High-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles as well as preparation method and application of high-dispersion-phase aqueous two-phase emulsion

Abstract

The invention relates to the technical field of functional food active substance delivery, in particular to a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles as well as a preparation method and application of the high-dispersion-phase aqueous two-phase emulsion. Chitosan reacts with phthalic anhydride to obtain a phthalic anhydride modified chitosan polymer; inducing the phthalic anhydride modified chitosan polymer to form a phthalic anhydride modified chitosan colloidal particle dispersion liquid; dialyzing the phthalic anhydride modified chitosan colloidal particle dispersion liquid in an aqueous solution, and removing the organic solvent to obtain a phthalic anhydride modified chitosan colloidal particle aqueous dispersion liquid; the preparation method comprises the following steps: dissolving polyethylene glycol in a phthalic anhydride modified chitosan colloidal particle aqueous dispersion to obtain a polyethylene glycol phase, dissolving dextran in ultrapure water to obtain a dextran phase, and mixing the polyethylene glycol phase with the dextran phase to obtain a dextran-polyethylene glycol aqueous two-phase emulsion. The problem of low loading capacity of the traditional emulsion is solved, and meanwhile, the stability of active substances is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of active substance delivery in functional foods, and particularly to a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, a preparation method thereof, and an application thereof. Background Art

[0002] In the contemporary consumer market, with the unprecedented popularization of the concept of healthy diet, the functional food field is facing unprecedented challenges. Active substances represented by vitamins, polyphenols, and flavonoids have significant biological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects. However, in practical applications, they encounter multiple difficulties due to their own physical and chemical properties. For example, the solubility of vitamin D in water is only at the μg / L level, and polyphenolic substances are easily oxidized and degraded in the gastrointestinal environment, resulting in a generally low bioavailability of less than 10%. These problems not only limit the efficacy of functional foods but also restrict the technological upgrading of the industry.

[0003] As an emerging green delivery carrier, aqueous two-phase emulsions (W / W emulsions) have shown great potential in the fields of food, cosmetics, and biomedicine in recent years. This system forms a dispersed structure based on the principle of immiscibility of two aqueous phases, and has unique advantages such as high biocompatibility, no organic solvent residue, and can be stored at room temperature. Compared with traditional oil-in-water emulsions, it can better retain the biological activity of active substances and avoid quality deterioration caused by oil oxidation. However, traditional W / W emulsions have significant technical bottlenecks. Traditional W / W emulsions are usually determined by the volume fraction of the aqueous two-phase system. The dispersed phase loaded with active substances is usually formed by the aqueous phase with a low volume fraction, resulting in limited loading capacity of active substances. Its low loading capacity requires taking a large dose of preparations daily, seriously affecting consumer compliance. Therefore, it is urgent to develop a high-dispersed-phase aqueous two-phase emulsion to increase the volume ratio of the dispersed phase and the encapsulation capacity of high-active substances. Summary of the Invention

[0004] The purpose of the present invention is to provide a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, a preparation method thereof, and an application thereof, which solves the problem of low loading capacity of traditional emulsions and improves the stability of active substances.

[0005] To achieve the above purpose, the present invention provides a preparation method of a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, including the following steps: S1. Prepare a dispersion of amphiphilic chitosan colloidal particles S1.1. Dissolve chitosan (CS) and phthalic anhydride (PA) in an organic solvent, and react under a nitrogen atmosphere to obtain a phthalic anhydride-modified chitosan polymer (PHCS polymer); S1.2. After dissolving the phthalic anhydride-modified chitosan polymer in an organic solvent, slowly add the poor solvent H2O dropwise under magnetic stirring to induce the formation of a dispersion of phthalic anhydride-modified chitosan colloidal particles (PHCS CPs). S2. Prepare a high-dispersion aqueous two-phase emulsion S2.1. After continuously stirring the dispersion of phthalic anhydride-modified chitosan colloidal particles in the mixed solution for 1 h, dialyze to remove the organic solvent in ultrapure water to obtain an aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles (aqueous dispersion of PHCSCPs). S2.2. Dissolve polyethylene glycol (PEG) in the aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles to obtain a polyethylene glycol phase (PEG phase), dissolve dextran (Dex) in ultrapure water to obtain a dextran phase (Dex phase), mix the polyethylene glycol phase and the dextran phase, and homogenize to obtain a dextran-polyethylene glycol aqueous two-phase emulsion, that is, a high-dispersion aqueous two-phase emulsion.

[0006] Preferably, in S1.1, based on the amount of chitosan in terms of the amount of substance of its repeating unit, the molar ratio of the chitosan repeating unit to phthalic anhydride is 1:3 to 1:5, the organic solvent is dimethylformamide, and the chitosan concentration is 0.5 to 8.0 mg / mL.

[0007] In the present invention, the molar ratio of chitosan to phthalic anhydride is controlled within the above range, so that the prepared phthalic anhydride-modified chitosan polymer has a good degree of substitution and appropriate hydrophobic properties, forms a stable adsorption layer at the aqueous two-phase interface, and improves the stability of the aqueous two-phase emulsion.

[0008] Preferably, in S1.1, the reaction conditions are as follows: after heating and stirring to raise the temperature to 110 - 130 °C, react for 7 - 9 h. After the reaction is completed, quickly precipitate the reactant in ice water, then wash with methanol for 0.5 - 1.5 h, filter by suction, and vacuum dry for 20 - 28 h to obtain the phthalic anhydride-modified chitosan polymer.

[0009] Preferably, in S1.2, the organic solvent is dimethylformamide (DMF), and the concentration of the phthalic anhydride-modified chitosan polymer in the organic solvent is 1.0 - 5.0 mg·mL -1 , and the dropping rate of the poor solvent is 10 - 100 μL / min.

[0010] Preferably, in S2.1, the mixed solution is a mixed solution of dimethylformamide and water.

[0011] Preferably, in S2.2, the concentration of polyethylene glycol in the polyethylene glycol phase is 10 - 30 wt%, and the concentration of dextran in the dextran phase is 10 - 30 wt%.

[0012] Preferably, in S2.2, the volume ratio of the dextran phase to the polyethylene glycol phase is 2:1, and the homogenization speed is 4000 - 10000 rpm.

[0013] In the present invention, the volume ratio of the dextran phase to the polyethylene glycol phase is controlled within the above range, so that the prepared aqueous two-phase emulsion has good solubility for the active substance, thereby increasing the loading amount of the active substance. At the same time, it avoids the enhancement of the interaction between solute molecules at high concentrations, which may lead to the degradation or precipitation of the active substance.

[0014] The present invention also provides a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, which is prepared by the preparation method of the above-mentioned high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles.

[0015] The present invention also provides an application of a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles in loading active substances. Before mixing the polyethylene glycol phase and the dextran phase, the active substance is dissolved in the dextran phase, and then the polyethylene glycol phase and the dextran phase dissolved with the active substance are mixed and homogenized to prepare a high-dispersion-phase aqueous two-phase emulsion loaded with the active substance.

[0016] Preferably, the active substance includes one of riboflavin, bovine serum albumin, and probiotics; the concentration of the active substance in the dextran phase is 0.01 - 0.10 mg·mL -1 .

[0017] The mechanism of the present invention: In the present invention, a hydrophobic phthaloyl group is introduced by the amidation reaction of phthalic anhydride and the amino group of chitosan, converting chitosan from hydrophilic to amphiphilic. The modified phthalic anhydride-modified chitosan colloidal particles have both a hydrophobic benzene ring structure and hydrophilic amino / hydroxyl groups, and can be adsorbed directionally at the interface of the dextran (Dex)-ethylene glycol (PEG) aqueous two-phase system to form a stable particle interfacial film.

[0018] The present invention adopts the above-mentioned high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, its preparation method and application, and has the following beneficial effects: (1) The high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles prepared by the present invention has the volume fraction of the dispersed phase increased from 25% of the traditional aqueous two-phase emulsion to 66.7%, and the encapsulation capacity of the active substance is increased by nearly 3 times; (2) In the high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles prepared by the present invention, the hydrophobic modification (substitution degree 1.05 - 1.10) of the phthalic anhydride-modified chitosan colloidal particles increases the three-phase contact angle from 86.5° of chitosan to 136.2°, enhancing the interfacial adsorption ability. The aqueous two-phase emulsion has a phase inversion point (V PEG :VDex = 1:3) forms a bicontinuous phase structure, enhancing its physical stability; (3) The high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles prepared by the present invention is a fully aqueous system without residual organic solvents and is applicable to the delivery of food-grade active substances.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of the synthesis mechanism of the PHCS polymer of the present invention; Figure 2 is an infrared spectrogram of chitosan (CS) and phthalic anhydride-modified chitosan (PHCS) in Example 1 of the present invention; Figure 3 is a nuclear magnetic resonance hydrogen spectrum of phthalic anhydride-modified chitosan (PHCS) in Examples 1-3 of the present invention; Figure 4 is the present invention's CS CPs and PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 schematic diagram of the physical properties of CPs; Figure 4 in which (a) is CS CPs and PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 schematic diagram of the average particle size of CPs, Figure 4 in which (b) is CS CPs and PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 schematic diagram of the Zeta potential of CPs, Figure 4 in which (c) is the SEM image of CSCPs, Figure 4 in which (d) is PHCS 1.08 SEM image of CPs; Figure 5 is the present invention's CS CPs and PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 schematic diagram of the three-phase contact angle of CPs at the interface between Dex solution and PEG solution; Figure 5 in which (a) is the schematic diagram of the three-phase contact angle of CS CPs at the interface between Dex solution and PEG solution, Figure 5 in which (b) is PHCS 1.05 schematic diagram of the three-phase contact angle of CPs at the interface between Dex solution and PEG solution,Figure 5 In (c), it is PHCS 1.08 Schematic diagram of the three-phase contact angle of CPs at the interface between Dex solution and PEG solution, Figure 5 In (d), it is PHCS 1.10 Schematic diagram of the three-phase contact angle of CPs at the interface between Dex solution and PEG solution; Figure 6 They are the CS CPs and PHCS of the present invention 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :V Dex Under the fluorescence microscope images, Figure 6 In (a), it is the aqueous two-phase emulsion of CS CPs at different volume ratios V PEG :V Dex Under the fluorescence microscope images, Figure 6 In (b), it is PHCS 1.05 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :V Dex Under the fluorescence microscope images, Figure 6 In (c), it is PHCS 1.08 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :V Dex Under the fluorescence microscope images, Figure 6 In (d), it is PHCS 1.10 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :V Dex Under the fluorescence microscope images; Figure 7 They are the CS CPs and PHCS of the present invention 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :V Dex Under the optical microscope images, Figure 7 In (a), it is the aqueous two-phase emulsion of CS CPs at different volume ratios V PEG :V Dex Under the optical microscope images, Figure 7 In (b), it is PHCS 1.05 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :V Dex Under the optical microscope images, Figure 7 In (c), it is PHCS 1.08 The aqueous two-phase emulsion of CPs at different volume ratios V PEG :VDex Optical microscope images below, Figure 7 where (d) in is PHCS 1.10 For the CPs aqueous two-phase emulsion at different volume ratios V PEG :V Dex Optical microscope images; Figure 8 are the fluorescence spectra of riboflavin in water, 20 wt% Dex aqueous solution, and D / P HDPEs of the present invention, Figure 8 where (a) in is the fluorescence spectrum of riboflavin in water after different UV irradiation times, Figure 8 where (b) in is a schematic diagram showing the change of the relative fluorescence retention rate of riboflavin in water, 20 wt% Dex aqueous solution, and D / P HDPEs over time. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the field to which the present invention belongs. The above-mentioned features mentioned in the present invention or the features mentioned in the specific examples can be combined arbitrarily. These specific embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.[[ID=**]] [[ID=**]]

[0022] Example 1 The present invention provides a method for preparing a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, comprising the following steps: S1. Prepare a dispersion of amphiphilic chitosan colloidal particles S1.1. Dissolve chitosan in 30 mL of DMF (99.8%) and swell for 12 h. The mass concentration of chitosan in DMF is 5 mg / mL. Then, according to the molar ratio of chitosan repeating units to phthalic anhydride of 1:3, add phthalic anhydride to the DMF solvent in which chitosan is dissolved. Under a nitrogen atmosphere, after dissolving for 40 min, heat and stir to raise the temperature to 120 °C, and then react for 8 h. After the reaction is completed, quickly sediment the reactant in ice water, then wash with methanol for 1 h, filter by suction, and vacuum dry for 24 h to obtain a phthalic anhydride-modified chitosan polymer (PHCS polymer).

[0023] S1.2. Dissolve the phthalic anhydride-modified chitosan polymer in DMF to obtain a PHCS solution with a concentration of -1 4.0 mg·mL

[0024] S2. Prepare a high-dispersed-phase aqueous two-phase emulsion S2.1. Continuously stir the phthalic anhydride modified chitosan colloidal particle dispersion in a mixed solution of DMF and water for 1 h, then dialyze it in ultrapure water for 24 h to remove DMF, and make the volume up to 1.0 mg·mL -1 , to obtain a phthalic anhydride modified chitosan colloidal particle aqueous dispersion (PHCS CPs aqueous dispersion).

[0025] S2.2. Dissolve polyethylene glycol in the phthalic anhydride modified chitosan colloidal particle aqueous dispersion to obtain a polyethylene glycol phase (PEG phase) with a concentration of 20 wt%, dissolve dextran in ultrapure water to obtain a dextran phase (Dex phase) with a concentration of 20 wt%, and mix them in a volume ratio of V Dex :V PEG =2:1. After homogenization at 8000 rpm, a dextran-polyethylene glycol aqueous two-phase emulsion, namely a high-dispersion-phase aqueous two-phase emulsion, is obtained.

[0026] Example 2 The present invention provides a method for preparing a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, comprising the following steps: S1. Prepare an amphiphilic chitosan colloidal particle dispersion S1.1. Disperse chitosan in 30 mL of DMF (99.8%) and swell it for 12 h. The mass concentration of chitosan in DMF is 5 mg / mL. Then, according to the molar ratio of chitosan repeating units to phthalic anhydride of 1:4, add phthalic anhydride to the DMF solvent dissolved with chitosan. Under a nitrogen atmosphere, after dissolving for 40 min, heat and stir to raise the temperature to 120 °C, and then react for 8 h. After the reaction is completed, quickly precipitate the reactant in ice water, then wash it with methanol for 1 h, filter by suction, and vacuum dry for 24 h to obtain a phthalic anhydride modified chitosan polymer (PHCS polymer). The synthesis mechanism is as Figure 1 shown.

[0027] S1.2. Dissolve the phthalic anhydride modified chitosan polymer in DMF to obtain a PHCS solution with a concentration of 4.0 mg·mL -1 . While stirring magnetically, dropwise add the poor solvent H2O at a dropping rate of 50 μL / min to induce the formation of a phthalic anhydride modified chitosan colloidal particle dispersion (PHCS CPs).

[0028] S2. Prepare a high-dispersion-phase aqueous two-phase emulsion S2.1. Continuously stir the phthalic anhydride modified chitosan colloidal particle dispersion in a mixed solution of DMF and water for 1 h, then dialyze it in ultrapure water for 24 h to remove DMF, and make the volume up to 1.0 mg·mL -1 , to obtain a phthalic anhydride modified chitosan colloidal particle aqueous dispersion (PHCS CPs aqueous dispersion).

[0029] S2.2. Dissolve polyethylene glycol in the aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles to obtain a polyethylene glycol phase (PEG phase) with a concentration of 20 wt%, dissolve dextran in ultrapure water to obtain a dextran phase (Dex phase) with a concentration of 20 wt%, and mix them in a volume ratio of V Dex :V PEG = 2:1. After homogenization at 8000 rpm, a dextran-polyethylene glycol aqueous two-phase emulsion, namely a high-dispersion-phase aqueous two-phase emulsion, is obtained.

[0030] Example 3 The present invention provides a method for preparing a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, which includes the following steps: S1. Prepare an amphiphilic chitosan colloidal particle dispersion S1.1. Disperse chitosan in 30 mL of DMF (99.8%) and swell for 12 h. The mass concentration of chitosan in DMF is 5 mg / mL. Then, according to the molar ratio of chitosan repeating units to phthalic anhydride of 1:5, add phthalic anhydride to the DMF solvent in which chitosan is dissolved. Under a nitrogen atmosphere, after dissolving for 40 min, heat and stir to raise the temperature to 120 °C, and then react for 8 h. After the reaction is completed, quickly sediment the reactants in ice water, then wash with methanol for 1 h, filter by suction, and vacuum dry for 24 h to obtain a phthalic anhydride-modified chitosan polymer (PHCS polymer).

[0031] S1.2. Dissolve the phthalic anhydride-modified chitosan polymer in DMF to obtain a PHCS solution with a concentration of 4.0 mg·mL -1 . While stirring magnetically, add the poor solvent H2O dropwise at a dropping rate of 50 μL / min to induce the formation of an aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles (PHCS CPs).

[0032] S2. Prepare a high-dispersion-phase aqueous two-phase emulsion S2.1. Continuously stir the aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles in a mixed solution of DMF and water for 1 h, then dialyze in ultrapure water for 24 h to remove DMF, and make up the volume to 1.0 mg·mL -1 to obtain an aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles (PHCS CPs aqueous dispersion).

[0033] S2.2. Dissolve polyethylene glycol in the aqueous dispersion of phthalic anhydride-modified chitosan colloidal particles to obtain a polyethylene glycol phase (PEG phase) with a concentration of 20 wt%, dissolve dextran in ultrapure water to obtain a dextran phase (Dex phase) with a concentration of 20 wt%, and the volume ratio is V Dex :V PEGMix in a ratio of 2:1 and homogenize at 8000 rpm to obtain a dextran - polyethylene glycol aqueous two - phase emulsion, that is, a high - dispersed - phase aqueous two - phase emulsion.

[0034] Example 4 The present invention provides a method for preparing a high - dispersed - phase aqueous two - phase emulsion based on amphiphilic chitosan colloidal particles, comprising the following steps: S1. Prepare an amphiphilic chitosan colloidal particle dispersion S1.1. Disperse chitosan in 30 mL of DMF (99.8%) and swell for 12 h. The mass concentration of chitosan in DMF is 5 mg / mL. Then, according to the molar ratio of chitosan repeating units to phthalic anhydride of 1:5, add phthalic anhydride to the DMF solvent in which chitosan is dissolved. Under a nitrogen atmosphere, after dissolving for 40 min, heat and stir to raise the temperature to 110 °C and react for 7 h. After the reaction is completed, quickly sediment the reactant in ice water, then wash with methanol for 0.5 h, filter by suction, and vacuum - dry for 20 h to obtain phthalic anhydride - modified chitosan polymer (PHCS polymer).

[0035] S1.2. Dissolve the phthalic anhydride - modified chitosan polymer in DMF to obtain a PHCS solution with a concentration of 3.0 mg·mL -1 , and dropwise add the poor solvent H2O at a dropping rate of 40 μL / min under magnetic stirring to induce the formation of a phthalic anhydride - modified chitosan colloidal particle suspension (PHCS CPs).

[0036] S2. Prepare a high - dispersed - phase aqueous two - phase emulsion S2.1. Continuously stir the phthalic anhydride - modified chitosan colloidal particle dispersion in a mixed solution of DMF and water for 1 h, then dialyze in ultrapure water for 24 h to remove DMF and make up the volume to 1.0 mg·mL -1 , to obtain a phthalic anhydride - modified chitosan colloidal particle aqueous dispersion (PHCS CPs aqueous dispersion).

[0037] S2.2. Dissolve polyethylene glycol in the phthalic anhydride - modified chitosan colloidal particle aqueous dispersion to obtain a polyethylene glycol phase (PEG phase) with a concentration of 15 wt%, dissolve dextran in ultrapure water to obtain a dextran phase (Dex phase) with a concentration of 15 wt%, and mix in a volume ratio of V Dex :V PEG =2:1 and homogenize at 7000 rpm to obtain a dextran - polyethylene glycol aqueous two - phase emulsion, that is, a high - dispersed - phase aqueous two - phase emulsion.

[0038] Example 5 The present invention provides a method for preparing a high - dispersed - phase aqueous two - phase emulsion based on amphiphilic chitosan colloidal particles, comprising the following steps: S1. Preparation of amphiphilic chitosan colloidal particle dispersion S1.1. Dissolve chitosan in 30 mL of DMF (99.8%) and swell for 12 h. The mass concentration of chitosan in DMF is 5 mg / mL. Then, according to the molar ratio of chitosan repeating units to phthalic anhydride of 1:5, add phthalic anhydride to the DMF solvent dissolving chitosan. Under a nitrogen atmosphere, after dissolving for 40 min, heat and stir to raise the temperature to 130 °C and react for 9 h. After the reaction is completed, quickly precipitate the reactant in ice water, then wash with methanol for 1.5 h, filter by suction, and vacuum dry for 28 h to obtain phthalic anhydride-modified chitosan polymer (PHCS polymer).

[0039] S1.2. Dissolve the phthalic anhydride-modified chitosan polymer in DMF to obtain a PHCS solution with a concentration of 5.0 mg·mL -1 . Dropwise add the poor solvent H2O at a dropping rate of 60 μL / min under magnetic stirring to induce the formation of phthalic anhydride-modified chitosan colloidal particle dispersion (PHCS CPs).

[0040] S2. Preparation of high-dispersion aqueous two-phase emulsion S2.1. Continuously stir the phthalic anhydride-modified chitosan colloidal particle dispersion in a mixed solution of DMF and water for 1 h, then dialyze in ultrapure water for 24 h to remove DMF and make up the volume to 1.0 mg·mL -1 to obtain phthalic anhydride-modified chitosan colloidal particle aqueous dispersion (PHCS CPs aqueous dispersion).

[0041] S2.2. Dissolve polyethylene glycol in the phthalic anhydride-modified chitosan colloidal particle aqueous dispersion to obtain a polyethylene glycol phase (PEG phase) with a concentration of 25 wt%. Dissolve dextran in ultrapure water to obtain a dextran phase (Dex phase) with a concentration of 25 wt%. Mix them in a volume ratio of V Dex :V PEG =2:1, and homogenize at a speed of 9000 rpm to obtain dextran-polyethylene glycol aqueous two-phase emulsion, that is, high-dispersion aqueous two-phase emulsion.

[0042] Application Example 1 Apply the high-dispersion aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles prepared in Example 1 to load active substances. The active substance is riboflavin. Specifically:[[]] Before mixing the polyethylene glycol phase and the dextran phase, dissolve riboflavin in the dextran phase. The concentration of riboflavin in the dextran phase is 0.05 mg·mL -1 , and then mix the polyethylene glycol phase and the dextran phase dissolved with the active substance in a volume ratio of V Dex :V PEGMix in a ratio of 2:1 and homogenize at 8000 rpm to prepare a double aqueous phase emulsion (D / P HDPEs) loaded with riboflavin.

[0043] Application Example 2 Apply a high-dispersion-phase double aqueous phase emulsion based on amphiphilic chitosan colloidal particles prepared in Example 2 to load an active substance, where the active substance is bovine serum albumin. Specifically: Before mixing the polyethylene glycol phase and the dextran phase, dissolve bovine serum albumin in the dextran phase, and the concentration of bovine serum albumin in the dextran phase is 0.05 mg·mL -1 , and then mix the polyethylene glycol phase and the dextran phase dissolved with the active substance in a volume ratio of V Dex :V PEG =2:1 and homogenize at 8000 rpm to prepare a double aqueous phase emulsion (D / P HDPEs) loaded with bovine serum albumin.

[0044] Application Example 3 Apply a high-dispersion-phase double aqueous phase emulsion based on amphiphilic chitosan colloidal particles prepared in Example 3 to load an active substance, where the active substance is probiotics. Specifically: Before mixing the polyethylene glycol phase and the dextran phase, dissolve the probiotics in the dextran phase, and the concentration of the probiotics in the dextran phase is 0.10 mg·mL -1 , and then mix the polyethylene glycol phase and the dextran phase dissolved with the active substance in a volume ratio of V Dex :V PEG =2:1 and homogenize at 8000 rpm to prepare a double aqueous phase emulsion (D / P HDPEs) loaded with probiotics.

[0045] Compare the use of traditional double aqueous phase emulsions to load active substances with Application Example 1. In the traditional double aqueous phase emulsion, V Dex :V PEG =1:3.

[0046] After testing, the volume fraction of the active substance loaded in the traditional double aqueous phase emulsion is 25%. The volume fraction of the active substance loaded in the high-dispersion-phase D / P emulsion of Application Example 1 increases to 66.7%.

[0047] Characterization Experiment Characterize the chemical structures of chitosan (CS) and phthalic anhydride-modified chitosan (PHCS) in Example 1 by Fourier transform infrared spectroscopy (FT-IR). The results are as Figure 2 shown. In the infrared spectrum of the PHCS polymer, at 1712 cm -1 and 1775 cm -1The imide absorption peak appears at a certain position, which is formed by the interaction and splitting of two carbonyl groups; the absorption peak at 750 cm -1 is the vibration peak of ortho-disubstituted benzene, indicating the occurrence of phthaloylation reaction. In addition, by comparing the spectra of CS and PHCS polymers, it can be found that the polymer spectra have relatively strong stretching vibration peaks around 158 cm -1 and vibration peaks at 1397 cm N-H , indicating that the reaction mainly occurs on the amino group and the product formed is an amide structure. -1 The v C-N vibration peak at

[0048] The molecular structure of phthalic anhydride modified chitosan (PHCS) in Examples 1 - 3 was characterized by nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR), and the results are as Figure 3 shown. Proton peaks of the chitosan backbone unit structure appear between δ = 3.0 - 5.2 ppm ( δ = 2.50 ppm is for the solvent DMSO-d6); proton absorption peaks of C on the carbon ring appear between δ = 7.2 - 7.7 ppm. Absorption peaks of the oxygen-substituted benzene ring on the phthaloyl group appear between δ = 7.3 - 7.5 ppm; Absorption peaks of the nitrogen-substituted benzene ring on the phthaloyl group appear between

[0049] δ = 7.5 - 7.9 ppm.

[0050] The degree of substitution of PHCS polymer was calculated by the integral ratio of the phthaloyl group of chitosan to the pyranose ring, and the results are shown in Table 1. ;

[0051] As can be seen from Table 1, with the increase of the PA feeding ratio, the degree of substitution of PHCS polymer gradually increases from 1.05 to 1.10, indicating that the higher the degree of substitution of chitosan amino group by phthaloyl group, the stronger the hydrophobic modification degree, ensuring the efficient loading and stable encapsulation of active substances in high-dispersed phase emulsions.

[0052] PHCS 1.05 , PHCS 1.08 and PHCS 1.10 are used to represent the polymers synthesized with the CS to PA feeding ratios of 1:3, 1:4, and 1:5 (i.e., the PHCS polymers in Examples 1 - 3). The unmodified chitosan colloidal particles (CS CPs) and PHCS 1.05CPs, PHCS 1.08 CPs, PHCS 1.10 The physical properties of CPs were characterized, and the results are as Figure 4 shown.

[0053] From Figure 4 in (a), it can be seen that the average particle size of unmodified chitosan colloidal particles is about 402.5 nm, while the average particle diameter of phthalic anhydride-modified chitosan colloidal particles is about 100 nm. PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 The average particle sizes of CPs are 100.7 nm, 109.8 nm, and 121.9 nm respectively, indicating that as the degree of substitution of PHCS increases, the average particle size of its colloidal particles increases synchronously.

[0054] From Figure 4 in (b), it can be known that the Zeta potential of unmodified chitosan is +28.3 mV, and the potentials of PHCS 1.05 , PHCS 1.08 , PHCS 1.10 drop to -10.0 mV, -13.7 mV, and -15.8 mV respectively. With the increase in the degree of hydrophobic modification of chitosan, the introduction of hydrophobic groups and the gradual increase in O-substitution, both the average particle size and the Zeta potential show a downward trend.

[0055] From Figure 4 in (c) and Figure 4 in (d), it can be known that both unmodified chitosan colloidal particles and PHCS 1.08 CPs are spherical in shape. Among them, the particle size of PHCS 1.08 CPs in the dry state is the same as that in the solution, indicating that the structure of phthaloylated chitosan is dense, while the particle size of unmodified chitosan colloidal particles is 198.6 nm, which is much smaller than that in the solution, indicating that unmodified chitosan is in a swollen state in aqueous solution.

[0056] Performance test The wettability at the interface of unmodified chitosan colloidal particles (CS CPs) and PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10 CPs was studied by measuring the three-phase contact angle, and the results are as Figure 5 shown. The three-phase contact angle of unmodified chitosan colloidal particles is 86.5°, and PHCS 1.05 CPs, PHCS 1.08 CPs, PHCS 1.10The three-phase contact angles of CPs are 117.3°, 126.5°, and 136.2° respectively, indicating that the three-phase contact angle increases with the increase of the degree of substitution.

[0057] The influence of different volume ratios (V PEG :V Dex ) of the aqueous two-phase emulsions of chitosan colloidal particles (CS CPs) and phthalic anhydride-modified chitosan colloidal particles (PHCS CPs) on the emulsion type was investigated by fluorescence microscopy (FITC-Dex staining), and the results are as Figure 6 shown.

[0058] As can be seen from (a) in Figure 6 , when the volume ratio V PEG :V Dex of the CS CPs aqueous two-phase emulsion is greater than 1:1, a D / P emulsion (an aqueous two-phase emulsion with the Dex phase as the dispersed phase (inner phase, discontinuous phase) and the PEG phase as the continuous phase (outer phase)) is formed. Conversely, a P / D emulsion (an aqueous two-phase emulsion with the PEG phase as the dispersed phase and the Dex phase as the continuous phase) is formed, indicating that the inversion point of the CS CPs aqueous two-phase emulsion is 1:1. As can be seen from (b) to (d) in Figure 6 , with the increase of the degree of substitution of PHCS CPs, the inversion point of its aqueous two-phase emulsion gradually migrates towards 1:4, which are 1:2, 1:3, and 1:3 respectively, consistent with the results of the three-phase contact angle characterization.

[0059] In addition, when the V 1.08 :V 1.10 of the PHCS PEG CPs aqueous two-phase emulsion and the PHCS Dex CPs aqueous two-phase emulsion are 1:2 and 1:3 respectively, a bicontinuous phase emulsion structure is observed. This indicates that in addition to changing the two-phase volume ratio, the emulsion type of the aqueous two-phase emulsion can also be regulated by adjusting the chemical properties and wettability of the colloidal particles. By regulating the degree of substitution of PHCS CPs, the microstructure and stability of the aqueous two-phase emulsion can be effectively regulated. High-substitution degree (≥1.08) particles break through the volume ratio limitation of traditional emulsions by enhancing interfacial adsorption and inducing a bicontinuous phase, providing a structural basis for the efficient loading of active substances in functional foods.

[0060] The droplet morphology and size changes of the aqueous two-phase emulsions of chitosan colloidal particles (CS CPs) and phthalic anhydride-modified chitosan colloidal particles (PHCS CPs) at different volume ratios (V PEG :V Dex ) were observed by optical microscopy, and the results are as Figure 7 shown.

[0061] As can be seen from Figure 7It can be seen that when the emulsion is at the inversion point, the emulsion droplet size is the largest; for the CS CPs aqueous two-phase emulsion, the droplet size of the P / D emulsion is smaller than that of the D / P emulsion, while the experimental results of the PHCS CPs aqueous two-phase emulsion are opposite to this. This is mainly because the affinity of chitosan for the external aqueous solution is stronger than that of the PHCS polymer, and the experimental results are consistent with the expected results of the air contact angle data. It shows that phthaloylation modification realizes the controllable migration of the inversion point of the aqueous two-phase emulsion and the optimization of the droplet size by adjusting the hydrophobicity of chitosan colloidal particles. High-substitution-degree PHCS CPs (≥1.08) break through the volume ratio limit of traditional emulsions by forming a strong adsorption interfacial film, providing a structural basis for the high loading and stable delivery of active substances in functional foods.

[0062] The photodegradation behaviors of riboflavin in aqueous solution, dextran (Dex) aqueous solution, and high-dispersed-phase aqueous two-phase emulsion (D / P HDPEs, with the polymer being PHCS 1.10 ) were compared by fluorescence spectroscopy and the relative fluorescence intensity retention rate, and the results are as Figure 8 shown.

[0063] As can be seen from (a) in Figure 8 , with the increase of the illumination time, the maximum absorption peak of riboflavin at 533 nm weakens due to the decomposition of riboflavin; when the time is extended to 120 min, the maximum absorption wavelength of riboflavin begins to blue-shift. As can be seen from (b) in Figure 8 , the final fluorescence retention rate of the riboflavin aqueous solution is the lowest, followed by the 20 wt% Dex aqueous solution. However, for the D / P HDPEs with PHCS 1.10 CPs, it slowly decreases within the first 120 min and then remains stable, and the relative fluorescence retention rate of D / P HDPEs is 55% at 480 min. This indicates that D / P HDPEs have a protective effect on delaying the photodegradation of riboflavin. It shows that the high-dispersed-phase aqueous two-phase emulsion significantly improves the photostability of riboflavin through the physical barrier and microenvironment isolation effects of the colloidal particle interfacial film.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, characterized in that, It includes the following steps: S1. Prepare an amphiphilic chitosan colloidal particle dispersion S1.

1. Dissolve chitosan and phthalic anhydride in an organic solvent, and react under a nitrogen atmosphere to obtain a phthalic anhydride-modified chitosan polymer; S1.

2. After dissolving the phthalic anhydride-modified chitosan polymer in an organic solvent, slowly add the poor solvent H2O under magnetic stirring to induce the formation of a phthalic anhydride-modified chitosan colloidal particle dispersion; S2. Prepare a high-dispersion aqueous two-phase emulsion S2.

1. Continuously stir the phthalic anhydride-modified chitosan colloidal particle dispersion in a mixed solution for 1 h, then dialyze in ultrapure water to remove the organic solvent to obtain a phthalic anhydride-modified chitosan colloidal particle aqueous dispersion; S2.

2. Dissolve polyethylene glycol in the phthalic anhydride-modified chitosan colloidal particle aqueous dispersion to obtain a polyethylene glycol phase, dissolve dextran in ultrapure water to obtain a dextran phase, mix the polyethylene glycol phase and the dextran phase, and homogenize to obtain a dextran-polyethylene glycol aqueous two-phase emulsion, that is, a high-dispersion aqueous two-phase emulsion.

2. The preparation method of a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 1, characterized in that: In S1.1, the dosage of chitosan is calculated by the amount of substance of its repeating unit. The molar ratio of the chitosan repeating unit to phthalic anhydride is 1:3 to 1:

5. The organic solvent is dimethylformamide, and the chitosan concentration is 0.5 to 8.0 mg / mL.

3. The preparation method of a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 1, characterized in that: In S1.1, the reaction conditions are as follows: heat and stir to raise the temperature to 110 to 130 °C, then react for 7 to 9 h. After the reaction is completed, quickly precipitate the reactant in ice water, then wash with methanol for 0.5 to 1.5 h, filter by suction, and vacuum dry for 20 to 28 h to obtain the phthalic anhydride-modified chitosan polymer.

4. The preparation method of a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 1, characterized in that: In S1.2, the organic solvent is dimethylformamide, and the concentration of phthalic anhydride-modified chitosan polymer in the organic solvent is 1.0 - 5.0 mg·mL -1 , and the dropping rate of the poor solvent is 10 - 100 μL / min.

5. The preparation method of a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 1, wherein: In S2.1, the mixed solution is a mixed solution of dimethylformamide and water.

6. The preparation method of a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 1, wherein: In S2.2, the concentration of polyethylene glycol in the polyethylene glycol phase is 10 to 30 wt%, and the concentration of dextran in the dextran phase is 10 to 30 wt%.

7. The preparation method of a high-dispersion-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 1, wherein: In S2.2, the volume ratio of the dextran phase to the polyethylene glycol phase is 2:1, and the homogenization speed is 4000 to 10000 rpm.

8. A high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles, characterized in that: It is prepared by the preparation method of a high-dispersion aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles described in any one of claims 1-7.

9. Use of a high-dispersed-phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles as described in claim 8, characterized in that: It is applied to load active substances. Before mixing the polyethylene glycol phase and the dextran phase, dissolve the active substance in the dextran phase, and then mix and homogenize the polyethylene glycol phase and the dextran phase dissolved with the active substance to prepare a high-dispersion aqueous two-phase emulsion loaded with the active substance.

10. Use of a highly dispersed phase aqueous two-phase emulsion based on amphiphilic chitosan colloidal particles according to claim 9, characterized in that: The active substance includes one of riboflavin, bovine serum albumin, and probiotics; the concentration of the active substance in the dextran phase is 0.01 - 0.10 mg·mL -1 .

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