Method for preparing astaxanthin-loaded ovomucin-pectin-epsilon-polylysine hydrochloride complex nanogel

Abstract

This invention belongs to the field of polymer technology, specifically relating to a method for preparing an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel. Under stirring conditions, a pectin solution is added to an ovomucoid solution; then an astaxanthin solution is added, followed by ultrasonic treatment to obtain a mixed solution; an ε-polylysine hydrochloride solution is added to the mixed solution, and the mixture is stirred to obtain a further mixture; the mixture is centrifuged to obtain a dispersion and a precipitate. After separation, the dispersion is dialyzed to obtain the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel. This invention uses naturally sourced ovomucoid, pectin, and ε-polylysine hydrochloride as raw materials, effectively avoiding the use of organic solvents and reducing the risk of organic residues. The preparation method is mild, simple to operate, uses widely available and inexpensive raw materials, requires no complex equipment or organic solvents, and has good prospects for large-scale production and application.

Description

Technical Field

[0001] This invention belongs to the field of polymer technology, specifically relating to a method for preparing an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel. Background Technology

[0002] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-carotene) is a fat-soluble carotenoid with significantly superior antioxidant capacity compared to other carotenoids and tocopherols, attracting widespread attention in the food and nutritional supplement industry. Astaxanthin's bioactivity stems from its unique molecular structure; its conjugated polyolefin chains and terminal ketone groups enable it to efficiently scavenge free radicals, thus offering potential health benefits in alleviating oxidative stress. However, astaxanthin's highly unsaturated structure also makes it highly susceptible to degradation by light, heat, and oxygen during processing and storage. Furthermore, its inherent hydrophobicity and crystallinity result in poor water solubility and low oral bioavailability, severely limiting its effective application in functional foods and beverages.Research progress on extraction, biological activities and delivery systems of natural astaxanthin. Zhao, T., Yan, XJ, Sun, L. J, etal. Trends in Food Science & Technology (2019), 91, 354-361 and NanocarrierSystem: State-of-the-Art in Oral Delivery of Astaxanthin. Wahab, NRA, Affandi, M., et al. Antioxidants (2022), 11(9), 23, Article 1676 disclosed that astaxanthin has excellent antioxidant activity; Ultrasonic Self-Emulsification Nanocarriers for Cellular Enhanced Astaxanthin Delivery. Zhang, XD, Zhao, X., Tie, SS, et al. Journal of Agricultural and Food Chemistry (2021), 69(9), 2719-2728 and Astaxanthin-Loaded Nanostructured Lipid Carriers for Preservation of Antioxidant Activity. Rodriguez-Ruiz, V., Salatti-Dorado, JA, Barzegari, A., et al. Molecules (2018), 23(10), 12, Article 2601 discloses the problem that astaxanthin's application is limited due to its poor stability and low bioavailability.

[0003] To address the issues of poor stability, insufficient water solubility, and low oral bioavailability of astaxanthin, encapsulation within colloidal drug delivery systems offers a promising solution. Among various carriers such as emulsions, liposomes, and nanoparticles, protein-polysaccharide-based nanogels stand out in the food industry. These systems combine the biocompatibility and functionality of food-grade biopolymers with the structural advantages of nanogels, enhancing the stability and bioavailability of hydrophobic bioactive substances while enabling controlled release. Proteins and polysaccharides can self-assemble into nanogels with stable network structures driven by electrostatic interactions. Compared to single-component systems containing only proteins or polysaccharides, composite protein-polysaccharide nanogels exhibit higher encapsulation efficiency, stronger environmental stability, and richer functional diversity due to the synergistic effects between the components. Preparation and Self-Assembly Mechanism of Bovine Serum Albumin-Citrus Peel Pectin Conjugated Hydrogel: A Potential Delivery System for Vitamin C. Peng, HL, Chen, S., Luo, M., et al. Journal of Agricultural and Food Chemistry (2016), 64(39), 7377-7384 and Enhancing theantitumor activity of tea polyphenols encapsulated in biodegradable nanogels by macromolecular self-assembly. Liu, C., Zhang, Z., Kong, QJ, et al. RscAdvances (2019), 9(18), 10004-10016 disclose the application of protein-polysaccharide nanogels in food.

[0004] Although existing protein-polysaccharide complex systems have shown excellent delivery potential, there is still a need to explore novel food-grade protein-polysaccharide complex systems. These systems should not only provide effective encapsulation for astaxanthin, but also have additional functions such as protecting the active ingredient and high stability, thereby achieving a synergistic effect of activity protection and functional enhancement. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel. The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel prepared by this method has the characteristics of small particle size, high water dispersibility, strong environmental stability and high biocompatibility, and can significantly improve the solubility, photothermal stability and in vitro antioxidant activity of astaxanthin.

[0006] The preparation method of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel of the present invention includes the following steps: (1) Under stirring conditions, pectin solution was added to ovomucoid solution; then astaxanthin solution was added, and the mixture was sonicated to obtain a mixed solution; ε-polylysine hydrochloride solution was added to the mixed solution, and the mixture was stirred to obtain a mixture. (2) The mixture was centrifuged to obtain a dispersion and a precipitate. After separation, the dispersion was dialyzed to obtain an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel.

[0007] The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel is a stable aqueous dispersion system formed by dispersing nanoscale colloidal particles in water.

[0008] The preparation method of ovomucoid solution in step (1) is as follows: Egg white is used as raw material. Egg white is diluted with 100mM NaCl aqueous solution and stirred thoroughly to obtain a mixture. The pH of the mixture is adjusted to 6 with 2M hydrochloric acid and left to stand overnight to obtain a precipitate containing ovomucoid. The precipitate containing ovomucoid is treated with 500mM NaCl solution to remove impurities and obtain ovomucoid. The obtained ovomucoid is placed in ultrapure water and soaked overnight at 4℃ to ensure complete hydration. The completely hydrated ovomucoid is ultrasonically treated for 5min with an ultrasonic cell disruptor at 0~4℃ ice water bath with 100W power and 1s / 1s pulse mode. The treated protein is centrifuged at 14000×g centrifugation force at 4℃ for 30min and the supernatant is collected to obtain ovomucoid solution. A concentration standard curve is established with bovine serum albumin (BSA). The concentration of the obtained ovomucoid solution is determined by BCA method and diluted as needed.

[0009] In step (1), the pectin solution is prepared by dissolving pectin in ultrapure water and stirring overnight at 4°C to ensure complete hydration.

[0010] In step (1), the volume ratio of astaxanthin solution, ovomucoid solution, pectin solution and ε-polylysine hydrochloride solution is 1:9.6~10.4:2.2~2.8:0.6~1.2.

[0011] In step (1), the solvent for the astaxanthin solution is dimethyl sulfoxide, and the concentration of the astaxanthin solution is 0.5~1 mg / mL.

[0012] In step (1), the solvents for the ovomucoid solution, pectin solution, and ε-polylysine hydrochloride solution are all ultrapure water.

[0013] In step (1), the concentration of ovomucoid solution is 1.5~3.5 mg / mL, and the concentration of pectin solution is 1~2.5 mg / mL.

[0014] In step (1), the concentration of the ε-polylysine hydrochloride solution is 1~2 mg / mL.

[0015] In step (1), the stirring speed is 150~200 rpm, the stirring speed during the reaction is 150~200 rpm, and the stirring reaction time is 20~40 min.

[0016] In step (1), the ultrasonic treatment temperature is 0~4℃, the ultrasonic treatment power is 60~150W, the ultrasonic treatment time is 3~7min, and the ultrasonic treatment is performed in pulse mode.

[0017] In step (2), the centrifugation temperature is 4~6℃, preferably 4℃; the centrifugation time is 30-40min, the centrifugation is carried out using a high-speed refrigerated centrifuge, the rotor type is an angle rotor (suitable for 50mL centrifuge tubes), and the centrifugation force is 13000~15000×g.

[0018] In step (2), dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5~4kDa and a dialysis temperature of 4~6℃, preferably 4℃. The dialysis fluid is ultrapure water, and the total dialysis time is 22~26h. The dialysis fluid is replaced every 4~6h during the dialysis process.

[0019] In step (2), the average particle size of the composite nanoparticles in the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin is 116~177nm, the zeta potential is -28~-23mV, and the encapsulation rate of astaxanthin in the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel reaches more than 85%.

[0020] Ovomucin is a high-molecular-weight glycoprotein in egg white that maintains its gelling properties. Its hydrolysates possess antibacterial, antiviral, and anti-inflammatory biological activities. Pectin is a natural anionic polysaccharide with anti-inflammatory and gut microbiota-regulating effects, and is widely used as a thickener and stabilizer. ε-Polylysine is a cationic polypeptide composed of 25-30 lysine residues. Its hydrochloride form (ε-polylysine hydrochloride) is widely used for preserving vegetables, fruits, and meats due to its broad-spectrum antibacterial activity. As a polycation, ε-polylysine hydrochloride can induce the self-assembly of ovomucin and pectin through electrostatic interactions to form ovomucin-pectin composite nanogels, which are suitable for the delivery of active ingredients.

[0021] The beneficial effects of this invention are as follows: (1) The ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin prepared in this invention has the characteristics of uniform particle size, stable structure and high ζ potential, which can significantly improve the water solubility of astaxanthin and the dispersion stability of astaxanthin in aqueous phase. The gel system exhibits excellent physical stability under harsh environmental conditions such as extreme pH, high ionic strength, ultraviolet irradiation and heat treatment, which can effectively protect astaxanthin from degradation induced by the external environment. At the same time, the astaxanthin encapsulated in the gel exhibits higher ABTS and DPPH free radical scavenging efficiency than free astaxanthin.

[0022] (2) The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel prepared in this invention has good intestinal delivery ability. In a simulated gastrointestinal fluid environment, the composite nanogel can achieve continuous release of astaxanthin. At the same time, the composite nanogel system shows excellent biocompatibility in the RAW 264.7 macrophage model and has no significant inhibitory effect on cell proliferation in a wide concentration range.

[0023] (3) The present invention uses natural sources of ovomucoid, pectin and ε-polylysine hydrochloride as raw materials, which can effectively avoid the use of organic solvents and reduce the risk of organic residues; the preparation method of composite nanogel is simple, the conditions are mild, the raw materials are widely available and inexpensive, no complicated equipment is required, and it is easy to scale up production and promote application.

[0024] (4) In this invention, positively charged ε-polylysine hydrochloride acts as an inducer, simultaneously interacting electrostatically with negatively charged pectin and ovomucin. ε-polylysine hydrochloride acts as an "electrostatic bridge" connecting pectin and ovomucin to form a ternary complex. Ovomucin, as the main component of the ternary complex, mainly achieves astaxanthin loading by interacting with astaxanthin through its hydrophobic domains. Pectin, as a high-charge-density anionic polysaccharide, can increase the total negative charge of the ternary complex system, enhance the electrostatic repulsion effect, prevent colloidal particles from agglomerating and precipitating, and thus improve the long-term dispersion stability of the astaxanthin-loaded ovomucin-pectin-ε-polylysine hydrochloride composite nanogel. In addition, pectin also acts as a structural regulator by providing steric hindrance, promoting the transformation of the ternary complex from a loose or easily aggregated structure to a compact and regular structure, and inhibiting the tendency of colloidal particles to aggregate and precipitate near the isoelectric point of proteins. As an encapsulated compound, astaxanthin provides itself with a physical barrier protection by binding to the hydrophobic domains of ovomucoid. At the same time, the extensive non-covalent interactions (such as hydrophobic interactions and hydrogen bonds) formed between astaxanthin and ovomucoid and pectin further stabilize the molecular structure of astaxanthin, thereby enhancing the tolerance of astaxanthin to environmental stresses such as ultraviolet radiation or high temperature, and achieving effective protection of astaxanthin.

[0025] Positively charged ε-polylysine hydrochloride interacts electrostatically with negatively charged pectin and ovomucin through multiple amino sites, building a stable "molecular electrostatic bridge" between the two mutually repulsive negatively charged components. This makes the three components a ternary complex, achieving stable binding of pectin and ovomucin and completing the nucleation and framework construction of a stable complex at the molecular level. In the ternary complex, the electrostatic bridging of ε-polylysine hydrochloride anchors pectin and ovomucoid, causing the pectin sugar chains to form a steric barrier around the nanoparticles. This not only restricts the formation of disordered aggregates between ovomucoid particles due to hydrophobic interactions but also enhances the repulsive effect between particles, avoiding the polarization of particle size caused by the Ostwald ripening effect. Ultimately, this results in a tightly structured and uniformly sized nanogel. The ternary complex formed by ovomucoid, pectin, and ε-polylysine hydrochloride has a stable core-shell structure of "hydrophobic core-hydrophilic shell." The core consists of ovomucoid anchoring the hydrophobic region of astaxanthin through hydrophobic interactions, while the shell consists of the hydrophilic water region of ovomucoid and the outward extension of hydrophilic sugar chains from pectin. This allows a continuous and dense hydration layer to form on the surface of the nanogel, significantly reducing interfacial tension and thus reducing the driving force for hydrophobic aggregation between particles. This results in excellent dispersion stability of the nanogel in the aqueous phase and good encapsulation of the internal astaxanthin. Attached Figure Description

[0026] Figure 1The graph shows the average particle size and encapsulation efficiency of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels obtained in Examples 1-4.

[0027] Figure 2 The average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin-loaded pectin-ε-polylysine hydrochloride complex, astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogel, and ovomucoid-pectin-astaxanthin mixed solution obtained in Comparative Examples 1-3 are shown in the figure.

[0028] Figure 3 The encapsulation efficiency diagrams are shown for the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin-loaded pectin-ε-polylysine hydrochloride complex, astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogel, and ovomucoid-pectin-astaxanthin mixed solution obtained in Comparative Examples 1-3.

[0029] Figure 4 The graph shows the average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 at different pH values.

[0030] Figure 5 The graph shows the average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 at different NaCl concentrations.

[0031] Figure 6 The graph shows the average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 at different storage times.

[0032] Figure 7 The graph shows the astaxanthin retention rates of the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4 at different time points after exposure to ultraviolet light.

[0033] Figure 8 The graph shows the astaxanthin retention rates at different temperatures for the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4.

[0034] Figure 9 The average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 after incubation at 80°C for different times are shown in the figure.

[0035] Figure 10The graph shows the ABTS radical scavenging rates of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin aqueous dispersion obtained in Comparative Example 5 at different astaxanthin concentrations. This indicates a highly significant difference (p<0.001). This indicates a significant difference (p<0.05).

[0036] Figure 11 The graph shows the DPPH free radical scavenging rates of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin aqueous dispersion obtained in Comparative Example 5 at different astaxanthin concentrations. This indicates a highly significant difference (p<0.001).

[0037] Figure 12 The diagram shows the in vitro simulated release behavior of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4.

[0038] Figure 13 The image shows the absorbance values ​​at 450 nm obtained by CCK-8 assay after treating RAW 264.7 cells with ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels loaded with astaxanthin obtained in Example 1 at different concentrations.

[0039] Figure 14 The images show the macroscopic morphology of ovomucoid solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin. In the figure, a is the macroscopic morphology without laser irradiation, and b is the macroscopic morphology under laser irradiation.

[0040] Figure 15 The UV-Vis spectra of ovomucoid solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel in the 350-600 nm range are shown.

[0041] Figure 16 The images show the microstructure of the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin, where a is a scanning electron microscope image and b is a transmission electron microscope image.

[0042] Figure 17The diagram shows the protein secondary structure composition of ovomucoid solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin. In the diagram, a, b, c and ab are Duncan multiple comparison / significance letter labels, that is, different letters indicate significant differences (p<0.05).

[0043] Figure 18 The intrinsic fluorescence spectra of proteins in ovomucoid solutions, ovomucoid-pectin mixed solutions, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels loaded with astaxanthin are shown.

[0044] Figure 19 Fourier transform infrared spectra of ε-polylysine hydrochloride finished solid powder, astaxanthin finished solid powder, pectin finished solid powder, ovomucoid lyophilized powder, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder loaded with astaxanthin.

[0045] Figure 20 X-ray diffraction patterns of astaxanthin solid powder, ovomucoid lyophilized powder, ovomucoid-pectin lyophilized powder, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder loaded with astaxanthin. Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments.

[0047] Example 1 (1) Under the conditions of room temperature and stirring at 200 rpm, 1.25 mL of pectin solution (concentration of 2 mg / mL, solvent of ultrapure water) was added to 5 mL of ovomucoid solution (concentration of 2 mg / mL, solvent of ultrapure water); then 500 μL of astaxanthin solution (concentration of 1 mg / mL, solvent of dimethyl sulfoxide) was added; the mixture was sonicated for 5 minutes at 100 W power and 5 s / 5 s pulse mode in an ice-water bath at 0 °C; then 0.4 mL of ε-polylysine hydrochloride solution (concentration of 1 mg / mL, solvent of ultrapure water) was added, and the mixture was stirred at 200 rpm for 30 min at room temperature to obtain a mixture; (2) At 4℃, the mixture was centrifuged at a centrifugal force of 14000×g for 30min using a high-speed refrigerated centrifuge to obtain a dispersion and a precipitate. After separation, the dispersion was dialyzed in a dialysis bag with a molecular weight cutoff of 3.5kDa in a 4℃ refrigerator to remove dimethyl sulfoxide. The dialysate was ultrapure water. The total dialysate time was 24 hours. The dialysate was replaced every 6 hours during the dialysate process to obtain an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, which was stored at 4℃.

[0048] Example 2 (1) Under the conditions of room temperature and stirring at 150 rpm, 1.1 mL of pectin solution (concentration of 2.5 mg / mL, solvent of ultrapure water) was added to 4.8 mL of ovomucoid solution (concentration of 3 mg / mL, solvent of ultrapure water); then 500 μL of astaxanthin solution (concentration of 0.75 mg / mL, solvent of dimethyl sulfoxide) was added; the mixture was sonicated for 3 minutes at 150 W power and 5 s / 5 s pulse mode in an ice-water bath at 4 °C; then 0.6 mL of ε-polylysine hydrochloride solution (concentration of 2 mg / mL, solvent of ultrapure water) was added, and the mixture was stirred at 150 rpm for 40 min at room temperature to obtain a mixture; (2) At 5℃, the mixture was centrifuged at a centrifugal force of 13000×g for 40min using a high-speed refrigerated centrifuge to obtain a dispersion and a precipitate. After separation, the dispersion was dialyzed in a 5℃ refrigerator using a dialysis bag with a molecular weight cutoff of 4kDa to remove dimethyl sulfoxide. The dialysate was ultrapure water. The total dialysate time was 26 hours. The dialysate was replaced every 4 hours during the dialysate process to obtain an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, which was stored at 4℃.

[0049] Example 3 (1) Under the conditions of room temperature and stirring at 180 rpm, 1.4 mL of pectin solution (concentration of 1 mg / mL, solvent of ultrapure water) was added to 5.2 mL of ovomucoid solution (concentration of 1.5 mg / mL, solvent of ultrapure water); then 500 μL of astaxanthin solution (concentration of 0.5 mg / mL, solvent of dimethyl sulfoxide) was added; the mixture was sonicated for 7 minutes at 60 W power and 5 s / 5 s pulse mode in an ice-water bath at 2℃; then 0.3 mL of ε-polylysine hydrochloride solution (concentration of 1.5 mg / mL, solvent of ultrapure water) was added, and the mixture was stirred at 180 rpm for 20 min at room temperature to obtain a mixture; (2) At 6℃, the mixture was centrifuged at a centrifugal force of 15000×g for 35min using a high-speed refrigerated centrifuge to obtain a dispersion and a precipitate. After separation, the dispersion was dialyzed in a dialysis bag with a molecular weight cutoff of 3.5kDa in a 6℃ refrigerator to remove dimethyl sulfoxide. The dialysate was ultrapure water. The total dialysate time was 22 hours. The dialysate was replaced every 5 hours during the dialysate process to obtain an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, which was stored at 4℃.

[0050] Example 4 (1) Under the conditions of room temperature and stirring at 200 rpm, 1.25 mL of pectin solution (concentration of 1.5 mg / mL, solvent of ultrapure water) was added to 5 mL of ovomucoid solution (concentration of 3.5 mg / mL, solvent of ultrapure water); then 500 μL of astaxanthin solution (concentration of 1 mg / mL, solvent of dimethyl sulfoxide) was added; the mixture was sonicated for 5 minutes at 100 W power and 5 s / 5 s pulse mode in an ice-water bath at 0 °C; then 0.5 mL of ε-polylysine hydrochloride solution (concentration of 1.75 mg / mL, solvent of ultrapure water) was added, and the mixture was stirred at 200 rpm for 30 min at room temperature to obtain a mixture; (2) At 4℃, the mixture was centrifuged at a centrifugal force of 14000×g for 30min using a high-speed refrigerated centrifuge to obtain a dispersion and a precipitate. After separation, the dispersion was dialyzed in a dialysis bag with a molecular weight cutoff of 3.5kDa in a 4℃ refrigerator to remove dimethyl sulfoxide. The dialysate was ultrapure water. The total dialysate time was 24 hours. The dialysate was replaced every 6 hours during the dialysate process to obtain an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, which was stored at 4℃.

[0051] Comparative Example 1 Without adding ovomucoid solution, the other operations were the same as in Example 1 to obtain astaxanthin-loaded pectin-ε-polylysine hydrochloride complex, which was stored at 4°C.

[0052] Comparative Example 2 Without adding pectin solution, the other operations were the same as in Example 1 to obtain ovomucoid-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin, which was stored at 4°C.

[0053] Comparative Example 3 Without adding ε-polylysine hydrochloride solution, the other operations were the same as in Example 1 to obtain a mixed solution of ovomucoid-pectin-astaxanthin, which was stored at 4°C.

[0054] Comparative Example 4 Astaxanthin was dissolved in 90 vol% dimethyl sulfoxide solution (the volume ratio of dimethyl sulfoxide to ultrapure water was 9:1), and shaken to fully dissolve the astaxanthin, resulting in an astaxanthin solution with a concentration of 1 mg / mL.

[0055] Comparative Example 5 Using the astaxanthin solution from Comparative Example 4 as the stock solution, accurately transfer 1000 μL of the astaxanthin stock solution into a 20 mL volumetric flask, dilute with ultrapure water and bring to volume, sonicate, and shake well to obtain an astaxanthin aqueous dispersion.

[0056] Performance testing 1. Particle size, polydispersity index, and encapsulation efficiency At 25°C, the particle size (average particle size) and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels in Examples 1-4 and the astaxanthin-loaded pectin-ε-polylysine hydrochloride complexes, astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogels, and ovomucoid-pectin-astaxanthin mixed solutions obtained in Comparative Examples 1-3 were determined using a Malvern nanoparticle size potentiometer. The encapsulation efficiency of astaxanthin in the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels of Examples 1-4 and the astaxanthin-loaded pectin-ε-polylysine hydrochloride complexes, astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogels, and ovomucoid-pectin-astaxanthin mixed solutions of Comparative Examples 1-3 was calculated in the following manner: Wash the precipitate from step (2) three times with ultrapure water, and add 3 mL of dimethyl sulfoxide to dissolve the precipitate. Centrifuge at 10000×g for 10 min at 25℃. Collect the supernatant and measure the absorbance of the supernatant at a wavelength of 475 nm. Substitute the absorbance value into the pre-established standard curve to calculate the astaxanthin concentration, and calculate the mass of astaxanthin in the precipitate based on the volume. The encapsulation efficiency (EE) is calculated according to the following formula: , Where W1 is the total mass of astaxanthin added in step (1), and W2 is the mass of astaxanthin in the precipitate.

[0057] The average particle size and encapsulation efficiency of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels obtained in Examples 1-4 are shown in the figure. Figure 1 ,like Figure 1As shown, the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels obtained in Examples 1-4 of this invention have an average particle size in the range of 116-177 nm and an encapsulation efficiency in the range of 88.4%-95.1%. The average particle size of the composite gels obtained in Examples 1-4 is less than 180 nm and the encapsulation efficiency is higher than 88.0%, indicating that the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels prepared in this invention have a small particle size and a high encapsulation efficiency.

[0058] The average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin-loaded pectin-ε-polylysine hydrochloride complex, astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogel, and ovomucoid-pectin-astaxanthin mixed solution are shown in the figure. Figure 2 See the encapsulation rate diagram. Figure 3 .like Figure 2 As shown, the average particle sizes of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1, the astaxanthin-loaded pectin-ε-polylysine hydrochloride complex obtained in Comparative Example 1, the astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogel obtained in Comparative Example 2, and the ovomucoid-pectin-astaxanthin mixed solution obtained in Comparative Example 3 are 128 nm, 350 nm, 209 nm, and 563 nm, respectively, and the polydispersity indices are 0.25, 0.44, 0.29, and 0.76, respectively. The average particle size and polydispersity index of Example 1 are lower than those of Comparative Examples 1 to 3, indicating that Example 1 has better dispersibility and uniformity compared with Comparative Examples 1 to 3.

[0059] like Figure 3 As shown, the encapsulation efficiencies of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1, the astaxanthin-loaded pectin-ε-polylysine hydrochloride complex obtained in Comparative Example 1, the astaxanthin-loaded ovomucoid-ε-polylysine hydrochloride composite nanogel obtained in Comparative Example 2, and the ovomucoid-pectin-astaxanthin mixed solution obtained in Comparative Example 3 were 95.1%, 10.5%, 89.1%, and 83.2%, respectively. The encapsulation efficiency of Example 1 was higher than that of Comparative Examples 1-3, indicating that Example 1 has better encapsulation ability compared with Comparative Examples 1-3.

[0060] 2. Stability Test pH stability: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was diluted with ultrapure water to half of its original concentration. The pH was adjusted to 2, 3, 4, 5, 6 and 7 respectively using 0.1M NaOH solution or HCl solution. After vortex mixing, the particle size and polydispersity index were measured using a Malvern nanoparticle size potentiometer.

[0061] Ionic strength stability: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was mixed with NaCl solutions of the same volume but different concentrations, so that the final NaCl concentrations of the mixed solutions were 0, 50, 100, 200, 300, 400 and 500 mM, respectively. The average particle size and polydispersity index of the above mixed solutions were measured using a Malvern nanoparticle size potentiometer.

[0062] Storage stability: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was stored at 4°C for 28 days. Equal amounts of solution were collected every 7 days and the particle size and polydispersity index were determined using a Malvern nanoparticle size potentiometer.

[0063] UV stability: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4 were placed in a 10 mm quartz cuvette and exposed to 365 nm UV light for irradiation times of 0, 30, 60, 90, 120, and 150 min. 1 mL of the UV-treated composite nanogel was mixed with 5 mL of dichloromethane / methanol solution (dichloromethane to methanol volume ratio of 2:1), shaken for 30 min, and then centrifuged to collect the dichloromethane layer. The absorbance of the dichloromethane layer and the astaxanthin solution was measured at 475 nm using a Shimadzu UV-Vis spectrophotometer. The mass of astaxanthin was calculated according to a pre-established standard curve, and the astaxanthin retention rate (%) was calculated using the following formula: , Where W1 is the mass of astaxanthin in the nanogel after ultraviolet irradiation, and W2 is the mass of astaxanthin in the nanogel before ultraviolet irradiation.

[0064] Thermal stability: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4 were placed in test tubes and incubated in water baths at 50, 60, 70, and 80°C for 1 h, respectively. 1 mL of the incubated composite nanogel was mixed with 5 mL of dichloromethane / methanol solution (dichloromethane to methanol volume ratio of 2:1), shaken for 30 min, and then centrifuged to collect the dichloromethane layer. The samples were processed according to the description under the UV stability section, and the retention rate was calculated. Furthermore, the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was incubated in a water bath at 80°C for 0, 30, 60, 90, 120, 150, and 180 min. After cooling, the particle size and polydispersity index were determined using a Malvern nanoparticle size potentiometer.

[0065] The average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 at different pH values ​​are shown in the figure. Figure 4 The average particle size and polydispersity index at different NaCl concentrations are shown in the figure. Figure 5 The average particle size and polydispersity index at different storage times are shown in the figure. Figure 6 .like Figure 4 As shown, as the pH decreased from 7 to 4, the surface charge of the colloidal particles decreased, weakening the electrostatic repulsion. The average particle size of Example 1 gradually increased, reaching a maximum (>800 nm) at pH 4. This was because hydrophobic interactions caused particle aggregation, resulting in an increase in the polydispersity index of Example 1 as the pH decreased from 7 to 4. Further acidification to pH 3 caused the electrostatic repulsion to rebound, leading to redispersibility of the aggregates and a decrease in both particle size and polydispersity index. Further pH reduction to 2 caused pectin deprotonation, weakening internal cross-linking and resulting in increased particle size. No precipitation occurred within the pH range of 7 to 4, indicating that the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 possesses high pH stability.

[0066] like Figure 5 As shown, when the sodium chloride concentration increased from 0 to 50 mM, the particle size of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 increased from 132 nm to 1165 nm, and the polydispersity index also increased accordingly, indicating that the composite nanogel particles aggregated. When the sodium chloride concentration was further increased, the average particle size of the composite nanogel gradually decreased and stabilized at about 155 nm, and the polydispersity index also decreased accordingly and remained stable. No precipitation occurred in the system within the range of ionic strength variation, indicating that the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 has good tolerance to changes in ionic strength, especially high stability under high ionic strength.

[0067] like Figure 6 As shown, the particle size of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 remained relatively stable over 28 days, and the polydispersity index of the system remained at around 0.25, demonstrating good storage stability.

[0068] The astaxanthin retention rates of the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4 at different time points after exposure to ultraviolet light are shown in the figure. Figure 7 The astaxanthin retention rates at different temperatures are shown in the graph. Figure 8 The average particle size and polydispersity index of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 after incubation at 80℃ for different times are shown in the figure. Figure 9 .like Figure 7 As shown, after 150 min of ultraviolet irradiation, the astaxanthin retention rate in the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin obtained in Example 1 was as high as 86.3%, while the astaxanthin retention rate in the astaxanthin solution obtained in Comparative Example 4 dropped to below 10%. This indicates that the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel can stabilize the molecular configuration of astaxanthin and slow down its degradation process by constructing physical barriers and secondary intermolecular interactions.

[0069] like Figure 8 As shown, at temperatures above 50°C, the astaxanthin retention rate in the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was higher than that in the astaxanthin solution obtained in Comparative Example 4. Compared to the initial astaxanthin content, at 80°C, the astaxanthin content in the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 decreased by only 10.7%, and the decrease in Example 1 was approximately one-third of the decrease in Comparative Example 4. Figure 9 As shown, slight protein denaturation induced by high temperature led to particle aggregation, increasing the particle size of the system. However, after the treatment time exceeded 60 min, the particle size no longer changed significantly, and the polydispersity index remained below 0.3, indicating that the system had reached a stable state. These results demonstrate that the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel can resist temperature changes and reduce the thermal loss of astaxanthin.

[0070] 3. Antioxidant activity ABTS scavenging activity: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin aqueous dispersion obtained in Comparative Example 5 were diluted with ultrapure water to astaxanthin concentrations of 10, 20, 30, 40, and 50 μg / mL, respectively, to obtain sample solutions. ABTS working solution and sample solutions were mixed at a volume ratio of 1:1 as test samples; ABTS working solution and ultrapure water were mixed at a volume ratio of 1:1 as blank samples; ultrapure water and sample solutions were mixed at a volume ratio of 1:1 as background correction samples. After 6 min, the absorbance of the supernatant of the above samples was measured at 734 nm using a microplate reader. The ABTS scavenging activity of the mixtures obtained in Example 1 and Comparative Example 4 at different astaxanthin concentrations was calculated and compared. The ABTS free radical scavenging activity (ABTS free radical scavenging rate, ABTS scavenging activity) of the samples was calculated according to the following formula: , Where A a A represents the absorbance of the test sample. b A represents the absorbance of the corresponding blank sample. c This indicates the absorbance of the background-corrected sample.

[0071] DPPH scavenging activity: The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin aqueous dispersion obtained in Comparative Example 5 were diluted with ultrapure water to astaxanthin concentrations of 10, 20, 30, 40, and 50 μg / mL, respectively, to obtain sample solutions. A 0.04 mM DPPH solution was mixed with the sample solutions at a volume ratio of 10:1 as the test sample; a 0.04 mM DPPH solution was mixed with ultrapure water at a volume ratio of 10:1 as the blank sample; and anhydrous ethanol was mixed with the sample solutions at a volume ratio of 10:1 as the background correction sample. All samples were incubated in the dark for 30 min, and the absorbance of the sample supernatant at 517 nm was measured using a microplate reader. The DPPH free radical scavenging activity (DPPH free radical scavenging rate) of the samples was calculated according to the following formula: , Where A a A represents the absorbance of the test sample. b A represents the absorbance of the corresponding blank sample. c This indicates the absorbance of the background-corrected sample.

[0072] The ABTS radical scavenging rates of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin aqueous dispersion obtained in Comparative Example 5 at different astaxanthin concentrations are shown in the figure. Figure 10 The DPPH free radical scavenging rate at different astaxanthin concentrations is shown in the figure. Figure 11 .like Figure 10 As shown, the astaxanthin aqueous dispersion obtained in Comparative Example 5 exhibited low ABTS free radical scavenging activity, with scavenging rates below 10% in the concentration range of 10-50 μg / mL. At the same concentration, the free radical scavenging rate of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was significantly improved, and the improvement was concentration-dependent.

[0073] like Figure 11 As shown, due to the solubilizing effect of ethanol in DPPH solution on astaxanthin, the astaxanthin aqueous dispersion obtained in Comparative Example 5 exhibited a scavenging rate exceeding 35% at the initial concentration (10 μg / mL); however, when the concentration increased to 30 μg / mL, the scavenging rate plateaued due to the solubility limit. Throughout the entire experimental concentration range, the free radical scavenging rate of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was higher than that of the astaxanthin aqueous dispersion obtained in Comparative Example 5, and increased in a concentration-dependent manner, further confirming that the composite nanogel enhanced the in vitro antioxidant activity of astaxanthin.

[0074] 4. In vitro release and biocompatibility In vitro release: The astaxanthin solution obtained in Comparative Example 4 and the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 were respectively dispersed in simulated gastric juice (SGF, pH=2.0, containing 1 mg / mL pepsin and 0.4 vol% Tween-80) to prepare dispersions with consistent astaxanthin concentrations. 10 mL of each dispersion was transferred to a dialysis bag with a molecular weight cutoff of 500 kDa, and the dialysis bag was fixed below the basket shaft of an Agilent dissolution tester equipped with a 250 mL TruAlign dissolution cup. The system parameters were set as follows: rotation speed 100 rpm, equilibration time 10 min. After 120 min of simulated gastric fluid, the pH of the dissolution medium outside the dialysis bag was adjusted to 7.0. Simultaneously, trypsin was added to the medium inside the dialysis bag to convert it into simulated intestinal fluid (SIF, pH=7.4, containing 2 mg / mL trypsin and 0.4 vol% Tween-80). After equilibration for 10 min, incubation continued for 240 min at the same rotation speed. 5 mL samples were taken at each preset time point, and an equal volume of simulated gastric / intestinal fluid was immediately added. The astaxanthin content in the samples was quantitatively analyzed using the dichloromethane / methanol extraction method: 5 mL of sample was mixed with 5 mL of dichloromethane / methanol (dichloromethane to methanol volume ratio 2:1), shaken for 30 min, and then centrifuged to collect the dichloromethane layer. The absorbance of the dichloromethane layer and the free astaxanthin solution was measured at 475 nm using a Shimadzu UV-Vis spectrophotometer, and the astaxanthin mass was calculated according to a pre-established standard curve. The release rate was calculated using the following formula: , Where W1 represents the total mass of astaxanthin in the external medium of the dialysis bag at the sampling time point, and W2 represents the total mass of astaxanthin in the dialysis bag at the initial time point.

[0075] Biocompatibility: RAW 264.7 cells were cultured at 3 × 10⁻⁶ cells per cell line. 4Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in DMEM medium supplemented with 10 vol% fetal bovine serum, 100 μg / mL penicillin, and 100 μg / mL streptomycin for 24 h at 37°C and 5 vol% CO2 to allow cell adhesion. The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 was mixed with ultrapure water to obtain stock solutions of different concentrations. These stock solutions were then mixed with an equal volume of serum-free medium to achieve final concentrations of 25, 50, 75, 100, 150, and 200 μg / mL for the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel. The original medium was discarded and replaced with an equal volume of medium containing the corresponding concentration of composite nanogel, and cultured for another 24 h. After culturing, the drug-containing medium was discarded, and 100 μL of serum-free medium containing 10 vol% CCK-8 reagent was added to each well, and the cells were incubated for 2 h. The absorbance of each well at 450 nm was measured using an ELISA reader.

[0076] The in vitro simulated release behavior of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1 and the astaxanthin solution obtained in Comparative Example 4 are shown in the figure. Figure 12 ,like Figure 12 As shown, in the simulated gastric juice (SGF) stage, the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel prepared in Example 1 released only 19.1% of the astaxanthin, indicating that the gel can effectively encapsulate and protect astaxanthin in the gastric environment. Upon entering the simulated intestinal juice (SIF) stage, astaxanthin exhibited rapid release characteristics, with the release rate rising to approximately 69.8% within 60 minutes and eventually approaching 80%. In contrast, the astaxanthin solution in Comparative Example 4, due to its poor water solubility, precipitated in the simulated gastric / intestinal juice, forming visible deposits, resulting in a release rate consistently below 20% throughout the digestion process. These results demonstrate that the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel of this invention can significantly improve the solubility and release behavior of astaxanthin.

[0077] The absorbance values ​​at 450 nm obtained by CCK-8 assay after treating RAW 264.7 cells with ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels loaded with astaxanthin obtained in Example 1 at different concentrations are shown in the figure. Figure 13 ,like Figure 13As shown, with the increase of the concentration of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel obtained in Example 1, the absorbance at 450 nm showed a trend of first increasing and then slightly decreasing. Based on the CCK-8 detection principle, the absorbance value at 450 nm wavelength is positively correlated with the mitochondrial dehydrogenase activity of live cells. The higher the absorbance, the more live cells there are, the less affected cell proliferation is, and the lower the cytotoxicity of the sample. Compared with the control group (without the composite nanogel and replaced with an equal volume of PBS solution, wherein the PBS solution is phosphate buffer with pH=7.4), the composite nanogel did not show obvious cytotoxicity throughout the entire test concentration range (25-200 μg / mL), and the cell proliferation activity remained at a high level, indicating that the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel of the present invention has good cell compatibility.

[0078] Study on the encapsulation mechanism of astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel Astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels, ovomucoid solutions, ovomucoid-pectin mixed solutions, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels were prepared in the following manner: Astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel: Astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel was prepared according to the method of Example 1 and stored at 4°C. Ovalbumin solution: Ultrapure water was used instead of pectin solution and ε-polylysine hydrochloride solution, and dimethyl sulfoxide was used instead of astaxanthin solution. Other operations were the same as in Example 1 to obtain ovomucoid solution, which was stored at 4°C. Ovalbumin-pectin mixed solution: Ultrapure water was used instead of ε-polylysine hydrochloride solution, and dimethyl sulfoxide was used instead of astaxanthin solution. Other operations were the same as in Example 1 to obtain ovomucin-pectin mixed solution, which was stored at 4°C. Ovalbumin-pectin-ε-polylysine hydrochloride composite nanogel: Dimethyl sulfoxide solution was used instead of astaxanthin solution, and other operations were the same as in Example 1 to obtain ovomucin-pectin-ε-polylysine hydrochloride composite nanogel, which was stored at 4°C.

[0079] Characterization analysis: I. Morphological Characterization Ovalbumin solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel were placed at room temperature (25°C) and diluted with ultrapure water to 0.5 mg / mL. They were then scanned using a UV-Vis spectrophotometer in the range of 350–600 nm.

[0080] Astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels were cooled to room temperature (25°C), diluted 200 times with ultrapure water, and dropped onto a silicon wafer. After drying, they were sputtered with gold and characterized using a Thermo Fisher Scientific field emission scanning electron microscope at an accelerating voltage of 2 kV. The astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogels were then cooled to room temperature (25°C), diluted 50 times with ultrapure water, dispersed on a copper grid, stained with 2% (w / v) phosphotungstic acid, and their morphology was observed using a Thermo Fisher Scientific transmission electron microscope at an accelerating voltage of 200 kV.

[0081] Macroscopic morphology images of ovomucoid solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel are shown below. Figure 14 The UV-Vis spectrum in the 350-600 nm range is shown below. Figure 15 The microstructure of the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel is shown in the figure. Figure 16 .like Figure 14 As shown, the macroscopic morphology of both the ovomucoid solution and the ovomucoid-pectin mixed solution is that of a colorless and transparent liquid. After the addition of ε-polylysine hydrochloride solution, the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel exhibits a slight white turbidity, indicating the formation of nanocolloids. The ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin is reddish-brown, consistent with the color of astaxanthin. Figure 15 As shown, compared with ovomucoid solution, ovomucoid-pectin mixed solution, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin showed a new absorption peak at about 450 nm in the UV-Vis spectrum, confirming that astaxanthin was successfully encapsulated in the nanogel matrix.

[0082] like Figure 16 As shown in Figure a, the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin exhibits approximately spherical particles with a relatively uniform morphology and no widespread aggregation; according to Figure 16Size analysis of the transmission electron microscope images in b shows that the average diameter of the colloidal particles is approximately 100-150 nm, which is basically consistent with the hydration dynamic diameter of the colloidal particles.

[0083] II. Structural Analysis Circular dichroism chromatography: Ovalbumin solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel were all diluted with ultrapure water to a concentration of 0.2 mg / mL and analyzed using a Japanese circular dichroism chromatograph. The parameters were set as follows: temperature 25℃, scan range 190~240 nm, scan speed 10 nm / s, and resolution 0.1 nm. Intrinsic fluorescence spectra: Ovalbumin solution, ovomucoid-pectin mixed solution, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel were all diluted with ultrapure water to a concentration of 0.5 mg / mL and scanned using a fluorescence spectrometer. The excitation wavelength was 274 nm, the scanning range was 280~500 nm, the slit width was 2 nm, and the step size was 0.5 nm. Fourier transform infrared spectroscopy: Ovumin solution, ovumin-pectin-ε-polylysine hydrochloride composite nanogel and astaxanthin-loaded ovumin-pectin-ε-polylysine hydrochloride composite nanogel were pre-frozen at -80℃ for 12 hours, and then dried in a vacuum freeze dryer for 24 hours to prepare ovumin freeze-dried powder, ovumin-pectin-ε-polylysine hydrochloride composite nanogel freeze-dried powder and astaxanthin-loaded ovumin-pectin-ε-polylysine hydrochloride composite nanogel freeze-dried powder, respectively. At 25℃, ε-polylysine hydrochloride solid powder, astaxanthin solid powder, pectin solid powder, ovomucoid lyophilized powder, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder, and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder were ground and uniformly mixed with KBr powder at a mass ratio of 1:150, and then pressed into thin films. Subsequently, the pressed films were placed in a scanning optical path and scanned and recorded at 4000-525 cm⁻¹ using a Thermo Fisher Fourier transform infrared spectrometer. -1 The transmission spectrum; X-ray diffraction: Ovumin solution, ovumin-pectin mixed solution, ovumin-pectin-ε-polylysine hydrochloride composite nanogel and astaxanthin-loaded ovumin-pectin-ε-polylysine hydrochloride composite nanogel were pre-frozen at -80°C for 12 hours, and then dried in a vacuum freeze dryer for 24 hours to prepare ovumin freeze-dried powder, ovumin-pectin freeze-dried powder, ovumin-pectin-ε-polylysine hydrochloride composite nanogel freeze-dried powder and astaxanthin-loaded ovumin-pectin-ε-polylysine hydrochloride composite nanogel freeze-dried powder, respectively. X-ray diffraction patterns of astaxanthin solid powder, ovomucoid lyophilized powder, ovomucoid-pectin lyophilized powder, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder, and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder were obtained in the range of 5-30° (2θ) using an X-ray diffractometer; the working voltage was set to 40kV, the current to 40mA, and the scanning speed to 5° / min.

[0084] See the protein secondary structure diagrams for ovomucin solution, ovomucin-pectin mixed solution, ovomucin-pectin-ε-polylysine hydrochloride composite nanogel, and astaxanthin-loaded ovomucin-pectin-ε-polylysine hydrochloride composite nanogel. Figure 17 The intrinsic fluorescence spectrum of the protein is shown in [reference needed]. Figure 18 .like Figure 17 As shown, the secondary structure compositions of ovomucoid solutions and ovomucoid-pectin mixed solutions are highly similar. The secondary structure composition of the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel also showed no significant change compared to the ovomucoid solutions and ovomucoid-pectin mixed solutions, indicating that the addition of ε-polylysine hydrochloride hardly altered the secondary structure of ovomucoid. ε-polylysine hydrochloride is more likely to aggregate proteins through electrostatic interactions with glycans rather than acting on peptide chains. Compared to the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin showed a decrease in α-helix content and an increase in β-sheet content. The binding of astaxanthin to the hydrophobic regions of the protein induced inward folding of these regions, leading to a rearrangement of the secondary structure.

[0085] like Figure 18 As shown, the ultrasonic treatment during the preparation process caused slight changes in the protein conformation, resulting in a slight increase in the fluorescence intensity of the ovomucoid-pectin mixed solution and the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel compared to the ovomucoid solution; while the ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin exhibited fluorescence quenching, indicating that astaxanthin and ovomucoid formed a non-fluorescent complex.

[0086] Fourier transform infrared spectra of ε-polylysine hydrochloride finished solid powder, astaxanthin finished solid powder, pectin finished solid powder, ovomucoid lyophilized powder, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder, and ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder loaded with astaxanthin are shown below. Figure 19 ,like Figure 19 As shown, ovomucoid is at 1636.79 cm. -1 and 1536.51 cm -1 The peak represents the amide I and amide II bands, while 3275.50 cm⁻¹ -1 The α-hydroxyl stretching vibration peak indicates its glycosylation characteristics. The ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel showed a peak at 1048.60 cm⁻¹. -1 and 1024.02cm -1 Two new peaks appeared at 1000-1200cm. -1 The pectin COC and COH stretching vibrational bands within the range were observed. Compared to ovomucin, the amide I band of the ovomucin-pectin-ε-polylysine hydrochloride composite nanogel showed no significant shift, indicating that electrostatic interactions may not involve the main peptide chain of ovomucin. The interaction between astaxanthin and the carrier in the astaxanthin-loaded ovomucin-pectin-ε-polylysine hydrochloride composite nanogel altered the dipole moment and vibrational intensity of the CO bonds, resulting in a stretching vibrational band within the 1020 cm⁻¹ range. -1 The nearby absorption peak shifted to 1015.82 cm⁻¹. -1 Furthermore, the intensity increased. A characteristic peak of astaxanthin at 952.66 cm⁻¹ was observed in the astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel. -1 This further supports the successful encapsulation of astaxanthin.

[0087] X-ray diffraction patterns of astaxanthin solid powder, ovomucoid lyophilized powder, ovomucoid-pectin lyophilized powder, ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder, and astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel lyophilized powder are shown below. Figure 20 ,like Figure 20As shown, the diffraction patterns of ovomucin, ovomucin-pectin, and ovomucin-pectin-ε-polylysine hydrochloride composite nanogels all exhibit amorphous broad peaks, indicating that they lack a specific crystal structure. Astaxanthin molecules spontaneously form an ordered crystal structure through strong π-π stacking interactions of conjugated double bonds, producing multiple characteristic peaks in the spectrum. However, no characteristic diffraction peaks of astaxanthin were detected in the diffraction pattern of the ovomucin-pectin-ε-polylysine hydrochloride composite nanogel loaded with astaxanthin. This indicates that astaxanthin molecules are dispersed within the carrier through non-covalent interactions, thereby significantly improving its solubility and bioavailability.

Claims

1. A method for preparing an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel, characterized in that... Includes the following steps: (1) Under stirring conditions, pectin solution was added to ovomucoid solution; then astaxanthin solution was added, and the mixture was sonicated to obtain a mixed solution; ε-polylysine hydrochloride solution was added to the mixed solution, and the mixture was stirred to obtain a mixture. (2) The mixture was centrifuged to obtain a dispersion and a precipitate. After separation, the dispersion was dialyzed to obtain an astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel.

2. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the volume ratio of astaxanthin solution, ovomucoid solution, pectin solution and ε-polylysine hydrochloride solution is 1:9.6~10.4:2.2~2.8:0.6~1.

2.

3. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the solvent for the astaxanthin solution is dimethyl sulfoxide, and the concentration of the astaxanthin solution is 0.5~1 mg / mL.

4. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the solvents for the ovomucoid solution, pectin solution, and ε-polylysine hydrochloride solution are all ultrapure water.

5. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the concentration of ovomucoid solution is 1.5~3.5 mg / mL, and the concentration of pectin solution is 1~2.5 mg / mL.

6. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the concentration of the ε-polylysine hydrochloride solution is 1~2 mg / mL.

7. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the stirring speed is 150~200 rpm, the stirring speed during the reaction is 150~200 rpm, and the stirring reaction time is 20~40 min.

8. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (1), the ultrasonic treatment temperature is 0~4℃, the ultrasonic treatment power is 60~150W, the ultrasonic treatment time is 3~7min, and the ultrasonic treatment is performed in pulse mode.

9. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (2), the centrifugation temperature is 4~6℃ and the centrifugation time is 30-40min.

10. The method for preparing astaxanthin-loaded ovomucoid-pectin-ε-polylysine hydrochloride composite nanogel according to claim 1, characterized in that... In step (2), dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5~4kDa and a dialysis temperature of 4~6℃. The dialysis fluid is ultrapure water, and the total dialysis time is 22~26h. The dialysis fluid is replaced every 4~6h during the dialysis process.

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