CN117770347A - Preparation of modified whey protein and application of modified whey protein in embedding apigenin - Google Patents
Preparation of modified whey protein and application of modified whey protein in embedding apigenin Download PDFInfo
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- CN117770347A CN117770347A CN202311811998.9A CN202311811998A CN117770347A CN 117770347 A CN117770347 A CN 117770347A CN 202311811998 A CN202311811998 A CN 202311811998A CN 117770347 A CN117770347 A CN 117770347A
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- 229940117893 apigenin Drugs 0.000 title claims abstract description 63
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to preparation and application of modified whey protein, and particularly shows good embedding effect in embedding apigenin. The preparation method comprises the following steps: (1) 49mL of deionized water was used to dissolve 0.5g of whey protein in a beaker, 1mL of 5M hydrogen peroxide solution prepared was added to the beaker after the protein was sufficiently dissolved, 0.25g of ascorbic acid was weighed and added thereto, and the mixture was allowed to stand at room temperature for 2 hours after being uniformly mixed. (2) After 2h, 0.35mM EGCG and 1mM NaCl were added to the protein solution 2 Stirring to obtain polyphenol and CaCl 2 After dissolution, the solution was allowed to stand at room temperature for reaction for 24 hours. (3) Dialyzing the reacted solution with 3500D dialysis bag to remove excessive unbound proteinThe solution was dialyzed at 4℃for 48h, with dialysate being changed every 6 h. The invention researches the method by adding Ca 2+ The grafting rate of WP-EGCG is improved, the functional characteristic of whey protein is enhanced, and the embedding rate of apigenin is improved better.
Description
Technical Field
The invention belongs to the technical field of food, relates to preparation and application of modified whey protein, and particularly shows good embedding effect in embedding apigenin.
Background
Proteins are natural vehicles that have the special ability to form protein-ligand complexes with small molecule compounds while providing protection to the small molecule compounds. In recent years, encapsulation and delivery of bioactive substances has been the subject of intense research in colloidal particles. Whey Protein (WP) is a high-quality animal protein, contains multiple active ingredients, has complete types of essential amino acids, is rich in content and high in nutritive value, and is a recognized 'protein king' based on the advantages, and the whey protein attracts great attention. It has been reported that whey utilization is low, and about half of whey is further processed, wasting resources and polluting the environment. Therefore, the utilization rate of the whey protein is improved by a protein modification technology, and the production of products with high added value has practical significance for fully and reasonably utilizing the whey protein. Whey protein plays an important role in the food industry due to its special physicochemical properties. The research shows that the whey protein can be used as a common food ingredient and can be used as food additives such as emulsifying agent, foaming agent, water binding agent and the like, so that the product has ideal characteristics. In addition, the physicochemical properties of whey proteins can be changed by molecular modification techniques, enhancing the feasibility of application of whey proteins as food ingredients, especially using whey proteins to enhance the rheological and structural properties of the product, e.g., application of whey proteins to edible films, coatings, hydrogels, nanoparticles, and the like. More and more research is directed to modifying WP to enhance functionality, thereby adding value to whey proteins.
Epigallocatechin gallate (EGCG) is the main active ingredient of flavanol in polyphenol, and is the catechin with highest content in green tea, and has antioxidant, antibacterial and antitumor effects. Free radical grafting is the reaction of the active groups on the protein side chains with hydroxyl radicals to form covalent bonds between the polyphenols and the proteins, the covalent bond formation involving irreversible interactions, thereby forming a more stable conjugate. Compared with the enzymatic method and the alkaline method, the free radical grafting method does not involve an organic solvent in the reaction process, has higher safety, and is widely applied to the food industry. How to increase the grafting rate of WP-EGCG is one of the problems to be solved by researchers. Apigenin (AP) is a potential drug for developing strategies for preventing and treating cancer. Research shows that apigenin can inhibit cancer cell proliferation, promote cell cycle arrest, and induce cancer cell apoptosis. However, the use of apigenin to improve human health is hampered by low water solubility. Therefore, much work is required to improve the solubility and bioavailability of apigenin.
The invention researches the method by adding Ca 2+ The grafting rate of WP-EGCG is improved, the functional characteristic of whey protein is enhanced, and the embedding rate of apigenin is improved better. The research provides a new thought and a new way for developing protein modification theory and technology.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a novel technology capable of improving the grafting rate of WP-EGCG so as to improve the functional characteristics of whey protein on the basis of the prior art, and researches the application of the technology in embedding apigenin.
The technical scheme adopted by the invention is as follows:
the preparation method of the modified whey protein comprises 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) And (3) dialyzing with a dialysis bag after the reaction is finished, and freeze-drying the solution after the dialysis is finished to obtain the modified whey protein.
Specifically, the preparation method comprises the following steps:
(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 dialyzed solution to obtain modified whey protein.
In the step (1), 1mL of a 5M hydrogen peroxide solution was added, 0.25g of ascorbic acid was added thereto, and after mixing uniformly, the mixture was allowed to stand at room temperature for 2 hours.
In step (2), 0.35mM EGCG and 1mM CaCl were added 2 。
In step (3), dialysis is performed with 3500D dialysis bags.
The steps of embedding apigenin by the modified whey protein are as follows:
(1) The modified whey protein is dissolved by adding Shui Fu,
(2) Apigenin is added into the solution and stirred,
(3) Freeze drying and storing for standby.
Specifically, the embedding method comprises the following steps:
(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.
In the step (2), the mixture is stirred on a magnetic stirrer for 2 hours and then is stored in a refrigerator at 4 ℃ for 12 hours.
In the step (3), 0.2,0.4,0.6,0.8mg/mL apigenin is added.
The invention has the advantages and positive effects that:
1. the invention creatively adds CaCl 2 The modified starch is nontoxic and harmless, can be added into food, and widens the application of the modified starch in the field of food processing.
2. At the time of adding CaCl 2 After that, the grafting rate of the modified whey protein is obviously improved, and compared with WP control, WP-EGCG and WP-EGCG-Ca 2+ The polyphenol binding equivalent of the covalent complex is increased and WP-EGCG-Ca 2+ The group polyphenols bind most. And the free amino group and the mercapto group content are reduced. In addition to these property changes, due to the polyhydroxy character of EGCGThe WP has enhanced antioxidant capacity, solubility, foamability, emulsifying property and other protein functions after being combined with EGCG. The theoretical basis is provided for improving the functional characteristics of WP in practical application.
3. Apigenin has high hydrophobicity, poor water solubility and poor photo-thermal stability. In use WP-EGCG-Ca 2+ After apigenin is embedded by taking the covalent compound as a wall material, the bioavailability is greatly improved. In this invention, we devised a stable, soluble and bioavailable structure to increase 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 that may find application in many fields.
Drawings
FIG. 1 shows WP and WP-EGCG, WP-EGCG-Ca in the invention 2+ Covalent complex group content.
FIG. 2 shows WP and WP-EGCG, WP-EGCG-Ca in the invention 2+ Endogenous fluorescence, tyrosine synchronous fluorescence spectrum and tryptophan synchronous fluorescence spectrum of the covalent complex;
FIG. 3 shows WP and WP-EGCG, WP-EGCG-Ca in the invention 2+ Fourier infrared spectrogram of covalent complex;
FIG. 4 shows a diagram of a WP, EGCG, caCl embodiment of the invention 2 、WP-P、WP-EGCG、WP-EGCG-Ca 2+ Scanning electron microscopy of the covalent complex;
FIG. 5 shows WP and WP-EGCG, WP-EGCG-Ca in the invention 2+ A covalent complex protein solubility profile;
FIG. 6 shows WP and WP-EGCG, WP-EGCG-Ca in the invention 2+ A graph of the emulsibility and emulsion stability of the covalent complex;
FIG. 7 shows WP and WP-EGCG, WP-EGCG-Ca in the invention 2+ An entrapment rate map of covalent complexes to apigenin;
FIG. 8 is a graph showing the retention of free apigenin and apigenin after embedding under 20W fluorescent lamp irradiation in the present invention
FIG. 9 is a graph showing the retention of free apigenin and apigenin at 60deg.C and 80deg.C after embedding in the present invention
FIG. 10 is a graph showing the retention of free apigenin and apigenin after entrapment in a simulated gastrointestinal digestion in accordance with the invention
Detailed Description
The invention will be further illustrated with reference to the following examples; the following examples are illustrative, not limiting, and are not intended to limit the scope of the invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Example 1
The preparation method of the WP-EGCG covalent complex comprises the following steps:
(1) 49mL of deionized water was used to dissolve 0.5g of whey protein in a beaker, 1mL of 5M hydrogen peroxide solution prepared was added to the beaker after the protein was sufficiently dissolved, 0.25g of ascorbic acid was weighed and added thereto, and the mixture was allowed to stand at room temperature for 2 hours after being uniformly mixed.
(2) After 2h, 0.35mM EGCG was added to the protein solution and dissolved with stirring, after which the solution was allowed to stand at room temperature for 24h.
(3) After the reaction is completed, the reacted solution is dialyzed by 3500D dialysis bags to remove superfluous polyphenol which is not combined with protein, the solution is dialyzed for 48 hours at the temperature of 4 ℃, the dialysate is changed every 6 hours, and the dialyzed solution is freeze-dried for standby.
Example 2
WP-EGCG-Ca 2+ The preparation of the covalent complex comprises the following steps:
(1) 49mL of deionized water was used to dissolve 0.5g of whey protein in a beaker, 1mL of 5M hydrogen peroxide solution prepared was added to the beaker after the protein was sufficiently dissolved, 0.25g of ascorbic acid was weighed and added thereto, and the mixture was allowed to stand at room temperature for 2 hours after being uniformly mixed.
(2) After 2h, 0.35mM EGCG and 1mM CaCl were added to the protein solution 2 Stirring to obtain polyphenol and CaCl 2 After dissolution, the solution was allowed to stand at room temperature for reaction for 24 hours.
(3) After the reaction is completed, the reacted solution is dialyzed by 3500D dialysis bags to remove superfluous polyphenol which is not combined with protein, the solution is dialyzed for 48 hours at the temperature of 4 ℃, the dialysate is changed every 6 hours, and the dialyzed solution is freeze-dried for standby.
Comparative example 1
50mL deionized water is taken to dissolve 0.5g of whey protein in a beaker, and the obtained material is the original blank whey protein.
The effect test was performed on sample preparation, characterization, functional properties, embedding, etc. of example 1, example 2, and comparative example 1.
1. Firstly accurately weighing 15mg of a freeze-dried sample, then adding 5mL of Tris-glycine buffer solution into the sample to fully dissolve the sample, then adding 50 mu L of Ellman reagent (4 mg of DTNB is dissolved in 1mL of Tris-glycine buffer solution), vibrating and uniformly mixing, performing light-shielding reaction at 25 ℃ for 1h, finally measuring the absorbance value of the solution to be measured at 412nm, and determining the mercapto content (mu mol/g) = 75.53 ×A according to the formula 412 Calculating the sulfhydryl content of the sample by using/C (C is protein concentration, mg/mL); firstly, the water bath kettle is opened to adjust the temperature to 35 ℃, then 200 mu L of sample solution is prepared in a centrifuge tube, 4mL of phthalaldehyde solution is added into the centrifuge tube, the mixture is immediately placed in the water bath with the temperature of 35 ℃ after being uniformly mixed by vortex for 2min, and then the solution is taken out to determine the absorbance value of the solution at 340 nm. Finally, analyzing the content of free amino in the sample according to a lysine standard curve; dissolving a proper amount of sample in deionized water, adding Fu Lin Fen reagent and Na 2 CO 3 And detecting the absorbance at 760nm after the solution, and finally calculating the binding equivalent of the protein and the polyphenol according to the prepared polyphenol standard curve.
2. 2.5mL of a sample having a protein sample concentration of 0.2mg/mL was prepared, and the sample was taken out by a pipette and placed in a 1cm quartz cuvette for fluorescence spectrum scanning at 37 ℃. The experimental parameters of the fluorescence spectrum are set to be in the range of 300-450nm of emitted light, and the bandwidth values of excitation and emission spectra are 5nm. The synchronous fluorescence spectra are scanned in a synchronous scanning mode with wavelength differences between excitation and emission wavelengths of Δλ=60 nm and Δλ=15 nm, respectively.
3. 1mg of sample and 150mg of dry KBr were weighed. Grinding uniformly by using a mortar, tabletting, performing Fourier infrared spectrum scanning, and subtracting an air background peak to obtain an infrared scanning spectrum of the sample. By 32 scans, fourier infrared spectrum with wave number range of 4000-500 cm < -1 > and resolution of 4cm < -1 > is obtained.
4. The freeze-dried sample was fixed on a copper sample stage with a conductive adhesive, subjected to metal spraying treatment, and observed with 500-fold magnification under an acceleration voltage of 20 kV.
5. 100mg of the sample was dissolved in 10mL of distilled water to prepare a 10mg/mL sample solution. The sample solution was then gently stirred at room temperature for 30min. Finally, the sample solution was centrifuged at 12000 Xg at 20℃for 20min, and the supernatant was collected. The solubility formula is:
6. 0.15g of the sample is weighed and dissolved in 15mL of deionized water, 5mL of soybean oil is added, the mixture is dispersed at a speed of 14000rmp for 5min, 100 mu L of the emulsion bottom sample which is kept stand for 0min and 10min is added into 10mL of 0.1% SDS solution, shaking and mixing are carried out, 0.1% SDS solution is used as a blank control, and the absorbance is measured at a wavelength of 500 nm. The formula for the Emulsion Activity (EAI) and Emulsion Stability (ES) is as follows:
A 0 and A 10 Absorbance of the diluted emulsion at 0 and 10 min; DF is the dilution factor, 100; c is the initial sample concentration, 10000g/m3; phi is the proportion of the oil phase in the emulsion, 0.25; l is the optical path, 0.0057m.
7. After 0.2,0.4,0.6,0.8mg/mL apigenin was added to the sample solutions, the mixture was stirred for another 30min, and centrifuged at 10000g for 10min to obtain a transparent dispersion. The pellet after centrifugation was dissolved in DMSO and absorbance at 337nm was measured to quantify unencapsulated apigenin according to a standard curve prepared with a standard solution of apigenin dissolved in DMSO. The embedding rate formula is as follows:
8. their thermal stability was tested in a water bath (60, 85 ℃) for 120min, the kinetics of absorbance decrease at 337nm was observed, and the measurement was performed every 20 min. For all cases, the initial absorbance was set to 100%. The retention of apigenin was calculated by the following formula:
9. their photostability was tested under 20W fluorescent lamp irradiation for 120min, and the kinetics of absorbance decrease at 337nm was observed, once every 20 min. For all cases, the initial absorbance was set to 100%.
10. The simulated gastric fluid consisted of 2.0g NaCl, 7.0mL 37% HCl and 1000mL double distilled water. The final pH was 1.2. Simulated intestinal juice consists of 6.8g KH 2 PO 4 Consists of and is dissolved in 250mL double distilled water plus 190mL0.2N NaOH and 400mL double distilled water. The pH was adjusted to 7.5 using 0.2N NaOH. Pepsin (3.2 g in simulated gastric fluid) or pancreatin (10.0 g in simulated intestinal fluid) is added before use, and volume is fixed by double distilled water to 1000mL, and simulated gastric fluid and simulated intestinal fluid are respectively prepared. The free apigenin and the embedded apigenin samples were mixed with simulated gastric fluid or simulated intestinal fluid medium (1:4, v/v) and incubated in a 37℃water bath with stirring at 120 rpm. Their gastrointestinal stability was tested for 180min, and the kinetics of absorbance decrease at 337nm was observed, once every 30min.
The related detection results of improving the functional characteristics of whey protein and embedding rate of apigenin are as follows:
1. in the free radical induction method, hydroxyl free radicals attack sensitive groups in protein side chains to generate active intermediate substances, and whether the protein and polyphenol are covalently bound or not can be reacted by detecting the free amino groups of covalent complexes. As can be seen from FIG. 1, WP-EGCG and WP-EGCG-Ca compared to the WP control 2+ The polyphenol binding equivalent of the covalent complex is increased and WP-EGCG-Ca 2+ The polyphenol binding equivalent of the covalent complex is up to 10.94.+ -. 0.32. And the free amino group and the sulfhydryl group content are reduced, which indicates that EGCG and WP are covalently combined by a free radical induction method.
2. Fluorescence spectra can detect the fluorescence intensity of some amino acid residues of proteins, and changes in the fluorescence intensity or shifts in the peak of WP at 343nm (excitation wavelength of 280 nm) can be used to evaluate structural changes in the protein after WP binds to polyphenols. From fig. 2A, it is known that as the grafting ratio of polyphenol increases, the quenching degree of endogenous fluorescence of whey protein increases, which is probably due to the specific interaction between whey protein and polyphenol, which affects the structure of whey protein, promotes the micro-environmental change of tryptophan (Trp) and tyrosine (Tyr) residues of whey protein, and results in the decrease of fluorescence quantum yield. Synchronous fluorescence spectroscopy further detects conformational changes in whey protein and ligand interactions, as well as changes in the microenvironment of Trp, tyr residues, by measuring emission spectral shifts. As can be seen from an examination of fig. 2b, c of the simultaneous fluorescence spectra results at Δλ=15 nm and Δλ=60 nm, fluorescence quenching at Δλ=60 nm is greater than Δλ=15 nm, indicating that the binding site is also near the tryptophan residue.
3. FIG. 3 is WP, WP-EGCG-Ca 2+ Is characterized by the Fourier infrared spectrum of (1) the amide I band (1600-1700 cm) -1 ) Mainly caused by C=O stretching vibration, amide II (≡1540 cm) -1 ) The band contains C-N stretching vibration and N-H bending vibration, and the two intervals are related to the secondary structure of the protein and are typical spectral characteristic peaks of the protein. As the grafting ratio of the polyphenols increased, the peak of the amide I band of the protein shifted to a lower wavenumber (1654.28 cm -1 To 1651.17cm -1 ) The main strip being movedThe phenomenon indicates that the whey protein in the complex has a certain change in structure. The absorption peak of the amide II band did not change significantly. Phenolic hydroxyl groups and hydrogen bonds are known to be 3500cm -1 -3200cm -1 As shown in FIG. 3, when phenolic acid is grafted into whey protein molecule, a broad peak appears in the infrared spectrum between 3500 and 3200cm < -1 >, so that it can be concluded that phenolic acid is grafted into whey protein and WP-EGCG-Ca 2+ The peak of the complex was the widest, and the maximum amount of grafted phenolic acid was inferred.
4. To further investigate the free radical grafted polyphenol pairs WP, WP-EGCG-Ca 2+ The microstructure of the sample is observed with a scanning electron microscope. The WP formed by the spray drying process is spherical in structure as shown in FIG. 4, but the freeze drying process promotes the formation of broken flakes of WP with relatively flat and smooth edges of the flakes. EGCG is branch-shaped. After grafting the phenolic acid on the whey protein, the protein is broken, and the dendritic phenolic acid is combined with the protein. WP-EGCG-Ca 2+ The particles of the covalent complex are smaller and more loosely than the particles of WP. The above demonstrates that when a complex is prepared, whey protein binds to polyphenols, eventually changing the morphology of the protein, the microstructure changes and thus the functional properties change.
5. The solubility of proteins is the basis for achieving their functional properties of emulsifying, antioxidancy and gelling properties and affects their use in food processing. WP, WP-EGCG-Ca 2+ Protein solubility profile of the complex. As shown in FIG. 5, after grafting phenolic acid, whey protein has good protein solubility, probably due to the fact that a large number of hydroxyl groups are introduced after covalent bonding, the hydrophilicity of WP is improved, and the protein solubility is further increased.
6. WP and WP-EGCG, WP-EGCG-Ca 2+ The emulsification ability index (EA) and emulsion stability index (ES) of the covalent complex are shown in fig. 6. The results show that, compared with the blank WP, ca 2+ The addition of (3) significantly improves the EA value (P < 0.05) of the WP-EGCG covalent complex from 7.46+/-0.04 m 2 The/g is increased to 8.10.+ -. 0.13m 2 And/g. Studies have shown that EA changes are related to surface hydrophobicity. Whey (whey)The change in conformational structure of the protein affects the surface hydrophobicity of the protein, thereby affecting the emulsifying properties of the protein. Increasing the exposed aromatic hydrocarbon residues will increase the affinity of the protein for the oil/water interface, which can increase the emulsifying activity of WP. ES and EA show the same trend, prove that the material not only improves the emulsifying activity of WP-EGCG, but also increases the emulsifying stability, and is an ideal emulsifying agent.
7. FIG. 7 shows WP, WP-EGCG-Ca 2+ Apigenin (AP) entrapment rate of the composite. WP-EGCG-Ca 2 + The complex has the highest embedding rate on the AP, and proves the success of whey protein modification. As the AP concentration increased, the entrapment rate decreased, indicating that at higher AP concentrations, a portion of the AP was not intercalated into the whey protein solution.
8. Fig. 8 shows the light stability of AP and its composite particles under a 20W fluorescent lamp for 120 min. After 20min, the stability of free AP was reduced to about 79.68%, after 100min about 60% of the AP molecules were degraded and tended to be relatively stable. The embedding of the protein AP composite particles significantly delayed AP degradation. The AP molecules are implanted into the protein hydrophobic cavity, most of visible light and invisible light energy can be blocked or absorbed by the composite particles, the photolysis ratio is higher, the stability is higher, and the ultraviolet protection effect is better.
9. The thermal stability of AP and its composite particles is shown in FIG. 9 in a 60℃water bath heated for 120min at 80 ℃. After 20min, the stability of free AP was reduced to about 71.69%, and after 120min, about 58% of the AP molecules degraded. In addition, the protein-AP composite particles significantly reduce AP degradation, and in addition, the embedded composite particles exhibit better stability under heat treatment. The thermal stability of free AP decreases with increasing temperature. After heat treatment, the AP thermal stability of the complex is higher than that of free AP.
10. The bioavailability of free or entrapped apigenin was assessed using an in vitro simulated digestion model. The experimental results are shown in fig. 10, and the embedded apigenin shows better stability in simulated gastric fluid during incubation at 37 ℃. At 90min, the apigenin retention of the embedded sample in simulated gastric fluid was 72.38%, but for the free apigenin sample, the retention was only 45.04%. The bioavailability after the whole digestion process is only 34.98%, and the retention rate of the embedded sample is 48.78%.
As described above, the WP-EGCG-Ca of the present invention 2+ The complex has excellent functional characteristics and shows excellent performance in embedding 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).
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