Preparation method of curcumin-loaded ultrasonic combined enzymatic glycosylation modified protein high internal phase emulsion
Through ultrasonic combination enzymatic glycosylation of sodium caseinate modified and curcumin embedded with curcumin, the problem of limited application of curcumin in food preparations is solved, and the effect of improving curcumin bioavailability and stability is achieved.
Patent Information
- Application Number
- CN202510049934.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-18
AI Technical Summary
The application of curcumin in food preparations is limited by its poor water solubility, low bioavailability and high photosensitiveness.
Ultrasonic-binding enzyme method is used to modify sodium caseinate to form a sodium caseinate-arabinxyllan crosslinker solution, and curcumin is embedded through high internal phase emulsion technology to improve its bioavailability.
The prepared high internal phase emulsion has the characteristics of good environmental stability, excellent viscoelasticity and high bioavailability, effectively improving the stability of curcumin in extreme environments and the sustained release transport ability of biologically active ingredients.
Smart Images
Figure CN119837258A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of food and biomedicine, and particularly relates to a method for preparing a high internal phase emulsion of ultrasound-combined enzymatic glycosylation-modified protein loaded with curcumin. Background Art
[0002] Curcumin, a natural flavonoid obtained from turmeric, is known to be a potent bioactive compound that protects against a variety of diseases, including cancer, obesity, infectious diseases, and cardiovascular diseases. In addition, curcumin has other beneficial health properties associated with reduced inflammation, metabolic syndrome, and pain. Given this wide range of bioactive properties, its incorporation into food matrices as a natural flavoring additive, yellow colorant, and preservative has attracted great interest from the food industry. However, its poor water solubility, reduced bioavailability, and high photosensitivity are the main drawbacks that limit its application in food formulations. Therefore, it is of great significance to apply curcumin in the development of nutritional supplements and functional foods and to improve its bioavailability.
[0003] High internal phase emulsions usually refer to emulsions with a dispersed phase volume of ≥74%. High internal phase emulsions are extremely important to the development of the food industry. They not only give products unique sensory properties, but also play an extremely important role in nutrient delivery. This special emulsion system is not prone to aggregation, Ostwald ripening and demulsification, resulting in excellent emulsion stability. In recent years, high internal phase emulsions have attracted great interest from the food processing industry due to their creamy appearance, high physical and chemical stability, unique rheological properties and a wide range of applications (such as encapsulation of lipophilic bioactive compounds and replacement of saturated fat in formulated foods).
[0004] Sodium caseinate is the sodium salt of casein, the main protein in milk. Because of its high safety and easy access, it is generally permitted to be used as a food additive in countries around the world. Although the emulsifying property of sodium caseinate has been widely recognized in a large number of studies, it still does not meet the requirements of high emulsifying protein in the actual production of the food industry. Moreover, because casein is rich in proline and does not have too many regular secondary or tertiary structures, this open structure makes them easily hydrolyzed by pepsin, causing the embedded active substances to be destroyed or degraded in a strong acidic environment, and cannot achieve the purpose of improving the bioavailability of bioactive substances. Therefore, its application range is relatively limited. Therefore, when sodium caseinate is used alone as an emulsifier or carrier wall material, it is generally necessary to improve its functional properties by modification, addition of polysaccharides, etc. Among the many modification methods, enzymatic modification is highly safe, mild in conditions, and has high product acceptability. It is a very potential protein modification method. Summary of the invention
[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with an enzymatic method loaded with curcumin. The prepared high internal phase emulsion encapsulating curcumin has the characteristics of good environmental stability, excellent viscoelasticity and high bioavailability.
[0006] In order to achieve the above object, the present invention adopts the following technical scheme:
[0007] A method for preparing a high internal phase emulsion of an ultrasound-enzymatically glycosylated protein loaded with curcumin, characterized in that it comprises the following steps:
[0008] (1) Preparation of sodium caseinate solution: sodium caseinate is added to phosphate buffer, stirred until dissolved, hydrated at 4° C. for a certain period of time, and then subjected to ultrasonic treatment to obtain a sodium caseinate solution;
[0009] (2) Preparation of arabinoxylan solution: adding arabinoxylan to phosphate buffer, stirring until dissolved, and hydrating at 4°C for a certain period of time to obtain an arabinoxylan solution;
[0010] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed evenly in a certain volume ratio, horseradish peroxidase and hydrogen peroxide solution were added, and the mixture was reacted at room temperature for a certain period of time. After the enzyme was inactivated at high temperature, an ultrasound-combined enzymatic glycosylation-modified sodium caseinate solution was obtained as the aqueous phase.
[0011] (4) Preparation of oil phase: Curcumin was added to corn oil, heated and magnetically stirred for 1-2 h to prepare the oil phase.
[0012] (5) Preparation of high internal phase emulsion: The oil phase and the water phase are mixed in a certain volume ratio and homogenized to obtain a high internal phase emulsion encapsulating curcumin.
[0013] Preferably, in step (1), the preparation method of the sodium caseinate solution is as follows: dissolving sodium caseinate powder in phosphate buffer, stirring at room temperature until completely dissolved, then placing in a 4°C refrigerator for hydration for 10-15 hours, and then taking out and returning to room temperature to obtain a sodium caseinate solution.
[0014] Preferably, in step (1), the mass fraction of sodium caseinate is 2%-6%, the concentration of phosphate buffer is 0.01M, the pH is 6.0-8.0, and the hydration time is 10-15h.
[0015] Preferably, in step (1), the ultrasonic treatment power is 100-400 W, and the ultrasonic treatment time is 8-15 min.
[0016] Preferably, in step (2), the mass fraction of arabinoxylan is 2%-6%, the concentration of phosphate buffer is 0.01M, the pH is 6.0-8.0, and the hydration time is 10-15h.
[0017] Preferably, in step (3), the sodium caseinate solution and the arabinoxylan solution are mixed in different volume ratios to a mass ratio of 1:1-4:1.
[0018] Preferably, in step (3), the amount of horseradish peroxidase added is 150-250 U / g (based on protein content), and the concentration of hydrogen peroxide solution is 0.1-1M.
[0019] Preferably, in step (3), the reaction time is 3-5 hours, the enzyme inactivation temperature is 85-100°C, and the time is 2-10 minutes.
[0020] Preferably, in step (4), the mass fraction of curcumin in the oil phase is 0.1%-0.2%.
[0021] Preferably, in step (5), the volume ratio of the oil phase is 70%-80%.
[0022] Preferably, in step (5), the homogenization condition is: homogenizing the solution at 10000-15000 rpm for 2-3 min.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] (1) The invention discloses a curcumin-loaded ultrasound-enzyme-glycosylated protein high internal phase emulsion, wherein natural sodium caseinate is subjected to ultrasound-enzyme-glycosylation modification to obtain a sodium caseinate-arabinoxylan crosslinked solution, thereby improving the limitations of sodium caseinate in practical applications. The modification method is simple in process, has few by-products, and is green and environmentally friendly.
[0025] (2) High internal phase emulsions usually refer to emulsions with a dispersed phase volume of ≥74%, which also play an extremely important role in nutrient delivery. This special emulsion system usually has excellent viscoelasticity and is not prone to aggregation, Ostwald ripening and demulsification, resulting in excellent emulsion stability, which can effectively improve the stability of curcumin in extreme environments, thereby better achieving the sustained release of bioactive ingredients to achieve the purpose of targeted transport, thereby improving the bioavailability of curcumin.
[0026] (3) This method uses food-grade proteins and polysaccharides as raw materials for high internal phase emulsions. The resulting high internal phase emulsions are more environmentally friendly, safer, and have a simple production process, which broadens the application of curcumin and high internal phase emulsions in industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a comparison diagram of particle size distribution and potential in Examples 1-3 of the present invention and Comparative Examples 1-3;
[0028] Figure 2 CLSM images of high internal phase emulsions at different oil phase volume fractions in Examples 1-3 of the present invention and Comparative Examples 1-3 (magnification: 40x);
[0029] Figure 3 It is a comparison chart of protein adsorption content in Examples 1-3 of the present invention and Comparative Examples 1-3;
[0030] Figure 4 It is a comparison chart of curcumin embedding efficiency in Examples 1-3 of the present invention and Comparative Examples 1-3;
[0031] Figure 5 It is a comparison chart of free fatty acid release rates in Examples 1-3 of the present invention and Comparative Examples 1-3;
[0032] Figure 6 It is a comparison chart of the bioaccessibility of curcumin in Examples 1-3 of the present invention and Comparative Examples 1-3. DETAILED DESCRIPTION
[0033] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.
[0034] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.
[0035] Example 1
[0036] A method for preparing a high internal phase emulsion of ultrasound-enzymatically glycosylated modified protein loaded with curcumin comprises the following steps:
[0037] (1) Preparation of sodium caseinate solution: sodium caseinate was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, hydrated at 4°C for 12 h, and then treated at 400 W ultrasonic power for 10 min in a 4°C ice water bath to obtain a sodium caseinate solution;
[0038] (2) Preparation of arabinoxylan solution: arabinoxylan was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, and hydrated at 4°C for 12 h to obtain an arabinoxylan solution;
[0039] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed in a volume ratio of 1:1, and horseradish peroxidase and hydrogen peroxide solution were added. The enzyme addition amount was 200 U / g protein, the concentration of the hydrogen peroxide solution was 0.1 M, and the volume ratio of horseradish peroxidase and hydrogen peroxide solution was 1:1. The mixture was reacted at room temperature for 4 h, and then heated in a water bath at 100°C for 5 min to inactivate the enzyme, thereby obtaining an ultrasound-combined enzymatic glycosylation-modified sodium caseinate solution as the aqueous phase.
[0040] (4) Preparation of oil phase: Curcumin was added to corn oil at a mass fraction of 0.1%, and magnetically stirred at 100°C for 1 h until completely dissolved to prepare the oil phase.
[0041] (5) Preparation of high internal phase emulsion: The oil phase and the water phase were mixed at a volume ratio of 70:30, and the solution was homogenized for 3 min at 12000 rpm using an IKA homogenizer to obtain a high internal phase emulsion encapsulating curcumin.
[0042] Example 2
[0043] A method for preparing a high internal phase emulsion of ultrasound-enzymatically glycosylated modified protein loaded with curcumin comprises the following steps:
[0044] (1) Preparation of sodium caseinate solution: sodium caseinate was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, hydrated at 4°C for 12 h, and then treated at 400 W ultrasonic power for 10 min in a 4°C ice water bath to obtain a sodium caseinate solution;
[0045] (2) Preparation of arabinoxylan solution: arabinoxylan was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, and hydrated at 4°C for 12 h to obtain an arabinoxylan solution;
[0046] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed in a volume ratio of 1:1, and horseradish peroxidase and hydrogen peroxide solution were added. The enzyme addition amount was 200 U / g protein, the concentration of the hydrogen peroxide solution was 0.1 M, and the volume ratio of horseradish peroxidase and hydrogen peroxide solution was 1:1. The mixture was reacted at room temperature for 4 h, and then heated in a water bath at 100°C for 5 min to inactivate the enzyme, thereby obtaining an ultrasound-combined enzymatic glycosylation-modified sodium caseinate solution as the aqueous phase.
[0047] (4) Preparation of oil phase: Curcumin was added to corn oil at a mass fraction of 0.1%, and magnetically stirred at 100°C for 1 h until completely dissolved to prepare the oil phase.
[0048] (5) Preparation of high internal phase emulsion: The oil phase and the water phase were mixed at a volume ratio of 75:25, and the solution was homogenized for 3 min at 12000 rpm using an IKA homogenizer to obtain a high internal phase emulsion encapsulating curcumin.
[0049] Example 3
[0050] A method for preparing a high internal phase emulsion of ultrasound-enzymatically glycosylated modified protein loaded with curcumin comprises the following steps:
[0051] (1) Preparation of sodium caseinate solution: sodium caseinate was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, hydrated at 4°C for 12 h, and then treated at 400 W ultrasonic power for 10 min in a 4°C ice water bath to obtain a sodium caseinate solution;
[0052] (2) Preparation of arabinoxylan solution: arabinoxylan was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, and hydrated at 4°C for 12 h to obtain an arabinoxylan solution;
[0053] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed in a volume ratio of 1:1, and horseradish peroxidase and hydrogen peroxide solution were added. The enzyme addition amount was 200 U / g protein, the concentration of the hydrogen peroxide solution was 0.1 M, and the volume ratio of horseradish peroxidase and hydrogen peroxide solution was 1:1. The mixture was reacted at room temperature for 4 h, and then heated in a water bath at 100°C for 5 min to inactivate the enzyme, thereby obtaining an ultrasound-combined enzymatic glycosylation-modified sodium caseinate solution as the aqueous phase.
[0054] (4) Preparation of oil phase: Curcumin was added to corn oil at a mass fraction of 0.1%, and magnetically stirred at 100° C. for 1 h until completely dissolved to prepare the oil phase.
[0055] (5) Preparation of high internal phase emulsion: The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min at 12000 rpm using an IKA homogenizer to obtain a high internal phase emulsion encapsulating curcumin.
[0056] Comparative Example 1
[0057] A method for preparing a high internal phase emulsion of ultrasound-modified protein loaded with curcumin comprises the following steps:
[0058] (1) Preparation of sodium caseinate solution: sodium caseinate was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, hydrated at 4°C for 12 h, and then treated at 400 W ultrasonic power for 10 min in a 4°C ice water bath to obtain a sodium caseinate solution;
[0059] (2) Preparation of arabinoxylan solution: arabinoxylan was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, and hydrated at 4°C for 12 h to obtain an arabinoxylan solution;
[0060] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed evenly in a volume ratio of 1:1, and a hydrogen peroxide solution was added. The concentration of the hydrogen peroxide solution was 0.1 M. The mixture was reacted at room temperature for 4 h, and then heated in a water bath at 100°C for 5 min to inactivate the enzyme. A mixed solution of ultrasonically modified sodium caseinate and arabinoxylan was obtained as the aqueous phase.
[0061] (4) Preparation of oil phase: Curcumin was added to corn oil at a mass fraction of 0.1%, and magnetically stirred at 100°C for 1 h until completely dissolved to prepare the oil phase.
[0062] (5) Preparation of high internal phase emulsion: The oil phase and the water phase were mixed at a volume ratio of 70:30, and the solution was homogenized for 3 min at 12000 rpm using an IKA homogenizer to obtain a high internal phase emulsion encapsulating curcumin.
[0063] Comparative Example 2
[0064] A method for preparing a high internal phase emulsion of ultrasound-modified protein loaded with curcumin comprises the following steps:
[0065] (1) Preparation of sodium caseinate solution: sodium caseinate was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, hydrated at 4°C for 12 h, and then treated at 400 W ultrasonic power for 10 min in a 4°C ice water bath to obtain a sodium caseinate solution;
[0066] (2) Preparation of arabinoxylan solution: arabinoxylan was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, and hydrated at 4°C for 12 h to obtain an arabinoxylan solution;
[0067] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed evenly in a volume ratio of 1:1, and a hydrogen peroxide solution was added. The concentration of the hydrogen peroxide solution was 0.1 M. The mixture was reacted at room temperature for 4 h, and then heated in a water bath at 100°C for 5 min to inactivate the enzyme. A mixed solution of ultrasonically modified sodium caseinate and arabinoxylan was obtained as the aqueous phase.
[0068] (4) Preparation of oil phase: Curcumin was added to corn oil at a mass fraction of 0.1%, and magnetically stirred at 100°C for 1 h until completely dissolved to prepare the oil phase.
[0069] (5) Preparation of high internal phase emulsion: The oil phase and the water phase were mixed at a volume ratio of 75:25, and the solution was homogenized for 3 min at 12000 rpm using an IKA homogenizer to obtain a high internal phase emulsion encapsulating curcumin.
[0070] Comparative Example 3
[0071] A method for preparing a high internal phase emulsion of ultrasound-modified protein loaded with curcumin comprises the following steps:
[0072] (1) Preparation of sodium caseinate solution: sodium caseinate was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, hydrated at 4°C for 12 h, and then treated at 400 W ultrasonic power for 10 min in a 4°C ice water bath to obtain a sodium caseinate solution;
[0073] (2) Preparation of arabinoxylan solution: arabinoxylan was added to 0.01 M phosphate buffer (pH 7.0) at a mass fraction of 3%, magnetically stirred at room temperature until completely dissolved, and hydrated at 4°C for 12 h to obtain an arabinoxylan solution;
[0074] (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution were mixed evenly in a volume ratio of 1:1, and a hydrogen peroxide solution was added. The concentration of the hydrogen peroxide solution was 0.1 M. The mixture was reacted at room temperature for 4 h, and then heated in a water bath at 100°C for 5 min to inactivate the enzyme. A mixed solution of ultrasonically modified sodium caseinate and arabinoxylan was obtained as the aqueous phase.
[0075] (4) Preparation of oil phase: Curcumin was added to corn oil at a mass fraction of 0.1%, and magnetically stirred at 100°C for 1 h until completely dissolved to prepare the oil phase.
[0076] (5) Preparation of high internal phase emulsion: The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min at 12000 rpm using an IKA homogenizer to obtain a high internal phase emulsion encapsulating curcumin.
[0077] The performance tests of the emulsions prepared in Examples 1-3 and Comparative Examples 1-3 were carried out as follows:
[0078] 1. Particle size and Zeta potential of high internal phase emulsion of protein modified by ultrasound combined with enzymatic glycosylation loaded with curcumin
[0079] Methods: Zeta potential and particle size distribution were measured at 25°C using a Zetasizer Nano ES90. Particle size was determined by dynamic light scattering (DLS), and the average particle size was calculated by the instrument based on the particle size distribution image. Samples were adjusted to 1 mg / mL with 0.01 M phosphate buffer (pH 7.0) for measurement.
[0080] The instrument parameters are: refractive index 1.45, absorptivity 0.001, and temperature controlled at 25.0±0.2℃.
[0081] Result description: Figure 1 A and B show the particle size visualization and Zeta potential diagram of the high internal phase emulsion of Examples 1-3 and Comparative Examples 1-3, respectively. Particle size is an important indicator for evaluating the stability of an emulsion. The smaller the particle size, the more concentrated the distribution, and the more stable the emulsion. Volume average diameter d 4,3 It can be used to characterize the average size of emulsion droplets and measure the degree of aggregation of emulsion droplets. Figure 1 As can be seen from A, as the volume of the oil phase increases, the d 4,3 The overall trend is downward (P<0.05), among which the particle size of Example 3 is the smallest, which is 16.95±0.04um, which is more conducive to forming a stable emulsion. Zeta potential is a physical property exhibited by emulsion particles, which reflects the properties of the electrostatic potential near the particle surface. Figure 4 As shown in Figure B, Examples 1-3 and Comparative Examples 1-3 are all negatively charged. As the volume of the oil phase increases, the absolute value of the emulsion potential also gradually increases, indicating that the system is more stable.
[0082] 2. Confocal laser scanning electron microscopy of high internal phase emulsion of protein modified by ultrasound combined with enzymatic glycosylation loaded with curcumin
[0083] Method: Take 1mL of sample and add 2uL of mixed dye (containing 0.1% fluorescein isothiocyanate and 0.1% Nile red mixed solution, dissolved in isopropanol). Place an appropriate amount of emulsion on a glass slide, with excitation wavelengths of 488nm and 553nm, oil phase signal set to red, and protein signal set to green. Figure 2 AF are 40x objective lens pictures of Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Example 2 and Example 3 respectively.
[0084] Result description: Figure 2It can be seen that the red fluorescence indicates the oil phase contained in the emulsion droplets, while the green fluorescence corresponds to the sodium caseinate on the surface of the emulsion droplets. The emulsion droplets are uniformly dispersed in the protein network, indicating that the prepared high internal phase emulsion has a typical water-in-oil structure. At the same time, as the volume of the oil phase increases, the emulsion droplets of Examples 1-3 tend to gradually decrease, and the distribution of the emulsion droplets is more compact. This is because the increase in the volume of the oil phase makes the network structure more compact, and the interfacial tension of the smaller droplets with a relatively large interfacial area increases.
[0085] 3. Protein adsorption content of high internal phase emulsion of protein modified by ultrasound combined with enzymatic glycosylation loaded with curcumin
[0086] Method: 1.5 g of the emulsion was centrifuged at 12000 rpm for 30 min. The aqueous phase at the bottom was collected by a syringe and further filtered through a 0.45 μm filter membrane. The protein concentration was determined using a BCA kit. The emulsion solution was diluted by a corresponding multiple, and the absorbance was measured at 562 nm according to the kit method. The raw serum albumin (BSA) solution was used as the protein standard, and the standard curve was drawn with its mass concentration (mg / mL) and absorbance as the horizontal and vertical coordinates. The protein concentration was calculated according to the regression equation.
[0087] Formula: AP (%) = (C0-C f) ) / C0×100%
[0088] Among them, C f refers to the protein concentration in the solution after membrane filtration, and (C0) refers to the initial protein concentration.
[0089] The results show that the adsorption layer at the interface (interface protein adsorption content and interface area) affects the aggregation and flocculation stability of the emulsion. When the interface is completely covered, the stability of the emulsion is maintained and the aggregation and flocculation of oil droplets are prevented. Figure 3 As shown, the interface protein adsorption (AP) content of Example 3 was the highest, reaching 83.05±3.87%.
[0090] 4. Encapsulation rate of curcumin
[0091] Methods: A certain amount of freshly prepared high internal phase emulsion was centrifuged at 10000rpm for 20min, and a certain amount of anhydrous ethanol solution was added to the supernatant to remove the unencapsulated curcumin. The absorbance of curcumin in the supernatant at 425nm was measured using a UV-2600 spectrophotometer (UnicoCorporation, Shanghai, China). The curcumin concentration was calculated from the curcumin standard curve (y = 0.1296 × + 0.007R2 = 0.9999). During the experiment, (high internal phase emulsion without curcumin) was used as the blank group.
[0092] Formula: Encapsulation rate (%) = (1-free curcumin / initial curcumin concentration) × 100%
[0093] Result description: The experimental results are as follows Figure 2 As shown, in the ultrasound-combined enzymatic glycosylation-modified protein high internal phase emulsion loaded with curcumin prepared by the present invention, the curcumin high internal phase emulsion prepared by using ultrasonically treated sodium caseinate solution and peroxidase-mediated arabinoxylan-sodium caseinate crosslinked product as emulsifier has good embedding rate and stability, among which the curcumin high internal phase emulsion of Example 3 has the highest embedding rate (reaching 90.01%).
[0094] 4. Free fatty acid release rate of curcumin
[0095] Methods: NaCl (40 mg) was dissolved in 20 mL of water at pH 1.2 to prepare simulated gastric fluid. The emulsion sample carrying 2 g of oil was mixed with 16 mL of simulated gastric fluid and stirred evenly in a 37°C water bath. After adding simulated gastric fluid (4 mL) containing pepsin to the above mixture, gastric digestion was carried out in a 37°C water bath for 2 h and terminated by adjusting the pH to 7.5. Subsequently, Tris, CaCl2, and ox bile salts were dissolved in 20 mL of water, and simulated intestinal fluid was prepared after adjusting the pH to 7.5 for intestinal digestion at 37°C. After the simulated intestinal fluid containing pancreatic enzymes was mixed with the chyme produced by gastric digestion, it was soaked in a water bath for 2 h. Due to the production of free fatty acids (FFAs) during digestion, the pH value of the entire system would decrease, and it was maintained at 7.5 by adding NaOH (0.25 mol / L). The release curve of FFAs was obtained by recording the amount of NaOH added. It was assumed that the digestion of 1 mol of triglyceride resulted in the release of 2 mol of FFAs and the consumption of 2 mol of NaOH, and the free fatty acid (FFA) release rate was calculated based on the volume consumed during simulated intestinal digestion.
[0096] Results: The release of free fatty acids (FFA) was measured to detect the degree of fat decomposition of the sample in simulated intestinal fluid. Figure 4 As shown, the fat of the three groups of samples was rapidly digested in the first 30 minutes, and then the digestion rate gradually slowed down during the whole digestion process. The results showed that the FFA release order was: Example 3 > Example 2 > Comparative Example 3 > Comparative Example 2 > Example 1 > Comparative Example 1. During the fat digestion process, the contact area between lipase and oil droplets affects the decomposition rate of fat. For Example 3, under the action of homogenization, more protein-polysaccharide cross-links were adsorbed to the oil-water interface to form a dense interface layer, so that the high internal phase emulsion has a smaller droplet size and a relatively large specific surface area, which improves the interaction between lipase and oil droplets, thereby improving the efficiency of lipase and accelerating the release of free fatty acids.
[0097] 5. Bioaccessibility of Curcumin
[0098] Method: The emulsion was centrifuged at 11000rpm for 45min to collect the intermediate micelle phase. An appropriate amount of the intermediate micelle was thoroughly mixed with ethanol at a volume ratio of 1:1. The above mixture was centrifuged at 11000rpm for 20min to obtain the supernatant. The absorbance value of the diluted supernatant at 426nm was tested using a UV-visible spectrophotometer. The bioaccessibility of curcumin was obtained based on the following formula:
[0099] Formula: Curcumin bioaccessibility = (curcumin content in micelles / curcumin content in emulsion) × 100%
[0100] Results show that curcumin is a fat-soluble nutrient that can enhance immune function and has excellent anti-inflammatory and antioxidant properties. It is of great significance to enhance the stability of curcumin in the digestive system to improve its application in nutritional supplements and functional foods. Therefore, the in vitro gastrointestinal simulation method was used to evaluate the protective effect of Examples 1-3 and Comparative Examples 1-3 on the bioaccessibility of curcumin. The experimental results are as follows Figure 6 As shown, the bioaccessibility of Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 is 45.97±0.63%, 33.41±1.32%, 17.95±2.01%, 9.88±1.70%, 27.16±0.66% and 33.49±0.44%, respectively. It can be found that the protective effect of curcumin in Example 3 better exerts the utilization value of curcumin. This result is positively correlated with the release rate of free fatty acids, and the release of free fatty acids can promote the formation of curcumin micelles. For comparative example 1, due to the large droplets, the specific surface area is very low, resulting in insufficient reaction of lipase with the oil droplets, thereby inhibiting the conversion of curcumin from oil to micelles; for example 3, ultrasonic treatment and combination with polysaccharides jointly promote the adsorption of sodium caseinate-arabinoxylan crosslinks on the surface of the oil droplets, so that the oil droplets are not easy to aggregate in the gastric digestion stage, thereby having a large specific surface area, thereby promoting the reaction with lipase in intestinal digestion, so that more curcumin can be detected in the micelles. Therefore, example 3 can be used as an effective carrier of bioactive substances to improve the aqueous solubility and gastrointestinal stability of fat-soluble nutrients and form controlled release in the intestine, so as to achieve the purpose of improving the bioavailability of nutrient molecules.
Claims
1. A method for preparing a high internal phase emulsion of an ultrasound-enzymatically glycosylated protein loaded with curcumin, characterized in that: The steps include: (1) Preparation of sodium caseinate solution: sodium caseinate is added to phosphate buffer, stirred until dissolved, hydrated at 4° C. for a certain period of time, and then subjected to ultrasonic treatment to obtain a sodium caseinate solution; (2) Preparation of arabinoxylan solution: adding arabinoxylan to phosphate buffer, stirring until dissolved, and hydrating at 4°C for a certain period of time to obtain an arabinoxylan solution; (3) Preparation of aqueous phase: The prepared sodium caseinate solution and arabinoxylan solution are uniformly mixed in a certain volume ratio, horseradish peroxidase and hydrogen peroxide solution are added, reacted at room temperature for a certain time, and the enzyme is inactivated at high temperature to obtain an ultrasound-combined enzymatic glycosylation-modified sodium caseinate solution as the aqueous phase; (4) Preparation of oil phase: Curcumin was added to corn oil, heated and magnetically stirred for 1-2 h to prepare the oil phase; (5) Preparation of high internal phase emulsion: The oil phase and the water phase are mixed in a certain volume ratio and homogenized to obtain a high internal phase emulsion encapsulating curcumin.
2. The method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with enzymatic method loaded with curcumin as described in claim 1, characterized in that: In step (1), the mass fraction of sodium caseinate is 2%-6%, the concentration of phosphate buffer is 0.01M, the pH is 6.0-8.0, and the hydration time is 10-15h.
3. The method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with enzymatic method loaded with curcumin as described in claim 1, characterized in that: In step (1), the ultrasonic treatment power is 100-400W, and the ultrasonic treatment time is 8-15min.
4. The method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with enzymatic method loaded with curcumin as described in claim 1, characterized in that: In step (2), the mass fraction of arabinoxylan is 2%-6%, the concentration of phosphate buffer is 0.01M, the pH is 6.0-8.0, and the hydration time is 10-15h.
5. The method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with enzymatic method loaded with curcumin as described in claim 1, characterized in that: In step (3), the sodium caseinate solution and the arabinoxylan solution are mixed in different volume ratios to a mass ratio of 1:1-4:
1.
6. The method for preparing a high internal phase emulsion of a glycosylated protein loaded with curcumin by ultrasound combined with enzymatic method as described in claim 1, characterized in that: In step (3), the amount of horseradish peroxidase added is 150-250 U / g (based on protein content), and the concentration of the hydrogen peroxide solution is 0.1-1 M.
7. The method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with enzymatic method loaded with curcumin as described in claim 1, characterized in that: In step (3), the reaction time is 3-5 hours, the enzyme inactivation temperature is 85-100°C, and the time is 2-10 minutes.
8. The method for preparing a high internal phase emulsion of a glycosylated protein loaded with curcumin by ultrasound combined with enzymatic method as described in claim 1, characterized in that: In step (4), the mass fraction of curcumin in the oil phase is 0.1%-0.2%.
9. The method for preparing a high internal phase emulsion of a protein glycosylated by ultrasound combined with enzymatic method loaded with curcumin as described in claim 1, characterized in that: In step (5), the volume ratio of the oil phase is 70%-80%.
10. The method for preparing a high internal phase emulsion of a glycosylated protein loaded with curcumin by ultrasound combined with enzymatic method as described in claim 1, characterized in that: In step (5), the homogenization condition is: homogenizing the solution at 10000-15000 rpm for 2-3 min.