Gelatin / antibacterial peptide covalent conjugate with antibacterial activity, preparation and application thereof

Abstract

The present application provides a kind of gelatin / antibacterial peptide covalent conjugate with antibacterial activity, preparation and application thereof.The preparation method comprises the following steps: adding reaction medium to gelatin, heating, and fully dissolving gelatin to obtain a gelatin solution;adding antibacterial peptide to the gelatin solution, and then adding or not adding functional reagent, adjusting the pH to 3-6.5 or 9-12 after fully dissolving to obtain a premix;adding coupling agent to the premix, carrying out coupling reaction, adjusting to neutral after reaction, dialysis, and freeze-drying to obtain the product.The preparation method has the characteristics of low cost, high efficiency and mild reaction.By simply adjusting the acidity and alkalinity of the reaction system, the activation of the amino or carboxyl groups of gelatin and antibacterial peptide can be realized, and then the covalent coupling of gelatin, antibacterial peptide and coupling agent can be realized, so that the antibacterial peptide molecules are coupled to the gelatin molecular chain, and the gelatin is endowed with excellent antibacterial performance.

Description

Technical Field

[0001] This invention belongs to the field of polymeric antimicrobial biomaterials technology, specifically including a gelatin / antimicrobial peptide covalent conjugate with antimicrobial activity, its preparation and application. Background Technology

[0002] Gelatin is a partially hydrolyzed product of collagen after treatment with acids, alkalis, or enzymes. It possesses excellent biocompatibility, biodegradability, and low immunogenicity, making it a widely used biopolymer material. It has been extensively applied in the production and daily life of photosensitive imaging, packaging materials, food, cosmetics, and animal feed. In the high-value-added biomedical field, gelatin also has wide applications, being used in therapeutic drugs and devices in dermatology, ophthalmology, orthopedics, and other departments.

[0003] In real-world production and daily life, microbial contamination has been a major problem plaguing humanity for thousands of years. During food storage and distribution, microbial contamination leads to food spoilage and deterioration, and ingesting contaminated food can cause foodborne illnesses. Simultaneously, harmful microbial infections cause various human diseases; surgical infections delay wound healing and, in severe cases, can even be life-threatening. Gelatin lacks antibacterial activity, and because it is composed of amino acids, it can also serve as a nutrient source for microorganisms, which greatly limits its applications. To impart antibacterial activity to gelatin, antibacterial agents are added to gelatin products, including organic, inorganic, and natural antibacterial materials.

[0004] Antimicrobial peptides are a class of polypeptides with antimicrobial activity, widely found in bacteria, plants, and animals, and are important effector molecules in the host cell's innate immunity. Antimicrobial peptides possess broad-spectrum antimicrobial properties, exhibiting good inhibitory effects against multidrug-resistant bacteria, while being less likely to induce drug resistance, making them a promising alternative to antibiotics and attracting widespread attention from scientists. Natural preservatives such as ε-polylysine, natamycin, and nisin are antimicrobial peptides produced by Streptomyces and Lactococcus, exhibiting good inhibitory effects against various bacteria and fungi.

[0005] Currently, there are two main methods for preparing gelatin / antimicrobial peptide composites: 1) physical mixing of gelatin and antimicrobial peptides; 2) covalent coupling of gelatin and antimicrobial peptides. In physical mixing, the gelatin and antimicrobial peptides lack strong chemical bonds, leading to easy loss of the antimicrobial peptides and thus the composite material losing its antimicrobial activity. Furthermore, antimicrobial peptides also suffer from drawbacks such as easy enzymatic degradation, unstable activity, low bioavailability, and some hemolytic and cytotoxic effects. For example, acidic polysaccharides, hydrochlorides, phosphates, and copper ions can reduce the antimicrobial activity of ε-polylysine. Additionally, when ε-polylysine decomposes and its molecular weight falls below 1300, it loses its antimicrobial activity. Covalent coupling of gelatin and antimicrobial peptides can overcome the drawbacks of physical mixing and contribute to the stability of the antimicrobial peptide activity. Current literature reports methods using gelatin and antimicrobial peptides as raw materials. The carboxyl groups of either gelatin or the antimicrobial peptides are activated via a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide (EDC / NHS) system, reacting with the amino group of another reactant to form an amide bond, thus generating a covalent coupling. However, the EDC / NHS reagents used in this method are expensive, and a large amount is required to activate the carboxyl groups, thereby increasing the cost of antimicrobial gelatin. Furthermore, this method is only suitable for generating couplings under low reactant concentrations; when the gelatin concentration is increased, the gelatin rapidly cross-links, forming a gel product.

[0006] Therefore, there is an urgent need to develop a method for preparing gelatin / antimicrobial peptide covalent conjugates that is low in cost, easy to operate, highly efficient, and suitable for large-scale preparation. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the first objective of this invention is to provide a method for preparing a gelatin / antimicrobial peptide covalent conjugate with antibacterial activity. This method is characterized by low cost, high efficiency, and mild conditions. It involves activating the amino groups of gelatin and the antimicrobial peptide under alkaline conditions, or activating the carboxyl groups of gelatin and the antimicrobial peptide under acidic conditions, thereby coupling them with the epoxy groups at both ends of the coupling agent molecule to form a gelatin / antimicrobial peptide covalent conjugate with antibacterial properties. Furthermore, this method allows for the production of gelatin raw materials from a few grams to several hundred grams, enabling large-scale preparation of gelatin / antimicrobial peptide covalent conjugates with antibacterial activity.

[0008] A second objective of this invention is to provide a gelatin / antimicrobial peptide covalent conjugate with antibacterial activity prepared by the method described above. This gelatin / antimicrobial peptide covalent conjugate exhibits high antibacterial activity, and further, by introducing functional reagents, it can be endowed with new properties, thereby broadening its application range.

[0009] A third objective of this invention is to provide an application of the gelatin / antimicrobial peptide covalently coupled as described above in the preparation of antimicrobial and / or bacteriostatic products.

[0010] To achieve the first objective mentioned above, the technical solution adopted by this invention includes:

[0011] This invention discloses a method for preparing a gelatin / antimicrobial peptide covalent conjugate with antibacterial activity, comprising the following steps:

[0012] 1) Add the reaction medium to the gelatin, heat it, and let the gelatin dissolve completely to obtain a gelatin solution;

[0013] 2) Add antimicrobial peptides to the gelatin solution, and add or not add functional reagents. After fully dissolving, adjust the pH to 3-6.5 or 9-12 to obtain a premixed solution.

[0014] 3) Add a coupling agent to the premixed solution to carry out the coupling reaction. After the reaction is complete, adjust to neutral, dialyze, and freeze dry to obtain the final product.

[0015] This invention activates the amino or carboxyl groups of gelatin and antimicrobial peptides by simply adjusting the acidity or alkalinity of the reaction system. The activated gelatin and antimicrobial peptides are then coupled through the epoxy groups at both ends of the coupling agent molecule, thereby coupling the antimicrobial peptide molecule to the gelatin molecular chain to form a gelatin / antimicrobial peptide covalently coupled with antimicrobial properties. From a cost perspective, this preparation method abandons the EDC / NHS activation system used in traditional technologies, resulting in lower cost and higher efficiency. It can easily and quickly prepare antimicrobial gelatin composite materials at high gelatin concentrations. Furthermore, by coordinating and controlling the concentration of the gelatin solution and the ratio of gelatin / polylysine / coupling agent, the rapid cross-linking of gelatin to form a gel is avoided, thus maintaining the solution state throughout the preparation process.

[0016] Furthermore, the gelatin used in this invention can be any gelatin commonly used in the art, and the source of the gelatin is not limited, including but not limited to one or more gelatin sources from pigs, cattle, fish and poultry.

[0017] Furthermore, the mass fraction of gelatin in the gelatin solution is 2-20%, preferably 5-15%.

[0018] Furthermore, the reaction medium includes, but is not limited to, one or more of distilled water, sodium chloride aqueous solution, carbonate buffer, Tris-HCl solution, 2-morpholine ethanesulfonic acid solution, and acetate buffer.

[0019] Furthermore, when dissolving the gelatin in step 1, the dissolution temperature is controlled at 35-60℃, preferably 40-55℃.

[0020] Furthermore, the antimicrobial peptides used in this invention can be natural antimicrobial peptides or artificially synthesized antimicrobial peptides, as long as they have free amino and / or carboxyl groups. The antimicrobial peptides include, but are not limited to, one or more of ε-polylysine or its salts, nisin or its salts, and natamycin or its salts.

[0021] Furthermore, the mass ratio of the antimicrobial peptide to gelatin is 0.05-2:1, preferably 0.1-1:1.

[0022] Furthermore, the functional reagents include, but are not limited to, one or more of the following: antioxidant enhancing agents, antibacterial enhancing agents, mechanical strength enhancing agents, and extensibility enhancing agents; exemplarily, the functional reagents include, but are not limited to, one or more of the following: water-soluble chitosan, tea polyphenols, citric acid, tannic acid, and hyaluronic acid. To aid in the dissolution of these functional reagents, appropriate amounts of solvents such as ethanol, ethylene glycol, and glycerol may be added. These solvents can be completely removed during dialysis and drying steps without affecting biosafety.

[0023] Furthermore, the functional reagent is 0-20 wt% of the gelatin mass.

[0024] Furthermore, considering that gelatin is easily decomposed under excessively acidic or alkaline conditions, and the impact of acidity and alkalinity on reaction vessels in future large-scale production, the more suitable pH range for step 2 is 4-6 or 10-11.

[0025] Furthermore, the coupling agent has multiple epoxy groups, including but not limited to ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, pentaerythritol diglycidyl ether, glycerol triglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, trimethylolpropane triglycidyl ether, tetra-arm polyethylene glycol diglycidyl ether, etc., preferably one or more of ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol triglycidyl ether, and 1,4-butanediol diglycidyl ether that have high water solubility.

[0026] Furthermore, the mass ratio of the coupling agent to gelatin is 0.01-0.10:1, preferably 0.02-0.05:1.

[0027] Furthermore, the reaction temperature of the coupling reaction is 35-60℃, preferably 40-55℃, and the reaction time is 8-48h, preferably 12-24h.

[0028] Furthermore, the dialysis process removes unreacted antimicrobial peptides and coupling agents, reducing their impact on subsequent applications. This can be controlled by the molecular weight cutoff of the dialysis bag. The temperature controlled during the dialysis process is room temperature - 50°C, preferably 35-45°C, and the dialysis time is 1-5 days. The molecular weight cutoff of the dialysis bag is 20-40 kDa.

[0029] To achieve the second objective mentioned above, the technical solution adopted by the present invention includes:

[0030] This invention discloses a gelatin / antimicrobial peptide covalent conjugate with antibacterial activity prepared by the preparation method described above.

[0031] To achieve the third objective mentioned above, the technical solution adopted by this invention includes:

[0032] This invention discloses the application of the gelatin / antimicrobial peptide covalently coupled as described above in the preparation of antimicrobial and / or bacteriostatic products.

[0033] Beneficial effects of this invention:

[0034] This invention discloses a method for preparing gelatin / antimicrobial peptide covalent conjugates. This method is characterized by low cost, high efficiency, and mild reaction. It involves activating the amino groups of gelatin and antimicrobial peptides under alkaline conditions or the carboxyl groups of gelatin and antimicrobial peptides under acidic conditions, and then coupling them with the epoxy groups at both ends of the coupling agent molecule to form a gelatin / antimicrobial peptide covalent conjugate with antimicrobial properties. At the same time, this method can achieve the production of gelatin raw materials from a few grams to several hundred grams, and can prepare gelatin / antimicrobial peptide covalent conjugates with antimicrobial activity on a large scale.

[0035] This invention can activate the amino or carboxyl groups of gelatin and antimicrobial peptides by simply adjusting the acidity or alkalinity of the reaction system. The activated gelatin and antimicrobial peptides are then coupled through the epoxy groups at both ends of the coupling agent molecule, thereby coupling the antimicrobial peptide molecule to the gelatin molecular chain to form a gelatin / antimicrobial peptide covalently coupled with antimicrobial properties. From a cost perspective, this preparation method abandons the EDC / NHS activation system used in traditional technology, resulting in lower cost and higher efficiency. It can easily and quickly prepare antimicrobial gelatin composite materials at high gelatin concentrations. Attached Figure Description

[0036] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0037] Figure 1 The images show the antibacterial activity of the gelatin / ε-polylysine covalent conjugates obtained in Examples 1-4 against Staphylococcus aureus and Escherichia coli.

[0038] Figure 2 The infrared spectra of the gelatin / ε-polylysine covalent conjugate obtained in Example 1 are shown in comparison with those of gelatin and ε-polylysine.

[0039] Figure 3 Photographs showing the antibacterial activity of the gelatin / ε-polylysine covalent conjugate obtained in Example 5 against Staphylococcus aureus and Escherichia coli are shown.

[0040] Figure 4 Photographs showing the antibacterial activity of the gelatin / nisin Z covalent conjugate obtained in Example 6 against Staphylococcus aureus and Escherichia coli are shown. Detailed Implementation

[0041] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0042] All numerical specifications in this invention (e.g., temperature, time, concentration, and weight, including ranges for each) are generally approximate values ​​that may be changed (+) or (-) in increments of 0.1 or 1.0. All numerical specifications are to be understood as being preceded by the term "about".

[0043] Example 1

[0044] 10g of gelatin was dissolved in 100mL of 2% NaCl aqueous solution at 40℃ for half an hour to form a gelatin solution. 10g of ε-polylysine hydrochloride was added to the obtained gelatin solution, and the mixture was stirred continuously until dissolved. The pH of the reaction system was adjusted to 10 to obtain a premix. 0.2g of ethylene glycol diglycidyl ether was added to the premix, and the mixture was stirred continuously at 40℃ for 24 hours. After the reaction, the pH of the solution was adjusted to neutral, and dialyzed using a dialysis bag with a molecular weight cutoff of 30kDa at 40℃ for 5 days to remove unreacted polylysine and coupling agent. The solution was then frozen at -80℃ and vacuum dried to obtain the final product.

[0045] Example 2

[0046] 10g of gelatin was dissolved in 100mL of 2% NaCl aqueous solution at 40℃ for half an hour to form a gelatin solution. 5g of ε-polylysine hydrochloride was added to the obtained gelatin solution, and the mixture was stirred continuously until dissolved. The pH of the reaction system was adjusted to 10 to obtain a premix. 0.2g of ethylene glycol diglycidyl ether was added to the premix, and the mixture was stirred continuously at 40℃ for 24 hours. After the reaction, the pH of the solution was adjusted to neutral, and dialyzed using a dialysis bag with a molecular weight cutoff of 30kDa at 40℃ for 3 days to remove unreacted polylysine and coupling agent. The solution was then frozen at -80℃ and vacuum dried to obtain the final product.

[0047] Example 3

[0048] 10g of gelatin was dissolved in 100mL of 2% NaCl aqueous solution at 40℃ for half an hour to form a gelatin solution. 2.5g of ε-polylysine hydrochloride was added to the obtained gelatin solution, and the mixture was stirred continuously until dissolved. The pH of the reaction system was adjusted to 10 to obtain a premix. 0.2g of ethylene glycol diglycidyl ether was added to the premix, and the mixture was stirred continuously at 40℃ for 24 hours. After the reaction, the pH of the solution was adjusted to neutral, and dialyzed using a dialysis bag with a molecular weight cutoff of 30kDa at 40℃ for 3 days to remove unreacted polylysine and coupling agent. The solution was then frozen at -80℃ and vacuum dried to obtain the final product.

[0049] Example 4

[0050] 10g of gelatin was dissolved in 100mL of 2% NaCl aqueous solution at 40℃ for half an hour to form a gelatin solution. 1.25g of ε-polylysine hydrochloride was added to the obtained gelatin solution, and the mixture was stirred continuously until dissolved. The pH of the reaction system was adjusted to 10 to obtain a premix. 0.2g of ethylene glycol diglycidyl ether was added to the premix, and the mixture was stirred continuously at 40℃ for 24 hours. After the reaction, the pH of the solution was adjusted to neutral, and dialyzed using a dialysis bag with a molecular weight cutoff of 30kDa at 40℃ for 3 days to remove unreacted polylysine and coupling agent. The solution was then frozen at -80℃ and vacuum dried to obtain the final product.

[0051] Test case

[0052] The KB disc method was used to investigate the antibacterial activity of gelatin / ε-polylysine covalent conjugates obtained with different raw material ratios against Staphylococcus aureus and Escherichia coli. PBS solution was used as the control. Figure 1 The gelatin / ε-polylysine covalent conjugate showed obvious inhibition zones against both *Escherichia coli* and *Staphylococcus aureus*. When the ratio of gelatin to ε-polylysine was changed, the resulting gelatin / ε-polylysine covalent conjugate exhibited different minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against *Staphylococcus aureus* and *Escherichia coli*. With increasing ε-polylysine dosage, both MICs decreased, indicating that even lower concentrations of antibacterial gelatin were sufficient to inhibit and kill *Escherichia coli* and *Staphylococcus aureus*.

[0053] Table 1

[0054]

[0055] Figure 2 The Fourier transform infrared spectrum of the sample prepared in Example 1 is shown in the figure. The gelatin undergoes a coupling reaction with ε-polylysine, located at 3767 cm⁻¹. -1The infrared absorption peak of NH at a lower wavenumber shifts towards lower wavenumbers, indicating that the NH in the gelatin molecule undergoes a covalent reaction. (Located at 1667 cm⁻¹) -1 1568cm -1 The absorption peak at a certain wavenumber shifts simultaneously to lower wavenumbers, indicating that the amide bonds in the gelatin molecule have reacted. (Located at 1541 cm⁻¹) -1 The absorption peak at the wavenumber is significantly enhanced, indicating that the primary amino group in the gelatin molecule is coupled with the epoxy group, and the primary amino group is converted into a secondary amide group.

[0056] Example 5

[0057] The preparation process was the same as in Example 4, except that the preparation system was scaled up to 500g of gelatin and 62.5g of ε-polylysine hydrochloride, with 10g of ethylene glycol diglycidyl ether as the coupling agent. The KB disc method showed that the gelatin / ε-polylysine covalent conjugate exhibited significant inhibition zones against both *Escherichia coli* and *Staphylococcus aureus*. Figure 3 This demonstrates that the preparation method provided by the present invention can successfully prepare gelatin materials with antibacterial activity at high concentrations and on a large scale.

[0058] Example 6

[0059] 10g of gelatin was dissolved in 200mL of 2-morpholinoethanesulfonic acid (MES) buffer at 40℃ for half an hour to form a gelatin solution. 100mL of an acidic solution containing 1.25g of nisin-Z was added to the obtained gelatin solution, and the mixture was stirred continuously until dissolved. The pH of the reaction system was adjusted to 5.5 to obtain a premix. 0.2g of polyethylene glycol diglycidyl ether (molecular weight 2000) was added to the premix, and the mixture was stirred continuously at 50℃ for 24 hours. After the reaction, the pH of the solution was adjusted to neutral, and the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 30kDa at 40℃ for 3 days to remove unreacted polylysine and coupling agent. The solution was then frozen at -80℃ and vacuum dried to obtain the final product. The KB disc method showed that the gelatin / nisin covalent conjugate exhibited a significant inhibition zone against Staphylococcus aureus, but no significant antibacterial effect against Escherichia coli. Figure 4 This is because nisin itself is only effective against Staphylococcus aureus and has no effect on Escherichia coli. When it forms a covalently coupled with gelatin, its properties are preserved.

[0060] Example 7

[0061] Dissolve 10g of gelatin in 100mL of MES buffer at 40℃ for half an hour to form a gelatin solution. Add 2g of ε-polylysine hydrochloride to the obtained gelatin solution, followed by 100mL of 2% water-soluble chitosan MES solution. Stir continuously until dissolved, and adjust the pH of the reaction system to 5.5 to obtain a premix. Add 0.3g of 1,4-butanediol diglycidyl ether to the premix, and stir continuously at 50℃ for 24 hours. After the reaction, adjust the pH of the solution to neutral, and dialyze using a dialysis bag with a molecular weight cutoff of 30kDa at 40℃ for 5 days to remove unreacted polylysine and coupling agent. Then freeze at -80℃ and vacuum dry to obtain the final product.

[0062] Comparative Example 1

[0063] 10g of gelatin was dissolved in 100mL of 2-morpholine ethanesulfonic acid buffer at 40℃ for half an hour to form a gelatin solution. 0.25g of ε-polylysine hydrochloride was added, and the solution was continuously stirred to dissolve. The pH of the reaction system was adjusted to 5.5. 1g-10g of EDC and 1g-10g of NHS were added for coupling. The system formed a gel within minutes, making it impossible to remove impurities and obtain powdered antibacterial gelatin material.

[0064] Comparative Example 2

[0065] 2 g of gelatin was dissolved in 100 mL of 2-morpholine ethanesulfonic acid buffer at 40 °C for half an hour to form a gelatin solution. 0.05 g of ε-polylysine hydrochloride was added, and the solution was continuously stirred until dissolved. The pH of the reaction system was adjusted to 5.5. 0.2 g-2 g of EDC and 0.2 g-2 g of NHS were added for coupling, and the reaction was carried out at 40 °C with continuous stirring for 24 hours. After the reaction, the pH of the solution was adjusted to neutral, and the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 30,000 to remove unreacted polylysine. The solution was then frozen at -80 °C and vacuum dried to obtain the final product. During this preparation process, the reaction system remained in a solution state and did not form a gel. This indicates that when using EDC / NHS coupling, a low concentration of the reaction system is necessary to maintain a solution state.

[0066] Comparative Example 3

[0067] The preparation process is the same as in Example 1, except that ε-polylysine hydrochloride is not added or 0.1g of ε-polylysine hydrochloride is added to participate in the reaction. Other conditions remain unchanged. After the coupling reaction, a large amount of gel-like substance is formed in the reaction system, which affects the next step of impurity removal and obtaining powdered antibacterial gelatin material.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing gelatin / antibacterial peptide covalent conjugate having antibacterial activity, characterized in that, Includes the following steps: 1) Add the reaction medium to the gelatin, heat it, and let the gelatin dissolve completely to obtain a gelatin solution; 2) Add antimicrobial peptides to the gelatin solution, and add or not add functional reagents. After fully dissolving, adjust the pH to 3-6.5 or 9-12 to obtain a premixed solution. 3) Add coupling agent to the premixed solution to carry out the coupling reaction. After the reaction is complete, adjust to neutral, dialyze, and freeze dry to obtain the product. The gelatin solution contains 2-20% gelatin by mass. The mass ratio of the antimicrobial peptide to gelatin is 0.05-2:1; The mass ratio of the coupling agent to gelatin is 0.01-0.10:1; The coupling agent is selected from one or more of the following: ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, pentaerythritol diglycidyl ether, glycerol triglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, trimethylolpropane triglycidyl ether, and tetra-arm polyethylene glycol diglycidyl ether. The antimicrobial peptides include one or more of ε-polylysine or its salts, nisin or its salts, and natamycin or its salts.

2. The preparation method according to claim 1, characterized in that, The gelatin includes one or more gelatin sources from pigs, cattle, fish, and poultry.

3. The preparation method according to claim 1, characterized in that, The mass fraction of gelatin in the gelatin solution is 5-15%.

4. The preparation method according to claim 1, characterized in that, The reaction medium includes one or more of distilled water, sodium chloride aqueous solution, carbonate buffer, Tris-HCl solution, 2-morpholine ethanesulfonic acid solution, and acetate buffer.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the antimicrobial peptide to gelatin is 0.1-1:

1.

6. The preparation method according to claim 1, characterized in that, The functional reagents include one or more of the following: antioxidant enhancing reagents, antibacterial enhancing reagents, mechanical strength enhancing reagents, and extensibility enhancing reagents.

7. The preparation method according to claim 1, characterized in that, The functional reagent is 0-20 wt% of the gelatin mass.

8. The preparation method according to claim 1, characterized in that, In step 2, the pH is adjusted to 4-6 or 10-11.

9. The preparation method according to claim 1, characterized in that, The mass ratio of the coupling agent to gelatin is 0.02-0.05:

1.

10. The preparation method according to claim 1, characterized in that, The coupling reaction is carried out at a temperature of 35-60℃ for 8-48 hours.

11. The preparation method according to claim 1, characterized in that, The dialysis temperature is room temperature - 50°C, and the dialysis time is 1-5 days; the molecular weight cutoff of the dialysis bag is 20-40 kDa.

12. The preparation method according to claim 1, characterized in that, The dialysis temperature is 35-45℃.

13. A gelatin / antimicrobial peptide covalent conjugate with antibacterial activity, characterized in that, It is prepared by the preparation method described in any one of claims 1-12.

14. The use of the gelatin / antimicrobial peptide covalent conjugate as described in claim 13 in the preparation of antimicrobial and / or bacteriostatic products.

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