Method for enriching whey protein main monomer from cow milk

Abstract

The invention discloses a method for enriching whey protein main monomers from cow milk. According to the method, whey protein subjected to membrane separation is taken as a raw material, interaction between main monomers of the whey protein and other milk proteins is weakened by adopting a pH conversion technology and an ultrasonic technology, mineral substances, lactose, bovine serum albumin and residual casein are removed through ion exchange chromatography, the main monomers of the whey protein are enriched, and the high-purity whey protein is obtained. And drying the alpha-lactalbumin and the beta-lactoglobulin to obtain a milk protein product rich in two main monomers of the whey protein, and the total content of the two monomers is up to 85% or above. According to the technology, cow milk serves as a raw material, efficient enrichment of two main monomers of whey protein can be achieved only through membrane separation, pH conversion, ultrasonic assistance and ion exchange chromatography, only pH needs to be adjusted in the separation process, other chemical reagents are not added, and the process is environmentally friendly, simple, easy to implement and suitable for large-scale processing; the prepared milk protein base material rich in the whey protein main monomer can be used as a main material and a base material of infant food, and is high in nutritional value and wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the field of food processing, and in particular relates to a method for enriching main monomers of whey protein from cow's milk. Technical Background

[0002] Whey protein is a type of protein that is soluble at pH 4.6. It is a mixture of multiple protein components, the main components of which are β-lactoglobulin and α-lactalbumin. Whey protein is rich in essential amino acids. Because the ratio of amino acids it contains meets the needs of the human body, it is recognized as one of the high-quality protein supplements for the human body. It has high digestibility, high bioavailability, and rich nutritional value. Whey protein is rich in branched-chain amino acids, such as leucine, isoleucine, and valine; these branched-chain amino acids play a key role in sports nutrition, especially leucine, which can directly stimulate the synthesis of muscle protein and promote muscle repair and growth. In addition, whey protein is the main protein in breast milk, accounting for more than 60% of the breast milk protein content. Therefore, whey protein is called the king of protein and is the main base material and nutritional enhancer for infant formula milk powder.

[0003] Whey protein in cow's milk is easy to combine with casein micelles during high-temperature sterilization, making it difficult to separate from the casein component. In addition, cow's milk is easy to spoil and has high requirements for the storage environment. The milk protein separation cycle is not easy to be too long, which makes it difficult to enrich and separate whey protein monomers. There is still a lack of efficient and convenient β-lactoglobulin and α-lactalbumin monomer enrichment technology. Relevant reports indicate that the isoelectric point of bovine serum albumin is about 5.8, and the isoelectric point of lactoferrin is about 8.0. β-lactoglobulin is sensitive to environmental pH. When pH>7, β-lactoglobulin becomes an expanded molecular form, and the intermolecular repulsion decreases; at this time, the repulsion between а-lactalbumin molecules is enhanced, and the strong repulsion leads to reduced aggregation and increased solubility. It can be seen that adjusting the appropriate pH is conducive to the separation of the main monomers of whey protein. αs-casein is sensitive to calcium ions; at neutral pH, calcium ions adsorb to αs1-casein. As the concentration of calcium ions increases, the surface charge of αs1-casein decreases, the Zeta potential decreases, the stability decreases, and even precipitation occurs. αs2-casein contains two cysteine ​​residues, which are easy to form disulfide bridges. It can bind to 10 phosphate ions on serine and is more sensitive to calcium ions. Under neutral pH conditions, each mole of αs2-casein can bind to 10 mol of calcium ions. Compared with αs1-casein and αs2-casein, β-casein is less sensitive to calcium ions. In addition, whey protein is also less sensitive to calcium ions. It can be seen that adjusting the calcium ion content in the system is beneficial to the separation of casein and whey protein.

[0004] Low-frequency ultrasound technology is a non-thermal processing technology that has emerged in recent years. Ultrasound can produce cavitation, thermal and microjet effects in liquid matrices, causing strong turbulence, generating high shear forces, and increasing the frequency of collisions between particles, thereby weakening particle agglomeration and changing their physical properties. The team's previous research found that ultrasound-assisted technology can promote the dissociation of casein and whey protein aggregates and reduce the difficulty of separating milk protein monomers.

[0005] Based on the above theoretical basis and research conclusions, the present invention uses membrane separation technology to separate casein micelles from cow's milk, and then uses membrane separation technology to remove lactose in the filtrate to achieve the enrichment and concentration of whey protein; then, according to the isoelectric points of lactoferrin and bovine serum albumin and the structural characteristics and solubility of β-lactoglobulin and α-lactalbumin in different pH environments, the pH of the system is adjusted to promote the full separation of the main monomers of whey protein from other protein components; then, using the characteristic of casein being sensitive to calcium ions, calcium chloride is added to promote casein aggregation, and then ultrasonic technology is used to weaken the interaction between casein and whey protein; finally, anion exchange resin is used to enrich and separate β-lactoglobulin and α-lactalbumin in whey protein to prepare a milk protein product rich in the two main monomers of whey protein, wherein the total content of the two monomers is as high as more than 85%.

[0006] The milk protein base prepared by the technology of the present invention is rich in β-lactoglobulin and α-lactalbumin, and can be used as a raw material for infant formula milk powder and infant food, and can also be used as a nutritional supplement for fitness and sports people. The preparation process of the milk protein base does not require any heat treatment, enzyme treatment and other processes, and does not add other chemical reagents. It has the advantages of simple process, low processing cost, and natural green. The milk protein base can also be used as a functional food base to strengthen protein, improve the nutritional properties of the product, and increase bioavailability. At the same time, the casein micelle product is obtained after the first membrane separation interception liquid is dried, which can be used as a protein enhancer in the food industry; the lactose crude product is obtained after the secondary membrane separation filtrate is dried, which can be used as a high-purity lactose processing raw material, and can also be used as a food auxiliary material for lactose-containing food processing. Summary of the invention

[0007] The technical problem to be solved by the present invention is, on the one hand, to achieve the enrichment of β-lactoglobulin and α-lactalbumin and reduce the casein content in the whey protein product; on the other hand, to simplify the whey protein separation process, provide a simple and feasible β-lactoglobulin and α-lactalbumin enrichment technology for industrial production, and achieve green and efficient preparation of the main monomers of whey protein.

[0008] The technical solution adopted by the present invention is: A method for enriching the main monomers of whey protein from cow's milk, the specific steps are as follows: (1) Milk membrane separation: sterilized milk is centrifuged and skimmed at 4000-6000g for 20-30min, the upper fat layer is discarded, the lower skim milk is collected, and the skim milk is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100kDa. The membrane inlet pressure is 0.5-1.0MPa, the concentration multiple is 3-5, and 3-6 times the volume of deionized water as the raw skim milk is added to dilute the retentate. After low-speed circulation balance for 20-60min, ultrafiltration is continued to concentrate 3-5 times to obtain whey protein filtrate. The retentate is casein micelles. (2) Preparation of whey protein concentrate: The filtrate in step (1) is used as a raw material, and is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa. The membrane inlet pressure is 0.5-1.0 MPa, and the concentration multiple is 3-5. Deionized water 3-6 times the volume of the raw material is added to dilute the retentate. After low-speed circulation equilibrium for 20-60 minutes, ultrafiltration is continued to concentrate 3-5 times. The retentate is a whey protein concentrate, and the filtrate is rich in lactose. (3) Ultrasonic-assisted alkalization treatment: Take 100-300 mL of whey protein concentrate, add 0.5-1.0 mol / L sodium hydroxide at a rate of 0.4-0.6 mL / min under magnetic stirring at 500-1000 r / min, adjust the pH to 8.0-9.0 and stabilize it, then perform ultrasonic treatment on the solution. The diameter of the ultrasonic probe is 12.7 mm, the depth of immersion in the center of the sample is 2-4 cm, the ultrasonic frequency is 20 kHz, the amplitude is 20-40%, the ultrasonic time is 6-10 min, and the pulse mode is 1:1. The process was started for 3-5 seconds and stopped for 2-4 seconds. Then, 5-10 mL of a 0.06-0.08 mol / L calcium chloride solution was slowly added at a rate of 0.4-0.6 mL / min under magnetic stirring at 500-1000 r / min. The solution was then ultrasonically treated. The diameter of the ultrasonic probe was 12.7 mm, the depth of immersion in the center of the sample was 2-4 cm, the ultrasonic frequency was 20 kHz, the amplitude was 20-40%, the ultrasonic time was 6-10 min, and the pulse mode was started for 3-5 seconds and stopped for 2-4 seconds. (4) Ion exchange chromatography: Weigh 500-1000 g of anion exchange resin and load it into a chromatography column. Rinse the resin with 0.5 mol / L sodium hydroxide solution until it is alkaline, then rinse it with distilled water until it is neutral, then rinse the resin with 0.5 mol / L hydrochloric acid until it is acidic, and then rinse it with distilled water until it is neutral. Take 100-200 mL of the solution treated in step (3) and pump it into the chromatography column. Elute with distilled water (liquid A) and 0.5 mol / L sodium chloride aqueous solution (liquid B). Each gradient The eluent is 0.4-0.6 times the column volume, the eluent flow rate is 5-10 mL / min, and the elution gradient is that the volume ratio of solution A to solution B is 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively; the eluents are collected respectively, and the absorbance of the eluents at 220 nm and 280 nm is measured by a UV-visible spectrophotometer, and the elution curve is drawn to collect the protein elution peak; (5) Spray drying: The collected elution peak is spray dried at an inlet temperature of 130-150° C., an atomization pressure of 0.05-0.1 MPa, a striker interval of 1 s, a striker execution time of 1 s, and a material flow rate of 200-300 mL / h. After drying, a milk protein base rich in whey protein monomer is obtained. The retentate in step (1) is dried to obtain casein micelle powder. The filtrate in step (2) is dried to obtain crude lactose.

[0009] Beneficial effects or advantages of the present invention: (1) The present invention utilizes membrane separation technology, uses cow milk as raw material, first realizes the separation of casein micelles and whey protein, then uses membrane separation technology to concentrate whey protein, and then slowly adjusts the pH to alkaline to weaken the interaction between whey protein monomers and casein; then slowly adds calcium ions to achieve casein aggregation. This technology utilizes the structural characteristics of each component of whey protein in different pH environments, cleverly utilizes pH changes, promotes the change of the main monomer structure of whey protein, and enhances solubility. Then, it utilizes the sensitivity of casein monomers to calcium ions, reduces its solubility by adding calcium ions, and changes the charge it carries, and then uses anion exchange resin to achieve the separation of casein and whey protein; the separation process does not add other chemical reagents, only fine-tunes the pH, and the process is green and pollution-free, the process is simple and feasible, and the cost is low.

[0010] (2) The present invention uses skim milk as a raw material, and while achieving the separation of the two main monomers of whey protein, obtains a casein micelle product, thereby simplifying the difficulty of milk protein separation. A milk protein base rich in casein micelles and two main monomers rich in whey protein can be obtained at the same time, and a crude lactose product can also be obtained, providing the food industry and the health care product industry with a selectable milk protein monomer base and lactose auxiliary material. The separation process is gentle, and the nutritional value of milk protein is retained to the maximum extent.

[0011] (3) The present invention uses ultrasound-assisted technology to promote structural changes in whey protein monomers, weaken the non-covalent interaction between whey protein and casein, and promote the separation of the two, so as to better enrich the two main monomers of whey protein. In addition, ultrasound treatment can effectively promote the interaction between calcium ions and casein, promote the cross-linking of casein and calcium ions to form aggregates, significantly reduce the surface charge of casein, and facilitate the elution of casein in the subsequent anion exchange chromatography process, thereby achieving efficient separation of casein from whey protein.

[0012] In summary, the present invention utilizes pH control, ultrasound combined with anion exchange chromatography technology to achieve the enrichment of two main monomers of whey protein, and develops a whey protein base rich in β-lactoglobulin and α-lactalbumin, providing high-quality milk protein base for the food and health care product industries, with a simple processing process, a wide range of product applications, and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Attached Figure 1 This is the elution curve of Example 1 of the present invention.

[0014] Attached Figure 2 This is the elution curve of Example 2 of the present invention.

[0015] Attached Figure 3 This is the elution curve of Example 3 of the present invention.

[0016] Attached Figure 4 The elution curves of the β-lactoglobulin standard product, the first membrane separation filtrate, and the separation obtained by the processes of Examples 1 to 3 in the present invention (see Appendix Figures 1 to 3 ) are the electrophoretic bands of the elution peaks marked on the graph. DETAILED DESCRIPTION

[0017] In order to better understand the present invention, the present invention is further described below in conjunction with the accompanying drawings and embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0018] The method for drawing the elution curve in the embodiment is as follows: the eluate is collected, and the absorbance of the eluate at 220nm and 280nm is measured by a UV-visible spectrophotometer, and the elution curve is drawn with the number of test tubes as the abscissa and the absorbance as the ordinate. The test tube solution corresponding to the higher elution peak in the elution curve is taken for gel electrophoresis experiment.

[0019] The gel electrophoresis method in the embodiment is as follows: the eluent and the electrophoresis buffer are mixed uniformly at a volume ratio of 2:1, boiled in a water bath for 4 minutes, centrifuged at 4000 r / min for 4 minutes, 10 μL of the supernatant is injected into the gel track and electrophoresed at a constant voltage of 130 V. The gel slice is stained in Coomassie Brilliant Blue R-250 staining solution, and then photographed after decolorization.

[0020] Example 1, a method for enriching the main monomers of whey protein from cow's milk, the specific steps are as follows: (1) Milk membrane separation: sterilized milk was centrifuged and skimmed at 4000g for 30 min, the upper fat layer was discarded, the lower skim milk was collected, and the skim milk was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The membrane inlet pressure was 0.5 MPa, the concentration factor was 3, and 3 times the volume of deionized water as the skim milk raw material was added to dilute the retentate. After low-speed circulation balance for 60 min, ultrafiltration and concentration were continued for 3 times to obtain whey protein filtrate. The retentate was casein micelles. (2) Preparation of whey protein concentrate: The filtrate in step (1) is used as a raw material and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa. The membrane inlet pressure is 0.5 MPa and the concentration factor is 3. Deionized water 3 times the volume of the raw material is added to dilute the retentate. After low-speed circulation balance for 60 minutes, ultrafiltration is continued to concentrate 3 times. The retentate is a whey protein concentrate and the filtrate is rich in lactose. (3) Ultrasonic-assisted alkalization treatment: Take 100 mL of whey protein concentrate, add sodium hydroxide with a concentration of 1.0 mol / L at a rate of 0.4 mL / min under magnetic stirring at 500 r / min, adjust the pH to 8.0 and stabilize, then perform ultrasonic treatment on the solution, the diameter of the ultrasonic probe is 12.7 mm, the depth of immersion into the center of the sample is 2 cm, the ultrasonic frequency is 20 kHz, the amplitude is 20%, the ultrasonic time is 10 min, and the pulse mode is on for 3 s and off for 2 s; then, under magnetic stirring at 500 r / min, slowly add 5 mL of calcium chloride solution with a concentration of 0.06 mol / L at a rate of 0.4 mL / min, then perform ultrasonic treatment on the solution, the diameter of the ultrasonic probe is 12.7 mm, the depth of immersion into the center of the sample is 2 cm, the ultrasonic frequency is 20 kHz, the amplitude is 20%, the ultrasonic time is 10 min, and the pulse mode is on for 3 s and off for 2 s; (4) Ion exchange chromatography: Weigh 500 g of D213 resin and load it into a chromatography column. Rinse the resin with 0.5 mol / L sodium hydroxide solution until it is alkaline, then rinse it with distilled water until it is neutral, then rinse the resin with 0.5 mol / L hydrochloric acid until it is acidic, and then rinse it with distilled water until it is neutral. Take 100 mL of the solution treated in step (3) and pump it into the chromatography column. Elute with distilled water (liquid A) and 0.5 mol / L sodium chloride aqueous solution (liquid B). The elution gradient is liquid A: The volume ratio of solution B was 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. Each gradient elution was 100 mL, and the flow rate of the elution was 5 mL / min. Every 15 mL of the elution was collected in one bottle. The absorbance of the elution at 220 nm and 280 nm was measured by a UV-visible spectrophotometer, and the elution curve was drawn to collect the protein elution peak. (5) Spray drying: The collected elution peak is spray dried with an inlet temperature of 130°C, an atomization pressure of 0.05 MPa, an impaction interval of 1 s, an impaction execution time of 1 s, and a material flow rate of 200 mL / h. After drying, a milk protein base rich in two monomers of whey protein is obtained.

[0021] See attached for elution curve Figure 1 The gel electrophoresis results of elution peaks 1 to 3 are shown in the attached Figure 4 . As can be seen from the figure, the separation effect of each protein peak is good. Discard the first large peak, which is the aggregated casein, and take the other peaks for gel electrophoresis test. It can be seen that they are all mixtures of β-lactoglobulin and а-lactalbumin. It was determined that the protein content in the whey protein product was 92.68±2.13%, and the total content of the two monomers was 86.45±0.54%.

[0022] Example 2, a method for enriching the main monomers of whey protein from cow's milk, the specific steps are as follows: (1) Milk membrane separation: sterilized milk was centrifuged and skimmed at 6000g for 20 min, the upper fat layer was discarded, the lower skim milk was collected, and the skim milk was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The membrane inlet pressure was 1.0 MPa, the concentration factor was 5, and 6 times the volume of deionized water as the raw skim milk was added to dilute the retentate. After low-speed circulation balance for 20 min, ultrafiltration was continued to concentrate 5 times to obtain whey protein filtrate. The retentate was casein micelles. (2) Preparation of whey protein concentrate: The filtrate in step (1) is used as a raw material and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa. The membrane inlet pressure is 1.0 MPa and the concentration factor is 5. Deionized water 6 times the volume of the raw material is added to dilute the retentate. After low-speed circulation balance for 20 minutes, ultrafiltration is continued to concentrate 5 times. The retentate is a whey protein concentrate and the filtrate is rich in lactose. (3) Ultrasonic-assisted alkalization treatment: 300 mL of whey protein concentrate was added with 0.5 mol / L sodium hydroxide at a rate of 0.6 mL / min under magnetic stirring at 1000 r / min. After the pH was adjusted to 9.0 and stabilized, the solution was ultrasonically treated with an ultrasonic probe of 12.7 mm in diameter, a depth of 4 cm in the center of the sample, an ultrasonic frequency of 20 kHz, an amplitude of 40%, an ultrasonic time of 6 min, and a pulse mode of 5 s on and 4 s off. Then, 10 mL of 0.08 mol / L calcium chloride solution was slowly added at a rate of 0.6 mL / min under magnetic stirring at 1000 r / min. Then, the solution was ultrasonically treated with an ultrasonic probe of 12.7 mm in diameter, a depth of 4 cm in the center of the sample, an ultrasonic frequency of 20 kHz, an amplitude of 40%, an ultrasonic time of 6 min, and a pulse mode of 5 s on and 4 s off. (4) Ion exchange chromatography: Weigh 1000 g of 213 resin and load it into a chromatography column. Rinse the resin with 0.5 mol / L sodium hydroxide solution until it is alkaline, then rinse it with distilled water until it is neutral, then rinse the resin with 0.5 mol / L hydrochloric acid until it is acidic, and then rinse it with distilled water until it is neutral. Take 200 mL of the solution treated in step (3) and pump it into the chromatography column. Elute with distilled water (liquid A) and 0.5 mol / L sodium chloride aqueous solution (liquid B). The elution gradient is: the volume ratio of liquid A to liquid B is 1:1. 00:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, each gradient elution was 200 mL, the eluent flow rate was 10 mL / min, and each 25 mL of eluent was collected in one bottle. The absorbance of the eluent at 220 nm and 280 nm was measured by UV-visible spectrophotometer, and the elution curve was drawn to collect the protein elution peak; (5) Spray drying: The collected elution peak is spray dried with an inlet temperature of 150°C, an atomization pressure of 0.1 MPa, an impactor interval of 1 s, an impactor execution time of 1 s, and a material flow rate of 300 mL / h. After drying, a milk protein base rich in the main monomers of whey protein is obtained.

[0023] See attached for elution curve Figure 2 The gel electrophoresis results of elution peaks 1 to 4 are shown in the attached Figure 4. As can be seen from the figure, the separation effect of each protein peak is good. Discard the first large peak, which is the aggregated casein, and take the other peaks for gel electrophoresis test. It can be seen that they are all mixtures of β-lactoglobulin and а-lactalbumin. It was determined that the protein content in the whey protein product was 93.21±1.45%, and the total content of the two monomers was 87.12±1.02%.

[0024] Example 3, a method for enriching the main monomers of whey protein from cow's milk, the specific steps are as follows: (1) Milk membrane separation: sterilized milk was centrifuged and skimmed at 5000g for 25 min, the upper fat layer was discarded, the lower skim milk was collected, and the skim milk was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The membrane inlet pressure was 0.75 MPa, the concentration factor was 4, and 4.5 times the volume of deionized water as the raw skim milk was added to dilute the retentate. After low-speed circulation balance for 40 min, ultrafiltration was continued to concentrate 4 times to obtain whey protein filtrate. The retentate was casein micelles. (2) Preparation of whey protein concentrate: The filtrate in step (1) is used as a raw material and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa. The membrane inlet pressure is 0.75 MPa and the concentration factor is 4. Deionized water 4.5 times the volume of the raw material is added to dilute the retentate. After low-speed circulation balance for 40 minutes, ultrafiltration is continued to concentrate 4 times. The retentate is a whey protein concentrate and the filtrate is rich in lactose. (3) Ultrasonic-assisted alkalization treatment: Take 200 mL of whey protein concentrate, add 0.7 mol / L sodium hydroxide at a rate of 0.5 mL / min under magnetic stirring at 750 r / min, adjust the pH to 8.5 and stabilize, then perform ultrasonic treatment on the solution, the diameter of the ultrasonic probe is 12.7 mm, the depth of immersion into the center of the sample is 3 cm, the ultrasonic frequency is 20 kHz, the amplitude is 30%, the ultrasonic time is 8 min, and the pulse mode is on for 4 s and off for 3 s; then, under magnetic stirring at 750 r / min, slowly add 7.5 mL of 0.05 mol / L calcium chloride solution at a rate of 0.5 mL / min, then perform ultrasonic treatment on the solution, the diameter of the ultrasonic probe is 12.7 mm, the depth of immersion into the center of the sample is 3 cm, the ultrasonic frequency is 20 kHz, the amplitude is 30%, the ultrasonic time is 8 min, and the pulse mode is on for 4 s and off for 3 s; (4) Ion exchange chromatography: Weigh 750 g of D301-III resin and load it into a chromatography column. Rinse the resin with 0.5 mol / L sodium hydroxide solution until it is alkaline, then rinse it with distilled water until it is neutral, then rinse the resin with 0.5 mol / L hydrochloric acid until it is acidic, and then rinse it with distilled water until it is neutral. Take 150 mL of the solution treated in step (3) and pump it into the chromatography column. Elute with distilled water (liquid A) and 0.5 mol / L sodium chloride aqueous solution (liquid B). The elution gradient is: the volume of liquid A and liquid B: The ratios were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. Each gradient elution was 150 mL, and the eluent flow rate was 7.5 mL / min. Every 20 mL of eluent was collected in one bottle. The absorbance of the eluent at 220 nm and 280 nm was measured by a UV-visible spectrophotometer, and the protein elution peak was collected; (5) Spray drying: The collected elution peak is spray dried with an inlet temperature of 140°C, an atomization pressure of 0.07 MPa, an impactor interval of 1 s, an impactor execution time of 1 s, and a material flow rate of 250 mL / h. After drying, a milk protein base rich in the main monomers of whey protein is obtained.

[0025] See attached for elution curve Figure 3 The gel electrophoresis results of elution peaks 1 to 3 are shown in the attached Figure 4 . As can be seen from the figure, the separation effect of each protein peak is good. Discard the first large peak, which is the aggregated casein, and take the other peaks for gel electrophoresis test. It can be seen that they are all mixtures of β-lactoglobulin and а-lactalbumin. It was determined that the protein content in the whey protein product was 92.87±1.55%, and the content of the two monomers was 86.21±0.87%.

Claims

1. A method for enriching the main monomers of whey protein from cow's milk, characterized in that The process includes the following steps and process conditions: (1) Milk membrane separation: The sterilized milk was centrifuged and skimmed at 4000-6000g for 20-30min, the upper fat layer was discarded, and the lower skim milk was collected. The skim milk was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100kDa. The membrane inlet pressure was 0.5-1.0MPa, and the concentration multiple was 3-5. Deionized water 3-6 times the volume of the skim milk raw material was added to dilute the retentate. After low-speed circulation balance for 20-60min, ultrafiltration was continued to concentrate 3-5 times to obtain whey protein filtrate. The retentate was casein micelles. (2) Preparation of whey protein concentrate: The filtrate in step (1) is used as a raw material, and is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa. The membrane inlet pressure is 0.5-1.0 MPa, and the concentration multiple is 3-5. Deionized water 3-6 times the volume of the raw material is added to dilute the retentate. After low-speed circulation balance for 20-60 minutes, ultrafiltration is continued to concentrate 3-5 times. The retentate is a whey protein concentrate; (3) Ultrasonic-assisted alkalization treatment: Take 100-300 mL of whey protein concentrate, slowly add 0.5-1.0 mol / L sodium hydroxide under magnetic stirring, adjust the pH to 8.0-9.0 and stabilize it, then treat the solution with ultrasound, then slowly add 5-10 mL of 0.06-0.08 mol / L calcium chloride solution, and then treat the solution with ultrasound; (4) Ion exchange chromatography: Take 100-200 mL of the solution treated in step (3), pump it into a chromatography column containing 500-1000 g of activated anion exchange resin, use an eluent for gradient elution, use a UV-visible spectrophotometer to measure the absorbance of the eluent at 220 nm and 280 nm, draw an elution curve, and collect the protein elution peak. Each gradient eluent is 0.4-0.6 times the column volume, and the flow rate is 5-10 mL / min; (5) Spray drying: The collected elution peak is spray dried, with an inlet temperature of 130-150° C., an atomization pressure of 0.05-0.1 MPa, a striker interval of 1 s, a striker execution time of 1 s, and a material flow rate of 200-300 mL / h. The filtrate is dried to obtain a milk protein base rich in the main monomers of whey protein. The filtrate in step (2) is dried to obtain a crude lactose product. The retentate in step (1) is dried to obtain a casein micelle powder.

2. A method for enriching main monomers of whey protein from milk according to claim 1, characterized in that: The sodium hydroxide, hydrochloric acid and calcium chloride described in step (3) are all added at a rate of 0.4-0.6 mL / min, and the magnetic stirring rate is 500-1000 r / min. The ultrasonic treatment conditions described in step (3) are as follows: the diameter of the ultrasonic probe is 12.7 mm, the depth of immersion in the center of the sample is 2-4 cm, the ultrasonic frequency is 20 kHz, the amplitude is 20-40%, the ultrasonic time is 6-10 min, and the pulse mode is on for 3-5 s and off for 2-4 s.

3. A method for enriching main monomers of whey protein from cow's milk according to claim 1, characterized in that: The anion exchange resin described in step (4) can be a macroporous styrene-based, acrylic acid-based strong base anion exchange resin, a gel-type styrene-based, acrylic acid-based strong base anion exchange resin, a macroporous styrene-based, acrylic acid-based weak base anion exchange resin, or a gel-type styrene-based, acrylic acid-based weak base anion exchange resin; the resin activation method is to use 0.5 mol / L sodium hydroxide solution to wash the resin to alkalinity, then wash it with distilled water to neutrality, then wash the resin with 0.5 mol / L hydrochloric acid to acidity, and then wash it with distilled water to neutrality; The eluents were distilled water (liquid A) and 0.5 mol / L sodium chloride aqueous solution (liquid B), and the elution gradient was the volume ratio of liquid A to liquid B of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively.

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