential amino acids and has high nutritional value. It plays an important role in the healthy growth and development of infants and young children. Milk powder is the main food source for infants and young children. If milk powder is removed from the diet of allergic patients, it will inevitably cause nutritional imbalance. Therefore, it is very important to develop a method for preparing low-allergenic whey protein, which can avoid allergic reactions in infants and young children, meet their nutritional needs, and promote their healthy growth.

[0003] At present, the methods for reducing protein allergenicity mainly include physical method, chemical method and enzymatic method. Among them, enzymatic hydrolysis is a more commonly used method, and the development of products with low allergenic milk formula is a very popular research. Among them, enzymatic hydrolysis is a more commonly used method, and these methods have certain limitations. First, moderate hydrolysis cannot completely reduce allergenicity, and moderate hydrolysis will also lead to the exposure of peptides that originally did not express allergenicity. Secondly, deep hydrolysis of whey protein will lead to excessive degree of hydrolysis and produce bitter peptides, which has a great impact on flavor. The glycosylation method can effectively modify the allergenic peptides, and the bitterness caused by excessive hydrolysis can also be avoided. At present, the method of reducing the allergenicity of whey protein by glycosylation has been applied. Therefore, the present invention adopts an enzymatic method combined with a glycosylation processing technology to prepare low allergenic whey protein.

[0004] Due to the good functional properties of glycosylated proteins and the high correlation between the functional properties of proteins and the quality of food. Whey protein is used as a substrate for moderate enzymatic hydrolysis, followed by a glycosylation reaction, and the effect of the glycosylation cross-linking reaction on the allergenicity of whey protein is analyzed at the same time. This method combines the advantages of both enzymatic and glycosylation methods. In summary, the development of a whey protein base with a simple preparation process that can not only efficiently and directionally destroy and modify residual original epitopes but also has low bitterness is a prerequisite for the development of low-allergenic formula products, which is very necessary for people allergic to cow's milk. Summary of the invention

[0005] In view of the existing method for preparing low-allergenic whey protein, the present invention establishes a method for preparing low-allergenic whey protein by combining enzymatic hydrolysis with glycosylation. This method ensures low allergenicity after digestion, avoids increased allergenicity due to digestion, and has low bitterness and good flavor. In order to achieve the above-mentioned purpose of the invention, the technical scheme of the present invention is as follows:

[0006] (1) The whey protein is dissolved by using a phosphate buffer solution, and the whey protein is hydrolyzed by using an alkaline protease, and then the enzyme is inactivated after the hydrolysis.

[0007] (2) Add allose to the whey protein hydrolysate, mix well, and perform freeze-drying.

[0008] (3) After freeze-drying, the whey protein hydrolysate is placed in an oven for glycosylation reaction. After the reaction is completed, the glycosylated whey protein hydrolysate is placed in an ice bath to terminate the reaction.

[0009] (4) Redissolve with phosphate buffer solution, then use ultrafiltration centrifuge tube to remove unreacted sugar and salt. In order to further remove unbound reducing sugar, transfer the mixed solution into a dialysis bag and dialyze with continuous stirring. After dialysis, freeze-dry and store.

[0010] (5) Conduct an in vitro simulation experiment on the product in (4). Simulate the digestion process of the two stages of the gastrointestinal environment of infants and young children in vitro, and measure the changes in allergenicity before and after digestion to determine its allergenicity.

[0011] The specific steps of the present invention are:

[0012] Preferably, in step (1), the raw material is whey protein isolate WPI, whose protein content (by dry weight) is not less than 90.0%, water content is less than 5.5%, and milk fat content is less than 5.0%.

[0013] Preferably, in step (1), the protease used is alkaline protease.

[0014] Preferably, in step (1), the phosphate buffer solution used is 0.01 mol / L and has a pH of 7.4.

[0015] Preferably, the substrate mass concentration in step (1) is 6%.

[0016] Preferably, the pH value in step (1) is 10.0.

[0017] Preferably, the amount of enzyme added in step (1) is 6000-7000 U of alkaline protease per 1 g of whey protein.

[0018] Preferably, in step (1), the hydrolysis time is 1.5 hours and the hydrolysis temperature is 55°C.

[0019] Preferably, in step (1), the enzyme inactivation temperature is 90° C. and the enzyme inactivation time is 10 min.

[0020] Preferably, in step (2), the solute mass ratio of allose to protein is 1:1.

[0021] Preferably, in step (3), the condition in the oven is to maintain the relative humidity at about 65% (saturated sodium chloride solution).

[0022] Preferably, in step (3), the glycosylation reaction temperature is 65° C. and the reaction time is 3 h.

[0023] Preferably, in step (4), the reconstitution concentration of the glycosylation reaction product is 5 mg / mL.

[0024] Preferably, the ultrafiltration centrifuge tube in step (4) is 3kDa.

[0025] Preferably, in step (4), the molecular weight cutoff of the dialysis bag is 3 kDa.

[0026] Preferably, in step (4), the dialysis temperature is 4° C., the stirring dialysis time is 24 h, and the ultrapure water is replaced 3 times a day.

[0027] The present invention utilizes the glycosylation reaction of allose and enzymatic whey protein to reduce the allergenicity, wherein the allergenic epitope of β-lactoglobulin contains more lysine, and the glycosylation is mainly the reaction of lysine in the protein and reducing sugar, so the reaction can effectively reduce the allergenicity of whey protein. After the sample is prepared, the antigen reduction rate is determined by ELISA method. The processing sample conditions are optimized by single factor and response surface, and the reduction of antigenicity is further analyzed by simulating the gastrointestinal digestion of infants in vitro. The results show that the glycosylation temperature is 55°C, the reaction time is 4h, and the mass ratio of sugar to whey protein is 1:1, which has a good allergenicity reduction rate. The allergenicity reduction rate of α-lactalbumin is 92.31%, and the allergenicity reduction rate of β-lactoglobulin is 89.37%. The preparation method is simple to operate, has low allergenicity, does not over-hydrolyze whey protein, and has more nutritional value.

[0028] The low-allergenic whey protein prepared by the above method has the following advantages: (1) The preparation method of the present invention avoids the previous method of reducing allergenicity by excessive enzymatic hydrolysis. First, moderate hydrolysis is performed by enzymes to open the spatial structure of whey protein, which is conducive to the occurrence of glycosylation reactions and reduces the formation of bitter peptides to a certain extent. At the same time, the nutritional value of whey protein is guaranteed and the loss of nutrition is avoided.

[0029] (2) The preparation method of the present invention has good adaptability and ensures the low allergenicity of whey protein after digestion. The glycosylation sites can be accurately located and mask the allergenic peptide segments during the glycosylation reaction, thereby achieving the reduction of allergenicity. It has the advantages of high accuracy and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram of the grafting degree of the enzymatically hydrolyzed glycosylated whey protein of the present invention.

[0031] Figure 2 This is a graph showing the reduction rate of α-lactalbumin antigen in the enzymatically glycosylated whey protein of the present invention.

[0032] Figure 3 This is a graph showing the antigen reduction rate of the enzymatically glycosylated whey protein β-lactalbumin of the present invention.

[0033] Figure 4 This is a graph showing the decrease rate of α-lactalbumin antigen after digestion of whey protein of the present invention.

[0034] Figure 5 This is a graph showing the decrease rate of β-lactoglobulin antigen after digestion of whey protein of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. It should be pointed out that if there is no special detailed description of the process below, it is realized or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, it is regarded as a conventional product that can be purchased commercially.

[0036] The raw materials used in the embodiment of the present invention are whey protein isolate WPI; alkaline protease: 2×10 5 , D-allose, ELISA kits (α-lactalbumin, β-lactoglobulin) were purchased from MacLean Biotechnology Co., Ltd.

[0037] Embodiment 1

[0038] 1. Enzymatic hydrolysis: Take 6g of whey protein isolate and dissolve it in 10mmol / L, pH7.4 phosphate buffer solution, dissolve it according to the substrate concentration of 6% to make it hydrated. Adjust the pH to 10.0, add 6000-7000U of alkaline protease per 1g of whey protein, and perform enzymatic hydrolysis for 1.5 hours at a hydrolysis temperature of 55°C. After the hydrolysis is completed, the temperature is raised to 90°C and the water bath is bathed for 10 minutes to inactivate the enzyme.

[0039] 2. Glycosylation: Add 6 g of xylose to the hydrolyzed whey protein in step 1, fully dissolve and mix, then freeze-dry, and then keep the relative humidity at about 65% (saturated sodium chloride solution), put it in an oven, and carry out glycosylation reaction at 65°C. Take out the mixture after 3 hours. After the reaction is completed, put the centrifuge tube in an ice bath to terminate the reaction.

[0040] 3. The glycosylated whey protein in step 2 was redissolved with phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then unreacted sugar and salt were removed using a 3 kDa ultrafiltration centrifuge tube. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4 ° C for 24 h with continuous stirring. The ultrapure water was replaced 3 times a day. After dialysis, it was freeze-dried.

[0041] Embodiment 2

[0042] 1. Enzyme hydrolysis: Take 6g of whey protein isolate and dissolve it in 10mmol / L, pH7.4 phosphate buffer solution, dissolve it according to the substrate concentration of 6% to make it hydrated. Adjust the pH to 10.0, add 6000-7000U of alkaline protease per 1g of whey protein, and perform enzymatic hydrolysis for 1.5 hours at a hydrolysis temperature of 55°C. After the hydrolysis is completed, the temperature is raised to 90°C and the water bath is bathed for 10 minutes to inactivate the enzyme.

[0043] 2. Glycosylation: Add 6 g of allose to the hydrolyzed whey protein in step 1, fully dissolve and mix, then freeze-dry, and then keep the relative humidity at about 65% (saturated sodium chloride solution), put it in an oven, and carry out glycosylation reaction at 65° C. After 3 hours, take out the mixture, and after the reaction is completed, put the centrifuge tube in an ice bath to terminate the reaction.

[0044] 3. The glycosylated whey protein in step 2 was redissolved with phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then unreacted sugar and salt were removed using a 3 kDa ultrafiltration centrifuge tube. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4 ° C for 24 h with continuous stirring. The ultrapure water was replaced 3 times a day. After dialysis, it was freeze-dried.

[0045] Embodiment 3

[0046] 1. Enzyme hydrolysis: Take 6g of whey protein isolate and dissolve it in 10mmol / L, pH7.4 phosphate buffer solution, dissolve it according to the substrate concentration of 6% to make it hydrated. Adjust the pH to 10.0, add 6000-7000U of alkaline protease per 1g of whey protein, and perform enzymatic hydrolysis for 1.5 hours at a hydrolysis temperature of 55°C. After the hydrolysis is completed, the temperature is raised to 90°C and the water bath is bathed for 10 minutes to inactivate the enzyme.

[0047] 2. Glycosylation: Add 6 g of fucose to the hydrolyzed whey protein in step 1, fully dissolve and mix, then freeze-dry. Then, maintain the relative humidity at about 65% (saturated sodium chloride solution), put it in an oven, and carry out glycosylation reaction at 65°C. After 3 hours, take out the mixture. After the reaction is completed, put the centrifuge tube in an ice bath to terminate the reaction.

[0048] 3. The glycosylated whey protein in step 2 was redissolved with phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then unreacted sugar and salt were removed using a 3 kDa ultrafiltration centrifuge tube. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4 ° C for 24 h with continuous stirring. The ultrapure water was replaced 3 times a day. After dialysis, it was freeze-dried.

[0049] Comparative Example 1

[0050] 1. Glycosylation: Take 6 g of whey protein isolate, dissolve it with phosphate buffer solution, add 6 g of xylose, fully dissolve and mix, then freeze-dry, then keep the relative humidity at about 65% (saturated sodium chloride solution), put it in an oven, and carry out glycosylation reaction at 65°C. After 3 hours, take out the mixture, and after the reaction is completed, put the centrifuge tube in an ice bath to terminate the reaction.

[0051] 2. The glycosylated whey protein in step 1 was redissolved with phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then unreacted sugar and salt were removed using a 3 kDa ultrafiltration centrifuge tube. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4 ° C for 24 h with continuous stirring. The ultrapure water was replaced 3 times a day. After dialysis, it was freeze-dried.

[0052] Comparative Example 2

[0053] 1. Glycosylation: Take 6 g of whey protein isolate, dissolve it with phosphate buffer solution, add 6 g of allose, fully dissolve and mix, then freeze-dry, then keep the relative humidity at about 65% (saturated sodium chloride solution), put it in an oven, and carry out glycosylation reaction at 65°C. After 3 hours, take out the mixture, and after the reaction is completed, put the centrifuge tube in an ice bath to terminate the reaction.

[0054] 2. The glycosylated whey protein in step 1 was redissolved with phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then unreacted sugar and salt were removed using a 3 kDa ultrafiltration centrifuge tube. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4 ° C for 24 h with continuous stirring. The ultrapure water was replaced 3 times a day. After dialysis, it was freeze-dried.

[0055] Comparative Example 3

[0056] 1. Glycosylation: Take 6 g of whey protein isolate, dissolve it with phosphate buffer solution, add 6 g of fucose, fully dissolve and mix, then freeze-dry, then keep the relative humidity at about 65% (saturated sodium chloride solution), put it in an oven, and carry out glycosylation reaction at 65°C. After 3 hours, take out the mixture, and after the reaction is completed, put the centrifuge tube in an ice bath to terminate the reaction.

[0057] 2. The glycosylated whey protein in step 1 was redissolved with phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then unreacted sugar and salt were removed using a 3 kDa ultrafiltration centrifuge tube. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4 ° C for 24 h with continuous stirring. The ultrapure water was replaced 3 times a day. After dialysis, it was freeze-dried.

[0058] The whey proteins prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were analyzed. The experimental methods for grafting degree, antigenicity and antigen analysis after digestion are as follows:

[0059] Grafting degree detection method:

[0060] Dilute the sample to a protein and hydrolyzate concentration of 2 mg / mL, take out 200 μL, add 4 mL of OPA reagent, mix well and place in a 35°C water bath for 2 min, and measure the absorbance of the sample at 340 nm. The blank control group is 4.0 mL OPA + 200 μL distilled water. Preparation method of OPA reagent: Take 40 mg OPA and dissolve it in 1 mL methanol, add 25 mL of 0.1 mol / L borax solution, 2.5 mL of 20% (mass fraction) SDS solution, 100 μL β-mercaptoethanol, and dilute to 50 mL with deionized water.

[0061] The calculation formula of grafting degree is as follows:

[0062]

[0063] Where: A t —The absorbance of the graft solution at a wavelength of 340nm; A0—The absorbance of a single protein or hydrolyzate solution at a wavelength of 340nm under the same reaction conditions.

[0064] Antigenicity test method:

[0065] Solution preparation:

[0066] (1) Coating buffer solution: A 0.05 M carbonate buffer solution with a pH of 9.6 was used. The specific formula is as follows: 1.59 g Na2CO3 and 2.94 g NaHCO3 were mixed and diluted to 1 L with ultrapure water.

[0067] (2) Phosphate buffer solution (PBS) was prepared according to the following formula: 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 1.44 g of disodium hydrogen phosphate (Na2HPO4), 0.24 g of potassium dihydrogen phosphate (KH2PO4), 800 mL of double distilled water, and the pH was adjusted to 7.0-7.4 with 1 M HCl or NaOH, and the volume was fixed to 1 L. Then, the solution was sterilized by high pressure steam and stored at room temperature.

[0068] (3) Standard diluent: PBS solution containing 10% NBS, 0.05% Tween-20, and 0.2% Proline 300 preservative. Sample diluent: PBS solution containing 10% bovine serum and 0.2% Proline 300 preservative.

[0069] (4) Washing solution: Prepare a Tween-20 washing solution with a mass concentration of 0.05% using PBS buffer.

[0070] (5) Blocking solution: Dissolve gelatin in coating buffer and prepare 1% gelatin as blocking solution.

[0071] (6) Color developing solution: In this experiment, color developing solution A is carbamide peroxide and color developing solution B is TMB color developing solution.

[0072] (7) Stop solution: Use H2SO4 with a concentration of 2 mol / L as the stop solution.

[0073] Antigenicity assay steps:

[0074] (1) Antigen coating: dilute the sample to 1 mg / mL, prepare 2.5 μg / mL of purified bovine β-LG or α-LA with coating buffer, add 100 μL to each well of a 96-well plate, and coat at 4°C for 12 h.

[0075] (2) Blocking: Wash the plate, pat dry, and repeat the operation three times to absorb the unabsorbed coated antigen. Then, add 1% gelatin blocking solution to each well, 250 μL per well, and incubate in a 37°C incubator for 1 hour. This is the prepared solid phase antibody.

[0076] (3) Antigen-antibody reaction: dilute the standard with different concentration gradients. Add 50 μL of the standard with different concentrations to each well, add 40 μL of sample diluent and then 10 μL of sample to each well, shake evenly and add HRP enzyme-labeled antibody to each well. Set up a blank well. Seal the plate with a sealing film and place in a 37°C incubator for 60 min.

[0077] (4) Color development: Discard the liquid and spin dry. Wash the plate with detergent, repeat several times and pat dry. Add 50 μL of color developer carbamide peroxide to each well, then add 50 μL of color developer TMB solution, shake gently and develop color at 37°C in the dark for 15 min.

[0078] (5) Reaction termination and measurement: Add 50 μL of stop solution to each well. Then adjust the blank well to zero and measure the absorbance of each sample at a wavelength of 450 nm using an ELISA reader.

[0079] (6) Antigenicity calculation formula: Draw a standard curve based on the standard sample, and quantify the content of the allergenic protein in the sample using the standard curve. Calculate the residual antigenicity according to the following formula. Figure 1 .

[0080] Antigenicity A = C × N × 5 μg / L

[0081]

[0082] Wherein: C—the BLG or ALA content (μg / mL) of the diluted sample is calculated by substituting the absorbance value of the above reaction into the standard curve; N—the dilution multiple during sample treatment; A1—the antigenicity of the original sample (μg / L); A2—the antigenicity of the treated sample (μg / L).

[0083] The samples were processed by in vitro simulation of infant gastrointestinal digestion to determine the changes in allergenicity after digestion. The specific method is as follows:

[0084] (1) Gastric digestion: Before the reaction, the sample protein and pepsin solution (pH = 2) were preheated at 37°C for 10 min, with the ratio of pepsin to protein being 182:1 (units / mg protein). The sample and pepsin solution were mixed. Simulated gastric digestion was performed in a water bath at 37°C, and samples were taken at 60 and 120 min. After the reaction time was up, an appropriate amount of 1 M NaHCO3 solution was quickly added to adjust the pH to 6.5 to terminate the reaction. After the gastric digestion was completed, the antigenicity of the gastric digestion fluid was determined.

[0085] (2) Intestinal digestion: Take the sample digestion solution after 120 minutes of gastric digestion, add trypsin solution (pH 6.5) and mix evenly, and continue gastrointestinal digestion. The concentration ratio of trypsin and gastric digestion sample is 35:1 (units / mg protein), and samples are collected and retained at 60 and 120 minutes of digestion time. After the reaction time is up, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride is quickly added to make the final concentration of 4mmol / L to terminate the intestinal digestion. After the gastrointestinal digestion is completed, the antigenicity of the digestion solution is determined.

Claims

1. A method for preparing low-allergenic whey protein, characterized in that: The samples were enzymatically hydrolyzed, glycosylated, and finally digested, and the reduction in antigenicity was determined by indirect enzyme-linked immunosorbent assay. (1) Enzymatic hydrolysis: Take 6 g of whey protein isolate and dissolve it in a 10 mmol / L, pH 7.4 phosphate buffer solution, dissolve it according to the substrate concentration of 6% to make it hydrated. Adjust the pH to 10.0, add 6000-7000 U of alkaline protease per 1 g of whey protein, and perform enzymatic hydrolysis for 2 hours. After the hydrolysis is completed, the temperature is raised to 90°C and the water bath is bathed for 10 minutes to inactivate the enzyme. (2) Glycosylation: 6 g of allose was added to the hydrolyzed whey protein at a protein to sugar mass ratio of 1:1, the mixture was fully dissolved and mixed, and then freeze-dried. The relative humidity was maintained at about 65% (saturated sodium chloride solution), and the mixture was placed in an oven at 55° C. for glycosylation reaction. After 4 hours, the mixture was taken out and the reaction was terminated by placing the centrifuge tube in an ice bath. (3) The glycosylated whey protein was re-dissolved in phosphate buffer solution (0.01 mol / L, pH 7.4) to a concentration of 5 mg / mL, and then a 3 kDa ultrafiltration centrifuge tube was used to remove unreacted sugars and salts. In order to further remove unbound reducing sugars, the mixed solution was transferred into a dialysis bag and dialyzed at 4°C for 24 h with continuous stirring. The ultrapure water was replaced three times a day. After dialysis, the mixture was freeze-dried.

2. The preparation method according to claim 1, characterized in that: The preparation method involves enzymatic hydrolysis conditions of 6% substrate concentration, 2 hours of enzymatic hydrolysis time and a pH value of 10.

3. The preparation method according to claim 1, characterized in that: The preparation method involves that the glycosylated sugar is allose, and the mass ratio of glycosylated protein to sugar is 1:

1.

4. The preparation method according to claim 1, characterized in that: Sugar removal was performed using a 3 kDa ultrafiltration centrifuge tube.

5. The preparation method according to claim 1, characterized in that: The preparation method is suitable for preparing whey protein from cow's milk and goat's milk.

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