Preparation method of resveratrol loaded on whey protein amyloid fibrils based on modification of moderate electric field

Abstract

This invention discloses a method for preparing resveratrol-loaded whey protein amyloid fibers based on moderate electric field modification. The method involves pretreating whey protein using a moderate electric field non-thermal effect, forming amyloid fibers through acid-thermal treatment, and further constructing a resveratrol delivery system based on protein fibrils. The resulting whey protein fiber-resveratrol complex exhibits excellent loading performance, antioxidant activity, and bioavailability, providing an effective carrier construction strategy for the efficient delivery and stabilization of hydrophobic and environmentally sensitive active molecules. This invention reveals the regulatory mechanism of the moderate-intensity electric field non-thermal effect in the protein fibrillation process, providing new technical ideas and theoretical references for the precise design of protein fiber structures and the development of functional protein-based delivery carriers, and providing technical support for the green preparation of protein fibrils and their in-depth development in functional foods.

Description

Technical Field

[0001] This invention belongs to the field of food and biomedicine technology, specifically relating to a method for preparing resveratrol-loaded whey protein amyloid fibers based on moderate electric field modification. Background Technology

[0002] Resveratrol is a polyphenolic compound naturally found in plants such as grapes and Japanese knotweed. It possesses various physiological activities, including antioxidant, anti-inflammatory, and cardiovascular protective effects, and shows great promise for application in the development of functional foods and beverages. However, resveratrol has extremely poor water solubility and is sensitive to environmental factors such as light, heat, and oxygen. It also exhibits poor stability and is easily degraded and inactivated during processing and storage, resulting in very low bioavailability and severely limiting its application in practical food systems.

[0003] Constructing efficient delivery systems is crucial for improving the bioavailability of bioactive ingredients. Whey protein, as a high-quality food-grade protein raw material, possesses excellent biocompatibility and ligand binding capacity. Studies have shown that whey protein can self-assemble under specific conditions (low pH, high temperature) to form amyloid fibrous structures with high aspect ratio and high surface activity. These protein fibers not only exhibit excellent emulsifying and gelling properties, but their unique hydrophobic regions and highly ordered structure also endow them with great potential as carriers of hydrophobic bioactive molecules. The formation pathway and morphological structure of protein fibrils are closely related to their functional properties, especially when they serve as carriers of bioactive substances, where structural differences directly affect their delivery efficiency. Therefore, precisely controlling protein assembly behavior and fiber structure through emerging technologies has become a core challenge and research frontier in this field.

[0004] Among various regulatory strategies, physical field processing technology is favored due to its green, efficient, and non-exogenous additive characteristics. Ohmic heating, as a processing method that utilizes an electric field to generate a thermal effect within a material, can be deconstructed into two parts: thermal effect and non-thermal effect (i.e., moderate electric field effect). However, existing research mainly focuses on the impact of its thermal effect on protein denaturation and fibrillation, while the specific mechanism of the single moderate electric field effect in driving protein unfolding, molecular orientation, and final fibrillation remains poorly elucidated. How to efficiently prepare structurally controllable whey protein fibers under mild conditions through process innovation and use them for efficient resveratrol loading remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as low resveratrol loading, a method for preparing resveratrol-loaded whey protein amyloid fibers based on moderate electric field modification is provided. The prepared complex exhibits high encapsulation efficiency and antioxidant activity, thus improving bioavailability.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing resveratrol-loaded whey protein amyloid fibers modified by a moderate electric field, the method comprising the following steps:

[0008] (1) Disperse whey protein in phosphate buffer, stir to obtain whey protein solution, subject the solution to electric field treatment, and cool after electric field treatment;

[0009] (2) Adjust the pH of the treated protein solution to acidic, heat it to carry out the fibrosis reaction, cool it, and obtain whey protein fiber solution;

[0010] (3) Adjust the pH of the obtained whey protein fiber solution to acidic beverage conditions, add resveratrol solution, stir in the dark, and obtain whey protein fiber-resveratrol complex.

[0011] Preferably, in step (1), the concentration of phosphate buffer is 1-100 mM, the pH value is 6.8-7.2, the stirring time is 1-3 h, and the mass fraction of whey protein is 0.5%-3%.

[0012] Preferably, in step (1), the electric field treatment uses sinusoidal alternating current with a frequency of 30-100 Hz.

[0013] Preferably, in step (1), the electric field strength of the electric field treatment is 1-15 V / cm, and the treatment time is 10-60 min.

[0014] Preferably, in step (1), the electric field treatment process is accompanied by an ice bath and magnetic stirring, and the treatment temperature is controlled to be below 35°C.

[0015] Preferably, in step (2), the pH is adjusted to 1.8-2.5, the heating temperature is 80-90℃, and the heating time is 4-24h.

[0016] Preferably, in step (2), the material is cooled to room temperature using an ice bath after heating.

[0017] Preferably, in step (3), the pH of the protein fiber solution is adjusted to 3.0-3.5.

[0018] Preferably, in step (3), resveratrol is added after being dissolved in ethanol, and the final concentration of resveratrol in the complex solution is 0.1-0.6 mg / mL, and the final volume fraction of ethanol is less than 2%.

[0019] Preferably, in step (3), the stirring time in the dark is 1-4 h and the stirring rate is 100-500 rpm.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method for preparing modified whey protein amyloid fibers loaded with resveratrol provided by this invention is simple and controllable, requires no addition of organic solvents or chemical cross-linking agents, is green and safe, has low energy consumption, and is suitable for industrial-scale production.

[0022] The whey protein fiber-resveratrol complex obtained in this invention has excellent loading capacity, antioxidant activity and bioavailability, providing an effective carrier construction strategy for the efficient delivery and stabilization protection of hydrophobic and environmentally sensitive active molecules.

[0023] This invention reveals the regulatory mechanism of the non-thermal effect of medium-intensity electric fields in the protein fibrillation process, providing new technical ideas and theoretical references for the precise design of protein fiber structures and the development of functional protein-based delivery carriers, and providing technical support for the green preparation of protein fibrils and their in-depth development in functional foods. Attached Figure Description

[0024] Figure 1 This is a summary figure;

[0025] Figure 2 This is a comparison chart of the encapsulation efficiency and loading of the complexes prepared in Examples 1-4 of this invention;

[0026] Figure 3 Comparison of particle size and polydispersity index of the complexes prepared in Examples 1-4 of this invention;

[0027] Figure 4 This is a comparison diagram of the antioxidant activities of the complexes prepared in Examples 1-4 of this invention;

[0028] Figure 5 This is a comparison chart of the bioavailability of the complexes prepared in Examples 1-4 of this invention. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the present invention. It should be noted that, unless otherwise specified, all chemical reagents involved in the present invention are purchased through commercial channels.

[0030] Example 1

[0031] (1) Accurately weigh 1 g of whey protein isolate, dissolve it in 100 mL of phosphate buffer (50 mM, pH 7.0), and stir for 2 h under a magnetic stirrer to obtain a whey protein isolate solution with a mass fraction of 1%.

[0032] (2) The pH of the protein solution was adjusted to 3.2 ± 0.01 using 2 M NaOH or 2 M HCl to simulate beverage conditions. Resveratrol was dissolved in ethanol to obtain a 25 mg / mL resveratrol solution. The stock resveratrol solution was slowly added to the sample solution to make the final concentration of resveratrol in the sample solution 0.5 mg / mL and the final ethanol content fixed below 2% (v / v). The mixture was magnetically stirred for 2 h under light-protected conditions to obtain the complex of whey protein isolate and resveratrol.

[0033] Example 2

[0034] (1) Accurately weigh 1 g of whey protein isolate, dissolve it in 100 mL of phosphate buffer (50 mM, pH 7.0), and stir for 2 h with a magnetic stirrer to obtain a 1% whey protein isolate solution. Take 80 mL of the whey protein isolate solution and place it in a quartz reaction cell, and treat it under an electric field strength of 6 V / cm for 30 min. During the treatment, the sample is kept under ice bath conditions with slow magnetic stirring to ensure the system is homogeneous, and K-type thermocouples are used for real-time monitoring to ensure that the treatment temperature is below 35℃;

[0035] (2) The pH of the protein solution was adjusted to 3.2 ± 0.01 using 2 M NaOH or 2 M HCl to simulate beverage conditions. Resveratrol was dissolved in ethanol to obtain a 25 mg / mL resveratrol solution. The stock resveratrol solution was slowly added to the sample solution to make the final concentration of resveratrol in the sample solution 0.5 mg / mL and the final ethanol content fixed below 2% (v / v). The mixture was magnetically stirred for 2 h under light-protected conditions to obtain a complex of whey protein isolate and resveratrol pretreated with a moderate electric field.

[0036] Example 3

[0037] (1) Accurately weigh 1 g of whey protein isolate, dissolve it in 100 mL of phosphate buffer (50 mM, pH 7.0), and stir for 2 h under a magnetic stirrer to obtain a whey protein isolate solution with a mass fraction of 1%.

[0038] (2) Adjust the pH of the protein solution to 2.0 ± 0.02 with 6M HCl, heat in a water bath at 85℃ with magnetic stirring for 16 h, and then immediately cool to room temperature with an ice bath to obtain whey protein cellulose solution.

[0039] (3) The pH of the protein fiber solution was adjusted to 3.2 ± 0.01 using 2 M NaOH or 2 M HCl to simulate beverage conditions. Resveratrol was dissolved in ethanol to obtain a 25 mg / mL resveratrol solution. The stock resveratrol solution was slowly added to the sample solution to make the final concentration of resveratrol in the sample solution 0.5 mg / mL and the final ethanol content fixed below 2% (v / v). The mixture was magnetically stirred for 2 h under light-protected conditions to obtain a complex of whey-separated protein fiber and resveratrol.

[0040] Example 4

[0041] (1) Accurately weigh 1 g of whey protein isolate, dissolve it in 100 mL of phosphate buffer (50 mM, pH 7.0), and stir for 2 h with a magnetic stirrer to obtain a 1% whey protein isolate solution. Take 80 mL of the whey protein isolate solution and place it in a quartz reaction cell, and treat it under an electric field strength of 6 V / cm for 30 min. During the treatment, the sample is kept under ice bath conditions with slow magnetic stirring to ensure the system is homogeneous, and K-type thermocouples are used for real-time monitoring to ensure that the treatment temperature is below 35℃;

[0042] (2) The pH of the WPI solution after electric field treatment was adjusted to 2.0 ± 0.02 with 6M HCl, heated in a water bath at 85℃ with magnetic stirring for 16 h, and then immediately cooled to room temperature with an ice bath to obtain whey protein cellulose solution.

[0043] (3) The pH of the protein fiber solution was adjusted to 3.2 ± 0.01 using 2 M NaOH or 2 M HCl to simulate beverage conditions. Resveratrol was dissolved in ethanol to obtain a 25 mg / mL resveratrol solution. The stock resveratrol solution was slowly added to the sample solution to make the final concentration of resveratrol in the sample solution 0.5 mg / mL and the final ethanol content fixed below 2% (v / v). The mixture was magnetically stirred for 2 h under light-protected conditions to obtain a complex of whey protein fiber and resveratrol pretreated with a moderate electric field.

[0044] The complexes prepared in Examples 1-4 above were characterized.

[0045] 1. Encapsulation efficiency and loading of the complex

[0046] Methods: Unencapsulated resveratrol was removed by centrifugation (5000 g, 10 min). The resulting supernatant was mixed with ethanol to extract resveratrol, and then centrifuged again (5000 g, 10 min) to obtain free resveratrol. The absorbance was then measured at 306 nm using a microplate reader. A standard curve for resveratrol concentration was established (y = 0.0739x + 0.1275, R0). 2=0.9993) Calculate the content of resveratrol in the solution, and calculate the encapsulation efficiency and loading of resveratrol according to the following formulas.

[0047] Encapsulation efficiency (%) = (Total resveratrol - Free resveratrol content) / Total resveratrol

[0048] Loading capacity (μg / mg) = (Resveratrol loading capacity - Total content of the complex) / Total amount of resveratrol

[0049] Results explanation: Figure 1 Regarding the encapsulation efficiency and loading of the composites prepared in Examples 1-4 of this invention, the loading performance of Examples 3 and 4 is significantly better than that of Examples 1 and 2. This may be due, on the one hand, to the fact that higher hydrophobicity is more conducive to the binding with resveratrol, and on the other hand, its higher aspect ratio provides a larger contact area, improving the interaction with resveratrol, thereby achieving a large amount of encapsulation of hydrophobic resveratrol molecules. In addition, Example 4 exhibits a higher encapsulation efficiency and loading than Example 3, indicating that moderate electric field pretreatment imparts higher surface hydrophobicity to the fibers.

[0050] 2. Particle size and polydispersity index of the complex

[0051] Methods: The particle size of the complex was determined using a particle size and potential analyzer. Before measurement, the sample was diluted to 1 mg / mL with PBS at pH 3.2. The refractive indices of the particles and the dispersant were set to 1.460 and 1.330, respectively. The average of three parallel measurements for each sample was taken.

[0052] Results explanation: Figure 2 The particle size and polydispersity index (PDI) of the complexes prepared in Examples 1-4 of this invention are shown below. The particle sizes of Examples 1 and 2 are 623.57 ± 2.65 nm and 722.4 ± 25.48 nm, respectively, with relatively high PDI values. This indicates that the addition of resveratrol triggered the formation of large aggregates from the entanglement of whey proteins and the formation of aggregates from the resveratrol complex. Compared to Example 1, the particle size of Example 2 increased significantly. This is because the moderate electric field pretreatment resulted in the moderate unfolding of the proteins, exposing more hydrophobic regions and thus promoting the formation of larger copolymers. However, the particle size of Example 3 increased to 1161 ± 72.17 nm, and the PDI decreased to 0.317 ± 0.074, indicating that the amyloid fibers were orderly bound to resveratrol, effectively inhibiting random aggregation. The particle size of Example 4 further increased to 1355 ± 27.84 nm. This phenomenon suggests that the moderate electric field pretreatment before acid heat treatment may further alter the protein conformation, promote fiber elongation, expose more binding sites on the fibers, and thus form a larger but more uniformly distributed resveratrol-loaded complex.

[0053] 3. Antioxidant activity of the complex

[0054] Methods: DPPH solution (2 mM, dissolved in ethanol) was mixed with the sample at a 1:1 ratio and reacted in the dark for 30 min. After the reaction, the absorbance of the mixture at 517 nm was measured using a UV spectrophotometer. The DPPH radical scavenging rate of the sample was calculated using a formula. Here, A0 and A are the absorbances of the DPPH solution and the sample solution after the reaction with DPPH, respectively, at 517 nm. ABTS solution (7 mM) was mixed with potassium persulfate solution (4.9 mM) at a 1:1 ratio and reacted in the dark for 12 h to generate ABTS radicals. The solution was diluted several times until its absorbance at 734 nm was 0.70 (±0.02) to obtain the ABTS working solution. 140 μL of sample was mixed with 4 mL of ABTS working solution and reacted in the dark for 6 min. After the reaction, the absorbance of the mixture at 734 nm was measured using a UV spectrophotometer. The ABTS radical scavenging rate of the sample was calculated using a formula. Wherein, A1 and A2 are the absorbances of the ABTS solution and the sample solution after reacting with ABTS at 734 nm, respectively.

[0055] DPPH radical scavenging rate (%) = (A0 - A) / A0

[0056] ABTS radical scavenging rate (%) = (A1-A2) / A1

[0057] Results explanation: Figure 4 The figures show the DPPH and ABTS radical scavenging rates of the complexes prepared in Examples 1-4 of this invention. As can be seen from the figures, the DPPH radical scavenging rates of Examples 3-4 are higher than those of Examples 1-2. This may be due to the higher specific surface area and ordered structure of the fiber carriers, which increases the resveratrol loading. Notably, the scavenging rate of Example 4 is higher than that of Example 3, indicating that the moderate electric field pretreatment, by increasing the orderliness of the fibers and reducing disordered entanglement between carriers, is more conducive to the penetration of DPPH radicals into the complex and their reaction with the embedded resveratrol. Consistent with the trend of the DPPH results, Examples 3 and 4 show the highest ABTS radical scavenging rates, further demonstrating the enhanced antioxidant activity of resveratrol after binding with proteoglycan fibers.

[0058] 4. Bioavailability of the complex

[0059] Methods: Simulated gastric juice (SGF) was prepared by mixing 3.2 mg / mL pepsin and 2.0 mg / mL sodium chloride. The sample was mixed with SGF at a 1:1 ratio, and the pH was adjusted to 2.0. The mixture was then subjected to simulated gastric digestion for 2 h with continuous stirring at 37 °C. The digested sample was then mixed with simulated intestinal fluid (SIF: containing 10 mg / mL trypsin, 36.67 mg / mL CaCl2, 218.7 mg / mL NaCl, and 20 mg / mL ox bile salt, dissolved in pH 7.0 phosphate buffer) at a 1:1 ratio. The mixture was then subjected to continuous stirring at 37 °C for 2 h, and the digestive fluid was collected.

[0060] Results explanation: Figure 5 The bioavailability of the complexes prepared in Examples 1-4 of this invention is shown. The bioavailability of Example 2 is higher than that of Example 1. The electric field causes the protein structure to unfold moderately, increasing its flexibility. Proteins in this state are more easily hydrolyzed by digestive enzymes after entering the small intestine. Rapid degradation of the protein matrix promotes the release of resveratrol, allowing it to better integrate into the mixed micelles formed by bile salts, thereby improving intestinal absorption potential. The bioavailability of Examples 3-4 is better than that of Examples 1-2. Amyloid protein fibers have a large specific surface area, which helps the active substances maintain a highly dispersed state in digestive juices and promotes the transport of mixed micelles to small intestinal epithelial cells. The bioavailability of Example 4 is higher than that of Example 3, indicating that moderate electric field pretreatment promotes the formation of a more uniform and regular protein fiber network. This ordered nanostructure exhibits a fairly stable protective effect in the gastrointestinal environment. The post-digestion product morphology also facilitates transmembrane absorption of resveratrol encapsulated within it by intestinal cells.

Claims

1. A method for preparing resveratrol-loaded whey protein amyloid fibrous material based on moderate electric field modification, characterized in that, The steps of the method are as follows: (1) Disperse whey protein in phosphate buffer, stir to obtain whey protein solution, pre-treat the solution with a moderate electric field, and then cool it; (2) Adjust the pH of the treated protein solution to acidic, heat it to carry out the fibrosis reaction, cool it, and obtain whey protein fiber solution; (3) Adjust the pH of the obtained whey protein fiber solution to acidic beverage conditions, add resveratrol solution, stir in the dark, and obtain whey protein fiber-resveratrol complex.

2. The method for preparing the whey protein fiber-resveratrol complex according to claim 1, characterized in that: In step (1), the concentration of phosphate buffer is 1-100 mM, the pH value is 6.8-7.2, the stirring time is 1-3 h, and the mass fraction of whey protein is 0.5%-3%.

3. The method for preparing whey protein fiber according to claim 1, characterized in that: In step (1), the electric field processing uses sinusoidal alternating current with a frequency of 30-100 Hz.

4. The method according to claim 1, characterized in that: The electric field strength for step (1) is 1-15 V / cm, and the treatment time is 10-60 min.

5. The method for preparing the whey protein fiber-resveratrol complex according to claim 1, characterized in that: In step (1), the electric field treatment process is accompanied by an ice bath and magnetic stirring, and the treatment temperature is controlled to be below 35°C.

6. The method for preparing whey protein fiber according to claim 1, characterized in that: In step (2), the pH is adjusted to 1.8-2.5, the heating temperature is 80-90℃, and the heating time is 4-24 h.

7. The method for preparing whey protein fiber according to claim 1, characterized in that: After heating in step (2), the mixture is cooled to room temperature using an ice bath.

8. The method for preparing the whey protein fiber-resveratrol complex according to claim 1, characterized in that: In step (3), adjust the pH of the protein fiber solution to 3.0-3.

5.

9. The preparation method according to claim 1, characterized in that: In step (3), resveratrol is added after being dissolved in ethanol. The final concentration of resveratrol in the complex solution is 0.1-0.6 mg / mL, and the final volume fraction of ethanol is less than 2%.

10. The method for preparing the whey protein fiber-resveratrol complex according to claim 1, characterized in that: In step (3), the stirring time in the dark is 1-4 h, and the stirring speed is 100-500 rpm.

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