Method for preparing resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel and application thereof

Abstract

The application discloses a preparation method and application of resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel, and first obtains amyloid fibril by heating ovalbumin under acid-heat conditions, then is combined with chitosan and resveratrol, adjusts the pH value, and the self-assembled hydrogel can be obtained in a static state. The method for preparing the composite hydrogel loaded with bioactive molecules through the self-assembly characteristics of amyloid fibril is simple in process operation, and no organic cross-linking reagent is additionally added in the preparation process, so that the safety is safe and no toxic side effect is caused; the prepared hydrogel has the advantages of good stability, high loading rate and good biocompatibility, and can be applied to the fields of food, medicine, health care product and cosmetic, etc.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of hydrogel, and particularly relates to a preparation method of resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel and application thereof. BACKGROUND

[0002] Resveratrol is a common dietary polyphenolic compound with a wide range of biological and pharmacological activities. Studies have found that it has the functions of anti-tumor, anti-virus, anti-platelet aggregation, anti-atherosclerosis, protection of cardiovascular and cerebrovascular system, and low toxicity and small adverse reactions. However, resveratrol has poor water solubility, unstable properties, is easy to isomerize under ultraviolet light, and is easy to oxidize under strong acid, strong base, light and high temperature, which seriously limits the bioavailability of resveratrol. Therefore, it is necessary to develop a drug delivery system that can protect and stabilize resveratrol to improve its bioavailability.

[0003] In recent years, nanoparticles, molecular complexes, microcapsules, hydrogels and emulsions have been widely used as delivery systems for nutrients in the food industry, biomedicine and light textile industry. Among them, hydrogel has a dense network structure and can accommodate drugs or nutrients, and is an ideal delivery carrier for drugs and nutrients. In addition, the swelling rate and permeability of protein gel at different pH values are different. When the hydrophilic gel embedding the bioactive substances enters the body, the active substances will be slowly released from the inside of the gel under the double power of self-diffusion and degradation of the protein gel, so as to achieve the effect of slow release. Natural biological macromolecules (such as ovalbumin and chitosan) not only have high nutritional value, but also have many functional properties, and have been widely used as raw materials in hydrogel-based delivery systems. Ovalbumin is a protein with good water solubility, good digestibility, excellent self-assembly and amphiphilic properties, and high nutritional value, accounting for 54%-69% of total egg white protein. The hydrophobic amino acids of ovalbumin account for 50% of the total, and 33% are charged, which is the reason why it can be used as a lipophilic carrier. In addition, ovalbumin can form protein nanofibers under certain conditions, which are linear structures formed by the spontaneous self-assembly of protein molecules or partial structures, also known as protein amyloid fibers. After the protein forms fibrous aggregates, the gelation, emulsification and foam stability of the protein are significantly improved. Chitosan is the only cationic polysaccharide in nature, and is a good stabilizer and thickener.

[0004] In food processing, the formation of protein gel is often induced by transglutaminase (MTGase), but the efficacy of MTGase often depends on the accessibility of glutamine and lysine residues, which can be buried inside the protein and are not conducive to the formation of protein hydrogel. Chemical crosslinking is another method to induce protein gel, but most high-performance chemical crosslinking agents, such as glutaraldehyde, are not suitable for application in the food industry due to potential toxicity. Residual organic solvents can easily lead to potential safety and health hazards. Therefore, it is necessary to design and develop a new method for preparing biocompatible and non-toxic hydrogels. SUMMARY

[0005] The present application aims to provide a preparation method of resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel and its application, which solves the problems of poor stability, low embedding rate and low bioavailability of resveratrol in existing loading technologies.

[0006] The preparation method of resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel of the present application first heats ovalbumin under acid-heat conditions for a long time to obtain amyloid fibrils, then composites with chitosan and resveratrol, adjusts the pH value, and obtains self-assembled hydrogel in a stationary state through molecular self-assembly.

[0007] The preparation method of resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel of the present application comprises the following steps:

[0008] Step 1: Dissolve ovalbumin powder in deionized water to obtain an ovalbumin solution with a concentration of 10-20 mg / mL, place it at 4℃ overnight for sufficient hydration, adjust the pH value of the system to 2.0 with 1 mol / L hydrochloric acid, then heat the ovalbumin solution at 85℃ under magnetic stirring at 200 rpm for 24 h at pH 2.0 to obtain an ovalbumin fibril solution;

[0009] Step 2: Mix chitosan with 2% (v / v) acetic acid solution, continuously stir uniformly under magnetic stirring at 200 rpm to obtain a chitosan solution with a mass concentration of 1%-3% (w / v);

[0010] Step 3: Dissolve resveratrol powder in a certain volume of absolute ethanol to obtain a resveratrol dispersion;

[0011] Step 4: mixing the egg white fibril solution and the chitosan solution to obtain a blending solution at a volume ratio of 5-15:1, adding a certain volume of resveratrol dispersion into the blending solution, the final blending solution has an ethanol concentration of not more than 2% (v / v) and a resveratrol concentration of 10-50 μM; then adjusting the pH value of the complex solution to 6-8, and standing overnight at 4-25℃ to obtain the resveratrol-loaded egg white fibril / chitosan composite hydrogel. In this step, the pH value has a great influence. Because the structure of the fibril is different in the pH range of 6-8, a higher pH value will cause the fibril to partially degrade, thus being not conducive to the structural stability thereof.

[0012] The resveratrol-loaded egg white fibril / chitosan composite hydrogel prepared by the method of the present application is used as a resveratrol-loaded composite hydrogel delivery system.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] 1. The present application first proposes to use egg white fibrils and chitosan to form a resveratrol-loaded hydrogel delivery system, which is conducive to the efficient use of egg white and improves the added value of egg white.

[0015] 2. The present application uses food protein as a raw material, forms amyloid fibrils by heating under acidic conditions, and the prepared fibrils have no obvious toxic side effects on cells; in addition, the method does not need to add additional chemical cross-linking agents, thus ensuring the safety of the product.

[0016] 3. The present application uses the self-assembly characteristics of egg white fibrils to form a gel structure without introducing any organic cross-linking agent, and adds cationic chitosan to stabilize the hydrogel, thereby significantly increasing the embedding rate and stability of resveratrol, realizing efficient encapsulation and controlled release of resveratrol; and the resveratrol delivery system prepared by the present application uses materials that are easy to obtain, the required equipment is easy to operate, and the product has superior safety performance, and is expected to be applied in the fields of food, medicine, health care products and cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The embedding rate of resveratrol in the hydrogels prepared in Example 1, Example 2, Example 3, Comparative Example 1 and the degradation rate of resveratrol after the hydrogels were stored at 4℃ for 4 weeks.

[0018] Figure 2 The release curve of resveratrol in the hydrogels prepared in Example 3 and Comparative Example 1 after simulated gastrointestinal digestion.

[0019] Figure 3 The microstructure of the hydrogels prepared in Example 3 and Comparative Example 1. DETAILED DESCRIPTION

[0020] Non-limiting embodiments are described as follows:

[0021] Example 1:

[0022] The ovalbumin powder was dissolved in deionized water to obtain a protein solution with a concentration of 10 mg / mL, which was placed at 4°C overnight for full hydration. The pH value of the protein solution was adjusted to 2.0 with 1 mol / L hydrochloric acid, and then the ovalbumin solution was heated at 85°C for 24 h under magnetic stirring at 200 rpm and at pH 2.0 to obtain an ovalbumin fibril solution. Chitosan was mixed with 2% (v / v) acetic acid solution under magnetic stirring at 200 rpm to obtain a chitosan solution with a mass concentration of 1% (w / v). The resveratrol powder was dissolved in a certain volume of absolute ethanol to obtain a resveratrol dispersion. The ovalbumin fibril solution and the chitosan solution were mixed at a volume ratio of 15:1 to obtain a blending solution, and a certain volume of resveratrol dispersion was added to the blending solution. The final ethanol concentration in the blending solution was not more than 2% (v / v), and the resveratrol concentration was 10 μM. Then the pH value of the composite solution was adjusted to 6, and the resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel was obtained after standing overnight at 25°C.

[0023] Example 2:

[0024] The ovalbumin powder was dissolved in deionized water to obtain a protein solution with a concentration of 15 mg / mL, which was placed at 4°C overnight for full hydration. The pH value of the protein solution was adjusted to 2.0 with 1 mol / L hydrochloric acid, and then the ovalbumin solution was heated at 85°C for 24 h under magnetic stirring at 200 rpm and at pH 2.0 to obtain an ovalbumin fibril solution. Chitosan was mixed with 2% (v / v) acetic acid solution under magnetic stirring at 200 rpm to obtain a chitosan solution with a mass concentration of 2% (w / v). The resveratrol powder was dissolved in a certain volume of absolute ethanol to obtain a resveratrol dispersion. The ovalbumin fibril solution and the chitosan solution were mixed at a volume ratio of 10:1 to obtain a blending solution, and a certain volume of resveratrol dispersion was added to the blending solution. The final ethanol concentration in the blending solution was not more than 2% (v / v), and the resveratrol concentration was 30 μM. Then the pH value of the composite solution was adjusted to 8, and the resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel was obtained after standing overnight at 4°C.

[0025] Example 3:

[0026] The ovalbumin powder was dissolved in deionized water to obtain a protein solution with a concentration of 20 mg / mL, which was placed at 4°C overnight for full hydration. The pH value of the protein solution was adjusted to 2.0 with 1 mol / L hydrochloric acid. The ovalbumin solution was then heated at 85°C for 24 hours under a magnetic stirring state at a pH of 2.0 at 200 rpm to obtain an ovalbumin fibril solution. Chitosan was mixed with a 2% (v / v) acetic acid solution and continuously stirred under a magnetic stirring state at 200 rpm to obtain a chitosan solution with a mass concentration of 3% (w / v). Resveratrol powder was dissolved in a certain volume of anhydrous ethanol to obtain a resveratrol dispersion. The ovalbumin fibril solution and the chitosan solution were mixed in a volume ratio of 5:1 to obtain a blended solution. A certain volume of resveratrol dispersion was added to the blended solution. The final ethanol concentration in the blended solution did not exceed 2% (v / v), and the resveratrol concentration was 50 μM. Then, the pH value of the composite solution was adjusted to 7, and the solution was allowed to stand at 4° C. overnight to obtain the resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel.

[0027] The preparation conditions of Examples 1-3 adopted in this application are based on the smooth formation of hydrogels. For the obtained hydrogel system, increasing the proportion of ovalbumin fibrils will improve the performance of the hydrogel.

[0028] Comparative Example 1:

[0029] Comparative Example 1 provides a method for preparing a resveratrol-loaded hydrogel, the formulation and preparation method of which are substantially the same as those of Example 3, except that no chitosan solution is added.

[0030] Depend on Figure 1 It can be seen that the resveratrol embedding efficiency in the ovalbumin fibril hydrogel prepared in Comparative Example 1 was 58.64%, and the degradation rate of resveratrol in the hydrogel was approximately 57.48% after storage at 4°C for 4 weeks. In contrast, the resveratrol embedding efficiency in the ovalbumin fibril / chitosan composite hydrogel prepared by the present invention reached a maximum of 94.33%. After storage at 4°C for 4 weeks, the degradation rate of resveratrol in the ovalbumin fibril / chitosan composite hydrogel decreased to approximately 13.74%. This indicates that the ovalbumin fibril / chitosan composite hydrogel prepared by the present invention can improve the embedding efficiency and stability of resveratrol.

[0031] Figure 2The release of resveratrol from the egg white fibrin hydrogel and the egg white fibrin / chitosan composite hydrogel during gastrointestinal digestion is shown. For the egg white fibrin hydrogel, about 45% of the resveratrol was released after 2 hours of digestion in simulated gastric fluid. Then after 2 hours of digestion in simulated intestinal fluid, the release rate of resveratrol was about 90%. While for the egg white fibrin / chitosan composite hydrogel, about 50% of the resveratrol was released from the composite hydrogel after 4 hours of digestion in simulated gastric fluid and simulated intestinal fluid. Therefore, the present application plays a sustained-release role for resveratrol in the in vitro simulated gastric fluid and intestinal fluid environment, which is conducive to the improvement of its bioavailability.

[0032] Figure 3 The microstructure images of the egg white fibrin hydrogel and the egg white fibrin / chitosan composite hydrogel are shown. It can be seen from the images that the microstructure of the egg white fibrin hydrogel prepared in Comparative Example 1 is loose, while when the cationic chitosan is added, it can play a role in stabilizing the structure of the egg white fibrin, so that the microstructure of the composite hydrogel is significantly different. Chitosan can change the gel properties of the gel by changing the structure of the gel, so that the network structure of the composite hydrogel is more compact. Therefore, the composite hydrogel with a dense structure can resist the degradation of hydrolytic enzymes, thereby achieving the protection and sustained release of resveratrol.

Claims

1.A method for preparing resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel, characterized in that: first, the ovalbumin is heated under acid-heat conditions to obtain amyloid fibrils, and then the amyloid fibrils are compounded with chitosan and resveratrol, and after adjusting the pH value, a self-assembled hydrogel is obtained in a static state, comprising the following steps: Step 1: Dissolve ovalbumin powder in deionized water to obtain an ovalbumin solution with a concentration of 10-20 mg / mL, and place it at 4 ℃ overnight to fully hydrate, then adjust the pH value of the system to 2.0 with hydrochloric acid, and then heat the ovalbumin solution under magnetic stirring to obtain an ovalbumin fibril solution; Step 2: Mix chitosan with acetic acid solution and continuously stir under magnetic stirring to obtain a chitosan solution; Step 3: Dissolve resveratrol powder in anhydrous ethanol to obtain a resveratrol dispersion; Step 4: Mix the ovalbumin fibril solution with the chitosan solution to obtain a blended solution, add a certain volume of resveratrol dispersion to the blended solution, then adjust the pH value of the composite solution to 6-8, and place it at 4-25 ℃ overnight to obtain a resveratrol-loaded ovalbumin fibril / chitosan composite hydrogel; In step 2, the volume concentration of the acetic acid solution is 2%, and the concentration of chitosan in the obtained chitosan solution is 1%-3%; In step 4, the volume ratio of the ovalbumin fibril solution to the chitosan solution is 5-15:1; In step 4, the concentration of ethanol in the obtained blended solution is not more than 2%, and the concentration of resveratrol is 10-50 μM. 2.The method according to claim 1, characterized in that: in step 1, the concentration of the ovalbumin solution is 10-20 mg / mL. 3.The method according to claim 1, characterized in that: in step 1, the ovalbumin solution is heated at 85 ℃ under magnetic stirring for 24 h at a pH value of 2.0 to obtain the ovalbumin fibril solution. Used as a resveratrol-loaded composite hydrogel delivery system. ​ ​ ​ ​ ​ ​ ​ ​ ​ 4. Use of the resveratrol-loaded ovalbumin fibrils / chitosan composite hydrogel prepared according to the method of any one of claims 1-3, characterized in that: ​

Images