Method for encapsulating anthocyanin based on modified protein nanofiber
Modified protein nanofibers were prepared by enzyme treatment and heating modification, and based on their embedding of anthocyanins, the problems of long preparation time, high energy consumption and browning in the prior art were solved, and anthocyanins delivery vectors with high efficiency load and improved stability were achieved.
Patent Information
- Application Number
- CN202510116756.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-24
- Publication Date
- 2025-05-06
AI Technical Summary
The existing preparation methods for protein nanofibers have a long time, high energy consumption and browning, which affects heating efficiency and makes it difficult to effectively build anthocyanin delivery vectors.
After the whey protein is treated with enzymes under specific conditions, it is heated and modified to obtain the modified protein fibers, and the anthocyanins are embedded based on the modified protein fibers to form an anthocyanins protective cavity to improve loading effect and stability.
The loading effect and stability of anthocyanins was significantly improved, and a new functional delivery vehicle of anthocyanins was successfully constructed, which improved the formation efficiency of protein fibers and reduced the energy consumption of the preparation process.
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Figure CN119924502A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of physical food processing, in particular to a method based on modified protein nanofibers encapsulating anthocyanins. Background Art
[0002] Due to the high aspect ratio, excellent mechanical properties and adjustable functional groups on the surface, self-assembly of food-derived proteins into amyloid fiber aggregates is an important means to improve and broaden the functional properties of food proteins, especially for constructing various delivery systems for bioactive compounds. Currently, whey protein with a purity of up to 90% can be obtained through ultrafiltration concentration and microfiltration defatting, which is mainly composed of α-lactalbumin, β-lactoglobulin and a small amount of other proteins. Compared with plant-derived proteins, whey protein has the advantages of high nutritional value, easy digestion and absorption, and can be used as capsule wall material. It is also gradually used as a carrier material to construct food delivery media for bioactive substances, and is used for the embedding and delivery of hydrophilic and hydrophobic functional factors.
[0003] Compared with natural whey protein, the conversion of whey protein into nanofibers by heat treatment can give it a high aspect ratio and significant amphiphilicity of the functional groups on the surface of the protein fiber. It can be used as a carrier for the delivery of functional factors and has multiple functional properties such as antioxidant and antibacterial activity. It is considered to be a modified milk protein raw material with development potential in the future food processing field. At present, the traditional method for preparing protein nanofibers generally adopts the heat-induced acid hydrolysis method. This method takes a long time to form fibers, has high energy consumption and is accompanied by severe browning, which affects the heating efficiency. Therefore, exploring a new method for preparing protein nanofibers is important for promoting the function of excellent delivery carriers. Summary of the invention
[0004] The purpose of the present invention is to provide a method for encapsulating anthocyanins based on modified protein nanofibers to solve the problems existing in the above-mentioned prior art. By enzymatically treating whey protein under specific conditions and then heating it for modification, modified protein fibers are obtained. Based on the embedding of anthocyanins in the modified protein fibers, anthocyanin protection cavities can be formed, which significantly improves the loading effect and stability of anthocyanins, and successfully constructs a new type of anthocyanin functionalized delivery carrier.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The present invention provides a method for encapsulating anthocyanins based on modified protein nanofibers, comprising the following steps:
[0007] The whey protein solution is enzymatically hydrolyzed with pancreatic enzyme under alkaline conditions, and the enzymatic hydrolysis supernatant is freeze-dried to obtain a whey protein hydrolyzate;
[0008] After the whey protein hydrolysate is dissolved, it is adjusted to be acidic, and the supernatant is taken and subjected to a heating treatment to perform protein modification to obtain whey protein nanofibers;
[0009] After anthocyanin is dissolved, whey protein nanofibers are added to react to prepare anthocyanin-loaded nanocomplexes.
[0010] Optionally, the enzymatic hydrolysis comprises the following steps:
[0011] The whey protein solution was adjusted to pH 7.5-8.5, pancreatin was added, and heated at 37°C for 1 h, after which the enzyme was inactivated.
[0012] Optionally, the concentration of the whey protein solution is 1.0-2.0% w / v, and the concentration of the pancreatic enzyme is 0.2% w / v.
[0013] Optionally, the whey protein hydrolysate is dissolved into a solution with a concentration of 3.0-4.0% w / v, and then the pH is adjusted to 1.0-2.0, the supernatant is taken and filtered with a filter membrane, and then heated in a water bath at 80-100°C for 12 hours for protein modification to obtain the whey protein nanofibers.
[0014] Optionally, the mass ratio of the anthocyanin to the whey protein nanofibers is 1:(3-6).
[0015] Optionally, the reaction includes the following steps: adding whey protein nanofibers after the anthocyanin solution, adjusting the pH to 3.0-4.0, stirring at room temperature for 1-2 hours, and then subjecting the solution to 10000-20000 high-speed shearing for 3-5 minutes and 500-600W ultrasonic treatment for 3-5 minutes to obtain the whey protein nanofibers.
[0016] The invention also provides a nanocomposite loaded with anthocyanin, which is prepared by adopting the method.
[0017] The present invention also provides application of the method in preparing a delivery carrier for improving anthocyanin loading effect and stability.
[0018] The invention also provides application of the anthocyanin-loaded nanocomposite in food processing.
[0019] The present invention discloses the following technical effects:
[0020] The present invention improves the efficiency of protein fiber formation by enzymatic hydrolysis of whey protein with pancreatic enzymes under specific conditions and then combining it with heating modification; based on the large number of hydrophilic groups on the surface of the modified protein fiber, its binding with anthocyanins is increased to form an anthocyanin protection cavity, which significantly improves the loading effect and stability of anthocyanins, successfully constructs a new anthocyanin functional delivery carrier, and provides a new method and strategy for the application of whey protein and anthocyanins in the field of food processing. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0022] Figure 1 TEM image of whey protein nanofibers prepared without enzymatic pretreatment;
[0023] Figure 2 Transmission electron microscopy image of whey protein nanofibers prepared by enzymatic pretreatment (trypsin concentration of 0.2%);
[0024] Figure 3 Transmission electron microscopy image of whey protein nanofibers prepared by enzymatic pretreatment (trypsin concentration is 2%);
[0025] Figure 4 Scanning electron microscopy image of whey protein nanofibers combined with anthocyanins prepared by enzymatic method (trypsin concentration of 0.2%);
[0026] Figure 5 Thermal stability determination results of anthocyanin-loaded nanocomposite samples prepared for different groups. DETAILED DESCRIPTION
[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0032] Example 1
[0033] 1. Enzymatic pretreatment method for preparing protein fiber
[0034] First, a whey protein solution (1.0%, w / v) was prepared, and the solution pH was adjusted to 7.5 using 1.0M NaOH, and heated in a 37°C water bath. After the temperature stabilized, a certain amount of pancreatin (0.2%, w / v) (250U / mg) was added for pretreatment, heated at 37°C for 1h, and then heated in an 80°C water bath for 15min to inactivate the pancreatin. The supernatant was cooled to room temperature, centrifuged at 3000r / min for 20min, and freeze-dried to obtain a whey protein hydrolysate. The whey protein hydrolysate was dissolved (3.0%, w / v), the solution pH was adjusted to 1.0 using 1.0MHCl, and placed at 4°C overnight to ensure full hydration. The solution was centrifuged at 8000g for 20min. The supernatant was aspirated and undissolved protein was removed using a 0.45μm regenerated cellulose filter under vacuum conditions. Subsequently, the whey protein nanofiber (WPN) solution was obtained by heating in a conventional water bath at 90°C for 12 h under gentle stirring conditions, and then the WPN solution was immediately cooled to room temperature using an ice bath. The obtained fibrillated WPN solution was stored at 4°C or freeze-dried at -20°C for later use.
[0035] 2. Preparation of anthocyanin nanocomposites
[0036] Anthocyanins were dissolved in deionized water and filtered through a 0.22 μm filter to remove large insoluble impurities. Then, they were added to the continuously stirred sample solution to make the mass ratio of anthocyanins to whey protein nanofibers 1:3. The pH value was adjusted to 3.0 using hydrochloric acid (0.1 M). The mixture was stirred for 1 h at room temperature and sheared at 10,000 rpm for 3 min. The sample was then ultrasonically treated for 3 min at 500 W using a 13 mm ultrasonic probe in an ice bath to obtain an anthocyanin-loaded nanocomposite.
[0037] Example 2
[0038] 1. Enzymatic pretreatment method for preparing protein fiber
[0039] First, a whey protein solution (1.5%, w / v) was prepared, and the solution pH was adjusted to 8.5 using 1.0M NaOH, and heated in a 37°C water bath. After the temperature stabilized, a certain amount of pancreatin (0.2%, w / v) (250U / mg) was added for pretreatment, heated at 37°C for 1h, and then heated in an 80°C water bath for 15min to inactivate the pancreatin. The supernatant was cooled to room temperature, centrifuged at 3000r / min for 20min, and freeze-dried to obtain a whey protein hydrolysate. The whey protein hydrolysate was dissolved (4.0%, w / v), the solution pH was adjusted to 1.5 using 1.0MHCl, and placed at 4°C overnight to ensure full hydration. The solution was centrifuged at 8000g for 20min. The supernatant was aspirated and undissolved protein was removed using a 0.45μm regenerated cellulose filter under vacuum conditions. Subsequently, the solution was heated in a conventional water bath at 100°C for 12 h under gentle stirring to obtain a whey protein nanofiber (WPN) solution, and then the WPN solution was immediately cooled to room temperature using an ice bath. The obtained fibrillated WPN solution was stored at 4°C or freeze-dried at -20°C for later use.
[0040] 2. Preparation of anthocyanin nanocomposites
[0041] Anthocyanins were dissolved in deionized water and filtered through a 0.22 μm filter to remove large insoluble impurities. Then, the solution was added to the stirred sample solution to make the mass ratio of anthocyanins to whey protein nanofibers 1:4. The pH value was adjusted to 3.5 using hydrochloric acid (0.1 M). The solution was stirred for 1 h at room temperature and sheared at 20,000 rpm for 5 min. The sample was then ultrasonically treated for 5 min at 600 W using a 13 mm ultrasonic probe in an ice bath to obtain an anthocyanin-loaded nanocomposite.
[0042] Example 3
[0043] 1. Enzymatic pretreatment method for preparing protein fiber
[0044] First, a whey protein solution (2.0%, w / v) was prepared, and the solution pH was adjusted to 7.8 using 1.0M NaOH, and heated in a 37°C water bath. After the temperature stabilized, a certain amount of pancreatin (0.2%, w / v) (250U / mg) was added for pretreatment, heated at 37°C for 1h, and then heated in an 80°C water bath for 15min to inactivate the pancreatin. The supernatant was cooled to room temperature, centrifuged at 3000r / min for 20min, and the supernatant was freeze-dried to obtain a whey protein hydrolysate. The whey protein hydrolysate was dissolved (3.5%, w / v), the solution pH was adjusted to 2.0 using 1.0M HCl, and placed at 4°C overnight to ensure full hydration. The solution was centrifuged at 8000g for 20min. The supernatant was drawn and undissolved protein was removed using a 0.45μm regenerated cellulose filter under vacuum conditions. Subsequently, the whey protein nanofiber (WPN) solution was obtained by heating in a conventional water bath at 80°C for 12 h under gentle stirring conditions, and then the WPN solution was immediately cooled to room temperature using an ice bath. The obtained fibrillated WPN solution was stored at 4°C or freeze-dried at -20°C for later use.
[0045] In addition, two control groups were set up:
[0046] (1) Blank group: whey protein nanofibers prepared without enzymatic pretreatment;
[0047] (2) Control group: 2.0% w / v pancreatic enzyme was used for enzymatic hydrolysis, and the other steps remained unchanged.
[0048] 2. Preparation of anthocyanin nanocomposites
[0049] Anthocyanins were dissolved in deionized water and filtered through a 0.22 μm filter to remove large insoluble impurities. Then, they were added to the continuously stirred sample solution to make the mass ratio of anthocyanins to whey protein nanofibers 1:6. The pH value was adjusted to 3.5 using hydrochloric acid (0.1 M), and the mixture was stirred for 1 h at room temperature and sheared at 10,000 rpm for 5 min. The sample was then ultrasonically treated for 5 min at 540 W using a 13 mm ultrasonic probe in an ice bath to obtain an anthocyanin-loaded nanocomposite.
[0050] At the same time, the following two groups were set up as control groups:
[0051] The anthocyanin solution was mixed with deionized water according to the above ratio to obtain a solution of the same concentration as the blank group;
[0052] The whey protein nanofibers loaded with anthocyanins prepared without 0.2% enzyme treatment were used as the control group.
[0053] 3. Determination of total anthocyanin content
[0054] The anthocyanin content was determined using the pH differential method. The anthocyanin-loaded nanocomposite samples were diluted to appropriate multiples in pH 1.0 (0.025 M) and pH 4.5 (0.4 M) buffers, respectively. After 10 min of equilibrium, the absorbance was measured at 520 nm and 700 nm, and distilled water was used as a control. The total anthocyanin content C (mg / L) was calculated according to formulas (1) and (2):
[0055] A=ApH 1.0 (A 520nm -A 700nm )-ApH 4.5 (A 520nm -A 700nm ) (1);
[0056]
[0057] Where:
[0058] ApH 1.0 The λmax of the sample to be tested is diluted into pH 1.0 buffer;
[0059] ApH 4.5 Dilute the λmax of the sample to be tested into pH 4.5 buffer;
[0060] Mw is the molar molecular weight of cyanidin-3-O-glucoside (C3G) (449.2 g / mol);
[0061] DF is the sample dilution factor;
[0062] ε is the molar extinction coefficient of cyanidin-3-O-glucoside (C3G) (26900 L / mol·cm -1 );
[0063] L is the optical path length (1 cm).
[0064] 4. Determination of anthocyanin encapsulation efficiency
[0065] The encapsulation efficiency (EE) was determined by ultrafiltration centrifugation. An appropriate amount of anthocyanin-loaded nanocomposite sample was placed in the inner tube of an ultrafiltration centrifuge tube with a molecular cutoff of 3 kDa, and centrifuged at 5000 g for 30 min at 4°C. After the centrifugation was completed, the free anthocyanin content in the filtrate was determined.
[0066] The encapsulation efficiency (%) is calculated as follows:
[0067]
[0068] Where:
[0069] TotalACNs: total amount of anthocyanins added to the prepared sample;
[0070] FreeACNs: amount of anthocyanins in the filtrate.
[0071] 5. Thermal stability determination of anthocyanin nanocomposites
[0072] The thermal stability test of anthocyanins was carried out at 60°C. The nanocomposite sample loaded with anthocyanins was placed in a glass sample bottle with a screw cap and heated in a 60°C water bath for 6 hours. Samples were taken every 1 hour to determine the anthocyanin retention rate in the sample. Free anthocyanins were used as blank controls. The anthocyanin content was determined by the pH differential method. The retention rate of anthocyanins was calculated according to formula (4):
[0073]
[0074] Among them, C0 and C t represent the anthocyanin contents at the initial stage and at time t, respectively.
[0075] 6. Transmission electron microscopy
[0076] 10 μL of diluted anthocyanin-loaded nanocomposite sample was dropped onto a common carbon support film (230 mesh), placed in a dry environment for 25 min, and then left overnight after absorbing excess moisture with filter paper. The microstructure of the sample was observed at an operating voltage of 80 kV. TEM images were analyzed using Nano Measurer software.
[0077] 7. Scanning electron microscope
[0078] The freeze-dried samples were observed using a field emission scanning electron microscope (FE-SEM). In order to better observe the sample morphology, the conductivity of the samples was enhanced by gold spraying. They were then fixed to the sample holder with conductive double-sided tape and analyzed in high vacuum mode.
[0079] 8. Results and Analysis
[0080] (1) Linear whey protein nanofibers were successfully prepared by enzymatic pretreatment. The entanglement between protein fibers was low and a relatively stable dispersion state was maintained (see Figure 2 However, excessive enzyme modification (2.0%) destroyed some fiber structures and formed a small amount of aggregates (see Figure 3 The preparation method provided by the present invention has a process efficiency that is 50% higher than that of the traditional heating treatment method (requiring treatment for 24 hours) in terms of the amount of protein fibers formed (see Figure 1 ).
[0081] (2) The hydrophilic functional groups on the surface of whey protein nanofibers help promote their binding with anthocyanins, with an encapsulation efficiency of 58.8%, further forming an anthocyanin protection cavity (see Figure 4 ), reducing the contact between anthocyanins and other food components in the external environment, delivering anthocyanins more efficiently, and the thermal degradation retention rate increased by 39.4% compared with blank anthocyanins (see Figure 5 ), which effectively improved the thermal stability of anthocyanins.
[0082] The method for preparing anthocyanin-loaded nanocomposite provided by the present invention utilizes the advantages of enzymatic modification, such as specificity, mildness and ease of control, and can specifically modify the molecular structure of whey protein within a moderate hydrolysis degree range through enzymatic hydrolysis, thereby shortening the preparation time and improving the efficiency of protein fiber formation. The hydrophilic functional groups on the surface of the protein fiber formed by enzymatic hydrolysis and modification help promote its combination with compounds such as anthocyanin, further forming an anthocyanin protection cavity, improving the loading effect and stability of anthocyanin, successfully constructing a new anthocyanin functionalized delivery carrier, and providing a new method for the utilization of whey protein and anthocyanin in the field of food processing.
[0083] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for encapsulating anthocyanins based on modified protein nanofibers, characterized in that: The following steps are involved: The whey protein solution is enzymatically hydrolyzed with pancreatic enzyme under alkaline conditions, and the enzymatic hydrolysis supernatant is freeze-dried to obtain a whey protein hydrolyzate; After the whey protein hydrolysate is dissolved, it is adjusted to be acidic, and the supernatant is taken and subjected to a heating treatment to perform protein modification to obtain whey protein nanofibers; After anthocyanin is dissolved, whey protein nanofibers are added to react to prepare anthocyanin-loaded nanocomplexes.
2. The method according to claim 1, characterized in that The enzymatic hydrolysis comprises the following steps: The whey protein solution was adjusted to pH 7.5-8.5, pancreatin was added, and heated at 37°C for 1 h, after which the enzyme was inactivated.
3. The method according to claim 2, characterized in that The concentration of the whey protein solution is 1.0-2.0% w / v, and the concentration of the pancreatic enzyme is 0.2% w / v.
4. The method according to claim 1, characterized in that The whey protein hydrolysate is dissolved into a solution with a concentration of 3.0-4.0% w / v, and then the pH is adjusted to 1.0-2.
0. The supernatant is taken and filtered with a filter membrane, and then placed in a water bath at 80-100° C. and heated for 12 hours for protein modification to obtain the whey protein nanofibers.
5. The method according to claim 1, characterized in that The mass ratio of the anthocyanin to the whey protein nanofibers is 1:(3-6).
6. The method according to claim 1, characterized in that The reaction comprises the following steps: adding whey protein nanofibers to the anthocyanin solution, adjusting the pH to 3.0-4.0, stirring at room temperature for 1-2 hours, and then subjecting the solution to 10000-20000 high-speed shearing for 3-5 minutes and 500-600W ultrasonic treatment for 3-5 minutes to obtain the whey protein nanofibers.
7. A nanocomposite loaded with anthocyanins, characterized in that: The invention discloses a novel novel nanostructured carbon foam. The nanostructured carbon foam is prepared by the method described in any one of claims 1 to 6.
8. Use of the method according to any one of claims 1 to 6 in preparing a delivery carrier that improves anthocyanin loading effect and stability.
9. Use of the anthocyanin-loaded nanocomposite as claimed in claim 7 in food processing.