Method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nano precipitation

Abstract

The invention discloses a method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nano precipitation, and belongs to the technical field of nano materials. According to the preparation method, hydrophobic zein and soybean lecithin are taken as raw materials, a method of combining anti-solvent precipitation and thermal induction is adopted, and three-phase interfacial tension of a system is adjusted by regulating and controlling parameters such as temperature and surfactant concentration in a rapid exchange process of a solvent, so that composite nanoparticles are converted into dumbbell-shaped structures; finally, the food-grade Janus nanoparticles with uniform particle size distribution and stable properties are formed. The method for constructing the food-grade Janus nano-particles is easy and convenient to operate, low in cost and suitable for large-scale industrial production, and meanwhile the constructed Janus nano-particles have good stability and are widely applied to the fields of food, medicine or cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nanoprecipitation. Background Art

[0002] Janus nanoparticles are nanoparticles with a dual-compartment structure exhibiting physical or chemical anisotropy. Their unique ability to simultaneously exhibit dual, or even opposing, properties, such as hydrophobicity and hydrophilicity, negative and positive charge, and polarity and nonpolarity, allows them to adapt to complex and diverse application scenarios. As a class of nanomaterials with a unique asymmetric structure, Janus nanoparticles can simultaneously load and deliver multiple active ingredients, act as emulsifiers to stabilize the oil-water interface, and respond anisotropically to their environment. They have played an important role in drug delivery, interfacial catalysis, bioimaging, and other fields. However, existing Janus nanoparticles are typically made from synthetic polymers, and their poor safety profile severely limits their application in the food industry. Proteins and lipids, as naturally occurring biopolymers, are not only abundant, readily available, and inexpensive, but also possess excellent biocompatibility, biodegradability, and edible safety. Therefore, proteins and lipids could be used as raw materials to develop food-grade Janus nanoparticles. Currently, Janus nanoparticles are typically prepared using methods such as interfacial masking, seeded emulsion polymerization, and block copolymer self-assembly. These methods are characterized by low efficiency, high cost, poor safety, and difficulty in achieving large-scale industrial production. Therefore, it is necessary to develop a method for preparing food-grade Janus nanoparticles that is high-yield, low-cost, simple to operate, and easy to scale.

[0003] Patent document No. CN 119856775 A discloses a method for constructing Janus droplets based on multiphase spatial reorganization. By manipulating the spatial phase behavior of a zein solution and a sodium alginate solution, the two mutually incompatible solutions are used as the dispersed phase, while an emulsifier is dissolved in an oil phase as the continuous phase. By manipulating the laminar flow of the two dispersed phases, food-grade Janus droplets are prepared under the shear action of the continuous phase. The difference between this patent and the present invention is that the former prepares a liquid, while the latter prepares solid particles. Furthermore, the two methods employ different raw materials and methods.

[0004] Patent document CN 117106139 A discloses a technology for preparing Janus particles based on seeded emulsion polymerization. This method uses magnetic nanoparticles (ferroferric oxide) and the hydrophobic monomer styrene as raw materials to prepare seed particles. The hydrophilic polymer monomer ethyl orthosilicate is then coated on one end of the seed particles via a seed swelling method to produce magnetically responsive, amphiphilic Janus nanoparticles. However, this method is complex, time-consuming, and costly, making it difficult to achieve continuous production. Furthermore, the solvents used in the preparation process are highly toxic and environmentally polluting, making them unsuitable for application in the food industry.

[0005] Patent document CN 113913198 A discloses a method for preparing Janus particles through interfacial masking. This method uses silica particles as raw material and asymmetric chemical modification via a paraffin wax protection-deprotection method to produce multi-responsive Janus particles. However, this method requires steps such as paraffin wax coating, zonal modification, and wax dissolution, making it cumbersome and unsuitable for large-scale industrial production. Furthermore, the wax dissolution process requires the use of organic solvents such as dichloromethane, which may pose a risk of solvent residue and poor food safety.

[0006] The article "Synthesis of amphiphilic dumbbell-like Janus nanoparticles through one-step coupling" discloses a method for synthesizing Janus nanoparticles through coupling. This method uses silica nanospheres as raw materials, modifies their surfaces to make them hydrophilic and hydrophobic, and then achieves coupling through covalent bonding between surface groups to form amphiphilic dumbbell-shaped Janus nanoparticles. This method relies on surface hydroxyl modification of the silica nanospheres, making it limited in applicability to other materials and restricting its application in other systems. Furthermore, the Janus nanoparticles are composed of silica and involve the introduction of multiple organic solvents during the preparation process, making them unsuitable for use in food.

[0007] As mentioned above, there is still a lack of a method for preparing food-grade Janus nanoparticles with simple steps, low cost, and easy large-scale industrial production. Currently, there are no reports on methods for constructing food-grade Janus nanoparticles using proteins and lipids as raw materials based on thermodynamic equilibrium and transient nanoprecipitation. Summary of the Invention

[0008] In order to overcome the deficiencies of the prior art, the primary technical problem to be solved by the present invention is to provide a food-grade Janus nanoparticle constructed based on thermodynamic equilibrium and instantaneous nanoprecipitation.

[0009] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned food-grade Janus nanoparticles.

[0010] The technical principle of the present invention is based on the principle of thermodynamic equilibrium and instantaneous nanoprecipitation technology. By controlling the thermodynamic parameters in the rapid solvent exchange process of the reaction system to regulate the three-phase interfacial tension, the precise construction of food-grade Janus nanoparticles is achieved. First, two water-insoluble food-grade polymers, zein and soy lecithin, are co-dissolved in ethanol. When the polymer solution is quickly injected into the aqueous solution, the ethanol rapidly diffuses into the aqueous phase. The polymer undergoes a nucleation, growth, and aggregation process due to a sudden drop in solubility, and co-precipitates to form composite nanoparticles. At the same time, by adjusting parameters such as temperature and surfactant during the instantaneous nanoprecipitation process, the three-phase interfacial tension of the control system is adjusted. When the interfacial tension of the zein-water solution and the soy lecithin-water solution is close, the composite nanoparticles transform into a dumbbell-shaped structure with the lowest system free energy, ultimately forming food-grade Janus nanoparticles with a uniform particle size distribution.

[0011] To achieve the above objectives, the technical solutions adopted by the present invention are as follows: 1) Dissolve zein in an ethanol-water solution, adjust the pH of the solution, and stir at 300 rpm for 2 h to completely dissolve the zein solution; 2) treating the zein solution described in step 1) with ultrasound at 300 W for 5 minutes to obtain an ultrasound-treated zein solution; 3) Dissolve soybean lecithin in an ethanol-water solution, adjust the pH of the solution, and stir at 300 rpm for 2 h to completely dissolve the soybean lecithin to obtain a soybean lecithin solution; 4) mixing equal volumes of the sonicated zein solution of step 2) and the soy lecithin solution of step 3) to obtain a zein-soy lecithin mixed solution; 5) Dissolve Tween 80 in ultrapure water, adjust the pH of the solution, and stir at 200 rpm for 0.5 h to completely dissolve it, to obtain an aqueous solution containing Tween 80; 6) heating the zein-soy lecithin mixed solution described in step 4) and the aqueous solution containing Tween 80 described in step 5) to a constant temperature in a constant temperature water bath; 7) Taking 1 mL of the zein-soy lecithin mixed solution described in step 6), injecting it into 10 mL of the aqueous solution containing Tween 80 described in step 6) under stirring at 300 rpm to obtain a dispersion of zein-soy lecithin Janus nanoparticles in the Tween 80 aqueous solution; 8) The dispersion of the zein-soy lecithin Janus nanoparticles in the Tween 80 aqueous solution described in step 7) is allowed to stand at room temperature for a period of time, and then purified by ultrafiltration to remove the surfactant, thereby obtaining a zein-soy lecithin Janus nanoparticle dispersion; 9) The zein-soy lecithin Janus nanoparticle dispersion described in step 8) is placed in a vacuum freeze dryer and freeze-dried for 48 hours to obtain solid zein-soy lecithin Janus nanoparticles.

[0012] Preferably, the ethanol concentration in the ethanol-water solution in step 1) is 90% (v / v), the solution pH is 4.5, and the zein concentration is 1.5% (w / v).

[0013] Preferably, the ethanol concentration in the ethanol-water solution in step 3) is 90% (v / v), the solution pH is 4.5, and the soy lecithin concentration is 1.5% (w / v).

[0014] Preferably, the concentration of Tween 80 in step 5) is 0.4% (w / v) and the pH of the solution is 4.5.

[0015] Preferably, the heating temperature in step 6) is 60°C.

[0016] Preferably, the injection speed in step 7) is 10 mL / min.

[0017] Preferably, the standing time in step 8) is 6 h, the ultrafiltration membrane has a molecular weight cut-off of 50 kDa and a pore size of 0.1 μm.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Zein, a natural plant protein extracted from corn endosperm, is abundant in resources and relatively inexpensive. It has good biocompatibility and self-assembly capabilities, and can synergize with other ingredients to form structurally stable nanoparticles, which have excellent encapsulation effects on fat-soluble active substances. Soy lecithin, a phospholipid extracted from soybeans, is naturally non-toxic, environmentally friendly, biodegradable, and has excellent interfacial activity. It can maintain the uniformity and stability of nanoparticles over a wide concentration range.

[0019] (2) The present invention prepares for the first time protein-lipid combined food-grade dumbbell-shaped Janus nanoparticles. The two parts of the nanoparticles are composed of protein and lipid respectively. They have the core characteristics and advantages of two biological macromolecules, such as the biocompatibility and active ingredient encapsulation ability of protein and the interfacial activity and emulsification and dispersibility of lipids.

[0020] (3) The food-grade Janus nanoparticles are prepared based on the thermodynamic equilibrium principle and the instantaneous nanometer precipitation technology for the first time, by dynamically regulating the nanometer coprecipitation and phase separation process. The preparation method is simple in operation, mild in condition, high in efficiency, low in cost, and suitable for large-scale industrial production.

[0021] (4) The ultrasonic treatment method is adopted in the preparation of the zein solution, which helps to improve the raw material dissolution effect, reduce the aggregation and precipitation, and improve the solution stability. The ultrasonic treatment can also promote the zein to expose more binding sites, enhance the tightness of the combination with soybean lecithin, and help to improve the stability of the food-grade Janus nanoparticles.

[0022] (5) The ultrafiltration method is adopted in the purification of the Janus nanoparticles, which can be continuously operated compared with the centrifugal method and dialysis method, avoiding the damage of high centrifugal force to the structure of the Janus nanoparticles. At the same time, the active separation mode of constant volume washing and filtration can efficiently remove the surfactant in a short time, greatly improving the purification efficiency and operation convenience.

[0023] (6) The Janus nanoparticles prepared by the application have small particle size, uniform distribution, and good stability, which is beneficial to maintain a stable state in various application scenarios in the food field, and can be used in the field of functional factor delivery system. The application also widens the research and application path for the development of new nanoparticles in the food industry. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The scanning electron microscope graph of the food-grade Janus nanoparticles prepared in Example 1.

[0025] Figure 2 The laser confocal microscope graph of the food-grade Janus nanoparticles prepared in Example 1.

[0026] Figure 3 The particle size and PDI graph of the nanoparticles prepared in Examples 1-4, Comparative Example 1 and Comparative Example 2.

[0027] Figure 4 The turbidity graph of the nanoparticles prepared in Examples 1, Comparative Example 1 and Comparative Example 2.

[0028] Figure 5 The ultraviolet-visible absorption spectrum graph of the nanoparticles prepared in Examples 1, Comparative Example 1 and Comparative Example 2.

[0029] Figure 6 The Fourier transform infrared spectrum graph of the nanoparticles prepared in Examples 1, Comparative Example 1 and Comparative Example 2.

[0030] Figure 7These are the X-ray diffraction patterns of the nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0031] Figure 8 These are thermogravimetric curves of the nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0032] Figure 9 These are differential thermogravimetric curves of the nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0033] Figure 10 The differential scanning calorimetry curves of the nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2 are shown.

[0034] Figure 11 This is the appearance of the food-grade Janus nanoparticles prepared in Example 5 after storage for 30 days.

[0035] Figure 12 Particle size and PDI of the food-grade Janus nanoparticles prepared in Example 5 after storage for 30 days.

[0036] Figure 13 The particle size and PDI diagram of the nanoparticles prepared in Comparative Example 1 after storage for 30 days.

[0037] Figure 14 These are TSI graphs of the nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the following examples. It should be noted that the description of these embodiments is intended to help understanding the present invention, but does not constitute a limitation of the present invention.

[0039] The experimental methods used in the present invention are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0040] Example 1 Zein was dissolved in 90% (v / v) ethanol, the pH of the solution was adjusted to 4.5, and the solution was stirred to dissolve, resulting in a 1.5% (w / v) zein solution. The zein solution was ultrasonically treated at 300 W for 5 minutes to obtain a sonicated zein solution. Soy lecithin was dissolved in 90% (v / v) ethanol, the pH of the solution was adjusted to 4.5, and the solution was stirred to dissolve, resulting in a 1.5% (w / v) soy lecithin solution. The sonicated zein solution and soy lecithin solution were mixed in equal volumes to obtain a zein-soy lecithin mixed solution. Tween 80 was dissolved in ultrapure water, the pH of the solution was adjusted to 4.5, and the solution was stirred to dissolve, resulting in an aqueous solution containing 0.4% (v / v) Tween 80. The zein-soy lecithin mixed solution and the Tween 80 aqueous solution were heated to 60°C separately. One mL of the mixed solution was injected into a 10 mL aqueous solution containing Tween 80 at a rate of 10 mL / min to obtain a dispersion of zein-soy lecithin Janus nanoparticles in the Tween 80 solution. The dispersion was allowed to stand at room temperature for 6 hours and then purified using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and a pore size of 0.1 μm to obtain a dispersion of zein-soy lecithin Janus nanoparticles. The dispersion was freeze-dried for 48 hours to obtain solid zein-soy lecithin Janus nanoparticles.

[0041] Example 2 The difference between the technical solution in this embodiment and the above-mentioned embodiment 1 is that the pH value of the solution and the concentration of Tween 80 are different. The specific method is as follows: Zein was dissolved in 90% (v / v) ethanol, the pH of the solution was adjusted to 4.0, and the solution was stirred to dissolve, resulting in a 1.5% (w / v) zein solution. The zein solution was ultrasonically treated at 300 W for 5 minutes to obtain a sonicated zein solution. Soy lecithin was dissolved in 90% (v / v) ethanol, the pH of the solution was adjusted to 4.0, and the solution was stirred to dissolve, resulting in a 1.5% (w / v) soy lecithin solution. The sonicated zein solution and soy lecithin solution were mixed in equal volumes to obtain a zein-soy lecithin mixed solution. Tween 80 was dissolved in ultrapure water, the pH of the solution was adjusted to 4.0, and the solution was stirred to dissolve, resulting in an aqueous solution containing 0.1% (v / v) Tween 80. The zein-soy lecithin mixed solution and the Tween 80 aqueous solution were heated to 60°C separately. One mL of the mixed solution was injected into a 10 mL aqueous solution containing Tween 80 at a rate of 10 mL / min to obtain a dispersion of zein-soy lecithin Janus nanoparticles in the Tween 80 solution. The dispersion was allowed to stand at room temperature for 6 hours and then purified using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and a pore size of 0.1 μm to obtain a dispersion of zein-soy lecithin Janus nanoparticles. The dispersion was freeze-dried for 48 hours to obtain solid zein-soy lecithin Janus nanoparticles.

[0042] Example 3 The difference between the technical solution in this embodiment and the above-mentioned embodiment 2 is that the concentration of the ethanol solution and the concentration of Tween 80 are different. The specific method is as follows: The zein was dissolved in 70% (v / v) ethanol solution, the pH value of the solution was adjusted to 4.0, and the solution was stirred and dissolved to obtain a zein solution with a mass concentration of 1.5% (w / v). The zein solution was subjected to ultrasonic treatment at 300 W for 5 min to obtain an ultrasonic-treated zein solution. The soybean lecithin was dissolved in 70% (v / v) ethanol solution, the pH value of the solution was adjusted to 4.0, and the solution was stirred and dissolved to obtain a soybean lecithin solution with a mass concentration of 1.5% (w / v). The ultrasonic-treated zein solution and the soybean lecithin solution were mixed in equal volumes to obtain a zein-soybean lecithin mixed solution. The Tween 80 was dissolved in ultrapure water, the pH value of the solution was adjusted to 4.0, and the solution was stirred and dissolved to obtain an aqueous solution containing 0.4% (v / v) Tween 80. The zein-soybean lecithin mixed solution and the aqueous solution containing Tween 80 were heated to 60°C, respectively. 1 mL of the mixed solution was injected into 10 mL of the aqueous solution containing Tween 80 at a speed of 10 mL / min to obtain a dispersion of zein-soybean lecithin Janus nanoparticles in the aqueous solution containing Tween 80. The dispersion was allowed to stand at room temperature for 6 h, and was purified by using an ultrafiltration membrane with a molecular weight cut-off of 50 kDa and a pore size of 0.1 μm to obtain a dispersion of zein-soybean lecithin Janus nanoparticles. The dispersion was freeze-dried for 48 h to obtain solid zein-soybean lecithin Janus nanoparticles.

[0043] Example 4 The technical scheme in this example is different from that of Example 3 above in that the reaction temperature is different, and the specific method is as follows: Zein was dissolved in 70% (v / v) ethanol, the pH of the solution was adjusted to 4.0, and the solution was stirred to dissolve, resulting in a 1.5% (w / v) zein solution. The zein solution was ultrasonically treated at 300 W for 5 minutes to obtain a sonicated zein solution. Soy lecithin was dissolved in 70% (v / v) ethanol, the pH of the solution was adjusted to 4.0, and the solution was stirred to dissolve, resulting in a 1.5% (w / v) soy lecithin solution. The sonicated zein solution and soy lecithin solution were mixed in equal volumes to obtain a zein-soy lecithin mixed solution. Tween 80 was dissolved in ultrapure water, the pH of the solution was adjusted to 4.0, and the solution was stirred to dissolve, resulting in an aqueous solution containing 0.4% (v / v) Tween 80. The zein-soy lecithin mixed solution and the Tween 80 aqueous solution were heated to 70°C separately. One mL of the mixed solution was injected into a 10 mL aqueous solution containing Tween 80 at a rate of 10 mL / min to obtain a dispersion of zein-soy lecithin Janus nanoparticles in the Tween 80 solution. The dispersion was allowed to stand at room temperature for 6 hours and then purified using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and a pore size of 0.1 μm to obtain a dispersion of zein-soy lecithin Janus nanoparticles. The dispersion was freeze-dried for 48 hours to obtain solid zein-soy lecithin Janus nanoparticles.

[0044] Example 5 The difference between the technical solution in this embodiment and the above-mentioned embodiment 4 is that the concentrations of zein and soybean lecithin are different. The specific method is as follows: The zein was dissolved in 70% (v / v) ethanol solution, the pH of the solution was adjusted to 4.0, and the solution was stirred and dissolved to obtain a 2.0% (w / v) zein solution. The zein solution was ultrasonically treated at 300 W for 5 min to obtain an ultrasonically treated zein solution. The soybean lecithin was dissolved in 70% (v / v) ethanol solution, the pH of the solution was adjusted to 4.0, and the solution was stirred and dissolved to obtain a 2.0% (w / v) soybean lecithin solution. The ultrasonically treated zein solution and the soybean lecithin solution were mixed in equal volumes to obtain a zein-soybean lecithin mixed solution. The Tween 80 was dissolved in ultrapure water, the pH of the solution was adjusted to 4.0, and the solution was stirred and dissolved to obtain a 0.4% (v / v) Tween 80-containing aqueous solution. The zein-soybean lecithin mixed solution and the Tween 80-containing aqueous solution were separately heated to 70°C. 1 mL of the mixed solution was injected into 10 mL of the Tween 80-containing aqueous solution at a speed of 10 mL / min to obtain a dispersion of zein-soybean lecithin Janus nanoparticles in the Tween 80 aqueous solution. The dispersion was left to stand at room temperature for 6 h, and was purified using an ultrafiltration membrane with a molecular weight cut-off of 50 kDa and a pore size of 0.1 μm to obtain a zein-soybean lecithin Janus nanoparticle dispersion. The dispersion was freeze-dried for 48 h to obtain solid zein-soybean lecithin Janus nanoparticles.

[0045] Comparative Example 1 This comparative example provides zein nanoparticles prepared from zein and Tween 80, according to the following specific method: The zein was dissolved in 90% (v / v) ethanol solution, the pH of the solution was adjusted to 4.5, and the solution was stirred and dissolved to obtain a 1.5% (w / v) zein solution. The zein solution was ultrasonically treated at 300 W for 5 min to obtain an ultrasonically treated zein solution. The Tween 80 was dissolved in ultrapure water, the pH of the solution was adjusted to 4.5, and the solution was stirred and dissolved to obtain a 0.4% (v / v) Tween 80-containing aqueous solution. The ultrasonically treated zein solution and the Tween 80-containing aqueous solution were separately heated to 60°C. 1 mL of the zein solution was injected into 10 mL of the Tween 80-containing aqueous solution at a speed of 10 mL / min to obtain a dispersion of zein nanoparticles in the Tween 80 aqueous solution. The dispersion was left to stand at room temperature for 6 h, and was purified using an ultrafiltration membrane with a molecular weight cut-off of 50 kDa and a pore size of 0.1 μm to obtain a zein nanoparticle dispersion. The dispersion was freeze-dried for 48 h to obtain solid zein nanoparticles.

[0046] Comparative Example 2 This comparative example provides soybean lecithin nanoparticles prepared from soybean lecithin and Tween 80, and the specific method is as follows: Soy lecithin was dissolved in 90% (v / v) ethanol, the pH of the solution was adjusted to 4.5, and the solution was stirred to obtain a 1.5% (w / v) soy lecithin solution. Tween 80 was dissolved in ultrapure water, the pH of the solution was adjusted to 4.5, and the solution was stirred to obtain an aqueous solution containing 0.4% (v / v) Tween 80. The soy lecithin solution and the Tween 80 aqueous solution were heated to 60°C separately. 1 mL of the soy lecithin solution was injected into 10 mL of the Tween 80 aqueous solution at a rate of 10 mL / min to obtain a dispersion of soy lecithin nanoparticles in the Tween 80 aqueous solution. The dispersion was allowed to stand at room temperature for 6 hours and then purified using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and a pore size of 0.1 μm to obtain a dispersion of soy lecithin nanoparticles. The dispersion was freeze-dried for 48 hours to obtain solid soy lecithin nanoparticles.

[0047] Experimental Example 1 Scanning electron microscope observation The food-grade Janus nanoparticle dispersion prepared in Example 1 was used as the experimental sample, and the sample's microstructure was observed using a field-emission scanning electron microscope. A small amount of the sample was dropped onto a silicon wafer, dried, and attached to a conductive adhesive. The wafer was then vacuum-coated with platinum in a vacuum sputtering apparatus for 20 seconds, and the sample's morphology was observed at an accelerating voltage of 5 kV.

[0048] pass Figure 1 The experimental results show that the zein-soy lecithin nanoparticles are composed of two hemispheres of similar size, forming a dumbbell-shaped whole. The nanoparticles have a smooth surface, a major diameter of approximately 250 nm, and a minor diameter of approximately 120 nm, indicating the successful preparation of food-grade Janus nanoparticles.

[0049] Experimental Example 2 Laser confocal microscopy observation Zein and soybean lecithin were labeled with FITC and Nile Blue A, respectively. Then, a food-grade Janus nanoparticle dispersion was prepared according to the method of Example 1. The structural composition of the sample was observed using a laser confocal microscope with the excitation wavelengths set to 488 nm and 633 nm.

[0050] pass Figure 2 The experimental results show that the two hemispheres of the Janus nanoparticle exhibit different colors. One hemisphere is green, representing zein, while the other is blue, representing soy lecithin. This phenomenon indicates that the two hemispheres of the nanoparticle are composed of two different materials, further demonstrating the successful preparation of the Janus nanoparticle.

[0051] Experimental Example 3 Particle Size Distribution Measurement The nanoparticle dispersions prepared in Examples 1-4, Comparative Example 1, and Comparative Example 2 were used as experimental samples. The particle size and PDI of the samples were measured using a nanoparticle size analyzer. The sample concentration was diluted to 1 mg / mL to minimize multiple scattering effects. The measurement temperature was 25°C, and the average of three measurements was taken.

[0052] pass Figure 3 From the experimental results, it can be seen that the particle sizes of the four Janus nanoparticles are all within the nanoscale range and are uniformly distributed. The particle size of the Janus nanoparticles prepared in Example 1 is 142.37 ± 6.48 nm, which is consistent with the results of scanning electron microscopy observation. The particle size of the zein nanoparticles is 121.87 ± 2.13 nm, and the particle size of the soy lecithin nanoparticles is 69.61 ± 1.04 nm, while the particle size of the Janus nanoparticles is significantly smaller than the sum of the two. This result shows that the Janus nanoparticles are not a simple superposition of a single component, but are jointly regulated by the interface coupling and spatial orientation of the two components, and ultimately present a size larger than the two single-component nanoparticles.

[0053] Experimental Example 4 Turbidity Measurement The nanoparticle dispersions prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used as experimental samples, and the turbidity of the samples was measured at 600 nm using an ultraviolet-visible spectrophotometer.

[0054] pass Figure 4 The experimental results show that soy lecithin nanoparticles have the lowest turbidity, which can be attributed to their smaller particle size and better dispersibility. Zein-soy lecithin Janus nanoparticles have significantly lower turbidity than zein nanoparticles, which can be attributed to the unique structure of Janus nanoparticles. The asymmetric dumbbell-shaped structure biases light scattering toward the side, reducing the scattering attenuation of forward transmitted light and exhibiting better light transmission compared to spherical structures.

[0055] Experimental Example 5 UV-Vis Absorption Spectrum Analysis The nanoparticle dispersions prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used as experimental samples, and the UV-visible absorption spectra of the samples were scanned in the wavelength range of 190 to 400 nm using a UV-visible spectrophotometer.

[0056] pass Figure 5Experimental results show that zein-soy lecithin Janus nanoparticles exhibit optical properties that differ significantly from those of single-component spherical nanoparticles. The UV-visible absorption spectrum of the Janus nanoparticles exhibits a bimodal characteristic, with the main peak absorption intensity near 197 nm and a characteristic shoulder peak at 227 nm, which may be due to the dumbbell-shaped structure of the Janus nanoparticles. The asymmetric structure causes differences in the interaction between light and different regions of the Janus nanoparticles, resulting in multiple absorption features during electronic transitions, further proving that the nanoparticles are not spherical, but rather have an optically anisotropic dumbbell-shaped morphology.

[0057] Experimental Example 6 Fourier Transform Infrared Spectroscopy Analysis The solid nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used as experimental samples, and the chemical structures of the samples were characterized using a Fourier transform infrared spectrometer. The samples were mixed with KBr at a ratio of 1:100 (w / w), pressed into tablets, and tested using a Fourier transform infrared spectrometer. The wavenumber range was 400–4000 cm -1 , the number of scans is 64 times, the resolution is 4cm -1 .

[0058] pass Figure 6 The experimental results show that zein has a peak at 3346 cm -1 (OH stretching vibration and NH stretching vibration), 2926 cm -1 (CH stretching vibration), 1657 cm -1 (amide I band, C=O stretching vibration), 1531 cm -1 (amide II band, NH bending coupled with CN stretching) and 1450 cm -1 (Amide III band, CN stretching vibration) shows a characteristic peak. Soybean lecithin shows a characteristic peak at 3421 cm -1 The characteristic broadband at 2925 cm -1 and 2854 cm -1 The characteristic peak at 1466 cm-1 belongs to the symmetric stretching vibration of C–H on the aliphatic chain. -1 The characteristic peak at 1736cm corresponds to CN stretching vibration. -1 、1246 cm -1 、1091 cm -1 and 969 cm -1correspond to the characteristic groups C=0, C-O-C, P-O-C and P=0 of the lipid and phosphorus-containing compounds, respectively. In the zein-soy lecithin Janus nanoparticles, the characteristic peak corresponding to the O-H group red-shifted to 3313 cm -1 , indicating that hydrogen bonds were formed between zein and soy lecithin. After the combination of zein and soy lecithin, the absorption peaks at Amide II and Amide III blue-shifted to 1541 cm -1 and 1456 cm -1 , respectively, and a new peak appeared at 1350 cm -1 , indicating that hydrophobic interaction and electrostatic interaction participated in the formation of the zein-soy lecithin Janus nanoparticles. Due to the stretching and bending of the soy lecithin molecular vibration after the combination with zein, the typical characteristic peaks of the lipid compound in the Janus nanoparticles were shifted to different degrees.

[0059] Experimental Example 7 X-ray Diffraction Analysis The solid-state nanoparticles prepared in Example 1, Comparative Example 1 and Comparative Example 2 were used as experimental samples, and the crystallinity of the samples was analyzed using an X-ray diffractometer. The scanning angle range was from 5° to 50°, the scanning rate was 2° / min, and the voltage and current were set to 40 kV and 40 mA, respectively.

[0060] From the experimental results of Figure 7 , it can be seen that zein had two moderate hump peaks at 9.32° and 19.72°, indicating that zein had an amorphous structure; there were weak diffraction peaks at 31.84° and 45.53°, which were attributed to the presence of NaCl crystals. Soy lecithin had two weak hump peaks at 12.97° and 29.25°, and no crystalline peaks were observed, indicating that lecithin was in an amorphous state. The Janus nanoparticles showed hump-shaped signals at 12.21° and 28.61°, and the peak shape was widened and the intensity was reduced, indicating that when zein and soy lecithin formed a Janus structure, the interaction between them reduced the molecular arrangement order of the system. The amorphous state with low order usually has higher oral bioavailability, which is beneficial to its application in the field of functional factor delivery.

[0061] Experimental Example 8 Thermogravimetric Analysis The solid-state nanoparticles prepared in Example 1, Comparative Example 1 and Comparative Example 2 were used as experimental samples, and the thermal behavior of the samples was determined using a thermogravimetric analyzer. The samples were sealed in a crucible and heated from 40°C to 550°C at a heating rate of 10°C / min, and the nitrogen flow rate was 20 mL / min.

[0062] From the experimental results of Figure 8The experimental results show that all samples show varying degrees of mass loss around 100°C, typically due to the release of bound water. Zein and soy lecithin begin to lose weight rapidly around 310.0°C and 343.3°C, respectively. The zein-soy lecithin Janus nanoparticles exhibit a higher initial decomposition temperature, with an onset temperature close to 378.9°C, indicating greater thermal stability.

[0063] pass Figure 9 From the experimental results, it can be seen that the temperatures of maximum mass loss rate of zein and soybean lecithin are 331.0℃ and 394.7℃ respectively, while the temperature of maximum mass loss rate of Janus nanoparticles is 411.5℃, indicating that the combination of zein and soybean lecithin effectively improves the thermal stability of the nanoparticles.

[0064] Experimental Example 9 Differential Scanning Calorimetry The thermal properties of the solid nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2 were analyzed using a differential scanning calorimeter. The samples were sealed in a crucible and heated from 40°C to 220°C at a heating rate of 10°C / min and a nitrogen flow rate of 20 mL / min.

[0065] pass Figure 10 The experimental results show that zein-soy lecithin Janus nanoparticles, zein and soy lecithin have characteristic endothermic peaks at 80.8°C, 80.6°C and 83.6°C, respectively, which are attributed to the energy absorption of the evaporation of bound water. In addition, the glass transition temperature (Tg) of zein is 155.1°C, and after combining with soy lecithin, the Tg decreases to 108.2°C, indicating that the flexible hydrophobic chain of soy lecithin breaks the aggregation state of zein, increases the chain segment freedom, and makes the Janus nanoparticles more sensitive to thermal responsiveness. Phospholipids have a characteristic main phase transition temperature (Tg). C ), in T C The following is a rigid and orderly arrangement, at T C The above is a liquid crystal phase. C The peak at 51.8 °C and the secondary peak at 57.6 °C may correspond to a secondary phase transition, such as fine-tuning of the crystal form. The Janus curve of nanoparticles does not show T C The peak indicates that after the combination of soy lecithin and zein, the movement of the phospholipid molecules is restricted by the steric hindrance and hydrophobic interactions of zein, which inhibits the main phase transition and results in a wider phase stability range. Therefore, Janus nanoparticles have higher thermal stability and can maintain a relatively stable structure even at high temperatures.

[0066] Experimental Example 10 Storage Stability Analysis The nanoparticle dispersions prepared in Example 5 and Comparative Example 1 were used as experimental samples and stored at 4° C. for 1 month. Their appearance was observed, and their particle size and PDI were measured to evaluate storage stability.

[0067] pass Figure 11 and Figure 12 The experimental results show that the visual appearance of zein-soy lecithin Janus nanoparticles did not change significantly before and after storage, and the particle size and PDI did not change significantly, indicating that Janus nanoparticles have good storage stability. Figure 13 The experimental results show that the particle size and PDI of zein nanoparticles increased significantly after one month of storage, indicating that the storage stability of zein nanoparticles is poor. Therefore, zein-soy lecithin Janus nanoparticles have excellent storage stability, improving the poor stability of single zein nanoparticles.

[0068] Experimental Example 11 Turbiscan Stability Analysis The nanoparticle dispersions prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used as experimental samples. The physical stability of the samples was determined using a Turbiscan LAB. 20 mL of sample was transferred to a sample bottle and tested at 30°C for 2 hours with 30-second intervals. The resulting Turbiscan Stability Index (TSI) was analyzed.

[0069] pass Figure 14 From the experimental results, it can be seen that the TSI value of zein-soy lecithin Janus nanoparticles is smaller than that of zein nanoparticles, indicating that Janus nanoparticles have better physical stability.

Claims

1. Application of zein and soy lecithin in the co-preparation of food-grade Janus nanoparticles.

2. A food-grade Janus nanoparticle, characterized in that: Contains zein and soy lecithin.

3. The food-grade Janus nanoparticles according to claim 2, characterized in that It is constructed based on thermodynamic equilibrium and instantaneous nanoprecipitation.

4. A method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nanoprecipitation, characterized in that: Using hydrophobic zein and soy lecithin as raw materials, a method combining thermal induction and antisolvent precipitation was adopted. By adjusting thermodynamic parameters such as temperature and surfactant concentration, the nanocoprecipitation and phase separation processes were dynamically controlled to obtain zein-soy lecithin Janus nanoparticles.

5. The method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nanoprecipitation according to claim 4, characterized in that: The specific steps include: 1) Dissolve zein in an ethanol-water solution, adjust the pH of the solution, and stir at 300 rpm for 2 h to completely dissolve the zein solution; 2) treating the zein solution described in step 1) with ultrasound at 300 W for 5 minutes to obtain an ultrasound-treated zein solution; 3) Dissolve soybean lecithin in an ethanol-water solution, adjust the pH of the solution, and stir at 300 rpm for 2 h to completely dissolve the soybean lecithin to obtain a soybean lecithin solution; 4) mixing equal volumes of the sonicated zein solution of step 2) and the soy lecithin solution of step 3) to obtain a zein-soy lecithin mixed solution; 5) Dissolve Tween 80 in ultrapure water, adjust the pH of the solution, and stir at 200 rpm for 0.5 h to completely dissolve it, to obtain an aqueous solution containing Tween 80; 6) heating the zein-soy lecithin mixed solution described in step 4) and the aqueous solution containing Tween 80 described in step 5) to a constant temperature in a constant temperature water bath; 7) Taking 1 mL of the zein-soy lecithin mixed solution described in step 6), injecting it into 10 mL of the aqueous solution containing Tween 80 described in step 6) under stirring at 300 rpm to obtain a dispersion of zein-soy lecithin Janus nanoparticles in the Tween 80 aqueous solution; 8) The dispersion of the zein-soy lecithin Janus nanoparticles in the Tween 80 aqueous solution described in step 7) is allowed to stand at room temperature for a period of time, and then purified by ultrafiltration to remove the surfactant, thereby obtaining a zein-soy lecithin Janus nanoparticle dispersion; 9) The zein-soy lecithin Janus nanoparticle dispersion described in step 8) is placed in a vacuum freeze dryer and freeze-dried for 48 hours to obtain solid zein-soy lecithin Janus nanoparticles.

6. The method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nanoprecipitation according to claim 5, characterized in that: The ethanol-water solution in step 1) is a 60%-95% (v / v) ethanol solution with a pH of 3.0-5.5, and the concentration of the zein solution is 0.5%-5% (w / v).

7. The method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and transient nanoprecipitation according to claim 5, characterized in that: In step 3), the ethanol concentration in the ethanol-water solution is 60%-95% (v / v), the solution pH is 3.0-5.5, and the soy lecithin solution concentration is 0.5%-5% (w / v).

8. The method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nanoprecipitation according to claim 5, characterized in that: The Tween 80 concentration in the aqueous solution containing Tween 80 in step 5) is 0.05%-1.5% (v / v), and the pH of the solution is 3.0-5.

5.

9. The method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and instantaneous nanoprecipitation according to claim 5, characterized in that: The heating temperature in step 6) is 50-70°C.

10. The method for constructing food-grade Janus nanoparticles based on thermodynamic equilibrium and transient nanoprecipitation according to claim 5, characterized in that: The injection rate described in step 7) is 1-15 mL / min.

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