A hyaluronic acid-micellar casein concentrate hybrid particle and its use in emulsion gels

Abstract

The application discloses a hyaluronic acid-micellar casein concentrate hybrid particle and application thereof in emulsion gels, and belongs to the field of new materials. The HA-MCC hybrid particle with excellent performance is prepared by using micellar casein concentrate and sodium hyaluronate with a certain molecular weight based on a green industrial technology which is very simple, fast and environment-friendly. The high internal phase emulsion gel with excellent rheological performance, mechanical performance and 3D printing performance is prepared by using the HA-MCC hybrid particle. The HA-MCC hybrid particle and the high internal phase emulsion gel prepared by the application have good application prospects in various fields such as food, health products, cosmetics, daily chemical products and biological medicines, and provide great potential for the combination of 3D printing and food science.

Description

Technical Field

[0001] This invention relates to a hyaluronic acid-micelle casein concentrate hybrid particle and its application in emulsion gels, belonging to the field of new materials. Background Technology

[0002] O / W emulsions and O / W high internal phase emulsion gels have broad development prospects in the food, pharmaceutical, daily chemical, and petrochemical industries. However, due to their thermodynamic instability, emulsion systems are prone to emulsification, flocculation, aggregation, and Ostwald ripening, leading to instability and hindering their application in various fields. To stabilize emulsion systems, it is necessary to rationally select high-performance particulate emulsifiers. The physicochemical and structural characteristics of particulate emulsifiers control their adsorption and assembly at the fluid interface, which is crucial for stabilizing emulsions and foams. Soft particles with excellent interfacial properties possess high surface activity and emulsifying characteristics, enabling them to quickly adsorb onto the fluid interface and deform and collapse to form a viscoelastic interfacial layer, providing a mechanical barrier and thus achieving the stability of foams and emulsions.

[0003] Milk, a natural biological fluid developed through biological evolution, can fix more calcium and inorganic phosphorus (Pi) without causing pathological calcification of the mammary glands. To prevent calcium phosphate precipitation, calcium-sensitive caseins (αs1-, αs2-, and β-casein) fix amorphous calcium phosphate (ACP) nanoclusters, forming a complex hierarchical assembly called casein micelles. Highly hydrated micelles are porous structures with numerous water-filled cavities and channels, exhibiting a polydisperse size distribution of 40% and an average diameter of 100–200 nm. α-casein is present throughout the entire structure of the casein micelles, β-casein is mainly located within the micelles, while trichotic κ-casein is primarily found on the surface region of the micelles to stabilize them. Casein, a traditional dairy ingredient, is produced by skimming fresh milk and then adding acid (lactic acid, acetic acid, hydrochloric acid, or sulfuric acid) to adjust the pH to 4.8. This causes the casein microparticles to lose their charge and coagulate and precipitate. Before use, it requires the addition of alkali to adjust the pH to an alkaline environment to dissolve the casein. During this process, most of the casein loses its micellar structure, becoming a mixture of different casein components (αs1-, αs2-, β-, and κ-casein). Compared to whey protein, which has superior emulsifying properties, the application and development of traditional dairy casein are relatively limited. With advancements in membrane separation technology, the industrialization of micellar casein has gradually reached a significant scale. Micellar casein retains the original micellar structure of milk and does not require pH adjustment to an alkaline environment to dissolve. It possesses emulsifying and foaming properties comparable to whey protein, and has a wider range of applications in food, pharmaceuticals, health products, and chemical industries. More importantly, compared to whey protein, a byproduct of cheese processing, milk protein concentrates (MPC) and casein micelles are gradually becoming better choices.

[0004] High-internal-phase emulsions (HIPEs) have broad development potential and application space in the daily chemical, food and pharmaceutical, and even petrochemical industries. HIPEs can be used to prepare porous materials with different properties and can be applied in some high-tech fields closely related to people's lives, such as filter membranes, organic semiconductors, and tissue engineering scaffolds. They can also serve as carriers for food and pharmaceuticals to deliver and sustain the release of active substances in the human body. With the increasing demand for "clean labels," the application of some inorganic and chemically synthesized high-internal-phase emulsions in the food industry has been greatly limited. However, using natural materials as particulate emulsifiers not only offers advantages such as low cost, low dosage, high safety, and low environmental pollution, but also endows HIPEs with excellent emulsification stability, resulting in a wide range of applications and high development potential. Currently, a key challenge is to find a more energy-efficient, environmentally friendly, simple, time-saving, and labor-saving method for preparing food-grade particulate emulsifiers, and the produced particulate emulsifiers should possess excellent performance, high safety, and the ability to achieve industrial-scale production and commercial application. Casein micelles are complex hierarchical assemblies with porous structures. After adsorption to the interface, they exhibit greater deformation potential, making them excellent particulate emulsifiers. Chinese patent application CN118476605A discloses MPC rich in casein micelles as a type of heterogeneously cross-linked gel particle (with regional differences in cross-linking density within the particle). It combines the advantages of both hard and soft particles (i.e., regions with higher cross-linking density resemble hard particles, while regions with lower cross-linking density resemble soft particles), while also exhibiting spontaneous adsorption and limited compliance (the stiffness / softness of the particles is adjustable). Since interfacial adsorption does not present a significant energy barrier, it is beneficial for improving foaming ability and emulsifying activity. Furthermore, limited compliance can counteract Ostwald ripening, thereby achieving long-lasting emulsion stability.

[0005] With the further integration of 3D printing technology and food science, the creation of personalized and structurally complex foods presents greater challenges to the performance of food materials. Among various printable materials, high-internal-phase emulsion gels (HIPE gels) stand out due to their high oil content and tunable rheological properties, making them promising candidates for food-grade 3D printing. However, the widespread adoption of HIPE emulsion gels in practical applications is hindered by mechanical weaknesses, rheological challenges, and environmental sensitivity. Protein-stabilized food-grade HIPE gels are highly valued for their natural emulsifying properties and compatibility with clean label formulations. However, their use in 3D printing is limited by insufficient mechanical strength, which compromises the stability of printed structures and leads to problems such as layer collapse. Balancing shear-thinning behavior during extrusion and rapid recovery after deposition remains difficult, further affecting structural integrity. In addition, environmental factors such as pH, temperature, and ionic strength can unpredictably affect gelation and stability.

[0006] Traditional strategies for improving the properties of HIPE emulsion gels, such as chemical modification, thermal processing, and ionic crosslinking, often compromise the material's natural functions or bioactivity. Furthermore, weak interlayer adhesion remains a significant challenge, hindering the fabrication of robust and visually appealing structures. HIPE emulsion gels offer a wide range of applications, superior performance, and a high internal phase (oil phase) content, which is particularly important for printing complex and finely shaped food products. The stability and plasticity of high-internal-phase emulsion gels make them ideal materials for 3D food printing, meeting the high performance requirements of food printing.

[0007] The prior art (Emulsions stabilized by casein and hyaluronic acid: Effects of high intensity ultrasound on the stability and digestive characteristics of vitamin E. Ultrasonics Sonochemistry, 94.) discloses an emulsion stabilized by an ultrasonically treated casein (CAS)-hyaluronic acid (HA) complex. This is achieved by preparing a CAS solution (3%, w / v), adjusting the pH to 7.0 with 1.0 mol / L NaOH, and then mixing the CAS solution with HA (194 kDa) powder to obtain a CAS-HA complex with an HA concentration of 0.5% (w / v) as the aqueous phase, and using soybean oil as the oil phase, dissolving vitamin E in the soybean oil. The existing technique of preparing emulsions with an oil / water volume ratio of 20 / 80 has the problem of inconsistent casein composition in traditional dairy ingredients. That is, the casein raw material needs to be dissolved under alkaline conditions. As mentioned earlier, the composition of such raw materials is uncontrollable in each batch in the industry. In addition, this study only reported an oil phase composition of 20%. The preparation of emulsions with a higher oil phase may require further cross-linking of the protein in the aqueous phase or the addition of more food stabilizers.

[0008] Existing technology (Fabrication of flavor oil high internal phase emulsions by casein / pectin hybrid particles: 3D printing The paper "performance.FoodChemistry.2022Mar;371:131349.DOI:10.1016 / j.foodchem.2021.131349" discloses the preparation of a 3D printable oil / water (O / W) high internal phase emulsion (75% internal phase fraction) using casein / pectin hybrid particles and flavor oil. The pH of a 3% (w / v) casein solution was adjusted to 11. Pectin was added to an aqueous phase containing 3% (w / v) casein and pectin at concentrations of 1%, 2%, 3%, 4%, and 5%, respectively. The pH of the solution was adjusted to 3. Flavor oil was added to the casein-pectin mixture to obtain a mixture with an aqueous phase:oil phase mass ratio of 1:3. After homogenization, the high internal phase emulsion was obtained. This existing technology still uses traditional dairy ingredient casein, requiring the raw material to be dissolved at pH 11. As mentioned earlier, the composition of such raw materials is uncontrollable in each batch in industrial applications. Furthermore, this existing technology only reports obtaining proteoglycan complexes in solutions at pH 3. In reality, pectin itself is extremely complex; it is a structurally complex anionic polysaccharide extracted from the cell walls of higher plants. Different plant sources and preparation processes significantly affect the molecular structure and functional properties of pectin, including monosaccharide composition, domain ratio, degree of esterification, and branching. Generally, pectin is more stable in acidic solutions than in alkaline solutions, and it is difficult to dissolve in pure water, requiring heating to around 60°C to dissolve. Furthermore, when this pectin is dissolved in pure water at a concentration of 1%, the solution pH is approximately 3.3. This is relatively unfavorable for research on proteoglycans. Given the isoelectric point of proteins, a polysaccharide solution at around pH 3 acts as a precipitant for proteins with isoelectric points of around 2-4. Although the pH can be adjusted to near neutral by adding alkali, acid-base neutralization will generate more salts. For protein research, the introduction of salts leads to more questions, such as whether it causes salting-out or salting-out effects. In protein research, the type (cations, anions), valence state (monovalent, divalent, trivalent, etc.), and strength of ions all have complex effects on the structure and properties of proteins. The casein-pectin solution reported in this prior art, which can stabilize 75% of the oil phase, may only be stable in a solution at pH 3. This overly narrow pH stability range severely limits the application of this system, even in the food industry.

[0009] Prior Art (Development and characterization of a casein-hyaluronic acidemulsion gel with high water-holding capacity and excellent rheologicalproperties for 3D printing.Food Hydrocolloids (https: / / doi.org / 10.1016 / j.foodhyd.2023.108632) discloses a method to enhance the rheological properties and printability of casein (CAS) emulsion gels by adding hyaluronic acid (HA). The method involves dispersing CAS powder in water, adjusting the pH to 7.0 with NaOH, and preparing a casein solution (8%, w / v). The CAS solution is then brought to 500 mL with water. Different amounts of HA powder are added to the CAS solution (30 mL) at concentrations of 0.02, 0.04, 0.06, 0.08, 0.1, and 0.2% w / v, forming an aqueous phase. The oil / water phase volume ratio is controlled at 20 / 80 to obtain an emulsion. Finally, GDL (4%, w / v) is added to obtain the emulsion gel. This prior art still uses traditional casein as a raw material, requiring the addition of NaOH to dissolve the raw material. As mentioned earlier, the composition of such a mixture is uncontrollable in each batch in industrial applications. In addition, in order to obtain stable interfacial emulsion particles, this prior art requires crosslinking with GDL to further stabilize the emulsion with an oil phase of 20%. The addition of the crosslinking agent will solidify the structure of the interfacial emulsion particles, thus limiting the adjustability of the emulsion interfacial properties.

[0010] In summary, current technologies primarily revolve around casein, a traditional dairy ingredient. As a raw material with difficult-to-control batch stability, it faces numerous technical challenges in use. Most studies either only stabilize a low oil phase ratio, or require conjugation with polysaccharides to achieve a higher oil phase ratio at specific pH values, or necessitate the addition of additional cross-linking agents to obtain better stability. Therefore, to facilitate the preparation of emulsion gels with broader applications, it is necessary to design a clean-label-grade food-grade soft particle capable of long-term stable high internal phase emulsion gels. Summary of the Invention

[0011] To address the aforementioned issues, this invention utilizes micellar casein concentrate (MCC) and sodium hyaluronate (HA) of a certain molecular weight to prepare HA-MCC hybrid particles with excellent properties using a very simple, rapid, and environmentally friendly green industrial technology. These HA-MCC hybrid particles are then used to prepare high-internal-phase emulsion gels with excellent structural, rheological, mechanical, and 3D printing properties. This invention offers significant potential for combining 3D printing with food science.

[0012] This invention provides a method for preparing hyaluronic acid-micelle casein concentrate hybrid particles (HA-MCC hybrid particles) using micellar casein concentrate (MCC) and sodium hyaluronate (HA). The method comprises:

[0013] Micellar casein concentrate (MCC) was dispersed in water, mixed, and homogenized; sodium hyaluronate (HA) was added and stirred to obtain HA-MCC hybrid particles.

[0014] In one embodiment of the present invention, the final concentration of the micelle casein concentrate (MCC) in water is 1% to 5% (w / w);

[0015] Preferably, the final concentration is 5% (w / w).

[0016] In one embodiment of the present invention, the mixing conditions are: mixing at 40–60°C for 0.5–3 hours;

[0017] Preferably, the mixture is mixed at 50°C for 2 hours.

[0018] In one embodiment of the present invention, the homogenization conditions are homogenization at a pressure of 0 to 150 MPa (but not 0), and homogenization is performed 3 to 6 times.

[0019] Preferably, the homogenization is performed three times at 90 MPa.

[0020] In one embodiment of the present invention, the molecular weight of the sodium hyaluronate is 100-1090 kDa;

[0021] Preferably, the molecular weight of the sodium hyaluronate is 200–600 kDa;

[0022] More preferably, the sodium hyaluronate has a molecular weight of 400 kDa.

[0023] In one embodiment of the present invention, the amount of sodium hyaluronate added is 0 to 1.0% (w / v) (but not 0);

[0024] Preferably, the amount of sodium hyaluronate added is 1.0% (w / v).

[0025] In one embodiment of the present invention, the stirring conditions are 200-800 rpm for 8-12 hours.

[0026] The present invention also provides HA-MCC hybrid particles prepared by the above preparation method.

[0027] This invention also provides the application of the above-mentioned HA-MCC hybrid particles in the fields of food, health products, cosmetics, daily chemical products or biomedicine. The application includes encapsulating and delivering bioactive substances, gelling liquid oils, or preparing emulsions, foams, emulsion gels or high internal phase emulsion gels, etc. It also includes further using these materials in the preparation of food, health products, biomedicine, daily chemical products, etc., which helps to create advanced materials with structural hierarchy and complex interface functions.

[0028] In one embodiment of the present invention, the food includes, but is not limited to, mayonnaise, salad dressing, etc.

[0029] This invention also provides a method for preparing emulsion gels using the above-mentioned HA-MCC hybrid particles, the method being:

[0030] Using the above-mentioned HA-MCC hybrid particles as the aqueous phase, the aqueous phase and oil phase were mixed and dispersed by high-speed shearing to obtain an emulsion gel.

[0031] In one embodiment of the present invention, the volume ratio of the oil phase to the water phase is (0:100) to (85:15), but not 0.

[0032] In one embodiment of the present invention, the rotational speed of the high-speed shearing is 10,000 to 12,000 rpm.

[0033] This invention also provides a method for preparing high internal phase emulsion gels using the above-mentioned HA-MCC hybrid particles, the method being:

[0034] Using the above-mentioned HA-MCC hybrid particles as the aqueous phase, the aqueous phase and oil phase are mixed and dispersed by high-speed shearing to obtain a high internal phase emulsion gel; the volume ratio of the aqueous phase to the oil phase is (0:100) to (15:85), but not 0.

[0035] In one embodiment of the present invention, the volume ratio of the aqueous phase to the oil phase is 1:3.

[0036] In one embodiment of the present invention, the aqueous phase and the oil phase are mixed by dripping the aqueous phase into the oil phase at a rate of 50-70 drops / minute.

[0037] In one embodiment of the present invention, the rotational speed of the high-speed shearing is 10,000 to 12,000 rpm.

[0038] In one embodiment of the present invention, the oil phase includes, but is not limited to, soybean oil, corn oil, rapeseed oil, sunflower seed oil, palm oil, castor oil, conjugated linoleic acid, and linolenic acid.

[0039] In one embodiment of the present invention, the oil phase may also contain lipophilic food nutrients such as curcumin, β-carotene, astaxanthin, lycopene, vitamin E, n-3 fatty acids, and vitamin A.

[0040] The present invention also provides an emulsion gel or a high internal phase emulsion gel obtained by the above preparation method.

[0041] The present invention also provides the application of the above-mentioned high internal phase emulsion gel in the carrier of lipid-soluble active ingredients.

[0042] In one embodiment of the present invention, the fat-soluble active ingredients include, but are not limited to, curcumin, β-carotene, astaxanthin, lycopene, vitamin E, n-3 fatty acids, vitamin A, resveratrol, and capsaicin.

[0043] The present invention also provides the application of the above-mentioned HA-MCC hybrid particles or the above-mentioned high internal phase emulsion gel in 3D printed food.

[0044] In one embodiment of the present invention, the application is to use a 3D printer to perform emulsion gel 3D printing using the above-mentioned high internal phase emulsion gel.

[0045] In one embodiment of the present invention, when using a 3D printer to perform emulsion gel 3D printing in the application, the 3D printing model is a cuboid (24.00mm×24.00mm×15.00mm); the nozzle diameter is 0.8mm, the printing speed is 28mm / s, and the printing is carried out at room temperature.

[0046] The present invention also provides a 3D printed food based on the above-mentioned high internal phase emulsion gel, wherein the food is obtained by 3D printing HA-MCC hybrid particles prepared by the above method or the above-mentioned high internal phase emulsion gel.

[0047] In one embodiment of the present invention, the food is an easily swallowable food.

[0048] Beneficial effects:

[0049] (1) The HA-MCC hybrid particles prepared by this invention have good interfacial properties.

[0050] (2) The present invention can prepare emulsion gels using HA-MCC hybrid particles, with an internal phase (oil phase) fraction of 0 to 85%; the present invention can prepare high internal phase emulsion gels using HA-MCC hybrid particles, with an internal phase (oil phase) fraction of up to 80%, which have excellent emulsifying and foaming properties.

[0051] (3) The emulsion gel prepared by the present invention does not require the addition of additional cross-linking agents and can be used to prepare clean label food; it is economical, low-cost, simple to implement and industrialized; the preparation method of the present invention is very simple, fast and environmentally friendly.

[0052] (4) The hybrid particles and emulsion gels prepared by the present invention using micellar casein concentrate (MCC) have wide applications in the food industry. They can be used not only in alkaline systems, but also in neutral systems or acidic conditions.

[0053] (5) This invention develops a HA-MCC-stabilized high internal phase emulsion gel with enhanced properties, capable of creating complex 3D printed shapes, enabling the creation of food products with cultural and aesthetic significance. This invention offers enormous potential for combining 3D printing with food science, making complex and customized food design possible.

[0054] (6) The high internal phase emulsion gel prepared by the present invention can be used as a carrier for encapsulating fat-soluble active ingredients such as curcumin, thereby improving bioavailability and having wide application value in food delivery systems.

[0055] (7) The high internal phase emulsion gel of the present invention shows potential application prospects in the care of dysphagia. Through food 3D printing technology, it solves the problem of easy-to-swallow food paste / mud, improves the eating pleasure of people with dysphagia, increases oral intake, and significantly improves quality of life. The gel of the present invention provides individuals with dysphagia with a safe, nutritionally balanced, and visually appealing choice, enhancing safety and dining experience. Attached Figure Description

[0056] Figure 1 This is a graph showing the FTIR test results; where MCC is micellar casein concentrate; L-HA is HA with a molecular weight of 100 kDa; H-LA is HA with a molecular weight of 400 kDa; L-HA 0.1 -MCC refers to the HA-MCC hybrid particles prepared in Example 4 using HA with a molecular weight of 100 kDa and an addition amount of 0.1%; L-HA 1.0 -MCC refers to the HA-MCC hybrid particles prepared in Example 2 using HA with a molecular weight of 100 kDa and an addition amount of 1.0%; H-HA 0.1 -MCC refers to the HA-MCC hybrid particles prepared in Example 3 using HA with a molecular weight of 400 kDa and an addition amount of 0.1%; H-HA 1.0 -MCC refers to the HA-MCC hybrid particles prepared in Example 1 using HA with a molecular weight of 400 kDa and an addition amount of 1.0%.

[0057] Figure 2 Scanning electron microscopy images of MCC lyophilized solution and HA-MCC hybrid particles; where M is micellar casein concentrate; L-HA / M 0.1 Example 4 uses HA with a molecular weight of 100 kDa, added at a concentration of 0.1%, to prepare HA-MCC hybrid particles; L-HA / M 1.0 Example 2 uses HA with a molecular weight of 100 kDa, added at a concentration of 1.0%, to prepare HA-MCC hybrid particles; H-HA / M 0.1 Example 3 uses HA with a molecular weight of 400 kDa, added at a concentration of 0.1%, to prepare HA-MCC hybrid particles; H-HA / M 1.0 The HA-MCC hybrid particles prepared in Example 1 were prepared using HA with a molecular weight of 400 kDa and an addition amount of 1.0%.

[0058] Figure 3 The image shows the visual appearance of the emulsion gel obtained in Comparative Example 4.

[0059] Figure 4 Figure 1 shows the rheological properties of HIPE gel; (a) the relationship between apparent viscosity and shear rate; (b) the three-stage incremental shear thinning test (3-ITT); (c) the creep recovery curve; (d) the scanning storage modulus (G′); (e) the scanning loss modulus (G″); and (f) the average loss tangent (tanδ). MCC is a high internal phase emulsion gel stabilized only with micellar casein concentrate; L-HA 0.1 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles from Example 4 as the aqueous phase; L-HA 1.0 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles (HA molecular weight of 100 kDa) from Example 2 as the aqueous phase; H-HA 0.1 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles from Example 3 as the aqueous phase; H-HA 1.0 -MCC is the emulsion gel prepared in Example 6.

[0060] Figure 5Figure 1 shows the stability analysis results of HIPE gels. Among them, (AE) are the transmittance curves of the high internal phase emulsion gel stabilized only with micellar casein concentrate (A), the emulsion gel prepared in Example 11 using HA-MCC hybrid particles from Example 4 as the aqueous phase (B), the emulsion gel prepared in Example 11 using HA-MCC hybrid particles from Example 3 as the aqueous phase (C), the emulsion gel prepared in Example 11 using HA-MCC hybrid particles from Example 2 (HA molecular weight of 100 kDa) as the aqueous phase (D), and the emulsion gel sample prepared in Example 6 (E) during centrifugation, reflecting their stability; (F) is a visual comparison of the appearance of HIPE gels captured by LUMiSizer before centrifugation (top row) and after centrifugation (bottom row).

[0061] Figure 6 Visual appearance of HIPE gel (a) and CLSM observation (b); where MCC is a high internal phase emulsion gel stabilized only with micellar casein concentrate; L-HA 0.1 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles from Example 4 as the aqueous phase; L-HA 1.0 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles (HA molecular weight of 100 kDa) from Example 2 as the aqueous phase; H-HA 0.1 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles from Example 3 as the aqueous phase; H-HA 1.0 -MCC is the emulsion gel prepared in Example 6.

[0062] Figure 7 Figure 1 shows the swallowing performance results of HIPE gel; where MCC is a high internal phase emulsion gel stabilized only by micellar casein concentrate; 0.1% 100HA-M is the emulsion gel prepared in Example 11 using HA-MCC hybrid particles from Example 4 as the aqueous phase; 1.0% 100HA-M is the emulsion gel prepared in Example 11 using HA-MCC hybrid particles from Example 2 (HA molecular weight is 100 kDa) as the aqueous phase; 0.1% 400HA-M is the emulsion gel prepared in Example 11 using HA-MCC hybrid particles from Example 3 as the aqueous phase; and 1.0% 400HA-M is the emulsion gel prepared in Example 6.

[0063] Figure 8 Visual appearance images of the different colored HIPE gels obtained in Example 12.

[0064] Figure 9Visual images of HIPE gel 3D printing; (a) the fresh appearance and appearance after standing for 30 and 60 minutes on the gel 3D printed blocks prepared in Examples 6 and 11; (b) images of yellow high internal phase emulsion gel 3D printed flowers and bows prepared in Example 12; wherein, MCC is a high internal phase emulsion gel stabilized only with micellar casein concentrate; L-HA 0.1 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles from Example 4 as the aqueous phase; L-HA 1.0 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles (HA molecular weight of 100 kDa) from Example 2 as the aqueous phase; H-HA 0.1 -MCC is the emulsion gel prepared in Example 11 using the HA-MCC hybrid particles from Example 3 as the aqueous phase; H-HA 1.0 -MCC is the emulsion gel prepared in Example 6. Detailed Implementation

[0065] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0066] The materials involved in the following embodiments are as follows:

[0067] Micellar casein concentrate (MCC) was purchased from Leprino Foods, Denver, USA, and its composition was: 91.81% protein, 0.00% carbohydrates, 6.46% ash, 5.76% moisture, and 1.64% fat.

[0068] Casein (CAS) was purchased from Shangri-La Kangmei Dairy Development Co., Ltd.

[0069] Pectin was purchased from Sigma-Aldrich Co (St. Louis, America), CAS No.: 9000-69-5;

[0070] The protein concentrate (MPC) was purchased from Fonterra Ltd., New Zealand, model MPC485;

[0071] The soybean oil was purchased from E Hai Kerry Alavana Holdings Limited (Shanghai, China);

[0072] Fast green was purchased from Sigma-Aldrich Co. (St. Louis, America);

[0073] Nile red was purchased from Aladdin Reagent Co., Ltd. (Jinan, China);

[0074] Curcumin was purchased from Shanghai Wokai Biotechnology Co., Ltd. (Shanghai, China).

[0075] The methods involved in the following embodiments are as follows:

[0076] 1. Fourier Transform Infrared Spectroscopy (FTIR)

[0077] The hybrid particles were freeze-dried at -80°C for 48 hours, and their FTIR spectra were recorded using a Frontier, Perkin Elmer spectrometer. Against an air background, all spectra were scanned 32 times in the range of 4000–400 cm⁻¹, and the average value was obtained.

[0078] 2. Measurement of particle size and zeta potential

[0079] Dynamic light scattering (DLS) technology was used to measure the particle size of the hybrid particle solution by performing dynamic light scattering (DLS) at a 90° angle using a 4mW He / Ne laser beam (λ=633nm).

[0080] During measurement, the solution was diluted 50 times with ultrapure water, the pH of the solution was adjusted to neutral, and the refractive indices of the dispersion medium and the protein were set to 1.33 and 1.57, respectively. The zeta potential was measured at 25°C.

[0081] 3. Scanning electron microscopy observation

[0082] The sample was rapidly frozen in liquid nitrogen and freeze-dried at -50°C for 48 hours. The dried sample was placed on a stub with double-sided tape and coated with a 15 nm gold-palladium layer using a sputtering coating machine. The morphology of the fracture section was observed under a scanning electron microscope (TM3030, Hitachi) at an accelerating voltage of 15.0 kV.

[0083] 4. Gel strength test

[0084] The gel strength was determined using a texture analyzer (TA).

[0085] In TPA mode, select the P / 25R probe, and set the pre-measurement, during-measurement, and post-measurement speeds to 1.0, 2.0, and 2.0 mm / s, respectively. Set the compressive strain to 50% and the trigger force to 5.0 g.

[0086] 5. Determination of rheological properties

[0087] The rheological properties of the emulsion gel were evaluated using a 25 mm parallel plate rheometer (MCR 92, Anton Paar).

[0088] The plate gap was set to 1000 μm; viscosity measurements were performed at shear rates from 0.1 to 100 seconds.

[0089] The apparent viscosity versus shear rate curve was fitted using a power-law model (Equation 1) (method reference: Improvement of 3D Printing Performance of Whey Protein Isolate Emulsion Gels by Regulating Rheological Properties: Effect of Polysaccharides Incorporation. Food and Bioprocess Technology. https: / / doi.org / 10.1007 / s11947-024-03488-9).

[0090] τ=Kγ n (1)

[0091] In the formula, τ is the shear force (Pa); K is the consistency coefficient (Pa·s); and γ is the shear rate s. -1 ; n is the mobility exponent (dimensionless), and n represents the deviation from the Newtonian model.

[0092] The linear viscoelastic region (LVR) was determined by scanning the emulsion gel at a frequency of 1.0 Hz with an amplitude of 0.01–10%.

[0093] Frequency scanning was performed in the range of 0.1-10 Hz under a constant strain of 0.1% to capture the change in dynamic modulus in the linear viscoelastic region;

[0094] For creep testing, a constant stress of 10 Pa is applied for 3 minutes, followed by stress removal, which is a 3-minute recovery phase during which shear strain is monitored; the 3-stage thixotropic test (3-ITT) includes a 1-second recovery period. -1 (60 seconds), 100 seconds -1 (5 seconds) and 1 second -1 During the 120-second shear rate phase, the viscosity changes over time.

[0095] Large amplitude oscillating shear (LAOS) test frequency was 1.0 Hz, temperature was constant at 25 ℃, and strain range was 0.1% to 1000%. Raw waveform data at different strains (10%, 100%, 1000%) at 1.0 Hz were collected, and Lissajous curves were obtained using the MITlaos program.

[0096] 6. Determination of water-holding capacity (WHC) and oil-holding capacity (OHC)

[0097] The WHC and OHC of the emulsion gel were measured according to the method described in the prior art (Rheological property, β-carotene stability and 3Dprinting characteristic of whey protein isolate emulsion gels by adding different polysaccharides Food Chemistry. 2023 Jul; 414:135702. DOI:10.1016 / j.foodchem.2023.135702.), with slight modifications:

[0098] The sample (20g) was placed in a 50mL centrifuge tube and centrifuged at 8000rpm for 30 minutes at 4℃. After centrifugation, the water and oil released due to centrifugal force were removed and weighed. The WHC and OHC of the emulsion gel were calculated using equations (2) and (3):

[0099]

[0100] Among them, W T W is the weight of the sample before centrifugation. F and W O These represent the weights of water and oil released after centrifugation.

[0101] 7. Determination of gel stability

[0102] The effects of temperature and centrifugal force on gel stability were investigated using a LUMiSizer 651 dispersion analyzer.

[0103] During the test, near-infrared light (865nm) passed through the sample, and the intensity of the transmitted light was measured as a function of position and time. The conditions were set at 25°C, 3000 rpm, and 150 minutes, with the transmission intensity recorded every 10 seconds. The transmission profile was analyzed using STEP View software (LUM GmbH), and the instability index was calculated.

[0104] 6. Gel microstructure

[0105] The microstructure of the emulsion gel was observed using a confocal laser scanning microscope (CLSM) (TCS SP8, Leica).

[0106] The oil phase (red) was stained with Nile Red, and the protein phase (green) was stained with Fast Green. Nile Red (1% w / v, ethanol) was mixed with the oil phase at a ratio of 1:100 (v / v), and Fast Green (1% w / v, water) was mixed with the protein solution (aqueous phase) at a ratio of 1:100 (v / v). The stained sample was placed in the dark for 30 minutes, and then the stained protein solution (aqueous phase) was mixed with the stained oil phase according to the method in Example 6 or Example 11 to prepare a gel. A small amount of the stained gel sample was placed on a glass slide, spread evenly, and sealed with a cap. The microscope objective was set to 20x, and the image was further magnified by 2x using LAS X software. The excitation wavelengths of Nile Red and Fast Green were 552 nm and 638 nm, respectively.

[0107] 7. Determination of Gel 3D Printing Performance

[0108] Emulsion gel 3D printing was performed using a 3D printer (FOODBOT-S2, Shiyin).

[0109] The 3D printed model is a cuboid (24.00mm×24.00mm×15.00mm); the nozzle diameter is 0.8mm, the printing speed is 28mm / s, and the printing is carried out at room temperature; after printing, the planar dimensions (Ls,mm) and height (H0,mm) of the 3D printed product are measured using vernier calipers, and the 3D printing accuracy (%) is calculated according to formula (4); then the printed product is placed at room temperature (25℃) for 60 minutes, the height (H60,mm) is measured, and the 3D printing stability (%) is calculated using formula (5):

[0110]

[0111] In the formula, L s L represents the planar dimensions of the 3D printed object, in millimeters (mm); m H is the expected size of the 3D printed model, in millimeters; H0 is the initial height of the 3D printed object at minute 0. 60 The height is measured in millimeters after standing for 60 minutes.

[0112] 8. Determination of IDSSI

[0113] Following the International Initiative for Standardization of Dietary Disorders for Dysphagia (IDDSI Framework), the IDDSI testing method was used to perform spoon tilt test, fork drip test and fork pressure test on the samples.

[0114] The spoon tilt test mainly tests the adhesiveness of food. IDDSI indicates that foods with high adhesiveness are not suitable for recommendation to the target population.

[0115] The fork drip test categorizes food into three levels: Level 3, Level 4, and Level 5. Level 3 (liquid) food drips slowly through the fork's slits. Level 4 (pure) food may accumulate on the fork or a small amount may flow through the slits, forming a tail, but it should not drip continuously. Level 5 (moist and finely chopped) food accumulates on the fork, does not easily or not at all flow through the slits, and does not form a tail.

[0116] The fork crush test can distinguish between food of grade 5 and above. In the fork crush test, grade 5 food can be crushed to a suitable size (<4mm) without requiring a force that turns the thumb white (<17kPa). For grade 6 (soft and bite-sized) and grade 7 (regular) food, a greater force is required to crush it to a suitable size and turn the thumb white (>17kPa).

[0117] Example 1: Preparation of HA-MCC hybrid particles

[0118] (1) Disperse micelle casein concentrate (MCC) in deionized water to a final concentration of 5% (w / w) to obtain an MCC solution; mix the MCC solution at 50°C for 2 hours, and then homogenize it 3 times in a high-pressure homogenizer (AMH-3, ATS) with an average pressure of 90 MPa.

[0119] (2) Sodium hyaluronate (HA) with a molecular weight of 400 kDa was added to the homogenized MCC solution obtained in step (1) at an addition amount of 1.0% (w / v, g / mL) and stirred at 500 rpm for 12 h at 4 °C to obtain HA-MCC hybrid particles.

[0120] Example 2: Optimization of the molecular weight of sodium hyaluronate

[0121] The specific implementation method is the same as in Example 1, except that the sodium hyaluronate (HA) with a molecular weight of 400kDa in step (2) is replaced with sodium hyaluronate (HA) with a molecular weight of 100kDa, 600kDa or 1090kDa; the remaining steps are the same as in Example 1, and HA-MCC hybrid particles are obtained.

[0122] Example 3: Optimization of Sodium Hyaluronate Addition Amount

[0123] The specific implementation method is the same as in Example 1, except that the amount of sodium hyaluronate (HA) added in step (2) is replaced by 0.1% (w / v, g / mL) instead of 1.0% (w / v, g / mL); the remaining steps are the same as in Example 1, and HA-MCC hybrid particles are obtained.

[0124] Example 4: Optimization of Sodium Hyaluronate Molecular Weight and Dosage

[0125] The specific implementation method is the same as in Example 1, except that the sodium hyaluronate (HA) with a molecular weight of 400 kDa in step (2) is replaced with sodium hyaluronate (HA) with a molecular weight of 100 kDa, and the amount of sodium hyaluronate (HA) added in step (2) is replaced from 1.0% (w / v, g / mL) to 0.1% (w / v, g / mL); the remaining steps are the same as in Example 1, and HA-MCC hybrid particles are obtained.

[0126] Example 5: Optimization of the amount of micellar casein concentrate (MCC) added

[0127] The specific implementation method is the same as in Example 1, except that the amount of micelle casein concentrate (MCC) added in step (1) is replaced by 1%, 2%, 3% and 4% (w / w) instead of 5% (w / w); the remaining steps are the same as in Example 1, and HA-MCC hybrid particles are obtained.

[0128] In addition, MCC with an addition of 6% (w / w) is difficult to dissolve, and the viscosity after dissolution is too high, which is not conducive to homogenization;

[0129] When the MCC concentration is less than 1% (w / w), even if particulate emulsifiers are formed, they cannot form a complete coverage at the interface.

[0130] Comparative Example 1: Preparation of HA-CAS Hybrid Particles

[0131] The specific implementation method is the same as in Example 1, except that the micelle casein concentrate (MCC) in step (1) is replaced with casein (CAS), and the remaining steps are the same as in Example 1, to obtain HA-CAS hybrid particles.

[0132] Comparative Example 2: Preparation of Pectin-MCC Hybrid Particles

[0133] The specific implementation method is the same as in Example 1, except that the sodium hyaluronate (HA) with a molecular weight of 400kDa in step (2) is replaced with pectin; the remaining steps are the same as in Example 1.

[0134] The results showed that when 1% (w / w) of pectin was added, the pH was 3.32 after dissolution. When the pectin was mixed with 5% (w / w) of MCC, flocculent material immediately appeared, and the pH of the solution was 5.81.

[0135] If a stable dispersion of pectin-MCC hybrid particle complex is required for particle size potential testing, the pectin needs to be diluted 50 times, and the MCC needs to be diluted 50 times accordingly. At this point, pectin-MCC hybrid particles are obtained, and the solution of the pectin-MCC hybrid particles has a pH of 6.96.

[0136] Comparative Example 3: Preparation of HA-MPC Hybrid Particles

[0137] The specific implementation method is the same as in Example 1, except that the micelle casein concentrate (MCC) in step (1) is replaced with milk protein concentrate (MPC); the remaining steps are the same as in Example 1, and HA-MPC hybrid particles are obtained.

[0138] The hybrid particles prepared in Examples 1-5 and Comparative Examples 1-3 were subjected to particle size and zeta potential measurements, Fourier transform infrared spectroscopy (FTIR) determinations, and scanning electron microscopy observations. The results are as follows:

[0139] 1. Measurement results of particle size and zeta potential

[0140] To assess whether the above proteoglycan complexes exhibit the expected particle characteristics and to evaluate their potential as Pickering emulsifiers, the particle size and surface potential of the MCC and HA-MCC complexes were measured.

[0141] The results are shown in Table 1:

[0142] Table 1. Particle size and zeta potential of hybrid particles

[0143]

[0144] As shown in Table 1, although the net surface charge of MCC and HA is negative, the addition of HA leads to an increase in particle size, indicating that the protein-polysaccharide interaction not only promotes complex formation but also affects the particle size distribution. The zeta potential of the complex increases, falling between that of pure MCC and HA. Notably, the zeta potential of the HA-MCC complex is primarily controlled by HA, suggesting that HA may interact with positively charged regions on the MCC. HA chains may extend to the surface of the MCC particles rather than being completely embedded or simply covering them. These findings highlight the role of hydrogen bonding and electrostatic interactions in the formation of HA-MCC hybrid particles, which hold promise as protein-polysaccharide Pickering emulsifiers.

[0145] Based on the particle size and distribution, samples with a particle size of about 250 nm were selected for further investigation of their interface characteristics, namely the HA-MCC hybrid particles obtained in Examples 1, 2 (HA molecular weight of 100 kDa), 3, and 4, and the pectin-MCC hybrid particles prepared in Comparative Example 2 (pectin, diluted 50 times).

[0146] 2. Results of Fourier Transform Infrared Spectroscopy (FTIR)

[0147] Figure 1 The results showed that the hydrogen bond correlation peaks of the -OH groups in the spectra of MCC, HA with a molecular weight of 100 kDa, and HA with a molecular weight of 400 kDa appeared at 3280.82 cm⁻¹.-1 3284.80cm -1 and 3261.03cm -1 When HA-MCC hybrid particles were prepared using HA with a molecular weight of 100 kDa at an addition amount of 0.1% (Example 4), HA-MCC hybrid particles were prepared using HA with a molecular weight of 100 kDa at an addition amount of 1.0% (Example 2), HA-MCC hybrid particles were prepared using HA with a molecular weight of 400 kDa at an addition amount of 0.1% (Example 3), and HA-MCC hybrid particles were prepared using HA with a molecular weight of 400 kDa at an addition amount of 1.0% (Example 1), the -OH stretching vibration peak in the composite spectrum shifted to 3280.62 cm⁻¹, respectively. -1 3274.16cm -1 3273.58cm -1 and 3271.79cm -1 This indicates that hydrogen bonds are formed between the amino and carboxyl groups of MCC and the hydroxyl groups of HA, as hydrogen bonds tend to lower the tensile vibrational frequency. Furthermore, the characteristic peaks of amide I (C=O stretching) and amide II (-NH2 bending and Cn stretching) of MCC are located at 1639.79 cm⁻¹. -1 and 1513.19cm -1 The formation of the HA-MCC complex shifted the peak of amide I to 1640.56 cm⁻¹. -1 1641.12cm -1 1644.52cm -1 and 1644.56cm -1 The peak shift of amide II was 1514.25 cm⁻¹. -1 1532.79cm -1 1515.62cm -1 and 1515.06cm -1 These changes indicate the existence of electrostatic interactions and hydrogen bonds between MCC and HA.

[0148] Figure 2 The results showed significant morphological differences between the lyophilized MCC solution and the HA-MCC hybrid particles. The lyophilized MCC solution exhibited a layered network structure, which gradually transitioned to a rod-like network structure with the addition of HA. This transformation became increasingly pronounced with increasing HA concentration. These findings suggest that the interaction between HA and MCC may lead to the formation of the HA-MCC complex, and that the polysaccharide chains of HA may form a protective layer on the surface of the MCC particles. The presence of HA not only enhances electrostatic repulsion but also introduces steric hindrance, thereby limiting the aggregation of MCC particles.

[0149] Example 6: Preparation of a high internal phase emulsion gel with 75% (v / v) oil phase

[0150] Using the HA-MCC hybrid particles obtained in Example 1 as the aqueous phase solution, 10 mL of the HA-MCC hybrid particle aqueous phase solution obtained in Example 1 was poured into a 100 mL plastic beaker and slowly added to 30 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). At the same time, high shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing, followed by a 10-second pause, and the operation was repeated. The entire process lasted for about 20 minutes, and a high internal phase emulsion gel containing 75% (v / v) oil phase was successfully obtained.

[0151] Example 7: Preparation of a high internal phase emulsion gel with 80% (v / v) oil phase

[0152] Using the HA-MCC hybrid particles obtained in Example 1 as the aqueous solution, 10 mL of the HA-MCC hybrid particle aqueous solution obtained in Example 1 was poured into a 100 mL plastic beaker and slowly added to 40 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). At the same time, high shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing, followed by a 10-second pause, and the operation was repeated. The entire process lasted for about 20 minutes, and a high internal phase emulsion gel containing 80% (v / v) oil phase was successfully obtained.

[0153] Example 8: Preparation of a high internal phase emulsion gel with 85% (v / v) oil phase

[0154] Using the HA-MCC hybrid particles obtained in Example 1 as the aqueous phase solution, 7.5 mL of the HA-MCC hybrid particle aqueous phase solution obtained in Example 1 was poured into a 100 mL plastic beaker and slowly added to 42.5 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). At the same time, high shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing, followed by a 10-second pause, and the operation was repeated. The entire process lasted for about 20 minutes, and a high internal phase emulsion gel containing 85% (v / v) oil phase was successfully obtained.

[0155] Example 9: Preparation of an emulsion gel with a 50% (v / v) oil phase

[0156] Using the HA-MCC hybrid particles obtained in Example 1 as the aqueous phase solution, 20 mL of the HA-MCC hybrid particle aqueous phase solution obtained in Example 1 was poured into a 100 mL plastic beaker and slowly added to 20 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). At the same time, high shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing, followed by a 10-second pause, and the operation was repeated. The entire process lasted for about 20 minutes, and an emulsion gel containing 50% (v / v) oil phase was successfully obtained.

[0157] Example 10: Preparation of 20% (v / v) oil phase emulsion gel

[0158] Using the HA-MCC hybrid particles obtained in Example 1 as the aqueous phase solution, 40 mL of the HA-MCC hybrid particle aqueous phase solution obtained in Example 1 was poured into a 100 mL plastic beaker and slowly added to 10 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). At the same time, high shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing, followed by a 10-second pause, and the operation was repeated. The entire process lasted for about 20 minutes, and an emulsion gel containing 20% ​​(v / v) oil phase was successfully obtained.

[0159] Example 11: Preparation of high internal phase emulsion gel

[0160] Using the hybrid particles prepared in Examples 2 (HA molecular weight 100 kDa), 3, and 4 as the aqueous phase solution, 10 mL of the hybrid particle aqueous phase solution was poured into a 100 mL plastic beaker and slowly added to 30 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). Simultaneously, high-shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing, followed by a 10-second pause, and was repeated. The entire process lasted approximately 20 minutes, yielding a high internal phase emulsion gel containing 75% (v / v) oil phase. Comparative Example 4: Preparation of an emulsion gel with 75% (v / v) oil phase using pectin-MCC hybrid particles as the particle emulsifier.

[0161] Using the pectin-MCC hybrid particles prepared in Comparative Example 2 (pectin, diluted 50 times) as the aqueous phase solution, 10 mL of the pectin-MCC hybrid particle aqueous phase solution from Comparative Example 2 was poured into a 100 mL plastic beaker and slowly added to 30 mL of soybean oil (oil phase) using a constant flow pump (HL-2S, Qingpu Huxi) (60 drops / min). Simultaneously, high-shear dispersion was performed using a disperser (T10, IKA) at a shear rate of 10,000 rpm. This process included 1 minute of shearing followed by a 10-second pause, which was repeated. The entire process lasted approximately 20 minutes, yielding an emulsion gel containing 75% (v / v) oil phase. The results are as follows... Figure 3 As shown.

[0162] from Figure 3 It can be seen that the emulsion gel containing 75% (v / v) oil phase obtained in Comparative Example 4 has the oil phase and aqueous phase in a separate state.

[0163] The emulsion gels prepared in Examples 6 and 11 above were subjected to tests for strength, rheological properties, water-holding capacity (WHC), oil-holding capacity (OHC), stability, and microstructure. The results are as follows:

[0164] 1. Results of gel strength determination, water-holding capacity (WHC), and oil-holding capacity (OHC) determination.

[0165] Table 2 shows the WHC values ​​of HA-MCC-stabilized HIPE gels formulated with different HA concentrations and molecular weights. The samples containing 1.0% HA have a WHC exceeding 90%, with the 1.0% 400kDa HA-MCC-stabilized HIPE gel exhibiting the highest WHC. This suggests that the HIPE gel with a 400kDa HA concentration of 1.0% may have better 3D printing performance compared to HIPE gels containing 0.1% 100kDa HA and pure MCC. The enhanced WHC can be attributed to the formation of HA-MCC composite particles, which have a larger specific surface area and surface-extended HA chains, promoting greater water-binding capacity. The increased WHC observed in HIPE gels with higher HA concentrations may be due to HA transforming from a single molecule in water to forming a three-dimensional network structure with increasing concentration. The strong water-binding capacity of HA further enhances the interaction between the HA-MCC complex and water molecules. Low molecular weight HA typically adopts a rigid rod-like structure, while high molecular weight HA produces a more flexible chain-like structure. For the flexible structure, each polysaccharide chain exhibits significant water molecule swelling. The chain entanglement of high molecular weight HA forms a network structure that can transmit tension under external forces, maintaining network integrity and thus inhibiting water migration, resulting in higher water content concentration (WHC). Furthermore, high molecular weight HA has many exposed hydrogen bond sites, allowing for strong interactions with water molecules, further reducing water migration and enhancing WHC.

[0166] Oil content analysis confirmed that all HIPE gels achieved 100% heat content without oil phase separation under centrifugation conditions, highlighting the superior emulsifying ability of MCC. Incorporation of HA into HA-MCC particles enhanced chain entanglement and interfacial viscoelasticity, significantly improving gel stability. Unlike single biopolymer adsorption, which forms a weak interfacial film, the HA-MCC complex generates a thicker, more robust network, providing excellent oil droplet protection during centrifugation and dehydration. This dense structure prevents oil phase separation, ensuring emulsion homogeneity and stability. The enhanced oil-water interface achieved by the HA-MCC complex is crucial for maintaining emulsion integrity, especially in applications such as 3D printing, where stability under mechanical stress directly impacts performance and reliability.

[0167] Table 2 Results of gel strength and water-holding capacity (WHC) determination

[0168]

[0169] 2. Rheological performance results

[0170] like Figure 4 As shown in Figure a, both MCC and HA-MCC stabilized HIPE gels exhibit shear thinning behavior in the linear viscoelastic region (LVR), with viscosity decreasing with increasing shear rate, classifying them as pseudoplastic fluids. The viscosity of the HA-MCC stabilized HIPE gel is higher than that of the MCC stabilized gel, and the viscosity increases with increasing HA concentration and molecular weight. The entanglement of hyaluronic acid chains on the MCC particles enhances gel stability and improves interlayer adhesion during printing.

[0171] The apparent viscosity of the HIPE gel was analyzed using a power-law model (Table 3), where K is the consistency coefficient reflecting viscosity and n is the flow behavior exponent representing pseudoplasticity. The n values ​​for all samples were well below 1, confirming their shear-thinning behavior. The high R² values ​​(0.948–0.997) indicate that the model accurately describes the rheological properties of the gel. Notably, K increases with increasing polysaccharide concentration and molecular weight, reflecting stronger cross-linking between the HA polysaccharide chains and MCC particles. This enhanced network structure significantly improves the apparent viscosity of the gel, consistent with experimental observations. These findings highlight the crucial role of molecular weight and concentration in regulating gel viscosity and stability.

[0172] Table 3 Apparent viscosity of HIPE gel

[0173]

[0174] Three-interval thixotropic testing (3-ITT) was used to evaluate the viscosity behavior of the emulsified gel under different shear conditions, simulating extrusion kinetics. The results showed that viscosity decreased under high shear, enabling smooth extrusion, and recovered after shear removal, indicating that robust structural modification is crucial for interlayer adhesion in 3D printing. Viscosity increased with increasing HA concentration and molecular weight. Figure 4 (b) This is due to the improved interfacial viscoelasticity resulting from HA chain entanglement and enhanced steric hindrance. These properties enable efficient layer stacking, rapid extrusion molding, and improved accuracy and structural integrity of printed structures.

[0175] Creep recovery testing evaluates the gel's resistance to deformation, a key characteristic for 3D printing applications. For example... Figure 4 As shown in Figure c, the MCC-stabilized HIPE gel exhibits rapid strain under stress and then stabilizes upon removal, demonstrating resilience. Increasing the polysaccharide concentration and molecular weight enhances the network robustness, reduces flowability, and lowers peak strain. In the HA-MCC-stabilized gel, these factors promote larger chain entanglements, strengthen the gel network, and further improve deformation resistance. The resulting lower peak strain indicates reduced flowability, which is beneficial for 3D printing by improving structural stability and accuracy. Compared to MCC alone, the HA-MCC gel exhibits superior mechanical strength and elasticity, minimizing permanent deformation under stress. These improvements are crucial for producing reliable and durable printed structures.

[0176] The self-supporting ability of 3D printing materials is crucial for maintaining the stability of layer-by-layer stacked structures. Dynamic viscoelasticity is a key indicator, in which the storage modulus (G′) reflects the elastic properties and post-printing support capacity of the material, while the loss modulus (G″) represents the flow resistance under stress. Figure 4 d and Figure 4Figure e shows the frequency-dependent responses of G′ and G″ at a constant shear strain of 0.1% (within the linear viscoelastic region, LVR). G′ consistently exceeds G″ at all tested frequencies, indicating that both MCC and HA-MCC stabilized HIPE gels exhibit solid-like elastic behavior. The G′ value of the HA-MCC stabilized HIPE gel increases with increasing polysaccharide concentration and molecular weight, and further with increasing frequency. These trends suggest enhanced self-support capabilities with increasing HA concentration. At the same polysaccharide concentration, longer HA chains produce the highest G′ values ​​due to their chain conformation and network formation, which strengthen the gel structure. Notably, the 1.0% 400 kDa HA-MCC stabilized HIPE gel exhibits the strongest self-supporting properties. This superior elasticity is crucial for optimizing 3D printing materials. High G′ values ​​improve interlayer adhesion and structural stability during printing, enhancing the quality and durability of the final product. The reinforcement provided by longer HA chains further underscores their role in molding materials suitable for reliable, high-precision 3D printing.

[0177] The viscoelasticity of a material is rigorously evaluated using the dynamic loss tangent (tan δ = G″ / G′), which reflects internal friction and energy dissipation under applied stress. In 3D printing, a lower tanδ value indicates greater internal friction and energy loss, thus affecting printing efficiency and the durability of the printed part. Figure 4 As shown in Figure f, MCC and HA-MCC stabilized HIPE gels exhibit tanδ values ​​below 1, with the storage modulus (G′) consistently exceeding the loss modulus (G″), indicating that elastic behavior dominates. Increasing the polysaccharide concentration in HA-MCC stabilized HIPE gels further reduces tanδ and enhances gel elasticity. This elasticity is crucial for maintaining the structural integrity of the printed object, as it ensures stability during and after printing. However, excessively low tanδ values ​​may lead to extrusion challenges, such as increased viscosity or filament breakage, potentially disrupting the printing process.

[0178] 3. Gel stability results

[0179] We further investigated the effect of HA on the stability of HIPE gel. Figure 5The composite transmittance curves of different HIPE gels under 865 nm near-infrared parallel light over time are shown in the image. The 130 mm position on these curves corresponds to the bottom of the sample, while approximately 105 mm is marked as the top. The transmittance of the emulsion is inversely proportional to the droplet concentration. Higher transmittance generally reflects lower droplet concentration, while lower transmittance indicates higher concentration. A generally low transmittance was observed when near-infrared light passed through the HIPE gel, indicating a compact and opaque gel structure. However, if the structure of the HIPE gel is disrupted by centrifugal force, oil droplets tend to rise, while the aqueous phase settles at the bottom. This structural disruption increases transmittance due to the concentration of the aqueous phase in the lower portion. Figure 5 In the intermediate AF curve, the initial profile (red line) shows very low bottom transmittance, indicating that the HIPE gel was initially uniform and opaque, except for the 1.0% 400kDa HA-MCC-stabilized HIPE gel. After centrifugation at 3000 rpm for 150 minutes, the final curve (green line) shows a significant increase in bottom transmittance, indicating that the gel structure was disrupted and the aqueous phase separation led to higher transmittance.

[0180] Figure 5 The results from the AF study clearly demonstrate that the difference in transmission spectra during centrifugation is reduced in HA-MCC-stabilized HIPE gels, indicating that HA significantly enhances the stability of the HIPE gel. This enhanced stability can be attributed to the network formed by entangled HA chains, which enhances the interfacial viscoelasticity of the emulsion droplets. When an external force is applied, the HA chains slide relatively, transmitting tension along the chains and dispersing it throughout the network through entanglement. This interfacial layer acts like an elastic net, tightly encapsulating the emulsion droplets and preventing them from breaking under compression or vibration, thus significantly improving the stability of the emulsion gel. Furthermore, at the same HA concentration, the HIPE gel stabilized by longer HA chains (i.e., 400 kDa HA-MCC) shows significantly smaller changes in strength under external force. Notably, the 1.0% 400 kDa HA-MCC-stabilized HIPE gel, with its longer HA chains and higher concentration, shows minimal change during accelerated centrifugation, with almost no change in transmittance. This indicates that these HIPE gels exhibit significant anti-agglomeration properties even under high-speed centrifugation.

[0181] 4. Results of the determination of gel morphology and microstructure

[0182] HA-MCC-stable gels adhered to sample vials at different HA concentrations and molecular weights, exhibiting a self-supporting structure. Figure 6 a). For example Figure 6As shown in Figure b, the microstructure of the emulsion gel indicates that MCC and HA interact in the continuous phase, forming a gel network. The red oil phase droplets are encapsulated by the green continuous phase composed of MCC or HA-MCC complexes. With increasing HA concentration and molecular weight, the oil droplet size in the gel decreases, and the distribution becomes more uniform. This effect is due to the entanglement of HA polysaccharide chains at higher concentrations and molecular weights, forming an intramolecular network that strengthens the cross-linking interactions and steric hindrance between polysaccharide chains around the droplets, resulting in a tighter interfacial structure. Furthermore, the negative charge of HA interacts with the surface charge of MCC, enhancing the electrostatic stability of the emulsion and effectively preventing droplet aggregation, leading to a more uniform droplet distribution. When the HA concentration is 1.0% (w / v), the droplets are even smaller, with each droplet completely encapsulated by the surrounding HA-MCC complex. At the same HA concentration, the molecular weight of HA also plays a crucial role in the gel structure. Among these, the emulsion gel prepared using the HA-MCC hybrid particles of Example 1 exhibits the smallest droplet size and the most uniform distribution. Figure 6 As shown in b. This is likely due to the longer chain length of H-HA, which allows for greater chain entanglement, resulting in a stronger network structure and greater steric hindrance, thus enhancing the stability of the emulsion.

[0183] 5. Swallowing performance

[0184] like Figure 7 As shown, for the 0.1% HA samples with two different molecular weights, both samples slid slowly when the spoon was tilted, with only a small amount of food residue observed. This indicates that both samples belong to IDDSI level 4 (pasty) food. Furthermore, the residue on the spoon for both samples was less than that for other samples, suggesting that the lower concentration (0.1%) HA sample had less residue during swallowing, resulting in better swallowing performance. This allows the food bolus to remain more compact during swallowing, preventing it from breaking down into fragments, making it more suitable for people with swallowing difficulties.

[0185] Example 12: High internal phase emulsion gels of different colors

[0186] 1. Preparation of yellow high internal phase emulsion gel

[0187] Curcumin (0.5 mg / mL) was dissolved in soybean oil, and a yellow HIPE gel was prepared according to Example 6. The results are as follows. Figure 8 As shown in the image above.

[0188] Curcumin (0.5 mg / mL) was dissolved in soybean oil, and three kinds of yellow HIPE gels were prepared according to Example 11.

[0189] 2. Preparation of pink high internal phase emulsion gel

[0190] Anthocyanins (0.1 g / mL) were dissolved in MCC, and a pink HIPE gel was prepared according to Example 6. The results are as follows. Figure 8 As shown in the image below.

[0191] The 3D printing performance of the emulsion gels prepared in Examples 6, 11, and 12 above was measured, and the results are as follows:

[0192] like Figure 9 As shown, the gel stabilized solely by MCC particles remains liquid after printing and cannot maintain its designed shape. When HA-MCC hybrid particles stabilize the gel at a low HA concentration (0.1% w / v), the gel initially can be extruded but lacks structural stability and eventually collapses. At a higher HA concentration (1.0% w / v), the gel can be printed into a stable, well-defined structure very similar to the design model, with enhanced texture and durability.

[0193] The accuracy and stability of 3D printing were determined for the high internal phase emulsion gel of Example 6, and the high internal phase emulsion gels obtained in Example 11 using the hybrid particles of Example 4, the hybrid particles of Example 3, or the hybrid particles of Example 2 (HA molecular weight of 100 kDa) as the aqueous phase.

[0194] The results are shown in Table 4. The high internal phase emulsion gel obtained in Example 6 had the highest printing accuracy and stability.

[0195] Table 4. Accuracy and stability of 3D printed samples

[0196]

[0197] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a sodium hyaluronate-micelle casein concentrate hybrid particle aqueous solution using micellar casein concentrate and sodium hyaluronate, characterized in that, The method is as follows: The micelle casein concentrate was dispersed in water, mixed, and homogenized; sodium hyaluronate was added and stirred to obtain an aqueous solution of HA-MCC hybrid particles. The sodium hyaluronate has a molecular weight of 100~1090 kDa; the amount of sodium hyaluronate added is 0.1% w / v or 1.0% w / v; The final concentration of the micelle casein concentrate in water is 1% to 5% w / w.

2. The method according to claim 1, characterized in that, The molecular weight of the sodium hyaluronate is 200~600kDa.

3. The method according to claim 1, characterized in that, The molecular weight of the sodium hyaluronate is 400 kDa.

4. The method according to claim 1, characterized in that, The amount of sodium hyaluronate added is 1.0% w / v.

5. An aqueous phase solution of HA-MCC hybrid particles prepared by any one of the methods described in claims 1 to 4.

6. The application of the HA-MCC hybrid particle aqueous solution according to claim 5 in the preparation of cosmetics or daily chemical products.

7. The application of the HA-MCC hybrid particle aqueous solution according to claim 6, characterized in that, The applications include encapsulating and delivering bioactive substances, gelling liquid oils; or preparing emulsions, foams, and emulsion gels.

8. The application according to claim 7, characterized in that, The emulsion gel is a high internal phase emulsion gel.

9. A method for preparing an emulsion gel using the HA-MCC hybrid particle aqueous solution according to claim 5, characterized in that, The method is as follows: Using the HA-MCC hybrid particle aqueous solution of claim 5 as the aqueous phase, the aqueous phase is mixed with the oil phase and dispersed by high-speed shearing to obtain an emulsion gel.

10. The method according to claim 9, characterized in that, The volume ratio of the oil phase to the water phase is (0:100) to (85:15), but not 0.

11. A method for preparing a high internal phase emulsion gel using the HA-MCC hybrid particle aqueous solution of claim 5, characterized in that, The method is as follows: Using the HA-MCC hybrid particle aqueous solution of claim 5 as the aqueous phase, the aqueous phase and oil phase are mixed and dispersed by high-speed shearing to obtain a high internal phase emulsion gel; the volume ratio of the aqueous phase to the oil phase is (0:100) to (15:85), but not 0; or the volume ratio of the aqueous phase to the oil phase is 1:

3.

12. The emulsion gel obtained by the method of claim 9 or 10.

13. The high internal phase emulsion gel obtained by the method of claim 11.

14. The use of the high internal phase emulsion gel of claim 13 in the preparation of a carrier for lipid-soluble active ingredients.

15. The application according to claim 14, characterized in that, The fat-soluble active ingredients include curcumin, beta-carotene, astaxanthin, lycopene, vitamin E, n-3 fatty acids, vitamin A, resveratrol, and capsaicin.

16. The application of the HA-MCC hybrid particle aqueous solution of claim 5 or the high internal phase emulsion gel of claim 13 in 3D printing.

17. The application according to claim 16, characterized in that, The application is to use a 3D printer to perform emulsion gel 3D printing using the high internal phase emulsion gel of claim 13.

18. A 3D printed product based on the high internal phase emulsion gel of claim 13, characterized in that, The 3D printed product is obtained by 3D printing using the high internal phase emulsion gel of claim 13.

Images