Preparation method of sodium caseinate-epigallocatechin gallate-iron ion ternary complex, product and application

Abstract

The invention relates to the technical field of compositions of high-molecular compounds, and particularly discloses a preparation method of a sodium caseinate-epigallocatechin gallate-iron ion ternary complex as well as a product and application thereof. The preparation method comprises the following steps: S1, adding EGCG (epigallocatechin gallate) into an SC solution, and stirring in a shading environment to obtain a binary compound; s2, adding a FeCl3. 6H2O solution into the binary compound, and stirring in a shading environment to obtain a ternary compound; wherein in the ternary complex, the mass ratio of SC to EGCG to Fe < 3 + > is (10-14): 1: (2-4). According to the preparation method disclosed by the invention, the stable ternary complex can be prepared, the ternary complex utilizes the metal phenolic network to modify the sodium caseinate, and a high internal phase emulsion formed after the sodium caseinate is loaded with the beta-carotene can show excellent stability and relatively high encapsulation efficiency, so that the bioavailability of the beta-carotene can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of polymer composition technology, specifically to a method for preparing a ternary complex composed of sodium caseinate, epigallocatechin gallate, and iron ions, and to the obtained product and its application in high internal phase emulsions. Background Technology

[0002] Beta-carotene is a carotenoid that, after being ingested by the human digestive system, is converted into vitamin A only when needed. Therefore, it is generally considered a safe source of vitamin A. Beta-carotene can be used to maintain healthy eyes and skin, improve night blindness and rough skin, and help protect the body from free radical damage, thus gaining widespread attention in the field of nutritional fortification.

[0003] However, beta-carotene is extremely sensitive to light, heat and oxygen, and is easily oxidized and degraded if not stored properly. As a fat-soluble component, the absorption of beta-carotene in the human body depends on dietary fat and bile secretion, and its bioavailability is low and varies greatly among different individuals.

[0004] Therefore, when applying β-carotene to the field of nutritional fortification, a β-carotene delivery system is urgently needed. This delivery system needs to be able to reduce the degradation of β-carotene to achieve a storage effect; at the same time, it needs to be able to mimic dietary fat and stimulate bile secretion, thereby improving the body's absorption of β-carotene and thus improving the overall bioavailability of β-carotene. Summary of the Invention

[0005] This invention provides a method for preparing a sodium caseinate-epigallocatechin gallate-iron ternary complex. This method can produce a stable ternary complex. The ternary complex utilizes a metallophenolic network to modify sodium caseinate. The high internal phase emulsion formed after loading β-carotene exhibits excellent stability and high encapsulation efficiency, thereby significantly improving the bioavailability of β-carotene.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a sodium caseinate-epigallocatechin gallate-iron ternary complex, comprising the following steps: S1. Adding epigallocatechin gallate to a sodium caseinate solution and stirring under a light-shielding environment to obtain a binary complex; S2. Adding FeCl3·6H2O solution to the binary complex and stirring under a light-shielding environment to obtain a ternary complex; wherein, in the ternary complex, the mass ratio of sodium caseinate:epigallocatechin gallate:iron ions is (10~14):1:(2~4).

[0008] As a further improvement of the present invention, the preparation of sodium caseinate solution includes the following steps: dissolving sodium caseinate in a phosphate buffer solution with a pH of 7 to 7.5 and stirring, then transferring the solution to an environment of 3 to 5°C for hydration for 12 to 16 hours to obtain sodium caseinate solution.

[0009] As a further improvement of the present invention, in S2, vortex stirring is used for mixing, with the speed controlled at 3000~3500 rpm and the duration at 5~10 min.

[0010] As a further improvement of the present invention, the following steps are also included: S3. After filtering the ternary compound, freeze-dry it to obtain a powdered ternary compound.

[0011] Secondly, the present invention provides a ternary composite material prepared by any of the above-described preparation methods.

[0012] Thirdly, the present invention provides a method for preparing a high internal phase emulsion, comprising the following steps: preparing a ternary complex solution with a concentration of 2-3 wt% as an aqueous phase; dissolving β-carotene in soybean oil as an oil phase; and adding the oil phase dropwise to the aqueous phase and homogenizing and emulsifying to obtain a high internal phase emulsion.

[0013] As a further improvement of the present invention, in the high internal phase emulsion, the volume fraction of the oil phase is 70-75%.

[0014] As a further improvement of the present invention, the oil phase is added dropwise to the aqueous phase at a rate of 1~1.5 mL / min.

[0015] Fourthly, the present invention provides a high internal phase emulsion loaded with β-carotene, which is prepared by any of the above-described methods for preparing high internal phase emulsions.

[0016] Fifthly, the present invention provides an application of the above-mentioned β-carotene-loaded high internal phase emulsion in functional foods.

[0017] This invention uses sodium caseinate as a matrix and optimizes its ratio with epigallocatechin gallate to initially construct a binary composite system. Subsequently, a stable ternary complex is finally obtained by introducing iron ions to form a stable ternary metal phenolic network structure.

[0018] The controlled ratio of sodium caseinate to epigallocatechin gallate ensures the composite exhibits good emulsifying activity and foaming ability. This ratio guarantees sufficient binding between sodium caseinate and epigallocatechin gallate while avoiding aggregation caused by excessive epigallocatechin gallate. Appropriate iron ions induce protein structure unfolding and steric hindrance, improving both molecular flexibility at the interface and enhancing interfacial film stability, thereby further improving the optimal emulsifying activity index (EAI) and emulsifying stability index (ESI) of the ternary composite. Overall, when epigallocatechin gallate is added to the sodium caseinate dispersion system, polyphenols spontaneously adsorb onto the surface of sodium caseinate through non-covalent interactions such as hydrogen bonding, forming a loose composite structure dominated by hydrogen bonds. The introduction of iron ions promotes the deprotonation of catechol groups, forming a stable five-membered ring coordination structure. The coordination layer in the gallic catechin gallate-iron ion system of this invention synergistically assembles with the sodium caseinate matrix through polyphenol metal chelation and protein surface affinity, ultimately constructing a stable ternary complex. In other words, this invention obtains a stable ternary complex through relatively simple processing steps by selecting and proportioning the three raw materials.

[0019] This invention utilizes a ternary complex to load β-carotene, forming a high internal phase emulsion. This emulsion system exhibits excellent stability and high encapsulation efficiency, reducing β-carotene degradation and thus improving its bioavailability. Specifically, the high internal phase emulsion in this invention exhibits elastomer characteristics, effectively inhibiting delamination, sedimentation, and Ostwald ripening, ensuring long-term storage stability. Simultaneously, the elastomer characteristics also ensure excellent shaping properties and mechanical shock resistance, maintaining structural integrity during application or transportation. Furthermore, the ternary complex used in the high internal phase emulsion forms a dense interface layer around the β-carotene, effectively blocking heat conduction and reducing thermal degradation of β-carotene, which is beneficial for its preservation. Attached Figure Description

[0020] The accompanying drawings are provided below to illustrate the preferred embodiments of the invention and to aid in understanding the objectives and advantages of the invention, wherein:

[0021] Figure 1 Statistical graphs showing the foaming properties and foam stability of the ternary composites in Examples 1-2 and Comparative Examples 1-3;

[0022] Figure 2 This is a statistical chart showing the emulsifying activity and emulsifying stability results of the ternary complexes in Examples 1-2 and Comparative Examples 1-3;

[0023] Figure 3 The following are statistical graphs showing the spectral characterization results of the ternary complexes in Examples 1-2 and Comparative Examples 1-3;

[0024] Figure 4 Fourier transform infrared spectra of the ternary complexes in Examples 1-2 and Comparative Examples 1-3;

[0025] Figure 5 Differential scanning calorimetry (DSC) curves of the ternary complexes in Examples 1-2 and Comparative Examples 1-3 are shown.

[0026] Figure 6 This is a summary graph of the rheological property test results of the emulsions in Example a and Comparative Examples a~b;

[0027] Figure 7 The graph shows the statistical results of the thermal stability test of β-carotene in the emulsions of Example a and Comparative Examples a~b.

[0028] Figure 8 The graphs show the release curves of free fatty acids from the emulsions in Example a and Comparative Examples a to b.

[0029] Figure 9 This is a statistical chart showing the bioavailability of β-carotene in the emulsions of Example a and Comparative Examples a-b.

[0030] exist Figure 3 Part A shows the fluorescence spectrum under 280 nm excitation, Part B shows the fluorescence spectrum under 295 nm excitation, Part C shows the UV-Vis spectrum, and Part D shows the CD spectrum.

[0031] Figure 6 Part A shows the frequency scan results, and Part B shows the flow scan results. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0034] Example 1:

[0035] This embodiment provides a sodium caseinate-epigallocatechin gallate-iron ion ternary complex, which is prepared by the following steps:

[0036] S1. Dissolve 1 g of sodium caseinate (hereinafter referred to as SC) in 100 mL of phosphate buffer (hereinafter referred to as PBS buffer) at pH 7 to prepare a 10 g / L SC solution. Under room temperature and light-protected conditions, set the magnetic stirring speed to 500 rpm and stir the SC solution until completely dissolved. Then, transfer the solution to a 4°C refrigerator for 12 h to ensure sufficient protein hydration and obtain a sodium caseinate solution. Add 0.1 g of epigallocatechin gallate (hereinafter referred to as EGCG) to the SC solution to ensure a SC:EGCG mass ratio of 10:1. Then, stir at 25°C in the dark for 30 minutes to form a binary complex. S2. Add FeCl3·6H2O solution to the binary complex to ensure a EGCG:Fe... 3+ The mass ratio was 1:2, and the mixture was then vortexed for 5 minutes at a controlled rotation speed of 3000 rpm under a light-shielding environment to obtain the ternary composite.

[0037] Preferably, if long-term storage of the prepared ternary complex is required, the preparation method further includes: S3. Filtering the obtained ternary complex solution through a 0.22 μm microporous membrane to remove insoluble matter, and finally freeze-drying to obtain a powdered ternary complex. The powdered substance obtained by this method can be stored for a long time in a light-protected environment at 4°C.

[0038] In summary, within the ternary complex of this embodiment, SC:EGCG:Fe 3+ The mass ratio is 10:1:2.

[0039] Example 2:

[0040] This embodiment provides an SC-EGCG-Fe 3+ The ternary complex differs from that in Example 1 in that, in this example, SC:EGCG:Fe 3+ The mass ratio is 10:1:4.

[0041] Comparative Example 1:

[0042] This embodiment provides an SC-EGCG-Fe 3+ The ternary complex differs from that in Example 1 in that, in this example, SC:EGCG:Fe 3+ The mass ratio is 10:1:0.25.

[0043] Comparative Example 2:

[0044] This embodiment provides an SC-EGCG-Fe3+ The ternary complex differs from that in Example 1 in that, in this example, SC:EGCG:Fe 3+ The mass ratio is 10:1:0.5.

[0045] Comparative Example 3:

[0046] This embodiment provides an SC-EGCG-Fe 3+ The ternary complex differs from that in Example 1 in that, in this example, SC:EGCG:Fe 3+ The mass ratio is 10:1:1.

[0047] Detection of ternary complexes:

[0048] ① Physicochemical property analysis

[0049] In this embodiment, the emulsifying activity index (EAI) and emulsifying stability index (ESI) of the ternary complexes in Examples 1-2 and Comparative Examples 1-3 were measured. Figures 1-2 As shown, higher Fe 3+ Concentration (e.g., EGCG:Fe in Example 1) 3+ When the ratio was 1:2, the foaming and emulsifying properties of the ternary compound system were significantly improved. The inventors speculate that this is mainly due to the appropriate amount of Fe. 3+ The induced protein structure unfolding and the resulting steric hindrance effect improve both the flexibility of the molecule at the interface and enhance the stability of the interfacial membrane. However, when Fe... 3+ When added in excess, excessive protein defolding leads to overly strong hydrophilicity, which in turn hinders molecular migration and adsorption at the oil-water interface, resulting in decreased emulsifying performance; correspondingly, low Fe... 3+ The formation of large protein aggregates at certain concentrations (such as the mass ratios in Comparative Examples 1-3) also significantly reduces the stability of the system, a phenomenon that corroborates the changing trend of foaming properties.

[0050] ② Interaction Analysis

[0051] In this embodiment, fluorescence spectroscopy, ultraviolet absorption spectroscopy, far-ultraviolet circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry were performed on the ternary complexes in Examples 1-2 and Comparative Examples 1-3. The specific steps and results are shown below.

[0052] (1) Fluorescence spectroscopy: A sample solution with an SC content of 1 mg / mL was prepared and analyzed by a fluorescence spectrometer. The excitation wavelength was set to 280 or 295 nm, and the emission spectrum measurement range was 300 to 500 nm. The widths of both the excitation and emission bands were set to 5 nm.

[0053] Fluorescence spectroscopy analysis indicates (e.g.) Figure 3As shown in parts A and B), the SC-EGCG binary complex exhibits a typical tryptophan fluorescence emission peak at 350 nm. After the formation of the ternary complex, the system shows significant fluorescence quenching, where EGCG:Fe... 3+ At a ratio of 2:1 (as in Comparative Example 2), the fluorescence intensity decreased by approximately 70% with a significant red shift, while EGCG:Fe 3+ When the ratio is 1:2, the quenching is less severe (approximately 45%). According to the inventors' analysis, this quenching is only achieved in high Fe... 3+ Under certain conditions, metal ions can act as crosslinking agents to promote the formation of stable three-dimensional network structures and generate metal-ligand charge transfer complexes with unique fluorescence properties.

[0054] (2) Ultraviolet-Vis Spectroscopy: The ultraviolet-visible (UV-Vis) spectra of the samples in the wavelength range of 250 to 800 nm were observed using a UV-Vis spectrophotometer (GENESYS 150, Thermo Fisher Scientific, USA). The final concentration of SC in each sample was adjusted to 1 mg / mL using PBS buffer (pH 7.0).

[0055] Ultraviolet absorption spectroscopy analysis (e.g.) Figure 3 As shown in section C), SC-EGCG-Fe 3+ The formation of the ternary complex significantly altered the electronic absorption characteristics of the system, manifested as a marked increase in the intensity of the maximum absorption peak (without a shift in the maximum absorption wavelength), and the maintenance of strong absorption in the 320–330 nm range accompanied by spectral band broadening. Furthermore, the ternary complex exhibited a new characteristic absorption band in the 520–600 nm range, confirming the presence of Fe. 3+ It coordinates with the EGCG catechol group to form a metal-ligand charge transfer (LMCT) complex, a feature completely absent in binary systems. Simultaneously, the detection results show that low Fe... 3+ The changes in absorption and fluorescence signals were more significant at certain concentrations, indicating a greater perturbation to the SC-EGCG structure at these concentrations; while sufficient Fe... 3+ Under these conditions, uniform dispersion of metal ions helps maintain the structural stability of the complex. The aforementioned spectral characteristics provide conclusive evidence for the formation of the ternary complex.

[0056] (3) Circular dichroism (CD): The secondary structure of protein samples was analyzed using far-ultraviolet circular dichroism. The SC concentration of the samples in Examples 1-2 and Comparative Examples 1-3 was adjusted and fixed at 0.2 mg / mL. The scanning temperature was set to 25℃ and the scanning rate was set to 100 nm / min. Multiple scans were performed in the wavelength range of 190-260 nm. The CD spectral data were smoothed and preprocessed using spectral management software, and the relative content of protein secondary structures was calculated using CDNN software.

[0057] Far-ultraviolet circular dichroism analysis (e.g.) Figure 3 (as shown in part D) indicates that Fe 3+ The addition of [a specific ingredient] significantly altered the secondary structure of the SC-EGCG complex, manifested as a decrease in the intensity of the α-helix characteristic negative peaks at 210 nm and 220 nm. [The specific ingredient] is EGCG:Fe. 3+ The complex with a 2:1 ratio (as in Comparative Example 2) exhibited the most significant conformational change: the β-sheet content increased by 16.1% (to 43.5% ± 0.07), while the α-helix content decreased by 5.0% (to 7.4% ± 0.09). This result demonstrates that Fe... 3+ It can both promote the formation of β-sheets between protein molecules and cause local unfolding of α-helical fragments; among them, lower Fe 3+ Concentration has a more significant effect on secondary structure, while higher concentrations maintain better protein structural integrity. These results indicate that Fe in the ternary complex... 3+ The addition of [a substance] further led to the unfolding of the protein structure, enhancing its flexibility.

[0058] (4) Fourier transform infrared spectroscopy (FTIR): using an FTIR spectrometer at 400~4000 cm⁻¹ -1 The lyophilized powder sample was scanned within the specified range, with 32 scans and a resolution of 4 cm⁻¹. -1 .

[0059] Fourier transform infrared spectroscopy analysis (e.g.) Figure 4 (As shown) illustrates the molecular interaction characteristics of different complexes. The SC-EGCG binary complex exhibits molecular interactions at 1000-1200 cm⁻¹. -1 The appearance of a new characteristic peak for phenolic hydroxyl groups in the region confirms that the two are bound together through hydrogen bonding and hydrophobic interactions. After the formation of the ternary complex, amide I shifts to a lower wavenumber (red shift) while amide II shifts to a higher wavenumber (blue shift), and the absorption intensity changes, indicating that Fe... 3+ Coordination significantly affects protein secondary structure and peptide bond vibrational modes. Among these, the OH / NH stretching vibration peak (3307.55 cm⁻¹) is particularly significant. -1 ) in Fe 3+ A blue shift occurs after the addition of a small amount of Fe. 3+ (As in Comparative Example 2) the peak is blue-shifted by 9 cm. -1 Furthermore, the strength decreased significantly, indicating that metal coordination weakened the original hydrogen bond network; while an appropriate amount of Fe... 3 + (As in Examples 1-2) only caused 2.21cm -1 The slight blue shift and small change in intensity indicate that the SC-EGCG hydrogen bonds and EGCG-Fe bonds are at this point. 3+Coordination bonds and electrostatic interactions work together to maintain the stable structure of the complex. These spectral variations reflect the appropriate Fe... 3+ Concentration allows for precise control of intermolecular forces.

[0060] (5) Differential Scanning Calorimetry (DSC): The modified protein sample was calorimetric using a differential scanning calorimeter. The specific procedure was to place 5 mg of the sample into a sealed aluminum DSC crucible. Under a nitrogen atmosphere, the temperature was heated to 200 °C at a rate of 10 °C / min, with an initial temperature of 30 °C.

[0061] Differential scanning calorimetry (as shown) Figure 5 (As shown) The transition temperatures of the ternary composites in Example 1 and Comparative Example 2 were 110.49℃ and 114.53℃, respectively, indicating that although the introduced Fe... 3+ Coordination weakens intermolecular hydrogen bonds and hydrophobic interactions, reducing the α-helix content, but appropriately increases Fe. 3+ (In the cases of Examples 1-2) thermal stability can be improved by enhancing coordination bonds and electrostatic interactions, which can partially counteract the instability effect of EGCG.

[0062] The above test results show that when EGCG is added to the SC dispersion system, polyphenols spontaneously adsorb onto the SC surface through non-covalent interactions such as hydrogen bonding, forming a loose composite structure dominated by hydrogen bonds. 3+ The introduction of this compound promotes the deprotonation of the catechol group in EGCG, forming a stable five-membered ring coordination structure. This EGCG-Fe 3+ The coordination layer synergistically assembles with the SC matrix through polyphenol metal chelation and protein surface affinity, ultimately constructing a stable ternary complex.

[0063] Overall, this invention obtains a stable ternary composite through simple processing steps, enabling its application and demonstrating high practicality.

[0064] Example a:

[0065] This embodiment provides a high internal phase emulsion loaded with β-carotene, which is prepared by the following steps:

[0066] First, 0.01 g of β-carotene was dissolved in 100 mL of refined soybean oil at 40 °C in the dark, with stirring for 30 min to ensure complete dissolution. The solution was then filtered through a 0.45 μm microporous membrane to obtain the oil phase. Next, the ternary complex from Example 1 was dissolved to obtain a 2 wt% aqueous phase. Finally, at room temperature, the oil phase was slowly added dropwise to the aqueous phase at a rate of 1 mL / min, followed by homogenization and emulsification using a high-speed shear emulsion at 12000 rpm for 3 min to prepare a high internal phase emulsion loaded with β-carotene. In this example, the volume fraction of the oil phase in the high internal phase emulsion was 75%.

[0067] Comparative example a:

[0068] This comparative example provides an emulsion that differs from that in Example a in that the aqueous phase used is prepared by the following steps:

[0069] Weigh 1.0 g of SC and dissolve it in 100 mL of PBS buffer (pH 7.0). Then, stir magnetically at 500 rpm until completely dissolved. Transfer the solution to a 4°C environment and hydrate for 12 h to obtain the SC solution as the aqueous phase.

[0070] The emulsion was then prepared using the preparation method described in Example a.

[0071] Comparative example b:

[0072] This comparative example provides an emulsion that differs from that in Example a in that the aqueous phase used is prepared by the following steps:

[0073] Using the same binary complex preparation method as in Example 1, 1.0 g of SC and 0.1 g of EGCG were mixed in PBS buffer at pH 7.0 to prepare a binary complex solution; and this binary complex solution was used as the aqueous phase.

[0074] The emulsion was then prepared using the preparation method described in Example a.

[0075] Detection of high internal phase emulsions:

[0076] Due to the addition of Fe 3+ The pH of the SC-EGCG complex system dropped to 2.0, while the pH of the SC or SC-EGCG binary complex system remained at 7.0. Therefore, in this embodiment, the emulsions in Example a and Comparative Examples a~b were each divided into two groups, and the pH of the emulsions was adjusted to 2.0 or 7.0 using NaOH or HCl solution, respectively. Thus, in this embodiment, a total of six groups of emulsions were finally tested.

[0077] ①Stability testing

[0078] (1) Rheological property testing

[0079] The rheological properties of each emulsion group were measured using a dynamic shear rheometer. The fixture consisted of parallel aluminum plates with a diameter of 40 mm and a gap of 1000 μm. The emulsion was subjected to an oscillation frequency scan within the range of 0.1–100 rad / s, and within the range of 0.1–100 s... -1 Flow scanning is performed within the range of shear rates.

[0080] Test results as follows Figure 6 As shown, in this embodiment, all emulsions exhibit elastomer characteristics (i.e., storage modulus G' > loss modulus G), and among them, SC-EGCG-Fe 3+ The system exhibits the highest G' value and viscosity, attributed to the robust colloidal network structure formed by its three components. While acidic conditions typically lead to interfacial layer thickening and increased G', the SC-EGCG system in the emulsion still demonstrates excellent gelling properties due to hydrogen bonding. Furthermore, all systems exhibit shear-thinning behavior, meaning their viscosity decreases with increasing shear rate, meeting the performance requirements for emulsions in the food industry.

[0081] In summary, the emulsion in Example a meets the basic requirements for the rheological properties of emulsions in the food industry.

[0082] (2) Thermal stability

[0083] In this embodiment, an additional control group is added, in which a pure β-carotene solution is used in the thermal stability test, and the heating and other steps involved are the same as those of the high internal phase emulsion.

[0084] Before heating, the initial concentration of β-carotene in the high internal phase emulsion loaded with β-carotene was determined and denoted as C0. For content determination, an appropriate amount of emulsion was added to a mixture of ethanol and n-hexane (2:3 volume ratio), shaken to mix, and the extraction was repeated three times. The upper extracts were then combined, and the absorbance was measured at 450 nm using a UV-Vis spectrophotometer. The β-carotene content was calculated based on a standard curve. Subsequently, the high internal phase emulsion loaded with β-carotene was placed in a serum bottle, heated at 85°C for 30 min, cooled to room temperature, and the β-carotene concentration was measured again and denoted as C. Finally, the retention rate of β-carotene was obtained by dividing C by C0 for thermal stability analysis.

[0085] Test results as follows Figure 7 As shown, in this embodiment, after heating at 80°C for 30 minutes, the retention rate of free β-carotene in soybean oil was only 60%, while the emulsion encapsulation system exhibited a significant protective effect on β-carotene: under pH 7 conditions, the retention rate of β-carotene in ordinary emulsions exceeded 85%, while SC-EGCG-Fe 3+The retention rate of β-carotene in the emulsion composed of the complex further reached 96%. The inventors analyzed that the protective effect stems from the dense interfacial layer formed by the protein and its complex, which effectively blocks heat conduction and reduces the thermal degradation of β-carotene. In contrast, the emulsion prepared under pH 2 conditions exhibited reduced β-carotene retention due to protein aggregation, increased droplet size, and EGCG-Fe... 3+ The weakening of coordination reduces its protective effect on β-carotene, but it still has a relatively higher β-carotene retention rate.

[0086] ②Analysis of in vitro digestion characteristics and bioaccessibility

[0087] To simulate human eating conditions, an in vitro gastrointestinal digestive model was established based on the INFOGEST methodology (an international consensus reached by the EU-funded COST Action FA1005INFOGEST (2011-2015) project). A three-step simulation of the oral cavity, stomach, and small intestine was used to evaluate the gastrointestinal digestive characteristics of different samples.

[0088] Before the experiment began, simulated digestive solutions for the oral (SSF), gastric (SGF), and intestinal (SIF) digestion stages were prepared at a concentration of 1.25× according to the INFOGEST method, and then the digestion process of the oral, gastric, and intestinal stages was simulated.

[0089] Oral digestion:

[0090] Weigh 10g of the emulsion sample and mix it with 10mL of SSF simulated digestion solution. Then adjust the pH of the mixture to 7.0 and incubate it in a water bath with magnetic stirring at 37℃ for 2min.

[0091] Stomach digestion:

[0092] Take 15 mL of the sample after oral digestion, add 15 mL of SGF simulated digestion solution, then adjust the pH of the sample to 3.0 with HCl solution, add pepsin solution (enzyme activity of 2000 U / mL in the final system), adjust the pH to 3.0 again, and digest magnetically in a 37 ℃ water bath for 2 h.

[0093] Intestinal digestion:

[0094] Take 20 mL of the sample after gastric digestion, add an equal volume of SIF simulated digestion solution, adjust the pH to 7.0, then add trypsin, lipase, and bile salt solutions, continue adjusting the pH to 7.0, and stir in a 37 ℃ water bath for 2 h. The trypsin activity is 100 U / mL, the lipase activity is 2000 U / mL, and the bile salt concentration is 10 mM. During this process, due to the release of free fatty acids (hereinafter referred to as FFA), the sample needs to be titrated with NaOH solution to maintain its pH value at 7.0.

[0095] During simulated intestinal digestion, the volume of NaOH consumed was recorded every 10, 20, 40, 60, 90, and 120 minutes. The amount of FFA released was calculated using the following formula:

[0096] FFA(%)=(V NaOH ×M NaOH ×M lipid ) / (2×W lipid ) ×100

[0097] 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 in this example); W lipid Mass of oil in the emulsion (g).

[0098] Test results as follows Figure 8 As shown, FFA is continuously released during digestion, indicating that the emulsion structure is gradually destroyed. Under the same pH conditions, the more complex the complex structure (such as SC-EGCG-Fe in Example a), the more likely it is to cause damage. 3+ The lower the FFA release rate, the more likely the system is to exhibit slow release characteristics. The inventors speculate that the dense three-dimensional network structure creates steric hindrance, while the thicker interface layer hinders enzyme contact, and the larger droplets also reduce the effective hydrolysis area. Furthermore, the emulsion in Example a maintains a slow release throughout the digestion process, demonstrating excellent sustained-release properties.

[0099] A certain amount of digestate from the final stage of simulated digestion was taken and centrifuged at 4000 rpm for 40 min. The intermediate micelle phase was taken and the concentration of β-carotene was determined. The bioavailability of β-carotene was calculated according to the following formula.

[0100] Bioaccessibility (%) = C Micelle / C Original ×100

[0101] Where C Micelle and C Original These represent the mass concentrations of β-carotene in the micelles and in the initial emulsion, respectively.

[0102] Test results as follows Figure 9As shown, the bioavailability of β-carotene in all three emulsions exceeded 45%. In particular, under neutral conditions (pH 7), the emulsion in Example a exhibited the best performance (56.1%). This is because the stable complex interfacial film it forms effectively protects oil droplets, reduces the damage of the gastric environment to the active ingredient, and promotes the release and absorption of β-carotene in the small intestine.

[0103] Overall, the high internal phase emulsion of this invention, while possessing the basic properties of an emulsion, can achieve stable storage of β-carotene, enable slow release of β-carotene during use, and greatly improve the bioavailability of β-carotene.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing sodium caseinate-epigallocatechin gallate-iron ion ternary complex, characterized by, Comprising the following steps: S1. Adding epigallocatechin gallate to sodium caseinate solution, stirring in the dark environment to obtain a binary complex; S2. Adding FeCl3·6H2O solution to the binary complex, stirring in the dark environment to obtain a ternary complex; Wherein, in the ternary complex, the mass ratio of sodium caseinate: epigallocatechin gallate: iron ion is (10-14): 1: (2-4).

2. A process for the preparation of sodium caseinate-epigallocatechin gallate-iron ion ternary complex as claimed in claim 1, wherein, The preparation of the sodium caseinate solution comprises the following steps: dissolving sodium caseinate in a phosphate buffer solution with pH 7-7.5 and stirring, then transferring the solution to a 3-5℃ environment for hydration for 12-16h to obtain the sodium caseinate solution.

3. The method for preparing sodium caseinate-epigallocatechin gallate-iron ion ternary complex according to claim 1, characterized in that, In S2, vortex stirring is used for mixing, the rotation speed is controlled at 3000-3500rpm, and the duration is 5-10min.

4. The method for preparing sodium caseinate-epigallocatechin gallate-iron ion ternary complex according to claim 1, characterized in that, Further comprising the following steps: S3. Filtering the ternary complex and freeze-drying to obtain a powdered ternary complex.

5. A ternary complex characterized in that, Prepared by the preparation method of any one of claims 1-4.

6. A process for the preparation of a high internal phase emulsion, characterized in that, Comprising the following steps: The ternary complex of claim 5 is configured as a solution with a concentration of 2-3wt% as the water phase; β-carotene is dissolved in soybean oil as the oil phase; the oil phase is added dropwise to the water phase and homogenously emulsified to obtain a high internal phase emulsion.

7. A process for the preparation of a high internal phase emulsion according to claim 6, characterized in that, In the high internal phase emulsion, the volume fraction of the oil phase is 70-75%.

8. A process for the preparation of a high internal phase emulsion according to claim 6, characterized in that, The oil phase is added dropwise to the water phase at a speed of 1-1.5mL / min.

9. A high internal phase emulsion loaded with beta-carotene, characterized in that, Prepared by the preparation method of any one of claims 1-8.

10. Use of the β-carotene-loaded high internal phase emulsion of claim 9 in functional food.

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