A ternary composite material and a preparation method and application thereof

Abstract

This invention provides a ternary composite material, its preparation method, and its application. The preparation method of the ternary composite material includes the following steps: (1) mixing sodium caseinate with epigallocatechin gallate to obtain a protein-polyphenol binary complex; (2) mixing the protein-polyphenol binary complex with tea polysaccharide to obtain a ternary composite material. This invention employs a molecular self-assembly strategy to construct an SC-EGCG binary composite system, utilizing the non-covalent interactions (hydrogen bonds, hydrophobic interactions, etc.) between tea polysaccharide (TPS) and the SC-EGCG complex to construct a stable SC-EGCG-TPS ternary composite system with a three-dimensional network structure.

Description

Technical Field

[0001] This invention relates to the field of food technology, and in particular to a ternary composite material, its preparation method, and its application. Background Technology

[0002] β-Carotene, an important fat-soluble nutrient, possesses provitamin A activity and various physiological functions, making it valuable for the development of functional foods. However, β-carotene is highly sensitive to environmental factors such as light, heat, and oxygen, exhibits extremely low solubility in aqueous systems, and has poor bioavailability. These characteristics severely limit its application in practical food systems. Therefore, constructing an efficient and stable delivery system to achieve the protection and controlled release of β-carotene has become a critical issue urgently needing to be addressed in the field of food science.

[0003] Emulsion delivery systems, especially high internal phase emulsions (HIPEs), exhibit significant advantages in encapsulating lipophilic active ingredients due to their unique structural characteristics. HIPEs are viscous emulsions with a dispersed phase volume fraction exceeding 74%. Their semi-solid structure, formed by tightly packed polygonal droplets, not only endows the system with excellent plastic rheological properties but also provides an effective environmental barrier for the encapsulated material, significantly improving the physicochemical stability and bioavailability of lipophilic active ingredients. Currently, stabilizing HIPEs using natural biomacromolecules has become a research hotspot in food science, with the development of safe and efficient natural emulsifiers being particularly important.

[0004] Sodium caseinate (SC) is an amphiphilic biomolecule derived from milk protein, possessing excellent emulsifying, film-forming, and surface-active properties, and is widely used in food emulsion systems. However, natural sodium caseinate has shortcomings in environmental stability (such as pH, ionic strength, and temperature) and antioxidant capacity, limiting its application in complex food matrices and functional foods, especially in the encapsulation and delivery of fat-soluble active ingredients (such as β-carotene), where problems include poor emulsion stability, easy oxidation and degradation of active ingredients, and low bioavailability.

[0005] In recent years, the functional modification of proteins through the interaction of polyphenolic compounds with proteins has become a research hotspot. Epigallocatechin gallate (EGCG) is the most abundant and bioactive catechin polyphenol in tea, possessing excellent antioxidant, antibacterial, and protein-binding abilities. Studies have shown that EGCG can bind to sodium caseinate through non-covalent interactions such as hydrogen bonding and hydrophobic interactions, improving its emulsifying properties and antioxidant activity. However, EGCG modification alone suffers from problems such as unstable binding, easy oxidation, and insufficient long-term stability in high internal phase emulsion systems, making it difficult to meet the dual requirements of β-carotene encapsulation systems for emulsion stability and antioxidant protection.

[0006] A Chinese patent document, CN120959390A, discloses a "sodium caseinate-epigallocatechin gallate-iron ternary complex". This patent introduces Fe... 3+ Metal-phenolic networks are constructed by coordinating EGCG with metal coordinate bonds. However, their practical application in food systems presents the following problems: First, the complex introduces exogenous metal ions, which may catalyze lipid oxidation and cause flavor degradation during long-term food storage, raising potential safety concerns. Second, although the complex is composed of EGCG and Fe... 3+ Coordination formation occurs, but this metal-polyphenol coordination complex is prone to proton competitive dissociation in acidic environments, leading to structural instability of the complex and thus limiting its stability in acidic food systems (such as juice and fermented dairy products). In addition, the stability of this complex is quite sensitive to pH changes, making it difficult to meet the actual needs of food processing with a wide pH range. While existing technologies report on the composite modification of sodium caseinate and polysaccharides, most studies focus on binary systems and primarily address emulsion stability (Liu Qiudie, Cao Yang, Zhang Yanpeng, et al. Physicochemical properties and emulsion stability of sodium caseinate-polysaccharide complex [J]. Food Research and Development, 2024, 45(11):54-61; Li Peiyuan. Preparation and structural characterization of composite emulsion gels of sodium caseinate and different polysaccharides [D]. Shaanxi Normal University, 2022. DOI:10.27292 / d.cnki.gsxfu.2022.000286.), without addressing the synergistic effect of simultaneously introducing EGCG and tea polysaccharides. Furthermore, both polyphenols and polysaccharides are active substances that interact with proteins, and they may compete for binding sites on the same or adjacent molecules of sodium caseinate, leading to structural disorder or mutual functional inhibition of the complex. Therefore, those skilled in the art would find it difficult to anticipate that the simultaneous introduction of EGCG and tea polysaccharides would produce a synergistic effect rather than mutual interference. This invention was made precisely to overcome this technical bias. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a ternary composite material and its preparation method and application, which solves the problems of the existing sodium caseinate single system in terms of environmental stability and antioxidant capacity, the unstable binding and poor long-term stability of single polyphenol modification, and the difficulty of the sodium caseinate and polysaccharide binary composite system in taking into account the interfacial stability, antioxidant activity and efficient encapsulation and protection of fat-soluble active ingredients.

[0008] To achieve the above and other related objectives, the present invention provides a method for preparing a ternary composite material, comprising the following steps: (1) Sodium caseinate and epigallocatechin gallate were mixed to obtain a protein-polyphenol binary complex; (2) The protein-polyphenol binary complex was mixed with tea polysaccharide to obtain a ternary composite material.

[0009] Preferably, the protein is sodium caseinate (SC), the polyphenol is epigallocatechin gallate (EGCG), and the polysaccharide is tea polysaccharide.

[0010] Tea polysaccharides (TPS), another important active ingredient in tea, possess excellent hydrophilicity, thickening properties, and emulsion stability. Polysaccharides can form stable conjugated compounds or complexes with proteins, synergistically improving the interfacial stability and rheological properties of emulsion systems. Current research reports on the composite modification of sodium caseinate with polysaccharides, but these studies mostly focus on single polysaccharide systems and primarily on improving emulsion stability. Systematic research on their synergistic antioxidant protective effects in encapsulating fat-soluble active ingredients (such as β-carotene) is lacking. Furthermore, both polyphenols and polysaccharides are active substances that interact with proteins; they may compete for binding sites on the same or adjacent binding sites on sodium caseinate molecules, leading to structural disorder or mutual functional inhibition of the complex. Therefore, those skilled in the art would find it difficult to anticipate that the simultaneous introduction of EGCG and tea polysaccharides would produce a synergistic effect rather than mutual interference.

[0011] This invention creatively breaks through this technical bias, discovering for the first time that a protein-polyphenol-polysaccharide ternary complex in a specific ratio can achieve a synergistic enhancement of interfacial activity, antioxidant properties, and thickening stability, achieving unexpected technical effects. In the prior art, there are no reports of simultaneously utilizing EGCG (polyphenol) and tea polysaccharide (polysaccharide) to synergistically modify sodium caseinate, constructing a ternary complex as a stabilizer for high internal phase emulsions, while simultaneously achieving efficient encapsulation and antioxidant protection of β-carotene. Existing single or binary modification systems are insufficient in terms of interfacial stability, antioxidant activity, and protection of active ingredients.

[0012] Preferably, the mass ratio of sodium caseinate to epigallocatechin gallate is (8~12):1.

[0013] Preferably, the weight-volume ratio of the tea polysaccharide in the ternary composite material mixture is 1-5% (w / v).

[0014] Based on the synergistic mechanism of polyphenols and polysaccharides, this application innovatively uses sodium caseinate (SC) as the structural matrix to construct a functional delivery system through multi-scale regulation technology. First, a molecular self-assembly strategy is employed to construct a binary SC-EGCG complex system. Then, utilizing the non-covalent interactions (hydrogen bonds, hydrophobic interactions, etc.) between tea polysaccharide (TPS) and the SC-EGCG complex, a stable SC-EGCG-TPS ternary complex system with a three-dimensional network structure is constructed. Finally, the optimal ratio of the complex is determined, and it is successfully applied to the construction of an internal phase emulsion for lipid-soluble active substances.

[0015] The compatibility relationships of the components in the ternary composite material of this application are as follows: Sodium caseinate, as an amphiphilic structural matrix, has hydrophobic regions, amino groups, and carboxyl groups distributed on its molecules, providing the basic interfacial activity and emulsifying ability for this invention, while also providing abundant binding sites for polyphenols and polysaccharides. EGCG binds to sodium caseinate through hydrogen bonds and hydrophobic interactions, first forming an SC-EGCG binary complex. This process endows the system with excellent antioxidant activity and initially enhances the compactness of the interfacial film. On this basis, tea polysaccharides further bind to the SC-EGCG complex through non-covalent interactions (mainly manifested as hydrogen bonds and hydrophobic interactions), forming an SC-EGCG-TPS ternary complex. The tea polysaccharide molecular chain contains a large number of hydroxyl groups, and its hydrophilic long-chain structure not only enhances the steric hindrance effect and hydration capacity of the complex, but also improves the thickening properties of the continuous phase, thereby synergistically enhancing the long-term stability of the emulsion.

[0016] The protein-polyphenol-polysaccharide ternary composite material of the present invention relies on the non-covalent interaction between natural macromolecules and does not introduce any additional exogenous metal ions. It can maintain excellent structural stability at high temperature, high salt and a wide pH range (pH 2.0~7.0). All components are from natural food sources, making it safer.

[0017] Preferably, in steps (1) and (2), before mixing, each component is dissolved in phosphate buffer solution, and the pH of the mixed system is 6.5~7.5.

[0018] The present invention also provides a ternary composite material prepared by the above preparation method.

[0019] Preferably, the ternary composite material has an average particle size of 250~300 nm in aqueous solution and an absolute value of 14~20 mV for the Zeta potential.

[0020] The present invention also provides an application of the above-mentioned ternary composite material in the preparation of high internal phase emulsion.

[0021] The present invention also provides a high internal phase emulsion comprising an aqueous phase and an oil phase, wherein the aqueous phase is the ternary composite material as described in claim 6; and the oil phase has a volume fraction of 70-85%.

[0022] The present invention also provides an application of the above-mentioned high internal phase emulsion in the encapsulation of lipid-soluble active substances.

[0023] In vitro simulated digestion experiments showed that the high internal phase emulsion stabilized by the ternary complex of this application can effectively protect the retention rate of fat-soluble active substances in the gastrointestinal environment, improve their bioavailability, and solve the problems of easy oxidation and degradation and low bioavailability of fat-soluble active ingredients in the prior art. The high internal phase emulsion system of this application also has the characteristics of a healthy fat substitute and can be widely used in the processing and manufacturing of low-fat foods.

[0024] Preferably, the fat-soluble active substance is one or more of β-carotene, lycopene, curcumin, and vitamin E.

[0025] The ternary composite material stable high internal phase emulsion constructed in this invention can not only be used for the encapsulation of β-carotene, but also be extended to other fat-soluble active substances such as lycopene, curcumin, and vitamin E. It can also be used as a healthy fat substitute in the processing and manufacturing of low-fat foods in neutral or wide pH ranges, and has stronger industrial applicability and broader application prospects.

[0026] As described above, the present invention has the following beneficial effects: (1) A molecular self-assembly strategy was adopted to construct a binary SC-EGCG complex system. The non-covalent interactions (hydrogen bonds, hydrophobic interactions, etc.) between tea polysaccharide (TPS) and the SC-EGCG complex were utilized to construct a stable SC-EGCG-TPS ternary complex system with a three-dimensional network structure. (2) The protein-polyphenol-polysaccharide ternary composite material of the present invention relies on the non-covalent interaction between natural macromolecules and does not introduce any additional exogenous metal ions. It can maintain excellent structural stability in high temperature, high salt and wide pH range (pH 2.0~7.0). All components are from natural food sources and are safer. (3) The high internal phase emulsion stabilized by the ternary complex of this application can effectively protect the retention rate of lipid-soluble active substances in the gastrointestinal environment, improve their bioavailability, and solve the problems of easy oxidation and degradation and low bioavailability of lipid-soluble active ingredients in the prior art. Attached Figure Description

[0027] Figure 1 The diagram shown is a schematic representation of the preparation process of this invention.

[0028] Figure 2 The particle size, PDI, and Zeta potential of the SC-EGCG-TPS ternary complex at different TPS concentrations are shown below: (A) Particle size and PDI; (B) Zeta potential. Different capital letters indicate significant differences (p<0.05).

[0029] Figure 3 The following are the thermal and centrifugal stability of high internal phase emulsions at different TPS concentrations: (A) Emulsion state before and after heating at 100 ℃ for 30 min; (B) Emulsion state before and after centrifugation at 4000 rpm for 30 min.

[0030] Figure 4 The following are rheological properties of high internal phase emulsions at different TPS concentrations: (A) Frequency scan; (B) Flow scan (bar graphs show shear rates of 1 s⁻¹). -1 (The viscosity of the emulsion at that time).

[0031] Figure 5 The protective effect of high internal phase emulsion on β-carotene at different TPS concentrations is shown: (A) retention rate after heating at 80 ℃ for 30 min; (B) retention rate after storage at room temperature for 7 days.

[0032] Figure 6 The following are rheological properties of high internal phase emulsions under different pH conditions: (A) Frequency scan; (B) Flow scan (bar graphs show shear rates of 1 s⁻¹). -1 (The viscosity of the emulsion at that time).

[0033] Figure 7 The following are rheological properties of high internal phase emulsions at different salt ion concentrations: (A) Frequency scan; (B) Flow scan (bar graphs show shear rates of 1 s⁻¹). -1 (The viscosity of the emulsion at that time).

[0034] Figure 8 The figures show the release curves of free fatty acids in the emulsion (A) and the bioavailability of β-carotene (B). Different letters indicate significant differences (p<0.05). Detailed Implementation

[0035] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0037] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0038] See Figure 1 This application provides a method for preparing a ternary composite material, comprising the following steps: (1) Preparation of SC solution Dissolve 1.0 g of sodium caseinate SC in 100 mL of phosphate-buffered saline (PBS, pH 7.0) to prepare a 10 g / L sodium caseinate SC solution. Stir magnetically (500 rpm) at room temperature until completely dissolved, then transfer to a 4 °C refrigerated environment for 12–16 h to ensure adequate protein hydration.

[0039] (2) Preparation of Epigallocatechin Gallate (EGCG) Solution Epigallocatechin gallate (EGCG) solutions of various concentrations were prepared using 10 mM PBS (pH 7.0–7.2) at concentration gradients of 5.00, 3.33, 2.50, 2.00, 1.67, 1.43, and 1.25 mg / mL. All EGCG solutions were sterilized by filtration through a 0.22 μm filter membrane.

[0040] (3) Construction of the SC-EGCG-TPS ternary complex The SC solution and EGCG solution were mixed in equal volumes (1:1), controlling the final mass ratio of SC to EGCG to be 10:1. The pH of the system was monitored during mixing to ensure it remained within the range of 7.0 ± 0.2. TPS solutions with concentrations of 2%, 3%, 4%, 5%, and 6% were prepared. The tea polysaccharide used in this example was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (product number: S25491). The preparation method for the tea polysaccharide solution was as follows: 0.2 g, 0.3 g, 0.4 g, 0.5 g, and 0.6 g of tea polysaccharide powder were weighed and dissolved in 10 mL of phosphate buffered saline (PBS, pH 7.0). The solution was magnetically stirred at room temperature until completely dissolved to obtain the corresponding concentration of TPS solution.

[0041] The above-mentioned SC-EGCG binary complex was mixed with TPS solutions of different concentrations at an equal volume ratio (1:1) to obtain mixed systems with final TPS concentrations of 1%, 1.5%, 2%, 2.5%, and 3%, respectively. After homogeneous mixing, the mixture was magnetically stirred at 500 rpm for 30 min at room temperature to ensure sufficient interaction between TPS and SC-EGCG, forming a stable SC-EGCG-TPS ternary complex through non-covalent interactions such as hydrogen bonding and hydrophobic interactions. The SC-EGCG binary complex without added TPS served as a control group. All samples were stored at 4 ℃ for subsequent performance characterization.

[0042] Performance characterization of the SC-EGCG-TPS ternary composite (1) Measurement of particle size, potential and polydispersity index (PDI) The particle size, potential, and polydispersity index (PDI) of the samples were measured using a Malvern laser particle size analyzer at 25 °C. To avoid multiple scattering, the samples were appropriately diluted and the measurements were repeated three times.

[0043] Depend on Figure 2 The measurement results show that the average particle size of the SC-EGCG binary complex (control group) is 262.4 nm, the PDI is 0.324, and the Zeta potential is -19.07 mV, indicating that SC and EGCG successfully formed a binary complex with good uniformity and negative surface charge.

[0044] Depend on Figure 2As can be seen from A, with the addition of TPS, the particle size of the ternary composite initially decreases slightly and then gradually increases. When the TPS concentration was 2%, the particle size was 273.9 nm, slightly higher than the control group. When the TPS concentration increased to 3%, the particle size was 263.3 nm, basically the same as the control group (262.4 nm), and the PDI was 0.337, which was not significantly different from the control group (0.324) (p>0.05), indicating that the introduction of 3% TPS did not cause significant changes in the structure of the complex, and the complex still maintained a good uniform dispersion. When the TPS concentration continued to increase to 4%, 5%, and 6%, the particle size increased to 277.6 nm, 287.4 nm, and 292.1 nm, respectively, which were significantly different from the control group (p<0.05). With the increase of TPS concentration, the particle size showed a gradual increasing trend. This may be because the high concentration of TPS and the SC-EGCG complex have stronger hydrogen bonding and hydrophobic interactions, resulting in a certain degree of aggregation or conformational change in the complex structure. The PDI values ​​of the complex at all concentrations were less than 0.5, indicating that the complex did not aggregate significantly in the solution and maintained a good dispersion.

[0045] Figure 2 The zeta potential measurements of B showed that all complexes carried a negative charge. The zeta potential of the control group was -19.07 mV, while that of the TPS group at 3% concentration was -18.5 mV, showing no significant difference from the control group (p>0.05), indicating that the introduction of low concentrations of TPS had little effect on the surface charge properties of the complexes. When the TPS concentration increased to 6%, the zeta potential was -14.29 mV, a significant decrease in absolute value (p<0.05). The phenomenon that the absolute value of the zeta potential decreased with increasing TPS concentration may be related to the structural characteristics of tea polysaccharide molecules: as a neutral or weakly acidic polysaccharide, tea polysaccharide has a relatively limited content of negatively charged groups in its molecules. When high concentrations of TPS bind to the SC-EGCG complex, it may shield some of the negative charge on the protein surface through hydrogen bonding, or cause conformational changes in the complex leading to alterations in charge distribution, thereby reducing the absolute value of the zeta potential. Nevertheless, the absolute values ​​of the zeta potentials of the complexes at all concentrations were greater than 14 mV, still exhibiting good electrostatic stability.

[0046] Considering the variations in particle size, PDI, and Zeta potential, a TPS concentration of 3% resulted in the SC-EGCG-TPS ternary complex exhibiting similar particle size distribution and dispersion characteristics to the control group, while maintaining a high absolute Zeta potential value, making it the optimal formulation. At this concentration, the complex retained the basic structural characteristics of the binary complex and successfully introduced tea polysaccharide components, providing an ideal aqueous phase stabilizer for the subsequent construction of high internal phase emulsions.

[0047] Preparation and performance characterization of SC-EGCG-TPS ternary composite stable high internal phase emulsion 1. Preparation of high internal phase emulsion Using the SC-EGCG-TPS ternary complexes with different TPS concentrations (2%, 3%, 4%, 5%, 6%) prepared above as the aqueous phase and soybean oil as the oil phase, a high internal phase emulsion was prepared by a one-step homogenization method. The specific steps are as follows: β-carotene was dissolved in soybean oil to prepare a β-carotene oil phase solution with a final concentration of 0.01% (w / w). The aqueous phase and oil phase were mixed at a volume ratio of 1:3 (i.e., the oil phase volume fraction was 75%), and homogenized using a high-speed homogenizer at 10,000 rpm for 2 min to obtain a high internal phase emulsion. An emulsion stable by the SC-EGCG binary complex was used as a control group.

[0048] 2. Performance characterization of high internal phase emulsions (1) High internal phase emulsion thermal stability and centrifugal stability The prepared fresh high internal phase emulsions were subjected to thermal stability and centrifugal stability tests. Thermal stability test: The emulsion was allowed to stand at 100 °C for 0.5 h, and the appearance changes (such as stratification, demulsification, oil separation, etc.) were observed. Centrifugal stability test: The emulsion was centrifuged at 8000 rpm for 30 min, and the stratification was observed. The stability test results of the high internal phase emulsions at different TPS concentrations are shown below. Figure 3 As shown.

[0049] Figure 3The thermal stability test results of A showed that different TPS concentrations had a significant impact on the heat resistance of the high internal phase emulsion. The control group (without TPS) emulsion, after heating at 100 °C for 30 min, rapidly flowed down when inverted, indicating significant demulsification and poor thermal stability. While the thermal stability of the emulsion improved slightly at a TPS concentration of 2%, it still flowed down rapidly after heating and inversion, indicating insufficient stability. The emulsion with a TPS concentration of 3% exhibited the best thermal stability. At this concentration, the emulsion maintained a good gel state after heating at 100 °C for 30 min, without significant flow, stratification, demulsification, or oil separation, indicating that appropriate TPS addition significantly enhanced the thermal stability of the emulsion. This is mainly attributed to the formation of a suitable three-dimensional network structure between 3% TPS and the SC-EGCG complex, which enhanced the density and heat resistance of the interfacial film and improved the gel strength of the emulsion. However, when the TPS concentration continued to increase to 4%, 5%, and 6%, the thermal stability of the emulsion decreased. These TPS concentration emulsions exhibited significant porosity and oil precipitation upon heating. This may be because excessively high TPS concentrations lead to over-thickening of the system, causing an imbalance in the interfacial tension between the aqueous and oil phases during heating. Additionally, excessive polysaccharides may have interfered with the orderly arrangement of the interfacial film, resulting in emulsion structural instability under high-temperature conditions.

[0050] Figure 3 The centrifugation stability test results of B showed that TPS concentration significantly affected the centrifugation stability of the emulsion. The control group emulsion exhibited obvious stratification after centrifugation, with an oil phase precipitating in the upper layer and an aqueous phase in the lower layer. When the TPS concentration was 3%, the stratification was least pronounced, and the emulsion maintained a relatively good homogeneous state, indicating that the emulsion at this concentration had the best centrifugation stability. This is mainly attributed to the fact that the three-dimensional network structure formed by 3% TPS and the SC-EGCG complex effectively resists the damage to the emulsion structure caused by centrifugal force. When the TPS concentration increased to 4%, 5%, and 6%, the stratification phenomenon gradually worsened after centrifugation, with varying degrees of oil and aqueous phase precipitation, indicating that excessively high TPS concentrations are actually detrimental to the centrifugation stability of the emulsion.

[0051] In summary, at a TPS concentration of 3%, the SC-EGCG-TPS ternary complex-stabilized high internal phase emulsion exhibits the best thermal and centrifugal stability, effectively resisting the damage to the emulsion structure caused by high temperature and centrifugal force, thus providing favorable conditions for its application in food processing and storage.

[0052] (2) Characterization of rheological behavior The rheological properties of high internal phase emulsions at different TPS concentrations were determined using a rotational rheometer. Measurement conditions: a plate-plate measurement system (plate diameter 40 mm, gap 1 mm), a fixed strain of 1% within the linear viscoelastic region, and an angular frequency range of 0.1–100 rad / s. The storage modulus (G'), loss modulus (G''), and apparent viscosity were measured as a function of angular frequency. The rheological properties of high internal phase emulsions at different TPS concentrations are shown below. Figure 4 As shown.

[0053] Dynamic oscillation test results show that the high internal phase emulsions prepared at all TPS concentrations exhibit typical gel behavior (G'>G''), and have solid-like elastic and plastic rheological properties.

[0054] As the TPS concentration increases, the G' value of the emulsion exhibits a regular change. In the low-frequency region, the G' value is highest at a TPS concentration of 3%, indicating that the emulsion has the optimal structural strength under static conditions at this concentration, forming the most suitable three-dimensional gel network structure. In the high-frequency region, the G' value is highest at a TPS concentration of 6%, followed by 3%, which is mainly attributed to the stronger resistance to high-frequency strain caused by the thickening effect of high-concentration TPS.

[0055] Steady-state shear test results showed that all emulsions exhibited shear-thinning behavior, with apparent viscosity decreasing with increasing shear rate, consistent with typical characteristics of non-Newtonian fluids. The highest viscosity was observed at a TPS concentration of 6%, followed by 3%. With increasing shear rate, the apparent viscosity of each emulsion gradually decreased, demonstrating good shear-thinning properties, which is beneficial for the emulsion's flowability during processing. Although the 6% TPS emulsion had the highest G' value in the high-frequency region, combined with thermal and centrifugal stability results, this concentration showed oil precipitation and stratification under high temperature and centrifugal conditions, indicating poor stability. The 3% TPS emulsion, on the other hand, had the highest G' value in the low-frequency region and remained the second highest in the high-frequency region, forming a moderately dense gel network structure with good flexibility, making it the optimal formulation choice.

[0056] (3) Evaluation of the encapsulation effect of high internal phase emulsion on β-carotene at different TPS concentrations Take an appropriate amount of β-carotene emulsion, add a mixture of ethanol and n-hexane at a volume ratio of 2:3, vortex to mix, repeat the extraction three times, combine the upper extracts, and measure the absorbance at 450 nm using a UV-Vis spectrophotometer. Plot a standard curve using β-carotene standards, and calculate the β-carotene content in the sample based on the standard curve. Take a certain amount of each β-carotene-loaded emulsion in a serum bottle, heat at 80 ℃ for 30 min, cool to room temperature, and determine the β-carotene content according to the above method. The retention rate of β-carotene was used for thermal stability analysis, and the result is expressed as C / C0, where C is the concentration of β-carotene after heating, and C0 is the initial concentration of β-carotene.

[0057] Retention rate (%) = (C / C0) × 100% The high internal phase emulsion containing β-carotene was stored at 25 °C in the dark for 7 days. Samples were taken on day 0 and day 7, and the β-carotene content was determined according to the method described above. The retention rate was calculated using the following formula: Retention rate (%) = (C7 / C0) × 100% Wherein, C0 is the β-carotene content in the initial emulsion, and C7 is the β-carotene content in the emulsion after 7 days of storage.

[0058] The measurement results are as follows Figure 5 As shown, different TPS concentrations significantly affected the protective effect of β-carotene in high internal phase emulsions. Under heat treatment conditions of 80 °C for 30 min, the retention rate of β-carotene in the control group (without TPS) emulsion was 86.13%, while the retention rate significantly increased to 93.19% with a TPS concentration of 3%, an increase of 7.06 percentage points. This indicates that the introduction of 3% TPS can effectively enhance the thermal stability of the emulsion and provide a better protective barrier for β-carotene. This is mainly attributed to the fact that the three-dimensional network structure formed by 3% TPS and the SC-EGCG complex enhances the compactness of the interfacial film, effectively blocking the oxidative degradation of β-carotene by high temperature.

[0059] Under 7 days of storage at room temperature, the retention rate of β-carotene in the control group emulsion was 70.46%, while the retention rate significantly increased to 83.13% when the TPS concentration was 3%, an increase of 12.67 percentage points. This indicates that the addition of 3% TPS can significantly prolong the storage stability of β-carotene and effectively slow down its degradation loss during storage. This is mainly attributed to the thickening effect of tea polysaccharides and the synergistic antioxidant effect of EGCG. The stable interface layer formed by the ternary complex reduces the contact between β-carotene and the external environment, and inhibits the destruction of β-carotene by lipid oxidation through the antioxidant activity of EGCG.

[0060] In summary, at a TPS concentration of 3%, the stable high internal phase emulsion of the SC-EGCG-TPS ternary complex exhibits the best protective effect on β-carotene, significantly improving its stability during heat treatment and long-term storage, thus providing strong support for its application in functional foods. Furthermore, without the addition of EGCG, the SC-TPS binary system composed only of sodium caseinate and tea polysaccharides struggles to form a stable high internal phase emulsion under the same conditions, and its encapsulation and storage stability for β-carotene is significantly lower than that of the SC-EGCG-TPS ternary system of this invention. Therefore, the introduction of EGCG plays a crucial role in achieving synergistic effects among the three components, enhancing emulsion stability, and improving encapsulation protection. The pH stability and salt ion stability of the high internal phase emulsion under the preferred formulation were evaluated. 1. Preparation of high internal phase emulsion According to the preferred ratio determined in Example 2, an SC-EGCG-TPS ternary complex was prepared by mixing the SC-EGCG binary complex and 3% TPS solution at an equal volume ratio (1:1) as the aqueous phase, and soybean oil (75% by volume) as the oil phase. A high internal phase emulsion was prepared using a one-step homogenization method, with the specific preparation method as described above. For ease of differentiation, the emulsions prepared using different stability systems were named as follows: SC-BC (stable in a single SC system), SC-EGCG-BC (stable in a binary SC-EGCG system), and SC-EGCG-TPS-BC (stable in a ternary SC-EGCG-TPS system), where "BC" represents β-carotene.

[0061] 2. pH stability determination The prepared high internal phase emulsion was adjusted to pH 2.0, 3.0, 6.0, and 7.0 with 1 mol / L HCl or 1 mol / L NaOH, respectively. After thorough mixing, the emulsions were allowed to stand for 24 h. The rheological properties of the emulsions under different pH conditions were measured using a rotational rheometer to evaluate the stability of the emulsions under different pH conditions. The test results are as follows: Figure 6 As shown.

[0062] Dynamic oscillation tests showed that the storage modulus (G') of the emulsions was higher than the loss modulus (G'') under all pH conditions, indicating that each emulsion exhibited typical gel-like rheological behavior and formed a stable three-dimensional network structure. The emulsions under acidic conditions (pH 2.0 and pH 3.0) showed larger G' values, indicating that an acidic environment is beneficial for enhancing the gel strength and structural stability of the emulsions. This is mainly attributed to the protonation of negatively charged amino acid residues in sodium caseinate molecules under acidic conditions, which weakens electrostatic repulsion and promotes hydrophobic interactions and hydrogen bond formation between protein molecules, thereby enhancing the compactness of the interfacial film.

[0063] Steady-state shear test results showed that the apparent viscosity of all emulsions gradually decreased with increasing shear rate until it plateaued, exhibiting typical shear-thinning behavior consistent with the characteristics of non-Newtonian fluids. Among them, the emulsion under acidic conditions exhibited the highest initial apparent viscosity, further confirming that an acidic environment contributes to enhancing the structural strength of the emulsion.

[0064] In summary, pH significantly affects the rheological properties of emulsions with a high internal phase. Under acidic conditions (pH 2.0–3.0), the emulsion exhibits higher storage modulus and apparent viscosity, demonstrating a stronger gel network structure and resistance to deformation, which is beneficial for the long-term stability of the emulsion. Under neutral conditions (pH 6.0–7.0), the emulsion still maintains good gelation properties and is suitable for applications in neutral food systems.

[0065] 3. Determination of salt ion stability NaCl was added to the prepared high internal phase emulsion to adjust the salt ion concentration to 0, 50, 100, 200, 300, and 400 mM, respectively. After thorough mixing, the emulsions were allowed to stand for 24 h. The rheological properties of the emulsions at different salt ion concentrations were measured using a rotational rheometer. The measurement conditions were the same as those for pH stability determination to evaluate the stability of the emulsions at different salt ion concentrations.

[0066] The rheological properties of high internal phase emulsions under different salt ion concentrations are as follows: Figure 7 As shown in the figure. Dynamic oscillation test results show that the storage modulus (G') of the emulsion is higher than the loss modulus (G'') at all salt ion concentrations, indicating that all emulsions maintain typical gel-like rheological behavior. When the salt ion concentration is 200~400 mM, the G' values ​​of the emulsions are relatively close, and the rheological curves at each concentration almost overlap, indicating that within this concentration range, the salt ions have little effect on the emulsion gel network structure, and the emulsions exhibit good salt ion tolerance.

[0067] Steady-state shear tests showed that the apparent viscosity of all emulsions gradually decreased with increasing shear rate, exhibiting typical shear-thinning behavior. The initial apparent viscosity of the emulsions gradually increased with increasing salt ion concentration, reaching its highest value at 400 mM. This is mainly attributed to the addition of salt ions shielding the negative charges on the protein molecule surface, promoting intermolecular interactions, and enhancing the three-dimensional network structure.

[0068] In summary, the stable high internal phase emulsion of the SC-EGCG-TPS ternary complex exhibits good stability in the range of 200–400 mM salt ion concentration, demonstrating strong salt ion tolerance.

[0069] In vitro simulated digestion and bioaccessibility assay In vitro digestion experimental methods Using the INFOGEST static in vitro digestive model, a three-step digestion method (oral-gastric-small intestine) was employed to evaluate the gastrointestinal digestive characteristics of different samples. Before the experiment, simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF) were prepared at 1.25 times the working concentration.

[0070] (1) Oral digestion Take 10 g of β-carotene-loaded high internal phase emulsion and mix it with 10 mL of SSF. Adjust the pH to 7.0 with 1 M HCl or NaOH. Incubate at 100 rpm for 2 min in a 37 ℃ water bath with magnetic stirring to simulate oral chewing and enzymatic digestion. Immediately after digestion, take 1 mL of sample for testing.

[0071] (2) Stomach digestion Mix 15 mL of oral digestive fluid with 15 mL of SGF, adjust the pH to 3.0 with 1 M HCl, add pepsin to achieve a final activity of 2000 U / mL, and confirm the pH is 3.0 again. Digest at 100 rpm for 2 h in a 37 ℃ water bath with magnetic stirring to simulate the acidic environment and enzymatic hydrolysis process of the stomach.

[0072] (3) Intestinal digestion 20 mL of gastric digestive fluid was mixed with an equal volume (20 mL) of SIF, and the pH was adjusted to 7.0 with 1 M NaOH. Trypsin (final activity 100 U / mL), lipase (final activity 2000 U / mL), and bile salts (final concentration 10 mM) were added sequentially, and the pH was adjusted to 7.0 again. Digestion was performed at 100 rpm for 2 h in a 37 ℃ water bath with magnetic stirring to simulate the enzymatic digestion and absorption environment of the small intestine. During digestion, fat breakdown released free fatty acids (FFAs), requiring continuous titration with 0.2 M NaOH to maintain a constant pH of 7.0. The release characteristics and bioavailability of β-carotene free fatty acids (FFAs) were determined. During simulated intestinal digestion, the volume of NaOH consumed was recorded every 10, 20, 40, 60, 90, and 120 minutes. The amount of free fatty acids (FFA) released was calculated using the following formula: FFA (%)=(V NaOH× M NaOH× M lipid ) / (2 × W lipid )×100 Where V NaOH : Volume of NaOH consumed over the corresponding time (L); M NaOH Molar concentration of NaOH solution (mol / L); M lipid Molar mass of oil (238.19 g / mol); W lipidMass of oil in the emulsion (g).

[0073] like Figure 8 As shown in Figure A, free fatty acids (FFA) are continuously released during digestion, reflecting the gradual disruption of the emulsion structure. The emulsion prepared at pH 2 exhibits a faster FFA release rate, primarily due to flocculation of the emulsifier, leading to a decrease in its encapsulation ability on the oil phase. Under the same pH conditions, the more complex the complex structure (e.g., the SC-EGCG-TPS system), the slower the FFA release rate. This is because: the dense three-dimensional network structure creates steric hindrance; a thicker interfacial layer hinders contact between the digestive enzyme and the oil droplets; and the larger droplet size reduces the interfacial area available for enzyme interaction. The SC-EGCG-TPS-BC system, in particular, maintains a slow release throughout the entire 2-hour digestion process, demonstrating excellent sustained-release characteristics.

[0074] A certain amount of digestate from the final stage of simulated digestion was centrifuged at 4000 rpm for 40 min. The intermediate micelle phase was collected, and the concentration of β-carotene was determined. The bioavailability of β-carotene was calculated according to the following formula.

[0075] Bioaccessibility (%) = C Micelle / C Original ×100 Where C Micelle and C Original The emulsions represent the mass concentrations of β-carotene in the micelles and in the initial emulsion, respectively.

[0076] like Figure 8As shown in Figure B, the bioavailability determination results of β-carotene in different systems indicate that, under the same pH conditions, the bioavailability ranking for the three systems is SC-EGCG-TPS > SC-EGCG > SC. At pH=2, the bioavailability of the SC single system was 42.68%, the SC-EGCG binary system increased to 44.82%, and the SC-EGCG-TPS ternary system further increased to 48.61%. The ternary complex showed an improvement of 5.93 percentage points compared to the SC single system and 3.79 percentage points compared to the SC-EGCG binary system. At pH=7, the bioavailability was 45.76% for the SC single system, 47.43% for the SC-EGCG binary system, and 51.09% for the SC-EGCG-TPS ternary system. The ternary complex showed an improvement of 5.33 percentage points compared to the SC single system and 3.66 percentage points compared to the SC-EGCG binary system. The above results indicate that the synergistic effect of EGCG and tea polysaccharides can effectively improve the bioavailability of β-carotene, with the SC-EGCG-TPS ternary complex system showing the best effect. All systems exhibited higher bioavailability at pH 7 than at pH 2, which may be related to the higher activity of digestive enzymes under neutral conditions. The SC-EGCG-TPS ternary complex reached 51.09% at pH 7, the highest among all samples. In summary, the stable high internal phase emulsion of the SC-EGCG-TPS ternary complex constructed in this invention can significantly improve the bioavailability of β-carotene, demonstrating excellent delivery performance.

[0077] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a ternary composite material, characterized in that, Includes the following steps: (1) Sodium caseinate and epigallocatechin gallate were mixed to obtain a protein-polyphenol binary complex; (2) The protein-polyphenol binary complex was mixed with tea polysaccharide to obtain a ternary composite material.

2. The preparation method according to claim 1, characterized in that: The mass ratio of sodium caseinate to epigallocatechin gallate is (8~12):

1.

3. The preparation method according to claim 1, characterized in that: The weight-volume ratio of the tea polysaccharide in the ternary composite mixture is 1-5% (w / v).

4. The preparation method according to claim 1, characterized in that: In steps (1) and (2), before mixing, each component is dissolved in phosphate buffer, and the pH of the mixed system is 6.5~7.

5.

5. A ternary composite material prepared by any one of claims 1 to 4.

6. The ternary composite material according to claim 5, characterized in that: The average particle size of the ternary composite material in aqueous solution is 250~300 nm, and the absolute value of the Zeta potential is 14~20 mV.

7. The application of the ternary composite material as described in claim 5 in the preparation of high internal phase emulsions.

8. A high internal phase emulsion, characterized in that: It includes an aqueous phase and an oil phase, wherein the aqueous phase is the ternary composite material as described in claim 5; and the oil phase has a volume fraction of 70-85%.

9. The application of the high internal phase emulsion as described in claim 8 in the encapsulation of lipid-soluble active substances.

10. The application according to claim 9, characterized in that: The fat-soluble active substance is one or more of β-carotene, lycopene, curcumin, and vitamin E.

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