A water-in-oil-in-water compound emulsion embedding lactoferrin and vitamin D3, a preparation method and application thereof

Abstract

The application belongs to the field of food functional factor processing, and particularly relates to a water-in-oil-in-water composite emulsion embedding lactoferrin, a preparation method and application. The W1 / O / W2 double emulsion system is constructed by a two-step method, and the leakage and inactivation risks of lactoferrin and VD3 in the preparation, storage and processing processes are effectively reduced through the double barrier effect of the oil phase film and the outer water phase film. Under the optimized sodium caseinate and gellan gum compounding conditions, the encapsulation rate of lactoferrin can be up to 82.52%, which proves that the structure has obvious advantages in improving the loading efficiency of active substances, and can significantly improve the encapsulation rate and structural stability of lactoferrin. The water-in-oil-in-water composite emulsion system prepared by the application can significantly improve the retention rate of lactoferrin in the digestion process, enhance the tolerance of lactoferrin to the gastrointestinal environment, and then enhance the arrival rate of lactoferrin in the intestinal tract, thereby improving the overall bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of food functional factor processing, specifically relating to a water-in-oil-in-water composite emulsion containing lactoferrin, its preparation method, and its application. Background Technology

[0002] A widespread nutritional problem in my country is insufficient dietary calcium intake. Long-term calcium deficiency can reduce the body's bone synthesis capacity, leading to decreased bone mass, reduced bone density, and an increased risk of osteoporosis. Lactoferrin (LF) is an important natural glycoprotein widely found in human and cow's milk, possessing the ability to bind and transport iron ions. Studies have shown that LF plays a crucial role in antibacterial, antiviral, immunomodulatory, anti-inflammatory, iron absorption promotion, and maintaining intestinal health. Therefore, LF is widely used as a food fortifier in infant formula, functional foods, nutritional supplements, and pharmaceutical preparations. Vitamin D3 (VD3) is a core substance for maintaining calcium and phosphorus homeostasis. It can increase serum calcium levels by upregulating the calcium ion absorption signaling pathway in intestinal epithelial cells, providing a raw material basis for bone matrix mineralization.

[0003] Studies show that lactoferrin (LF) and vitamin D3 have a synergistic effect, which can further promote calcium absorption and bone health. However, LF still faces technical bottlenecks in food applications. On the one hand, LF is easily degraded in the acidic environment of the stomach, and its bioactivity decreases rapidly, resulting in low oral bioavailability. On the other hand, LF is sensitive to temperature, pH, and enzymatic hydrolysis during processing and storage, and is prone to conformational changes or inactivation, thus affecting the functional effects of the final product. In addition, LF and lipid-soluble vitamin D3 differ significantly in physicochemical properties and interfacial behavior, making it difficult to achieve stable coexistence and efficient delivery in conventional systems. Therefore, there is an urgent need to develop a novel delivery system that can effectively protect the activity of lactoferrin in food or pharmaceutical applications, significantly improve its stability and bioavailability in the gastrointestinal tract, and thus better realize its nutritional and functional value. Patent document CN118844639A discloses such a water-in-oil single emulsion system for the delivery of lactoferrin; however, it often fails to effectively protect its structure in vivo, has a limited encapsulation rate, and is difficult to achieve targeted release in the intestine. Summary of the Invention

[0004] This invention aims to overcome the shortcomings of existing technologies where lactoferrin and vitamin D3 are difficult to achieve stable coexistence and efficient delivery in conventional systems during oral administration, resulting in poor stability, low bioavailability, and easy inactivation in the gastrointestinal environment. The invention provides a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, a preparation method thereof, and applies it to the preparation processes of functional foods, nutritional supplements, and pharmaceutical preparations.

[0005] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, wherein the water-in-oil-in-water composite emulsion has a water-in-oil-in-water structure (W1 / O / W2), comprising: An internal aqueous phase solution, wherein the internal aqueous phase solution comprises lactoferrin; The oil phase includes edible oil, vitamin D3, and emulsifier; An external aqueous phase comprising a protein-polysaccharide complex formed by combining a protein and a polysaccharide, wherein the protein comprises sodium caseinate and the polysaccharide comprises gellan gum.

[0006] This invention improves the encapsulation efficiency and emulsion structure stability of active ingredients by optimizing the ratio and concentration of gellan gum (GG) and sodium caseinate (NaCas) in the outer aqueous phase, thereby significantly enhancing the bioavailability of lactoferrin (LF) and vitamin D3 and further leveraging their nutritional and functional advantages in promoting bone growth. Furthermore, this invention employs a W1 / O / W2 dual emulsion system, where the inner aqueous phase (W1) contains a lactoferrin solution, the oil phase (O) consists of edible oil with added vitamin D3 and an emulsifier, and the outer aqueous phase (W2) consists of a protein-polysaccharide complex. This structural design significantly improves the encapsulation efficiency of LF and enhances its tolerance to gastric acid and digestive enzymes, thereby increasing the reach and functional activity of lactoferrin in the intestine. Simultaneously, this invention utilizes a water-in-oil-in-water (W1 / O / W2) dual emulsion delivery system to simultaneously load and protect both hydrophilic and hydrophobic components, achieving synergistic controlled release and significantly enhancing its stability and absorption efficiency in the digestive tract. Compared with existing single emulsions or other carrier systems, the W1 / O / W2 dual emulsion of the present invention has higher physical stability and bioavailability, and can be widely used in functional foods, nutritional supplements and pharmaceutical preparations.

[0007] Specifically, this invention utilizes a two-step method to prepare a W1 / O / W2 dual emulsion, achieving highly efficient loading and delivery of lactoferrin. The final product does not require freeze-drying and exhibits a high effective concentration of lactoferrin. The multi-layered complex emulsion structure helps lactoferrin resist the damaging effects of gastric acid, thereby improving bioavailability and enabling targeted intestinal delivery. The final product can be directly applied to various wet-based foods such as moisturizing milk, coffee, and dairy beverages, demonstrating significant economic benefits and market potential.

[0008] Preferably, the volume ratio of the inner aqueous phase solution to the oil phase is 1:3~5; the volume ratio of the sum of the volumes of the inner aqueous phase solution and the oil phase to the volume of the outer aqueous phase is 1:1~3.

[0009] Preferably, the protein-polysaccharide complex is formed by combining sodium caseinate and gellan gum.

[0010] Preferably, the concentration of the sodium caseinate solution prepared from the sodium caseinate in the external aqueous phase is 0.8-3.2%, and the concentration of the gellan gum solution prepared from the gellan gum is 0.2-1.0%.

[0011] As a further preferred embodiment, the concentration of the sodium caseinate solution prepared from the sodium caseinate in the external aqueous phase is 2.4%, and the concentration of the gellan gum solution prepared from the gellan gum is 1.0%.

[0012] Sodium caseinate and gellan gum, when combined, form a stable gel network structure. The inventors of this invention have demonstrated through extensive experimentation that the protein-polysaccharide complex prepared from sodium caseinate and gellan gum is beneficial for improving the encapsulation efficiency of lactoferrin. This is because sodium caseinate acts as an emulsifier and provides film protection at the oil-water interface, while gellan gum forms a gel network in the continuous phase. The combination of these two components significantly improves the physical stability of water-in-oil-in-water composite emulsions. Simultaneously, this system imparts higher viscosity and better texture to the emulsion, making it more suitable for applications in food systems such as dairy beverages, functional yogurts, and solid drinks.

[0013] Preferably, the edible oil is any one or a combination of medium-chain triglycerides, soybean oil, corn oil, olive oil, and fish oil.

[0014] Preferably, the emulsifier is any one or a combination of polyglycerol ricinoleate, glyceryl monostearate, sucrose fatty acid ester, and soybean lecithin.

[0015] The method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 as described above includes the following steps: S1: Dissolve lactoferrin in water to obtain an internal aqueous phase solution; S2: Add vitamin D3 and emulsifier to edible oil and stir well to obtain the oil phase; S3: Add the aqueous phase solution obtained in step S1 to the oil phase obtained in step S2, stir and sonicate to obtain a water-in-oil primary emulsion. S4: Dissolve sodium caseinate and gellan gum in water, stir and hydrate to obtain an external aqueous phase; S5: Add the water-in-oil primary emulsion obtained in step S3 to the external aqueous phase, stir and sonicate to obtain the water-in-oil-in-water composite emulsion.

[0016] The method of this invention employs a multi-compartmental structure of "three-phase, two-membrane (inner aqueous phase, oil phase, outer aqueous phase, oil phase membrane, and outer aqueous phase membrane)" to encapsulate lactoferrin. This effectively isolates lactoferrin from the effects of external temperature and light, maintaining its stability and activity. The final product obtained by this method has a lactoferrin encapsulation rate of up to 82.52%, making it directly applicable to wet-based foods. Furthermore, the preparation process is simple, the method is easy to operate, and it is convenient for widespread application, enabling industrial-scale production.

[0017] Preferably, the volume ratio of the inner aqueous phase solution to the oil phase in step S3 is 1:3~5; and the volume ratio of the water-in-oil primary emulsion to the outer aqueous phase in step S5 is 1:1~3.

[0018] As a further preferred embodiment, the volume ratio of the inner aqueous phase solution to the oil phase in step S3 is 1:4; and the volume ratio of the water-in-oil primary emulsion to the outer aqueous phase in step S5 is 2:3.

[0019] Preferably, in steps S3 and S5, the mixture is stirred and sheared at 6000 rpm at room temperature for 2 min.

[0020] Preferably, the water-in-oil-in-water composite emulsion obtained in step S5 is stored at 4°C.

[0021] The application of the water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 as described above in the preparation of food, health food or pharmaceuticals.

[0022] As a preferred application, the water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 as described above is used in the preparation of infant formula, dairy beverages, functional yogurt, nutritional supplements, and oral iron supplements.

[0023] Therefore, the present invention has the following beneficial effects: (1) This invention employs a two-step method to construct a W1 / O / W2 dual emulsion system. Through the dual barrier effect of the oil phase membrane and the external aqueous phase membrane, the risk of leakage and inactivation of lactoferrin and VD3 during preparation, storage and processing is effectively reduced. Experimental results show that under optimized conditions of sodium caseinate and gellan gum, the encapsulation efficiency of lactoferrin can reach up to 82.52%, which is significantly higher than that of existing single emulsion or ordinary carrier systems. This demonstrates that this structure has a significant advantage in improving the loading efficiency of active substances and can significantly improve the encapsulation efficiency and structural stability of lactoferrin. (2) Because lactoferrin is embedded in a W1 / O / W2 multi-compartment structure, the outer layer, composed of sodium caseinate and gellan gum, forms a protein-polysaccharide complex network that effectively buffers the gastric acid environment and blocks the direct action of digestive enzymes, thereby significantly reducing the degradation rate of lactoferrin in the stomach. In vitro simulated digestion experiments show that the water-in-oil-in-water composite emulsion system prepared in this invention can significantly improve the retention rate of lactoferrin during digestion, enhance its tolerance to the gastrointestinal environment, and thus enhance its reach in the intestine, thereby improving overall bioavailability. (3) The external aqueous phase used in this invention is formed by a combination of sodium caseinate and gellan gum. Sodium caseinate forms a dense emulsion film at the oil-water interface, while gellan gum constructs a three-dimensional gel network structure in the continuous phase. The synergistic effect of the two significantly improves the physical stability of the emulsion and effectively inhibits oil droplet aggregation and phase separation. At the same time, this system endows the emulsion with high viscosity and good textural properties, improves the rheological properties of the emulsion system, and is beneficial to its application in actual food systems. (4) The water-in-oil-in-water composite emulsion prepared by this invention does not require freeze-drying and can be directly applied in liquid form to dairy beverages, functional yogurts, coffee, and other wet-based foods. This helps reduce production costs and process complexity, further expanding the application forms of lactoferrin, and has good industrialization feasibility and economic benefits. At the same time, this technology provides a new technical path to improve the utilization efficiency of lactoferrin in functional foods and nutritional supplements, and has positive social health value and broad market application prospects. Attached Figure Description

[0024] Figure 1 Images showing the appearance of emulsions prepared from NaCas-GG complexes of different concentrations.

[0025] Figure 2 Optical and fluorescence confocal micrographs of emulsions prepared from NaCas-GG complexes of different concentrations are shown. Figure 2 In the image, 'a' represents an optical micrograph of emulsions prepared from NaCas-GG complexes of different concentrations. Figure 2 b in the image represents a fluorescence confocal micrograph of emulsions prepared from NaCas-GG complexes of different concentrations.

[0026] Figure 3 The figure shows the encapsulation efficiency of LF and VD3 by emulsions prepared from NaCas-GG complexes of different concentrations.

[0027] Figure 4 The rheological properties of emulsions prepared from NaCas-GG complexes of different concentrations are shown in the figure. Figure 4In the figure, 'a' represents the surface viscosity of emulsions prepared from NaCas-GG complexes of different concentrations as a function of shear rate. Figure 4 In the figure, b is a graph showing the elastic modulus of emulsions prepared from NaCas-GG complexes of different concentrations as a function of angular frequency.

[0028] Figure 5 Particle size and zeta potential of emulsions prepared from NaCas-GG complexes of different concentrations are shown in the figure; where, Figure 5 In the figure, 'a' represents the particle size distribution of emulsions prepared from NaCas-GG complexes of different concentrations. Figure 5 In the diagram, b represents the zeta potential of emulsions prepared from NaCas-GG complexes of different concentrations.

[0029] Figure 6 The encapsulation efficiency and appearance of lactoferrin obtained by heating an emulsion prepared from a compound of NaCas (2.4%) and GG (1.0%) at 40–90 °C for 1 h are shown. Figure 6 Figure 'a' shows the encapsulation efficiency of lactoferrin obtained by heating an emulsion prepared from NaCas (2.4%) and GG (1.0%) at 40-90℃ for 1 hour. Figure 6 Figure b shows the appearance of an emulsion prepared by combining NaCas (2.4%) and GG (1.0%) after heating at 40-90℃ for 1 hour.

[0030] Figure 7 The appearance of emulsions prepared from different concentrations of NaCas-GG complexes after 30 days of storage at 4°C.

[0031] Figure 8 The graph shows the free fatty acid release rate and bioavailability of emulsions prepared from NaCas-GG complexes at different concentrations after 120 min of simulated in vitro digestion. Figure 8 In the figure, 'a' represents the free fatty acid release rate of emulsions prepared from NaCas-GG complexes at different concentrations after 120 min of simulated in vitro digestion. Figure 8 Figure b in the figure shows the bioavailability of emulsions prepared from different concentrations of NaCas-GG complex after 120 min of simulated in vitro digestion. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0033] Example 1 This embodiment provides a water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3, and its preparation method.

[0034] A method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 includes the following steps: S1: Preparation of the internal aqueous phase (W1) solution: Dissolve 0.1 g lactoferrin (LF) in 100 mL of ultrapure water to obtain an internal aqueous phase solution for later use; S2: Preparation of the oil phase (O): Add 0.01g of vitamin D3 (VD3) to 20mL of medium-chain triglycerides, stir well, then add 0.8g of polyglycerol ricinoleate, stir well to obtain the oil phase, and set aside. S3: Preparation of water-in-oil primary emulsion: The aqueous phase solution obtained in step S1 was added dropwise to the oil phase obtained in step S2 at a volume ratio of 1:4. After standing for 10 minutes, the mixture was stirred and sheared at 6000 rpm at room temperature for 2 minutes to obtain an emulsion. The emulsion was then placed at 4°C for 30 minutes to obtain a water-in-oil primary emulsion. S4: Preparation of the external aqueous phase (W2): S4.1: Preparation of sodium caseinate (NaCas) stock solution: Dissolve 2.4 g of sodium caseinate in 100 mL of ultrapure water to prepare a 2.4% (w / w) NaCas stock solution, and stir for 60 min for later use. S4.2: Preparation of sodium caseinate-gellan gum (NaCas-GG) stock solution: Dissolve 0.2 g of gellan gum (GG) in NaCas stock solution and stir for 120 min for later use; S4.3: Store the NaCas-GG stock solution at 4℃ for 12h to allow the NaCas-GG complex to fully hydrate and obtain the external aqueous phase; S5: Add the water-in-oil primary emulsion obtained in step S3 to the external aqueous phase obtained in step S4.3 at a volume ratio of 2:3, let stand for 10 minutes, stir at 6000 rpm for 2 minutes to obtain a water-in-oil composite emulsion, and store at 4℃.

[0035] A water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 was prepared by the method described above.

[0036] The application of the water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 as described above in the preparation of food, health food or pharmaceuticals.

[0037] Example 2 The difference between this embodiment and Embodiment 1 is that: A method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, wherein in step S4.2, 0.4 g of gellan gum (GG) is dissolved in NaCas stock solution. All other steps are the same as in Example 1.

[0038] Example 3 The difference between this embodiment and Embodiment 1 is that: A method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, wherein in step S4.2, 0.6 g of gellan gum (GG) is dissolved in NaCas stock solution. All other steps are the same as in Example 1.

[0039] Example 4 The difference between this embodiment and Embodiment 1 is that: A method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, wherein in step S4.2, 0.8 g of gellan gum (GG) is dissolved in NaCas stock solution. All other steps are the same as in Example 1.

[0040] Example 5 The difference between this embodiment and Embodiment 1 is that: A method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, wherein in step S4.2, 1.0 g of gellan gum (GG) is dissolved in NaCas stock solution. All other steps are the same as in Example 1.

[0041] [Comprehensive Performance Testing and Analysis] 1. A water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 was prepared according to the methods in Examples 1 to 5, and the emulsion was observed by the naked eye, optical microscope, and fluorescence confocal microscope, respectively.

[0042] The microstructure of the W1 / O / W2 water-in-oil-in-water composite emulsion was observed using the white light path of an inverted fluorescence microscope. The prepared emulsion was diluted 5 times with distilled water and then dropped onto a grooved glass slide, which was then covered with a coverslip. Images were obtained at 40x magnification.

[0043] The microstructure of each emulsion was observed using a bio-laser confocal microscope. 30 μL of emulsion was transferred to a glass slide, and the protein and oil phases were stained with Nile Red (0.1% w / v, dissolved in DMSO), respectively. 5 μL of each staining agent was mixed with the emulsion for staining. CLSM images of the W1 / O / W2 emulsion were obtained under a bio-laser confocal microscope at 20x magnification, with excitation wavelengths of 633 nm and 488 nm, respectively, to observe the microstructure. The appearance of emulsions prepared from different concentrations of the NaCas-GG complex is shown in the figures below. Figure 1 As shown in the figure. Optical and fluorescence confocal micrographs of emulsions prepared from NaCas-GG complexes of different concentrations are shown in the figure. Figure 2 As shown. Among them, Figure 2 In the image, 'a' represents an optical micrograph of emulsions prepared from NaCas-GG complexes of different concentrations. Figure 2 b in the image represents a fluorescence confocal micrograph of emulsions prepared from NaCas-GG complexes of different concentrations.

[0044] Microstructure of emulsions under different concentrations of GG, as follows Figures 1-2 As shown in the figure, this invention uses Nile Red for specific fluorescent staining of the oil phase and characterizes it using confocal laser microscopy (CLSM). From the CLSM images, it can be clearly observed that aqueous droplets without fluorescence (appearing black in the image) are dispersed within oil droplets with strong fluorescence (appearing red in the image), while the oil droplets are dispersed in a continuous outer aqueous background. This morphological feature intuitively confirms that the system successfully constructs a typical water-in-oil-in-water (W1 / O / W2) hierarchical structure.

[0045] Furthermore, a distinct double emulsion morphology was observed in all samples with different concentrations of GG (0.2%–1.0%), indicating that the addition of GG to the external aqueous phase did not disrupt the original framework structure of the double emulsion. However, it is noteworthy that as the GG concentration gradually increased, more small "empty" oil droplets (i.e., monolayer O / W droplets that did not encapsulate the W1 internal aqueous phase solution) began to appear in the system.

[0046] 2. A water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 was prepared according to the methods in Examples 1 to 5, and the LF encapsulation rate was tested on each emulsion.

[0047] LF encapsulation efficiency is used to evaluate the encapsulation effect of a dual emulsion delivery system on active ingredients. This invention refers to the high-performance liquid chromatography (HPLC) method for determining LF content in the national standard, quantitatively analyzing the total and free LF content in the emulsion system, and calculating the encapsulation efficiency accordingly.

[0048] In the determination of total LF content in the emulsion, 10 mL of emulsion sample was taken, and 10 mL of anhydrous ethanol was added as a demulsifier. The mixture was thoroughly shaken to disrupt the W1 / O / W2 double emulsion structure, ensuring complete release of LF from the system. The sample was then centrifuged (4℃, 10000 r / min, 10 min), and the supernatant was collected and filtered through a 0.45 μm microporous membrane for HPLC analysis. Anhydrous ethanol has good demulsifying ability and good compatibility with the chromatographic system, achieving complete destruction of the emulsion structure without significantly affecting the quantitative analysis of LF. The determination of free LF content was performed without disrupting the overall emulsion structure. 10 mL of emulsion sample was taken, diluted 5-fold, centrifuged, and the aqueous phase was collected. This aqueous phase was also filtered through a 0.45 μm microporous membrane, and the LF content was determined by HPLC.

[0049] LF content was determined using high-performance liquid chromatography (HPLC), with chromatographic conditions performed according to national standard methods. The chromatographic column was a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile-water (20:80 v / v), with isocratic elution; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelength was 280 nm; and the injection volume was 20 μL. Standard curves were plotted using LF standard solutions of different concentrations, and the LF content was calculated based on the peak area of ​​the samples. This method exhibits good linearity, repeatability, and accuracy, and is suitable for the quantitative analysis of LF in emulsion systems.

[0050] The LF encapsulation efficiency (EE) is calculated according to formula (1): (1); In the formula, m1 is the total LF content in the emulsion (mg), and m2 is the content of free LF in the aqueous phase outside the emulsion (mg).

[0051] VD3 encapsulation efficiency is used to evaluate the encapsulation effect of a dual emulsion delivery system on lipid-soluble active ingredients. This invention uses ultraviolet-visible spectrophotometry (UV-Vis) to determine the total and free VD3 content in the emulsion system and calculates the encapsulation efficiency accordingly.

[0052] In the determination of total VD3 content, 10 mL of emulsion sample was taken, and 10 mL of anhydrous ethanol was added as a demulsifier. The mixture was thoroughly shaken to disrupt the W1 / O / W2 double emulsion structure, allowing complete release of VD3 from the system. Subsequently, 5 mL of n-hexane was added for extraction. After shaking and mixing, the mixture was allowed to stand for separation. The upper organic phase was collected, dehydrated with anhydrous sodium sulfate, and filtered to obtain the test solution. The determination of free VD3 content was performed without disrupting the overall structure of the emulsion. 10 mL of emulsion sample was taken, centrifuged, and the aqueous phase was collected. The same extraction procedure as for the total content determination was used to extract free VD3 from the aqueous phase with n-hexane. The resulting test solution was then subjected to UV absorbance measurement.

[0053] The UV detection wavelength for VD3 was set to 265 nm. Standard curves were plotted using VD3 standard solutions of different mass concentrations, and the VD3 content was calculated based on the sample absorbance values. This method is simple to operate, has good repeatability, and is suitable for the quantitative analysis of VD3 in emulsion systems. The VD3 encapsulation efficiency (EE) is calculated according to formula (2): (2); In the formula, m3 represents the total VD3 content in the emulsion (μg), and m4 represents the free VD3 content in the aqueous phase outside the emulsion (μg). The encapsulation efficiency of LF and VD3 in emulsions prepared from NaCas-GG complexes of different concentrations is shown in the figure below. Figure 3 As shown.

[0054] from Figure 3 Analysis shows that GG concentration has a significant impact on the encapsulation efficiency of LF in W1 / O / W2 emulsions. When the GG concentration increases from 0 to 1.0 wt%, the encapsulation efficiency of LF increases from approximately 61% to 83%. This increase in LF encapsulation efficiency with increasing GG concentration may be related to the formation of a denser and more stable protein-polysaccharide composite coating around the W1 / O droplets. This structure effectively inhibits the diffusion and leakage of LF during emulsification and storage. Normally, fluid exchange easily occurs between the inner and outer aqueous phases of a dual emulsion due to osmotic pressure gradients, which is the main driving force for the leakage of water-soluble encapsulated materials. The dense composite layer reduces the permeability of the interface, overcoming the weakness of traditional single-phase interfaces. Simultaneously, the thickened outer aqueous phase also reduces the osmotic mass transfer dynamics between the inside and outside of the system to some extent, delaying the transmembrane migration of water molecules, thereby more effectively locking in water-soluble components in the inner aqueous phase. In contrast, changes in GG concentration did not significantly affect the encapsulation efficiency of VD3 in the W1 / O / W2 emulsion. This may be because VD3, as a lipid-soluble active substance, is mainly distributed and stably embedded in the oil phase (O), and its encapsulation behavior is less sensitive to changes in GG content in the external aqueous phase.

[0055] 3. A water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 was prepared according to the methods in Examples 1 to 5, and the rheological properties of the emulsion were tested.

[0056] The rheological properties of the W1 / O / W2 emulsion were detected at 25°C using a dynamic shear rheometer. Approximately 2 mL of the water-in-oil-in-water composite emulsion sample was placed on the instrument's platform, using a 40 mm diameter clamp (equilibrium time: 120 s, temperature: 25°C, gap height: 1000 μm). A small amount of silicone oil was applied to the outer edge of the sample to prevent water evaporation. The linear viscoelastic region was determined by increasing the strain from 0.1% to 100% at a fixed frequency of 1 Hz. The apparent viscosity of the sample was measured by flow scanning, with shear rates ranging from 0.1 to 100 s⁻¹. -1 The measurements were repeated three times. The rheological properties of emulsions prepared from NaCas-GG complexes of different concentrations are shown in the following figures. Figure 4 As shown. Among them, Figure 4 In the figure, 'a' represents the surface viscosity of emulsions prepared from NaCas-GG complexes of different concentrations as a function of shear rate. Figure 4 In the figure, b is a graph showing the elastic modulus of emulsions prepared from NaCas-GG complexes of different concentrations as a function of angular frequency.

[0057] from Figure 4 Analysis reveals that under steady-state shear conditions, the emulsion system exhibits more typical shear-thinning behavior with increasing GG content, and the apparent viscosity in the low shear rate range increases significantly. This indicates that GG molecules construct a relatively dense spatial network in the continuous phase through segment entanglement and interactions, and this physically cross-linked network will disentangle or break down under an applied shear field. Dynamic rheological analysis shows that the storage modulus (G') of the system increases with increasing GG concentration, reflecting the enhanced elasticity of the emulsion. Higher GG concentrations promote the formation of a weak gel network with certain mechanical strength within the system, thus endowing the emulsion with superior structural stability. Furthermore, this continuous phase three-dimensional gel network dominated by polysaccharides significantly increases the steric hindrance of Brownian motion and upward floating of internal droplets, and synergistically works with the composite adsorption layer at the oil-water interface to jointly construct a physical barrier that hinders droplet aggregation. As expected by Stokes' Law, the high-viscosity continuous phase restricts droplet buoyancy caused by density differences; while the enhanced elastic network allows the emulsion to better maintain its structural framework in a static state, resisting phase separation caused by Ostwald ripening.

[0058] 4. A water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 was prepared according to the methods in Examples 1 to 5, and its particle size distribution and zeta potential were measured respectively.

[0059] The particle size distribution of the prepared W1 / O / W2 emulsion was determined using a laser diffraction particle size analyzer. This instrument uses the Fraunhofer method to calculate the particle size distribution from the light scattering pattern and can detect particles with a size range of 0.04–1000 μm. The sample was diluted with distilled water to obtain a measurable signal and avoid multiple scattering.

[0060] Zeta potential was determined using a Malvern laser nanoparticle size analyzer to measure the zeta potential values ​​of the W1 / O / W2 emulsion. Measurements were repeated three times at room temperature. The particle size and zeta potential of emulsions prepared from different concentrations of the NaCas-GG complex are shown in the figure below. Figure 5 As shown. Among them, Figure 5 In the figure, 'a' represents the particle size distribution of emulsions prepared from NaCas-GG complexes of different concentrations. Figure 5 In the diagram, b represents the zeta potential of emulsions prepared from NaCas-GG complexes of different concentrations.

[0061] from Figure 5 Analysis revealed that the average particle size of the W1 / O / W2 emulsion gradually decreased with increasing GG concentration. This result suggests that NaCaS and GG molecules may form a stable composite interface layer on the oil droplet surface through electrostatic interactions or steric hindrance, thereby inhibiting droplet aggregation and coalescence, and thus reducing the emulsion particle size. During the secondary shearing process of the dual emulsion, the introduction of polysaccharide macromolecules increased the viscosity of the continuous phase, delaying the collision frequency of newly formed droplets; simultaneously, the thickened composite interface provided stronger steric repulsion, effectively preventing secondary aggregation of droplets after the removal of high shear force.

[0062] Zeta potential analysis further revealed that the absolute value of the Zeta potential of the water-in-oil-in-water composite emulsion droplets gradually increased with increasing GG concentration. This change can be attributed to the adsorption of anionic GG molecules on the surface of the NaCaS-coated oil droplets, resulting in a greater negative charge on the droplet surface. Simultaneously, the presence of free GG molecules in the external aqueous phase may also enhance the overall electronegativity of the system. Notably, the further increase in the absolute value of the Zeta potential when the GG concentration increased from 0.6 wt% to 1.0 wt% may be related to the dominance of unadsorbed GG molecules in the solution, thus contributing more to the electrical signal during the Zeta potential measurement.

[0063] 5. A water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 was prepared according to the method in Example 5. The encapsulation rate of lactoferrin and its appearance were tested and observed after heating in a water bath at 40~90℃ for 1 hour.

[0064] 10 mL of freshly prepared W1 / O / W2 emulsions containing LF and VD3 were placed in sample vials and heated in water baths at 40, 50, 60, 70, 80, and 90 °C for 60 min, respectively. After cooling to room temperature and standing for 1 h, the LF encapsulation efficiency of the emulsions was measured. The encapsulation efficiency and appearance of lactoferrin in emulsions prepared from NaCas (2.4%) and GG (1.0%) after heating at 40–90 °C for 1 h are shown in the figures. Figure 6 As shown. Among them, Figure 6 Figure 'a' in the figure represents the encapsulation efficiency of LF prepared by combining NaCas (2.4%) and GG (1.0%) and heated at 40-90℃ for 1 h. Figure 6 Figure b shows the appearance of an emulsion prepared by combining NaCas (2.4%) and GG (1.0%) after heating at 40~90℃ for 1h.

[0065] from Figure 6 Analysis revealed that under lower temperature treatment conditions, the W1 / O / W2 emulsion maintained good dispersion and a uniform appearance, with no obvious emulsion separation or phase separation observed, indicating good structural stability under mild heat treatment. As the heating temperature further increased, the LF encapsulation efficiency gradually decreased, and the emulsion sample exhibited stratification and aggregation under high-temperature treatment; however, the overall dual emulsion structure remained intact, demonstrating the system's tolerance to heat treatment.

[0066] 6. A water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 was prepared according to the methods in Examples 1-5, and each emulsion was stored at 4°C for 30 days, with the state changes recorded during this period. The appearance of emulsions prepared from different concentrations of the NaCas-GG complex after 30 days of storage at 4°C is shown in the figures below. Figure 7 As shown.

[0067] from Figure 7 Analysis showed that the emulsion with only 0.2% GG had the worst stability and demulsified. By day 12 of storage, only the emulsions coated with 0.8% and 1.0% GG remained stable. The GG-stabilized emulsions underwent phase separation and had a minimal emulsion layer. On day 7, only the emulsion with 1.0% GG showed a uniform appearance. Demulsification began until day 15. After day 25, all emulsions showed obvious separation. Overall, the complex prolonged the stability of the emulsions.

[0068] 7. A water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 was prepared according to the methods in Examples 1 to 5, and a simulated in vitro digestion test was performed on each emulsion for 120 min.

[0069] The simulated digestion process included oral, gastric, and intestinal digestion. The experiment was conducted in a shaker at 37°C and 100 rpm. 10 mL of fresh emulsion was mixed with 10 mL of simulated saliva (SSF) by shaking for 2 min. Simulated gastric digestion was performed by mixing the emulsion digested with SSF and simulated gastric juice (SGF) at a volume ratio of 1:3, with a total gastric digestion time of 120 min. The pH of the composite solution was adjusted to 7.0 with 0.1 mol / L NaOH to end the tail-end simulated digestion. Then, the emulsion digested with SGF was mixed with simulated intestinal juice (SIF) at a volume ratio of 1:1, and the intestinal simulated digestion lasted for 120 min. The pH of the composite solution was adjusted to 7.0 with 0.1 mol / L NaOH every 20 min, and the amount of NaOH required for the release of free fatty acids (FFA) from lipid hydrolysis was recorded. The release rate of FFA was calculated according to formula (3): (3); Where: V NaOH M is the molar concentration of NaOH consumed. NaOH It is the concentration of NaOH consumed (mol / L), M Lipid W is the mass (g) of medium-chain triglycerides (MCT) in the sample. Lipid It is the molar mass of MCT (g / mol).

[0070] The bioavailability of LF after the small intestine digests milk is calculated by equation (4): (4); Among them: W S For LF(U) released in SIF, W Initial The free fatty acid release rate and bioavailability of emulsions prepared from different concentrations of NaCas-GG complex after 120 min of simulated in vitro digestion are shown in the figure below. Figure 8 As shown. Among them, Figure 8 In the figure, 'a' represents the free fatty acid release rate of emulsions prepared from NaCas-GG complexes at different concentrations after 120 min of simulated in vitro digestion. Figure 8 Figure b in the figure shows the bioavailability of emulsions prepared from different concentrations of NaCas-GG complex after 120 min of simulated in vitro digestion.

[0071] from Figure 8Analysis revealed that the lipid digestion of each group of samples exhibited a biphasic characteristic: FFA release was rapid in the initial stage of digestion (0-40 min), followed by a gradual slowdown and plateau. The study found that the introduction of GG inhibited the lipid digestion process of the emulsion; as the GG concentration increased from 0.2% to 1.0%, the final FFA release rate showed a significant dose-dependent decrease. This inhibitory effect was mainly attributed to the high-viscosity three-dimensional network structure formed by GG in the continuous phase (W2): on the one hand, this network increased the steric hindrance of the system, limiting the diffusion and adsorption of lipases and bile salts to the oil-water interface; on the other hand, the polysaccharide network restricted the movement of emulsion droplets, reducing the effective specific surface area for lipase action, thereby delaying the interfacial hydrolysis reaction.

[0072] Based on the in vitro release results of the encapsulated material, the delayed hydrolysis of the intermediate oil phase (O) prolonged its effective time as a physical barrier, reduced premature leakage of LF from the inner aqueous phase (W1) into the gastrointestinal fluid, and increased its in vitro bioavailability to approximately 86%. Simultaneously, the reduced lipid hydrolysis rate allowed lipid-soluble vitamin D3 to slowly and continuously enter the mixed micelles, maintaining an absorption rate of 60%–64%. These results indicate that the appropriately GG-modified W1 / O / W2 water-in-oil-in-water composite emulsion not only slowed down the overall digestion process but also achieved synergistic controlled release of LF and vitamin D3, providing a material basis for these two active factors to exert their calcium absorption-promoting effects in the intestine. For LF encapsulated in the inner aqueous phase (W1), its bioavailability increased significantly with increasing GG concentration, reaching a maximum (approximately 86%) at a GG concentration of 0.8%. This improvement is mainly attributed to the dense polymeric gel network constructed by GG in the continuous phase (W2). Rheological and digestive kinetic data show that the enhanced outer aqueous phase (W2) physical barrier effectively resists the disruption of the gastrointestinal environment, slows down the rate of lipid digestion, and thus effectively prevents premature rupture of the inner aqueous phase (W1) and pepsin degradation of LF, achieving targeted and protective delivery of LF to the small intestine. In contrast, the bioavailability (approximately 60%–64%) of VD3 dissolved in the intermediate oil phase (O) showed no significant difference among different GG concentration groups. This indicates that although the high viscosity network of GG slows down the release rate of free fatty acids, it does not affect the final degree of lipid micellization or the intestinal absorption efficiency of VD3. In conclusion, the W1 / O / W2 water-in-oil-in-water composite emulsion modified with an appropriate amount of GG (0.6%–1.0%) can significantly improve the bioavailability of LF without sacrificing the absorption of fat-soluble vitamins.

[0073] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3, characterized in that, include: An internal aqueous phase solution, wherein the internal aqueous phase solution comprises lactoferrin; The oil phase includes edible oil, vitamin D3, and emulsifier; An external aqueous phase comprising a protein-polysaccharide complex formed by combining a protein and a polysaccharide, wherein the protein comprises sodium caseinate and the polysaccharide comprises gellan gum.

2. The water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 according to claim 1, characterized in that, The volume ratio of the internal aqueous phase solution to the oil phase is 1:3~5; the volume ratio of the sum of the volumes of the internal aqueous phase solution and the oil phase to the volume of the external aqueous phase is 1:1~3.

3. The water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 according to claim 1, characterized in that, The concentration of the sodium caseinate solution prepared from the sodium caseinate in the external aqueous phase is 0.8-3.2%, and the concentration of the gellan gum solution prepared from the gellan gum is 0.2-1.0%.

4. The water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 according to claim 1, characterized in that, The edible oil is any one or a combination of medium-chain triglycerides, soybean oil, corn oil, olive oil, and fish oil.

5. The water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 according to claim 1, characterized in that, The emulsifier is any one or a combination of polyglycerol ricinoleate, glyceryl monostearate, sucrose fatty acid ester, and soybean lecithin.

6. A method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Dissolve lactoferrin in water to obtain an internal aqueous phase solution; S2: Add vitamin D3 and emulsifier to edible oil and stir well to obtain the oil phase; S3: Add the aqueous phase solution obtained in step S1 to the oil phase obtained in step S2, stir and sonicate to obtain a water-in-oil primary emulsion. S4: Dissolve sodium caseinate and gellan gum in water, stir and hydrate to obtain an external aqueous phase; S5: Add the water-in-oil primary emulsion obtained in step S3 to the external aqueous phase, stir and sonicate to obtain the water-in-oil-in-water composite emulsion.

7. The method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 according to claim 6, characterized in that, In step S3, the volume ratio of the inner aqueous phase solution to the oil phase is 1:3~5; in step S5, the volume ratio of the water-in-oil primary emulsion to the outer aqueous phase is 1:1~3.

8. The method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 according to claim 6, characterized in that, In steps S3 and S5, the mixture is stirred and sheared at 6000 rpm at room temperature for 2 minutes.

9. The method for preparing a water-in-oil-in-water composite emulsion encapsulating lactoferrin and vitamin D3 according to claim 6, characterized in that, The water-in-oil-in-water composite emulsion obtained in step S5 is stored at 4°C.

10. The use of a water-in-oil-in-water composite emulsion containing lactoferrin and vitamin D3 as described in any one of claims 1 to 5 in the preparation of food, health food, or pharmaceuticals.

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