Preparation method of laccase-mediated whey protein-arabinoxylan covalent stable high internal phase emulsion
Through ultrasonic-assisted laccase-mediated method, whey protein and arabinoxican were covalently modified, which solved the problem of poor emulsification ability of whey protein, and prepared a stable high internal phase emulsion with excellent viscoelastic properties and stability, suitable for the delivery of small biologically active molecules.
Patent Information
- Application Number
- CN202510049993.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-15
AI Technical Summary
Whey protein has poor emulsification ability, resulting in unstable high-phase emulsion prepared.
Using an ultrasonic-assisted laccase-mediated method, whey protein and arabinoxican are covalently reacted to form a stable high internal phase emulsion. The method involves hydrating whey protein in phosphate buffer and sonicating, followed by reaction with laccase, H2O2 and arabinoxican under specific conditions, and finally undergoing enzyme detoxification and homogenization to obtain a high internal phase emulsion.
The prepared high internal phase emulsion has excellent viscoelastic properties, storage stability and environmental stability. It is suitable for the delivery of small biologically active molecules. It has a simple production process, is safe, and has no harmful by-products, and is low in cost. It is suitable for large-scale industrial promotion.
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Figure CN119817790A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food science and engineering, and relates to a method for preparing a high internal phase emulsion stabilized by whey protein-arabinoxylan covalent compounds formed by ultrasound-assisted enzymes. Background Art
[0002] High internal phase emulsions usually refer to emulsions with an internal phase volume fraction greater than 74%. Compared with traditional dilute emulsions, the tightly packed state inside high internal phase emulsions makes the emulsions more stable, so they have unique advantages in the delivery of bioactive substances and the protection of probiotics. In addition, high internal phase emulsions form a three-dimensional network structure on the surface of droplets, limit the contact between the external environment and the droplets, and inhibit the degradation of active ingredients in the digestive tract, thereby achieving the purpose of improving digestion stability and targeted sustained release.
[0003] The growing consumer demand for food ingredients that do not contain animal-derived or synthetic ingredients has led to a growing interest in natural emulsifiers such as proteins and polysaccharides. Protein-polysaccharides have shown great potential in stabilizing high internal phase emulsions due to their good functional properties, and have therefore received extensive research and attention. Glycosylation modification has become the most direct and promising method for modifying the functional properties of food proteins due to its outstanding effects on protein solubility and emulsification. There are many studies on the glycosylation of proteins and the relationship between glycosylation and functional properties using the Maillard reaction. However, when the Maillard reaction is applied to protein modification, the reaction process is complicated. In addition to the desired protein-sugar condensation reaction, there are other side reactions, leading to food safety issues.
[0004] Compared with the Maillard glycosylation reaction, enzymatic glycosylation modification based on laccase catalysis has become an ideal means of protein glycosylation modification due to its advantages such as simple operation, no harmful byproducts and high efficiency. Ultrasonic treatment, as a common physical modification method, can effectively expose laccase cross-linking sites, promote the improvement of cross-linking degree and increase the amount of cross-linked products. Ultrasonic treatment has great potential in protein modification due to its advantages of fast, efficient, safe, reliable, safe and non-toxic. The preparation of high internal phase emulsions with excellent viscoelastic properties, storage stability and environmental stability by combining ultrasound with enzymatic glycosylation provides an effective delivery carrier construction strategy for the construction of hydrophobic and thermosensitive bioactive small molecule delivery systems. Summary of the invention
[0005] In order to solve the problem that whey protein has poor emulsifying ability and the prepared high-internal-phase emulsion is unstable, the present invention provides an ultrasound-assisted laccase-mediated preparation method and application of a whey protein-arabinoxylan covalently stabilized high-internal-phase emulsion.
[0006] The present invention provides a method for preparing a high internal phase emulsion, wherein the high internal phase emulsion is prepared according to the following steps:
[0007] (1) Dissolve whey protein in phosphate buffer, stir to dissolve, hydrate at 4°C for a certain period of time, and obtain a whey protein solution after appropriate ultrasonic treatment. Dissolve arabinoxylan in phosphate buffer, stir to dissolve, and refrigerate to obtain an arabinoxylan solution;
[0008] (2) whey protein, laccase and H2O2 were mixed, and then arabinoxylan was added, and then the mixture was reacted at 40°C for a certain time. After the reaction, the mixture was kept at a certain temperature for 15 minutes to inactivate the enzyme;
[0009] (3) mixing curcumin and corn oil to obtain a curcumin / corn oil solution (oil phase);
[0010] (4) mixing the oil phase and the water phase in a volume-to-volume ratio, and homogenizing them to obtain a high internal phase emulsion encapsulating curcumin.
[0011] Preferably, in step (1), the stirring time is 1-3 h, the stirring speed is 800-1500 rpm, and the hydration time is 6-12 h.
[0012] Preferably, the pH of the phosphate buffer in step (1) is 6-8.
[0013] Preferably, in step (1), the concentration of whey protein is 1%-4% (w / w), and the mass ratio of whey protein to arabinoxylan is 1:2-2:1.
[0014] Preferably, the ultrasonic treatment in step (1) is carried out in an ice bath, and the ultrasonic time is 10-40 min.
[0015] Preferably, in step (2), the amount of laccase added is 70U / g-90U / g (based on protein content), the incubation time at 40°C is 4-12h, and the enzyme inactivation temperature is 70-80°C.
[0016] Preferably, in step (3), an ultrasonic dissolution-promoting operation is performed when curcumin and corn oil are mixed.
[0017] Preferably, in step (4), the volume fraction of the oil phase in the high internal phase emulsion system is 70%-85%.
[0018] Preferably, the homogenization treatment in step (4) is performed by homogenizing the solution at a rotation speed of 8000-15000 rpm for 2-3 minutes.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] (1) The present invention prepares a whey protein-arabinoxylan covalent compound mediated by ultrasound-assisted laccase and a high internal phase emulsion stabilized by the same. The preparation method is simple, no harmful substances are generated during the preparation process, the production cost is low, the production cycle is short, the nutrition is rich, the product is green and pollution-free, and the economic utilization value is high, and the product is suitable for large-scale industrial promotion.
[0021] (2) The ultrasound-assisted laccase-formed whey protein-arabinoxylan covalently stabilized high internal phase emulsion obtained in the present invention has excellent viscoelastic properties, storage stability and environmental stability, and provides an effective delivery carrier construction strategy for the construction of hydrophobic and thermosensitive bioactive small molecule delivery systems.
[0022] (3) The present invention addresses the problems of poor water solubility, poor environmental stability and low bioavailability of curcumin by using a high internal phase emulsion stabilized by a whey protein-arabinoxylan covalent compound for encapsulation, thereby improving its bioavailability and digestion stability, and has broad application prospects in the fields of biomedicine, cosmetics and food. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the summary diagram;
[0024] Figure 2 The droplet size diagram and potential diagram of the high internal phase emulsion prepared in Examples 1-6 of the present invention;
[0025] Figure 3 The rheological characteristic property diagram of the high internal phase emulsion prepared in Examples 1-6 of the present invention;
[0026] Figure 4 This is a diagram of the droplet size of the high internal phase emulsion prepared in Examples 1-6 of the present invention before and after storage;
[0027] Figure 5 This is a laser confocal microstructure image of the high internal phase emulsion prepared in Examples 1-6 of the present invention. DETAILED DESCRIPTION
[0028] The present invention is further described in detail below by specific preferred embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.
[0029] Embodiment 1:
[0030] (1) Accurately weigh 2g of whey protein concentrate, dissolve it in 100mL phosphate buffer (10mM, pH7.4), place it under a stirrer at 1200rpm for 2h, and place it at 4℃ for 10h. Then use an ultrasonic device to ultrasonicate the protein solution in an ice bath, with an ultrasonic power of 600W, an ultrasonic time of 3s, an interval of 4s, and an ultrasonic time of 10min to obtain a whey protein concentrate solution with a concentration of 2%. Accurately weigh 3g of arabinoxylan, dissolve it in 100mL phosphate buffer (10mM, pH 7.4), place it under a stirrer at 1200rpm for 2h, and place it at 4℃ for 10h to obtain a 3% arabinoxylan solution.
[0031] (2) Laccase was added to the protein solution at 80 U / g, and H2O2 was added to the protein solution at 17 μL / mL, and the solution was equilibrated at 25°C for 30 min. An equal volume of arabinoxylan solution was added at intervals of 5 min over 50 min to make the mass ratio of whey protein to arabinoxylan 3:2, and then the mixture was kept at 40°C for 10 h and 75°C for 15 min.
[0032] (3) Accurately weigh 0.1 g of curcumin, dissolve it in 100 mL of corn oil, and dissolve it by ultrasonication to obtain a curcumin / soybean oil emulsion (oil phase);
[0033] (4) The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min using an IKA homogenizer at a rotation speed of 12000 rpm to obtain a high internal phase emulsion encapsulating curcumin.
[0034] Embodiment 2:
[0035] The differences between this embodiment and embodiment 1 are as follows: the rotation speed in step (1) is 1000 rpm, the hydration time is 12 h, the protein is not subjected to ultrasonic treatment, and the reaction time of the mixture at 40° C. in step (2) is 12 h.
[0036] (1) Accurately weigh 2 g of whey protein concentrate, dissolve it in 100 mL of phosphate buffer (10 mM, pH 7.4), stir it under a stirrer at 1000 rpm for 2 h, and place it at 4°C for 12 h to obtain a whey protein concentrate solution with a concentration of 2%. Accurately weigh 3 g of arabinoxylan, dissolve it in 100 mL of phosphate buffer (10 mM, pH 7.4), stir it under a stirrer at 1000 rpm for 2 h, and place it at 4°C for 12 h to obtain a 3% arabinoxylan solution.
[0037] (2) Laccase was added to the protein solution at 80 U / g, and H2O2 was added to the protein solution at 17 μL / mL, and the solution was equilibrated at 25°C for 30 min. An equal volume of arabinoxylan solution was added at intervals of 5 min over 50 min to make the mass ratio of whey protein to arabinoxylan 3:2, and then the mixture was kept at 40°C for 12 h and 75°C for 15 min.
[0038] (3) Accurately weigh 0.1 g of curcumin, dissolve it in 100 mL of corn oil, and dissolve it by ultrasonication to obtain a curcumin / soybean oil emulsion (oil phase);
[0039] (4) The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min using an IKA homogenizer at a rotation speed of 12000 rpm to obtain a high internal phase emulsion encapsulating curcumin.
[0040] Embodiment 3:
[0041] The difference between this embodiment and embodiment 1 is that laccase is not added in step (2).
[0042] (1) Accurately weigh 2g of whey protein concentrate, dissolve it in 100mL of phosphate buffer (10mM, pH 7.4), place it under a stirrer and stir it at 1200rpm for 2h, and place it at 4℃ for 10h. Then use an ultrasonic device to ultrasonicate the protein solution in an ice bath, with an ultrasonic power of 600W, an ultrasonic time of 3s, an interval of 4s, and a total ultrasonic time of 10min to obtain a whey protein concentrate solution with a concentration of 2%. Accurately weigh 3g of arabinoxylan, dissolve it in 100mL of phosphate buffer (10mM, pH 7.4), place it under a stirrer and stir it at 1200rpm for 2h, and place it at 4℃ for 12h to obtain a 3% arabinoxylan solution.
[0043] (2) H2O2 was added to the protein solution at 17 μL / mL and equilibrated at 25°C for 30 min. An equal volume of arabinoxylan solution was added at intervals of 5 min over 50 min to make the mass ratio of whey protein to arabinoxylan 3:2, and then the mixture was kept at 40°C for 10 h and 75°C for 15 min.
[0044] (3) Accurately weigh 0.1 g of curcumin, dissolve it in 100 mL of corn oil, and dissolve it by ultrasonication to obtain a curcumin / soybean oil emulsion (oil phase);
[0045] (4) The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min using an IKA homogenizer at a rotation speed of 12000 rpm to obtain a high internal phase emulsion encapsulating curcumin.
[0046] Embodiment 4:
[0047] The differences between this example and Example 1 are as follows: the rotation speed in step (1) is 1000 rpm, the hydration time is 12 h, the protein is not subjected to ultrasonic treatment, and laccase is not added in step (2).
[0048] (1) Accurately weigh 2 g of whey protein concentrate, dissolve it in 100 mL of phosphate buffer (10 mM, pH 7.4), stir it at 1000 rpm for 2 h under a stirrer, and place it at 4 ° C for 12 h to obtain a whey protein concentrate solution with a concentration of 2%. Accurately weigh 3 g of arabinoxylan, dissolve it in 100 mL of phosphate buffer (10 mM, pH 7.4), stir it at 1000 rpm for 2 h under a stirrer, and place it at 4 ° C for 12 h to obtain a 3% arabinoxylan solution.
[0049] (2) H2O2 was added to the protein solution at 17 μL / mL and equilibrated at 25°C for 30 min. An equal volume of arabinoxylan solution was added at intervals of 5 min over 50 min to make the mass ratio of whey protein to arabinoxylan 3:2, and then the mixture was kept at 40°C for 10 h and 75°C for 15 min.
[0050] (3) Accurately weigh 0.1 g of curcumin, dissolve it in 100 mL of corn oil, and dissolve it by ultrasonication to obtain a curcumin / soybean oil emulsion (oil phase);
[0051] (4) The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min using an IKA homogenizer at a rotation speed of 12000 rpm to obtain a high internal phase emulsion encapsulating curcumin.
[0052] Embodiment 5:
[0053] The difference between this example and Example 1 is that no laccase and arabinoxylan solution are added in step (2).
[0054] (1) Accurately weigh 2 g of whey protein concentrate, dissolve it in 100 mL of phosphate buffer (10 mM, pH 7.4), stir it under a stirrer at 1200 rpm for 2 h, and place it at 4° C. for 10 h. Then use an ultrasonic device to ultrasonicate the protein solution in an ice bath, with an ultrasonic power of 600 W, an ultrasonic time of 3 s, an interval of 4 s, and a total ultrasonic time of 10 min to obtain a whey protein concentrate solution with a concentration of 2%.
[0055] (2) H2O2 was added to the protein solution at 17 μL / mL and equilibrated at 25°C for 30 min. An equal volume of phosphate buffer was added at intervals of 5 min over 50 min, and the mixture was then kept at 40°C for 10 h and 75°C for 15 min.
[0056] (3) Accurately weigh 0.1 g of curcumin, dissolve it in 100 mL of corn oil, and dissolve it by ultrasonication to obtain a curcumin / soybean oil emulsion (oil phase);
[0057] (4) The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min using an IKA homogenizer at a rotation speed of 12000 rpm to obtain a high internal phase emulsion encapsulating curcumin.
[0058] Embodiment 6:
[0059] The differences between this example and Example 1 are that the rotation speed in step (1) is 1000 rpm, the protein is not subjected to ultrasonic treatment, and laccase and arabinoxylan solution are not added in step (2).
[0060] (1) Accurately weigh 2 g of whey protein concentrate, dissolve it in 100 mL of phosphate buffer (10 mM, pH 7.4), stir it under a stirrer at 1000 rpm for 2 h, and place it at 4°C for 12 h to obtain a whey protein concentrate solution with a concentration of 2%.
[0061] (2) H2O2 was added to the protein solution at 17 μL / mL and equilibrated at 25°C for 30 min. An equal volume of phosphate buffer was added at intervals of 5 min over 50 min, and the mixture was then kept at 40°C for 10 h and 75°C for 15 min.
[0062] (3) Accurately weigh 0.1 g of curcumin, dissolve it in 100 mL of corn oil, and dissolve it by ultrasonication to obtain a curcumin / soybean oil emulsion (oil phase);
[0063] (4) The oil phase and the water phase were mixed at a volume ratio of 80:20, and the solution was homogenized for 3 min using an IKA homogenizer at a rotation speed of 12000 rpm to obtain a high internal phase emulsion encapsulating curcumin.
[0064] The performance of the mixed high internal phase emulsion system prepared in the above example was characterized:
[0065] Figure 2 The droplet size diagram and potential diagram of the high internal phase emulsion prepared in Examples 1-6 of the present invention. It can be seen from Examples 1 and 2 that after ultrasound, the D 4,3 The absolute value of the potential is significantly reduced. From Examples 1, 3 and 5, it can be seen that the D of Example 1 prepared by enzymatic glycosylation4,3 The absolute value of the potential increases significantly.
[0066] Figure 3 The rheological characteristic property diagram of the high internal phase emulsion prepared in Examples 1-6 of the present invention shows that G' of Examples 1-6 is greater than G", which means that all examples have elastic gel behavior. From Examples 1 and 2, it can be seen that G' and G" of Example 1 increase after ultrasound, which indicates that ultrasound strengthens the network structure of the high internal phase emulsion and makes it denser. From Examples 1, 3 and 5, it can be seen that Example 1 prepared by enzymatic glycosylation has the densest structure, the flow of the emulsion is restricted, and phase separation is not easy to occur.
[0067] Figure 4 The droplet size diagram of the high internal phase emulsion prepared in Examples 1-6 of the present invention before and after storage shows that all Examples 1, 2, 5 and 6 have strong resistance to droplet aggregation during storage, and the particle size does not change much, indicating that the present invention has good storage stability.
[0068] Figure 5 The laser confocal microscopic structure diagram of the high internal phase emulsion prepared in Examples 1-6 of the present invention shows that Examples 1-6 are all composed of dark green spheres embedded in a red matrix, indicating that they are O / W type high internal phase emulsions composed of tightly arranged oil droplets wrapped in a rich protein complex. Compared with Examples 2-6, the oil droplets in Example 1 are smaller and more uniform, indicating that the whey protein-arabinoxylan covalent compound prepared in Example 1 by ultrasound-assisted enzyme formation has excellent viscoelastic properties, excellent storage stability and environmental stability.
Claims
1. A method for preparing a high internal phase emulsion stabilized by laccase-mediated whey protein-arabinoxylan covalent compounds, wherein the high internal phase emulsion is prepared according to the following steps: (1) Dissolve whey protein in phosphate buffer, stir to dissolve, hydrate at 4°C for a certain period of time, and obtain a whey protein solution after appropriate ultrasonic treatment. Dissolve arabinoxylan in phosphate buffer, stir to dissolve, and refrigerate to obtain an arabinoxylan solution; (2) whey protein, laccase and H2O2 were mixed, and then arabinoxylan was added, and then the mixture was reacted at 40°C for a certain time. After the reaction, the mixture was kept at a certain temperature for 15 minutes to inactivate the enzyme; (3) mixing curcumin and corn oil to obtain a curcumin / corn oil solution (oil phase); (4) 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 high internal phase emulsion according to claim 1, characterized in that In step (1), the stirring time is 1-3 hours, the stirring speed is 800-1500 rpm, and the hydration time is 6-12 hours.
3. The high internal phase emulsion according to claim 1, characterized in that The pH of the phosphate buffer in step (1) is 6-8.
4. The high internal phase emulsion according to claim 1, characterized in that In step (1), the concentration of whey protein is 1%-4% (w / w), and the mass ratio of whey protein to arabinoxylan is 1:2-2:
1.
5. The high internal phase emulsion according to claim 1, characterized in that The ultrasonic treatment in step (1) is carried out in an ice bath, and the ultrasonic time is 10-40 min.
6. The high internal phase emulsion according to claim 1, characterized in that In step (2), the amount of laccase added is 70U / g-90U / g (based on protein content), the incubation time at 40°C is 4-12h, and the enzyme inactivation temperature is 70-80°C.
7. The high internal phase emulsion according to claim 1, characterized in that: In step (3), an ultrasonic dissolution-promoting operation is performed when curcumin is mixed with corn oil.
8. The high internal phase emulsion according to claim 1, characterized in that: In step (4), the volume fraction of the oil phase in the high internal phase emulsion system is 70%-85%.
9. The high internal phase emulsion according to claim 1, characterized in that: The homogenization treatment condition in step (4) is to homogenize the solution at a rotation speed of 8000-15000 rpm for 2-3 minutes.