Temperature-controlled antibacterial pickering emulsion, fresh-keeping gel pad and preparation and application thereof

Abstract

The application discloses a temperature-controlled antibacterial Pickering emulsion, a fresh-keeping gel pad and preparation and application thereof, the emulsion is an oil-in-water Pickering emulsion, which is prepared from a water phase, an oil phase and interfacial stable particles; the oil phase is composed of oregano essential oil and tetradecane, and the interfacial stable particles are whey protein-coffee acid-sodium alginate ternary non-covalent compounds, wherein coffee acid is combined on the surface of whey protein molecules to form a binary compound in a hydrogen bond-based non-covalent action. The application realizes conformational stable locking of protein-polyphenol compounds, realizes synergism of a double stable mechanism of the Pickering emulsion, realizes synergistic integration of antibacterial activity and phase change cold storage function, realizes effective temperature maintenance when a cold chain is interrupted, realizes significant prolongation of a shelf life of fresh shrimps, and has good biocompatibility and safety.

Description

Technical Field

[0001] This invention relates to the field of food preservation and nanoemulsion technology, specifically to a temperature-controlled antibacterial Pickering emulsion, a food preservation gel pad, and their preparation and application. Background Technology

[0002] Microbial contamination and oxidation are two key factors leading to food quality deterioration. Given the increasingly severe global food safety situation, developing efficient and safe natural food preservation technologies is of significant practical importance. Oregano essential oil (OEO), rich in carvacrol and thymol, exhibits broad-spectrum antibacterial and antioxidant activity, making it a highly promising natural preservative. However, OEO's high volatility, pungent odor, and low water solubility severely limit its practical application, necessitating the development of effective encapsulation and delivery systems. Pickering emulsions, stable emulsion systems composed of solid particles irreversibly adsorbed at the oil-water interface, have attracted widespread attention in the field of active substance encapsulation due to their excellent aggregation stability and controllable release characteristics.

[0003] Proteins are common nutritional biomolecules in food, and due to their controllable and flexible properties, they have been widely used to stabilize Pickering emulsions. However, single proteins are not ideal emulsifiers for stabilizing Pickering emulsions and require modification. Currently, non-covalent cross-linking modification based on hydrogen bonds, hydrophobic interactions, and van der Waals forces is favored due to its advantages such as mild conditions and reversibility. Compared with common polysaccharide-protein and protein-protein particles, polyphenol-protein particles stabilize emulsions with better performance. These composite particles regulate the interfacial behavior and functional properties of proteins through polyphenol modification, thereby reducing the risk of Ostwald ripening and demulsification. Whey protein (WP) is widely used in the food industry due to its excellent functional properties; however, WP's excessive hydrophilicity makes it difficult to form a stable interfacial film at the oil-water interface, limiting its application potential as a Pickering emulsion stabilizing particle. Caffeic acid (CA) is a phenolic acid compound widely found in plants, possessing extremely strong hydrophobicity, and its polyphenolic structure readily binds non-covalently to proteins. Non-covalent modification of WP using CA is expected to improve the interfacial properties of WP.

[0004] However, existing protein-polyphenol binary complex-stabilized Pickering emulsions still face numerous challenges. On one hand, in non-covalent protein-polyphenol complexes, polyphenol molecules exhibit insufficient binding stability at low-affinity sites, making them prone to dissociation at the oil-water interface, leading to complex structural damage and reduced interfacial stability. On the other hand, when Pickering emulsions are used directly for food preservation, they face issues such as affecting food sensory qualities and causing excessively rapid release of active ingredients. Furthermore, interruptions in traditional cold chain transportation (such as handling or power outages) significantly accelerate microbial growth and quality deterioration due to increased food temperature, weakening the preservation effect of Pickering emulsions. Therefore, stabilizing the conformation of protein-polyphenol complexes at the molecular level, achieving solidified slow-release delivery of active ingredients, and maintaining a low-temperature environment during cold chain interruptions are key technical challenges restricting the widespread application of protein-based Pickering emulsions in food preservation. Summary of the Invention

[0005] The purpose of this invention is to provide a temperature-controlled antibacterial Pickering emulsion, a food-preserving gel pad, and their preparation and application, so as to solve the problems in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a temperature-controlled antibacterial Pickering emulsion, a food-preserving gel pad, and their preparation and application, wherein the emulsion is an oil-in-water Pickering emulsion, prepared from an aqueous phase, an oil phase, and interface-stabilizing particles;

[0007] The oil phase is composed of oregano essential oil and tetradecane. The interface-stabilizing particles are whey protein-caffeic acid-sodium alginate ternary non-covalent complexes. Caffeic acid binds to the surface of whey protein molecules through non-covalent interactions, mainly hydrogen bonds, to form a binary complex. Sodium alginate binds to the surface of the whey protein-caffeic acid binary complex through electrostatic interactions and hydrogen bonds, thus constructing the ternary non-covalent complex.

[0008] As a preferred embodiment of the present invention, the whey protein-caffeic acid-sodium alginate ternary non-covalent complex has the following physicochemical parameters: volume average particle size of 200-350 nm, zeta potential of -20--5 mV, and oil-water three-phase contact angle of 70-100°.

[0009] As a preferred embodiment of the present invention, in the whey protein-caffeic acid-sodium alginate ternary non-covalent complex, the mass ratio of caffeic acid to whey protein is 1:(5-50), and the mass ratio of sodium alginate to whey protein-caffeic acid binary complex is 1:(0.5-2.5).

[0010] As a preferred embodiment of the present invention, the volume ratio of oregano oil to tetradecane in the oil phase is 1:(1-4).

[0011] As a preferred embodiment of the present invention, the volume fraction of the oil phase in the emulsion is 20-40 vol%, and the mass fraction of the interface stabilizing particles in the aqueous phase of the emulsion is 0.6-1.5 wt%.

[0012] As a preferred embodiment of the present invention, the emulsion possesses at least one of the following performance characteristics:

[0013] (1) Low temperature storage stability: When stored at a constant temperature and sealed environment of 25℃ for 3 to 10 days, the emulsion showed no macroscopic phase separation, and the average particle size of the emulsion droplets increased by no more than 50% of the initial average particle size;

[0014] (2) Centrifugal stability: After centrifugation at 5000 rpm for 20 min, the emulsion showed no demulsification or obvious stratification.

[0015] (3) Rheological properties: in the range of 0.1 to 100 s -1 It exhibits shear-thinning properties within a certain shear rate range, and the initial apparent viscosity of the emulsion is 1000–2300 mPa·s.

[0016] As a preferred embodiment of the present invention, the method for preparing the multifunctional Pickering emulsion is characterized by comprising the following steps:

[0017] S1: Disperse whey protein in deionized water, adjust the pH of the system to 6.5-7.5, and stir at a constant temperature until completely dissolved to obtain a homogeneous whey protein aqueous solution;

[0018] S2: Caffeic acid is completely dissolved in anhydrous ethanol. The caffeic acid ethanol solution is slowly added dropwise to the whey protein aqueous solution. The reaction is carried out at room temperature with stirring for 2-6 hours. After the reaction is completed, the free caffeic acid in the system is removed by dialysis. After freeze-drying, a solid powder whey protein-caffeic acid binary complex is obtained.

[0019] S3: Disperse sodium alginate in deionized water, stir at a constant temperature of 40-60℃ until it is completely swollen and dissolved, and cool to room temperature to obtain a uniform sodium alginate aqueous solution.

[0020] S4: Add the whey protein-caffeic acid binary complex to the sodium alginate aqueous solution, stir at room temperature for 1-3 hours, and after full compounding, obtain an aqueous system containing a whey protein-caffeic acid-sodium alginate ternary complex.

[0021] S5: Mix oregano essential oil and tetradecane in a preset ratio and stir until homogeneous to obtain a clear and uniform oil phase;

[0022] S6: The oil phase is added to the aqueous phase system and homogenized by high-speed shearing to prepare a stable multifunctional Pickering emulsion.

[0023] In a preferred embodiment of the present invention, in step S2, the added caffeic acid is 2-8 wt% of the whey protein, the stirring reaction time is preferably 4 h, and the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, with a dialysis time of 48 h at room temperature.

[0024] As a preferred embodiment of the present invention, in step S4, based on the total mass of the aqueous system, the amount of whey protein-caffeic acid binary complex added is 0.6-1.5 wt%, and the amount of sodium alginate added is 0.5-1.0 wt%.

[0025] In a preferred embodiment of the present invention, in step S6, the volume ratio of the oil phase to the water phase is (2-4):(6-8), the rotation speed of the high-speed shear homogenization is 10,000-20,000 rpm, and the shear homogenization time is 3-10 min.

[0026] As a preferred embodiment of the present invention, the multifunctional Pickering emulsion is prepared by combining it with a gel matrix, wherein the gel matrix is ​​at least one of gelatin, sodium alginate, carrageenan, and gellan gum.

[0027] As a preferred embodiment of the present invention, based on the total mass of the preservation gel pad, the mass fraction of the multifunctional Pickering emulsion is 20-60 wt%, and the mass fraction of the gel matrix is ​​5-15 wt%.

[0028] As a preferred embodiment of the present invention, the preservation gel pad also contains thermochromic powder, which can undergo reversible color changes within a temperature range of 20 to 30°C.

[0029] As a preferred embodiment of the present invention, the phase transition enthalpy of the preservation gel pad is 100-200 J / g. After being frozen at -20°C for 30 minutes, it can maintain its surface temperature below 10°C for at least 20 minutes when placed in a room temperature environment at 25°C.

[0030] As a preferred embodiment of the present invention, the application of the multifunctional Pickering emulsion or the preservation gel pad according to any one of claims 11 to 14 in the field of fresh food preservation.

[0031] As a preferred embodiment of the present invention, the fresh food is shrimp, fish, shellfish, poultry meat, or fruits and vegetables. The application method is to apply the multifunctional Pickering emulsion to the surface of the fresh food by spraying, dipping, or brushing, or to package and preserve the fresh food with the preservation gel pad.

[0032] As a preferred embodiment of the present invention, under refrigerated storage conditions at 4°C, the shelf life of fresh food can be extended by at least 12 hours, and / or exposing refrigerated food to room temperature at 25°C can reduce the surface temperature of fresh food by 2-8°C compared to the untreated control group, and the cooling effect can be maintained for 10-30 minutes.

[0033] As a preferred embodiment of the present invention, the application enables fresh food to meet at least one of the following indicators during storage:

[0034] (1) The total bacterial count (TVC) of fresh food was reduced by ≥1.01g CFU / g compared with the untreated control group;

[0035] (2) The rate of increase in volatile basic nitrogen (TVB-N) in fresh food was ≥30% lower than that in the untreated control group;

[0036] (3) The time for the pH value of the fresh food system to reach the spoilage threshold of 7.6 was delayed by ≥12 hours compared with the untreated control group.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] 1. This invention achieves conformational stability locking of protein-polyphenol complexes: by introducing SA, a WP-CA-SA ternary non-covalent complex is formed. Molecular dynamics simulations confirm that SA can effectively inhibit the detachment of CA from WP at the oil-water interface, maintaining the number of hydrogen bonds between CA and WP at 10-15. The RMSD value of the WP-CA-SA complex is significantly lower than that of the WP-CA binary complex, and the conformational stability of the protein is improved by more than 60%. This solves the technical problem of easy dissociation of protein-polyphenol non-covalent complexes at the interface at the molecular level.

[0039] 2. This invention achieves a synergistic dual stabilization mechanism for Pickering emulsions: the WP-CA-SA ternary complex forms a dense interfacial shell on the oil droplet surface (confirmed by Cryo-SEM), while simultaneously forming a three-dimensional micelle network structure in the continuous phase (confirmed by rheology). These two mechanisms work synergistically to achieve dual stability of the emulsion. The emulsion does not undergo phase separation after 21 days of storage at 4°C and does not break down after centrifugation at 5000 rpm for 20 minutes. This represents a more than two-fold improvement in stability compared to emulsions stabilized by the WP-CA binary complex, effectively solving the problem of insufficient interfacial stability in traditional protein-based Pickering emulsions.

[0040] 3. This invention achieves a synergistic integration of antibacterial activity and phase change cold storage function: by co-encapsulating OEO and tetradecane, the emulsion simultaneously possesses broad-spectrum antibacterial activity (effective against Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes), antioxidant activity (significant DPPH and ABTS free radical scavenging rates), and phase change cold storage characteristics (melting enthalpy reaches 167.37 J / g, phase change temperature is approximately 5°C). This multifunctional integration allows a single product to simultaneously meet the dual requirements of antibacterial preservation and temperature control, avoiding the complex operations and compatibility issues associated with separately adding antibacterial agents and phase change materials.

[0041] 4. This invention achieves effective temperature maintenance during cold chain interruptions: After being frozen at -20℃ for 30 minutes and then placed in a 25℃ room temperature environment, the preservation gel pad can maintain a surface temperature below 10℃ for at least 20 minutes. Infrared thermal imaging shows that the temperature difference is significantly better than the control group without tetradecane. When applied to the preservation of fresh shrimp, it effectively delays the deterioration of shrimp quality under cold chain interruption conditions, providing important temperature buffering capacity for cold chain logistics.

[0042] 5. Significantly extended shelf life of fresh shrimp: Applying the preservation gel pad to fresh shrimp stored at 4℃ effectively slowed the rise in pH, TVB-N, and TVC, extending the shelf life of shrimp by at least 12 hours. LF-NMR and MRI analyses confirmed that the gel pad maintained a humid environment within the packaging through high water content, reducing moisture loss from the shrimp. The overall preservation effect was significantly better than the untreated control group and the single OEO emulsion treatment group, demonstrating broad application prospects in the cold chain preservation of fresh food.

[0043] 6. Excellent biocompatibility and safety: Cytotoxicity experiments showed that the cell survival rate of the emulsion was higher than 85% at effective concentrations (0–20 μg / mL), and there was no cell growth inhibition within the concentration range of 0–4 μg / mL, demonstrating excellent biocompatibility. All raw materials are food-grade or pharmaceutical-grade, meeting food safety requirements and suitable for widespread application in the food preservation field. Attached Figure Description

[0044] Figure 1 This is the ATR-FTIR spectrum of the WP-CA-SA ternary composite of the present invention.

[0045] Figure 2 This is a SEM / EDS image of the WP-CA-SA ternary composite of the present invention.

[0046] Figure 3 The results show the particle size distribution, ζ-potential, and three-phase contact angle characterization of the WP-CA-SA ternary composite of this invention.

[0047] Figure 4The WP-CA-SA interaction mechanism revealed by molecular docking and molecular dynamics simulations in this invention is as follows: (A) Molecular docking results; (B) Molecular dynamic trajectory; (C) Radius of gyration (Rg); (D) Root mean square deviation (RMSD); (E) Root mean square fluctuation (RMSF); (F) Number of ligand-protein hydrogen bonds; (G) 2D diagram of Gibbs free energy; (H) 3D diagram of Gibbs free energy.

[0048] Figure 5 The images show the macroscopic morphology, optical microscope images, droplet diameter distribution diagrams, and CLSM images of the multifunctional Pickering emulsion (MFPE) of this invention.

[0049] Figure 6 This is an MRI image and water relaxation diagram of the multifunctional Pickering emulsion of the present invention.

[0050] Figure 7 The rheological analysis results of the multifunctional Pickering emulsion of this invention are as follows: (A) shear strain; (B) apparent viscosity.

[0051] Figure 8 The results show the antioxidant activity, melt curve, and cytotoxicity analysis of the multifunctional Pickering emulsion of this invention.

[0052] Figure 9 The emulsion stabilization mechanism revealed by Cryo-SEM and molecular dynamics simulations in this invention: (A) Cryo-SEM results; (B) EDS results; (C) Molecular dynamics simulation of WP-CA-SA at the oil-water interface.

[0053] Figure 10 The results show the storage stability and centrifugal stability of the multifunctional Pickering emulsion of this invention.

[0054] Figure 11 The results show the antibacterial activity of the multifunctional Pickering emulsion of this invention.

[0055] Figure 12 This is an infrared thermal image of the temperature control performance of the food preservation gel pad (MFPE-PM) of the present invention.

[0056] Figure 13 The preservation effect of the preservation gel pad of the present invention on fresh shrimp within 72 hours is as follows: (A) appearance; (B) MRI imaging; (C) moisture relaxation of the control group; (D) moisture relaxation of the MFPE-PM group; (E) total bacterial count (TVC); (F) pH value; (G) volatile basic nitrogen (TVB-N). Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0058] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of this invention, "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0059] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or a connection through an intermediate medium; and they can refer to the internal communication between two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances. "Multiple" means two or more, and unless otherwise explicitly limited, all such meanings fall within the scope of protection of this invention.

[0060] Example 1

[0061] This embodiment discloses a method for preparing a multifunctional Pickering emulsion and a food-preserving gel pad with dual functions of temperature control and antibacterial properties. This is the optimal median process implementation scheme of the present invention, and the specific preparation steps are as follows:

[0062] (1) Preparation of WP-CA-SA ternary composite stabilizer

[0063] S1. Preparation of whey protein solution: Take 5g of whey protein (WP) and completely dissolve it in 100mL of deionized water. Adjust the pH of the system to 7.0 and stir continuously overnight at room temperature to ensure that the whey protein is fully dissolved and obtain a homogeneous whey protein aqueous solution.

[0064] S2. Preparation of modified whey protein-caffeic acid complex: 0.2 g of caffeic acid (CA) was dissolved in 5 mL of anhydrous ethanol to prepare a caffeic acid ethanol solution. This solution was slowly added dropwise to the whey protein solution, and the reaction was continued for 4 h at room temperature and with stirring at 1000 rpm. After the reaction, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 48 h. The dialysate was changed every 2 h to completely remove ungrafted free caffeic acid from the system. After dialysis, the dialysate was freeze-dried to obtain WP-CA composite powder. In this step, the amount of caffeic acid added was 4 wt% of the whey protein mass.

[0065] S3. Preparation of sodium alginate solution: Take 150 mg of sodium alginate (SA), add it to 20 mL of deionized water, stir at 50 °C for 1 h until the solute is completely dissolved, and prepare an aqueous solution of sodium alginate with a mass fraction of 0.75 wt%.

[0066] S4. Preparation of the aqueous phase of the WP-CA-SA ternary complex: Accurately weigh 180 mg of the WP-CA composite powder prepared above and add it to the sodium alginate aqueous solution. Stir at room temperature for 2 h to ensure that the system is fully mixed and homogeneous, and obtain a uniform and stable aqueous phase of the WP-CA-SA ternary complex; in this aqueous phase, the mass fraction of the WP-CA composite is 0.9 wt% and the mass fraction of the sodium alginate is 0.75 wt%.

[0067] (2) Preparation of multifunctional Pickering emulsion (MFPE)

[0068] S5. Oil phase preparation: Oregano essential oil (OEO) and tetradecane are mixed in a volume ratio of 1:2 and stirred evenly to prepare a composite oil phase.

[0069] S6. High-speed shear emulsification: 7 mL of the aqueous phase of the WP-CA-SA ternary composite prepared in step S4 was placed in a centrifuge tube, and 3 mL of the above composite oil phase (the volume fraction of the oil phase in the system was 30 vol%) was added. The mixture was homogenized by high-speed shearing at 16000 rpm for 5 min to obtain a stable multifunctional Pickering emulsion (MFPE).

[0070] (3) Preparation of temperature-controlled antibacterial preservation gel mat (MFPE-PM)

[0071] Gelatin was placed in deionized water and heated in a 60°C water bath with stirring until completely dissolved, yielding a 10 wt% gelatin-based solution. The prepared MFPE emulsion was added to the gelatin-based solution at a ratio of 40 wt%, along with 0.5 wt% thermochromic powder (critical color change temperature 25°C), and stirring continued until the system was completely homogeneous. The mixture was poured into a custom mold and refrigerated at 4°C for 2 hours to cure. After demolding, the target preservation gel pad (MFPE-PM) was obtained.

[0072] Product performance parameters in this embodiment

[0073] The WP-CA-SA ternary composite prepared in this embodiment has a volume-average particle size of 291.1 nm, a PDI of 0.25, a ζ-potential of -13.17 mV, and a three-phase contact angle of 80.44 ± 9.53°. The prepared MFPE emulsion is a water-in-oil Pickering emulsion with an average droplet size of approximately 4 μm. It exhibits typical shear-thinning rheological properties, with an initial apparent viscosity of 2255.71 mPa·s. After 21 days of storage at 4°C, the system showed no stratification or demulsification, demonstrating excellent storage stability. The prepared preservation gel pad has a phase transition enthalpy of 167.37 J / g. After being frozen at -20°C for 30 min, the gel pad can maintain a surface temperature below 10°C for at least 20 min when placed in a 25°C room temperature environment, demonstrating good temperature-controlled preservation effect.

[0074] Technical features and application scenarios of this embodiment

[0075] This embodiment uses the median parameter combination within the process parameter range of the present invention. The amount of caffeic acid added, the concentration of the WP-CA complex, the concentration of sodium alginate, the oil phase ratio, and the oil phase volume fraction are all at the median values ​​of the optimal parameter range. The technical route has strong stability and good process repeatability, which can effectively ensure the consistency of batch product quality. The process of this embodiment is suitable for medium-scale industrial mass production, especially for production enterprises that are applying this technology for the first time. It can minimize the risk of process fluctuations and ensure the stability of large-scale product production.

[0076] Example 2

[0077] This embodiment provides a method for preparing a multifunctional Pickering emulsion and preservation gel pad with dual functions of temperature control and antibacterial properties. The overall preparation process is the same as in Example 1, except that the amount of active material added and the oil phase ratio are optimized and adjusted. The specific differences are as follows:

[0078] 1. In step S2, the amount of caffeic acid added is adjusted to 6 wt% of the whey protein (corresponding to 0.3 g of caffeic acid added).

[0079] 2. In step S4, the amount of WP-CA complex added is adjusted to 240 mg, corresponding to a mass fraction of WP-CA complex in the aqueous phase of 1.2 wt%, while the mass fraction of sodium alginate remains unchanged at 0.75 wt%.

[0080] 3. In step S5, the volume ratio of oregano essential oil to tetradecane is adjusted to 1:1.5;

[0081] 4. In step S6, the volume fraction of the oil phase in the system is adjusted to 35 vol.

[0082] Product performance parameters in this embodiment

[0083] The WP-CA-SA ternary composite prepared in this embodiment has a volume-average particle size of 275.65 nm, a PDI of 0.28, a ζ-potential of -15.42 mV, and a three-phase contact angle of 85.37 ± 7.21°. The prepared MFPE emulsion droplets have an average droplet size of approximately 3.5 μm, an initial apparent viscosity of 2178.56 mPa·s, and an emulsion phase transition enthalpy increased to 172.58 J / g. It exhibits stable performance and no demulsification or stratification after 21 days of storage at 4°C.

[0084] Technical features and application scenarios of this embodiment

[0085] This embodiment focuses on enhancing the antibacterial and antioxidant properties of the product. By increasing the grafting amount of caffeic acid and the concentration of the WP-CA composite stabilizer, the loading of active functional substances in the system is increased. Simultaneously, the oil phase ratio is optimized to increase the relative proportion of oregano essential oil, further strengthening the antibacterial and antioxidant activity of the emulsion. Increasing the oil phase volume fraction effectively improves the emulsion's solid content and phase transition enthalpy, significantly enhancing the temperature-controlled slow-release capability of the gel pad. This embodiment is suitable for long-distance cold chain transportation of fresh food, meeting the requirements for high antibacterial and high-temperature preservation performance.

[0086] Example 3

[0087] This embodiment provides a method for preparing a multifunctional Pickering emulsion and preservation gel pad with dual functions of temperature control and antibacterial properties. The overall preparation process is the same as in Example 1, except that the proportion of active substances is reduced and the oil phase component ratio is optimized. The specific differences are as follows:

[0088] 1. In step S2, the amount of caffeic acid added is adjusted to 2wt% of the whey protein mass (corresponding to 0.1g of caffeic acid added).

[0089] 2. In step S4, the amount of WP-CA complex added was adjusted to 120 mg, corresponding to a WP-CA complex mass fraction of 0.6 wt% in the aqueous phase;

[0090] 3. In step S5, the volume ratio of oregano essential oil to tetradecane is adjusted to 1:3;

[0091] 4. In step S6, the volume fraction of the oil phase in the system is adjusted to 25 vol.

[0092] Product performance parameters in this embodiment

[0093] The WP-CA-SA ternary composite prepared in this embodiment has a volume-average particle size of 331.6 nm, a PDI of 0.29, a ζ-potential of -18.07 mV, and a three-phase contact angle of 78.26 ± 6.84°. The prepared MFPE emulsion droplets have an average droplet size of approximately 4.5 μm, and the initial apparent viscosity is reduced to 1157.57 mPa·s, indicating a significant improvement in system fluidity. It can be stably stored at 4°C for 18 days.

[0094] Technical features and application scenarios of this embodiment

[0095] This embodiment employs mild, low-load process parameters, reducing the addition of caffeic acid and composite stabilizers while increasing the relative proportion of tetradecane, the temperature-controlled substrate, to prioritize and enhance the system's temperature control performance. This makes it suitable for applications requiring low antibacterial properties and high-temperature control. The low viscosity characteristic gives the emulsion excellent spray and brush application performance, making it widely applicable in the preservation of fresh fruits and vegetables. It specifically addresses the spoilage problems caused by temperature fluctuations during storage and transportation, and is compatible with various flexible coating application processes.

[0096] Example 4

[0097] This embodiment provides a method for preparing a multifunctional Pickering emulsion and preservation gel pad with dual functions of temperature control and antibacterial properties. The overall preparation process is the same as in Example 1, and this embodiment serves as a verification example of the upper limit boundary of the parameters of the present invention. The specific differences in parameters are as follows:

[0098] 1. In step S2, the amount of caffeic acid added is adjusted to 8 wt% of the whey protein content (corresponding to 0.4 g of caffeic acid added, which is close to the upper limit of the process parameters of this invention).

[0099] 2. In step S4, the amount of WP-CA complex added is adjusted to 300 mg, corresponding to a mass fraction of WP-CA complex in the aqueous phase of 1.5 wt% (close to the upper limit of the process parameters of this invention).

[0100] 3. In step S5, the volume ratio of oregano essential oil to tetradecane is adjusted to 1:1 to maximize the proportion of antibacterial components;

[0101] 4. In step S6, the volume fraction of the oil phase in the system is adjusted to 20 vol.

[0102] Product performance parameters in this embodiment

[0103] The WP-CA-SA ternary composite prepared in this embodiment has a volume-average particle size of 258.43 nm, a PDI of 0.23, excellent particle size uniformity, a ζ-potential of -11.35 mV, and a three-phase contact angle of 91.44 ± 5.37°, indicating significantly improved interfacial activity. The prepared MFPE emulsion droplets have an average droplet size of only 2.8 μm, significantly improved interfacial film density, an initial apparent viscosity of 2058.14 mPa·s, and good system stability after 21 days of storage at 4 °C.

[0104] Technical features and application scenarios of this embodiment

[0105] This embodiment demonstrates a process boundary verification scheme. By employing a maximum concentration of caffeic acid, a composite stabilizer, and a high proportion of oregano essential oil, the antibacterial performance of the system is maximized. Smaller emulsion droplet size and a higher three-phase contact angle prove that the composite stabilizer has stronger interfacial adsorption capacity, forming a dense and stable interfacial protective film. The product in this embodiment is suitable for the preservation of fresh aquatic products and livestock and poultry meat with high microbial loads. It can provide long-lasting and high-strength antibacterial protection under conditions of cold chain interruption and temperature fluctuations, preventing microbial contamination and spoilage of fresh products.

[0106] Comparative Example 1

[0107] This comparative example is used to verify the synergistic effect of sodium alginate (SA) on the stability of Pickering emulsion systems.

[0108] The preparation process of this comparative example is basically the same as that of Example 1, except that the sodium alginate preparation and compounding steps in the preparation of the ternary complex are omitted (steps S3 and S4 are deleted). The 180 mg WP-CA complex powder prepared in Example 1 is directly dispersed in 20 mL of deionized water and stirred at room temperature for 2 h to obtain a single WP-CA aqueous phase. The subsequent emulsification and gel pad preparation processes are exactly the same as those in Example 1.

[0109] Comparative Example 2

[0110] This comparative example is used to verify the core contribution of tetradecane to the temperature control performance of Pickering emulsion and food preservation gel pad.

[0111] The preparation process of this comparative example is basically the same as that of Example 1, except that the tetradecane component is removed in step S5, and pure oregano essential oil (OEO) is used in the oil phase. The other preparation parameters and process steps are exactly the same as those of Example 1.

[0112] Comparative Example 3

[0113] This comparative example is used to verify the core role of oregano essential oil (OEO) in the antibacterial properties of the system.

[0114] The preparation process of this comparative example is basically the same as that of Example 1, except that the oregano essential oil component is removed in step S5, and pure tetradecane is used in the oil phase. The other preparation parameters and process steps are exactly the same as those of Example 1.

[0115] Comparative Example 4

[0116] This comparative example is used to verify the negative impact of ultra-high caffeic acid addition on the structure of the composite stabilizer and the overall performance of the emulsion, and to determine the upper limit threshold of the process parameters.

[0117] The preparation process of this comparative example is basically the same as that of Example 1, except that the amount of caffeic acid added in step S2 is increased to 10wt% of the whey protein (corresponding to an added mass of 0.5g). The other preparation parameters and process steps are exactly the same as those of Example 1.

[0118] Comparative Example 5

[0119] This comparative example was used to verify the effect of excessively high WP-CA complex concentration on emulsion rheological properties and storage stability.

[0120] The preparation process of this comparative example is basically the same as that of Example 1, except that the amount of WP-CA complex added in step S4 is increased to 360 mg, which corresponds to an aqueous phase mass fraction of 1.8 wt%. The other preparation parameters and process steps are exactly the same as those of Example 1.

[0121] Comparative Example 6

[0122] This comparative example was used to verify the weakening effect of low oregano essential oil ratio on the antibacterial properties of the system and to clarify the lower limit threshold of the oil phase composition ratio.

[0123] The preparation process of this comparative example is basically the same as that of Example 1, except that the volume ratio of oregano oil to tetradecane in step S5 is adjusted to 1:6, which significantly reduces the relative content of oregano oil. The other preparation parameters and process steps are exactly the same as those of Example 1.

[0124] Comparative Example 7

[0125] This comparative example is used to verify the destructive effect of ultra-high oil phase volume fraction on the interfacial stability and storage stability of Pickering emulsions.

[0126] The preparation process of this comparative example is basically the same as that of Example 1, except that the volume fraction of the oil phase in step S6 is increased to 45 vol%, and the other preparation parameters and process steps are exactly the same as those of Example 1.

[0127] Comparative Example 8

[0128] This comparative example is used to verify the core role of caffeic acid modification in optimizing the whey protein structure and improving the interface properties of the complex.

[0129] The preparation process of this comparative example is basically the same as that of Example 1, except that: in step S2, whey protein is treated with pure anhydrous ethanol solution without caffeic acid and no caffeic acid grafting modification is performed. The other preparation parameters and process steps are exactly the same as those of Example 1.

[0130] Product performance testing methods

[0131] 1. Methods for determining particle size and ζ-potential

[0132] This test uses the WP-CA-SA ternary composite solution as the test sample. Based on the principle of dynamic light scattering (DLS), it utilizes the intensity fluctuation signal of scattered light caused by the Brownian motion of particles to accurately calculate the hydrodynamic diameter and ζ-potential of the sample particles, thereby characterizing the dispersion stability of the sample.

[0133] The specific testing steps are as follows: The WP-CA-SA ternary complex sample was completely dissolved in deionized water to prepare a homogeneous sample solution with a final concentration of 0.5 mg / mL; a NanoBrook Omni particle size analyzer was used, with the test environment temperature set to 25℃, scattering angle to 173°, and sample equilibration time to 2 min. All test samples were measured in parallel for a total of n≥3 times, and the final test result was the average value ± standard deviation of the multiple measurements.

[0134] 2. Three-phase contact angle measurement method

[0135] This test uses WP-CA-SA ternary composite lyophilized tablets as the test sample. Based on the seat drop method, the hydrophilicity and hydrophobicity of the sample surface are quantitatively evaluated by detecting the contact angle of deionized water droplets on the sample tablet surface.

[0136] The specific testing steps are as follows: Accurately weigh 200 mg of the lyophilized WP-CA-SA ternary composite sample, and continuously compress it under 0.8T pressure for 30 seconds to prepare a flat and uniform sample pellet. Fix the sample pellet on the sample stage of the contact angle analyzer, keeping the test stage horizontal and the ambient temperature constant at 25℃. Accurately add 10 μL of deionized water to the center of the pellet. Collect and analyze the droplet contact angle data using CAST 3.0 software. All samples were measured in parallel (n≥3 times) to ensure the reliability of the test data. Key fixed parameters: droplet volume 10 μL, test temperature 25℃.

[0137] 3. Method for determining the droplet size of emulsions

[0138] This test uses freshly prepared MFPE emulsion as the test sample. Based on the principle of optical microscopy image analysis, the droplet size of the emulsion is statistically analyzed to characterize the particle size and distribution uniformity of the emulsion.

[0139] The specific test steps are as follows: Accurately transfer 5 μL of fresh MFPE emulsion sample and evenly drop it onto a clean glass slide, smoothly covering it with a coverslip to avoid air bubbles; place the sample under an upright fluorescence microscope, and acquire microscopic morphology images using a 40x objective lens. Measure the droplet size in the images using ImageJ software. The number of effective droplets in a single test should be no less than 200. Calculate the volume-average particle size and particle size distribution coefficient of the emulsion. Key fixed parameters: objective lens magnification 40×, effective droplet count n≥200.

[0140] 4. Methods for determining the storage stability of emulsions

[0141] This test used MFPE emulsions stored at 25°C for different durations as test samples. The long-term storage stability of MFPE emulsions was comprehensively evaluated by observing the macroscopic morphology and detecting the microscopic droplet morphology and particle size.

[0142] The specific testing steps are as follows: Freshly prepared MFPE emulsion was uniformly dispensed into sterile glass bottles, sealed, and stored in a 25°C incubator after being protected from light. Samples were taken and observed at preset time points on days 0, 3, 6, 9, 12, 15, 18, and 21. After each sampling, the macroscopic state of the emulsion was first observed. Then, 50 μL of the emulsion sample was accurately transferred, diluted 3 times, and the microscopic droplet morphology and particle size changes were observed using an optical microscope. At the same time, 2 mL of fresh MFPE emulsion was placed in a centrifuge tube and centrifuged at 5000 rpm for 20 min. After dilution, the microscopic morphology was observed.

[0143] Stability assessment criteria: The MFPE emulsion is deemed to have satisfactory storage stability if, during the storage period, there is no stratification, demulsification, or flocculation / precipitation, and the maximum droplet size increase does not exceed 50% of the initial fresh emulsion droplet size. Key fixed parameters: storage temperature 25℃, centrifugation speed 5000 rpm, centrifugation time 20 min, and a total of 8 observation points.

[0144] 5. Rheological property analysis methods

[0145] This test uses MFPE emulsion as the test sample. The rheological properties of the sample are detected by a rotational rheometer to evaluate the viscoelasticity, shear flowability and mechanical stability of MFPE emulsion.

[0146] The specific test steps are as follows: Take a sufficient amount of uniform MFPE emulsion sample and place it in the parallel plate test system of the AR2000 rotational rheometer. Set the parallel plate gap to 1 mm, and the test temperature to a constant 25 ± 0.1 °C. After the sample has been allowed to stand for 5 minutes to reach equilibrium, start the test. Set the shear rate scan range to 0.1–100 s. -1The apparent viscosity of the sample was examined to determine the variation with shear rate. With a frequency scanning range of 1–10 Hz and a constant strain of 1%, the storage modulus G′ and loss modulus G″ of the sample were examined to determine the variation with frequency. All tests were performed in parallel for at least three times (n ≥ 3), and the average value was taken to ensure data accuracy.

[0147] 6. DSC Differential Scanning Calorimetry Method

[0148] This test uses MFPE emulsion and preservation gel pads as test samples. Based on the principle of differential scanning calorimetry, the phase transition temperature and phase transition enthalpy of the samples are determined by detecting the heat flow changes of the samples during the temperature rise process, thus characterizing the thermal stability and phase transition characteristics of the samples.

[0149] The specific testing steps are as follows: Accurately weigh 5 mg of the sample to be tested, place it in a standard aluminum crucible, seal and compact it, using an empty aluminum crucible as a test reference; use a DSC Q2000 differential scanning calorimeter, introduce high-purity nitrogen as a protective gas, set the gas flow rate to 50 mL / min, set the heating rate to 10℃ / min, and the temperature scan range to -25℃~50℃ to complete the programmed temperature scan test. After the test, use TA Universal Analysis software to fit and analyze the heat flow curve, obtain the sample melting peak temperature, phase transition enthalpy, and other core parameters. All samples were tested in parallel for n≥3 times, and the test results were taken as the average of multiple tests.

[0150] 7 Cryo-SEM Analysis Method

[0151] This test used MFPE emulsion as the test sample and employed cryo-scanning electron microscopy to rapidly fix the microstructure of the emulsion at low temperature, allowing for in-situ observation of the micromorphology, droplet structure, and interface stabilization mechanism of the MFPE emulsion.

[0152] The specific testing steps are as follows: A precise 5 μL sample of uniform MFPE emulsion was transferred and rapidly frozen in liquid nitrogen (-196℃) for 30 seconds. The frozen sample was then transferred to a cryogenic preparation chamber and sublimated at -90℃ for 5 minutes, followed by gold sputtering for conductivity. The treated sample was then transferred to a Sigma-300 field emission scanning electron microscope (FET), with an accelerating voltage of 5 kV and a working distance of 10 mm, to observe the microstructure and acquire images. Key fixation parameters: freezing temperature -196℃, sublimation temperature -90℃, sublimation time 5 min, accelerating voltage 5 kV, working distance 10 mm.

[0153] 8. Methods for determining antibacterial activity (inhibition zone method)

[0154] This test used MFPE emulsion and its raw material components (pure OEO, pure tetradecane) as test samples and employed the agar plate diffusion inhibition zone method to evaluate the broad-spectrum antibacterial activity of the samples against a variety of pathogenic bacteria.

[0155] The specific testing steps are as follows: Staphylococcus aureus ATCC 29213, Escherichia coli CICC 21525, and Listeria monocytogenes ATCC 19118, the three test bacterial strains, were cultured to a bacterial concentration of 10... 8 CFU / mL; 100 μL of bacterial suspension was evenly spread onto the corresponding solid culture medium plate. Sterile filter paper discs (6 mm in diameter) were attached to the culture medium surface in a triangular arrangement. 10 μL of the MFPE emulsion, pure OEO, and pure tetradecane sample were added to each plate. The culture medium plates were sealed and incubated at 37℃ in the dark for 12 h. After incubation, the diameter of the inhibition zone (including the diameter of the filter paper disc) was measured for each group. All experimental groups were measured in parallel (n≥3). The final results were taken as the mean ± standard deviation. Key fixed parameters: bacterial suspension concentration 10... 8 CFU / mL, culture temperature 37℃, culture time in the dark 12h, filter paper diameter 6mm.

[0156] 9. Methods for determining antioxidant activity (DPPH and ABTS dual methods)

[0157] This test used MFPE emulsion as the test sample and employed the DPPH radical scavenging method and ABTS radical scavenging method to characterize the hydrogen atom transfer ability and electron transfer ability of the sample, respectively, and comprehensively evaluated the antioxidant activity of the sample.

[0158] DPPH test procedure: Dilute the MFPE emulsion sample 50 times with deionized water, take 50 μL of the diluted sample and mix it thoroughly with 10 mL of 0.2 mM DPPH-ethanol solution, place it in a dark environment at room temperature of 25℃ for 30 min, and measure the absorbance of the system at a wavelength of 517 nm.

[0159] ABTS test procedure: Dilute the MFPE emulsion sample 50 times with deionized water, take 20 μL of the diluted sample and mix it thoroughly with 8 μL of ABTS⁺ working solution, place it in a dark environment at room temperature of 25℃ for 20 min, and measure the absorbance of the system at a wavelength of 734 nm.

[0160] Using deionized water as a blank control, the free radical scavenging rate was calculated according to the following formula: Scavenging rate = (1 - (A1 - A2) / A3) × 100%; where A1 is the absorbance of the sample group, A2 is the absorbance of the blank sample, and A3 is the absorbance of the blank control group. All tests were performed in parallel at least three times to ensure data reproducibility. Key fixed parameters were: reaction temperature 25℃, DPPH reaction time 30 min, and ABTS reaction time 20 min.

[0161] 10. Cytotoxicity assay (CCK-8 assay)

[0162] This test used MFPE emulsion as the test sample and employed the CCK-8 cell proliferation toxicity assay to evaluate the biocompatibility and cell safety of MFPE emulsion by detecting the activity of mitochondrial dehydrogenases in live cells.

[0163] The specific testing steps are as follows: RAW264.7 macrophages were seeded at a density of 8000 cells / well in 96-well cell culture plates and cultured for 24 hours to allow cell adhesion. Then, MFPE emulsion sample solutions at concentrations of 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 14 μg / mL, 16 μg / mL, 18 μg / mL, and 20 μg / mL were added, and the cells were treated for 24 hours. The drug solution in the wells was discarded, and the cells were thoroughly washed with PBS buffer. CCK-8 working solution diluted 1:50 was added, and the cells were incubated at a constant temperature until the absorbance at 450 nm was within the effective detection range of 0.3–1.2. The OD value of each well was measured, and cell viability was calculated based on the OD value to evaluate the cytotoxicity of the samples. All concentration groups were tested in parallel (n≥3). Key fixation parameters: The test cell line was RAW264.7, the sample processing time was 24 hours, and the detection wavelength was 450 nm.

[0164] 11. Method for Determining the Preservation Effect of Fresh Shrimp

[0165] This test uses fresh shrimp treated with preservation gel pads as the test subject. By detecting changes in the shrimp's pH value, total bacterial count (TVC), volatile basic nitrogen (TVB-N), and moisture state during storage, the freshness preservation effect of the preservation gel pads is comprehensively evaluated.

[0166] The specific testing steps are as follows: Fresh shrimp of uniform size and consistent freshness were selected, washed, and dried. They were then randomly divided into two groups: a blank control group and an MFPE preservation gel pad experimental group. The shrimp in the blank control group were stored in a regular food storage container. In the MFPE experimental group, the shrimp and the preservation gel pad were placed together in a food storage container, with the gel pad positioned under and in contact with the shrimp. Both groups of samples were stored at a constant temperature of 4℃. Samples were taken and tested at seven preset time points: 0h, 12h, 24h, 36h, 48h, 60h, and 72h of storage.

[0167] The shrimp were tested according to national standards: pH value was determined according to GB 5009.237-2016, total bacterial count (TVC) was determined according to GB 4789.2-2022, and volatile basic nitrogen (TVB-N) was determined according to GB 5009.228-2016. Simultaneously, low-field nuclear magnetic resonance (LF-NMR) technology was used to detect the shrimp's water relaxation characteristics, and MRI imaging was used to observe the water distribution in the shrimp. All tests were performed in parallel (n≥3). Key fixed parameters: storage temperature 4℃, 7 sampling time points.

[0168] 12. Method for determining temperature-controlled cold storage performance (infrared thermal imaging method)

[0169] This test uses a food preservation gel pad as the test sample and employs infrared thermal imaging technology to detect the temperature change of the sample with the environment, and evaluates the phase change cold storage, constant temperature maintenance and temperature control buffering performance of the food preservation gel pad.

[0170] The specific test steps are as follows: The preservative gel pads were placed in a -20℃ low-temperature environment for freezing pretreatment for 30 minutes. Immediately after removal, they were placed in a 25℃ constant-temperature environment. A FLIR E8 infrared thermal imager was used to acquire thermal images of the sample surface, recording the sample temperature data every 3 minutes for 30 minutes. Another preservative gel pad from the same batch was placed on a 37℃ heating plate for constant-temperature treatment for 5 minutes, and the temperature change of the sample surface was observed. Thermochromic powder was added to the test system as a visual indicator medium; the color change of the powder from pink to white indicated that the sample surface temperature had risen to approximately 25℃. All tests were performed in parallel (n≥3 groups) to ensure stable and reliable data. Key fixed parameters: pretreatment freezing temperature -20℃, freezing time 30 min, ambient temperature 25℃, image acquisition interval 3 min, high-temperature treatment temperature 37℃, and treatment time 5 min.

[0171] 13. Water relaxation and MRI imaging analysis methods (LF-NMR)

[0172] This test used fresh shrimp samples with different storage times as the test objects. Based on low-field nuclear magnetic resonance (LF-NMR) technology, the transverse relaxation time T2 of shrimp water was detected to analyze the migration state, binding form and distribution changes of shrimp water during storage, and to reveal the preservation mechanism.

[0173] The specific testing steps are as follows: The fresh shrimp samples to be tested were placed in an NMI20-040 LF-NMR analyzer, and the test temperature was controlled at 25℃. The transverse relaxation signal of water was measured using a Q-CPMG sequence, with the following core parameters set: main frequency 21MHz, 90° pulse width 14.50μs, echo time 0.12ms, and echo number 10000. The shrimp body water was imaged using an SE sequence, with parameters TR=500ms and TE=20ms. After the test, the relaxation curves were fitted using MultiExp Inv Analysis software, and the bound water (T) was statistically analyzed. 21 ,0.01~10ms), non-flowing water (T 22 ,10~100ms), free water (T 23 The relaxation time and peak area ratio (>100 ms) of all samples were measured in parallel (n≥3). As a conceptual design of this invention, sodium alginate (SA) was used to design conformational locking of the whey protein-caffeic acid (WP-CA) complex, primarily to enhance the interfacial stability of the WP-CA complex and prevent CA detachment at the oil-water interface. Molecular dynamics simulations showed that CA binds to the main component of WP, β-lactoglobulin, through hydrogen-bonded non-covalent interactions. The binding energy of a small amount of CA with β-LG is -5.8 kcal / mol. In the presence of a large amount of CA, CA forms hydrogen bonds with residues such as ASN-109, THR-4, GLN-5, LYS-69, ASN-90, GLN-119, and LYS-141, with a total binding energy of -19.171 kcal / mol. However, some CA molecules bound to low-affinity sites are prone to detach from WP and further detach in the oil-water interface environment. The introduction of SA can bind to the WP-CA complex through electrostatic and hydrogen bonding interactions, forming a WP-CA-SA ternary complex. This effectively locks the conformation of WP, maintaining the number of hydrogen bonds between CA and WP at 10–15, significantly inhibiting CA detachment. Simultaneously, the long-chain structure of SA forms a hydrophilic shell on the complex surface, increasing the complex's hydrophilicity and steric hindrance, further stabilizing the overall conformation of the WP-CA complex. By controlling the mass ratio of caffeic acid to whey protein to 1:(5–50) and the mass ratio of sodium alginate to the WP-CA complex to 1:(0.5–2.5), the volume-average particle size of the WP-CA-SA ternary complex is 250–350 nm, and the three-phase contact angle is 70–100°. This achieves conformational stability while ensuring a suitable hydrophilic-hydrophobic balance, improving the interfacial adsorption stability by more than 60% compared to the WP-CA binary complex. This effectively solves the technical challenge of easy CA detachment at the oil-water interface, demonstrating the synergistic effect of molecular conformational locking and interfacial stability.

[0174] As another aspect of this invention, a WP-CA-SA ternary complex is used as the interface stabilizing particle. A multifunctional Pickering emulsion (MFPE) is constructed using a mixture of oregano oil (OEO) and tetradecane as the oil phase, primarily to enhance the emulsion's interface stability, antibacterial activity, and temperature regulation capabilities. Combined studies using cryo-scanning electron microscopy (Cryo-SEM) and molecular dynamics simulations show that the WP-CA-SA ternary complex spontaneously adsorbs at the oil-water interface, beginning contact with the oil phase at approximately 2.5 ns and forming a stable interfacial adsorption layer by 7.5 ns. On the oil droplet surface, WP-CA-SA particles form a dense interfacial shell structure, preventing droplet aggregation through steric hindrance and electrostatic repulsion. In the continuous phase, WP-CA-SA particles form a three-dimensional micelle network structure through the entanglement and cross-linking of SA segments, restricting the Brownian motion of the oil droplets. These two mechanisms synergistically achieve a dual stabilization mechanism for the emulsion. Rheological analysis showed that the emulsion exhibited shear-thinning properties, with an initial apparent viscosity of 1000–2300 mPa·s. The storage modulus G′ and loss modulus G″ increased with frequency, and G″ was consistently higher than G′, indicating weak gel behavior. The co-encapsulated OEO endowed the emulsion with broad-spectrum antibacterial activity (inhibiting Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes) and antioxidant activity (significant DPPH and ABTS free radical scavenging rates). Tetradecane endowed the emulsion with phase transition cooling properties (melting enthalpy reaching 167.37 J / g, phase transition temperature approximately 5 °C). By controlling the volume ratio of OEO to tetradecane in the oil phase to be 1:(1-4), the volume fraction of the oil phase to be 20-40 vol%, and the mass fraction of interfacial stable particles in the aqueous phase to be 0.6-1.5 wt%, the emulsion does not undergo phase separation after being stored at 25°C for 21 days and does not break down after centrifugation at 5000 rpm for 20 min. This achieves a synergistic integration of long-lasting antibacterial effect, intelligent temperature control, and excellent stability, improving the functionality by more than 2 times compared to single OEO emulsions or single tetradecane emulsions.

[0175] As another aspect of this invention, a food preservation gel pad (MFPE-PM) is designed by combining a multifunctional Pickering emulsion with gelatin. This design primarily aims to achieve solidified, slow-release delivery of active ingredients and maintain temperature during cold chain disruptions. OEO in the MFPE is secondary encapsulated within the gel network, delaying its evaporation rate through the barrier effect of the gel matrix, thus achieving long-lasting antibacterial effects. Tetradecane maintains its phase change properties within the gel pad; when the ambient temperature rises, tetradecane absorbs heat through a solid-liquid phase change, maintaining a low-temperature environment for the gel pad and surrounding food. Infrared thermal imaging analysis shows that after being frozen at -20°C for 30 minutes and then placed in a 25°C room temperature environment, the gel pad can maintain a surface temperature below 10°C for at least 20 minutes, a significantly better temperature difference than the control group without tetradecane. Experiments applied to the preservation of fresh shrimp showed that the gel pad effectively delayed the rise in shrimp pH, volatile basic nitrogen (TVB-N), and total bacterial count (TVC), extending the shelf life of shrimp by at least 12 hours. Simultaneously, the high moisture content of the gel pad maintained a humid environment within the packaging, reducing moisture loss from the shrimp. Low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) analyses further confirmed the gel pad's effectiveness in retaining moisture in shrimp. By controlling the amount of emulsion added to 20–60 wt% of the gel pad mass and the gel matrix mass fraction to 5–15 wt%, the gel pad possesses suitable mechanical strength, phase transition enthalpy (100–200 J / g), and active substance release characteristics. This achieves multiple synergies of sustained release of active substances, temperature maintenance, and moisture retention, solving the dual problems of excessively rapid release of active substances and decreased preservation effect when using Pickering emulsions directly and the interruption of the cold chain. It will be apparent to those skilled in the art that this invention is not limited to the details of the above exemplary embodiments, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of the equivalents of the claims are intended to be included within the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0176] Table 1 Performance summary of Examples and Comparative Example 1

[0177] Example 1 291.1 -13.17 80.44 4.0 Stablize 78.5 Example 2 275.7 -15.42 85.37 3.5 Stablize 84.2 Example 3 331.6 -18.07 78.26 4.5 Stablize 71.3 Example 4 258.4 -11.35 91.44 2.8 Stablize 86.7 Comparative Example 1 275.7 -8.52 65.13 8.5 Milk breaking on the ninth day 76.2 Comparative Example 2 290.5 -13.05 79.86 4.2 Stablize 77.9 Comparative Example 3 288.3 -13.42 81.22 3.8 Stablize 12.4 Comparative Example 4 245.8 -8.85 95.72 6.5 Breast breaking on day 12 88.5 Comparative Example 5 310.6 -9.74 83.15 7.2 Milk breaking on the sixth day 80.3 Comparative Example 6 292.4 -13.28 81.63 4.3 Stablize 52.7 Comparative Example 7 315.8 -10.82 76.54 9.8 The third day, the milk broke. 75.6 Comparative Example 8 487.3 -12.40 33.26 12.5 The third day, the milk broke. 45.3

[0178] Note: "—" indicates that the sample was not tested or does not possess the corresponding properties; "stable" indicates that there was no demulsification during the 21-day storage period.

[0179] As can be seen from the performance of the examples and comparative examples in Table 1, Examples 1-4 achieved a good balance in terms of particle size distribution, emulsion stability, and antioxidant activity, fully meeting the product quality requirements. Comparative Example 1, due to the lack of SA, resulted in conformational instability of the WP-CA complex, and the emulsion demulsified on day 9. Comparative Example 3, due to the lack of OEO, resulted in loss of antioxidant activity. Comparative Example 4, due to excessive CA addition, resulted in decreased complex stability. Comparative Example 5, due to excessively high WP-CA complex concentration, resulted in premature emulsion demulsification. Comparative Example 7, due to excessively high oil phase volume fraction, resulted in emulsion demulsification on day 3. Comparative Example 8, due to the absence of CA, resulted in unmodified WP, ​​extremely poor emulsification performance, emulsion demulsification on day 3, and low antioxidant activity. The above comparative example data fully demonstrate the scientific validity and necessity of the component synergy of the WP-CA-SA ternary complex, the bifunctional co-encapsulation of OEO and tetradecane, and the optimized design of key process parameters in this invention.

Claims

1. A multifunctional Pickering emulsion with temperature control and antibacterial functions, characterized in that, The emulsion is an oil-in-water Pickering emulsion, prepared from an aqueous phase, an oil phase, and interfacial stabilizing particles. The oil phase is composed of oregano essential oil and tetradecane. The interface-stabilizing particles are whey protein-caffeic acid-sodium alginate ternary non-covalent complexes. Caffeic acid binds to the surface of whey protein molecules through non-covalent interactions, mainly hydrogen bonds, to form a binary complex. Sodium alginate binds to the surface of the whey protein-caffeic acid binary complex through electrostatic interactions and hydrogen bonds, thus constructing the ternary non-covalent complex.

2. The multifunctional Pickering emulsion according to claim 1, characterized in that, The whey protein-caffeic acid-sodium alginate ternary non-covalent complex has the following physicochemical parameters: volume average particle size of 200-350 nm, zeta potential of -20--5 mV, and oil-water three-phase contact angle of 70-100°.

3. The multifunctional Pickering emulsion according to claim 1, characterized in that, In the whey protein-caffeic acid-sodium alginate ternary non-covalent complex, the mass ratio of caffeic acid to whey protein is 1:(5-50), and the mass ratio of sodium alginate to whey protein-caffeic acid binary complex is 1:(0.5-2.5).

4. The multifunctional Pickering emulsion according to claim 1, characterized in that, In the oil phase, the volume ratio of oregano oil to tetradecane is 1:(1-4).

5. The multifunctional Pickering emulsion according to claim 1, characterized in that, The oil phase in the emulsion has a volume fraction of 20–40 vol, and the interfacial stabilizing particles have a mass fraction of 0.6–1.5 wt% in the aqueous phase of the emulsion.

6. The multifunctional Pickering emulsion according to claim 1, characterized in that, The emulsion possesses at least one of the following performance characteristics: (1) Storage stability: When stored at a constant temperature and in a sealed environment at 25℃ for 3 to 10 days, the emulsion showed no macroscopic phase separation, and the average droplet size of the emulsion increased by no more than 50% of the initial average droplet size; (2) Centrifugal stability: After centrifugation at 5000 rpm for 20 min, the emulsion showed no demulsification or obvious stratification. (3) Rheological properties: in the range of 0.1 to 100 s -1 It exhibits shear-thinning properties within a certain shear rate range, and the initial apparent viscosity of the emulsion is 1000–2300 mPa·s.

7. A method for preparing the multifunctional Pickering emulsion according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Disperse whey protein in deionized water, adjust the pH of the system to 6.5-7.5, and stir at a constant temperature until completely dissolved to obtain a homogeneous whey protein aqueous solution; S2: Caffeic acid is completely dissolved in anhydrous ethanol. The caffeic acid ethanol solution is slowly added dropwise to the whey protein aqueous solution. The reaction is carried out at room temperature with stirring for 2-6 hours. After the reaction is completed, the free caffeic acid in the system is removed by dialysis. After freeze-drying, a solid powder whey protein-caffeic acid binary complex is obtained. S3: Disperse sodium alginate in deionized water, stir at a constant temperature of 40-60℃ until it is completely swollen and dissolved, and cool to room temperature to obtain a uniform sodium alginate aqueous solution. S4: Add the whey protein-caffeic acid binary complex to the sodium alginate aqueous solution, stir at room temperature for 1-3 hours, and after full compounding, obtain an aqueous system containing a whey protein-caffeic acid-sodium alginate ternary complex. S5: Mix oregano essential oil and tetradecane in a preset ratio and stir until homogeneous to obtain a clear and uniform oil phase; S6: The oil phase is added to the aqueous phase system and homogenized by high-speed shearing to prepare a stable multifunctional Pickering emulsion.

8. The preparation method according to claim 7, characterized in that, In step S2, the amount of caffeic acid added is 2-8 wt% of the whey protein, the stirring reaction time is preferably 4 h, and the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, with a dialysis time of 48 h at room temperature.

9. The preparation method according to claim 7, characterized in that, In step S4, based on the total mass of the aqueous system, the amount of whey protein-caffeic acid binary complex added is 0.6-1.5 wt%, and the amount of sodium alginate added is 0.5-1.0 wt%.

10. The preparation method according to claim 7, characterized in that, In step S6, the volume ratio of the oil phase to the water phase is (2-4):(6-8), the rotation speed of the high-speed shear homogenization is 10,000-20,000 rpm, and the shear homogenization time is 3-10 min.

11. A food preservation gel mat, characterized in that, It is prepared by combining the multifunctional Pickering emulsion according to any one of claims 1 to 6 with a gel matrix, wherein the gel matrix is ​​at least one of gelatin, sodium alginate, carrageenan, and gellan gum.

12. The food preservation gel pad according to claim 11, characterized in that, Based on the total mass of the preservation gel pad, the mass fraction of the multifunctional Pickering emulsion is 20-60 wt%, and the mass fraction of the gel matrix is ​​5-15 wt%.

13. The food preservation gel pad according to claim 11, characterized in that, The preservation gel pad also contains thermochromic powder, which can undergo reversible color changes within a temperature range of 20 to 30°C.

14. The food preservation gel pad according to claim 11, characterized in that, The preservation gel pad has a phase change enthalpy of 100-200 J / g. After being frozen at -20℃ for 30 min, it can maintain its surface temperature below 10℃ for no less than 20 min when placed in a 25℃ room temperature environment.

15. The application of the multifunctional Pickering emulsion according to any one of claims 1 to 6, or the preservation gel pad according to any one of claims 11 to 14, in the field of fresh food preservation.

16. The application according to claim 15, characterized in that, The fresh food is shrimp, fish, shellfish, poultry meat, or fruits and vegetables. The application method is to apply the multifunctional Pickering emulsion to the surface of the fresh food by spraying, dipping, or brushing, or to package and preserve the fresh food with the preservation gel pad.

17. The application according to claim 15, characterized in that, Under refrigerated storage conditions at 4°C, the shelf life of fresh food can be extended by at least 12 hours, and / or exposing refrigerated food to room temperature at 25°C can reduce the surface temperature of fresh food by 2–8°C compared to the untreated control group, and this cooling effect can be maintained for 10–30 minutes.

18. The application according to claim 15, characterized in that, The application enables fresh food to meet at least one of the following indicators during storage: (1) The total bacterial count (TVC) of fresh food was reduced by ≥1.01g CFU / g compared with the untreated control group; (2) The rate of increase in volatile basic nitrogen (TVB-N) in fresh food was ≥30% lower than that in the untreated control group; (3) The time for the pH value of the fresh food system to reach the spoilage threshold of 7.6 was delayed by ≥12 hours compared with the untreated control group.

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