A transparent high internal phase emulsion and a preparation method and application thereof

Abstract

This invention relates to the field of food-grade high internal phase emulsion preparation, specifically to a transparent high internal phase emulsion, its preparation method, and its applications. The invention involves dispersing myofibrillar protein (MP) in pure water and stirring to prepare a modified myofibrillar protein base solution; then adding glycerol to prepare an MP-water-glycerol mixed solution; finally, adding medium-chain triglycerides to the MP-water-glycerol mixed solution to obtain a transparent high internal phase emulsion. This transparent high internal phase emulsion possesses excellent aesthetic value, reversible stability under high temperature and freeze-thaw conditions, good environmental tolerance, and a good taste. It can be used as a 3D printing food ink, a food for special environments, and a carrier for functional ingredients.

Description

Technical Field

[0001] This invention relates to the field of food-grade high internal phase emulsion preparation, specifically to a transparent high internal phase emulsion, its preparation method, and its application. Background Technology

[0002] High internal phase emulsions (HIPEs) exhibit viscoelastic and rheological properties similar to solids due to their tightly packed droplets. They are widely used in the food science field as fat substitutes or encapsulation carriers and matrices for bioactive substances.

[0003] Although research on HIPEs has been relatively in-depth, existing food-grade HIPEs technologies and related fat alternatives still have many significant defects in practical applications, specifically in the following three aspects: (1) Defects in optical performance and environmental stability: Existing food-grade HIPEs usually exhibit an opaque, milky-white appearance due to the mismatch of the refractive index (RI) between the oil and water phases, which causes light to scatter at the interface, limiting their application in transparent foods or products with specific visual effects. Although transparent HIPEs exist in the chemical industry, they often involve toxic reagents and are not edible. In addition, traditional protein-stabilized HIPEs mainly rely on the physical barrier of the interfacial protein film, and their thermal stability and freeze-thaw stability are poor. After undergoing heating that causes protein denaturation and flocculation, or freeze-thaw cycles that cause ice crystals to pierce the interfacial film, the emulsion structure is prone to irreversible damage, resulting in demulsification or oil-water separation, thus failing to meet the requirements for use in environments such as heat sterilization, cold chain transportation, or extreme temperature changes, such as space and polar regions. (2) Limitations of lipid carrier nutrition and metabolism: Existing technologies mostly use long-chain triglycerides (LCTs) such as soybean oil and corn oil as the dispersion phase. LCTs have a long digestive pathway in the human body, slow metabolism, and are easily stored in adipose tissue, which can easily lead to obesity with long-term intake. In contrast, medium-chain triglycerides (MCTs) can provide energy quickly through the portal vein and are not easy to store, but pure liquid MCTs are greasy and poorly tolerated when taken orally. Existing technologies lack a carrier technology that can effectively solidify high proportions of MCTs into a gel, which can both improve the taste and provide rapid energy. (3) Insufficient adaptability to extreme environments and aerospace applications: Existing food matrices pose safety hazards for weightlessness and long-term mission environments. First, traditional baked goods or low-viscosity foods are prone to producing debris when consumed. In a microgravity environment, the debris floating may be inhaled into the lungs or damage precision instruments; second, existing high-protein food inks usually have high water activity (α). w The risk of microbial contamination is high, and without relying on high-strength preservatives or ultra-low temperature freezing, it is difficult to meet the shelf-life requirements of space food that can last for several years.

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a food-grade transparent high internal phase emulsion based on pH control and polyol system, its preparation method and application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a transparent high internal phase emulsion, which addresses the shortcomings of existing HIPEs such as opaque appearance, poor environmental tolerance, poor taste, and poor adaptability to extreme environments and aerospace applications.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the transparent high internal phase emulsion.

[0007] The final technical problem to be solved by this invention is to provide the application of the transparent high internal phase emulsion.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a transparent high internal phase emulsion, comprising the following steps:

[0010] (1) Disperse myofibrillar protein MP in pure water and stir to prepare a modified myofibrillar protein base solution;

[0011] (2) Add glycerol to the modified myofibrillar protein base solution obtained in step (1), stir, and prepare an MP-water-glycerol mixed solution;

[0012] (3) Medium-chain triglycerides were added to a mixed solution of MP-water-glycerol, and after homogenization, a transparent high internal phase emulsion was prepared.

[0013] Wherein, the refractive index RI of the medium-chain triglyceride at 25℃ is 1.4520, and the refractive index error between the MP-water-glycerol mixed solution at 25℃ and the refractive index of the medium-chain triglyceride is ≤0.006%, calculated as: (n-1.4510) / 1.4510×100%≤0.006%, n=1.4500~1.4520.

[0014] In step (1), the protein concentration of myofibrillar protein MP in pure water is 20~80 mg / mL.

[0015] Preferably, the myofibrillar protein MP has a protein concentration of 60 mg / mL in pure water.

[0016] In step (1), after the myofibrillar protein MP is dispersed in pure water, the pH is adjusted to 3.0±0.2. At this time, the myofibrillar protein can partially unfold, exposing hydrophobic groups and carrying a high density of positive charges. Electrostatic repulsion is used to prevent protein aggregation, which prepares for the subsequent formation of a stable interface film.

[0017] In step (2), the mass concentration of the glycerol is 80-84%.

[0018] Preferably, the glycerol has a mass concentration of 84%.

[0019] At this point, the refractive index of the MP-water-glycerol mixture is close to or equal to that of the medium-chain triglyceride (1.4510), eliminating interfacial light scattering and achieving a transparent appearance.

[0020] In steps (1) and (2), the stirring is carried out at 4°C for 3 to 12 hours.

[0021] In step (3), the volume fraction of the medium-chain triglycerides is φ = 0.60~0.80 based on the total volume of the transparent high internal phase emulsion; preferably, the volume fraction of the medium-chain triglycerides is φ = 0.70~0.75; more preferably, the volume fraction of the medium-chain triglycerides is φ = 0.75.

[0022] In step (3), the homogenization process is performed at 10,000-12,000 rpm for 120 seconds. High shear force breaks the medium-chain triglycerides into tiny droplets, and acidic myofibrillar protein (MP) particles are adsorbed onto the oil-water interface to form a robust Pickering interfacial film, locking a high proportion of the oil phase within the gel network, ultimately forming a transparent emulsion with high internal phase properties.

[0023] In step (3), the medium-chain triglycerides may also contain ergosterol, such as 0.1 wt%, or 0.2 wt%, or 0.3 wt%, or 0.4 wt%, or 0.5 wt%, or 0.6 wt%, or 0.7 wt%, or 0.8 wt%, or 0.9 wt%, or 1.0 wt% ergosterol.

[0024] In some embodiments of the present invention, 1.0 wt% ergosterol is embedded in the medium-chain triglycerides. The ergosterol is completely soluble and stably confined within continuous MCT oil droplets without affecting the stability and functional properties of the transparent high internal phase emulsion.

[0025] In existing technologies, the preparation of transparent emulsions typically relies on non-food-grade surfactants or organic solvents (microemulsion method), or the resulting food-grade HIPEs are milky white due to the large difference in refractive index between oil and water. This invention abandons the traditional neutral pH environment, specifically placing MP at pH 3.0±0.2 to achieve high protonation (positive charge), and introducing up to 84% glycerol as a continuous phase solvent. This constructs a dual-regulated continuous phase system of "pH 3.0± strong electrostatic repulsion + high-concentration glycerol refractive index matching," achieving a balance between "fully food-grade" and "high transparency."

[0026] Specifically,

[0027] Optical transparency: By precisely adjusting the refractive index of the continuous phase to 1.4510 with glycerol, light scattering at the oil-water interface is eliminated, making the emulsion with up to 75% oil phase appear as a transparent gel, solving the problem of poor appearance of traditional food HIPEs.

[0028] Interface enhancement: The strong electrostatic repulsion at pH 3.0 synergistically with the high viscosity of glycerol prevents the aggregation of protein particles, forming a more stable interfacial film than that under pure water at pH 7.0.

[0029] Salt Reduction: Traditionally, protein solubles (MPs), as typical "salt-soluble proteins," are considered to require a certain salt concentration to remain stable and function properly. This study proposes a pure water dissolution process that achieves precise regulation of MP functionality under extremely low salt concentrations, truly realizing the goal of "reducing salt without compromising function," and providing crucial technical support for the development of low-sodium, healthy meat products.

[0030] In a second aspect, the present invention provides a transparent high internal phase emulsion, which is prepared by the preparation method described in the first aspect.

[0031] The transparent high internal phase emulsion is in a gel state.

[0032] The transparent high internal phase emulsion has an oil-in-water (O / W) structure, with a dispersed phase of medium-chain triglycerides (MCT) and a continuous phase of an aqueous solution containing myofibrillar protein (MP) and glycerol.

[0033] Wherein, the volume fraction of the medium-chain triglyceride φ is 0.60~0.80; preferably, the volume fraction of the medium-chain triglyceride φ is 0.70~0.75; more preferably, the volume fraction of the medium-chain triglyceride φ is 0.75.

[0034] The protein concentration of the myofibrillar protein MP is 20-80 mg / mL, with a preferred protein concentration of 60 mg / mL.

[0035] The glycerol has a mass concentration of 80-84%; preferably, the glycerol has a mass concentration of 84%.

[0036] Specifically, the transparent high internal phase emulsion exhibits a microscopic structure of tightly packed polyhedral oil droplets with myofibril proteins adsorbed at the oil droplet interfaces by Pickering particles; macroscopically, the emulsion is optically transparent or translucent and can spontaneously recover its gel structure after undergoing heating or freeze-thaw cycles.

[0037] Existing protein-based HIPEs primarily rely on physical adsorption. Once heated, the protein undergoes thermal denaturation, or frozen, ice crystals rupture, leading to irreversible structural damage (oil-water separation). This invention modifies the phase transition behavior and intermolecular forces of the system by binding high concentrations of glycerol (antifreeze / humectant / refractive index modifier / sweetener / thickener) to unfolded and unfolded proteins. A heat / freeze-thaw "intelligent" reversible stabilization mechanism based on a polyol-protein network is established.

[0038] In some embodiments of the present invention, by verifying the antifreeze and thermal reversibility of the transparent high internal phase emulsion prepared in the first aspect, the transparent high internal phase emulsion of the present invention endows the emulsion with self-healing ability under extreme temperature changes.

[0039] Specifically,

[0040] Freeze resistance: The system of this invention significantly improves freeze resistance by interfering with water molecule crystallization through glycerol. Even when treated in an extreme low temperature environment of -80℃, only a trace amount of liquid oil is released after thawing (slight demulsification), without overall phase separation or structural collapse. Its gel network skeleton is still preserved, and its stability is significantly better than the traditional control group that is completely demulsified.

[0041] Thermal reversibility: A reversible cycle of "heating to become cloudy - cooling to become clear" is achieved. Even after heating disrupts some weak interactions, the system can spontaneously recombine upon cooling to room temperature using the hydrogen bond network between glycerol and protein, as well as electrostatic interactions. On the one hand, the gel structure can be restored; on the other hand, during cooling to room temperature, the system changes from a state of cloudiness upon heating to transparency upon cooling. This is an unexpected effect in existing food emulsion technologies.

[0042] In existing technologies, medium-chain triglycerides (MCTs) are mostly liquid oils, which have poor tolerability when taken orally directly (greasy, causing gastrointestinal discomfort), while solid fats usually contain harmful trans fatty acids or highly saturated long-chain fats. This invention locks 75% of the volume of liquid MCTs in a protein-glycerol network, achieving solid gelation delivery of high-concentration medium-chain triglycerides (MCTs), thus improving the efficiency and acceptability of energy delivery.

[0043] Specifically,

[0044] Improved taste: By changing the way you drink oil to "eat gel", the greasy taste of MCT is significantly improved.

[0045] Rapid energy supply without accumulation: It fully retains the metabolic characteristics of MCT, which is oxidized directly to the liver via the portal vein, providing astronauts or people who engage in high-intensity exercise with a new type of food matrix that provides a feeling of fullness like solid food and provides rapid energy supply like glucose.

[0046] Thirdly, the present invention provides the application of the aforementioned transparent high internal phase emulsion as a carrier in the preparation of special environment foods;

[0047] The special environmental food mentioned above is aerospace food, polar scientific research food, or emergency rescue food.

[0048] Fourthly, the present invention provides the application of the aforementioned transparent high internal phase emulsion as a 3D printing ink in the preparation of 3D food.

[0049] Existing aerospace and emergency food products typically extend shelf life by drying (which makes them fragile) or adding preservatives (which are unhealthy). This invention utilizes the strong water-binding ability of glycerol to significantly reduce water activity without drying, while simultaneously leveraging the stacking effect of high internal phase oil droplets to create solid rheological characteristics, thus developing a "low water activity (a)" product. w The MCT gel carrier formula with "high thixotropy" solves the contradiction between "debri-free" and "long-lasting antibacterial" properties in aerospace food and other products.

[0050] Specifically,

[0051] Natural antibacterial properties: No artificial preservatives required, low in ammonia. w The environment naturally inhibits the growth of microorganisms, meeting the shelf-life requirements of long-term space missions.

[0052] Printing safety: The ink is endowed with excellent viscoelasticity and interlayer bonding force, resulting in a compact structure after 3D printing. It does not produce floating debris when consumed, eliminating the risk of inhalation in microgravity environments.

[0053] Applications as 3D printing food inks: Utilizing the excellent shear-thinning behavior (thixotropy), high viscoelasticity, and low water activity of this emulsion, it can be used as a 3D printing ink. This gives it the combination of low water activity (natural antibacterial properties), high adhesion (no risk of debris), and suitable rheological properties, thereby meeting the food safety and nutritional supply requirements of extreme environments such as aerospace and polar regions. This allows for the preparation of various personalized foods with high fidelity, no risk of debris, and long shelf life.

[0054] Applications in aerospace and extreme environment food: Utilizing the high energy density (high MCT content), freeze-thaw stability, and natural antibacterial properties (low water activity) of this emulsion, it can be prepared into special medical food or energy supplement food suitable for aerospace, polar scientific research, or emergency rescue scenarios.

[0055] Applications as a carrier of functional ingredients: Utilizing its high oil phase properties, it can be used as a transparent encapsulation carrier for fat-soluble vitamins or bioactive substances. Beneficial effects:

[0056] (1) By controlling the mass concentration of glycerol in the continuous phase (preferably 84%) and the pH of the system (preferably 3.0 ± 0.2), this invention successfully raised the refractive index of the aqueous phase to a level highly matched with that of the oil phase (MCT, RI≈1.4510), thus constructing a dual-controlled continuous phase system of "strong electrostatic repulsion at pH 3.0 + high concentration of glycerol refractive index matching". The HIPEs prepared in this way can still exhibit excellent optical transparency even with an oil phase volume fraction as high as 75%, which greatly expands the aesthetic value and application range of HIPEs in the food industry, realizes the optical transparency of the entire food-grade system, and breaks through the appearance limitations of traditional HIPEs.

[0057] (2) This invention utilizes the antifreeze and moisturizing properties of high-concentration glycerol and the electrostatic repulsion mechanism at pH 3.0±0.2 to construct a "smart" gel network with heat / freeze-thaw reversibility. Regarding antifreeze properties, HIPEs do not freeze at -20℃ or even lower temperatures; after thawing, they undergo slight demulsification and can recover their structure through re-emulsification. Regarding heat reversibility, HIPEs, after becoming turbid upon heating (e.g., at 90℃), can spontaneously recover to their original transparent gel state upon cooling to room temperature. This characteristic enables them to withstand pasteurization, high-temperature cooking, and cold chain transportation during food processing, significantly outperforming existing technologies.

[0058] (3) This invention transforms liquid oil into a solid gel by "locking" 75% by volume of MCT oil within a gel network, thus fundamentally altering the physical state of the oil and achieving solid-state delivery of liquid oil, significantly improving the greasy feeling during consumption. Simultaneously, it retains the nutritional advantages of MCT, which rapidly enters the liver via the portal vein for oxidation and energy supply, and is less prone to accumulation as body fat. This provides athletes, Foods for Special Medical Purposes (FSMP), and astronauts with an ideal carrier that is high in energy density, easy to consume, and has a low metabolic burden.

[0059] (4) The HIPEs prepared by this invention possess both low water activity and excellent printability, perfectly meeting the food requirements of extreme environments. They also exhibit antibacterial properties and a long shelf life: the high-concentration glycerol system significantly reduces the system's water activity (α). w(Only 0.43). Utilizing the "fence effect," it provides natural antibacterial and preservative effects, allowing for long-term storage at room temperature (e.g., over 1 year) without the need for additional artificial preservatives. It also prevents moisture loss in vacuum or dry environments, perfectly meeting the long-term mission requirements of aerospace and polar scientific expeditions, solving the food safety and preservation challenges in extreme environments such as aerospace. 3D printing safety: This system exhibits excellent shear-thinning behavior and rapid structural recovery ability (thixotropy), resulting in high-precision printing lines and strong interlayer bonding. The formed gel structure has excellent integrity, producing no debris during consumption and eliminating the risk of inhalation in microgravity environments. Sensory comfort and payload optimization: For long-cycle missions such as deep space exploration, this system not only doubles the heat density, greatly optimizing the mass efficiency of the payload, but its highly transparent gel network can also realistically simulate the visual and juicy chewiness of animal adipose tissue. This effectively overcomes the "menu fatigue" and insufficient nutrient intake caused by the lack of texture and juiciness in traditional space soft canned food, perfectly balancing nutritional preservation and sensory comfort in extreme environments. It provides key technical support for the development of functional meat products in long-cycle missions. Attached Figure Description

[0060] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0061] Figure 1 This is a process flow diagram for preparing a transparent high internal phase emulsion.

[0062] Figure 2 Multiple light scattering analysis of myofibrillar protein (MP) solutions was performed. (a) shows the Turbiscan stability index (TSI) of MP solutions prepared in deionized water (W) and high-salt solutions (S) and treated for 3 hours at different pH values ​​(pH 3–11). (b) shows the backscattered light intensity change (ΔBS) of different MP solutions as a function of time and sample height (tube height), illustrating spatially and temporally resolved instability (sedimentation). (c) shows photographs of MP samples (pH 3, pH 7, and pH 11) after 3 hours of storage, acquired and exported using TowerSoft 1.3.1.85 software (representative images). (d) shows the dissolution of myofibrillar protein solutions at different pH values ​​under water and high-salt conditions. (e) shows laser confocal microscopy images of myofibrillar protein solutions at different pH values ​​under water and high-salt conditions (white scale bar is 50 μm).

[0063] Figure 3The refractive index of MP and glycerol mixed solutions with different mass ratios was matched. Among them, (a) shows the appearance of MP and glycerol mixed solutions with different mass ratios; (b) shows the refractive index of MP and glycerol mixed solutions with different mass ratios as measured by Abbe refractometer.

[0064] Figure 4 The appearance and rheological properties of different MP-glycerol-based HIPEs are shown. Among them, (a) represents viscosity; (b) represents frequency scanning.

[0065] Figure 5 MP-glycerol-based HIPEs (MP 0.96 G 84 (a) has a transparent appearance. Among them, (a) is a stable gel network structure that does not flow when inverted; and (b) has excellent transparency and clearly visible text.

[0066] Figure 6 Macroscopic appearance and IDSI swallowing criteria of MP-glycerol-based HIPEs with different ratios were evaluated.

[0067] Figure 7 The effect of different glycerol addition amounts on the rheological properties and thixotropic behavior of HIPEs under constant MP concentration (0.96 wt%) was investigated. (a) shows the apparent viscosity-shear rate (0.1~100 s⁻¹) obtained from steady-state shear tests. -1 (a) Flow curve. (b) Changes in storage modulus (G', solid symbol) and loss modulus (G'', hollow symbol) during dynamic strain scanning (0.01%~1000%), used to determine the linear viscoelastic region (LVR) and network yield behavior. (c) Shear stress-shear strain curve, used to characterize the change in yield stress of the system. (d) Dynamic frequency scanning (0.1~100 rad s). -1 (e) The results reflect the solid-sample gel characteristics of the system under small deformation conditions. (e) is a continuous three-stage step shear test (3ITT), which evaluates the instantaneous failure and reconstruction recovery ability of the system structure by alternately applying low shear (simulating post-extrusion rest) and high shear (simulating nozzle extrusion).

[0068] Figure 8 The appearance and characterization results of MP-glycerol-based HIPEs with different oil phase volume fractions (φ=0.20~0.85) are shown. Among them, (a) is the inverted flow test of MP-glycerol-based HIPEs; (b) is the laser confocal image of MP-glycerol-based HIPEs with different internal phase volume fractions; (c) is the blockage diagram of MP-glycerol-based HIPEs with different internal phase volume fractions; (d) is the laser confocal image of the negatively stained aqueous phase; (e) is the apparent viscosity; and (f) is the change of storage modulus with shear strain under strain scanning. Figure 9Appearance and infrared images of MP-glycerol-based HIPEs (0G and 84G) after being restored to room temperature under different temperature conditions. Among them, (a) shows (i) heating at 85°C for 30 minutes and then naturally cooling to room temperature; (ii) freezing at -20°C for 24 hours and then naturally warming to room temperature; (iii) freezing at -80°C for 24 hours and then naturally warming to room temperature; (b) shows infrared images of the products after heating at 85°C for 30 minutes and then naturally cooling to room temperature; and (c) shows infrared images of the products after freezing at -20°C for 24 hours and then naturally warming to room temperature.

[0069] Figure 10 The temperature-responsive rheological behavior and conformational stability of MP-glycerol-based HIPEs (0G and 84G) are shown. (a) shows the evolution of storage modulus (G') and loss modulus (G'') of the 0.96MP-0G system during three consecutive heating-cooling cycles. (b) shows the modulus evolution of the 0.96MP-84G system during three consecutive heating-cooling cycles, exhibiting high thermal reversibility. (c) shows the rheological changes of the 0.96MP-0G and 0.96MP-84G systems after freezing (-20℃) as the temperature rises to room temperature. The blue shaded area represents the ice crystal melting region. (d~e) show the evolution of the fluorescence centroid (BCM) of the intrinsic fluorescence spectra of myofibrillar proteins in the 0.96MP-0G (d) and 0.96MP-84G (e) systems and their corresponding first derivatives (dashed lines) during continuous heating (25~95℃). The shaded areas indicate the temperature ranges where significant conformational changes occur. (f) represents the difference in total fluorescence centroid shift (ΔBCM) between the 0G and 84G systems throughout the heating process (25–95 °C). Different lowercase letters indicate significant differences between groups (p < 0.05).

[0070] Figure 11 The water activity of MP-glycerol-based HIPEs (0G and 84G).

[0071] Figure 12 Appearance and rheological properties of transparent MP-glycerol-based HIPEs after ergosterol addition. (a) shows MP-glycerol-based HIPEs prepared using pure MCT (left) and MCT loaded with 1.0 wt% ergosterol (right) as the oil phase; (b) shows the apparent viscosity of the system before and after ergosterol loading as a function of shear rate (0.1~100 s⁻¹). -1 (a) is the flow curve of the system before and after loading ergosterol; (b) is the dynamic scanning curve of the system storage modulus (G') and loss modulus (G'') with angular frequency (0.1~100 rad / s) before and after loading ergosterol.

[0072] Figure 13This section describes transparent MP-glycerol-based HIPEs and their multicolor 3D printing applications. Image a shows various complex geometric shapes (pyramids, pentagrams) and custom patterns ("NJAU" lettering, a puppy model) 3D printed using transparent ink. Image B shows the physical structure and a magnified view of a coaxial 3D printing system, demonstrating the internal piping connections and operational status of the dual barrels (Ink A and Ink B) and coaxial extrusion nozzles (Nozzle 1 and Nozzle 2). Image C shows mesh and tubular structures with different core-shell ratios prepared using coaxial 3D printing technology. Ink A (red ink) serves as the core, and Ink B (transparent ink) as the shell. From left to right, the diameter ratios of the core and shell (A:B) are 0.1:0.9, 0.3:0.7, and 0.9:0.1, respectively. Image d shows a mixture of MCT oils loaded with different food-grade pigments (addition ratio: 1 μL food-grade pigment / 20 mL MCT oil). e shows the appearance of high internal phase emulsions (HIPEs) with different food-grade pigments after being left at room temperature for one month, demonstrating the excellent long-term physical stability of this colored emulsion system. F shows the three-dimensional letters "H" (green) and "A" (yellow) 3D printed using colored HIPE inks, showcasing their application potential in the color printing of complex structures. G demonstrates the system's excellent plasticity and shape memory: the slurry can perfectly adapt to centrifuge tube containers with different bottom shapes; after being left at room temperature for 3 days and then demolded, it still maintains the three-dimensional shape inside the container without collapsing. H shows a layered alternating cuboid structure prepared using dual-nozzle 3D printing technology. Ink A is black and ink B is red, demonstrating the precise deposition capability of multi-material composite structures. I shows a heart-shaped structure prepared using single-nozzle 3D printing technology, exhibiting a three-layer color gradient effect from top to bottom (green-cyan-yellow). J shows a biomimetic "streaky pork" structure constructed using dual-nozzle 3D printing. The "fat" part (ink A) uses transparent / white HIPEs, while the "lean meat" part (ink B) uses HIPEs with added red food coloring, successfully achieving macroscopic customization of complex biomimetic food structures. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0074] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0075] In the following examples, w / v refers to g / mL.

[0076] Example 1: Optimization and preparation of modified myofibrillar protein (MP) base solution

[0077] Fresh chicken breast was selected, and after removing fat and connective tissue, a standard salt solution (0.1 M KCl, 20 mM KH2PO4 / K2HPO4, 2 mM MgCl2, 1 mM EGTA, pH 7.0) was added at a ratio of 1:4 (w / v). The homogenate was homogenized twice at 6900 rpm for 30 seconds each time using an IKA digital display high-speed homogenizer. After filtering through double-layered gauze, the homogenate was centrifuged at 2000×g for 10 minutes at 4 °C to collect the initial precipitate. The collected initial precipitate was resuspended in the standard salt solution at a ratio of 1:4 (w / v). The homogenization, filtration, and centrifugation steps were repeated twice to remove impurities. Subsequently, the precipitate, after impurities were removed, was washed with 0.1 M KCl solution at a ratio of 1:4 (w / v), filtered, and centrifuged at 2500×g for 10 minutes at 4 °C. The purification process of resuspending the precipitate in 0.1 M KCl, filtering, and centrifuging was repeated three times to finally obtain high-purity myofibrillar protein (MP) precipitate. All extraction and purification operations were performed at 4 °C to prevent protein denaturation. The concentration of extracted MP was determined using the biuret method, and its absorbance at 540 nm was measured using a standard curve constructed with bovine serum albumin (BSA) to calculate the actual protein concentration.

[0078] The purified protein protein (MP) was initially dispersed in pure water, and the protein concentration was adjusted to an optimal 60 mg / mL (6 wt%). This concentration provides sufficient protein to stabilize the complex interfacial structure of high internal phase emulsions (HIPEs) while also imparting excellent rheological properties and overall stability to the system. Considering that MP is extremely unstable when directly dissolved in pure water and prone to thermodynamic aggregation, the pH of the protein solution was further systematically optimized to determine the optimal preparation parameters of the base solution to meet the stability requirements of subsequent high internal phase emulsions (HIPEs). A pH gradient of 3.0, 5.0, 7.0, 9.0, and 11.0 was set using acid-base adjusters, and the protein was dissolved in pure water and a high-salt solution (the high-salt solution served as a control group, with a formulation of 0.6 M KCl, 50 mM K2HPO4·3H2O, and 50 mM KH2PO4, pH=7) to explore the deeper effects of pH and ionic strength on protein hydration and dispersion behavior. The adjusted protein solution was placed in a 4°C environment and magnetically stirred for 3 h to ensure that the protein was fully hydrated and did not denature or aggregate, thus obtaining different modified myofibrillar protein base solutions.

[0079] Multiple light scattering analysis was performed on the obtained modified myofibrillar protein base solutions, and the results are as follows: Figure 2As shown. Analysis of the stability index and instability of the solution revealed that by using 2 M HCl as an acid regulator to adjust the pH of the protein solution to 3.0 ± 0.2, under these extremely acidic conditions, the protein exhibited excellent structural unfolding and steric repulsion, underwent extensive protonation, and generated a strong net positive charge on its surface. The resulting modified myofibrillar protein base solution possessed high stability and high clarity.

[0080] Specifically, in a low-ionic-strength pure water system, precise pH control successfully achieved efficient dispersion of myofibrillar proteins (MPs), overcoming the industry-wide challenge of their tendency to thermodynamically aggregate in low-salt environments. Its core advantages lie in:

[0081] (1) Stability driven by electrostatic repulsion: Under the extreme acidity of pH 3.0, MP molecules undergo extensive protonation, accumulating a high density of net positive charge on their surface. This long-range electrostatic repulsion, combined with the excellent structural unfolding and steric hindrance effect of the protein molecule in a low-salt environment, effectively inhibits hydrophobic association and thermodynamic aggregation between molecules, enabling MP to exhibit excellent colloidal stability in pure water.

[0082] (2) Breaking through the traditional limitation of "salt solubility": The traditional view holds that MP, as a typical "salt-soluble protein", must be under a certain salt concentration to remain stable and function. The pure water dissolution process proposed in this embodiment realizes the precise control of MP functionality under extremely low salt concentration environment, truly achieving the process goal of "reducing salt without reducing function", and providing key technical support for the development of low-sodium healthy meat products.

[0083] (3) Interface and optical advantages: By adjusting the pH of the protein solution to 3.0, the transparency and clarity of the MP solution were significantly improved. This not only facilitates the rapid unfolding and adsorption of protein molecules on the interface, but also achieves excellent refractive index matching effect, laying the foundation for subsequent microstructure observation and interface characteristic analysis.

[0084] Example 2: Construction of a refractive index-matched continuous phase (aqueous phase)

[0085] This embodiment, based on the modified myofibrillar protein base solution (pH 3.0, concentration 60 mg / mL, i.e., 6wt% MP solution, hereinafter referred to as "6MP solution") selected in Example 1, continues to optimize the continuous phase (aqueous phase) formulation of MP-based high internal phase emulsions (MP-HIPEs) by using two complementary experimental design strategies (formulation screening and critical concentration exploration based on gradient substitution strategy, and mechanism verification and solvent environment adjustment based on the single variable principle). Based on the refractive index matching principle (RIM), food-grade glycerol is introduced to regulate the refractive index of the aqueous phase in order to eliminate the milky white appearance of the emulsion system caused by light scattering at the oil-water interface, thereby obtaining a transparent and stable continuous phase, and revealing the mechanism of glycerol's influence on the physicochemical properties and stability of the system.

[0086] I. Formulation Screening and Critical Concentration Investigation Based on Gradient Substitution Strategy

[0087] 1. Formulation screening and critical concentration investigation

[0088] Under the condition of a fixed oil phase MCT volume fraction of 75% v / v, the final absolute concentration (C0) of the film-forming matrix MP was adjusted by dynamic mass substitution of glycerol with 6MP solution. MP The concentration of glycerol was gradually reduced from 6.0 wt% to 0 wt% to systematically evaluate the maximum tolerance of the emulsion system to glycerol. On the other hand, the aim was to find the "critical protein concentration" that maintains the stability of the three-dimensional network of the high internal phase emulsion, thereby screening out the optimal glycerol-protein balance ratio that can impart the target properties of the system (such as high transparency, excellent freeze-thaw resistance and thermal reversibility) while maintaining the structure without demulsification.

[0089] The specific process is as follows:

[0090] (1) Formulation design: As shown in Table 1, the total aqueous phase mass was fixed at 100 g (mass of 6MP solution + mass of glycerol = 100 g). Formulations of 6MP solution and glycerol with different mass ratios were designed, and the mass fraction C of MP in the aqueous phase was calculated. MP = (6MP solution mass × 6%) / total aqueous phase mass, unit: wt%.

[0091] (2) Preparation of the aqueous continuous phase: According to the formulation in Table 1, the modified myofibrillar protein base solution (6MP solution) and food-grade glycerol of different mass concentrations were added to beakers and magnetically stirred for 3 h at 4℃ and 300 rpm to ensure uniform mixing and removal of air bubbles, resulting in 9 groups of gradient substitution MP-water-glycerol mixed solutions with different glycerol mass concentrations as the aqueous continuous phase. Figure 3 (a) The refractive index (RI) of each mixed solution was determined using a binocular Abbe refractometer at room temperature (25°C).

[0092] Table 1. Myofibrillar protein solutions (6 wt.%) and glycerol formulations at different mass ratios

[0093]

[0094] Traditional emulsions have a significant refractive index difference between the oil and water phases (ΔRI = RI). oil -RI water This results in strong multiple scattering of light at the droplet interface. Figure 3 As shown in b, when the mass fraction of food-grade glycerol in the continuous phase is 80-84%, the refractive index of the aqueous phase is close to or equal to the refractive index of the oil phase MCT (1.4510), eliminating interfacial light scattering and forming a transparent continuous phase solution.

[0095] 2. Results of investigation on the appearance and rheological properties of MP-glycerol-based HIPEs with different ratios

[0096] from Figure 4 As can be visually observed in Figure 'a', with the increase of glycerol (G) and myofibrillar protein concentration (C), the effect of glycerol (G) increases. MP With the reduction of [amount], the optical transparency of the HIPEs system significantly improves, gradually transforming from a milky white to a highly transparent jelly-like state. Regarding self-supporting properties, from MP [amount]... 6.0 G0 up to MP 0.96 G 84 All samples did not flow in the inverted glass vials, exhibiting a firm gel state; however, when the protein concentration was reduced to 0.60 wt% (MPa), the flow rate decreased. 0.60 G 90 When the protein-free pure glycerol aqueous phase (MPOG) was used, the system began to exhibit fluidity; while the protein-free pure glycerol aqueous phase (MPOG) showed the same fluidity. 100 If glycerol is added, gelation will not occur. The improved transparency is mainly attributed to the addition of glycerol, which greatly matches the refractive index of the oil and water phases, reducing light scattering at the interface. More importantly, the inverted experiment defined the "gelation critical point" of the system. MP0G 100 The inability to gel proves that MP is an indispensable core emulsifier and network framework for stabilizing high internal phase emulsions; while MP 0.96 G 84 The ability to maintain a self-supporting structure even at an extremely low protein concentration of only 0.96 wt% reveals a strong synergistic network enhancement effect between high concentrations of glycerol and a small amount of protein.

[0097] Further quantification of this synergistic effect revealed that all gelling systems exhibited significant shear-thinning behavior. Figure 4 a) and the solid-like elastic characteristic G'>G'' ( Figure 4(b) satisfies the rheological prerequisites required for 3D printing. It is noteworthy that the system's viscosity and storage modulus (G') did not decrease monotonically with decreasing protein concentration, but rather exhibited a trend of first increasing and then decreasing. Specifically, when the formulation changed from MP... 6.0 G0 changes to MP 2.4 G 60 At that time, although the amount of film-forming proteins decreased by more than half, the viscosity and G' of the system reached their peak. This non-monotonic rheological evolution revealed the fierce competition between two opposing mechanisms within the system: (1) a strengthening mechanism dominated by glycerol: Glycerol, as a polyhydroxy cosolvent, significantly increased the viscosity of the continuous phase. At the same time, a large number of glycerol molecules formed a dense hydrogen bond network with water and polar groups on the protein surface, forcing the limited MP molecules to be arranged more tightly on the oil-water interface through the "crowding effect," thus enhancing the mechanical strength of the interfacial film. (2) a weakening mechanism dominated by protein depletion: As glycerol continued to increase, when it crossed 60G, the number of protein molecules maintaining the three-dimensional network structure became extremely scarce. 2.4 G 60 Previously, glycerol's enhancement mechanism was absolutely dominant; while in MP 0.96 G 84 In subsequent formulations, although the extreme lack of protein led to a slight decrease in modulus, the strong high-glycerol solvent network ensured that its mechanical properties remained sufficient to prevent structural collapse. 0.96 G 84 It combines high transparency, freeze-thaw resistance, and good 3D printability. This also facilitated the subsequent selection of MP. 0.96 G 84 This formulation, which offers the best overall performance, provides a solid theoretical basis in rheology.

[0098] Figure 5 The visual effect of introducing 84% glycerol into the continuous phase of the HIPEs system is shown. It can be seen that when the refractive index of the aqueous phase increases from 1.333 to about 1.4510, that is, when ΔRI approaches 0, light can pass through the oil droplets without deflection, thus giving the HIPEs system a unique transparent glassy visual effect on a macroscopic scale.

[0099] 3. Investigation of the physical state of oils in MP-glycerol-based HIPEs with different ratios

[0100] Low viscosity can easily lead to liquid flowing into the trachea and causing aspiration, while high viscosity and excessive adhesion can easily leave residues in the pharynx, posing a risk of suffocation. Referring to the IDDSI standard, the physical state of lipids in different MP-glycerol-based HIPEs was examined, and the macroscopic appearance and IDDSI swallowing grade of different MP-glycerol-based HIPEs were systematically evaluated.

[0101] (1) IDSSI Injector Flow Test

[0102] The IDDSI syringe flow test is a standard method for evaluating fluid consistency. For example... Figure 6 As shown, each group of samples (MP) 6.0 G0 up to MP 0.96 G 84 Each sample was loaded into a 10 mL syringe, and none of them exhibited free dripping or rapid outflow, demonstrating extremely strong self-support. This result indicates that the system has a high initial yield stress, fully meeting the IDDSI grade 4 (extremely viscous / pasty) standard. Even at film-forming protein concentrations (C... MP Even in the extreme case where the concentration of glycerol drops sharply to 0.96 wt%, the co-solvent thickening effect brought about by the high concentration of glycerol still maintains the network skeleton of the emulsion, ensuring that food will not spread uncontrollably before entering the oral cavity and pharynx, effectively avoiding the risk of aspiration caused by excessive flow rate.

[0103] (2) IDDSI spoon tilt test

[0104] The spoon tilt test is primarily used to assess the cohesiveness and surface adhesion of food. From Figure 6 As can be seen from the data, when the spoon is tilted, the MP values ​​of each group of samples (MP) 6.0 G0 up to MP 0.96 G 84 All of them can maintain an intact shape. Especially the high-glycerol group (such as MP) 1.08 G 82 and MP 0.96 G 84 The sample slides off completely with minimal residue on the spoon surface. This clumping yet non-sticky characteristic is a hallmark of ideal dysphagia-related foods, perfectly meeting IDDSI Level 4, and even approaching Level 5 in terms of fineness and moisture. Glycerin, as a powerful hydrating agent, locks a large amount of water in a three-dimensional network through hydrogen bonds, forming an excellent surface lubricating layer. This endows the system with appropriate shear-thinning behavior and excellent cohesion, ensuring that the food bolus can smoothly pass through the pharynx during swallowing, while avoiding dangerous food residue on the palate or esophageal wall.

[0105] Figure 6 The appearance on the right visually reflects the impact of formulation evolution on optical properties. The sample evolved from a traditional milky white, thick paste (MPa). 6.0 G0), gradually turning into a jelly-like, translucent state (MP). 0.96 G 84 )change.

[0106] II. Mechanism verification and solvent environment adjustment based on the single variable principle

[0107] 1. Mechanism verification based on the single variable principle

[0108] To eliminate the potential interference of the variable "decreased MP concentration" in the formulation screening experiment on the characterization of the system's macroscopic physicochemical properties, a control group with a constant MP concentration was further designed, fixing the absolute concentration C of MP. MP =0.96 wt%, the concentration gradient of glycerol in the aqueous phase was adjusted by only increasing or decreasing the mass of pure water, following the strict single variable principle, to verify the driving effect of glycerol concentration on the system properties.

[0109] The specific process is as follows:

[0110] (1) Formulation design: As shown in Table 2, weigh 8 g of 6MP solution. At this time, the mass of MP = 8 × 6% = 0.48 g. Weigh the corresponding masses of glycerol and water. The total mass of the aqueous phase is fixed at 50 g (mass of 6MP solution + mass of glycerol + mass of water = 50 g) to ensure a constant C MP =0.48 g / 50 g = 0.96 wt%.

[0111] (2) Preparation of continuous phase: According to the formula in Table 2, 6MP solution, food-grade glycerol and water were added to a beaker and magnetically stirred for 3 h at 4℃ and 300 rpm to ensure that the solution was mixed evenly and to remove air bubbles, resulting in 7 groups of aqueous continuous phases with single variable adjustment. The refractive index (RI) of each mixed solution was measured at room temperature of 25℃ using a binocular Abbe refractometer.

[0112] Table 2. Solution formulations for mixing with glycerol of different mass concentrations at the same MP concentration.

[0113]

[0114] By measuring the refractive index (RI) of each mixed solution, it was found that as the mass fraction of glycerol in the system (from 0 to 42 g) gradually replaced the volume of pure water, the refractive index of the aqueous continuous phase showed a significant and regular upward trend. Since the refractive index of pure water (approximately 1.333) is much lower than that of the MCT oil phase (approximately 1.44–1.45), in low-glycerol or glycerol-free systems (such as 0.96 MP-0 G), the large difference in refractive index between oil and water leads to strong interfacial light scattering, resulting in a cloudy and whitish appearance of the emulsion. However, when the glycerol addition reaches the 0.96 MP-84 G formulation, the refractive index of the high-concentration glycerol aqueous phase rich in polyhydroxyl groups is successfully increased to a level nearly equal to that of the MCT oil phase. This precise refractive index matching effect allows light to penetrate thousands of internal phase oil droplets, thus providing the most direct optical evidence for the excellent transparent macroscopic phase state of this high internal phase emulsion.

[0115] 2. Effects of different glycerol addition amounts on the rheological properties and thixotropic behavior of HIPEs

[0116] At constant C MP The effects of different glycerol additions on the rheological properties and thixotropic behavior of HIPEs were determined at a concentration of 0.96 wt%. The results are as follows: Figure 7 As shown in Figure a, all groups exhibited typical non-Newtonian fluid characteristics and strong shear-thinning behavior, which is a primary prerequisite for the successful extrusion of 3D printing ink from the nozzle. Crucially, at the same shear rate, the apparent viscosity of the system increased by orders of magnitude as the glycerol content increased from 0G to 84G. Since the number of protein network backbones in the system remained consistent, the increase in viscosity is directly attributed to the physicochemical properties of glycerol. Glycerol, as a small polyhydroxy molecule, inherently possesses high viscosity. The replacement of water molecules with a large number of glycerol molecules not only significantly increased the hydrodynamic viscosity of the continuous phase (aqueous phase), but its abundant hydroxyl groups (-OH) also formed a dense intermolecular hydrogen bond network with water molecules and dispersed protein residues, significantly increasing the internal frictional resistance of the fluid during shearing. Dynamic oscillation tests further confirmed the structural strengthening effect of glycerol. Frequency scan ( Figure 7 As shown in d), G' is always greater than G'' in all systems, and its dependence on frequency is extremely low, exhibiting typical characteristics of elastic soft solids. In strain scanning (… Figure 7 In (b) and (c), with the increase of glycerol addition, the initial storage modulus in the linear viscoelastic region (LVR) of the system is significantly increased, and the critical strain and yield stress required for structural collapse (G' crossover) are also increased. Figure 7 The turning point (c) in the equation shifts significantly to the right and upward. This indicates that glycerol does not simply increase liquid viscosity, but rather substantially participates in and strengthens the physical cross-linking network of the high internal phase emulsion. At high glycerol concentrations (e.g., 0.96 MP-84 G), strong solvent polarity changes and steric congestion effects force MP molecules at the interface to undergo a more compact conformational arrangement and intermolecular interactions. This enhanced interfacial film thickness and strength enable the emulsion droplets to withstand higher mechanical stresses without demulsification or macroscopic network collapse when resisting large external deformations. Figure 7The continuous step shear test perfectly simulates the dynamic process of 3D printing. When high shear is applied, the simulated ink undergoes shear liquefaction within the nozzle, causing a sudden drop in viscosity across all systems. When the high shear is removed and low shear is restored, the viscosity of the system recovers almost 100% to its initial level within seconds. The high-glycerol group, especially the 0.96MP-84G group, exhibited the highest absolute value of recovered viscosity and excellent repeatability in multiple high and low shear cycles. This superior thixotropic recovery capability is a core indicator for evaluating whether 3D printing ink can maintain its three-dimensional shape after deposition. The hydrogen bond network strengthened by high-concentration glycerol endows the system with extremely strong "structural memory" properties: high shear force only temporarily breaks the non-covalent physical cross-linking points; once the external force disappears, the dense hydrogen bond network drives rapid microscopic recombination between the interfacial proteins and the solvent, causing the deposited lines to solidify instantly. This provides a solid rheological guarantee for the subsequent printing of extremely complex biomimetic structures, such as multi-layered pyramids and suspended meshes.

[0117] In summary, through optimization and verification using two complementary experimental design strategies, it was found that: (1) Glycerol concentration is the core factor driving the improvement of system transparency. When the mass fraction of glycerol in the aqueous phase is 80-84%, preferably 84%, the refractive index matching effect is optimal, and a transparent and stable MP-glycerol-based HIPEs continuous phase can be obtained. (2) By introducing glycerol to achieve refractive index matching, a new form with extremely high transparency can be given to food without sacrificing the swallowing safety properties. This visual presentation, similar to "transparent jelly" or "hydrogel," greatly enhances the sensory appeal of the product and provides an ideal matrix material for the diversified visual design of swallowing-friendly foods, such as 3D printing of customized shapes and colors. (3) The evolution of the macroscopic properties of the system, such as the improvement of transparency, the change of rheological behavior and the enhancement of thermodynamic stability, is purely driven by the increase of glycerol concentration and the change of solvent polarity environment. High glycerol concentration, such as 0.96MP-84G group, can strengthen the physical cross-linking network of high internal phase emulsion and has excellent thixotropic recovery ability, which can provide a solid rheological guarantee for subsequent printing of extremely complex biomimetic structures.

[0118] Example 3: High-shear assembly of transparent high internal phase emulsions (HIPEs)

[0119] To determine the optimal conditions for preparing high internal phase emulsions, this embodiment systematically evaluated the effects of different oil phase volume fractions (φ=0.20~0.85) on the macroscopic morphology, rheological properties, and microstructure of the emulsions.

[0120] The specific process is as follows:

[0121] (1) Oil phase volume fraction gradient design

[0122] With the total emulsion volume fixed at 16 mL, the volume ratio of medium-chain triglycerides (MCT) in the oil phase to the aqueous phase (MP-glyceroyl HIPEs containing 84% glycerol by mass) was set according to Table 3, so that the volume fraction of the oil phase φ was in the range of 0.20~0.85.

[0123] Table 3 Oil Phase Volume Fraction Gradient Design Table

[0124]

[0125] (2) Preparation of high internal phase emulsion by high shear homogenization

[0126] According to the proportions in Table 3, the corresponding volumes of oil phase were added to the aqueous phase, and homogenization was performed using a high-speed shear mill. The shear force was used to break up the oil phase and adsorb protein particles at the interface. The homogenization speed was set to 10,000~12,000 rpm, and the homogenization time was 120 s. After the shearing was completed, different high internal phase emulsions were obtained.

[0127] From the macroscopic appearance of high internal phase emulsions ( Figure 8 In section a), the significant phase transition of the system with increasing φ can be visually observed. When φ = 0.50, the emulsion exhibits obvious fluidity, belonging to the fluid-like region where proteins are enriched. When φ increases to the range of 0.60–0.80, the emulsion can overcome gravity and stabilize at the bottom of the inverted vial, forming a typical self-supporting gel network. However, when φ further increases to 0.85, the system regains its fluidity and even experiences structural collapse. Therefore, 0.60–0.80 is the basic parameter range for forming macroscopically stable gels.

[0128] Further characterization of different high internal phase emulsions was performed, and the results are as follows:

[0129] Peak modulus: With the increase of φ, the apparent viscosity and storage modulus (G') of the emulsion both show a trend of first increasing and then decreasing, reaching the highest value in the range of φ=0.70~0.75 (at which point G' exceeds 1000 Pa), indicating that the system has formed the most dense and robust elastic three-dimensional network. When φ>0.80, the viscosity and modulus decline significantly.

[0130] Structural significance: The excellent high storage modulus and significant shear-thinning behavior at φ=0.75 provide excellent structural fidelity and gel self-support for subsequent processing and molding (such as gel extrusion or 3D printing), effectively preventing deformation and collapse after extrusion. The high-strength gel behavior exhibited in the φ=0.70~0.75 range is mainly attributed to the high spatial crowding effect within the system (e.g., Figure 8(As shown in the c-physical model). Because the internal phase volume fraction exceeds the maximum close-packing limit of spheres, the oil droplets are forced to compress tightly against each other, transforming their morphology from a traditional spherical shape to a polyhedral structure. This extreme crowding leads to significant interfacial stretching. The intense deformation between droplets and the extremely high steric hindrance effect together endow the emulsion with solid-like elastic characteristics and high yield stress, thus exhibiting strong gelation behavior on a macroscopic scale. Laser confocal scanning microscopy (CLSM) Figure 8 (b and d) reveal the law that the droplet size increases with the increase of the internal phase volume, which is mainly determined by the game between the interfacial area and the emulsifier concentration: Specific surface area control: Under the premise that the total amount of protein (i.e., interfacial stabilizer) in the aqueous phase is fixed, as the oil phase volume φ continues to increase, the total oil-water interfacial area required by the system expands sharply. Steady-state shear and dynamic rheological tests ( Figure 8 The results of e and f further confirm that the system has the best mechanical strength when φ=0.75.

[0131] Interfacial starvation: As φ increases, the number of protein particles that can be adsorbed per unit interfacial area decreases. In order to minimize the interfacial free energy of the system under limited emulsifier conditions, oil droplets tend to coalesce to a certain extent or maintain a large size, thereby reducing the total specific surface area.

[0132] Structural collapse critical point: At φ=0.75, although the interface is highly stretched, it can still be effectively encapsulated by the protein network and maintain stability. However, when φ exceeds 0.80 (especially at 0.85), severe "interfacial starvation" occurs (e.g., ...). Figure 8 (As shown by the white arrow in the image with φ=0.85 in d), this leads to interfacial film rupture, large-scale abnormal aggregation of oil droplets, and even local phase separation. This is also the fundamental microscopic reason for the sharp drop in rheological properties and the disintegration of the gel network after φ=0.80.

[0133] In summary, φ=0.60~0.80 is preferred, φ=0.70~0.75 is more preferred, and φ=0.75 is the optimal process parameter.

[0134] Example 4: Verification of freeze-thaw resistance and thermal reversibility of transparent high internal phase emulsion

[0135] This embodiment investigates the stability of myofibrillar protein-glycerol-based high internal phase emulsions (MP-glycerol-based HIPEs) under extreme temperature conditions and the role of glycerol in the thermal reversibility and freeze-thaw resistance of the system by comparing the appearance evolution and heat transfer behavior of the glycerol-free group (0G, i.e., 0.96MP-0G) and the high concentration glycerol group (84G, i.e., 0.96MP-84G).

[0136] 1. Verification of thermal reversibility

[0137] MP-glycerol-based HIPEs in the glycerol-free group (0G, i.e., 0.96MP-0G) and MP-glycerol-based HIPEs in the high-concentration glycerol group (84G, i.e., 0.96MP-84G) were heated at 85°C for 30 minutes and then naturally cooled to room temperature.

[0138] The results are as follows Figure 9 As shown in a(i), after high-temperature heating and cooling, the 0G system exhibits a cloudy, milky-white appearance, while the 84G system perfectly recovers and maintains its original high transparency and gel-like appearance, without significant phase separation. This phenomenon confirms the excellent thermal reversibility of the 84G system. During heating, the high temperature may temporarily weaken non-covalent interactions such as hydrogen bonds between protein networks; however, after cooling, the high concentration of glycerol, through its abundant hydroxyl groups (-OH), reforms a dense hydrogen bond network with MP and water molecules, assisting in the rapid reorganization of the interfacial membrane. This strong intermolecular force effectively reduces the aggregation of oil droplets under thermal shock, endowing the system with excellent thermal stability and structural memory function.

[0139] 2. Freeze-thaw resistance verification

[0140] MP-glycerol-based HIPEs in the glycerol-free group (0G, i.e., 0.96MP-0G) and the high-glycerol group (84G, i.e., 0.96MP-84G) were frozen at -20°C for 24 hours and then restored to room temperature. The results showed that the stability of the system exhibited a significant freezing temperature dependence in the freeze-thaw cycle test. Figure 9 As shown in a(ii), after freezing at -20°C for 24 hours and then restoring to room temperature, the 0G system experienced severe demulsification and oil-water separation, while the 84G system also showed some degree of demulsification and yellowing.

[0141] Further freezing conditions were lowered to -80°C for 24 hours, then restored to room temperature, and the results were as follows: Figure 9As shown in a(iii), the 0G system still completely demulsified, but the 84G system maintained a good transparent appearance and gel properties after thawing. This difference is mainly attributed to the change in ice crystal growth kinetics. During the relatively slow freezing process at -20°C, the system remained in the "maximum ice crystal formation zone" for a longer time, resulting in the formation of large ice crystals. The mechanical piercing force of the large ice crystals physically destroyed the adsorption layer of MP at the oil-water interface, triggering irreversible demulsification, which even 84G could not completely resist. However, at ultra-low temperature freezing at -80°C, the system could pass through the maximum ice crystal formation zone very quickly, thus forming only tiny, uniformly distributed ice crystals. At this time, the "cryoprotectant" effect of glycerol in the 84G system was amplified. Glycerol not only significantly lowered the freezing point of the system and reduced the proportion of freezeable water, but its strong water-binding ability also further inhibited the recrystallization growth of ice crystals, thereby maximally protecting the interfacial structure and gel network integrity of the emulsion.

[0142] 3. Infrared thermal imaging technology reveals the heat transfer behavior of glycerol in the system during freeze-thaw resistance verification.

[0143] Through infrared thermal imaging technology ( Figure 9 (b in 9 and c in 9) intuitively reveal the alteration of glycerol's effect on the system's heat transfer behavior. During the cooling process from 85°C to room temperature ( Figure 9 (b) In this context, the 84G system exhibits stronger heat retention than the 0G system, with a more gradual temperature drop. Conversely, during the rewarming process from -20°C to room temperature ( Figure 9 (c) The heating rate of the 84G system is significantly faster than that of the 0G system, and it maintains better internal temperature uniformity at room temperature. This bidirectional temperature regulation phenomenon indicates that the introduction of high-concentration glycerol significantly alters the specific heat capacity and thermal conductivity of the system. Glycerol, as an excellent heat buffer, can effectively slow down the internal transfer rate of heat or cold when the system faces drastic external temperature fluctuations. This heat buffering effect effectively prevents the internal oil droplets and protein network from generating strong mechanical stress due to rapid thermal expansion and contraction, further explaining from a thermodynamic perspective why the 84G system has excellent resistance to thermal shock and freeze-thaw cycles.

[0144] 4. Investigation of the temperature-response rheological behavior and protein conformational stability of MP-glycerol-based HIPEs (0G and 84G)

[0145] Figure 10 Figures a and b in the table show the rheological response of the system after three repeated thermal shocks (cycles from 25 to 85°C). For the glycerol-free control group (0G, ... Figure 10In the first heating cycle (a), G' and G'' experienced an irreversible sharp drop; in subsequent cooling and the second and third cycles, the modulus could not recover to the initial level, and there was a significant hysteresis between the cycle curves. Conversely, the 84G system with added high concentrations of glycerol (a) showed a significant improvement. Figure 10 In (b), G' and G'' decrease slightly upon heating, exhibiting thermal softening, but recover almost perfectly to their initial values ​​upon cooling. The rheological curves of the three heating-cooling cycles highly overlap. The rheological failure of the glycerol-free system indicates that high temperature leads to irreversible excessive denaturation and aggregation of myofibril proteins at the oil-water interface, resulting in the complete collapse of the physical cross-linking network of the interfacial film and causing macroscopic destruction of the emulsion structure. The perfect thermal reversibility exhibited by the 84G system is attributed to the robust hydrogen-bonded network constructed by glycerol as a co-solvent in the system. The introduction of glycerol increases the viscosity of the continuous phase and strengthens the hydration layer at the interface. This solvent effect effectively inhibits the intense thermal motion and excessive unfolding of proteins at high temperatures, allowing the interfacial network to undergo only reversible segment relaxation upon heating and rapid reorganization upon cooling, thus endowing the soft matter material with excellent thermomechanical fatigue resistance.

[0146] Figure 10 Figure 'c' reveals the structural evolution of the system during the freezing process. At -20°C, the 0G system exhibits an extremely high G', behaving as a hard, solid frozen substance. When the temperature crosses the melting zone (near 0°C), its modulus undergoes a catastrophic decrease, dropping by several orders of magnitude. After thawing, G' falls to an extremely low level, indicating complete emulsion demulsification. In contrast, the 84G system has a significantly lower modulus at -20°C than the 0G group, and after crossing the 0°C melting zone, G' only decreases slightly and remains at a high level (G'>G''), maintaining a stable elastic gel network after thawing. This rheological difference directly reflects the fatal impact of ice crystal formation on the physical stability of the emulsion. During freezing, the 0G system experiences a large amount of free water crystallization, forming sharp, large ice crystals that physically pierce the protein membrane adsorbed at the interface. After thawing, the unprotected oil droplets rapidly coalesce, leading to rheological liquefaction and macroscopic demulsification. In the 84G system, glycerol, through its extremely strong water-holding capacity, forms tight hydrogen bonds with water molecules, significantly lowering the freezing point of the system and converting a large amount of freezeable water into unfrozen bound water. This not only inhibits the nucleation and growth of macroscopic ice crystals but also reduces the osmotic pressure impact of the freeze-concentration effect on interfacial proteins, thus perfectly preserving the microstructure and macroscopic rheological properties of the high internal phase emulsion after thawing.

[0147] The fluorescence centroid (BCM) of intrinsic fluorescence spectra reflects the polarity changes of the tryptophan microenvironment in proteins. With increasing temperature, the OG system ( Figure 10The BCM of d) in the model exhibits a significant redshift, with its first derivative curve showing multiple distinct peaks near 35℃, 52℃, 62℃, and 72℃. Meanwhile, the 84G system ( Figure 10 The redshift trend of (e) in the graph is significantly weakened, and the derivative peak is significantly reduced and becomes flat. The final △BCM histogram ( Figure 10 f) further confirms that the total redshift of the 84G system (approximately 3 nm) during the entire heating process is significantly lower than that of the 0G system (approximately 4.5 nm). The redshift of the BCM represents the transition of the protein microenvironment from hydrophobic to hydrophilic, i.e., the protein unfolds, exposing internal hydrophobic residues to the polar aqueous phase. The multi-peak phenomenon of the derivative curve of the 0G system reveals that myofibrillar proteins undergo complex, multi-stage structural dissociation and denaturation in an unprotected state. In the 84G system, glycerol, through preferential hydration, repels itself from the protein surface, forcing the protein to maintain a more compact and folded native conformation. This thermodynamic conformational stabilization greatly limits the exposure of hydrophobic domains and subsequent irreversible hydrophobic aggregation of interfacial proteins caused by heat. This spectroscopic evidence at the molecular level also echoes and explains... Figure 10 The macroscopic rheological thermal reversibility and extremely high structural stability of b and c in the model.

[0148] Example 5: Effect of glycerol substitution rate on storage stability and microbial safety of the system

[0149] To further evaluate the impact of high glycerol substitution rates on the storage stability and microbiological safety of the system, the water activity (α) of the control group (0.96 MP-0 G) without glycerol addition and the optimal formulation transparent experimental group (0.96 MP-84 G) was compared. w Quantitative determination was performed.

[0150] The results are as follows Figure 11 As shown, the control group (0.96 MP-0G) without added glycerol had a w A value of 0.99 indicates that almost all of its internal water is in a free state. In contrast, when the mass fraction of glycerol in the aqueous phase reaches 84%, the water activity of the transparent group (0.96 MP - 84 G) decreases significantly (p < 0.05). w The value dropped to approximately 0.43. This significant decrease is attributed to the formation of a highly dense hydrogen bond network between the abundant hydroxyl groups in the high-concentration glycerol molecules and water molecules and myofibrillar proteins. A large amount of free water is tightly bound and converted into bound water, thus greatly reducing the amount of water available for biochemical reactions and microbial utilization in the system. From a practical application perspective in food science, water activity is a key thermodynamic parameter determining the shelf life and safety of food. The vast majority of spoilage bacteria grow in water activity at an a... w The lower limit is 0.90, while the proliferation of conventional molds and osmotic-tolerant yeasts usually requires a wAbove 0.60. The extremely low water activity of approximately 0.43 in the 0.96MP-84G system means that this system constructs an extremely secure barrier through the control of water activity without relying on external chemical preservatives. In this microenvironment, putrefactive microorganisms and pathogens are virtually unable to carry out any form of metabolism or proliferation.

[0151] In summary, the 0.96MP-84G transparent system not only possesses excellent rheological properties and the ability to encapsulate hydrophobic active substances, but also exhibits natural antiseptic and antibacterial characteristics. This is of core value for developing this transparent system into a functional 3D printing food ink, effectively overcoming the spoilage defects of traditional high-moisture emulsion gel inks, achieving long-term stable storage under room temperature conditions, and facilitating its future large-scale commercial application.

[0152] Example 6: Preparation of MP-glyceroyl HIPEs with ergosterol embedded in the oil phase

[0153] Based on the optimal formulation (0.96MP-84G aqueous phase, φ=0.75 oil phase MCT), 1.0 wt% of the functional component ergosterol (EGT) was encapsulated in the oil phase to prepare MP-glycerol-based HIPEs loaded with EGT. MP-glycerol-based HIPEs without encapsulated EGT were used as a blank group to evaluate the effect of encapsulation on the stability and functional properties of the emulsion.

[0154] Specifically, weigh 0.1000 g of ergosterol and dissolve it in 10 mL of MCT. Stir magnetically at 300 rpm for 30 min at 4°C until completely dissolved to obtain a 1.0 wt% EGT-MCT stock solution. Keep the total emulsion volume at 16 mL, and slowly inject 12 mL of EGT-MCT stock solution (or blank MCT) into 4 mL of 0.96MP-84G aqueous phase using a syringe (aqueous phase at the bottom, oil phase above aqueous phase). Avoid vigorous shaking to prevent air bubbles from being introduced. Homogenize using a high-speed shear press at 10000~12000 rpm for 120 s, and let stand at 25°C for 30 min until the air bubbles are completely eliminated.

[0155] The results are as follows Figure 12As shown in Figure a, after embedding 1.0 wt% ergosterol in the oil phase, the HIPEs maintained extremely high transparency and homogeneity, without any turbidity caused by solute precipitation or phase separation. In the inverted test, the loaded group also adhered tightly to the bottom of the tube, exhibiting excellent self-supporting solid gel characteristics consistent with the blank group. This result indicates that ergosterol can be completely dissolved and stably confined within continuous MCT oil droplets. 0.96 MP-84 G of HIPEs can effectively load 1.0 wt% ergosterol while retaining its original excellent optical transparency and gel rheological properties to a large extent. This structural inertness demonstrates that this system is an ideal, highly stable hydrophobic bioactive substance delivery carrier.

[0156] Further rheological characterization provided quantitative evidence for the structural stability of the system. Figure 12 Steady-state shear tests of b in the figure show that, after loading ergosterol, the shear-thinning behavior and apparent viscosity curve of the system almost perfectly coincide with those of the unloaded blank group. Similarly, in Figure 12 In the dynamic frequency scan of c, the G' and G'' curves of the two groups of samples highly overlapped, and the modulus values ​​did not show significant shifts or fluctuations. The high consistency of viscosity and dynamic viscoelasticity before and after embedding has important rheological and engineering significance.

[0157] From a colloid chemistry perspective, this demonstrates that the hydrophobicity of ergosterol allows it to spontaneously locate at the core of the oil droplet, without migrating to the oil-water interface to interfere with the conformational arrangement of MP, nor disrupting the tight hydrogen bond network between glycerol, water, and protein.

[0158] Example 7: Application of transparent high internal phase emulsions (MP-glycerol-based HIPEs) in the food industry

[0159] This embodiment focuses on evaluating the molding ability and long-term stability of transparent high internal phase emulsions (MP-glycerol-based HIPEs) as 3D printing "inks", as well as their application potential in complex structures and biomimetic food customization.

[0160] like Figure 13 As shown in Figure a, MP-glycerol-based HIPEs inks can successfully print three-dimensional structures with complex geometric features (such as pyramids and pentagrams) as well as intricate custom patterns (“NJAU”, a puppy model). The macroscopic dimensions and outlines of all printed products closely match the preset digital models, with no obvious edge collapse or deformation. Furthermore, this system exhibits excellent plasticity (…). Figure 13The glycerol-based HIPEs (g) can perfectly fill containers with different bottom shapes; after demolding and leaving at room temperature for 3 days, they still maintain their three-dimensional morphology without collapsing. This high fidelity is attributed to the excellent rheological properties of MP-glycerol-based HIPEs. During extrusion, the ink exhibits significant shear-thinning behavior, allowing it to pass smoothly through the fine printing nozzle; after extrusion, the system can quickly undergo structural reorganization, restoring its high storage modulus (G') and yield stress. It is this rapid solid-liquid state switching and strong self-supporting network that endow the deposited layer with the ability to resist gravity and the extrusion of the upper material, thereby ensuring the accurate construction of complex shapes and long-term shape memory. To meet the visual requirements in food 3D printing, different food-grade pigments are loaded into the system. Inverted bottle test ( Figure 13 (d) indicates that the stained system also did not flow, exhibiting a gel-like self-supporting characteristic. More importantly, the stained MP-glycerol-based HIPEs did not show any layering or fading after being left at room temperature for one month. Figure 13 The letter 'e' in the name of the letter can accurately print vivid 3D letters. Figure 13 (f in the figure). This result indicates that the addition of pigments did not disrupt the microstructure of the internal micronetwork of MP-glycerol-based HIPEs. Its one-month physical stability confirms that myofibrillar proteins formed a dense and strong interfacial film at the oil-water interface, effectively inhibiting droplet aggregation and Oswald ripening. This provides a stable material basis for subsequent multi-color, multi-material combination printing. Using a coaxial 3D printing system ( Figure 13 (b) In this study, we successfully achieved efficient construction of core-shell structures. By adjusting the extrusion parameters, the diameter ratio of the red core to the transparent shell (0.1~0.9:0.1~0.9) can be precisely controlled, resulting in highly customized mesh and tubular structures. Figure 13 (c) Furthermore, alternating dual-nozzle printing successfully constructed a clear, layered structure of alternating black and red colors. Figure 13 (h in the text), while a single nozzle achieves a continuous gradient transition of color (h). Figure 13 (i) The core-shell ratio in coaxial printing is precisely adjustable, demonstrating the highly controllable fluid behavior of this MP-glycerol-based HIPEs system during laminar extrusion. This is significant in the food industry. In multi-material alternating printing, the boundaries between layers are distinct, with no obvious color penetration or fusion, further confirming that this gel-like ink system possesses sufficiently high viscosity and yield stress to limit material diffusion. Based on the above high-precision multi-material printing capabilities, a biomimetic "pork belly" model was successfully constructed using a dual-nozzle system. Figure 13(j) Transparent MP-glycerol-based HIPEs perfectly simulate the visual texture of the fat layer, while MP-glycerol-based HIPEs with added red pigment realistically reproduce the lean meat layer, achieving integrated molding of multiphase biomimetic foods. The combination of 3D printing and MP-glycerol-based HIPEs not only visually achieves the alternating distribution of "fat-lean meat," but also provides a feasible technical path for customizing different nutritional components, textural properties, and even flavor substances in different layers in the future, demonstrating its enormous application potential in the development of engineered meat products.

[0161] This invention provides a transparent high internal phase emulsion, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a transparent emulsion with a high internal phase, characterized in that, Includes the following steps: (1) Disperse myofibrillar protein MP in pure water and stir to prepare a modified myofibrillar protein base solution; (2) Add glycerol to the modified myofibrillar protein base solution obtained in step (1), stir, and prepare an MP-water-glycerol mixed solution; (3) Medium-chain triglycerides were added to a mixed solution of MP-water-glycerol, and after homogenization, a transparent high internal phase emulsion was prepared. The medium-chain triglyceride has a refractive index RI of 1.4520 at 25°C, and the refractive index of the MP-water-glycerol mixed solution at 25°C has an error of ≤0.006% compared with that of the medium-chain triglyceride.

2. The preparation method according to claim 1, characterized in that, In step (1), the protein concentration of myofibrillar protein MP in pure water is 20~80 mg / mL; preferably, the protein concentration of myofibrillar protein MP in pure water is 60 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step (1), the myofibrillar protein MP is dispersed in pure water and the pH is adjusted to 3.0±0.

2.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass concentration of the glycerol is 80-84%; preferably, the mass concentration of the glycerol is 84%.

5. The preparation method according to claim 1, characterized in that, In steps (1) and (2), the stirring is carried out at 4°C for 3 to 12 hours.

6. The preparation method according to claim 1, characterized in that, In step (3), the volume fraction of the medium-chain triglycerides is φ = 0.60~0.80 based on the total volume of the transparent high internal phase emulsion; preferably, the volume fraction of the medium-chain triglycerides is φ = 0.70~0.75; more preferably, the volume fraction of the medium-chain triglycerides is φ = 0.

75.

7. The preparation method according to claim 1, characterized in that, In step (3), the homogenization process is performed at 10,000 to 12,000 rpm for 120 seconds.

8. A transparent emulsion with a high internal phase, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the transparent high internal phase emulsion of claim 8 as a carrier in the preparation of special environmental foods; in, The special environmental foods mentioned are aerospace foods, polar research foods, or emergency rescue foods.

10. The application of the transparent high internal phase emulsion of claim 8 as a 3D printing ink in the preparation of 3D food.

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