A tannic acid nanoparticle with whey protein isolate as a carrier, and a preparation method and application thereof

Abstract

The application discloses a kind of ellagic acid nanoparticles with whey protein isolate as carrier and its preparation method and application, belong to food processing technical field.The preparation method includes the following steps: ellagic acid is added to alkaline whey protein isolate solution, after being mixed uniformly, adjust pH to neutral;After ultrasonic auxiliary treatment, the ellagic acid nanoparticles are obtained.The application combines pH driving method and ultrasonic auxiliary treatment method, and combines the characteristics of whey protein isolate WPI itself, prepares WPI / EA nanoparticles with high EA encapsulation efficiency and strong stability, realizes the effect of improving EA solubility, stability and bioavailability, provides new preparation approach and theoretical support for EA preparation and functional activity application.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to ellagic acid nanoparticles with whey protein isolate as a carrier, their preparation method and application. Background Technology

[0002] Ellagic acid (EA) is a natural polyphenol dilactone widely found in plants such as blueberries, pomegranates, and green tea. It possesses hypoglycemic, lipid-lowering, anti-inflammatory, antioxidant, and anti-aging effects. However, its poor water and lipid solubility, along with low stability in gastrointestinal fluids, limit its bioavailability and thus restrict its functional efficacy. Previous studies have used soy protein isolate (SPI) to encapsulate EA. This technique disperses EA in an SPI solution using pH to construct nanocarriers. However, the encapsulation amount and efficiency were below expectations. The core reasons are twofold: firstly, the structure of the chosen carrier protein is relevant; different proteins have different conformations and different binding interactions with small molecules, resulting in varying encapsulation effects. Secondly, different treatments can alter the conformation of the carrier protein. Additionally, there are reports of using antisolvent precipitation to construct nanocarriers for other small molecules, but the calculation methods for encapsulation amount are flawed, leading to overestimation of the calculated encapsulation effect. Therefore, it is necessary to provide a method that has high encapsulation efficiency, strong stability, standardized quantitative calculation, and can improve the bioavailability of ellagic acid. Summary of the Invention

[0003] The purpose of this invention is to provide ellagic acid nanoparticles with whey protein isolate as a carrier, their preparation method, and applications, thereby addressing the problems existing in the prior art. This invention combines a pH-driven method and an ultrasound-assisted treatment method, taking advantage of the inherent properties of whey protein isolate (WPI), to prepare WPI / EA nanoparticles with high EA encapsulation efficiency and strong stability. This achieves improved EA solubility, stability, and bioavailability, providing a new preparation route and theoretical support for EA formulation development and functional applications.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing ellagic acid nanoparticles using whey protein isolate as a carrier, comprising the following steps: Ellagic acid was added to an alkaline whey protein isolate solution, mixed thoroughly, and the pH was adjusted to neutral. After ultrasonic-assisted treatment, the ellagic acid nanoparticles were obtained.

[0005] Furthermore, the alkalinity refers to pH=12.

[0006] Furthermore, the whey protein isolate solution has a mass-volume percentage concentration of 1.5%-3%.

[0007] Furthermore, the concentration of ellagic acid in the mixed solution is 0.025-0.3 mg / mL.

[0008] Furthermore, the mixing speed is 500-800 rpm and the mixing time is 1.5-2.5h.

[0009] Furthermore, the power of the ultrasound-assisted treatment is 50W, with a working time of 5 seconds and a stop time of 5 seconds, for a total duration of 15-25 minutes.

[0010] The present invention also provides ellagic acid nanoparticles prepared by the above preparation method.

[0011] This invention also provides the application of the above-mentioned ellagic acid nanoparticles in the preparation of functional products.

[0012] Optionally, the functional products include foods, pharmaceuticals, or nutritional supplements that help with antioxidation.

[0013] The present invention also provides a product that helps with anti-oxidation, the product comprising the above-mentioned ellagic acid nanoparticles.

[0014] The present invention discloses the following technical effects: This invention employs a pH-driven principle, adjusting the pH of the whey protein isolate (WPI) solution to alkaline. Utilizing the excellent aqueous dispersibility of ellagic acid (EA) in an alkaline environment, and the molten globular structure of WPI in an alkaline environment, ellagic acid is dispersed within the protein WPI. The pH is then adjusted to neutral, allowing the protein WPI structure to return to its native state, achieving effective encapsulation of EA. Simultaneously, moderate-intensity ultrasonic pretreatment is used to appropriately open the protein structure, further enhancing the interaction with EA and increasing the amount and efficiency of EA encapsulation.

[0015] Ultrasonic physical field treatment can modulate the interaction between whey protein isolate and insoluble small-molecule ellagic acid, thereby increasing the encapsulation capacity of EA by the protein carrier and improving the stability, antioxidant properties, and bioavailability of EA under different environmental conditions. This invention, through multispectral analysis, reveals that the specific interaction between whey protein isolate (the protein carrier) and small-molecule EA is achieved through spontaneous assembly driven by van der Waals forces and hydrogen bonds, forming a more stable composite system. This lays a theoretical foundation for the preparation and industrial application of small-molecule EA nanoparticles using protein as a carrier. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The images show the physical composition of WPI / EA nanoparticles (A), EA encapsulation efficiency and drug loading (B), protein solubility (C), and particle size distribution (D). Figure 2 The structural characterization results of WPI / EA nanoparticles are shown below; where A is the intrinsic fluorescence spectrum; B is the ultraviolet absorption spectrum; C is the synchronous fluorescence spectrum at Δλ=15nm; D is the synchronous fluorescence spectrum at Δλ=60nm; E is the circular dichroism spectrum; and F is the X-ray dichroism pattern. Figure 3 The results show the interaction force analysis of WPI / EA nanoparticles; where A is the fluorescence spectrum of WPI / EA nanoparticles treated at 298.15 K; B is the fluorescence spectrum of WPI / EA nanoparticles treated at 310.15 K; C is the fluorescence spectrum of WPI / EA nanoparticles treated at 318.15 K; D is the quenched Stern-Volemr plot; E is the static quenching plot; and F is the Van't Hoff plot. Figure 4 The results of thermal stability analysis of WPI / EA nanoparticles are shown below; where A represents the DPPH radical scavenging rate at 60℃; B represents the DPPH radical scavenging rate at 70℃; C represents the DPPH radical scavenging rate at 80℃; D represents the ABTS radical scavenging rate at 60℃; E represents the ABTS radical scavenging rate at 70℃; F represents the ABTS radical scavenging rate at 80℃; G represents the EA retention rate at 60℃; H represents the EA retention rate at 70℃; I represents the EA retention rate at 80℃; Capsulation represents WPI / EA nanoparticles, EA represents free EA, and VC represents the positive control; Figure 5The results show the salt ion stability analysis of WPI / EA nanoparticles; where A represents the DPPH radical scavenging rate under 50 mM NaCl treatment; B represents the DPPH radical scavenging rate under 100 mM NaCl treatment; C represents the DPPH radical scavenging rate under 200 mM NaCl treatment; D represents the ABTS radical scavenging rate under 50 mM NaCl treatment; E represents the ABTS radical scavenging rate under 100 mM NaCl treatment; F represents the ABTS radical scavenging rate under 200 mM NaCl treatment; G represents the EA retention rate under 50 mM NaCl treatment; H represents the EA retention rate under 100 mM NaCl treatment; I represents the EA retention rate under 200 mM NaCl treatment; Capsulation represents WPI / EA nanoparticles, EA represents free EA, and VC represents the positive control. Figure 6 The results of the storage stability analysis of WPI / EA nanoparticles; Figure 7 The results show the antioxidant activity of WPI / EA nanoparticles after in vitro simulated digestion; where A represents the DPPH radical scavenging rate of WPI / EA nanoparticles after in vitro digestion; B represents the DPPH radical scavenging rate of free EA after in vitro digestion; C represents the ABTS radical scavenging rate of WPI / EA nanoparticles after in vitro digestion; and D represents the ABTS radical scavenging rate of free EA after in vitro digestion. Figure 8 The results of EA release rate and bioaccessibility analysis of WPI / EA nanoparticles after simulated in vitro digestion are shown; where A is the EA release rate of WPI / EA nanoparticles after in vitro digestion; B is the EA release rate of free EA after in vitro digestion; and C is bioaccessibility. Figure 9 The graph shows the comparison results of EA encapsulation efficiency and drug loading of WPI / EA nanoparticles in Comparative Example 1 and Example 1; where A represents WPI / EA nanoparticles in Comparative Example 1 and B represents WPI / EA nanoparticles in Example 1. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0024] Example 1 1. Experimental materials EA standard (96%) and whey protein isolate (90%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0025] 2. Experimental Methods 2.1 Preparation of whey protein isolate-ellagic acid nanoparticles Whey protein isolate (WPI) was prepared into a 2% (w / v) protein solution and stirred thoroughly at 600 rpm at room temperature for 2 h, then hydrated overnight at 4°C. After centrifugation at 10000×g for 10 min, the supernatant was adjusted to pH=12 using 1M NaOH standard solution. Different masses of EA were rapidly added to the WPI solution and stirred for 2 h. The mixture was adjusted to pH=7 using 1M HCl standard solution and sonicated for 20 min (50 W, 5 s interval) to obtain WPI / EA nanoparticles encapsulated with different concentrations of EA. The obtained WPI / EA nanoparticles were named WPI, WPEA1, WPEA2, WPEA3, WPEA4, and WPEA5 according to the added EA concentrations, corresponding to EA concentrations of 0, 0.025, 0.05, 0.1, 0.2, and 0.3 mg / mL, respectively.

[0026] 2.2 Determination of Encapsulation Efficiency (EE) and Drug Loading (LA) WPI / EA nanoparticle solutions of different concentrations (0–0.3 mg / mL) were centrifuged at 10,000 × g for 10 min to remove unencapsulated free EA and insoluble aggregates. The supernatant was mixed with N-methylpyrrolidone (1:4; V / V), and extracted by sonication at 600 W for 15 min followed by vortexing for 5 min to extract the encapsulated EA. The EA content in the supernatant was then determined at 254 nm using an EA standard curve.

[0027] The encapsulation efficiency (EE) of WPI-encapsulated EA is calculated using the following formula: ; The drug loading (LA) of the EA packaged in WPI is calculated using the following formula: .

[0028] 2.3 Determination of protein solubility WPI protein solution and WPI / EA nanoparticles of different concentrations were transferred to centrifuge tubes and centrifuged. The protein content in the supernatant was determined by the Lowry method, and the solubility of the sample was calculated according to the following formula: ; In the formula, C0 represents the protein concentration in the sample before centrifugation; while C represents the protein concentration in the supernatant after centrifugation, in mg / mL.

[0029] 2.4 Characterization of WPI / EA nanoparticle structure 2.4.1 Ultraviolet-Vis Spectrophotometry (UV-Vis) The interaction between WPI and EA was investigated by scanning samples in the 200-400 nm wavelength range using a UV-Vis spectrophotometer. Before scanning, both the WPI protein solution and the WPI / EA nanoparticle solution were diluted 200-fold with ultrapure water. The prepared WPI / EA nanoparticles were named WPI, WPEA1, WPEA2, WPEA3, WPEA4, and WPEA5, with EA concentrations of 0, 0.000125, 0.00025, 0.0005, 0.001, and 0.0015 mg / mL, and the spectra were corrected using ultrapure water.

[0030] 2.4.2 Determination of Circular Dichroism (CD) Spectrum Secondary structure of proteins was detected in the 180-260 nm range using far-ultraviolet circular dichroism (CD) spectroscopy. Before scanning, WPI protein solution and WPI / EA nanoparticle solution were diluted 10-fold with ultrapure water. Samples were placed in 10 mm diameter quartz dishes for measurement using a circular dichroism spectrometer. The sample injection volume was 0.2 mL, the detection resolution was 0.5 mm, the scan rate was 100 nm / min, the detection sensitivity was adjusted to 20 mdeg, the signal response time was set to 0.25 s, and the slit width was 1 nm. Ultrapure water without protein samples was used as baseline correction. Data are expressed as molar ellipticity. CD spectral data were plotted using Graphpad V10.0, and secondary structure content was calculated using CDNN software.

[0031] 2.4.3 Fluorescence Spectroscopy To investigate the binding between WPI and EA, a fluorescence spectrophotometer (RF-6000, Shimadzu Corporation, Japan) was used. The sample was diluted 2000-fold with ultrapure water. The excitation wavelength was 280 nm, and the emission wavelength range was 300-500 nm. The spectral scan rate was 600 nm / min, and the excitation and emission wavelength slit widths were set to 5 nm. Automatic zeroing was performed using ultrapure water. Synchronous fluorescence parameters: At 298 K, the synchronous fluorescence spectra of the WPI and WPEA complex in the 200-400 nm range were recorded at wavelength intervals Δλ = 15 nm and Δλ = 60 nm, with the excitation and emission wavelength slit widths set to 2 nm.

[0032] 2.4.4 X-ray diffraction (XRD) X-ray diffraction was used to analyze the crystal structures of whey protein isolate (WPI) and whey protein isolate-ellagic acid composite nanoparticles (WPEA) at different EA concentrations. Detection conditions: voltage 50 kV, current 30 mA, scan range 5-60°, scan speed 4° / min, step size 0.02°.

[0033] 2.4.5 Fluorescence Quenching Analysis Fluorescence spectra of WPI protein and WPI / EA nanoparticles were measured using a fluorescence spectrophotometer (RF-6000, Shimadzu Corporation, Japan) at different temperatures (298.15 K: room temperature, 310.15 K: body temperature, 318.15 K: storage temperature). The excitation wavelength was set to 280 nm, the emission wavelength to 300-500 nm, the slit width for both excitation and emission wavelengths to 5 nm, and the scan rate to 600 nm / min. In all fluorescence experiments, the samples were diluted 2000-fold with ultrapure water. Fluorescence quenching analysis was performed based on the Stern-Volmer equation. ; In the formula, F and F0 represent the fluorescence intensity with and without EA, respectively, and K SV Let [Q] be the Stem-Volmer quenching constant, [Q] be the molar concentration of EA, and K be the molar concentration of EA. q Fluorescence quenching constant, The average lifetime of a biomolecule in the absence of a quencher is 10⁻⁸ s.

[0034] In studying the interactions between bioactive molecules and proteins, the binding constant (Ka) and binding site (n) are key parameters. These can be calculated using the Stern-Volmer double logarithmic equation: ; In the formula, F and F0 represent the fluorescence intensity in the presence and absence of EA, respectively. Q is the molar concentration of EA. Ka represents the binding affinity, and n represents the number of binding sites. When Kq is greater than the maximum dynamic diffusion quenching constant (2.0 × 10¹⁰ L mol), the quenching intensity is determined by the concentration of EA. -1 s -1 When the quenching rate is 60%, it is considered static quenching; otherwise, it is considered dynamic quenching.

[0035] Furthermore, by analyzing fluorescence data at different temperatures, thermodynamic parameters (including enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) were calculated using the Van't Hoff equation: ; ; ; In the formula, R is the gas constant (8.314 J·mol⁻¹). -1 ·K -1 T represents absolute temperature (298.15 K, 310.15 K, 318.15 K), and ΔH, ΔS, and ΔG represent enthalpy change, entropy change, and Gibbs free energy change, respectively.

[0036] 2.5 Antioxidant Analysis 2.5.1 Determination of DPPH free radical scavenging rate Mix 2 mL of sample solution with an equal volume of DPPH solution, and incubate the mixture at room temperature in the dark for 1 h. Then, measure the absorbance at 517 nm. Calculate the DPPH radical scavenging rate. VC was used as a positive control.

[0037] 2.5.2 Determination of ABTS free radical scavenging rate Mix 1 mL of sample solution with 3 mL of ABTS working solution and react for 1 h. Use distilled water as a blank control and measure the absorbance at 734 nm to calculate the ABTS free radical scavenging rate. Simultaneously, use vitamin C as a positive control.

[0038] 2.6 Salt Ion Stability Sodium chloride (NaCl) solutions with concentrations of 50, 100, and 200 mM were added to the samples, the mixtures were vortexed, and then centrifuged. The EE (%), DPPH, and ABTS of the nanoparticles at different salt ion concentrations were calculated. + Free radical scavenging rate.

[0039] 2.7 Thermal stability The samples were heated at 60°C, 70°C, and 80°C, the mixture was vortexed, and then centrifuged. The EE (%), DPPH, and ABTS of the nanoparticles were calculated at different temperatures. + Free radical scavenging rate.

[0040] 2.8 In vitro digestion simulation experiment of WPI / EA nanoparticles (1) Determination of antioxidant capacity Preparation of simulated digestive fluids: Based on the internationally recognized INFOGEST static in vitro digestive simulation model (Minekus et al., 2014), electrolyte stock solutions of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared, and the in vitro digestion simulation process was carried out.

[0041] Gastric phase: 20 mL of sample was added to an equal volume of SGF stock solution, and pepsin was added to achieve a concentration of 2000 U / mL. The mixture was then incubated at 37°C with shaking at 100 r / min for 120 min.

[0042] Intestinal phase: An equal volume of SIF stock solution was added to the remaining gastric digestion mixture. Trypsin was added to achieve a final concentration of 100 U / mL, and the mixture was incubated at 37°C with shaking at 100 r / min for 120 min.

[0043] After digestion, the scavenging rates of DPPH and ABTS free radicals in the samples were measured.

[0044] (2) Release rate of ellagic acid The release rates of ellagic acid and free ellagic acid in the sample were assessed by measuring changes in ellagic acid content during simulated gastric and intestinal digestion. The release rate of ellagic acid was calculated using the following equation: ; Where Ar is the content of ellagic acid released, and At is the total content of ellagic acid.

[0045] (3) Bioavailability of ellagic acid After SIF digestion, the sample solution was centrifuged to remove the precipitate, yielding mixed micelles. The EA content of the centrifuged digested sample was measured using high-performance liquid chromatography (HPLC), and bioaccessibility was calculated. ; Among them, C M and C D The concentrations of ellagic acid in the mixed micelle phase and in the small intestine are respectively.

[0046] 3. Experimental Results 3.1 Morphology of WPI / EA nanoparticles like Figure 1 As shown in Figure A, the WPI / EA nanoparticles prepared by this invention have good dispersibility and can be uniformly dispersed in the solvent, indicating that WPI encapsulation improves the dispersibility of EA.

[0047] 3.2 Encapsulation efficiency (EE) and drug loading (LA) of WPI / EA nanoparticles like Figure 1 As shown in B, the WPI / EA nanoparticles prepared by this invention have the effects of high EA encapsulation efficiency and controllable drug loading. Among them, the encapsulation efficiency and drug loading of nanoparticles WPEA3, WPEA4, and WPEA5 with EA concentrations of 0.1, 0.2, and 0.3 mg / mL are 82.84%, 90.97%, and 92.88% and 4.06, 9.07, and 13.81 (μg / mg), respectively.

[0048] 3.3 Protein solubility of WPI / EA nanoparticles like Figure 1As shown in Figure C, the solubility of WPEA3 and WPEA5 is better than that of pure WPI, indicating that encapsulation with EA not only improves the dispersibility of EA, but also significantly improves the solubility of nanoparticles.

[0049] 3.4 Particle size distribution of WPI / EA nanoparticles like Figure 1 As shown in Figure D, after WPI and EA are combined, the overall particle size of the nanoparticles shows a decreasing trend, and the uniformity becomes better.

[0050] 3.5 Structural characterization of WPI / EA nanoparticles like Figure 2 As shown, after WPI binds to EA, the amino acid residue microenvironment (polarity, conformation) of the protein changes, and the UV signal of EA is also affected, further proving that the two form nanoparticles. EA and WPI exhibit fluorescence quenching, indicating that EA enters the hydrophobic pocket of WPI (near tryptophan or tyrosine residues), altering the microenvironment of these amino acids and leading to a weakened fluorescence signal, proving that the two bind through hydrophobic interactions to form nanoparticles. Pure EA exists in a highly ordered crystalline form, exhibiting multiple extremely sharp and significant diffraction peaks. In all WPEA samples, the core crystalline feature of EA (28° peak) almost completely disappears. This proves that the crystal structure of ellagic acid is destroyed, and it is dispersed in an amorphous state within the protein matrix, providing direct structural evidence of "embedding." When EA is embedded inside the protein, it no longer exists in its original macroscopic crystalline form, but rather disperses in a molecular or amorphous state within the three-dimensional network structure of WPI. like Figure 3 As shown, the Kq values ​​at the three temperatures (298.15K, 310.15K, and 318.15K) are all greater than the maximum diffusion collision quenching constant (2×10⁻⁶). 10 The L / mol·L·s ratio indicates that the quenching effect of EA on WPI is static quenching. Combined with parameter fitting, it shows that the two form a stable complex, and the number of binding sites (n) is close to 1 at different temperatures, indicating that there is a major EA binding site on each WPI molecule. The enthalpy change ΔH and entropy change ΔS of the system are both significantly negative, which, according to classical thermodynamic laws, conclusively proves that hydrogen bonding and van der Waals forces are the core forces driving the non-covalent assembly of WPI and EA. The Gibbs free energy change (ΔG) at all temperatures is negative, confirming that the process of WPI encapsulating EA to form nanoparticles is thermodynamically completely spontaneous.

[0051] 3.5 Thermal stability analysis of WPI / EA nanoparticles like Figure 4As shown in the AF, the DPPH and ABTS radical scavenging rates of nanoparticles were significantly improved compared with free EA under three different temperature treatments (60, 70, and 80 °C).

[0052] like Figure 4 As shown in the GI, WPI / EA nanoparticles exhibit excellent protection of EA. Under high-temperature heating conditions of 80°C, nanoparticles containing high concentrations of EA can still maintain an EA retention rate of over 80%, while free EA has settled.

[0053] 3.6 Salt Ion Stability Analysis of WPI / EA Nanoparticles like Figure 5 As shown in the AF, in a NaCl environment of 50mM-200mM, the DPPH and ABTS free radical scavenging abilities of the nanoparticles are significantly better than those of free EA at the same concentration, confirming that the nanoparticles effectively improve the antioxidant activity of EA.

[0054] like Figure 5 As shown in the GI, with increasing ionic strength, the EA retention rate of nanoparticles (especially high concentrations of WPEA4 and WPEA5) can still be maintained at 50%-60%, while free EA is almost completely degraded and destabilized in a salt ion environment. This indicates that the three-dimensional protein network constructed by WPI in the nanoparticles can effectively resist the electrostatic shielding and aggregation effects caused by high salt, providing an excellent physical barrier for EA ionic stability.

[0055] 3.7 Storage stability analysis of WPI / EA nanoparticles like Figure 6 As shown, compared with the sedimentation phenomenon of free EA in water and the storage instability of low-concentration nanoparticles, high-concentration nanoparticles (such as WPEA3 and WPEA4) exhibit better stability over storage time.

[0056] 3.8 Results of in vitro digestion simulation experiment of WPI / EA nanoparticles 3.8.1 Antioxidant activity analysis like Figure 7 As shown, the antioxidant activity (DPPH and ABTS free radical scavenging rate) of nanoparticles after gastrointestinal digestion showed an increasing trend, and compared with free EA, it showed a significantly higher trend at different digestion stages.

[0057] 3.8.2 Release rate analysis like Figure 8 As shown in A and B, the nanoparticles are mainly released in the small intestine after being digested in the gastrointestinal tract, and the release rate is significantly improved compared to free EA.

[0058] 3.8.3 Biological Accessibility Analysis like Figure 8 As shown in C, for high-concentration nanoparticles, their bioaccessibility shows a significantly improved trend compared to free EA.

[0059] Comparative Example 1 This comparative example prepared a WPI / EA nanoparticle. The only difference between this preparation method and that of Example 1 is that the ultrasonic treatment step was omitted.

[0060] The encapsulation efficiency (EE) and drug loading (LA) of the WPI / EA nanoparticles in this comparative example were determined according to the experimental method in Example 1, and compared with the encapsulation efficiency and drug loading of the WPI / EA nanoparticles in Example 1. The results are as follows: Figure 9 As shown, after omitting the ultrasound-assisted treatment, the EA encapsulation efficiency and drug loading of the nanoparticles in Comparative Example 1 were lower than those in Example 1; and after omitting the ultrasound-assisted treatment, the EA encapsulation efficiency of the high-concentration EA nanoparticles (WPEA5) in Comparative Example 1 was even lower, indicating that 0.2 mg / mL had reached the encapsulation limit, while ultrasound-assisted treatment helped to improve the EA encapsulation concentration.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing ellagic acid nanoparticles using whey protein isolate as a carrier, characterized in that, Includes the following steps: Ellagic acid was added to an alkaline whey protein isolate solution, mixed thoroughly, and the pH was adjusted to neutral. After ultrasonic-assisted treatment, the ellagic acid nanoparticles were obtained.

2. The preparation method according to claim 1, characterized in that, The alkalinity refers to pH=12.

3. The preparation method according to claim 1, characterized in that, The whey protein isolate solution has a mass-volume percentage concentration of 1.5%-3%.

4. The preparation method according to claim 1, characterized in that, In the mixed solution, the concentration of ellagic acid is 0.025-0.3 mg / mL.

5. The preparation method according to claim 1, characterized in that, The mixing speed is 500-800 rpm, and the time is 1.5-2.5h.

6. The preparation method according to claim 1, characterized in that, The ultrasonic-assisted treatment has a power of 50W, operates for 5 seconds, stops for 5 seconds, and has a total duration of 15-25 minutes.

7. Ellagic acid nanoparticles prepared by the preparation method according to any one of claims 1-6.

8. The application of the ellagic acid nanoparticles according to claim 7 in the preparation of functional products.

9. The application according to claim 8, characterized in that, The functional products include foods, medicines, or nutritional supplements that help with antioxidation.

10. A product that helps fight oxidation, characterized in that, The product contains the ellagic acid nanoparticles as described in claim 7.

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