Method for enhancing oxidation resistance and emulsibility of whey protein isolate through cooperation of enzymolysis and polyphenol modification
By modifying whey protein isolate through enzymatic hydrolysis and polyphenol combination, the problems of insufficient antioxidant and emulsifying properties in existing technologies have been solved, achieving better modification effects and safety, and broadening its application in the food industry.
Patent Information
- Application Number
- CN202511303592.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-12
AI Technical Summary
Existing technologies are insufficient to effectively improve the antioxidant and emulsifying properties of whey protein isolate. The use of enzymatic hydrolysis or polyphenol-based methods alone has failed to fully leverage the synergistic effect, resulting in unsatisfactory modification results.
By enzymatically hydrolyzing whey protein isolate and then combining it with polyphenols, enzymatically hydrolyzed and polyphenol-modified whey protein isolate was prepared. This process included optimizing the enzymatic hydrolysis conditions and mixing with polyphenols, followed by dialysis and vacuum freeze-drying.
This significantly enhances the antioxidant and emulsifying properties of whey protein isolate, increasing its application potential in the food industry, while ensuring the safety and simplicity of the method.
Smart Images
Figure BDA0005593640080000051 
Figure BDA0005593640080000062 
Figure HDA0005593640090000011
Abstract
Description
Technical Field
[0001] This invention belongs to the field of food processing technology, specifically relating to a method for enhancing the antioxidant and emulsifying properties of whey protein isolate through enzymatic hydrolysis and polyphenol modification. Background Technology
[0002] Whey protein (WP) is a byproduct of cheese and casein production, accounting for 18–20% of total milk protein, and possesses a favorable amino acid distribution and functional properties. Whey protein isolate (WPI) is widely used and researched as a commercial product of WP due to its high protein purity (≥90%). However, the limitations of natural WPI in terms of antioxidant and emulsifying properties restrict its application in food systems.
[0003] Currently, researchers have tried various methods to improve the functional properties of proteins. For example, physical modification, such as ultrasonic treatment and high-pressure homogenization, can change the structure and function of proteins to some extent, but the effect is limited. Chemical modification methods, such as cross-linking or modification with chemical reagents, may introduce safety hazards, and the reaction conditions are difficult to control precisely, which can easily lead to over-modification of proteins and affect their nutritional value.
[0004] Enzymatic hydrolysis is a common method for protein modification. Through the action of enzymes, protein molecules are hydrolyzed into smaller peptides, which can improve the protein's solubility, emulsification, and other functional properties. Meanwhile, studies have found that polyphenols possess strong antioxidant activity; combining polyphenols with proteins can form complexes with even stronger antioxidant properties. However, existing methods often use either enzymatic hydrolysis or polyphenol combination alone, failing to fully utilize the synergistic effect of both, resulting in unsatisfactory modification results. Summary of the Invention
[0005] The purpose of this invention is to establish a method for improving the antioxidant and emulsifying properties of whey protein isolate, thereby broadening its application in the food industry.
[0006] This invention utilizes enzymatic hydrolysis to fully unfold the whey protein structure, and then leverages polyphenols to fully bind with it, thereby enhancing its antioxidant and emulsifying properties.
[0007] The technical solution of the present invention is as follows:
[0008] Step 1: Enzymatically hydrolyze the whey protein isolate to obtain the corresponding protease hydrolysate.
[0009] Step 2: Prepare a solution by mixing the protease hydrolysate and polyphenols at room temperature according to the specified ratio.
[0010] Step 3: After thorough dialyzing of the reaction solution, perform vacuum freeze-drying to obtain the modified whey protein isolate product.
[0011] In this invention, the whey protein isolate (protein content ≥90%) is composed of epigallocatechin gallate (EGCG), a polyphenol.
[0012] In this invention, the whey isolate enzymatic hydrolysis conditions are as follows: a mother liquor of 10 mg / mL is prepared using sodium carbonate buffer (pH 9), and an alkaline protease with a substrate concentration of 15% (w / v) is added. The whey isolate enzymatic hydrolysate is obtained by enzymatic hydrolysis at 55°C for 90 min.
[0013] In step two, whey protein isolate and polyphenols are mixed thoroughly at a ratio of 5:1 at room temperature. A product with good antioxidant and emulsifying properties was prepared by the above-mentioned method based on enzymatic hydrolysis and synergistic polyphenol modification of whey protein isolate.
[0014] The beneficial effects of this invention are:
[0015] (1) Synergistic effect: By first modifying the structure of whey protein isolate through enzymatic hydrolysis to expose more active sites, and then combining it with polyphenols, the synergistic effect between the two can be significantly enhanced. Compared with the method of using enzymatic hydrolysis or polyphenol combination alone, the modified whey protein isolate prepared by this invention has more significant improvements in antioxidant and emulsifying properties.
[0016] (2) High safety: The entire process of this invention does not introduce toxic or harmful chemical reagents, which meets the requirements of modern food engineering for green and safe food raw materials.
[0017] (3) Simple process: The method of the present invention is simple, the reaction conditions are mild and easy to promote and apply in industrial production. Attached Figure Description
[0018] Figure 1 These are scanning electron microscope images of each example.
[0019] Figure 2 The changes in the secondary structure of each instance
[0020] Figure 3 The surface hydrophobicity of each example
[0021] Figure 4 These are the fluorescence spectra of each example.
[0022] Figure 5 This is a graph showing the ABTS clearing capabilities of each instance.
[0023] Figure 6 This is a graph showing the DPPH removal capabilities of each instance.
[0024] Figure 7 The emulsion stability index and activity index of the emulsions prepared in each example are shown.
[0025] Figure 8 The particle size of the emulsions prepared in each example Detailed Implementation
[0026] The technical solution of the present invention will be further described below with reference to the embodiments of the present invention. The described embodiments are used to explain the present invention and do not limit the present invention.
[0027] Example 1
[0028] Dissolve whey protein isolate in phosphate buffer solution to prepare a stock solution with a concentration of 10 mg / mL for later use.
[0029] Example 2
[0030] Preparation of whey protein isolate hydrolysate: Dissolve whey protein isolate in 0.1M sodium carbonate buffer (pH 9) to prepare a 10 mg / mL stock solution, add 15% (w / v) alkaline protease substrate, and enzymatically hydrolyze at 55℃ for 90 min to obtain whey protein isolate hydrolysate.
[0031] Example 3
[0032] Preparation of whey protein isolate and EGCG complex: Prepare a 5 mg / mL stock solution of whey protein isolate and store at 4°C overnight for full hydration. Prepare a 1 mg / mL stock solution of EGCG (prepare fresh before use). Mix the protein solution and EGCG solution thoroughly (stir for 2 hours).
[0033] Example 4
[0034] The preparation of whey protein hydrolysate is the same as in Example 2.
[0035] Preparation of the complex: The steps are the same as those in Example 3, except that the whey protein hydrolysate is separated into a 5 mg / mL stock solution.
[0036] The solution after the reaction was dialyzed to remove unreacted small molecules and impurities, and then the complex was freeze-dried under vacuum to obtain WPI modified by enzymatic hydrolysis and EGCG.
[0037] In this invention, the method for determining the structural changes of whey protein isolate is as follows:
[0038] (1) Scanning electron microscope image
[0039] The surface of the freeze-dried sample was coated with gold by sputtering and observed using a tungsten filament scanning electron microscope.
[0040] (2) Circular dichroism spectroscopy analysis
[0041] Changes in the secondary structure of proteins in the samples were determined using a circular dichroism spectroscopy (CDS). A 0.2 mg / mL sample solution was prepared, and measurements were taken at 298 K and 180–260 nm.
[0042] (3) Determination of surface hydrophobicity
[0043] The surface hydrophobicity of proteins was determined using the fluorescent probe ANS. Protein solutions were diluted with ultrapure water to five concentration gradients (0.05, 0.1, 0.15, 0.2, and 0.25 mg / mL). The relative fluorescence intensity of the sample was measured after adding 0.025 mL of ANS solution (8 mM) to 4 mL of sample solution. The excitation and emission wavelengths were 390 nm and 470 nm, respectively. The initial slope of fluorescence intensity versus protein concentration represents the surface hydrophobicity of the protein. Endogenous fluorescence detection of tryptophan was performed. The excitation wavelength was set to 280 nm, and the emission spectrum in the 300-400 nm range was scanned.
[0044] (3) Fluorescence spectrum The sample was scanned at 298 K using a fluorescence spectrophotometer. The excitation wavelength was set to 280 nm, the emission spectrum range of the excitation and emission monochromators was 300–500 nm, and the slit width was 5 nm.
[0045] In this invention, the methods for determining the antioxidant and emulsifying properties of each sample are as follows:
[0046] ABTS radical scavenging activity: An ABTS (7 mM) stock solution containing 2.45 mM potassium persulfate was incubated overnight in the dark and diluted with ethanol before use to produce a working solution with an absorbance of 0.70 ± 0.02 at 734 nm. Subsequently, 0.1 ml of sample (1 mg / ml) was mixed with 2 ml of diluted ABTS stock solution, and the mixture was reacted at room temperature in the dark for 10 min before measuring the absorbance at 734 nm. The ABTS scavenging rate was calculated using the following formula:
[0047]
[0048] This is A b and A s These represent the absorbance values of the blank and the sample, respectively.
[0049] DPPH free radical scavenging activity: 2 mL of sample solution was mixed with 2 mL of 0.015 mM DPPH solution (dissolved in methanol) and incubated for 30 min. 2 mL of sample solution was mixed with 2 mL of methanol solution as a blank group, and 2 mL of DPPH solution was mixed with 2 mL of distilled water as a control group. The absorbance of different components at 517 nm was measured, and the DPPH scavenging rate was calculated using the following formula:
[0050] Here, As, Ab, and Ac represent the absorbance values of the sample, blank, and control group, respectively.
[0051] Emulsion preparation: 13.5 mL of the complex (10 mg / mL protein) was mixed with 1.5 mL of soybean oil, sheared at 13000 rpm for 3 min to prepare a crude emulsion, and sonicated at 400 W for 6 min. 5 mL of 9% SDS was added to the emulsion, and the absorbance at 500 nm was recorded. The emulsion stability index (ESI, %) and emulsifying activity index (EAI, m) of the protein complex solution were calculated. 2 / g):
[0052] ESI (%) = A 30 ×100 / A0
[0053] A0 and A 30 The absorbance was measured at 0 and 30 minutes.
[0054]
[0055] Where N is the dilution factor (100), φ is 0.1, C is the initial protein concentration (g / mL), and A... 500 The absorbance of the sample at 500 nm at 0 min is denoted as 0.
[0056] In this invention, the emulsion particle size was determined as follows:
[0057] The droplet size and particle distribution of the fresh emulsion were measured using a laser diffraction particle size distribution analyzer. All samples were diluted prior to analysis and then stirred for 2 minutes to ensure complete dispersion. All measurements were performed at room temperature.
[0058] Microstructural changes of whey protein isolate, such as Figure 1 As shown, whey protein isolate has a globular structure with slight surface depressions, and after hydrolysis, it exhibits a layered structure with a smooth surface. After the addition of polyphenols, the surface of the complex becomes rough and small particles adhere to it.
[0059] Changes in the secondary and tertiary structure of proteins, such as Figure 2 , 3 As shown in Figure 4, enzymatic hydrolysis disrupts the secondary structure of whey protein isolate from an ordered to a disordered state. The addition of polyphenols leads to a rearrangement of the secondary structure of both whey protein isolate and its hydrolysate. After hydrolysis, whey protein isolate becomes more hydrophilic, and the addition of polyphenols increases surface hydrophobicity, i.e., the number of hydrophobic groups increases. The hydrolysis-induced structural depolymerization of the protein enhances the polarity of the surrounding tryptophan and tyrosine residues, reducing hydrophobicity and increasing hydrophilicity, consistent with the surface hydrophobicity analysis. The addition of polyphenols exhibits static fluorescence quenching, inducing an increase in protein surface hydrophobicity.
[0060] pass Figure 5 , 6 It was found that the ABTS and DPPH scavenging abilities of enzymatically hydrolyzed and polyphenol-modified whey protein isolate reached 93.031±0.756% and 72.006±0.690%, respectively. The antioxidant capacity was higher than that of untreated whey protein isolate, whey protein isolate that was only enzymatically hydrolyzed, or whey protein isolate that was only bound to polyphenols.
[0061] The emulsion stability index, activity index, and particle size of the emulsions prepared in each example are as follows: Figure 7 , 8 As shown, enzymatic hydrolysis combined with polyphenol modification results in whey protein isolate with a smaller average particle size and better emulsifying properties. The emulsion activity and stability of the emulsion were 25.422 ± 0.126 m. 2 / g, 95.370±0.252%.
Claims
1. A method for enhancing the antioxidant and emulsifying properties of whey protein isolate through enzymatic hydrolysis and polyphenol modification, characterized in that, Includes the following steps: Step 1: Enzymatically hydrolyze the whey protein isolate to obtain the corresponding protease hydrolysate; Step 2: Prepare a solution by mixing the protease hydrolysate and polyphenols at room temperature according to the specified ratio; Step 3: After thorough dialyzing of the reaction solution, perform vacuum freeze-drying to obtain the modified whey protein isolate product.
2. The method according to claim 1, characterized in that, In step one, the concentration of whey protein isolate (protein content ≥90%) is 10 mg / mL, and the enzymatic hydrolysis conditions are: alkaline protease with a substrate concentration of 15% (w / v), and enzymatic hydrolysis at 55℃ for 90 min.
3. The method according to claim 1, characterized in that, In step two, the polyphenol is epigallocatechin gallate (EGCG), and the mixing ratio of protein to polyphenol is 5:
1.
4. The method according to claim 1, characterized in that, Step 3: Select a 500Da dialysis bag and perform dialysis for 24 hours.
5. A method for enhancing the antioxidant and emulsifying properties of whey protein isolate through enzymatic hydrolysis and polyphenol modification, implemented by the method described in any one of claims 1 to 4.