CN117770347A - Preparation of modified whey protein and application of modified whey protein in embedding apigenin - Google Patents

Abstract

The invention relates to the preparation and application of a modified whey protein, which particularly exhibits good embedding effect in embedding apigenin. The steps of the preparation method are as follows: (1) Take 49 mL of deionized water and dissolve 0.5 g of whey protein in a beaker. After the protein is fully dissolved, add 1 mL of prepared 5M hydrogen peroxide solution to the beaker, and weigh 0.25 g of ascorbic acid. Add it, mix well and let it stand at room temperature for 2 hours. (2) After 2 hours, add 0.35mM EGCG and 1mM CaCl ₂ to the protein solution, stir to dissolve the polyphenols and CaCl ₂ , and then let the solution stand at room temperature for 24 hours. (3) After the reaction is completed, use a 3500D dialysis bag to dialyze the reacted solution to remove excess polyphenols that are not bound to protein. The solution is dialyzed at 4°C for 48 hours, and the dialysate is changed every 6 hours. The present invention studies how adding Ca ²⁺ improves the grafting rate of WP-EGCG, enhances the functional properties of whey protein, and improves the embedding rate of apigenin.

Description

Preparation of a modified whey protein and its application in encapsulating apigenin

Technical Field

The invention belongs to the field of food technology and relates to the preparation and application of modified whey protein, and in particular shows a good embedding effect in embedding apigenin.

Background technique

Proteins are natural delivery vehicles and have the special ability to form protein-ligand complexes with small molecule compounds, and at the same time provide protection for small molecule compounds. In recent years, encapsulation and delivery of bioactive substances have been a popular research direction in colloidal particles. Whey protein (WP) is a high-quality animal protein with many active ingredients, a complete range of essential amino acids, rich content, and high nutritional value. It is recognized as the "King of Protein". Based on the above advantages, whey protein has attracted widespread attention. Reports indicate that the utilization rate of whey is low, with only about half of the whey being further processed, which wastes resources and pollutes the environment. Therefore, it is of practical significance to improve the utilization rate of whey protein through protein modification technology and produce products with high added value for the full and reasonable utilization of whey protein. Whey protein plays an important role in the food industry because of its special physical and chemical properties. Research shows that whey protein, a common food ingredient, can be used as a food additive such as an emulsifier, foaming agent and water binder to give products desirable properties. In addition, the physical and chemical properties of whey protein can be changed through molecular modification technology to enhance the application feasibility of whey protein as a food ingredient, especially the use of whey protein to enhance the rheological and structural properties of products, e.g. In edible films, coatings, hydrogels and nanoparticles, etc. Increasing research involves modifying WP to enhance functionality and thereby add value to whey protein.

Epigallocatechin gallate (EGCG) is the main active ingredient of flavanols in polyphenols and the catechin with the highest content in green tea. It has multiple effects such as antioxidant, antibacterial, and antitumor. Free radical grafting is a reaction between the active groups on the side chains of proteins and hydroxyl radicals to form covalent bonds between polyphenols and proteins. The formation of covalent bonds involves irreversible interactions, thereby forming a more stable conjugate. Compared with the enzymatic and alkaline methods, the free radical grafting method does not involve organic solvents during the reaction process and has higher safety. It is widely used in the food industry. How to improve the grafting rate of WP-EGCG is one of the problems that researchers urgently need to solve. .Apigenin (AP) is a potential drug for the development of strategies for the prevention and treatment of cancer. Studies have shown that apigenin can inhibit cancer cell proliferation, promote cell cycle arrest, and induce cancer cell apoptosis. However, the application of apigenin in improving human health is hindered by its low water solubility. Therefore, a lot of work needs to be done to improve the solubility and bioavailability of apigenin.

The present invention studies how to increase the grafting rate of WP-EGCG by adding Ca ²⁺ , enhance the functional properties of whey protein, and improve the embedding rate of apigenin. This research provides new ideas and new ways for the development of protein modification theory and technology.

Through searching, no published patent documents related to the patent application of the present invention have been found.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide a new technology based on the prior art that can improve the grafting rate of WP-EGCG, thereby improving the functional properties of whey protein, and study its application in encapsulating apigenin.

The technical solution adopted by the present invention is:

Preparation of modified whey protein, the steps of the preparation method are as follows:

(1) Dissolve whey protein in water, add hydrogen peroxide solution, then add ascorbic acid, mix well and let stand.

(2) After letting it stand, add EGCG and CaCl ₂ , stir and let it stand for reaction.

(3) After the reaction is completed, use a dialysis bag to dialyze, and the solution after dialysis is freeze-dried to obtain modified whey protein.

Specifically, the steps of the preparation method are as follows:

(1) Take 30-60mL of deionized water and dissolve 0.2-1g of whey protein in a beaker. After the protein is fully dissolved, add the prepared hydrogen peroxide solution to the beaker, weigh the ascorbic acid and add it, mix evenly and then Let stand at room temperature for 1-3h.

(2) After standing, add EGCG and CaCl ₂ to the protein solution, stir, and then let the solution stand at room temperature for 28-30 hours.

(3) After the reaction is completed, use a dialysis bag to dialyze, dialyze at 4°C for 48 hours, change the dialysate every 6 hours, and freeze-dry the modified whey protein in the solution after dialysis.

In step (1), add 1 mL of 5M hydrogen peroxide solution and 0.25 g of ascorbic acid, mix evenly and let stand at room temperature for 2 hours.

In step (2), add 0.35mM EGCG and 1mM CaCl ₂ .

In step (3), use a 3500D dialysis bag for dialysis.

The steps for entrapping apigenin in modified whey protein are as follows:

(1) Redissolve the modified whey protein in water.

(2) Add apigenin to the solution and stir.

(3) Freeze dry and store for later use.

Specifically, the steps of the embedding method are as follows:

(1) Dissolve the modified whey protein in deionized water at a concentration of 10 mg/mL, stir to fully hydrate the protein, and let it come to room temperature.

(2) Add apigenin and stir for 30 minutes.

(3) Freeze-drying the solution.

In step (2), the mixture was stirred on a magnetic stirrer for 2 h and then stored in a 4° C. refrigerator for 12 h.

In step (3), add 0.2, 0.4, 0.6, 0.8mg/mL apigenin.

The advantages and positive effects achieved by the present invention are:

1. The present invention creatively adds CaCl ₂ , which is non-toxic and harmless and can be added to food, thus broadening its application in the field of food processing.

2. After adding CaCl ₂ , the grafting rate of modified whey protein was significantly improved. Compared with the WP control, the polyphenol binding equivalents of WP-EGCG and WP-EGCG-Ca ²⁺ covalent complexes increased. , and the WP-EGCG-Ca ²⁺ group has the most polyphenol binding. The content of free amino groups and sulfhydryl groups decreased. In addition to these property changes, due to the polyhydroxyl properties of EGCG, the antioxidant capacity, solubility, foaming, emulsifying and other protein functions of WP are enhanced after being combined with EGCG. This provides a theoretical basis for WP to improve functional characteristics in practical applications.

3. Apigenin has high hydrophobicity, poor water solubility, and poor photothermal stability. After using WP-EGCG-Ca ²⁺ covalent complex as wall material to embed apigenin, its bioavailability was greatly improved. In this invention, we design a stable, soluble and bioavailable structure to improve the solubility and bioaccessibility of apigenin. This research aims to extend the modification and functionalization of animal proteins into value-added and cost-effective food and nutritional materials that may find applications in many fields.

Description of drawings

Figure 1 shows the group content of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ covalent complexes in the present invention.

Figure 2 shows the endogenous fluorescence, tyrosine synchronized fluorescence spectrum, and tryptophan synchronized fluorescence spectrum of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ covalent complexes in the present invention;

FIG3 is a Fourier transform infrared spectrum of the covalent complex of WP and WP-EGCG, WP-EGCG-Ca ²⁺ in the present invention;

FIG4 is a scanning electron micrograph of the covalent complex of WP, EGCG, CaCl ₂ , WP-P, WP-EGCG, and WP-EGCG-Ca ²⁺ in the present invention;

Figure 5 is a graph showing the protein solubility of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ covalent complexes in the present invention;

Figure 6 is a diagram of the emulsification and emulsification stability of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ covalent complexes in the present invention;

Figure 7 is a diagram of the entrapment efficiency of apigenin by WP, WP-EGCG, and WP-EGCG-Ca ²⁺ covalent complexes in the present invention;

Figure 8 is a diagram showing the retention rate of free apigenin and embedded apigenin under 20W fluorescent lamp irradiation in the present invention.

Figure 9 is a diagram showing the retention rates of free apigenin and embedded apigenin at 60°C and 80°C in the present invention.

Figure 10 is a diagram showing the retention rates of free apigenin and embedded apigenin in simulated gastrointestinal digestion in the present invention.

Detailed ways

The present invention will be further described below in conjunction with the examples; the following examples are illustrative, not restrictive, and the protection scope of the present invention cannot be limited by the following examples.

The raw materials used in the present invention, unless otherwise specified, are all conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the field.

Example 1

Preparation of WP-EGCG covalent complex, the steps of the preparation method are as follows:

(1) Dissolve 0.5g of whey protein in 49mL of deionized water in a beaker. After the protein is fully dissolved, add 1mL of prepared 5M hydrogen peroxide solution into the beaker. Weigh 0.25g of ascorbic acid and add it into the beaker. Mix evenly. Let stand at room temperature for 2h.

(2) After 2 hours, add 0.35mM EGCG to the protein solution, stir to dissolve, and then let the solution stand at room temperature for 24 hours.

(3) After the reaction is completed, use a 3500D dialysis bag to dialyze the reacted solution to remove excess polyphenols that are not bound to the protein. The solution is dialyzed at 4°C for 48 hours, and the dialysate is changed every 6 hours. The solution after the dialysis is completed is Freeze dry to preserve for later use.

Example 2

Preparation of WP-EGCG-Ca ²⁺ covalent complex, the steps of the preparation method are as follows:

(1) Dissolve 0.5g of whey protein in 49mL of deionized water in a beaker. After the protein is fully dissolved, add 1mL of prepared 5M hydrogen peroxide solution into the beaker. Weigh 0.25g of ascorbic acid and add it into the beaker. Mix evenly. Let stand at room temperature for 2h.

(2) After 2 hours, add 0.35mM EGCG and 1mM CaCl ₂ to the protein solution, stir to dissolve the polyphenols and CaCl ₂ , and then let the solution stand at room temperature for 24 hours.

(3) After the reaction is completed, use a 3500D dialysis bag to dialyze the reacted solution to remove excess polyphenols that are not bound to the protein. The solution is dialyzed at 4°C for 48 hours, and the dialysate is changed every 6 hours. The solution after the dialysis is completed Freeze dry to preserve for later use.

Comparative example 1

Dissolve 0.5g of whey protein in 50mL of deionized water in a beaker, and the resulting material is the original blank whey protein.

The sample preparation, characterization, functional properties, embedding, etc. of Example 1, Example 2 and Comparative Example 1 were tested for effects.

1. First, accurately weigh 15 mg of the freeze-dried sample, then add 5 mL of Tris-Glycine buffer solution to the sample to fully dissolve the sample, and then add 50 μL of Ellman reagent (4 mg DTNB dissolved in 1 mL of Tris-Glycine buffer) to the solution. solution), shake and mix evenly and react in the dark at 25°C for 1 hour. Finally, measure the absorbance value of the solution to be tested at 412 nm. According to the formula, thiol content (μmol/g) = 75.53 × A ₄₁₂ /C (C is Protein concentration, mg/mL) to calculate the sulfhydryl content of the sample; first open the water bath and adjust the temperature to 35°C, then prepare 200 μL sample solution in a centrifuge tube, add 4 mL of o-phthalaldehyde solution to the centrifuge tube, and mix by vortexing After uniformity, immediately place it in a 35°C water bath for 2 minutes, and then take out the solution and measure the absorbance value of the solution at 340 nm. Finally, analyze the content of free amino groups in the sample according to the lysine standard curve; take an appropriate amount of the sample, dissolve it in deionized water, add Folin phenol reagent and Na ₂ CO ₃ solution, and detect the absorbance value at 760 nm. Finally, according to the polyphenol produced Standard curve to calculate protein and polyphenol binding equivalents.

2. Prepare 2.5 mL of a protein sample with a concentration of 0.2 mg/mL, use a pipette to take out the sample, place it in a 1cm quartz cuvette, and perform a fluorescence spectrum scan at 37°C. The fluorescence spectrum experimental parameters were set to an emission range of 300-450nm, and an excitation and emission spectrum bandwidth of 5nm. The synchronous fluorescence spectrum of the protein was scanned in the synchronous scanning mode with the wavelength difference between the excitation wavelength and the emission wavelength being Δλ = 60 nm and Δλ = 15 nm respectively.

3. Weigh 1 mg of sample and 150 mg of dry KBr. Grind evenly with a mortar, press into tablets and perform Fourier transform infrared spectrum scanning. After subtracting the air background peak, the infrared scanning spectrum of the sample is obtained. Through 32 scans, a Fourier transform infrared spectrum with a wave number range of 4000 to 500 cm-1 was obtained with a resolution of 4 cm-1.

4. Fix the freeze-dried sample on the copper sample stage with conductive glue. After spraying with gold, observe the sample with 500 times magnification under the condition of an acceleration voltage of 20kV.

5. Dissolve 100 mg of sample in 10 mL of distilled water to prepare a 10 mg/mL sample solution. The sample solution was then gently stirred at room temperature for 30 min. Finally, the sample solution was centrifuged at 12000 × g and 20°C for 20 min, and the supernatant was collected. The solubility formula is:

6. Weigh 0.15g of the sample and dissolve it in 15mL of deionized water, add 5mL of soybean oil, disperse at a high speed of 14000rmp for 5min, then take 100μL of the emulsion bottom sample that has been left standing for 0min and 10min, add it to 10mL of 0.1% SDS solution, and shake Mix well, use 0.1% SDS solution as a blank control, and measure the absorbance at a wavelength of 500 nm. The formulas for emulsifying activity (EAI) and emulsifying stability (ES) are as follows:

A ₀ and A ₁₀ are the absorbance of the diluted emulsion at 0 and 10 minutes; DF is the dilution factor, 100; c is the initial sample concentration, 10000g/m3; φ is the proportion of the oil phase in the emulsion, 0.25; L is the optical path length, 0.0057m.

7. After adding 0.2, 0.4, 0.6, and 0.8 mg/mL apigenin to the sample solution, stir the mixture for another 30 minutes, and centrifuge at 10,000g for 10 minutes to obtain a transparent dispersion. The precipitate after centrifugation was dissolved with DMSO, and the absorbance at 337 nm was measured to quantify unencapsulated apigenin based on a standard curve prepared with a standard solution of apigenin dissolved in DMSO. The embedding rate formula is as follows:

8. Test their thermal stability in a water bath (60, 85°C) for 120 min, and observe the kinetics of the decrease in absorbance at 337 nm, measuring every 20 min. For all cases, the initial absorbance was set to 100%. The retention rate of apigenin was calculated by the following formula:

9. Test their photostability under 20W fluorescent lamp irradiation for 120 minutes, observe the kinetics of absorbance reduction at 337nm, and measure every 20 minutes. For all cases, the initial absorbance was set to 100%.

10. Simulated gastric juice consists of 2.0g NaCl, 7.0mL 37% HCl and 1000mL double-distilled water. The final pH value is 1.2. The simulated intestinal fluid consisted of 6.8 g KH ₂ PO ₄ dissolved in 250 mL double-distilled water, plus 190 mL 0.2 N NaOH and 400 mL double-distilled water. Adjust pH to 7.5 using 0.2N NaOH. Before use, add pepsin (3.2g in simulated gastric juice) or pancreatin (10.0g in simulated intestinal juice), dilute with double-distilled water to make the volume reach 1000mL, and prepare simulated gastric juice and simulated intestinal juice respectively. Free apigenin and embedded apigenin samples were mixed with simulated gastric juice or simulated intestinal juice culture medium (1:4, v/v), and incubated in a 37°C water bath with stirring at 120 rpm. Their gastrointestinal stability was tested for 180 min, and the kinetics of absorbance decrease at 337 nm was observed, measured every 30 min.

The present invention improves the functional properties of whey protein and the relevant detection results of apigenin entrapment rate as follows:

1. In the free radical induction method, hydroxyl radicals will attack the sensitive groups in the protein side chain to produce active intermediates. By detecting the free amino group and thiol content of the covalent complex, it can be reflected whether the protein and polyphenol are covalently bonded. As shown in Figure 1, compared with the WP control, the polyphenol binding equivalents of WP-EGCG and WP-EGCG-Ca ²⁺ covalent complexes increased, and the polyphenol binding equivalent of WP-EGCG-Ca ²⁺ covalent complexes was as high as 10.94±0.32. The free amino group and thiol content decreased, indicating that EGCG and WP were covalently bonded to WP through the free radical induction method.

2. Fluorescence spectrum can detect the fluorescence intensity of some amino acid residues of the protein. The change in the fluorescence intensity or peak shift of WP at 343nm (excitation wavelength is 280nm) can be used to evaluate the structural changes of the protein after WP is combined with polyphenol. It can be seen from Figure 2A that as the polyphenol grafting rate increases, the degree of quenching of the endogenous fluorescence of whey protein increases. This may be due to the specific interaction between whey protein and polyphenols, which affects the whey protein. The structure of the protein promotes changes in the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues in whey protein, resulting in a decrease in fluorescence quantum yield. Synchronous fluorescence spectroscopy analysis further detects the conformational changes in the interaction between whey protein and ligands and the changes in the microenvironment of Trp and Tyr residues by measuring the emission spectrum shift. Simultaneous fluorescence spectrum results under the conditions of Δλ = 15nm and Δλ = 60nm. Looking at Figure 2B and C, it can be seen that the fluorescence quenching when Δλ = 60nm is greater than Δλ = 15nm, indicating that the binding site is also at the tryptophan residue. nearby.

3. Figure 3 is the Fourier transform infrared spectrum of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ . The amide I band (1600~1700cm ^-1 ) of the protein is mainly caused by the stretching vibration of C=O. The amide II (≈1540cm ^-1 ) band contains the stretching vibration of CN and the bending vibration of NH. These two intervals are related to the secondary structure of the protein and are typical spectral characteristic peaks of proteins. As the polyphenol grafting rate increases, the peak value of the amide I band of the protein moves to a lower wave number (1654.28cm ^-1 to 1651.17cm ^-1 ). The phenomenon of the main band shifting indicates that the structure of the whey protein in the complex Certain changes have occurred above. The absorption peak of the amide II band did not change significantly. It is known that phenolic hydroxyl groups and hydrogen bonds have characteristic peaks in the range of 3500cm ^-1 -3200cm ^-1 . As shown in Figure 3, when phenolic acid is grafted into whey protein molecules, the range of 3500 to 3200cm-1 in the infrared spectrum Broad peaks appear, from which it can be concluded that phenolic acids are grafted onto the whey protein, and the peak of the WP-EGCG-Ca ²⁺ complex is the broadest, indicating that the amount of grafted phenolic acids is the largest.

4. In order to further study the effect of free radical grafted polyphenols on the microstructure of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ , the microstructure of the samples was observed using a scanning electron microscope. As shown in Figure 4, the WP formed by the spray drying process is a spherical structure, but the freeze-drying process will promote the formation of a broken flaky structure of WP, and the edges of the fragments are relatively flat and smooth. EGCG is branched. After whey protein is grafted with phenolic acid, the protein is broken, and the branched phenolic acid and protein are combined. The particles of the WP-EGCG-Ca ²⁺ covalent complex are smaller and looser than those of WP. The above shows that when preparing the complex, whey protein combines with polyphenols, which ultimately changes the morphology of the protein, and the change in microstructure leads to changes in its functional properties.

5. The solubility of protein is the basis for realizing its functional properties such as emulsification, antioxidant and gelling properties, and affects its application in food processing. Protein solubility plots of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ complexes. As shown in Figure 5, after grafting phenolic acid, whey protein has good protein solubility. This may be due to the introduction of a large number of hydroxyl groups after covalent bonding, which improves the hydrophilicity of WP, thereby increasing the solubility of the protein.

6. The emulsifying ability index (EA) and emulsion stability index (ES) of WP, WP-EGCG, and WP-EGCG-Ca ²⁺ covalent complexes are shown in Figure 6. The results showed that compared with blank WP, the addition of Ca ²⁺ significantly increased the EA value of WP-EGCG covalent complex (P<0.05), from 7.46±0.04m ² /g to 8.10±0.13m ² /g. Studies have shown that changes in EA are related to surface hydrophobicity. Changes in the conformational structure of whey protein will affect the surface hydrophobicity of the protein, thereby affecting the emulsification properties of the protein. Increasing the exposed aromatic residues will increase the protein's affinity for the oil/water interface, which can enhance the emulsifying activity of WP. ES and EA show the same trend, proving that this material not only improves the emulsifying activity of WP-EGCG, but also increases its emulsifying stability, making it an ideal emulsifier.

7. Figure 7 shows the entrapment rate of apigenin (AP) by WP, WP-EGCG, and WP-EGCG-Ca ²⁺ complexes. The WP-EGCG-Ca ²⁺ complex has the highest embedding rate of AP, ^proving the success of whey protein modification. As the AP concentration increased, the embedding rate decreased, indicating that at higher AP concentrations, a portion of AP was not embedded into the whey protein solution.

8. Figure 8 shows the photostability of AP and its composite particles under 20W fluorescent lamp for 120min. After 20 minutes, the stability of free AP decreased to about 79.68%. After 100 minutes, about 60% of AP molecules were degraded and became relatively stable. Entrapment of protein AP complex particles significantly delayed AP degradation. By implanting AP molecules into the hydrophobic cavity of the protein, the composite particles can block or absorb most of the visible and invisible light energy, have a higher photolysis ratio, higher stability, and better ultraviolet protection effect.

9. In water baths heated at 60 and 80°C for 120 minutes, Figure 9 shows the thermal stability of AP and its composite particles. After 20 min, the stability of free AP dropped to about 71.69%, and after 120 min, about 58% of AP molecules were degraded. In addition, the protein-AP composite particles significantly reduced AP degradation. In addition, the embedded composite particles showed better stability under heat treatment. The thermal stability of free AP decreases with increasing temperature. After heat treatment, the thermal stability of AP in the composite is higher than that of free AP.

10. Use an in vitro simulated digestion model to evaluate the bioavailability of free or entrapped apigenin. The experimental results are shown in Figure 10. The embedded apigenin showed good stability in simulated gastric juice during incubation at 37°C. At 90 minutes, the apigenin retention rate of the embedded sample in simulated gastric juice was 72.38%, but for the free apigenin sample, the retention rate was only 45.04%. The bioavailability rate after the entire digestion process was only 34.98%, and the sample retention rate after embedding was 48.78%.

In summary, the WP-EGCG-Ca ²⁺ complex of the present invention has excellent functional properties and shows excellent performance in encapsulating apigenin.

Claims (9)

1. A method for preparing modified whey protein, which is characterized by comprising the following steps:

(1) Dissolving whey protein in water, adding hydrogen peroxide solution, adding ascorbic acid, mixing, standing,

(2) Standing, adding EGCG and CaCl ₂ Stirring, standing for reaction,

(3) After the reaction is completed, the solution is dialyzed by a dialysis bag and freeze-dried after the dialysis is completed.

2. The method for producing a modified whey protein according to claim 1, characterized by comprising the steps of:

(1) Dissolving 0.2-1g of whey protein in 30-60mL of deionized water in a beaker, adding the prepared hydrogen peroxide solution into the beaker after fully dissolving the protein, weighing ascorbic acid, adding the ascorbic acid, uniformly mixing, and standing for 1-3h at room temperature.

(2) Adding EGCG and CaCl into the protein solution after standing ₂ After stirring, the solution was allowed to stand at room temperature for reaction for 28-30h.

(3) Dialyzing with dialysis bag after the reaction is completed, dialyzing at 4deg.C for 48 hr, changing dialysate every 6 hr, and lyophilizing the solution after the dialysis is completed.

3. A process for the preparation of a modified whey protein according to any of claims 1-2, characterized in that: in the step (1), 1mL of a 5M hydrogen peroxide solution, 0.25g of ascorbic acid was added, and the mixture was allowed to stand at room temperature for 2 hours after being uniformly mixed.

4. Modified milk according to any one of claims 1-2Albumin, characterized in that: in step (2), 0.35mM EGCG and 1mM CaCl were added ₂ 。

5. The method for producing a modified whey protein as defined in any one of claims 1 to 2, wherein in the step (3), dialysis is performed with 3500D dialysis bags.

6. A modified whey protein produced by the process for producing a modified whey protein according to any one of claims 1 to 4.

7. The preparation method of the embedded apigenin is characterized by comprising the following steps:

(1) Dissolving the modified whey protein of claim 5 in Shui Fu,

(2) Apigenin is added into the solution and stirred,

(3) Freeze drying and storing for standby.

8. The method for preparing the embedded apigenin according to claim 6, wherein:

(1) The modified whey protein was dissolved in deionized water at a concentration of 10mg/mL, stirred to fully hydrate its mass, and allowed to stand to room temperature.

(2) After apigenin is added, stirring is carried out for 30min.

(3) The solution was freeze-dried.

9. The process for preparing apigenin according to any one of claims 6 to 7, wherein in step (2), apigenin is added in an amount of 0.2,0.4,0.6,0.8mg/mL after stirring for 2 hours on a magnetic stirrer and then stored in a refrigerator at 4 ℃ for 12 hours, and in step (3).