A SPI-Que-Fe for loading β-carotene 3+ Preparation methods and applications of ternary complex high internal phase emulsions

Abstract

The purpose of this invention is to provide an SPI-Que-Fe for loading β-carotene. 3+ This invention relates to a method for preparing ternary complex high internal phase emulsions and their applications, belonging to the field of bio-food technology. Based on the SPI-Que binary complex, this invention introduces Fe... 3+ Constructing SPI-Que-Fe 3+ A ternary complex was used as an interface stabilizer to prepare a high internal phase emulsion loaded with β-carotene. This method synergistically regulates the interfacial adsorption and droplet protection capabilities of the complex through non-covalent complexation and metal-phenol coordination, thereby increasing the loading rate of β-carotene in the high internal phase emulsion, slowing down its degradation induced by environmental factors such as light, heat, and oxygen, and improving its low bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of bio-food technology, specifically relating to an SPI-Que-Fe for loading β-carotene. 3+ Preparation method and application of ternary complex high internal phase emulsion. Background Technology

[0002] Soy protein isolate (SPI) is widely available and high in protein, possessing functional properties such as emulsification, foaming, and gelling, making it a commonly used protein ingredient in plant-based and functional foods. However, natural SPI often exhibits a dense spherical structure, with insufficient molecular flexibility and interfacial rearrangement capabilities, which limits its emulsification stability and delivery capacity under complex conditions such as high oil phase, heat treatment, salt ions, and storage.

[0003] Quercetin (Que) is a natural flavonoid polyphenol with antioxidant activity, but it suffers from poor water solubility, limited stability, and low bioavailability. Constructing a non-covalent SPI-Que complex via a pH-driven isothermal approach allows for the regulation of SPI conformation and interfacial properties through hydrogen bonding and hydrophobic interactions, while avoiding the introduction of chemical cross-linking agents, making it more suitable for food applications. However, the interfacial strength and environmental stability of single-protein-polyphenol binary complexes remain limited.

[0004] Ferric ions (Fe) 3+ It can undergo metal-phenol coordination with the phenolic hydroxyl groups in Que, and in SPI, Que and Fe 3+ The formation of bridging structures between these structures helps enhance the structural compactness, interfacial adsorption capacity, and emulsion stability of the complex. β-carotene is a typical fat-soluble bioactive substance with poor water dispersibility, is easily degraded by light, heat, and oxygen, and has low bioavailability. High internal phase emulsions, with their high oil phase content, strong loading capacity, and tunable rheological properties, are suitable as β-carotene loading systems. Therefore, the construction of SPI-Que-Fe... 3+ Ternary complex-stabilized high internal phase emulsions can provide a new plant protein-based carrier for the efficient loading, stable protection, and digestion of β-carotene. Summary of the Invention

[0005] This invention provides an SPI-Que-Fe for loading β-carotene. 3+ This invention relates to a method for preparing ternary complex high internal phase emulsions and their applications, belonging to the field of bio-food technology. Based on the SPI-Que binary complex, this invention introduces Fe... 3+ Constructing SPI-Que-Fe 3+A ternary complex was used as an interface stabilizer to prepare a high-internal-phase emulsion loaded with β-carotene. This method synergistically regulates the interfacial adsorption and droplet protection capabilities of the complex through non-covalent complexation and metal-phenol coordination, thereby increasing the loading rate of β-carotene in the high-internal-phase emulsion, slowing down its degradation induced by environmental factors such as light, heat, and oxygen, and improving its low bioavailability.

[0006] The present invention adopts the following technical solution: A SPI-Que-Fe for loading β-carotene 3+ The preparation method of ternary complex high internal phase emulsion includes the following steps: S1. Preparation of soy protein isolate (SPI) dispersion: SPI was dispersed in deionized water, the pH was adjusted to 12.0, and the mixture was stirred at room temperature for 2 hours to allow the protein to be fully hydrated and expanded, thus obtaining the SPI dispersion. S2. Preparation of SPI-Que binary complex: Quercetin (Que) was first dissolved in anhydrous ethanol, and then added dropwise to the SPI dispersion to prepare a mixture of SPI and Que. The mixture was stirred in the dark for 20 min. The pH of the system was then adjusted to 7.0. The residual ethanol was removed by rotary evaporation at 40 °C to obtain the SPI-Que binary complex dispersion. S3, Preparation of SPI-Que-Fe 3+ Ternary complex: Take the SPI-Que binary complex dispersion and add FeCl3•5H2O aqueous solution dropwise to it. Vortex mix for 10 min to obtain SPI-Que-Fe 3+ The ternary complex dispersion was stored at 4°C. S4. Preparation of a high-internal-phase emulsion of a ternary complex: Adjusting SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion was used as the continuous aqueous phase; MCT was used as the oil phase; the oil phase was added dropwise to the continuous aqueous phase; and homogenization was performed at 10,000 rpm for 1 min to obtain SPI-Que-Fe. 3+ Ternary complex high internal phase emulsion; S5. Preparation of a high internal phase emulsion loaded with β-carotene: Adjusting SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion was used as the continuous aqueous phase; β-carotene was dissolved in MCT as the oil phase; the oil phase was added dropwise to the continuous aqueous phase, and homogenization was performed at 10,000 rpm for 1 min to obtain SPI-Que-Fe loaded with β-carotene. 3+ Ternary complex high internal phase emulsion, stored at 4°C protected from light.

[0007] Further, in S1, the SPI is dispersed in deionized water at a concentration of 10 mg / mL; The pH was adjusted using a 0.5 mol / L NaOH solution.

[0008] Further, in S2, the Que is dissolved in anhydrous ethanol at a concentration of 10 mg / mL; In the mixture, the mass ratio of SPI to Que is 25:1; The pH was adjusted using a 0.5 mol / L HCl solution.

[0009] Furthermore, in S3, the SPI-Que-Fe 3+ In the ternary composite dispersion, Que and Fe 3+ The mass ratio is any one of 10:1, 20:1, and 30:1; The concentration of the FeCl3•5H2O aqueous solution is 10 mg / mL.

[0010] Furthermore, in S4, SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion was adjusted to any one of 10 mg / mL, 30 mg / mL and 50 mg / mL; The oil phase is added dropwise to the continuous aqueous phase, and the volume fraction of the oil phase is controlled to be 75%.

[0011] In S5, the SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion is 30 mg / mL; the concentration of β-carotene in the oil phase is 1 mg / mL; the oil phase is added dropwise to the continuous aqueous phase, and the volume fraction of the oil phase is controlled to be 75%.

[0012] Another objective of this invention is to provide a SPI-Que-Fe³⁺ ternary complex that can be used as a plant protein-based interface stabilizer.

[0013] Another objective of this invention is to provide a ternary complex-stabilized high internal phase emulsion to improve emulsion dispersion stability and environmental tolerance.

[0014] The present invention also aims to provide a high internal phase emulsion loaded with β-carotene to improve the β-carotene loading rate, slow down its light, heat and oxygen degradation, and improve its bioavailability.

[0015] The present invention also aims to provide the application of this high internal phase emulsion in the delivery of fat-soluble active substances, plant protein-based functional foods, and nutritional emulsions.

[0016] The beneficial effects of this invention are as follows: 1. The complex structure is more stable. In this invention, the SPI-Que binary complex is first constructed by the pH-driven method, and then Fe is introduced 3+ to form metal-phenol coordination and bridging effects, resulting in a denser ternary assembly structure among proteins, polyphenols, and iron ions. This effect stems from the Que:Fe 3+ mass ratio regulation in the "ternary complex preparation".

[0017] 2. The interfacial stability ability is enhanced. When the ternary complex is used to stabilize high internal phase emulsions, it can increase the absolute value of the droplet surface charge, enhance the electrostatic repulsion and steric hindrance between droplets, reduce the emulsion particle size, improve the dispersibility, and form a denser and continuous interfacial film. This effect is due to the coordination network between Fe 3+ and the phenolic hydroxyl groups of Que, as well as the adsorption and rearrangement of SPI molecules at the oil-water interface.

[0018] 3. The protection and delivery effects of β-carotene are improved. β-carotene is dissolved in the MCT oil phase and encapsulated in a high internal phase emulsion with a 75% oil phase volume fraction. The ternary complex can form a stronger interfacial barrier, reducing the degradation of β-carotene during storage; after in vitro digestion, the bioaccessibility of β-carotene generally shows S < S-Q < S-QFE, indicating that Fe 3+ further improves the lipid digestion and the micellization transfer efficiency of β-carotene.

[0019] 4. The preparation process is mild and suitable for food system expansion. This method mainly uses conventional food processing operations such as aqueous phase dispersion, pH regulation, ethanol-assisted dissolution, rotary evaporation to remove ethanol, and high-speed homogenization. It does not require toxic cross-linking agents, has a simple process, and is suitable for further scale-up preparation of plant protein-based emulsions for delivering lipophilic active substances. Description of the Drawings

[0020] Figure 1 are the fluorescence spectra of the dispersion liquids of SPI, SPI-Que, and SPI-Que-Fe 3+ complexes; Figure 2 are the Fourier infrared spectra of SPI, SPI-Que, and SPI-Que-Fe 3+ complexes; Figure 3 are the electron micrographs of SPI, SPI-Que, and SPI-Que-Fe 3+ complexes; Figure 4 are the particle size diagrams of the high internal phase emulsions of SPI, SPI-Que, and SPI-Que-Fe 3+ complexes; Figure 5For SPI, SPI-Que and SPI-Que-Fe 3+ Zeta potential diagram of complex high internal phase emulsion; Figure 6 For SPI, SPI-Que and SPI-Que-Fe 3+ Environmental stress stability diagram of complex high internal phase emulsion; Figure 7 For SPI, SPI-Que and SPI-Que-Fe 3+ Image showing the storage appearance of the complex high internal phase emulsion; Figure 8 This is a graph showing the loading rate of β-carotene. Figure 9 Particle size distribution for simulated external digestion of a high-internal-phase emulsion loaded with β-carotene; Figure 10 Zeta-potential diagram for simulated external digestion of high-internal-phase emulsion loaded with β-carotene; Figure 11 A diagram showing the bioavailability of a high-internal-phase emulsion loaded with β-carotene after simulated digestion in an externally controlled manner. Figure 12 This is a stability diagram of a high internal phase emulsion loaded with β-carotene. Detailed Implementation

[0021] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] Example A SPI-Que-Fe for loading β-carotene 3+ The method for preparing a ternary complex high internal phase emulsion is characterized by comprising the following steps: S1. Preparation of SPI dispersion: Disperse SPI in deionized water to a concentration of 10 mg / mL, and adjust the pH to 12.0 with 0.5 mol / L NaOH solution; stir at room temperature for 2 h to allow the protein to fully hydrate and expand, and obtain the SPI dispersion. S2. Preparation of SPI-Que binary complex: Que was pre-dissolved in anhydrous ethanol to a concentration of 10 mg / mL, and then added dropwise to the SPI dispersion to make the mass ratio of SPI to Que 25:1; stirred in the dark for 20 min; then the pH of the system was adjusted to 7.0 with 0.5 mol / L HCl solution; residual ethanol was removed by rotary evaporation at 40℃. S3, Preparation of SPI-Que-Fe 3+Ternary complex: Take the SPI-Que binary complex dispersion and add 10 mg / mL FeCl3•5H2O aqueous solution dropwise to it, controlling the reaction of Que with Fe. 3+ The mass ratios were 30:1, 20:1, and 10:1; vortex mixing was performed for 10 min to obtain SPI-Que-Fe. 3+ Ternary complex dispersion; sample stored at 4℃ for later use; S4. Preparation of high internal phase emulsion of ternary complex: The above ternary complex dispersions were adjusted to protein concentrations of 10, 30, and 50 mg / mL, serving as the continuous aqueous phase; MCT was used as the oil phase; the oil phase was added dropwise to the continuous aqueous phase, controlling the oil phase volume fraction to 75%; homogenization was performed at 10000 rpm for 1 min using a high-speed homogenization method to obtain SPI-Que-Fe. 3+ Ternary complex high internal phase emulsion; S5. Preparation and application of β-carotene-loaded high internal phase emulsion: The above ternary complex dispersion was adjusted to a protein concentration of 30 mg / mL as the continuous aqueous phase; β-carotene was dissolved in MCT as the oil phase, with a concentration of 1 mg / mL; the oil phase was added dropwise to the continuous aqueous phase, controlling the oil phase volume fraction to 75%; homogenization was performed at 10,000 rpm for 1 min using a high-speed homogenization method to obtain β-carotene-loaded SPI-Que-Fe. 3+ The ternary complex high internal phase emulsion was stored at 4°C protected from light and used for loading, protection and in vitro digestion simulation evaluation of β-carotene.

[0023] Performance testing 1. Fluorescence spectrum The intrinsic fluorescence of SPI and its complex with Que (SPI-Que) was measured using a fluorescence spectrophotometer. SPI, SPI-Que, and SPI-Que-Fe were also studied. 3+ The complex dispersion was diluted with deionized water to a concentration of 0.2 mg / mL, with deionized water used as a blank control. The excitation wavelength was set to 280 nm, and the emission spectra in the range of 300 to 500 nm were recorded at a scan rate of 1000 nm / min. The excitation and emission slit widths were both maintained at 5 nm.

[0024] The results are as follows Figure 1 As shown, Figure 1 In this context, 25:1 indicates that the mass ratio of SPI to Que is 25:1; SQFE-10, SQFE-20, and SQFE-30 respectively indicate that the mass ratio of SPI to Que is 25:1, and that iron ions are introduced on this basis, with Que and Fe... 3+ The mass ratios are 10:1, 20:1, and 30:1, respectively.

[0025] Depend on Figure 1 It can be seen that compared with SPI alone, the fluorescence intensity of the 25:1 complex is significantly reduced, and the maximum emission wavelength undergoes a slight red shift. This indicates that Que can interact with aromatic amino acid residues such as tryptophan and tyrosine in the SPI molecule, change its surrounding microenvironment, adjust the conformation of the SPI molecule, and form an SPI-Que composite carrier structure.

[0026] Fe was further introduced into the SPI-Que (25:1) binary composite system. 3+ Subsequently, the fluorescence intensity of the system continued to decrease, and with the decrease of Fe... 3+ The addition amount showed a gradually decreasing trend, with the intensity order being SPI > SPI-Que (25:1) > SQFE-30 > SQFE-20 > SQFE-10. This result indicates that Fe... 3+ The addition of SPI, Que and Fe can further promote 3+ The interaction between them enhances the molecular assembly degree of the ternary composite system. With the interaction of Que:Fe 3+ As the mass ratio decreased from 30:1 to 10:1, the fluorescence quenching effect was further enhanced, indicating that the higher Fe content... 3+ It can serve as a coordination bridging node, forming a metal-phenol coordination structure with the phenolic hydroxyl group in Que, and further inducing conformational rearrangement and structural compaction of the SPI-Que complex.

[0027] In summary, the fluorescence spectroscopy results, from the perspective of changes in the microenvironment of the protein's endogenous fluorescent groups, indicate that the introduction of Que can induce conformational adjustments in the SPI molecule, while the further addition of Fe³⁺ enhances the fluorescence quenching effect of the system, suggesting that intermolecular interactions in the ternary system are further strengthened. These results demonstrate that the formation of the SPI-Que-Fe³⁺ composite system is accompanied by enhanced protein conformational rearrangement and molecular assembly behavior, providing a conformational basis for the subsequent formation of a stable interfacial film and improved emulsion stability.

[0028] 2. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy was used. The lyophilized powders of each sample were pressed into thin sheets using potassium bromide and then spectrophotometers were used at 500-4000 cm⁻¹. -1 Scanning was performed within the wavenumber range, with a resolution of 4 cm. -1 A total of 64 scans were performed, with blank potassium bromide tablets as the background.

[0029] Figure 2 In this context, 25:1 indicates that the mass ratio of SPI to Que is 25:1; SQFE-10, SQFE-20, and SQFE-30 respectively indicate that the mass ratio of SPI to Que is 25:1, and that iron ions are introduced on this basis, with Que and Fe... 3+The mass ratios are 10:1, 20:1, and 30:1, respectively.

[0030] Depend on Figure 2 It can be seen that: SPI is at 3420.15 cm -1 1640.58 cm -1 and 1552.97 cm -1 Distinct characteristic absorption peaks appear nearby, corresponding to the OH / NH stretching vibration, the C=O stretching vibration of amide I, and the NH bending vibration and CN stretching vibration of amide II in the protein molecule, respectively.

[0031] Compared to SPI alone, the characteristic peaks of the SPI-Que (25:1) complex all exhibited varying degrees of low wavenumber shifts, with the OH / NH stretching vibration peak shifting from 3420.15 cm⁻¹. -1 Moved to 3412.06 cm -1 The amide I band is 1640.58 cm. -1 Moved to 1637.19 cm -1 The amide II band is 1552.97 cm. -1 Moved to 1548.59 cm -1 This result indicates that Que interacts with hydroxyl, amino, and carbonyl groups in the SPI molecule, altering the internal and intermolecular hydrogen bond network of the SPI molecule, causing a protein conformational rearrangement of SPI, and forming an SPI-Que binary complex structure.

[0032] Further introduction of Fe into the SPI-Que composite system 3+ Subsequently, the characteristic peaks continued to shift towards lower wavenumbers. With the change in Que:Fe 3+ When the mass ratio decreased from 30:1 to 10:1, the OH / NH stretching vibration peak decreased from 3408.38 cm⁻¹. -1 Moved to 3405.01 cm -1 The amide I band is 1636.17 cm. -1 Moved to 1633.03 cm -1 The amide II band is 1545.66 cm. -1 Moved to 1539.72 cm -1 This change indicates that Fe 3+ The addition of Fe further enhanced the intermolecular interactions within the system. 3+ It is possible that SPI, Que, and Fe can be promoted through coordination with the phenolic hydroxyl groups in Que and bridging effects with polar groups such as carbonyl and amino groups in the SPI molecule. 3+ A more stable ternary composite structure is formed between them.

[0033] In summary, the Fourier transform infrared spectroscopy results further confirm, from the perspective of functional group vibrational changes, the presence of SPI, Que, and Fe. 3+ Effective intermolecular interactions exist. The continuous shift of amide-related characteristic peaks and OH / NH absorption peaks indicates that hydroxyl, amino, and carbonyl groups in the protein molecule participate in the construction of the complex structure; among them, the peak position change of the SQFE-10 group is the most obvious, indicating that the interaction of the ternary complex system is stronger and the degree of structural assembly is higher under this condition, providing a molecular basis for its use as a β-carotene load carrier or emulsion stabilizer.

[0034] 3. Scanning electron microscope The morphological characteristics of the sample were observed using a scanning electron microscope, with the accelerating voltage set to 10.0 kV and the magnification set to 30000.

[0035] Depend on Figure 3 It can be seen that pure SPI exhibits a relatively typical dense blocky aggregate structure with a relatively flat surface and clear particle boundaries, indicating that there is a strong self-aggregation effect between natural SPI molecules, and the overall structure is relatively compact.

[0036] Upon introduction of Que, the surface morphology of the SPI-Que (25:1) complex underwent significant changes. The originally dense, blocky structure was disrupted, forming a more loose, rough, and continuous porous honeycomb network structure. This result indicates that the addition of Que can interact with SPI molecules, weakening the original tight association between protein molecules and causing a certain degree of unfolding and reorganization of the SPI molecular structure, thereby forming a composite structure with a large specific surface area and porous characteristics.

[0037] Further introduction of Fe into the SPI-Que composite system 3+ Subsequently, the microstructure of the SQFE-10 group changed again. Compared with the 25:1 group, the porous structure of SQFE-10 was significantly reduced, and the overall structure exhibited a more continuous and dense lamellar or film-like structure, with some wrinkles and cracks visible on the surface. This indicates that Fe... 3+ The addition of SPI, Que and Fe 3+ Further assembly between Fe 3+ It may act as a coordination bridging node, interacting with the phenolic hydroxyl groups of Que through metal-phenol coordination, and further enhancing the degree of cross-linking within the composite system.

[0038] Scanning electron microscopy results showed that the introduction of Que helped to disrupt the original dense aggregated structure of SPI and form a loose porous network, while Fe... 3+ The further addition of [a specific element] promotes the transformation of the network structure into a more compact, continuous, and stable ternary composite structure. This morphological change illustrates the SPI-Que-Fe [structure / structure] constructed in this invention.3+ The composite system has stronger structural assembly capabilities and a more complete matrix morphology, which can provide a good microstructural basis for its use as a β-carotene load carrier or emulsion stabilizer.

[0039] 4. Particle size and zeta potential The particle size distributions of SPI, SPI-Que binary composite and SPI-Que-Fe were determined using a nanoparticle size analyzer. 3+ Particle size and Zeta potential of ternary complex-stabilized high internal phase emulsions. Before measurement, each emulsion sample was diluted with deionized water to a protein concentration of 0.01 mg / mL, and the refractive indices of protein and water were set to 1.45 and 1.33, respectively.

[0040] Particle size results as follows Figure 4 As shown, the Zeta potential results are as follows: Figure 5 As shown, Figure 4 and Figure 5 In the figure, the horizontal axes S10, S30, and S50 represent the protein concentrations of SPI, which are 10, 30, and 50 mg / mL, respectively.

[0041] S represents SPI; SQ represents a SPI to Que mass ratio of 25:1; SQFE-10 represents a SPI to Que mass ratio of 25:1, with the introduction of iron ions, and Que to Fe... 3+ The mass ratio is 10:1.

[0042] Depend on Figure 4 It was found that as the SPI concentration increased from 10 mg / mL to 50 mg / mL, the average particle size of all three emulsion groups showed a significant decreasing trend, indicating that increasing the protein concentration is beneficial for forming smaller emulsion droplets. Among them, the S group consistently had the largest particle size, followed by the SQ group, and the SQFE-10 group had the smallest, with the overall order being S>SQ>SQFE-10. This suggests that SPI alone has relatively limited interfacial adsorption and droplet protection capabilities when stabilizing emulsions, easily leading to the formation of larger droplets; the introduction of Que improves the interfacial adsorption capacity of protein molecules, reducing the emulsion particle size; further introduction of Fe... 3+ Subsequently, the particle size of the system further decreased, indicating that Fe 3+ It can promote SPI, Que and Fe 3+ This forms a more stable ternary composite structure, enhancing the adsorption and covering ability of the complex at the oil-water interface, thereby inhibiting droplet aggregation and forming a finer, more uniform emulsion system. Especially at 50 mg / mL, the SQFE-10 group exhibited the lowest particle size, indicating that higher protein concentration and Fe... 3+ Coordination synergistically improves the dispersion stability of the emulsion.

[0043] Depend on Figure 5 The Zeta potential results show that the absolute value of the Zeta potential of each emulsion group increases with increasing SPI concentration, indicating enhanced droplet surface charge and improved electrostatic repulsion between droplets, which helps prevent droplet aggregation and flocculation. At the same SPI concentration, the SQFE-10 group had the highest absolute Zeta potential, followed by the SQ group, while the S group had a relatively lower absolute value, indicating that Que and Fe... 3+ The introduction of [a specific ingredient] can further modulate the surface charge distribution of emulsion droplets. After Que forms a complex with SPI, it can alter the spatial conformation of the protein molecules and the exposure state of surface groups, resulting in stronger charge stability on the emulsion droplet surface; Fe [another ingredient] 3+ Upon further addition, metal-phenol coordination can occur between the phenolic hydroxyl groups in Que, and a more compact composite interface layer can be constructed together with the polar groups in the SPI molecule. This interface layer not only enhances the electrostatic repulsion of the droplet surface but also improves the droplets' resistance to aggregation and coalescence. Therefore, the SQFE-10 emulsion exhibits a smaller particle size and a higher absolute potential value, demonstrating the effectiveness of the SPI-Que-Fe constructed in this invention. 3+ The complex has superior emulsifying and stabilizing properties and can be used as an emulsifying and stabilizing carrier for β-carotene loading.

[0044] 5. Environmental stress stability of the emulsion (1) Heating stability: SPI-Que-Fe 3+ The ternary complex high internal phase emulsion was heated at 20, 30, 50 and 70 °C for 2 h, respectively. After treatment, the particle size of the emulsion was measured and its stability was evaluated.

[0045] (2) pH stability: SPI-Que-Fe was adjusted using 0.5 mol / L HCl or NaOH. 3+ After the ternary complex high internal phase emulsion was pHed to 5, 7, and 9, it was allowed to stand for 2 h. After the treatment, the particle size of the emulsion was measured to evaluate its stability.

[0046] (3) Salt ion stability: NaCl was added to the emulsion to a final concentration of 0, 100, 300, and 500 mmol / L, and the mixture was allowed to stand for 2 h to evaluate its stability.

[0047] (4) Centrifugal stability: The centrifugal stability of the emulsion is characterized by measuring the emulsification index. The specific steps are as follows: place the emulsion in a centrifuge tube, centrifuge at 13000 rpm for 2 min, and then use a vernier caliper to measure the height of the emulsion before and after centrifugation. Calculate the emulsification index (CI) value according to formula (1).

[0048] CI=[1-(H w -H e )]×100%, Equation (1); In the formula, H w H represents the height of the upper aqueous phase after centrifugation of a high internal phase emulsion. e The total height of the high internal phase emulsion before centrifugation.

[0049] (5) Flocculation stability: The flocculation index (FI, %) of the emulsion was calculated by measuring the volume-weighted average diameter in distilled water and 1% (w / v) SDS solution, respectively. The flocculation index was calculated by equation (2): Flocculation index (%) = [(D w / D s )-1]×100%, Equation (2); In the formula, D w D represents the particle size of the emulsion in distilled water. s This represents the particle size of the emulsion in a 1% SDS solution. This calculation method assumes that anionic SDS molecules adsorb onto the surface of oil droplets, displacing the original emulsifier and breaking up agglomerates through strong electrostatic repulsion. If there is no agglomeration, FI = 0%; the greater the degree of agglomeration, the higher the FI value.

[0050] (6) Storage stability: Freshly prepared emulsions were stored at 4°C in the dark for 28 days. Samples were taken at 0, 7, 14, 21 and 28 days to measure the particle size and observe the appearance changes in order to evaluate the stability of the emulsions during storage.

[0051] Figure 6 and Figure 7 In this text, S10, S30, and S50 represent the protein concentrations of SPI as 10, 30, and 50 mg / mL, respectively. S represents SPI, SQ indicates a SPI to Que mass ratio of 25:1, and S-QFE indicates a SPI to Que mass ratio of 25:1, with the addition of iron ions, resulting in a Que to Fe ratio... 3+ The mass ratios are 10:1.

[0052] S10-QFE, S30-QFE, and S50-QFE represent the corresponding SPI:Que ​​mass ratios of 25:1, Que:Fe, etc., at different SPI concentrations, respectively. 3+ A ternary complex with a mass ratio of 10:1.

[0053] (7) Thermal stability analysis: like Figure 6 As shown in Figure A, the overall particle size of the emulsions in each group increased with increasing treatment temperature, indicating that high-temperature treatment promotes droplet aggregation or interfacial film structure disruption. However, at the same temperature, the particle size consistently showed the order S>SQ>S-QFE, indicating that Que and Fe... 3+The introduction of [the substance] can enhance the interfacial film strength of the composite and reduce the impact of heat treatment on emulsion stability. Among them, the S-QFE group maintained a small particle size at different temperatures, exhibiting good thermal stability.

[0054] (8) pH stability analysis: like Figure 6 As shown in Figure B, the particle size of each emulsion group changed under different pH conditions. The overall particle size was smaller at pH 7.0, indicating that neutral conditions were more conducive to the stability of the emulsion system. Compared with group S, the particle size of group SQ decreased, indicating that the introduction of Que improved the interfacial stabilization effect of SPI; further introduction of Fe... 3+ Afterwards, the S-QFE group had the smallest particle size, indicating that SPI-Que-Fe 3 + Ternary complexes can improve the tolerance of emulsions to changes in acid and alkaline environments.

[0055] (9) Salt ion stability analysis: like Figure 6 As shown in Figure C, the particle size of the emulsions in all groups increased with increasing NaCl concentration, indicating that salt ions shield the surface charge of the droplets, reducing electrostatic repulsion between droplets and thus promoting droplet aggregation. Compared to groups S and SQ, the particle size of group S-QFE was smaller at all salt concentrations, indicating that SPI-Que-Fe 3+ The composite interface layer can effectively resist salt ion-induced aggregation and improve the ionic stability of the emulsion.

[0056] (10) Centrifugal stability analysis: like Figure 6 As shown in Figure D, the centrifugal stability index of each emulsion group increased with increasing SPI concentration, indicating that higher protein concentrations can form a more complete interfacial protective layer and improve the emulsion's resistance to centrifugal stratification. At the same concentration, the S-QFE group had the highest centrifugal stability index, followed by the SQ group, and the S group had the lowest, indicating that SPI-Que-Fe... 3+ Ternary complexes can significantly enhance the structural stability of emulsions and reduce aqueous phase precipitation and stratification.

[0057] (11) Flocculation Index Analysis: like Figure 6 As shown in Figure E, the flocculation index of all three emulsion groups decreased with increasing SPI concentration, indicating that increasing the protein concentration helps enhance interfacial coverage and reduce droplet flocculation. At the same SPI concentration, the flocculation index showed the order S>SQ>S-QFE, indicating that Que and Fe... 3+ The synergistic effect of these components can reduce the degree of droplet aggregation. Among them, the S-QFE group had the lowest flocculation index, indicating that it had the strongest anti-flocculation ability.

[0058] (12) Storage stability analysis: like Figure 6 As shown in Figure F, during 28 days of storage at 4℃ in the dark, the particle size of all emulsion groups gradually increased with the extension of storage time, indicating that long-term storage causes a certain degree of droplet aggregation. Compared with group S, the particle size increase of groups SQ and S-QFE was smaller. Among them, group S-QFE maintained the lowest particle size throughout the entire storage period, indicating that the ternary complex can effectively delay the aggregation and instability of emulsions during storage and improve the long-term storage stability of emulsions.

[0059] Based on the combined results of temperature, pH, salt ion concentration, centrifugal stability, flocculation index, and storage stability, the S-QFE group emulsion exhibits the best overall stability. This demonstrates that the SPI-Que-Fe emulsion constructed in this invention... 3+ Ternary complexes can form a more stable composite interface layer through non-covalent interactions and metal-phenol coordination, thereby improving the stability of emulsions under processing, storage and complex environmental conditions.

[0060] (13) Appearance of emulsion after 28 days of storage Depend on Figure 7 It can be seen that, in terms of appearance, the freshly prepared emulsions are uniformly dispersed and do not show obvious oil-water separation. The S group emulsion is milky white, the SQ group is light yellow due to the introduction of Que, and the S-QFE group is yellow due to Fe. 3+ When Que coordinates with Fe, the color deepens further, turning from yellow to brownish-yellow, indicating that Que and Fe... 3+ We have successfully participated in the construction of emulsion systems.

[0061] With prolonged storage, all emulsion groups exhibited varying degrees of appearance changes. Group S, especially the low-concentration S10 group, showed relatively poor stability, easily exhibiting emulsion heterogeneity, sedimentation, or slight stratification after storage. Group SQ showed a more uniform overall appearance than Group S, indicating that the introduction of Que helped improve the storage stability of the SPI emulsion. Group S-QFE maintained good overall dispersion after 28 days of storage, with no obvious oil phase precipitation, indicating that Fe... 3+ This further enhances the protective effect of the SPI-Que complex on emulsion droplets.

[0062] Comparison of different protein concentrations shows that as the SPI concentration increased from 10 mg / mL to 50 mg / mL, the overall appearance and stability of the emulsion improved. Higher protein concentrations provide more surfactants to cover the oil-water interface, reducing droplet aggregation and stratification; simultaneously, Que and Fe... 3+ The synergistic effect of these components can form a denser composite interface layer. Therefore, the S-QFE group, especially the S30-QFE and S50-QFE at higher protein concentrations, exhibits better storage appearance stability.

[0063] Visual observations indicate that the storage stability of SPI-stabilized emulsions alone is limited, while the introduction of Que and Fe... 3+ Further coordination assembly can improve the homogeneity and anti-stratification ability of the emulsion system, indicating that the SPI-Que-Fe constructed in this invention... 3+ The complex can effectively improve the stability of emulsions during long-term storage.

[0064] 6. β-Carotene loading rate Take SPI-Que-Fe loaded with β-carotene 3+ The ternary complex high internal phase emulsion sample was mixed with a mixed extractant (n-hexane: anhydrous ethanol = 2:1, v / v) at a ratio of 1:4 (v / v), and centrifuged after thorough mixing. The upper organic phase was collected. The absorbance was measured at 450 nm, and the β-carotene encapsulation rate in the emulsion was calculated according to formula (3).

[0065] EE (%) = (entrapped β-carotene) / (total mass of input β-carotene) × 100%; formula (3); In the formula, entrapped β-carotene represents the actual mass of β-carotene encapsulated in the emulsion system after extraction and determination, in mg; total mass of input β-carotene represents the total mass of β-carotene initially added during emulsion preparation, in mg; and β-carotene represents β-carotene.

[0066] The results are as follows Figure 8 As shown, Figure 8 In the diagram, the horizontal axis represents the concentration of SPI as 30 mg / mL, S30-Q represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1, and S30-QFE represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1. Based on this, iron ions are introduced, and the ratio of Que to Fe... 3+ The quality ratios are 10:1; the vertical axis represents the retention rate. Depend on Figure 8It can be seen that there are significant differences in the loading rates of β-carotene in different emulsion systems, and generally S30 < S30-Q < S30-QFE. Among them, the loading rate of the S30 emulsion stabilized by SPI alone is relatively low, about 54%, indicating that the interfacial film formed only by SPI has limited embedding and retention capabilities for β-carotene. After introducing Que, the loading rate of the S30-Q group increased to about 66%, indicating that after Que forms a complex with SPI, it can improve the interfacial adsorption ability of protein molecules and the stability of the emulsion structure, thereby reducing the loss of β-carotene during the preparation and extraction processes.

[0067] After further introducing Fe 3+ , the loading rate of the S30-QFE group increased significantly, reaching about 83%, which is the highest among the three groups. This result shows that Fe 3+ can undergo metal–phenol coordination with the phenolic hydroxyl groups in Que and further promote the formation of the ternary complex structure of SPI-Que-Fe 3+ , making the emulsion interfacial layer denser and more stable. The stronger composite interfacial film can more effectively encapsulate the oil-phase droplets, restrict the migration or loss of β-carotene to the outer phase, and thus improve its retention ability in the high internal phase emulsion.

[0068] In summary, the SPI-Que-Fe 3+ ternary complex has a better β-carotene loading effect than the single SPI and the SPI-Que binary complex, indicating that the ternary complex constructed in this invention can be used as an effective carrier for loading lipophilic active substances.

[0069] 7. In vitro simulated digestion Mouth: Mix the high internal phase emulsion sample loaded with β-carotene with artificial saliva at a volume ratio of 1:1 (v / v), and oscillate and digest at 37 °C and 100 rpm. The oral digestion time is set to 10 min. After the digestion ends, take samples and immediately measure the particle size and Zeta-potential of the samples.

[0070] Stomach: After the oral digestion ends, mix the solution after oral digestion with artificial gastric juice at a volume ratio of 1:1 (v / v), and adjust the final pH of the system to 2.0 with 0.5 mol / L HCl solution. Then continue to oscillate and digest at 37 °C and 100 rpm for 2 h. After the gastric digestion ends, take samples and immediately measure the particle size and Zeta-potential of the samples.

[0071] Small Intestine: After gastric digestion, the pH of the digested solution was adjusted to 7.0 with 0.5 mol / L NaOH solution. This solution was then mixed with artificial small intestinal fluid at a volume ratio of 10:3 (v / v) and subjected to continued shaking digestion at 37℃ and 100 rpm for 2 h. During digestion, 1M NaOH solution was used to adjust the system in real time to neutralize the released free fatty acids and maintain the pH constant between 7.0 and 7.2. Samples were taken immediately after small intestinal digestion, and the particle size and zeta potential were immediately measured.

[0072] Particle size results as follows Figure 9 As shown, the Zeta-potential results are as follows: Figure 10 As shown.

[0073] Figure 9 and Figure 10 In the diagram, the horizontal axis represents the concentration of SPI as 30 mg / mL, S30-Q represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1, and S30-QFE represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1. Based on this, iron ions are introduced, and the ratio of Que to Fe... 3+ The mass ratios are 10:1; Depend on Figure 9 It can be seen that the particle size of the β-carotene-loaded high internal phase emulsions changed significantly during in vitro simulated digestion. In the oral cavity stage, the particle size of all emulsion groups was relatively small, indicating that short-term oral digestion had a weak impact on the emulsion structure, and the system maintained a good dispersion state. After entering the gastric digestion stage, the particle size of all groups increased significantly, indicating that under the acidic environment of pH 2.0 and the action of gastric juice, the emulsion interface layer was damaged to some extent, and aggregation or flocculation occurred between the droplets. Among them, the particle size increase was more significant in the S30 and S30-Q groups, while the particle size of the S30-QFE group was relatively small, indicating that the SPI-Que-Fe group... 3+ The interfacial membrane formed by the ternary complex exhibits stronger resistance to gastric acid damage. Upon entering the small intestine, the emulsion particle size decreases compared to the gastric stage, possibly due to the replacement and dispersion of the emulsion droplet interfacial layer by digestive components such as bile salts and pancreatic enzymes. After small intestinal digestion, the overall particle size distribution is S30 > S30-Q > S30-QFE, with the S30-QFE group showing the lowest particle size, indicating the presence of Que and Fe. 3+ The resulting ternary composite interface structure can effectively slow down the aggregation of milk droplets during digestion, which is beneficial to maintaining the digestive stability of β-carotene-loaded emulsions.

[0074] Depend on Figure 10It can be seen that the Zeta potentials of all emulsion groups were negative at different digestion stages, indicating that the surface of the emulsion droplets carried a certain negative charge. In the oral cavity stage, the S30, S30-Q, and S30-QFE groups all exhibited high absolute values ​​of negative potential, indicating a certain electrostatic repulsion between the droplets, which helps maintain system stability. After entering the gastric digestion stage, the absolute values ​​of the Zeta potentials of all groups decreased significantly, mainly because the low pH environment of the stomach weakened the negative charge on the surface of the droplets and compressed the electrostatic repulsion between them, making aggregation more likely. Compared to the S30 and S30-Q groups, the S30-QFE group maintained a higher absolute value of negative potential in the gastric stage, indicating that Fe... 3+ The ternary composite interface layer involved in its formation can enhance the emulsion's resistance to gastric acid. Upon entering the small intestine, the absolute values ​​of the zeta potential in each group increased again, which may be related to the adsorption of bile salts, free fatty acids, and digestive products onto the surface of the emulsion droplets, thus increasing the negative charge on the droplet surface. Overall, the S30-QFE group exhibited good charge stability at all stages of digestion, indicating that the SPI-Que-Fe... 3+ The ternary complex can enhance the structural stability of the emulsion interface, thereby improving the stability of β-carotene-loaded emulsions during in vitro digestion.

[0075] 8. Biological accessibility After small intestinal digestion is completed, the liquid centrifugation is cancelled; the intermediate micelle phase is collected, and the content of β-carotene in it is determined according to the β-carotene loading rate method, and its bioavailability is calculated according to formula (4).

[0076] Bioaccessibility (%) = C micelle / C original ×100%; Equation (4); In the formula, C micelle This indicates the concentration (g / mL) of (β-carotene / active ingredient) in the intermediate micelle phase. C original This indicates the initial concentration (g / mL) of (β-carotene / active ingredient) in the digestive fluid.

[0077] The results are as follows Figure 11 As shown in the figure, the horizontal axis represents the concentration of SPI as 30 mg / mL, S30-Q represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1, and S30-QFE represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1. Based on this, iron ions are introduced, and the ratio of Que to Fe... 3+ The mass ratios are 10:1; the vertical axis, Bioaccessibility, represents biological accessibility.

[0078] Depend on Figure 11It can be seen that there are differences in the bioaccessibility of β-carotene in different emulsion systems, and the overall trend is S30 < S30-Q < S30-QFE. Among them, the bioaccessibility of the S30 group is the lowest, at 19.76%, indicating that the high internal phase emulsion stabilized by SPI alone has relatively limited ability to release and micellarize β-carotene during digestion. After introducing Que, the bioaccessibility of the S30-Q group increased to 29.68%, suggesting that the SPI-Que complex can improve the emulsion interfacial structure and dispersion state during digestion, which is beneficial to the release of β-carotene from the oil phase and its entry into the mixed micelle phase.

[0079] After further introducing Fe 3+ , the bioaccessibility of the S30-QFE group further increased to 35.35%, which is the highest among the three groups. This result shows that after Fe 3+ forms a metal–phenol coordination with Que, it can promote the formation of the SPI-Que-Fe 3+ ternary composite interfacial layer, enabling the emulsion to maintain better structural stability during gastrointestinal digestion, reducing excessive droplet aggregation, and thus providing more favorable conditions for lipid digestion and β-carotene micellarization transfer in the small intestine stage.

[0080] In summary, the high internal phase emulsion stabilized by the SPI-Que-Fe 3+ ternary complex can improve the bioaccessibility of β-carotene after in vitro digestion, indicating that this ternary composite system not only has good loading capacity but also can improve the release and utilization effects of lipophilic active substances during digestion.

[0081] 9. Stability of High Internal Phase Emulsion Loaded with β-Carotene (1) Light stability: The high internal phase emulsion loaded with β-carotene was placed under ultraviolet light treatment, and samples were taken at 2, 4, and 6 h of irradiation. The determination of β-carotene content was carried out using the method described for the loading rate of β-carotene.

[0082] (2) Thermal stability: The high internal phase emulsion loaded with β-carotene was placed in a water bath at 30, 50, and 70 °C for 2 h. After the treatment, it was cooled to room temperature and sampled, and the determination of β-carotene content was carried out using the method described for the loading rate of β-carotene.

[0083] (3) Storage stability: The high internal phase emulsion loaded with β-carotene was stored at 4 °C, and samples were taken at 10, 20, and 30 d of storage. The determination of β-carotene content was carried out using the method described for the loading rate of β-carotene.

[0084] The results are as Figure 12As shown in the figure, the horizontal axis represents the concentration of SPI as 30 mg / mL, S30-Q represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1, and S30-QFE represents the concentration of SPI as 30 mg / mL with a SPI to Que mass ratio of 25:1. Based on this, iron ions are introduced, and the ratio of Que to Fe... 3+ The quality ratios are 10:1; the vertical axis represents the retention rate. (4) Illumination stability analysis: like Figure 12 As shown in Figure A, with the UV irradiation time increasing from 2 h to 6 h, the residual β-carotene in all emulsion groups decreased significantly, indicating that β-carotene is sensitive to light and easily degrades. Under the same irradiation time, the overall residual β-carotene level showed the order of S30-QFE > S30-Q > S30. Among them, the S30-QFE group maintained the highest residual level after 2, 4, and 6 h of irradiation, indicating that SPI-Que-Fe... 3+ The emulsion interface layer formed by the ternary complex can more effectively block the damage of β-carotene to light and improve its light stability.

[0085] (5) Thermal stability analysis: like Figure 12 As shown in Figure B, the residual β-carotene content in each group gradually decreased as the treatment temperature increased from 30℃ to 70℃, indicating that high temperature accelerates β-carotene degradation. Compared with group S30, groups S30-Q and S30-QFE showed higher residual β-carotene content after treatment at different temperatures. Among them, group S30-QFE had the highest residual content, indicating that the introduction of Fe³⁺ enhanced the stability and density of the SPI-Que composite interfacial film, thereby improving the emulsion's resistance to heat treatment and reducing the thermal degradation of β-carotene.

[0086] (6) Storage stability analysis: like Figure 12 As shown in Figure C, the residual β-carotene levels in all groups decreased as the storage time increased from 10 days to 30 days, indicating that β-carotene is lost to some extent during long-term storage. Under the same storage time, the S30-QFE group had the highest residual level, followed by the S30-Q group, and the S30 group had the lowest. This result indicates that the introduction of Que can enhance the protective effect of the emulsion on β-carotene, while Fe... 3+ After further participation in coordination assembly, it can form a more stable ternary complex interface structure, thereby delaying the degradation of β-carotene during storage and improving its long-term storage stability.

[0087] In summary, SPI-Que-Fe 3+The ternary complex-stabilized high internal phase emulsion exhibited higher β-carotene residues under light, high temperature, and storage conditions, indicating that the ternary complex system can effectively improve the environmental stability of β-carotene.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of the present invention.

Claims

1. A SPI-Que-Fe for loading beta-carotene 3+ Process for the preparation of a ternary complex high internal phase emulsion, characterized in that: Includes the following steps: S1. Preparation of SPI dispersion: SPI was dispersed in deionized water, the pH was adjusted to 12.0, and the mixture was stirred at room temperature for 2 hours to allow the protein to be fully hydrated and expanded, thus obtaining the SPI dispersion. S2. Preparation of SPI-Que binary complex: Que was pre-dissolved in anhydrous ethanol, and then SPI dispersion was added dropwise to prepare a mixture of SPI and Que. The mixture was stirred in the dark for 20 min. The pH of the system was then adjusted to 7.

0. Residual ethanol was removed by rotary evaporation at 40 °C to obtain the SPI-Que binary complex dispersion. S3, Preparation of SPI-Que-Fe 3+ Ternary complex: Take the SPI-Que binary complex dispersion and add FeCl3•5H2O aqueous solution dropwise to it. Vortex mix for 10 min to obtain SPI-Que-Fe 3+ The ternary complex dispersion was kept at 4°C. S4. Preparation of a high-internal-phase emulsion of a ternary complex: Adjusting SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion was used as the continuous aqueous phase; MCT was used as the oil phase; the oil phase was added dropwise to the continuous aqueous phase; and homogenization was performed at 10,000 rpm for 1 min to obtain SPI-Que-Fe. 3+ Ternary complex high internal phase emulsion; S5. Preparation of a high internal phase emulsion loaded with β-carotene: Adjusting SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion was used as the continuous aqueous phase; β-carotene was dissolved in MCT as the oil phase; the oil phase was added dropwise to the continuous aqueous phase, and homogenization was performed at 10,000 rpm for 1 min to obtain SPI-Que-Fe loaded with β-carotene. 3+ Ternary complex high internal phase emulsion, stored at 4°C protected from light.

2. A SPI-Que-Fe for loading β-carotene according to claim 1 3+ Process for the preparation of a ternary complex high internal phase emulsion, characterized in that: In S1, the SPI is dispersed in deionized water at a concentration of 10 mg / mL; The pH was adjusted using a 0.5 mol / L NaOH solution.

3. A SPI-Que-Fe for loading β-carotene according to claim 1 3+ Process for the preparation of a ternary complex high internal phase emulsion, characterized in that: In S2, the Que is dissolved in anhydrous ethanol at a concentration of 10 mg / mL; In the mixture, the mass ratio of SPI to Que is 25:1; The pH was adjusted using a 0.5 mol / L HCl solution.

4. The SPI-Que-Fe for loading β-carotene according to claim 1 3+ A method for preparing a ternary complex high internal phase emulsion, characterized in that: In S3, the SPI-Que-Fe 3+ In the ternary complex dispersion, the mass ratio of Que to Fe 3+ is any one of 10:1, 20:1 and 30:1; The concentration of the FeCl3•5H2O aqueous solution is 10 mg / mL.

5. The SPI-Que-Fe according to claim 1 3+ A method for preparing a ternary complex high internal phase emulsion, characterized in that: In S4, SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion was adjusted to any one of 10 mg / mL, 30 mg / mL, and 50 mg / mL; The oil phase is added dropwise to the continuous aqueous phase, and the volume fraction of the oil phase is controlled to be 75%.

6. The SPI-Que-Fe for loading β-carotene according to claim 1 3+ A method for preparing a ternary complex high internal phase emulsion, characterized in that: In S5, the SPI-Que-Fe 3+ The protein concentration of the ternary complex dispersion is 30 mg / mL; the concentration of β-carotene in the oil phase is 1 mg / mL; the oil phase is added dropwise to the continuous aqueous phase, and the volume fraction of the oil phase is controlled to be 75%.

7. A SPI-Que-Fe prepared using the preparation method according to any one of claims 1-6 3+ Ternary complex.

8. SPI-Que-Fe prepared by the method of any one of claims 1-6 3+ Triple complex high internal phase emulsion.

9. A stable SPI-Que-Fe loaded with β-carotene prepared by the method of any one of claims 1-6 3+ Ternary complex high internal phase emulsions.

10. A SPI-Que-Fe as claimed in claim 8. 3+ Use of a ternary complex high internal phase emulsion in the delivery of a fat-soluble active, a plant protein based functional food or a nutritional emulsion.

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