Modified phytosterol nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses modified phytosterol nanoparticles and a preparation method thereof, and the preparation method comprises the following steps: (1) dissolving phytosterol in ethanol to form a transparent phytosterol solution; (2) adding zein into the phytosterol solution, and stirring until the zein is uniformly dispersed; (3) dropwise adding the dispersion liquid obtained in the step (2) into an excessive alkaline aqueous solution, and simultaneously mixing, inducing and precipitating by using a homogenizer; and (4) centrifuging to obtain a suspension with uniformly distributed particles, evaporating to remove residual ethanol in the suspension, and drying to obtain the modified phytosterol nanoparticles. The invention also provides an application of the modified phytosterol nano-particles in the construction of Pickering emulsion. The modified phytosterol nano-particles can effectively regulate and control the rheological property and stability of Pickering emulsion, and keep good dispersity under different environmental conditions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food colloids and cosmetic delivery systems, and specifically relates to a modified phytosterol nanoparticle, a preparation method and application thereof. BACKGROUND

[0002] Emulsification is a process in which a liquid is uniformly dispersed in the form of minute droplets in another liquid with which it is immiscible. Emulsification is a liquid-liquid interfacial phenomenon. Two immiscible liquids, such as oil and water, are separated into two layers in a container. The oil, which has a lower density, is on the upper layer, and the water, which has a higher density, is on the lower layer. Traditional emulsification requires the addition of a suitable surfactant, and then, under strong stirring, the oil is dispersed in the water to form an emulsion.

[0003] Pickering emulsion is an emulsion stabilized by colloidal solid particles instead of traditional surfactants at the water-oil interface. As a kind of emulsion system stabilized by solid particles, the stability mechanism of Pickering emulsion is that solid particles are adsorbed on the oil-water interface to form a monolayer or multilayer film, thereby stabilizing the emulsion. Compared with traditional emulsions stabilized by surfactants, Pickering emulsions have the advantages of strong stability, good safety, and many other advantages, and show wide application potential in the fields of food, cosmetics, and the like.

[0004] Food-grade Pickering emulsion can effectively solve the problems of poor stability, low safety, biological incompatibility, and difficulty in degradation of traditional emulsions, and has the functions of slow release and delivery of functional substances and as a substitute for traditional fats. In the field of food science and technology, it is of great significance to develop nanoparticles with adjustable hydrophilic and hydrophobic properties for the construction of stable Pickering emulsions.

[0005] The existing nanoparticle preparation technology has many shortcomings in regulating the rheological properties and stability of Pickering emulsion. For example, the nanoparticles prepared by traditional emulsification and spray drying methods are difficult to accurately control the hydrophilic and hydrophobic properties, and are prone to aggregation under high ionic strength or extreme pH conditions, resulting in a decrease in stability. Furthermore, the nanoparticles based on natural biological macromolecules (such as proteins, polysaccharides, etc.) have weak hydrophilic and hydrophobic property adjustment ability, which is difficult to meet the needs of different application scenarios. Further, in the existing technology, even if the nanoparticles are modified by natural biological macromolecules, the correlation between "particle hydrophobicity adjustment" and "emulsion gelation behavior and spreading performance" has not been established, and most systems have the problem of "high modulus and high viscosity". In the fields of cosmetics (such as face cream and essence milk) and food (such as spreadable nutritional emulsion), it is easy to have a strong sticky feeling and poor spreadability. Moreover, the transition of emulsion to gel mostly depends on external stimuli (such as high temperature and chemical additives), which not only destroys the "green and safe" property, but also cannot accurately regulate the rheological parameters to adapt to the spreading demand.

[0006] Developing nanoparticles with adjustable hydrophobicity can achieve precise control of Pickering emulsion properties. By adjusting the hydrophobicity of nanoparticles, their adsorption behavior at the oil-water interface can be optimized, thereby affecting the rheological properties and stability of the emulsion. This control ability is crucial for designing Pickering emulsions with specific functions, such as adjusting the mouthfeel and texture of emulsions in the food industry, and improving the spreadability and stability of products in the cosmetics industry. Specifically, if natural and edible biomacromolecules (such as zein) are used as modifiers to modify hydrophobic active ingredients (such as phytosterols), nanoparticles with controllable hydrophobicity can be prepared. Not only can this solve the problem of low solubility and poor bioavailability of phytosterols, but also can achieve a gentle transition from emulsion to low modulus and low viscosity gel through the ordered arrangement of particles at the oil-water interface, thereby improving the spreadability of the system and meeting the core needs of the food and cosmetics industries.

[0007] Therefore, developing nanoparticles with adjustable hydrophobicity is of great significance for promoting the application of Pickering emulsions in various fields. SUMMARY

[0008] The present application provides a modified phytosterol nanoparticle and a preparation method thereof. The preparation method can not only flexibly control the hydrophobicity of phytosterol nanoparticles, but also maintain the stable dispersion of modified phytosterol nanoparticles under wide environmental conditions. When used to construct Pickering emulsions, the modified phytosterol nanoparticles of the present application can significantly optimize the rheological properties of the emulsions, improve their flowability and spreadability, and solve the problem of weak hydrophobicity adjustment ability of phytosterol nanoparticles in the prior art, as well as the problem of easy aggregation and poor spreadability of Pickering emulsions constructed.

[0009] The technical solution of the present application is as follows: A preparation method of a modified phytosterol nanoparticle, comprising the following steps: (1) dissolving phytosterol in ethanol to form a transparent phytosterol solution; (2) adding zein to the phytosterol solution and stirring until uniformly dispersed; (3) adding the dispersion obtained in step (2) dropwise to an excess of alkaline aqueous solution, and simultaneously using a homogenizer to mix and induce precipitation; (4) obtaining a suspension with uniform particle distribution by centrifugation, evaporating the residual ethanol in the suspension, and then drying to obtain modified phytosterol nanoparticles.

[0010] Preferably, the concentration of phytosterol in the phytosterol solution is 1-7 g / L. This concentration can ensure that phytosterol is fully dissolved, avoiding incomplete dissolution due to high concentration, which affects the uniformity of the subsequent nanoparticles.

[0011] Preferably, in step (2), the amount of zein added is 1-20% based on the mass of phytosterols.

[0012] The addition amount of zein is selected to be 1-20% because when it is less than 1%, only a small amount of protein adheres to the surface of phytosterols, and the effect on the hydrophilicity modification of phytosterols is limited; when it is more than 20%, zein is prone to excessive stacking, which will cover some hydrophilic groups, resulting in a slowdown in the improvement of hydrophilicity and an increase in raw material costs.

[0013] The particle size of the modified phytosterol nanoparticles showed a trend of "first increasing and then decreasing" with the content of zein. This trend is related to the particle aggregation state: at low zein content, the protein only partially adheres to the surface of the phytosterol, which easily leads to particle stacking and increased particle size; at high content, the protein uniformly coats the phytosterol, forming a dense nanostructure, inhibiting aggregation, and reducing particle size. Meanwhile, the polymer molecular weight distribution index (PDI) of all samples was less than 0.3, indicating good particle dispersibility and no obvious agglomeration.

[0014] Preferably, step (2) includes: adding 1-10 mL of ethanol-water solution with a volume ratio of 1:1 to the phytosterol solution, stirring until the solution becomes clear and transparent again, and then adding zein and stirring to disperse it evenly.

[0015] The purpose of adding an ethanol-water solution is to adjust the polarity of the system, providing a suitable mesoscopic environment for the subsequent dispersion and anti-solvent precipitation of zein, and avoiding the rapid precipitation of phytosterols and the formation of large particles caused by direct addition of the aqueous phase.

[0016] Preferably, in step (3), the pH of the alkaline aqueous solution is 8-12; more preferably 9-10.

[0017] When the pH is below 8, the hydrophilic groups (such as carboxyl groups) of zein are not fully ionized, and the hydrophilic modification effect is weakened; when the pH is above 12, phytosterols may be partially hydrolyzed, affecting the structural stability of nanoparticles.

[0018] In step (3), the dispersion obtained in step (2) is slowly added dropwise to an excess of alkaline aqueous solution at a rate of 5-15 mL / min, while being mixed with a homogenizer for 1-10 min to induce precipitation of nanoparticles.

[0019] Preferably, in step (4), the centrifugation speed is 2000~5000×g and the centrifugation time is 5~15 min, so as to retain a suspension with uniform particle distribution.

[0020] Preferably, step (4) includes centrifuging the mixture obtained in step (3) at a centrifugation speed of 2000~5000×g for 5~15min to remove unreacted large particle impurities and retain a suspension with uniform particle distribution; then evaporating the suspension at 30~50℃ to remove residual ethanol; and finally drying the suspension by vacuum freeze drying to obtain dried modified phytosterol nanoparticles.

[0021] The present invention also provides modified phytosterol nanoparticles prepared by the preparation method described above.

[0022] The contact angle of the modified phytosterol nanoparticles is 91.15~113.07°.

[0023] Unmodified phytosterols exhibit strong hydrophobicity. Modification with 1-20% zein enhances the hydrophilicity of the modified phytosterols. This increased hydrophilicity stems from the amphiphilic nature of zein; its hydrophobic domains bind to phytosterols through hydrophobic interactions, exposing hydrophilic groups (such as amino and carboxyl groups) on the particle surface. Simultaneously, hydrogen bonding (phytosterol -OH and zein -NH) further stabilizes the interfacial structure, reducing the exposure of hydrophobic groups. This invention allows for a wide range of adjustment in the hydrophilicity and hydrophobicity of modified phytosterols by varying the amount of zein used for modification.

[0024] The modified phytosterol nanoparticles have a particle size of 120~160 nm.

[0025] The modified phytosterol nanoparticles of this invention have uniform particle size, good particle dispersibility, and no obvious agglomeration.

[0026] The absolute value of the Zeta potential on the surface of modified phytosterol nanoparticles showed a trend of "first decreasing and then increasing" with the increase of zein content. The change in Zeta potential reflects the adjustment of the surface charge density of the particles: at low zein content, the protein coverage is insufficient and the surface charge is partially blocked; at high content, the protein fully exposes the charged groups, the charge density increases, and the electrostatic repulsion is enhanced, which can improve the stability of the particles under wide pH (3~10) and high ionic strength (1.0 mol / L NaCl) conditions and avoid aggregation.

[0027] The addition of zein disrupts the regular crystal structure of phytosterols, forming partially amorphous characteristics, which can improve the water solubility and bioavailability of phytosterol particles. The thermal decomposition temperature of the modified phytosterol nanoparticles is maintained at 330~360℃, meeting the thermal stability requirements in food and cosmetic processing.

[0028] This invention also provides the application of the modified phytosterol nanoparticles in the construction of Pickering emulsions.

[0029] In the application of this invention, by adjusting the degree of modification of the surface of the modified phytosterol nanoparticles (the content of zein) and the water-oil phase ratio, the rheological properties and stability of the emulsion can be precisely controlled.

[0030] The applications include: (I) The modified phytosterol nanoparticles are added to water to form a particle dispersion; the mass-volume concentration of the modified phytosterols in the particle dispersion is 1-5%; (II) Mix the particulate dispersion with the oil phase at a water ratio (water phase volume / total emulsion volume) of 0.15-0.75 and stir evenly with a high-speed homogenizer to obtain the Pickering emulsion.

[0031] The water-to-oil ratio is defined as the volume of the aqueous phase divided by the total emulsion volume. For example, a water-to-oil ratio of 0.15 means that the volume of the aqueous phase accounts for 15% of the total emulsion volume, and the volume of the oil phase accounts for 85% of the total emulsion volume.

[0032] The Pickering emulsion constructed in this invention is a water-in-oil emulsion.

[0033] Unmodified phytosterol nanoparticles form gels at low water concentrations, exhibiting high viscosity and poor flowability; while modified phytosterol nanoparticles maintain good flowability and stable emulsion state even at higher water concentrations. This difference stems from the increased hydrophilicity of the modified phytosterol nanoparticles. The reduced adsorption capacity of phytosterol nanoparticles at the oil-water interface prevents excessive accumulation and the formation of a dense gel network, thus slowing down the gelation rate.

[0034] Within a water content range of 0.15–0.6, Pickering emulsions prepared from modified phytosterol nanoparticles all exhibited "shear-thinning" characteristics (viscosity decreased with increasing shear rate), and their viscosity was significantly lower than that of the unmodified group, resulting in a substantial improvement in spreadability. Simultaneously, the storage modulus (G') and loss modulus (G'') of the emulsion decreased with increasing zein content, indicating weakened gel strength and improved flowability and spreadability, making it more suitable for applications in food (e.g., spreads) and cosmetics (e.g., lotions and creams).

[0035] At particle concentrations of 1–5%, freshly prepared emulsions of modified phytosterol nanoparticles showed no demulsification and remained in an emulsion or soft gel state after standing, without significant stratification. The modified phytosterol nanoparticles form a dense gel network due to hydrogen bonding between proteins, which enhances interfacial membrane strength and improves the long-term storage stability of the emulsion.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Green and efficient preparation process: The modified phytosterol nanoparticles of the present invention are prepared by antisolvent coprecipitation method, which does not require complex equipment (such as high pressure homogenizer), and the process steps are simple (dissolution-dispersion-precipitation-drying). Ethanol, zein and phytosterol are all food-grade raw materials, which are safe and cost less than chemical modification methods (such as transesterification modification).

[0037] (2) Adjustable hydrophilicity and hydrophobicity: The modified phytosterol nanoparticles of the present invention can achieve continuous adjustment of the contact angle from 91.15 to 113.07° by controlling the content of zein (1~20%), which can meet the emulsion preparation requirements of different water-oil systems and solve the problem of narrow adjustment range of hydrophilicity and hydrophobicity of traditional natural macromolecular nanoparticles (such as pure protein and polysaccharide particles).

[0038] (3) Strong environmental adaptability: The modified phytosterol nanoparticles of the present invention remain stable under pH 3~10 and 1.0 mol / L NaCl conditions (particle size <450 nm, PDI <0.4), avoiding the defects of traditional nanoparticles that are prone to aggregation under extreme pH or high ionic strength, and are suitable for complex application scenarios such as food (such as acidic beverages) and cosmetics (such as salt-containing washing and care products).

[0039] (4) Good adaptability of emulsion performance: When constructing Pickering emulsion, the modified phytosterol nanoparticles of the present invention can adjust the degree of particle modification and the water-oil ratio to achieve the state control of "emulsion-soft gel-hard gel". The modulus and viscosity of the modified emulsion are significantly reduced, and the application performance is improved, which breaks through the limitations of unmodified phytosterol emulsions that are easy to gel and difficult to process. Attached Figure Description

[0040] Figure 1 The images shown are scanning electron microscope images of the PZ particles prepared in Example 1. (A), (B), (C), (D), and (E) correspond to PZ1, PZ5, PZ9, PZ13, and PZ17, respectively.

[0041] Figure 2 The water-air contact angle (A), particle size, and zeta potential (B) of the PZ particles prepared in step 1 of Example 1 are given.

[0042] Figure 3 XRD patterns of two raw materials (zein and phytosterols Ps) (A) and PZ particles (B).

[0043] Figure 4 The images show the FTIR spectra of two raw materials (zein and phytosterols Ps) and PZ particles.

[0044] Figure 5DSC spectra of two raw materials (zein and phytosterols Ps) (A) and PZ particles (B).

[0045] Figure 6 Thermogravimetric curves (A) and micro-thermogravimetric curves (B) are shown for the two raw materials and PZ particles.

[0046] Figure 7 Particle size, PDI and Zeta potential of PZ1 (A) and PZ17 (B) at pH 2 to 10, and particle size and PDI of PZ1 (C) and PZ17 (D) at different ionic intensities.

[0047] Figure 8 The oil-water contact angle of the PZ particles.

[0048] Figure 9 A macroscopic photograph of Pickering emulsion.

[0049] Figure 10 The graph shows the relationship between viscosity and shear rate of phytosterol particles (PZ0(i), PZ1(ii), PZ9(iii), PZ17(iv)) modified with different zein at particle concentrations of 1.5% (A), 3.0% (B), and 4.5% (C) under different water ratios.

[0050] Figure 11 The relationship between storage modulus, loss modulus and angular velocity of phytosterol particles (PZ0(i), PZ1(ii), PZ9(iii), PZ17(iv)) modified with zein at different water ratios for particle concentrations of 1.5% (A), 3.0% (B) and 4.5% (C).

[0051] Figure 12 Phase diagrams of freshly prepared emulsions (A) and emulsions (B) after being left to stand for one day at different particle concentrations (1.5% (i), 3.0% (ii) and 4.5% (iii)). Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0053] Example 1: Preparation of zein-modified phytosterol nanoparticles (PZ) (1) Weigh 0.2 g of phytosterol, add 35 mL of 99.9% anhydrous ethanol, place in a 45°C constant temperature water bath, and stir with a magnetic stirrer until completely dissolved, ensuring the solution is clear and transparent without any suspended matter or precipitate. Then, add 5 mL of ethanol-water solution (50%, v / v) to the above solution and continue stirring until the solution becomes clear and transparent again. Next, weigh 0.002 g (PZ1), 0.01 g (PZ5), 0.018 g (PZ9), 0.026 g (PZ13), and 0.034 g (PZ17) of zein according to 1%, 5%, 9%, 13%, and 17% of the phytosterol mass, respectively, and add them to the solution, and continue stirring until uniformly dispersed.

[0054] (2) Subsequently, 40 mL of the above dispersion was slowly added dropwise to 10 times the volume (400 mL) of deionized water adjusted to pH 10 with NaOH, while simultaneously homogenizing rapidly at 15000 rpm using a homogenizer, with the entire process controlled within 5 min. Afterward, the mixture was centrifuged at 3000 × g for 10 min to remove large particles, resulting in a suspension with uniform particle distribution. Residual ethanol was removed by continuous evaporation at 40°C for 30 min using a rotary evaporator. Finally, the resulting suspension was freeze-dried under vacuum to obtain dried nanoparticle powders PZ1 (1% zeinyl soluble protein), PZ5 (5% zeinyl soluble protein), PZ9 (9% zeinyl soluble protein), PZ13 (13% zeinyl soluble protein), and PZ17 (17% zeinyl soluble protein), which were stored at -80°C for later use.

[0055] The nanoparticle powder prepared in Example 1 was subjected to the following tests: 1. The surface morphology of the nanoparticles was observed using a scanning electron microscope. The sample was cast onto a silicon wafer and attached to the sample stage with conductive tape. The sample was sputtered with gold film for 180 s under high vacuum, with a working voltage of 10.0 kV and a magnification of 15 k to 30 k.

[0056] 2. The particle size of the nanoemulsion was tested using a Zeta-nanoparticle size analyzer. The data were presented in the form of average particle size, PDI, and Zeta potential, and the frequency of particle size distribution was statistically analyzed.

[0057] 3. Contact angle testing is used to evaluate the surface hydrophilicity and hydrophobicity of nanoparticles. Natural sterols or lyophilized nanoparticles are pressed into smooth discs and placed on a glass slide. 5 μL of deionized water is added to the sample surface using a syringe. After stabilizing for 2 min, the wetting of the water droplet on the sample surface is recorded using the instrument's built-in camera. Each sample is measured three times.

[0058] 4. The crystallinity of the nanoparticles was analyzed by X-ray diffraction. Under the conditions of 30 kV voltage and 10 mA current, diffraction patterns in the angular range of 5 to 50° were recorded with a step size of 0.02° and a scanning speed of 0.1 ° / s.

[0059] 5. Fourier transform infrared spectroscopy was used to analyze the chemical structure of the sample. The lyophilized sample was mixed with 100 mg of potassium bromide powder, pressed into transparent tablets, and the scanning wavenumber range was 4000 to 400 cm⁻¹. -1 Spectral resolution 4 cm -1 .

[0060] 6. The thermal stability of the nanoparticles was determined by thermogravimetric analysis and differential scanning calorimetry. Measurements were performed under a dry nitrogen atmosphere, with the temperature increased from 30 °C to 500 °C at a flow rate of 20 mL / min and a heating rate of 10 °C / min.

[0061] 7. The dispersive stability of the nanoparticles was evaluated using pH stability and ionic strength stability tests. For the pH stability test, the pH of the nanoparticle solution was adjusted to the range of 2.0–10.0 using 0.1 M hydrochloric acid and 0.1 M sodium hydroxide solution. After storing the samples overnight at 4 °C, the particle size, zeta potential, and PDI were measured. For the ionic strength stability test, the nanoparticles were mixed with sodium chloride solutions of different concentrations (0, 0.25, 0.50, 0.75, 1.0 mol / L), stored overnight at 4 °C, and then the particle size and PDI were evaluated.

[0062] like Figure 1 As shown, phytosterols mainly appear as pointed, sheet-like or elongated shapes, while zein adheres as small, round particles. At low zein concentrations (1%), phytosterols form larger sheet-like structures, with only a small number of zein particles embedded on the nanosheets through hydrophobic interactions. As the zein concentration increases, the phytosterol sheet-like structures shrink, and the number of attached particles increases. At a concentration of 9%, sheet-like structures of different sizes and multiple stackings appear. At a concentration of 17%, even smaller sheet-like structures are formed, with more pronounced overlapping and stacking. This indicates that increased zein concentration enhances the electrostatic, hydrophobic, and hydrogen bonding interactions with phytosterols, promoting the formation and stacking of smaller, more uniform sheet-like structures.

[0063] like Figure 2As shown in (A), the contact angle of unmodified phytosterols was 113.26°, exhibiting hydrophobicity. With increasing zein concentration, the contact angle first decreased, then increased, and then decreased again. At low concentrations, only a small amount of protein adhered, resulting in limited modification; above 9% concentration, hydrophilicity significantly increased. The contact angle slightly increased at 13% concentration, but dropped to 91.15° at 17% concentration. This may be related to changes in the plate-like structure of phytosterols. As zein concentration increased, the plate-like structure shrank, more protein adhered, and hydrophilic groups were exposed; at 13% concentration, due to the stacking effect and larger plate-like structure, some hydrophilic groups were covered, leading to decreased hydrophilicity.

[0064] Figure 2 As shown in Figure (B), the particle size of PZ particles initially increases and then decreases with increasing zein content, reaching its maximum at 5% zein content (150.85 nm) and its minimum at 17% zein content (123.73 nm). This indicates that zein content affects particle aggregation. The PDI values ​​are all less than 0.3, indicating good particle dispersibility. The absolute value of the zeta potential initially decreases and then increases with increasing zein content, reaching its minimum at 9% zein content (-38.53 mV) and its minimum at 17% zein content (-40.17 mV). Changes in zeta potential reflect changes in particle surface charge, affecting particle stability and dispersibility. Adjusting the zein content can optimize the particle size, dispersibility, and surface charge properties of PZ particles.

[0065] Figure 3 XRD analysis results in (A) showed that unmodified phytosterols had obvious crystallization peaks (PZ0 had strong crystallization peaks at 2θ = 7.19°, 15.11°, 16.70°, 17.57°, and 18.60°~19.29°), while Figure 3 In the PZ particles (B), the 16.70° and 17.57° crystallization peaks disappeared, and the intensity of the remaining peaks decreased significantly with increasing zein content. This indicates that the addition of zein disrupted the crystal structure of phytosterols, reducing their crystallinity and causing them to form amorphous or hypocrystalline structures. The change in crystallinity may be related to the interaction between zein and phytosterols, affecting the physicochemical properties of the particles, such as solubility and stability.

[0066] Figure 4 FTIR analysis results showed that unmodified phytosterols were present at 3425 cm⁻¹. -1 There is a hydroxyl (-OH) stretching vibration peak at 3309 cm⁻¹, and zein has a peak at 3309 cm⁻¹. -1 A peak of NH stretching vibration is present. With increasing zein content, the characteristic peaks of PZ particles shift: at 1% zein, the hydroxyl peak shifts to 3421 cm⁻¹. -1The NH peak shifted to 3319 cm⁻¹ -1 At 17% zein, the hydroxyl peak shifted to 3412 cm⁻¹. -1 The NH peak remained at 3319 cm⁻¹ -1 This indicates that there are hydrogen bond interactions between zein and phytosterols, and the strength of these hydrogen bonds decreases with increasing zein content. The amide I band of zein (1680–1630 cm⁻¹) -1 (C=O stretching vibration) and amide II band (1655~1590 cm) -1 The NH bending and CN stretching vibrations also change in PZ particles. The amide I and II bands of unmodified zein are located at 1656 cm⁻¹. -1 and 1536 cm -1 In PZ particles, the amide I and II bands of PZ1 shifted to 1741 cm⁻¹, respectively. -1 and 1647 cm -1 The amide I and II bands of PZ17 are stable at 1655 cm⁻¹. -1 and 1542 cm -1 This indicates that there is a hydrophobic interaction between zein and phytosterols, and that this interaction weakens with increasing zein content. The results suggest that the addition of zein alters the molecular structure of phytosterols, and the changes in hydrogen bonds and hydrophobic interactions have a significant impact on the physicochemical properties of PZ particles.

[0067] Figure 5 (A) and Figure 5 DSC analysis (B) showed that the thermal stability of PZ particles varied with the content of zein. The melting peak temperature of unmodified phytosterols was 139.83℃, while the melting peak temperature of PZ particles decreased, and the thermal decomposition peak temperature also changed, with the melting peak temperature of PZ particles being 137℃.

[0068] Figure 6 (A) and Figure 6(B) shows the TGA and DTG (micro-quotient thermogravimetric analysis) curves, respectively. The results show that the maximum weight loss rate temperatures of PZ1, PZ5, PZ9, PZ13, and PZ17 are 354℃, 366℃, 357℃, 359℃, and 360℃, respectively, indicating that the thermal stability of the samples from high to low is PZ5>PZ17>PZ13>PZ9>PZ1. Compared with natural phytosterols, its decomposition peak temperature and maximum weight loss rate temperature (371℃) are both lower, mainly due to the decrease in crystallinity. The main thermogravimetric stage of PZ0 is in the range of 250~360℃ (weight loss rate of about 60%), corresponding to the decomposition of phytosterols; the thermogravimetric onset temperature of the PZ series samples is slightly lower (240~245℃), but the maximum weight loss rate temperature (366℃ for PZ5) is higher than that of PZ1 (354℃), indicating that zein can improve thermal stability.

[0069] Figure 7 Figures (A), (B), (C), and (D) show the significant differences in the stability of PZ particles under different pH values ​​and ionic strengths. PZ17 particles maintained a particle size below 450 nm within the pH range of 3–10, exhibiting good dispersibility and stability; however, at an ionic strength of 1.0 mol / L, the particle size increased but no significant aggregation occurred. In contrast, PZ1 particles showed a significant increase in particle size at pH 2 and 7, and aggregation occurred at an ionic strength of 0.75 mol / L. The addition of zein significantly improved the stability of PZ particles in different environments, especially under neutral and weakly alkaline conditions and in environments with high ionic strength.

[0070] Example 2: Construction of Pickering emulsion PZ0, PZ1, PZ9, and PZ17 nanoparticle powders prepared in Example 1 were respectively used to prepare formulations with concentrations of 1.5%, 3%, and 4.5% (w / v). 水 The particle dispersion was prepared; the particle dispersion was mixed with camellia oil in proportions of 0.15, 0.3, 0.45, 0.6, and 0.75 (total system 50 mL) and stirred for 2 min using a high-speed homogenizer (8000 rpm).

[0071] The Pickering emulsion prepared in Example 2 was subjected to the following tests: 1. Oil-water contact angle test: A contact angle meter was used to evaluate the wettability of nanoparticles at the oil-water interface to clarify their interfacial mechanism for stabilizing emulsions. The test method is as follows: PZ0, PZ1, PZ9, and PZ17 nanoparticle powders were pressed into smooth discs with a diameter of 5 mm and a thickness of 1 mm using a tablet press (10 MPa pressure, holding pressure for 30 s). The discs were placed on the instrument stage, and a uniform oil film was spread on the sample surface. 5 μL of deionized water was drawn up with a microsyringe and slowly added to the surface of the nanoparticle discs. After the droplets stabilized for 2 min, the droplet morphology was photographed using the instrument's built-in camera. Each sample was measured three times.

[0072] The results are as follows Figure 8 As shown in the figure, the oil-water contact angle test results show that the unmodified nanoparticles have a larger contact angle, indicating that they have a stronger oleophilicity; while the modified nanoparticles have a smaller contact angle and a stronger hydrophilicity.

[0073] 2. Observation of emulsion appearance: The appearance and flowability of different formulations of Pickering emulsions are evaluated by visual observation combined with macroscopic flowability test. The test method is as follows: Pour freshly prepared emulsion and emulsion that has been stored for 1 day into transparent glass bottles (20 mL), tilt the glass bottle and keep it for 30 s, and observe whether the emulsion shows layering, precipitation or gelation.

[0074] The results are as follows Figure 9 As shown, the appearance of freshly prepared Pickering emulsions reveals that unmodified particles (zein) formed gels at a water ratio of 0.45, while modified particles (17%) maintained good flowability at a water ratio of 0.6. After a period of time, except for the PZ1 groups with water ratios of 0.15 and 0.3 which did not gel, all other groups formed emulsion gels. This indicates that unmodified particles gelled quickly and exhibited high modulus and viscosity, demonstrating high macroscopic viscosity; while modified nanoparticles slowed down the gelation rate and viscosity, improved flowability, and facilitated processing.

[0075] 3. Rheological property testing: A rotational rheometer with 50 mm parallel plates and a 1 mm gap was used to measure the steady-state rheology of the emulsion (shear rate 0.01~100 s⁻¹). -1 ) and dynamic rheology (angular frequency 0.1~100 rad / s, strain 0.1%).

[0076] Steady-state rheological results are as follows Figure 10As shown, all Pickering emulsions exhibited shear-thinning characteristics. The higher the particle concentration, the higher the overall viscosity of the emulsion. Furthermore, the viscosity increased more significantly with increasing aqueous content (≤0.6). Under the same aqueous phase and particle concentration, pure phytosterol particles (PZ0) formed a high-viscosity structure at various concentrations and most aqueous phase contents due to their strong interfacial assembly ability. In contrast, protein-modified particles, due to their increased surface hydrophilicity and weakened interfacial adsorption ability, improved the viscosity of the emulsion and reduced the strength of the "particle network." Although it was difficult to form a stable emulsion gel under ultra-high internal phase conditions, it could effectively avoid the high viscosity problem caused by excessive particle aggregation in a wide range of aqueous phases from 0.3 to 0.6, significantly improving the emulsion's spreadability and extensibility, and meeting the processing and usage needs of food spreads, cosmetic emulsions, and other scenarios.

[0077] Dynamic rheological results as follows Figure 11 As shown, the higher the particle concentration, the higher the overall G' and G'' of the emulsion. Moreover, when the aqueous phase content increases (≤0.75), the trend of G' and G'' increasing with the aqueous phase content becomes more significant. Protein modification weakens the viscoelasticity of the emulsion, causing the strength of the "particle network" to decrease. All samples have G'>G'', indicating that the emulsion is in a gel state. However, the gel strength of the modified group is significantly reduced, resulting in better fluidity and making it more suitable for the needs of topical products.

[0078] 4. Emulsion Phase Diagram Analysis: The emulsion phase diagram was drawn by visually observing the emulsion state (demulsification, emulsion, gel) and combining this with the G' value measured by a rheometer. Results showed: Figure 12 Figure (A) shows the phase diagram of a fresh emulsion. The demulsification zone is mainly concentrated in the high aqueous phase (water content 0.75) and covers the entire protein content range. This is because in the ultra-high aqueous phase, nanoparticles cannot fully encapsulate the aqueous phase, resulting in poor system stability. The emulsion zone is distributed in the low aqueous phase (water content ≤0.3). At this point, the aqueous phase is small, and nanoparticles can effectively adsorb at the oil-water interface to form a stable liquid emulsion. The gel zone appears in the medium aqueous phase (water content 0.45-0.6) and is mainly composed of low protein content (such as PZ0). This indicates that in the fresh state, pure particles or low-protein modified particles can quickly assemble into a gel structure in the medium aqueous phase, while the gel formation region of the protein-modified group is relatively narrow.

[0079] Figure 12 Figure B shows the phase diagram of the emulsion after one day of storage. The demulsification area is smaller than that of the fresh emulsion, but it still exists in the high aqueous phase (0.75) and some low aqueous phases (such as 0.15-0.3 for the protein-modified group). In the low aqueous phase, the emulsion is mainly viscous, and the particle network has developed to some extent after storage, with increased elasticity. The medium aqueous phase region tends to form gels, and even some gel points appear in the high aqueous phase region, indicating that the particle network has developed sufficiently after storage.

[0080] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing modified phytostanol nanoparticles, characterized by, The method comprises the following steps: (1) dissolving phytosterol in ethanol to form a transparent phytosterol solution; (2) adding zein to the phytosterol solution and stirring until uniformly dispersed; (3) adding the dispersion obtained in step (2) dropwise into an excess of an alkaline aqueous solution while mixing using a homogenizer to induce precipitation; (4) obtaining a suspension with uniformly distributed particles by centrifugation, evaporating the residual ethanol in the suspension, and drying to obtain modified phytosterol nanoparticles.

2. The method of preparing modified phytostanol nanoparticles according to claim 1, characterized in that, In step (2), the amount of zein added is 1-20% based on the mass of phytosterol.

3. The method of making modified phytostanol nanoparticles according to claim 1, wherein, In step (3), the pH of the alkaline aqueous solution is 8-12.

4. The method of making modified phytostanol nanoparticles according to claim 1, wherein, In step (3), the dropwise addition rate is 5-15 mL / min.

5. The method of making modified phytostanol nanoparticles of claim 1, wherein, In step (4), the centrifugation speed is 2000-5000 xg, and the centrifugation time is 5-15 min, and the suspension with uniformly distributed particles is retained.

6. Modified phytosterol nanoparticles prepared by the method of any one of claims 1-5.

7. The modified phytostanol nanoparticle of claim 6, wherein, The contact angle thereof is 91.15-113.07°.

8. The modified phytostanol nanoparticle of claim 6, wherein, The particle size thereof is 120-160 nm.

9. Use of the modified phytosterol nanoparticles of any one of claims 6-8 in the construction of a Pickering emulsion.

10. Use according to claim 9, characterized in that, The method comprises the following steps: (I) adding the modified phytosterol nanoparticles to water to form a particle dispersion; in the particle dispersion, the mass-volume concentration of modified phytosterol is 1-5%; (II) mixing the particle dispersion with an oil phase at a water phase ratio of 0.15-0.75, and stirring uniformly using a high-speed homogenizer to obtain a Pickering emulsion.

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