An ovalbumin-zein complex protein high internal phase emulsion and a preparation method and application thereof

Abstract

The application discloses an ovalbumin-zipper complex protein high internal phase emulsion and a preparation method and application thereof, and relates to the technical field of protein-based food emulsion processing. The application takes ovalbumin and zipper as raw materials, regulates the mass ratio of the two proteins, and combines pH offset, heat treatment, ultrasonic and microwave synergistic regulation technology to promote the interaction between protein molecules and structure recombination, improve the dispersibility and interfacial activity of the complex protein system. Then, the modified complex protein system is used as the water phase, and the oil is used as the oil phase to prepare the complex protein high internal phase emulsion with good structure stability and rheological properties under high-speed shearing. When the mass ratio of ovalbumin to zipper is 5:1, the stability and rheological properties of the obtained high internal phase emulsion are relatively close to those of the pure ovalbumin system, the effective partial replacement of ovalbumin by zipper is realized, the use amount of animal protein can be reduced, and the utilization efficiency of plant protein can be improved.

Description

Technical Field

[0001] This invention relates to the field of protein-based food emulsion processing technology, specifically to an ovalbumin-zein complex protein high internal phase emulsion and its preparation method and application. Background Technology

[0002] High internal phase emulsions (HIPEs) are a special type of emulsion system with a high dispersed phase volume fraction, typically referring to emulsions with an internal phase volume fraction exceeding 74%. Due to their high internal phase ratio, dense droplet packing structure, and gel-like three-dimensional network structure, HIPEs exhibit high viscosity, good viscoelastic properties, and structural plasticity, making them promising for applications in food processing, lipid structuring, functional ingredient encapsulation and delivery, and low-fat food development.

[0003] In food emulsion systems, proteins, due to their amphiphilic structure, can adsorb at the oil-water interface and form a protective interfacial film, thus being widely used as natural emulsifiers in the construction of high internal phase emulsions. Currently, proteins used for food emulsion stabilization mainly include animal proteins such as whey protein, casein, and ovalbumin, as well as plant proteins such as soy protein isolate, pea protein, and zein. Among these, animal proteins typically exhibit better solubility and interfacial activity, demonstrating a stronger advantage in emulsion formation and stabilization.

[0004] Ovalbumin is the most abundant protein in egg white, accounting for more than half of its total mass. It possesses excellent water solubility, interfacial adsorption capacity, and film-forming properties, effectively stabilizing the oil-water interface, and is therefore widely used in food emulsion systems. However, ovalbumin is derived from animal protein, and its application is limited by factors such as raw material supply, production costs, and increasing consumer demand for plant-based foods. Therefore, reducing the amount of ovalbumin used and introducing plant-derived proteins while ensuring the stability of the emulsion system has become an important research direction in the field of food emulsions.

[0005] Zein is a plant storage protein derived from corn, characterized by its abundant source, high edibility, and good biocompatibility. Due to the high proportion of hydrophobic amino acid residues in its molecular structure, it possesses strong hydrophobic interactions and interfacial adsorption capabilities. Simultaneously, zein exhibits some film-forming ability, showing potential application in food emulsification systems and functional delivery systems. Therefore, introducing zein into ovalbumin emulsification systems is expected to improve the utilization efficiency of plant protein resources while reducing the amount of animal protein used. However, compared to ovalbumin, zein has relatively poor water solubility and a more compact molecular structure, resulting in relatively weaker emulsifying performance. This makes its addition ratio a key factor affecting the structure and stability of high internal phase emulsions. When the zein substitution ratio is too high, it may lead to a decrease in protein interfacial adsorption capacity, resulting in increased emulsion particle size, reduced stability, and decreased rheological properties.

[0006] Currently, there are no reports in existing technologies regarding the use of zein to replace ovalbumin in the construction of high internal phase emulsions. In particular, the changes in emulsion structural properties, stability, and rheological behavior under different substitution ratios are unclear and lack systematic research. How to achieve complementary advantages through a reasonable blending of zein and ovalbumin, and determine the optimal ratio of zein to ovalbumin, are pressing problems to be solved in the field of high internal phase emulsion preparation.

[0007] Therefore, developing a high internal phase emulsion preparation method based on zein partially replacing ovalbumin and determining a reasonable substitution ratio is of great significance for reducing the use of animal protein, expanding the application range of plant protein, and promoting the development of plant-based food emulsion systems. Summary of the Invention

[0008] This invention addresses the problems of high proportion of animal protein and decreased emulsion structural stability after plant protein substitution in the preparation of high internal phase emulsions. It proposes an ovalbumin-zein complex high internal phase emulsion, its preparation method, and its application.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention proposes a method for preparing an ovalbumin-zein complex protein high internal phase emulsion, comprising the following steps: (1) Add ovalbumin and zein to deionized water at a mass ratio of 5:1 to 1:1 to prepare a composite protein nanoparticle solution with a total protein concentration of 1-3%; (2) Adjust the pH to 12.0 with 1 M NaOH solution, stir at 800 rpm for 1 h, then adjust the pH to 7.0 with 1 M HCl solution, and modify the composite protein nanoparticle solution in step (1) by ultrasonic treatment, wherein the ultrasonic power is 100-400 W and the ultrasonic time is 10-30 min. (3) Adjust the pH to 2.0 with 1 M HCl solution and treat the complex protein with microwave power of 100-300 W and microwave time of 30-90 s; (4) Stir at 200 rpm for 8-16 h at 70-90 ℃, and then immediately cool the sample to room temperature to obtain a composite protein dispersion; (5) After adjusting the pH of the composite protein dispersion to 6.0-9.0, mix it with soybean oil at a volume ratio of 1:3 and shear it at 10000 rpm for 2 min to form a composite protein high internal phase emulsion.

[0010] Furthermore, in step (1), the ratio of ovalbumin to zein is 5:1, and the total protein concentration is 2%.

[0011] Furthermore, in step (2), the ultrasonic power is 300 W and the ultrasonic time is 15 min.

[0012] Furthermore, in step (3), the microwave power is 200W and the microwave time is 60s.

[0013] Furthermore, in step (4), the stirring temperature is 85 ℃ and the stirring time is 10 h.

[0014] Furthermore, in step (5), the composite protein dispersion is adjusted to pH 8.0 and then mixed with soybean oil.

[0015] Furthermore, in step (5), the high-speed shearing time is 2 min.

[0016] In a second aspect, the present invention provides an ovalbumin-zein complex protein high internal phase emulsion, which is prepared by any of the methods described above.

[0017] Thirdly, the present invention also provides the application of the above-mentioned ovalbumin-zein complex protein high internal phase emulsion in the preparation of protein-based foods.

[0018] This invention proposes a method for preparing an ovalbumin-zein complex high internal phase emulsion. Different mass ratios of zein were used to replace ovalbumin, and the effects on the particle size, stability, and rheological properties of the high internal phase emulsion were investigated to screen for suitable protein substitution ratios. Specifically, when the mass ratio of ovalbumin to zein was 5:1, the resulting high internal phase emulsion exhibited structural characteristics and stability close to those of a pure ovalbumin system while reducing the amount of ovalbumin used. The method provided by this invention can achieve partial substitution of animal protein while maintaining the good performance of the high internal phase emulsion, improving the utilization efficiency of plant protein, and providing a new technical solution for the development of plant protein-based food emulsion systems.

[0019] Compared with the prior art, the technical effects of the present invention are as follows: (1) To achieve partial substitution of ovalbumin by zein. This invention improves the structure and interfacial properties of the composite protein by combining ovalbumin and zein and using synergistic regulation methods such as pH shift, heat treatment, ultrasound and microwave. While maintaining the basic properties of high internal phase emulsion, it achieves partial replacement of ovalbumin, thereby reducing the amount of animal protein used and improving the utilization rate of plant protein resources such as zein.

[0020] (2) Promotes intermolecular interactions and structural reorganization of complex proteins This invention utilizes pH shift and heat treatment to induce protein molecules to unfold, rearrange, and self-assemble. Combined with the physical effects of ultrasound and microwave treatment, it further promotes the interaction between ovalbumin and zein, regulates the spatial structure of the complex protein, and enables it to form complex protein aggregates with good dispersibility and interfacial activity. This is beneficial for the adsorption of the complex protein at the oil-water interface and the formation of high internal phase emulsions.

[0021] (3) Improve the dispersion and interfacial adsorption capacity of the complex protein system The above synergistic treatment can improve the solubility and dispersion characteristics of the zein-ovalbumin complex system, promote protein adsorption and interfacial film formation at the oil-water interface, and thus provide a stable interfacial basis for the construction of high internal phase emulsions.

[0022] (4) Determine the appropriate ratio of zein to ovalbumin. This invention compares the effects of different ovalbumin to zein mass ratios on the particle size, stability, and rheological properties of high internal phase emulsions, determining that an ovalbumin to zein mass ratio of 5:1 exhibits better overall performance. At this ratio, while reducing the amount of ovalbumin used, the particle size, stability, and rheological properties of the high internal phase emulsion are relatively close to those of the pure ovalbumin system, achieving a good balance between the degree of protein substitution and emulsion performance.

[0023] (5) The preparation method has good application potential. This invention combines protein self-assembly induction with physical regulation techniques such as ultrasound and microwave. The preparation process does not require complex chemical modification and has the advantages of wide availability of raw materials and controllable process. The resulting high internal phase emulsion can be used as a lipid structuring material in food systems, providing technical support for the food application of plant protein to replace animal protein and high internal phase emulsion. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the preparation method of the ovalbumin-zein complex protein high internal phase emulsion provided by the present invention.

[0025] Figure 2 The images show the appearance of composite protein high internal phase emulsions with different mass ratios provided in the embodiments of the present invention.

[0026] Figure 3 Optical microscope images of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0027] Figure 4 Laser confocal scanning microscope images of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0028] Figure 5 Particle size distribution of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0029] Figure 6 Gel strength diagrams of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0030] Figure 7 The water-holding capacity of composite protein high internal phase emulsions with different mass ratios provided in the embodiments of the present invention.

[0031] Figure 8 Frequency scan curves of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0032] Figure 9 Viscosity curves of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0033] Figure 10 Creep recovery curves of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0034] Figure 11 The creep compliance curves of composite protein high internal phase emulsions with different mass ratios provided in the embodiments of the present invention are shown.

[0035] Figure 12The backscattering curve of the OVA provided in the embodiment of the present invention.

[0036] Figure 13 The backscattering curve of the composite protein high internal phase emulsion OZ5:1 provided in the embodiment of the present invention.

[0037] Figure 14 The backscattering curve of the composite protein high internal phase emulsion OZ4:1 provided in the embodiment of the present invention.

[0038] Figure 15 The backscattering curve of the composite protein high internal phase emulsion OZ3:1 provided in the embodiment of the present invention.

[0039] Figure 16 The backscattering curve of the composite protein high internal phase emulsion OZ2:1 provided in the embodiment of the present invention.

[0040] Figure 17 The backscattering curve of the composite protein high internal phase emulsion OZ1:1 provided in the embodiment of the present invention.

[0041] Figure 18 Turbiscan stability index (TSI) graphs of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention.

[0042] Figure 19 The images show the appearance of composite protein high internal phase emulsions with different mass ratios after heat treatment at 80 and 95°C for 30 min, as provided in the embodiments of the present invention.

[0043] Figure 20 Frequency scan curves of composite protein high internal phase emulsions with different mass ratios provided in the embodiments of the present invention after heat treatment at 80 and 95°C for 30 min.

[0044] Figure 21 Viscosity curves of composite protein high internal phase emulsions with different mass ratios provided in embodiments of the present invention after heat treatment at 80 and 95°C for 30 min. Detailed Implementation

[0045] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0047] A method for preparing an ovalbumin-zein complex protein high internal phase emulsion, such as... Figure 2 As shown, it includes the following steps: (1) Add ovalbumin and zein to deionized water at a mass ratio of 5:1 to 1:1 to prepare a solution with a total protein concentration of 1-3%; preferably, the mass ratio of ovalbumin to zein is 5:1 and the total protein concentration is 2%.

[0048] The mass ratio of ovalbumin to zein is used to regulate the composition of the complex protein system and the interaction between the two proteins. A mass ratio of 5:1 to 1:1 balances the emulsifying properties of ovalbumin with the structural regulatory role of zein. When the ovalbumin ratio increases further, such as to 6:1 or higher, the zein content in the system decreases further, and the complex system gradually approaches a single ovalbumin protein system, reducing its substitution effect and complex regulatory function. Further increases in the zein ratio may increase the tendency for aggregation and sedimentation due to its relatively weak water dispersibility. This invention primarily focuses on the 5:1-1:1 range.

[0049] Total protein concentration is used to ensure that there are sufficient proteins in the system to participate in particle formation and oil-water interface stability, while also taking into account the dispersibility and processing performance of the system. Too low a protein concentration (less than 1%) can lead to insufficient interfacial proteins, affecting the effective coverage of oil droplets and emulsion stability; too high a concentration (greater than 3%) will increase the viscosity of the system and enhance the interaction and aggregation tendency between proteins, thereby increasing the difficulty of dispersion and subsequent emulsion preparation.

[0050] (2) Adjust the pH to 12.0 with 1 M NaOH solution, stir at 800 rpm for 1 h, then adjust the pH to 7.0 with 1 M HCl solution, and modify the composite protein nanoparticles by ultrasonic treatment. The ultrasonic power is 100-400 W and the ultrasonic time is 10-30 min; preferably, the ultrasonic power is 300 W and the ultrasonic time is 15 min.

[0051] In this invention, the system is first adjusted to pH 12.0 using a 1 M NaOH solution to improve the solubility and dispersion of zein in the aqueous phase, allowing it to fully contact ovalbumin and form a complex protein system. A pH that is too low (e.g., less than 11) will result in insufficient water dispersibility of zein; a pH that is too high (e.g., greater than 13) can cause excessive conformational changes in the protein, affecting the subsequent structure of the complex protein. Therefore, this invention selects pH 12.0 as the alkaline treatment condition.

[0052] After alkali treatment, the system is adjusted to pH 7.0, mainly to restore near-neutral conditions, so that the composite protein nanoparticles can maintain a suitable dispersion state before subsequent treatment, while avoiding the system being in a strongly alkaline environment for a long time. Too low a pH (less than 6.8) or too high a pH (greater than 7.2) may change the charge state of the protein and the intermolecular interactions, thereby affecting the dispersion and stability of the composite protein particles.

[0053] Ultrasonic treatment primarily promotes the dispersion of complex protein particles, reduces aggregation, and improves particle uniformity through cavitation and mechanical shearing, providing a more stable protein dispersion system for subsequent acid-heat treatment and emulsion interface construction. Insufficient power or treatment time may lead to inadequate dispersion; excessive power or treatment time may cause excessive temperature rise and excessive changes in protein structure, thus increasing the risk of aggregation. This invention uses an ultrasonic treatment range of 100-400 W for 10-30 min, achieving good dispersion results.

[0054] (3) Adjust the pH to 2.0 with 1 M HCl solution and treat the complex protein with microwave power of 100-300 W and microwave time of 30-90 s; preferably, the microwave power is 200 W and the microwave time is 60 s.

[0055] Adjusting the pH of the complex protein dispersion to 2.0 primarily provides a suitable acidic environment for subsequent acid-thermal treatment, promoting conformational changes, intermolecular interactions, and aggregation and reassembly of proteins, thereby forming complex protein aggregates with a certain degree of structural stability. Insufficient acidity may lead to inadequate structural reorganization, while excessive acidity may cause excessive protein denaturation or aggregation, affecting the system's dispersibility. Microwave treatment mainly serves as a pretreatment before acid-thermal treatment, rapidly providing energy to promote protein conformational adjustment and intermolecular interactions, thus creating conditions for subsequent acid-thermal aggregation and reassembly. Insufficient microwave power or treatment time may result in insufficient pretreatment; excessive power or time may cause excessively rapid temperature rise and excessive protein aggregation, affecting the subsequent system's dispersibility. This invention selects pH 2.0 as the preferred acidic treatment condition and uses 100-300 W and 30-90 s as the microwave treatment conditions, obtaining a complex protein aggregate with good dispersibility and stable structure.

[0056] (4) Stir at 200 rpm for 8-16 h at 70-90 ℃, and then immediately cool the sample to room temperature (e.g., ice bath) to obtain a composite protein dispersion; preferably, the stirring temperature is 85 ℃ and the stirring time is 10 h.

[0057] This step is the main acid-heat treatment process. Under acidic conditions, heat treatment at a specific temperature and time promotes conformational changes, intermolecular interactions, aggregation, and reassembly of proteins, thereby forming complex protein aggregates. Stirring at 200 rpm promotes mixing and heat transfer, making the treatment process more uniform. Too low a temperature or too short a treatment time may lead to insufficient protein structural changes and reassembly; too high a temperature or too long a treatment time may lead to excessive protein aggregation, affecting subsequent dispersibility and emulsion properties. Therefore, this invention uses 70-90 ℃ for 8-16 h as the acid-heat treatment range, with 200 rpm as the preferred stirring condition to obtain good dispersibility and emulsion properties.

[0058] The purpose of immediate cooling is to rapidly reduce the system temperature after acid heat treatment, thereby terminating or significantly slowing down heat-induced further protein aggregation and reducing structural changes caused by continuous heating.

[0059] (5) After adjusting the pH of the composite protein dispersion to 6.0-9.0, mix it with soybean oil at a volume ratio of 1:3 and shear it at 10000 rpm for 1-5 min to form a composite protein high internal phase emulsion.

[0060] Adjusting the pH of the composite protein dispersion to 6.0-9.0 is primarily used to regulate the charge state and intermolecular interactions of the proteins, ensuring good aqueous dispersibility and facilitating their adsorption at the oil-water interface to form a stable interfacial layer. Excessively low or high pH may alter the charge state and interactions of the proteins, leading to protein aggregation or decreased dispersion stability, thus affecting emulsion formation and stability. This invention currently primarily prepares emulsions under pH conditions of 6.0-9.0, resulting in emulsions with stable interfaces. Preferably, the composite protein dispersion is adjusted to pH 8.0 and then mixed with soybean oil. Soybean oil serves as the oil phase of the emulsion, providing oil phase volume for the high internal phase emulsion and forming an oil-water interface with the composite protein. Theoretically, other edible oils suitable for food systems, such as corn oil, sunflower oil, and peanut oil, can also be used from the perspective of emulsion formation principles. However, differences in viscosity, fatty acid composition, and interfacial properties among different oil phases can affect emulsion particle size, rheological properties, and storage stability. This invention uses soybean oil as the oil phase of the emulsion, which exhibits the best emulsion particle size, rheological properties, and storage stability. Simultaneously, the 1:3 volume ratio of the aqueous phase to the oil phase ensures an oil phase volume fraction of approximately 75%, meeting the basic structural requirements of a high internal phase emulsion while guaranteeing effective stabilization of the oil droplet interface by the composite protein. If the oil phase ratio is too low, a typical high internal phase structure cannot be formed; if the oil phase ratio is too high, the amount of protein required to stabilize the interface per unit oil phase decreases, potentially leading to insufficient interface coverage and increasing the possibility of droplet coalescence and emulsion instability. Therefore, this invention currently uses a 1:3 volume ratio as the preferred ratio. The main function of high-speed shearing is to rapidly break down soybean oil into smaller droplets and promote the adsorption of the composite protein at the newly formed oil-water interface, thereby forming a more stable high internal phase emulsion. Too low a rotation speed or too short a time may result in insufficient emulsification; too high a rotation speed or too long a time may lead to system temperature rise and excessive shearing, affecting the emulsion structure. 10000 rpm for 2 minutes is the preferred condition for this invention.

[0061] Example 1: Preparation of a composite protein high internal phase emulsion 1. Preparation of composite protein dispersion Ovalbumin and zein were dispersed in deionized water and magnetically stirred for 1 h to prepare a composite protein dispersion with a total protein concentration of 2%. The mass ratios of ovalbumin to zein were 5:1, 4:1, 3:1, 2:1, and 1:1. The pH of the composite protein dispersion was adjusted to 12.0 with 1M NaOH solution and stirred at 800 rpm for 1 h. The pH was then adjusted to 7.0 with 1M HCl solution. The composite protein nanoparticles were ultrasonically treated at 300 W for 15 min. The pH was then adjusted to 2.0 with 1M HCl solution and microwaved at 200 W for 60 s. Subsequently, the mixture was magnetically stirred at 200 rpm and heated at 85 °C for 10 h. After rapid cooling in an ice bath, composite protein dispersions with different mass ratios were obtained and stored at 4 °C.

[0062] 2. Preparation of complex protein high internal phase emulsion Different mass ratios of the composite protein dispersions were adjusted to pH 8.0 and mixed with soybean oil at a volume ratio of 1:3. The mixtures were then subjected to high-speed shearing at 10,000 rpm for 2 min to form composite protein high-internal-phase emulsions with different mass ratios, denoted as OZ5:1, OZ4:1, OZ3:1, OZ2:1, and OZ1:1, respectively. Simultaneously, a high-internal-phase emulsion sample (denoted as OVA) prepared using the same process with ovalbumin alone was used as a control sample.

[0063] The appearance of composite protein high internal phase emulsions with different mass ratios (5:1-1:1) are shown in the figure. Figure 2 As shown, all samples were milky white and did not flow when inverted, maintaining the integrity of their structure and appearance. This is mainly due to the high volume fraction of oil phase in the high internal phase emulsion. No oil leakage occurred in the high internal phase emulsion as the proportion of zein increased, indicating that all samples formed stable, self-supporting high internal phase emulsions.

[0064] Figure 3 Optical micrographs of complex protein high internal phase emulsions with different mass ratios are shown. All emulsion droplets are spherical and uniformly distributed.

[0065] Example 2: Confocal Laser Scanning Microscopy (CLSM) Testing Laser confocal microscopy (CLSM) was used to observe the morphology of emulsions and the properties of oil-water interface films. The microstructure of high internal phase emulsions (OZ5:1, OZ4:1, OZ3:1, OZ2:1, OZ1:1, OVA) was observed using CLSM. Before observation, the protein and oil phases of 1 g of high internal phase emulsions were stained with 20 μL (0.01%, w / v) Nile blue and 20 μL (0.01%, w / v) Nile red, respectively. The fluorescence excitation wavelengths of Nile blue and Nile red were 640 nm and 488 nm, respectively, and the objective lens magnification was 40x.

[0066] Laser confocal microscopy images of complex protein high internal phase emulsions with different mass ratios are shown below. Figure 4 As shown in the figure, green represents the Nile Red-stained oil phase, and red represents the Nile Blue-stained ovalbumin or ovalbumin-zein continuous phase. It can be seen from the figure that the green oil droplets are surrounded by the red ovalbumin or ovalbumin-zein edges, indicating that the prepared high internal phase emulsion is an O / W type emulsion. With the increase of the zein proportion, the droplet size of the high internal phase emulsion increases and the distribution becomes uneven, which is consistent with the results of the optical microscope images. Figure 3 The increased concentration of zein may be due to the strong hydrophobicity of zein, which drives the ovalbumin-zein complex to further self-assemble into larger aggregates as the proportion increases. This leads to increased interfacial adsorbed particle size, reduced arrangement efficiency, and difficulty in forming a complete and dense interfacial film. The weakened interfacial protection cannot effectively inhibit droplet aggregation and flocculation, ultimately resulting in a significant increase in emulsion droplet size and a tendency towards uneven distribution. The droplet sizes of OZ 5:1 high internal phase emulsion and OVA high internal phase emulsion are almost identical, indicating that ovalbumin can effectively regulate the molecular conformation of zein. An appropriate amount of zein and ovalbumin form a stable complex through non-covalent interactions, which helps maintain the strength and density of the interfacial film, making it close to the state when ovalbumin alone stabilizes the high internal phase emulsion.

[0067] Example 3: Measurement of particle size of composite protein high internal phase emulsion The average particle size of high internal phase emulsions (OZ5:1, OZ4:1, OZ3:1, OZ2:1, OZ1:1, OVA) was measured using a laser particle size analyzer (BT-9300HT) at a refractive index of 12-14%.

[0068] Particle sizes of complex protein high internal phase emulsions with different mass ratios are as follows: Figure 5 As shown. From Figure 5 As can be seen, the particle size of the complex protein high internal phase emulsion gradually increases with the increase of the Zein ratio. Smaller particle size indicates a more uniform distribution of emulsion droplets, allowing for better dispersion in the continuous phase and preventing droplet aggregation. Generally, smaller particle size results in stronger emulsion stability. The figure shows that the OZ5:1 and OVA high internal phase emulsions have the smallest particle sizes with no significant difference.

[0069] Example 4: Gel Strength Test In the high internal phase emulsion prepared by this invention, protein complexes adsorb at the oil-water interface and construct a continuous three-dimensional network structure during the formation process, giving the system both emulsion and gel characteristics. Therefore, the resulting high internal phase emulsion is a high internal phase emulsion gel. The gel strength is measured directly using the prepared high internal phase emulsion gel sample.

[0070] The gel strength was determined using a TA-XT2 texture analyzer. A high internal phase emulsion gel sample (50 mm high, 40 mm diameter) was placed on the measuring stage, and a P / 0.5 probe was used for measurement. The weak gel test mode was selected, with a compression rate of 2.0 mm / s, a downward displacement of 10 mm, and a trigger force of 5 g. The instrument automatically calculated the gel strength after measurement.

[0071] The gel strength of high internal phase emulsions of composite proteins with different mass ratios is as follows: Figure 6 As shown, the gel strength of the complex protein high internal phase emulsion gradually decreased with increasing Zein ratio. The OZ5:1 and OVA high internal phase emulsions showed no significant difference in gel strength and exhibited the highest gel strength, which may be related to the lower particle size of the complex protein, consistent with the results for high internal phase emulsion particle size. Figure 5 ).

[0072] Example 5: Water-holding capacity test The prepared high internal phase emulsions (OZ5:1, OZ4:1, OZ3:1, OZ2:1, OZ1:1, OVA) were transferred to 15 mL centrifuge tubes and centrifuged at 10,000 rpm for 15 min. The released water was carefully removed with a syringe, and the weight of the sample was recorded. The water-holding capacity of the high internal phase emulsion was calculated using the following formula:

[0073] In the formula: m1 is the total mass (g) of water in the high internal phase emulsion and m0 is the mass (g) of water released from the high internal phase emulsion after centrifugation.

[0074] Water-holding capacity of complex protein high internal phase emulsions with different mass ratios, such as Figure 7 As shown. For complex protein high internal phase emulsions, although the WHC of OZ4:1, OZ5:1, and OVA samples did not differ significantly, it still showed an increasing trend. This may be closely related to the more stable structure of the complex protein, which is related to the gel strength results. Figure 6 During centrifugation, high gel strength ensures a stronger network structure and stability of the high internal phase emulsion, which is beneficial for water retention and improved water holding capacity. This indicates that high gel strength helps to strengthen the emulsion structure and improve its textural properties.

[0075] Example 6 Rheological Test All high internal phase emulsions (OZ5:1, OZ4:1, OZ3:1, OZ2:1, OZ1:1, OVA) were relaxed at 25°C for 30 min before rheological testing was performed using an MCR 302 rheometer at 25°C with a parallel plate spacing of 1 mm. Amplitude scanning experiments were used to determine the 1% strain within the linear viscoelastic region. Measurements were taken at angular frequencies (ω) of 0.1–100 rad / s. Shear viscosity changes were determined within a shear rate range of 0.1–100 s. Creep recovery testing examined strain and creep yield changes under a stress of 8 Pa for 300 s, followed by a 300 s recovery period after stress removal.

[0076] 1. Dynamic viscoelasticity Dynamic oscillation testing can reflect the rheological properties of emulsions. By testing the elasticity and viscosity of the emulsion through small-amplitude oscillations or the application of stress, the gel structure and stability of the emulsion can be determined. The storage modulus (G') obtained from emulsion frequency scanning reflects the energy stored in the emulsion fluid due to elastic deformation, while the loss modulus (G″) reflects the energy lost in the emulsion fluid due to viscous deformation. The relative magnitudes of these two moduli and their changes with frequency can intuitively characterize the ability of high-internal-phase emulsions to form a continuous three-dimensional network supported by droplet interaction. Dynamic viscoelastic curves of high-internal-phase emulsions with different OVA-Zein mass ratios are shown. Figure 8 The figure shows that the G' of the high internal phase emulsions stabilized by the composite protein is greater than G″ for all ratios, indicating that the high internal phase emulsions have solid gel properties. G' gradually increases with increasing angular frequency. With increasing Zein ratio, the G' of the high internal phase emulsion gradually decreases, indicating a reduction in mechanical strength. The OVA high internal phase emulsion has the highest G', and it is closest to the G' of the OZ 5:1 high internal phase emulsion, indicating good elasticity, dense network structure, and strong mechanical properties.

[0077] 2. Apparent viscosity Apparent viscosity (η) is a key indicator for evaluating the flowability of emulsions and the network strength of high internal phase emulsions in steady-state shear rheology. Figure 9 The apparent viscosity of composite protein high internal phase emulsions with different mass ratios is shown. The viscosity of all high internal phase emulsions decreased with increasing shear rate, exhibiting significant shear-thinning behavior. The viscosity of the composite protein high internal phase emulsions gradually decreased with increasing Zein ratio. The OVA high internal phase emulsion had the highest viscosity, and its viscosity was closest to that of the OZ 5:1 high internal phase emulsion, which is consistent with dynamic viscoelasticity (…). Figure 8 The results are consistent.

[0078] 3. Creep characteristics Creep recovery curves of high internal phase emulsions of composite proteins with different mass ratios are shown below. Figure 10As shown in the figure, in the creep recovery test, continuous stress is applied to the high internal phase emulsion, and the strain gradually increases. Once the stress is removed, instantaneous deformation occurs, exhibiting the characteristics of a viscoelastic network structure. The lower the strain value, the higher the strength of the high internal phase emulsion, and the higher its elastic performance in resisting deformation under instantaneous stress changes. Figure 9 The results showed that with increasing Zein content, the deformation of the complex protein high internal phase emulsion increased, indicating a weakening of its deformation resistance. Compared with the OVA high internal phase emulsion, the OZ5:1 high internal phase emulsion exhibited similar deformable structure and deformation resistance.

[0079] Creep compliance is negatively correlated with the strength and structural stability of high internal phase emulsions. The creep compliance curves of high internal phase emulsions with different mass ratios of composite proteins are shown in the figure below. Figure 11 As shown, the compliance of all high internal phase emulsions gradually increases with the increase of the Zein ratio. High internal phase emulsions stabilized by ovalbumin alone exhibit lower creep compliance, and the creep compliance of the OZ5:1 high internal phase emulsion is similar. This indicates that increasing the proportion of OVA in the complex protein helps to form a denser network structure, increasing emulsion strength. At higher OVA ratios, its performance can approach the level of OVA-stable emulsions, which is related to gel strength (…). Figure 6 The results were consistent.

[0080] Example 7 Physical Stability Evaluation The stability of high internal phase emulsions (OZ5:1, OZ4:1, OZ3:1, OZ2:1, OZ1:1, OVA) was analyzed using multiple light scattering (PSS). Backscattering (BS) intensity and Turbiscan stability index (TSI) were used to evaluate the stability of the high internal phase emulsions. The high internal phase emulsions were injected into flat-bottomed cylindrical glass containers and monitored at 25 °C for 48 h to obtain ΔBS(BSt-BS0) and TSI.

[0081] BS curves of complex protein high internal phase emulsions with different mass ratios are shown below. Figure 12-17 As shown, the BS fluctuation (ΔBS) reflects changes in the internal microstructure and distribution of the high internal phase emulsion; a smaller ΔBS indicates stronger physical stability. With increasing Zein proportion, the ΔBS of the composite protein high internal phase emulsion gradually increases, indicating a gradual decrease in stability. The stability of the OVA high internal phase emulsion is similar to that of the OZ5:1 and OZ4:1 high internal phase emulsions. The ΔBS of the OVA (ΔBS = -0.11%), OZ5:1 (ΔBS = -0.11%), and OZ4:1 (ΔBS = -0.15%) high internal phase emulsions are all less than 0.2%, indicating that the internal structure and distribution of the high internal phase emulsions have not undergone drastic changes and exhibit high stability. Furthermore, the OVA and OZ5:1 samples possess the most stable structures.

[0082] The instability mechanisms caused by droplet migration (floating, flocculation, and settling) were further evaluated by calculating the TSI using Turbiscan software. Figure 18 As shown, the TSI gradually increases with the increase of the Zein ratio, with the OVA and OZ 5:1 high internal phase emulsions exhibiting the lowest TSI, indicating that they have the highest physical stability. This is because the OVA and OZ 5:1 high internal phase emulsions have higher elasticity (…). Figure 8 ) and viscosity ( Figure 9 This inhibited droplet migration and improved physical stability.

[0083] Example 8 Thermal stability evaluation Food products undergo temperature changes during production, transportation, and storage, which can affect the stability of emulsions. Therefore, the effect of different heating temperatures on the stability of high internal phase emulsions was investigated. High internal phase emulsion samples (OZ5:1, OZ4:1, OZ3:1, OZ2:1, OZ1:1, OVA) were placed in water baths at 80 and 95 ℃ for 30 min, then removed and allowed to return to 25 ℃. The appearance and rheological properties of the heat-treated high internal phase emulsions were analyzed to evaluate their thermal stability.

[0084] The appearance and rheological properties of composite protein high internal phase emulsions with different mass ratios after heat treatment at 80 and 95℃ for 30 min are shown in the figures below. Figure 19-21 As shown.

[0085] Figure 19 The results showed that all high internal phase emulsions were milky white after heat treatment, with no oil leakage, unlike the high internal phase emulsions without heat treatment. Figure 2 Compared to the previous results, there were no significant changes. When the high internal phase emulsion samples treated at different temperatures were inverted, all samples did not flow to the bottom and maintained a stable high internal phase emulsion structure, exhibiting strong gelation properties.

[0086] Based on the oscillation frequency scanning results of the composite protein high internal phase emulsion after heating at different temperatures ( Figure 20 As can be seen, at different heat treatment temperatures, the G' of all high internal phase emulsion samples was higher than G'', indicating that the high internal phase emulsions maintained a highly viscoelastic gel state and exhibited solid gel characteristics. With increasing angular frequency, G' gradually showed an upward trend. After heat treatment at 80℃ and 95℃, the G' and G'' of the OVA and OZ 5:1 high internal phase emulsions remained high, indicating that they maintained the strength of the interface and network structure and exhibited excellent thermal stability. These results demonstrate that the composite protein high internal phase emulsion formed by Zein and a high proportion of OVA can maintain good structural and physical stability during thermal processing.

[0087] Apparent viscosity of composite protein high internal phase emulsions after heating at different temperatures ( Figure 21The results showed that all emulsions exhibited typical shear-thinning behavior, with viscosity decreasing significantly with increasing shear rate. This indicates that the samples were all pseudoplastic fluids, suggesting that temperature changes did not alter the fluid type of the emulsions. The OVA high internal phase emulsion had the highest viscosity, and its viscosity was closest to that of the OZ 5:1 high internal phase emulsion.

[0088] In summary, as the substitution ratio of zein increases, the structural stability and rheological properties of high internal phase emulsions show a certain downward trend. Specifically, when the ratio of ovalbumin to zein is 5:1, the gel strength, water-holding capacity, rheological properties, physical stability, and thermal stability of the resulting high internal phase emulsion are all quite close to those of the OVA high internal phase emulsion system, achieving partial substitution of ovalbumin while maintaining good emulsion performance. Therefore, a 5:1 ratio of ovalbumin to zein strikes a balance between the degree of substitution and emulsion performance, making it a suitable composite ratio.

[0089] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an ovalbumin-zein complex protein high internal phase emulsion, characterized in that, Includes the following steps: (1) Add ovalbumin and zein to deionized water at a mass ratio of 5:1 to 1:1 to prepare a composite protein nanoparticle solution with a total protein concentration of 1-3%. (2) Adjust the pH to 12.0 with 1 M NaOH solution, stir at 800 rpm for 1 h, then adjust the pH to 7.0 with 1 M HCl solution, and modify the composite protein nanoparticle solution in step (1) by ultrasonic treatment, wherein the ultrasonic power is 100-400W and the ultrasonic time is 10-30 min. (3) Adjust the pH to 2.0 with 1 M HCl solution and treat the complex protein with microwave power of 100-300 W and microwave time of 30-90 s; (4) Stir at 200 rpm for 8-16 h at 70-90 ℃, and then immediately cool the sample to room temperature to obtain a composite protein dispersion; (5) After adjusting the pH of the composite protein dispersion to 6.0-9.0, mix it with soybean oil at a volume ratio of 1:3 and shear it at 10,000 rpm for 1-5 min to form a composite protein high internal phase emulsion.

2. The method for preparing the ovalbumin-zein complex protein high internal phase emulsion according to claim 1, characterized in that, In step (1), the ratio of ovalbumin to zein is 5:1, and the total protein concentration is 2%.

3. The method for preparing the ovalbumin-zein complex protein high internal phase emulsion according to claim 1, characterized in that, In step (2), the ultrasonic power is 300 W and the ultrasonic time is 15 min.

4. The method for preparing the ovalbumin-zein complex protein high internal phase emulsion according to claim 1, characterized in that, In step (3), the microwave power is 200W and the microwave time is 60s.

5. The method for preparing the ovalbumin-zein complex protein high internal phase emulsion according to claim 1, characterized in that, In step (4), the stirring temperature is 85 ℃ and the stirring time is 10 h.

6. The method for preparing the ovalbumin-zein complex protein high internal phase emulsion according to claim 1, characterized in that, In step (5), the composite protein dispersion is adjusted to pH 8.0 and then mixed with soybean oil.

7. The method for preparing the ovalbumin-zein complex protein high internal phase emulsion according to claim 1, characterized in that, In step (5), the high-speed shearing time is 2 min.

8. A high internal phase emulsion of ovalbumin-zein complex protein, characterized in that, It is made by the method described in any one of claims 1 to 7.

9. The application of the ovalbumin-zein complex protein high internal phase emulsion according to claim 8 in the preparation of protein-based foods.

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