A whey protein isolate-polyphenol complex, and a method of preparing and using the same

Abstract

This invention provides a whey protein isolate-polyphenol complex, its preparation method, and its application, relating to the food industry. The preparation method includes the following steps: S1, weighing whey protein isolate, dissolving it in phosphate buffer solution, and fully hydrating it to obtain a protein solution; S2, adding H2O2 solution and ascorbic acid to the protein solution, mixing thoroughly, and adding polyphenols to perform a grafting reaction to obtain a product solution; S3, dialyzing the product solution to remove unreacted polyphenols, and freeze-drying the retained solution to obtain the whey protein isolate-polyphenol complex. Compared with natural whey protein isolate, the whey protein isolate-polyphenol complex exhibits significantly enhanced ABTS free radical scavenging ability, iron reducing power, emulsifying activity index, emulsifying stability index, foaming ability, and foam stability, indicating that the covalent grafting of polyphenols successfully endows the whey protein isolate with superior antioxidant properties, emulsifying properties, and foaming properties.

Description

Technical Field

[0001] This invention relates to the food industry, specifically to a whey protein isolate-polyphenol complex, its preparation method, and its application. Background Technology

[0002] Whey protein isolate (WPI) is a high-purity protein product obtained from whey, a byproduct of cheese production, through separation and purification processes such as ultrafiltration and dialysis. Its protein content typically reaches over 80%. As a mixed protein system, WPI is mainly composed of various globular proteins, with β-lactoglobulin accounting for approximately 50% of the total protein and α-lactalbumin for approximately 20%. It also contains active protein components such as bovine serum albumin, immunoglobulins, and lactoferrin. These components together constitute the complex and stable protein network of WPI. From a nutritional perspective, WPI has outstanding nutritional value, with a biological value as high as 88, significantly superior to most common edible proteins. Furthermore, WPI is rich in essential branched-chain amino acids and sulfur-containing amino acids, and exhibits high digestibility and absorption, making it a high-quality protein supplement. At the structural level, the main protein components in WPI all exhibit a typical globular conformation. Their secondary structures are mainly α-helices and β-sheets. The three-dimensional structure is maintained relatively stably by hydrogen bonds, hydrophobic interactions and van der Waals forces, which enable it to maintain conformational integrity within a certain pH and temperature range.

[0003] WPI, due to its superior molecular structure and amphiphilic nature, exhibits excellent functional properties such as emulsifying, gelling, foaming, water-holding, and oil-holding properties, and is widely used in the food industry for emulsion construction, gel formation, foam stabilization, and delivery of bioactive substances. However, natural WPI is susceptible to structural changes during processing due to environmental factors, and its functional stability needs to be improved. How to endow WPI with more stable functional properties through chemical modification has become an urgent problem to be solved in this field. Summary of the Invention

[0004] Therefore, the purpose of this invention is to chemically modify whey protein isolate to improve its functional properties (antioxidant properties, emulsifying properties, foaming properties), thereby providing a whey protein isolate-polyphenol complex, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a whey protein isolate-polyphenol complex, comprising the following steps: S1. Weigh whey protein isolate, dissolve it in phosphate buffer solution, and fully hydrate it to obtain a protein solution; S2. Add H2O2 solution and ascorbic acid to the protein solution, mix thoroughly, add polyphenols to carry out grafting reaction, and obtain product solution; S3. Dialyze the product solution to remove unreacted polyphenols, freeze-dry the retained solution to obtain whey protein isolate-polyphenol complex.

[0007] Furthermore, the polyphenol is at least one of p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid.

[0008] Further, in step S1, the mass-to-volume ratio of the whey protein isolate to the phosphate buffer solution, expressed in mg / mL, is 500:45~55, preferably 500:49.

[0009] Further, in step S1, the concentration of the phosphate buffer solution is 0.1~0.3 mol / L, and the pH value is 6~7; preferably, the concentration is 0.1 mol / L, and the pH value is 6.75.

[0010] Furthermore, in step S1, the conditions for sufficient hydration are: hydration overnight at 3~5 ℃, preferably hydration overnight at 4 ℃.

[0011] Further, in step S2, the mass-to-volume ratio of the whey protein isolate to the H2O2 solution, expressed in mg / mL, is 500:1 to 3, preferably 500:1.

[0012] Further, in step S2, the mass ratio of the whey protein isolate to the ascorbic acid is 2:1 to 1.5, preferably 2:1.

[0013] Further, in step S2, the conditions for thorough mixing are constant temperature stirring at 24~26 ℃ for 100~150 min; preferably, constant temperature stirring at 25 ℃ for 120 min.

[0014] Further, in step S2, the mass ratio of the whey protein isolate to the polyphenols is 5:1 to 3, preferably 5:2.

[0015] Further, in step S2, the grafting reaction is carried out under the following conditions: stirring at room temperature for 18-24 h, preferably stirring at room temperature for 24 h.

[0016] Further, in step S3, the dialysis conditions are as follows: the dialysis bag retention is 6~8 kDa; dialysis with distilled water at 3~5 ℃ for 48~72 h, preferably dialysis with distilled water at 4 ℃ for 72 h; and the water is changed every 4~8 h, preferably every 6 h.

[0017] Furthermore, in step S3, the freeze-drying time is 36-48 hours, preferably 48 hours.

[0018] Secondly, the present invention provides a whey protein isolate-polyphenol complex obtained by the preparation method described above.

[0019] Thirdly, the present invention provides the application of the whey protein isolate-polyphenol complex obtained by the preparation method described above in the preparation of emulsified foods, gel foods or aerated foods.

[0020] The technical solution of this invention has the following advantages: This invention utilizes whey protein isolate and polyphenols in an ascorbic acid (Asc) / hydrogen peroxide (H2O2) redox system for free radical grafting. This system generates Asc or hydroxyl radicals, which induce oxidative cleavage of sensitive groups (such as amino and thiol groups) on the protein side chains, generating protein free radicals. These free radicals can covalently couple with polyphenols, ultimately yielding a stable whey protein isolate-polyphenol complex. This method not only features mild reaction conditions, effectively avoiding thermal degradation of polyphenols, but more importantly, it achieves targeted modification through free radical activation of the protein, rather than relying on polyphenol auto-oxidation. It can stably bind polyphenols to the protein molecular chain via covalent bonds.

[0021] Compared with natural whey protein isolate, the whey protein isolate-polyphenol complex showed significantly enhanced ABTS free radical scavenging ability, iron reducing power, emulsifying activity index (EAI), emulsifying stability index (ESI), foaming ability (FC), and foam stability (FS). This indicates that the covalent grafting of polyphenols successfully endowed whey protein isolate with superior antioxidant, emulsifying, and foaming properties, making it suitable for the preparation of emulsified or gel-type foods, such as ice cream, dairy beverages, salad dressings, mousses, jellies, puddings, and gummies. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a comparison chart of the antioxidant properties of WPI and WPI-polyphenol complex in Experimental Example 1 of this invention; Figure 2 This is a comparison chart of the emulsifying properties of WPI and WPI-polyphenol complex in Experimental Example 2 of this invention; Figure 3This is a comparison chart of the foaming properties of WPI and WPI-polyphenol complex in Experimental Example 3 of this invention. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0025] The materials and reagents used in the examples and experimental cases are shown in Table 1.

[0026] Table 1 Materials and Reagents

[0027] The instruments and equipment involved in the embodiments and experimental examples are shown in Table 2.

[0028] Table 2 Instruments and Equipment

[0029] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0030] Example 1 This embodiment provides a whey protein isolate-coumaric acid complex, and the specific preparation steps are as follows: Accurately weigh 500 mg of whey protein isolate (WPI) and dissolve it in 49 mL of phosphate buffer solution (0.1 mol / L, pH 6.75), hydrating overnight at 4 °C. Add 1 mL of H2O2 solution (5 mol / L) and 0.25 g of ascorbic acid (Asc), and stir in a 25 °C water bath for 120 min. Then, add 200 mg of p-coumaric acid (pCA) to the above system and stir at room temperature for 24 h. Transfer the resulting solution to a dialysis bag (6-8 kDa) and dialyze it with distilled water at 4 °C for 72 h, changing the water every 6 h to remove unreacted polyphenols. Collect the retentate and freeze-dry for 48 h to obtain the whey protein isolate-p-coumaric acid complex (WPI-pCA).

[0031] Example 2 This embodiment provides a whey protein isolate-caffeic acid complex, and the specific preparation steps are as follows: Accurately weigh 500 mg of whey protein isolate (WPI) and dissolve it in 49 mL of phosphate buffer solution (0.1 mol / L, pH 6.75), hydrating overnight at 4 °C. Add 1 mL of H2O2 solution (5 mol / L) and 0.25 g of ascorbic acid (Asc), and stir in a 25 °C water bath for 120 min. Then, add 200 mg of caffeic acid (CA) to the above system and stir at room temperature for 24 h. Transfer the resulting solution to a dialysis bag (6-8 kDa) and dialyze it with distilled water at 4 °C for 72 h, changing the water every 6 h to remove unreacted polyphenols. Collect the retentate and freeze-dry for 48 h to obtain the whey protein isolate-caffeic acid complex (WPI-CA).

[0032] Example 3 This embodiment provides a whey protein isolate-ferulic acid complex, and the specific preparation steps are as follows: Accurately weigh 500 mg of whey protein isolate (WPI) and dissolve it in 49 mL of phosphate buffer solution (0.1 mol / L, pH 6.75), hydrating overnight at 4 °C. Add 1 mL of H2O2 solution (5 mol / L) and 0.25 g of ascorbic acid (Asc), and stir in a 25 °C water bath for 120 min. Then, add 200 mg of ferulic acid (FA) to the above system and stir at room temperature for 24 h. Transfer the resulting solution to a dialysis bag (6-8 kDa) and dialyze it with distilled water at 4 °C for 72 h, changing the water every 6 h to remove unreacted polyphenols. Collect the retentate and freeze-dry for 48 h to obtain the whey protein isolate-ferulic acid complex (WPI-FA).

[0033] Example 4 This embodiment provides a whey protein isolate-sinapic acid complex, and the specific preparation steps are as follows: Accurately weigh 500 mg of whey protein isolate (WPI) and dissolve it in 49 mL of phosphate buffer solution (0.1 mol / L, pH 6.75), hydrating overnight at 4 °C. Add 1 mL of H2O2 solution (5 mol / L) and 0.25 g of ascorbic acid (Asc), and stir in a 25 °C water bath for 120 min. Then, add 200 mg of sinapic acid (SA) to the above system and stir at room temperature for 24 h. Transfer the resulting solution to a dialysis bag (6-8 kDa) and dialyze it with distilled water at 4 °C for 72 h, changing the water every 6 h to remove unreacted polyphenols. Collect the retentate and freeze-dry for 48 h to obtain the whey protein isolate-sinapic acid complex (WPI-SA).

[0034] Experimental Example 1: Antioxidant Performance Test I. Experimental Objective The antioxidant properties (ABTS free radical scavenging ability and iron reducing power) of WPI-pCA, WPI-CA, WPI-FA and WPI-SA prepared in Examples 1 to 4 were tested, with WPI as a comparison.

[0035] II. Experimental Methods 1. Determination of ABTS free radical scavenging ability 10 mg of ABTS powder was dissolved in 2.6 mL of potassium persulfate solution (2.45 mmol / L) and incubated at room temperature in the dark for 16 h. The resulting ABTS stock solution was diluted with distilled water to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Then, 1 mL of the sample solution (0.5 mg / mL) was mixed with 3 mL of the diluted ABTS solution and incubated at room temperature in the dark for 1 h. The absorbance of the mixed solution was recorded at 734 nm using a UV-Vis spectrophotometer. The ABTS radical scavenging capacity (SA%) of the sample was calculated using the following formula:

[0036] Where AS, AB, and AR represent the absorbance of the sample with ABTS solution, the sample with no ABTS solution, and the sample solution with only ABTS solution, respectively.

[0037] 2. Determination of the reducing power of iron Add 1 mL of sample solution (1 mg / mL) to a test tube, then add 2.5 mL of phosphate buffer (0.2 mol / L, pH 6.6) and 2.5 mL of potassium ferricyanide solution (1 g / 100 mL). React at 50 °C for 30 min, cool to room temperature in an ice-water bath, add 2.5 mL of trichloroacetic acid (10 g / 100 mL), vortex to mix, and let stand for 30 s. Measure 2.5 mL of the supernatant of the above mixture and mix it with 2.5 mL of distilled water and 0.5 mL of ferric chloride solution (0.1 g / 100 mL). After reacting at 25 °C for 10 min, measure the absorbance of the solution at 700 nm using a UV-Vis spectrophotometer. The iron reducing power of the sample is calculated using the following formula:

[0038] Among them, A 测试样品 A represents the absorbance of the sample measured at 700 nm after processing according to the experimental procedure. 空白样品 The absorbance was measured at 700 nm using distilled water instead of the sample solution, following the same procedure.

[0039] All experiments were repeated three times, and data are expressed as mean ± standard deviation. Microsoft Excel and SPSS 27.0 were used for data processing and statistical analysis. Origin 2022 software was used for graphing, and Duncan's multiple range test was selected to analyze significant differences between data. Different letters indicate significant differences (P < 0.05).

[0040] III. Experimental Results The effects of polyphenols on the antioxidant properties of WPI were evaluated using ABTS radical scavenging rate and ferric reducing power. The results are as follows: Figure 1 As shown in the figure, compared with natural WPI, all WPI-polyphenol complexes exhibited significantly enhanced antioxidant activity (P<0.05), indicating that the covalent grafting of polyphenols successfully endowed the protein with superior antioxidant properties. Furthermore, significant differences in antioxidant activity were observed among the different complexes. The order of ferric reducing power was: WPI-pCA < WPI-SA / WPI-FA < WPI-CA (P<0.05); the order of ABTS radical scavenging rate was: WPI-FA < WPI-SA / WPI-pCA < WPI-CA (P<0.05). Among these, the WPI-CA complex exhibited the strongest ferric reducing power and the highest ABTS radical scavenging rate. This may be attributed to the synergistic effect of the number of phenolic hydroxyl groups and the type of substituents in the polyphenol molecule; polyhydroxy polyphenols are more likely to donate hydrogen atoms in solution to quench free radicals. In addition, the WPI-CA complex had the highest polyphenol grafting rate, which can indirectly enhance the antioxidant performance of the complex.

[0041] Experimental Example 2: Emulsification Performance Testing I. Experimental Objective The emulsifying properties of WPI-pCA, WPI-CA, WPI-FA, and WPI-SA prepared in Examples 1-4 were tested, specifically characterized by the emulsifying activity index (EAI) and the emulsifying stability index (ESI), with WPI as a comparison.

[0042] II. Experimental Methods Prepare a 1 mg / mL sample solution using phosphate buffer (0.2 mol / L, pH 7.0). Mix 2 mL of soybean oil with 6 mL of the sample solution. Disperse the sample solution for 1 min at a high-speed disperser with a shear speed of 10000 rpm. Dilute 100 μL of the bottom sample solution with 4.9 mL of SDS solution (0.1 g / 100 mL) at 0 min and 10 min. Measure the absorbance of the solution at 500 nm using a UV spectrophotometer, with the SDS solution used as a blank reference. Calculate the EAI and ESI of the sample using the following formulas:

[0043]

[0044] In the formula: C is the concentration of the protein solution (g / mL), θ is the proportion of oil used to form the emulsion (0.25), L is the optical path length of the cuvette (1 cm), D is the dilution factor (50), A0 is the absorbance at 0 minutes, A 10 It is the absorbance at 10 min.

[0045] All experiments were repeated three times, and data are expressed as mean ± standard deviation. Microsoft Excel and SPSS 27.0 were used for data processing and statistical analysis. Origin 2022 software was used for graphing, and Duncan's multiple range test was selected to analyze significant differences between data. Different letters indicate significant differences (P < 0.05).

[0046] III. Experimental Results The effects of polyphenols on the emulsifying properties of WPI were evaluated using EAI and ESI, and the results are as follows: Figure 2 As shown in the figure, compared with natural WPI, the EAI and ESI of WPI-CA and WPI-pCA complexes were significantly enhanced (P<0.05), while only the ESI of the WPI-FA complex increased, and the emulsifying properties of the WPI-SA complex remained unchanged. This phenomenon may be attributed to the differential regulation of protein structure rearrangement and interfacial adsorption behavior by the type of polyphenol substituent: hydroxyl-substituted polyphenols covalently bind to WPI, which can partially unfold the WPI structure, exposing hydrophobic regions, thereby promoting protein adsorption at the oil-water interface, thus improving interfacial stability and preventing phase separation. In contrast, methoxy-substituted polyphenols have lower binding efficiency with WPI due to the steric hindrance of the ortho-methoxy group, and their regulatory effect on protein structure is limited, thus their effect on improving emulsifying properties is weaker.

[0047] Experiment Example 3: Foaming Performance Test I. Experimental Objective The foaming properties of WPI-pCA, WPI-CA, WPI-FA, and WPI-SA prepared in Examples 1-4 were tested, specifically characterized by foaming capacity (FC) and foaming stability (FS), with WPI as a comparison.

[0048] II. Experimental Methods Prepare a 30 mL sample solution (1 mg / mL) using phosphate buffer (0.01 mol / L, pH 7.0). Homogenize at 10,000 rpm for 2 min, then quickly transfer the solution to a graduated cylinder and record the total volume. Calculate the FC (Fluid Content). Allow the solution to stand at room temperature for 30 min, and record the total volume again. Calculate the FC and FS (Fluid Content) of the sample using the following formulas:

[0049]

[0050] Where V1 is the initial solution volume (mL), V2 is the solution volume measured immediately after high-speed homogenization (mL), and V3 is the solution volume measured after standing at room temperature for 30 min (mL).

[0051] All experiments were repeated three times, and data are expressed as mean ± standard deviation. Microsoft Excel and SPSS 27.0 were used for data processing and statistical analysis. Origin 2022 software was used for graphing, and Duncan's multiple range test was selected to analyze significant differences between data. Different letters indicate significant differences (P < 0.05).

[0052] III. Experimental Results The foaming ability of proteins depends on their rapid adsorption at the air-water interface, while foam stability is closely related to the mechanical strength and resistance to deformation of the interfacial film. This experiment compared the foaming ability and foam stability of natural WPI and four WPI-polyphenol complexes with different substituent types. The results are as follows: Figure 3 As shown in the figure, compared with natural WPI, all WPI-polyphenol complexes exhibited significantly increased foaming ability and foam stability (P<0.05), indicating that the covalent bonding of polyphenols to WPI can improve its foaming performance. This may be attributed to the moderate unfolding of the protein structure, which enhances its adsorption efficiency at the air-water interface, thereby strengthening foaming performance. Significant differences in foaming performance were observed among the different complexes, in the order: WPI-CA>WPI-pCA>WPI-FA>WPI-SA, indicating that the covalent bonding of hydroxyl-substituted polyphenols to WPI can more significantly enhance the foaming performance of WPI compared to methoxy-substituted polyphenols. Among them, the WPI-CA complex exhibited the highest foaming ability and foam stability. This may be attributed to the fact that polyphenols with multiple hydroxyl substituents can induce more favorable structural rearrangements in WPI through multi-site crosslinking, exposing more hydrophobic regions and improving interfacial adsorption capacity.

[0053] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a whey protein isolate-polyphenol complex, characterized in that, Includes the following steps: S1. Weigh whey protein isolate, dissolve it in phosphate buffer solution, and fully hydrate it to obtain a protein solution; S2. Add H2O2 solution and ascorbic acid to the protein solution, mix thoroughly, add polyphenols to carry out grafting reaction, and obtain product solution; S3. Dialyze the product solution to remove unreacted polyphenols, freeze-dry the retained solution to obtain whey protein isolate-polyphenol complex.

2. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, The polyphenol is at least one of p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid.

3. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, Step S1 satisfies at least one of the following conditions: The mass-to-volume ratio of the whey protein isolate to the phosphate buffer solution is 500:45~55, expressed in mg / mL. The concentration of the phosphate buffer solution is 0.1~0.3 mol / L, and the pH value is 6~7; The conditions for complete hydration are: hydration overnight at 3~5℃.

4. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, Step S1 satisfies at least one of the following conditions: The mass-to-volume ratio of the whey protein isolate to the phosphate buffer solution is 500:49, expressed in mg / mL. The phosphate buffer solution has a concentration of 0.1 mol / L and a pH value of 6.75; The conditions for complete hydration are: hydration overnight at 4 °C.

5. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, Step S2 satisfies at least one of the following conditions: The mass-to-volume ratio of the whey protein isolate to the H2O2 solution is 500:1~3, calculated in mg / mL. The mass ratio of the whey protein isolate to the ascorbic acid is 2:1~1.5; The conditions for thorough mixing are constant temperature stirring at 24~26 ℃ for 100~150 min; The mass ratio of the whey protein isolate to the polyphenols is 5:1~3; The grafting reaction was carried out under the following conditions: stirring at room temperature for 18-24 h.

6. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, Step S2 satisfies at least one of the following conditions: The mass-to-volume ratio of the whey protein isolate to the H2O2 solution is 500:1, expressed in mg / mL. The mass ratio of the whey protein isolate to the ascorbic acid is 2:1; The conditions for thorough mixing are constant temperature stirring at 25 °C for 120 min; The mass ratio of the whey protein isolate to the polyphenols is 5:2; The grafting reaction was carried out under the following conditions: stirring at room temperature for 24 hours.

7. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, Step S3 satisfies at least one of the following conditions: The dialysis conditions are as follows: the dialysis bag retainer is 6-8 kDa; dialysis with distilled water at 3-5 °C for 48-72 h; and the water is changed every 4-8 h. The freeze-drying time is 36-48 hours.

8. The method for preparing whey protein isolate-polyphenol complex according to claim 1, characterized in that, Step S3 satisfies at least one of the following conditions: The dialysis conditions are as follows: the dialysis bag cutoff is 6-8 kDa; dialysis with distilled water at 4°C for 72 h; and the water is changed every 6 h. The freeze-drying time is 48 hours.

9. The whey protein isolate-polyphenol complex obtained by the preparation method according to any one of claims 1 to 8.

10. The use of the whey protein isolate-polyphenol complex obtained by the preparation method according to any one of claims 1 to 8 in the preparation of emulsified foods or gel-type foods.

Images