Electrostatic self-assembled protein fiber hydrogel and preparation method thereof

Abstract

The invention discloses an electrostatic self-assembled protein fiber hydrogel and a preparation method thereof. The invention adopts a subcritical hydrothermal synthesis method to fibrosize whey protein isolate to form whey protein isolate nanofibers, which are then mixed with curcumin to form a curcumin-whey protein isolate nanofiber mixed solution, and then a pectin solution is added and stirred to obtain an electrostatic self-assembled protein fiber hydrogel. The preparation method of the invention is simple, the average particle size of the obtained hydrogel is small, the transmittance is moderate, the stability is high, and the efficient embedding and delivery of the active substance curcumin can be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of protein hydrogel preparation, and relates to an electrostatic self-assembled protein fiber hydrogel and a preparation method thereof. Background Art

[0002] Curcumin (Cur) is a hydrophobic polyphenol isolated from the root of traditional herbal medicine, which has multiple physiological activities such as antioxidant, anticancer, antibacterial, antifungal and anti-inflammatory. However, curcumin has low bioavailability due to its low chemical stability, poor water solubility and poor storage stability. In order to improve the water solubility, stability and bioavailability of curcumin, many researchers have encapsulated curcumin in delivery systems for gastrointestinal delivery.

[0003] Hydrogel is a polymer material that can retain a large amount of water or biological fluids within its structure. It is a three-dimensional cross-linked network structure that is not easily soluble. Natural hydrogels have poor mechanical properties, while protein-synthesized hydrogels have good mechanical properties, but they are fragile and easily degraded during gastrointestinal digestion.

[0004] Whey Protein Isolate Nanofiber (WPINs) is a material prepared by subcritical water device of whey protein isolate (WPI), which has obvious positive charge. In order to improve the stability of whey protein isolate-based hydrogel, some researchers use polysaccharides and proteins with different charges to form stable polymers to prepare hydrogels. Literature 1 prepares whey protein isolate-chitosan hydrogel by cross-linking whey protein isolate and chitosan with lignin. The release rate of the hydrogel in intestinal fluid is too slow, which is not conducive to the absorption of embedded bioactive substances (Food Hydrocolloids, 2020, 103: 105619). Literature 2 reports an elastic hydrogel made of whey protein isolate and xanthan gum, which has strong high thermal stability, but its consistency is too high and cannot be applied to mass production in food factories (Journal of Food Process Engineering, 2021, 44 (8): e13751). Summary of the invention

[0005] The purpose of the present invention is to provide an electrostatic self-assembled protein fiber hydrogel and a preparation method thereof. The present invention utilizes electrostatic self-assembly technology to assemble positively charged whey protein isolate nanofibers and negatively charged pectin to form a polymer hydrogel, thereby solving the problems of low mechanical strength and easy degradation of single protein hydrogels. The obtained hydrogel has a stable structure and encapsulates the active substance curcumin, thereby achieving efficient embedding of curcumin and having good stability, thereby achieving safe and stable delivery of curcumin.

[0006] The technical solution for achieving the purpose of the present invention is as follows:

[0007] The preparation method of electrostatic self-assembled protein fiber hydrogel, the specific steps are as follows:

[0008] (1) dissolving whey protein isolate in deionized water at a mass ratio of 2% to 5%:1, adjusting the pH to 2.0 to 4.0, and filtering to obtain a whey protein isolate solution;

[0009] (2) heating the whey protein isolate solution in a subcritical water apparatus for 120 min to obtain a whey protein isolate nanofiber (WPINs) solution;

[0010] (3) adding a curcumin ethanol solution having a concentration of 1 to 5 mg / mL to the whey protein isolate nanofiber solution at a volume ratio of 1:40 to 1:50 to obtain a curcumin-whey protein isolate nanofiber mixed solution;

[0011] (4) dissolving pectin (P) in deionized water at a mass ratio of 2±0.5%:1 to obtain a pectin solution;

[0012] (5) The curcumin-whey protein isolate nanofiber mixed solution and the pectin solution were mixed and stirred for 1 to 5 hours at a volume ratio of 1:3 to 1:4 to obtain an electrostatically self-assembled protein fiber hydrogel.

[0013] Preferably, in step (1), the pore size of the filter membrane used for filtration is 0.45 μm.

[0014] Preferably, in step (2), the temperature at which the subcritical water device is heated is 110°C.

[0015] Preferably, in step (1), the mass ratio is 5%:1, and the pH is adjusted to 2.0; in step (3), the volume ratio is 1:50, and the ethanol solution of curcumin is 1 mg / mL; in step (4), the mass ratio is 2%:1; in step (5), the volume ratio is 1:4, and the stirring time is 2 hours.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The whey protein isolate, pectin and curcumin used in the present invention are all biodegradable green materials, safe and environmentally friendly, and the protein fiber hydrogel is prepared by electrostatic self-assembly, which is simple and easy. The protein fiber hydrogel prepared by the present invention has a small average particle size (nanometer level), moderate light transmittance, high stability, and the embedding rate of curcumin can reach up to 96.17%, realizing stable and efficient embedding delivery of active substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a transmittance diagram of the hydrogel prepared in Example 1;

[0019] Figure 2 The particle size potential diagram of the hydrogels prepared in Example 2 and Comparative Examples 1-3;

[0020] Figure 3 Transmission electron microscopy (TEM) images of the hydrogels prepared in Example 2 and Comparative Examples 1-3;

[0021] Figure 4 The XRD patterns of the hydrogels prepared in Example 2 and Comparative Examples 1-3;

[0022] Figure 5 The thermal stability change diagram of the hydrogels prepared in Example 2 and Comparative Examples 1-3;

[0023] Figure 6 The figure is a graph showing the encapsulation efficiency of curcumin in the hydrogels prepared in Example 3 and Comparative Example 4. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0025] Example 1

[0026] Whey protein isolate was dissolved in deionized water at a mass ratio of 5%:1 to prepare a solution with a concentration of 5%. The pH value of the solution was adjusted to 2.0 and filtered through a 0.45μm filter membrane to obtain a whey protein isolate solution. The whey protein isolate solution was placed in a subcritical water device and heated at 110°C for 90, 120, and 180 minutes to obtain a whey protein isolate nanofiber solution. Pectin was dissolved in deionized water at a mass ratio of 0% to 5%:1 to prepare pectin solutions with a mass concentration of 0% to 5%. The whey protein isolate nanofiber solution and the pectin solution were mixed and stirred for 2 hours at a volume ratio of 1:4 to obtain a protein fiber hydrogel, which was named WPINs-90-PH, WPINs-120-PH, and WPINs-180-PH series according to the heating time. The transmittance of each protein fiber hydrogel was tested, and the results are as follows Figure 1As shown in the figure, it can be seen that with the increase of pectin concentration, the transmittance of the hydrogel increases first and then decreases, reaching the highest when the pectin concentration is 1%. Since the subsequent embedding experiment of curcumin is required, the transmittance should not be too high or too low, so a pectin concentration of 2% is selected for subsequent experiments.

[0027] Example 2

[0028] The whey protein isolate solution was dissolved in deionized water at a mass ratio of 5%:1 to prepare a 5% solution, the pH value of the solution was adjusted to 2.0, and the solution was filtered through a 0.45 μm filter membrane to obtain a whey protein isolate solution. The whey protein isolate solution was placed in a subcritical water device and heated at 110°C for 120 minutes to obtain a whey protein isolate nanofiber solution. Pectin was dissolved in deionized water at a mass ratio of 2%:1 to prepare a 2% pectin solution. The whey protein isolate nanofiber solution and the pectin solution were mixed and stirred for 2 hours at a volume ratio of 1:4 to obtain the protein fiber hydrogel WPINs-120-PH.

[0029] The particle size and potential of the hydrogel were tested, and the results are as follows: Figure 2 As shown in the figure, it can be seen that the particle size of WPINs-120-PH is 659nm and the potential is -30mV. Figure 3 From the transmission electron microscopy image, it can be seen that the fibrosis state is multi-branched, moderately long, and has the largest binding capacity with pectin. Figure 4 The XRD pattern shows that WPINs-120-PH forms two distinguishable peaks at 2θ, which are 26.7-27.1° and 39.2-40.8°, indicating the partial crystalline structure of whey protein isolate nanofibers and pectin. The protein fiber hydrogel WPINs-120-PH was heated at 30℃, 60℃, and 90℃ for 30min to test its thermal stability. The results are shown in Figure 5 As shown in the figure, it can be seen that the overall particle size has the smallest variation range and the best stability at different temperatures.

[0030] Comparative Example 1

[0031] This comparative example is basically the same as Example 2, except that the whey protein isolate solution is placed in a subcritical water device and heated at 110° C. for 90 min, and the obtained protein fiber hydrogel is WPINs-90-PH.

[0032] The particle size and potential of the hydrogel were tested, and the results are as follows: Figure 2 As shown in the figure, it can be seen that the particle size of WPINs-90-PH is 728nm and the potential is -17.2mV. Figure 3From the transmission electron microscopy images, it can be seen that the protein fibers of WPINs-90-PH are curved, have few branches, and have the least binding with pectin. Figure 4 The XRD pattern shows that WPINs-90-PH forms a distinguishable peak at 2θ, which is 26.7-27.1°, indicating the partial crystalline structure of whey protein isolate nanofibers. The protein fiber hydrogel WPINs-90-PH was heated at 30℃, 60℃, and 90℃ for 30min to test its thermal stability. The results are as follows Figure 5 As shown in the figure, it can be seen that after heating, the particle size increases and the stability is poor.

[0033] Comparative Example 2

[0034] This comparative example is basically the same as Example 2, except that the whey protein isolate solution is placed in a subcritical water device and heated at 110° C. for 180 min, and the obtained protein fiber hydrogel is WPINs-180-PH.

[0035] The particle size and potential of the hydrogel were tested, and the results are as follows: Figure 2 As shown in the figure, it can be seen that the particle size of WPINs-180-PH is 683nm and the potential is -24.4mV. Figure 3 From the transmission electron microscopy image, it can be seen that the fibrosis state is too branched, but the amount of pectin combined is small. Figure 4 The XRD pattern shows that WPINs-180-PH forms two distinguishable peaks at 2θ, which are 26.7-27.1° and 39.2-40.8°, indicating the partial crystalline structure of whey protein isolate nanofibers and pectin. The protein fiber hydrogel WPINs-180-PH was heated at 30℃, 60℃, and 90℃ for 30min to test its thermal stability. The results are shown in Figure 5 As shown in the figure, it can be seen that after heating, the particle size increases and the stability is poor.

[0036] Comparative Example 3

[0037] This comparative example is basically the same as Example 2, except that the whey protein isolate solution is not subjected to subcritical hydrothermal treatment, and the whey protein isolate solution and the pectin solution are directly mixed and stirred at a volume ratio of 1:4 for 2 hours to obtain the protein hydrogel WPI-PH.

[0038] The particle size and potential of the hydrogel were tested, and the results are as follows: Figure 2 As shown in the figure, it can be seen that the particle size of WPI-PH is 691nm and the potential is -21.2mV. Figure 3 From the transmission electron microscopy image, it can be seen that the whey protein isolate is in the form of dots and has a small amount of binding with pectin. Figure 4The XRD pattern shows that there is no distinguishable peak at 2θ. The protein fiber hydrogel WPI-PH was heated at 30℃, 60℃, and 90℃ for 30min to test its thermal stability. Figure 5 As shown in the figure, it can be seen that after heating at 90°C, the particle size increases significantly and the stability is poor.

[0039] Example 3

[0040] Dissolve whey protein isolate in deionized water at a mass ratio of 5%:1 to prepare a 5% solution, adjust the pH value of the solution to 2.0, and filter through a 0.45μm filter to obtain a whey protein isolate solution. After the whey protein isolate solution is placed in a subcritical water device and heated at 110°C for 120 minutes, a whey protein isolate nanofiber solution is obtained. Dissolve curcumin in ethanol to prepare a curcumin-ethanol solution with a concentration of 1 mg / mL. Add the curcumin-ethanol solution to the whey protein isolate nanofiber solution at a volume ratio of 1:50 to obtain a curcumin-whey protein isolate nanofiber mixed solution. Dissolve pectin in deionized water at a mass ratio of 2%:1 to prepare a pectin solution with a concentration of 2%. The curcumin-whey protein isolate nanofiber mixed solution and the pectin solution were mixed and stirred for 2 h at a volume ratio of 1:4 to obtain a protein fiber hydrogel loaded with curcumin (WPINs-120-PH-Cur). The embedding efficiency of curcumin was measured to be 96.17%. Figure 6 As shown, it is shown that the electrostatic self-assembled protein fiber hydrogel prepared by the present invention can be used as a delivery carrier of bioactive substances to improve the solubility and bioavailability of bioactive substances.

[0041] Comparative Example 4

[0042] This comparative example is basically the same as Example 3, except that the whey protein isolate solution was not subjected to subcritical hydrothermal treatment, and the curcumin-whey protein isolate mixed solution and the pectin solution were directly mixed and stirred at a volume ratio of 1:4 for 2 hours to obtain a protein hydrogel loaded with curcumin (WPI-PH-Cur). The embedding efficiency of curcumin was measured to be 31.02%, as shown in FIG. Figure 6 As shown, the embedding rate is low.

Claims

1. A method for preparing an electrostatically self-assembled protein fiber hydrogel, characterized in that: The specific steps are as follows: (1) dissolving whey protein isolate in deionized water at a mass ratio of 2% to 5%:1, adjusting the pH to 2.0 to 4.0, and filtering to obtain a whey protein isolate solution; (2) heating the whey protein isolate solution in a subcritical water device for 120 min to obtain a whey protein isolate nanofiber solution; (3) adding a curcumin ethanol solution having a concentration of 1 to 5 mg / mL to the whey protein isolate nanofiber solution at a volume ratio of 1:40 to 1:50 to obtain a curcumin-whey protein isolate nanofiber mixed solution; (4) dissolving pectin in deionized water at a mass ratio of 2±0.5%:1 to obtain a pectin solution; (5) The curcumin-whey protein isolate nanofiber mixed solution and the pectin solution were mixed and stirred for 1 to 5 hours at a volume ratio of 1:3 to 1:4 to obtain an electrostatically self-assembled protein fiber hydrogel.

2. The preparation method according to claim 1, characterized in that: In step (1), the pore size of the filter membrane used for filtration is 0.45 μm.

3. The preparation method according to claim 1, characterized in that: In step (2), the temperature of the subcritical water device heating is 110°C.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio is 5%:1, and the pH is adjusted to 2.0; in step (3), the volume ratio is 1:50, and the ethanol solution of curcumin is 1 mg / mL; in step (4), the mass ratio is 2%:1; in step (5), the volume ratio is 1:4, and the stirring time is 2 hours.

5. The electrostatic self-assembled protein fiber hydrogel prepared according to the preparation method according to any one of claims 1 to 4.

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