A dual milk protein and fucoidan complex system and its application in astaxanthin delivery

Abstract

This invention provides a complex system of dual milk proteins and fucoidan and its application in astaxanthin delivery. The complex system is an LGF-CN-FD conjugate. It utilizes a dielectric barrier discharge (DBD) plasma generator to induce a glycosylation reaction between dual natural milk proteins (β-LGF / β-CN) and FD, forming the LGF-CN-FD conjugate. This significantly improves the encapsulation efficiency, thermal stability, photostability, gastric tolerance, and intestinal bioavailability of astaxanthin, demonstrating its great potential as a multifunctional emulsifier and delivery carrier.

Description

Technical Field

[0001] This invention relates to a complex system of dual-lactate protein and fucoidan and its application in astaxanthin delivery, particularly a complex system of dual-lactate protein and fucoidan constructed by the self-assembly of β-lactoglobulin filament (β-LGF) and β-casein (β-CN) and its application in astaxanthin delivery. Background Technology

[0002] Protein-based nanomaterials, due to their excellent biocompatibility, biodegradability, and functional diversity, show broad application prospects in food, medicine, cosmetics, and other fields. Milk, as a natural protein source, is mainly composed of whey protein (20%) and casein (80%). Among them, β-lactoglobulin (β-LG) is the core component of whey protein. Under low pH conditions and heating, it can self-assemble to form semi-flexible β-lactoglobulin fibers (β-LGF). These fibers possess stable physicochemical properties, such as emulsifying, gelling, and thickening properties, as well as chemical resistance, cell compatibility, antioxidant properties, and biosensing functions, showing great application potential in antimicrobial agent carriers and nutrient delivery. Furthermore, β-LGF prepared by acid heating induction exhibits excellent functional properties and low allergenicity, further expanding its application scope in the functional food field.

[0003] Casein is a mixture of αs1-, αs2-, β-, and κ-casein, with β-casein (β-CN) having a molecular weight of approximately 24 kDa and accounting for 30% of total casein. It is a typical amphipathic protein, with a low positive charge at its hydrophobic C-terminus and a high negative charge at its hydrophilic N-terminus. β-CN molecules are rich in proline residues, and their unique cyclic structure promotes β-sheet formation, endowing them with excellent interfacial activity. As a linear protein, the flexible structure of β-CN effectively reduces interfacial tension, exhibiting superior emulsifying properties, making it an ideal carrier for essential nutrient delivery. However, β-casein has poor structural stability; changes in the external physicochemical environment, such as heat treatment and pH fluctuations, easily lead to its aggregation, thus limiting the formation of irreversible adhesive films and further applications in functional foods.

[0004] Based on this, researchers proposed to combine β-LGF with β-CN, using the fibrous structure of β-LGF as a "bridge" to combine with β-CN in the form of nanoparticles, to construct a novel self-assembled carrier with a multi-network structure. This composite system is expected to synergize the advantages of the two proteins and improve the functional properties of the carrier. However, the interaction mechanism between β-LGF and β-CN is not yet clear, and the functional properties of the composite carrier still need to be further optimized.

[0005] Glycosylation is an environmentally friendly and simple method for protein chemical modification. It involves the covalent bonding of amino groups in protein molecules with carbonyl groups in polysaccharide molecules, achieving the regulation of protein structure and function without the addition of additional chemical reagents. Proteins modified by glycosylation exhibit significantly improved thermal stability, emulsifying properties, and antioxidant capacity. For example, peanut protein isolates, after glycosylation modification, show altered spatial structure, increased flexibility, and significantly improved emulsifying properties. Simultaneously, glycosylation can enhance the protein's ability to scavenge reactive oxygen species, break free radical chains, and decompose hydrogen peroxide. However, the interaction mechanism between β-LGF and β-CN remains unclear, and the functional properties of the composite carrier require further optimization. Furthermore, astaxanthin (AST), as a keto-oxidized carotenoid derivative, possesses highly efficient antioxidant activity and also exhibits various physiological functions such as anti-inflammation, vascular protection, and metabolism promotion, and has been widely applied in food health, drug research, and aquaculture. However, due to the presence of numerous conjugated double bonds in the AST molecule, its water solubility is poor, and its stability under the influence of external factors such as oxygen, light, and heat is also poor, which greatly limits its effective application in food systems. Summary of the Invention

[0006] The purpose of this invention is to provide a complex system of dual milk proteins and fucoidan and its application in astaxanthin delivery. It utilizes a dielectric barrier discharge (DBD) plasma generator to induce glycosylation between dual natural milk proteins (β-LGF / β-CN) and FD, forming an LGF-CN-FD conjugate. This significantly improves the encapsulation efficiency, thermal stability, photostability, gastric tolerance, and intestinal bioavailability of astaxanthin, demonstrating its great potential as a multifunctional emulsifier and delivery carrier.

[0007] The technical solution of the present invention is a complex system of dual-milk protein and fucoidan, wherein the complex system is an LGF-CN-FD conjugate.

[0008] In the aforementioned dual-milk protein and fucoidan complex system, the preparation process of the LGF-CN-FD conjugate is as follows: S1. β-LGF was prepared by CP treatment of β-LG solution using a dielectric barrier discharge (DBD) plasma generator. S2. LGF-CN complex was prepared by self-assembly using the pH-cycling method; S3. The LGF-CN-FD conjugate was prepared by inducing the glycosylation reaction between LGF-CN and FD using a dielectric barrier discharge plasma generator (CP).

[0009] In the aforementioned complex system of double-lactal protein and fucoidan, the specific step S1 is as follows: under room temperature stirring conditions, β-LG powder is dissolved in deionized water to prepare a solution of 10 mg / mL. -1 The β-LG solution was stirred continuously for 2 h and then hydrated at 4 ℃ for 12 h. It was then centrifuged at 8000 rpm for 20 min. The supernatant was taken and the pH was adjusted to 2.0 with 1 M HCl. 5 mL of the obtained β-LG solution was placed between the two electrodes of a quartz reaction vessel with an inner diameter of 50 mm. The electrode diameter was 30 mm and the discharge gap was 5 mm. The CP treatment power was set to 60 W and the treatment time was 80 s.

[0010] In the aforementioned dual-milk protein and fucoidan complex system, the specific steps of S2 are as follows: β-CN powder is dispersed in deionized water at a total solids content of 1% (w / v), the pH is adjusted to 11.5 with 1.0 M NaOH, and the mixture is stirred continuously at 600 rpm for 2 h to completely dissolve it. Subsequently, the prepared β-LGF solution and β-CN solution are mixed at a mass ratio of 1-7.5:5, and the pH is adjusted to 7.0 with 1 M HCl. Then, the reaction is continued for 30 min under magnetic stirring at 800 rpm to promote the self-assembly of LGF-CN complex driven by electrostatic and hydrophobic interactions. Finally, the free salt ions are removed by dialysis to obtain the LGF-CN complex.

[0011] In the aforementioned dual-milk protein and fucoidan complex system, the β-LGF solution and β-CN solution are mixed at a mass ratio of 1:1.

[0012] In the aforementioned dual-milk protein and fucoidan complex system, the specific step S3 is as follows: Prepare a 1.0% (w / v) LGF-CN complex solution; at room temperature, mix the LGF-CN complex solution with 10 mg / mL... -1 The FD solution was magnetically stirred and mixed for 2 h to form an LGF-CN-FD dispersion. The LGF-CN-FD dispersion was subjected to CP treatment using a dielectric barrier discharge plasma generator. Specifically, the reaction was carried out between two electrodes in a quartz reactor with an inner diameter of 50 mm, a discharge gap of 5 mm, an input power of 40 W, and a treatment time of 10 min. After CP treatment, the LGF-CN-FD solution was cooled to room temperature and centrifuged at 3000 rpm for 20 min using a pre-cleaned 100 kDa ultrafiltration centrifuge tube to remove unreacted LGF-CN and FD molecules. The LGF-CN-FD conjugate was obtained after freeze-drying.

[0013] The application of a complex system of dual-milk protein and fucoidan in astaxanthin delivery includes the following steps: S1. Dissolve the above LGF-CN-FD conjugate in water to prepare an aqueous dispersion of LGF-CN-FD conjugate with a concentration of 2.5% as the aqueous phase; S2. Dissolve astaxanthin in camellia oil as the oil phase, with an astaxanthin concentration of 1 mg / mL. -1 ; S3. Mix the aqueous phase and oil phase at a volume ratio of 3:1, and homogenize them for 3 min at 25 ℃ and 22000 rpm using a high-speed disperser to obtain the finished product.

[0014] Compared with the prior art, the present invention has the following advantages: This application describes the development of a novel protein-polysaccharide conjugate for astaxanthin (AST) delivery by constructing a dual natural milk protein complex through the self-assembly of β-lactoglobulin filaments (β-LGF) and β-casein (β-CN), followed by dielectric barrier discharge cold plasma (DBD-CP)-assisted fucoidation of fucoidan (FD). The β-LGF / β-CN complex exhibits optimal properties at a mass ratio of 1:1, including reduced particle size, enhanced thermal stability, increased apparent viscosity, and the highest DPPH radical scavenging activity (21.05%). CP-assisted glycosylation significantly improved the degree of glycosylation from 9.83% to 18.07%, accompanied by increased β-sheet content, enhanced intermolecular interactions, and the formation of high molecular weight conjugates. The LGF-CN-FD conjugate demonstrates superior antioxidant capacity and structural stability. The AST-loaded emulsion stabilized by this conjugate achieved the highest encapsulation efficiency (80.57%) and loading capacity (10.73%). In vitro digestion experiments showed enhanced gastric protective capacity (AST retention rate of 63.25%) and improved bioavailability (51.07%). Attached Figure Description

[0015] Figure 1 shows the morphological analysis of β-LG, β-LGF, and LGF-CN complexes with different mass ratios according to the present invention. In the figure, A is the TEM image of β-LG, B is the TEM image of β-LGF, C is the SEM image of β-LG, D is the SEM image of β-LGF with a mass ratio of 1:0, E is the SEM image of β-CN with a mass ratio of 0:1, F is the SEM image of the LGF-CN complex with a mass ratio of 0.2:1, G is the SEM image of the LGF-CN complex with a mass ratio of 0.5:1, H is the SEM image of the LGF-CN complex with a mass ratio of 1:1, I is the SEM image of the LGF-CN complex with a mass ratio of 1.2:1, and J is the SEM image of the LGF-CN complex with a mass ratio of 1.5:1. Figure 2 shows the FTIR spectra and amide I band correlation analysis of the complexes with different β-LGF:β-CN mass ratios of the present invention; where A is the FTIR spectrum of the complex, B is the proportion of secondary structures obtained by deconvolution of the second derivative of the amide I band of the complex, and C is the peak fitting curve of the amide I band of the complex. Figure 3 shows the relevant performance test results of LGF-CN composites with different mass ratios according to the present invention; where A is the test result of particle size and ζ-potential of the composite, B is the intrinsic fluorescence spectrum of the composite, C is the DSC heat flow curve of the composite, D is the test result of apparent viscosity of the composite, E is the test result of surface hydrophobicity of the composite, and F is the test result of DPPH free radical scavenging ability of the composite. Figure 4 shows the AFM test results of the complexes with different β-LGF:β-CN mass ratios of the present invention; where A is the AFM height map of the complex, B is the AFM three-dimensional image of the complex, C is the height distribution map of the complex, and D is the length distribution map of the complex. Figure 5 shows the AFM morphology evolution diagram of the LGF-CN-FD mixture of the present invention (including height map, three-dimensional map, height distribution and length distribution); Figure 6 shows the AFM morphology evolution diagram of the LGF-CN-FD conjugate of the present invention (including height map, three-dimensional map, height distribution and length distribution); Figure 7 shows the FTIR spectrum of the LGF-CN-FD mixture and its conjugate, and the fitting diagram of the amide I band peaks of the present invention. Figure 8 shows the test results of the performance and structural differences of the samples of the present invention; where A is a comparison of the browning degree and grafting degree of the LGF-CN-FD mixture and the conjugate, B is the SDS-PAGE spectrum of the three groups of samples, C is the ultraviolet absorption spectrum of the three groups of samples, and D is a comparison of the absorbance values ​​of the three groups of samples at 294 nm and 420 nm. Figure 9 shows a comparison of the relevant performance of the three groups of samples in this invention; where A is a comparison of the intrinsic fluorescence spectra of the three groups of samples, B is a comparison of the DSC heat flow curves of the three groups of samples, C is a comparison of the surface hydrophobicity of the three groups of samples, and D is a comparison of the antioxidant capacity of the three groups of samples. Figure 10 shows the stability and related performance test results of the AST-loaded emulsion of the present invention; wherein, A is the test results of the encapsulation efficiency (EE) and loading capacity (LC) of the emulsion for AST, B is the test results of the stability of the emulsion, C is the test results of the thermal stability of the emulsion, D is the test results of the light stability of the emulsion, E is the test results of the AST retention rate of the emulsion during simulated gastrointestinal digestion, and F is the test results of the bioavailability of AST of the emulsion during simulated gastrointestinal digestion. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0017] Example. A complex system of dual-milk protein and fucoidan, wherein the complex system is an LGF-CN-FD conjugate, and the preparation process of the LGF-CN-FD conjugate is as follows: S1. β-LG solution is subjected to CP treatment using a dielectric barrier discharge plasma generator to obtain β-LGF. The specific steps of S1 are as follows: Under stirring at room temperature, β-LG powder is dissolved in deionized water to prepare a solution of 10 mg / mL. -1 The β-LG solution was stirred continuously for 2 h and then hydrated at 4 ℃ for 12 h. Then it was centrifuged at 8000 rpm for 20 min. The supernatant was taken and the pH was adjusted to 2.0 with 1 MHCl. 5 mL of the obtained β-LG solution was placed between two electrodes in a quartz reaction vessel with an inner diameter of 50 mm. The electrode diameter was 30 mm and the discharge gap was 5 mm. The CP treatment power was set to 60 W and the treatment time was 80 s. S2. LGF-CN complex was prepared by self-assembly using the pH-cycling method. The specific steps of S2 are as follows: β-CN powder was dispersed in deionized water at a total solids content of 1% (w / v), and the pH was adjusted to 11.5 with 1.0 M NaOH. The mixture was stirred at 600 rpm for 2 h to ensure complete dissolution. Then, the prepared β-LGF solution and β-CN solution were mixed at a mass ratio of 1-7.5:5, and the pH was adjusted to 7.0 with 1 M HCl. The reaction was then continued for 30 min under magnetic stirring at 800 rpm to promote the self-assembly of LGF-CN complex driven by electrostatic and hydrophobic interactions. Finally, free salt ions were removed by dialysis to obtain the LGF-CN complex. The β-LGF solution and β-CN solution were mixed at a mass ratio of 1:1. S3, CP-induced glycosylation reaction of LGF-CN and FD to prepare LGF-CN-FD conjugate. The specific steps of S3 are as follows: prepare a 1.0% (w / v) LGF-CN complex solution, and at room temperature, mix the LGF-CN complex solution with 10 mg / mL... -1The FD solution was magnetically stirred and mixed for 2 h to form an LGF-CN-FD dispersion. The LGF-CN-FD dispersion was subjected to CP treatment using a dielectric barrier discharge plasma generator. Specifically, the reaction was carried out between two electrodes in a quartz reactor with an inner diameter of 50 mm, a discharge gap of 5 mm, an input power of 40 W, and a treatment time of 10 min. After CP treatment, the LGF-CN-FD solution was cooled to room temperature and centrifuged at 3000 rpm for 20 min using a pre-cleaned 100 kDa ultrafiltration centrifuge tube to remove unreacted LGF-CN and FD molecules. The LGF-CN-FD conjugate was obtained after freeze-drying.

[0018] Application of a complex system of dual-milk protein and fucoidan in astaxanthin delivery.

[0019] S1. Dissolve the prepared LGF-CN-FD conjugate in water to obtain an aqueous dispersion of LGF-CN-FD conjugate with a concentration of 2.5% as the aqueous phase; S2. Dissolve astaxanthin in camellia oil as the oil phase, with an astaxanthin concentration of 1 mg / mL. -1 ; S3. Mix the aqueous phase and oil phase at a volume ratio of 3:1, and homogenize them for 3 min at 25 ℃ and 22000 rpm using a high-speed disperser to obtain the finished product.

[0020] I. Characterization and analysis of the LGF-CN complex.

[0021] 1.1 Microscopic Observation: To elucidate the microscopic characteristics of β-LGF, transmission electron microscopy (TEM) was performed. Figure 1A shows that natural β-LGF is a dispersed spherical structure with a particle size of 5–10 nm. After CP-assisted acid-heat treatment, Figure 1B shows the coexistence of short fibers (200–900 nm) and long fibers (1.0–1.2 μm), forming a loosely interwoven network on a copper grid. Subsequently, scanning electron microscopy (SEM) was used to analyze the microstructure of LGF-CN complexes with different mass ratios (1:0, 0:1, 0.2:1, 0.5:1, 1:1, 1.2:1, 1.5:1). In this text, LGF-CN1:1 indicates a mass ratio of LGF to CN of 1:1. Figure 1C shows that natural β-LGF is an irregular spherical structure with depressions; β-LGF in Figure 1D is fibrous, and β-CN in Figure 1E is a dense mass. After self-assembly, the morphology of the complexes with different ratios showed significant differences: with the increase of the β-LGF ratio, LGF-CN 0.2:1 and LGF-CN 0.5:1 exhibited flexible amyloid structures with varying outline lengths, and some fibers aggregated to form dense long fiber aggregates, with a large number of β-CN particles bound to the fibers; when LGF:CN>1, the β-CN particles gradually became invisible, attributed to the excessively high β-LGF ratio and its loosely interwoven network structure. SEM qualitative results showed that the LGF to CN mass ratio had a significant impact on the self-assembly morphology.

[0022] 1.2 FTIR Analysis: FTIR was used to analyze the chemical composition and secondary structure changes of the LGF-CN complex. Figure 2 A shows 3400–3200 cm -1 The region at 3304 cm⁻¹ corresponds to the amide A band, representing the hydrogen bond stretching vibration of the NH and OH groups in the peptide bond. β-CN is located at 3304 cm⁻¹. -1 A broad peak appears, corresponding to OH stretching; with increasing β-LGF ratio, this peak significantly blue-shifts to 3445, 3451, 3452, 3453, and 3452 cm⁻¹. -1 (Corresponding to LGF-CN 0.2:1–1.5:1 respectively), reflecting the conformational rearrangement of the hydrophobic groups in β-CN micelles and the decrease in polarity of the aliphatic side chains. This is similar to the 3346 cm⁻¹ of β-LGF. -1 In contrast, the complex peaks show a blue shift and gradual broadening, indicating changes in intermolecular hydrogen bonds, corresponding to a decrease in the β-sheet ratio and an increase in the β-turn ratio. Amide I band (1700–1600 cm⁻¹) -1The peaks mainly correspond to C=O stretching, NH bending, and CN stretching. Because CN bond vibrations contribute little, the peak broadens after non-covalent binding of β-LGF and β-CN, attributed to enhanced electrostatics and hydrogen bonding. Figure 2B shows the Peakfit fitting of the amide I band, revealing a decrease in the β-sheet proportion and an increase in the β-turn proportion compared to β-LGF (38.10%), further confirming that hydrogen bond changes drive the β-sheet to β-turn transition; hydrogen bonds are considered the main force stabilizing protein fiber β-sheets. Figure 2C shows sub-peak assignments: 1660–1700 cm⁻¹. -1 (β-turn), 1650–1660 cm -1 (α-helix), 1640–1650 cm -1 (Random curl), 1600–1640 cm -1 (β-sheet). The proportions of each secondary structure differed significantly, but no dose-response relationship was observed between protein mass ratio and structure.

[0023] 1.3 Particle size, ζ-potential, and intrinsic fluorescence analysis: Figure 3A shows that the particle sizes of β-LGF and β-CN are approximately 276 nm and 181 nm, respectively, with good PDI (Potential Induced Discharge). As the proportion of β-LGF increases, the particle size of the complex gradually decreases, indicating the formation of uniform nanoparticles. Excess β-CN, on the other hand, leads to aggregated composites and increased particle size. Regarding the zeta potential, β-LGF exhibits a potential of +23.9 mV, while β-CN and all binary mixtures show negative potentials. With increasing β-LGF proportion, the absolute value of the zeta potential of the composite decreases, attributed to the hydrophilic region of β-CN encapsulating β-LGF. The electrostatic interaction between the protonated amino group and the ionized group of the acidic side chain of β-CN compresses the electrical double layer, reducing the effective surface charge. Both particle size and potential results indicate that sufficient β-CN encapsulation of β-LGF can form stable nanoparticles.

[0024] Intrinsic fluorescence originates from aromatic hydrophobic amino acids (phenylalanine, tryptophan, and tyrosine). Its fluorescence properties are influenced by the polarity of the microenvironment and can be used to monitor protein conformational changes or aggregation states. Figure 3B shows that the maximum emission wavelength (λmax) of β-LGF is approximately 340 nm, indicating that the aromatic residues are located in the hydrophobic core; the peak position of β-CN is approximately 350 nm, suggesting that tryptophan is exposed to a hydrophilic environment. The decrease in fluorescence intensity after recombination indicates that the hydrophobic tyrosine and tryptophan residues are buried, and β-CN can inhibit the aggregation and misfolding of β-LGF during refolding. The change in the polarity of the tryptophan environment and its masking are attributed to the formation of the LGF-CN complex, similar to the formation of the WPN-CA complex, which involves photoinduced electron transfer from aromatic groups to electron-deficient side chains.

[0025] 1.4. DSC, apparent viscosity, and H0 analysis: Figure 3C shows the DSC heat flow curves of each sample, from which the denaturation enthalpy (ΔH) and denaturation temperature (T_d) can be read. All samples showed obvious endothermic peaks, corresponding to protein denaturation. The T_d for β-LGF and β-CN were 76.6℃ and 83.0℃, respectively. After self-assembly, as the mass ratio of β-LGF:β-CN increased from 0:2:1 to 1.2:1, the T_d of the LGF-CN complex gradually increased, indicating improved conformational thermal stability. Regarding ΔH, the complex values ​​were all higher than β-CN (176.6 J / g), indicating that protein-protein filament interactions require more energy to disrupt the structure. Hydrogen bonds, hydrophobicity, disulfide bonds, and electrostatic interactions are the main forces maintaining the globular protein conformation, and the enhanced thermal stability after complexation...

[0026] Apparent viscosity is related to intermolecular forces; the stronger the forces, the higher the viscosity. Compared to β-CN, as the β-LGF:β-CN ratio increases from 0:2:1 to 1.5:1, the composite exhibits shear-thinning behavior, indicating that the formation of the fiber network and the increase in particle size lead to increased viscosity. During the composite process, interactions increase fluid resistance; the LGF-CN 1:1 sample exhibits a viscosity peak due to the synergistic enhancement of the network.

[0027] ANS fluorescent probes can assess the degree of exposure of hydrophobic groups on the protein surface, and the H0 value reflects conformational changes. Figure 3E shows that β-LGF has the highest H0, which is attributed to the moderate oxidation of CP, which exposes hydrophobic regions buried at the interfaces of oligomeric subunits or domains. As the β-LGF:β-CN ratio increases, the H0 of the complex decreases significantly (p<0.05) because the hydrophilic region of β-LGF encapsulates β-CN, and the hydrophobic residues are buried inside the complex, resulting in a lower H0 than that of pure β-LGF.

[0028] 1.5 Antioxidant Activity Analysis: The antioxidant activity of the white sample is of great significance for its application as a functional food additive. DPPH free radical scavenging ability measures antioxidant capacity by converting free radicals into diamagnetic molecules. Figure 3F shows that the scavenging rates of β-LGF and β-CN were 7.05% and 7.12%, respectively; the scavenging rate of the self-assembled complex increased significantly (p<0.05), with the LGF-CN 1:1 reaching the highest value of 21.05%. Antioxidant activity is related to sequence, structure, and aromatic amino acids. Treatments such as CP and ultrasound can enhance activity through structural transformation; the scavenging rate increased when the β-LGF ratio increased from 0.2:1 to 1:1 because the high aspect ratio of CP-induced fibers increases the contact opportunities between amino acids and free radicals. When the ratio continued to increase to 1.5:1, the activity decreased, attributed to the excessive adsorption of β-CN on the fiber surface, which inhibited peptide chain oxidation.

[0029] 1.6 AFM Visualization: LGF-CN1:1 was selected for subsequent experiments. AFM was used to compare the morphological changes of LGF-CN1:1 and LGF-CN1.5:1 (Figure 4). CP pretreatment promoted β-LG amyloid fibrillation, forming distinctly long, soft, worm-like, curved, and mature fibers. β-LGF had a height of 1.23–1.96 nm and a length of 0–100 nm; β-CN was a regular sphere with a height of 0.88–1.55 nm and a diameter of 0–100 nm. After self-assembly, the fiber morphology disappeared, forming aggregates; the structure of LGF-CN1:1 was more compact and amorphous than that of LGF-CN1.5:1, with a height increasing to 0.62–2.77 nm, attributed to the hydrophobic accumulation between the folded backbones of β-LGF and β-CN forming a "fuzzy" core. With increasing β-LGF addition, non-uniformly discrete spherical particles appeared, indicating that partial refolding / unfolding promoted assembly.

[0030] II. Characterization and analysis of LGF-CN-FD conjugate.

[0031] 2.1 Microscopic observation and infrared spectroscopy analysis.

[0032] AFM was used to observe the morphological changes of LGF-CN-FD mixtures and their conjugates. Figure 5 , Figure 6 The mixture exhibits a heterogeneous, rugged structure with a height of 0.51–2.62 nm and a length mainly distributed between 0–100 nm. After CP treatment, an LGF-CN-FD conjugate is formed, with a height increasing to 0.46–6.25 nm and a length mainly distributed between 100–200 nm. This is attributed to the steric hindrance created by the glycosylated covalent bonds on the surface of the two proteins. CP induces the depolymerization of the polypeptide chains, and the size of the conjugate aggregates increases, indicating that glycosylation can disrupt protein-protein interactions and inhibit aggregation.

[0033] The FTIR analysis of chemical bond changes is shown in Figure 7. (3000–3500 cm⁻¹) -1 The enhanced bond (C=O and NH) indicates that FD covalent bonding increases the proportion of hydrophilic groups and improves the secondary structure order; 1500–1550 cm -1 Enhanced amide II bands indicate covalent cross-linking between the protein and polysaccharide. 900–1500 cm⁻¹ -1 Characteristic polysaccharide absorptions were observed (COC, CC / C=O stretching and CH bending), 1238 cm⁻¹ -1 Enhanced peptide bond vibration peak, 1639 cm⁻¹ -1 The redshift is attributed to the partial breaking of peptide bonds and the unfolding of protein structures. 1630–1650 cm⁻¹ -1It can identify Maillard primary products (Schiff base imine). Peakfit fitting of amide I bands showed that after glycosylation, the β-sheet ratio increased from 12.55% to 17.19%, while the proportions of random coils, α-helices, and β-turns all decreased, indicating that glycosylation promotes hydrogen bond formation and enhances ordered structure; CP has the potential to induce protein-polysaccharide glycosylation by altering secondary structure.

[0034] 2.2 Changes in DG and DB: Grafting degree (DG) is calculated by measuring the free amino content. Specifically, the grafting degree (DG) is determined using the o-phthalaldehyde (OPA) method to measure the free amino ratio. The DG calculation formula is: DG % = (A0– A t ) / A0× 100 % Where A0 and A t The values ​​represent the relative contents of free amino groups in the self-assembled LGF-CN-FD complex and the LGF-CN-FD conjugate, respectively.

[0035] Browning degree (DB): Absorbance at 420 nm was measured using a Jasco V-650 spectrophotometer. The sample was first centrifuged at 6000 g for 10 min, then diluted with ultrapure water to a final concentration of 10 mg / mL. -1 The absorbance was measured.

[0036] Browning intensity and UV absorption spectrum: conjugate solution (protein concentration 0.25 mg / mL) -1 Browning intensity was measured at 294 nm and 420 nm; the UV spectral scanning range was 200–450 nm, and the instrument was a Jasco V-650 spectrophotometer. Figure 8 A shows that the DG of the LGF-CN-FD mixture under simple stirring was 9.83 ± 0.25%, indicating that glycosylation can occur even without covalent bonding between protein and starch under untreated conditions. After CP treatment, the DG increased significantly to 18.07 ± 0.70%, because the high-energy electrons generated by the discharge dissociate oxygen molecules to generate reactive oxygen species such as •OH, ¹O2, and O3, which oxidize and relax the peptide chains, providing abundant binding sites. The high-energy particles introduce additional kinetic energy, breaking weak chemical bonds (hydrogen bonds and secondary bonds) on the protein surface, promoting glycosylation.

[0037] Browning degree (DB) reflects the amount of Maillard end products (such as melanoidins) generated and is a key indicator of reaction progress. After CP treatment, DB decreased significantly from 0.13 ± 0.02% (p<0.05), which was attributed to the aggregation of protein molecules that buried melanoidins.

[0038] 2.3 SDS-PAGE Analysis: The effect of DBD CP-assisted glycosylation on molecular weight distribution was analyzed using reduced SDS-PAGE (Figure 8B). The molecular weights of β-LG, β-CN, and FD were 18.4, 25.3, and 41.0 kDa, respectively. The bands of both the LGF-CN-FD mixture and the conjugate shifted upwards and became more diffuse, indicating the formation of a higher molecular weight complex. The enhanced band signal after CP treatment confirmed covalent cross-linking, consistent with the DG results, indicating that CP successfully regulated glycosylation.

[0039] 2.4 UV absorption spectroscopy, intrinsic fluorescence and DSC analysis.

[0040] The protein-polysaccharide binding originates from the reaction between the free amino group of the protein and the reducing end of the polysaccharide. Figure 8 Cc shows enhanced absorption at 275–300 nm, with peaks at 270–280 nm corresponding to tyrosine and tryptophan residues. The enhanced UV absorption due to glycosylation is attributed to the exposure of aromatic amino acids and the increased energy of π→π* transitions induced by CP. The polysaccharide introduces numerous hydrophilic groups, increasing environmental polarity. Significantly enhanced absorption at 294 nm and 420 nm (p<0.05) indicates that CP accelerates browning and promotes the formation of intermediate and final products, stemming from the energy transfer of CP breaking surface hydrogen bonds and secondary bonds, thus stimulating glycosylation.

[0041] Intrinsic fluorescence (Figure 9A) showed that the LGF-CN-FD conjugate had the lowest fluorescence intensity, attributed to the FD shielding effect and the burial or oxidation of aromatic amino acids. DSC ( Figure 9 B) indicates that the thermal stability and tertiary conformational stability of the complex are enhanced because the FD covalent connection increases molecular rigidity and strengthens the hydrogen bond network.

[0042] 2.5 Analysis of HO groups, disulfide bonds and antioxidant activity.

[0043] The tertiary structure was analyzed by changes in H0, free / total thiol groups (-SH), and disulfide bonds (SS). Figure 9C shows that the LGF-CN-FD conjugate has the highest H0, which is attributed to the partial burial of hydrophobic groups due to high-energy particle-driven covalent bonding, reducing the accessibility of ANS; excessive CP oxidation leads to structural damage and aggregation of glycoproteins, which in turn exposes the hydrophobic regions.

[0044] Table 1. Content of free thiol groups, total thiol groups, and disulfide bonds in samples.

[0045] The dynamic transformation of thiol groups reflects subtle changes in protein structure and affects function (emulsification, gelation). Table 1 shows that the free -SH content is: LGF-CN1:1 complex (38.13 ± 0.04 μmol / g) > LGF-CN-FD mixture (36.83 ± 0.07 μmol / g) > LGF-CN-FD conjugate (31.77 ± 0.09 μmol / g); the SS trend is opposite, indicating that glycosylation oxidizes free -SH to generate SS. CP free radicals react with proteins to form hydroperoxides, oxidizing -SH to S•, promoting the formation of intermolecular SS.

[0046] Glycosylated products exhibit enhanced antioxidant activity and have potential for food applications. Figure 9D shows that the LGF-CN-FD conjugate has the strongest DPPH scavenging ability. The reducing sugar in FD promotes glycosylation, generating antioxidants such as melanoidins; melanoidins can convert DPPH into stable DPPH-H in intermediate and final stages.

[0047] III. Application Analysis of LGF-CN-FD Conjugates

[0048] 3.1 Encapsulation efficiency and loading analysis of AST-loaded emulsions.

[0049] As shown in Figure 10A, after CP-induced glycosylation, the encapsulation efficiency (EE) and loading capacity (LC) of AST in the emulsion both showed an increasing trend. Among them, the emulsion stabilized by the LGF-CN-FD conjugate had the highest EE and LC, at 80.57% and 10.73%, respectively. Glycosylated covalents significantly improve the EE and LC of the active substance by increasing the steric hindrance of the molecular surface and optimizing the hydrophilic-hydrophobic balance. The complex-stabilized emulsion is yellow-orange to orange-red. Figure 10 B). Glycosylation can enhance the encapsulation efficiency (EE) of biomolecules by regulating molecular interactions and structural stabilization; CP-treated glycosylated proteins exhibit increased hydrophobicity on their surface, forming a dense three-dimensional network, which further enhances the encapsulation efficiency (EE) and loading capacity (LC) of emulsions.

[0050] 3.2 Stability test of LGF-CN-FD conjugate.

[0051] Heat treatment is a common method for food sterilization. The thermal stability of LGF-CN-FD conjugate-stabilized emulsions was evaluated using AST retention rate as an indicator. Figure 10C shows that the AST retention rate in camellia oil CAO was only 31.35%, while the emulsion systems all exceeded 30.48%, with the LGF-CN-FD conjugate-stabilized emulsion exhibiting the highest retention rate (63.52%). This improved heat tolerance is attributed to the dense gel network slowing heat conduction, and the tightly packed droplet interface layer resisting heat damage and protecting against AST degradation.

[0052] After 4 hours of UV irradiation (Figure 10D), the AST retention rate in camellia oil CAO decreased to 29.51%, significantly lower than that of the emulsion system. The LGF-CN-FD conjugate-stabilized emulsion, due to its thicker and denser interfacial layer, effectively blocked UV penetration and provided excellent radiation protection. With prolonged irradiation time, the retention rate decreased, indicating instability caused by UV damage to the emulsion network; the protein-polysaccharide conjugate-stabilized emulsion achieved superior stability through a synergistic electro-steric hindrance mechanism.

[0053] 3.3. In vitro simulated digestion.

[0054] Astaxanthin (AST) is readily oxidized and degraded in the intestine. Sustained release and stability are crucial for enhancing its bioactivity, and its retention rate and bioaccessibility depend on the structure and function of the carrier. Figure 10E shows that during gastric digestion, the degradation rates of astaxanthin AST in the LGF-CN1:1 complex, the LGF-CN-FD mixture, and the LGF-CN-FD conjugate stable emulsion were all lower than the control, providing better gastric protection. Among them, the LGF-CN-FD conjugate stable emulsion showed the highest astaxanthin AST retention rate (84.31%) at 1 h, due to the dense interfacial layer delaying pepsin hydrolysis. The steric hindrance provided by glycosylation and the CP-induced interfacial densification produced a synergistic gastric protective effect.

[0055] Release is accelerated upon entering the intestinal tract, and the final astaxanthin AST retention rate of the LGF-CN-FD conjugate stable emulsion still reaches 63.25%, indicating that the intelligent interface structure constructed by synergistic modification is resistant to gastric digestion but highly efficient in intestinal hydrolysis. Its superior gastric protection stems from covalently grafted polysaccharide chains: the dextran chains grafted onto the protein surface form a dynamic hydrophilic high-hydration layer, which blocks pepsin (a macromolecular enzyme) from approaching, adsorbing, and contacting the protein substrate through strong steric hindrance, significantly reducing the effective enzyme-substrate collision frequency.

[0056] Micelles can dissolve encapsulated fat-soluble nutrients, affecting their bioavailability. Figure 10F shows that the bioavailability of astaxanthin (AST) in camellia oil CAO is only 7.83%, far lower than that of the emulsion system (41.33%–51.07%); chemical degradation of camellia oil CAO during digestion further reduces bioavailability. The LGF-CN-FD conjugate stabilized emulsion has the highest bioavailability (51.07%), attributed to CP-induced glycosylation modification of protein molecular structure, enhancing amphiphilicity: covalent cross-linking exposes hydrophobic regions and directionally distributes hydrophilic groups. The high-efficiency protein carrier developed by CP-co-glycosylation modification broadens the application prospects of functional food production and novel delivery system design.

[0057] In summary, this study successfully constructed a novel AST delivery system based on β-LGF / β-CN self-assembly combined with dielectric barrier discharge plasma (CP) treatment to assist glycosylation. The LGF-CN complex (1:1 mass ratio) exhibited enhanced structural stability, antioxidant activity, and interfacial properties through the synergistic effects of hydrogen bonding, hydrophobic interactions, and electrostatic attraction. Further CP treatment using a dielectric barrier discharge plasma significantly promoted protein-polysaccharide glycosylation, increased grafting degree, and induced favorable conformational rearrangements, including increased β-sheet content and enhanced intermolecular networks. The resulting LGF-CN-FD conjugate significantly improved the encapsulation efficiency, thermal stability, photostability, gastric tolerance, and intestinal bioaccessibility of astaxanthin AST, demonstrating its great potential as a multifunctional emulsifier and delivery carrier.

Claims

1. A complex system of dual-milk protein and fucoidan, characterized in that, The composite system is an LGF-CN-FD conjugate.

2. The dual-milk protein and fucoidan complex system according to claim 1, characterized in that: The preparation process of the LGF-CN-FD conjugate is as follows: S1. β-LGF was prepared by CP treatment of β-LG solution using a dielectric barrier discharge plasma generator. S2. LGF-CN complex was prepared by self-assembly using the pH-cycling method; S3. The LGF-CN-FD conjugate was prepared by inducing the glycosylation reaction between LGF-CN and FD using a dielectric barrier discharge plasma generator (CP).

3. The dual-milk protein and fucoidan complex system according to claim 2, characterized in that, The specific steps of S1 are as follows: under room temperature stirring conditions, β-LG powder is dissolved in deionized water to prepare a solution of 10 mg / mL. -1 The β-LG solution was stirred continuously for 2 h and then hydrated at 4 ℃ for 12 h. It was then centrifuged at 8000 rpm for 20 min. The supernatant was taken and the pH was adjusted to 2.0 with 1 M HCl. 5 mL of the obtained β-LG solution was placed between the two electrodes of a quartz reaction vessel with an inner diameter of 50 mm. The electrode diameter was 30 mm and the discharge gap was 5 mm. The CP treatment power was set to 60 W and the treatment time was 80 s.

4. The dual-milk protein and fucoidan complex system according to claim 2, characterized in that, The specific steps of S2 are as follows: β-CN powder is dispersed in deionized water with a total solids content of 1% (w / v), the pH is adjusted to 11.5 with 1.0 M NaOH, and the mixture is stirred continuously at 600 rpm for 2 h to completely dissolve it. Then, the prepared β-LGF solution and β-CN solution are mixed at a mass ratio of 1-7.5:5, and the pH is adjusted to 7.0 with 1 M HCl. The reaction is then continued for 30 min under magnetic stirring at 800 rpm to promote the self-assembly of LGF-CN complex driven by electrostatic and hydrophobic interactions. Finally, the free salt ions are removed by dialysis to obtain the LGF-CN complex.

5. The dual-milk protein and fucoidan complex system according to claim 3, characterized in that: The β-LGF solution and the β-CN solution were mixed at a mass ratio of 1:

1.

6. The dual-milk protein and fucoidan complex system according to claim 2, characterized in that, The specific steps of S3 are as follows: prepare a 1.0% (w / v) LGF-CN complex solution, and at room temperature, mix the LGF-CN complex solution with 10 mg / mL... -1 The FD solution was magnetically stirred and mixed for 2 h to form an LGF-CN-FD dispersion. The LGF-CN-FD dispersion was subjected to CP treatment using a dielectric barrier discharge plasma generator. Specifically, the reaction was carried out between two electrodes in a quartz reactor with an inner diameter of 50 mm, a discharge gap of 5 mm, an input power of 40 W, and a treatment time of 10 min. After CP treatment, the LGF-CN-FD solution was cooled to room temperature and centrifuged at 3000 rpm for 20 min using a pre-cleaned 100 kDa ultrafiltration centrifuge tube to remove unreacted LGF-CN and FD molecules. The LGF-CN-FD conjugate was obtained after freeze-drying.

7. The application of a complex system of dual-milk protein and fucoidan in astaxanthin delivery, characterized in that, Includes the following steps: S1. Dissolve the LGF-CN-FD conjugate as described in any one of claims 1-6 in water to obtain an aqueous dispersion of LGF-CN-FD conjugate with a concentration of 2.5% as the aqueous phase; S2. Dissolve astaxanthin in camellia oil as the oil phase, with an astaxanthin concentration of 1 mg / mL. -1 ; S3. Mix the aqueous phase and oil phase at a volume ratio of 3:1, and homogenize them for 3 min at 25 ℃ and 22000 rpm using a high-speed disperser to obtain the finished product.

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