A Pickering emulsion stabilized by WG-DNPs and its application in loading β-carotene

Abstract

A WG-DNPs-stabilized Pickering emulsion and its application in β-carotene loading are disclosed. The emulsion is prepared by the following method: S1: Gluten protein and dextran form a covalent complex through Maillard reaction. The gluten protein complex is then prepared into a solution, and its pH is adjusted to neutral. The ionic strength of the solution is then adjusted to 15-25 mM using NaCl to obtain an intermediate solution. The intermediate solution is subjected to water bath heat treatment, ice bath cooling, and freeze drying sequentially to obtain WG-DNPs. S2: WG-DNPs are dispersed in distilled water to obtain a WG-DNPs dispersion. The WG-DNPs dispersion is mixed and homogenized with an oil phase to obtain the Pickering emulsion. This invention uses glycosylated gluten protein nanoparticles as a stabilizer for Pickering emulsions, giving the Pickering emulsions good thermal and storage stability. Furthermore, by loading β-carotene onto the Pickering emulsion stabilized by WG-DNPs, the bioavailability and digestive stability of β-carotene can be significantly improved, thus aiding in the human body's intake of β-carotene.

Description

Technical Field

[0001] This invention relates to the field of Pickering emulsion preparation and application, specifically a WG-DNPs-stabilized Pickering emulsion and its application in loading β-carotene. Background Technology

[0002] Pickering emulsions are based on particulate-stabilized emulsion systems. Common particulates include natural food-grade particles such as starch, protein, and cellulose. These particles cannot adequately meet the requirements for stable emulsions. Therefore, it is necessary to modify the natural particles to improve their emulsifying properties and stability, thereby enabling their wider application in the food industry.

[0003] Existing technologies also utilize gluten protein as a raw material to stabilize Pickering emulsions. There are two main methods for stabilizing Pickering emulsion systems with gluten protein: 1) preparing gliadin into water-dispersible protein nanoparticles using an antisolvent method, and then using them to stabilize the Pickering emulsion; 2) modifying gluten protein chemically to improve its functional properties. However, these methods may disrupt the inherent properties of gluten protein, such as aggregation, self-assembly, and covalent cross-linking, failing to fully utilize the unique protein structure and functional properties of gluten protein. Currently, there are no research reports on using glycosylated gluten protein nanoparticles (WG-DNPs) to stabilize Pickering emulsions as active substance delivery systems. Summary of the Invention

[0004] The present invention aims to provide a WG-DNPs-stabilized Pickering emulsion and its application in β-carotene loading, by using glycosylated gluten protein nanoparticles as a Pickering emulsion stabilizer to improve the stability of the Pickering emulsion.

[0005] To solve the above technical problems, the specific solution adopted in this invention is as follows: a WG-DNPs-stabilized Pickering emulsion, prepared by the following method:

[0006] S1: Prepare a glycosylated gluten protein solution, adjust its pH value to neutral, and then use NaCl to adjust the ionic strength of the solution to 15-25 mM to obtain an intermediate solution. The intermediate solution is subjected to water bath heat treatment, ice bath cooling, and freeze drying in sequence to obtain WG-DNPs.

[0007] S2: WG-DNPs are dispersed in distilled water to obtain a WG-DNPs dispersion. The WG-DNPs dispersion is then mixed and homogenized with the oil phase to obtain a Pickering emulsion.

[0008] As a further optimization of the above technical solution, the specific preparation process of the glycosylated gluten protein solution is as follows: under magnetic stirring, the glycosylated gluten protein is dispersed in distilled water, and then placed at 3-5℃ to obtain the glycosylated gluten protein solution.

[0009] As a further optimization of the above technical solution, gluten protein and dextran were added to a 0.1M phosphate buffer solution with a pH of 12.0 at a mass ratio of 1:1.25 to 1.35, and after being fully dissolved, the mixture was freeze-dried to obtain the glycosylated gluten protein.

[0010] As a further optimization of the above technical solution, the concentration of the glycosylated gluten protein solution is 1.5% to 3%.

[0011] As a further optimization of the above technical solution, the temperature of the water bath heat treatment in step S1 is 85-95℃.

[0012] As a further optimization of the above technical solution, in step S2, the particle concentration of the WG-DNPs dispersion is 1-5%, and the oil ratio is 20-50%.

[0013] As a further optimization of the above technical solution, the mixing and homogenizing speed is 11,000 to 13,000 rpm, and the mixing and homogenizing time is 2.5 to 3.5 min.

[0014] As a further optimization of the above technical solution, the particle concentration of the WG-DNPs dispersion is 4%, and the oil content is 40%.

[0015] As a further optimization of the above technical solution, the emulsion is an oil-in-water emulsion with a particle size of 11.8 μm.

[0016] Application of a Pickering emulsion stabilized with the above-mentioned WG-DNPs in loading β-carotene.

[0017] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses thermally induced glycosylated gluten protein nanoparticles as a stabilizer for Pickering emulsions, providing a theoretical basis for the application of glycosylated gluten protein nanoparticles in Pickering emulsions. By adjusting the ionic strength of the glycosylated gluten protein solution to 20 mM, glycosylated gluten protein nanoparticles with good thermal and storage stability are prepared. Using these glycosylated gluten protein nanoparticles as a stabilizer for Pickering emulsions gives the Pickering emulsions good thermal and storage stability.

[0018] Pickering emulsions loaded with oil-soluble bioactive substance β-carotene were prepared using thermally aggregated glycosylated nanoparticles. The controlled-release characteristics of β-carotene and the stability of the emulsion were investigated, providing theoretical support for the encapsulation of hydrophobic bioactive substances in O / W type Pickering emulsions. Loading β-carotene with Pickering emulsions stabilized by WG-DNPs can effectively improve the bioavailability and digestive stability of β-carotene, which is conducive to the human body's intake of β-carotene. Attached Figure Description

[0019] Figure 1 Microscopic images of Pickering emulsions prepared under different conditions;

[0020] Figure 2 CLSM images of Pickering emulsions prepared under different conditions;

[0021] Figure 3 A graph showing the interfacial protein adsorption content of Pickering emulsions prepared under different conditions;

[0022] Figure 4 Viscosity diagrams of Pickering emulsions prepared under different conditions;

[0023] Figure 5 Plots showing the variation of G' and G” with frequency for Pickering emulsions prepared under different conditions;

[0024] Figure 6 The loss tangent of Pickering emulsions prepared under different conditions varies with frequency.

[0025] Figure 7 For the particle size and PDI variation of WG-DNPs with different ionic strengths;

[0026] Figure 8 The three-phase contact angle (θ) of WG-DNPs with different ionic intensities;

[0027] Figure 9 Particle size distribution of Pickering emulsion loaded with β-carotene;

[0028] Figure 10 Laser confocal microscopy image of Pickering emulsion loaded with β-carotene;

[0029] Figure 11 Rheological dynamics analysis of Pickering emulsion loaded with β-carotene;

[0030] Figure 12Rheological steady-state results analysis of Pickering emulsion loaded with β-carotene;

[0031] Figure 13 This refers to the change in the retention rate of β-carotene in the emulsion;

[0032] Figure 14 The effect of heat treatment at 70℃ on the average particle size and β-carotene retention of Pickering emulsion loaded with β-carotene;

[0033] Figure 15 The effect of heat treatment at 100℃ on the average particle size and β-carotene retention of Pickering emulsion loaded with β-carotene;

[0034] Figure 16 To simulate the release rate of β-carotene at different times during gastrointestinal digestion in vitro;

[0035] Figure 17 To ensure the bioavailability and digestive stability of β-carotene;

[0036] Figure 18 Particle size variation with temperature for Pickering emulsions prepared under different conditions; Detailed Implementation

[0037] This invention discloses a WG-DNPs-stabilized Pickering emulsion, which is prepared by the following method:

[0038] S1: Preparation of WG-DNPs: Prepare a glycosylated gluten protein solution, adjust its pH value to neutral, and then use NaCl to adjust the ionic strength of the solution to 20mM to obtain an intermediate solution. The intermediate solution is subjected to water bath heat treatment, ice bath cooling, and freeze drying in sequence to obtain WG-DNPs.

[0039] S101: Take gluten protein and dextran in a mass ratio of 1:1.25 to 1.35, add 0.1M phosphate buffer solution with a pH of 12.0, mix, and freeze-dry after complete dissolution to obtain the glycosylated gluten protein.

[0040] S102: Under magnetic stirring, disperse glycosylated gluten in distilled water and then place it at 3-5℃ to obtain a glycosylated gluten solution. Control the concentration of the glycosylated gluten solution to be 1.5%-3%. Adjust the pH of the glycosylated gluten solution to 7.0 with 1M NaOH or 1M HCl.

[0041] S103: Add NaCl to the glycosylated gluten protein solution to adjust the ionic strength, centrifuge at 8000 r / min for 15 min to remove insoluble matter, incubate the solution in a water bath at 85-95℃ for 15 min, immediately cool it to room temperature in an ice bath, and then freeze dry it to form WG-DNPs, which are then stored in a refrigerator at 4℃.

[0042] S2: Preparation of Pickering emulsion: WG-DNPs are dispersed in distilled water to obtain a WG-DNPs dispersion with a particle concentration of c = 1-5%. The WG-DNPs dispersion is then mixed and homogenized with an oil phase with a phase ratio of φ = 20-50% to obtain the Pickering emulsion.

[0043] The technical solution of the present invention will be further described below with reference to specific embodiments:

[0044] Example 1

[0045] S1: Preparation of WG-DNPs

[0046] Weigh out gluten protein and dextran in a mass ratio of 1:1.35, mix with 0.1M pH 12.0 phosphate buffer solution, and stir magnetically for 2 hours to fully dissolve. Pour the mixture into an aluminum box and freeze-dry. Place the freeze-dried powder, along with the aluminum box, in a desiccator (relative humidity 79%) with saturated potassium bromide solution at the bottom, and react in a constant temperature oven at 60℃ for 50 hours. The reaction product is glycosylated surface protein (WG-D). Store the glycosylated surface protein in a refrigerator for later use.

[0047] Under magnetic stirring, lyophilized WG-D was dispersed in distilled water for 4 hours, then left to stand overnight at 4°C to allow for complete protein hydration, thus preparing a glycosylated gluten protein solution (2%, w / v). After overnight standing, the supernatant was retained, and the bottom insoluble matter was discarded. The pH was adjusted to 7.0 with 1M NaOH or 1M HCl, and the ionic strength was adjusted to 20 mM with the appropriate amount of NaCl. The solution was centrifuged at 8000 r / min for 15 min to remove insoluble matter. The solution was then placed in a 90°C water bath for 15 min, immediately cooled to room temperature in an ice bath, and then freeze-dried to form WG-DNPs. The prepared WG-DNPs were stored at 4°C.

[0048] S2: Preparation of Pickering emulsion:

[0049] WG-DNPs were dispersed in distilled water to prepare a WG-DNPs dispersion with a particle concentration of 4%. The WG-DNPs dispersion and a certain volume of oil phase were placed in a 30 mL vial (oil-to-dissolve ratio φ = 40%) and homogenized at 12000 rpm for 3 min to obtain 20 mL of Pickering emulsion. It should be noted that the oil-to-dissolve ratio refers to the volume ratio of the WG-DNPs dispersion to the oil phase.

[0050] Example 2-20

[0051] The preparation methods of Examples 2-20 are generally the same as those of Example 1, except that the particle concentration of the oil is different from that of the WG-DNPs dispersion.

[0052] The oil concentrations and particle concentrations in the different embodiments are shown in Table 1 below:

[0053] Table 1

[0054]

[0055]

[0056] Example 21

[0057] S1: Preparation of WG-DNPs

[0058] Weigh out gluten protein and dextran in a mass ratio of 1:1.25, mix with 0.1M pH 12.0 phosphate buffer solution, and stir magnetically for 2 hours to fully dissolve. Pour the mixture into an aluminum box and freeze-dry. Place the freeze-dried powder, along with the aluminum box, in a desiccator (relative humidity 79%) with saturated potassium bromide solution at the bottom, and react in a constant temperature oven at 60℃ for 50 hours. The reaction product is glycosylated surface protein (WG-D). Store the glycosylated surface protein in a refrigerator for later use.

[0059] Under magnetic stirring, lyophilized WG-D was dispersed in distilled water for 4 hours, then left to stand overnight at 3°C ​​to allow for complete protein hydration, thus preparing a glycosylated gluten protein solution (1.5%, w / v). The supernatant of the overnight solution was retained, and the bottom insoluble matter was discarded. The pH was adjusted to 7.0 with 1M NaOH or 1M HCl, and the ionic strength was adjusted to 15 mM with the appropriate amount of NaCl. The solution was centrifuged at 8000 r / min for 15 min to remove insoluble matter. The solution was then placed in a 90°C water bath for 15 min, immediately cooled to room temperature in an ice bath, and then freeze-dried to form WG-DNPs. The prepared WG-DNPs were stored at 4°C.

[0060] S2: Preparation of Pickering emulsion:

[0061] WG-DNPs were dispersed in distilled water to prepare a WG-DNPs dispersion. The particle concentration of the WG-DNPs dispersion was controlled at 4%. The WG-DNPs dispersion and a certain volume of oil phase were placed in a 30 mL vial with an oil phase ratio of φ = 40%. The mixture was homogenized at 11000 rpm for 3.5 min to obtain 20 mL of Pickering emulsion.

[0062] Example 22

[0063] S1: Preparation of WG-DNPs

[0064] Weigh out gluten protein and dextran in a mass ratio of 1:1.35, mix with 0.1M pH 12.0 phosphate buffer solution, and stir magnetically for 2 hours to fully dissolve. Pour the mixture into an aluminum box and freeze-dry. Place the freeze-dried powder, along with the aluminum box, in a desiccator (relative humidity 79%) with saturated potassium bromide solution at the bottom, and react in a constant temperature oven at 60℃ for 50 hours. The reaction product is glycosylated surface protein (WG-D). Store the glycosylated surface protein in a refrigerator for later use.

[0065] Under magnetic stirring, lyophilized WG-D was dispersed in distilled water for 4 hours, then left to stand overnight at 5°C to allow for complete protein hydration, thus preparing a glycosylated gluten protein solution (3%, w / v). The supernatant of the overnight solution was retained, and the bottom insoluble matter was discarded. The pH was adjusted to 7.0 with 1M NaOH or 1M HCl, and the ionic strength was adjusted to 25 mM with the appropriate amount of NaCl. The solution was centrifuged at 8000 r / min for 15 min to remove insoluble matter. The solution was then placed in a 90°C water bath for 15 min, immediately cooled to room temperature in an ice bath, and then freeze-dried to form WG-DNPs. The prepared WG-DNPs were stored at 4°C.

[0066] S2: Preparation of Pickering emulsion:

[0067] WG-DNPs were dispersed in distilled water to prepare a WG-DNPs dispersion. The particle concentration of the WG-DNPs dispersion was controlled at 4%. The WG-DNPs dispersion and a certain volume of oil phase were placed in a 30 mL vial with an oil phase ratio of φ = 40%. The mixture was homogenized at 13000 rpm for 2.5 min to obtain 20 mL of Pickering emulsion.

[0068] Comparative Examples 1-5

[0069] The preparation methods of Comparative Examples 1-5 are generally the same as those of Example 1, except that:

[0070] The ionic strength of the glycosylated gluten protein solution in Comparative Example 1 was 0 mM.

[0071] The ionic strength of the glycosylated gluten protein solution in Comparative Example 2 was 50 mM.

[0072] The ionic strength of the glycosylated gluten protein solution in Comparative Example 3 was 100 mM.

[0073] The ionic strength of the glycosylated gluten protein solution in Comparative Example 4 was 200 mM.

[0074] The ionic strength of the glycosylated gluten protein solution in Comparative Example 5 was 300 mM.

[0075] Results analysis:

[0076] I. The Pickering emulsions prepared in Examples 1-20 were analyzed, and the results are as follows:

[0077] <Emulsion Droplet Morphology>

[0078] Figure 1 Microscopic images of Pickering emulsions prepared under different conditions were provided. The microstructure of the droplets was observed using an optical microscope, and all droplets were spherical. At low WG-DNP particle concentrations (c < 2%), the particle size distribution was uneven, and droplet aggregation occurred. The droplet size increased with increasing φ. This is because when the WG-DNP concentration is too low, the WG-DNPs adsorbed at the oil-water interface cannot completely cover all oil droplets, resulting in a thin interfacial layer that cannot effectively inhibit droplet aggregation, thus leading to large particle sizes. When the WG-DNP concentration exceeds 3%, the emulsion particle size decreases with increasing WG-DNP particle concentration, exhibiting a good droplet distribution. This is because as the WG-DNP concentration increases, the WG-DNPs adsorbed at the oil-water interface gradually cover the entire oil droplet; the more adsorbed particles, the smaller the droplet size. When the particle concentration is sufficiently high (c = 4%, 5%), the droplet size no longer decreases, and the oil phase loading capacity increases. The results show that c and φ affect the size of the emulsion droplets. Pickering emulsions prepared with higher c and φ have smaller particle sizes. The WG-DNPs adsorbed on the surface of the oil droplets interact to form a dense network structure. The oil phase also participates in the formation of the network structure, which is equivalent to the fillers being tightly packed together, thus giving the Pickering emulsion a certain degree of viscoelasticity.

[0079] <Particle Size Analysis>

[0080] Particle size analysis was performed on the Pickering emulsions prepared in Examples 1-20, and the particle size was determined using a Malvern laser particle size analyzer. Parameter settings: relative refractive index 1.095, absorption index 0.001. D was calculated. 4,3 (Weighted average volume diameter) represents the particle size, and all measurements were performed in triplicate.

[0081]

[0082] In the formula: ni represents the number of emulsion droplets, and di represents the diameter of the emulsion droplets.

[0083] The particle sizes of Pickering emulsions prepared under different conditions are shown in Table 2 below:

[0084] Table 2

[0085] Particle size of Pickering emulsions prepared under different conditions

[0086]

[0087] Note: Different letters in the same column indicate statistically significant differences (P < 0.05).

[0088] Table 2 shows that with increasing WG-DNP concentration, the oil phase loading capacity increases, resulting in smaller Pickering emulsion particle size and enhanced stability. When c ≤ 2%, under the same c condition, the particle size increases with increasing φ; when c > 2%, under the same c condition, the droplet size decreases with increasing φ; when c ≥ 4%, the droplet size no longer changes with increasing φ (P > 0.05), consistent with optical microscopy results. At low WG-DNP concentrations (c ≤ 2%), the WG-DNP content is insufficient to cover the entire oil droplet, leading to larger droplet size. As the WG-DNP particle concentration increases (c > 2%), more WG-DNPs participate in the formation of the interfacial film and network structure, resulting in smaller particle size. When the particle concentration is sufficiently high (c ≥ 4%), the interfacial coverage increases, organizing oil aggregation, and the droplet size no longer decreases. This indicates that the oil-to-water ratio affects the emulsion droplet size by changing the number of adsorbable particles at the oil-water interface; with the same oil-to-water ratio, the emulsion particle size decreases with increasing particle concentration.

[0089] <Microstructure Characterization of Emulsions>

[0090] WG-DNPs were stained in aqueous phase with fluorescein isothiocyanate (FITC, 0.5 mg / mL) and in oil phase with Nile Red (0.5 mg / mL) according to Pickering emulsions prepared in Examples 1-20. 10 μL of each sample resuspension was added to a 24 mm × 60 mm coverslip, covered with another coverslip, and placed under a confocal fluorescence microscope. The samples were excited with FITC using a 488 nm laser and with Nile Red using a 514 nm laser, observed at 100x magnification.

[0091] Figure 2CLSM images of Pickering emulsions prepared under different conditions. A laser confocal microscope can characterize the microstructure of Pickering emulsions. The aqueous phase of WG-DNPs emits green fluorescence after being stained with FITC, and soybean oil emits red fluorescence after being stained with Nile red. As can be seen from Figure 2 it, the red oil droplets are wrapped inside the green WG-DNPs, proving that the type of emulsion prepared in this experiment is O / W type Pickering emulsion. There is a slightly yellowish ring structure outside the red oil droplets, which is caused by the superposition of the green fluorescence emitted by FITC and the red fluorescence of Nile red at the interface, indicating that WG-DNPs form a relatively thick protein adsorption layer at the oil-water interface. This composite dense packing layer helps the stability of the emulsion, effectively inhibits droplet coalescence, indicating that WG-DNPs stabilizes the Pickering emulsion well as a stabilizer.

[0092] <Interfacial protein adsorption amount of Pickering emulsion>

[0093] When proteins are used as emulsifiers for emulsions, they are quickly adsorbed on the oil-water interface to form an interfacial adsorption layer. The stability of the emulsion system is maintained by the protein particles firmly adsorbed on the oil-water interface and the reduction of interfacial tension. Protein particles form a dense three-dimensional network structure by adsorbing on the oil-water interface, and the increase in the thickness of the interfacial particle layer inhibits droplet aggregation, so the formed droplet size is smaller. The interfacial protein content can characterize the thickness and stability of the interfacial protein film. This protocol measures the interfacial protein adsorption content of Pickering emulsions prepared at different WG-DNPs concentrations (c = 1 - 5%) and oil volume ratios (φ = 20 - 50%), as shown in Figure 3 the figure. When c ≥ 3%, the interfacial protein adsorption content of the Pickering emulsion is significantly higher than that of the emulsion when c < 3%, indicating that the ability of high-concentration WG-DNPs to stabilize the Pickering emulsion is stronger.

[0094] <Rheological properties of emulsions>

[0095] Rheological behavior analysis can reflect the physical properties of emulsions (such as elasticity, viscosity, strength, etc.), and can effectively reflect the stability and functionality of emulsions, which is very important for the relationship between food production and processing and product quality. This protocol uses the test methods of flow scanning mode and oscillation frequency mode to characterize the bulk viscosity and structural characteristics of emulsions.

[0096] From Figure 4As can be seen from the results, the apparent viscosity decreases and eventually approaches 0 with the increase of shear rate, indicating that the emulsion exhibits shear thinning. Studies have shown that the network structure of droplets is maintained by interactions such as hydrogen bonds and van der Waals forces. Therefore, with the increase of external force (shear rate), the interaction forces between droplets weaken, the emulsion droplets rearrange, and disorder transforms into order, the emulsion flow resistance decreases, and the apparent viscosity decreases. Particle size analysis shows that with the increase of c, the droplet size decreases, the oil / water interface area is larger, the resistance to deformation is stronger, and the apparent viscosity is greater. Within the shear rate measurement range, the apparent viscosity increases with the increase of φ. This is because: (1) with the increase of φ, the oil droplets are equivalent to fillers that enhance the viscoelastic properties of the emulsion, increasing the degree of bridging and flocculation of the emulsion; (2) the apparent viscosity is negatively correlated with the droplet size. With the increase of c and φ, the apparent viscosity of the emulsion increases, indicating that the Pickering emulsion prepared at higher c and φ has better stability and can form an emulsion with a stronger gel network structure.

[0097] Calculation of apparent viscosity and shear rate:

[0098] η=Kγ n-1

[0099] In the formula: η is the apparent viscosity, Pa·s; K is the consistency coefficient, Pa·s n γ is the shear rate, s -1 n is the fluidity index (a constant, a measure of non-Newtonian fluids; n<1 indicates shear-thinning fluids, and n=1 indicates Newtonian fluids).

[0100] The Ostwald-de Waele model was used to fit all emulsions, and the fitting parameters R for all emulsion models were [not specified]. 2 ≥0.99. As can be seen from Tables 3 and 4, all emulsions have n<0.5, indicating that they are within the range of 0.1–100 s. -1 Within the shear rate range, it exhibits strong shear-thinning behavior, exhibiting a non-Newtonian fluid state. With increasing c and φ, the smaller the n value, the greater the deviation from Newtonian fluid behavior. The K value increases with increasing c and φ, indicating an increase in the system viscosity of the emulsion, consistent with the apparent viscosity trend. The increase in c leads to more particles being tightly adsorbed and arranged on the droplet surface, increasing the interfacial film thickness and contributing to the formation of more uniform and fine droplets (consistent with the emulsion particle size results). Therefore, the interaction between emulsion droplets is enhanced, forming a gel network structure with a certain strength. The increase in φ leads to an increase in the number of droplets, resulting in a larger collisional matrix between droplets. Droplets accumulate to form a tight elastic network, enhancing the elastic gel structure of the emulsion. The strength of the emulsion droplet network spatial structure is affected by the effective interfacial area. When c ≥ 4% and φ = 40%, the K value is the largest and the n value is the smallest, indicating that the Pickering emulsion prepared under these conditions has the best deformation resistance and good stability.

[0101] Table 3

[0102] Ostwald-de Waele model coefficients of Pickering emulsions prepared under different conditions (strain = 0.1%)

[0103]

[0104] Note: R 2 ≥0.99, different lowercase letters in the same column indicate significant differences (P < 0.05), different uppercase letters in the same row indicate significant differences (P < 0.05), the same below

[0105] Table 4

[0106] Flow index of Pickering emulsions prepared under different conditions

[0107]

[0108] (2) Vibration frequency mode

[0109] The relationship between the microstructure and macroscopic rheology of emulsions can be constructed through the vibration frequency mode. Figure 5 、 6 are stress frequency sweep diagrams of Pickering emulsions stabilized at different c and φ. At low particle concentrations (c = 1%, 2%), for too high or too low φ (20% or 50%), at low frequency scanning, G' (stored energy) < G'' (loss modulus), at high frequency scanning, G'' > G', and the tangent value of the loss angle has a large fluctuation range, indicating that it is not tolerant of high frequency scanning because its c / φ is at the lowest / highest value. When the external force exceeds its tolerance range, the gel network will be disordered. The yield stress is the critical stress point for the occurrence of structural damage transformation, and the yield stress increases with the increase of c and φ, indicating that the gel network structure of Pickering emulsions at higher c and φ is stronger. At higher particle concentrations (c > 2%), within the scanning range, for all emulsions, G' (stored energy) > G'' (loss modulus), the gel network structure of the emulsion is mainly elastic, confirming the formation of emulsion gels. G', G'', show an obvious dependence on the scanning frequency, indicating that non-covalent physical interactions are the main forces for the formation of the gel network structure. G' can reflect the relationship between the particle layer at the oil-water interface and the strength of the gel network structure, and G'' reflects the viscous behavior of the fluid characteristics of the emulsion, reflecting the relative strength of the viscous and elastic components in the viscoelastic system. With the increase of c and φ, G' is higher, The smaller the value, the lower the likelihood of deformation of the emulsion under external force, and the more stable the three-dimensional network structure. This is due to the increased number of particles adsorbed at the oil / water interface, the increased interfacial film thickness, and the tighter packing between droplets, forming a dense three-dimensional gel network structure with enhanced resistance to deformation. In summary, with the increase of c and φ in WG-DNPs, the apparent viscosity increases, and G' and G” increase. The decrease indicates that higher c and φ can form emulsions with stronger gel network structures, and that bridging flocculation structures exist within the emulsion, forming a gel network structure dominated by elasticity. When c = 4% and φ = 40%, G' > G". High apparent viscosity indicates that the emulsion is stable. At this point, the particle size is small, the droplets are tightly bound together, the interaction force is increased, and the resistance to deformation is strong.

[0110] <Thermal stability of emulsions>

[0111] Thermal stability was determined by heating the prepared Pickering emulsion in a water bath at 70°C and 100°C for 15 min, cooling it to room temperature, and then measuring the change in emulsion particle size using the particle size analysis method described above.

[0112] Figure 18 The graph shows the particle size variation of Pickering emulsions prepared under different conditions with temperature. Heating the emulsion increases the particle size. This is because high temperature disrupts the structure of WG-DNPs as stabilizers adsorbed at the interface, damaging the protein film at the oil-water interface and leading to increased emulsion droplet size. Overall, the particle size shows an increasing trend, but the change in particle size with temperature decreases with increasing particle concentration. When c = 4% and φ = 40%, under water bath heating conditions of 70℃ and 100℃, the particle size of the emulsion increased by 5.06% and 10.20%, respectively, showing the smallest change in particle size among all experimental groups, indicating the best thermal stability.

[0113] Storage stability

[0114] Storage stability was characterized by the stability index. After emulsion preparation, the volume fraction of the emulsion phase decreased due to gravity. The stability index was tested after 4 hours, and the results are shown in Table 5.

[0115] Table 5

[0116] different Stability index of Pickering emulsions at (20%–50%) and (1%–5%)

[0117]

[0118] As shown in Table 5, the stability index of Pickering emulsions prepared with higher c and φ values ​​is significantly higher than that prepared with lower c and φ values. The reasons for the better storage stability of Pickering emulsions with higher c and φ values ​​are as follows: (1) The higher the c value, the higher the coverage of oil droplets and the larger the interfacial area, thus reducing the probability of oil droplet aggregation and the less likely the emulsion will become unstable. In addition, the G' of the emulsion prepared with high particle concentration increases with increasing concentration, indicating that the droplets are tightly packed, forming a denser network structure with stronger resistance to deformation and better stability. (2) The larger the φ value, the more adsorbable particles at the oil-water interface become, which is conducive to the accumulation of droplets to form a dense three-dimensional network structure and improves stability. In addition, the apparent viscosity of the emulsion increases with increasing φ value, indicating that the gel network structure formed is stronger. When c = 4% and φ = 40%, the stability index is the highest, indicating that its storage stability is the best.

[0119] This invention successfully prepared a Pickering emulsion using WG-DNPs as a stabilizer. The emulsion was characterized by droplet morphology and microstructure, and the WG-DNPs particle concentration (c = 1%–5%) and oil-to-emulsion ratio (φ = 20%–50%) were obtained by analyzing the emulsion’s rheological behavior and stability. Experimental results showed that (1) CLSM results indicated that the prepared emulsion was an O / W type Pickering emulsion. Analysis of droplet morphology and microstructure showed that when c = 4% and φ = 40%, the droplets were evenly distributed, forming a good emulsion particle size of 11.8 μm; rheological behavior analysis showed that all emulsions exhibited shear thinning, and when c = 4% and φ = 40%, G' > G”. With the highest G', the highest viscosity, and the strongest resistance to deformation, the droplet size of 11.8 μm is moderate, and the Pickering emulsion prepared under these conditions is more stable.

[0120] (2) Thermal stability studies showed that the particle size of all emulsions increased after heating. In Example 1, the particle size change with temperature was the smallest when c = 4% and φ = 40%. The particle size increased by 5.06% and 10.20% at 70℃ and 100℃, respectively, showing good thermal stability. Storage stability studies showed that the Pickering emulsion prepared at higher c (c = 4%) and φ (φ = 40%) had the highest stability index of 86.95%, and the Pickering emulsion prepared under these conditions had good storage stability.

[0121] The WG-DNPs-stabilized Pickering emulsion prepared by this invention exhibits good thermal and storage stability, which is conducive to the widespread application of Pickering emulsions. Wheat gluten (WG) can be classified into four types according to its solubility characteristics: albumin, globulin, gliadin, and glutenin, with gliadin and glutenin being the most abundant, accounting for approximately 72%–85%. Wheat gluten is not only rich in nutrients but also possesses excellent emulsifying, gelling, and viscoelastic properties. However, due to its high content of uncharged and hydrophobic amino acids (glutamic acid 29.2%, proline 9.78%), and the fact that gliadin is non-covalently bonded to glutenin via disulfide bonds, its solubility is low, limiting its practical application in food processing. This invention, by glycosylating gluten and then preparing WG-DNPs to stabilize the Pickering emulsion, also broadens the application of gluten in food processing.

[0122] II. Analysis of the results of Example 1 and Comparative Examples 1-5

[0123] Depend on Figure 7 It can be seen that the particle size of WG-DNPs in Example 1 and Comparative Examples 1-5 is at the nanometer level. As the ionic strength increases from 0 mM to 300 mM, the particle size of WG-DNPs decreases to a certain extent, showing a trend of first decreasing and then increasing. This is because salt dissolution easily occurs at low ionic strength, leading to a decrease in the particle size of WG-DNPs. As the ionic strength increases (50-300 mM), the gradually increasing particle size reflects the salting-out effect, with increased interparticle interaction forces leading to the formation of large aggregates. At high ionic strength, the salting-out effect is more pronounced, resulting in a large amount of protein precipitation or aggregation.

[0124] Table 6 shows the changes in potential and surface hydrophobicity of WG-DNPs with different ionic strengths.

[0125] Table 6

[0126] Potential and surface hydrophobicity changes of WG-DNPs with different ionic strengths

[0127]

[0128] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), the same applies below.

[0129] As shown in Table 6, the absolute value of the Zeta-potential initially increases and then decreases with increasing ionic strength. This is because NaCl, upon binding to the protein, shields the electrostatic charge on the protein surface, weakening the electrostatic repulsion and enhancing the electrostatic shielding effect. Consequently, the amount of negative charge on the gluten protein surface decreases, leading to the aggregation of protein particles into larger particles with increased particle size. The unfolding level of a protein / the number of hydrophobic residues on its surface can be characterized by surface hydrophobicity (H0), which can indicate changes in protein conformation. As shown in Table 6, H0 increases with increasing ionic strength, indicating that increased ionic strength causes the protein conformation to loosen, resulting in the exposure of hydrophobic groups on the surface. It is generally believed that increased ionic strength makes the protein conformation more compact, thus causing hydrophobic groups to be encapsulated internally. However, the results of this experiment are exactly the opposite. This is because the -ANS probe is an anionic probe, which binds to cationic groups (such as lysine, histidine, arginine, etc.) on the gluten protein surface in the form of ion pairs. The degree of binding increases as the electrostatic repulsion of the solution system decreases.

[0130] <Surface wettability of glycosylated gluten protein nanoparticles>

[0131] The three-phase contact angle (θ) is used to characterize the wettability of nanoparticles. Wettability is an important factor in determining whether nanoparticles can serve as pickering emulsion stabilizers. Therefore, selecting suitable glycosylated nanoparticles as pickering emulsion stabilizers can be determined by assessing the type and stability of the resulting pickering emulsion through θ. Figure 8 It can be seen that the nanoparticles exhibit the smallest θ value (72.7°) at an ionic strength of 0 mM, demonstrating good hydrophilicity. With increasing ionic strength, the θ value of WG-DNPs gradually increases from 72.7° to 98.5°. When θ approaches 90°, the particles exhibit optimal amphiphilic properties, forming a stable Pickering emulsion at the oil-water interface. At an ionic strength of 20 mM, the contact angle is 89.6° (closest to 90°). Nanoparticles prepared under these conditions demonstrate superior wettability and are ideal materials for preparing oil-in-water (O / W) Pickering emulsions.

[0132] As can be seen from Example 1 and Comparative Examples 1-5, different ionic strengths affect the particle properties of WG-DNPs. When the ionic strength is 20 mM, the particle size of WG-DNPs is 301.73 ± 0.34 nm, the maximum absolute value of the Zeta- potential is -12.63 ± 0.93 mV, and the wettability is θ = 89.6°. The prepared WG-DNPs are Pickering emulsion stabilizer materials.

[0133] This invention also discloses the application of a WG-DNPs-stabilized Pickering emulsion in loading β-carotene. β-carotene is an important precursor for vitamin A synthesis and an essential nutrient for human health. Adequate intake of β-carotene can improve immunity, prevent cardiovascular disease, cataracts, and provide antioxidant benefits. However, the β-carotene molecule has two ionone rings at both ends of its conjugated polyhedral bonds, making it susceptible to degradation, oxidation, photosensitization, and isomerization, thus reducing its biological activity. Furthermore, β-carotene has extremely low water solubility, limiting its application in the food industry. This presents a challenge to the application of β-carotene in the food industry. The WG-DNPs-stabilized Pickering emulsion of this invention can load β-carotene, thereby improving its bioavailability and digestible stability, and facilitating human intake of β-carotene.

[0134] First, 4 mg / mL WG-DNPs were prepared, and the pH was adjusted to 7.0 using 1M HCl or NaOH. Pickering emulsion (containing 0.1% β-carotene in soybean oil) was prepared with an oil-water volume ratio of 4:6, and a Pickering emulsion without β-carotene loading was used as a blank control. The total volume of the emulsion was 10 mL.

[0135] Characterization of Pickering emulsions loaded with β-carotene

[0136] like Figure 9 As shown, the particle size distribution of the Pickering emulsion loaded with β-carotene did not change significantly compared to the unloaded Pickering emulsion, remaining within the range of 10–100 μm, exhibiting a unimodal distribution and perfectly conforming to a normal distribution trend, indicating a good particle size distribution. To observe the microstructural changes of the Pickering emulsion after β-carotene loading, the oil and protein phases were stained, and the distribution and size of oil droplets were observed, as shown in the figure. Figure 10 As shown, after loading β-carotene, the droplets are evenly distributed, and the green WG-DNPs are tightly adsorbed onto the surface of the red oil droplets, indicating that the emulsion is an O / W type Pickering emulsion. A light yellow ring structure is present on the surface of the oil droplets. Comparison with the confocal image above shows that the emulsion structure did not change after loading β-carotene, indicating that β-carotene does not affect the stabilization mechanism of WG-DNPs in the Pickering emulsion. This also demonstrates the reliability of the WG-DNPs prepared in this invention as a Pickering emulsion stabilizer.

[0137] <Rheological behavior of Pickering emulsions loaded with β-carotene>

[0138] like Figure 11 , 12 As shown, the Pickering emulsion loaded with β-carotene still exhibits shear thinning, with the apparent viscosity gradually decreasing and approaching 0 with increasing shear rate. From Figure 12 As can be seen from the steady-state results analysis, the apparent viscosity of the Pickering emulsion loaded with β-carotene is higher than that of the control group, indicating that after loading with β-carotene, the interaction between droplets is enhanced, the emulsion structure becomes more compact, the resistance to deformation is enhanced, and the emulsion is more stable.

[0139] Figure 11 The dynamic oscillation results before and after β-carotene loading show that G' > G”, indicating that the emulsion possesses an elastic gel structure. The frequency dependence of G' and G” within the scanning range indicates the formation of a non-physical covalent network structure in the emulsion gel. After β-carotene loading, G', G”, and the initial values ​​all increase, indicating increased gel strength and a lower likelihood of deformation under external forces. The results demonstrate that the addition of β-carotene leads to a more stable three-dimensional network structure in the emulsion, enhancing its stability.

[0140] <Stability of β-carotene>

[0141] I. Storage Stability

[0142] β-Carotene is chemically unstable and easily degraded by external factors. This invention investigated the degradation of β-carotene in an emulsion stored at room temperature (25°C) for one month.

[0143] Depend on Figure 13 It was found that the encapsulation efficiency of β-carotene decreased over time, and the half-life (degradation rate reaching 50%) was reached after 168 hours. In contrast, soybean oil loaded with β-carotene, as a blank control group, reached its half-life in approximately 72 hours. At this point, the retention rate of β-carotene in soybean oil was 51.32%, which was 19.08% lower than the retention rate of the Pickering emulsion loaded with β-carotene. This may be because β-carotene dissolved in soybean oil is more easily oxidized and degraded. The Pickering emulsion prepared in this invention, with its dense ring-shaped protein layer formed at the oil-water interface, can effectively isolate factors that easily degrade β-carotene (light, oxygen, metal ions, etc.), thus resulting in a higher retention rate of β-carotene. This indicates that the Pickering emulsion prepared in this invention, using WG-DNPs as a stabilizer, has an ideal anti-degradation effect on the oil-soluble active substance β-carotene.

[0144] II. Thermal stability

[0145] Using β-carotene dissolved in soybean oil as a control group, the effect of heating on the retention rate of encapsulated β-carotene was investigated. The results are as follows: Figure 14 , 15 The retention rate of β-carotene decreased with increasing temperature. The retention rates of β-carotene at 70℃ and 100℃ were 85.3% and 61.3%, respectively, while the retention rates of the control group were 71.7% and 37.2%, respectively. This indicates that the Pickering prepared by the present invention, with WG-DNPs as a stabilizer, can effectively isolate external factors that cause oxidative decomposition of β-carotene and has a good protective effect on β-carotene.

[0146] After a short heat treatment, the droplet size of the emulsion did not change significantly. The Pickering emulsion loaded with β-carotene showed a slight increase in droplet size after heating, exhibiting a bimodal distribution, but the overall particle size distribution was uniform. Figure 14 , 15 It can be seen that although the Pickering emulsion loaded with β-carotene shifted to the right after heat treatment compared with the control group, the particle size distribution curve of the emulsion loaded with β-carotene was high and narrow, indicating that the droplet distribution was more uniform and there were more droplets of similar size. It was more stable under heat treatment, indicating that loading with β-carotene helps to improve the thermal stability of the emulsion.

[0147] <Simulated in vitro digestion characteristics>

[0148] Highly hydrophobic active substances typically require dissolution in gastrointestinal fluids before absorption. Studies have shown that during gastric digestion, pepsin, under acidic conditions, causes the breakdown of both the emulsion and the active ingredient. β-carotene can only be dissolved when it enters the mixed micelles containing bile salts, phospholipids, and free fatty acids in gastrointestinal fluids, or binds to nonpolar polypeptides of proteins. β-carotene is beneficial to the human body because it is stable in the gastrointestinal tract and can be absorbed. However, in reality, due to its structural characteristics, the physiological properties and physicochemical structure of β-carotene often change before it even reaches the gastrointestinal tract upon ingestion. The stability of emulsion droplets in the gastric juice environment is an ideal property for delivery systems. Therefore, this study used soybean oil loaded with β-carotene as a control group for Pickering, and analyzed the release kinetics, digestive stability, and bioavailability of β-carotene through simulated digestion experiments.

[0149] Depend on Figure 16It was observed that during gastric digestion, the β-carotene release rate of the control group was consistently higher than that of the Pickering emulsion loaded with β-carotene, indicating that the Pickering emulsion containing β-carotene prepared by this invention, stabilized by WG-DNPs, can reduce the release of active substances under the harsh gastric environment. After entering the intestines for digestion, the release rate of β-carotene from the Pickering emulsion significantly increased and remained higher than that of the control group. This is because bile salts and polypeptides are present in the simulated intestinal fluid, and β-carotene forms complex micelles with them, thereby increasing the release rate in the intestines. Combined with the results of simulated gastrointestinal digestion, the Pickering emulsion containing β-carotene can be well preserved in the gastric environment. The rapid decomposition and release in the intestinal environment is due to the strong decomposition effect of trypsin, which disrupts the interfacial properties of the emulsion, causing β-carotene to leak out. This result is as expected, since most nutrients are usually absorbed in the intestines.

[0150] Bioavailability refers to the ease with which a substance is absorbed by an organism. This study aimed to determine the bioavailability and digestive stability of β-carotene encapsulated in a WG-DNPs-stabilized Pickering emulsion to further evaluate the effectiveness of the delivery system. An in vitro simulated digestion model was used to investigate the effects of using a WG-DNPs-stabilized Pickering emulsion as a β-carotene delivery system on the bioavailability and digestive stability of the active substance. Soybean oil-loaded β-carotene was used as a control group. The results are as follows: Figure 17 As shown, compared with the control group, the bioavailability and digestive stability of β-carotene in Pickering emulsion were improved by 29.18% and 35.67%, respectively. This indicates that the Pickering emulsion of the present invention has a rapid lipolysis rate. The greater the degree of lipolysis of the emulsion, the higher the bioavailability, because more micelles are generated by lipolysis, and more active substances are encapsulated, making them easier to release into the intestine.

[0151] As can be seen from the technical solution of the present invention, the stability of Pickering emulsion with β-carotene-loaded glycosylated gluten protein nanoparticles is enhanced.

[0152] In this invention, the retention rate of β-carotene in the emulsion after one month of storage was 19.08% higher than that of the control sample, and the half-life was extended by 96 hours, indicating that the Pickering emulsion with WG-DNPs as a stabilizer has an ideal anti-degradation effect on the oil-soluble active substance β-carotene. When heated to 70℃ and 100℃, the retention rates of β-carotene were 85.3% and 61.3%, respectively, demonstrating the good thermoprotective effect of the Pickering emulsion on β-carotene. Through in vitro simulated digestion, the bioavailability and digestive stability of β-carotene in the emulsion were increased by 29.18% and 35.67%, respectively. The Pickering emulsion stabilized by WG-DNPs can effectively inhibit the release of β-carotene under simulated gastric juice conditions, exhibiting a strong protective effect on β-carotene.

[0153] The Pickering emulsion stabilized by WG-DNPs of this invention can load β-carotene to improve the bioavailability and digestive stability of β-carotene, thereby helping the human body to absorb β-carotene.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A WG-DNPs-stabilized Pickering emulsion, characterized in that, It is prepared by the following method: S1: Prepare a glycosylated gluten protein solution, adjust its pH to neutral, and then use NaCl to adjust the ionic strength of the solution to 15~25 mM to obtain an intermediate solution. The intermediate solution is subjected to water bath heat treatment, ice bath cooling, and freeze drying in sequence to obtain WG-DNPs. The glycosylated gluten protein is prepared by adding gluten protein and dextran in a mass ratio of 1:1.25~1.35 to a 0.1M phosphate buffer solution with a pH of 12.0, dissolving them completely, and then freeze-drying. The glycosylated gluten protein is then dispersed in distilled water under magnetic stirring and placed at 3~5℃ to obtain a glycosylated gluten protein solution. The water bath heat treatment temperature is 85~95℃. S2: WG-DNPs are dispersed in distilled water to prepare a WG-DNPs dispersion. The WG-DNPs dispersion is then mixed and homogenized with the oil phase to obtain a Pickering emulsion. The particle concentration of the WG-DNPs dispersion is 1-5%, the oil content is 20-50%, the mixing and homogenization speed is 11000-13000 rpm, and the mixing and homogenization time is 2.5-3.5 min.

2. The WG-DNPs-stabilized Pickering emulsion according to claim 1, characterized in that, The concentration of the glycosylated gluten protein solution is 1.5% to 3%.

3. The WG-DNPs-stabilized Pickering emulsion according to claim 1, characterized in that, The particle concentration of the WG-DNPs dispersion was 4%, and the oil content was 40%.

4. A WG-DNPs-stabilized Pickering emulsion according to claim 1, characterized in that, The emulsion is an oil-in-water emulsion with a particle size of 11.8 μm.

5. The use of a WG-DNPs-stabilized Pickering emulsion as described in claim 1 in loading β-carotene.

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