Binary composite carrier for delivery of bioactive substances as well as preparation method and application of binary composite carrier

Abstract

The invention discloses a binary composite carrier for delivery of bioactive substances as well as a preparation method and application thereof, and relates to the technical field of biological materials. According to the invention, the YP-Sur binary composite carrier is successfully prepared by compounding yeast protein and lipopeptide. Through detection, the solubility of the YP-Sur binary composite carrier is remarkably improved and reaches 13.38 + / -0.14%, the method not only effectively improves the solubility of the yeast protein, but also synchronously improves the oxidation resistance and the Zeta potential absolute value of the yeast protein, and builds a high-quality carrier basis for subsequent construction of a high-efficiency delivery system. According to the invention, a YP-Sur-Zn < 2 + >-TP composite delivery system is further constructed, and inductively coupled plasma mass spectrometry and load rate analysis data show that the composite delivery system realizes efficient loading of the two active components Zn < 2 + > and TP.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a binary composite carrier for the delivery of bioactive substances, its preparation method, and its application. Background Technology

[0002] Yeast protein (YP), a novel natural protein resource derived from microbial fermentation, is gaining increasing attention from academia and industry due to its unique nutritional advantages and environmental properties. As a natural protein resource, it boasts a balanced amino acid composition that aligns with human protein requirements and is rich in bioactive components such as B vitamins. However, despite these advantages, the inherent functional limitations of natural yeast protein severely restrict its application in high-end fields. Numerous studies have confirmed that natural yeast protein exhibits strong hydrophobicity and a tendency to aggregate, resulting in poor emulsification properties and low solubility in aqueous solutions. It is particularly prone to precipitation in neutral and alkaline environments, making it unsuitable for processing liquid foods and functional beverages. More importantly, when used as a carrier for active ingredients, natural yeast protein suffers from low drug loading capacity, poor encapsulation stability, and a tendency for carrier structure disintegration during gastrointestinal digestion. This leads to premature release of active ingredients, significantly reducing delivery efficiency.

[0003] Zinc ions, as an essential trace element for the human body, are indispensable in promoting growth and development and maintaining immune function. Tea polyphenols, a type of natural polyphenol widely found in tea, possess strong antioxidant, anti-inflammatory, and anti-tumor biological activities, but are easily oxidized and degraded, resulting in low bioavailability. Both have the drawback of poor stability, and both deliver Zn... 2+ No system of tea polyphenols has been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a binary composite carrier for delivering bioactive substances, its preparation method, and its application, thereby addressing the problems existing in the prior art. This binary composite carrier can effectively improve the emulsification stability and encapsulation ability of yeast protein carriers.

[0005] To address some inherent limitations of yeast protein, overcome application bottlenecks, enhance functional properties, and expand application scenarios, this invention utilizes yeast protein and lipopeptides (Sur), which possess excellent surface activity, biocompatibility, and antibacterial and anti-inflammatory bioactivities, to construct a high-performance composite carrier—the YP-Sur binary complex. As a biosurfactant produced by microbial metabolism, lipopeptides exhibit outstanding surface activity, excellent biocompatibility, and significant antibacterial and anti-inflammatory bioactivities. Their safety has been verified through multiple toxicological studies, fully meeting the requirements for food-grade carriers. The unique amphiphilic structure of lipopeptide molecules makes them an ideal partner for enhancing the functional properties of yeast protein. Their hydrophilic groups can form stable hydrogen bonds or electrostatic interactions with the polar groups on the surface of yeast protein, effectively disrupting intermolecular aggregation and improving dispersibility; their hydrophobic groups can be directionally adsorbed at the oil-water interface, significantly enhancing the interfacial activity of yeast protein and improving its emulsification stability and encapsulation ability of active ingredients.

[0006] Building upon binary composite carriers, this invention further focuses on the delivery of active ingredients. Currently, research on protein-based delivery carriers largely concentrates on using single modified proteins as delivery matrices. To address the challenge of synergistic delivery of two high-value active ingredients and further enhance the application value of composite carriers, this invention differs from previous single-component delivery studies by innovatively employing a stepwise assembly method. Using the YP-Sur binary complex as the base carrier, Zn is constructed separately... 2+ Load type (YP-Sur-Zn) 2+ ), tea polyphenol (TP) supported type (YP-Sur-TP) and Zn 2+ / TP Dual Delivery Type (YP-Sur-Zn) 2+ -TP) Three types of multi-component composite systems.

[0007] This invention cleverly designs Zn 2+ By specifically coordinating with the phenolic hydroxyl groups in tea polyphenol molecules, a "metal ion-polyphenol" synergistic binding system is constructed. On the one hand, the binding strength between the two active ingredients and the carrier is enhanced through coordination, thereby improving the loading capacity and encapsulation stability. On the other hand, the dynamic response characteristics of the coordination bond are utilized to achieve the controllable release of the active ingredients at the target site, thereby significantly improving the bioavailability and delivery efficiency of the active ingredients.

[0008] Based on this, the present invention provides the following solution: This invention provides a method for improving the emulsification stability and encapsulation ability of yeast protein, comprising the step of mixing yeast protein and lipopeptide to obtain a binary composite carrier.

[0009] Furthermore, the lipopeptide is Surfactin C. Furthermore, the mass ratio of the yeast protein to the lipopeptide is 1 g: 50 mg.

[0010] The present invention also provides a binary composite carrier prepared according to the above method.

[0011] The present invention also provides the application of the above-mentioned binary composite carrier in the preparation of delivery systems for bioactive substances.

[0012] The present invention also provides a Zn 2+ The preparation method of the complex delivery system of tea polyphenols includes the following steps: The above-mentioned binary composite carrier is combined with Zn 2+ A mixed reaction was carried out to obtain YP-Sur-Zn 2+ complex; The YP-Sur-Zn 2+ The complex was mixed with tea polyphenols to produce YP-Sur-Zn. 2+ -The tea polyphenol complex is the composite delivery system.

[0013] Furthermore, the binary composite carrier and the Zn 2+ The ratio is 1 g: 0.1 mol.

[0014] Furthermore, the YP-Sur-Zn 2+ The specific steps for the mixed reaction of the complex and tea polyphenols include: The YP-Sur-Zn 2+ The complex was dissolved in phosphate buffer to achieve complete hydration, and the tea polyphenols were added. The mixture was stirred and reacted at pH 9.0. Uncomplexed tea polyphenols were removed by dialysis to obtain the YP-Sur-Zn complex. 2+ -Tea polyphenol complex.

[0015] The present invention also provides a composite delivery system prepared according to the above preparation method.

[0016] This invention also provides the above-described composite delivery system for the preparation of Zn-simultaneous supplementation. 2+ Applications of tea polyphenols in products.

[0017] The present invention discloses the following technical effects: This invention utilizes a solution blending method to successfully prepare a YP-Sur binary complex by combining yeast protein (YP) with a lipopeptide (Sur). The effects of the composite modification strategy were analyzed and discussed from multiple dimensions, including Fourier transform infrared spectroscopy, particle size and zeta potential, thermogravimetric analysis, and solubility determination. Solubility data showed that the solubility of the YP-Sur binary complex was significantly improved, reaching 13.38 ± 0.14%. This method not only effectively improved the solubility of yeast protein but also simultaneously improved its antioxidant properties and absolute zeta potential, laying a solid foundation for the subsequent construction of an efficient delivery system.

[0018] This invention also uses performance-optimized YP-Sur as the core carrier and employs a step-by-step assembly method to further construct three types of functionalized ternary delivery systems, including YP-Sur-Zn. 2+ The -TP dual delivery system exhibits the most outstanding performance advantages. Inductively coupled plasma mass spectrometry and loading rate analysis data show that the dual delivery system (YP-Sur-Zn) demonstrates superior performance. 2+ -TP) implements Zn 2+ Highly efficient loading of two active ingredients, TP and Zn. 2+ The loading rate reached 808.90±13.74 mg / 100g, and the tea polyphenol loading rate reached 81.36±1.6%, both significantly higher than those of single-component delivery systems (P<0.05), fully verifying the value of the synergistic effect between the carrier and the active ingredient. The hierarchical design of binary composite modification and ternary system assembly in this invention successfully overcomes the application bottleneck of yeast protein, providing valuable and feasible technical support for the large-scale application of yeast protein in fortified foods, functional health products, and pharmaceutical active ingredient delivery. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Fourier transform infrared spectrum (a), circular dichroism spectrum (b), ultraviolet-visible absorption spectrum (c), and fluorescence spectrum (d) of YP and YP-Sur; Figure 2 The results of particle size (a) and zeta potential (b) detection for YP and YP-Sur are shown in the figure. Figure 3 Statistical graphs of the number of thiol groups (a), disulfide bonds (b), surface hydrophobicity (c), and solubility (d) of YP and YP-Sur; Figure 4 This is a diagram showing the molecular docking of YP with lipopeptides. Figure 5 The diagram (a) and XRD pattern (b) show the polyphenol loading / release efficiency of the complex delivery system. Figure 6 Images of the complex delivery system are shown in the scanning electron microscope (SEM), transmission electron microscope (TEM), and laser confocal microscope (DFM) images; where ac represents the SEM, TEM, and DFM images of YP-Sur, respectively; and df represents the YP-Sur-Zn... 2+ The scanning electron microscope (SEM) image, transmission electron microscope (TEM) image, and laser confocal microscope (LCM) image of YP-Sur-TP are shown; gi are the SEM images, transmission electron microscope (TEM) images, and laser confocal microscope (LCM) images of YP-Sur-TP are shown; jl are the images of YP-Sur-Zn are shown. 2+ -Scanning electron microscope images, transmission electron microscope images, and laser confocal microscope images of TP; Figure 7 The graphs show the radical scavenging capabilities of DPPH (a) and ABTS (b) in the complex delivery system. Figure 8 Changes in tea polyphenol concentration (A) and Zn concentration in a digestion simulation experiment of a complex delivery system 2+ Statistical graphs of concentration changes (B), digestibility (C), and zeta potential (D); where different lowercase letters indicate significant differences between groups (p<0.05). Figure 9 The diagram shows the changes in rheological behavior of the system after digestion of the complex; where ad represents the changes in storage modulus, loss modulus, storage modulus, and loss modulus of the digestive fluid after gastric digestion, respectively. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The main experimental materials involved in this invention are as follows: Yeast protein (YP) of type 75 was purchased from Angel Yeast Co., Ltd., lipopeptide Surfactin C (CAS: 24730-31-2) was purchased from Shanghai Maclean's Biochemical Technology Co., Ltd., tea polyphenols were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and ZnCl2 reagent was purchased from Sinopharm Chemical Reagent Co., Ltd. All other reagents and chemicals used in this invention were of analytical grade.

[0027] Example 1 1. Construction of yeast protein-lipopeptide binary complex (YP-Sur): 1 g YP and 50 mg of lipopeptide Surfactin C were dissolved in 100 mL of phosphate buffer solution (0.01 M, pH 7.2-7.4) and mixed at room temperature on a magnetic stirrer for 2.5 h. After stirring, the mixture was removed and allowed to stand for 40 min. Then, it was dialyzed using a dialysis bag (molecular weight 1000 Da) for 24 h. The mixture was then poured onto a plate and placed at -20 °C overnight. Finally, it was freeze-dried to obtain the YP-Sur complex.

[0028] 2. Characterization of the YP-Sur complex 2.1 Rapid determination of lipopeptide content using CPC-BTB colorimetric method Equal volumes of 0.2 mM hexadecylpyridine chloride (CPC) and 0.2 mM bromothymol blue (BTB) were mixed in phosphate buffer (0.1 M, pH 8.0) to prepare a CPC-BTB solution, which was then incubated for two hours. The ratio of CPC-BTB solution to test sample was 8:1 (V / V). The reaction was carried out at 25°C for 5 min, and the A value was measured using a microplate reader. 600nm The yield was determined using the standard curve (y = 0.6311x + 0.4945, R²). 2 = 0.9921).

[0029] The amount of lipopeptide bound to the YP-Sur complex was rapidly determined by the CPC-BTB colorimetric method, and the result was 7.63±1.02 mg / 100mL, confirming the successful binding of lipopeptide to yeast protein.

[0030] 2.2 Fourier Transform Infrared Spectroscopy Analysis The functional groups of yeast protein and yeast protein-lipopeptide complex were analyzed using Fourier Transform Infrared Spectrometer (FT-IR). 150 mg of dry KBr powder was mixed with 1 mg of protein sample, ground, and pressed for 1 min, then analyzed at 4000-400 cm⁻¹. -1 Measurements were performed within the wavenumber range, at 4 cm intervals. -1 The scanning frequency is 16.

[0031] like Figure 1 As shown in Figure a, 1700-1600 cm -1 The characteristic absorption peak within the band is the amide I band, mainly originating from the stretching vibration of C=O. The absorption of the amide II band (the in-plane bending vibration of NH) is at 1533 cm⁻¹. -1 At this location, compared to YP, both the amide I and amide II bands in the YP-Sur band show a slight redshift towards lower wavenumbers, with the value redshifted to 1647.23 cm⁻¹. -1 1533.52cm -1 This indicates that the addition of lipopeptides altered the electron cloud distribution around the C=O amide bond of yeast proteins, as well as the NH bending vibration and CN stretching vibration. This confirms the interaction between lipopeptides and yeast proteins, which changes the secondary structure environment of yeast proteins.

[0032] 2.3 Circular dichroism After dissolving the lyophilized complex powder in PBS buffer solution to a concentration of 0.5 mg / mL, and hydrating for 2 h, the protein solution was then centrifuged at 10000 g for 15 min at room temperature. The CD spectrum was recorded in the wavelength range of 190-260 nm using a CD spectrometer. The buffer spectrum was subtracted to correct the baseline.

[0033] like Figure 1 As shown in Figure b and Table 1, compared with YP, the proportion of β-sheets decreased, while the proportions of β-turns and α-helices increased, indicating that the addition of lipopeptides can interact with the amino acid residues of yeast proteins. This interaction disrupts the hydrogen bond network that originally stabilized the β-sheet structure, causing some β-sheets to unfold. Simultaneously, these unfolded peptide segments rearrange to form more β-turns and α-helices.

[0034] Table 1 Secondary structure of YP and YP-Sur

[0035] Note: Data are expressed as mean ± standard deviation (n=3). Different lowercase letters indicate significant differences between samples (P<0.05).

[0036] 2.4 Ultraviolet-Visible Absorption Spectrum The spectral characteristics of the yeast protein-lipopeptide complex were analyzed by UV-Vis spectrophotometer. 6 mg of sample was fully dissolved in a 10 mL plastic centrifuge tube containing 6 mL of distilled water, and the yeast protein solution was scanned across the entire wavelength range of 200-800 nm using a UV-Vis spectrophotometer.

[0037] like Figure 1 As shown in Figure c, the UV absorption intensity of YP-Sur bound to lipopeptides is significantly increased compared to ordinary yeast protein (YP). This is because the interaction between lipopeptide molecules and yeast protein alters the state of aromatic amino acid chromophores in the yeast protein, thereby enhancing UV absorption. The difference in UV absorption peak intensity further confirms the occurrence of complexation.

[0038] 2.5 Fluorescence spectrum The intrinsic fluorescence changes of yeast protein-lipopeptide complexes were analyzed by fluorescence spectroscopy. Complex samples were dissolved in PBS (10 mmol / L; pH 7.2–7.4) to a concentration of 0.8 mg / mL. Fluorescence spectra were measured at excitation wavelengths of 280 nm and emission wavelengths of 300–450 nm, with a slit width of 5 nm and a moderate velocity.

[0039] like Figure 1As shown in Figure d, the fluorescence signal of yeast protein bound to lipopeptides (YP-Sur) was significantly reduced compared to untreated yeast protein (YP). This phenomenon indicates that yeast protein undergoes endogenous fluorescence quenching upon binding to lipopeptides. At the molecular level, the binding of lipopeptides to yeast protein may alter the protein's conformation, affecting the microenvironment of its fluorophores, thereby leading to fluorescence quenching.

[0040] 2.6 Zeta potential 10 mg of yeast protein-lipopeptide complex was fully dissolved in plastic centrifuge tubes containing 10 mL of ultrapure water, and the zeta potential of the protein sample was measured using a nanoparticle size and zeta potential analyzer.

[0041] like Figure 2 As shown in Figure a, the isoelectric point of the YP-Sur treatment group shifted towards the acidic direction compared to the untreated group (YP), indicating that the addition of lipopeptides enhanced the tendency of yeast proteins to carry a negative charge. This change may be due to the negatively charged groups carried by the lipopeptide molecules themselves. After the interaction between the protein and lipopeptides, the absolute value of the zeta potential of the yeast protein was reduced, thereby regulating the distribution of surface charge on the protein.

[0042] 2.7 Particle size Accurately weigh 80 mg of the sample to be tested, add ultrapure water and let it dissolve. Then, use a 10 mL volumetric flask to dilute it to the mark to obtain a protein solution with a concentration of 8 mg / mL. Sonicate for 10 min to dissolve the sample as much as possible, and then use a laser particle size analyzer to determine the particle size of the complex sample solution.

[0043] like Figure 2 As shown in Figure b, the average particle size of YP at a concentration of 8 mg / mL is 34.13 μm. The addition of lipopeptides reduces the particle size to D50 = 10.66 μm. Untreated yeast protein (YP) particles are larger and unevenly distributed; YP-Sur formed by binding with lipopeptides has a significantly smaller particle size and a more concentrated distribution; combined with the Zeta potential results, it can be seen that the addition of lipopeptide molecules promotes the formation of a more compact aggregate structure of YP-Sur.

[0044] 2.8 Free thiol groups and total thiol groups Prepare a 2 mg / mL complex sample accurately, dissolve it in Tris-HCl, and place 200 μL of the prepared sample in a 2 mL centrifuge tube. Add 1 mL of Tris-glycine and 20 μL of Ellman's reagent to the tube. Incubate at 25 °C for 1 h, then centrifuge at 8000 r / min for 10 min. Measure the absorbance of the supernatant at 412 nm using a microplate reader to calculate the free thiol value. Use Tris-HCl as a blank throughout the process. For the determination of total thiol, add 8 M urea to the system. The rest of the method is the same as above.

[0045] Where: D—dilution factor (F-SH: 3.05; T-SH: 10.01); C —Sample concentration, mg / mL.

[0046] from Figure 3 As shown in Figures a and b, compared to the untreated yeast protein (YP) group, the YP-Sur group bound to lipopeptides exhibited significantly increased total and free thiol content, with the sum of both being much higher than that of the YP group. This indicates that lipopeptides can promote the exposure or generation of more thiol groups in yeast protein molecules, altering the state of thiol groups within the protein molecule. Furthermore, it is evident that the number of disulfide bonds in the Sur-YP group is significantly increased compared to the YP group. This suggests that the addition of Sur causes reorganization of intramolecular / intermolecular chemical bonds in YP molecules. Thiol groups originally hidden within the protein may be exposed due to conformational unfolding, resulting in a significant increase in the number of free thiol groups. These exposed thiol groups can then further re-pair to form new disulfide bonds, ultimately leading to the reconstruction of the protein's secondary and tertiary structures. This demonstrates that Sur can regulate the structural state of yeast proteins by acting on the balance between thiol groups and disulfide bonds within the protein molecule, thereby positively influencing the physicochemical properties of the yeast protein itself.

[0047] 2.9 Surface hydrophobicity determination Surface hydrophobicity was determined using ANS as a fluorescent probe: PBS buffer containing 8 mmol / L ANS was precisely prepared, and proteins were diluted to concentrations of 0.05, 0.10, 0.20, 0.50, and 0.80 mg / mL to construct a series of detection samples. 6 mL of the prepared sample was added to 20 μL of ANS solution and reacted in the dark for 15 min, followed by detection. Fluorescence spectroscopy was performed on a fluorescence spectrophotometer with the following parameters: excitation wavelength (λex) = 390 nm, emission wavelength (λem) = 470 nm, and both excitation and emission slit widths were 5 nm. The fluorescence intensity values ​​corresponding to each concentration of yeast protein, lipopeptide, and zinc complex were recorded. A linear regression model was established to determine the relationship between fluorescence intensity and protein concentration, and the slope of this model was used to quantitatively represent the surface hydrophobicity of the protein.

[0048] like Figure 3 As shown in Figure c, the surface hydrophobicity (H0) of the YP-Sur complex significantly decreases after yeast protein binds to Sur. This is because yeast protein (YP) has high surface hydrophobicity, which is related to the large number of hydrophobic groups exposed on its molecular surface. The complexation with the lipopeptide (Sur) can significantly reduce the surface hydrophobicity (H0) of yeast protein, possibly because the amphiphilic structure of the lipopeptide interacts with the yeast protein molecule, altering the conformation of the protein molecule. This causes the hydrophobic groups originally exposed on the protein surface to be masked or redistributed, thereby reducing the hydrophobic region on the protein surface.

[0049] 2.10 Solubility Solubility assay: 6 mg of yeast protein-lipopeptide complex sample was dissolved in 6 mL of distilled water to prepare a concentration of 1 mg / mL, and hydrated for 2 h. The sample solution was then centrifuged at 10000 g for 15 min at room temperature. The protein content in the supernatant was determined using the biuret assay, and a standard curve was established using bovine serum albumin (BSA) (y = 0.0295x + 0.1122, R0). 2 =0.9986). Protein solubility is expressed as the ratio of protein concentration in the supernatant to the total protein content, as shown in the following formula: In the formula: C 1 —Protein concentration in the supernatant; C 0 —The concentration of protein in the solution.

[0050] Figure 3Figure d shows the solubility of yeast protein (YP) and yeast protein bound to lipopeptides (YP-Sur). The results showed that the solubility of YP was low, only 5.68±0.3%; after lipopeptide treatment, the solubility of YP-Sur was significantly improved, reaching 13.38±0.14%; there were significant differences in solubility between groups.

[0051] The solubility of proteins is closely related to the exposure of hydrophilic groups (such as free sulfhydryl groups) on their surface. This aligns with section 2.8, where the number of free sulfhydryl groups in yeast proteins increases significantly after binding to lipopeptides, indicating an increase in hydrophilic sites on the surface and a substantial improvement in protein solubility. It is precisely because the amphiphilic structure of lipopeptides (Sur) improves the dispersibility of yeast protein molecules and reduces intermolecular aggregation that their solubility in solvents is increased.

[0052] 2.11 Molecular docking of the complex Molecular docking is a computational technique for determining the binding affinity and interaction energy of receptor-ligand complexes. Docking studies were performed using AutoDock Vina software. Prior to docking, all water of crystallization and coarse-grained contact material were removed from the receptor. Kollman charges were calculated, and hydrogen atoms were added to the polar contact of the receptor using AutoDock tools. The gradient optimization algorithm remained constant throughout the docking process. This invention used software to perform docking analysis on a yeast protein domain (RPL3-60S ribosomal protein uL3 RPL3) and a lipopeptide (Surfactin C). Docking interaction studies were performed using grid sizes of 33, 33, and 33 dimensions in the x, y, and z directions, with a docking center (x, y, z) of (7.2260, -7.7783, -9.3345). All ligand and receptor binding sites studied were confirmed using AutoDock Tools software and visualized using Pymol and LigPlot+.

[0053] This invention demonstrates molecular docking between a yeast protein domain (RPL3-60S ribosomal protein uL3 RPL3) and a lipopeptide (Surfactin C). The binding energy of their interaction is -6.6 kcal / mol, indicating that they can form a stable complex. Figure 4As shown, Surfactin C is embedded in the hydrophobic pocket of RPL3 in a folded conformation, achieving stable binding through multi-site interactions. This conformational matching provides the structural basis for their functional interactions. The basic amino acid residues of RPL3, such as LYS-222, ARG-275, and ARG-19, form hydrogen bonds with the carboxyl and hydroxyl groups of Surfactin C, while the hydrophobic amino acid residues of RPL3 form hydrophobic stacks with the lipid chain portion of Surfactin C, further enhancing the stability of the complex.

[0054] Example 2 1. Yeast protein-lipopeptide / Zn 2+ Construction of TP ternary / quaternary complex delivery system 1.1 YP-Sur-metal (Zn) 2+） Preparation of the complex 1 g of YP-Sur complex was dissolved in 100 mL of ZnCl2 solution (1 mM) and mixed at room temperature on a magnetic stirrer for 2 h. After standing for 40 min, it was dialyzed (molecular weight 1000 Da) for 24 h, then the mixture was poured, placed at -20℃ overnight, and freeze-dried to obtain YP-Sur-Zn. 2+ Complex.

[0055] 1.2 Preparation of YP-Sur-Tea Polyphenol (TP) Complex First, 1 g of the YP-Sur complex sample was dissolved in 100 mL of phosphate buffer (0.01 mol / L, pH 7.0) and stirred overnight to ensure complete hydration. The pH of the complex solution was adjusted to 9.0 using 2 mol / L NaOH. Then, TP (10 mg / 100 mL) was added to the complex solution, maintaining the pH at 9.0. After magnetic stirring for 2 h at room temperature, the solution was allowed to stand for 1 h, then transferred to dialysis and dialyzed at 4 °C for 24 h to remove uncomplexed tea polyphenols. The solution was then plated, incubated at -20 °C overnight, and freeze-dried to obtain the YP-Sur-TP complex.

[0056] 1.3 YP-Sur-Zn 2+ Preparation of tea polyphenol (TP) complex First, add 1 g of YP-Sur-Zn 2+The complex sample was dissolved in 100 mL of phosphate buffer (0.01 mol / L, pH 7.0) and stirred overnight to ensure complete hydration. The pH of the complex solution was adjusted to 9.0 using 2 mol / L NaOH. Then, TP (10 mg / 100 mL) was added to the complex solution, maintaining the pH at 9.0. After magnetic stirring for 2 h at room temperature, the solution was allowed to stand for 1 h, then transferred to dialysis and dialyzed at 4 °C for 24 h to remove uncomplexed tea polyphenols. The solution was then plated, incubated at -20 °C overnight, and freeze-dried to obtain YP-Sur-Zn. 2+ -TP complex.

[0057] 2. Yeast protein-lipopeptide / Zn 2+ Characterization of TP ternary / quaternary complex delivery systems 2.1 Metal Binding Content Accurately weigh 50 mg of YP-Sur-Zn 2+ With YP-Sur-Zn 2+ -TP was placed in a clean digestion tube, and 6 mL of HNO3 was added. Microwave digestion was then performed for 1.5 h. All the digested liquid was then transferred to a colorimetric tube and diluted to 50 mL with ultrapure water. The sample was filtered through a membrane and diluted to an appropriate factor (10-100 times) for determination using inductively coupled plasma mass spectrometry (ICP-MS). The metal-bound content was calculated as follows: in: m 1 It is the total metal content (mg) in the chelate and m 0 It represents the mass (g) of the chelate.

[0058] Table 2 shows the YP-Sur-Zn 2+ With YP-Sur-Zn 2+ The metal-bound content of -TP confirmed that Zn 2+ In YP-Sur-Zn 2+ With YP-Sur-Zn 2+ Successful coupling in the -TP complex.

[0059] Table 2 YP-Sur-Zn 2+ With YP-Sur-Zn 2+ - TP metal bonding content

[0060] 2.2 Quantitative analysis of complexed tea polyphenols Use YP-Sur-TP and YP-Sur-Zn 2+The concentrations of free and bound tea polyphenols in the YP-Sur-TP composite material were determined. Free tea polyphenols in the complexed tea polyphenol material were extracted using 97% ethanol. The suspension was then centrifuged (4392 g, 15 min), and the supernatant was collected for the determination of free catechins. Subsequently, 90% DMSO was added to the precipitate and stirred until YP-Sur-TP and YP-Sur-Zn were mixed. 2+ The -TP composite material was completely dissolved, and after centrifugation (2657 g, 10 min), the supernatant was collected to determine the bound catechins. The absorbance was measured at 290 nm using a microplate reader. A linear calibration curve (R²) was prepared. 2 =0.974), the standard concentrations of tea polyphenols were selected as follows: 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL.

[0061] like Figure 5 As shown in Figure a, tea polyphenols (TP) were successfully loaded onto YP-Sur-TP and YP-Sur-Zn. 2+ -TP complex, in which YP-Sur-Zn 2+ -TP has a slightly higher loading efficiency than YP-Sur-TP, but in terms of the release efficiency of tea polyphenols (TP), YP-Sur-Zn is worth studying. 2+ The release efficiency of -TP was slightly lower than that of YP-Sur-TP, indicating that the introduction of Zn changed the surface properties of the sample, thus improving the sample's ability to load TP. This is because of the interaction between Zn and Zn. 2+ As a metallic element, its addition forms coordination bonds with N and O atoms in the sample matrix, constructing more hydrophilic active sites on the surface of the composite sample, enhancing the interaction with TP, and thus increasing the loading capacity. However, YP-Sur-Zn 2+ -TP has a certain inhibitory effect on the release of TP, possibly because there is an "anchoring" effect between Zn and TP, which increases the energy barrier for the desorption of the loaded material from the sample surface and slows down its release rate into the external environment.

[0062] 2.3 XRD YP-Sur-Zn was analyzed using an X-ray diffractometer (DMAX2500, Japan) under 40 kV voltage and 40 mA Cu-Kα radiation. 2+ YP-Sur-TP and YP-Sur-Zn 2+ Crystal structure of the TP composite material. XRD spectra were recorded in the range of 4–45° (2θ) at a scan rate of 2° / min.

[0063] like Figure 5As shown in Figure b, all four samples exhibited distinct characteristic diffraction peaks near 2θ = 31.7. Among them, YP-Sur-Zn... 2+ The intensity of the characteristic diffraction peaks of the sample was significantly higher than that of YP-Sur and YP-Sur-Zn. 2+ -TP and YP-Sur-TP samples, with a slight shift in diffraction peak positions, indicate that YP-Sur-Zn 2+ The sample exhibits higher crystallinity, possibly due to the presence of metallic Zn. 2+ The synergistic effect with the TP component led to subtle changes in the interplanar spacing. YP-Sur-Zn 2+ The relatively low intensity and broad peak shapes of the characteristic diffraction peaks in the YP-Sur-TP and YP-Sur-TP samples indicate a lower degree of crystallinity, potentially suggesting the presence of more amorphous phases or smaller crystal particles. Samples with higher crystallinity imply stronger intermolecular forces, maintaining the integrity of the support, compared to YP-Sur-Zn. 2+ -TP, Zn 2+ The synergistic effect with TP not only enhances the bilayer effect and targeting of the dual delivery complex system, but also maintains a certain degree of crystal regularity to ensure carrier strength, while its moderate crystallinity results in better drug dispersibility.

[0064] 2.4 Scanning Electron Microscopy Analysis Evaluation of YP-Sur-Zn by scanning electron microscopy (SEM) 2+ YP-Sur-TP and YP-Sur-Zn 2+ - Surface morphology characteristics of TP composite materials. Dry protein samples were coated on conductive adhesive, sprayed with gold, and observed by photography at 3.0 kV. The microscopic appearance of the protein samples was recorded at 100x, 200x, 300x, 500x, and 1000x magnification.

[0065] SEM images of the four complex samples are shown below. Figure 6 a, d, g, and j. Among them, compared to the YP-Sur complex, YP-Sur-Zn... 2+ The particles exhibit relatively regular, spherical aggregates with relatively uniform particle size, which is due to the Zn... 2+ A stable complex structure is formed with the carrier molecules, enhancing the repulsive force between particles, reducing aggregation, and making the particle size more uniform. However, in YP-Sur-TP, due to the strong hydrogen bonding between tea polyphenol (TP) molecules, self-aggregation easily occurs, resulting in irregular particle morphology and a high degree of aggregation. However, when tea polyphenols (TP) react with zinc ions (Zn... 2+Simultaneous participation of zinc ions (Zn) results in a uniform, near-spherical particle shape in the YP-Sur-Zn-TP dual delivery system, with reduced aggregation compared to YP-Sur-TP. 2+ A complexation reaction was formed between Zn and tea polyphenols (TP). 2+ As a "bridge," it inhibits severe aggregation of hydrogen bonds between TP molecules, while its own complexing effect optimizes the dispersibility and regularity of the particles.

[0066] 2.5 Transmission electron microscope Samples were prepared by dropping a 1 mg / mL complex solution onto a copper grid coated with a support film. The samples remained in the copper grid for 2–3 min. Then, the samples were immersed in a 1%–3% phosphotungstic acid (PTA) solution, stained with a staining agent adjusted to pH 6.4–7.0 using sodium hydroxide, and stained for 2–3 min. After air drying, the samples were observed under a transmission electron microscope.

[0067] TEM images of the four samples are shown below. Figure 6 In the samples b, e, h, and k, the pure YP-Sur complex particles are loosely distributed with a particle size of 703.1 nm, lacking a continuous network structure. Compared to YP-Sur, the YP-Sur-TP sample shows a discrete granular distribution with better overall dispersion and a greater number of small aggregates. This is because the loading of tea polyphenols (TP) on the surface of yeast protein-lipopeptide (YP-Sur) did not induce large-scale aggregation, indicating that the interaction between TP and YP-Sur has a dispersive guiding effect, maintaining the nanoscale particle morphology of the complex; YP-Sur-Zn 2+ The samples appeared as large aggregates, with aggregate sizes exceeding 1 μm. This is likely due to the strong aggregation behavior of zinc ions on the YP-Sur surface, possibly originating from Zn. 2+ Electrostatic attraction and chemical complexation lead to intermolecular cross-linking, forming large-sized aggregates. However, when TP and Zn... 2+ When simultaneously loaded with YP-Sur, TP and Zn 2+ Through dual interactions, the morphology of the composite system (YP-Sur-Zn-TP) is intermediate between that of YP-Sur-TP (discrete) and YP-Sur-Zn. 2+ Between these, the single-particle cluster size is approximately 947.9 nm, effectively suppressing Zn. 2+ The tendency of TP and Zn to aggregate leads to the formation of more uniform nanoparticle clusters. 2+ The YP-Sur surface exhibits a synergistic assembly effect, which plays an important role in controlling the morphology, structural equilibrium dispersibility, and component loading of the composite system.

[0068] 2.6 Laser confocal microscope Using a confocal laser scanning microscope (CLSM) LSM 980 and an Airyscan2 (Zeiss, Germany) YP-Sur-Zn 2 + YP-Sur-TP and YP-Sur-Zn 2+ -TP complex structure. The complex protein was stained with Nile blue, while tea polyphenols exhibit autofluorescence and do not require additional staining solution. 1 g of sample was added to a 2.00 mL plastic centrifuge tube, followed by 1 mL of distilled water and thorough mixing. Then, 20 μL of Nile blue staining solution was added and stained for 10 min. The sample was then dropped onto a glass microscope slide, covered with a coverslip, and finally fixed onto a plate. Stimulation was performed using 610 nm (Nile blue) and 510 nm (TP) lasers, and fluorescence signals were detected at room temperature. In the obtained images, the protein complex appeared red, and TP appeared green.

[0069] CLSM plots of the four complex samples are shown below. Figure 6 Among c, f, i, and l, YP-Sur showed only weak and scattered green fluorescence signals in the image; while YP-Sur-Zn... 2+ Compared to YP-Sur, YP-Sur-TP exhibited significantly enhanced and locally aggregated green fluorescent particles in the field of view. YP-Sur-TP showed a specific interaction between tea polyphenols (TP) and YP-Sur, manifested in a distinct yellow co-localization signal in the confocal image. This indicates that TP is not randomly dispersed but actively binds to the surface or interior of the YP-Sur complex particles, forming a stable YP-Sur-TP complex. Furthermore, the high overlap between the green fluorescence of TP and the red fluorescence of YP-Sur suggests that no significant phase separation occurred during the composite process, indicating high composite efficiency. When Zn 2+ Adding it to the sample did not block the interaction between YP and TP: a yellow colocalization signal could still be observed in the composite system, indicating that Zn... 2+ By coordinating with TP, it integrates into the complex system, thereby affecting the binding density of TP and YP and the complex morphology, ultimately forming YP-Sur-Zn. 2+ -TP complex.

[0070] Example 3 YP-Sur-Zn 2+ YP-Sur-TP and YP-Sur-Zn 2+ The oxidation resistance of the TP composite material was tested using the following method: (1) DPPH free radical scavenging ability YP-Sur-Zn was analyzed according to previous methods. 2+ YP-Sur-TP and YP-Sur-Zn2+ The DPPH free radical scavenging rate of the -TP composite material was determined to evaluate the in vitro antioxidant capacity of the composite sample.

[0071] Sample determination: Complex sample solutions of different concentrations (0.25, 0.50, 0.75, 1.00 mg / mL) were mixed 1:1 (v / v) with DPPH solution (0.04 mg / mL) prepared with anhydrous ethanol. After reacting for 30 min in the dark, the mixture was centrifuged at 5000 r / min for 10 min. The absorbance of the supernatant at 517 nm was measured. Anhydrous DPPH solution was used as the control group, anhydrous ethanol as the blank group, and tea polyphenols as the positive control. Three parallel groups were set up for each group. The calculation formula is as follows: In the formula: A s —Absorbance of the sample group; A b —Absorbance of the blank group; A c —Absorbance of the control group.

[0072] (2) ABTS free radical scavenging ability YP-Sur-Zn 2+ YP-Sur-TP and YP-Sur-Zn 2+ The ABTS free radical scavenging rate of the -TP composite material was determined to evaluate the in vitro antioxidant capacity of the composite.

[0073] ABTS + Preparation of mother liquor: 0.384 g ABTS + 0.134 g of potassium persulfate was dissolved separately in 100 mL of distilled water, then the two solutions were mixed in equal volumes and allowed to react in the dark at room temperature for 12–16 h to obtain ABTS. + The free radical mother liquor can then be transferred to a 4°C refrigerator for storage.

[0074] Sample determination: Before use, dilute ABTS with 80% ethanol solution. + The free radical mother liquor was diluted to an absorbance of 0.700–0.702 at 734 nm. YP-Sur-Zn 2+ YP-Sur-TP and YP-Sur-Zn 2+ -TP composite material samples at various concentrations (0.25, 0.50, 0.75, 1.00 mg / mL) and ABTS +The free radical working solution was mixed thoroughly at a volume ratio of 1:3, reacted in the dark for 10 min, and its absorbance was measured at 734 nm. The blank control was an 80% ethanol aqueous solution.

[0075] The free radical scavenging ability analysis diagram of the complex delivery system is shown in the figure. Figure 7 From 7a, we know that YP-Sur and YP-Sur-Zn 2+ YP-Sur-TP and YP-Sur-Zn 2+ The DPPH radical scavenging activity of YP-Sur-TP increased significantly with increasing concentration, exhibiting a relatively clear dose-dependent relationship. The YP-Sur complex itself possesses weak oxidizing activity. In comparison, the scavenging activity of YP-Sur-TP remained consistently high, exceeding 80% at a concentration of 2.0 mg / mL; YP-Sur-Zn... 2 + -TP scavenging activity increases with increasing concentration, eventually approaching a level comparable to YP-Sur-TP; while YP-Sur-Zn 2+ The scavenging activity was relatively low, with a scavenging rate of approximately 76.71% at 2.0 mg / mL, slightly higher than that of YP-Sur. This indicates that for the YP-Sur matrix, tea polyphenols (TP) significantly enhance the DPPH free radical scavenging ability, while Zn... 2+ The synergistic effect of YP-Sur with TP is only achieved at higher concentrations. The mechanism may be that the phenolic hydroxyl groups of tea polyphenols (TP) readily donate hydrogen atoms to combine with DPPH free radicals, while Zn... 2+ By participating in redox reactions, YP-Sur's peptide chain structure can assist in the scavenging of free radicals, providing a stable carrier environment for the active ingredients and promoting their contact with free radicals.

[0076] Depend on Figure 7 From b, we can see that YP-Sur-TP and YP-Sur-Zn 2+ The ABTS radical scavenging activity of TP remained stable and high within the concentration range (0.25–1.00 mg / mL), indicating a strong ability to scavenge water-soluble free radicals. This demonstrates the high efficiency and stability of the tea polyphenol complex (TP) in scavenging water-soluble free radicals. However, in stark contrast, the YP-Sur complex system and the YP-Sur complex system delivering zinc ions (Zn) showed significantly better performance. 2+ The ABTS free radical scavenging activity of tea polyphenols (TP) was low, with scavenging rates of 25.25±0.66% and 31.40±0.33% at 1.0 mg / mL, respectively. This further confirms that tea polyphenols (TP) are far superior to zinc ions (Zn) in scavenging water-soluble ABTS free radicals. 2+The YP-Sur complex system is suitable for simultaneous delivery of Zn. 2+ The presence of TP did not reduce the scavenging effect of the complex system on ABTS free radicals, indicating that the two do not have an antagonistic effect in the process of scavenging water-soluble free radicals.

[0077] Example 4 1. For composite delivery systems (YP-Sur-TP, YP-Sur-Zn) 2+ -TP) to conduct static simulation experiments on GI food digestion 1.1 In vitro digestion simulation In vitro digestion simulation was performed according to the standardized INFOGEST protocol. Stock solutions containing simulated saliva (SSF), gastric juice (SGF), and intestinal juice (SIF) were prepared and stored at 4°C. All solutions were preheated to 37°C before use. Oral administration phase: 200 mg sample (YP-Sur-Zn) 2+ YP-Sur-TP and YP-Sur-Zn 2+ α-Amylase (120 U / mL) and 6 beads were placed in an Erlenmeyer flask, and then α-amylase (120 U / mL) was added to 5 mL of SSF solution to achieve an amylase activity of 75 U / mL. The flask was then incubated at 37°C with continuous shaking for 5 min to simulate oral digestion.

[0078] Gastric phase: During the gastric digestion phase, SGF (containing 6.9 mmol / L KCl, 25 mmol / L NaHCO3, and 47.2 mmol / L NaCl) was added to the oral cavity samples to achieve a final volume ratio of 1:1. The mixture was adjusted to pH 3.0, and then 15000 U / mg pepsin was added to achieve a final activity of 2000 U / mL. The samples were then incubated at 37°C with shaking for 2 hours to simulate gastric digestion. Finally, when digestion was complete, the enzymatic reaction needed to be terminated in different ways depending on the measurement requirements. For samples measuring particle size, zeta potential, and rheological properties, they must be placed on ice and measured immediately. For samples measuring in vitro digestibility and the content of substances in the protein digestion solution, the pH must be adjusted to 8.0 to inactivate pepsin.

[0079] Intestinal phase: During the small intestinal digestion stage, gastric chyme is mixed with SIF (10 mL) and supplemented with bile salts (10 mM). After adjusting the pH to 7.0 using NaOH, trypsin is incorporated to achieve an activity of 100 U / mL. This stage also requires incubation at 37°C with shaking for 2 h to simulate intestinal conditions. After intestinal digestion is complete, the enzymatic reaction needs to be terminated in different ways depending on the measurement parameters. For samples measuring particle size, zeta potential, and rheological properties, they need to be placed on ice and immediately assessed. For samples measuring in vitro digestibility, a water bath is used for 5 min.

[0080] 1.2 Yeast protein-lipopeptide / Zn 2+ The property of the / TP complex to release tea polyphenols (TP). Digested YP-Sur-TP and YP-Sur-Zn 2+ -TP gastric and intestinal digestive fluids were centrifuged (10000 r / min, 5 min), and the supernatant was collected to determine the content of free tea polyphenols in the sample. The determination method was the same as in Example 2.

[0081] 1.3 Yeast protein-lipopeptide / Zn 2+ / TP complex releases Zn 2+ Characteristics (ICP-MS) After digestion, YP-Sur-Zn 2+ With YP-Sur-Zn 2+ -TP's gastric and intestinal digestive fluids were centrifuged (10000 r / min, 5 min), and 500 μL of the supernatant was collected for metal Zn analysis. 2+ The content of the bound element was determined using the same method as in Example 2.

[0082] 1.4 Protein digestibility YP-Sur, YP-Sur-Zn 2+ YP-Sur-TP and YP-Sur-Zn 2+ The digestion products of the -TP composite material, after simulating intestinal digestion, were mixed with ethanol (1:3, v / v) and incubated at 4°C for 2 h. Subsequently, the solution was centrifuged at 10000 r / min for 20 min. The amount of undigested protein was determined using the precipitate. The protein digestibility was calculated using the formula: In the formula, M 0 It represents the protein content before digestion, in grams. M 1 It represents the content of undigested protein, in grams (g).

[0083] 1.5 Zeta potential After cancellation, YP-Sur and YP-Sur-Zn 2+ YP-Sur-TP and YP-Sur-Zn 2+ The gastric and intestinal digestive fluids of the TP composite sample were analyzed using a nanoparticle size and zeta potential analyzer (BeNano 90 Zeta) to determine the zeta potential of the composite sample.

[0084] 1.6 Rheological properties In short, 0.2 mL of gastric and intestinal protein digestion fluid was collected after 2 hours and loaded onto the test platform. The upper plate was then lowered to contact the sample. The oscillation frequency was scanned using a rotational rheometer at 25°C and within a frequency range of 0.1–10 Hz with a constant strain of 0.1% (within the LVR). The changes in storage modulus (G') and loss modulus (G'') with frequency were recorded.

[0085] Temperature scanning was performed by heating from 25℃ to 90℃ at a heating rate of 5℃ / min. During the temperature scanning, a constant frequency of 1 Hz and a strain of 0.5% were set to investigate the relationship between the storage modulus (G') and loss modulus (G'') of the composite solution and temperature.

[0086] 2. Experimental Results 2.1 Changes in the concentration of free tea polyphenols at different stages of digestion The effects of two composite delivery systems (YP-Sur-TP and YP-Sur-Zn) on the concentration changes of free tea polyphenols were analyzed through experiments. 2+ -TP) The release behavior of tea polyphenols at different time points during gastric and intestinal digestion.

[0087] like Figure 8 As shown in Figure A, during the gastric digestion stage, YP-Sur-TP and YP-Sur-Zn 2+ The free tea polyphenol concentrations of YP-Sur-Zn were all at low levels (≤0.01 mg / mL), with no significant differences between different time points. This indicates that both delivery systems can effectively inhibit the release of tea polyphenols in the acidic environment of the stomach, demonstrating good gastric controlled-release effects. After entering the intestinal digestion stage, the free tea polyphenol concentrations increased significantly. Specifically, YP-Sur-Zn... 2+ The concentration of free tea polyphenols in YP-Sur-TP was significantly higher than that of YP-Sur-TP after 240 minutes of intestinal digestion. This is because, under the acidic environment of the stomach, YP-Sur-TP and YP-Sur-Zn... 2+-TP forms a stable aggregated structure with a positively charged surface. This structural feature allows tea polyphenols (TP) to be tightly embedded within the complex, making release in the stomach difficult and thus achieving a controlled-release effect. This is consistent with the results of rheological analysis showing a high elastic modulus and low viscous modulus in the gastric system. However, in the neutral to slightly alkaline environment of the intestine, the aggregated structure of the complex dissociates, and the surface charge becomes negative. For YP-Sur-Zn... 2+ -TP, Zn 2+ The coordination with TP gradually dissociates in the intestinal environment, while the yeast protein-lipopeptide carrier (YP-Sur) is slowly degraded by digestive enzymes, thus promoting the continuous release of tea polyphenols. In contrast, YP-Sur-TP releases tea polyphenols solely through the enzymatic hydrolysis of the carrier, therefore its release rate and final concentration are lower than those of YP-Sur-Zn. 2+ -TP.

[0088] 2.2 Digestion of Zn at Each Stage 2+ Concentration change Digestion products make a very important contribution to understanding changes in the concentration of elements in food. Figure 8 China B showcased YP-Sur-Zn 2+ and YP-Sur-Zn 2+ -TP concentration changes during gastric and intestinal digestion. During gastric digestion, the acidic environment of the stomach causes Zn... 2+ Coordination structure dissociation, YP-Sur-Zn 2+ Because there is no synergistic binding of tea polyphenols, the dissociation efficiency of Zn is higher. 2+ The concentration is YP-Sur-Zn 2+ Significantly higher than YP-Sur-Zn 2+ -TP. When the gastric digested sample enters the intestinal digestion stage, the neutral to slightly alkaline environment causes the Zn content in both systems to... 2+ The high release levels indicate that the promoting effect of the intestinal environment on zinc ion release is not affected by tea polyphenols. This complex delivery system demonstrates the characteristics of differential release in the stomach and highly efficient release in the intestine. YP-Sur-Zn 2+ -The sustained release of TP in the intestine ensures bioavailability and provides a scientific basis for the optimization of nutrient delivery carriers.

[0089] 2.3 Changes in the digestibility of the complex delivery system In vitro digestibility is a key indicator reflecting the breakdown and absorption of substances in the digestive tract. After digestion in the stomach and intestines, the digestibility of all four complexes was greater than 80%. Figure 8(C). However, the digestibility of different complex samples differed significantly (P<0.05). Yeast protein-lipopeptide (YP-Sur) is a digestible biomolecule that can be degraded by gastric and intestinal digestive enzymes, which is the basis for the high digestibility of all four systems. Figure 8 From C, we can see that YP-Sur-Zn 2+ -TP has a slightly higher digestibility than YP-Sur-Zn. 2+ And YP-Sur-TP, presumably Zn 2+ The synergistic effect with TP optimizes the vector; on the one hand, Zn 2+ It can act as a cofactor for certain digestive enzymes, promoting enzymatic reactions. On the other hand, the phenolic hydroxyl group of TP can form a weak interaction with the amino group of the carrier protein. This interaction gradually dissociates during digestion, which not only ensures the digestibility of the carrier, but may also improve digestibility by regulating the binding efficiency of enzymes and substrates.

[0090] 2.4 Zeta potentials of the digestion complex delivery system at each stage Zeta potential is mainly determined by the surface charge distribution and ionic strength of protein molecules. It is a key parameter for measuring the electrostatic interaction between particles, reflecting their aggregation and system stability, and thus affecting the digestibility of nutrients. Figure 8 The diagram in Figure D shows the dynamic fluctuations of the system's Zeta potential during digestion. During the gastric digestion phase, YP-Sur and YP-Sur-Zn... 2+ YP-Sur-TP and YP-Sur-Zn 2+ The zeta potentials of all three YP-Sur-TP complexes were positive and showed no significant difference. This is because the acidic environment of the stomach causes protonation of the amino groups of yeast proteins and lipopeptides, resulting in a positive charge on the complex surface. This positive charge contributes to the stability of the complex in the stomach. Upon entering the intestine, due to the influence of bile salts and FFA, the zeta potentials of all three complexes turned negative, with significant differences. YP-Sur-TP exhibited the largest negative zeta potential, indicating its strongest colloidal stability in the intestine; YP-Sur-Zn... 2+ The zeta potential is the least negative, and its stability is relatively weak; while YP-Sur-Zn 2+ The zeta potential of -TP lies between the two. Combining this with particle size data, it can be found that YP-Sur-Zn... 2+ -TP exhibits strong aggregation in the stomach and moderate dispersion in the intestine. This stability is jointly regulated by charge repulsion (Zeta potential) and particle size, which avoids rejection by intestinal epithelial cells that may be caused by excessive negative charge, while maintaining the system's dispersibility through appropriate particle size and charge.

[0091] 2.5 Changes in the rheological behavior of the complex delivery system at each stage of digestion The viscosity of the digestive system can reflect particle entanglement to some extent. The apparent viscosity of a sample significantly affects the digestibility of proteins within the sample. Figure 9 The relationship between apparent viscosity and frequency of samples after simulated gastric digestion is shown. Under gastric digestion conditions, the elastic modulus of the four complexes increases with increasing frequency. The viscous modulus of YP-Sur-TP peaks at 7 Hz and then decreases; the viscous modulus of YP-Sur-Zn... 2+ The viscous modulus increases slowly with increasing frequency; YP-Sur-Zn 2+ -TP has a generally low viscosity modulus. Combined with the change in elastic modulus (G'), it can be seen that YP-Sur-Zn 2+ During gastric digestion, TP exhibits elasticity as the dominant mechanism, a rheological conclusion consistent with the previous results regarding particle size and zeta potential. (YP-Sur-Zn) 2+ -TP in the stomach forms a stable structure with elasticity due to strong aggregation.

[0092] After entering the intestinal digestive environment, the rheological properties of the four complex systems changed significantly: the elastic modulus of YP-Sur-TP reached its peak at 7 Hz and then decreased; YP-Sur-Zn 2+ With YP-Sur-Zn 2+ The elastic modulus of -TP showed a process-wise increasing trend, and the viscosity modulus of the composite system all peaked at 7 Hz, but YP-Sur and YP-Sur-Zn... 2+ and YP-Sur-Zn 2+ -TP exhibits relatively small changes in apparent viscosity with frequency after gastrointestinal digestion, likely due to the hydrolysis of most proteins into small peptides and amino acids, resulting in reduced interparticle interactions. YP-Sur-Zn 2+ -TP exhibits increased elastic modulus in the intestine with increasing frequency, while maintaining a moderate viscous modulus. This rheological behavior allows it to maintain a certain structural integrity under the shearing action of intestinal peristalsis, while also ensuring uniform dispersion of the system in the intestine through moderate viscosity, thereby increasing the probability of contact with intestinal epithelial cells and optimizing absorption efficiency.

[0093] Rheological results show that all four complex systems can undergo structural dissociation in the gastric and intestinal environments, providing sufficient sites for enzymatic hydrolysis and ensuring the smooth progress of the digestion process.

[0094] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the emulsion stability of a yeast protein and the encapsulation capacity of an active ingredient, characterized in that, The step of mixing the yeast protein and the lipopeptide to obtain the binary complex carrier.

2. The production method according to claim 1, characterized by, The lipopeptide is Surfactin C.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the yeast protein and the lipopeptide is 1 g:50 mg.

4. The binary complex carrier prepared by the method according to any one of claims 1-3.

5. Use of the binary complex carrier according to claim 4 in the preparation of a delivery system for a bioactive substance.

6. A Zn 2+ A method of preparing a complex delivery system of Zn The step of mixing the yeast protein and the lipopeptide to obtain the binary complex carrier. The binary composite support according to any one of claims 1 to 3 is mixed with Zn 2+ to obtain YP-Sur-Zn 2+ composite; The YP-Sur-Zn 2+ The complex is mixed with tea polyphenols to obtain YP-Sur-Zn 2+ - tea polyphenol complex, which is the complex delivery system.

7. The preparation method according to claim 6, characterized in that, The ratio of the binary composite carrier to the Zn 2+ is 1 g:0.1 mol.

8. The preparation method according to claim 6, characterized in that, The YP-Sur-Zn 2+ The specific steps of the mixed reaction of the complex with tea polyphenols include: The YP-Sur-Zn 2+ The complex was dissolved in phosphate buffer, made completely hydrated, the tea polyphenols were added, the reaction was stirred at pH 9.0, the uncomplexed tea polyphenols were removed by dialysis, and the YP-Sur-Zn 2+ - tea polyphenol complex.

9. The complex delivery system prepared by the method according to any one of claims 6-8.

10. Use of a composite delivery system as claimed in claim 9 in the manufacture of a product for the simultaneous supplementation of Zn 2+ and tea polyphenols.

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