Preparation method and application of ovalbumin fibril-resveratrol self-assembled hydrogel

Abstract

The application discloses a preparation method and application of an egg white protein fibril-resveratrol self-assembled hydrogel. First, egg white protein is heated under acid-heat conditions to obtain amyloid protein fibrils, then the amyloid protein fibrils are mixed with hydrophobic resveratrol, and after pH adjustment, the self-assembled hydrogel can be obtained by standing. The hydrogel is prepared by using egg white protein fibrils and resveratrol as raw materials and through non-covalent interaction self-assembly. The application firstly proposes that hydrophobic polyphenols and egg white protein fibrils are used to form a hydrogel through self-assembly, and the embedded resveratrol can trigger the formation of the fibril hydrogel; meanwhile, due to the good anti-digestive enzyme hydrolysis characteristics of the fibril hydrogel, the resveratrol is effectively self-released. The hydrogel prepared by the method can be applied to the fields of food and medicine such as tissue engineering, bioactive substance delivery carrier and wound healing.

Description

Technical Field

[0001] This invention belongs to the field of protein hydrogels, specifically relating to a method for preparing an ovalbumin fibrils-resveratrol self-assembled hydrogel and its application. Background Technology

[0002] Resveratrol (trans-3,4',5-trihydroxystilbene) is a non-flavonoid polyphenol mainly found in grapes, peanuts, and medicinal plants. It possesses various bioactive functions, including antioxidant, anti-aging, antibacterial, immunomodulatory, cardiovascular disease prevention, and anticancer effects. However, resveratrol is easily oxidized and destroyed during food production, storage, and digestion, resulting in extremely low bioavailability. Furthermore, its poor water solubility and chemical stability limit its application in functional foods.

[0003] In recent years, domestic and international research has shown that proteins are natural carriers of polyphenols. Proteins and polyphenols form stable complexes through interaction forces, which can improve the chemical stability and biological activity of polyphenols. The interaction between proteins and polyphenols is mainly through non-covalent forces such as hydrogen bonds, hydrophobic interactions, and ionic interactions, as well as covalent forces mediated by enzymatic or non-enzymatic oxidation of ortho-quinones. However, the binding efficiency of natural proteins to polyphenols is relatively weak, requiring protein modification to improve their polyphenol-binding capacity. Protein fibrillation aggregates, also known as protein amyloid fibrils, are highly ordered linear structures spontaneously formed by protein molecules or parts of their structures, and are widely found in nature. In vitro protein fibrillation aggregation can effectively enhance various protein properties, including antioxidant properties, emulsifying properties, foaming properties, and gelling properties. Furthermore, protein fibrils are also novel carriers for stabilizing and solubilizing nutrients, showing broad application prospects.

[0004] Various naturally derived proteins, such as β-lactoglobulin, whey protein, ovalbumin, and soy protein, form amyloid fibrils after acid-heat treatment and are used in nutrient delivery and food-grade emulsion preparation. Ovalbumin is a typical high-quality globulin, accounting for 54%-69% of total egg white protein. Composed of 385 amino acids, 50% are hydrophobic, while 33% are charged, which is why it can serve as a highly efficient lipophilic carrier. Ovalbumin is a protein with excellent water solubility, good digestibility, excellent self-assembly and amphiphilic properties, and high nutritional value, making it a highly promising biomaterial for the food industry.

[0005] Hydrogels are an important drug delivery system, exhibiting pH responsiveness and controlled-release properties. In food processing, glutaminase (MTG) enzymes are often used to induce protein gel formation; however, the efficacy of MTG enzymes often depends on the accessibility of glutamine and lysine residues, which may be deeply embedded within the protein, hindering hydrogel formation. Chemical cross-linking is a direct and effective method to improve protein gel properties; however, most high-performance chemical cross-linking agents, such as glutaraldehyde, are unsuitable for use in the food industry due to their potential toxicity. Therefore, it is necessary to design and develop novel hydrogel preparation methods that are biocompatible and non-toxic. Molecular self-assembly is an effective method for preparing hydrogels without the need for additional inducing agents or methods. Furthermore, food-derived amyloid protofibrils, with their high aspect ratio and non-cytotoxicity, are excellent materials for hydrogel preparation. Due to the abundant functional groups on the surface of the protofibrils, individual fibers can form supramolecular structures through molecular self-assembly. However, forming a stable hydrogel network from individual protein protofibrils remains challenging. Proteins, bioactive peptides, polyphenols, and nucleic acids are all potential self-assemblers in nature. The self-assembly properties of proteins and bioactive peptides have been extensively studied. However, the self-assembly properties of small-molecule polyphenols with complex supramolecular structures are often overlooked in materials science. The presence of catechol or pyrogallol moieties in polyphenols allows for strong intermolecular interactions with protein fibrils. However, utilizing the self-assembly of hydrophobic polyphenols with protein amyloid fibrils to form hydrogels, thereby achieving hydrogel network structure reconstruction and effective polyphenol release, remains a research gap. Summary of the Invention

[0006] To meet consumer demand for healthy and nutritious foods and to solve the aforementioned technical challenges, this invention provides a method for preparing an ovalbumin fibrillary-resveratrol self-assembled hydrogel and its applications. This invention utilizes the self-assembly of hydrophobic polyphenols with ovalbumin fibrillary fibers to form a hydrogel. The addition of the hydrophobic polyphenols triggers the formation of the ovalbumin fibrillary hydrogel; simultaneously, the resveratrol encapsulated in the hydrogel is effectively protected and released. Therefore, the hydrogel prepared by this method can be widely used in the food and pharmaceutical fields, including tissue engineering, bioactive substance delivery carriers, and wound healing.

[0007] This invention discloses a method for preparing an ovalbumin fibrils-resveratrol self-assembled hydrogel. First, ovalbumin is heated under acidic and hot conditions for an extended period to obtain amyloid fibrils. These are then mixed with hydrophobic resveratrol, and after pH adjustment, allowed to stand to obtain the self-assembled hydrogel. In the self-assembled hydrogel obtained by this invention, the protein fibrils have a large specific surface area and numerous surface-active groups, enabling them to stably bind with hydrophobic polyphenols through non-covalent interactions, thereby improving the water solubility, stability, and bioavailability of the hydrophobic polyphenols. The addition of resveratrol triggers the formation of the ovalbumin fibrils hydrogel; simultaneously, due to the good resistance to digestive enzyme hydrolysis in the fibrils hydrogel, resveratrol in the hydrogel can be effectively released spontaneously.

[0008] The present invention discloses a method for preparing an ovalbumin fibrils-resveratrol self-assembled hydrogel, comprising the following steps:

[0009] Step 1: Weigh the ovalbumin powder and mix it with deionized water to obtain an ovalbumin solution with a concentration of 10-30 mg / mL. Let it stand overnight at 4℃ to fully hydrate. Adjust the pH of the solution to 2-6 with 1 mol / L hydrochloric acid. Then, heat the ovalbumin solution at 85℃ and pH 2 for 24 hours under magnetic stirring at 200 rpm to obtain an ovalbumin fibrillary solution.

[0010] Step 2: Take resveratrol, add a certain amount of anhydrous ethanol, and stir until the resveratrol is completely dissolved to prepare a resveratrol dispersion;

[0011] Step 3: Mix the resveratrol dispersion with the ovalbumin fibrillary solution at a volume ratio of 5-20:1 to obtain a complex solution. Adjust the pH of the complex solution to 6-7 and let it stand overnight at 4-25℃ to obtain an ovalbumin fibrillary-resveratrol self-assembled hydrogel.

[0012] In step 3, the complex solution obtained by mixing the resveratrol dispersion with the egg white protein fibrillary solution has an ethanol concentration of 2% (v / v) and a resveratrol concentration of 10-50 μM.

[0013] The ovalbumin fibrils-resveratrol self-assembled hydrogel prepared in this invention is used as a composite hydrogel delivery system loaded with resveratrol.

[0014] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0015] 1. This invention uses food protein as raw material and forms amyloid protein fibrils by heating under acidic conditions. The prepared fibrils have no obvious toxic side effects on cells. In addition, the hydrogel prepared by this method does not require the addition of additional chemical cross-linking agents, thus ensuring the safety of the product.

[0016] 2. This invention utilizes hydrogen bonding and hydrophobic resveratrol to form a stable hydrogel through self-assembly, thereby solving the problems of low solubility and poor stability of resveratrol, and effectively overcoming the technical difficulties of easy oxidation and low bioavailability of resveratrol in food applications. The ratio of egg albumin fibrils to resveratrol and the pH value of the system have a significant impact on the formation of a stable hydrogel.

[0017] 3. The ovalbumin fibrils-resveratrol self-assembled hydrogel disclosed in this invention can trigger the formation of fibril hydrogels by embedding resveratrol (fibrils alone cannot form stable hydrogels; only by adding a certain amount of resveratrol can the two interact to form a stable hydrogel); at the same time, since the fibril hydrogel has good resistance to digestive enzyme hydrolysis, the resveratrol encapsulated in the hydrogel can be effectively released, ultimately achieving controlled drug release.

[0018] 4. The method disclosed in this paper for preparing ovalbumin fibrils-resveratrol self-assembled hydrogels is simple, low-cost, environmentally friendly, commercially viable, and suitable for large-scale production. It is expected to be applied in the food and pharmaceutical fields such as tissue engineering, bioactive substance delivery carriers, and wound healing. Attached Figure Description

[0019] Figure 1 Example 1 illustrates the effect of protein concentration on the rheological properties of ovalbumin fibrils-resveratrol self-assembled hydrogels.

[0020] Figure 2 Example 2 illustrates the effect of resveratrol concentration on the rheological properties of ovalbumin fibrils-resveratrol self-assembled hydrogels.

[0021] Figure 3 This is an image of the appearance of the ovalbumin fibrils-resveratrol self-assembled hydrogel from Example 3.

[0022] Figure 4 This refers to the non-covalent interactions involved in the assembly of the ovalbumin fibrils-resveratrol hydrogel in Example 3.

[0023] Figure 5 Example 3 shows the microstructure of the ovalbumin fibrils-resveratrol complex and hydrogel under pH 6.5 conditions.

[0024] Figure 6 The degradation curve of ovalbumin fibrils-resveratrol hydrogel in Example 3 during simulated gastrointestinal digestion is shown.

[0025] Figure 7The release curve of resveratrol in ovalbumin fibrils-resveratrol hydrogel during simulated gastrointestinal digestion is shown in Example 3. Detailed Implementation

[0026] The non-limiting implementation method is described below:

[0027] Example 1:

[0028] Ovalbumin powder was weighed and mixed with deionized water to obtain protein solutions with concentrations of 10, 15, 20, 25, and 30 mg / mL. These solutions were left to stand overnight at 4°C for full hydration. The pH of the solution was adjusted to 6 with 1 mol / L hydrochloric acid. The ovalbumin solution was then heated at 85°C and pH 2 for 24 hours under magnetic stirring at 200 rpm to obtain an ovalbumin fibrils solution. Resveratrol was taken, and a certain amount of anhydrous ethanol was added. The mixture was stirred until the resveratrol was completely dissolved to prepare a resveratrol dispersion. The resveratrol dispersion was mixed with the ovalbumin fibrils solution at a volume ratio of 20:1 to obtain a complex solution. The final complex solution had an ethanol concentration of 2% (v / v) and a resveratrol concentration of 50 μM. The pH of the complex solution was then adjusted to 6, and the solution was left to stand overnight at 4°C to obtain an ovalbumin fibrils-resveratrol self-assembled hydrogel.

[0029] The effect of protein concentration on the rheological properties of ovalbumin fibrils-resveratrol self-assembled hydrogels is shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that the G' and G" values ​​of all hydrogel samples increase with increasing angular frequency, exhibiting frequency-dependent behavior; in addition, the G' and G" values ​​of ovalbumin fibrils-resveratrol hydrogel increase with increasing ovalbumin concentration.

[0030] Example 2:

[0031] Ovalbumin powder was weighed and mixed with deionized water to obtain a protein solution with a concentration of 20 mg / mL. The solution was left to stand overnight at 4°C for full hydration. The pH of the solution was adjusted to 4 with 1 mol / L hydrochloric acid. The ovalbumin solution was then heated at 85°C for 24 hours with magnetic stirring at 200 rpm to obtain an ovalbumin fibrils solution. Resveratrol was taken and a certain amount of anhydrous ethanol was added, and the mixture was stirred until the resveratrol was completely dissolved to prepare a resveratrol dispersion. The resveratrol dispersion was mixed with the ovalbumin fibrils solution at a volume ratio of 10:1 to obtain a complex solution. The final complex solution had an ethanol concentration of 2% (v / v) and resveratrol concentrations of 10, 20, 30, 40, and 50 μM. The pH of the complex solution was then adjusted to 7, and the solution was left to stand overnight at 25°C to obtain an ovalbumin fibrils-resveratrol self-assembled hydrogel.

[0032] The effect of resveratrol concentration on the rheological properties of ovalbumin fibrils-resveratrol self-assembled hydrogels is shown in the results. Figure 2 .Depend on Figure 2 It can be seen that the G' and G" values ​​of all hydrogel samples increase with increasing angular frequency, exhibiting frequency-dependent behavior; in addition, the G' and G" values ​​of ovalbumin fibrils-resveratrol hydrogel increase with increasing resveratrol concentration.

[0033] Example 3:

[0034] Ovalbumin powder was weighed and mixed with deionized water to obtain a protein solution with a concentration of 20 mg / mL. The solution was left to stand overnight at 4°C for full hydration. The pH of the solution was adjusted to 2 with 1 mol / L hydrochloric acid. The ovalbumin solution was then heated at 85°C and pH 2 for 24 hours under magnetic stirring at 200 rpm to obtain an ovalbumin fibrils solution. Resveratrol was taken, and a certain amount of anhydrous ethanol was added. The mixture was stirred until the resveratrol was completely dissolved to prepare a resveratrol dispersion. The resveratrol dispersion was mixed with the ovalbumin fibrils solution at a volume ratio of 5:1 to obtain a complex solution. The final complex solution had an ethanol concentration of 2% (v / v) and a resveratrol concentration of 50 μM. The pH of the complex solution was then adjusted to 6.5, and the solution was left to stand overnight at 4°C to obtain an ovalbumin fibrils-resveratrol self-assembled hydrogel.

[0035] The appearance of the egg white protein fibrils-resveratrol hydrogel is shown in the image. Figure 3 .like Figure 3 As shown, the results indicate that a stable hydrogel (non-flowing after 12 hours of inversion) can only be formed when ovalbumin fibrils and resveratrol are present simultaneously. Figure 3 It can be seen that the protofibrils alone (20 mg / mL) cannot form a stable hydrogel and will flow to the bottom of the tube when inverted; however, after adding resveratrol, a stable hydrogel can be formed (it does not flow when inverted). Natural ovalbumin cannot self-assemble with resveratrol to form a hydrogel. At the same time, ovalbumin protofibrils alone cannot form a stable hydrogel, which proves that ovalbumin protofibrils and resveratrol have excellent self-assembly properties. They can self-assemble into a stable hydrogel through non-covalent interactions.

[0036] The non-covalent interactions involved in the assembly of ovalbumin fibrils-resveratrol hydrogels are described in [the following text is incomplete and requires further context]. Figure 4 .like Figure 4 As shown, the results indicate that hydrophobic interactions and hydrogen bonds are the main driving forces in the self-assembly process of hydrogels, while only a small number of electrostatic interactions are involved in this process.

[0037] The microstructure of the ovalbumin fibrils-resveratrol complex and hydrogel at pH 6.5 is shown in [reference needed]. Figure 5 .like Figure 5 As shown, transmission electron microscopy (TEM) images reveal that small-molecule resveratrol is adsorbed onto the surface of ovalbumin fibrils. Furthermore, the addition of resveratrol promotes the aggregation of fibril molecules, demonstrating the interaction between ovalbumin fibrils and resveratrol. Scanning electron microscopy (SEM) images show that the ovalbumin fibril-resveratrol self-assembled hydrogel possesses a regular and dense network structure, with tight connections between protein particles.

[0038] The degradation curves of ovalbumin fibrils-resveratrol hydrogel during simulated gastrointestinal digestion are shown in the figure. Figure 6 The degradation characteristics of the hydrogel were reflected by the decrease in ThT fluorescence intensity. The results showed that the ThT fluorescence intensity of the hydrogel decreased rapidly within the first 30 minutes of digestion by gastric and intestinal fluids. The ThT fluorescence intensity of the hydrogel after pancreatic digestion was 69.6%, indicating that the hydrogel retained some of its fibrous structure after gastrointestinal digestion, which is related to its resistance to proteolytic digestion. This demonstrates that ovalbumin fibrils-resveratrol self-assembled hydrogels have advantages in nutrient delivery and controlled release.

[0039] Release curves of resveratrol in ovalbumin fibrils-resveratrol hydrogel during simulated gastrointestinal digestion are shown below. Figure 7 .like Figure 7 As shown, 28.1% of resveratrol was released from the hydrogel during gastric digestion, providing an opportunity for its subsequent release in the intestines. Following gastrointestinal digestion, the resveratrol release rate from the hydrogel was 66.1%, indicating that more than half of the resveratrol in the hydrogel was released during gastrointestinal digestion. Therefore, the ovalbumin fibrils-resveratrol self-assembled hydrogel can be used as a drug delivery carrier or bioactive substance delivery system, exhibiting good sustained-release properties.

Claims

1. A method for preparing an ovalbumin fibrils-resveratrol self-assembled hydrogel, characterized in that: First, ovalbumin is heated under acidic and hot conditions to obtain amyloid proficiency. Then, it is mixed with hydrophobic resveratrol, the pH is adjusted, and it is allowed to stand to obtain a self-assembled hydrogel. Includes the following steps: Step 1: Weigh the ovalbumin powder and mix it with deionized water to obtain an ovalbumin solution with a concentration of 10-30 mg / mL. Let it stand at 4℃ for 8-12 h to fully hydrate. Adjust the pH of the system with hydrochloric acid. Then, heat the ovalbumin solution at pH 2 and 85℃ for 24 h under magnetic stirring to obtain an ovalbumin fibrillary solution. Step 2: Take resveratrol, add anhydrous ethanol, and stir until the resveratrol is completely dissolved to prepare a resveratrol dispersion; Step 3: Mix resveratrol dispersion and ovalbumin fibrillary solution at a volume ratio of 5-20:1 to obtain a complex solution. The resveratrol concentration in the complex solution is 10-50 μM. Adjust the pH of the complex solution to 6-7 and let it stand at 4-25℃ for 8-12 h to obtain ovalbumin fibrillary-resveratrol self-assembled hydrogel.

2. The preparation method according to claim 1, characterized in that: In step 3, the ethanol volume concentration in the complex solution obtained by mixing the resveratrol dispersion with the egg albumin fibrillary solution is 2%.

3. The application of the ovalbumin fibrils-resveratrol self-assembled hydrogel prepared by the method according to claim 1 or 2, characterized in that: The ovalbumin fibrils-resveratrol self-assembled hydrogel is used to prepare a bioactive substance delivery carrier as a composite hydrogel delivery system loaded with resveratrol.

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