Anthocyanin double emulsion gel, preparation method and application thereof

Abstract

This invention relates to the field of functional food carriers and nutrient delivery technology, specifically to an anthocyanin dual emulsion gel, its preparation method, and its application. The invention provides an anthocyanin dual emulsion gel with a W1 / O / W2 dual emulsion structure; W1 is the inner aqueous phase, O is the oil phase, and W2 is the outer aqueous phase, all of which are gelled; the inner aqueous phase is loaded with anthocyanins; and W2 is loaded with zein-high methoxyl pectin composite particles. This anthocyanin dual emulsion gel exhibits high anthocyanin encapsulation efficiency, good shear stability, and storage stability, while also possessing both gastric protection and small intestinal sustained release delivery functions, which is beneficial for improving the bioavailability of anthocyanins and solves the problems of heat / light instability and premature gastric release of anthocyanins in traditional food ingredients.

Description

Technical Field

[0001] This invention relates to the field of functional food carriers and nutrient delivery technology, specifically to anthocyanin dual emulsion gel, its preparation method, and its application. Background Technology

[0002] Anthocyanins are natural polyphenol pigments with antioxidant and anti-inflammatory physiological activities. However, they are easily degraded by light, heat, neutral pH and the gastrointestinal environment, resulting in low bioavailability and utilization, which limits their application in the food industry.

[0003] Current carrier systems used for oral delivery of polyphenolic active substances such as anthocyanins, such as ordinary emulsions, microcapsules, spray-dried powders, and liposomes, generally suffer from problems such as insufficient interfacial protection, poor stability in gastric acid and bile salt environments, and rapid release. They are difficult to simultaneously meet the requirements of thermal stability, shear stability, and sustained release in the gastrointestinal tract during food processing.

[0004] In particular, some systems rely on small-molecule surfactants or non-natural polymers, which limits their food-grade safety and ingredient applicability. Furthermore, it is difficult to achieve precise interfacial protection and site-specific release based on the structural characteristics of anthocyanins. Therefore, there is an urgent need to develop an anthocyanin delivery carrier that is entirely composed of food-grade ingredients, possesses excellent processing adaptability, and can provide sustained release. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of anthocyanins in the prior art, such as early release, thermal instability and light instability, thereby providing an anthocyanin dual emulsion gel, preparation method and application.

[0006] Therefore, the present invention provides the following technical solution: This invention provides an anthocyanin dual emulsion gel, which has a W1 / O / W2 dual emulsion structure; W1 is the inner aqueous phase, O is the oil phase, and W2 is the outer aqueous phase, all of which are gelled; the inner aqueous phase is loaded with anthocyanins, and the W2 is loaded with zein-high methoxyl pectin composite particles (Zein-HMP composite particles) and low methoxyl pectin.

[0007] Zein possesses naturally strong hydrophobicity, but it readily undergoes irreversible self-aggregation in aqueous phases due to hydrophobic interactions. Introducing HMPs (high-methoxyl pectin) allows for the formation of composite particles through electrostatic attraction, regulating the hydrophilic-hydrophobic balance and enabling stable adsorption at the oil-water interface. Compared to modified starch, which requires complex hydrophobic modifications to effectively stabilize emulsions, and soy protein isolate, which is susceptible to isoelectric point and ionic strength and often requires enzymatic hydrolysis and glycosylation, zein's core advantage lies in its natural amphiphilic structure, which spontaneously assembles into nanoparticles. It can efficiently adsorb at the oil-water interface without complex chemical modifications, forming physically and thermodynamically stable Pickering emulsions. This invention utilizes zein-high-methoxyl pectin composite particles as a natural emulsifier to construct a W1 / O / W2 anthocyanin dual emulsion gel. Through the synergistic effect of "interfacial stability + bulk support," the encapsulation efficiency, environmental stability, and controlled-release performance of anthocyanins can be improved.

[0008] The anthocyanin dual emulsion gel of this invention can achieve precise interfacial protection and site-specific release of anthocyanins based on their structural characteristics. This gel significantly improves the stability of anthocyanins under heat treatment and light exposure conditions, resulting in high retention rates of the dual emulsion and gel system under pasteurization and continuous light exposure—the heat treatment retention rate is approximately 88%–92%. Figure 8 (Among A), the retention rate after 8 hours of light exposure is approximately 90%-94%. Figure 8 (B) effectively avoids significant loss of anthocyanin color and activity during processing and storage, and successfully solves the technical problem that traditional anthocyanins are mostly added directly or through simple emulsification, which easily lead to fading and degradation under heating, light, and neutral pH conditions, and lack gastrointestinal controlled release function.

[0009] Preferably, the preparation method of the zein-high methoxyl pectin composite particles includes: mixing and drying a zein solution with a pectin stock solution to obtain zein-high methoxyl pectin composite particles; The mass ratio of the zein solution to the pectin stock solution in this invention is (1-3):(1-3).

[0010] Preferably, the ratio of corn protein to aqueous ethanol solution added during the preparation of the corn gliadin solution is 1g:(50-150)mL, more preferably 1g:100mL; The ratio of pectin to water added during the preparation of the pectin stock solution is 1g:(50-150)mL, more preferably 1g:100mL; The mixing method includes high-speed shear homogenization; The drying method includes freeze drying.

[0011] Preferably, the rotation speed of the high-speed shear homogenization is 5000-10000 rpm, more preferably 7000 rpm, and the time is 3-7 min, more preferably 5 min; The freeze-drying temperature is -90 to -50°C; in a specific embodiment of the present invention, the freeze-drying temperature is any one of -90°C, -80°C, -70°C, -60°C, and -50°C, or a range between any two of these values.

[0012] The volume fraction of ethanol in the aqueous ethanol solution is 50%-90%, more preferably 70%; the mixture also includes rotary evaporation.

[0013] In a specific embodiment of the present invention, Zein is dissolved in a 70% (v / v) aqueous ethanol solution, and HMP is dissolved in deionized water; the Zein solution is slowly added to the HMP solution at a mass ratio (Zein:HMP) of (3:1)-(1:3), and the mixture is sheared at high speed of 7000 rpm for 7 min using a homogenizer and the ethanol is evaporated by a rotary evaporator to obtain a composite particle dispersion, which is then frozen in a refrigerator at -78~-80℃, more preferably at -80℃, and freeze-dried using a freeze dryer to obtain Zein-HMP composite particles.

[0014] Preferably, the volume ratio of the inner aqueous phase to the oil phase is (1-5):(5-9); the total volume ratio of the inner aqueous phase and the oil phase to the outer aqueous phase is (1-5):(5-9); and the gelation is achieved by calcium salt induction. In specific embodiments of the present invention, the volume ratio of the inner aqueous phase to the oil phase is 1:9, 2:8, 3:7, 4:6, and 5:5.

[0015] In specific embodiments of the present invention, the total volume ratio of the internal aqueous phase and the oil phase to the external aqueous phase is 1:9, 2:8, 3:7, 4:6, and 5:5.

[0016] The anthocyanin dual emulsion gel of this invention is a food-grade anthocyanin stable delivery system. It stabilizes the external aqueous phase of the W1 / O / W2 dual emulsion using Zein-HMP composite particles and introduces Ca... 2+ Ion-induced formation of a three-dimensional gel network significantly improves the retention rate of anthocyanins under heat treatment and light irradiation, and achieves gastric retention and slow release in the small intestine during in vitro simulated digestion.

[0017] Preferably, the internal aqueous phase includes an anthocyanin-containing solution; the concentration of anthocyanins in the anthocyanin-containing solution is 0.1%-0.9% (w / v), more preferably 0.5% (w / v); And / or, the oil phase comprises vegetable oil and an emulsifier; the vegetable oil comprises at least one of soybean oil, corn oil, peanut oil and olive oil, and the emulsifier is selected from at least one of Zein-HMP composite particles, polyglycerol ricinoleate and Tween-80; the emulsifier accounts for 2%-4% of the mass of the vegetable oil, more preferably 3%; and / or, the final concentration of the external aqueous phase zein-high methoxyl pectin composite particles is 0.5%-2.5% (w / v). And / or, the final concentration of the external aqueous phase low-methoxyl pectin is 0.5%-2.5% (w / v). And / or, the anthocyanin dual emulsion gel also includes calcium salts, the final concentration of which is 1%-5% (w / v).

[0018] In a specific embodiment of the present invention, W1 is an anthocyanin-containing solution; O is corn oil containing polyglycerol ricinoleate (PGPR) as an oil phase emulsifier; W2 is an external aqueous phase, which is a dispersion medium containing Zein-HMP composite particles as an external interface stabilizer, and contains a certain proportion of low-methoxyl pectin. CaCl2 is subsequently added for ionic crosslinking to obtain a double emulsion gel.

[0019] This invention provides a method for preparing anthocyanin dual emulsion gel, comprising: S1, mix the inner aqueous phase and the oil phase to prepare the primary emulsion W1 / O; mix zein-high methoxyl pectin composite particles and low methoxyl pectin to obtain the outer aqueous phase solution; S2, the primary emulsion W1 / O and the external aqueous solution are mixed to obtain W1 / O / W2 anthocyanin dual emulsion gel; the final concentration of zein-high methoxyl pectin composite particles in the external aqueous solution is 0.5%-2.5% (w / v). The final concentration of low-methoxyl pectin in the external aqueous phase solution is 0.5%-2.5% (w / v), more preferably 1.5% (w / v). Preferably, in step S2, the anthocyanin dual emulsion gel further includes calcium salt, and the final concentration of the calcium salt is 1%-5% (w / v). The calcium salt includes at least one of CaCl2, calcium lactate, and calcium carbonate.

[0020] This invention provides the application of anthocyanin dual emulsion gel as a microcarrier system.

[0021] The anthocyanin dual emulsion gel of the present invention can be used as an oral nutrient delivery carrier for anthocyanins, providing effective protection in the stomach and achieving sustained release of anthocyanins in the small intestine, thereby improving the utilization rate of anthocyanins.

[0022] Preferably, the microcarrier system is applied in the fields of food, health products, medicine, or cosmetics, and has a sustained-release delivery function; the products of the microcarrier system include powder microcapsules.

[0023] The technical solution of the present invention has the following advantages: 1. This invention prepares an anthocyanin dual emulsion gel, which is loaded with zein-high methoxyl pectin composite particles. This invention uses Zein-HMP composite particles as an external phase emulsion stabilizer to construct a W1 / O / W2 dual emulsion, embedding anthocyanins in the inner aqueous phase of the W1 / O / W2 dual emulsion. The Zein-HMP composite particles construct a dense interface in the external aqueous phase, forming a three-dimensional gel network, achieving dual protection of "dense interface of composite particles + gel spatial network". This gives the anthocyanin dual emulsion gel high encapsulation efficiency, good shear stability and storage stability, solving the problems of thermal instability, light instability and early gastric release of anthocyanins in traditional food ingredients.

[0024] Furthermore, all components of the anthocyanin dual emulsion gel are food-grade raw materials, belonging to a natural protein-polysaccharide complex carrier. It has the characteristics of being safe, edible, and palatable, making it more suitable as an oral delivery carrier in the field of functional foods and nutritional ingredients.

[0025] Furthermore, in this invention, Zein and HMP form composite particles through electrostatic interactions and hydrogen bonds, with a Zein to HMP mass ratio of (3:1-1:3). The resulting composite particle size ranges from 285.67 to 579.07 nm. When the Zein to HMP mass ratio is 1:2, the composite particle size is the smallest, approximately 285.67 ± 32.89 nm, with a high absolute zeta potential (approximately -30.62 mV), exhibiting optimal emulsifying activity and stability. The resulting dual emulsion, with 1.5% (w / v) composite particles, has an average particle size of approximately 11-13 μm and anthocyanin encapsulation efficiency ≥90%. This optimized ratio and structural design significantly inhibits droplet aggregation and leakage, ensuring the colloidal stability and efficient encapsulation capability of the system during processing and storage.

[0026] Furthermore, by controlling the amount of CaCl2 added within the final concentration range of 1%-5% (w / v), this invention constructs a dense and uniform three-dimensional gel network in the external aqueous phase, significantly improving the water-holding capacity and viscosity of the system. G′ / G″ indicates the formation of a gel structure with high strength. In in vitro simulated digestion, this network structure can effectively inhibit the rapid release in the gastric segment (pH 2.0, 0-2 h), ensuring that the cumulative release ratio of the gel group at the end of the gastric segment is ≤25%, while achieving slow and continuous release in the small intestine segment (pH 7.0, 2-6 h). Thus, it has the delivery function of "gastric segment protection + small intestine sustained release", which is beneficial to improving the bioavailability of anthocyanins.

[0027] 2. This invention provides a method for preparing anthocyanin dual emulsion gel, which has mild process conditions and is easy to operate and implement.

[0028] 3. This invention provides the application of anthocyanin dual emulsion gel as a microcarrier system. The anthocyanin dual emulsion gel provided by this invention can be flexibly transformed according to the end product form. On the one hand, it can be directly added to systems such as yogurt and fermented milk to impart stable natural color and antioxidant function; on the other hand, it can be prepared into powder microcapsules through spray drying or freeze drying, and then applied to products such as solid beverages, tablets, or capsules, exhibiting good industrial feasibility and shelf-life stability. Industrial feasibility means large-scale production, good reproducibility, and stable effects.

[0029] This invention first prepares Zein-HMP composite particles and disperses them in an aqueous phase as particulate natural emulsifiers. Then, it undergoes secondary emulsification with anthocyanin-encapsulated W1 / O colostrum and ion-induced gelation to form a W1 / O / W2 anthocyanin dual emulsion gel delivery system that combines interface protection and three-dimensional network support. This system has significant advantages in terms of thermal / light stability and gastrointestinal sustained release, and is suitable for the processing and storage of functional foods and beverages. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 Characterization of composite particles with different mixing methods, where A represents the particle size and PDI results of Zein-HMP and Zein-LMP with different mixing methods, and B represents the FTIR results; Figure 2 Characterization of composite particles with different Zein:HMP ratios, where A represents the potential results, B represents the particle size and PDI results, C represents the emulsification activity index and emulsification stability index results, and D is the emulsification effect appearance diagram; Figure 3 Particle size / distribution and encapsulation efficiency diagrams for different protein-polysaccharide combinations are shown. A represents the particle size and PDI results of different protein-HMP combinations, B represents the particle size and PDI results of Zein-different polysaccharides, and C represents the encapsulation efficiency results of different systems. Figure 4 The encapsulation efficiency of the W1 / O emulsion is given by A, where A represents the encapsulation efficiency results for different anthocyanin concentrations and B represents the encapsulation efficiency results for different oil phases. Figure 5 The image shows the characterization of the dual emulsion. In the image, A represents the laser confocal microscopy results for different composite particle concentrations, B represents the encapsulation efficiency of different Zein-HMP composite particles, C represents the viscosity results for different LMP additions, D represents the storage modulus results for different LMP additions, and E represents the loss modulus results for different LMP additions. Figure 6 Characterization diagrams of gels at different CaCl2 concentrations are shown, where A represents morphology, B represents moisture retention, C represents viscosity, D represents intermolecular forces (solubility), E represents hardness, and F represents elasticity. Figure 7 Characterization diagrams of gels at different CaCl2 concentrations are shown, where A represents the storage modulus and loss modulus results, and B represents the microstructure results. Figure 8 The graph shows the anthocyanin retention rates of the free anthocyanin group, the double emulsion group, and the emulsion gel group under different thermal and photostability conditions, where A represents the thermal stability result and B represents the photostability result. Figure 9 The diagram shows the structural evolution and cumulative release curves of free anthocyanins in in vitro simulated digestion, double emulsions, and emulsion gels. In this diagram, A represents the structural change result and B represents the release rate result. In the above attached diagram, the different letters a, b, and c represent... p The difference was statistically significant at levels <0.05. Detailed Implementation

[0032] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0033] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0034] Unless otherwise specified, the raw materials and equipment used in this invention can be purchased from the market or are commonly used in the field.

[0035] Unless otherwise specified, all quantities in this application are mass quantities.

[0036] The freeze-drying parameters in the following examples are: temperature -80℃, time 24h, and vacuum degree 1 Pa.

[0037] Blackberry extract anthocyanins: Fufeng Snowt Biotechnology Co., Ltd.; High-methoxyl pectin [galacturonic acid (dry basis) ≥74.0%], molecular weight 100,000~300,000 Da; Low-methoxyl pectin (degree of esterification <50.0%), molecular weight 50,000–200,000 Da; Polyglycerol ricinoleate (PGPR) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P902442-100g; Calcium chloride, sodium chloride: Shanghai Aladdin Biochemical Technology Co., Ltd.; Porcine pepsin, trypsin, and porcine bile salts: Shanghai Yuanye Biotechnology Co., Ltd. Anhydrous ethanol: concentration ≥99.7%, Shanghai Maclean Biochemical Technology Co., Ltd. SA: Sodium alginate; CMC-Na: Sodium carboxymethyl cellulose; WPI: Whey protein isolate; Gli (wheat protein) and SPI (soy protein isolate) were both purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0038] The measurement methods used in the following examples are as follows: (1) Fourier transform infrared (FTIR) analysis Fourier transform infrared (FTIR) analysis was performed using the KBr particle method to determine the interactions between components. The instrument used was a NICOLET iS50FT-IR (Thermo Nicolet Ltd., Waltham, MA, USA), with a wavenumber range of 4000–400 cm⁻¹. -1 Each sample was subjected to a 4 cm test. -1 A uniform 32 scans.

[0039] (2) Particle size, PDI and potential measurement and analysis The particle size, polydispersity index (PDI), and zeta potential of the newly prepared particles were determined using a 25℃ dynamic light scattering particle size analyzer.

[0040] (3) Analysis of emulsification characteristics The emulsifying activity index (EAI) and emulsifying stability index (ESI) are used to reflect the emulsifying properties of the dual emulsion. Freshly prepared emulsion (50 μL) was transferred to a 0.1% SDS solution using a pipette. The absorbance of the mixture was measured at 500 nm using a UV-1800 spectrophotometer (Shimadzu, Japan), with the 0.1% SDS solution serving as a control blank. The ESI and EAI values ​​were then calculated.

[0041]

[0042] Among them, A 500 The absorbance value was measured at a wavelength of 500 nm, and DF represents the dilution factor. C Protein concentration, g / mL; φ: oil phase volume fraction (0.7 in this experiment).

[0043] Among them, A 20 A1 is the absorbance value measured at 20 min, and A20 is the absorbance value measured at 0 min.

[0044] (4) Microstructure The microstructure of the emulsion gel was observed by FE-SEM (GeminiSEM 300, ZEISS, Oberkochen, Germany) at 30x magnification.

[0045] (5) Encapsulation efficiency test The mass concentration of anthocyanins in the samples was determined using the pH difference method. 1 mL of the sample was mixed with 4 mL of distilled water and stirred for 2 min. After thorough mixing, the mixture was centrifuged at 4,000 rpm for 10 min. 1 mL of the supernatant was collected and diluted with potassium chloride-hydrochloric acid buffer (pH 4.5) and potassium hydrogen phthalate-sodium hydroxide buffer (pH 1.0), respectively. The samples were equilibrated in the dark for 15 min. Using distilled water as a blank control, the absorbance of the samples was measured at 520 nm and 700 nm. Ht This refers to the original amount of anthocyanins added. Hf The content of unencapsulated free anthocyanins is represented by EE (%), which indicates the encapsulation rate (%).

[0046]

[0047] (6) Laser confocal microscopy measurement Add 1 mL of anthocyanin W1 / O / W2 double emulsion to a centrifuge tube. Stain corn oil with Nile Red for 15 min. Then, place 10 μL of the stained sample on a glass slide and cover with thin paper to prepare a thin sample. Observe and image using an inverted fluorescence microscope.

[0048] (7) Rheological property determination The rheological properties of rhodioloside W1 / O / W2 double emulsion samples were detected using a DHR-2 rheometer. For the frequency sweep model, storage modulus and loss modulus were recorded in the frequency range of 0.1–10 Hz at a strain of 0.16% determined by the oscillating stress sweep model. The experimental temperature was 25 °C, and the gap was 1 mm. Shear rates ranged from 0.1 to 100 s⁻¹. -1 The viscosity curve was measured at a temperature of 25℃.

[0049] (8) Water Holding Capacity (WHC) Measurement The sample was centrifuged at 4000 rpm for 20 min at 25°C. Excess water was drained, and the sample surface was carefully dried using a paper filter. The emulsion gel was then weighed in a centrifuge tube. W 0 represents the mass of the gel before centrifugation. W 1 represents the mass of the gel after centrifugation. The formula for calculating WHC is as follows:

[0050] (9) Measurement of intermolecular forces The samples were treated with denaturing solvents, which can disrupt different intermolecular interactions. Specifically, 0.6 mol / L sodium chloride solution can disrupt ionic bonds, 1.5 mol / L urea solution can disrupt hydrogen bonds, 8 mol / L urea solution can disrupt both hydrogen bonds and hydrophobic interactions, and 0.5 mol / L β-mercaptoethanol (β-ME) solution can disrupt disulfide bonds.

[0051] The specific solvent to be prepared is as follows: Solvent A1: 0.6 mol / L sodium chloride solution; Solvent A2: 0.6 mol / L sodium chloride + 1.5 mol / L urea; A3 solvent: 0.6 mol / L sodium chloride + 8 mol / L urea; A4 solvent: 0.6 mol / L sodium chloride + 8 mol / L urea + 0.5 mol / L β-mercaptoethanol.

[0052] The samples were dissolved in four solvents, A1-A4, respectively, and the contributions of different interactions to the fermentation-induced gel formation process were analyzed. The protein concentration in the supernatant was determined using the Coomassie Brilliant Blue method, with bovine serum albumin as the standard. The solubility differences of A1, (A2-A1), (A3-A2), and (A4-A3) represent the contributions of ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bonds, respectively.

[0053] (10) Texture determination The texture of the emulsion gel was determined using a texture analyzer. The deformation parameter was set to 50%, and the hardness and elasticity of the emulsion gel were measured.

[0054] Photostability test method: To ensure consistent anthocyanin content across the three sample groups, weigh each group and seal them in stoppered test tubes. Place the samples in a simulated light irradiation device and continuously irradiate them with simulated natural light at a constant temperature of 10℃ for 8 hours. Immediately after the irradiation period, take samples to determine the anthocyanin retention rate in each group to evaluate their photostability.

[0055] Thermal stability test method: To ensure consistent anthocyanin content in the three sample groups, weigh out the samples and seal them in stoppered test tubes. Place the samples in a 65℃ constant temperature water bath in the dark for 30 min (simulating pasteurization conditions). After the reaction, immediately remove the samples and cool them to room temperature in an ice-water bath to terminate the thermal effect. Subsequently, measure the anthocyanin retention rate in each sample group to evaluate its thermal stability.

[0056] Example 1 Zeat gliadin-pectin composite particles were prepared using an antisolvent precipitation method. The specific steps are as follows: Treatment 1: Dissolve 1g of corn gluten in 100mL of 70% (v / v) ethanol aqueous solution to obtain a zein solution. Dissolve 1g of high-methoxyl pectin (HMP) in 100mL of deionized water to obtain a high-methoxyl pectin stock solution.

[0057] Zein solution and high-methoxyl pectin stock solution were mixed at a mass ratio of 3:1, i.e., the Zein solution was slowly introduced into the high-methoxyl pectin solution under continuous stirring, and homogenized in a high-speed shear homogenizer at 7000 rpm for 5 minutes to ensure thorough mixing and particle formation. Then, ethanol was removed by rotary evaporation to obtain a dispersion. The dispersion was frozen at -80°C and freeze-dried to obtain zein-pectin composite nanoparticle powder, i.e., Zein-HMP complex particles.

[0058] Treatment 2: Same as Treatment 1, except that the corn gliadin solution and high methoxyl pectin solution are mixed at a mass ratio of 2:1.

[0059] Treatment 3: Same as Treatment 1, except that the corn gliadin solution and high methoxyl pectin solution are mixed at a mass ratio of 1:1.

[0060] Treatment 4: Same as Treatment 1, except that the corn gliadin solution and high methoxyl pectin solution are mixed at a mass ratio of 1:2.

[0061] Specifically, 1g of corn gluten was dissolved in 100mL of 70% (v / v) ethanol aqueous solution to obtain a zein solution, i.e., a Zein solution. 1g of high-methoxyl pectin (HMP) was dissolved in 100mL of deionized water to obtain a high-methoxyl pectin stock solution. The Zein solution and the high-methoxyl pectin stock solution were mixed at a mass ratio of 1:2, i.e., the Zein solution was slowly introduced into the high-methoxyl pectin solution under continuous stirring, and homogenized at 7000 rpm for 5 min in a high-speed shear homogenizer to ensure thorough mixing and particle formation. Afterwards, the ethanol was removed by rotary evaporation to obtain a dispersion. The dispersion was frozen at -80℃ and then freeze-dried to obtain zein-pectin composite nanoparticle powder, i.e., Zein-HMP composite particles.

[0062] Treatment 5: Same as Treatment 1, except that the corn gliadin solution and high methoxyl pectin solution are mixed at a mass ratio of 1:3.

[0063] Treatment 6 (Zein): Dissolve 1g of corn gluten in 100 mL of 70% (v / v) ethanol aqueous solution to obtain a zein solution, i.e., Zein solution.

[0064] Treatment 7 (HMP): Dissolve 1g of HMP in 100 mL of deionized water to obtain a high-methoxyl pectin solution.

[0065] Process 8: Same as Process 4, except that the mixing method is simple mixing. Simple mixing is done using a magnetic stirrer with the following parameters: speed of 500 rpm and time of 5 min, to obtain Zein-HMP(S).

[0066] Treatment 9: Same as treatment 4, except that high methoxyl pectin (HMP) is replaced with low methoxyl pectin (LMP) in equal amounts to obtain Zein-LMP(H).

[0067] Treatment 10: Same as treatment 8, except that high methoxyl pectin (HMP) is replaced with low methoxyl pectin in equal amounts to obtain Zein-LMP (S).

[0068] Zein-HMP composite particles prepared under high-speed shearing conditions are referred to as Zein-HMP(H).

[0069] The particle size and PDI of the products obtained from treatments 4 and 8-10 were determined, and the results are shown in the figure. Figure 1 A. Figure 1 As shown in Figure A, the Zein-HMP composite particles have the smallest particle size under high-speed shear conditions.

[0070] FTIR analysis was performed on the results of treatments 6, 7, 4, and 8. The results are shown in [Figure number missing]. Figure 1 From B, it can be seen that compared with the simple mixed group (Zein-HMP (S)), the high-speed shear group (Zein-HMP (H)) in the OH / NH tensile vibration region (3000-3600cm) -1 The peak value is 3339.95 cm. -1 Up to 3411.80 cm -1 The amplitude gradually decreases, and the peak shape may be smoother, indicating a more uniform interaction between Zein and HMP. In the simple mixed group, the characteristic peaks of pectin and the C=O peak of ester carbonyl (1730-1750 cm⁻¹) are also present. -1 ) and carboxylate COO peak (1600-1650 cm) -1 The change in pectin concentration was smaller than that of pure pectin, indicating that the structure of pectin did not change during the simple mixing process, and the intermolecular interactions were uneven. In homogeneous groups, the characteristic peak of pectin, namely the C=O peak of the ester carbonyl group (1730-1750 cm⁻¹), is visible. -1 The strength of HMP may be weakened. This is because the ester carbonyl group of HMP is more likely to react with the protonated amino group (-NH3) of corn protein. + Hydrogen bonds are formed between the hydroxyl group (-OH) and the hydroxyl group (-OH), causing a change in the vibrational frequency and a gradual decrease in absorption intensity.

[0071] The carboxyl group of HMP is partially ionized, forming a carboxylate ion at (1600-1650 cm⁻¹). -1 The asymmetric stretching vibration peak at () may be shifted due to the formation of hydrogen bonds with the amide group of zein, further confirming the hydrogen bond interaction between Zein and HMP.

[0072] Potential, particle size, and PDI analyses were performed on the Zein-HMP complex particles obtained from treatments 1-6 to characterize their emulsification properties.

[0073] The emulsification properties were characterized as follows: Zein solution and high-methoxyl pectin (HMP) stock solution were mixed at different mass ratios (treatments 1-6), and Zein-HMP composite particles were prepared by antisolvent co-precipitation. The composite particles were used as the emulsifier. An oil phase (containing 1.5% (w / v) of the composite particles) and an oil phase (corn oil) with a volume ratio of 7:3 were prepared using a high-speed shear press at 15000 rpm for 3 min to produce an oil-in-water emulsion. The changes in emulsion stratification, precipitation, and homogeneity were observed, and the emulsification properties of different mass ratios of the composite particles were analyzed.

[0074] See results Figure 2 China A Figure 2 B, Figure 2 C and Figure 2From D, it can be seen that the optimal mass ratio of Zein to HMP is Zein:HMP=1:2, which has the smallest particle size distribution and the best emulsifying activity and emulsifying stability. Figure 2 In the image, D represents the image taken 0 minutes after the preparation of the oil-in-water emulsion, and 20 minutes represents the image taken 20 minutes after the preparation of the oil-in-water emulsion.

[0075] Comparative Example 1 Preparation method of Zein / SA: 1g of Zein was dissolved in 100mL of 70% (v / v) ethanol aqueous solution to obtain Zein solution; 2g of SA was dissolved in 100mL of deionized water to obtain SA solution; the Zein solution and SA solution were mixed at a mass ratio of 1:1, the Zein solution was slowly added to the SA solution, the mixture was homogenized at 7000rpm for 5min and the ethanol was removed by rotary evaporator to obtain composite particle dispersion, which was then frozen in a -80℃ freezer and freeze-dried to obtain Zein / SA composite particles.

[0076] Comparative Example 2 Preparation method of Zein / CMC-Na: Same as the preparation of Zein / SA (sodium alginate) in Comparative Example 1, the only difference is that SA is replaced with an equal amount of CMC-Na, and the mass ratio of Zein to CMC-Na is kept at 1:2.

[0077] Comparative Example 3 Preparation method of WPI / HMP: The mass ratio of WPI to HMP is 1:2. WPI is dissolved in a 70% (v / v) aqueous ethanol solution, and HMP is dissolved in deionized water. The WPI solution is slowly added to the HMP solution, and the mixture is homogenized at 7000 rpm for 5 min. The ethanol is then evaporated by a rotary evaporator to obtain a composite particle dispersion. The dispersion is then frozen at -80℃ and freeze-dried using a freeze dryer to obtain WPI / HMP composite particles.

[0078] Comparative Example 4 Preparation method of Gli / HMP: Same as the preparation of WPI / HMP in Comparative Example 3, except that WPI is replaced with Gli, and the mass ratio of Gli to HMP is kept at 1:2.

[0079] Comparative Example 5 Preparation method of SPI / HMP: Same as the preparation of WPI / HMP in Comparative Example 3, except that WPI is replaced with SPI, while maintaining the mass ratio of SPI to HMP at 1:2.

[0080] The encapsulation efficiency and particle size / distribution of the Zein-HMP composite particles obtained in treatment 4 of Example 1 and the Zein / SA, Zein / CMC-Na, WPI (whey protein isolate) / HMP, Gli (gliadin) / HMP and SPI (soy protein isolate) / HMP obtained in Comparative Examples 1-5 were determined as follows: Figure 3 .

[0081] The method for determining the effect of different composite particles on the encapsulation efficiency of anthocyanins in double emulsions is as follows: S1, Preparation of anthocyanin W1 / O emulsion: The inner aqueous phase W1 contains 0.5% (w / v) anthocyanins, i.e., W1 is a 0.5% (w / v) anthocyanin aqueous solution. The oil phase O contains corn oil and PGPR, with PGPR accounting for 3% (w / w) of the corn oil. After adding the inner aqueous phase W1 and oil phase O at a volume ratio of 3:7, the mixture is sheared at 15000 rpm for 3 min to form the W1 / O primary emulsion.

[0082] S2, Preparation of composite particle solution: The Zein-HMP composite particles obtained from treatment 4 of Example 1 and the Zein / SA, Zein / CMC-Na, WPI (whey protein isolate) / HMP, Gli (wheat gliadin) / HMP and SPI (soy protein isolate) / HMP composite particles obtained from Comparative Examples 1-5 were dissolved in deionized water to obtain a composite particle solution with a final concentration of 1.5% (w / v), and allowed to stand at 4°C for overnight hydration.

[0083] 40 mL of W1 / O primary emulsion was mixed with 60 mL of different types of composite particle solutions, and the mixture was sheared at 20,000 rpm for 3 min to obtain emulsions. The anthocyanin encapsulation rate of the obtained emulsions was then determined.

[0084] according to Figure 3 It can be seen that the Zein-HMP composite particles constructed in this invention, when compared with nanocomposite particles prepared from various common proteins (such as wheat gliadin, soy protein isolate (SPI), and whey protein (WPI)) and polysaccharides (such as CMC-Na, LMP, and sodium alginate), show that the Zein-HMP system of this invention exhibits a significantly smaller average particle size (285.67 nm) and a highly uniform polydispersity index (PDI=0.16), achieving a high anthocyanin encapsulation efficiency (92.4%). The results demonstrate the advantages of the Zein-HMP system in constructing acid-stable active substance delivery carriers. The combination of zein and high-methoxyl pectin (HMP), with its excellent natural amphiphilicity and dense film-forming ability, improves the encapsulation efficiency of combinations with other common plant proteins (such as soy protein isolate and wheat gliadin) and polysaccharides (such as CMC-Na and sodium alginate).

[0085] Example 2 Treatment 1: Preparation of anthocyanin W1 / O emulsion: The aqueous phase W1 contains 0.5% (w / v) anthocyanins, i.e., W1 is a 0.5% (w / v) anthocyanin aqueous solution. The oil phase O contains corn oil and PGPR, with PGPR accounting for 3% (w / w) of the corn oil. After adding the aqueous phase W1 and oil phase O at a volume ratio of 3:7, the mixture is sheared at 15000 rpm for 3 min to form the W1 / O primary emulsion.

[0086] Treatment 2 is the same as Treatment 1, except that corn oil is replaced with soybean oil.

[0087] Process 3 is the same as Process 1, except that corn oil is replaced with peanut oil.

[0088] Process 4 is the same as Process 1, except that corn oil is replaced with olive oil.

[0089] Treatment 5 was the same as treatment 1, except that W1 was a 0.1% (w / v) anthocyanin aqueous solution.

[0090] Treatment 6 was the same as treatment 1, except that W1 was a 0.3% (w / v) anthocyanin aqueous solution.

[0091] Treatment 7 was the same as treatment 1, except that W1 was a 0.7% (w / v) anthocyanin aqueous solution.

[0092] Treatment 8 was the same as treatment 1, except that W1 was a 0.9% (w / v) anthocyanin aqueous solution.

[0093] The encapsulation efficiency of the products obtained from treatments 1-8 was determined, and the results are shown in the figure. Figure 4 .according to Figure 4 It can be seen that as the anthocyanin concentration increases, the encapsulation efficiency first increases and then decreases, with the maximum encapsulation efficiency at 0.5% (w / v) anthocyanin.

[0094] Example 3 S1, Preparation of anthocyanin W1 / O emulsion: The inner aqueous phase W1 contains 0.5% (w / v) anthocyanins, i.e., W1 is a 0.5% (w / v) anthocyanin aqueous solution. The oil phase O contains corn oil and PGPR, with PGPR accounting for 3% (w / w) of the corn oil. After adding the inner aqueous phase W1 and oil phase O at a volume ratio of 3:7, the mixture is sheared at 15000 rpm for 3 min to form the W1 / O primary emulsion.

[0095] S2, Preparation of anthocyanin W1 / O / W2 dual emulsion: The Zein-HMP composite particles prepared in treatment 4 of Example 1 were dissolved in deionized water to obtain a Zein-HMP composite particle solution with a final concentration of 1.5% (w / v). The solution was allowed to stand at 4°C overnight for hydration to obtain the W2 external aqueous phase solution. Then, 40 mL of the W1 / O primary emulsion was mixed with 60 mL of the W2 external aqueous phase solution, and sheared at 20,000 rpm for 3 min to obtain the W1 / O / W2 dual emulsion.

[0096] Example 4 Same as Example 3, except that in S2, a Zein-HMP composite particle solution with a final concentration of 0.5% (w / v) was obtained.

[0097] Example 5 Same as Example 3, except that in S2, a Zein-HMP composite particle solution with a final concentration of 1.0% (w / v) was obtained.

[0098] Example 6 Same as Example 3, except that in S2, a Zein-HMP composite particle solution with a final concentration of 2.0% (w / v) was obtained.

[0099] Example 7 Same as Example 3, except that in S2, a Zein-HMP composite particle solution with a final concentration of 2.5% (w / v) was obtained.

[0100] Comparative Example 6 Same as Example 3, except that in S2, Zein-HMP composite particle solution is not added.

[0101] Laser confocal microscopy was used to measure the encapsulation efficiency of the W1 / O / W2 dual emulsions obtained in Examples 3-7 and Comparative Example 6. The results are shown in [Figure 1]. Figure 5 A and B in the middle, Figure 5 When the concentration of the emulsifier Zein-HMP composite particles in the middle A emulsion increased from 0% to 1.5%, the particle size of the emulsion decreased significantly from 18.85±1.17μm. p The particle size ranged from <0.05 μm to a minimum of 11.56 ± 0.95 μm; this phenomenon follows the classical theory of colloidal stability: in the low concentration range (0%-1.0%), the concentration of the emulsifier is the key factor limiting interfacial coverage. Increasing the concentration ensures that a sufficient number of composite particles can be rapidly adsorbed onto the newly formed oil-water interface. By reducing interfacial tension, an interfacial film with high mechanical strength is formed, effectively inhibiting droplet aggregation during homogenization, thereby significantly reducing the particle size.

[0102] Figure 5Figure B shows that as the concentration of Zein-HMP composite particles increases, the encapsulation efficiency of the dual emulsion initially rises and then stabilizes, reaching a maximum of 93.4 ± 0.7% at 1.5%. This trend can be attributed to the gradual saturation of the oil-water interface by the composite particles. At low concentrations, insufficient interfacial coverage leads to partial leakage of the internal aqueous phase, resulting in lower encapsulation efficiency. Increasing the particle concentration enhances interfacial adsorption, forming a denser and more cohesive interfacial film that effectively retains the internal phase. However, once the interface is saturated, excess particles disperse in the continuous phase, increasing viscosity and thus limiting further improvements in encapsulation efficiency.

[0103] Example 8 S1, Preparation of anthocyanin W1 / O emulsion: Aqueous phase W1 contains 0.5% (w / v) anthocyanins, i.e., W1 is a 0.5% (w / v) anthocyanin aqueous solution. Oil phase O contains corn oil and PGPR, with PGPR accounting for 3% (w / w) of the corn oil. After adding aqueous phase W1 and oil phase O at a volume ratio of 3:7, the mixture is sheared at 15000 rpm for 3 min to form the W1 / O primary emulsion.

[0104] S2, Preparation of anthocyanin W1 / O / W2 dual emulsion: The Zein-HMP composite particles prepared in treatment 4 of Example 1 were dissolved in deionized water to obtain a Zein-HMP composite particle solution with a final concentration of 1.5% (w / v). The solution was allowed to stand at 4°C overnight for hydration. Low-methoxyl pectin was then added to the resulting solution to achieve a final mass-volume concentration of 1.5% (w / v), resulting in the W2 outer aqueous phase solution. The W2 outer aqueous phase solution serves as the outer emulsifier for the W2 outer layer.

[0105] Then, mix 40 mL of the W1 / O primary emulsion with 60 mL of the W2 external aqueous phase solution and shear at 20,000 rpm for 3 min to obtain the W1 / O / W2 double emulsion.

[0106] Example 9 Same as Example 8, except that in S2, the final mass volume concentration of low-methoxyl pectin is 0.5% (w / v).

[0107] Example 10 Same as Example 8, except that in S2, the final mass volume concentration of low methoxyl pectin is 1.0% (w / v).

[0108] Example 11 Same as Example 8, except that in S2, the final mass volume concentration of low methoxyl pectin is 2.0% (w / v).

[0109] Example 12 Same as Example 8, except that in S2, the final mass volume concentration of low-methoxyl pectin is 2.5% (w / v).

[0110] Comparative Example 7 Same as Example 8, except that in S2, the final mass volume concentration of low-methoxyl pectin is 0% (w / v).

[0111] The viscosity, storage modulus, and loss modulus of the W1 / O / W2 anthocyanin dual emulsion gels obtained in Examples 8-12 and Comparative Example 7 were measured, and the results are shown in the figure. Figure 5 C, D, E.

[0112] Figure 5 C, Figure 5 D, Figure 5 The study investigated the effect of introducing low-methoxyl pectin into the external aqueous phase on the rheological properties of dual emulsions. Within the scanning frequency range of 0.1–10 Hz, the loss modulus G” of all emulsion samples consistently exceeded the storage modulus G', indicating that the system was in a liquid state and exhibited prominent viscous characteristics. Polysaccharides are often used as stabilizers in the continuous phase to achieve emulsification and enhance emulsion stability by increasing viscosity. The results showed that all samples exhibited shear thinning behavior. This phenomenon may be due to the disruption of the aggregation network between droplets during shearing, indicating that the emulsion samples are non-Newtonian fluids. As non-Newtonian fluids, the emulsion samples exhibit typical shear thinning behavior, combining high stability at rest with easy transport and application under high shear, resulting in a more stable system, more process-friendly design, and a wider range of applications.

[0113] Example 13 S1, Preparation of anthocyanin W1 / O emulsion: Aqueous phase W1 contains 0.5% (w / v) anthocyanins, i.e., W1 is a 0.5% (w / v) anthocyanin aqueous solution. Oil phase O contains corn oil and PGPR, with PGPR accounting for 3% (w / w) of the corn oil. After adding aqueous phase W1 and oil phase O at a volume ratio of 3:7, the mixture is sheared at 15000 rpm for 3 min to form the W1 / O primary emulsion.

[0114] S2, Preparation of anthocyanin W1 / O / W2 dual emulsion: The Zein-HMP composite particles prepared in treatment 4 of Example 1 were dissolved in deionized water to obtain a Zein-HMP composite particle solution with a final concentration of 1.5% (w / v). The solution was allowed to stand at 4°C overnight for hydration. Low-methoxyl pectin was then added to the resulting solution to achieve a final mass-volume concentration of 1.5% (w / v), resulting in the W2 outer aqueous phase solution. The W2 outer aqueous phase solution serves as the outer emulsifier for the W2 outer layer.

[0115] Then, mix 40 mL of the W1 / O primary emulsion with 60 mL of the W2 external aqueous phase solution and shear at 20,000 rpm for 3 min to obtain the W1 / O / W2 double emulsion.

[0116] S3, CaCl2 was added to the W1 / O / W2 double emulsion to make the final concentration of CaCl2 1%, and the mixture was allowed to stand in a refrigerator at 4°C to form a gel, forming a dense three-dimensional network to fix the double emulsion droplets and inhibit phase separation and leakage, thus obtaining a stable W1 / O / W2 anthocyanin double emulsion gel.

[0117] Example 14 Same as Example 13, except that CaCl2 is added in S3 to make the final concentration of CaCl2 2%.

[0118] Example 15 Same as Example 13, except that CaCl2 is added in S3 to make the final concentration of CaCl2 3%.

[0119] Example 16 Same as Example 13, except that CaCl2 is added in S3 to make the final concentration of CaCl2 4%.

[0120] Example 17 Same as Example 13, except that CaCl2 is added in S3 to make the final concentration of CaCl2 5%.

[0121] Comparative Example 8 Same as Example 13, except that CaCl2 is not added in S3.

[0122] The W1 / O / W2 anthocyanin dual emulsion gels obtained in Examples 13-17 and Comparative Example 8 were characterized by their appearance, moisture retention, loss modulus G”, storage modulus G’, viscosity, solubility, hardness, elasticity, and characterization. The results are shown in the figure. Figure 6 and Figure 7 ,according to Figure 6 It can be seen that, Figure 6 Image A is a visual representation of the emulsion gel, showing that the emulsion gel prepared at a concentration of 3% CaCl2 appears uniform and dense. Figure 6 China B and Figure 6 The values ​​in C show that the emulsion gel prepared with 3% CaCl2 concentration has the highest water retention rate and the strongest viscosity. Figure 6 The D-axis shows that the intermolecular forces in the emulsion gel are mainly ionic bonds, and the ionic bonds are strongest at a 5% concentration of CaCl2. According to... Figure 7 It can be seen that as the calcium salt concentration increases, the gel G” first increases and then decreases, and is always greater than G’ to maintain the gel state.

[0123] The concentration of calcium salts reaches its maximum at 3%, indicating that the emulsion has the strongest gel strength at this concentration. Figure 7(A). Furthermore, viscosity state analysis revealed that the viscosity remained at its highest level at 3%, which corresponds to both. The emulsion gel exhibits a porous structure; at 3%, the gel's microstructure evolves into a high-density, uniform, and continuous three-dimensional network structure with fine pores (…). Figure 7 (B)

[0124] Application Example 1: Stability Analysis Free anthocyanins: 0.5% anthocyanin aqueous solution; Double emulsion: The preparation method is the same as in Example 8, wherein the final mass volume concentration of low methoxyl pectin is 1.5%.

[0125] Emulsion gel group: The preparation method is the same as in Example 15.

[0126] The free anthocyanin group, the double emulsion group, and the emulsion gel group were measured. The specific measurement methods are as follows: Photostability test method: To ensure consistent anthocyanin content across the three sample groups, weigh each group and seal them in stoppered test tubes. Place the samples in a simulated light irradiation device and continuously irradiate them with simulated natural light at a constant temperature for 8 hours. Immediately after the irradiation period, take samples to determine the anthocyanin retention rate in each group to evaluate their photostability.

[0127] Thermal stability test method: To ensure consistent anthocyanin content in the three sample groups, weigh out the samples and seal them in stoppered test tubes. Place the samples in a 65℃ constant temperature water bath in the dark for 30 min (simulating pasteurization conditions). After the reaction, immediately remove the samples and cool them to room temperature in an ice-water bath to terminate the thermal effect. Subsequently, measure the anthocyanin retention rate in each sample group to evaluate its thermal stability.

[0128] See results Figure 8 It can be seen that the three-dimensional structure of the gel network in the emulsion gel group can provide additional physical barriers under high temperature and light, restricting water migration and molecular diffusion, thereby effectively reducing the degradation rate of anthocyanins, reducing the aggregation between emulsion droplets and component migration, and forming a "spatial shielding effect" on active substances, thus effectively reducing the degradation rate of anthocyanins, reducing the release rate of anthocyanins, and improving the retention rate of anthocyanins; when the heat treatment retention rate is 30 min: the retention rate of anthocyanins in the double emulsion group is 83.60±0.24%, and the retention rate of anthocyanins in the emulsion gel group is 88.70±0.22% (…). Figure 8 (A); Retention rate after 8 hours of light exposure: Anthocyanin retention rate in the double emulsion group was 87.30±0.23%, and anthocyanin retention rate in the emulsion gel group was 93.20±0.28% ( Figure 8 (B)

[0129] Application Example 2: Simulated Digestion Analysis Free anthocyanins: 0.5% anthocyanin aqueous solution; Double emulsion: The preparation method is the same as in Example 8, wherein the final mass volume concentration of low methoxyl pectin is 1.5%.

[0130] Emulsion gel: prepared in the same manner as in Example 15.

[0131] Simulated digestion analysis was performed on three treatment groups: the free anthocyanin group, the dual-emulsion group, and the latex gel group. Specifically: Prepare 6 centrifuge tubes, labeled 1, 2, 3, 4, 5, and 6, each containing 0.5g of sample. Centrifuge tubes 1, 2, 3, 4, 5, and 6 correspond to digestion times of 1h, 2h, 3h, 4h, 5h, and 6h, respectively. Digestion time 1-2h simulates the gastric digestion stage, and digestion time 3-6h simulates the intestinal digestion stage.

[0132] Table 1 Simulated Digestive Fluid Formula

[0133] Artificial gastric juice: Weigh porcine pepsin and mix it with simulated gastric juice to make the concentration of porcine pepsin in the mixture 0.2% (w / v), then adjust the pH to 2.5, and shake at 37℃ to obtain artificial gastric juice; the preparation method of simulated gastric juice is shown in Table 1.

[0134] Artificial intestinal fluid: Weigh trypsin and mix with 5% porcine bile salt solution (5% porcine bile salt solution is 53.6 mg / mL porcine bile salt solution, which is obtained by dissolving porcine bile salt in 5 mM, pH 7 PBS buffer) to make the concentration of trypsin in the mixture 1% (w / v), then adjust the pH to 7.0, shake at 37℃ to obtain artificial intestinal fluid.

[0135] See results Figure 9It can be seen that during the simulated gastric digestion stage, the release rate of anthocyanins from both the double emulsion and the emulsion gel was relatively low. After gastric digestion was completed, the release rates of anthocyanins from the double emulsion and the emulsion gel were 46.5% and 24.3%, respectively, indicating a high retention rate of anthocyanins in the stomach. However, during the small intestinal digestion stage, the release rate increased significantly. This may be because the emulsion and gel structures were disrupted under neutral environmental conditions, leading to the release of anthocyanins. During the simulated small intestinal digestion process, pectin was gradually degraded by trypsin, and the network structure was partially disrupted, resulting in the release of some anthocyanins. After small intestinal digestion was completed, the release rate of anthocyanins from both the double emulsion and the emulsion gel reached approximately 70%. This indicates that both the double emulsion and the emulsion gel still have a good protective effect on anthocyanins, with the emulsion gel showing a better protective effect.

[0136] The goal of this invention is to construct a delivery system that is "stable in the gastric phase and controlled intestinal release". This invention controls the release rate during the gastric digestion stage to a low level of 20%-30%, thereby increasing the release rate in the intestine to 40%-50%, protecting anthocyanins so that they can reach the intestinal absorption zone, and achieving the goal of stable gastric phase and intestinal release.

[0137] In summary, this invention prepares zein / high-methoxyl pectin (Zein-HMP) composite particles via an antisolvent precipitation method, and uses them as a natural emulsifier in the external aqueous phase, combining two-step emulsification and Ca²⁺. + An ion-induced gelation process was used to construct a W1 / O / W2 anthocyanin dual emulsion-gel composite delivery system. Compared with emulsion systems stabilized by a single protein or a single polysaccharide, and with non-gelling dual emulsions, the dual emulsion-gel system obtained in this invention not only forms a dense and stable "protein-polysaccharide composite film" at the oil-water interface, but also forms a uniform and continuous three-dimensional gel network structure within the bulk phase. This significantly improves the emulsifying activity and stability of the system, reducing the particle size of the dual emulsion from approximately 18.85 μm to approximately 11.56 μm. Figure 5 The anthocyanin encapsulation rate (A) reaches over 90%, and the water holding capacity of the gel is around 70%. This system exhibits excellent stability under both heat treatment and light irradiation conditions, with a significantly higher anthocyanin retention rate than the dual emulsion of free anthocyanins and ungelled anthocyanins. In in vitro simulations of gastrointestinal digestion, it effectively inhibits rapid release in the gastric segment, achieving slow and controlled release in the small intestine, thereby improving the overall stability and bioavailability of anthocyanins.

[0138] The Zein-HMP composite particle-stabilized anthocyanin dual emulsion-gel system constructed in this invention uses only food-grade raw materials and excipients, exhibiting excellent safety and biocompatibility. It can be flexibly applied as a functional ingredient in various food systems such as yogurt. This multi-level delivery system combines the benefits of "dense interface protection of composite particles and Ca²⁺..." +The dual advantages of "cross-linked three-dimensional gel support" can significantly mitigate the adverse effects of temperature and light changes on anthocyanin degradation during processing and storage, and achieve delayed release and effective delivery in the gastrointestinal tract during digestion. This provides a technical solution with industrial scale-up potential and formulation versatility for developing highly stable and highly available natural pigments and polyphenolic active substances.

[0139] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A dual emulsion gel containing anthocyanins, characterized in that, The anthocyanin dual emulsion gel has a W1 / O / W2 dual emulsion structure; W1 is the inner aqueous phase, O is the oil phase, and W2 is the outer aqueous phase, all of which are gelled; the inner aqueous phase is loaded with anthocyanins; and the outer aqueous phase is loaded with zein-high methoxyl pectin composite particles and low methoxyl pectin.

2. The anthocyanin dual emulsion gel according to claim 1, characterized in that, The preparation method of the zein-high methoxyl pectin composite particles includes: mixing and drying a zein solution with a high methoxyl pectin stock solution to obtain zein-high methoxyl pectin composite particles; the mass ratio of the zein solution to the high methoxyl pectin stock solution is (1-3):(1-3).

3. The anthocyanin dual emulsion gel according to claim 2, characterized in that, The ratio of corn protein to ethanol aqueous solution added during the preparation of the corn gliadin solution is 1g:(50-150)mL; And / or, the ratio of high methoxyl pectin to water added during the preparation of the high methoxyl pectin stock solution is 1:g (50-150)mL; And / or, the mixing method includes high-speed shear homogenization; And / or, the drying method includes freeze drying.

4. The anthocyanin dual emulsion gel according to claim 3, characterized in that, The high-speed shear homogenization process involves a rotation speed of 5000-10000 rpm and a time of 3-7 minutes. And / or, the freeze-drying temperature is -90 to -50°C; And / or, the volume fraction of ethanol in the aqueous ethanol solution is 50%-90%; and / or, the mixture further includes rotary evaporation.

5. The anthocyanin dual emulsion gel according to claim 1, characterized in that, The volume ratio of the inner aqueous phase to the oil phase is (1-5):(5-9); the total volume ratio of the inner aqueous phase and the oil phase to the outer aqueous phase is (1-5):(5-9); the gelation is achieved by calcium salt induction.

6. The anthocyanin dual emulsion gel according to claim 1, characterized in that, The concentration of anthocyanins loaded in the internal aqueous phase is 0.1%-0.9% (w / v). And / or, the oil phase comprises vegetable oil and an emulsifier; optionally, the vegetable oil comprises at least one selected from soybean oil, corn oil, peanut oil, and olive oil; optionally, the emulsifier is selected from at least one selected from Zein-HMP composite particles, polyglycerol ricinoleate, and Tween-80; the emulsifier accounts for 2%-4% of the mass of the vegetable oil; And / or, the final concentration of the external aqueous phase zein-high methoxyl pectin composite particles is 0.5%-2.5% (w / v). And / or, the final concentration of the external aqueous phase low-methoxyl pectin is 0.5%-2.5% (w / v). And / or, the anthocyanin dual emulsion gel also includes calcium salts, the final concentration of which is 1%-5% (w / v).

7. The method for preparing the anthocyanin dual emulsion gel according to any one of claims 1-6, characterized in that, include: S1, mix the inner aqueous phase and the oil phase to prepare the primary emulsion W1 / O; mix zein-high methoxyl pectin composite particles and low methoxyl pectin to obtain the outer aqueous phase solution; S2, the primary emulsion W1 / O and the external aqueous phase solution are mixed to obtain W1 / O / W2 anthocyanin dual emulsion gel.

8. The preparation method according to claim 7, characterized in that, In step S2, the mixture further includes the addition of calcium salt; Optionally, the calcium salt includes at least one of CaCl2, calcium lactate, and calcium carbonate.

9. The use of an anthocyanin dual emulsion gel as described in any one of claims 1-6 or an anthocyanin dual emulsion gel obtained by the preparation method described in claim 7 or 8 as a microcarrier system.

10. The application according to claim 9, characterized in that, The microcarrier system is applied in the fields of food, health products, medicine, or cosmetics, and has a sustained-release delivery function; the product types of the microcarrier system include powder microcapsules.

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