A microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel and its preparation method

Abstract

This invention discloses a microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel and its preparation method. The technical solution involves using casein as a substrate, with lysozyme adsorbed onto the surface of casein micelles via electrostatic forces, and chitosan coating the outer layer of the lysozyme. The casein micelle solution, lysozyme solution, and chitosan solution are uniformly mixed through physical blending. Electrostatic forces physically cross-link the carboxyl groups on the casein micelle surface with the cations of lysozyme and chitosan amino groups, forming a microsphere-filled lysozyme-chitosan / casein composite hydrogel. This material exhibits excellent sustained-release performance, is environmentally friendly, has good biocompatibility, stable performance, and good antibacterial function.

Description

Technical Field

[0001] This invention belongs to the field of polymer biomaterials technology, and specifically relates to a microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel and its preparation method. Background Technology

[0002] Casein is the main protein in mammalian milk, existing in the form of micelles. It consists of four proteins bound together by colloidal calcium phosphate to form spherical particles. Its molecular structure and charge depend on the pH of the solution. Changes in pH affect the electrostatic and hydrophobic interactions between casein molecules, thus altering the structure of casein micelles. As the pH increases, the negative charge of casein micelles increases, attracting more water molecules and increasing hydration, thereby increasing the micelle size.

[0003] Lysozyme primarily works by disrupting the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bacterial cell wall, thereby breaking down the cell wall and causing the contents to leak out, thus lysing the bacteria. It exhibits significant antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Bacillus licheniformis, among others. It has good biocompatibility with humans and is non-immunogenic, making it widely used clinically for its antibacterial, antiviral, hemostatic, immune-enhancing, and tissue-recovery-accelerating effects.

[0004] Chitosan is an alkaline amino polysaccharide obtained by hydrolyzing chitin under alkaline conditions. It is the only polycationic weakly basic polysaccharide in nature possessing active amino groups. The intramolecular and intermolecular hydrogen bonding of chitosan gives it high crystallinity, thus it cannot be directly dissolved in neutral or alkaline solvents. Under acidic conditions, the amino groups protonate, weakening the hydrogen bonding on chitosan, reducing its crystallinity, and making it soluble in water. Chitosan possesses excellent biocompatibility, biodegradability, mucosal adsorption, non-toxicity, and antibacterial properties, making it one of the most common materials for preparing biomedical hydrogels. Summary of the Invention

[0005] The purpose of this invention is to provide a microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel material and its preparation method. The prepared microsphere-filled composite antibacterial hydrogel has a stable structure and can effectively encapsulate lysozyme, giving it good sustained-release antibacterial properties.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] 1) Casein solubility

[0008] Take deionized water and adjust the pH to 9-11 with an alkaline solution. Take 52-54.5 parts by mass of the alkaline-adjusted deionized water and add 0.5-3 parts by mass of casein powder. Mix the mixture with stirring and add it to a three-necked flask. Stir in a water bath at 30-55°C until dissolved. Then add an acidic solution to adjust the pH to 6.5-7 to obtain a uniform and stable casein dispersion.

[0009] 2) Lysozyme addition

[0010] Prepare a phosphate buffer solution with a pH of 6.3–7.4. Take 8.5–9.9 parts by weight of the phosphate buffer solution and add 0.1–1.5 parts by weight of lysozyme to it to obtain a mixture. While stirring, add the mixture to the casein dispersion solution and stir at 30–45°C to disperse the lysozyme evenly in the casein micelle solution to obtain a lysozyme / casein dispersion.

[0011] 3) Chitosan addition

[0012] Take 0.4 to 1 part by weight of chitosan and add it to 34 to 34.6% deionized water. Adjust the pH value to 5 to 6.5 with acidic solution and stir mechanically at room temperature until it is evenly dispersed to obtain a chitosan solution. Add the chitosan solution to the lysozyme / casein dispersion within 20 to 35 minutes while stirring at 30 to 45°C.

[0013] 4) Preparation of composite hydrogels

[0014] The dispersion obtained in step 3) was uniformly dispersed at 35–45°C and a stirring speed of 400–600 r / min for 50–80 min to obtain a milky white microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel.

[0015] The alkaline solution in step 1) is a sodium hydroxide, sodium carbonate, or sodium bicarbonate solution.

[0016] The casein powder mentioned herein is made from one or both of bovine casein and sheep casein.

[0017] The stirring speed in step 1) is 250-500 r / min, and the stirring is continued for 40-70 min.

[0018] The acidic solution in steps 1) and 3) is a sulfuric acid, hydrochloric acid, or acetic acid solution.

[0019] The phosphate buffer solution described in step 2) is a phosphate buffer solution with a pH of 6.3-7.4 prepared from disodium hydrogen phosphate and potassium dihydrogen phosphate.

[0020] The lysozyme used in step 2) is recombinant human lysozyme.

[0021] The chitosan has a relative molecular mass of 5000-50000 and a degree of deacetylation greater than 90%.

[0022] The microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel was prepared according to the above method.

[0023] The advantages of this invention are:

[0024] 1) This invention utilizes the unique natural micelle structure of casein, the bio-antibacterial properties of lysozyme, and the natural antibacterial properties of chitosan to prepare a composite hydrogel that is green and environmentally friendly and has good biodegradability.

[0025] 2) Within the pH range of 6 to 7, casein, lysozyme, and chitosan form a hydrogel through charge adsorption, which achieves the sustained release of lysozyme without changing its conformation and antibacterial function.

[0026] 3) The composite hydrogel preparation method is simple, has broad application prospects, and is suitable for industrial production. The microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel method proposed in this invention can be fully met by the current equipment of biopharmaceutical companies. The composite hydrogel can be used in dressings on substrates such as paper, textiles, and nonwoven materials, giving the substrates excellent medical and hygiene properties and increasing the added value of products. Attached Figure Description

[0027] Figure 1 This is a scanning electron microscope (SEM) image of the lysozyme-chitosan / casein composite antibacterial hydrogel prepared in Example 4 of the present invention.

[0028] Figure 2 The image shows the antibacterial effect (Staphylococcus aureus) of the lysozyme-chitosan / casein composite antibacterial hydrogel prepared in Example 4 of this invention. Detailed Implementation

[0029] The following embodiments are used to further illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above description of the present invention.

[0030] Example 1

[0031] 1) Casein solubility

[0032] Take deionized water and adjust the pH to 9 with sodium hydroxide solution. Take 52 parts by mass of alkaline-adjusted deionized water and add 0.5 parts by mass of bovine casein powder. Mix and add to a three-necked flask. Stir continuously at 250 r / min for 40 min in a 40℃ water bath. Then add sulfuric acid solution to adjust the pH to 6.8 to obtain a uniform and stable casein dispersion.

[0033] 2) Lysozyme addition

[0034] A phosphate buffer solution with a pH of 7.4 was prepared using disodium hydrogen phosphate and potassium dihydrogen phosphate. Nine parts by mass of the phosphate buffer solution were taken and 0.1 parts of recombinant human lysozyme were added to it to obtain a mixture. While stirring, the mixture was added to the casein dispersion and stirred at 30°C to disperse the lysozyme evenly in the casein micelle solution to obtain a lysozyme / casein dispersion.

[0035] 3) Chitosan addition

[0036] Take 0.4 parts by weight of chitosan with a relative molecular mass of 5000-50000 and a degree of deacetylation greater than 90% and add it to 34% deionized water. Adjust the pH value to 6 with sulfuric acid and mechanically stir at room temperature until it is evenly dispersed to obtain a chitosan solution. Add the chitosan solution to the lysozyme / casein dispersion within 20 minutes under stirring at 30°C.

[0037] 4) Preparation of composite hydrogels

[0038] The dispersion obtained in step 3) was uniformly dispersed at 35°C and a stirring speed of 400 r / min for 50 min to obtain a milky white microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel.

[0039] Example 2

[0040] 1) Casein solubility

[0041] Take deionized water and adjust the pH to 10 with sodium carbonate solution. Take 53 parts by mass of alkaline-adjusted deionized water and add 1 part of sheep milk casein powder to it. Mix and add to a three-necked flask. Stir continuously at 300 r / min for 40, 50, 60 and 70 min in a 30℃ water bath. Then add hydrochloric acid solution to adjust the pH to 6.5 to obtain a uniform and stable casein dispersion.

[0042] 2) Lysozyme addition

[0043] A phosphate buffer solution with a pH of 7 was prepared using disodium hydrogen phosphate and potassium dihydrogen phosphate. 8.5 parts by weight of the phosphate buffer solution were taken and 0.5 parts by weight of recombinant human lysozyme were added to it to obtain a mixture. While stirring, the mixture was added to the casein dispersion and stirred at 35°C to disperse the lysozyme evenly in the casein micelle solution to obtain a lysozyme / casein dispersion.

[0044] 3) Chitosan addition

[0045] Take 0.6 parts by weight of chitosan with a relative molecular mass of 5000-50000 and a degree of deacetylation greater than 90% and add it to 34.2% deionized water. Adjust the pH value to 5 with hydrochloric acid solution and stir mechanically at room temperature until it is evenly dispersed to obtain a chitosan solution. Add the chitosan solution to the lysozyme / casein dispersion within 25 minutes under stirring at 35℃.

[0046] 4) Preparation of composite hydrogels

[0047] The dispersion obtained in step 3) was uniformly dispersed at 40°C and a stirring speed of 450 r / min for 60 min to obtain a milky white microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel.

[0048] Example 3

[0049] 1) Casein solubility

[0050] Take deionized water and adjust the pH to 10 with sodium bicarbonate solution. Take 54 parts by mass of alkaline-adjusted deionized water and add 2 parts by mass of mixed powder of bovine casein and sheep casein. After mixing, add the mixture to a three-necked flask and stir continuously at 500 r / min for 60 min in a 50℃ water bath. Then add acetic acid solution to adjust the pH to 7 to obtain a uniform and stable casein dispersion.

[0051] 2) Lysozyme addition

[0052] A phosphate buffer solution with a pH of 6.5 was prepared using disodium hydrogen phosphate and potassium dihydrogen phosphate. 9.5 parts by weight of the phosphate buffer solution were taken and 1 part of recombinant human lysozyme was added to it to obtain a mixture. The mixture was added to the casein dispersion while stirring. The mixture was stirred and dispersed at 40°C to ensure that the lysozyme was evenly dispersed in the casein micelle solution to obtain a lysozyme / casein dispersion.

[0053] 3) Chitosan addition

[0054] Take 0.8 parts by weight of chitosan with a relative molecular mass of 5000-50000 and a degree of deacetylation greater than 90% and add it to 34.4% deionized water. Adjust the pH value to 6.5 with acetic acid solution and stir mechanically at room temperature until it is evenly dispersed to obtain a chitosan solution. Add the chitosan solution to the lysozyme / casein dispersion within 30 minutes under stirring at 40℃.

[0055] 4) Preparation of composite hydrogels

[0056] The dispersion obtained in step 3) was uniformly dispersed at 45°C and a stirring speed of 500 r / min for 70 min to obtain a milky white microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel.

[0057] Example 4

[0058] 1) Casein solubility

[0059] Take deionized water and adjust the pH to 11 with sodium hydroxide solution. Take 54.5 parts by mass of alkaline-adjusted deionized water and add 3 parts by mass of bovine casein powder. Mix and add to a three-necked flask. Stir continuously at 400 r / min for 70 min in a 55℃ water bath. Then add sulfuric acid solution to adjust the pH to 7 to obtain a uniform and stable casein dispersion.

[0060] 2) Lysozyme addition

[0061] A phosphate buffer solution with a pH of 6.3 was prepared using disodium hydrogen phosphate and potassium dihydrogen phosphate. 9.9 parts by weight of the phosphate buffer solution were taken and 1.5 parts by weight of recombinant human lysozyme were added to it to obtain a mixture. The mixture was added to the casein dispersion while stirring. The mixture was stirred and dispersed at 45°C to ensure that the lysozyme was evenly dispersed in the casein micelle solution to obtain a lysozyme / casein dispersion.

[0062] 3) Chitosan addition

[0063] Take 1 part by weight of chitosan with a relative molecular mass of 5000-50000 and a degree of deacetylation greater than 90% and add it to 34.6% deionized water. Adjust the pH to 5.5 with sulfuric acid solution and stir mechanically at room temperature until it is evenly dispersed to obtain a chitosan solution. Add the chitosan solution to the lysozyme / casein dispersion within 35 minutes while stirring at 45°C.

[0064] 4) Preparation of composite hydrogels

[0065] The dispersion obtained in step 3) was uniformly dispersed at 45°C and a stirring speed of 600 r / min for 80 min to obtain a milky white microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel.

[0066] The lysozyme-chitosan / casein composite antibacterial hydrogel prepared in Example 4 was analyzed by SEM, and the results were as follows: Figure 1 The SEM image shown is illustrated. As shown, the material exhibits a dense three-dimensional network structure, with casein microspheres dispersed within it.

[0067] The antibacterial properties of the lysozyme-chitosan / casein composite antibacterial hydrogel prepared in Example 4 were tested, and the results were as follows: Figure 2 The image shows the antibacterial effect. From... Figure 2 It can be seen that the addition of composite antibacterial materials has a significant inhibitory effect on Staphylococcus aureus. The antibacterial effect increases significantly with the increase of the concentration of lysozyme-chitosan / casein composite antibacterial hydrogel, and the minimum inhibitory concentration is 0.8 mg / mL.

[0068] Based on the surface morphology and antibacterial effect of the lysozyme-chitosan / casein composite antibacterial hydrogel, combined with theoretical analysis, it can be determined that lysozyme and chitosan cross-link with casein through electrostatic attraction, thus successfully preparing the lysozyme-chitosan / casein composite antibacterial hydrogel. The results prove that this preparation method is effective.

Claims

1. A method for preparing a microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel, characterized in that... Includes the following steps: 1) Casein dissolution Take deionized water and adjust the pH to 9-11 with an alkaline solution. Take 52-54.5 parts by mass of the alkaline-adjusted deionized water and add 0.5-3 parts by mass of casein powder. Mix the mixture with stirring and add it to a three-necked flask. Stir in a water bath at 30-55°C until dissolved. Then add an acidic solution to adjust the pH to 6.5-7 to obtain a uniform and stable casein micelle dispersion. 2) Lysozyme addition Prepare a phosphate buffer solution with a pH of 6.3–7.

4. Take 8.5–9.9 parts by weight of the phosphate buffer solution and add 0.1–1.5 parts by weight of lysozyme to it to obtain a mixture. While stirring, add the mixture to the casein micelle dispersion and stir at 30–45°C to disperse the lysozyme evenly in the casein micelle dispersion to obtain a lysozyme / casein micelle dispersion. 3) Chitosan addition Take 0.4 to 1 part by weight of chitosan and add it to 34 to 34.6% deionized water. Adjust the pH value to 5 to 6.5 with acidic solution and stir mechanically at room temperature until it is evenly dispersed to obtain a chitosan solution. Add the chitosan solution to the lysozyme / casein micelle dispersion within 20 to 35 minutes while stirring at 30 to 45°C. The chitosan has a relative molecular mass of 5000-50000 and a degree of deacetylation greater than 90%. 4) Preparation of composite hydrogels The dispersion obtained in step 3) was uniformly dispersed at 35–45°C and a stirring speed of 400–600 r / min for 50–80 min to obtain a milky white microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel.

2. The method for preparing the microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel according to claim 1, characterized in that: The alkaline solution in step 1) is a sodium hydroxide, sodium carbonate, or sodium bicarbonate solution.

3. The method for preparing the microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel according to claim 1, characterized in that: The casein powder mentioned herein is made from one or both of bovine casein and sheep casein.

4. The method for preparing the microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel according to claim 1, characterized in that: The stirring speed in step 1) is 250-500 r / min, and the stirring is continued for 40-70 min.

5. The method for preparing the microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel according to claim 1, characterized in that: The acidic solution in steps 1) and 3) is a sulfuric acid, hydrochloric acid, or acetic acid solution.

6. The method for preparing the microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel according to claim 1, characterized in that: Step 2) The phosphate buffer solution is a phosphate buffer solution with a pH of 6.3-7.4 prepared from disodium hydrogen phosphate and potassium dihydrogen phosphate.

7. The method for preparing the microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel according to claim 1, characterized in that: The lysozyme used in step 2) is recombinant human lysozyme.

8. A microsphere-filled lysozyme-chitosan / casein composite antibacterial hydrogel prepared by the preparation method according to any one of claims 1 to 7.

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