Preparation method and application of antibacterial water-soluble chitosan derivative

Abstract

The invention provides a preparation method and application of an antibacterial water-soluble chitosan derivative, chitosan is used as a raw material, the chitosan has active sites of two aldehyde groups through a chemical reaction mode, lysine is grafted to a chitosan carbon skeleton, and the antibacterial water-soluble chitosan derivative is prepared. The chitosan derivative has good antibacterial activity and has no obvious cytotoxicity, and the application range of chitosan in food preservation is widened.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive polysaccharides, and specifically relates to a preparation method and application of an antibacterial water-soluble chitosan derivative. Background Art

[0002] Food preservation is a key link to ensure that food remains fresh, edible and safe during production, transportation, storage and sales. With the growth of people's demand for food, the continuous expansion of the scale of the food industry, the increasing expansion of the food supply chain and the deepening of international trade, food preservation faces many problems. The core position of food preservation technology has become increasingly prominent. Its application is not only a prerequisite for ensuring the quality of food, but also meets consumers' expectations for healthy food, laying a solid foundation for the sustainable development of the food industry. Therefore, finding safe and effective preservation methods is of great significance to the food processing industry and food safety. Natural preservation is the general trend. At present, chitosan, as a cationic polysaccharide, has excellent antibacterial effect. Therefore, the preparation of chitosan with good antibacterial effect can effectively solve the problem of food preservation. Chitosan is a biocompatible and biodegradable macromolecular polysaccharide obtained by deacetylation of chitin. This polysaccharide is distributed on shrimp and crab shells, insect epidermis and plant cell walls, and is the only alkaline polysaccharide found so far. However, due to its large molecular weight, chitosan is not easy to dissolve in water and alkaline solutions, and can only be dissolved in weak acidic solutions such as acetic acid, which greatly restricts its widespread use. Therefore, the preparation of chitosan compounds with good water solubility is the focus of current research at home and abroad. Chitosan not only has significant advantages in antibacterial degradation, but is also widely used in fruits, vegetables, meat and aquatic products. However, due to its poor water solubility, chitosan is limited in a wider range of applications. How to modify it to make it more water-soluble is a hot topic of research at home and abroad. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for preparing an antibacterial water-soluble chitosan derivative and its application, using chitosan as a raw material, through chemical reaction, making it have two aldehyde active sites, lysine (Lys) grafted onto the chitosan carbon skeleton, to prepare a chitosan derivative. The chitosan derivative has good antibacterial activity and no obvious cytotoxicity, and has good development and utilization prospects.

[0004] Technical solution: A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution, stirred until completely dissolved, sodium periodate solution was added, nitrogen was filled, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, and reacting at 60-80° C. for 6-8 hours to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added to obtain a solution containing a lysine-modified chitosan derivative; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Furthermore, in step S1, the material-liquid ratio of chitosan to buffer solution is 1:(5-10); and the mass ratio of sodium periodate to chitosan is 1:(1-1.5). Furthermore, in step S3, the mass ratio of chitosan to lysine is 1:(2-4). Furthermore, in step S4, the mass ratio of chitosan to sodium cyanoborohydride is 1:(0.3-0.6). Furthermore, the preparation method provides an antibacterial water-soluble chitosan derivative. Furthermore, the antibacterial water-soluble chitosan derivative is used in food preservation. Beneficial effects: 1. The present invention uses chitosan as a raw material, and through a chemical reaction, it is made to have two aldehyde active sites, and lysine is successfully grafted onto the chitosan carbon skeleton to prepare a chitosan derivative (DLC); 2. The antibacterial mechanism of chitosan depends on the interaction between the positive charge on its own C2 amino group and the negative charge on the bacterial cell membrane, thereby causing bacterial death and playing an antibacterial role; the present invention uses chemical modification to convert the original chitosan C2 amino group into two aldehyde groups, so that the amino group of lysine and the aldehyde group undergo Schiff base reaction and are grafted onto the chitosan carbon skeleton. Due to the special structure of lysine, it not only contains α-NH2 but also ε-NH2. Therefore, it is equivalent to grafting two amino groups onto the chitosan carbon skeleton, thereby enhancing the positive charge of the chitosan derivative and enhancing the antibacterial property of the prepared chitosan derivative; 3. The chitosan derivatives obtained by the present invention have enhanced water solubility, good thermal stability, biodegradability, and no obvious cytotoxicity; 4. The chitosan derivatives obtained by the present invention have a good preservation effect on refrigerated chicken, can significantly inhibit the growth of foodborne microorganisms, have a broad-spectrum antibacterial activity against foodborne bacteria, and have good prospects in the field of food preservation. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is a reaction schematic diagram of the present invention; Figure 2 The infrared spectra of chitosan, lysine, the dialdehyde chitosan derivative prepared in Comparative Example 2, and the chitosan derivative prepared in Example 6; Figure 3 is the hydrogen nuclear magnetic resonance spectrum of chitosan and the chitosan derivative prepared in Example 6; Figure 4 are scanning electron microscope images of chitosan and the chitosan derivative prepared in Example 6; Figure 5 The scanning and transmission electron micrographs are of chitosan and the chitosan derivative prepared in Example 6 after treating bacteria, wherein A is a scanning electron micrograph; B is a transmission electron micrograph; Figure 6 Thermogravimetric analysis diagrams of chitosan and chitosan derivatives prepared in Example 6; Figure 7 This is a graph showing the effect of chitosan derivatives on cell viability. DETAILED DESCRIPTION The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are intended to explain the present invention but the present invention is not limited to the following embodiments. Example 1 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:5, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:2, reacting at 60°C for 8h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.3, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 2 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:5, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:2, reacting at 62°C for 8h to obtain a reaction product; S4. The reaction product is dissolved in a PBS buffer solution, sodium cyanoborohydride is added, and the mass ratio of chitosan to sodium cyanoborohydride is 1:0.3 to obtain a solution containing a lysine-modified chitosan derivative; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 3 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:6, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.1, nitrogen was introduced, and the mixture was reacted at 4°C for 12 h; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:2, reacting at 65°C for 7.5h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.4, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 4 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:6, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.2, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:3, reacting at 65°C for 7.5h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.4, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 5 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:7, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.3, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:3, reacting at 70°C for 7h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.5, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 6 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:8, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.3, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:3, reacting at 70°C for 7h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.5, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 7 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:9, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.4, nitrogen was introduced, and the mixture was reacted at 4°C for 12 h; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:4, reacting at 75°C for 6.5h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.6, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Example 8 A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:10, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.5, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, the mass ratio of chitosan to lysine being 1:4, reacting at 80°C for 6h to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added, the mass ratio of chitosan to sodium cyanoborohydride was 1:0.6, and a solution containing a lysine-modified chitosan derivative was obtained; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative. Comparative Example 1 The difference between this comparative example and Example 6 is that only the dialdehyde chitosan derivative is prepared, which is as follows: A method for preparing an antibacterial water-soluble chitosan derivative comprises the following steps: S1. At pH 3-4, chitosan was added to PBS buffer solution at a solid-liquid ratio of 1:8, stirred until completely dissolved, sodium periodate solution was added at a mass ratio of sodium periodate to chitosan of 1:1.3, nitrogen was introduced, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, remove the unreacted chitosan, precipitate with ethanol, and filter to obtain a filter cake, which is the dialdehyde chitosan derivative. Performance test: (1) Infrared spectroscopy Chitosan, lysine, the dialdehyde chitosan derivative (NaIO4-M) prepared in Comparative Example 1 and the chitosan derivative prepared in Example 6 were analyzed by a Nicolet iS10 Fourier transform infrared spectrometer using a potassium bromide tablet method. Scanning range 500-4000 cm -1 , scan times 128 times, resolution 4cm -1 . like Figure 2 As shown in the infrared spectrum of chitosan, 3200-3500 cm -1 The broad peak at 2923 cm represents the overlap of multiple stretching vibration absorption peaks of -OH and amino groups in chitosan molecules; -1 The weak absorption peak at 1656cm -1and 1600cm -1 The stretching vibrations at 1159 cm-1 correspond to the methylene group and the methyl group on the acetamide group, representing the stretching vibrations of amide I and amide II, respectively; -1 The peak at 1159 cm was determined to be the characteristic absorption peak of the entire polysaccharide structure of the chitosan carbon skeleton. After chitosan was oxidized with sodium periodate, the pyranose ring opened, resulting in the introduction of a dialdehyde group, 1159 cm -1 The disappearance of the peak indicates that the polysaccharide structure of the chitosan carbon skeleton has changed. -1 The appearance of the peak indicates the presence of aldehyde groups, forming dialdehyde chitosan. For the infrared spectrum of lysine, 3200-2500cm -1 The broad peaks in the region are carboxyl groups, while the peaks in the range of 1300 to 1000 cm -1 The peak in the region is the CO absorption peak, which is characteristic of the carboxyl group of lysine; 2125cm -1 The peak at 3400-3300 cm is the absorption peak of the stretching vibration of the amino acid hydrocarbon chain CH. -1 The peak at 549 cm is the antisymmetric stretching peak of amino NH. -1 The peak at 1739 cm is the bending peak of the amino NH plane. The infrared spectrum of the target product shows -1 The shoulder peak at 4° disappeared, indicating the formation of Schiff base. In addition, the characteristic peak of lysine appeared in the infrared spectrum of the product, indicating that lysine was grafted onto dialdehyde chitosan. (2) Nuclear magnetic resonance spectroscopy analysis Chitosan and the chitosan derivative prepared in Example 6 of the present invention were analyzed by using a JNM-ECP600 fully digital nuclear magnetic resonance spectrometer from JEOL, Japan. 1 H NMR spectrum analysis. The detection temperature was 40°C and the sample was dissolved in D2O. like Figure 3 As shown, the chemical shift of the sugar skeleton in the chitosan molecule is mainly between 2.0 and 5.0 ppm. Among them, the peak at 4.51 ppm corresponds to the hydrogen proton of [H1], the peaks at 3.44 to 4.01 ppm correspond to the hydrogen protons of the chitosan carbon skeleton [H3] to [H6], and the peak at 3.03 ppm is attributed to the hydrogen proton connected to the C2 atom, recorded as [H2]. In the lysine-modified chitosan derivatives, the C2 and C3 positions of chitosan are converted to methylene after the chitosan is connected to the N atom, and the 3.03 ppm hydrogen proton connected to [H2] on the C2 atom disappears. The chemical shifts of these modified derivatives are between 2.7 and 2.9 ppm. In addition, the characteristic absorption peaks of the protons on the lysine carbon skeleton were observed in the derivatives, ranging from 1.41 to 1.91 ppm. These results provide further evidence for the successful preparation of lysine-modified chitosan derivatives. (3) Scanning electron microscopy characterization The morphology and microstructure of chitosan and the chitosan derivative prepared in Example 6 were studied using a scanning electron microscope, and the samples were gold sputtered at an accelerating voltage of 3.0 kV. Figure 4 The microstructure of chitosan and lysine-modified chitosan was observed by scanning electron microscopy. The results showed that the surface of unmodified chitosan had a smooth membrane structure without pores, while for the chitosan derivatives modified with lysine, the smooth membrane structure was decomposed into a more broken structure, and a dense particle structure was observed on its surface. This is because sodium periodate is a strong oxidant that can destroy the ether bonds between chitosans and increase the degree of breakage of the long-chain structure of chitosan. The dense particles on the surface of lysine-modified chitosan may be due to lysine grafting. The SEM results further confirmed the successful synthesis of lysine-modified chitosan derivatives. (4) Antibacterial activity analysis First, the E. coli bacterial cells were cultured in a shaker until the logarithmic phase, and chitosan derivatives were added to the bacterial solution to fully bind to the E. coli bacterial solution. PBS was added to the blank group instead of chitosan derivatives, and incubated in a 37°C incubator for 2 hours; then centrifuged to remove the supernatant, and glutaraldehyde was added for fixation, and then suspended and sealed. Scanning electron microscopy and transmission electron microscopy analysis were performed to observe its antibacterial activity. like Figure 5 To further analyze the antibacterial activity of lysine-modified chitosan, the effect of lysine-modified chitosan on the appearance of bacteria was studied using scanning electron microscopy ( Figure 5 A), the results showed that PBS treatment did not cause significant damage to the visual properties of bacteria. However, in the presence of lysine-modified chitosan, the cell structures of Salmonella typhimurium, Escherichia coli, Lactobacillus monocytogenes, and Pseudomonas aeruginosa were substantially distorted and damaged, resulting in unclear edges. Transmission electron microscopy was used to analyze the effects of lysine-modified chitosan on the ultrastructure of bacterial cells ( Figure 5 B), the cells in the untreated group showed typical morphology, intact cell membranes, and uniform cytoplasmic electron density. However, after exposure to lysine-modified chitosan for 2 hours, these cells showed distorted morphology, release of cellular components, and uneven cytoplasmic electron density. Scanning electron microscopy and transmission electron microscopy showed that lysine-modified chitosan did not destroy the integrity of the cells, but instead caused leakage of contents and bacterial death. These results indicate that lysine-modified chitosan has the ability to target and disrupt bacterial membranes, ultimately leading to the destruction of bacteria. (5) Thermogravimetric analysis The thermal stability of chitosan and chitosan derivatives was evaluated using a Hitachi DSC7000 thermal stability instrument in a temperature range of 25-500°C, a heating rate of 10°C / min, and a nitrogen flow rate of 20 mL / min. like Figure 6As shown in the figure, the thermogravimetric analysis spectra of unmodified chitosan and lysine-modified chitosan exhibited similar weight loss processes, which were divided into two stages. Initially, due to the evaporation of water during heating, both materials had a slight weight loss before 100°C; the second stage was thermal decomposition, with chitosan degrading at 217.6°C and lysine-modified chitosan degrading at 191.5°C. During the experiment, the mass loss rate of chitosan was 55.19%, and the mass loss rate of lysine-modified chitosan was 15.06%. The DTG curve showed that the lysine-modified chitosan derivative had a lower degradation temperature, which may be because the introduction of lysine changed the crystal form of the chitosan structure itself, thereby causing a decrease in thermal stability. Although the lysine-modified chitosan derivative is slightly inferior to the unmodified chitosan in thermal stability, there is no obvious difference within the normal use temperature range. (6) Toxicity evaluation Take Caco-2 cells in logarithmic phase and adjust the cell concentration to 2×10 4 After culturing in a 96-well plate for 24 hours (37°C, 5% CO2), chitosan and DLC solutions were added to reach different gradient concentrations (100, 200, 300, 400, 500 μg / mL), and a control group was set up with the culture medium as the control. After culturing for 24 hours, the culture medium was discarded, and then 200 μL of culture medium containing 0.5 mg / mL MTT was added and cultured for another 4 hours. The culture was terminated, 150 μL of dimethyl sulfoxide was added, and the absorbance at 570 nm was measured with an ELISA reader after slight shaking for 10 minutes. MTT method is the most commonly used method for detecting cell activity. Its principle is that succinate dehydrogenase in living cells can reduce exogenous MTT to insoluble blue crystals and deposit in cells, while dead cells do not have this function. The cell survival rate is determined by measuring the absorbance value compared with the control group. In the entire test concentration range, chitosan and its derivatives showed a significant effect on the survival rate of the tested cells ( Figure 7 ), chitosan is considered to be a non-toxic food additive. Combined with the experimental results, we can believe that the chitosan derivatives modified with lysine do not have obvious cytotoxicity within a certain concentration range (≤500μg / mL). Therefore, the introduction of lysine does not significantly increase the toxicity of chitosan derivatives, which provides a basis for it to become a potential safe antibacterial agent. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing an antibacterial water-soluble chitosan derivative, characterized in that: The following steps are involved: S1. At pH 3-4, chitosan was added to PBS buffer solution, stirred until completely dissolved, sodium periodate solution was added, nitrogen was filled, and the reaction was carried out at 4°C for 12 hours; S2. After the reaction is completed, unreacted chitosan is removed, ethanol precipitation is performed, and a filter cake is obtained by filtration to obtain a dialdehyde chitosan derivative; S3. dissolving the dialdehyde chitosan derivative in a PBS buffer solution, adding lysine, and reacting at 60-80° C. for 6-8 hours to obtain a reaction product; S4. The reaction product was dissolved in a PBS buffer solution, sodium cyanoborohydride was added to obtain a solution containing a lysine-modified chitosan derivative; S5. Add ethanol for precipitation to obtain a crude lysine-modified chitosan derivative, dissolve the crude lysine-modified chitosan derivative in water, dialyze for 48 hours, and freeze-dry to obtain an antibacterial water-soluble chitosan derivative.

2. The method for preparing an antibacterial water-soluble chitosan derivative according to claim 1, characterized in that: In the step S1, the material-liquid ratio of chitosan to buffer solution is 1:(5-10); the mass ratio of sodium periodate to chitosan is 1:(1-1.5).

3. The method for preparing an antibacterial water-soluble chitosan derivative according to claim 1, characterized in that: In step S3, the mass ratio of chitosan to lysine is 1:(2-4).

4. The method for preparing an antibacterial water-soluble chitosan derivative according to claim 1, characterized in that: In step S4, the mass ratio of chitosan to sodium cyanoborohydride is 1:(0.3-0.6).

5. An antibacterial water-soluble chitosan derivative prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the antibacterial water-soluble chitosan derivative according to claim 5 in food preservation.

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