A modified zein delivery carrier and its preparation method and application

Abstract

The present invention discloses a modified zein delivery vehicle, its preparation method, and application. The modified zein delivery vehicle comprises solid or hollow zein nanoparticles prepared using a Na2CO3 solution or a Na2CO3 sacrificial template solution, followed by phosphorylation. Compared to nanoparticles prepared from unmodified zein, the phosphorylated zein nanoparticles exhibit improved solubility, greater storage stability, and significantly enhanced emulsification, foaming, and encapsulation properties, significantly enhancing their performance as a delivery vehicle.

Description

Technical Field

[0001] The invention relates to a modified zein delivery carrier and a preparation method and application thereof, belonging to the field of delivery carriers. Background Art

[0002] Zein, primarily derived from maize endosperm, is a common natural plant-based biopolymer. However, the high level of α-helices in its three-dimensional structure gives zein unique self-assembly properties, making it an ideal material for delivering bioactive substances, particularly hydrophobic ones. Zein-based delivery systems can be divided into two main categories based on droplet or particle size: microscale (e.g., microparticles, microcapsules, Pickering emulsions), and nanoscale (e.g., nanoparticles, nanofiber films, nanoemulsions). Nanoparticles, owing to their simple manufacturing process and excellent oral availability, have become a commonly used delivery system. However, zein itself has poor solubility, stability, and dispersibility, necessitating modification of zein or zein particles to obtain nanoparticles with superior delivery efficacy. Summary of the Invention

[0003] Purpose of the invention: The first purpose of the present invention is to provide a modified zein delivery carrier, the second purpose of the present invention is to provide a method for preparing the modified zein delivery carrier, and the third purpose of the present invention is to provide the application of the modified zein delivery carrier in the preparation of drugs or functional food ingredients.

[0004] Technical solution: A modified zein delivery vector of the present invention comprises zein nanoparticles prepared with a Na2CO3 solution or a Na2CO3 sacrificial template solution and then modified by phosphorylation.

[0005] Furthermore, the zein nanoparticles are hollow or solid structures.

[0006] Furthermore, the phosphorylation modification is carried out by sodium tripolyphosphate.

[0007] Furthermore, the preparation of the Na2CO3 sacrificial template solution includes the following steps: mixing a Na2CO3 aqueous solution with anhydrous ethanol.

[0008] Furthermore, the concentration of the Na2CO3 aqueous solution is 0.5-2.5% (w / v).

[0009] Furthermore, the volume ratio of the Na2CO3 aqueous solution to anhydrous ethanol is 1-5:9-5.

[0010] The method for preparing the modified zein delivery carrier of the present invention comprises the following steps:

[0011] (1) Mixing a zein stock solution with a Na2CO3 sacrificial template solution and stirring to obtain a mixed solution. Mixing the mixed solution with ultrapure water, continuing to stir, and rotary evaporating to obtain hollow zein nanoparticles (HZn);

[0012] Alternatively, a zein stock solution is mixed with a Na2CO3 solution, stirred to obtain a mixed solution, the mixed solution is mixed with ultrapure water, further stirred, and rotary evaporated to obtain zein nanoparticles (SZn) with a solid structure;

[0013] (2) Mix the sodium tripolyphosphate solution with the hollow or solid zein nanoparticle solution and stir.

[0014] Furthermore, in step (1), the concentration of the zein stock solution is 0.5-2.5% (w / v), and the volume ratio of the zein stock solution to the Na2CO3 sacrificial template solution is 1:1-5.

[0015] Furthermore, in step (1), the stirring time is 2-10 min, and the volume ratio of the mixed liquid to ultrapure water is 1:1-5.

[0016] Furthermore, in step (1), the stirring time is continued for 10-30 minutes, the rotary evaporation temperature is 35-42° C., and the rotary evaporation time is 25-40 minutes.

[0017] Furthermore, in step (2), the mass ratio (w / w) of sodium tripolyphosphate to zein nanoparticles is 0.75-1.5:1.

[0018] Application of the modified zein delivery carrier of the present invention in the preparation of medicines or functional food ingredients.

[0019] Furthermore, the drug or functional factor is puerarin.

[0020] Generally, phosphorylation-modified proteins refer to protein molecules that are phosphorylated on specific amino acid residues, i.e., a phosphate group is added. This modification is usually catalyzed by kinases and is a reversible post-translational modification process that has a significant impact on the function, activity, stability, and subcellular localization of proteins. Phosphorylated proteins are widely present in cells and are involved in various biological processes such as cell signaling, cell cycle regulation, and metabolic regulation. The phosphorylation-modified protein particles of the present invention are prepared by first preparing zein protein into protein particles through a Na2CO3 or Na2CO3 sacrificial template and then phosphorylation-modified, utilizing the special properties of nanoparticles (such as large specific surface area, special force, electrical, magnetic, optical, and thermal properties) and the biological properties of phosphorylated proteins. By regulating the construction of nanoparticles and further phosphorylation modification conditions, nano delivery carriers with excellent comprehensive performance are obtained. Protein particles modified by phosphorylation can not only improve the stability and bioavailability of hydrophobic drugs or functional factors, but also regulate the release kinetics of drugs by regulating the surface properties and sustained-release properties of nanoparticles, thereby playing an important role in drug delivery systems. Compared with phosphorylated proteins, preparing particles first and then phosphorylating them mainly changes the surface modification sites, while some potential phosphorylation sites inside are less likely to be phosphorylated because they participate in the interaction between protein molecules in the particles. Therefore, a particle structure with a hydrophilic exterior and a hydrophobic interior can be formed, which is conducive to the encapsulation of hydrophobic active substances.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0022] The modified zein delivery carrier provided by the present invention is a phosphorylated zein nanoparticle. Compared with the zein nanoparticles prepared from unmodified zein, the modified zein nanoparticles have the advantages of better solubility, stronger storage stability, and significantly improved emulsification, foaming and encapsulation properties, which greatly improves its performance as a delivery carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 is a graph showing the phosphorylation degree of PHZn and PSZn prepared in Example 1;

[0024] Figure 2 Particle size and potential diagrams of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 were prepared for Example 1;

[0025] Figure 3 The free amino group content graph of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1;

[0026] Figure 4 The free thiol content graph of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1;

[0027] Figure 5 SEM images of HZn, SZn, PHZn1, and PSZn1 prepared in Example 1;

[0028] Figure 6 TEM images of HZn and SZn prepared in Example 1;

[0029] Figure 7 The infrared spectra of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 were prepared for Example 1;

[0030] Figure 8 Circular dichroism spectra of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 were prepared for Example 1;

[0031] Figure 9 Fluorescence emission spectra of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1;

[0032] Figure 10 A comparative chart of the protein solubility of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared for Example 1;

[0033] Figure 11 The particle size change diagram of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 at different times was prepared for Example 1;

[0034] Figure 12 The foaming results of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1 are shown;

[0035] Figure 13 The emulsification results of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1 are shown;

[0036] Figure 14The encapsulation efficiency and loading rate of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 encapsulating puerarin were prepared in this example. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0038] Example 1

[0039] 1. Sample preparation

[0040] (1) Hollow zein nanoparticles (HZn) were prepared using a Na2CO3 sacrificial template.

[0041] First, a 5% (w / v) zein stock solution was prepared using freshly prepared 70% (v / v) ethanol solution. A 1% (w / v) aqueous solution of Na2CO3 and anhydrous ethanol were mixed in a ratio of 3:7 to prepare a Na2CO3 sacrificial template solution. The 5% (w / v) zein stock solution and the Na2CO3 sacrificial template solution were then thoroughly mixed in a 1:1 ratio and magnetically stirred for 5 minutes to obtain a mixture. This mixture of zein stock solution and Na2CO3 sacrificial template solution was then mixed with ultrapure water in a 1:4 (v / v) ratio and magnetically stirred for 10 minutes to induce Na2CO3 precipitation, leaving a cavity within the zein nanoparticles. Finally, the ethanol was removed using a rotary evaporator at 38°C for 35 minutes, and the evaporation volume was made up by adding ultrapure water to obtain a hollow zein nanoparticle (HZn) solution.

[0042] (2) The preparation process is the same as step (1), and the other sample preparation steps remain unchanged. The difference is that an equal amount of Na2CO3 aqueous solution is used instead of the Na2CO3 sacrificial template solution to prepare a solid structure zein nanoparticle (SZn) solution as a control.

[0043] (3) 5 mg, 12.5 mg, 25 mg, 27.5 mg, 50 mg, 62.5 mg, and 75 mg of sodium tripolyphosphate (STP) were dissolved in 40 mL of ultrapure water to form STP solutions of different mass concentrations. 10 mL of hollow zein nanoparticle solution or solid zein nanoparticle solution was mixed with the solution and magnetically stirred for 30 min to allow the phosphorylation reaction to proceed. At this point, the mass ratios (w / w) of STP to zein were 0.1, 0.25, 0.5, 0.75, 1, 1.25, and 1.5, respectively. After the phosphorylation reaction, the nanoparticle solution was dispensed into dialysis bags at 4°C and dialyzed continuously with deionized water for 72 h. Finally, the dialyzed solution was freeze-dried to obtain powder samples, respectively, to obtain phosphorylated hollow zein nanoparticles (PHZn) and phosphorylated solid zein nanoparticles (PSZn), which were then sealed and placed in a refrigerator at 4°C for use.

[0044] 2. Phosphorylation degree test

[0045] 5 mL of each sample of phosphorylated hollow zein nanoparticles or phosphorylated solid zein nanoparticles prepared in Example 1 was added with an excess of 10% triacetic acid (TCA) to precipitate the protein, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. Then, an excess of 1 mol / L zinc acetate solution was added to the supernatant to adjust the pH to 3.8 to form a precipitate, and then a pH 10 ammonia-ammonium chloride buffer was added to dissolve it. Finally, using Eriochrome Black T as an indicator, the solution was titrated with a 0.1 mol / L disodium ethylenediaminetetraacetic acid (EDTA-Na2) standard solution. The titration was stopped when the solution turned from purple-red to blue, and the volume was recorded. The results are as follows: Figure 1 shown.

[0046] Figure 1 The comparison of the phosphorylation degree of PHZn and PSZn prepared in Example 1 is shown in FIG. Figure 1As can be seen, the phosphorylation levels of both PHZn and PSZn first increased and then decreased with the addition of STP, with the phosphorylation levels of PHZn and PSZn consistently higher than those of PSZn. When the STP / zein (w / w) ratio was 1, the phosphorylation levels of the two phosphorylated zein nanoparticles reached their highest values, reaching 50.79 mg / g and 48.83 mg / g, respectively. Based on this, samples with STP / zein (w / w) ratios of 0.1, 0.5, and 1 were selected as low-phosphorylated zein nanoparticles (PHZn0.1, PSZn0.1), medium-phosphorylated zein nanoparticles (PHZn0.5, PSZn0.5), and high-phosphorylated zein nanoparticles (PHZn1, PSZn1), respectively, for subsequent studies. If phosphate is added in excess, it will lead to overphosphorylation of protein particles, increasing electrostatic interactions between particles, causing proteins to aggregate into larger particles. Excessive phosphorylation may cause irreversible changes in the structure of protein particles, thereby affecting their biological functions, such as enzyme activity and binding properties. Excessive phosphate may lead to excessive surface charge of nanoparticles, increasing repulsive forces between particles and affecting their stability. In addition, in drug delivery systems, the stability and targeting of nanoparticles are crucial. Excessive phosphorylation modification may change the surface properties of nanoparticles, affecting their interactions with biomolecules, thereby reducing the efficiency and targeting of drug delivery.

[0047] 3. Particle size and potential test

[0048] About 1.5 mL of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1 were placed in a quartz dish for particle size analysis and Zeta potential measurement using a potential cell. The results are shown in Figure 2. Figure 2 shown.

[0049] Figure 2 The particle size and potential comparison diagram of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared for Example 1, where A is the particle size and B is the potential. Figure 2As shown, with increasing phosphate concentration, the particle sizes of PHZn and PSZn increased from 138.2 nm and 170.5 nm to 186.5 nm and 222.4 nm, respectively, with the particle size of HZn consistently smaller than that of SZn. In the absence of STP, both HZn and SZn possessed negative surface charges, with zeta potentials of -27.7 mV and -25.7 mV, respectively. With the gradual addition of STP, the negatively charged phosphate groups gradually bound to the protein, increasing the electronegativity of PHZn and PSZn. When the mass ratio of STP to zein was 1, the zeta potentials reached maximum values ​​of -53.2 mV and -50.2 mV, respectively, nearly double those of the unphosphorylated samples. The absolute zeta potential values ​​of HZn were greater than those of SZn before and after phosphorylation and in samples with varying degrees of phosphorylation.

[0050] 4. Determination of free amino groups

[0051] The free amino group contents of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 prepared in Example 1 were determined by o-phthalaldehyde (OPA) method. Figure 3 shown.

[0052] Figure 3 Comparative graph of free amino group contents of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1; Figure 3 As can be seen, the initial free amino group content of HZn and SZn was 0.202mmol / g and 0.193mmol / g, respectively. As the phosphorylation reaction proceeded, it decreased to 0.134mmol / g and 0.143mmol / g, a decrease of 0.068mmol / g and 0.050mmol / g, respectively. Without phosphorylation, the free amino group content of HZn was higher than that of SZn.

[0053] 5. Determination of free thiol groups

[0054] 5,5'-dithiobis(2-nitrobenzoic acid) was dissolved in Tris-Gly buffer (pH 8.0, containing 0.086mol / LTris, 0.09mol / L Gly, 0.004mol / L EDTA, 8mol / L Urea) to prepare Ellman's reagent at a concentration of 4mg / mL. 1mL of sample solution was added to 2mL of Tris-Gly buffer solution and mixed with 100μL of Ellman's reagent, incubated in the dark for 30min at room temperature, and the absorbance was measured at 412nm. An equal amount of ultrapure water was used instead of the sample solution and mixed with 2mL of Tris-Gly buffer solution as a blank control. The above sample solutions were HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1, and the content of free thiol groups was determined respectively. The results are as shown in FIG. Figure 4 shown.

[0055] Figure 4 Comparative diagram of free thiol content of HZn solution, SZn solution, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1; Figure 4 It can be seen that with the increase of phosphorylation degree, the free thiol content of PHZn and PSZn showed a downward trend, decreasing by 14.36 μmol / g and 12.25 μmol / g, respectively.

[0056] 6. Scanning electron microscope test

[0057] Take appropriate amounts of freeze-dried samples HZn, SZn, PHZn1, and PSZn1 and place them on a metal conductive surface and plate them with gold. Use S-4800Ⅱ field emission scanning electron microscope to observe the surface morphology of the samples at an accelerating voltage of 10.0 kV. The results are shown in Figure 2. Figure 5 shown. Figure 5 The SEM images of HZn, SZn, PHZn1 and PSZn1 prepared in Example 1 are as follows: Figure 5 It can be seen from the result diagram of low magnification (magnification 1k) that all samples are in the state of lamellar aggregates; from the result diagram of high magnification (magnification 50k), it can be seen that the individual particles of all samples are spherical, and the average particle size is between 100-200nm, which is the same as the particle size measurement results. Compared with particles with solid structures, particles with hollow structures have smaller particle sizes (magnification 50k) and more uniform distribution (magnification 10k). When the phosphate group is inserted, the sample particles become uneven (magnification 50k), obvious small holes appear on the surface of the aggregate (magnification 1k), and a network structure is observed (magnification 10k). Transmission electron microscopy analysis was performed on the freeze-dried samples HZn and SZn, and the results are as follows. Figure 6shown. Figure 6 TEM images of HZn and SZn prepared in Example 1; Figure 6 It can be seen that the prepared HZn particles have a hollow structure, while the SZn particles have a solid structure.

[0058] 7. Fourier infrared test

[0059] The freeze-dried sample powders of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1 were thoroughly ground and mixed with dry potassium bromide powder at a ratio of 1:150, pressed into tablets, and then measured for infrared spectroscopy. The measurement parameters were set as scanning range: 4000-400 cm-1, resolution: 4 cm-1, and number of scans: 32 times. The results are as follows. Figure 7 shown.

[0060] Figure 7 The infrared spectra of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 were prepared for Example 1; Figure 7 It can be seen that all samples have the -1 The absorption peak near 2960cm is mainly due to the stretching vibration of OH and the asymmetric stretching vibration of NH. -1 The absorption peak near 1650cm is mainly due to the asymmetric stretching vibration of -CH2; -1 The absorption peak near 1540cm is mainly due to the stretching vibration of C=O; -1 The absorption peaks near 1135cm-1 are mainly due to the stretching vibration of CN and the bending vibration of NH. The new characteristic peaks at 1135cm-1, 990cm-1, and 895cm-1 of the phosphorylated zein nanoparticle sample are attributed to the stretching vibrations of PO and P=O, indicating that the phosphate group has been successfully incorporated into the protein side chain.

[0061] 8. Circular dichroism test

[0062] Take appropriate amount of deionized water to dilute the HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 solutions prepared in Example 1 to 0.2% (w / v), then add the diluted sample solutions to quartz microcuvettes and measure their spectra at 190-250nm using a circular dichroism spectrometer. The results are as follows: Figure 8 shown.

[0063] Figure 8The circular dichroism spectra of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 were prepared for Example 1. Figure 8 All samples exhibit two negative peaks at approximately 210 nm and 225 nm, and a positive peak at approximately 195 nm, typical of zein, which is rich in α-helical structures. At the same phosphorylation level, the negative peak intensity of HZn is higher than that of SZn, indicating that HZn contains a higher α-helical content than SZn. The negative peak intensities of both PHZn and PSZn increase to varying degrees with increasing phosphorylation levels, indicating that phosphorylation can increase the α-helical content of zein.

[0064] 9. Intrinsic fluorescence spectroscopy

[0065] The fluorescence emission spectra of the HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 samples prepared in Example 1 were measured using a fluorescence spectrophotometer. The sample concentration was fixed at 1 mg / mL, the excitation wavelength was set at 280 nm, the spectrum scanning range was 290-400 nm, and the excitation and emission slit widths were both 5 nm. The results are shown in Figure 2. Figure 9 shown.

[0066] Figure 9 The fluorescence emission spectra of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 were prepared for Example 1. Figure 9 It can be seen that the fluorescence intensity of both hollow and solid structure samples increases with the increase of STP concentration, and the fluorescence intensity of HZn is significantly higher than that of SZn.

[0067] 10. Solubility

[0068] The HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 protein samples prepared in Example 1 were diluted to 0.5 mg / mL with ultrapure water and then centrifuged at 12,000 rpm for 3 minutes at room temperature. The supernatant was assayed using a BCA protein concentration kit and the dissolved protein concentration was calculated. Native zein was used as a control. Protein solubility was calculated according to the following formula:

[0069]

[0070] Where C1 is the measured supernatant protein concentration, and C0 is the diluted protein sample concentration: 0.5 mg / mL. Figure 10 shown.

[0071] Figure 10 The protein solubility comparison chart of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 was prepared for Example 1. Figure 10 As can be seen, the solubility of native zein is only 4.61%, significantly limiting its application as a delivery vehicle. When zein was prepared into solid and hollow particles, the solubility increased to 21.88% and 26.08%, respectively. Phosphorylation with STP further enhanced the solubility of SZn and HZn, reaching a maximum of 36.34% and 43.45% when the STP addition level was equal to the zein mass. At the same phosphorylation level, the solubility of HZn was consistently higher than that of SZn. It is precisely because HZn exhibits superior physical and chemical properties that HZn ultimately outperforms SZn in terms of functional properties.

[0072] 11. Storage stability

[0073] The HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn10.1% (w / v) aqueous dispersions prepared in Example 1 were stored at room temperature for 21 days. The particle sizes of the samples were measured at 25°C using a Malvern Zetasizer ES90 Nano laser particle size analyzer on the 1st, 7th, and 21st days, respectively. The storage stability of the samples was analyzed by the changes in the particle size. The results are shown in Figure 2. Figure 11 shown.

[0074] Figure 11 The particle size change diagram of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 at different times was prepared in Example 1. Figure 11 As can be seen, the particle size of all samples remained stable over 7 days, indicating that zein nanoparticles maintain good stability even after one week of storage at room temperature. After 21 days of storage, the particle sizes of HZn and SZn significantly expanded to 774.9 nm and 872.4 nm, respectively, representing increases of 636.7 nm and 701.9 nm compared to day 1. The particle size change of the phosphorylated samples was significantly smaller than that of the unphosphorylated samples, and the higher the degree of phosphorylation, the smaller the particle size change.

[0075] 12. Foamability and foam stability

[0076] 5 mL of the HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 solutions prepared in Example 1 were placed in clearly graduated centrifuge tubes. The samples were sheared at 10,000 rpm using a high-speed homogenizer for 2 minutes, and the total volume was recorded. The samples were then allowed to stand at room temperature for 30 minutes, and the total volume was recorded again. The formulas for calculating FC and FS are as follows:

[0077]

[0078] Where V0 is the initial volume of the sample, V1 is the volume of the sample after high-speed shearing, and V2 is the volume of the sample after standing for 30 minutes. Figure 12 shown.

[0079] Figure 12 The steam drum results of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1 and PSZn1 prepared in Example 1 are shown, where A represents foaming property and B represents bubble stability. Figure 12 As can be seen, the foaming properties and foaming stability of both solid and hollow zein nanoparticles increased with increasing phosphorylation degree, with hollow particles consistently slightly higher than solid particles. The foaming properties of SZn increased from 14.13% to 35.76%, and the foaming stability improved from 75.86% to 90.65%. The foaming properties of HZn increased from 17.53% to 38.60%, and the foaming stability increased from 76.34% to 92.67%.

[0080] 13. Emulsification and emulsion stability

[0081] 15 mL of a 0.1% (w / v) aqueous dispersion of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 sample nanoparticles prepared in Example 1 was thoroughly mixed with 5 mL of edible peanut oil. The mixture was then sheared at 10,000 rpm for 4 min using a high-speed homogenizer to prepare an emulsion. The prepared emulsion was allowed to stand for 20 min. At 0 and 20 min, 50 μL of the emulsion was drawn from the bottom and thoroughly mixed with 5 mL of 0.1% (w / v) sodium dodecyl sulfate (SDS) solution. The absorbance was measured at 580 nm. EAI and ESI were calculated according to the following formulas.

[0082]

[0083] Where N represents the dilution factor; C represents the protein concentration before emulsification, g / mL; Ψ represents the volume fraction of the oil phase; A0 and A20 represent the absorbance of the dilution at 580nm at 0min and 20min, respectively. Figure 13 shown.

[0084] Figure 13 The emulsification results of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 prepared in Example 1 are shown in Figure 1, where A represents emulsification and B represents emulsification stability. Figure 13 It can be seen that with the increase of phosphorylation degree, the EAI and ESI of both hollow and solid structures increased significantly. The EAI of SZn and HZn increased from 19.25 m2 / g and 25.09 m2 / g before phosphorylation to 103.77 m2 / g after phosphorylation, respectively. 2 / g and 137.88m 2 / g, both increased by more than 5 times; and the ESI of SZn and HZn also increased from 19.2% and 25.09% before phosphorylation to 73.58% and 78.74% after phosphorylation, respectively.

[0085] 14. Encapsulation rate and loading rate

[0086] To investigate the effect of phosphorylation modification on nanoparticle encapsulation of hydrophobic active substances, the encapsulation efficiency and loading efficiency of puerarin were measured. 10 mg of puerarin was added to 1 mL of a 5% (w / v) zein stock solution and magnetically stirred for 15 minutes, replacing the 5% (w / v) zein stock solution in Example 1. Puerarin-loaded sample particles were prepared according to the sample preparation procedures of Example 1.

[0087] The content of puerarin encapsulated in the nanoparticles was determined by ultraviolet-visible spectrophotometry. The sample was centrifuged at 2000r for 20 minutes to remove unstable large particles and unencapsulated puerarin. 300 μL of supernatant was added to 700 μL of anhydrous ethanol and appropriately diluted with 70% ethanol. The absorbance of the sample dilution at 250 nm was then measured using an ultraviolet spectrophotometer. The absorbance value obtained was substituted into the puerarin standard curve: y = 0.1123x + 0.0206 to calculate the content of successfully encapsulated puerarin. In the formula, y represents the absorbance value and x represents the puerarin concentration (μg / mL). The encapsulation efficiency (EE) and loading capacity (LC) of puerarin were calculated according to the following formula:

[0088]

[0089] Among them, M2 is the content of embedded puerarin (mg), M1 is the initial addition amount of puerarin (mg), and M0 is the mass of zein (mg). The results are as follows Figure 14 shown.

[0090] Figure 14 The encapsulation efficiency and liability ratio of HZn, SZn, PHZn0.1, PSZn0.1, PHZn0.5, PSZn0.5, PHZn1, and PSZn1 encapsulating puerarin were prepared for this example, where A is the encapsulation efficiency and B is the liability ratio. Figure 14 It can be seen that, using puerarin as a model, the effects of Na2CO3 template method for preparing hollow particles and STP-induced phosphorylation modification on the EE and LC of zein nanoparticles were investigated. Figure 14 As shown. Figure 14 As can be seen, compared to SZn, the EE of HZn increased significantly, from 60.24% to 69.39%, and the LC increased from 12.05% to 13.88%. This is due to the larger surface area provided by the hollow particle preparation and the cavities formed after Na2CO3 diffusion, which leave more capture sites for bioactive substances, thereby improving the encapsulation efficiency and loading capacity of puerarin. After STP-induced phosphorylation, the EE and LC of both SZn and HZn steadily increased with increasing phosphorylation degree. This is attributed to the phosphorylation modification exposing the hydrophobic groups within the protein, increasing the surface hydrophobicity of zein.

Claims

1. A modified zein delivery carrier, characterized in that: The modified zein delivery carrier comprises zein nanoparticles prepared with a Na2CO3 solution or a Na2CO3 sacrificial template solution and then modified by phosphorylation. The phosphorylation modification is performed by sodium tripolyphosphate, and the mass ratio of sodium tripolyphosphate to zein nanoparticles is 0.75-1.5:

1.

2. The modified zein delivery carrier according to claim 1, characterized in that Zein nanoparticles are hollow or solid structures.

3. The modified zein delivery carrier according to claim 1, characterized in that The preparation of the Na2CO3 sacrificial template solution includes the following steps: mixing a Na2CO3 aqueous solution with anhydrous ethanol.

4. The modified zein delivery carrier according to claim 3, characterized in that The volume ratio of Na2CO3 aqueous solution to anhydrous ethanol is 1-5:9-5.

5. The method for preparing the modified zein delivery carrier according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Mixing a zein stock solution with a Na2CO3 sacrificial template solution, stirring to obtain a mixed solution, mixing the mixed solution with ultrapure water, continuing to stir, and rotary evaporating to obtain hollow zein nanoparticles; Alternatively, a zein stock solution is mixed with a Na2CO3 solution, stirred to obtain a mixed solution, the mixed solution is mixed with ultrapure water, further stirred, and rotary evaporated to obtain zein nanoparticles with a solid structure; (2) Mix the sodium tripolyphosphate solution with the hollow or solid zein nanoparticle solution and stir.

6. The preparation method according to claim 5, characterized in that In step (1), the concentration of the zein stock solution is 0.5-2.5% (w / v), and the volume ratio of the zein stock solution to the Na2CO3 solution or the Na2CO3 sacrificial template solution is 1:1-5.

7. The preparation method according to claim 5, characterized in that In step (1), the stirring time is 2-10 min, the volume ratio of the mixed solution to ultrapure water is 1:1-5, the stirring time is continued for 10-30 min, the rotary evaporation temperature is 35-42 ° C, and the rotary evaporation time is 25-40 min.

8. Use of the modified zein delivery carrier according to any one of claims 1 to 4 in the preparation of a drug or functional food ingredient.

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