Multi-active metal-polyphenol network-protein compound, medicine and application

Abstract

The invention belongs to the technical field of protein compounds, and particularly relates to a multi-active metal-polyphenol network-protein compound as well as a product and application thereof. The metal-polyphenol network-protein compound is a compound of a Fe < 3 + >-EGCG metal polyphenol network and isolated whey protein, has good oxidation resistance and alpha-glucosidase inhibitory activity, has obvious comprehensive advantages of various properties or activity, and is suitable for being used as an active ingredient of medicines for resisting oxidation, reducing blood sugar and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein complexes, and specifically relates to a multi-active metal-polyphenol network-protein complex, drugs and uses thereof. Background Art

[0002] Whey protein is a protein isolated from milk, accounting for about 20% of milk protein. The main constituent proteins in whey protein are β-lactoglobulin (accounting for about 50% - 55%), α-lactalbumin (accounting for about 20% - 25%), as well as immunoglobulins (accounting for about 10% - 15%), serum proteins (accounting for about 5%) and a small amount of lactoferrin. In addition, it also contains high concentrations of branched-chain amino acids leucine, isoleucine, and valine. Isolated whey protein (isolated whey protein or Whey protein isolate; also known as whey protein isolate) is a protein obtained by further processing of whey protein. Whey protein or its degraded bioactive peptide segments have excellent functional properties, such as enhancing human immunity, reducing the risk of cardiovascular diseases, regulating blood sugar, antioxidant, foaming and emulsifying properties, etc. Therefore, it has attracted much attention and is increasingly used in the pharmaceutical and food industries. However, whey protein is also a major allergen, which can trigger allergic reactions by binding to antibodies on mast cells, leading to symptoms such as dermatitis, vomiting, diarrhea, etc., and in severe cases, even endangering life. Currently, the allergenicity is usually reduced or its emulsifying properties are changed by degrading the protein and / or preparing a covalent or non-covalent complex with plant polyphenols.

[0003] Patent document CN118633744A provides a method for obtaining whey protein peptide segments by means of ultrasonic treatment and enzymatic hydrolysis, and then adding polyphenols to prepare a complex to obtain a low-allergenic whey protein product. CN113045768A provides a whey protein isolate-polyphenol complex-stabilized oil-in-water emulsion and its preparation method. However, degradation methods such as hydrolysis and enzymatic hydrolysis have a direct and essential impact on the primary structure of whey protein, shortening its peptide segments, resulting in the influence of its original activity and the unpredictability of activity due to the complexity of degradation sites and products. Protein-polyphenol complexes are prone to changing the secondary and tertiary structures of proteins, and the characteristics of the changes in secondary and tertiary structures are unpredictable, resulting in the unpredictable efficacy of the complexes. For example, the interaction between polyphenols and proteins has a masking effect on the free radical scavenging activity, which is likely to lead to a decrease in antioxidant potential or the masking of essential amino acids, resulting in a decrease in availability (Yang Hui, et al. Research progress on the biological activities and applications of plant polyphenol-protein complexes. Food Science, Vol. 43, No. 3, 2022, pp. 258 - 266); polyphenols may also reduce the in vivo hydrolysis of proteins (Zhao Qian. Research on the effects of protein, starch digestion and polyphenol antioxidant based on molecular interactions. Jiangsu University, 2020 doctoral dissertation).

[0004] The metal-polyphenol network is an emerging material processing technology with potential application value in fields such as water treatment (e.g., patent document CN118267979A), drug carriers (e.g., patent documents CN119033957A, CN119385980A, CN119385980A, etc.). However, there is still a lack of systematic research and reports on the impact of the metal-polyphenol network on the activity of active pharmaceutical proteins. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a multi-active protein complex, its products and uses. The protein complex is a metal-polyphenol network-whey protein isolate complex, which is composed of whey protein isolate (WPI), Fe 3+ , (-)-epigallocatechin-3-gallate (EGCG), and has higher antioxidant activity, higher α-glucosidase inhibitory activity and lower allergenicity. The technical solution of the present invention is as follows:

[0006] The present invention first provides a multi-active protein complex. The protein complex is a metal-polyphenol network-protein complex. The preparation method of the protein complex includes the following steps:

[0007] S1: Dissolve whey protein isolate in deionized water to prepare an aqueous solution of whey protein isolate;

[0008] S2: Add an iron salt to the aqueous solution of whey protein isolate obtained in step S1 and adjust the pH to alkaline to obtain a first mixed solution. The iron salt is a salt of iron ions (Fe 3+ ) or a hydrate of a salt of iron ions (Fe 3+ );

[0009] S3: Add (-)-epigallocatechin-3-gallate to the first mixed solution obtained in step S2 and adjust the pH to alkaline to obtain a second mixed solution. The second mixed solution is stirred and dialyzed with a molecular weight cut-off of 3000 Da.

[0010] Preferably, in step S2, the weight ratio of whey protein isolate to the iron salt is 1000:(0.67 - 5.4) based on the weight ratio of whey protein isolate to Fe 3+ . Here, the molecular weight of Fe 3+ is calculated as 55.845 g / mol.

[0011] Preferably, in the protein complex, the molar ratio of Fe 3+ to (-)-epigallocatechin-3-gallate is 1:(0.2 - 5).

[0012] Preferably, in the step S1, the mass-volume concentration of the whey protein separated from the whey protein aqueous solution is 1% to 50% (i.e., 1 g / 100 mL to 50 g / 100 mL);

[0013] Preferably, in the step S1, the preparation method of the whey protein aqueous solution includes the following steps: adding whey protein to deionized water, stirring for 1 to 3 h, and then hydrating at 2°C to 8°C for 10 to 16 h.

[0014] Preferably, in the step S2, the pH of the first mixed solution is 8 to 9.

[0015] Preferably, in the step S2, NaOH is used to adjust the pH.

[0016] Preferably, in the step S2, the iron salt is FeCl3·6H2O.

[0017] Preferably, in the step S3, the pH of the second mixed solution is 8 to 9.

[0018] Preferably, in the step S3, NaOH is used to adjust the pH.

[0019] Preferably, in the step S3, the second mixed solution is dialyzed at 2°C to 8°C for 20 to 72 h to obtain a metal-polyphenol network-whey protein complex.

[0020] The product obtained in the step S3 can be further freeze-dried into a freeze-dried powder.

[0021] In the second aspect of the present invention, a health-regulating preparation containing the aforementioned protein complex is provided.

[0022] Preferably, the health-regulating preparation includes a drug or a health food with a health-regulating effect, and the preparation is a liquid or solid preparation. When the preparation is a drug, the preparation of the drug is a liquid or solid preparation.

[0023] In the third aspect of the present invention, the use of the aforementioned protein complex is provided, including:

[0024] (1) The use of the aforementioned protein complex in the preparation of a health-regulating preparation for antioxidant use; the health-regulating preparation includes a drug or a health food with a health-regulating effect, and the preparation is a liquid or solid preparation. When the preparation is a drug, the preparation of the drug is a liquid or solid preparation.

[0025] (2) Use of the aforementioned protein complex in the preparation of a health regulatory formulation for lowering blood glucose. The protein complex has α-glucosidase inhibitory activity. The health regulatory formulation includes a drug or a health food with health regulatory effects, and the formulation is a liquid or solid formulation. When the formulation is a drug, the preparation of the drug is a liquid or solid preparation.

[0026] (3) Use of the aforementioned protein complex in the preparation of a low-allergenic whey protein food; preferably, the food is a liquid or solid beverage.

[0027] The aforementioned "health regulatory formulation" and "health regulatory formulation" include drugs, health foods, etc. that regulate certain health-related indicators of the human body.

[0028] In the fourth aspect of the present invention, a method for reducing the allergenicity of isolated whey protein is provided. The method is to prepare isolated whey protein into a metal-polyphenol network-protein complex, and the method includes the following steps:

[0029] S1: Dissolve isolated whey protein in deionized water to prepare an aqueous solution of isolated whey protein.

[0030] S2: Add an iron salt to the aqueous solution of isolated whey protein obtained in step S1 and adjust the pH to alkaline; obtain a first mixture. The iron salt is a salt of iron ion (Fe 3+ ) or a hydrate of a salt of iron ion (Fe 3+ ).

[0031] S3: Add (-)-epigallocatechin-3-gallate to the first mixture obtained in step S2 and adjust the pH to alkaline to obtain a second mixture; stir and dialyze the second mixture with a molecular weight cut-off of 3000 Da to obtain an allergenic protein complex.

[0032] In the protein complex, the weight ratio of isolated whey protein to the iron salt is 1000:(0.67 - 5.4) based on the weight ratio of isolated whey protein to Fe 3+ . In the protein complex, the molar ratio of Fe 3+ to (-)-epigallocatechin-3-gallate is 1:1.

[0033] Further preferably, in the protein complex, the weight ratio of isolated whey protein to the iron salt is 1000:(2.6 - 5.4) based on the weight ratio of isolated whey protein to Fe 3+ .

[0034] Still further preferably, in the protein complex, the weight ratio of isolated whey protein to the iron salt is 1000:(4 - 5.4) based on the weight ratio of isolated whey protein to Fe 3+ .

[0035] Advantages of the present invention:

[0036] The protein complex of the present invention not only has excellent foaming properties, but also has significantly improved antioxidant properties and α-glucosidase inhibitory activity, and has lower allergenicity.

[0037] The preparation method of the protein complex of the present invention is simple, which is conducive to industrial production and promotion. Description of the drawings

[0038] Figure 1 For the turbidity (A), particle size (B), Zeta potential (C) test results and energy dispersive spectroscopy (D) test results of the product of Example 1;

[0039] Figure 2 For the Fourier transform infrared spectrum (A) and microstructure micrograph (B) of the product of Example 1;

[0040] Figure 3 For the intrinsic fluorescence spectrum (A), ultraviolet absorption spectrum (B), surface hydrophobicity (C) and total sulfhydryl content (D) test results of the product of Example 1;

[0041] Figure 4 For the total antioxidant capacity (A) and reducing capacity (B) measurement results of Example 2;

[0042] Figure 5 For the α-glucosidase inhibitory activity measurement result of Example 3;

[0043] Figure 6 For the allergenicity measurement result of Example 4. Detailed implementation manners

[0044] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments and the accompanying drawings. In the embodiments, "WPI" is whey protein isolate, purchased from Hilmar Ingredients, USA, and the product model is Hilmar 9410 (marked with the production date of March 12, 2023); "EGCG" is (-)-epigallocatechin-3-gallate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; FeCl3·6H2O is purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; the serum of milk allergic patients and goat anti-human IgE-horseradish peroxidase (HRP) conjugate are purchased from Chongqing Wokaway Biotechnology Co., Ltd. The reagents used in the embodiments are of analytical pure grade or above. Room temperature usually refers to 10-30°C, and 25°C is used in the specific operations of the embodiments.

[0045] Preparation and Characterization of Metal-Polyphenol Network-Whey Protein Complexes in Example 1

[0046] 1. Preparation of Metal-Polyphenol Network-Whey Protein Complexes

[0047] Add WPI into deionized water, stir continuously at room temperature for 2 h, then hydrate overnight for 12 h at 4 °C to dissolve and obtain a WPI aqueous solution with a concentration of 1% (1 g / 100 mL).

[0048] Then, under magnetic stirring, slowly add 1 mL of FeCl3·6H2O aqueous solution into 100 mL of WPI aqueous solution, and adjust the pH value to 8.5 with 1 M NaOH to obtain a first mixed solution.

[0049] After stirring the first mixed solution for 30 min, add 1 mL of polyphenol (EGCG) aqueous solution. Adjust the pH value to 8.5 to obtain a second mixed solution; stir the second mixed solution at room temperature for 1 h, then dialyze at 4 °C for 48 h with a molecular weight cut-off of 3000 Da to prepare a metal-polyphenol network-WPI complex.

[0050] Freeze-dry the obtained metal-polyphenol network-WPI complex by dialysis into a powder for standby, and store the sample at -20 °C.

[0051] The concentrations of the above-mentioned FeCl3·6H2O aqueous solution and polyphenol (EGCG) aqueous solution correspond to 12 - 96 mM (i.e., the molar ratio of FeCl3·6H2O to EGCG is 1:1). After conversion, the weight ratio of WPI to iron salt (FeCl3·6H2O) in the preparation of the metal-polyphenol network-WPI complex is 1000:(3.2 - 25.95); after conversion, the weight ratio of WPI to iron salt (FeCl3) in the preparation of the metal-polyphenol network-WPI complex is 1000:(1.9 - 15.58); after conversion, the weight ratio of WPI to Fe 3+ (with a molecular weight calculated as 55.845 g / mol) in the preparation of the metal-polyphenol network-WPI complex is 1000:(0.67 - 5.4).

[0052] Among them, the metal-polyphenol network-WPI complex samples prepared with the concentrations of FeCl3·6H2O aqueous solution and polyphenol aqueous solution corresponding to 12 mM, 24 mM, 48 mM, 72 mM, and 96 mM are represented by WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 respectively. That is, the FeCl3·6H2O aqueous solution and polyphenol aqueous solution are taken at a molar ratio of Fe 3+ to polyphenol of 1:1, and metal-polyphenol network-WPI complexes are prepared with concentrations of 12 mM, 24 mM, 48 mM, 72 mM, and 96 mM respectively.

[0053] The sample prepared with the concentration of FeCl3·6H2O aqueous solution and polyphenol aqueous solution being 0 mM is denoted as WEF-0 (that is, only the corresponding volume of deionized water is added, without adding FeCl3·6H2O and EGCG).

[0054] 2. Characterization of Metal-Polyphenol Network-Whey Protein Complexes

[0055] 2.1 Turbidity

[0056] The absorbance of the sample (1 mg / mL) at a wavelength of 600 nm was measured using a SpectraMax i3x multi-functional microplate reader (Molecular Devices Co., Ltd.) to represent the turbidity.

[0057] 2.2 Particle Size and Zeta Potential

[0058] The particle size and zeta potential of the sample (0.2 mg / mL) were measured using a Nano Zetasizer (Malvern Instruments Ltd).

[0059] 2.3 Energy Dispersive Spectrometer (EDS)

[0060] The carbon, nitrogen, oxygen, and iron elements in the sample were observed and analyzed using an EX-250 EDS (HORIBA).

[0061] 2.4 Fourier Transform Infrared Spectroscopy (FTIR)

[0062] The freeze-dried powder was thoroughly mixed with dry KBr powder and pressed. The spectrum of the sample in the range of 4000 - 400 cm -1 was measured using a Fourier transform infrared spectrometer (Nicolet iS5, Thermo Fisher).

[0063] 2.5 Scanning Electron Microscope (SEM)

[0064] After the sample was sputter-coated with gold, its structure was observed using a scanning electron microscope (Hitachi SU 8020).

[0065] 2.6 Intrinsic Fluorescence Spectrum

[0066] The spectrum of the sample (0.1 mg / mL) at 300 - 400 nm under excitation at 280 nm was measured using a fluorescence spectrophotometer (Shimadzu RF6000).

[0067] 2.7 Ultraviolet Absorption Spectrum

[0068] The absorbance of the sample at wavelengths of 280 - 400 nm was measured using an ultraviolet spectrophotometer (Shimadzu UV-2600).

[0069] 2.8 Surface hydrophobicity (H0)

[0070] Mix 4 mL of 1 mg / mL sample with 100 μL of 8 mmol / L 8 - anilino - 1 - naphthalene sulfonic acid (ANS) solution and react in the dark at room temperature for 20 min. The fluorescence intensity of the mixture is measured at an excitation wavelength of 390 nm and an emission wavelength of 470 nm.

[0071] 2.9 Total thiol content

[0072] Dissolve the sample in Tris - glycine buffer (pH 8.0) containing 8 M urea at a concentration of 1 mg / mL. Add Ellman's reagent and react in the dark at room temperature for 15 min. Measure the absorbance (A412) of the mixture with a microplate reader at a wavelength of 412 nm. The calculation formula for thiol content is as follows:

[0073] SH (μM / g) = 73.53×A 412 ×D / C

[0074] D is the dilution factor; C is the protein concentration (mg / mL).

[0075] 2.10 Liquid chromatography - tandem mass spectrometry (LC - MS / MS)

[0076] The sample is filtered through a 10 kDa ultrafiltration tube (Merck, Germany), and then hydrolyzed with trypsin overnight. Subsequently, desalting is performed using a C18 desalting column. Peptide separation and characterization are carried out using an EASY - nLC 1200 and a Q - Exactive system (Thermo Fisher Scientific), respectively. Data is analyzed using Proteome Discoverer 2.2 software (Thermo Fisher). Allergenic peptides in the sample are identified using the Biopep database (https: / / www.uwm.edu.pl / biochemia).

[0077] 2.11 Foaming properties

[0078] Homogenize 10 mL of 1 mg / mL (V0) sample at 10,000 rpm for 1 min, and then measure its volume (Vt). Then, place the sample at room temperature for 10 min and measure its volume (VT). The calculation formulas for foaming capacity (FC) and foaming stability (FS) are as follows.

[0079]

[0080] 3. Characterization results

[0081] (1) As Figure 1As shown in A, the purple color of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 gradually deepens in turn ( Figure 1 The insets in the upper left corner of A are the physical pictures of the samples of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 from left to right). The turbidity of the product also increased significantly (p < 0.05). This indicates that EGCG and Fe 3+ successfully formed a coordination bond.

[0082] (2) The particle sizes of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 are as Figure 1 shown in B. There was no significant difference in the particle sizes between WEF-0 and WEF-1 (p > 0.05). The particle sizes of WEF-2, WEF-3, WEF-4, and WEF-5 were all significantly higher than that of WEF-0 (p < 0.05). This further indicates the successful modification of WPI. The particle sizes of WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 first increased, then decreased, and then increased again. Among them, the particle size of WEF-3 was lower than that of WEF-2, WEF-4, and WEF-5, probably because the metal-polyphenol network modification at this concentration (48 mM) would cause the protein volume to shrink, resulting in protein aggregation.

[0083] (3) The zeta potential results of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 are as Figure 1 shown in C. There was no significant difference in the zeta potentials of WEF-0, WEF-1, and WEF-2 (p > 0.05), indicating that the change in the complex particle size might be due to the increase in the metal-polyphenol network. Further increase in the concentration led to a decrease in the complex zeta potential.

[0084] (4) The elemental distribution on the surface of WEF-5 was measured by EDS energy spectrum analysis ( Figure 1 D), and it was found that the iron element was evenly distributed on the surface of WEF-5.

[0085] (5) The Fourier transform infrared spectra of the samples were measured. The amide A region (3700 - 3000 cm -1 ) represents the change of hydrogen bonds in the protein structure, the amide I band (1700 - 1600 cm -1 ) represents the C=O stretch, the amide II band (≈1540 cm -1 ) represents the C-N stretch and N-H bending modes, and the amide III band (1300 - 1200 cm -1 ) represents the C-N stretching vibration. As Figure 2As shown in A, the modification led to changes in the peaks of WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 compared to WEF-0 (the peaks had redshifts or blueshifts of varying degrees).

[0086] (6) As Figure 2 shown in B, the surface structure of WEF-0 remained intact, and the surface structures of WEF-1 and WEF-2 changed less, but showed more obvious fragmentation compared to WEF-0. The microstructures of WEF-3, WFE-4, and WEF-5 were significantly more fragmented, presenting an irregular lamellar shape. This lamellar structure may be due to the modification of the WPI surface by the five-membered ring network structure formed by EGCG and Fe 3+ ions.

[0087] (7) Intrinsic fluorescence spectroscopy can reflect the exposure of tyrosine (Tyr) and tryptophan (Trp) in WPI, thus reflecting the conformational changes of proteins. As Figure 3 shown in A, the intensities of the fluorescence spectra of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 gradually decreased and redshifted. This indicates that the aromatic amino acids of WPI were exposed to a more polar environment. This may be because the metal-polyphenol network changed the conformation of WPI.

[0088] (8) Ultraviolet absorption spectroscopy is an important tool for evaluating the changes in the tertiary structure of proteins. As Figure 3 shown in B, the ultraviolet absorption spectra of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 gradually increased in turn, indicating that the microenvironment of the aromatic amino acid residues of WPI changed, reflecting the conformational changes of WPI due to the presence of the metal-polyphenol network.

[0089] (9) H0 (surface hydrophobicity) can provide important information for evaluating the interaction between WPI and the metal-polyphenol network. As Figure 3 shown in C, the metal-polyphenol network significantly reduced the H0 of WPI (p < 0.05). The H0 of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 showed a gradually decreasing trend.

[0090] (10) The sulfhydryl group contents of WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 were also significantly lower than that of WEF-0, and the sulfhydryl group contents of WEF-3, WEF-4, and WEF-5 decreased particularly significantly (as Figure 3 shown in D).

[0091] (11) LC-MS / MS can provide peptide information of trypsin-hydrolyzed WPI and its complexes, thereby revealing the structural changes of WPI and the binding sites of the metal-polyphenol network. β-Lg (P02754) and α-La (Q9TSN6) are the main allergens in WPI.

[0092] The results of LC-MS / MS analysis showed that the metal-polyphenol network changed the peptide profile distribution of β-Lg and α-La in WPI. With the increase of the metal-polyphenol network concentration, the spectral distribution and intensity of the peptide segments of P02754 changed greatly. Among them, the peptide segments of 60-80 amino acids and about 150-160 amino acids were not found in WEF-5, which may be related to the metal-polyphenol network causing WPI to resist trypsin hydrolysis.

[0093] Cluster heat map analysis was performed on the peptide segments identified by LC-MS / MS. WEF-1 and WEF-5 in α-La and β-Lg were grouped together, indicating that WEF-1 and WEF-5 had similar peptide segment distribution and intensity. It is worth noting that the abundance of the ALKALPMHI peptide segment in the complex was always lower than that in WEF-0. This indicates that the metal-polyphenol network has a stronger binding ability to the amino acid residues near this peptide. In addition, the abundances of the two peptide segments of TKIPAVFK and VAGTWYSLAMAASDISLLDAQSAPLR were similar in WEF-0 and WEF-1, but higher than those in other complexes.

[0094] (12) Foaming agents are used in foods such as cakes, cookies, and chocolate mousses. Natural protein foaming agents have received increasing attention. After measurement and calculation, the FCs of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 were 64%, 75.33%, 75.67%, 93.33%, 102.33%, and 104% respectively. Compared with WEF-0, the FCs of WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 were all significantly increased (p < 0.05). This may be due to the metal-polyphenol network improving the flexibility of WPI and accelerating the adsorption of WPI at the gas-liquid interface. There was no significant difference in the FS of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5.

[0095] Example 2 Investigation of the antioxidant activity of the metal-polyphenol network-whey protein complex

[0096] 1. Samples

[0097] The freeze-dried samples prepared in Example 1 were used. See the specific detection method for the preparation of the test solution.

[0098] 2. Determination method

[0099] 2.1 Determination of total antioxidant capacity

[0100] The total antioxidant capacity was determined according to the instructions of the total antioxidant capacity detection kit (Nanjing Jiancheng Bioengineering Institute, China; ABTS method). The sample (2 mg / mL), ABTS working solution and peroxidase application solution were mixed and incubated at room temperature in the dark for 6 min. Subsequently, the absorbance of the mixture was read at a wavelength of 405 nm.

[0101] 2.2 Determination of ferric ion reducing ability

[0102] 100 μL of 5 mg / mL sample, 250 μL of deionized water and 250 μL of K3[Fe(CN)6] (1%, w / v) were mixed thoroughly and reacted at 50 °C for 20 min. 250 μL of trichloroacetic acid (10%, w / v) was added at room temperature and centrifuged at 1500 g for 10 min. Then, 500 μL of the supernatant, 500 μL of water and 100 μL of ferric chloride (0.1%, w / v) were mixed and incubated at room temperature for 10 min. The absorbance of the mixture was measured and read at a wavelength of 700 nm.

[0103] 3. Determination results

[0104] As Figure 4 shown in A, the total antioxidant capacity of WEF-0 was 1.70%, and the total antioxidant capacities of WEF-1, WEF-2, WEF-3, WEF-4 and WEF-5 were 10.30%, 15.23%, 28.09%, 35.54% and 44.01% respectively, all significantly higher than that of WEF-0 (p < 0.05). As Figure 4 shown in B, the reducing abilities of WEF-1, WEF-2, WEF-3, WEF-4 and WEF-5 were all significantly higher than that of WEF-0 (p < 0.05).

[0105] Although Fe 3+ is an oxidant, and previous reports have shown that the Fe 3+ -EGCG network belongs to oxidase mimetics. This study shows that the total antioxidant effect of Fe 3+ -EGCG is significantly improved after forming a complex with WPI. It indicates that there is a complex complexation behavior between the Fe 3+ -EGCG network and proteins, and the formation of the Fe 3+ -EGCG-WPI complex changes the activity of the Fe 3+ -EGCG network.

[0106] Example 3 Investigation of α-glucosidase inhibitory activity of metal-polyphenol network-whey protein complex

[0107] 1. Samples

[0108] The metal-polyphenol network-whey protein complex freeze-dried sample prepared in Example 1 was used. In addition, an EGCG-WPI complex was prepared and freeze-dried as an EGCG-WPI control. The EGCG-WPI complex was prepared according to the method of Example 1, except that FeCl3·6H2O was not added (i.e., only a corresponding volume of deionized water was added without FeCl3·6H2O), and the EGCG concentration of the polyphenol aqueous solution was 96 mM.

[0109] 2. Determination method

[0110] 50 μL of 2 mg / mL sample was mixed with 50 μL of 0.06 U / mL α-glucosidase solution and reacted at 37°C for 10 min. Then 50 μL of 2.5 mM 4-nitrophenyl-β-D-pyranoglucoside was added and reacted at 37°C for 30 min. 100 μL of 0.5 M Na2CO3 was added and the absorbance of the mixture was measured at a wavelength of 405 nm.

[0111] 3. Measurement results

[0112] α-Glucosidase can release glucose by hydrolyzing glucosidic bonds, leading to an increase in postprandial blood glucose levels. EGCG is an important α-glucosidase inhibitor, but it has been reported that the combination of WPI and EGCG reduces its inhibitory ability against α-glucosidase. Therefore, this study evaluated the inhibitory ability of the prepared complex against α-glucosidase by evaluating the inhibitory ability of α-glucosidase.

[0113] like Figure 5 As shown in the figure, there is no significant difference in the α-glucosidase inhibitory ability between WEF-0 and WEF-1 (p>0.05); the α-glucosidase inhibitory ability of WEF-2, WEF-3, WEF-4, and WEF-5 is significantly higher than that of WEF-0 (p<0.05), among which the α-glucosidase inhibitory ability of WEF-4 and WEF-5 is significantly higher than that of EGCG-WPI complex (p<0.05). However, since the amount of EGCG in WEF-2 and WEF-3 is several times lower than that of EGCG-WPI, the α-glucosidase inhibitory ability of WEF-2 and WEF-3 is not lower than that of EGCG-WPI at the same EGCG concentration, especially the α-glucosidase inhibitory ability of WEF-3 is not significantly different from that of EGCG-WPI with high EGCG concentration (p>0.05).

[0114] As can be seen from Example 1, in the preparation of WEF-1, WEF-2, WEF-3, WEF-4 and WEF-5, the weight ratio of isolated whey protein to iron salt is as follows: 3+By weight ratio calculated based on 55.845 g / mol, they are respectively 1000:0.67, 1000:1.34, 1000:2.68, 1000:4.02, 1000:5.36.

[0115] Example 4 Investigation of the Allergenicity of Metal-Polyphenol Network-Whey Protein Complex

[0116] 1. Samples

[0117] The lyophilized samples prepared in Example 1 were used. Additionally, an EGCG-WPI complex was formulated and lyophilized as an EGCG-WPI control. The preparation method of the EGCG-WPI complex was the same as that in Example 1, except that FeCl3·6H2O was not added (i.e., only the corresponding volume of deionized water was added without FeCl3·6H2O), and the EGCG concentration in the polyphenol aqueous solution was 96 mM.

[0118] 2. Measurement Method

[0119] The samples (100 μL; 5 μg / mL) were incubated overnight at 4 °C in a 96-well plate, and then the wells were sealed with a 3 g / 100 mL gelatin solution. After washing the plate 3 times with phosphate-buffered saline containing 0.05% Tween 20 (PBST buffer), allergic patient serum (1:20 v / v) was added and incubated at 37 °C for 1 hour; after washing the plate, IgE-HRP (1:3000 v / v) or IgG-HRP (1:500000 v / v) was added and incubated at 37 °C for 1 h; finally, the plate was washed, TMB single-component chromogenic solution was added, and incubated at 37 °C for 15 min. 50 μL of 2 M sulfuric acid was added to terminate the reaction, and the absorbance of the chromogenic solution was measured at a wavelength of 450 nm.

[0120] 3. Measurement Results

[0121] The allergenic epitopes on the allergen bind to specific antibodies on mast cells, triggering an allergic reaction. The allergic reaction caused by WPI is mediated by IgE and can occur within minutes to hours. In addition, some epitopes that bind to IgE also bind to IgG, thus triggering an allergic reaction. Therefore, in this study, a serum pool was constructed using the sera of 10 milk protein allergic patients to measure the IgE and IgG binding abilities of the prepared complexes.

[0122] As Figure 6As shown in

[0123] Figure Figure 6 A, the IgE-binding capacities of WEF-0, WEF-1, WEF-2, WEF-3, WEF-4, and WEF-5 decreased successively, being 100%, 88.48%, 88.20%, 76.96%, 76.40%, and 74.24%, respectively. This indicates that the metal-polyphenol network significantly reduced the IgE-binding capacity of WPI (p < 0.05), thus significantly reducing its allergenicity, especially for WEF-3, WEF-4, and WEF-5, which showed more significant decreases.

[0124] Compared with the EGCG-WPI complex, it was also found that the IgE-binding and IgG-binding capacities of WEF-3, WEF-4, and WEF-5 were lower than those of the EGCG-WPI complex. Among them, the IgE and IgG-binding capacities of WEF-4 and WEF-5 were significantly lower than those of the EGCG-WPI complex; the IgE-binding capacity of WEF-3 was significantly lower than that of the EGCG-WPI complex, and there was no significant difference in the IgG-binding capacity compared with the EGCG-WPI complex. However, the EGCG concentration in WEF-3 was also much lower than that in the EGCG-WPI complex. Therefore, a comprehensive evaluation showed that the allergenicity of WEF-3, WEF-4, and WEF-5 was significantly lower than that of the EGCG-WPI complex.

[0125] The low allergenicity of the complex of the present invention may be related to its disruption of the epitopes of WPI. It is worth noting that in the LC-MS / MS analysis in Example 1, it was also found that compared with other samples, WEF-5 was significantly lacking in the peptides ALKALPMHIR and VYVEELKPTPEGDLEILLQK, which may be the reason for its low allergenicity.

[0126] Example 5 Investigation of the Antioxidant and Allergenic Properties of Different Metal-Polyphenol Network-Whey Protein Complexes

[0127] Before carrying out Examples 2 to 4, the method of Example 1 was also used to replace polyphenols with chlorogenic acid (CHA), caffeic acid (CA), gallic acid (GA), and quercetin (QE) at the same molar concentration, respectively, to prepare Fe 3+ -CHA-WPI complex, Fe 3+ -CA-WPI complex, Fe 3+ -GA-WPI complex, Fe 3+ -QE-WPI complex.

[0128] The antioxidant capacity of the complex was determined by the method of Example 2. The results showed that under the same conditions, the antioxidant capacity of Fe 3+ -CHA-WPI complex, Fe 3+ -CA-WPI complex, Fe 3+ -GA-WPI complex, Fe 3+ -QE-WPI complex was significantly lower than that of Fe 3+ -EGCG-WPI complex.

[0129] The allergenicity of the complex was determined by the method of Example 4. The results showed that under the same conditions, only the IgE-binding ability of Fe 3+ -QE-WPI complex was slightly higher than that of Fe 3+ -EGCG-WPI complex in Example 1 (p > 0.05), and the IgE-binding ability of other complexes was significantly higher than that of Fe 3+ -EGCG-WPI complex (p < 0.05). And the IgE-binding ability of Fe 3+ -CHA-WPI complex, Fe 3+ -CA-WPI complex, Fe 3+ -GA-WPI complex, Fe 3+ -QE-WPI complex was not significantly different from that of WPI.

[0130] The above research suggests that the interaction sites of metal-polyphenol-WPI complexes are relatively complex, resulting in unpredictable changes in the activity of the complexes.

Claims

1. A multi-active metal-polyphenol network-protein complex, characterized in that, The preparation method of the protein complex comprises the following steps: S1: Dissolve whey protein isolate in deionized water to prepare an aqueous solution of whey protein isolate; S2: Add an iron salt to the separated whey protein aqueous solution obtained in step S1 and adjust the pH to alkaline to obtain a first mixture. The iron salt is Fe 3+ salt or Fe 3+ hydrate of the salt; S3: Add (-)-epigallocatechin-3-gallate to the first mixture obtained in step S2, adjust the pH to alkaline to obtain a second mixture; Stir and dialyze the second mixture, with a molecular weight cut-off value of 3000 Da; In the step S2, the weight ratio of separated whey protein to iron salt for separating whey protein and Fe 3+ is 1000:(0.67 - 5.4) based on the weight ratio; in the protein complex, the molar ratio of Fe 3+ to (-)-epigallocatechin-3-gallate is 1:(0.2 - 5).

2. A multi-active metal-polyphenol network-protein complex according to claim 1, wherein In step S1, the mass-volume concentration of whey protein isolate in the aqueous solution of whey protein isolate is 1% - 50%.

3. A multi-active metal-polyphenol network-protein complex according to claim 1, characterized in that, In step S1, the preparation method of the aqueous solution of whey protein isolate comprises the following steps: Add whey protein isolate to deionized water, stir for 1 - 3 h, and then hydrate at 2°C - 8°C for 10 - 16 h.

4. A multi-active metal-polyphenol network-protein complex according to claim 1, wherein In step S2, the pH of the first mixture is 8 - 9.

5. A multi-active metal-polyphenol network-protein complex according to claim 1, characterized in that, In step S2, the iron salt is FeCl3·6H2O.

6. The multi-active metal-polyphenol network-whey protein complex according to claim 1, characterized in that, In step S3, the pH of the second mixture is 8 - 9.

7. A multi-active metal-polyphenol network-protein complex according to claim 1, characterized in that, In step S3, dialyze the second mixture at 2°C - 8°C for 20 - 72 h to obtain a metal-polyphenol network-whey protein complex.

8. A drug, characterized in that Containing a multi-active metal-polyphenol network-protein complex according to any one of claims 1 to 7.

9. The drug according to claim 8, characterized in that, The preparation of the drug is a liquid or solid preparation.

10. Use of a multi-active metal-polyphenol network-protein complex according to any one of claims 1 to 7 or the drug according to claim 8 in the preparation of a drug for antioxidation.

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