An emulsion gel for delivering fat-soluble vitamins, methods and uses

Abstract

The present application relates to the technical field of food processing, and particularly relates to an emulsion gel for delivering fat-soluble vitamins, a method and application, the method comprising: subjecting a compound emulsion containing soybean protein isolate, fenugreek gum and an oil phase loaded with fat-soluble vitamins to ultrasonic treatment at a frequency of 15 kHz to 25 kHz for 10 min to 20 min, to obtain an emulsion gel with improved fat-soluble vitamin delivery performance. The present application reduces the average particle size of the obtained emulsion gel from 2.829 mu m to 0.937 mu m through ultrasonic treatment, and improves the water retention, vitamin D3 retention rate and bioavailability of the emulsion gel.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and more specifically to an emulsion gel, method, and application for delivering fat-soluble vitamins. Background Technology

[0002] Vitamin D3, an essential fat-soluble vitamin, plays a crucial physiological role in calcium and phosphorus metabolism, bone health, and immune regulation, and has been widely used in functional foods, dietary supplements, and pharmaceuticals. However, vitamin D3 has extremely poor water solubility (Log P ≈ 10) and is highly sensitive to light, heat, and oxygen, making it easily degraded and inactivated during food processing, storage, and gastrointestinal transit, resulting in actual utilization efficiency far lower than the theoretical intake. Furthermore, the oral absorption of vitamin D3 is highly dependent on lipid digestion in the gastrointestinal tract and its transfer to the bile salt-phospholipid mixed micelle phase; this complex process is significantly regulated by the structural characteristics of the formulation carrier. Therefore, how to construct an efficient delivery carrier that can both protect vitamin D3 from processing and environmental stresses and promote its gastrointestinal digestion, release, and micellar transfer remains a core issue of long-term concern in this field.

[0003] Emulsion systems are a common strategy for improving the aqueous dispersibility and oral absorption efficiency of fat-soluble nutrients. However, traditional liquid emulsions are prone to oil droplet aggregation, flocculation, phase separation, and interfacial instability during processing, transportation, and storage, making it difficult to precisely control the release behavior of encapsulated active substances and resulting in unstable delivery efficiency. To overcome these limitations, emulsion-gel systems, which fix emulsion structures within gel networks, have attracted widespread attention in recent years. Emulsion gels combine the encapsulation ability of emulsions for fat-soluble active substances with the three-dimensional fixation of gel networks, exhibiting unique advantages in improving physical stability, delaying lipid digestion, and regulating release behavior. Among these, emulsion gels constructed using plant proteins and polysaccharides have become an important research direction in the field of food colloids and nutrient delivery due to their wide availability of raw materials, high sustainability, high processing adaptability, and good consumer acceptance.

[0004] Soy protein isolate is widely used in the construction of emulsion gel systems due to its excellent emulsifying properties, gel-forming ability, and nutritional value. However, in its natural state, soy protein isolate molecules have a relatively compact conformation, with sulfhydryl and hydrophobic groups mostly embedded within the molecule. Under actual processing conditions, this often results in slow interfacial adsorption rates and insufficient gel network formation ability, limiting its structural regulation precision and functional performance as a delivery carrier. Adding polysaccharides is a common method to improve the performance of protein-based emulsion gels. Fenugreek gum, a natural polysaccharide of the galactomannan class, possesses excellent thickening, water-retaining, and emulsifying stabilizing effects. Existing research shows that fenugreek gum can interact with soy protein to improve the solubility and emulsifying properties of protein dispersion systems. However, existing work has largely focused on the complex emulsion level; a systematic understanding of the emulsion gel network formed after further gelation of this complex system, and how this network regulates the delivery behavior of fat-soluble nutrients, is still lacking.

[0005] Ultrasonic treatment, as a green physical modification method, has been explored for regulating the microstructure and functional properties of protein-polysaccharide complexes. Existing technologies have reported studies on how ultrasonic treatment improves the stability and gel properties of protein-based emulsions. For example, Chinese patent CN109288065A discloses an emulsion using plant protein and polysaccharide complexes loaded with fat-soluble vitamins and its preparation method, achieving active ingredient encapsulation through high-pressure homogenization and heat treatment. However, existing technologies generally treat ultrasonic treatment as an intensity-adjustable process, focusing on a monotonic enhancement logic of "higher ultrasonic intensity and longer treatment time lead to better system performance," primarily concerned with improving single indicators such as gel strength and emulsion stability, which has significant shortcomings.

[0006] Since the ultimate bioavailability of vitamin D3 depends on its comprehensive performance in multiple stages such as processing protection, gastric retention and intestinal release and transfer, and the requirements of these stages for carrier structural characteristics are not entirely consistent or even contradictory, the optimization of a single performance index often fails to bring about a comprehensive improvement in delivery efficiency. Summary of the Invention

[0007] To develop a plant-based emulsion gel delivery system capable of on-demand differentiated control of delivery performance through green physical means, while also possessing dynamic structural recombination characteristics, this invention provides an emulsion gel, method, and application for delivering fat-soluble vitamins. This invention utilizes ultrasonic treatment to reduce the average particle size of the obtained emulsion gel from 2.829 μm to 0.937 μm, thereby improving the water-holding capacity, vitamin D3 retention rate, and bioavailability of the emulsion gel.

[0008] The present invention also provides a method for improving the delivery performance of fat-soluble vitamins in emulsion gels, the method comprising: subjecting a composite emulsion containing soy protein isolate, fenugreek gum and an oil phase loaded with fat-soluble vitamins to ultrasonic treatment at a frequency of 15 kHz to 25 kHz for a time of 10 min to 20 min, thereby obtaining an emulsion gel with improved fat-soluble vitamin delivery performance.

[0009] This invention reduces the average particle size of the emulsion gel from 2.829 μm to 0.937 μm through ultrasonic treatment, thereby improving the water retention, vitamin D3 retention, and bioavailability of the emulsion gel.

[0010] Furthermore, the ultrasonic treatment conditions are: a frequency of 20 kHz and a time of 15 min.

[0011] Furthermore, the improvement of the delivery performance of fat-soluble vitamins in emulsion gels includes: increasing the water retention capacity of the emulsion gel, and increasing the retention rate, release rate, and bioavailability of fat-soluble vitamins in the emulsion gel.

[0012] The present invention also provides an emulsion gel for delivering fat-soluble vitamins, the emulsion gel comprising soy protein isolate and fenugreek gum, and having a gel network structure composed of reversible non-covalent bonds; The emulsion gel is formed by ultrasonic treatment at 15 kHz to 25 kHz for 10 min to 20 min, acid induction and / or heat induction gelation of a composite emulsion containing soy protein isolate, fenugreek gum and oil phase. The mass ratio of soy protein isolate to fenugreek gum is 20-100:1.

[0013] The SPI-FG emulsion gel constructed in this invention has an average particle size of 0.937 μm, a water holding capacity of 69.16%, and a vitamin D3 retention rate as high as 93.6%. The interfacial self-healing behavior of the SPI-FG emulsion gel demonstrates its dynamic network structure. This gel network exhibits reversible non-covalent interactions and dynamic segment rearrangement capabilities. It also improves the retention, release, and bioavailability of fat-soluble vitamins in the emulsion gel.

[0014] Furthermore, the average particle size of the emulsion gel is 0.5 μm to 2.0 μm; the water retention capacity of the emulsion gel is 60% to 80%.

[0015] Furthermore, the fat-soluble vitamin is vitamin D3.

[0016] Furthermore, the average particle size of the emulsion gel is 0.937 μm.

[0017] Furthermore, the emulsion gel has interfacial self-healing ability after the cross-sections are bonded, and the self-healing occurs within 12 to 24 hours after bonding.

[0018] The present invention also provides the application of the emulsion gel in the preparation of delivery carriers with interfacial self-healing capabilities.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention reveals for the first time the regulatory mechanism of ultrasound time on delivery performance. Through experiments, this invention demonstrates that ultrasound induces time-dependent structural remodeling in the SPI-FG system, reducing the average particle size from 2.8291 μm to 0.9373 μm. Simultaneously, it alters the non-covalent interactions between the protein and polysaccharide and the gel network structure, resulting in significant changes in emulsion gel properties and vitamin D3 delivery behavior. Specifically, 15 min of ultrasound significantly improved the water-holding capacity of the emulsion gel, facilitated vitamin D3 retention, and achieved the highest bioavailability. Furthermore, the SPI-FG emulsion gel treated with moderate ultrasound exhibited significant interfacial healing after cross-sectional re-contact, indicating that reversible non-covalent interactions in the system can promote gel network reconnection. This invention reveals the structure-activity relationship between ultrasound-regulated SPI-FG emulsion gel structural evolution and vitamin D3 delivery performance, and demonstrates that the SPI-FG emulsion gel possesses potential interfacial healing capabilities, providing a new theoretical basis for constructing efficient lipid-soluble nutrient factor delivery systems using plant-based emulsion gels. This breakthrough breaks through the conventional understanding and technical bias held by those skilled in the art that "the longer the ultrasound time, the greater the processing intensity, and the better the performance," making it possible to achieve "on-demand customization" for different delivery stages using the same green physical means, and providing a brand-new strategy for the precise design of fat-soluble nutrient delivery carriers.

[0020] The SPI-FG emulsion gel constructed by ultrasonic treatment in this invention reduces the average particle size from 2.829 μm to 0.937 μm, increases water retention to 69.16% (more than 6.62% higher than the non-ultrasonicated group), and achieves a vitamin D3 retention rate of up to 93.6%, significantly improving bioavailability compared to the non-ultrasonicated group. This system exhibits excellent performance in multiple delivery stages, including processing protection, gastrointestinal controlled release, and micellar transfer, overcoming the technical challenge of existing technologies where optimizing a single indicator is insufficient to improve overall delivery efficiency.

[0021] This invention is the first to discover the interfacial self-healing behavior of SPI-FG emulsion gels, demonstrating their dynamic network structure. Specifically, it reveals that SPI-FG emulsion gels possess macroscopic interfacial healing capabilities after cross-sectional adhesion, strongly suggesting the existence of reversible nonvalent interactions and dynamic segment rearrangement capabilities within the gel network. This discovery not only provides a novel perspective for understanding the structural response behavior of this type of gel in the digestive environment but also suggests that its release kinetics and micellar transfer efficiency can be modulated through dynamic network recombination.

[0022] The soy protein isolate and fenugreek gum used in this invention are both food-grade natural raw materials, widely available, inexpensive, highly safe, and readily accepted by consumers. Ultrasonic treatment, as a physical modification method, eliminates the need for chemical cross-linking agents or organic solvents, aligning with the trends of green manufacturing and clean labeling. The entire preparation process is simple to operate, operates under mild conditions, and is easily scaled up for production, showing broad industrial application prospects in plant-based functional foods, precision nutritional supplements, and fat-soluble nutrient-fortified foods. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This invention illustrates the effect of different ultrasonic treatment times on the macroscopic appearance of SPI-FG emulsions and emulsion gels. In the image, A represents the inverted appearance of the SPI-FG emulsion; B represents the inverted appearance of the SPI-FG emulsion gel.

[0025] Figure 2 This invention illustrates the effect of different ultrasonic treatment times on the intermolecular interactions and structural characteristics of SPI-FG emulsion gels. In the figure, A represents intermolecular forces; B represents the X-ray diffraction pattern.

[0026] Figure 3 The effect of different ultrasonic treatment times on the properties of SPI-FG emulsion gel network: Figure A shows the effect of different ultrasonic treatment times on the water-holding capacity of SPI-FG emulsion gel; B. Effect of different ultrasonic treatment times on the distribution of transverse relaxation time (T2) measured by low-field nuclear magnetic resonance of SPI-FG emulsion gel; C. Effect of different ultrasonic treatment times on the gel strength of SPI-FG emulsion gel; The effect of different ultrasonic treatment times on the frequency dependence of the storage modulus (G′) and loss modulus (G″) of SPI-FG emulsion gel.

[0027] Figure 4 This is an observation diagram of the interfacial self-healing behavior of the SPI-FG emulsion gel with different ultrasonic treatment times according to the present invention.

[0028] Figure 5 Laser confocal microscopy images of SPI-FG emulsion gels with different ultrasonic treatment times according to the present invention; red fluorescent labels are for oil droplets, green fluorescent labels are for protein phases, and the scale bar is 20 μm. In the figure, A is the fluorescence image of the oil phase; B is the fluorescence image of the protein phase; C is a fused image of the oil phase and the protein phase.

[0029] Figure 6 This is a diagram showing the droplet size distribution of SPI-FG emulsion gels with different ultrasonic treatment times according to the present invention; In the diagram, A is U0; B is U5; C is U10; D is U15; and E is U20.

[0030] Figure 7 This invention illustrates the effect of different ultrasonic treatment times on the vitamin D3 delivery performance of SPI-FG emulsion gel. In the figure, A represents the effect of different ultrasonic treatment times on the vitamin D3 retention rate of SPI-FG emulsion gel; B represents the effect of different ultrasound treatment times on the cumulative release rate of vitamin D3 during in vitro simulated gastrointestinal digestion of SPI-FG emulsion gel; C represents the effect of different ultrasonic treatment times on the bioavailability of vitamin D3 in SPI-FG emulsion gel. Detailed Implementation

[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0032] Example 1: I. Experimental Materials and Methods 1. Experimental Materials Soy protein isolate (SPI) (food grade, protein content ≥90%) was purchased from Henan Wanbang Industrial Co., Ltd.; fenugreek gum (FG) (food grade) was purchased from Zhongkai Food Ingredients Co., Ltd.; non-GMO soybean oil (food grade) was purchased from Jiusan Food Co., Ltd. Vitamin D3 standard (cholecalciferol, purity ≥98%), pepsin, trypsin, and bile salts were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Glucono-δ-lactone (GDL), Tris, β-mercaptoethanol, anhydrous ethanol, n-hexane, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide, hydrochloric acid, urea, sodium dodecyl sulfate (SDS), glycine, disodium ethylenediaminetetraacetate, rhodamine B, Fast Green, and Nile Red were all purchased from Sinopharm Chemical Reagent Co., Ltd. Unless otherwise specified, all chemical reagents used were of analytical grade. Deionized water was used in the experiments.

[0033] 2. Preparation of Vitamin D3-Loaded SPI-FG Emulsion Soy protein isolate (SPI) was dispersed in 0.02 mol / L, pH 7.0 phosphate buffer to a final concentration of 5% (w / v) and magnetically stirred at room temperature for 2 h to ensure complete hydration. Fenugreek gum (FG) was then added to a final concentration of 0.1% (w / v), and stirring continued for 1 h until complete dispersion, yielding the SPI-FG composite dispersion. Vitamin D3 was pre-dissolved in soybean oil, mixed thoroughly in the dark, and used as the oil phase, achieving a vitamin D3 concentration of 20 μg / mL. The vitamin D3-containing soybean oil was added to the SPI-FG composite dispersion to a volume fraction of 6% (v / v). The mixture was then homogenized using a high-speed homogenizer (IKA-T18, IKA Werke, Staufen, Germany) at 10000 r / min for 3 min under ice bath conditions to prepare a vitamin D3-loaded SPI-FG emulsion.

[0034] 3. Ultrasonic treatment and preparation of SPI-FG emulsion gel The prepared vitamin D3-loaded SPI-FG emulsion was placed in an ice bath to maintain the sample temperature below 25 °C. Ultrasonic treatment was performed using a probe-type ultrasonic processor (Scientz-IID, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). The ultrasonic frequency was 20 kHz, the rated power was 400 W, the working amplitude was 40%, the probe diameter was 6 mm, the probe was inserted approximately 2 cm below the sample surface, the sample volume was 20 mL, and the pulse mode was 4 s operation followed by a 1 s interval. Ultrasonic treatment times were set to 0, 5, 10, 15, and 20 min, and the resulting samples were designated U0, U5, U10, U15, and U20, respectively. Gluconate-δ-lactone was added to the vitamin D3-loaded SPI-FG emulsions after different ultrasonic treatments to a final concentration of 2% (w / v), and after thorough mixing, acid-induced gelation was performed. The sample was then heated in an 80 °C water bath for 10 min, removed and cooled to room temperature, and stored at 4 °C for 12 h to obtain a vitamin D3-loaded SPI-FG emulsion gel.

[0035] 4. Characterization of the molecular structure and interactions of SPI-FG emulsion gel (1) Measurement of intermolecular forces Four dissociation solutions were prepared: S1 was ultrapure water, S2 was a Tris-Gly buffer at pH 8.0, S3 was a Tris-Gly buffer containing 2% (w / v) SDS and 8 mol / L urea, and S4 was a dissociation solution prepared by adding 1% (v / v) β-mercaptoethanol to S3. The Tris-Gly buffer was mainly used to weaken electrostatic interactions, SDS and urea were used to disrupt hydrophobic interactions and non-covalent interactions such as hydrogen bonds, and β-mercaptoethanol was used to reduce disulfide bonds. 2.0 g of each ultrasonically treated emulsion gel sample was weighed and added to 30 mL of the above dissociation solutions. The samples were homogenized at 10000 r / min for 1 min, followed by stirring for 1 h. The treated samples were centrifuged at 5000 r / min for 30 min, and the absorbance of the supernatant was measured at 500 nm. The relative turbidity was calculated using the following formula:

[0036] ; In the formula, T represents the relative turbidity, A represents the absorbance at 500 nm, and l represents the optical path length (1 cm). The relative contributions of electrostatic interactions, non-covalent interactions, and disulfide bonds to the gel network are represented by T2−T1, T3−T2, and T4−T3, respectively, where T1, T2, T3, and T4 are the relative turbidities of the samples after S1, S2, S3, and S4 treatments, respectively.

[0037] (2) Fourier transform infrared spectroscopy analysis Freshly prepared SPI-FG emulsion gels were pre-frozen in liquid nitrogen for 30 min, followed by vacuum freeze-drying for at least 48 h to thoroughly remove moisture from the samples. The freeze-dried samples were ground into powder and analyzed using a Fourier transform infrared spectrometer (IS50, Thermo Nicolet, USA). The scanning wavenumber range was 4000–400 cm⁻¹, with a resolution of 4 cm⁻¹, and each sample was scanned 64 times. Baseline correction, smoothing, deconvolution, and curve fitting were performed on the amide I band in the 1600–1700 cm⁻¹ range, and the relative contents of α-helices, β-sheets, β-turns, and random coils were calculated based on the integrated area of ​​each sub-peak.

[0038] 5. Determination of physicochemical and rheological properties of SPI-FG emulsion gel (1) Determination of textural properties The textural properties of SPI-FG emulsion gels subjected to different ultrasonic treatments were determined using a texture analyzer (TA.XT Plus, Stable Micro Systems, Surrey, UK). Before testing, the gel samples were removed from 4 °C and equilibrated to room temperature. Compression tests were performed using a 5 mm diameter cylindrical probe with a range of 1000 N, a deformation of 75%, a trigger force of 0.5 N, and a test speed of 60 mm / min, for two consecutive compressions.

[0039] (2) Water holding capacity test The water-holding capacity (WHC) of emulsion gels treated with different ultrasonic methods was determined

[24] . A certain mass of emulsion gel sample was weighed and recorded as W1; then the sample was placed in a centrifuge tube and centrifuged at 5000 r / min for 15 min. After centrifugation, the precipitated water was removed, and the residual water on the tube wall was gently absorbed with filter paper. The mass of the sample was weighed again and recorded as W2. The water-holding capacity was calculated by the following formula:

[0040] ; In the formula, W1 is the mass of the emulsion gel before centrifugation, and W2 is the mass of the emulsion gel after centrifugation.

[0041] (3) Rheological property determination The dynamic viscoelastic properties of SPI-FG emulsion gels subjected to different ultrasonic treatments were determined using a rotational rheometer (Discovery HR-1, TA Instruments, New Castle, DE, USA). A 40 mm diameter stainless steel parallel plate fixture with a 500 μm gap was used, and the temperature was 25 °C. After the samples were placed on the testing platform, a strain scan was first performed at 1 Hz, with a strain range of 0.01%–100%, to determine the linear viscoelastic region. Subsequently, a fixed strain (1.0%) within the linear viscoelastic region was selected for frequency scanning, with a frequency range of 0.1–100 Hz, and the storage modulus (G′) and loss modulus (G″) were recorded.

[0042] 6. CLSM observation of oil droplet distribution in SPI-FG emulsion gel The oil droplet distribution and protein phase structure of SPI-FG emulsion gels treated with different ultrasonic methods were observed using laser confocal microscopy. Nile Red dye solution (2 mg / mL) was added to soybean oil at a concentration of 10 μL / mL and mixed thoroughly in the dark. The stained emulsion gel was then prepared using the method described above. An appropriate amount of emulsion gel was placed on a glass slide, and Fast Green solution (2 mg / mL) was added for staining for 5 min to label the protein phase. Excess dye was then carefully blotted away with filter paper, and a coverslip was applied. The microstructure of the samples was observed using a laser confocal microscope (TCS SP8, Leica Microsystems, Wetzlar, Germany). The Nile Red channel was excited at 488 nm, and the emission wavelength was 500–600 nm; the Fast Green channel was excited at 645 nm, and the emission wavelength was 650–750 nm. After image acquisition, ImageJ was used for channel color adjustment and oil droplet size analysis.

[0043] 7. Observation of macroscopic interface healing behavior Two identical SPI-FG emulsion gel samples were taken. One sample was stained with Rhodamine B, while the other remained unstained. The two gels were cut open, and their fresh cut surfaces were allowed to align fully. They were then placed in a sealed environment to minimize the impact of moisture evaporation on the interface healing process. Interface changes were observed and photographed at 0, 12, and 24 hours. This experiment was used to evaluate the macroscopic interface healing and dynamic recovery behavior of emulsion gels treated with different ultrasound techniques.

[0044] 8. Determination of Vitamin D3 Retention Rate Weigh 2g of vitamin D3-supported emulsion gel sample, add 10 mL of anhydrous ethanol to disrupt the gel structure and release vitamin D3 from the sample, then extract with 20 mL of n-hexane. The sample was protected from light throughout the extraction process to minimize vitamin D3 degradation. After extraction, the upper n-hexane phase was collected, and its absorbance was measured at 264 nm. A blank emulsion gel without vitamin D3 was treated in the same manner as a matrix blank control. A series of standard solutions were prepared using vitamin D3 standards, and a standard curve was established under the same solvent system and measurement wavelength. The vitamin D3 content in the sample was calculated based on the absorbance corrected for the matrix blank. The retention rate was calculated using the following formula:

[0045] ; In the formula, Mr is the actual measured mass of vitamin D3 in the emulsion gel, and M0 is the theoretical mass of vitamin D3 added during sample preparation.

[0046] 9. In vitro simulated gastrointestinal digestion and determination of vitamin D3 release rate and bioavailability A precise mass of vitamin D3-loaded SPI-FG emulsion gel sample was weighed and dispersed in 10 mL of distilled water. During the gastric digestion phase, the sample was mixed with simulated gastric juice containing 3.2 mg / mL pepsin at a 1:1 (v / v) ratio, and the pH was adjusted to 2.5. Digestion was then carried out at 37 °C with shaking for 1 h. Subsequently, the pH was adjusted to 7.5, and simulated intestinal juice containing 20 mg / mL bile salts and 10 mg / mL pancreatic enzymes was added and mixed at a 1:1 (v / v) ratio. Digestion was continued at 37 °C with shaking for 2 h. The total digestion time was 180 min, with the gastric phase lasting 0–60 min and the intestinal phase lasting 60–180 min.

[0047] In the simulated digestion process, independent digestion samples were prepared at 30, 60, 90, 120, 150, and 180 min, respectively. 30 and 60 min corresponded to the gastric digestion stage, while 90, 120, 150, and 180 min corresponded to the intestinal digestion stage. Upon reaching the set digestion time, anhydrous ethanol was immediately added to disrupt the emulsion structure and terminate the digestion process. Vitamin D3 was then extracted with n-hexane. A blank emulsion gel without vitamin D3 was treated with the same digestion and extraction steps as a matrix blank. The absorbance of the extract was measured at 264 nm, and the mass of vitamin D3 in the digest at different time points was calculated based on the vitamin D3 standard curve. The release rate at each time point was calculated using the following formula:

[0048] ; In the formula, Mr is the mass of vitamin D3 released at time point t after digestion, and Me is the mass of vitamin D3 actually embedded in the emulsion gel.

[0049] After intestinal digestion, the digestive fluid was centrifuged, and the supernatant was collected as a mixed micelle phase. The mixed micelle phase sample was processed using the above-described anhydrous ethanol demulsification and n-hexane extraction methods, and a blank digest matrix was used as a control for calibration. Vitamin D3 bioavailability was calculated using the following formula:

[0050] ; In the formula, Mm is the mass of vitamin D3 in the mixed micelle phase, and Me is the actual mass of vitamin D3 embedded in the emulsion gel.

[0051] 10. Data Processing All experiments were repeated at least three times, and results are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS software, and Duncan's multiple comparison test was used to analyze significant differences between treatment groups; p < 0.05 was considered statistically significant. Graphs were generated using Origin software.

[0052] II. Experimental Results 1. Appearance and macroscopic gel state To understand the effect of ultrasonic treatment on SPI-FG emulsion gel, the phenomena observed in homogenized and acid-induced gels under different ultrasonic times were investigated.

[0053] like Figure 1 As shown, the homogenized sample easily slips off when inverted in the tube, indicating that the sample forms an emulsion rather than a monolithic gel. Figure 1 (A) All homogenized emulsions exhibited gelation after ultrasonic treatment, indicating that ultrasonic treatment promoted the formation of SPI-FG emulsion gel. This phenomenon is consistent with the results of emulsion rheological properties and gel strength.

[0054] 2. Effects of ultrasonic treatment on the molecular structure and interactions of SPI-FG emulsion gel To further elucidate the mechanism by which ultrasonic treatment affects the formation of the SPI-FG emulsion gel network, intermolecular forces, FTIR spectra, and XRD patterns of samples treated with different ultrasonic methods were analyzed.

[0055] like Figure 2As shown in Figure A, the intermolecular forces in the SPI-FG emulsion gel mainly include electrostatic interactions, non-covalent interactions, and disulfide bonds, with non-covalent interactions contributing the most. With increasing sonication time, non-covalent interactions and disulfide bonds generally showed a trend of first increasing and then decreasing, reaching a relatively high level in sample U15, while electrostatic interactions showed relatively little change. This suggests that moderate sonication may promote SPI conformation unfolding, allowing more groups to participate in hydrophobic interactions, hydrogen bonding, and disulfide bond-related network structures, thereby altering the relative contributions of different intermolecular forces in the SPI-FG emulsion gel. However, when the sonication time was further extended to 20 min, the contributions of non-covalent interactions and disulfide bonds decreased, possibly related to re-aggregation or excessive rearrangement of the network structure after excessive protein unfolding.

[0056] This invention further analyzes the effect of ultrasonic treatment on the secondary structure of proteins in SPI-FG emulsion gel by performing peak fitting on amide I bands.

[0057] Table 1. Effects of different ultrasonic treatment times on the secondary structural properties of SPI-FG emulsion gels α-helix (%) β-folding (%) β-turn angle (%) Irregular curl (%) U0 <![CDATA[17.66 ± 0.42 a ]]> <![CDATA[53.68 ± 0.85 b ]]> <![CDATA[24.63 ± 0.51 a ]]> <![CDATA[4.03 ± 0.16 a ]]> U5 <![CDATA[16.79 ± 0.36 a ]]> <![CDATA[54.19 ± 0.92 b ]]> <![CDATA[25.35 ± 0.48 a ]]> <![CDATA[3.67 ± 0.12 ab ]]> U10 <![CDATA[14.52 ± 0.31 b ]]> <![CDATA[60.83 ± 1.05 a ]]> <![CDATA[21.54 ± 0.63 b ]]> <![CDATA[3.11 ± 0.10 b ]]> U15 <![CDATA[16.96 ± 0.44 a ]]> <![CDATA[55.16 ± 0.78 b ]]> <![CDATA[24.25 ± 0.57 a ]]> <![CDATA[3.63 ± 0.14 ab ]]> U20 <![CDATA[17.57 ± 0.39 a ]]> <![CDATA[52.94 ± 0.81 b ]]> <![CDATA[25.67 ± 0.46 a ]]> <![CDATA[3.82 ± 0.15 a ]]> As shown in Table 1, β-sheets were the predominant secondary structure in all treated samples, indicating that protein molecules in the SPI-FG emulsion gel system mainly participate in gel network formation via β-sheet structures. Compared with the control group, moderate sonication treatment increased the β-sheet content while decreasing the α-helix content, suggesting that sonication may promote partial unfolding of SPI molecules and induce the transformation of α-helices into β-sheet structures. The increase in β-sheet content is beneficial for enhancing intermolecular interactions between protein molecules and promoting the co-construction of a more stable continuous gel network by SPI and FG. However, with further increases in sonication intensity, the β-sheet content decreased while the β-turn content rebounded, indicating that excessive sonication may disrupt some ordered aggregate structures, causing protein conformational rearrangement, which is detrimental to the further stability of the gel network. Overall, moderate sonication treatment can regulate the secondary structure of proteins in SPI-FG emulsion gels, promoting protein conformational rearrangement and network structure formation.

[0058] like Figure 2As shown in Figure B, all SPI-FG emulsion gels exhibited broad, diffuse peaks around 20°, without any obvious sharp diffraction peaks, indicating that the gels were predominantly amorphous. With prolonged sonication, the main peak gradually shifted from 19.62° in group U0 to 22.30° in group U20. According to Bragg's law, an increase in diffraction angle implies a decrease in the characteristic spacing of the corresponding structural units, indicating that sonication promoted short-range molecular rearrangement and localized close packing in the SPI-FG system. This change is related to sonication-induced dissociation and conformational unfolding of SPI aggregates, allowing exposed active groups to further participate in protein-protein and protein-polysaccharide interactions. All samples maintained broad peak characteristics, indicating that sonication mainly altered the degree of local order and did not induce significant long-range crystal structure formation.

[0059] 3. Effect of ultrasonic treatment on the properties of SPI-FG emulsion gel network Water retention and textural properties can reflect the ability of the SPI-FG emulsion gel network to bind water and its mechanical response at a macroscopic level.

[0060] like Figure 3 As shown in Figure A, the water-holding capacity first increased and then decreased with increasing sonication time, reaching a maximum of 69.16% at U15, an increase of 6.62 percentage points compared to U0. This result indicates that 15 min of sonication effectively promotes SPI conformational unfolding and SPI-FG interactions, enabling proteins, polysaccharides, and interfacial oil droplets to form a more continuous, dense, and homogeneous composite network, thereby reducing water migration and precipitation during centrifugation. In contrast, insufficient treatment time resulted in inadequate network reconstruction, while extending the treatment time to 20 min led to excessive cavitation and shearing-induced protein re-aggregation and local structural defects, weakening the network's water binding capacity. Therefore, U15 exhibited the best water retention capacity, further supporting 15 min as a suitable treatment condition for optimizing emulsion gel structure.

[0061] LF-NMR characterizes water migration in gels using the transverse relaxation time T2; a shorter relaxation time indicates a tighter binding of water to the gel network. Figure 3 As shown in Figure B, the main relaxation peaks of each sample are concentrated in the T21 region, indicating that the water in the system mainly exists in the form of fixed water. The main relaxation signal of U15 shifts to a shorter time region, indicating that its water migration is most strongly restricted, consistent with the highest water holding capacity of U15. This change can be attributed to the formation of a more uniform and highly connected SPI-FG continuous phase network by 15 min of ultrasound, and the reduction of network porosity and local water channels by the uniform filling of fine oil droplets. The peak broadening of U20 reflects the microenvironment inhomogeneity caused by excessive ultrasound. Combining the water holding capacity and LF-NMR results, U15 has the optimal water confinement state.

[0062] The textural results further indicate that the effect of ultrasonic treatment on the mechanical properties of SPI-FG emulsion gels is not a unidirectional enhancement. For example... Figure 3 As shown in Figure C, the gel strength increased with treatment time, reaching approximately 3.42 N·mm at U15, an increase of about 27.6% compared to U0; no further significant increase was observed at U20. This enhancement can be attributed to the moderate cavitation and shearing promoting partial unfolding of the SPI, entanglement of FG segments, and the establishment of protein-protein and proteoglycan interactions, thereby increasing network connectivity density and resistance to deformation. Considering the increase in particle size and decrease in molecular forces observed at U20, U15 represents the optimal balance between network strengthening and avoiding excessive reaggregation.

[0063] The rheological results further reflect the viscoelastic characteristics of emulsion gels treated with different ultrasonic methods. For example... Figure 3 As shown in Figure D, all samples exhibited a higher G′ than G″ within the test frequency range, indicating the formation of a three-dimensional network dominated by elastic response. U15 showed the highest or generally higher G′ within the test frequency range, corresponding to the highest gel strength, smallest oil droplets, highest non-covalent interaction, and best water retention. This indicates that 15 min of sonication can enhance the coupling between the protein continuous phase, FG segments, and interfacial oil droplets, allowing the oil droplets to participate in stress bearing as active filling units, thereby improving network elasticity and structural stability. With further extension of the processing time, excessive unfolding and re-aggregation led to a reduction in effective network connections, therefore U20 did not show further gains. Considering the rheological, textural, and moisture distribution results, U15 formed the most stable composite gel network.

[0064] like Figure 4 As shown, all treated samples exhibited some degree of interfacial healing after contact with fresh fracture surfaces. With the standing time increasing from 0 h to 24 h, the interface gradually blurred and macroscopically fused, indicating that reversible non-covalent interactions, segment diffusion, and dynamic cross-linking can drive fracture surface remodeling. Among them, U15 showed the most significant interfacial fusion and fracture surface continuity, consistent with its highest non-covalent interactions and relatively intact gel network. Moderate sonication increased the number of rebinding active sites at the fracture surface while maintaining sufficient segment migration capacity, allowing hydrogen bonds, hydrophobic interactions, and other reversible interactions to be re-established at the contact interface. The weakened interfacial recovery of U20 may be due to protein re-aggregation and a reduction in migratory segments caused by excessive sonication. Therefore, U15 achieved the optimal balance between mechanical stability and dynamic network remodeling capacity.

[0065] 4. Effects of ultrasonic treatment on the microstructure of SPI-FG emulsion gel CLSM dual-channel images were used to observe the distribution of oil droplets and changes in protein phase structure in SPI-FG emulsion gels treated with different ultrasound.

[0066] CLSM dual-channel images showed uneven distribution of oil droplets in U0, with localized large oil droplets and aggregated areas, indicating limited interfacial coverage and continuous phase organization of the unultrasonicated emulsion precursor. Figure 5 As the sonication time increased, the oil droplets gradually became finer, and the distribution of the protein phase around the oil droplets and in the continuous phase became more continuous. Among them, U15 exhibited the most uniform oil droplet distribution, the most continuous protein phase, and the tightest oil droplet-network coupling structure. This microstructure not only reduced internal structural defects in the gel but also improved the interfacial membrane coverage and continuous phase connectivity, consistent with U15's highest water-holding capacity, gel strength, and non-covalent interactions. Simultaneously, the larger effective interfacial area can improve the accessibility of lipases and bile salts to the oil droplets during intestinal digestion, thus providing a structural basis for U15's highest vitamin D3 bioavailability. 5. Effect of Ultrasonic Treatment on Oil Droplet Size Distribution of SPI-FG Emulsion Gel

[0067] from Figure 6 It can be seen that the droplet size of all SPI-FG emulsion gel samples is mainly distributed in the range of 0.1–15 μm. The average droplet size continuously decreases with increasing ultrasonic time from 0 min to 15 min, from 2.8291±1.2983 μm for U0 to 0.9373±0.6287 μm for U15, with U15 exhibiting the narrowest distribution peak. Ultrasonic cavitation, shearing, and microjets can break up large oil droplets and promote the rapid adsorption of the SPI-FG complex onto the newly formed interface, thereby inhibiting aggregation. When the treatment time is extended to 20 min, the average droplet size rebounds to 1.6196 μm and the distribution widens, indicating that excessive unfolding and re-aggregation weaken the interfacial film stability. Therefore, U15 performs best in terms of droplet refinement, distribution uniformity, and inhibition of re-aggregation, and is the key structural basis for subsequent delivery performance improvement.

[0068] 6. Effects of ultrasound treatment on vitamin D3 retention, release, and bioavailability like Figure 7 As shown in Figure A, the vitamin D3 retention rate first increased and then decreased with increasing sonication time, reaching a maximum of 93.6% at U15. This result indicates that the fine, uniform oil droplets, intact interfacial film, and dense SPI-FG gel network formed by sonication for 15 min synergistically limit the migration and loss of vitamin D3 during homogenization, sonication, and gel formation. At shorter treatment times, protein conformation unfolding and interfacial rearrangement are insufficient, resulting in relatively weak oil droplet interfacial coverage and network confinement. However, after extending the treatment time to 20 min, protein re-aggregation and excessive interfacial film rearrangement increase local structural defects, thereby reducing vitamin D3 retention. Therefore, U15 exhibits the best performance in terms of interfacial stability and gel network protection, as shown in the in vitro release results. Figure 7As shown in Figure B, different samples exhibited staged release characteristics during simulated gastrointestinal digestion, indicating that vitamin D3 release behavior is jointly influenced by the emulsion gel network structure, the oil droplet interface layer, and the digestive environment. During gastric digestion, the emulsion gel network can, to some extent, limit the rapid release of vitamin D3; upon entering the intestinal digestion stage, pancreatic enzymes and bile salts promote oil droplet digestion and interface structure disruption, leading to further vitamin D3 release and transfer to the mixed micelle phase. Compared to the non-ultrasound group, the release behavior of the ultrasound-treated group changed, indicating that ultrasound-regulated SPI-FG emulsion gel network reconstruction can affect the vitamin D3 release pathway during the gastrointestinal process.

[0069] The vitamin D3 bioavailability results further reflect the impact of different emulsion gel structures on its potential absorption efficiency. For example... Figure 7As shown in Figure C, the bioavailability of vitamin D3 first increases and then decreases with prolonged ultrasound time, reaching its highest value at U15. The small, uniform oil droplets formed at U15 increase the oil-water interface area. The stable SPI-FG network, which can gradually dissociate under intestinal digestion conditions, can limit premature release of vitamin D3 in the stomach and promote the approach of lipases, bile salts, and digestive products to the oil droplet interface in the intestine, thereby enhancing lipid hydrolysis and the transfer of vitamin D3 to the mixed micelle phase. Similar studies on emulsion-filled gels also show that the gel network structure and the transfer behavior of fat-soluble nutrients during digestion jointly affect its delivery effect. In summary, this invention investigates the regulation of the structural characteristics of SPI-FG emulsion gels and their vitamin D3 delivery performance by short-term ultrasound treatment. First, under the action of ultrasonic cavitation, shearing, and microfluidics, the oil droplets in the emulsion are further broken down and dispersed, with the average particle size decreasing from 2.8291 μm to 0.9373 μm, and the emulsion system gradually forms a finer and more uniform dispersion structure. Simultaneously, some SPI molecules undergo conformational unfolding under ultrasound, altering the protein microenvironment, exposing more active sites for interaction, and enhancing the non-covalent interaction between SPI and FG. Another portion of the protein-polysaccharide complex may participate in oil droplet interface stabilization and the construction of a continuous gel network, thereby restricting the free movement of oil droplets and improving the structural stability of the emulsion gel system. Therefore, moderate ultrasound can significantly improve the dispersion state of the emulsion system, reducing the average oil droplet size from 2.8291 μm to 0.9373 μm, and regulating the intermolecular interactions and gel network structure of the SPI-FG system, thus significantly altering the water-holding capacity, embedding stability, and in vitro delivery performance of the emulsion gel. Further research shows that the regulation of SPI-FG emulsion gel by ultrasound treatment has a significant time dependence; ultrasound for 15 min is beneficial for improving water-holding capacity, vitamin D3 retention, and bioavailability. This result reveals a close structure-activity relationship between the microstructure evolution of the emulsion gel and the delivery performance of lipid-soluble nutrients. Furthermore, the SPI-FG emulsion gel, after moderate sonication, exhibited a certain degree of interfacial healing upon re-contact of the cross-sections, indicating that reversible non-covalent interactions within the system facilitate the reconnection of the gel network, providing a new research approach for the structural regulation of plant-based emulsion gels. Overall, this study reveals the structure-activity relationship between short-term sonication-regulated SPI-FG emulsion gel structural evolution and vitamin D3 delivery performance, providing a theoretical basis for constructing efficient fat-soluble nutrient delivery systems using plant-based emulsion gels. It also offers a new technical strategy for the development of ultrasound-assisted plant-based functional foods and fortified foods. Future research could further integrate microscopic characterization and long-term storage evaluation to elucidate the mechanism of ultrasound-induced network evolution and its application potential in complex food systems.Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0070] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for improving the delivery performance of fat-soluble vitamins in emulsion gels, characterized in that, The method includes: subjecting a composite emulsion containing soy protein isolate, fenugreek gum, and an oil phase loaded with fat-soluble vitamins to ultrasonic treatment at a frequency of 15 kHz to 25 kHz for 10 min to 20 min to obtain an emulsion gel with improved fat-soluble vitamin delivery performance.

2. The method for improving the delivery performance of fat-soluble vitamins in emulsion gels according to claim 1, characterized in that, The ultrasonic treatment conditions are: frequency of 20 kHz and time of 15 min.

3. The method for improving the delivery performance of fat-soluble vitamins in emulsion gels according to claim 2, characterized in that, The improvement of the delivery performance of fat-soluble vitamins in emulsion gels includes: increasing the water retention capacity of the emulsion gel, and increasing the retention rate, release rate, and bioavailability of fat-soluble vitamins in the emulsion gel.

4. An emulsion gel for delivering fat-soluble vitamins, characterized in that, The emulsion gel contains soy protein isolate and fenugreek gum, and has a gel network structure composed of reversible non-covalent bonds. The emulsion gel is formed by ultrasonic treatment at 15 kHz to 25 kHz for 10 min to 20 min, followed by acid induction and / or heat induction gelation of a composite emulsion containing soy protein isolate, fenugreek gum and oil phase. The mass ratio of soy protein isolate to fenugreek gum is 20-100:

1.

5. The emulsion gel for delivering fat-soluble vitamins according to claim 4, characterized in that, The emulsion gel has an average particle size of 0.5 μm to 2.0 μm and a water-holding capacity of 60% to 80%.

6. The emulsion gel according to claim 4, characterized in that, The fat-soluble vitamin mentioned is vitamin D3.

7. The emulsion gel according to claim 4, characterized in that, The average particle size of the emulsion gel is 0.937 μm.

8. The emulsion gel according to claim 4, characterized in that, The emulsion gel has an interface self-healing ability after the cross-sections are bonded, and the self-healing occurs within 12 h to 24 h after bonding.

9. The use of the emulsion gel according to any one of claims 4 to 8 in the preparation of a fat-soluble vitamin delivery carrier with interfacial self-healing ability.

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