Preparation of modified whey protein and application thereof in embedding apioin

Abstract

The application relates to preparation and application of modified whey protein, especially showing good embedding effect in embedding apigenin. The steps of the preparation method are as follows: (1) 49 mL of deionized water is used to dissolve 0.5 g of whey protein in a beaker, 1 mL of 5M hydrogen peroxide solution which has been prepared is added into the beaker after the protein is fully dissolved, 0.25 g of ascorbic acid is weighed and added, uniform mixing is carried out, and then the mixture is placed at room temperature for 2 h. (2) 0.35 mM of EGCG and 1 mM of CaCl2 are added into the protein solution after 2 h, the polyphenol and CaCl2 are stirred to be dissolved, and then the solution is placed at room temperature for reaction for 24 h. (3) after the reaction is completed, the solution after the reaction is dialyzed by using a 3500D dialysis bag to remove the polyphenol which is not combined on the protein, and the solution is dialyzed at 4 DEG C for 48 h, and the dialysis liquid is replaced every 6 h. 2+ The grafting rate of WP-EGCG is improved, the functional properties of the whey protein are enhanced, and the embedding rate of apigenin is preferably improved.

Description

Technical Field

[0001] This invention belongs to the field of food technology and relates to the preparation and application of modified whey protein, especially showing a good encapsulation effect in the encapsulation of apigenin. Background Technology

[0002] Proteins are natural transport vehicles, possessing the unique ability to form protein-ligand complexes with small molecules, while simultaneously providing protection for these compounds. In recent years, the encapsulation and delivery of bioactive substances has become a hot research area for colloidal particles. Whey protein (WP) is a high-quality animal protein, rich in active ingredients, a complete range of essential amino acids, and high nutritional value, widely recognized as the "king of proteins." Based on these advantages, whey protein has attracted widespread attention. However, reports indicate that whey utilization is low, with only about half undergoing further processing, resulting in resource waste and environmental pollution. Therefore, improving whey protein utilization through protein modification technology and producing high-value-added products is of practical significance for the full and rational utilization of whey protein. Due to its unique physicochemical properties, whey protein plays an important role in the food industry. Studies have shown that whey protein, as a common food ingredient, can be used as an emulsifier, foaming agent, and water-binding agent, thereby giving products ideal properties. Furthermore, molecular modification techniques can alter the physicochemical properties of whey protein, enhancing its feasibility as a food ingredient. This is particularly true for using whey protein to enhance the rheological and structural properties of products, such as in edible films, coatings, hydrogels, and nanoparticles. Increasing research focuses on modifying whey protein to enhance its functionality, thereby adding value to the product.

[0003] Epigallocatechin gallate (EGCG) is the main active component of flavanols in polyphenols and the most abundant catechin in green tea, possessing various effects such as antioxidation, antibacterial, and antitumor activity. Free radical grafting involves the reaction of active groups and hydroxyl radicals on the side chains of proteins, forming covalent bonds between polyphenols and proteins. The formation of these covalent bonds involves irreversible interactions, resulting in more stable compounds. Compared to enzymatic and alkaline methods, free radical grafting does not involve organic solvents and has higher safety, making it widely used in the food industry. Improving the grafting rate of WP-EGCG is one of the problems researchers urgently need to solve. Apigenin (AP) is a potential drug for developing strategies to prevent and treat cancer. Studies have shown that apigenin can inhibit cancer cell proliferation, promote cell cycle arrest, and induce apoptosis in cancer cells. However, the application of apigenin in improving human health is hindered by its low water solubility. Therefore, much work is needed to improve the solubility and bioavailability of apigenin.

[0004] This invention investigates the effect of adding Ca 2+This study improved the grafting rate of WP-EGCG and enhanced the functional properties of whey protein, while also significantly improving the encapsulation rate of apigenin. This research provides new ideas and approaches for the development of protein modification theories and technologies.

[0005] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and, based on the prior art, to provide a new technology that can improve the grafting rate of WP-EGCG, thereby improving the functional properties of whey protein, and to study its application in encapsulating apigenin.

[0007] The technical solution adopted in this invention is:

[0008] The preparation method for a modified whey protein includes the following steps:

[0009] (1) Dissolve whey protein in water, add hydrogen peroxide solution, then add ascorbic acid, mix well and let stand.

[0010] (2) After standing, add EGCG and CaCl2, stir, and let the reaction stand.

[0011] (3) After the reaction is complete, dialyze the solution using a dialysis bag. After the dialysis is complete, freeze-dry the solution to obtain modified whey protein.

[0012] Specifically, the preparation method involves the following steps:

[0013] (1) Dissolve 0.2-1g of whey protein in 30-60mL of deionized water in a beaker. After the protein is fully dissolved, add the prepared hydrogen peroxide solution to the beaker, weigh out ascorbic acid and add it to the beaker. Mix well and let stand at room temperature for 1-3 hours.

[0014] (2) After standing, add EGCG and CaCl2 to the protein solution, stir, and then let the solution stand at room temperature for 28-30 hours.

[0015] (3) After the reaction is complete, dialyze with a dialysis bag at 4°C for 48 hours. Change the dialysate every 6 hours. After the dialysis is complete, freeze-dry the solution to modify whey protein.

[0016] In step (1), 1 mL of 5M hydrogen peroxide solution and 0.25 g of ascorbic acid were added, mixed evenly, and then left to stand at room temperature for 2 hours.

[0017] In step (2), 0.35 mM EGCG and 1 mM CaCl2 are added.

[0018] In step (3), dialysis is performed using a 3500D dialysis bag.

[0019] The steps for encapsulating apigenin in modified whey protein are as follows:

[0020] (1) Redissolve the modified whey protein in water.

[0021] (2) Add apigenin to the solution and stir.

[0022] (3) Freeze-dry and store for later use.

[0023] Specifically, the steps of the embedding method are as follows:

[0024] (1) Dissolve the modified whey protein in deionized water at a concentration of 10 mg / mL, stir to fully hydrate it, and let it stand at room temperature.

[0025] (2) Add apigenin and stir for 30 minutes.

[0026] (3) Freeze-dry the solution.

[0027] In step (2), after stirring on a magnetic stirrer for 2 hours, the mixture is stored in a 4°C refrigerator for 12 hours.

[0028] In step (3), 0.2, 0.4, 0.6, and 0.8 mg / mL of apigenin were added.

[0029] The advantages and positive effects of this invention are as follows:

[0030] 1. This invention creatively adds CaCl2, which is non-toxic and harmless, and can be added to food, thus broadening its application in the food processing field.

[0031] 2. After adding CaCl2, the grafting rate of modified whey protein was significantly improved. Compared with the WP control, WP-EGCG and WP-EGCG-CaCl2 showed significantly higher grafting rates. 2+ The polyphenol binding equivalent of the covalent complex increased, and WP-EGCG-Ca 2+ The polyphenol group showed the highest binding concentration, while the content of free amino groups and thiol groups decreased. In addition to these changes in properties, the polyhydroxy properties of EGCG enhance the antioxidant capacity, solubility, foaming properties, and emulsifying properties of WP after binding with EGCG. This provides a theoretical basis for improving the functional properties of WP in practical applications.

[0032] 3. Apigenin is highly hydrophobic, poorly water-soluble, and has poor photothermal stability. (The text then abruptly shifts to a seemingly unrelated topic: "In the use of WP-EGCG-Ca...") 2+Encapsulating apigenin with a covalent complex as a wall material significantly improves its bioavailability. In this invention, we designed a stable, soluble, and bioavailable structure to enhance the solubility and bioavailability of apigenin. This research aims to extend the modification and functionalization of animal proteins to value-added and cost-effective food and nutritional materials with potential applications in numerous fields. Attached Figure Description

[0033] Figure 1 In this invention, WP and WP-EGCG, WP-EGCG-Ca 2+ Content of covalent complex groups.

[0034] Figure 2 In this invention, WP and WP-EGCG, WP-EGCG-Ca 2+ (A) Intrinsic fluorescence spectrum of the covalent complex, (B) Synchronous fluorescence spectrum of tyrosine, (C) Synchronous fluorescence spectrum of tryptophan;

[0035] Figure 3 In this invention, WP and WP-EGCG, WP-EGCG-Ca 2+ Fourier transform infrared spectrum of covalent complex;

[0036] Figure 4 In this invention, (A) WP, (B) EGCG, (C) CaCl2, (D) WP-P, (E) WP-EGCG, and (F) WP-EGCG-Ca 2+ Scanning electron microscope image of the covalent complex;

[0037] Figure 5 In this invention, WP and WP-EGCG, WP-EGCG-Ca 2+ Covalent complex protein solubility diagram;

[0038] Figure 6 In this invention, WP and WP-EGCG, WP-EGCG-Ca 2+ Emulsifiable properties and emulsion stability of covalent complexes;

[0039] Figure 7 In this invention, WP and WP-EGCG, WP-EGCG-Ca 2+ Encapsulation efficiency of apigenin by the covalent complex;

[0040] Figure 8 The free apigenin and WP-EGCG-Ca in this invention 2+ Retention rate of apigenin after encapsulation under 20W fluorescent lamp irradiation

[0041] Figure 9The free apigenin and WP-EGCG-Ca in this invention 2+ Retention rate of apigenin at 60℃ and 80℃ after encapsulation

[0042] Figure 10 The free apigenin and WP-EGCG-Ca in this invention 2+ Retention rate of apigenin after encapsulation during simulated gastrointestinal digestion. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments; the following embodiments are illustrative and not limiting, and should not be used to limit the scope of protection of the present invention.

[0044] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0045] Example 1

[0046] The preparation steps of the WP-EGCG covalent complex are as follows:

[0047] (1) Dissolve 0.5g of whey protein in 49mL of deionized water in a beaker. After the protein is fully dissolved, add 1mL of the prepared 5M hydrogen peroxide solution to the beaker, weigh 0.25g of ascorbic acid and add it to the beaker. Mix well and let stand at room temperature for 2h.

[0048] (2) After 2 hours, add 0.35 mM EGCG to the protein solution, stir to dissolve, and then let the solution stand at room temperature for 24 hours.

[0049] (3) After the reaction is completed, the solution is dialyzed with a 3500D dialysis bag to remove excess polyphenols that have not been bound to proteins. The solution is dialyzed at 4°C for 48 hours, and the dialysate is changed every 6 hours. The solution after dialysis is completed is freeze-dried for storage.

[0050] Example 2

[0051] WP-EGCG-Ca 2+ The preparation steps of the covalent complex are as follows:

[0052] (1) Dissolve 0.5g of whey protein in 49mL of deionized water in a beaker. After the protein is fully dissolved, add 1mL of the prepared 5M hydrogen peroxide solution to the beaker, weigh 0.25g of ascorbic acid and add it to the beaker. Mix well and let stand at room temperature for 2h.

[0053] (2) After 2 hours, add 0.35 mM EGCG and 1 mM CaCl2 to the protein solution and stir to dissolve the polyphenols and CaCl2. Then, let the solution stand at room temperature for 24 hours.

[0054] (3) After the reaction is completed, the solution is dialyzed with a 3500D dialysis bag to remove excess polyphenols that have not been bound to proteins. The solution is dialyzed at 4°C for 48 hours, and the dialysate is changed every 6 hours. The solution after dialysis is completed is freeze-dried for storage.

[0055] Comparative Example 1

[0056] Dissolve 0.5g of whey protein in 50mL of deionized water in a beaker. The resulting material is the original blank whey protein.

[0057] The sample preparation, characterization, functional properties, and embedding of Examples 1, 2, and Comparative Example 1 were tested.

[0058] 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 it. Next, add 50 μL of Ellman's reagent (4 mg DTNB dissolved in 1 mL of Tris-glycine buffer solution), shake to mix thoroughly, and react at 25°C in the dark for 1 hour. Finally, measure the absorbance of the test solution at 412 nm. The thiol content (μmol / g) is calculated using the formula: thiol content = 75.53 × A. 412 The thiol content of the sample was calculated using C / C (where C is the protein concentration, mg / mL). First, the water bath was set to 35℃. Then, 200 μL of sample solution was placed in a centrifuge tube, and 4 mL of phthalaldehyde solution was added. After vortexing and mixing thoroughly, the tube was immediately placed in a 35℃ water bath for 2 minutes. The absorbance at 340 nm was then measured. Finally, the content of free amino groups in the sample was analyzed according to the lysine standard curve. An appropriate amount of sample was dissolved in deionized water, and then Folin-Ciocalteu reagent and Na₂CO₃ solution were added. The absorbance at 760 nm was then measured. Finally, the protein-polyphenol binding equivalent was calculated based on the prepared polyphenol standard curve.

[0059] 2. Prepare 2.5 mL of a protein sample with a concentration of 0.2 mg / mL. Use a pipette to transfer the sample into a 1 cm quartz cuvette and perform fluorescence spectroscopy scanning at 37°C. The fluorescence spectroscopy parameters are set as follows: emission range 300-450 nm, excitation and emission bandwidth 5 nm. Simultaneous fluorescence spectroscopy is performed using wavelength differences between excitation and emission wavelengths of Δλ = 60 nm and Δλ = 15 nm, respectively, scanning the protein's synchronous fluorescence spectrum in synchronous scanning mode.

[0060] 3. Weigh 1 mg of sample and 150 mg of dry KBr. Grind them evenly in a mortar, compress them into a tablet, and perform Fourier transform infrared spectroscopy. After subtracting the air background peak, the infrared spectrum of the sample is obtained. Through 32 scans, a Fourier transform infrared spectrum with a wavenumber range of 4000–500 cm⁻¹ and a resolution of 4 cm⁻¹ was obtained.

[0061] 4. After freeze-drying, the sample was fixed on a copper sample stage with conductive adhesive and then sputtered with gold. The sample was then observed at 500x magnification under an acceleration voltage of 20kV.

[0062] 5. Dissolve 100 mg of sample in 10 mL of distilled water to prepare a 10 mg / mL sample solution. Then, gently stir the sample solution at room temperature for 30 min. Finally, centrifuge the sample solution at 12000 × g at 20 °C for 20 min and collect the supernatant. The solubility formula is:

[0063]

[0064] 6. Weigh 0.15g of the sample and dissolve it in 15mL of deionized water. Add 5mL of soybean oil and disperse at a high speed of 14000rpm for 5min. Then, take 100μL of the bottom layer sample of the emulsion after standing for 0min and 10min and add it to 10mL of 0.1% SDS solution. Shake to mix well. Use 0.1% SDS solution as a blank control and measure its absorbance at a wavelength of 500nm. The formulas for emulsifying activity (EAI) and emulsifying stability (ES) are as follows:

[0065]

[0066] A0 and A 10 1 is the absorbance of the diluted emulsion at 0 and 10 min; DF is the dilution factor, 100; c is the initial sample concentration, 10000 g / m3; φ is the proportion of the oil phase in the emulsion, 0.25; L is the optical path length, 0.0057 m.

[0067] 7. After adding 0.2, 0.4, 0.6, and 0.8 mg / mL of apigenin to the sample solutions, respectively, the mixtures were stirred for 30 min and centrifuged at 10000 g for 10 min to obtain transparent dispersions. The precipitate after centrifugation was dissolved in DMSO, and the absorbance at 337 nm was measured to quantify the unencapsulated apigenin according to a standard curve prepared using a standard solution of apigenin dissolved in DMSO. The encapsulation efficiency formula is shown below:

[0068]

[0069] 8. Test their thermal stability in a water bath (60, 85℃) for 120 min, observing 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 using the following formula:

[0070]

[0071] 9. Test their photostability for 120 min under 20W fluorescent lamp illumination, 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%.

[0072] 10. Simulated gastric fluid consisted of 2.0 g NaCl, 7.0 mL 37% HCl, and 1000 mL double-distilled water. The final pH was 1.2. Simulated intestinal fluid consisted of 6.8 g KH₂PO₄ dissolved in 250 mL double-distilled water, with 190 mL 0.2 N NaOH and 400 mL double-distilled water added. The pH was adjusted to 7.5 using 0.2 N NaOH. Before use, pepsin (3.2 g in simulated gastric fluid) or trypsin (10.0 g in simulated intestinal fluid) was added, and the volume was brought up to 1000 mL with double-distilled water to prepare simulated gastric and intestinal fluids respectively. Free apigenin and embedded apigenin samples were mixed with simulated gastric or intestinal fluid culture medium (1:4, v / v) and incubated at 120 rpm in a 37°C water bath. Their gastrointestinal stability was tested for 180 min, and the kinetics of the decrease in absorbance at 337 nm were observed, with measurements taken every 30 min.

[0073] The relevant detection results of this invention regarding the improved functional properties of whey protein and the encapsulation rate of apigenin are as follows:

[0074] 1. In the free radical induction method, hydroxyl radicals attack sensitive groups in the protein side chain to produce reactive intermediates. By detecting the free amino and thiol content of the covalent complex, it can be determined whether the protein and polyphenol are covalently bonded. Figure 1 It can be seen that, compared with the WP control, WP-EGCG and WP-EGCG-Ca 2+ The polyphenol binding equivalent of the covalent complex increased, and WP-EGCG-Ca 2+ The polyphenol binding equivalent of the covalent complex was as high as 10.94 ± 0.32. The reduced content of free amino groups and thiol groups indicates that EGCG and WP were covalently bound via a free radical-induced method.

[0075] 2. Fluorescence spectroscopy can detect the fluorescence intensity of some amino acid residues in proteins. Changes in the fluorescence intensity or peak shift of WP at 343 nm (excitation wavelength 280 nm) can be used to evaluate structural changes in proteins after WP binds to polyphenols. Figure 2 A indicates that as the polyphenol grafting rate increases, the quenching of the intrinsic fluorescence of whey protein intensifies. This may be due to the specific interaction between whey protein and polyphenols, which affects the structure of whey protein and promotes changes in the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues, leading to a decrease in fluorescence quantum yield. Synchronous fluorescence spectroscopy analysis further detects conformational changes in the interaction between whey protein and ligands, as well as changes in the microenvironment of Trp and Tyr residues, by measuring the emission spectral shift. The synchronous fluorescence spectral results at Δλ = 15 nm and Δλ = 60 nm were observed. Figure 2 As can be seen from B and C, the fluorescence quenching at Δλ = 60 nm is greater than that at Δλ = 15 nm, indicating that the binding site is also near the tryptophan residue.

[0076] 3. Figure 3 It is WP, WP-EGCG, WP-EGCG-Ca 2+ Fourier transform infrared spectrum, amide I band of protein (1600-1700 cm⁻¹) -1 It is mainly caused by the stretching vibration of C=O, amide II (≈1540cm) -1 The amide I band contains the stretching vibration of CN and the bending vibration of NH, both of which are related to the secondary structure of proteins and are typical spectral characteristic peaks of proteins. With increasing polyphenol grafting rate, the peak value of the amide I band of the protein shifts to a lower wavenumber (1654.28 cm⁻¹). -1 Up to 1651.17cm -1 The shift in the main band indicates a structural change in the whey protein within the complex. The absorption peak of the amide II band did not change significantly. It is known that phenolic hydroxyl groups and hydrogen bonds are most prominent at 3500 cm⁻¹. -1 -3200cm -1 This interval contains characteristic peaks, such as Figure 3 As shown, when phenolic acids are grafted into whey protein molecules, a broad peak appears in the 3500–3200 cm⁻¹ region of the infrared spectrum. This confirms that phenolic acids are grafted onto whey protein, and that WP-EGCG-Ca... 2+ The complex peak is the broadest, suggesting the highest amount of grafted phenolic acid.

[0077] 4. To further investigate the effects of free radical grafted polyphenols on WP, WP-EGCG, and WP-EGCG-Ca 2+ The influence of the microstructure is observed using a scanning electron microscope. For example... Figure 4The WP formed by spray drying is spherical, while freeze drying promotes the formation of fragmented, sheet-like structures with relatively smooth and even edges. EGCG, on the other hand, is branched. After phenolic acids are grafted onto whey protein, the protein breaks down, and the branched phenolic acids bind to the protein. WP-EGCG-Ca 2+ The covalent complex particles are smaller and more loosely distributed than WP particles. This illustrates that during the preparation of the complex, whey protein and polyphenols combine, ultimately altering the protein's morphology and microstructure, which in turn leads to changes in its functional properties.

[0078] 5. The solubility of proteins is fundamental to their functional properties such as emulsification, antioxidant properties, and gelling properties, and influences their application in food processing. WP, WP-EGCG, WP-EGCG-Ca 2+ Protein solubility diagram of the complex. (See attached image.) Figure 5 As shown, whey protein exhibits good protein solubility after grafting with phenolic acids. This may be due to the introduction of a large number of hydroxyl groups after covalent bonding, which increases the hydrophilicity of WP and thus increases the solubility of the protein.

[0079] 6. WP and WP-EGCG, WP-EGCG-Ca 2+ The emulsifying power index (EA) and emulsion stability index (ES) of covalent complexes are as follows: Figure 6 As shown. The results indicate that, compared to the blank WP, Ca 2+ The addition of [a specific ingredient] significantly increased the EA value of the WP-EGCG covalent complex (P<0.05), from 7.46±0.04m. 2 / g increased 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 affect its surface hydrophobicity, thus influencing its emulsifying properties. Increasing the exposure of aromatic residues will increase the protein's affinity for the oil / water interface, which can improve the emulsifying activity of WP. ES and EA show the same trend, demonstrating that this material not only improves the emulsifying activity of WP-EGCG but also increases its emulsifying stability, making it an ideal emulsifier.

[0080] 7. Figure 7 It shows WP, WP-EGCG, WP-EGCG-Ca 2+ Encapsulation efficiency of the complex for apigenin (AP). WP-EGCG-Ca 2 + The complex exhibited the highest AP encapsulation rate, demonstrating the success of whey protein modification. The encapsulation rate decreased with increasing AP concentration, indicating that at higher AP concentrations, some AP was not incorporated into the whey protein solution.

[0081] 8. Figure 8 The photostability of AP and its composite particles under a 20W fluorescent lamp for 120 min was demonstrated. After 20 min, the stability of free AP decreased to approximately 79.68%, and after 100 min, approximately 60% of the AP molecules were degraded and tended to be relatively stable. Encapsulation of protein AP composite particles significantly delayed AP degradation. By embedding AP molecules into the hydrophobic cavity of the protein, the composite particles can block or absorb most of the visible and invisible light energy, resulting in a higher photolysis ratio, higher stability, and better UV protection.

[0082] 9. In a water bath at 60 or 80°C for 120 minutes, Figure 9 The thermal stability of AP and its composite particles was demonstrated. After 20 min, the stability of free AP decreased to approximately 71.69%, and after 120 min, approximately 58% of AP molecules degraded. Furthermore, the protein-AP composite particles significantly reduced AP degradation, and the encapsulated composite particles exhibited better stability under heat treatment. The thermal stability of free AP decreased with increasing temperature. After heat treatment, the AP in the composite had higher thermal stability than free AP.

[0083] 10. The bioavailability of free or encapsulated apigenin was evaluated using an in vitro simulated digestion model. Experimental results are as follows: Figure 10 As shown, the encapsulated apigenin exhibited good stability in simulated gastric fluid during incubation at 37°C. At 90 min, the apigenin retention rate of the encapsulated sample in simulated gastric fluid was 72.38%, while the retention rate of free apigenin was only 45.04%. The bioavailability after the entire digestion process was only 34.98%, with the encapsulated sample retaining 48.78%.

[0084] In summary, the present invention WP-EGCG-Ca 2+ The complex exhibits excellent functional properties and demonstrates superior performance in encapsulating apigenin.

Claims

1. A method for preparing modified whey protein, characterized in that, Prepared by the following method: (1) Dissolve whey protein in water, add hydrogen peroxide solution, then add ascorbic acid, mix well and let stand. (2) After standing, add EGCG and CaCl2, stir, and let the reaction stand. (3) After the reaction is complete, dialyze the solution using a dialysis bag, and freeze-dry the solution after dialysis. In step (1), add 1 mL of 5M hydrogen peroxide solution and 0.25 g of ascorbic acid; In step (2), 0.35 mM EGCG and 1 mM CaCl2 are added; In step (3), dialysis is performed using a 3500D dialysis bag.

2. The method for preparing modified whey protein according to claim 1, characterized in that, Prepared by the following method: (1) Dissolve 0.2-1g of whey protein in 30-60mL of deionized water in a beaker. After the protein is fully dissolved, add the prepared hydrogen peroxide solution to the beaker, and weigh out ascorbic acid and add it to the solution. After mixing thoroughly, let stand at room temperature for 1-3 hours. (2) After standing, add EGCG and CaCl2 to the protein solution, stir, and then let the solution stand at room temperature for 28-30 hours. (3) After the reaction is complete, dialyze with a dialysis bag at 4°C for 48 hours. Change the dialysate every 6 hours. After the dialysis is complete, freeze-dry the solution.

3. The method for preparing modified whey protein according to claim 1, characterized in that: In step (1), add 1 mL of 5M hydrogen peroxide solution and 0.25 g of ascorbic acid, mix well and let stand at room temperature for 2 h.

4. The method for preparing modified whey protein according to claim 2, characterized in that: In step (1), add 1 mL of 5M hydrogen peroxide solution and 0.25 g of ascorbic acid, mix well and let stand at room temperature for 2 h.

5. Modified whey protein prepared by the method for preparing modified whey protein according to any one of claims 1-4.

6. A method for preparing apigenin-encapsulated apigenin, characterized in that, The steps are as follows: (1) The modified whey protein according to claim 5 is reconstituted with water. (2) Add apigenin to the solution and stir. (3) Freeze-dry and store for later use.

7. The method for preparing encapsulated apigenin according to claim 6, characterized in that: (1) Dissolve the modified whey protein according to claim 5 in deionized water at a concentration of 10 mg / mL, stir to fully hydrate it, and let it stand at room temperature. (2) Add apigenin and stir for 30 minutes. (3) Freeze-dry the solution.

8. The method for preparing encapsulated apigenin according to any one of claims 6-7, characterized in that, In step (2), after stirring on a magnetic stirrer for 2 hours, the mixture is stored in a refrigerator at 4°C for 12 hours. In step (3), 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, or 0.8 mg / mL apigenin is added.

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