Application of SPI-PD-EGCG in improving bioaccessibility of beta-carotene

Abstract

The invention relates to the technical field of nutrient delivery systems, in particular to application of SPI-PD-EGCG to improvement of bioaccessibility of beta-carotene, the SPI-PD-EGCG is a ternary covalent complex, the SPI-PD-EGCG and the beta-carotene are prepared into Pickering emulsion, the SPI-PD-EGCG is prepared through a wet Maillard reaction in combination with alkali treatment, and the SPI-PD-EGCG is prepared into the beta-carotene. The reaction sequence is that the SPI-PD covalent complex is prepared firstly and then the EGCG is covalently bound. The SPI-PD-EGCG stable emulsion disclosed by the invention generates the highest bioaccessibility, and the excellent performance possibly derives from a synergistic mechanism of a reaction sequence: a physical barrier derived from PD regulates interfacial displacement and lipase contact, and the grafted EGCG protects beta-carotene from oxidative degradation in the whole digestion process, and finally, optimal micellization is achieved. In conclusion, the SPI-PD-EGCG ternary covalent composite stable emulsion not only has a good embedding effect and physical and chemical stability, but also has the advantage of promoting absorption of beta-carotene.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of nutrient component delivery systems, and particularly relates to application of SPI-PD-EGCG in improving the bioaccessibility of beta-carotene. BACKGROUND

[0002] Pickering emulsion refers to an emulsion stabilized by solid colloidal particles through irreversible adhesion on the oil-water interface, rather than a small molecule or a biopolymer stabilized emulsion, and the particles used to stabilize the Pickering emulsion can be partially wetted by the oil phase and the water phase at the same time, so as to stabilize the oil-water interface. Therefore, the Pickering emulsion is widely used as a carrier for effectively encapsulating bioactive substances and improving the bioaccessibility of the bioactive substances, and proteins, polysaccharides, polyphenols and their complexes are usually used to stabilize the Pickering emulsion.

[0003] Chinese published patent document (CN121040618A) discloses a protein-polysaccharide-polyphenol ternary complex with excellent emulsifying performance and the use of the ternary complex as an interfacial active agent of Pickering emulsion. First, whey protein is modified to prepare protein nanofibers and a covalent complex of polyphenol and pectin is synthesized, then the whey protein nanofibers are combined with the pectin-polyphenol covalent complex in a non-covalent interaction mode to form a final product. Experimental results show that the ternary complex has very excellent interfacial stability.

[0004] Chinese published patent document (CN121336993A) discloses a preparation method of a protein-polyphenol-polysaccharide novel emulsifier and application of the novel emulsifier in the field of Pickering emulsion. A complex formed by whey protein isolate, grape seed oligomeric proanthocyanidin and lycium barbarum polysaccharide is formed through interaction, and through covalent crosslinking of the whey protein isolate and the grape seed oligomeric proanthocyanidin under alkaline conditions, and the space steric hindrance effect of the lycium barbarum polysaccharide, a novel emulsifier with high emulsifying activity and antioxidant capacity is formed, which is beneficial to preparation of stable Pickering emulsion rich in various functional components.

[0005] Chinese published patent document (CN118525916A) discloses a preparation method of a ternary covalent complex. First, a quinone substance formed by catalyzing gallic acid (GA) by laccase is reacted with nucleophilic groups (free amino groups, free sulfhydryl groups and free tryptophan) on a protein to form a soybean protein isolate-gallic acid complex (SPI-GA); then the complex is induced to occur a Maillard reaction with guar gum (GG) to form a ternary covalent complex (SPI-GA-GG), and the ternary complex can improve the stability of emulsion gels and provide strong antioxidant activity.

[0006] Beta-carotene is a fat-soluble carotenoid, which is prone to isomerization and oxidative degradation due to the presence of unsaturated double bonds, resulting in poor stability. The above prior art focuses on how to construct a ternary complex of protein, polysaccharide and polyphenol as an emulsifier for Pickering emulsion and play a role in emulsion stability and antioxidant activity, which is sufficient to achieve the purpose of emulsion stability.

[0007] However, the in vivo bioavailability of beta-carotene is very low, and its absorption and transport mechanism is as follows: after chewing by teeth in the oral cavity and catalysis by related enzymes, it reaches the stomach, during which beta-carotene is gradually released and dispersed and dissolved in oil droplets under the action of gastric peristalsis, enzymes and emulsification; after entering the duodenum, the oil droplets are hydrolyzed into free fatty acids and monoglycerides under the dual digestion of pancreatic juice and bile, beta-carotene is released from the oil droplets, and mixed micelles are formed together with free fatty acids, phospholipids, cholesterol, bile salts, etc.; the mixed micelles pass through the mucus layer on the surface of the intestinal cells and reach the surface of the small intestinal epithelial cells by simple diffusion, the fatty acids in the micelles are protonated under acidic conditions and fall off from the micelles, the micelles dissociate, and the combined beta-carotene is released and absorbed by the small intestinal cells. As can be seen, the emulsification and micellization of beta-carotene in the digestive tract will greatly affect its bioavailability.

[0008] Therefore, it is crucial to design a suitable drug delivery system to enhance the bioactivity of beta-carotene. Soybean protein isolate (SPI) is an optional protein in protein delivery systems, such as Chinese published patent document (CN118525916A), but all focus on the application of improving emulsion stability and antioxidant activity, and there is no application of ternary complex constructed by soybean protein isolate in improving the bioavailability of beta-carotene. SUMMARY

[0009] The purpose of the present application is to provide an application of SPI-PD-EGCG in improving the bioavailability of beta-carotene, to explore the influence of emulsion stability and digestion characteristics, and to exhibit high bioavailability of beta-carotene in the in vitro digestion process.

[0010] The application of SPI-PD-EGCG in improving the bioavailability of beta-carotene, wherein the SPI-PD-EGCG is a ternary covalent complex, and the SPI-PD-EGCG is prepared into a Pickering emulsion with beta-carotene.

[0011] Preferably, the SPI-PD-EGCG is prepared by wet Maillard reaction combined with alkali treatment, and the reaction sequence is to prepare SPI-PD covalent complex first and then covalently combine EGCG.

[0012] Preferably, the SPI and PD in the SPI-PD covalent complex preparation step have a mass ratio of 4±0.5:1, and the amount of EGCG in the EGCG covalent binding step is 0.015±0.005wt% of the SPI-PD solution.

[0013] Preferably, the oil phase and the water phase in the SPI-PD-EGCG and β-carotene preparation Pickering emulsion step are mixed according to a mass ratio of 9±0.5:1, the concentration of SPI-PD-EGCG in the water phase is 1±0.5% (w / v), and the mass fraction of β-carotene in the Pickering emulsion is 0.1±0.05%.

[0014] Compared with the prior art, the present application has the following beneficial effects: Bioaccessibility is determined by measuring the β-carotene content in the mixed micelles produced after the digestion of the emulsion ends. With the enzymatic hydrolysis of oil droplets during digestion, the β-carotene embedded inside is released and can be dissolved in micelles formed by the mutual combination of monoglycerides, free fatty acids, bile salts and phospholipids, etc. The β-carotene existing in the form of micelles can be absorbed by the human body. The SPI-PD-EGCG stabilized emulsion produced by the present application has the highest bioaccessibility, and this excellent performance may be due to the synergistic mechanism of the reaction sequence: the physical barrier derived from PD regulates the interface displacement and the contact of lipase, and the grafted EGCG protects the β-carotene from oxidative degradation during the entire digestion process to achieve optimal micellization. In summary, the SPI-PD-EGCG ternary covalent complex stabilized emulsion not only has good embedding effect and physicochemical stability, but also has the advantage of promoting the absorption of β-carotene. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1-1 The SPI backscattering BS intensity change curve of the present application.

[0016] Figure 1-2 The SPI-EGCG backscattering BS intensity change curve of the present application.

[0017] Figure 1-3 The SPI-PD backscattering BS intensity change curve of the present application.

[0018] Figure 1-4 The SPI-EGCG-PD backscattering BS intensity change curve of the present application.

[0019] Figure 1-5 The SPI-PD-EGCG backscattering BS intensity change curve of the present application.

[0020] Figure 2 The stability index TSI curve of the five emulsions of the present application.

[0021] Figure 3-1 Figure 5 is a graph showing the change in particle size of the five emulsions of the present application under different pH conditions.

[0022] Figure 3-2 Figure 6 is a graph showing the change in zeta potential of the five emulsions of the present application under different pH conditions.

[0023] Figure 4-1 Figure 7 is a graph showing the change in particle size of the five emulsions of the present application after heat treatment.

[0024] Figure 4-2 Figure 8 is a graph showing the change in zeta potential of the five emulsions of the present application after heat treatment.

[0025] Figure 4-3 Figure 9 is a graph showing the retention rate of β-carotene of the five emulsions of the present application after heat treatment.

[0026] Figure 5-1 Figure 10 is a graph showing the change in particle size of the five emulsions of the present application under different sodium ion concentrations.

[0027] Figure 5-2 Figure 11 is a graph showing the change in zeta potential of the five emulsions of the present application under different sodium ion concentrations.

[0028] Figure 6-1 Figure 12 is a graph showing the change in particle size of the five emulsions of the present application after storage for different days.

[0029] Figure 6-2 Figure 13 is a graph showing the change in zeta potential of the five emulsions of the present application after storage for different days.

[0030] Figure 6-3 Figure 14 is a graph showing the retention rate of β-carotene of the five emulsions of the present application after storage for different days.

[0031] Figure 6-4 Figure 15 is a photograph showing the storage stability of the five emulsions of the present application after storage for different days.

[0032] Figure 7-1 Figure 16 is a graph showing the lipid peroxide curve of the five emulsions of the present application after storage for different days.

[0033] Figure 7-2 Figure 17 is a graph showing the TBARS curve of the five emulsions of the present application after storage for different days.

[0034] Figure 8-1 Figure 18 is a graph showing the change in particle size of the five emulsions of the present application during simulated gastrointestinal digestion.

[0035] Figure 8-2 Figure 19 is a graph showing the change in zeta potential of the five emulsions of the present application during simulated gastrointestinal digestion.

[0036] Figure 9 Figure 20 is a graph showing the bioaccessibility of the five emulsions of the present application.

[0037] Figure 10 Free fatty acid release rate control chart of five emulsions of the present application.

[0038] Figure 11 Laser confocal scanning microscope chart of five emulsions of the present application. DETAILED DESCRIPTION

[0039] 1. Materials Soybean protein isolate (SPI) was extracted by ourselves, with a purity of 92.8 ± 0.31%. Epigallocatechin gallate (EGCG) ≥ 98%, polydextrose (PD) ≥ 90%, β-carotene (96%), soybean oil, thiobarbituric acid, methanol, n-butanol, n-hexane, isopropyl, anhydrous ethanol, Nile red, pepsin, pancreatin, porcine bile salt, isooctane, isopropyl alcohol, ammonium thiocyanate, BaCl2, FeSO4 were purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0040] 2. Preparation of covalent complexes 2.1. Preparation of SPI-EGCG covalent complex The SPI solution (10 mg / mL) was stirred at 4°C for 24 h to fully hydrate; the pH of the solution was adjusted to 9.0; 0.015 wt% EGCG was added to the protein solution, and the pH was maintained at 9.0; after stirring for 24 h, the pH of the mixed solution was adjusted to 7.0; the unbound polyphenol was removed by dialysis at 4°C for 48 h, and freeze-dried into a powder; the freeze-dried sample was stored at 4°C for standby.

[0041] 2.2. Preparation of SPI-PD covalent complex The SPI-PD covalent complex was prepared by a wet Maillard reaction, SPI (1.0 g) was dissolved in deionized water (100 mL), and the solution was stirred at 4°C for 24 h to fully hydrate; SPI: PD was dissolved in the SPI hydrated solution at a mass ratio of 4:1, and stirred magnetically for 4 h, the pH value of the system was adjusted to 7, after stirring at 95°C for 1 h, the reaction was quickly cooled to room temperature, and freeze-dried into a powder.

[0042] 2.3. Preparation of SPI-PD-EGCG covalent complex Referring to the preparation method of 2.1, SPI-EGCG-PD ternary covalent complex emulsifier was prepared by replacing SPI in the alkali treatment with SPI-PD covalent complex.

[0043] 2.4. Preparation of SPI-EGCG-PD covalent complex Referring to the preparation method of 2.2, SPI-EGCG-PD ternary covalent complex emulsifier was prepared by replacing SPI in the Maillard reaction with SPI-EGCG covalent complex.

[0044] 3. Preparation of β-carotene emulsion SPI, SPI-EGCG, SPI-PD, SPI-EGCG-PD, and SPI-PD-EGCG covalent complexes were dispersed in deionized water to prepare a 1% (w / v) protein concentration sample solution. The solution was stirred overnight at 4°C to ensure complete hydration and served as the aqueous phase. β-carotene was dispersed in soybean oil and stirred at room temperature until the β-carotene was completely dissolved to prepare the oil phase. The oil phase and aqueous phase were mixed at a mass ratio of 9:1 for 2 min at 12000 rpm to obtain a crude emulsion. The crude emulsion was homogenized three times under high pressure at an operating pressure of 60 MPa to obtain a refined emulsion. The mass fraction of β-carotene in the refined emulsion was 0.1%.

[0045] 4. Detection indicators 4.1 Multiple light scattering The emulsion (20 mL) was placed in a flat-bottomed cylindrical glass tube and analyzed using a stability analyzer at a wavelength of 880 nm. Backscattering (BS) and transmission (T) data were recorded every 2 minutes for 2 hours. The stability index (TSI) was calculated based on the changes in T and BS intensities. The TSI was calculated using equation (1).

[0046]

[0047] 4.2 Standard Curve of β-Carotene 5 mg of β-carotene standard was dissolved in n-hexane and diluted to 25 mL with n-hexane to prepare a β-carotene standard solution. Then, 500 μL of the above standard solution was diluted to 10 mL with n-hexane. 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of this solution were taken and diluted to 10 mL with n-hexane again to obtain standard solutions with β-carotene concentrations of 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, and 5 μg / mL, respectively. Using deionized water as a blank control, the absorbance at 450 nm was measured to obtain a standard curve of absorbance versus β-carotene.

[0048] 4.3 Retention rate of β-carotene Take 20 μL of β-carotene emulsion, dilute it 50 times, and extract it three times with a mixture of 1 mL of ethanol and n-hexane (1:1, v / v). Combine the upper extracts and measure their absorbance at 450 nm. Calculate the β-carotene content according to the standard curve. The retention rate of β-carotene was used for chemical stability analysis, and the results are expressed as C / C0.

[0049]

[0050] C represents the actual concentration of β-carotene at any stage, and C0 represents its original concentration.

[0051] 4.4 Particle size and Zeta potential The particle size and zeta potential of the emulsion were accurately determined using a dynamic optical nanoparticle size potentiometer. Samples were diluted 1000-fold before testing to reduce measurement errors caused by multiple light scattering. Each sample was analyzed three times, and the results are expressed as the average value.

[0052] 4.5 Determination of Environmental Stability 4.5.1 pH stability The pH of the emulsions prepared from different complexes was adjusted to 2.0-9.0 using appropriate concentrations of NaOH and HCl solutions, respectively. They were then allowed to stand at room temperature in the dark for 24 hours before their particle size and potential were measured.

[0053] 4.5.2 Ion stability NaCl solution was added to 9 mL of fresh emulsion at salt ion concentrations of 50 mM, 100 mM, and 200 mM for β-carotene emulsions. The samples were stored at 4 °C for 24 h, and then their particle size, potential, and appearance were measured.

[0054] 4.5.3 Thermal stability Take 10 mL of emulsions prepared with different complex emulsifiers and put them into centrifuge tubes. After heating the samples in a 90℃ water bath for 30 min, cool them to room temperature and centrifuge them to determine the particle size, potential, and β-carotene retention rate of the emulsions.

[0055] 4.5.4 Storage stability Different stable β-carotene emulsions were dispensed into tubes, with 10 mL of emulsion in each tube. The tubes were stored at 4°C, and the layering of the emulsions was observed at 1, 7, 14, and 21 days. The particle size, zeta potential, appearance, and β-carotene retention rate were measured to reflect the flocculation and aggregation of different emulsions.

[0056] 4.6 Oxidative stability 4.6.1 Determination of peroxide value Take 0.3 mL of the emulsion, mix it with 1.5 mL of isooctane / isopropanol (3:1, v / v), and vortex for 1 min. After centrifugation at 5000 rpm for 5 min, collect the organic solvent phase. Take 200 μL of the supernatant and add it to 2.8 mL of methanol:n-butanol mixture (2:1, v / v). Then add 50 μL of 3.94 mol / L ammonium thiocyanate and 50 μL of ferrous solution (prepared by mixing equal volumes of 0.132 mol / L BaCl2 and 0.144 mol / L FeSO4). Measure the absorbance at 510 nm. Calculate the concentration of hydrogen peroxide in the sample based on a standard curve prepared using hydrogen peroxide.

[0057] 4.6.2 Determination of Thiobarbituric Acid Value Disperse 0.3 mL of the emulsion in 1.8 mL of deionized water, and add 4.0 mL of thiobarbituric acid (TBA) solution. React the resulting mixture in boiling water for 15–30 min, then rapidly cool it in ice water. Finally, centrifuge the mixture at 10,000 rpm for 15 min, collect the supernatant, and measure the absorbance at 532 nm. Calculate the TBARS value of the sample based on the standard curve prepared using 1,1,3,3-tetraethoxypropane.

[0058] 4.7 In vitro digestion experiment The gastrointestinal digestive properties of a ternary covalent complex-stabilized β-carotene emulsion were studied using a two-stage digestion method.

[0059] Simulated gastric digestion stage: Take 10 mL of fresh β-carotene emulsion, dilute it with an equal volume of deionized water, heat it to 37°C, add 20 mL of gastric juice containing 64 mg of pepsin (preheated to 37°C), mix well, and adjust the pH to 2.0 with 0.1 mol / L HCl; then place the sample in a 37°C constant temperature shaking incubator and shake at 100 r / min for 2 h for digestion; after gastric digestion, add NaOH dropwise to adjust the pH to 7.0 to inactivate pepsin and terminate the reaction.

[0060] Simulated intestinal digestion stage: Add 2 mL of small intestinal fluid, 4.7 mL of 53.5 mg / mL bile salts, and 3.3 mL of 24 mg / mL pancreatic enzyme suspension (all solutions were preheated to 37°C) to the above gastric digestion fluid; adjust the pH to 7.0, and place in a 37°C constant temperature shaking incubator to digest at a speed of 100 r / min for 2 h; maintain the pH of the digestion fluid at 7.0 ± 0.1 during the reaction; after the reaction, add hydrochloric acid to inactivate the enzymes and terminate the digestion.

[0061] 4.7.1 Particle size and zeta potential of emulsions during in vitro digestion The particle size and zeta potential of the emulsion were accurately determined using a dynamic optical nanoparticle size potentiometer. Before measurement, the emulsion was diluted 1000 times with buffer solutions of different pH values ​​to reduce measurement errors caused by multiple light scattering. Each sample was analyzed in triplicate, and the results are expressed as averages. The particle refractive index was 1.460, and the dispersant refractive index was 1.330.

[0062] 4.7.2 Free fatty acid release rate During simulated small intestinal digestion, 0.1 mol / L NaOH was used every 20 minutes to maintain the pH at 7.0. The amount of NaOH consumed was recorded, and the free fatty acid release rate (FFA) was measured. The formula for calculating the free fatty acid release rate is as follows:

[0063] Among them, V NaOH m is the volume of NaOH consumed (mL); NaOH M is the molar concentration of NaOH (mol / mL); lipid W represents the average molecular weight of soybean oil (g / mol). lipid The mass (g) of oil in the initial emulsion.

[0064] 4.7.3 Biological Accessibility The small intestinal digestive fluids from each group were centrifuged at 10,000 rpm for 40 min, and the middle layer containing dissolved β-carotene, i.e., the "micelle" fraction formed during digestion, was collected. Any crystalline β-carotene was removed using a filter, and approximately 1 mL of the emulsion was collected. Bioavailability was defined as the concentration of β-carotene in the micelle fraction after digestion divided by the concentration of β-carotene in the original emulsion. β-carotene in the initial emulsion and micelles was extracted and determined. The formula for calculating the bioavailability of β-carotene is as follows:

[0065] In the formula ρmicelle and ρinitial The emulsions represent the mass concentrations of β-carotene in the micelles and in the initial emulsion, respectively.

[0066] 4.7.4 Laser confocal The microstructure of the emulsion in different stages of simulated gastrointestinal digestion was observed using confocal fluorescence scanning microscopy. Lipids were labeled with Nile Red (dissolved in ethanol, concentration 1 mg / mL) and excited at 543 nm. 2 mL of digested sample was mixed with 100 μL of Nile Red staining agent, and then the mixture was dropped onto a glass slide, covered with a coverslip, and observed under a confocal microscope.

[0067] 5. Discussion and Analysis 5.1 Multiple light scattering The stability of different emulsions during storage was evaluated using changes in backscattered radiation (BS) intensity and the Total Stability Index (TSI). For example... Figure 1-1 , Figure 1-2 , Figure 1-3 , Figure 1-4 , Figure 1-5 and Figure 2 As shown, compared to emulsions stable with single SPI and binary covalent complexes (SPI-EGCG, SPI-PD), emulsions stable with ternary covalent complexes (SPI-PD-EGCG, SPI-EGCG-PD) exhibit more stable BS curves, while their TSI values ​​increase more slowly and reach a lower final value. This indicates that ternary complexes have significant advantages in inhibiting droplet flocculation, aggregation, and gravity separation, and their stability is significantly better than that of binary systems.

[0068] The stability of the ternary covalent complexes varied significantly depending on the reaction sequence. The emulsion stabilized by the complex prepared in the SPI-PD-EGCG sequence showed the smallest change in the BS curve and the lowest and slowest increase in TSI value throughout the testing period, indicating that the ternary covalent complex emulsion prepared in the SPI-PD-EGCG sequence was the most stable. This may be because this reaction sequence is more conducive to the formation of a dense, cohesive, and elastic interfacial film, thus more effectively preventing droplet aggregation. In contrast, while the SPI-EGCG-PD-stabilized emulsion was also superior to the binary system, its BS and TSI curves fluctuated slightly more, indicating that the reaction sequence affects the interfacial structure and spatial arrangement of the complex, thereby modulating the physical stability of the emulsion.

[0069] In summary, ternary covalent complexes can significantly improve the physical stability of β-carotene emulsions, and the reaction sequence has a significant impact on its stability. In the system studied, the ternary complex prepared according to the reaction sequence of SPI-PD-EGCG exhibited the best stability effect, providing a basis for its application in efficient delivery systems.

[0070] 5.2 pH stability The pH stability of the emulsion in the gastrointestinal environment was evaluated by measuring the particle size and zeta potential of the emulsion under different pH conditions. Figure 3-1 and Figure 3-2As shown, within the pH range of 2.0-9.0, the emulsion stabilized by SPI exhibited the largest particle size and the lowest overall absolute potential value, indicating the worst stability. Emulsions stabilized by binary covalent complexes (SPI-EGCG, SPI-PD) showed improved performance, but still exhibited significant particle size increase and potential approaching zero near the isoelectric point (pH 4-5), leading to aggregation. In contrast, emulsions stabilized by ternary covalent complexes (SPI-EGCG-PD and SPI-PD-EGCG) showed smaller particle size and higher absolute potential values ​​across the entire pH range. This is due to the interfacial film formed by the rapid adsorption and synergistic cross-linking of the ternary covalent complexes, as well as their excellent ability to reduce interfacial tension, which helps to disperse oil droplets more uniformly, reducing particle size. Furthermore, the ternary covalent complexes provide stronger electrostatic repulsion and steric hindrance, effectively mitigating the aggregation tendency in the isoelectric point region, thus resulting in wider pH adaptability and better dispersion stability.

[0071] The two ternary complexes with different reaction sequences exhibited differences in pH stability. The emulsion with the SPI-PD-EGCG sequence exhibited the smallest variation in particle size across the entire pH range, with relatively low peak particle sizes at extremely acidic conditions (e.g., pH 2.0) and the isoelectric point region (pH 4.0), while maintaining a higher absolute potential value under neutral to alkaline conditions. This may be because this sequence is more conducive to constructing a synergistic and efficient interfacial structure, where the polysaccharide layer provides excellent physical anti-agglomeration capabilities, and the polyphenol layer enhances chemical stability, thus resulting in superior overall stability over a wide pH range. In contrast, the interfacial structure constructed by the protein-polyphenol-polysaccharide reaction sequence may have less integrity and thickness of the physical barrier than the protein-polysaccharide-polyphenol sequence, thus exhibiting slightly insufficient physical stability in response to charge neutralization and protein denaturation caused by extreme pH conditions.

[0072] In summary, the ternary covalent complex significantly improves the pH stability of β-carotene emulsions, effectively broadening their adaptability. This is mainly due to the covalent introduction of polysaccharide (PD) and polyphenol (EGCG), which synergistically enhance the steric hindrance and electrostatic repulsion of the interfacial layer. The SPI-PD-EGCG sequence in this system demonstrates the potential to construct a superior interfacial structure and achieve better pH stability.

[0073] 5.3 Thermal stability like Figure 4-1 , Figure 4-2 , Figure 4-3As shown, the thermal stability of the emulsions after heat treatment was evaluated by measuring the particle size, zeta potential, and β-carotene retention rate. After heat treatment, the particle size of all emulsions increased significantly, while the absolute value of the potential decreased, indicating droplet aggregation and enhanced surface charge shielding. Among them, the SPI-stabilized emulsion had the worst thermal stability, the largest increase in particle size, and the lowest β-carotene retention rate (69%). The binary covalent complexes (SPI-EGCG, SPI-PD) stabilized emulsions showed improvements in all indicators, with the β-carotene retention rate increasing to 74-76%, indicating that covalent modification alleviated thermal aggregation and protected the active substances to some extent. The ternary covalent complex stabilized emulsion showed the slowest particle size increase, relatively good potential retention, and the highest β-carotene retention rate, exhibiting the best thermal stability. This may be due to the increased thermal stability resulting from the covalent bonding of protein, polyphenols, and polysaccharides, which raises their thermal denaturation temperature, and the steric hindrance that prevents droplet aggregation. This suggests that the ternary complex can more effectively resist protein denaturation and droplet aggregation caused by heat treatment and provides better protection for β-carotene.

[0074] Among the ternary complexes prepared with different reaction sequences, the SPI-PD-EGCG-stabilized emulsion exhibited relatively superior thermal stability. It showed the smallest increase in particle size, the least decrease in absolute potential, and the highest β-carotene retention rate, reaching 85%. This is likely due to the protein-polysaccharide-polyphenol reaction sequence constructing a structure with the polysaccharide layer acting as a physical barrier and the polyphenol layer serving as an anchoring layer. This structure more effectively resists droplet aggregation caused by protein denaturation during heat treatment, thus resulting in the smallest increase in particle size and the least decrease in absolute potential in its stable emulsion. In contrast, the structure constructed with the protein-polyphenol-polysaccharide reaction sequence exhibited relatively lower synergistic efficiency between the physical barrier and conformational stability under thermal stress.

[0075] In summary, ternary covalent complexes can significantly improve the thermal stability of emulsions and their protective effect against β-carotene, and the reaction sequence is a key factor affecting their performance. The SPI-PD-EGCG sequence in this system constructs a more synergistic interfacial structure between the physical barrier (PD) and the antioxidant (EGCG) in terms of both space and function, thereby exhibiting superior stability under heat treatment conditions.

[0076] 5.4 Salt Ion Stability The salt ion stability of the emulsion was evaluated by analyzing the particle size, zeta potential, and macroscopic appearance at different Na⁺ concentrations. Figure 5-1 , Figure 5-2As shown, with increasing Na⁺ concentration, the particle size of emulsions stabilized by SPI and SPI-EGCG binary complexes significantly increased, while the absolute value of the electrostatic potential decreased sharply. Severe stratification occurred at lower salt concentrations (50-100 mM), indicating that the electrostatic shielding effect severely damaged their stability. The SPI-PD binary complex, due to the strong steric hindrance provided by the grafted polysaccharide, was less affected by salt ions, resulting in significantly improved stability. Emulsions stabilized by the two ternary covalent complexes (SPI-EGCG-PD and SPI-PD-EGCG) exhibited the best salt resistance: their particle size and electrostatic potential changed minimally, maintaining a uniform appearance at all tested concentrations without significant phase separation. This is because, compared to the binary covalent complex, the protein, polyphenol, and polysaccharide work synergistically to provide a certain electrostatic repulsion. The ternary complex, through synergistic integration of electrostatic repulsion and steric hindrance, can more effectively resist the destructive effects of salt ions on the emulsion structure.

[0077] Among the ternary complexes prepared with different reaction sequences, the SPI-PD-EGCG-stabilized emulsion exhibited relatively superior stability under high-salt conditions. It showed the smallest increase in particle size and decrease in potential at the highest Na⁺ concentration (200 mM), and the most thorough suppression of visual phase separation. This is likely due to the more optimized interfacial structure constructed by the reaction sequence, where the polysaccharide chains form a protective layer, effectively shielding the droplets from the effects of changes in ionic strength. In contrast, the SPI-EGCG-PD-sequentially stabilized emulsion performed slightly worse, especially at extremely high salt concentrations where its macroscopic homogeneity was slightly weaker. It is speculated that in this sequence, EGCG binds to SPI first, which may affect the full extension and encapsulation of the subsequent PD polysaccharide chains at the interface, resulting in a relatively lower efficiency of the spatial barrier formed to resist high salt stress.

[0078] In summary, the ternary covalent complex significantly improves the salt ion stability of β-carotene emulsions, with the introduction of polysaccharide PD playing a crucial role in resisting salt-induced flocculation. The reaction sequence further modulates the salt tolerance of the emulsion by influencing the conformation and spatial arrangement of the polysaccharide chains in the interfacial structure. In this system, the SPI-PD-EGCG sequence exhibits the best physical stability under high ionic strength conditions.

[0079] 5.5 Storage stability The long-term storage stability of the emulsion was evaluated by measuring its particle size, zeta potential, β-carotene retention rate, and appearance during storage. Figure 6-1 , Figure 6-2 , Figure 6-3 , Figure 6-4As shown, during the 21-day storage period, the particle size of all emulsions increased over time, the absolute value of the potential decreased, and the β-carotene content gradually decreased. Among them, the SPI-stabilized emulsion had the worst stability, with a sharp increase in particle size and obvious stratification in the later stage of storage, and the lowest β-carotene retention rate. The stability of the binary covalent complexes SPI-EGCG and SPI-PD-stabilized emulsions improved, but the SPI-EGCG group still showed phase separation earlier. In contrast, the two ternary covalent complex-stabilized emulsions exhibited significantly optimized storage performance: their particle size increased slowly, the potential remained well, the β-carotene retention rate was the highest, and they maintained a uniform macroscopic appearance throughout the entire storage period. This indicates that their stabilized emulsions have a stronger interfacial layer and stability, and therefore are less prone to aggregation and oxidative degradation of active ingredients during storage.

[0080] Among the two ternary complexes, the SPI-PD-EGCG sequentially stabilized emulsion exhibited the best long-term storage stability. It showed the smallest particle size increase, the slowest potential decrease, and the highest β-carotene retention rate after 21 days. This is likely due to the reaction sequence of SPI-PD-EGCG constructing a physical protective layer with a polysaccharide backbone, synergistically with antioxidants. This allows it to more effectively inhibit the physical aggregation and sedimentation of droplets during long-term storage, and also more directly protect the core active substance from oxidative loss. In summary, the SPI-PD-EGCG ternary covalent complex stabilized emulsion has better storage stability than emulsions stabilized by single proteins and binary covalent complexes. Its stabilized emulsion has a stronger interfacial layer and greater stability, making it an effective β-carotene delivery system.

[0081] 5.6 Oxidative stability The oxidative stability of β-carotene-loaded emulsions during storage is significantly affected by the properties of the stabilizer. For example... Figure 7-1 , Figure 7-2As shown, emulsions stabilized by natural SPI exhibited rapid and significant increases in both lipid peroxides (primary oxidation products) and TBARS values ​​(secondary oxidation products), indicating poor lipid oxidation protection. Covalent coupling with EGCG (SPI-EGCG) significantly inhibited peroxide formation and the increase in TBARS values, highlighting the direct antioxidant effect of the grafted polyphenols. The SPI-PD conjugate also showed a reduction in peroxide and TBARS values ​​compared to SPI alone, but its effect was mainly manifested in delaying the late stage of oxidation (TBARS), possibly due to its enhanced physical barrier properties. Notably, the ternary covalent complexes SPI-EGCG-PD and SPI-PD-EGCG-stabilized emulsions showed the most effective lipid oxidation inhibition during 21 days of storage. This superior performance can be attributed to a synergistic mechanism: the physical barrier provided by the covalently linked polysaccharide chains hinders the penetration of pro-oxidants, while the grafted EGCG actively quenches free radicals at the interface.

[0082] Under different reaction sequences, the slightly better performance of the SPI-PD-EGCG-stabilized emulsion is consistent with data on salt ion stability. This stable emulsion possesses a stronger and denser interfacial layer acting as a physical barrier, preventing water droplets from contacting the co-oxidants in the aqueous phase. The study indicates that solid particles present at the water-oil interface hinder the emulsion's contact with oxygen; therefore, the more stable emulsion exhibits better antioxidant properties. This further supports the specific reaction sequence of SPI-PD-EGCG, optimizing the interfacial structure to achieve maximum antioxidant protection. In conclusion, the application of the SPI-PD-EGCG ternary covalent complex can delay lipid oxidation in emulsion systems.

[0083] 5.7 In vitro simulated digestion experiment Emulsion delivery systems must enter the human digestive system to be absorbed by the body, and the encapsulated bioactive components are released in the most suitable region. β-carotene is mainly absorbed by the human body in the small intestine. This study investigated the particle size, potential, bioavailability of β-carotene, free fatty acid release rate, and microstructure of protein- and covalently complex-stabilized emulsions during digestion using in vitro simulated gastrointestinal digestion experiments.

[0084] 5.7.1 Particle size potential like Figure 8-1 , Figure 8-2As shown, the gastrointestinal stability of emulsions was assessed by measuring changes in particle size and zeta potential during simulated gastrointestinal digestion. During gastric digestion, the particle size of all emulsions significantly increased, while the absolute value of the zeta potential decreased. This was mainly attributed to the strong acidity and high ionic strength of gastric juice, as well as the disruption of interfacial protein structures by pepsin. Among them, the SPI and SPI-EGCG binary complexes showed the largest increase in particle size and the worst stability; the SPI-PD binary complex showed a reduced increase in particle size due to the steric hindrance provided by the covalently linked polysaccharide. The two ternary covalent complexes (SPI-EGCG-PD and SPI-PD-EGCG) maintained the smallest particle size and a higher absolute value of zeta potential in the gastric stage, possibly because they effectively protected the droplets from the gastric digestive environment by increasing the interfacial layer thickness. Their final particle size was also significantly smaller than that of the other groups after entering the intestinal stage. This indicates that the ternary complexes can more effectively maintain the integrity of the interfacial layer in the digestive environment, reduce droplet aggregation, and thus exhibit better digestive stability.

[0085] Among the ternary complexes with different reaction sequences, the SPI-PD-EGCG-stabilized emulsion exhibited the best stability throughout digestion, consistently exhibiting the smallest particle size and the highest relative absolute potential. This is likely because the polysaccharide more effectively provides steric hindrance and charge stabilization in the gastric environment, resisting erosion by acids, enzymes, and ions; the subsequently bound EGCG further enhances the interfacial membrane's tightness and antioxidant capacity, jointly maintaining the integrity of the droplets in the intestinal stage. In contrast, the SPI-EGCG-PD-sequentially stabilized emulsion performed slightly worse. It is speculated that in this sequence, EGCG binds to SPI first, which may affect the full extension and encapsulation efficiency of the PD polysaccharide chains at the interface, resulting in a relatively weaker steric barrier resistance to the digestive environment, thus leading to slightly insufficient particle size control and potential maintenance.

[0086] In summary, the ternary covalent complex significantly enhances the stability of β-carotene emulsions in a simulated gastrointestinal environment, effectively inhibiting droplet aggregation and interfacial disruption during digestion. The sequential flow of SPI-PD-EGCG achieves optimized synergy between steric hindrance and chemical stability, thereby better protecting the emulsion structure throughout digestion.

[0087] 5.7.2 Biological Accessibility Bioavailability is determined by measuring the β-carotene content in the mixed micelles produced after emulsion digestion. During digestion, the enzymatic breakdown of oil droplets releases the embedded β-carotene, which can dissolve in micelles formed by the binding of substances such as monoglycerides, free fatty acids, bile salts, and phospholipids. β-carotene in micelle form can be absorbed by the human body. Figure 9As shown, emulsions stabilized by natural SPI exhibit the lowest bioavailability because the interface is easily replaced by digestive surfactants, leading to rapid lipid digestion and potentially degrading released β-carotene in the harsh intestinal environment. Covalent binding of EGCG / PD improves bioavailability because the covalently formed network structure enhances the environmental stability of β-carotene, minimizing its loss during gastric digestion, increasing the contact area between lipases and oil droplets in the intestine, and improving the reaction rate, thus ensuring more fat particles enter the intestine. Emulsions stabilized by ternary covalent complexes exhibit the highest bioavailability, possibly due to the relatively small particle size and large specific surface area, enabling faster digestion and the formation of mixed micelles.

[0088] Under different reaction sequences, the SPI-PD-EGCG-stabilized emulsion exhibited the highest bioavailability. This superior performance may stem from a synergistic mechanism of reaction sequence: the PD-derived physical barrier regulates interfacial displacement and lipase contact, while the grafted EGCG protects β-carotene from oxidative degradation throughout digestion, ultimately achieving optimal micellization. In summary, the SPI-PD-EGCG ternary covalently stabilized emulsion, in addition to its excellent encapsulation effect and physicochemical stability, also has the advantage of promoting β-carotene absorption, making it a potentially efficient β-carotene delivery system.

[0089] 5.7.3 Free fatty acid release rate Studies have shown that the release rate of free fatty acids is controlled by the surface accessibility of lipases and influenced by the properties of the emulsion interface and the properties of surfactants. For example... Figure 10 As shown, all samples exhibited similar trends. In the initial 20 minutes, the release rate of free fatty acids (FAs) from all emulsions was faster due to the absorption of bile salts and lipases at the oil-water interface. With increasing digestion time (20–40 minutes), the release rate of free fatty acids decreased, stabilizing after 60 minutes. This may be due to the saturation of bile salts at the oil-water interface and the accumulation of lipolysis products on the emulsion surface, which hindered the contact between lipases and lipids. Furthermore, the gradual digestion of lipids reduced the lipolysis rate. Emulsions stabilized by the ternary covalent complex showed higher free fatty acid release rates than those stabilized by the SPI and binary covalent complexes. Their superior ability to maintain a stable, non-aggregated state of small lipid droplets during gastrointestinal transport maximizes the lipid-water interface area for enzymatic action. This efficient digestion is a key factor in the highest bioavailability of β-carotene in these systems.

[0090] 5.7.4 Laser confocal The microstructure changes of SPI and its covalently complexed β-carotene emulsions during digestion were observed using laser confocal scanning microscopy.Figure 11 As shown, in the initial stage, all emulsions exhibited a uniform and separated droplet distribution. Compared to the covalently stabilized emulsion, the SPI-stabilized emulsion exhibited large-sized oil droplets, indicating its limited emulsifying ability. After covalently binding EGCG / PD, the particle size of the emulsions formed by SPI-EGCG and SPI-PD decreased, and the size distribution became more uniform, indicating that covalent modification effectively improved the interfacial adsorption and droplet stabilization ability of SPI. The droplet size of the ternary covalently stabilized emulsion further decreased, indicating a significant improvement in spatial distribution uniformity and showing superior emulsifying performance. Notably, the droplets of the SPI-PD-EGCG covalently stabilized emulsion were the smallest and most uniformly distributed. The smaller droplets have a larger interfacial area, thus providing more sites for protein adsorption, indicating that this complex can efficiently adsorb at the interface and form a stable interfacial film. During gastric digestion, due to the low pH and high ionic strength in gastric juice leading to weak electrostatic interactions, all emulsions exhibited flocculation, and the particle size increased sharply. Relatively large aggregations were observed in emulsions stabilized by SPI, SPI-EGCG, and SPI-PD, while smaller aggregations were observed in emulsions stabilized by the ternary covalent complex. After the small intestinal digestion stage, the number and size of emulsion droplets decreased, indicating that the oil droplets were partially digested by enzymes. This result is consistent with... Figure 8-1 The results were consistent. Compared to SPI-EGCG-PD, the emulsion stabilized by the ternary covalent complex constructed sequentially by SPI-PD-EGCG exhibited a superior and more stable microstructure at all stages of digestion. This directly confirms the superior ability of its interfacial structure to resist aggregation and maintain the integrity of lipid droplets.

[0091] 6. Conclusion This study constructed monovalent, binary, and ternary covalent complex emulsions loaded with β-carotene by regulating the covalent reaction sequence of SPI, EGCG, and PD. The effects of the covalent complex on emulsion stability, protection of β-carotene, and digestibility were compared. Experiments showed that, compared to proteins and binary covalent complexes, the ternary covalent complex-loaded β-carotene emulsion exhibited good stability under pH, heat treatment, salt ion concentration, and storage conditions. Compared to SPI-EGCG-PD, the interface structure constructed using the SPI-PD-EGCG reaction sequence showed the best overall performance. The stable β-carotene emulsion exhibited excellent physical stability and the highest β-carotene retention rate under all tested environmental stresses (pH, salt ion concentration, heat treatment, and storage). In in vitro simulated digestion, the SPI-PD-EGCG stable emulsion achieved the highest β-carotene bioavailability, confirming that it not only protects the active substance during storage and transportation but also achieves efficient delivery within the digestive tract. This study provides a theoretical reference for the targeted design and construction of safe and stable functional delivery systems.

[0092] The foregoing has shown and described the basic principles, main features and advantages of this invention. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of this invention. Various changes and modifications can be made to this invention without departing from the spirit and scope of this invention. All such changes and modifications fall within the scope of this invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. The application of SPI-PD-EGCG in improving the bioavailability of β-carotene, characterized by: The SPI-PD-EGCG is a ternary covalent complex, and SPI-PD-EGCG and β-carotene are used to make Pickering emulsion.

2. The application of SPI-PD-EGCG according to claim 1 in improving the bioavailability of β-carotene, characterized in that: The SPI-PD-EGCG was prepared by a wet Maillard reaction combined with alkali treatment. The reaction sequence was to first prepare the SPI-PD covalent complex and then covalently bind EGCG.

3. The application of SPI-PD-EGCG according to claim 2 in improving the bioavailability of β-carotene, characterized in that: In the step of first preparing the SPI-PD covalent complex, the mass ratio of SPI to PD is 4±0.5∶1, and in the step of covalently binding EGCG, the amount of EGCG used is 0.015±0.005wt% of the SPI-PD solution.

4. The application of SPI-PD-EGCG according to claim 2 in improving the bioavailability of β-carotene, characterized in that: In the step of preparing the Pickering emulsion from SPI-PD-EGCG and β-carotene, the oil phase and the aqueous phase are mixed at a mass ratio of 9±0.5∶1, the concentration of SPI-PD-EGCG in the aqueous phase is 1±0.5% (w / v), and the mass fraction of β-carotene in the Pickering emulsion is 0.1±0.05%.

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