Emulsion gel based on modified WalPI-PA compound as well as preparation method and application of emulsion gel

Abstract

The invention discloses emulsion gel based on a modified WalPI-PA compound as well as a preparation method and application of the emulsion gel, and belongs to the technical field of food processing. The emulsion gel is Pickering emulsion gel which is prepared by taking the modified WalPI-PA compound as a stabilizer and medium-chain triglyceride MCT as an oil phase and has a three-dimensional network structure. Wherein the modified WalPI is obtained by treating a walnut protein isolate WalPI solution through at least one modification means of low-temperature plasma or high-pressure microjet. According to the preparation method disclosed by the invention, the WalPI is subjected to composite physical modification through synergistic cooperation of high-pressure microjet and low-temperature plasma, the preparation process is simple and efficient, the dispersity and the stability of a modified WalPI-PA compound system are effectively ensured, and then the modified WalPI-PA compound is used as a stabilizer to prepare the Pickering emulsion gel; and the influence of the particle concentration and the oil phase volume fraction on the emulsifying capacity and the interface characteristics is systematically explored. Therefore, an important theoretical basis is provided for deeply understanding the formation mechanism of the stable emulsion gel of the protein-polyphenol composite particles and the application of the stable emulsion gel in food.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to emulsion gels based on modified WalPI-PA complexes, their preparation methods, and applications. Background Technology

[0002] Pickering emulsion gels are a class of emulsions with a gel-like network structure and solid mechanical properties, exhibiting significant advantages in food structure design and loading of bioactive ingredients. Proteins have attracted widespread attention for stabilizing emulsion gels due to their unique amphiphilic properties and film-forming abilities. Proteins can form continuous and stable interfacial films, and protein fragments on the film can radiate ring-like or tail-like structures, spatially restricting the aggregation between oil droplets. Simultaneously, unadsorbed proteins in the continuous phase of the emulsion can increase viscosity, preventing oil droplet aggregation due to contact caused by flow. In recent years, plant-derived proteins have attracted considerable interest due to their wide availability, good biocompatibility and degradability, and ability to impart mechanical and sensory properties to emulsion gels.

[0003] Walnut protein isolate (WalPI) has a high gluten content (70.11%), resulting in poor solubility, emulsifying properties, and other functional characteristics, limiting its application in the food industry. Modification of WalPI can improve its solubility, emulsifying properties, water-holding capacity, and oil-holding capacity, which is currently a hot research topic. Enzymatic hydrolysis and glycosylation are commonly used chemical modification methods for proteins. These methods may promote the loosening of the rigid structure of proteins, increasing the efficiency of protein unfolding and rearrangement at the interface, thereby reducing interfacial tension. However, chemical modification methods are complex, time-consuming, and involve multiple chemical reagents and reactions, making it difficult to meet the requirements of industrial production and food safety standards. In comparison, physical modification methods are relatively safer.

[0004] Proanthocyanidins (PA), as natural polyphenols, possess strong antioxidant and anti-inflammatory activities. However, their instability under conditions of high temperature, high oxygen, and high light limits their application in food. Existing reports indicate that combining PA with proteins can improve PA's stability and bioavailability. Furthermore, the abundant phenolic hydroxyl groups in PA molecules can interact with proteins, inducing protein cross-linking and regulating their interfacial behavior. In addition, the combination of polyphenols and proteins can enhance the nutritional value of food and promote health. Walpis polyphenols (PA) are rich in nutrients, containing high levels of essential amino acids, especially arginine, glutamic acid, histidine, and tyrosine. Combining them with PA has positive implications for the development of health foods.

[0005] However, the high gluten content of WalPI leads to significantly different interfacial behavior compared to soybean protein. Therefore, the emulsification mechanism and adsorption behavior of the modified WalPI-PA complex at the oil-water interface remain unclear. Furthermore, existing technologies do not address the optimization of system parameters and the specific mechanisms of the WalPI-PA complex in Pickering emulsion gels. Therefore, to gain a deeper understanding of the formation mechanism of protein-polyphenol composite particle-stabilized emulsion gels and their applications in food processing, this invention proposes an emulsion gel based on a modified WalPI-PA complex, its preparation method, and its applications. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned technical problems by providing an emulsion gel based on a modified WalPI-PA complex, its preparation method, and its application.

[0007] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, an emulsion gel based on a modified WalPI-PA complex is provided, the emulsion gel being a Pickering emulsion gel with a three-dimensional network structure prepared using a modified walnut protein isolate-proanthocyanidin complex MWalPI-PA as a stabilizer and medium-chain triglycerides (MCT) as the oil phase; wherein the modified walnut protein isolate MWalPI is obtained by treating a walnut protein isolate WalPI solution with at least one modification method, namely, low-temperature plasma or high-pressure microfluidic jet.

[0008] As a further optimization of the present invention, the walnut protein isolate WalPI is obtained by alkaline solubilization and acid precipitation.

[0009] As a further optimization of the present invention, the modified walnut protein isolate MWalPI is obtained by first treating the walnut protein isolate WalPI solution with high-pressure microfluidic jet and then treating it with low-temperature plasma.

[0010] As a second aspect of the present invention, the application of the emulsion gel based on the modified WalPI-PA complex as described in any of the above claims in the field of food processing is also provided.

[0011] As a third aspect of the present invention, a method for preparing an emulsion gel based on a modified WalPI-PA complex as described in any one of the foregoing claims is also provided, comprising the following steps: (1) Disperse WalPI isolated protein in phosphate buffer solution to obtain WalPI solution; (2) The WalPI solution obtained in step (1) is treated by at least one of the modification methods, namely low-temperature plasma or high-pressure microfluidic, to obtain a modified walnut protein isolate MWalPI solution. (3) Add proanthocyanidins PA to the modified walnut protein isolate MWalPI solution, stir magnetically, dialyze at 4°C for 24 h, and then freeze dry under vacuum to obtain the modified WalPI-PA complex. (4) The modified WalPI-PA complex obtained in step (3) was used as a stabilizer and mixed with medium chain triglyceride MCT. After high-speed homogenization, a mixture was obtained. The mixture was subjected to ultrasonic cell disruption treatment at 400 W power in a pulse mode with 2 working seconds and 3 intermittent seconds to obtain Pickering emulsion gel.

[0012] As a further optimization of the present invention, in step (1), the mass concentration of WalPI in the WalPI solution is 1%, w / v.

[0013] As a further optimization of the present invention, in step (2), the WalPI solution is first subjected to high-pressure microjets with a microjets pressure of 50 MPa and 5 cycles, and then subjected to low-temperature plasma treatment with a treatment voltage of 80 V and a treatment time of 90 s.

[0014] As a further optimization of the present invention, in step (3), the concentration of proanthocyanidins added to the MWalPI solution is 4 mg / mL.

[0015] As a further optimization of the present invention, in step (4), the mass concentration of the modified WalPI-PA complex in the Pickering emulsion gel is 1-5% w / v, and the volume fraction of the medium-chain triglyceride is 10-70%.

[0016] As a further optimization of the present invention, in step (4), the process parameters of the high-speed homogenization are 11,000 rpm, 5 min, and the ultrasonic cell disruption treatment time is 10 min.

[0017] The working principle of this invention is as follows: High-pressure microfluidics is a processing technology that uses extreme high pressure to drive fluid through micrometer-level channels, achieving nanoscale dispersion and homogenization of materials through the synergistic effect of multiple physical effects. When the fluid moves at high speed within the extremely narrow microchannel, it generates a huge velocity gradient, forming extremely strong interlayer shear forces that directly act on the particle aggregate structure, resulting in highly fragmented liquid particles. During this homogenization process, intense processing conditions such as high-speed liquid impact, high-speed shear, and high-speed oscillation can lead to changes in the macromolecular structure. The secondary structure of proteins is maintained by hydrogen bonds within and between peptide bonds, and high pressure promotes the formation of hydrogen bonds; therefore, high pressure is beneficial to the stability of protein secondary structures. Furthermore, some tertiary globular proteins combine to form quaternary structures, which are maintained by covalent interactions and are very sensitive to high pressure. Therefore, after dynamic high-pressure microfluidic treatment, the structure of proteins will inevitably undergo changes in their physicochemical properties. Cold plasma (CP) is formed by ionizing a neutral gas by applying energy. Energy is stored within active particles in the form of kinetic energy or internal excitation, granting them multiple degrees of freedom. When particle collisions reach thermal equilibrium, the velocity distribution follows the Maxwell-Boltzmann distribution law. However, in non-thermal plasma, different types of particles exhibit the characteristics of multiple superimposed Maxwell-Boltzmann distributions, reflecting that CP is in a non-thermal equilibrium state. In this state, electrons possess significantly higher energy than other heavy particles in the gas (such as ions, neutral molecules, and free radicals). Despite the extremely high electron temperature, the temperature of heavy particles can still be maintained at near-room temperature. This characteristic makes CP a safe and effective technique for processing thermosensitive materials. In protein modification, CP primarily mediates RONS to disrupt the natural protein's closed structure, making it more active and allowing it to fully expand at the interface. High-energy particles generated by CP bombard proteins expose hydrophobic groups buried in hydrophobic centers to the protein surface, firmly fixing the protein at the interface from both the aqueous and oil phases. Simultaneously, the exposure of more residues also means more interactions and a more robust interfacial network.

[0018] The beneficial effects of this invention are as follows: This invention modifies WalPI using different methods and then prepares a WalPI-PA complex with PA. Analysis of particle size, polydispersity index (PDI), and zeta potential shows that high-pressure microfluidic treatment followed by low-temperature plasma treatment of WalPI can ensure the dispersibility and stability of the complex system. In addition, it can ensure the degree of binding between protein and PA, stabilize the protein structure, and the process is simple and efficient. Furthermore, this invention prepared an emulsion gel with a three-dimensional network structure using the MWalPI-PA complex as an emulsifier and MCT as the oil phase, and investigated the effects of different MWalPI-PA complex concentrations and MCT volume fractions on the properties of the emulsion gel. The results showed that at a MWalPI-PA complex concentration of 5% and an MCT volume fraction of 60%, the emulsion gel exhibited the smallest droplet size (560.32 nm) and the largest zeta potential (-46.0 mV), demonstrating the best overall performance. Rheological testing showed that the emulsion gel exhibited the highest G' and G″ values ​​and significant thixotropic behavior. CLSM confirmed that the emulsion gel formed the most uniform and dense microstructure. These findings provide a theoretical basis for constructing stable MWalPI-PA emulsion gels and offer insights into their applications in food processing fields such as dairy products and baked goods, as well as in bioactive delivery systems. Attached Figure Description

[0019] Figure 1 The particle size, polydispersity index (PDI), and zeta potential of MWalPI-PA composites prepared by treating MWalPI with different modification methods are provided by this invention. Figure 2 The PA binding equivalent of MWalPI-PA composites prepared by treating MWalPI with different modification methods is provided by the present invention. Figure 3 The average particle size (A), zeta potential (B), particle size distribution (C), AP (D), interfacial tension (E), and adsorption mechanism of MWalPI-PA at the oil-water interface (F) of Pickering emulsion gels prepared with different concentrations of MWalPI-PA complex provided by this invention are shown in the diagram. Figure 4 The appearance (A), optical microscope image (scale bar: 100 μm) (B) and CLSM image (scale bar: 100 μm) (C) of Pickering emulsion gels prepared with different concentrations of MWalPI-PA complex provided in this invention. Figure 5 Pickering emulsion gels prepared with different concentrations of MWalPI-PA composites provided in this invention: viscosity-shear rate curves (A), changes of G' and G″ as strain functions during the scanning process (B), changes of the tangent of the loss angle (tanδ) with strain scanning (C), and thixotropic properties (D). Figure 6 The average particle size (A), zeta potential (B), particle size distribution (C), and AP rate (D) of Pickering emulsion gels prepared with different MCT volume fractions provided in this invention. Figure 7 The appearance (A), optical microscope image (scale bar: 100 μm) (B), and CLSM image (scale bar: 100 μm) (C) of Pickering emulsion gels prepared with different MCT volume fractions provided for this invention. Figure 8 Pickering emulsion gels prepared with different MCT volume fractions provided for this invention: viscosity-shear rate curves (A), changes of G' and G″ as strain functions during scanning (B), changes of the tangent of the loss angle (tanδ) with strain scanning (C), and thixotropic properties (D). Detailed Implementation

[0020] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] Unless otherwise specified, all reagents and materials used below are commercially available products. Unless otherwise specified, all methods used below are conventional methods known to those skilled in the art.

[0022] 1. Preparation of MWalPI-PA complex (1) Preparation of composite modified walnut protein isolate (MWalPI) Walnut protein isolate (WalPI) was obtained by alkaline solubilization and acid precipitation. The average particle size of WalPI was 187.6 mm, the PDI was 0.61, and the zeta potential was 18.55. First, WalPI (1%, w / v) was dispersed in PBS (0.1 mol / L, pH=7.4) to obtain a WalPI solution. Then, the WalPI solution was treated with the following modification method AD to obtain modified WalPI (denoted as MWalPI).

[0023] Modification method A: Low-temperature plasma treatment, the WalPI solution is placed in CTP-2000K low-temperature plasma for low-temperature plasma treatment, the treatment voltage is 80V, and the treatment time is 90s; Modification method B: High-pressure microjets treatment. The WalPI solution was placed in an FB-110T5 ultra-high pressure microjets homogenizer for high-pressure microjets treatment. The microjets pressure was 50 MPa and the cycle was 5 times.

[0024] Modification method C: First, high-pressure microjets are treated, followed by low-temperature plasma treatment; Modification method D: First, treat with low-temperature plasma, then treat with high-pressure microjets.

[0025] (2) Preparation of MWalPI-PA complex 4 mg / mL proanthocyanidins (PA, purity >98%) were added to MWalPI (1%, w / v) solutions obtained by four different modification methods, respectively. The solutions were magnetically stirred for 2 h (25°C, 500 rpm), dialyzed at 4°C for 24 h, and then freeze-dried under vacuum to obtain four MWalPI-PA complexes.

[0026] In addition, WalPI-PA was prepared from an unmodified WalPI solution using the same preparation method described above.

[0027] 2. Characterization of the MWalPI-PA complex 2.1 Determination of particle size, polydispersity index (PDI), and zeta potential The average droplet size, polydispersity index (PDI), and zeta potential of the MWalPI-PA composite were measured using a NANO-ZS nanoparticle size and zeta potential meter. The equilibration time was set to 120 s and the temperature to 25 °C.

[0028] The results are as follows Figure 1 As shown, compared with WalPI, the average particle size and PDI of MWalPI-PA are significantly increased, indicating that WalPI and PA combine to form large particles, and the addition of PA affects the dispersibility of WalPI in water.

[0029] Compared to the unmodified WalPI-PA complex, the MWalPI obtained through high-pressure microfluidic treatment and low-temperature plasma treatment exhibits a smaller average particle size and PDI, indicating better dispersibility. Furthermore, applying the two methods sequentially to WalPI further enhances dispersibility compared to applying them individually, demonstrating a temporal difference. First, high-pressure microfluidic treatment mechanically unfolds the protein structure, altering protein-protein interactions and creating a more rigid interfacial layer. Then, low-temperature plasma treatment mediates RONS to disrupt the native protein's closed structure, making it more active and allowing for further unfolding at the interface. The high-energy particles generated by the low-temperature plasma bombard the protein, exposing hydrophobic groups buried in the hydrophobic centers to the protein surface, firmly fixing the protein at the interface from both the aqueous and oil phases. Simultaneously, increased residue exposure signifies more interactions and a more robust interfacial network. This improved dispersibility of MWalPI-PA is beneficial for its further application in Pickering emulsion gels.

[0030] 2.2 PA binding equivalent PA standard solutions with concentrations of 0, 0.05, 0.10, 0.15, 0.20, and 0.25 mg / mL were prepared. 2.5 mL of 0.2 mol / L Folin-Phenol reagent was added to each 0.5 mL PA standard solution. After standing in the dark for 5 minutes, 2.0 mL of 7.5% sodium carbonate solution was added, and the mixture was allowed to stand for 1 hour. The absorbance was measured at 765 nm using a multi-functional microplate reader (Hangzhou Ausen Instruments Co., Ltd., Hangzhou, China), and a standard curve was obtained (y = 3.9437x + 0.0454 (R²)). 2 =0.9994). Sample solutions were analyzed using the same method, and the results were applied to a standard curve to determine the PA binding equivalent. The protein content of the MWalPI-PA complex was determined using the BCA method, and the PA binding equivalent was expressed as nmol / mg protein.

[0031] The results are as follows Figure 2 As shown, PA binding equivalent reflects the degree of binding between protein and PA, which affects protein structure, function, and bioavailability. In the preparation of MWalPI-PA complexes, using the same amount of PA, different modification methods of WalPI affected the PA binding equivalent. Compared to the WalPI-PA complex prepared from unmodified WalPI, the MWalPI-PA complex prepared from WalPI treated with high-pressure microfluidic jet and low-temperature plasma showed increased binding equivalent. Furthermore, applying both methods sequentially to WalPI further enhanced the PA binding equivalent compared to applying them individually.

[0032] 3. Preparation of Pickering emulsion gel To further explore the application of the MWalPI-PA complex in food-grade Pickering emulsion gels, a MWalPI-PA complex prepared from MWalPI and PA (obtained by high-pressure microfluidic treatment followed by low-temperature plasma treatment) was selected as a stabilizer and medium-chain triglycerides (MCTs) to prepare the Pickering emulsion gel. First, the two compounds were mixed and homogenized at 11,000 rpm for 5 min. Subsequently, the sample was treated with ultrasonic cell disruption at 400 W in pulse mode (2 s on, 3 s off) for 10 min to obtain the Pickering emulsion gel.

[0033] 3.1 Optimization of MWalPI-PA Composite Particle Concentration With a fixed oil volume ratio (MCT = 0.6, v / v), the MWalPI-PA complex particle size was set at 1%, 2%, 3%, 4%, and 5%, w / v, to investigate the effect of MWalPI-PA complex particle size on the gel properties of Pickering emulsion.

[0034] (3.1.1) Particle size and zeta potential The average droplet size and zeta potential of the emulsion gel were measured using a nanoparticle size analyzer. The equilibration time was set to 120 s, and the temperature to 25 °C. The results are as follows: Figure 3 The AC diagram is shown.

[0035] Figure 3 A shows the average droplet size of emulsion gels prepared using different concentrations of the MWalPI-PA complex at a fixed oil volume ratio (MCT = 0.6, v / v). The average droplet size first decreased and then increased with increasing MWalPI-PA complex concentration. The emulsion had the smallest average droplet size (560.32 nm) at a MWalPI-PA complex concentration of 5%, significantly lower than other concentrations (P < 0.05). This is likely due to the extremely high stability of the MWalPI-PA complex, which allows it to rapidly cover the newly formed oil droplet interface, effectively preventing droplet aggregation during homogenization. Figure 3 A).

[0036] like Figure 3 As shown in Figure B, the zeta potentials of emulsion gels prepared with different concentrations of the MWalPI-PA complex exhibit a strong negative charge. The zeta potential of the emulsion gel reaches its maximum (-46.0) when the MWalPI-PA concentration is 5%. This is mainly attributed to the ability of more particles to closely pack together to form a dense interfacial layer, resulting in strong stability of the droplets through electrostatic interactions and the mechanical stability provided by the particles.

[0037] Figure 3 C shows the droplet size distribution of emulsion gels prepared with different concentrations of the MWalPI-PA complex. At a 5% MWalPI-PA complex concentration, the droplet size peak is centered in the 440-530 nm range, exhibiting a single-peak distribution. This may be because a given MCT volume fraction provides sufficient particle coverage, thereby promoting the formation of a more stable system.

[0038] (3.1.2) Determination of protein adsorption rate (AP%) The protein concentration in the emulsion gel was determined using the Coomassie Brilliant Blue method, and the protein adsorption rate was calculated using the following formula.

[0039] ; In the formula, C0 is the original concentration of the protein solution (before emulsification), C f The concentration of unadsorbed protein in the filtrate after filtration is expressed in mg / mL.

[0040] The results are as follows Figure 3As shown in Figure D, the AP of the emulsion gel first decreases and then increases with increasing MWalPI-PA concentration. When the MWalPI-PA concentration is 5%, the AP value of the emulsion gel decreases to 89.25%. This is attributed to the fact that at this particle concentration, protein molecules can fully unfold and rearrange at the interface, thereby forming a denser and more uniform three-dimensional interfacial membrane network.

[0041] (3.1.3) Measurement of interfacial surface pressure Aqueous phase was slowly injected into oil phase using a microinjection system to form droplets, and droplet profile images were acquired. The Young-Laplace equation was used to analyze the droplet shape.

[0042] The results are as follows Figure 3 The EF plot shows the change in γ at the oil-water interface with adsorption time for MWalPI-PA at different concentrations. γ is lowest at a MWalPI-PA complex concentration of 5%. This indicates that minimizing γ promotes molecular rearrangement at the interface and facilitates the formation of a more stable emulsion gel.

[0043] Figure 3 F shows a schematic diagram illustrating the adsorption behavior of the MWalPI-PA complex at the oil-water interface as a function of concentration. When the concentration of the MWalPI-PA complex reaches 5%, the MWalPI-PA complex exhibits maximum conformational unfolding and forms a tightly ordered arrangement at the interface.

[0044] (3.1.4) Observation using optical and fluorescence microscopes The droplet morphology of the emulsion gel was observed under a 10x magnifying eyepiece using an optical microscope.

[0045] The oil phase and protein phase were labeled with Nile Red and Nile Blue, respectively. The excitation wavelengths were set to 488 nm and 633 nm, and the image magnification was 100x.

[0046] The results are as follows Figure 4 As shown, Figure 4 A shows the appearance of emulsion gels prepared with different concentrations of the MWalPI-PA complex. When the concentration of the MWalPI-PA complex was 1% and 2%, the emulsion gels were fluid and could not withstand the inversion test. This may be because the particle concentration was too low to form a gel structure with the oil phase. After being placed at 25°C for 30 days, the emulsion gels at concentrations of 1%, 2%, and 3% of the MWalPI-PA complex exhibited stratification.

[0047] like Figure 4As shown in Figure B, a negative correlation was observed between droplet size and protein concentration. With increasing MWalPI-PA complex concentration, the emulsion droplets became more uniform, and the droplet size decreased. At a 5% MWalPI-PA complex concentration, the emulsion gel exhibited the most uniform droplet distribution. This is mainly attributed to the formation of a uniform, dense particle network at the oil-water interface, which hindered droplet-droplet contact and aggregation.

[0048] Figure 4 C shows CLSM images of emulsion gels with different concentrations of the MWalPI-PA complex, where green represents the oil phase and red represents the aqueous phase. With increasing MWalPI-PA complex concentration, the droplet size decreases and the distribution becomes more uniform. At a 5% MWalPI-PA complex concentration, the emulsion gel exhibits a uniform droplet distribution, with a tightly packed, uniformly arranged interfacial layer surrounding the oil droplets.

[0049] (3.1.5) Rheological properties Using a parallel plate with a diameter of 40 mm, the measurement gap and temperature were set to 1 mm and 25 ℃, respectively. The apparent viscosity, storage modulus (G′), loss modulus (G″), and thixotropic properties of the emulsion gel were determined by rheometer.

[0050] Figure 5 A shows the viscosity-shear rate curves of emulsion gels prepared with different concentrations of MWalPI-PA composites. With increasing shear rate, the viscosity of all emulsion gels decreases, exhibiting typical shear-thinning behavior and classifying them as non-Newtonian fluids. The viscosity reaches its maximum at a concentration of 5% MWalPI-PA, mainly due to the smaller particle size of WalPI after modification by low-temperature plasma combined with high-pressure microjets, resulting in more thorough bonding with PA. Therefore, the obtained MWalPI-PA composite exhibits denser and stronger adsorption at the interface. At this concentration, the emulsion gel forms a dense network, leading to reduced droplet flowability.

[0051] The effects of different concentrations of the MWalPI-PA complex on the emulsion gel G′ and G″ are as follows: Figure 5 As shown in Figure B, within the scanning frequency range of 0.1-100 Hz, when the concentration of MWalPI-PA is 2-6%, the G′ of the emulsion gel is greater than G″, and no yield point is observed (G′=G″), indicating that the system has high viscoelasticity. G′ and G′′ are higher at 4% and 5% than at other concentrations, possibly due to smaller droplet size and more compact droplet packing.

[0052] Figure 5C is the tanδ curve of emulsion gel loss angle for different concentrations of MWalPI-PA complex. Tanδ = G'' / G′ represents the ratio of viscous response to elastic response; a smaller value indicates a stronger elastic response. At a 1% MWalPI-PA complex concentration, tanδ > 1, indicating the presence of a weak gel network where the viscous response dominates at higher frequencies. At 2-6% MWalPI-PA complex concentrations, tanδ < 1, consistent with smaller droplet sizes and a denser particle network, thus conferring elastic advantage.

[0053] Figure 5 D shows the thixotropic properties of emulsion gels prepared with different concentrations of the MWalPI-PA complex. In the low-shear phase (0–60 s), the viscosity values ​​of all gels remained essentially constant. In the high-shear phase (60–120 s), the viscosity value of the emulsion gel containing 1% MWalPI-PA complex decreased rapidly, indicating that the gel network was subjected to strong shear stretching. When the MWalPI-PA content was 2–6%, the viscosity value of the emulsion gel decreased slowly, exhibiting shear-thinning behavior. When returning to the low-shear phase (120–180 s), the viscosity values ​​of all emulsion gels partially recovered, exhibiting typical thixotropic behavior.

[0054] 3.2 Effect of MCT component on Pickering emulsion gel With the MWalPI-PA complex particles fixed at 5% (w / v), different volume fractions of MCT (30%, 40%, 50%, 60%, and 70%, v / v) were set to investigate the effect of MCT on the properties of Pickering emulsion gels. The resulting Pickering emulsion gels were subjected to particle size and zeta potential, protein adsorption rate (AP%), optical microscopy, fluorescence microscopy observation, and rheological property determination, as described above.

[0055] (3.2.1) Particle size and zeta potential The results are as follows Figure 6 As shown in the AC diagram. Figure 6 A shows the emulsion gels prepared by varying the volume fraction of MCT while maintaining a constant MWalPI-PA complex concentration of 5% (w / v). At a 60% MCT volume fraction, the emulsion gel exhibited the smallest average droplet size, a significant difference (P<0.05). This is because sufficient oil phase and emulsifier bind well, forming a denser, mechanically stronger interfacial film that prevents droplet aggregation and coalescence.

[0056] like Figure 6As shown in Figure B, the zeta potential initially increases and then decreases with increasing MCT volume fraction. The zeta potential reaches its peak at an MCT volume fraction of 60%, indicating that an appropriate MCT volume fraction can enhance the electrostatic stability of the emulsion gel. This may be attributed to stronger electrostatic repulsion between droplets and the permeated particle network, which enhances the stability of the emulsion gel.

[0057] Figure 6 C shows the effect of different MCT volume fractions on the droplet size distribution of the emulsion gel. With increasing MCT volume fraction, the droplet size distribution peak first shifts to the left and then to the right. When the MCT content is between 30% and 60%, the droplet distribution exhibits a single peak with a narrow peak width, gradually shifting to the left. This is consistent with the stronger Brownian motion exhibited by smaller droplets, which inhibits droplet aggregation.

[0058] (3.2.2) Protein adsorption rate The results are as follows Figure 6 As shown in Figure D, protein adsorption (AP) initially decreases and then increases with increasing MCT volume fraction. The lowest AP is observed at an MCT volume fraction of 60%. This reflects optimized interfacial coverage and a dense interfacial layer that restricts multilayer adsorption. However, increasing the MCT volume fraction may lead to steric hindrance in the adsorption layer, thus preventing protein adsorption at the interface.

[0059] (3.2.3) Observation using optical and fluorescence microscopes The surface appearance of emulsion gels at different MCT volume fractions is as follows: Figure 7 As shown in Figure A. Except for the emulsion gel with an MCT volume fraction of 60%, all others were liquids and failed the inversion test. Furthermore, after storage at 25 °C for 30 days, all samples exhibited phase separation.

[0060] like Figure 7 As shown in Figure B, large droplets aggregated at MCT volume fractions of 20% and 30%, consistent with the droplet size study. At an MCT volume fraction of 60%, the droplets were smaller and more uniformly distributed, and no aggregation occurred. This is mainly due to the excellent coverage of MWalPI-PA on the droplet surface, effectively preventing aggregation and deformation.

[0061] CLSM images of emulsion gels with different MCT volume fractions are shown below. Figure 7 As shown in Figure C, the droplet size increases slightly with increasing MCT volume fraction. At MCT volume fractions of 50% and 60%, the droplet size increases further, possibly due to the higher bulk viscosity reducing the fragmentation efficiency during homogenization.

[0062] (3.2.4) Rheological properties Figure 8Figure A shows the viscosity-shear rate curves of the emulsion gels at different MCT volume fractions. With increasing shear rate, the viscosity of all emulsion gels decreases, indicating shear-thinning behavior. Viscosity initially increases and then decreases with increasing MCT volume fraction. The maximum viscosity was observed at an MCT volume fraction of 60%. This is likely due to the near-close packing of oil droplets, which inhibits droplet flow and leads to the peak viscosity.

[0063] like Figure 8 As shown in Figure B, within the scanning range, at MCT volume fractions of 20%, 30%, 50%, 60%, and 70%, G' > G″, indicating a predominantly elastic gel-like behavior. At an MCT volume fraction of 60%, G′ and G″ are highest in the low-frequency scanning range. The MWalPI-PA complex, with sufficient oil phase, likely forms a tight three-dimensional network, restricting droplet movement and collisions, thus enhancing its stability.

[0064] like Figure 8 As shown in Figure C, at low scan frequencies, the tanδ of MWalPI-PA emulsion gels with MCT fractions of 20-60% is <1, and G' > G″, mainly exhibiting elastic behavior. With increasing scan frequency, the tanδ of MWalPI-PA emulsion gels with MCT volume fractions of 20%, 30%, and 70% decreases sharply. This may be because at high scan frequencies, the applied oscillatory deformation is faster, leading to a rapid increase in G' and a sharp decrease in tanδ.

[0065] like Figure 8 As shown in Figure D, all samples exhibited thixotropic behavior in the 3ITT test. At lower shear rates (0.1 s⁻¹), [they showed thixotropic behavior]. −1 The viscosity of the emulsion gel did not change significantly with increasing shear time. At high shear rates (10 s⁻¹), the viscosity remained relatively stable. −1 The viscosity values ​​of emulsion gels with MCT volume fractions of 20%, 30%, and 70% showed a significant decrease. When the shear rate returned to (0.1 s⁻¹), the viscosity decreased. −1 When the viscosity of all emulsion gels is partially restored, it reflects the reconstruction of the thixotropic structure.

[0066] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An emulsion gel based on a modified WalPI-PA complex, characterized in that: The emulsion gel is a Pickering emulsion gel with a three-dimensional network structure prepared using modified walnut protein isolate-proanthocyanidin complex MWalPI-PA as a stabilizer and medium-chain triglyceride MCT as the oil phase; wherein, the modified walnut protein isolate MWalPI is obtained by treating a walnut protein isolate WalPI solution with at least one modification method in low-temperature plasma or high-pressure microfluidic.

2. The emulsion gel based on the modified WalPI-PA complex according to claim 1, characterized in that, WalPI, the walnut protein isolate, was obtained using an alkaline solubilization and acid precipitation method.

3. The emulsion gel based on the modified WalPI-PA complex according to claim 1, characterized in that the modified walnut protein isolate MWalPI is obtained by first treating the walnut protein isolate WalPI solution with high-pressure microfluidic jet and then treating it with low-temperature plasma.

4. The application of an emulsion gel based on a modified WalPI-PA complex as described in any one of claims 1-3 in the field of food processing.

5. A method for preparing an emulsion gel based on a modified WalPI-PA complex as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Disperse WalPI isolated protein in phosphate buffer solution to obtain WalPI solution; (2) The WalPI solution obtained in step (1) is treated by at least one of the modification methods, namely low-temperature plasma or high-pressure microfluidic, to obtain a modified walnut protein isolate MWalPI solution. (3) Add proanthocyanidins PA to the modified walnut protein isolate MWalPI solution, stir magnetically, dialyze at 4 °C for 24 h, and then freeze dry under vacuum to obtain the modified WalPI-PA complex. (4) The modified WalPI-PA complex obtained in step (3) was used as a stabilizer and mixed with medium chain triglyceride MCT. After high-speed homogenization, a mixture was obtained. The mixture was subjected to ultrasonic cell disruption treatment at 400 W power in a pulse mode with 2 working seconds and 3 intermittent seconds to obtain Pickering emulsion gel.

6. The preparation method and application of the emulsion gel based on the modified WalPI-PA complex according to claim 5, characterized in that, In step (1), the WalPI solution has a WalPI mass concentration of 1%, w / v.

7. The emulsion gel based on the modified WalPI-PA complex according to claim 5, its preparation method, and its application, characterized in that, In step (2), the WalPI solution is first subjected to high-pressure microjets with a microjets pressure of 50 MPa and 5 cycles, and then subjected to low-temperature plasma treatment with a treatment voltage of 80 V and a treatment time of 90 s.

8. The method for preparing the emulsion gel based on the modified WalPI-PA complex according to claim 5, characterized in that, In step (3), the concentration of proanthocyanidins added to the MWalPI solution is 4 mg / mL.

9. The method for preparing the emulsion gel based on the modified WalPI-PA complex according to claim 5, characterized in that, In step (4), the modified WalPI-PA complex in the Pickering emulsion gel has a mass concentration of 1-5% w / v and the medium-chain triglyceride has a volume fraction of 10-70%.

10. The method for preparing the emulsion gel based on the modified WalPI-PA complex according to claim 5, characterized in that, In step (4), the process parameters for high-speed homogenization are 11,000 rpm, 5 min, and 10 min for ultrasonic cell disruption.

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