Preparation method of hyperoside-loaded Pickering emulsion

Abstract

The invention discloses a preparation method of hyperoside-loaded Pickering emulsion, which comprises the following steps: weighing whey protein, dissolving the whey protein in deionized water, adjusting the pH value to 6.5-7.5, stirring, cooling, and fully hydrating to obtain a whey protein solution; weighing xanthan gum powder, adding water to disperse, cooling, and fully hydrating to obtain a xanthan gum solution; mixing the whey protein solution with the xanthan gum solution, and then homogenizing to obtain a whey protein and xanthan gum compound solution; hyperoside is dissolved in edible oil, a hyperoside solution is obtained, the hyperoside solution and a whey protein xanthan gum compound solution are mixed and homogenized, and the hyperoside-loaded Pickering emulsion is obtained. According to the invention, a carrier platform with excellent physical stability, high loading efficiency, intelligent controlled release characteristic and good processing performance is constructed by utilizing multiple interaction among natural macromolecules.

Description

Technical Field

[0001] The invention relates to the field of Pickering emulsions, and in particular to a method for preparing a Pickering emulsion loaded with hyperoside. Background Art

[0002] The negative impacts of traditional emulsions on the environment and human health have limited their application. Consequently, interest in the development of novel emulsions has grown in recent years. Pickering emulsions offer an alternative in this field. Unlike traditional emulsions, they use solid particles rather than conventional surfactants to stabilize the emulsion. These solid particles are irreversibly adsorbed at the oil-water interface, forming a barrier that prevents aggregation and maintains the stability of the Pickering emulsion. Due to their environmental friendliness, resistance to aggregation, resistance to Ostwald ripening, and high stability, Pickering emulsions are considered promising delivery systems for bioactive compounds. With the increasing preference for natural ingredients in the food industry, the development of naturally derived solid particles as emulsifiers in Pickering emulsions has gained widespread recognition. Currently, proteins, polysaccharides, and their complexes stabilize Pickering emulsions. The forces involved in complex formation between proteins and polysaccharides are primarily hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Proteins and polysaccharides can adsorb at the oil-water interface, thereby enhancing the stability of the emulsion. For example, potato protein isolate (PoPI) and κ-carrageenan (κC) form a protein-polysaccharide complex through electrostatic interactions. The long-term stability of emulsions prepared with the PoPI-κC complex is slightly improved compared to emulsions prepared with PoPI alone. At pH 3-4.5, casein and soy polysaccharides form complex aggregates through electrostatic and hydrophobic interactions, forming an emulsion that encapsulates curcumin. This encapsulation significantly improves the oral bioavailability of curcumin. Therefore, there is a need to investigate protein-polysaccharide complexes as novel emulsifiers.

[0003] Whey protein isolate (WPI), a byproduct of cheese production, is not only a high-quality protein source used as an ingredient in sports nutrition products, but also, due to its excellent emulsifying properties and versatility, has been used in various food systems to improve product texture, flavor, and shelf life. The whey protein molecule, composed of hydrophilic amino acid residues and hydrophobic fatty acid chains, exhibits amphiphilic properties. This structure can reduce the surface tension between the two phases, forming a stable adsorption layer, preventing droplet aggregation and coagulation, and achieving uniform dispersion of the emulsion. Therefore, WPI has the potential to serve as a Pickering stabilizer.

[0004] Xanthan gum (XG) is an anionic polysaccharide produced by the microorganism Xanthomonas campestris. It consists of repeating pentasaccharide units composed of D-glucosyl, D-mannosyl, and D-glucuronic acid residues in a molar ratio of 2:2:1. The main chain of XG consists of glucose (Glc) linked by β-1,4-glycosidic bonds, with trisaccharide side chains containing glucuronic acid (GlcA) residues attached to the C(3) position of each replacing glucose residue between two mannose (Man) units. The terminal β-D-mannose is linked to GlcA via a β-1,4-glycosidic bond and in turn to α-D-mannose via an α-1,2-glycosidic bond. In its natural state, XG has an ordered conformation in solution. The native conformation of XG is a five-fold helical molecule stabilized by intermolecular and intramolecular hydrogen bonds, with a diameter of 1.9 nm and a pitch of 4.7 nm. In aqueous solution, XG easily forms a network structure due to the spontaneous formation of an ordered double helical structure. XG is frequently used as a stabilizer and thickener due to its high viscosity and excellent stability at high temperatures and in both acidic and alkaline conditions. When added to emulsions, it improves the stability of the emulsion by increasing the viscosity of the system and preventing droplet migration. Studies have shown that the aggregation and structural changes of lysozyme / XG nanoparticles can significantly enhance the stability of Pickering emulsions. The addition of XG improves the apparent viscosity and storage modulus of emulsions stabilized by pea protein isolate microgel particles / XG. Furthermore, emulsions at concentrations above 0.2 wt% exhibit significant storage stability.

[0005] Hyperoside (Hyp) is a plant flavonoid compound found in a variety of edible and medicinal plants, including hawthorn, Zanthoxylum bungeanum, and Hypericum perforatum. Recently, Hyp has received increasing attention due to its beneficial health effects. However, its application in food and pharmaceutical industries is limited due to its poor water solubility and low bioavailability. It has been found that encapsulating hydrophobic compounds in Pickering delivery systems can effectively improve their water dispersibility and oral bioavailability, thereby enabling the incorporation of these hydrophobic substances into commercial products. However, research on its Pickering delivery system is limited. Therefore, it is important to develop an effective Pickering delivery system to expand the application of Hyp in the food and pharmaceutical industries. Summary of the Invention

[0006] The invention discloses a method for preparing a hyperoside-loaded Pickering emulsion.

[0007] A method for preparing a hyperoside-loaded Pickering emulsion comprises the following steps:

[0008] 1) weighing whey protein and dissolving it in deionized water to prepare an initial whey protein solution, then adjusting the pH to 6.5-7.5, magnetically stirring until visually clear, cooling, and fully hydrating to obtain a fully hydrated whey protein solution;

[0009] 2) weighing xanthan gum powder, adding water to disperse it to obtain a xanthan gum solution, cooling it, and fully hydrating it to obtain a fully hydrated xanthan gum solution;

[0010] 3) mixing the fully hydrated whey protein solution obtained in step 1) with the fully hydrated xanthan gum solution obtained in step 2), and then homogenizing to obtain a whey protein xanthan gum complex solution;

[0011] 4) dissolving hyperoside in edible oil to obtain a hyperoside solution, and homogenizing the hyperoside solution and the whey protein xanthan gum complex solution to obtain a hyperoside-loaded Pickering emulsion.

[0012] In step 1), the mass fraction of the initial whey protein solution is 0.5% to 6%. The solution is stirred magnetically at 45 to 55°C for 60 to 180 minutes and then placed at 0 to 6°C for 8 to 12 hours to allow for full hydration.

[0013] In step 1), the whey protein solution is prepared by adjusting the pH (6.5-7.5) to a neutral environment away from the isoelectric point (pH ≈ 5.2). Charge stabilization: When the pH is above 5.2, whey protein becomes negatively charged, enhancing intermolecular electrostatic repulsion and preventing aggregation and precipitation. Structural stretching: The molecular conformation stretches, exposing hydrophobic regions and improving interfacial adsorption capacity. Solubility optimization: Magnetic stirring and sufficient hydration ensure complete dissolution of the protein, forming a homogeneous, clear solution, laying the foundation for subsequent complex formation.

[0014] In step 2), water at 55-85°C is added and dispersed for 30-60 minutes. The mass fraction of the xanthan gum solution is 0.05%-2%. The mixture is placed at 0-6°C to fully hydrate for 8-12 hours.

[0015] In step 2), during the preparation of the xanthan gum solution, the xanthan gum powder is dispersed and fully hydrated to activate its thickening and stabilizing properties. Avoiding micelle formation: Slowly adding water to the dispersion prevents the outer layer from absorbing water and swelling, which blocks water penetration, ensuring complete dissolution. Pseudoplastic activation: After hydration, the xanthan gum forms a high-viscosity solution that is stable upon standing and experiences a sharp drop in viscosity upon shear, facilitating subsequent homogenization. Wide pH / thermal stability: The viscosity is stable within the pH range of 3-11 and 10-80°C, adapting to subsequent processing conditions.

[0016] In step 3), the volume ratio of the fully hydrated whey protein solution to the fully hydrated xanthan gum solution is 1:1-4. Homogenize at 8000-16000 rpm for 2-5 minutes. Adjust the pH to 5-7 during the homogenization process.

[0017] In step 3), during the formation of the whey protein-xanthan gum complex, homogenization allows the whey protein and xanthan gum to form composite nanoparticles through electrostatic interactions and hydrogen bonding. This enhances particle stability: the complex combines the interfacial adsorption of whey protein with the steric hindrance of xanthan gum, enhancing the particle's anchoring ability at the oil-water interface. A synergistic stabilization mechanism: the xanthan gum's network structure encapsulates the whey protein, inhibiting its thermal aggregation and improving the emulsion's tolerance to pH and ionic strength. Particle size uniformity: homogenized shear forces refine the complex size, optimizing the interface coverage of the Pickering particles.

[0018] In step 4), during Pickering emulsion construction and hyperoside loading, hydrophobic hyperoside is dissolved in the oil phase and homogenized with the complex solution to form an O / W Pickering emulsion. Interface armoring: The composite particles adsorb onto the surface of the oil droplets, forming a dense barrier that prevents droplet coalescence. Active ingredient protection: Hyperoside is encapsulated within the oil core, reducing light and oxygen degradation and improving bioavailability. Efficient loading and stabilization: The pseudoplasticity of xanthan gum maintains a high viscosity during storage (inhibiting delamination), while shear thinning facilitates application.

[0019] Hyperoside-loaded Pickering emulsion is a novel delivery system that stabilizes the oil-water interface through solid particles (whey protein-xanthan gum complex). Hyperoside is easily inactivated by light, oxygen, and pH. The oil phase core of the Pickering emulsion isolates it from environmental factors, reducing degradation. Furthermore, the dense interface layer formed by the solid particles delays the release of hyperoside, prolonging its duration of action. It can replace traditional surfactants, reducing skin irritation and cytotoxicity.

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

[0021] The present invention reduces the loss and degradation of bioactive substances during the preparation and storage process. WPI-XG PEK has good emulsification stability and can replace some emulsifiers in food processing. WPI-XG PEK loaded with Hyp has an encapsulation efficiency of 85.47% and can be released continuously. At the same time, it achieved a free fatty acid release efficiency of 66% in an in vitro simulated digestion experiment, effectively solving its poor water solubility and low bioavailability. By utilizing the multiple interactions between natural macromolecules, a carrier platform with excellent physical stability, high loading efficiency, intelligent controlled release characteristics and good processing performance was constructed, which significantly improved the stability of hydrophobic active substances (such as Hyp) during processing, storage and transportation, as well as the targeted delivery efficiency and bioavailability in the body. The raw materials are safe and the process is feasible. It has broad application prospects in the fields of food, health products and pharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 (A) Particle size and polydispersity index (PDI) of WPI, (B) Zeta potential of WPI;

[0023] Figure 2 (A) XG particle size; (B) XG Zeta potential;

[0024] Figure 3 (A) Particle size and polydispersity index (PDI) of WPI-XG complex; (B) Zeta potential of WPI-XG complex;

[0025] Figure 4 is the viscosity of WPI-XG complex with different concentrations of xanthan gum;

[0026] Figure 5 FTIR spectra of WPI, WPI-XG with different XG concentrations, and XG;

[0027] Figure 6 is θo / w of WPI, XG, and WPI-XG with different XG concentrations;

[0028] Figure 7 Particle size (A) and zeta potential (B) of WPI-XG Pickering emulsions with different XG concentrations;

[0029] Figure 8 Optical microscope images of WPI-XG Pickering emulsions with different XG concentrations at 20×;

[0030] Figure 9 (A) Viscosity of WPI-XG Pickering emulsions with different XG concentrations; (B) Storage modulus and loss modulus of WPI-XG Pickering emulsions;

[0031] Figure 10 The microstructure of Pickering emulsion in CLSM;

[0032] Figure 11 The microstructures of Pickering emulsions with different XG concentrations at 4°C and 25°C on day 0 and day 21;

[0033] Figure 12 The emulsification properties of Pickering emulsions with different XG concentrations at 4°C on days 0, 7, 14, and 21;

[0034] Figure 13 is the centrifugal stability of Pickering emulsions with different XG concentrations;

[0035] Figure 14 is the encapsulation efficiency of Hpy in Pickering emulsions with different XG concentrations;

[0036] Figure 15 is the FFA release rate during intestinal digestion of Hyp emulsions loaded with different XG concentrations;

[0037] Figure 16 The release rate of Hyp loaded PEK at different XG concentrations. DETAILED DESCRIPTION

[0038] Example 1

[0039] 2.2 Preparation and characterization of WPI-XG complex

[0040] 2.2.1 Preparation of different raw material solutions

[0041] (1) Preparation of WPI solution: A certain amount of isolated whey protein was weighed and dissolved in deionized water to prepare WPI solutions with different mass fractions (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%). The pH value of the WPI solution was adjusted to 6.5 by adding NaOH dropwise. The solution was magnetically stirred at 50°C for 120 min until visually clear. The solution was then quickly cooled to room temperature (25°C) in an ice-water bath and placed at 4°C for 10 h to be fully hydrated.

[0042] (2) Preparation of XG solution: A certain mass of xanthan gum powder was weighed and dispersed in deionized water at 80°C for 30 minutes. The mixture was mechanically stirred to prepare XG solutions with different mass fractions (0.05%, 0.10%, 0.15%, 0.20%, and 0.25%). The water lost due to evaporation during the dissolution of XG at 80°C was compensated by adding deionized water. The solution was then quickly cooled to room temperature (25°C) in an ice-water bath and then placed at 4°C for 10 hours to fully hydrate.

[0043] (3) Preparation of WPI-XG binary nanocomposites: WPI and XG solutions were mixed at different ratios at 20°C and homogenized in a beaker using an IKA homogenizer at 14,000 rpm for 2 min. Citric acid was added during the homogenization process to adjust the pH to 5.5, and the mass fractions of the components were preliminarily determined (Table 1).

[0044] Table 1. Final composition of composite dispersion

[0045]

[0046] 2.2.2 Particle Size Distribution and Zeta Potential of WPI, XG, and Their Composites

[0047] According to the method of Zhao et al. [S. Matsuyama et al., "Stabilization of whey protein isolate-based emulsions via complexation with xanthan gum under acidic conditions", Food Hydrocolloids, Vol. 111, p. 106365, February 2021, doi: 10.1016 / j.foodhyd.2020.106365.] and [S. Zhao et al., "Bacteriostatic pickering emulsions stabilized by whey protein isolate–vanillin nanoparticles: fabrication, characterization and stability in vitro", Food Chem., Vol. 429, p. 136871, December 2023, doi: 10.1016 / j.foodchem.2023.136871.], WPI, XG, and their binary complexes were diluted 20-fold, and their particle sizes were measured using a particle size analyzer. The measurement parameters are: the material absorptivity is set to 0.001, the refractive indices of the particles and the dispersant are 1.450 and 1.330, respectively. All measurements are performed at 25°C.

[0048] The particle size of the composite samples was determined according to the method of Yang et al. [T. Yang, J. Zheng, B.-S. Zheng, F. Liu, S. Wang, and C.-H. Tang, "High internal phase emulsions stabilized by starch nanocrystals," Food Hydrocolloids, Vol. 82, pp. 230–238, September 2018, doi:10.1016 / j.foodhyd.2018.04.006]. To avoid the influence of aggregation, the composite samples were diluted 1:100 with ultrapure water before measuring the zeta potential.

[0049] 2.2.3 Rheological properties of WPI-XG composites

[0050] The steady-state flow shear viscosity of aqueous dispersions of the WPI-XG composite was measured at 25°C using a rheometer equipped with parallel plate geometry (diameter = 50 mm, gap = 1.0 mm) at shear rates of 0.1-100 s. Viscosity measurements were performed one day after sample preparation. To minimize the effects of emulsification, the test samples were gently mixed without foaming prior to viscosity measurement.

[0051] 2.2.4 Three-phase contact angle measurement

[0052] WPI-XG nanocomplexes with different ratios were pre-frozen at -80°C for 24 hours and then freeze-dried in a freeze dryer for 48 hours. The freeze-dried powder was sealed in a desiccating dish for storage. The method was slightly modified as described by Li et al. [L. Li, W. Wang, S. Ji, and Q. Xia, "Soy protein isolate-xanthan gum complexes to stabilize pickering emulsions for quercetin delivery," Food Chem., Vol. 461, p. 140794, Dec. 2024, doi:10.1016 / j.foodchem.2024.140794.]. The θ / w values ​​of the samples were determined using contact angle measurement. Briefly, the freeze-dried samples were pressed into thin tablets, immersed in corn oil in a transparent container, and a drop of distilled water was gently applied to the surface of the tablet using a high-precision syringe. After equilibrium was reached, a photograph of the drop was taken, and the contact angle was determined. At least three measurements were performed for each sample at 25°C.

[0053] 2.2.5 Fourier transform infrared spectroscopy (FTIR)

[0054] The experimental method of Fourier transform infrared spectroscopy was referred to Di [X.Di, Y.Li, X.Qin, Q.Wang and G.Liu, "Investigating the effect of whey protein isolate:proanthocyanidin complex ratio on the stability and antioxidant capacity of pickering emulsions", Int.J.Biol.Macromol., Vol. 279, p. 135342, November 2024, doi:10.1016 / j.ijbiomac.2024.135342.] et al., with slight modifications. The freeze-dried powder of WPI, XG, WPI-XG and KBr powder were mixed at a mass ratio of 1:100, and the mixed powder was placed in an agate dish and evenly ground, and then pressed into uniform thin sheets using a tablet press, and the functional group structure was further analyzed by FTIR spectroscopy. The test parameters are as follows: at 400-4000cm -1 16 scans were performed within the wavenumber range with a resolution of 2 cm -1 , the test temperature is 25℃.

[0055] 2.3 Preparation and characterization of WPI-XG nanocomposite Pickering emulsion

[0056] 2.3.1 Preparation of Pickering emulsions using WPI-XG complex

[0057] Different ratios of WPI-XG nanocomposites were added to a beaker and degassed under vacuum before being mixed with corn oil and homogenized to form a Pickering emulsion. Briefly, corn oil was slowly added to the WPI-XG nanocomposites. After the corn oil was completely incorporated, 0.05% sodium azide was added, and the mixture was further homogenized at 14,000 rpm for 3 minutes using an IKA high-speed homogenizer to obtain a Pickering emulsion (WPI-XG PEK).

[0058] 2.3.2 Particle size and potential of emulsion

[0059] The droplet size of WPI-XG PEK was measured as previously cited in the literature with some modifications [S. Wang et al., "pH-induced conformational changes and interfacial dilatationalrheology of soy protein isolated / soy hull polysaccharide complex and its effects on emulsion stabilization", Food Hydrocolloids, Volume 109, Page 106075, December 2020, doi:10.1016 / j.foodhyd.2020.106075.]. In short, the droplet size of WPI-XG PEK was determined by laser diffraction particle size analyzer. The test angle detection range was 0.015-144. The refractive index of corn oil and the dispersion medium (deionized water) was set to 1.467 and 1.330, respectively. The sample was dispersed into a sample jar and measured after reaching the light shielding degree. WPI-XG PEK samples were diluted 1:50 with deionized water. The diluted samples were placed in a folded capillary Zeta cell to measure their potential. All samples were diluted 50-fold, and the results were recorded and measured in triplicate.

[0060] 2.3.3 Observation and comparison of emulsion morphology

[0061] The microstructure of WPI-XG PEK was observed using an inverted optical microscope. Diluted WPI-XG PEK was placed on a microscope slide and covered with a coverslip. The slide was then observed using a 20× objective lens at 25°C.

[0062] 2.3.4 Rheological analysis of different emulsions

[0063] According to the method of Zhang Xinyue et al. [Zhang Xinyue, "Study on the Extraction Process of Anthocyanins from Purple Potatoes and Analysis of the Characteristics of Pickering Emulsions," Master's thesis, Chengdu University, Chengdu, 2024.], the rheological properties of different emulsion samples were tested using a rheometer. A 25mm diameter PP25 probe was used, and the plate gap was set to 1mm. The test conditions were set to a shear rate setting of 0.1-100s. -1 , constant strain 0.1%, frequency 0.001~10Hz, angular frequency from 0.1 to 10rad / s, record the viscosity value, storage modulus G' and loss modulus G" during the measurement process.

[0064] 2.3.5 Confocal laser scanning microscopy (CLSM)

[0065] The morphology of WPI-XG PEK at different ratios was investigated using CLSM. Nile Blue A (1%) and Nile Red (0.1%) were added to freshly prepared WPI-XG PEK. WPI-XG was stained with Nile Blue A at an excitation wavelength of 633 nm, and corn oil was stained with Nile Red at an excitation wavelength of 488 nm. Pickering emulsion images were recorded at excitation wavelengths of 488 nm and 633 nm, respectively.

[0066] 2.3.6 Emulsion stability analysis

[0067] 2.3.6.1 Storage stability of emulsion

[0068] WPI-XG PEK was stored at 25°C and 4°C for 21 days to evaluate storage stability. The appearance and optical microscopic images of WPI-XG PEK were observed and the emulsification index (CI) was measured at 0, 7, 14, and 21 days.

[0069] Calculation method: CI (%) = H S / H T ×100

[0070] H S : Serum layer height

[0071] H T : Total height of emulsion

[0072] 2.3.6.2 Centrifugal stability of emulsions

[0073] Take appropriate amount of WPI-XG PCK in different proportions, put them into a centrifuge tube, centrifuge at 10000r / min for 20min, and then remove the water layer with a syringe.

[0074] According to the following formula:

[0075]

[0076] M0 is the mass of the empty centrifuge tube;

[0077] M1 is the mass of the emulsion and centrifuge tube before centrifugation;

[0078] M2 is the mass of the emulsion and centrifuge tube after centrifugation with the aqueous layer discarded.

[0079] 2.4 Application of WPI-XG PEK for Hyp Delivery

[0080] 2.4.1 Preparation of Hyp-loaded WPI-XG emulsion

[0081] Hyp was dissolved in corn oil (0.01%, w / w) at 25°C with constant stirring. Hyp-loaded corn oil WPI-XG 0.05, WPI-XG 0.15, and WPI-XG 0.25 were mixed and homogenized to yield Hyp-loaded WPI-XG PEK with an oil mass fraction of 0.6. The Hyp-loaded WPI PEK with an oil mass fraction of 0.6 served as a control sample.

[0082] 2.4.2 Loading Efficiency (EE) of WPI-XG Emulsion

[0083] An appropriate amount of Hyp-loaded corn oil emulsions of WPI-XG 0.05, WPI-XG 0.15, and WPI-XG 0.25 were added to a centrifuge tube and centrifuged at 15,000 rpm for 20 min. The Hyp loading efficiency (EE) in the emulsion was measured at 374 nm using a UV-visible spectrophotometer.

[0084] According to the following formula:

[0085]

[0086] M T is the total mass of Hyp;

[0087] M R is the mass of free Hyp in the serum layer;

[0088] 2.4.3 Simulated in vitro digestion

[0089] Simulated in vitro digestion was performed according to the method of Chen et al. [S. Chen et al., "The lipid digestion behavior of oil-in-water pickering emulsions stabilized by whey protein microgels of various rigidities," Food Hydrocolloids, Vol. 130, p. 107735, Sept. 2022, doi:10.1016 / j.foodhyd.2022.107735.]. Simulated gastric fluid (SGF) contained 0.32 g pepsin and 0.2 g sodium chloride per 100 mL, with a pH of 2. 100 mL of simulated intestinal fluid (SIF) was prepared by adding 1 g pancreatin and 0.68 g KH2PO4 to ultrapure water, and the pH was adjusted to 6.8. SGF was mixed with the loaded Hyp emulsion at a ratio of 1:14 and incubated at 37°C and 100 rpm for 2 h. Afterwards, SIF was mixed with gastric digestion products in a 1:1 ratio, the pH value was quickly adjusted to 7.5 with 1 mol / L NaOH, and then incubated at 37 °C for 2 h at 100 rpm with continuous stirring.

[0090] 2.4.3.1 Free fatty acid (FFA) release

[0091] During intestinal digestion, the pH of the emulsion was maintained at 7.5 by continuously adding 0.1 mol / L NaOH. The amount of NaOH was recorded and used to calculate the release of FFA, which was then calculated according to the equation.

[0092]

[0093] Where: V NaOH is the volume of NaOH required to maintain pH (mL);

[0094] C NaOH is the NaOH concentration required for titration (mol / L);

[0095] M lipid is the average molecular weight of corn oil, recorded as 850 g / mol;

[0096] W lipid is the total mass of corn oil in the system (g).

[0097] 2.4.3.2 Digestion Release Rate of Hyp-WPI-XG Emulsion

[0098] The release rate of Hyp was determined during in vitro digestion according to the method of Yang et al. [J. Yang et al., "Mechanisms of in vitro controlled release of astaxanthin from starch-based double emulsion carriers", Food Hydrocolloids, Volume 119, Page 106837, October 2021, doi:10.1016 / j.foodhyd.2021.106837.]. A 1 mL aliquot was taken every 20 minutes and replaced with the same volume of fresh medium. After centrifugation at 12,000 rpm for 30 minutes, the release rate of Hyp was measured using a UV-visible spectrophotometer at 374 nm.

[0099] The release rate of Hyp was obtained according to the formula

[0100]

[0101] Among them: Hyp released is the release amount of Hyp;

[0102] Hyp total is the amount of Hyp released.

[0103] Results and Discussion

[0104] 3.1 Characterization of WPI, XG, and Their Complexes

[0105] 3.1.1 Particle Size Distribution and Zeta Potential of WPI and XG

[0106] like Figure 1 (A) It can be seen that the particle size range of WPI at all concentrations (0.5%-2.5%) is between 250-300nm, with a small fluctuation range (about ±50nm), indicating that the whey protein molecules do not significantly aggregate or dissociate at different concentrations and have good structural stability. The polydispersity index (PDI) of WPI continues to decrease from 0.34 to 0.25. The decrease in PDI indicates that the uniformity of the solution is enhanced, especially when the concentration is ≥1.0%, PDI <0.3, reaching the standard of a monodisperse system (PDI <0.3 is good monodispersity), indicating that the protein particles are more evenly distributed at high concentrations. Figure 1 As shown in Figure (B), the absolute value of the zeta potential of WPI gradually increases from 28.18 mV to 34.87 mV. This is likely due to the increased steric hindrance between molecules as the WPI concentration increases, while the electrostatic shielding effect of negatively charged groups on the whey protein surface (such as carboxylates) decreases, resulting in a better electrostatic stabilization mechanism and enhanced anti-aggregation ability of the colloidal structure. Therefore, subsequent experiments were conducted using a 2.0% WPI concentration.

[0107] like Figure 2 (A) It can be seen that as the concentration of XG increases, its particle size increases significantly from 141.8nm to 315.3nm, showing a concentration-dependent linear growth. Figure 2 As shown in (B), as the concentration of XG continues to increase, its zeta potential gradually decreases from -37.95 mV to -48.73 mV. This is because as the concentration increases, the distance between the molecular chains decreases, and hydrophobic interactions (acetyl groups) and hydrogen bonds promote the local aggregation of the molecular chains or the formation of a network structure. This process increases the rigidity of the chains and stabilizes their conformation, exposing more of the previously encapsulated carboxyl groups to the surface. This leads to an increase in the surface negative charge density and a significant increase in the absolute value of the zeta potential.

[0108] 3.1.2 Particle Size and Zeta Potential of WPI-XG Complex

[0109] Depend on Figure 3 (A) As shown, as the xanthan gum content in the WPI-XG composite increases from 0% to 0.25%, the particle size of the WPI-XG composite increases continuously from 265.17 nm to 295.78 nm. The polydispersity index (PDI) is in the range of 0.3-0.4, indicating that the composite has a narrow particle size distribution, close to monodispersity.

[0110] 3.1.3 Rheological properties of WPI-XG composites

[0111] Figure 4 The results showed that the viscosity of all WPI-XG composites at different concentrations decreased significantly with increasing shear rate, exhibiting typical pseudoplastic (shear-thinning) characteristics. Higher xanthan gum concentrations resulted in higher initial viscosities and greater sensitivity to shear rate changes. This may be because low-concentration composites have sparse molecular chains, resulting in only localized alignment adjustments and a gradual viscosity change. High-concentration composites, on the other hand, have dense networks, resulting in a large number of free molecular chains released upon shear disruption, leading to a sharp drop in viscosity.

[0112] 3.1.4 Fourier transform infrared spectroscopy (FTIR)

[0113] FTIR analysis can help to gain a deeper understanding of the interaction between proteins and polysaccharides. The FTIR spectra of WPI, WPI-XG and XG are shown in Figure 2. Figure 5 As shown. 3200-3500cm in FTIR -1 Peaks in the range are usually associated with hydrogen bonds [J. Yu, X. Wang, D. Li, L. Wang and Y. Wang, "Development of soy protein isolate emulsion gels as extrusion-based 3D food printing inks: effect of polysaccharides incorporation", Food Hydrocolloids, vol. 131, p. 107824, October 2022, doi:10.1016 / j.foodhyd.2022.107824.]. In the XG spectrum, 3400.4 cm -1 The peak at 2927.8cm is caused by the strong hydrogen bonding of polysaccharide hydroxyl groups (OH). -1 The peak corresponds to the stretching vibration of the C-H bond, 1725 cm -1 The peak is the stretching vibration of C=O. 1700-1500cm -1The peaks at 1617.3 cm represent amide I and amide II [Y.Xiong, Q.Li, S.Miao, Y.Zhang, B.Zheng and L.Zhang, "Effect of ultrasound on physicochemical properties of emulsion stabilized byfish myofibrillar protein and xanthan gum", Innovative Food Sci. Emerg. Technol., Vol. 54, pp. 225–234, June 2019, doi:10.1016 / j.ifset.2019.04.013.]. -1 and 1393.5cm -1 The peak corresponds to the symmetrical stretching vibration of the carboxylic acid group (-COO-). The spectrum of WPI shows that its peak at 3298.2 cm -1 The absorption peak at 2924.7cm is strong, mainly due to the NH stretching vibration of amide I band in protein and the OH vibration of water molecules. -1 Corresponding to aliphatic CH vibration in protein side chains. 1653.0cm -1 and 1540.1cm -1 The peak at 347.7 cm indicates the NH-bending vibration of the amide group and the β-sheet structure stabilized by WPI [S.Ahmadian, R.E.Kenari, Z.R.Amiri, F.Sohbatzadeh and M.H.Khodaparast, "Fabrication of double nano-emulsions loaded with hyssop (hyssopus officinalis L.) extract stabilized with soy protein isolate alone and combined with chia seed gumin controlling the oxidative stability of canola oil", Food Chem., Vol. 430, p. 137093, Jan. 2024, doi:10.1016 / j.foodchem.2023.137093.]. Compared with WPI, the O—H bond stretching vibration peaks of WPI-XG 0.05, WPI-XG 0.15 and WPI-XG 0.25 shifted from 3296.2 cm to 3311.3 cm, respectively. -1 to 3298.2cm -1 、3300.1cm -1 and 3301.1cm-1 , indicating that WPI and XG form a hydrogen bond network through hydroxyl, amino and carboxyl groups. The C—H stretching vibration of WPI-XG increases from 2925.1 cm -1 Offset to 2940.1cm -1 Compared with WPI, it shows that hydrophobic interaction is involved in the formation of WPI-XG. -1 The peaks of WPI-XG0.05, WPI-XG 0.15 and WPI-XG 0.25 are 1542.1cm -1 、1540.1cm -1 and 1538.1cm -1 This may be due to the interaction between the carboxylic acid group (-COO-) of XG and the positively charged groups of WPI (such as -NH3 of lysine). + ) may cause electrostatic attraction, resulting in a decrease in the intensity of the carboxylic acid peak.

[0114] 3.1.5 Three-phase contact angle

[0115] The interfacial wettability of solid particles is an important factor affecting their distribution at the oil-water interface. It plays a crucial role in the formation and stability of Pickering emulsions. Assessing the interfacial wettability of solid particles is crucial for preparing stable Pickering emulsions [X. Zhang, H. Liang, J. Li, X. Wei, and B. Li, "Improving the emulsifying property of gliadin nanoparticles as a stabilizer of pickering emulsions: modification with sodium carboxymethyl cellulose," Food Hydrocolloids, vol. 107, p. 105936, October 2020, doi:10.1016 / j.foodhyd.2020.105936.]. θo / w is a valuable characterization method. Generally, when the θo / w of solid particles is close to 90°, it promotes the effective adsorption of solid particles at the oil-water interface and inhibits the aggregation of oil droplets [Y. Zong et al., "All-natural protein-polysaccharide conjugates with bead-on-a-string nanostructures as stabilizers of high internal phase emulsions for 3D printing", Food Chem., Vol. 388, p. 133012, Sept. 2022, doi: 10.1016 / j.foodchem.2022.133012.]. Therefore, if Figure 5As shown in the figure, the θo / w of WPI is 67.3±1.94°, indicating that WPI can be preferentially wetted by the water phase due to its hydrophilicity. The θo / w of XG is 111.73±2.90°, indicating that XG can be preferentially wetted by the oil phase. The θo / w of WPI-XG 0.05 is 81.73±2.50°, the θo / w of WPI-XG 0.15 is 84.83±2.21°, and the θo / w of WPI-XG 0.25 is 91.67±1.55°. The results show that the addition of XG improves the wettability of WPI-XG in the oil phase. This change may be due to the formation of hydrogen bonds between WPI and XG through hydroxyl groups, which reduces the number of free hydroxyl groups in WPI that absorb water

[15] . This result is similar to the study of Li et al. [4]. When the XG concentration increases from 0.05% to 0.25%, XG has a smaller effect on WPI unfolding, partially burying the hydrophobic groups and slightly decreasing θo / w. In summary, WPI-XG has a θo / w of approximately 91.67°, indicating biphasic wettability, which makes WPI-XG more suitable than WPI for forming stable Pickering emulsions.

[0116] 3.2 Characterization of WPI-XG Nanocomposite Pickering Emulsion

[0117] 3.2.1 Particle size and potential of emulsion

[0118] Droplet size and zeta potential are important indicators for evaluating the stability of Pickering emulsions. Figure 7 (A) shows that as the XG concentration increases from 0% to 0.25%, the average emulsion droplet size decreases significantly from 315.3 nm to 52.6 nm, a decrease of 83.3%. The results indicate that at a certain oil mass fraction, the droplet size of WPI-XG PEK decreases with increasing XG concentration, consistent with optical microscopy observations. At XG concentrations between 0% and 0.15%, the particle size decreases rapidly to 88.1 nm, indicating that XG binds to whey protein (WPI) through electrostatic attraction to form a dense composite interfacial film, inhibiting droplet coalescence. At concentrations between 0.15% and 0.25%, the particle size decrease slows, likely due to saturation of interfacial adsorption. At this point, the weak gel network formed by XG in the continuous phase further restricts droplet collisions through steric hindrance. The Zeta potential (Figure B) continuously decreased from -37.4 mV to -42.0 mV, indicating that the carboxylic acid group (COO-) of XG dominated the interfacial charge characteristics, and its ionization degree increased with the increase of XG concentration, significantly increasing the negative charge density on the droplet surface.

[0119] 3.2.2 Observation and comparison of emulsion morphology

[0120] The microstructure of xanthan gum (XG) and whey protein isolate (WPI) composite emulsions with different concentrations was observed by microscope. Figure 8), it was found that the addition of XG significantly affected the size, distribution and stability of the emulsion droplets. The droplet size in the WPI emulsion was large, unevenly distributed and there was obvious agglomeration, indicating that the stability of the single protein interface film was poor. As the XG concentration increased (0.05%, 0.15%, 0.25%), the droplets gradually tended to be uniform: the droplet size range of the WPI-XG 0.05% emulsion narrowed, and local agglomeration decreased; the droplets in the WPI-XG 0.15% emulsion were further reduced, and the distribution uniformity was significantly improved; and the WPI-XG 0.25% emulsion showed the best characteristics, with the smallest droplet size and a close to monodisperse distribution.

[0121] 3.2.3 Rheological analysis of different emulsions

[0122] In food processing applications, the rheological properties of emulsions are crucial. The apparent viscosity of WPI-XGPickering emulsions with different XG concentrations varies with shear rate. Figure 9 As shown in Figure 2 (A), all six emulsions with different XG concentrations exhibited shear-thinning and non-Newtonian behavior during shearing, indicating that the flow properties changed with the external force. Increasing shear rate disrupted the internal structure of the Pickering emulsion, resulting in a decrease in viscosity. The apparent viscosity of the Pickering emulsion at 0.25% XG concentration was higher than at other concentrations, likely due to the xanthan gum in the system acting as a thickener, resulting in increased viscosity.

[0123] The storage modulus G' and loss modulus G" of Pickering emulsion are as follows: Figure 9 (B). The G' of all Pickering emulsions is greater than G", indicating that Pickering emulsions have gel-like properties and the two moduli are weakly dependent on frequency. It is worth noting that when the XG concentration is higher than 0.10%, the G' and G" of WPI-XG PEK are greater than those of WPIPEK, which may be due to the gelling properties of XG. High concentrations of XG form a viscoelastic interfacial layer with a gel network structure on the surface of the oil droplets, preventing the aggregation of oil droplets and maintaining the physical stability of WPI-XG PEK.

[0124] 3.2.4 Confocal laser scanning microscopy

[0125] Figure 10The microstructure of a Pickering emulsion. The oil phase and protein phase were stained with Nile Red and Nile Blue A, represented in green and red in the image, respectively. The Pickering emulsion is uniformly distributed with no apparent aggregation, exhibiting a water-in-oil structure. The red-labeled WPI and WPI-XG cover the green-labeled oil droplets. The results show that WPI and WPI-XG successfully adsorb at the oil-water interface and form an interfacial layer around the oil droplets. After the addition of XG, the droplet size of WPI-XG PEK is smaller than that of SPIPEK. WPI-XG forms an interfacial layer on the surface of the oil droplets, which gives WPI-XG PEK greater physical stability.

[0126] 3.2.5 Emulsion stability analysis

[0127] Depend on Figure 12 It can be seen that the stability of WPIPEK decreased after storage at 4℃ and 25℃ for 21 days. However, the storage stability of Pickering emulsions with different XG concentrations was improved to a certain extent, and the particle size could be maintained at a smaller size. Figure 11 To some extent, the same characteristics are exhibited. The density of the Pickering emulsion increases after the addition of XG. This phenomenon can be attributed to the fact that higher XG concentrations facilitate saturation of the oil-water interface, leading to increased particle density at the oil-water interface and wider interfacial coverage, thereby limiting droplet aggregation and enhancing the physical stability of the WPI-XG PEK.

[0128] like Figure 13 As shown in the figure, as the XG concentration increases from 0% to 0.25%, the water retention rate continues to rise from 74.4% to 91.3%. In the low concentration range (0-0.15%), the water retention rate increases rapidly, while in the high concentration range (0.15-0.25%), the WPI-XG emulsion has good centrifugal stability. This may be because low-concentration XG forms a composite interfacial film with WPI through electrostatic attraction, enhancing the interfacial rigidity of the emulsion and reducing the loss of water from the gaps between oil droplets during centrifugation. At the same time, XG forms a loose network in the continuous phase, binding free water through hydrogen bonds. As the XG concentration increases, a dense three-dimensional gel network is constructed in the bulk phase, significantly improving the viscoelasticity of the continuous phase and inhibiting water migration.

[0129] 3.3WPI-XG PEK for Hyp delivery

[0130] 3.3.1 Loading Rate of WPI-XG PEK for Hyp

[0131] To determine the encapsulation efficiency of bioactive substances, the entrapment rate of Hpy in freshly prepared Pickering emulsions with different XG concentrations was determined. Among all samples, the Pickering emulsion stabilized by only 0.1% WPI particles had the lowest EE ( Figure 14 ). As the XG concentration continues to increase, the EE value in the Pickering emulsion increases by 85.47%. XG concentration is a key factor affecting encapsulation efficiency, and as the XG concentration gradually increases, the encapsulation efficiency also shows an upward trend. This phenomenon shows that the XG concentration is crucial to the feasibility of Pickering emulsion as an effective carrier of bioactive substances. Low concentrations of particles are not enough to cover the surface of the droplets, resulting in the formation of an emulsion with large-sized droplets, which further leads to more loss of bioactive substances during the emulsion preparation process. In addition, low concentrations of particles will cause the oil phase located in the inner core to be more exposed to the external environment, resulting in chemical degradation of bioactive substances such as hyperoside due to light, heat, oxygen and other external stresses.

[0132] 3.3.2 Lipid Digestion of Pickering Emulsions

[0133] Within 2 h of intestinal digestion, the FFA released from the Hyp-loaded Pickering emulsion was as follows: Figure 15 As shown in the figure, the release of FFA from each emulsion gradually increased as the digestion process progressed. Throughout the intestinal digestion process, FFA release from corn oil loaded with Hyp was lower, likely due to the larger oil droplets reducing the contact area with lipase, resulting in reduced lipid digestion. The final degree of FFA release from the Hyp-loaded Pickering emulsions was ranked as WPI-0.25% XG-PEK > WPI-0.15% XG-PEK > WPI-0.05% XG-PEK > WPI-PEK. This may be due to the smaller droplet size of the Pickering emulsion. As the XG concentration increases, the smaller droplets are able to increase the contact area with lipase, resulting in increased FFA release.

[0134] 3.3.3 Release rate of Hyp in Pickering emulsion

[0135] The release rate of Hyp in in vitro simulated digestion, such as Figure 16 As shown, all curves show sustained release of Hyp. During SGF digestion, the release rate of Hyp from WPI-PEK was greater than that from WPI-XG PEK, which was mainly due to the poor stability of WPI-PEK and its easy emulsification and rupture under pepsin digestion. In contrast, the gel network structure formed by WPI-XG resisted pepsin digestion. During SIF digestion, the release of Hyp increased with increasing XG concentration, which may be because the droplet size of WPI-XG-PEK decreased with increasing XG concentration, thereby increasing the contact area with lipase and promoting lipid hydrolysis, releasing more Hyp.

Claims

1. A method for preparing a Pickering emulsion loaded with hyperoside, characterized in that: The following steps are involved: 1) weighing whey protein and dissolving it in deionized water to prepare an initial whey protein solution, then adjusting the pH to 6.5-7.5, magnetically stirring until visually clear, cooling, and fully hydrating to obtain a fully hydrated whey protein solution; 2) weighing xanthan gum powder, adding water to disperse it to obtain a xanthan gum solution, cooling it, and fully hydrating it to obtain a fully hydrated xanthan gum solution; 3) mixing the fully hydrated whey protein solution obtained in step 1) with the fully hydrated xanthan gum solution obtained in step 2), and then homogenizing to obtain a whey protein xanthan gum complex solution; 4) dissolving hyperoside in edible oil to obtain a hyperoside solution, and homogenizing the hyperoside solution and the whey protein xanthan gum complex solution to obtain a hyperoside-loaded Pickering emulsion.

2. The method for preparing a hyperoside-loaded Pickering emulsion according to claim 1, wherein In step 1), the mass fraction of the initial whey protein solution is 0.5% to 6%.

3. The method for preparing the hyperoside-loaded Pickering emulsion according to claim 1, wherein In step 1), the mixture is magnetically stirred at 45-55° C. for 60-180 min.

4. The method for preparing a hyperoside-loaded Pickering emulsion according to claim 1, wherein In step 1), the mixture is placed at 0-6°C to be fully hydrated for 8-12 hours.

5. The method for preparing the hyperoside-loaded Pickering emulsion according to claim 1, wherein In step 2), add water at 55-85° C. and disperse for 30-60 minutes.

6. The method for preparing the hyperoside-loaded Pickering emulsion according to claim 1, wherein In step 2), the mass fraction of the xanthan gum solution is 0.05% to 2%.

7. The method for preparing a hyperoside-loaded Pickering emulsion according to claim 1, wherein: In step 2), the mixture is placed at 0-6°C to be fully hydrated for 8-12 hours.

8. The method for preparing a hyperoside-loaded Pickering emulsion according to claim 1, wherein: In step 3), the mixing volume ratio of the fully hydrated whey protein solution to the fully hydrated xanthan gum solution is 1:1-4.

9. The method for preparing a hyperoside-loaded Pickering emulsion according to claim 1, wherein: In step 3), homogenize at 8000-16000 rpm for 2-5 minutes.

10. The method for preparing a hyperoside-loaded Pickering emulsion according to claim 1, wherein: In step 3), the pH is adjusted to 5-7 during the homogenization process.

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