Lactoferrin antibacterial slow-release hydrogel constructed based on natural source materials and preparation method of lactoferrin antibacterial slow-release hydrogel

Abstract

The invention discloses lactoferrin antibacterial slow-release hydrogel constructed based on natural source materials and a preparation method thereof.The hydrogel comprises sodium alginate, deferrited lactoferrin, gluconic acid-delta-lactone, calcium carbonate and a natural source phenolic antibacterial agent.The preparation method comprises the steps that 1, deferrited lactoferrin is prepared through an iron depletion method; step 2, obtaining an iron-removed lactoferrin solution; step 3, obtaining a sodium alginate solution; step 4, forming a phenol antibacterial agent ethanol solution; step 5, obtaining a hydrogel precursor solution; step 6, preparing a gluconic acid-delta-lactone solution; step 7, forming a calcium carbonate suspension; 8, adding the solutions obtained in the step 6 and the step 7 into the precursor solution obtained in the step 5; and step 9, obtaining the deferrited lactoferrin composite hydrogel loaded with the phenolic natural antibacterial agent. The method has the beneficial effects that the mechanical property and uniformity of the gel are enhanced; and good biocompatibility is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactoferrin antibacterial sustained-release hydrogel and a preparation method thereof, in particular to a lactoferrin antibacterial sustained-release hydrogel based on natural source materials and a preparation method thereof. BACKGROUND

[0002] At present, with the increasing demand for environmentally friendly, biodegradable and high safety functional materials, natural hydrogels have attracted widespread attention in many application fields such as active ingredient sustained release, tissue repair, food preservation and biological antibacterial dressing due to their good biocompatibility, high water content and structural adjustability. Hydrogel materials have good hydrophilicity and material transport capacity, and can slowly and stably release functional components in complex environments, effectively improving their biological utilization rate, action duration and reducing the unstable reaction caused by the initial concentration peak.

[0003] Sodium alginate is a natural source of anionic polysaccharide, which is widely used to construct ion cross-linked hydrogel structure. It forms an "egg-box" structure with calcium ions to realize the construction of physical gel network. However, when calcium chloride is used as a calcium source, the cross-linking process is too fast, which often leads to uneven gel structure, poor mechanical properties and rapid initial release (burst effect), affecting the stability of the material's sustained release performance.

[0004] In order to regulate the cross-linking rate and improve the structural density, researchers introduced a combination system of gluconic acid-delta-lactone and calcium carbonate. Gluconic acid-delta-lactone can slowly hydrolyze into gluconic acid in water, releasing H⁺, which in turn promotes the release of calcium ions from calcium carbonate, achieving a slow and mild gel formation process. However, the current related system is mostly used in food gels, and its application in controlled release antibacterial systems is still rare.

[0005] On the other hand, natural phenolic compounds, especially monoterpenes, are widely studied for use in food, medicine and biomedical materials due to their natural origin, high safety and good antibacterial spectrum. Such compounds include but are not limited to thymol, carvacrol, eugenol, etc., which are usually derived from essential oil components of plants such as thyme, oregano, and clove. Among them, thymol, as a representative component, has been listed as a GRAS (Generally Recognized As Safe) substance by the US FDA, and has been proven to have significant inhibitory effect on common pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Escherichia coli O157:H7, and Salmonella.

[0006] However, these phenolic antibacterial agents generally suffer from common problems such as strong hydrophobicity, poor water solubility, volatility, and low biostability, which limit their widespread application in aqueous systems and multifunctional materials. Previous studies have attempted to load these substances into hydrogels to delay their release and improve their stability and sustained antibacterial activity, but these efforts still face challenges such as low encapsulation efficiency, difficulty in releasing the substance, and insufficient antibacterial persistence. Furthermore, current research is mostly based on single essential oil components, lacking systematically designed natural composite controlled-release systems.

[0007] In recent years, studies have attempted to introduce protein-based substances, such as whey protein and gelatin, into hydrogel systems to enhance material functionality. Proteins, as natural functional macromolecules, have been extensively studied in hydrogel construction, participating in network structure formation and improving gel toughness, bioactivity, and loading capacity. However, most current research uses iron-saturated lactoferrin, which primarily acts as an iron carrier, contributing only limited to hydrogel structural stability and antibacterial activity. Furthermore, there are no clear reports on constructing hydrogel systems with synergistic antibacterial and controlled-release effects using deferriferrin. Deferriferrin, as a natural protein, possesses excellent metal ion chelating ability and broad-spectrum antibacterial activity, with enhanced antibacterial capacity under iron-deficient conditions. Its synergistic function in gel carriers has not yet been effectively utilized.

[0008] Therefore, there is an urgent need to develop a functional hydrogel system that is structurally stable, safe and controllable, has sustained-release capability and significant antibacterial activity, in order to solve the key technical bottlenecks of existing materials in the stable loading and sustained release of natural antibacterial agents. Summary of the Invention

[0009] The main objective of this invention is to address the numerous problems existing in current hydrogel products, such as poor stability of antibacterial agents, uneven hydrogel structure, burst release effect, and low material safety. This invention provides a lactoferrin antibacterial sustained-release hydrogel constructed from naturally derived materials and its preparation method.

[0010] The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials provided by this invention includes sodium alginate, defermented lactoferrin, glucono-δ-lactone, calcium carbonate, and naturally derived phenolic antibacterial agents. The defermented lactoferrin is cross-linked with sodium alginate and forms a slow-cross-linked natural hydrogel through glucono-δ-lactone and calcium carbonate. Sodium alginate, defermented lactoferrin, glucono-δ-lactone, calcium carbonate, and naturally derived phenolic antibacterial agents are dissolved, mixed, stirred, and subjected to a slow cross-linking reaction to finally form a three-dimensional antibacterial sustained-release hydrogel.

[0011] Sodium alginate, as a polysaccharide framework material, provides the three-dimensional network basis required for gel formation.

[0012] Deferroferrin, as a protein auxiliary agent, synergistically constructs a gel structure with polysaccharides, enhancing network density and imparting synergistic antibacterial activity.

[0013] Glucono-δ-lactone slowly hydrolyzes in water to produce hydrogen ions, which lowers the pH of the system.

[0014] Calcium carbonate slowly releases calcium ions under acidic conditions, forming an ionic cross-linked structure with sodium alginate;

[0015] Naturally derived phenolic antibacterial agents are hydrophobic natural aromatic phenolic substances. These agents are thymol, carvacrol, or eugenol. They are embedded in the gel network in a non-covalent manner to achieve continuous release and broad-spectrum antibacterial effects.

[0016] The present invention provides a method for preparing lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials, the method comprising the following steps:

[0017] Step 1: Prepare deferroferrin using the iron depletion method. Place a 40-60 mg / ml solution of natural lactoferrin in deionized water and dialyze it with 0.1 M sodium citrate solution in a citrate buffer solution with a pH of 4.0 for 24 hours. Change the dialysate every 8 hours for a total of 3 times. Then, dialyze the dialyzed solution again with deionized water for 24 hours, changing the dialysate every 8 hours, repeating this process 3 times to completely remove residual salts. Finally, freeze-dry the solution and store it in powder form at 4°C for later use.

[0018] Step 2: Dissolve the aborted lactoferrin in water, let it stand to fully hydrate, and obtain an aborted lactoferrin solution with a mass concentration of 5–30 mg / mL.

[0019] Step 3: Dissolve sodium alginate in water, stir well, let stand overnight to remove bubbles, and obtain a sodium alginate solution with a mass concentration of 5–30 mg / mL.

[0020] Step 4: Select at least one naturally sourced phenolic antibacterial agent. The naturally sourced phenolic antibacterial agent is a hydrophobic monoterpenoid phenolic compound. The naturally sourced phenolic antibacterial agent is thymol, carvacrol, or eugenol. Dissolve the naturally sourced phenolic antibacterial agent in food-grade anhydrous ethanol to form a phenolic antibacterial agent ethanol solution with a mass concentration of 300–500 mg / mL.

[0021] Step 5: Mix the solutions from Steps 1, 2 and 3 in a ratio, wherein the volume ratio of the defermented lactoferrin solution to the sodium alginate solution is 100:(50–100), and ensure that the final concentration of the added phenolic antibacterial agent in the mixed system is 2 mg / mL or 4 mg / mL, to obtain the hydrogel precursor solution.

[0022] Step 6: Dissolve gluconate-δ-lactone in water to prepare a gluconate-δ-lactone solution with a mass concentration of 400–600 mg / mL;

[0023] Step 7: Dissolve calcium carbonate in water, mix well, and form a calcium carbonate suspension with a mass concentration of 40–70 mg / mL;

[0024] Step 8: Add the solutions obtained in Step 6 and Step 7 to the precursor solution obtained in Step 5, wherein the volume ratio of gluconate-δ-lactone to calcium carbonate is 5:4-2:1, and the total amount added accounts for 2-10% of the volume of the precursor solution;

[0025] Step 9: Stir the mixture obtained in Step 8 at room temperature until homogeneous, and then let it stand under sealed conditions for 6–20 hours to complete the slow ionic cross-linking and gelation, thereby obtaining a lactoferrin composite hydrogel loaded with phenolic natural antibacterial agents.

[0026] The beneficial effects of this invention are:

[0027] (1) Strong sustained-release stability: The gluconic acid-δ-lactone-calcium carbonate system provides a gradual release of Ca²⁺, achieving mild gelation and significantly reducing the initial release rate;

[0028] (2) Excellent structural density: Deferoxated lactoferrin and sodium alginate synergistically construct a three-dimensional structural network, enhancing the mechanical properties and uniformity of the gel;

[0029] (3) Excellent broad-spectrum antibacterial properties: It can effectively inhibit Gram-positive and Gram-negative bacteria and is suitable for food preservation, packaging, medical antibacterial dressings and other scenarios;

[0030] (4) The raw materials are natural and safe: all components are food-grade or GRAS certified substances with good biocompatibility.

[0031] (5) The system has good adjustability: the release behavior and antibacterial performance can be adjusted according to different phenolic antibacterial agents, concentrations and cross-linking ratios. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the strain scanning curve of the hydrogel loaded with thymol described in this invention in rheology.

[0033] Figure 2 This is a schematic diagram of the frequency sweep curve of the hydrogel loaded with thymol described in this invention in rheology.

[0034] Figure 3 This is a schematic diagram of the viscosity curve of the hydrogel loaded with thymol described in this invention in rheology.

[0035] Figure 4 This is a TEM image of the deferroferrin-sodium alginate hydrogel described in this invention.

[0036] Figure 5 This is a TEM image of the defermented lactoferrin-sodium alginate-thymol-2 hydrogel described in this invention.

[0037] Figure 6 This is a TEM image of the defermented lactoferrin-sodium alginate-thymol-4 hydrogel described in this invention.

[0038] Figure 7 The diagram shows the inhibition zones of the four hydrogels described in this invention—sodium alginate, defermented lactoferrin-sodium alginate, defermented lactoferrin-sodium alginate-thymol-2, and defermented lactoferrin-sodium alginate-thymol-4—against Escherichia coli.

[0039] Figure 8 The diagram shows the inhibition zones of the four hydrogels described in this invention—sodium alginate, defermented lactoferrin-sodium alginate, defermented lactoferrin-sodium alginate-thymol-2, and defermented lactoferrin-sodium alginate-thymol-4—against Staphylococcus aureus.

[0040] Figure 9 This image shows the inhibition zone widths of the four hydrogels described in this invention: sodium alginate, defermented lactoferrin-sodium alginate, defermented lactoferrin-sodium alginate-thymol-2, and defermented lactoferrin-sodium alginate-thymol-4, against Escherichia coli.

[0041] Figure 10 This image shows the inhibition zone widths of the four hydrogels described in this invention—sodium alginate, aferroferrin-sodium alginate, aferroferrin-sodium alginate-thymol-2, and aferroferrin-sodium alginate-thymol-4—against Staphylococcus aureus.

[0042] Figure 11 The graph shows the cumulative release rate of thymol from two hydrogels, lactoferrin-sodium alginate-thymol-2 and lactoferrin-sodium alginate-thymol-4, at pH 7.4, as described in this invention.

[0043] Figure 12 The graph shows the cumulative release rate of thymol from two hydrogels, lactoferrin-sodium alginate-thymol-2 and lactoferrin-sodium alginate-thymol-4, at pH 5, as described in this invention. Detailed Implementation

[0044] Please see Figures 1 to 12 As shown:

[0045] The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials provided by this invention includes sodium alginate, defermented lactoferrin, glucono-δ-lactone, calcium carbonate, and naturally derived phenolic antibacterial agents. The defermented lactoferrin is cross-linked with sodium alginate and forms a slow-cross-linked natural hydrogel through glucono-δ-lactone and calcium carbonate. Sodium alginate, defermented lactoferrin, glucono-δ-lactone, calcium carbonate, and naturally derived phenolic antibacterial agents are dissolved, mixed, stirred, and subjected to a slow cross-linking reaction to finally form a three-dimensional antibacterial sustained-release hydrogel.

[0046] Sodium alginate, as a polysaccharide framework material, provides the three-dimensional network basis required for gel formation.

[0047] Deferroferrin, as a protein auxiliary agent, synergistically constructs a gel structure with polysaccharides, enhancing network density and imparting synergistic antibacterial activity.

[0048] Glucono-δ-lactone slowly hydrolyzes in water to produce hydrogen ions, which lowers the pH of the system.

[0049] Calcium carbonate slowly releases calcium ions under acidic conditions, forming an ionic cross-linked structure with sodium alginate;

[0050] Naturally derived phenolic antibacterial agents are hydrophobic natural aromatic phenolic substances. These agents are thymol, carvacrol, or eugenol. They are embedded in the gel network in a non-covalent manner to achieve continuous release and broad-spectrum antibacterial effects.

[0051] The present invention provides a method for preparing lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials, the method comprising the following steps:

[0052] Step 1: Prepare deferroferrin using the iron depletion method. Place a 40-60 mg / ml solution of natural lactoferrin in deionized water and dialyze it with 0.1 M sodium citrate solution in a citrate buffer solution with a pH of 4.0 for 24 hours. Change the dialysate every 8 hours for a total of 3 times. Then, dialyze the dialyzed solution again with deionized water for 24 hours, changing the dialysate every 8 hours, repeating this process 3 times to completely remove residual salts. Finally, freeze-dry the solution and store it in powder form at 4°C for later use.

[0053] Step 2: Dissolve the aborted lactoferrin in water, let it stand to fully hydrate, and obtain an aborted lactoferrin solution with a mass concentration of 5–30 mg / mL.

[0054] Step 3: Dissolve sodium alginate in water, stir well, let stand overnight to remove bubbles, and obtain a sodium alginate solution with a mass concentration of 5–30 mg / mL.

[0055] Step 4: Select at least one naturally sourced phenolic antibacterial agent. The naturally sourced phenolic antibacterial agent is a hydrophobic monoterpenoid phenolic compound. The naturally sourced phenolic antibacterial agent is thymol, carvacrol, or eugenol. Dissolve the naturally sourced phenolic antibacterial agent in food-grade anhydrous ethanol to form a phenolic antibacterial agent ethanol solution with a mass concentration of 300–500 mg / mL.

[0056] Step 5: Mix the solutions from Steps 1, 2 and 3 in a ratio, wherein the volume ratio of the defermented lactoferrin solution to the sodium alginate solution is 100:(50–100), and ensure that the final concentration of the added phenolic antibacterial agent in the mixed system is 2 mg / mL or 4 mg / mL, to obtain the hydrogel precursor solution.

[0057] Step 6: Dissolve gluconate-δ-lactone in water to prepare a gluconate-δ-lactone solution with a mass concentration of 400–600 mg / mL;

[0058] Step 7: Dissolve calcium carbonate in water, mix well, and form a calcium carbonate suspension with a mass concentration of 40–70 mg / mL;

[0059] Step 8: Add the solutions obtained in Step 6 and Step 7 to the precursor solution obtained in Step 5, wherein the volume ratio of gluconate-δ-lactone to calcium carbonate is 5:4-2:1, and the total amount added accounts for 2-10% of the volume of the precursor solution;

[0060] Step 9: Stir the mixture obtained in Step 8 at room temperature until homogeneous, and then let it stand under sealed conditions for 6–20 hours to complete the slow ionic cross-linking and gelation, thereby obtaining a lactoferrin composite hydrogel loaded with phenolic natural antibacterial agents.

[0061] The specific implementation method is as follows:

[0062] Example 1

[0063] Preparation method of deferroferrin:

[0064] Abolished lactoferrin was prepared using the iron depletion method. A 50 mg / ml solution of natural lactoferrin was placed in deionized water and dialyzed against a 0.1 M sodium citrate solution in a citrate buffer solution at pH 4.0 for 24 h, with the dialysate changed every 8 h for a total of 3 times. Subsequently, the dialyzed solution was dialyzed again against deionized water for 24 h, again with the dialysate changed every 8 h, repeated 3 times to completely remove residual salts. Finally, the solution was freeze-dried and stored as a powder at 4°C for later use.

[0065] Example 2

[0066] Preparation of deferriferrin hydrogel loaded with phenolic antibacterial agents:

[0067] Weigh the deferoxated lactoferrin prepared in Example 1 and dissolve it in pure water to prepare a protein solution with a concentration of 20 mg / mL, then hydrate it overnight. Dissolve sodium alginate in water, stir well, and let it stand overnight to remove bubbles, obtaining a sodium alginate solution with a mass concentration of 20 mg / mL. Add thymol to anhydrous ethanol and mix well to obtain a thymol solution with a mass concentration of 400 mg / mL. Dissolve gluconate-δ-lactone in water to prepare a gluconate-δ-lactone solution with a mass concentration of 600 mg / mL. Dissolve calcium carbonate in water and mix well to form a calcium carbonate suspension with a mass concentration of 60 mg / mL.

[0068] Add 10 μL and 20 μL of thymol solution to 1 ml of aferric lactoferrin solution, respectively. After mixing, add 1 ml of sodium alginate solution to achieve final thymol concentrations of 2 mg / ml and 4 mg / ml, respectively. Then add 50 μL of gluconate-δ-lactone and 40 μL of calcium carbonate suspension, and let stand at room temperature for 12 h to allow hydrogel formation. This will eventually form aferric lactoferrin-sodium alginate-thymol-2 and aferric lactoferrin-sodium alginate-thymol-4 hydrogels. The aferric lactoferrin-sodium alginate hydrogel was used as a control group.

[0069] Example 3

[0070] Rheological property testing of hydrogels

[0071] The rheological properties of the hydrogel were characterized using a TA rheometer equipped with a parallel plate geometry (40 mm in diameter, 1 mm gap). The hydrogel sample from Example 2 was placed on the rheometer plate and equilibrated for 2 min before testing. Strain scan and dynamic oscillatory frequency scan tests were performed at a constant temperature of 25°C, with an angular frequency range of 1–10 rad / s and a strain set to 1% (linear viscoelastic region). The changes in storage modulus (G') and loss modulus (G'') with strain (0.1–10%) and angular frequency (1–10 rad / s) were recorded. In addition, steady-state shear viscosity tests were performed at 25°C and shear rates of 0.01–10 s⁻¹, and the viscosity as a function of shear rate was recorded.

[0072] Figure 1 and Figure 2The storage modulus (G') of all lactoferrin-sodium alginate-thymol hydrogels is higher, likely due to the interaction of phenolic hydroxyl groups in the thymol structure, which enhances the viscoelasticity of the hydrogel. The newly formed hydrogen bonds and hydrophobic interactions between thymol and lactoferrin result in a cross-linked structure, enabling the hydrogel to withstand higher stresses and loads. However, lactoferrin-sodium alginate-thymol-2 exhibits a higher storage modulus than lactoferrin-sodium alginate-thymol-4, indicating that the addition of high concentrations of thymol leads to a decrease in the overall structural strength of the hydrogel. Furthermore, in... Figure 3 In the study, as the concentration of thymol increased, the viscosity of the hydrogel first increased and then decreased, which further confirms that an appropriate concentration of thymol can provide some support for the cross-linking of the hydrogel.

[0073] Example 4

[0074] Scanning electron microscopy observation of hydrogel microstructure

[0075] The microstructure of the hydrogel was observed using field emission scanning electron microscopy. After vacuum freeze-drying, the hydrogel sample from Example 2 was sputtered with gold under vacuum using an E-1010 ion sputtering system. The cross-sectional morphology of the sample was observed at accelerating voltages of 5 kV or 10 kV. The pore area of ​​the hydrogel was analyzed using ImageJ software.

[0076] Figure 4 , Figure 5 and Figure 6 The results showed that the addition of thymol significantly altered the uniform cross-linked network structure of sodium alginate and lactoferrin induced by calcium ions. At a thymol concentration of 2 mg / mL, intermolecular interactions occurred between thymol and lactoferrin, filling the original gaps and promoting structural rearrangement to form an ordered layered morphology. However, from... Figure 5 and Figure 6 As can be seen, with further increases in thymol concentration, the observed pore structure becomes uneven, leading to a decrease in gel strength.

[0077] Example 5

[0078] Antibacterial properties test of hydrogel

[0079] Cryopreserved solutions of *Escherichia coli* and *Staphylococcus aureus* were incubated at 37°C in their respective solid media for 24 hours. Single colonies were then collected and incubated again at 37°C for another 24 hours. Finally, they were added to their respective liquid media, and the absorbance values ​​were adjusted to 0.45 and 0.42, respectively. The solutions were then diluted 100-fold with sterile PBS. The diluted bacterial suspensions (10... 6 The hydrogel (CFU / mL) was evenly spread onto an agar plate using a sterile cotton swab. Then, a piece of hydrogel from Example 2 was cut to a diameter of 1 cm, placed in the center of the agar plate, and incubated at 37°C for 24 h. After incubation, its antibacterial activity was assessed based on the inhibition zone formed around the hydrogel. Sodium alginate hydrogel served as a blank control under the same conditions. The formula for calculating the width of the inhibition zone is:

[0080] Inhibition zone width = (Hd) / 2

[0081] Where H represents the diameter of the inhibition zone and d represents the diameter of the hydrogel.

[0082] Figure 7 The images, in order, are the control group, the aborted lactoferrin experimental group, the aborted lactoferrin-thymol-2 experimental group, and the aborted lactoferrin-thymol-4 experimental group, showing the inhibition zone diagram against Escherichia coli. Figure 8 The groups, in order, are the control group, the aferroferrin experimental group, the aferroferrin-thymol-2 experimental group, and the aferroferrin-thymol-4 experimental group, showing the inhibition zones against Staphylococcus aureus. From Figure 8 It can be seen that the higher the concentration of thymol, the stronger the antibacterial effect and the larger the inhibition zone. Comparing the antibacterial effects of hydrogels on the two bacteria, the effect on Staphylococcus aureus is better than that on Escherichia coli.

[0083] Figure 9 and Figure 10 These are inhibition zone widths of the control group and lactoferrin-sodium alginate hydrogels loaded with different concentrations of thymol against *Escherichia coli* and *Staphylococcus aureus*, respectively. Figure 9 and Figure 10 As can be seen, the lactoferrin-sodium alginate hydrogel exhibits a certain antibacterial effect compared to sodium alginate hydrogel. Furthermore, the addition of thymol continuously expands the width of the inhibition zone. In the inhibition zone experiment against *E. coli*, when the thymol concentration was 4 mg / ml, the inhibition zone width was approximately four times the original. In the inhibition zone experiment against *Staphylococcus aureus*, when the thymol concentration was 4 mg / ml, the inhibition zone width almost reached 30 times the original.

[0084] Example 6

[0085] Sustained-release performance test of phenolic antibacterial agents

[0086] First, PBS solutions (pH 5.0 and 7.4, respectively) were placed into centrifuge tubes containing the hydrogel samples from Example 2, with the lactoferrin-sodium alginate hydrogel used as a control group. The centrifuge tubes were then placed in a 37°C constant-temperature shaker. Subsequently, at predetermined time points (0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h), 2 ml of release medium was removed from the centrifuge tubes, and 2 ml of fresh PBS buffer was added to each tube. Finally, the absorbance of the release medium was measured using a UV spectrophotometer (274 nm), and the thymol concentration was calculated based on the standard curve.

[0087] Figure 11 and Figure 12 This describes the release process of thymol-loaded hydrogels in PBS at pH 7.4 and 5. From Figure 11 and Figure 12 As can be seen, the release rate is relatively similar regardless of whether the pH is near neutral or slightly acidic, indicating good pH stability suitable for systemic release. Within 0-8 hours, thymol rapidly reaches its inhibitory concentration in the initial stage, exhibiting good antibacterial effect, and maintains a stable and slow release over the following 48 hours, sustaining the therapeutic concentration rather than releasing it all at once, demonstrating a sustained-release effect. Furthermore, at the same pH, the total release at 4 mg / mL is consistently higher than at 2 mg / mL, with the high-drug-load group releasing more, indicating a positive correlation between drug loading and cumulative release. The release in the high-concentration group is also very stable, indicating that the colloid still has a stable control capability under high drug loading conditions.

Claims

1. A lactoferrin antibacterial sustained-release hydrogel constructed from naturally derived materials, characterized in that: It includes sodium alginate, defermented lactoferrin, glucono-δ-lactone, calcium carbonate, and naturally derived phenolic antibacterial agents. Among them, defermented lactoferrin and sodium alginate are cross-linked, and glucono-δ-lactone and calcium carbonate together form a natural hydrogel with a slow cross-linking structure. Sodium alginate, defermented lactoferrin, glucono-δ-lactone, calcium carbonate and naturally derived phenolic antibacterial agents are dissolved, mixed, stirred and subjected to a slow cross-linking reaction to finally form a three-dimensional antibacterial sustained-release hydrogel.

2. The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials according to claim 1, characterized in that: The sodium alginate described herein serves as a polysaccharide framework material, providing the three-dimensional network foundation required for gel formation.

3. The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials according to claim 1, characterized in that: The deferroferrin described herein serves as a protein auxiliary agent, synergistically constructing a gel structure with polysaccharides to enhance network density and impart synergistic antibacterial activity.

4. The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials according to claim 1, characterized in that: The gluconate-δ-lactone slowly hydrolyzes in water to produce hydrogen ions, thus lowering the pH of the system.

5. The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials according to claim 1, characterized in that: The calcium carbonate slowly releases calcium ions under acidic conditions, forming an ionic cross-linked structure with sodium alginate.

6. The lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials according to claim 1, characterized in that: The naturally derived phenolic antibacterial agent is a hydrophobic natural aromatic phenolic substance. The naturally derived phenolic antibacterial agent is thymol, carvacrol, or eugenol. The naturally derived phenolic antibacterial agent is embedded in the gel network in a non-covalent manner to achieve continuous release and broad-spectrum antibacterial effect.

7. A method for preparing a lactoferrin antibacterial sustained-release hydrogel based on naturally derived materials, characterized in that: The method includes the following steps: Step 1: Prepare deferroferrin using the iron depletion method. Place a 40-60 mg / ml solution of natural lactoferrin in deionized water and dialyze it with 0.1 M sodium citrate solution in a citrate buffer solution with a pH of 4.0 for 24 hours. Change the dialysate every 8 hours for a total of 3 times. Then, dialyze the dialyzed solution again with deionized water for 24 hours, changing the dialysate every 8 hours, repeating this process 3 times to completely remove residual salts. Finally, freeze-dry the solution and store it in powder form at 4°C for later use. Step 2: Dissolve the aborted lactoferrin in water, let it stand to fully hydrate, and obtain an aborted lactoferrin solution with a mass concentration of 5–30 mg / mL. Step 3: Dissolve sodium alginate in water, stir well, let stand overnight to remove bubbles, and obtain a sodium alginate solution with a mass concentration of 5–30 mg / mL. Step 4: Select at least one naturally sourced phenolic antibacterial agent. The naturally sourced phenolic antibacterial agent is a hydrophobic monoterpenoid phenolic compound. The naturally sourced phenolic antibacterial agent is thymol, carvacrol, or eugenol. Dissolve the naturally sourced phenolic antibacterial agent in food-grade anhydrous ethanol to form a phenolic antibacterial agent ethanol solution with a mass concentration of 300–500 mg / mL. Step 5: Mix the solutions from Steps 1, 2 and 3 in a ratio, wherein the volume ratio of the defermented lactoferrin solution to the sodium alginate solution is 100:(50–100), and ensure that the final concentration of the added phenolic antibacterial agent in the mixed system is 2 mg / mL or 4 mg / mL, to obtain the hydrogel precursor solution. Step 6: Dissolve gluconate-δ-lactone in water to prepare a gluconate-δ-lactone solution with a mass concentration of 400–600 mg / mL; Step 7: Dissolve calcium carbonate in water, mix well, and form a calcium carbonate suspension with a mass concentration of 40–70 mg / mL; Step 8: Add the solutions obtained in Step 6 and Step 7 to the precursor solution obtained in Step 5, wherein the volume ratio of gluconate-δ-lactone to calcium carbonate is 5:4-2:1, and the total amount added accounts for 2-10% of the volume of the precursor solution; Step 9: Stir the mixture obtained in Step 8 at room temperature until homogeneous, and then let it stand under sealed conditions for 6–20 hours to complete the slow ionic cross-linking and gelation, thereby obtaining a lactoferrin composite hydrogel loaded with phenolic natural antibacterial agents.

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