ESP-Zein-lutein composite nanoparticle delivery system and its preparation method

Abstract

This invention discloses an apple pomace ESP-Zein-lutein composite nanoparticle delivery system and its preparation method, belonging to the field of functional food carrier and bioactive substance delivery technology. ESP is purified from apple pomace through hot water extraction, TCA deproteinization, and dialysis decolorization. Combined with antisolvent precipitation and electrostatic adsorption technology, composite nanoparticles with uniform particle size and high stability are prepared. The optimized ratio is Zein:ESP:lutein = 1:0.4:0.08. In this system, Zein and lutein form a hydrophobic core, and ESP forms a shell through hydrogen bonding and electrostatic interactions. The lutein encapsulation rate reaches 90.5%, significantly higher than the 54.7% of the single Zein system, making it suitable for targeted delivery of functional food additives, antioxidants, and eye health-related products.

Description

Technical Field

[0001] This invention belongs to the field of functional food carriers and bioactive substance delivery technology, and in particular relates to a nanoparticle delivery system and its preparation method, which is prepared by extracting alcohol-soluble polysaccharide (ESP) from agricultural waste apple pomace and combining it with zein and lutein. The system is suitable for the stable encapsulation and efficient delivery of hydrophobic active ingredients such as lutein, as well as for the development of eye health-related products. Background Technology

[0002] Lutein, a natural carotenoid, is a key component of the macular region of the retina. It possesses potent antioxidant properties, protects vision, and helps prevent cardiovascular diseases, making it an important ingredient in functional foods and health supplements. However, lutein molecules contain multiple conjugated double bonds, resulting in extremely poor water solubility (only 2.67 μg / mL). It is easily damaged by light, heat, and oxygen during processing and storage. Furthermore, after oral administration, it is readily hydrolyzed by enzymes in the gastrointestinal tract, resulting in an actual bioavailability of less than 10%, severely limiting its industrial application.

[0003] Zein, a food-grade natural protein, is rich in hydrophobic amino acids (approximately 75%), which can self-assemble into nanoparticles through antisolvent precipitation, providing hydrophobic encapsulation space for lutein and exhibiting good biocompatibility and biodegradability. However, single Zein nanoparticles have significant drawbacks: their isoelectric point is pH 5.6-6.4, near which they are prone to aggregation due to hydrophobic interactions, and they are sensitive to ionic strength, making them difficult to tolerate food processing and the gastrointestinal environment.

[0004] Meanwhile, apple pomace, a byproduct of the apple processing industry, is produced in huge quantities and is rich in alcohol-soluble polysaccharides (ESPs), which have good water solubility and bioactivity. Existing research has confirmed that ESP molecules contain a large number of functional groups such as hydroxyl and carboxyl groups, which can interact with proteins. However, there are no reports on its use in modifying Zein nanoparticles to improve the stability of lutein encapsulation. This invention aims to utilize apple pomace ESPs to modify Zein nanoparticles, construct a novel composite delivery system, and solve the problems of poor stability and bioavailability in the lutein delivery process. Summary of the Invention

[0005] To address the problems of poor water solubility and low bioavailability of lutein, insufficient stability of single Zein nanoparticles, waste of ESP resources from apple pomace, and difficulties in binding with lutein and Zein, this invention provides an ESP-Zein-lutein composite nanoparticle delivery system, along with a method for preparing the ESP-Zein-lutein composite nanoparticle delivery system and its applications.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: On the one hand, the present invention provides an ESP-Zein-lutein composite nanoparticle delivery system, which is a core-shell structure composed of Zein, ESP and lutein. The Zein and lutein form a hydrophobic core, and the ESP is coated to form a shell through hydrogen bonding and electrostatic interaction, thus forming the delivery system.

[0007] Preferably, the mass ratio of Zein, ESP, and lutein is 1:0.2-0.6:0.05-0.1, that is, 1 part by mass of Zein is mixed with 0.2-0.6 parts by mass of ESP and 0.05-0.1 parts by mass of lutein.

[0008] Preferably, the mass ratio of Zein, ESP, and lutein is 1:0.4:0.08.

[0009] Furthermore, the nanoparticles have a particle size of 150-250 nm, a Zeta potential of +10 to +30 mV, a PDI of <0.3, and a lutein encapsulation rate of ≥85%.

[0010] Furthermore, the ESP is extracted from apple pomace.

[0011] On the other hand, the present invention provides a method for preparing an ESP-Zein-lutein composite nanoparticle delivery system, which includes the following steps: (1) Extraction and purification of ESP: After drying and crushing apple pomace, remove enzymes by boiling water and wash with distilled water to remove impurities. Mix with distilled water and heat for a certain time. Centrifuge and take the supernatant. Add TCA to the supernatant and let it stand at low temperature to remove protein. Then precipitate polysaccharides with ethanol. After removing ethanol by rotary evaporation, decolorize through a dialysis bag and freeze dry to obtain pure ESP. (2) Preparation of Zein solution: Dissolve Zein in an aqueous ethanol solution and stir thoroughly to form a Zein solution with a concentration of 4-6 mg / mL (0.4-0.6% w / v); (3) Preparation of Zein-lutein complex solution: Add lutein to the Zein solution in step (2) and stir until completely dispersed to obtain Zein-lutein complex solution; (4) Preparation of composite nanoparticles: The Zein-lutein composite solution from step (3) was added dropwise to the ESP aqueous solution, stirred and rotary evaporated to remove ethanol, and after centrifugation and filtration, the composite nanoparticle delivery system was obtained.

[0012] Furthermore, in step (3), the mass ratio of Zein to lutein is 1:(0.05-0.1); in step (4), the mass ratio of Zein to ESP is 1:(0.2-0.6).

[0013] Preferably, the mass ratio of Zein to lutein in step (3) is 1:0.08; and the mass ratio of Zein to ESP in step (4) is 1:0.4.

[0014] Specifically, the extraction and purification process of ESP is as follows: after drying and pulverizing apple pomace, the enzyme is removed by boiling water and the impurities are removed by washing with distilled water. The pomace is mixed with distilled water at a material-to-liquid ratio of 1:20 (g:mL) and extracted at 80℃ for 3 hours. The supernatant is collected by centrifugation. An equal volume of 20% trichloroacetic acid (TCA) is added to the supernatant and the mixture is allowed to stand at 4℃ to remove proteins (repeated twice). Polysaccharides are precipitated with 95% ethanol. After removing the ethanol by rotary evaporation, the polysaccharide is decolorized through a 14kDa dialysis bag and then freeze-dried to obtain pure ESP.

[0015] Specifically, the specific process for preparing the Zein solution is as follows: dissolve Zein in an 85% ethanol aqueous solution and stir magnetically for 30 minutes to form a Zein solution with a concentration of 4-6 mg / mL (0.4-0.6% w / v).

[0016] Specifically, the specific process for preparing the Zein-lutein composite solution is as follows: add lutein powder to the Zein solution, control the mass ratio of Zein to lutein to be 1:(0.05-0.1), and stir for 30 minutes until the lutein is completely dispersed (no visible particles); when the ratio is lower than 1:0.05, the lutein encapsulation rate is less than 70%, and when it is higher than 1:0.1, lutein precipitation is likely to occur.

[0017] Specifically, the specific process for preparing the composite nanoparticles is as follows: the Zein-lutein composite solution from step (3) is added dropwise to an ESP aqueous solution of 10-12 mg / mL (1-1.2% w / v), stirred at 300 rpm for 10 min; ethanol is removed by rotary evaporation at 45°C for 35 min, large particles are removed by centrifugation at 4000 r / min for 10 min, and the mixture is filtered through a 0.45 μm filter membrane to obtain the composite nanoparticle delivery system.

[0018] On another front, this invention provides an application of the ESP-Zein-lutein composite nanoparticle delivery system in the stable encapsulation and efficient delivery of hydrophobic active ingredients, which is particularly suitable for the targeted delivery of functional food additives, antioxidants and eye health-related health products.

[0019] Furthermore, the hydrophobic active ingredients include, but are not limited to, lutein. They can also be applied to naturally occurring hydrophobic active ingredients found in apple pomace and other exogenous hydrophobic functional substances, such as curcumin, fat-soluble vitamins, and functional peptides. Specifically, they can be used as hydrophobic carriers to encapsulate fat-soluble vitamins, ensuring uniform dispersion of fat-soluble vitamins in water-based foods such as beverages and yogurt, preventing stratification; for the stable encapsulation and efficient delivery of functional peptides containing hydrophobic amino acid residues and hydrophilic groups; and for the stable encapsulation and efficient delivery of curcumin. This system, through the synergistic effect of hydrophobic core encapsulation and shell protection, addresses the pain points of poor water solubility, easy oxidation, and low bioavailability of such substances.

[0020] This application utilizes ESP extracted from apple pomace to prepare nanoparticles, successfully applying this natural resource to the functional food field, achieving effective resource utilization and sustainable development. It also broadens the application scope of ESP, provides reference and inspiration for the development and utilization of other natural products, and offers important theoretical basis for understanding the interaction mechanism between ESP and lutein, optimizing nanoparticle preparation conditions, and improving the bioavailability of lutein. The ESP of this invention can combine with Zein through electrostatic interactions to form core-shell structured composite particles, significantly improving the stability of the system. This overcomes the technical pain points of existing technologies, such as poor water solubility and low bioavailability of lutein, insufficient stability of single Zein nanoparticles, and waste of apple pomace ESP resources. Through innovative ESP extraction processes and composite ratios, it achieves the synergistic goals of high-value utilization of waste, high carrier stability, and efficient delivery of active ingredients.

[0021] Specifically, the beneficial effects compared with the prior art of this invention are as follows: (1) This application converts apple pomace waste into high-value ESP raw materials at a cost of only RMB 20 / kg, which is about 40% lower than commonly used commercial beet pulp polysaccharides, tremella polysaccharides, etc. At the same time, it solves the problem of apple pomace pollution and meets the needs of rural revitalization and sustainable development. (2) Because ESP has a high carboxyl density, a small amount can cover the positive charge sites of Zein through electrostatic interaction to form a stable electrostatic layer. Therefore, the optimal composite ratio of ESP and Zein (1:0.4) is lower than that of beet pulp polysaccharide (1:0.6) and tremella polysaccharide (1:0.5), and the amount of polysaccharide used is less. Moreover, the acidic anionic properties of ESP, combined with hydrogen bonding and hydrophobic interaction, strengthen the shell density, which can effectively enhance the Zein particles' resistance to ion interference, resistance to thermal damage and storage stability. The particle size is still stable within 200nm after heating at 80℃ for 150min. (3) The lutein encapsulation rate (90.5%) was about 65.4% higher than that of the single Zein system (54.7%) and about 11% higher than that of the soybean alcohol polysaccharide-Zein system (about 80%), which greatly improved bioavailability. Attached Figure Description

[0022] Figure 1 The particle size of the composite nanoparticles and their PDI (A) and Zeta potential (B) are shown for different Zein-ESP mass ratios. in, Figure 1 In Figure A, the horizontal axis represents the Zein:ESP mass ratio (1:0, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6), and the vertical axis represents the particle size (nm) on the left and the PDI on the right. The results show that the particle size is the smallest (129.1nm) at a ratio of 1:0.4, and increases to 252.9nm at a ratio of 1:0.6. Figure 1 The x-coordinate in B is the same Figure 1 A, the vertical axis represents the Zeta potential (mV); the results show that as the ESP ratio increases, the potential decreases from +28.5mV to -0.2mV, and at 1:0.3 the potential is +18.7mV, indicating suitable electrostatic repulsion; Figure 2 Fourier transform infrared (FTIR) spectra of apple pomace ESP and Zein-ESP composite particles. Figure 2 In the figure, the horizontal axis represents the wavenumber (cm). -1 The vertical axis represents absorbance; ESP is at 3430 cm⁻¹. -1 (OH stretching), 950-1200cm -1 (Carbohydrates) have characteristic peaks; complex particles show peaks at 2900 cm⁻¹. -1 1320cm -1 The peak shift confirms the presence of hydrogen bonding and electrostatic interactions between ESP and Zein; Figure 3 The encapsulation efficiency of lutein by composite nanoparticles at different Zein-ESP mass ratios; Figure 3 In the figure, the horizontal axis represents the Zein:ESP mass ratio (1:0, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6), and the vertical axis represents the lutein encapsulation rate (%). The encapsulation rate reached 90.5% at 1:0.3, only 54.7% at 1:0, decreased to 37.4% at 1:0.5, and continued to decline at 1:0.6. Combining particle size and PDI data, the optimal ratio was determined to be 1:0.4. Figure 4 The particle size of the composite nanoparticles with different ESP addition amounts are related to PDI (A) and Zeta potential (B). in Figure 4 In A, the horizontal axis represents the ratio of ESP to Zein (0.0-0.6), and the vertical axis represents the particle size (nm) on the left and PDI on the right. Figure 4 In B, the x-axis is the same as... Figure 4A, with the vertical axis representing the Zeta potential (mV); Figure 5 Infrared spectral analysis (A) and molecular weight (B) of alcohol-soluble polysaccharides from apple pomace; Figure 5A: The horizontal axis represents wavenumber (cm) -1 The vertical axis represents absorbance; the results show that ESP is at 3430 cm⁻¹. -1 (OH stretching vibration), 980cm -1 The presence of typical polysaccharide characteristic peaks at the (COC stretching vibration) position confirms that the polysaccharide structure is intact and free from obvious impurity interference.

[0023] Figure 5B: The horizontal axis represents the molecular weight reference value (kDa), and the vertical axis represents the signal response intensity. The results show that the elution peaks of ESP are symmetrical, without obvious impurity peaks, and the molecular weight distribution is uniform, which meets the requirements of composite nanoparticle carriers for raw material structure.

[0024] Figure 6 This is a curve showing the composition of each component in an apple pomace alcohol-soluble polysaccharide sample determined by ion chromatography. The x-axis represents retention time (min), and the y-axis represents the detection response value (μS). The results show that the main monosaccharide component in the sample is arabinose, which shows a strong response peak at the corresponding retention time. Calculation based on peak area integration shows that arabinose accounts for 83.8%, while the proportions of other monosaccharides (such as glucose and galactose) are extremely low, consistent with the characteristics of highly hydrophobic groups. This provides structural support for its hydrophobic interaction with Zein and lutein encapsulation. Detailed Implementation

[0025] The technical solution of the present invention will be further described in a non-limiting manner below with reference to the accompanying drawings and specific embodiments; the reagents used in the implementation process are all food grade or analytical grade, and the instruments include Malvern Nano ZS90 particle size analyzer, Thermo IS10 FTIR spectrometer, and UV-Vis spectrophotometer.

[0026] Example 1: Extraction and purification of ESP from apple pomace 1) Raw materials: Apple pomace was provided by Shandong Xinruijie Biotechnology Co., Ltd.; 2) Pretreatment: Weigh the apple pomace, crush it through an 80-mesh sieve; add 5 times the volume of distilled water, boil in a water bath for 10 minutes to remove pectinase; after cooling, add 10 times the volume of distilled water, centrifuge at 3000 r / min for 15 minutes, and discard the precipitate; repeat the washing twice, and dry at 70℃ to a moisture content of 7-9% to obtain pretreated apple pomace. 3) Hot water extraction: Mix pretreated apple pomace with distilled water at a material-to-liquid ratio of 1:20 (g:mL), extract in a constant temperature water bath at 80℃ for 3 hours, centrifuge at 5000r / min for 20 minutes, and collect the supernatant; 4) Purification: Add an equal volume of 20% TCA solution to the supernatant, let stand at 4℃ for 12 h, centrifuge at 3000 r / min for 10 min, and collect the supernatant (deproteinize); repeat the deproteinization once to obtain the supernatant; add 3 volumes of 95% ethanol to the supernatant, let stand at 25℃ for 12 h, centrifuge at 12000 r / min for 20 min and collect the precipitate; reconstitute the precipitate with 10 mL of ultrapure water, put it into a 14 kDa dialysis bag, dialyze with ultrapure water for 11 h (changing the water every 2 h), and freeze-dry at -40℃ to obtain pure ESP; 5) Detection: The yield of pure ESP was 0.36%, the protein removal rate was 89.5%, and the polysaccharide purity was 86.2%; FTIR characterization showed a concentration of 3430 cm⁻¹. -1 (OH), 980cm -1 The characteristic peak of (COC) confirmed the integrity of the polysaccharide structure; ion chromatography analysis showed that arabinose accounted for 83.8%, which is consistent with the characteristics of highly hydrophobic groups.

[0027] Example 2: Sorting of Zein-ESP composite nanoparticles (ZENPs) 1) Solution preparation: 5 mg / mL (0.5% w / v) Zein solution (dissolved in 85% ethanol), 10 mg / mL (1% w / v) ESP aqueous solution; 2) Setting the ratio gradient: Take 10 mL of Zein solution according to the Zein:ESP mass ratios of 1:0, 1:0.2, 1:0.3, 1:0.4, 1:0.5, and 1:0.6, and add the corresponding volume of ESP aqueous solution dropwise. 3) Preparation: Stir at 300 rpm for 10 min, rotary evaporate at 45℃ for 35 min to remove ethanol, centrifuge at 4000 rpm for 10 min, and filter through a 0.45 μm filter membrane; 4) Thermal stability test procedure: Take 5 mL of the prepared ZENPs dispersion and place it in an 80℃ constant temperature water bath with an accuracy of ±1℃. Keep it at this temperature for 150 min, and gently stir it once every 30 min at 50 rpm (to avoid local particle sedimentation and agglomeration). After heating, immediately place it in an ice water bath to cool to room temperature. Use a Malvern NanoZS90 nanoparticle size analyzer to determine the particle size and PDI (each sample was measured in parallel 3 times, and the average value was taken).

[0028] 4) Physicochemical performance testing (Malvin NanoZS90):

[0029] 5) Particle size and PDI after heating at 80℃

[0030] 6) Conclusion: ZENPs maintained excellent stability: the particle size of the 1:0.4 group was stable at 163.4 nm (<200 nm), and the particle size of the 1:0.3 group was 174.6 nm (<200 nm). The core reason is that the carboxyl group density of ESP is as high as 3.86 mmol / g, which forms a dense electrostatic layer with the positively charged sites on the Zein surface through strong electrostatic interaction. The synergistic reinforcement of hydrogen bonding and hydrophobic interaction effectively resists the damage to the particle structure caused by thermal motion and avoids irreversible aggregation of particles. The Zein:ESP ratio of 1:0.4 has the smallest particle size, the lowest PDI, and the best stability; the potential of 1:0.3 is moderate, which can balance stability and encapsulation efficiency when used for lutein encapsulation.

[0031] Example 3: Preparation and Encapsulation Efficiency Determination of Zein-ESP-Lutein Composite Nanoparticles (ZE-LNPs) 1) Zein-lutein complex solution: Take 100mL of 5mg / mL Zein solution, add 0.04g of lutein (Zein:lutein=1:0.08), stir at 750rpm for 30min until the lutein is completely dispersed and there are no visible particles; 2) Compounding: Add 30 mL of 10 mg / mL ESP aqueous solution at a ratio of Zein:ESP = 1:0.3, and stir at 300 rpm for 10 min; 3) Post-treatment: Ethanol was removed by rotary evaporation at 45℃ for 35 min, centrifugation at 4000 r / min for 15 min, and filtration through a 0.45 μm filter membrane to obtain ZE-LNPs solution; 4) Encapsulation rate determination: a) Take 1 mL of ZE-LNPs solution, add 9 mL of n-hexane-acetone (1:1 v / v), and sonicate for 30 min to destroy the particles; b) Centrifuge at 4000 r / min for 20 min, collect the supernatant, and measure the absorbance at 445 nm using a UV spectrophotometer; c) According to the standard curve Y=5.6965x+0.03002 (R²=0.9888, Chang Ying, Jiao Yan, Liu Qingsong, et al. Structural characterization and release performance study of corn peptide-loaded lutein nanoparticles [J]. Food Science and Technology, 2021, 46(2):238-243.) Calculate the lutein content; d) Encapsulation efficiency (EE) = (mass of lutein in granules / mass of added lutein) × 100% = (0.0362g / 0.04g) × 100% = 90.5%; e) Loading rate (LE) = (mass of lutein in particles / total mass of nanoparticles) × 100% = 5.82%; 5) Structural characterization: FTIR analysis showed that ZE-LNPs were located at 1650 cm⁻¹.-1 (Zein's C=O), 3430cm -1 (ESP's OH) peak shift, and lutein's 1520cm peak -1 The disappearance of the (C=C) characteristic peak confirms that lutein was successfully encapsulated in the hydrophobic core of the Zein-ESP complex particles.

[0032] The system prepared by the above method has a core-shell structure: Zein and lutein form a hydrophobic core with a diameter of 80-120 nm through hydrophobic interactions, and ESP forms a shell layer with a thickness of 30-50 nm through hydrogen bonds (the OH bond of ESP and the C=O bond of Zein) and electrostatic interactions (the carboxyl group of ESP and the amino group of Zein, with Zein being positively charged at pH 4.0). The system has stable physicochemical properties: particle size 150-250 nm, Zeta potential +10 to +30 mV, PDI < 0.3, lutein retention rate ≥ 96% after 15 days of storage at 4℃, and particle size change ≤ 20% within the pH range of 5-9 (food processing and gastrointestinal environment).

[0033] Comparative Example 1: Preparation and Encapsulation Efficiency Determination of Zein-Lutein Composite Nanoparticles (Z-LNPs) 1) Zein-lutein complex solution: Take 100mL of 5mg / mL Zein solution, add 0.04g of lutein (Zein:lutein=1:0.08), stir at 750rpm for 30min until the lutein is completely dispersed and there are no visible particles; 2) Combining: Add 30 mL of aqueous solution and stir at 300 rpm for 10 min; 3) Post-treatment: Ethanol was removed by rotary evaporation at 45℃ for 35 min, centrifugation at 4000 r / min for 15 min, and filtration through a 0.45 μm filter membrane to obtain Z-LNPs solution; 4) Encapsulation rate determination: a) Take 1 mL of Z-LNPs solution, add 9 mL of n-hexane-acetone (1:1 v / v), and sonicate for 30 min to destroy the particles; b) Centrifuge at 4000 r / min for 20 min, collect the supernatant, and measure the absorbance at 445 nm using a UV spectrophotometer; c) Calculate the lutein content using the standard curve Y=5.6965x+0.03002 (R²=0.9888, Chang Ying, 2021); d) Encapsulation efficiency (EE) = (mass of lutein in granules / mass of added lutein) × 100% = (0.0219g / 0.04g) × 100% = 54.7%; e) Loading rate (LE) = (mass of lutein in particles / total mass of nanoparticles) × 100% = (0.0219g / (0.5g Zein + 0.0219g lutein)) × 100% ≈ 4.19%; 5) Structural characterization: FTIR analysis showed that Z-LNPs were located at 1650 cm⁻¹. -1 (Zein's C=O), 3430cm -1 (ESP's OH) peak shift, and lutein's 1520cm peak -1 The reduction in the (C=C) characteristic peaks confirms that some lutein is embedded in a hydrophobic core.

[0034] 6) Results Analysis: The encapsulation efficiency of single Zein nanoparticles was 54.7%, which was much lower than the 90.5% of ZE-LNPs in Example 3; the initial particle size was 98 nm, and near the isoelectric point (pH 6.0), it agglomerated violently due to hydrophobic interactions, with a particle size change rate of 89.3%; after heating at 80℃ for 150 min, the particle size increased to 386.5 nm (>200 nm), PDI=0.45 (outside the stable range), and the lutein retention rate was 74.1%; after storage at 4℃ for 15 days, the lutein retention rate was less than 70%, confirming that the addition of ESP is the key to improving encapsulation efficiency and stability.

[0035] Comparative Example 2: Preparation and Encapsulation Efficiency Determination of Beetroot Pulp Polysaccharide-Lutein Composite Nanoparticles (B-LNPs) 1) Preparation of beet pulp polysaccharide (BESP) solution: Pure BESP was prepared according to the ESP extraction process in Example 1, and a 10 mg / mL BESP aqueous solution (pH 4.0) was prepared. 2) BESP-lutein complex solution: Take 100mL of 10mg / mL BESP aqueous solution, add 0.04g of lutein (BESP:lutein = 1:0.004), stir at 750rpm for 30min until the lutein is completely dispersed and there are no visible particles; 3) Post-treatment: No rotary evaporation is required to remove ethanol (no Zein-ethanol solution), centrifuge at 4000 r / min for 15 min, filter through a 0.45 μm filter membrane to obtain B-LNPs solution; 4) Encapsulation rate determination: a) Take 1 mL of B-LNPs solution, add 9 mL of n-hexane-acetone (1:1 v / v), and sonicate for 30 min to destroy the particles; b) Centrifuge at 4000 r / min for 20 min, collect the supernatant, and measure the absorbance at 445 nm using a UV spectrophotometer; c) Calculate the lutein content using the standard curve Y=5.6965x+0.03002 (R²=0.9888, Chang Ying, 2021); d) Encapsulation efficiency (EE) = (mass of lutein in granules / mass of added lutein) × 100% = (0.0095g / 0.04g) × 100% = 23.7%; e) Loading rate (LE) = (mass of lutein in particles / total mass of nanoparticles) × 100% = (0.0095g / (1.0g BESP + 0.0095g lutein)) × 100% ≈ 0.94%; 5) Structural characterization: FTIR analysis showed that BESP has a 3430 cm³ structure. -1 The (OH) peak showed no significant shift, while the lutein peak was at 1520 cm⁻¹. -1 (C=C) The characteristic peak intensity did not decrease significantly, confirming that there was no effective embedding effect.

[0036] 6) Results Analysis: Single beet pulp polysaccharide nanoparticles have obvious defects: the lutein encapsulation rate is only 23.7%, far lower than the 90.5% of ZE-LNPs; the particles are severely aggregated, with an initial particle size of 896.2 nm; the thermal stability is extremely poor, with the particle size increasing to 1187.3 nm (far exceeding 200 nm) after heating at 80℃ for 150 min, and the lutein retention rate is 42.3% (lower than the thermal stability retention rate of the composite system); the lutein degrades rapidly during storage, with a retention rate of less than 40%.

[0037] Comparative Example 3: Preparation and Encapsulation Efficiency Determination of Tremella Polysaccharide-Lutein Composite Nanoparticles (T-LNPs) 1) Preparation of Tremella polysaccharide (TESP) solution: Pure TESP was prepared by deproteinization using the Sevag method, and a 10 mg / mL TESP aqueous solution (pH 4.0) was prepared. 2) TESP-lutein complex solution: Take 100mL of 10mg / mL TESP aqueous solution, add 0.04g of lutein (TESP:lutein = 1:0.004), stir at 750rpm for 30min until the lutein is completely dispersed and there are no visible particles; 3) Post-treatment: No rotary evaporation is required to remove ethanol. Centrifuge at 4000 r / min for 15 min, filter through a 0.45 μm filter membrane to obtain T-LNPs solution; 4) Encapsulation rate determination: a) Take 1 mL of T-LNPs solution, add 9 mL of n-hexane-acetone (1:1 v / v), and sonicate for 30 min to destroy the particles; b) Centrifuge at 4000 r / min for 20 min, collect the supernatant, and measure the absorbance at 445 nm using a UV spectrophotometer; c) Calculate the lutein content using the standard curve Y=5.6965x+0.03002 (R²=0.9888, Chang Ying, 2021); d) Encapsulation efficiency (EE) = (mass of lutein in granules / mass of added lutein) × 100% = (0.0076g / 0.04g) × 100% = 18.9%; e) Loading rate (LE) = (mass of lutein in particles / total mass of nanoparticles) × 100% = (0.0076g / (1.0g TESP + 0.0076g lutein)) × 100% ≈ 0.75%; 5) Structural characterization: FTIR analysis showed that TESP has a 3430 cm³ structure. -1 (OH) Peak shape showed no shift; lutein peak was at 1520 cm⁻¹. -1 The intensity of the characteristic peak (C=C) did not decrease significantly, confirming that no effective embedding structure was formed.

[0038] 6) Results Analysis: The performance of single Tremella fuciformis polysaccharide nanoparticles was inferior to that of the single Zein system: the lutein encapsulation rate was only 18.9%, lower than the 54.7% of Z-LNPs; and far lower than the 88.59% of the Tremella fuciformis polysaccharide-Zein composite system; the particle dispersibility was poor, with an initial particle size of 947.6 nm (far exceeding the optimal particle size of 164.0 nm in the composite system); the thermal stability was extremely poor, with the particle size increasing to 1328.5 nm (far exceeding 200 nm) after heating at 80℃ for 150 min, and the lutein retention rate was 40.5% (far lower than the 90.74% of the composite system); the lutein retention rate during storage was less than 35%, because the side chains of Tremella fuciformis polysaccharide were loose and lacked hydrophobic encapsulation sites, so it could only physically adsorb a small amount of lutein and could not effectively deliver lutein.

[0039] Comparative Example 4: Preparation and Encapsulation Efficiency Determination of ESP-Lutein Composite Nanoparticles (E-LNPs) 1) Preparation of ESP solution: Prepare pure ESP according to the method in Example 1, and prepare an aqueous solution of 10 mg / mL ESP (pH 4.0). 2) ESP-lutein complex solution: Take 100mL of 10mg / mL ESP aqueous solution, add 0.04g of lutein (ESP:lutein = 1:0.004), stir at 750rpm for 30min until the lutein is completely dispersed and there are no visible particles; 3) Post-treatment: No rotary evaporation is required to remove ethanol. Centrifuge at 4000 r / min for 15 min, filter through a 0.45 μm filter membrane to obtain E-LNPs solution; 4) Encapsulation rate determination: a) Take 1 mL of E-LNPs solution, add 9 mL of n-hexane-acetone (1:1 v / v), and sonicate for 30 min to destroy the particles; b) Centrifuge at 4000 r / min for 20 min, collect the supernatant, and measure the absorbance at 445 nm using a UV spectrophotometer; c) Calculate the lutein content using the standard curve Y=5.6965x+0.03002 (R²=0.9888, Chang Ying, 2021); d) Encapsulation efficiency (EE) = (mass of lutein in granules / mass of added lutein) × 100% = (0.0061g / 0.04g) × 100% = 15.3%; e) Loading rate (LE) = (mass of lutein in particles / total mass of nanoparticles) × 100% = (0.0061g / (1.0g ESP + 0.0061g lutein)) × 100% ≈ 0.61%; 5) Structural characterization: FTIR analysis showed that the ESP has a 3430 cm³ structure. -1 (OH), 950-1200cm -1 (Carbohydrate) Characteristic peaks showed no shift; lutein's peak was at 1520 cm⁻¹. -1 The (C=C) peak intensity did not decrease significantly, confirming that ESP alone cannot form an effective encapsulation system.

[0040] 6) Results Analysis: Single ESP nanoparticles could not form an effective encapsulation system: the lutein encapsulation rate was only 15.3%, far lower than the 90.5% of ZE-LNPs and 54.7% of Z-LNPs; consistent with the low encapsulation rate trend of other anionic polysaccharides (beet pulp polysaccharide, tremella polysaccharide) used alone; severe particle aggregation, with an initial particle size of 912.8 nm (far exceeding the 156.8 nm of ZE-LNPs); extremely poor thermal stability, with the particle size increasing to 1513.7 nm (far exceeding 200 nm) after heating at 80℃ for 150 min; and less than 30% lutein retention rate during storage.

[0041] The above description is only an explanation of the present invention and is not a limitation thereof. The scope of the present invention is defined by the claims. Within the scope of protection of the present invention, any form of modification may be made.

Claims

1. An ESP-Zein-lutein composite nanoparticle delivery system, characterized in that: The delivery system consists of a core-shell structure composed of Zein, ESP, and lutein. Zein and lutein form a hydrophobic core, while ESP forms a shell through hydrogen bonding and electrostatic interactions.

2. The delivery system according to claim 1, characterized in that: The mass ratio of Zein, ESP, and lutein is 1:0.2-0.6:0.05-0.1, that is, 1 part by mass of Zein is mixed with 0.2-0.6 parts by mass of ESP and 0.05-0.1 parts by mass of lutein.

3. The delivery system according to claim 2, characterized in that: The mass ratio of Zein, ESP, and lutein is 1:0.4:0.

08.

4. The delivery system according to claim 1, characterized in that: The nanoparticles have a particle size of 150-250 nm, a zeta potential of +10 to +30 mV, a PDI of <0.3, and a lutein encapsulation rate of ≥85%.

5. The delivery system according to any one of claims 1-4, characterized in that: The ESP was extracted from apple pomace.

6. A method for preparing an ESP-Zein-lutein composite nanoparticle delivery system, characterized in that, Includes the following steps: (1) Extraction and purification of ESP: After drying and crushing apple pomace, remove enzymes by boiling water and wash with distilled water to remove impurities. Mix with distilled water and heat for a certain time. Centrifuge and take the supernatant. Add TCA to the supernatant and let it stand at low temperature to remove protein. Then precipitate polysaccharides with ethanol. After removing ethanol by rotary evaporation, decolorize through a dialysis bag and freeze dry to obtain pure ESP. (2) Preparation of Zein solution: Dissolve Zein in an aqueous ethanol solution and stir thoroughly to form a Zein solution with a concentration of 4-6 mg / mL (0.4-0.6% w / v); (3) Preparation of Zein-lutein complex solution: Add lutein to the Zein solution in step (2) and stir until completely dispersed to obtain Zein-lutein complex solution; (4) Preparation of composite nanoparticles: The Zein-lutein composite solution from step (3) was added dropwise to the ESP aqueous solution, stirred and rotary evaporated to remove ethanol, and after centrifugation and filtration, the composite nanoparticle delivery system was obtained.

7. The preparation method according to claim 6, characterized in that: In step (3), the mass ratio of Zein to lutein is 1:(0.05-0.1); in step (4), the mass ratio of Zein to ESP is 1:(0.2-0.6).

8. The preparation method according to claim 7, characterized in that: In step (3), the mass ratio of Zein to lutein is 1:0.08; in step (4), the mass ratio of Zein to ESP is 1:0.

4.

9. The application of the delivery system according to any one of claims 1-8 in the stable encapsulation and efficient delivery of hydrophobic active ingredients.

10. The application according to claim 9, characterized in that: The hydrophobic active ingredient is lutein.

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