Preparation method of whey protein isolate-acetylated glucan composite carrier steady-state emulsion with glucose responsiveness

Abstract

The invention discloses a preparation method of a WPI-AcDEX composite carrier steady-state emulsion with glucose responsiveness, and belongs to the technical field of emulsion preparation. The method comprises the following steps: S1, carrying out simple reaction on DEX to generate AcDEX; s2, WPI and water are mixed, GOx and CAT are added, and a mixed protein solution is prepared; s3, mixing AcDEX with water to prepare an AcDEX solution, and then mixing the AcDEX solution with the mixed protein solution prepared in the step S2 to prepare a water phase; s4, fat-soluble active substances are added into edible oil, and an oil phase is prepared; and S5, mixing the water phase and the oil phase, and carrying out high-speed dispersion and high-pressure homogenization to obtain the steady-state emulsion. The preparation method disclosed by the invention is simple and convenient to operate, and the prepared emulsion has good emulsion stability, storage stability, relatively high capability of protecting active substances and relatively strong glucose responsiveness.

Description

Technical Field

[0001] This invention belongs to the field of emulsion preparation technology, specifically relating to a method for preparing a glucose-responsive whey protein isolate-acetylated dextran composite carrier stabilized emulsion. Background Technology

[0002] With technological advancements and social development, the pace of life has accelerated. Obesity, high-fat, high-sugar diets, and a lack of exercise have prevented the body from effectively regulating blood sugar, gradually leading to a loss of insulin sensitivity and ultimately hyperglycemia. Therefore, blood sugar control has both social and economic significance. While currently widely used chemical hypoglycemic drugs (such as acarbose and voglibose) are highly effective, frequent injections can easily cause acute hypoglycemia and adverse reactions such as nausea and muscle stiffness. Long-term use of these drugs can also lead to kidney damage, and excessive use may cause hypoglycemia. Dextran (DEX) is a natural polysaccharide mainly composed of α-1,6 glycosidic bonds. Under the catalysis of pyridine p-methylsulfonate, it reacts with 2-methoxypropylene, causing the dangling hydroxyl groups on the dextran to react and form acetal bonds, yielding pH-sensitive acetylated dextran (AcDEX).

[0003] AcDEX is an FDA-approved chemically modified dextran product that typically binds to enzyme systems containing glucose oxidase (GOx) and catalase (CAT) to create a pH-triggered delivery system. When the body's glucose level is high, GOx, which specifically responds to glucose, converts it into gluconolactone and further hydrolyzes it into gluconic acid, thereby lowering the ambient pH. The acetal bonds on AcDEX break rapidly under acidic conditions, dissolving the polysaccharide and achieving a high-speed release of the active ingredient. Therefore, AcDEX possesses biodegradability, sensitivity to acidic pH, high encapsulation efficiency, the ability to chemically bind to the encapsulated material, and the ability to penetrate mucosal layers, demonstrating significant application potential in the precise regulation of blood glucose.

[0004] However, AcDEX has poor physical stability. Current research strategies focus on constructing composite systems of AcDEX and multi-source bioactive components (including proteins, polysaccharides, and polyphenols), but no significant progress has been made so far.

[0005] In addition, Chinese patent CN 120226765 A discloses a polysaccharide-protein complex stable high bioavailability ferrous microemulsion and its preparation method and application, which uses whey protein isolate and polysaccharide for compounding, but it does not conduct research on AcDEX.

[0006] Chinese patent CN 111772028 A discloses a method for preparing a protein ingredient with high oil absorption, high emulsification, and low-temperature gelation. This method uses horseradish peroxidase and glucose oxidase to cross-link whey protein, resulting in a novel protein ingredient with high oil absorption, high emulsification, and low-temperature gelation. However, the enzymes used in this patent operate on a different principle than those in this invention, and the polysaccharide used is different from AcDEX and lacks pH sensitivity. Summary of the Invention

[0007] Technical issues AcDEX possesses both pH sensitivity and biodegradability. Under the combined action of GOx and CAT, it reacts glucose to form glucuronic acid, causing the pH to drop. This breaks the acetal bonds on AcDEX, releasing active substances that regulate blood sugar, thus achieving precise blood sugar control. However, due to its inherent instability, it may not exert its maximum effect when used alone. Therefore, it is necessary to explore composite systems composed of AcDEX and multi-source bioactive components to improve its stability.

[0008] Technical solution To address the aforementioned issues, this invention employs whey protein isolate (WPI) as a functional carrier, as its nutritional and functional properties make it a commonly used stabilizer and emulsifier in emulsion preparation. Through electrostatic interactions, hydrophobic interactions, and hydrogen bonding, WPI can form a stable complex with AcDEX, significantly improving the emulsification stability of the system. Furthermore, using WPI and AcDEX as a shell to encapsulate volatile, low-water-soluble, and poorly stable lipid-soluble active substances with blood sugar-regulating functions (such as benzyl isothiocyanate (BITC)) can achieve precise delivery of active ingredients, thereby optimizing their bioavailability and better exerting their blood sugar control effect. This innovative technical approach of this invention also provides new possibilities for the development of precisely released functional foods.

[0009] The first objective of this invention is to provide a stabilized emulsion using WPI and AcDEX as a composite carrier, wherein the stabilized emulsion contains 0.8-1 wt% WPI, 0.02-0.3 wt% AcDEX, 0.01-0.03 wt% GOx, 0.01-0.03 wt% CAT, 9-10 wt% edible oil, and 89-90 wt% water.

[0010] In one embodiment of the present invention, the stabilized emulsion is composed of 0.8-1 wt% WPI, 0.02-0.3 wt% AcDEX, 0.01-0.03 wt% GOx, 0.01-0.03 wt% CAT, 9-10 wt% edible oil and 89-90 wt% water.

[0011] Preferably, the stabilized emulsion is composed of 0.8-1 wt% WPI, 0.1-0.15 wt% AcDEX, 0.01-0.03 wt% GOx, 0.01-0.03 wt% CAT, 9-10 wt% edible oil and 89-90 wt% water.

[0012] As one embodiment of the present invention, the edible oil includes one or more of corn oil, peanut oil, sunflower seed oil, soybean oil, and rapeseed oil.

[0013] As one embodiment of the present invention, the stabilized emulsion may further contain 0.04~0.06 wt% of fat-soluble active substances.

[0014] As one embodiment of the present invention, the fat-soluble active substances include one or more of benzyl isothiocyanate, proanthocyanidins, carotenoids, phytosterols, and polyunsaturated fatty acids.

[0015] In one embodiment of the present invention, the enzyme activity of GOx is 100~500 u / mg.

[0016] In one embodiment of the present invention, the enzyme activity of CAT is 3000~5000 u / mg.

[0017] The second objective of this invention is to provide a method for preparing a stabilized emulsion using WPI and AcDEX as a composite carrier, comprising the following steps: S1. Preparation of protein solution: Mix WPI with water and stir to prepare a protein solution; S2, Aqueous phase preparation: Mix AcDEX with water to prepare an AcDEX solution, then add GOx and CAT to obtain a mixed solution. Then mix the mixed solution with the protein solution prepared in step S2 and stir to prepare an aqueous phase. S3. Oil phase preparation: Use edible oil as the oil phase; S4. Mixing: The aqueous phase prepared in step S2 is mixed with the oil phase prepared in step S3, dispersed at high speed, and then homogenized under high pressure to obtain a stabilized emulsion.

[0018] In one embodiment of the present invention, the stirring time in step S1 is 1 to 3 hours.

[0019] In one embodiment of the present invention, in step S1, the concentration of WPI in the protein solution is 2-5 wt%.

[0020] In one embodiment of the present invention, in step S2, the concentration of AcDEX in the mixed solution is 0.5~1wt%.

[0021] In one embodiment of the present invention, in step S2, the concentration of GOx in the mixed solution is 0.02~0.05wt%.

[0022] In one embodiment of the present invention, in step S2, the concentration of CAT in the mixed solution is 0.02~0.05wt%.

[0023] In one embodiment of the present invention, the stirring time in step S2 is 1 to 5 hours.

[0024] In one embodiment of the present invention, in step S3, the edible oil includes one or more of corn oil, peanut oil, sunflower seed oil, soybean oil, and rapeseed oil.

[0025] In one embodiment of the present invention, in step S4, the volume ratio of the oil phase to the water phase is 1:4 to 1:15.

[0026] In one embodiment of the present invention, in step S4, the parameters for high-speed dispersion are 10000~14000 r / min and the time is 2~4 min.

[0027] In one embodiment of the present invention, in step S4, the parameters of high-pressure homogenization are 5000~10000 psi and the number of times is 6~8.

[0028] In one embodiment of the present invention, in step S4, the final concentration of WPI in the stabilized emulsion is 0.8~1wt%.

[0029] In one embodiment of the present invention, in step S4, the final concentration of AcDEX in the stabilized emulsion is 0.02~0.3wt%.

[0030] In one embodiment of the present invention, in step S4, the final concentration of GOx in the stabilized emulsion is 0.01~0.03wt%.

[0031] In one embodiment of the present invention, in step S4, the final concentration of CAT in the stabilized emulsion is 0.01~0.03wt%.

[0032] In one embodiment of the present invention, in step S4, the final concentration of edible oil in the stabilized emulsion is 9-10 wt%.

[0033] In one embodiment of the present invention, in step S4, the final concentration of water in the stabilized emulsion is 89-90 wt%.

[0034] In one embodiment of the present invention, in step S3, a fat-soluble active substance may be added to the edible oil.

[0035] In one embodiment of the present invention, in step S3, the fat-soluble active substance includes one or more of benzyl isothiocyanate, proanthocyanidins, carotenoids, phytosterols, and polyunsaturated fatty acids.

[0036] In one embodiment of the present invention, in step S3, the mass concentration of the fat-soluble active substance in the oil phase is 1~10 mg / mL.

[0037] In one embodiment of the present invention, in step S4, the final concentration of the fat-soluble active substance in the stabilized emulsion is 0.04~0.06wt%.

[0038] The present invention provides a stabilized emulsion prepared based on the above method.

[0039] The application of the stabilized emulsion provided by this invention in the food industry.

[0040] The present invention also provides the application of the above-mentioned stabilized emulsion in the field of health products.

[0041] The beneficial effects of this invention are: (1) The composite carrier of WPI and AcDEX prepared in this invention can significantly improve the stability of the emulsion through the interaction between the two and improve the utilization rate of AcDEX. When the blood glucose concentration increases, under the action of GOx and CAT, the pH in the body decreases and the acetal bond of AcDEX breaks, thereby realizing the precise release of fat-soluble active substances.

[0042] (2) The emulsion interface of the present invention has high stability. When the concentration of AcDEX is 0.15%, the angle of the oil-water interface tends to the critical value of -90°, and the prepared oil-in-water emulsion is the most stable. Furthermore, after measuring the average particle size, Zeta potential, appearance structure, and oil droplet distribution of the WPI and AcDEX composite emulsion stored at 4 °C for 14 days, it was found that the composite system did not undergo obvious stratification during storage, and the Zeta potential changed little throughout the storage process. Figure 13 This indicates that the emulsion has good storage stability; finally, when the AcDEX concentration is 0.15%, the BITC retention rate and glucose responsiveness are the highest. p The concentration of glucose precipitate (<0.05) indicates that the composite emulsion possesses high bioactive substance (BITC) protection capability and strong glucose responsiveness. In summary, the emulsion of this invention exhibits multiple superior effects and is a product with broad market application prospects. Attached Figure Description

[0043] Figure 1 This is the 1H NMR spectrum of AcDEX in this invention; Figure 2This is a graph showing the cytotoxicity assay of AcDEX in this invention (different lowercase letters indicate significant differences between groups). p <0.05)); Figure 3 These are the ultraviolet spectra of Comparative Example 1 and Examples 1-6 in this invention; Figure 4 These are the fluorescence spectra of Comparative Example 1 and Examples 1-6 in this invention; Figure 5 These are the X-ray diffraction patterns of Comparative Example 1 and Examples 1-6 in this invention; Figure 6 These are the Fourier transform infrared spectra of Comparative Example 1 and Examples 1-6 in this invention; Figure 7 This is a diagram showing the measurement of the contact angle in Comparative Example 1 of this invention; Figure 8 This is a diagram showing the measurement of the contact angle in Example 1 of this invention; Figure 9 This is a diagram showing the measurement of the contact angle in Example 2 of this invention; Figure 10 This is a diagram showing the contact angle measurement in Example 3 of this invention; Figure 11 This is a diagram showing the measurement of the contact angle in Example 4 of this invention; Figure 12 This is a diagram showing the contact angle measurement in Example 5 of this invention; Figure 13 This is a diagram showing the contact angle measurement in Example 6 of this invention; Figure 14 This is a particle size change graph of Comparative Examples 2 and Examples 7-12 of this invention after being stored at 4°C for 0, 1, 4, 7, and 14 days (different lowercase letters represent significant differences between different series at the same number of days, and different uppercase letters represent significant differences between the same series at different numbers of days). p <0.05)); Figure 15 This is a potential change graph of Comparative Examples 2 and Examples 7-12 of this invention stored at 4°C for 0, 1, 4, 7, and 14 days (different lowercase letters represent significant differences between different series at the same number of days, and different uppercase letters represent significant differences between the same series at different numbers of days). p <0.05)); Figure 16 These are actual images of Comparative Examples 2 and Examples 7-12 of this invention stored at 4°C for 0 days; Figure 17 These are actual images of Comparative Examples 2 and Examples 7-12 of this invention stored at 4°C for 1 day; Figure 18These are actual images of Comparative Examples 2 and Examples 7-12 of this invention stored at 4°C for 4 days; Figure 19 These are actual images of Comparative Examples 2 and Examples 7-12 of this invention stored at 4°C for 7 days; Figure 20 These are actual images of Comparative Examples 2 and Examples 7-12 stored at 4°C for 14 days in this invention; Figure 21 These are fluorescence microscope images of Comparative Examples 2 and Examples 7-12 of this invention stored at 4 °C for 0 days; Figure 22 These are fluorescence microscope images of Comparative Examples 2 and Examples 7-12 of this invention stored at 4°C for 14 days; Figure 23 This is a graph showing the encapsulation rate of BITC stored at 4°C for 0 days and 14 days in Comparative Examples 2 and Examples 7-12 of this invention. (Different uppercase letters represent significant differences between different series at the same number of days, and different lowercase letters represent significant differences between the same series at different numbers of days.) p <0.05)); Figure 24 These are glucose response measurement graphs for Comparative Example 2 and Examples 7-12 in this invention (different lowercase letters indicate significant differences between groups). p <0.05). Detailed Implementation

[0044] Source of raw materials Dextran (DEX) was purchased from Sigma with a molecular weight of 70,000; glucose oxidase was purchased from Maclean's with an enzyme activity of 100 u / mg; catalase was purchased from Maclean's with an enzyme activity of 3500 u / mg; other materials and reagents used in the following examples were commercially available unless otherwise specified.

[0045] Preparation of AcDEX: 1 g of DEX was placed in a three-necked flask and purged with N2 to obtain a dry powder. The powder was mixed with 10 mL of anhydrous dimethyl sulfoxide and stirred to obtain a clear solution. 15.6 mg of p-toluenesulfonic acid pyridine and 3.4 mL of dimethoxypropylene were added and reacted for 1 to 1.5 h. 1 mL of triethylamine was added to quench the reaction. The mixture was rinsed several times with water, and the remaining solid was freeze-dried to obtain AcDEX.

[0046] Comparative Example 1 S1. Prepare a WPI solution with a mass concentration of 1%. Add 0.012% GOx and 0.0017% CAT to the WPI solution and stir with a magnetic stirrer at room temperature for 1 h to obtain a composite WPI emulsion.

[0047] Comparative Example 2 S1. Preparation of protein solution: Prepare a 1% WPI solution, add 0.012% GOx and 0.0017% CAT to the WPI solution, and stir with a magnetic stirrer at room temperature for 1 h to obtain a composite WPI emulsion. S2. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase. S3. Mix the oil phase prepared in step S2 with the composite WPI solution prepared in step S1 at a volume ratio of 1:10 and disperse at 12000r / min for 2 min. S4. The emulsion dispersed at high speed in S3 was then homogenized under high pressure at 7252 psi six times to obtain the control group emulsion with only WPI.

[0048] Comparative Example 3 S1. Preparation of protein solution: Prepare a fish skin gelatin (FSG) solution with a mass concentration of 2% and stir it thoroughly with a magnetic stirrer for 1 h at room temperature to obtain the FSG solution; S2. Preparation of aqueous phase: Prepare an AcDEX solution with a mass concentration of 0.5%, add GOx with a mass concentration of 0.024% and CAT with a mass concentration of 0.0034%, and stir thoroughly until completely dissolved; mix the FSG solution prepared in step S1, the mixed solution prepared in step S2 and deionized water in a certain proportion, stir for 2 h, and prepare a solution with a final FSG concentration of 1 wt%, a final AcDEX concentration of 0.025 wt%, a final GOx concentration of 0.012 wt%, and a final CAT concentration of 0.017 wt%, which is the aqueous phase.

[0049] S3. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase. S4. Mix the oil phase prepared in step S3 with the aqueous phase prepared in step S2 at a volume ratio of 1:10 and disperse at 12000 r / min for 2 min. S5. The emulsion dispersed at high speed in S4 is then homogenized at 7252 psi for 6 times to obtain an emulsion with WPI and AcDEX as composite carriers.

[0050] After 14 days of storage, severe stratification was observed on the surface of the resulting emulsion, indicating that FSG is not conducive to improving the stability of AcDEX.

[0051] Comparative Example 4 S1. Preparation of protein solution: Prepare a 2% (w / w) pea protein isolate (PPI) solution and stir it thoroughly with a magnetic stirrer at room temperature for 1 h to obtain the PPI solution; S2. Preparation of aqueous phase: Prepare an AcDEX solution with a mass concentration of 0.5%, add GOx with a mass concentration of 0.024% and CAT with a mass concentration of 0.0034%, and stir thoroughly until completely dissolved; mix the solution prepared in step S1, the mixed solution prepared in step S2, and deionized water in a certain proportion, stir for 2 h, and prepare a solution with a final concentration of 1 wt%, a final concentration of AcDEX of 0.025 wt%, a final concentration of GOx of 0.012 wt%, and a final concentration of CAT of 0.017 wt%, which is the aqueous phase.

[0052] S3. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase. S4. Mix the oil phase prepared in step S3 with the aqueous phase prepared in step S2 at a volume ratio of 1:10 and disperse at 12000 r / min for 2 min. S5. The emulsion dispersed at high speed in S4 is then homogenized at 7252 psi for 6 times to obtain an emulsion with WPI and AcDEX as composite carriers.

[0053] After 14 days of storage, the resulting emulsion showed severe stratification on the surface, indicating that PPI is not conducive to improving the stability of AcDEX.

[0054] Comparative Example 5 S1. Preparation of protein solution: Prepare an ovalbumin (OVA) solution with a mass concentration of 2% and stir it thoroughly with a magnetic stirrer at room temperature for 1 h to obtain the OVA solution; S2. Preparation of aqueous phase: Prepare an AcDEX solution with a mass concentration of 0.5%, add GOx with a mass concentration of 0.024% and CAT with a mass concentration of 0.0034%, and stir thoroughly until completely dissolved; mix the solution prepared in step S1, the mixed solution prepared in step S2, and deionized water in a certain proportion, stir for 2 h, and prepare a solution with a final concentration of 1 wt%, a final concentration of AcDEX of 0.025 wt%, a final concentration of GOx of 0.012 wt%, and a final concentration of CAT of 0.017 wt%, which is the aqueous phase.

[0055] S3. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase. S4. Mix the oil phase prepared in step S3 with the aqueous phase prepared in step S2 at a volume ratio of 1:10 and disperse at 12000 r / min for 2 min. S5. The emulsion dispersed at high speed in S4 is then homogenized at 7252 psi for 6 times to obtain an emulsion with WPI and AcDEX as composite carriers.

[0056] After 14 days of storage, the resulting emulsion showed severe stratification on the surface, indicating that OVA is not conducive to improving the stability of AcDEX.

[0057] Example 1 S1. Preparation of protein solution: Prepare a 2% WPI solution and stir it thoroughly with a magnetic stirrer at room temperature for 1 hour to obtain the WPI solution. S2. Preparation of aqueous phase: Prepare an AcDEX solution with a mass concentration of 0.5%, add GOx with a mass concentration of 0.024% and CAT with a mass concentration of 0.0034%, and stir thoroughly until completely dissolved; mix the WPI solution prepared in step S1, the mixed solution prepared in step S2 and deionized water in a certain proportion, stir for 2 h, and prepare a solution with a final WPI concentration of 1 wt%, a final AcDEX concentration of 0.025 wt%, a final GOx concentration of 0.012 wt%, and a final CAT concentration of 0.017 wt%, which is the aqueous phase.

[0058] Example 2 The difference from Example 1 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.05 wt%.

[0059] Example 3 The difference from Example 1 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.1 wt%.

[0060] Example 4 The difference from Example 1 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.15 wt%.

[0061] Example 5 The difference from Example 1 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.2 wt%.

[0062] Example 6 The difference from Example 1 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.25 wt%.

[0063] Example 7 S1. Preparation of protein solution: Prepare a 2% WPI solution and stir it thoroughly with a magnetic stirrer at room temperature for 1 hour to obtain a mixed WPI solution. S2. Aqueous phase preparation: Prepare an AcDEX solution with a mass concentration of 0.5%, add GOx with a mass concentration of 0.024% and CAT with a mass concentration of 0.0034%, and stir thoroughly until completely dissolved; mix the WPI solution prepared in step S1, the mixed solution prepared in step S2, and deionized water in a certain proportion, stir for 2 hours, and prepare a solution with a final WPI concentration of 1 wt%, a final AcDEX concentration of 0.025 wt%, a final GOx concentration of 0.012 wt%, and a final CAT concentration of 0.017 wt%, which is the aqueous phase. S3. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase. S4. Mix the oil phase prepared in step S3 with the aqueous phase prepared in step S2 at a volume ratio of 1:10 and disperse at 12000 r / min for 2 min. S5. The emulsion dispersed at high speed in S4 is then homogenized at 7252 psi for 6 times to obtain an emulsion with WPI and AcDEX as composite carriers.

[0064] Example 8 The difference from Example 7 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.05 wt%.

[0065] Example 9 The difference from Example 7 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.1 wt%.

[0066] Example 10 The difference from Example 7 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.15 wt%.

[0067] Example 11 The difference from Example 7 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.2 wt%.

[0068] Example 12 The difference from Example 7 is that the final concentration of AcDEX in the aqueous phase obtained in step S2 is 0.25 wt%.

[0069] Test methods 1H NMR spectroscopy Weigh 10-20 mg of the dried powder after the reaction and dissolve it completely in deuterated dimethyl sulfoxide to obtain a clear and transparent solution. Then, place the solution in an NMR tube for 1H NMR spectroscopy.

[0070] Cytotoxicity assayLogarithmic growth phase Caco-2 cells were digested and a single-cell suspension was prepared, with a cell concentration of 1×10⁻⁶. 5 The prepared cell suspension was seeded into the required well plates and slowly placed in an incubator for culture. After the cells had completely adhered to the plate, the cytotoxicity was determined using the CCK-8 assay.

[0071] Ultraviolet fluorescence spectrum Comparative Example 1 and Examples 1-6 were diluted 8-fold. Ultraviolet (UV) spectroscopy analysis was performed using a UV spectrometer, with all samples scanned in the wavelength range of 200-700 nm.

[0072] Fluorescence spectroscopy determination Comparative Example 1 and Examples 1-6 were diluted 3 times. The fluorescence spectra of the samples were measured using a fluorescence spectrometer with a scanning range of 300-500 nm, an excitation wavelength of 280 nm, and a scan rate of 50 nm / min.

[0073] Infrared spectroscopy measurement Fourier transform infrared spectroscopy was used to measure the interactions of Comparative Example 1 and Examples 1-6. 10 mg of sample was mixed with dry potassium bromide powder at a 100-fold ratio and ground to prepare thin films. Wavenumbers were measured in the range of 4000–400 cm⁻¹. -1 The scan.

[0074] X-ray diffraction measurement Comparative Example 1 and Examples 1-6 lyophilized powder samples were uniformly filled into the sample cell of the X-ray diffractometer. The diffraction angle was 10-50° at a voltage of 40 kV and a current of 40 mA. The scanning speed was set to 10 ° / min.

[0075] Determination of contact angle of composite system Comparative Example 1 and Examples 1-6 lyophilized powder samples were prepared into circular sample pieces with a thickness of 1-2 mm in a tableting tank. Different sample pieces were immersed in corn oil and thoroughly oil-bathed. After being removed, they were placed on a glass slide. Ultrapure water was slowly added to the oil-bathed sample piece using the seated drop method. The angle between the droplet and the plane was measured by photographing the sample. The specific analytical basis is that a contact angle of 90° is the critical value for amphiphilicity. When the contact angle is greater than the critical value, the oil-water interface tends to be hydrophobic, and vice versa. When the contact angle approaches the critical value, the prepared oil-in-water emulsion is the most stable.

[0076] Observation of the appearance and structure of the emulsion Appropriate amounts of the emulsions prepared in Examples 7-12 and the emulsion in Comparative Example 2 (stored at 4 ℃ for 0 days, 1 day, 4 days, 7 days, and 14 days, respectively) were taken and their appearance and structure were observed. The specific analysis was based on the fact that the appearance of the emulsions did not change significantly, indicating strong system stability.

[0077] Emulsion Particle Size and Potential MeasurementA suitable amount of the emulsions prepared in Examples 7-12 were diluted 200 times with the emulsion in Comparative Example 2 (stored at 4 ℃ for 0 days, 1 day, 4 days, 7 days, and 14 days, respectively). The average particle size and zeta potential were then measured using a nanoparticle size analyzer. Specific analysis and theoretical basis include: (1) The smaller the average particle size of the emulsion, the more stable the system.

[0078] (2) The larger the absolute value of the emulsion potential, the more stable the system.

[0079] Observation of oil droplet distribution in emulsion Appropriate amounts of the emulsions prepared in Examples 7-12 and Comparative Example 2 (stored at 4 ℃ for 0 days and 14 days respectively) were taken. WPI and AcDEX were labeled with fluorescein isothiocyanate as wall materials, and corn oil was labeled with Nile Red as the core material. The dyes and emulsion samples were mixed evenly at a ratio of 1:25. 5 μL of the stained sample was placed on a glass slide, and images of each sample were obtained using a fluorescence microscope. The specific analytical basis was that the more uniform the oil droplet distribution in the fluorescence microscope image, the more stable the system.

[0080] BITC retention rate determination Take appropriate amounts of the product emulsions prepared in Examples 7-12 and the emulsion of Comparative Example 2 (stored at 4 ℃ for 0 days and 14 days), and extract BITC from the emulsions using n-hexane and methanol via vortex extraction. Finally, analyze the BITC content by high performance liquid chromatography. Based on the positive correlation between the concentration of BITC in the solution and the peak content, calculate the retention rate of BITC in the emulsion: Retention rate (%) = Peak area of ​​BITC in the emulsion / Peak area of ​​BITC in the standard.

[0081] Glucose Response Assay When glucose concentration increases, glucose oxidase reacts glucose to form glucuronic acid. The pH decreases, causing the acetal bonds in AcDEX to break, resulting in larger nanoparticles. 4 mg / mL glucose was mixed with 1 mL each from Examples 7-12 and Comparative Example 2, stirred for 4 h, diluted 200 times, and the average particle size was measured using a nanoparticle size analyzer.

[0082] Results Analysis This invention constructs emulsions simultaneously encapsulating BITC using pure WPI as a carrier and different proportions of WPI-AcDEX composite carriers. The study found that: After modification, DEX exhibits characteristic peaks at corresponding chemical shifts for the methyl hydrogen atom on the acetyl group, with new peaks appearing at δ 1.39 and 3.45 ppm. Figure 1 This indicates that the DEX has been modified into AcDEX.

[0083] Figure 2This is a cytotoxicity assay for AcDEX. As shown in the figure, cell viability is high at lower concentrations (0.1–0.4%), indicating that AcDEX has a relatively small impact on cells within this concentration range. Cell viability decreases significantly with increasing concentration, reaching its lowest point at a concentration of 0.5%.

[0084] All samples showed peaks around 280 nm. Figure 3 WPI contains tryptophan and tyrosine. With increasing AcDEX concentration, the maximum UV absorption peak of WPI gradually increases, exhibiting a hyperchromic effect accompanied by a red shift. The addition of AcDEX causes a conformational change in the WPI protein, exposing the previously embedded aromatic amino acids. This phenomenon indicates an interaction between WPI and AcDEX. Compared to Comparative Example 1, the position of the maximum absorption peak at different AcDEX concentrations shows a slight red shift after the addition of AcDEX. Figure 4 The results were consistent with those obtained using UV light, and the fluorescence intensity decreased, indicating a dose-response relationship. This suggests that WPI interacted with AcDEX, resulting in a change in its tertiary conformation. This may be due to the alteration of the microenvironment for tryptophan and tyrosine in WPI following the addition of AcDEX.

[0085] Figure 5 The X-ray diffraction pattern shows that all samples exhibit peaks around 20°, indicating the presence of a β-plate-like structure. Furthermore, the peak value decreases after WPI is combined with AcDEX. With the addition of different concentrations of AcDEX, the diffraction peak intensity first decreases and then increases, indicating that the addition of AcDEX alters the tertiary conformation of WPI, causing rearrangement of larger aggregates and the formation of a more stable structure. After the addition of AcDEX, the WPI-AcDEX complex system exhibits a peak in the amide A band (3100–3500 cm⁻¹). -1 The characteristic peaks of unsaturated carbon (CH stretching) underwent varying degrees of vibrational shift. Figure 6 These changes are due to the formation of hydrogen bonds through condensation between the functional groups of WPI and AcDEX. The WPI-AcDEX complex system is located in the amide I band (1600~1700 cm⁻¹). -1 The characteristic peak of WPI shifts to different degrees with increasing AcDEX concentration, indicating an electrostatic interaction between WPI and AcDEX.

[0086] Contact angle ( Figures 7-13The results showed that, compared with Comparative Example 1, the contact angles of Examples 1-6 all increased, exhibiting a trend of first increasing and then decreasing with increasing AcDEX concentration. This is attributed to the binding of proteins with low-concentration polysaccharides, leading to the exposure of hydrophobic amino acids in the WPI. This, in turn, increases the affinity of the particles for oil, enhancing their amphiphilicity and thus increasing the contact angle. Example 4 showed the largest contact angle, closest to 90°. When excessive polysaccharides bind to proteins, the hydrophobic interaction between them is enhanced, resulting in an overall hydrophobic state and a decrease in the contact angle. These results indicate that an AcDEX concentration of 0.15% can alter the oil-water interface adsorption capacity of the system and improve the stability of the WPI-AcDEX complex. Depend on Figure 14 It can be seen that as the concentration of AcDEX increases, the particle size of Examples 7-12 shows a trend of first decreasing and then increasing. In Example 12, when AcDEX was added in excess, the strong electrostatic repulsion led to the formation of an electrically neutral complex, which further inhibited the protein-polysaccharide molecule interaction, resulting in an increase in particle size. Compared with Comparative Example 2 and Examples 7-12 after 0 days of storage, the average particle size of Comparative Example 2 and Examples 7-12 did not change significantly after 14 days of storage, while Comparative Example 10 had a smaller particle size and a larger absolute potential value after 14 days of storage, and there was no significant difference. Figure 15 This indicates that the emulsion corresponding to Example 10 is the most stable.

[0087] Figures 16-20 The image shows the appearance and structure of the WPI-AcDEX composite emulsion stored at 4 °C for 14 days. The results show that all samples remained stable during these 14 days, with good appearance and structure, and no stratification, flocculation or precipitation occurred, demonstrating excellent physical stability.

[0088] Fluorescence microscopy revealed that during storage at 4 ℃ for 14 days ( Figures 21-22 No large-scale oil droplet aggregation was observed in any of the samples; the distribution of emulsified oil droplets was uniform. Compared to Comparative Example 2 and Examples 7-11, the emulsified oil droplets in Example 12 were relatively larger. After 14 days of storage, the microstructure size of all compound emulsions increased compared to freshly prepared emulsions. This result is consistent with the trend of emulsion particle size change. Figure 14 ).

[0089] Depend on Figure 23 It can be seen that the BITC encapsulation rate on the first day of storage in Example 10 was 93.74 ± 5.90%, which was significantly higher than that of other compound concentrations ( p <0.05), after 14 days of storage, the highest BITC encapsulation rate (72.19 ± 0.85%) was observed in the compound emulsion with AcDEX concentration of 0.15% (Example 10), indicating that the BITC encapsulation effect of Example 10 was optimal after 14 days of storage.

[0090] Compared with Comparative Example 2, the average particle size of the compound systems in Examples 6-10 first increased and then decreased. Figure 24 The reason for this is that when the glucose concentration increases, GOx reacts with glucose to form glucuronic acid, causing the pH to drop. The acetal bonds of AcDEX decompose in acid, leading to the rupture of the nanoemulsion and an increase in particle size. In Example 10, when the AcDEX concentration was 0.15%, the emulsion rupture was significant, indicating the most pronounced glucose responsiveness. When the AcDEX concentration is too high, it forms large, irregular, and highly cross-linked aggregates with WPI. These aggregates have a very dense structure, making it difficult for glucose molecules to diffuse into their interior. This prevents the internal AcDEX from being effectively triggered by glucose hydrolysis, resulting in a decrease in glucose responsiveness.

[0091] The results above indicate that, under the condition of 1% WPI and 0.15% AcDEX compounding, the compound emulsion has good storage stability, high bioactive substance (BITC) protection ability and strong glucose responsiveness.

[0092] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A stabilized emulsion characterized in that, The steady emulsion comprises 0.8-1 wt% of whey protein isolate, 0.02-0.3 wt% of acetylated dextran, 0.01-0.03 wt% of glucose oxidase, 0.01-0.03 wt% of catalase, 9-10 wt% of edible oil and 89-90 wt% of water.

2. The self- stabilizing emulsion as claimed in claim 1, wherein, The steady emulsion comprises 0.8-1 wt% of whey protein isolate, 0.02-0.3 wt% of acetylated dextran, 0.01-0.03 wt% of glucose oxidase, 0.01-0.03 wt% of catalase, 9-10 wt% of edible oil and 89-90 wt% of water.

3. The self- stabilizing emulsion according to claim 1 or 2, characterized in that The enzyme activity of GOx is 100-500 u / mg; the enzyme activity of CAT is 3000-5000 u / mg.

4. The self- stabilizing emulsion as claimed in claim 1 or 2, characterized in that, The edible oil comprises one or more of corn oil, peanut oil, sunflower oil, soybean oil and rapeseed oil.

5. The self- stabilizing emulsion as claimed in claim 1 or 2, characterized in that, The steady emulsion contains a fat-soluble active substance; the fat-soluble active substance comprises one or more of benzyl isothiocyanate, proanthocyanidin, carotenoid, phytosterol and polyunsaturated fatty acid.

6. The emulsion of claim 5, wherein the emulsion is a stable emulsion. The mass fraction of the fat-soluble active substance in the steady emulsion is 0.04-0.06 wt%.

7. A process for the preparation of the steady emulsion according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1, protein solution preparation: mixing whey protein isolate and water to prepare a protein solution; S2, water phase preparation: mixing acetylated dextran and water to prepare an acetylated dextran solution, then adding glucose oxidase and catalase to obtain a mixed solution, and then mixing the mixed solution with the protein solution prepared in step S2 to prepare a water phase; S3, oil phase preparation: taking edible oil as the oil phase; S4, mixing: mixing the water phase prepared in step S3 and the oil phase prepared in step S4, performing high-speed dispersion, and then high-pressure homogenization to prepare a steady emulsion.

8. The production method according to claim 7, characterized by, In step S4, the parameters of high-speed dispersion are 8000-12000 r / min, the time is 2-4 min, the parameters of high-pressure homogenization are 5000-10000 psi, and the number of times is 6-8; in step S5, the volume ratio of the oil phase to the water phase is 1:4-1:

15.

9. The production method according to claim 7, characterized by, In the S4, the fat-soluble active substance is added to the edible oil as the oil phase; the mass concentration of the fat-soluble active substance in the oil phase is 1-10 mg / mL.

10. The steady emulsion according to any one of claims 1-3 for use in the field of food or health care products.

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