An amino acid-modified quaternized chitosan, its preparation method, and its application in the preparation of antibacterial products.

Abstract

This invention discloses an amino acid-modified quaternized chitosan, its preparation method, and its application in the preparation of antibacterial products, relating to the field of antibacterial materials technology. The amino acid-modified quaternized chitosan comprises a quaternized chitosan backbone and amino acids covalently grafted onto the quaternized chitosan backbone. The degree of quaternization of the amino acid-modified quaternized chitosan is 0.2–1.6, the degree of amino acid grafting is 0.1–0.8, and the weight-average molecular weight is 5 × 10⁻⁶. 3 ~8×10 5 g / mol. The degree of quaternization substitution, degree of amino acid grafting, and weight-average molecular weight of the amino acid-modified quaternized chitosan provided by this invention are all controlled within the above range, which ensures that the material has good antibacterial and bactericidal effects, and also has good biocompatibility and low cytotoxicity.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, and in particular to an amino acid-modified quaternized chitosan, its preparation method, and its application in the preparation of antibacterial products. Background Technology

[0002] Chitosan is widely available, biodegradable, and biocompatible, but its poor solubility under neutral and weakly alkaline conditions limits its direct application in water-based antibacterial coatings.

[0003] Currently, quaternization modification is a common method used to improve the water solubility of chitosan. Quaternization modification can improve the water solubility of chitosan and enhance its cationic properties, thereby improving its inhibitory effect on microorganisms. However, chitosan modified by quaternization alone has a narrow antibacterial spectrum and limited activity, and the distribution of quaternization groups in the structure is uneven, making it difficult to precisely control the degree of substitution. Summary of the Invention

[0004] The main objective of this invention is to propose an amino acid-modified quaternized chitosan, its preparation method, and its role in the preparation of antibacterial products, aiming to solve the problem of weak antibacterial strength of quaternized modified chitosan in the prior art.

[0005] To achieve the above objectives, the present invention proposes an amino acid-modified quaternized chitosan, wherein the amino acid-modified quaternized chitosan comprises a quaternized chitosan backbone and amino acids covalently grafted onto the quaternized chitosan backbone;

[0006] The degree of quaternization of the amino acid-modified quaternized chitosan is 0.2–1.6, the degree of amino acid grafting is 0.1–0.8, and the weight-average molecular weight is 5 × 10⁻⁶. 3 ~8×10 5 g / mol.

[0007] In one embodiment, the degree of quaternization of the amino acid-modified quaternized chitosan is 0.2 to 0.9; and / or, The amino acids include one or more of arginine, lysine, and histidine.

[0008] In one embodiment, the minimum inhibitory concentration (MIC) of the amino acid-modified quaternized chitosan against Staphylococcus aureus is 6.25–12.5 μg / mL; and / or, The minimum bactericidal concentration of the amino acid-modified quaternized chitosan against Staphylococcus aureus is 50-100 μg / mL; and / or, The minimum inhibitory concentration of the amino acid-modified quaternized chitosan against Candida albicans is 50-200 μg / mL.

[0009] This invention also provides a method for preparing amino acid-modified quaternized chitosan, the method comprising the following steps: S1. Mix chitosan and an alkaline solution containing urea, and stir the mixture at a temperature below 5°C to obtain a mixed solution. S2. Mix the mixed solution with the quaternizing agent to carry out the quaternization reaction and obtain quaternized chitosan; S3. The quaternized chitosan, a buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and amino acids are mixed and subjected to an amino acid grafting reaction. The mixture is then purified by dialysis and freeze-dried to obtain the amino acid-modified quaternized chitosan.

[0010] In one embodiment, step S1 includes: Chitosan raw material is oxidized and degraded in water to obtain chitosan; Chitosan and an alkaline solution containing urea are mixed and stirred at a temperature below 5°C to obtain a mixed solution.

[0011] In one embodiment, in step S1: The urea-containing alkaline solution has a urea mass fraction of 4% to 16%; and / or, The alkaline solution containing urea has an alkali mass fraction of 2% to 16%; and / or, The alkaline solution containing urea includes one or more of the following: KOH, NaOH, and LiOH.

[0012] In one embodiment, in step S2: The molar ratio of the free amino groups of chitosan to the quaternizing agent in the mixed solution is 4~30:1; and / or, The quaternizing agent includes at least one of glycidyltrialkylammonium chloride and 3-chloro-2-hydroxypropyltrimethylammonium chloride; and / or, The quaternization reaction is performed at a temperature of -10 to 40°C; and / or, The quaternization reaction takes 6 to 96 hours.

[0013] In one embodiment, in step S3: The buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide has a pH of 4.5 to 6.5; and / or, The solvent in the buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is MES buffer or phosphate buffer; and / or, The amino acid grafting reaction is performed at a temperature of 20-25°C; and / or, The amino acid grafting reaction takes 36-48 hours.

[0014] The present invention also provides an application of amino acid-modified quaternized chitosan in the preparation of antibacterial products, wherein the amino acid-modified quaternized chitosan includes the aforementioned amino acid-modified quaternized chitosan or the amino acid-modified quaternized chitosan prepared by the aforementioned method.

[0015] In one embodiment, the antibacterial product includes at least one of antibacterial liquid, antibacterial coating, antibacterial film, and antibacterial composite material.

[0016] In the technical solution of this invention, covalent grafting of amino acids onto quaternized chitosan enhances its positive charge, thereby reducing the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for bacteria, and enhancing the material's ability to bind to fungal cell membranes, thus improving the antibacterial effect. The amino acid-modified quaternized chitosan provided by this invention, with its quaternization degree, amino acid grafting degree, and weight-average molecular weight all controlled within the aforementioned ranges, ensures that the material exhibits good antibacterial and bactericidal effects, while also possessing good biocompatibility and low cytotoxicity. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 The chemical reaction formulas for the preparation of amino acid-modified quaternized chitosan in one embodiment of the present invention are as follows: (1) is the quaternization reaction formula; (2) is the amino acid substitution reaction formula. Figure 2 a is the FTIR image of the materials and chitosan raw material prepared in Examples 2, 4 and Comparative Example 1 of this invention; Figure 2 b represents the material prepared in Comparative Example 1 and Example 5 of this invention. 1 H NMR spectrum; Figure 2 c represents the material prepared in Examples 1 and 2 of this invention. 1 H NMR spectrum; Figure 2 d represents the material prepared in Examples 3 and 4 of this invention. 1 H NMR spectrum; Figure 3a is a graph showing the effect of the materials prepared in Comparative Example 1 and Example 5 of this invention on the bacterial survival rate at different concentrations; Figure 3 b is a graph showing the minimum inhibitory concentration of the materials prepared in Comparative Example 1 and Example 5 of this invention against Gram-positive Staphylococcus aureus; Figure 3 c is a graph showing the minimum bactericidal concentration of the materials prepared in Comparative Example 1 and Example 5 of this invention against Gram-positive Staphylococcus aureus; Figure 3 d is a graph showing the effect of the materials prepared in Examples 1 and 2 of this invention on the bacterial survival rate at different concentrations; Figure 3 e is a graph showing the minimum inhibitory concentration of the materials prepared in Examples 1 and 2 of this invention against Gram-positive Staphylococcus aureus; Figure 3 f is a graph showing the minimum bactericidal concentration of the materials prepared in Examples 1 and 2 of this invention against Gram-positive Staphylococcus aureus; Figure 3 h is a graph showing the effect of the materials prepared in Examples 3 and 4 of this invention on the bacterial survival rate at different concentrations; Figure 3 i is a graph showing the minimum inhibitory concentration of the materials prepared in Examples 3 and 4 of this invention against Gram-positive Staphylococcus aureus; Figure 3 j is a graph showing the minimum bactericidal concentration of the materials prepared in Examples 3 and 4 of this invention against Gram-positive Staphylococcus aureus; Figure 4 The left image of a is a scanning electron microscope image of Staphylococcus aureus treated without any antibacterial materials. Figure 4 The right figure of a is Figure 4 A magnified view of a portion of the drawing; Figure 4 The left image of b is a scanning electron microscope image of Staphylococcus aureus after treatment with the material prepared in Example 4 for 12 hours. Figure 4 The right figure of b is Figure 4 b. A magnified view of a portion of the plot; Figure 4 The left image in c is a scanning electron microscope image of Staphylococcus aureus after treatment with the material prepared in Example 3 for 12 hours. Figure 4 The right figure of c is Figure 4 A magnified view of a portion of the plot; Figure 5 a is a graph showing the minimum inhibitory concentration of the materials prepared in Comparative Example 1 and Example 5 of this invention against Candida albicans; Figure 5 b is a graph showing the minimum bactericidal concentration of the materials prepared in Comparative Example 1 and Example 5 of this invention against Candida albicans; Figure 5 c is a graph showing the minimum inhibitory concentration of the materials prepared in Examples 1 and 2 of this invention against Candida albicans; Figure 5 d is a graph showing the minimum bactericidal concentration of the materials prepared in Examples 1 and 2 of this invention against Candida albicans; Figure 5e is a graph showing the minimum inhibitory concentration of the materials prepared in Examples 3 and 4 of this invention against Candida albicans; Figure 5 f is a graph showing the minimum bactericidal concentration of the materials prepared in Examples 3 and 4 of this invention against Candida albicans; Figure 6 The figure shows the effect of the quaternized chitosan material prepared in this invention on the cytotoxicity of L929 cells. Figure 7 The figure shows the effect of the amino acid-modified quaternized chitosan material prepared in this invention on the cytotoxicity of L929 cells.

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Chitosan is widely available, biodegradable, and biocompatible, but its poor solubility under neutral and weakly alkaline conditions limits its direct application in water-based antibacterial coatings.

[0022] Currently, quaternization modification is a common method used to improve the water solubility of chitosan. Quaternization modification can improve the water solubility of chitosan and enhance its cationic properties, thereby improving its inhibitory effect on microorganisms. However, chitosan modified by quaternization alone has a narrow antibacterial spectrum and limited activity, and the distribution of quaternization groups in the structure is uneven, making it difficult to precisely control the degree of substitution.

[0023] In view of this, the present invention provides an amino acid-modified quaternized chitosan, wherein the amino acid-modified quaternized chitosan comprises a quaternized chitosan backbone and amino acids covalently grafted onto the quaternized chitosan backbone; wherein the degree of quaternization of the amino acid-modified quaternized chitosan is 0.2~1.6, the degree of amino acid grafting of the amino acid-modified quaternized chitosan is 0.1~0.8, and the weight-average molecular weight of the amino acid-modified quaternized chitosan is 5×10⁻⁶. 3 ~8×10 5 g / mol.

[0024] In the technical solution of this invention, covalent grafting of amino acids onto quaternized chitosan enhances its positive charge, thereby reducing the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for bacteria and enhancing its interaction with fungal cell membranes, thus improving the antibacterial effect. The degree of quaternization substitution, degree of amino acid grafting, and weight-average molecular weight of the amino acid-modified quaternized chitosan provided by this invention are simultaneously controlled within the aforementioned ranges, ensuring that the material exhibits good antibacterial and bactericidal effects, while also possessing good biocompatibility and low cytotoxicity. It should also be noted that, compared to existing antibiotics, the material provided in this application is less likely to induce drug resistance.

[0025] In this invention, the degree of quaternization substitution, molecular weight and amino acid grafting degree can be controlled simultaneously to achieve controllability of each parameter, which is convenient for adaptive improvement for different application scenarios. The amino acid-modified quaternized chitosan provided by this invention can be directly used in water-based systems such as antibacterial coatings.

[0026] For example, the degree of quaternization substitution can be a numerical range consisting of any two values ​​of 0.2, 0.6, 1.0, 1.4, 1.6 or higher; the degree of amino acid grafting can be a numerical range consisting of any two values ​​of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or higher; and the weight-average molecular weight can be 5 × 10⁻⁶. 3 g / mol, 2×10 4 g / mol, 4×10 4 g / mol, 6×10 4 g / mol, 8×10 4 g / mol, 1×10 5 g / mol, 2×10 5 g / mol, 4×10 5 g / mol, 6×10 5 g / mol, 8×10 5 A numerical range consisting of any two values ​​of g / mol or above.

[0027] Preferably, the degree of quaternization of the amino acid-modified quaternized chitosan is 0.2 to 0.9. Within this range, the degree of quaternization significantly improves water solubility and facilitates subsequent amino acid grafting.

[0028] In some embodiments, the amino acid includes one or more of arginine, lysine, and histidine. The side chains of these amino acids contain basic groups (positively charged amino or imidazole groups), which can further enhance the cationic properties of the material, thereby improving its ability to disrupt bacterial and fungal cell membranes.

[0029] Within the aforementioned weight-average molecular weight range, the smaller the molecular weight of the amino acid-modified quaternized chitosan provided by this invention, the stronger its antibacterial ability. In some embodiments, the minimum inhibitory concentration (MIC) of the amino acid-modified quaternized chitosan against Staphylococcus aureus is 6.25–12.5 μg / mL; and / or, the minimum bactericidal concentration (MBC) of the amino acid-modified quaternized chitosan against Staphylococcus aureus is 50–100 μg / mL; and / or, the minimum inhibitory concentration (MIC) of the amino acid-modified quaternized chitosan against Candida albicans is 50–200 μg / mL.

[0030] This invention also provides a method for preparing amino acid-modified quaternized chitosan, the method comprising the following steps: Step S1: Mix chitosan and an alkaline solution containing urea, and stir the mixture at a temperature below 5°C to obtain a mixed solution. Step S2: Mix the mixed solution with the quaternizing agent to carry out the quaternization reaction and obtain quaternized chitosan; Step S3: Mix the quaternized chitosan, a buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and amino acids, perform an amino acid grafting reaction, dialysis purification, and freeze-drying to obtain the amino acid-modified quaternized chitosan.

[0031] In the technical solution of this invention, in step S1, chitosan is dissolved at low temperature in an alkaline solution containing urea to obtain a homogeneous reaction system, which is beneficial for large-scale production and stable parameter control; the glucose units of chitosan have free amino and free hydroxyl groups, such as Figure 1 As shown, in step S2, the quaternizing reagent and the mixed solution are mixed and then subjected to a quaternization reaction, so that the free amino group at position 2 and / or the free hydroxyl group at position 6 of the chitosan in the mixed solution reacts with the quaternizing reagent to form a chitosan derivative with a positively charged quaternary ammonium salt group. The chitosan derivative retains some unreacted free amino and free hydroxyl groups. The quaternization reaction enhances the water solubility of chitosan under neutral and alkaline conditions; in step S3, as... Figure 1As shown, in a buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide activates the carboxyl group of amino acid (EDC), N-hydroxysuccinimide (NHS) stabilizes the activated intermediate, and the carboxyl group of the activated intermediate can undergo a grafting reaction with the free amino group remaining in the quaternized chitosan, thereby obtaining amino acid-modified quaternized chitosan.

[0032] It should be noted that in this invention, chitosan can be derived from chitosan or chitin, but the chitosan in the final mixed solution undergoing the quaternization reaction has a certain degree of deacetylation, preferably 10% or more, to ensure that there are more free amino groups in the chitosan participating in the amino acid grafting reaction.

[0033] In some embodiments, step S1 includes: oxidatively degrading chitosan raw material in water to obtain chitosan; mixing chitosan with an alkaline solution containing urea and stirring at a temperature below 5°C to obtain a mixed solution. The above method is suitable for cases where the chitosan raw material has a high molecular weight. Firstly, the chitosan raw material is degraded by hydrogen peroxide as an oxidant. The hydroxyl radicals generated by hydrogen peroxide randomly cleave the β-(1,4)-glycosidic bonds on the chitosan backbone, thereby effectively reducing the molecular weight of chitosan, significantly improving its water solubility, and exposing more active reaction sites.

[0034] In some embodiments, in step S1: the mass fraction of urea in the alkaline solution containing urea is 4% to 16%; and / or, the mass fraction of alkali in the alkaline solution containing urea is 2% to 16%; and / or, the type of alkali in the alkaline solution containing urea includes one or more of KOH, NaOH, and LiOH. Simultaneously controlling the mass fraction of urea, the type of alkali, and their mass fraction within the above ranges ensures good low-temperature dissolution of chitosan, facilitating the subsequent quaternization reaction.

[0035] In some embodiments, in step S2: the molar ratio of the free amino group of chitosan to the quaternizing agent in the mixed solution is 4~30:1; and / or, the quaternizing agent includes at least one of glycidyltrialkylammonium chloride and 3-chloro-2-hydroxypropyltrimethylammonium chloride; and / or, the temperature of the quaternization reaction is -10~40℃; and / or, the time of the quaternization reaction is 6~96 h. Simultaneously controlling the molar ratio of the amino group of chitosan to the quaternizing agent, the type of quaternizing agent, the quaternization reaction temperature, and the time within the above ranges ensures a more complete quaternization reaction, allowing the free hydroxyl groups or free amino groups on the chitosan to be quaternized more rapidly, forming a quaternized chitosan backbone, improving the water solubility of the backbone, and ensuring the presence of a large number of free amino groups, facilitating subsequent amino acid grafting reactions.

[0036] In some embodiments, in step S3: the pH of the buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 4.5~6.5; and / or, the solvent in the buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is MES buffer (i.e., 2-(N-morpholino)ethanesulfonic acid buffer) or phosphate buffer; and / or, the temperature of the amino acid grafting reaction is 20-25℃; and / or, the time of the amino acid grafting reaction is 36~48h. Simultaneously controlling the above parameters within the above ranges can ensure that a large number of amino acids are covalently grafted onto the quaternized chitosan backbone, forming amino acid-modified quaternized chitosan.

[0037] This invention also provides an application of amino acid-modified quaternized chitosan in the preparation of antibacterial products. The amino acid-modified quaternized chitosan includes the aforementioned amino acid-modified quaternized chitosan or the amino acid-modified quaternized chitosan prepared by the aforementioned preparation method. Therefore, it possesses all the beneficial effects of the aforementioned amino acid-modified quaternized chitosan or the aforementioned preparation method of amino acid-modified quaternized chitosan, which will not be elaborated further here.

[0038] In some embodiments, the antibacterial product includes at least one of an antibacterial liquid, an antibacterial coating, an antibacterial film, and an antibacterial composite material. The specific form of the antibacterial product may be changed according to the actual application scenario.

[0039] For example, amino acid-modified quaternized chitosan can be dissolved in deionized water to prepare a 0.05%~2% (w / v) solution as an antibacterial coating liquid. This solution can be applied to the surface of substrates such as plastics, paper, glass, or metals using spraying, dip coating, or blade coating methods and then dried to form a film. Alternatively, an antibacterial film can be obtained by casting. The resulting coating or film can be used for antibacterial applications such as the protection of the inner surface of food packaging or the surface of utensils.

[0040] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0041] Example 1 An amino acid-modified quaternized chitosan was prepared by the following steps: (1) Oxidative degradation: Take chitosan raw material (CS, molecular weight 1.21×10 5 Chitosan was prepared by mixing chitosan (g / mol, degree of deacetylation 80%) into a 1% aqueous solution, placing it in a 60℃ water bath, adding hydrogen peroxide to achieve a final concentration of 0.5%, and stirring for 8 h to obtain chitosan.

[0042] (2) Quaternized chitosan: The chitosan prepared in (1) is added to a KOH solvent containing urea (the mass fraction of urea is 8% and the mass fraction of KOH is 16%) and dissolved at -15℃. The mixture is stirred until it is completely dissolved to obtain a homogeneous mixed solution.

[0043] Glycidyltrialkylammonium chloride (i.e., quaternizing agent) was added to the mixed solution to make the molar ratio of chitosan to quaternizing agent 1:20, and the reaction was stirred at 0°C for 24 h. After the reaction was completed, the pH was adjusted to neutral. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for more than 7 days until small molecules and ions were completely removed. Then it was frozen and freeze-dried to obtain quaternized chitosan, denoted as D1-QCS-1:20 (1:20 is the molar ratio of chitosan to quaternizing agent).

[0044] (3) Amino acid modification: 45 mg of quaternized chitosan was weighed and added to 9 mL of MES in HCl solution (MES concentration 25 mmol / L, pH=5.0) and stirred for 2 h to obtain mixed solution 1. NHS and EDC were weighed and dissolved in 2 mL of MES buffer (pH=5.5) to make the molar ratio of NHS to EDC 1:1 to obtain mixed solution 2. 45 mg of arginine was added to mixed solution 2 and stirred for 2 h to obtain mixed solution 3. Mixed solution 3 was added to mixed solution 1 and subjected to amino acid grafting reaction at 25 °C for 48 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for about one week until complete. The solution was then freeze-dried to obtain amino acid-modified quaternized chitosan, denoted as D1-QCS 1:20 / AA.

[0045] Example 2 The difference between Example 2 and Example 1 is that: Step (3) is not performed. The obtained quaternized chitosan is designated as D1-QCS 1:20.

[0046] Example 3 The difference between Example 3 and Example 1 is that: In step (1), hydrogen peroxide is added to bring the final concentration to 1%; The final amino acid-modified quaternized chitosan is denoted as D2-QCS 1:20 / AA.

[0047] Example 4 The difference between Example 4 and Example 3 is that: Step (3) is not performed. The obtained quaternized chitosan is designated as D2-QCS 1:20.

[0048] Example 5 The difference between Example 5 and Example 1 is as follows: No hydrogen peroxide is added in step (1); The obtained amino acid-modified quaternized chitosan is denoted as QCS 1:20 / AA.

[0049] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that: No hydrogen peroxide is added in step (1), and step (3) is not performed. The obtained quaternized chitosan is denoted as QCS 1:20.

[0050] Performance testing 1. The molecular weight of chitosan, the degree of quaternization substitution of quaternized chitosan, and the amino acid grafting rate of amino acid-modified quaternized chitosan in Examples 1-5 and Comparative Example 1 were determined. The test methods are as follows: (1) Molecular weight of chitosan: The sample was separated by size by the chromatographic column and the absolute molecular weight was directly measured by the light scattering detector without the need for standard calibration. The weight average molecular weight and molecular weight distribution can be accurately obtained.

[0051] (2) Degree of quaternization substitution of quaternized chitosan: determined by the precipitation reaction of quaternary ammonium salt groups with silver nitrate. Specifically, after dissolving the quaternized chitosan sample, excess standard silver nitrate solution is added to allow the quaternary ammonium salt anions (such as Cl-) to precipitate. - ) and Ag + The reaction proceeds completely to form a precipitate. After filtration to remove the precipitate, the remaining silver nitrate in the filtrate is titrated with a standard ammonium thiocyanate solution. By calculating the amount of silver nitrate consumed, the content of the quaternary ammonium salt can be deduced, and thus the degree of quaternization substitution can be calculated.

[0052] (3) Amino acid grafting rate of amino acid modified quaternized chitosan: Based on the total nitrogen content variation method, the total nitrogen content of the samples before and after modification was determined by the Kjeldahl method. The amount of introduced amino acids was calculated by the difference in nitrogen content, thereby determining the amino acid grafting rate.

[0053] The test results are shown in Table 1.

[0054] Table 1. Material information for Examples 1-5 and Comparative Example 1

[0055] As shown in Table 1, the higher the hydrogen peroxide concentration, the smaller the molecular weight of the resulting chitosan.

[0056] 2. The materials prepared in Examples 1-5 and Comparative Example 1, and the chitosan raw material (CS), were subjected to FTIR and... 1 H NMR characterization, results as follows Figure 2 a, Figure 2 b、 Figure 2 c. Figure 2 As shown in d.

[0057] Depend on Figure 2 From a, we can know that at 1479 cm -1 N appeared at the location + The characteristic absorption peak of the bending vibration of the CH bond in (CH3)3 confirms that quaternary ammonium groups were successfully introduced into the QCS 1:20, D1-QCS 1:20 and D2-QCS 1:20 samples.

[0058] Depend on Figure 2 As shown in b-2d, the characteristic peak at 2 ppm belongs to the acetyl proton (-COCH3) of the N-acetylglucosamine residue in QCS (quaternized chitosan, including Comparative Example 1, Example 2, and Example 4). After grafting, the signal peak at this position in QCS / AA (amino acid-modified quaternized chitosan, including Example 5, Example 1, and Example 3) remains clearly visible, indicating that the chemical structure of the QCS backbone is maintained after the coupling reaction. The significant signal peak at 3.17 ppm should be attributed to the quaternary ammonium group –N. + The characteristic resonance of the methyl proton in (CH3)3. Furthermore, the new multiplet appearing in the 1.90–1.50 ppm range corresponds to the proton signals of the α- and β-methylene groups (–CH2–CH2–) in the arginine side chain, directly confirming the successful grafting of arginine. The above analysis shows that, compared to the ungrafted sample, the amino acid-grafted sample exhibits characteristic changes related to the amino acid side chain in its spectrum, while retaining quaternization-related features, indicating successful quaternization substitution and amino acid grafting.

[0059] 3. In vitro antibacterial experiments were conducted on the materials prepared in Examples 1-5 and Comparative Example 1. The specific steps are as follows: (1) Determination of minimum inhibitory concentration (MIC): Staphylococcus aureus was selected (S. aureus) As the target bacterium, the minimum inhibitory concentration (MIC) was determined using the standard broth dilution method. Specifically, the material prepared according to this invention was serially diluted with broth culture medium, and then a standard concentration of fresh bacterial suspension (approximately 5 × 10⁻⁶) was inoculated into each well. 5 The culture was incubated at 37°C for 12 hours (CFU / mL) and then observed. The lowest drug concentration at which bacterial growth was inhibited by more than 90% was determined as the MIC value, and wells containing culture medium without the material prepared in this invention were used as positive controls, while wells containing sterile culture medium were used as negative controls. The results are as follows: Figure 3 a, Figure 3 b、 Figure 3 d、 Figure 3 e Figure 3 h、 Figure 3 As shown in i; (2) Minimum Bactericidal Concentration (MBC) Determination: Based on the MIC determination results above, 100 μL of bacterial suspension was taken from each well where no visible bacterial growth was observed, and evenly spread onto fresh solid agar plates. The plates were then incubated at 37°C for 18-24 hours. The minimum concentration that could kill more than 99.9% of the inoculated bacteria (i.e., a reduction of ≥3 logarithmic orders in colony count compared to the original inoculum) was determined as the MBC value of the material. Results are as follows: Figure 3 c. Figure 3 f、 Figure 3 As shown in j.

[0060] Depend on Figure 3 a to Figure 3It can be seen that the MIC of QCS 1:20 / AA is 12.5 μg / mL, the MIC of D1-QCS 1:20 / AA is 12.5 μg / mL, and the MIC of D2-QCS 1:20 / AA is 6.25 μg / mL, indicating that the antibacterial ability is enhanced after amino acid grafting, and the MIC can be further reduced by decreasing the molecular weight within a certain range. Furthermore, the MBC of QCS 1:20 / AA was 100 μg / mL, that of D1-QCS 1:20 / AA was 100 μg / mL, and that of D2-QCS 1:20 / AA was 50 μg / mL, indicating that the bactericidal ability was enhanced after amino acid grafting. Within a certain range, the MBC could be further reduced by decreasing the molecular weight. The reason for this may be that, compared with the intramolecular entanglement and huge steric hindrance that are easily generated by high molecular weight long chains, low molecular weight short chains have better dispersibility in aqueous solution and can more efficiently expose active positively charged sites such as arginine guanidine groups and quaternary ammonium groups, thereby strengthening the electrostatic adsorption with bacterial cell membranes. More importantly, small molecular chains can easily cross the thick cell walls of bacteria (such as peptidoglycan layers) and penetrate the cytoplasmic membrane to enter the cell and directly bind to DNA, RNA or proteins, thus killing bacteria synergistically from two dimensions: "external physical damage" and "internal biochemical interference".

[0061] Adding 2MIC concentrations of D2-QCS 1:20 and D2-QCS 1:20 / AA to a Staphylococcus aureus bacterial suspension and incubating at 37℃ for 12 h followed by scanning electron microscopy observation, using Staphylococcus aureus without any antibacterial materials (… S. aureu s) served as the control group. Results were as follows: Figure 4 a, Figure 4 b、 Figure 4 As shown in c.

[0062] SEM images show untreated Staphylococcus aureus (S. aureus) S. aureu s) The cell membrane structure is intact and the surface is smooth. Figure 4 a); After treatment with D2 QCS at a 1:20 ratio, the bacterial cell membrane showed obvious damage, shrinkage, and even disintegration, ultimately leading to bacterial death. Figure 4 b). After treatment with D2-QCS 1:20 / AA, large aggregates formed by the interaction of this material with the cell membrane and intracellular substances could be observed around the bacteria. Figure 4 c). Furthermore, the cell membranes of most treated bacteria ruptured or perforated, and the bacterial structure disintegrated into scattered fragments. This indicates that QCS / AA can target negatively charged microbial membranes through electrostatic interactions, thereby leading to bacterial death.

[0063] 4. In vitro antifungal experiments were conducted on the materials prepared in Examples 1-5 and Comparative Example 1. The specific steps are as follows: (1) Determination of minimum inhibitory concentration (MIC): Candida albicans ( C. albicans As the target bacterium, the minimum inhibitory concentration (MIC) was determined using the standardized broth microdilution method. Specifically, the material prepared according to this invention was serially diluted with RPMI 1640 medium in a 96-well plate, and then each well was inoculated with a standard concentration of fresh fungal spore suspension (final concentration approximately 2.5 × 10⁻⁶). 3 After incubation at 35°C for 24 hours, the absorbance of each well at 530 nm was measured using a microplate reader. The lowest concentration at which the absorbance value decreased by more than 90% compared to the growth control well was determined as the MIC value of the material. Results are as follows: Figure 5 a, Figure 5 c. Figure 5 As shown in e; (2) Minimum fungicidal concentration (MFC) determination: Candida albicans ( C. albicans The target bacterium was selected as [a specific bacterium]. Based on the MIC determination described above, 100 μL of bacterial suspension was taken from each well that was determined to be sterile by optical detection and evenly spread onto Sabouraud dextrose agar (SDA) plates. The plates were then incubated at 35°C for 48 hours. The minimum drug concentration (MFC) that could kill more than 99.9% of the inoculum fungi (i.e., a reduction of ≥3 logarithmic orders in colony count compared to the original inoculum) was determined using the plate colony counting method. The results are as follows: Figure 5 b、 Figure 5 d、 Figure 5 As shown in f.

[0064] Depend on Figure 5 a to Figure 5 As shown in f, the MIC of amino acid-modified quaternized chitosan against Candida albicans is in the range of 50–200 μg / mL, and the MFC is not less than 100 μg / mL. The molecular weight of the sample affects the antifungal effect differently, and it can be optimized synergistically with the degree of substitution / grafting to suit different application requirements.

[0065] Arginine-modified quaternized chitosan exhibited different molecular weight-dependent effects on bacteria and fungi. Lower molecular weight samples showed stronger inhibition against bacteria, which is related to their higher diffusion capacity, less steric hindrance, and easier access to and penetration of the bacterial cell membrane, thereby enhancing membrane permeability and intracellular interference. In contrast, higher molecular weight samples showed stronger inhibition against fungi, which is due to the difference in cell structure between fungi and arginine. Fungi possess a cell wall-cell membrane double barrier, with the cell wall containing chitin, β-glucan, mannoproteins, etc., and the membrane containing the ergosterol system. High molecular weight arginine-modified quaternized chitosan is more likely to form multi-site adsorption on the surface of fungal cell walls / cell membranes, inducing membrane structural instability and leakage of intracellular substances, thus exerting its antifungal advantage through perturbation of the cell wall-membrane interface.

[0066] 5. Cytotoxicity test (1) The cytotoxicity of different materials to L929 cells was evaluated by the CCK-8 assay, and the results are as follows: Figure 6 As shown. Figure 6 The difference between D2-QCS 1:16, D2-QCS 1:12, and D2-QCS 1:8 and the materials in Example 4 is that the molar ratio of chitosan to quaternizing agent is 1:16, 1:12, and 1:8, respectively. like Figure 6 As shown, even at high concentrations of up to 1000 μg / mL, the cell viability of all material groups remained above 60%, demonstrating that the materials prepared in this invention have good cell compatibility over a wide concentration range.

[0067] (2) Furthermore, a more precise toxicity assessment was conducted on the amino acid grafted modified samples, and the results are as follows: Figure 7 As shown.

[0068] like Figure 7 As shown, the IC20 values ​​(concentrations at which cell viability is reduced to 80%) of QCS 1:20 / AA, D1-QCS 1:20 / AA, and D2-QCS 1:20 / AA were 200, 400, and 400 μg / mL, respectively, with corresponding cell viability of 82.49%, 84.15%, and 79.81%. This clearly demonstrates that these modified materials can maintain high cell viability within the subtoxic concentration range, further confirming their good cell compatibility and providing safety support for their potential applications in the biomedical field.

[0069] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. An amino acid modified quaternized chitosan, characterized in that, The amino acid-modified quaternized chitosan includes a quaternized chitosan backbone and amino acids covalently grafted onto the quaternized chitosan backbone; The amino acid modified quaternized chitosan has a quaternization degree of 0.2-1.6, an amino acid grafting degree of 0.1-0.8, and a weight average molecular weight of 5x10 3 ~8x10 5 g / mol.

2. The amino acid modified quaternized chitosan of claim 1, wherein, The degree of quaternization of the amino acid-modified quaternized chitosan is 0.2 to 0.9; and / or, The amino acids include one or more of arginine, lysine, and histidine.

3. The amino acid modified quaternized chitosan of claim 1, wherein, The minimum inhibitory concentration (MIC) of the amino acid-modified quaternized chitosan against Staphylococcus aureus is 6.25–12.5 μg / mL; and / or, The minimum bactericidal concentration of the amino acid-modified quaternized chitosan against Staphylococcus aureus is 50-100 μg / mL; and / or, The minimum inhibitory concentration of the amino acid-modified quaternized chitosan against Candida albicans is 50-200 μg / mL.

4. A process for the preparation of an amino acid modified quaternized chitosan according to any one of claims 1 to 3, characterized in that, The preparation method of the amino acid-modified quaternized chitosan includes the following steps: S1. Mix chitosan and an alkaline solution containing urea, and stir the mixture at a temperature below 5°C to obtain a mixed solution. S2. Mix the mixed solution with the quaternizing agent to carry out the quaternization reaction and obtain quaternized chitosan; S3. The quaternized chitosan, a buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and amino acids are mixed and subjected to an amino acid grafting reaction. The mixture is then purified by dialysis and freeze-dried to obtain the amino acid-modified quaternized chitosan.

5. The method for preparing the amino acid-modified quaternized chitosan according to claim 4, characterized in that, Step S1 includes: Chitosan raw material is oxidized and degraded in water to obtain chitosan; Chitosan and an alkaline solution containing urea are mixed and stirred at a temperature below 5°C to obtain a mixed solution.

6. The method for preparing amino acid-modified quaternized chitosan as described in claim 4, characterized in that, In step S1: The urea-containing alkaline solution has a urea mass fraction of 4% to 16%; and / or, The alkaline solution containing urea has an alkali mass fraction of 2% to 16%; and / or, The alkaline solution containing urea includes one or more of the following: KOH, NaOH, and LiOH.

7. The method for preparing amino acid-modified quaternized chitosan as described in claim 4, characterized in that, In step S2: The molar ratio of the free amino groups of chitosan to the quaternizing agent in the mixed solution is 4~30:1; and / or, The quaternizing agent includes at least one of glycidyltrialkylammonium chloride and 3-chloro-2-hydroxypropyltrimethylammonium chloride; and / or, The quaternization reaction is performed at a temperature of -10 to 40°C; and / or, The quaternization reaction takes 6 to 96 hours.

8. The method for preparing amino acid-modified quaternized chitosan as described in claim 4, characterized in that, In step S3: The buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide has a pH of 4.5 to 6.5; and / or, The solvent in the buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is MES buffer or phosphate buffer; and / or, The amino acid grafting reaction is performed at a temperature of 20-25°C; and / or, The amino acid grafting reaction takes 36-48 hours.

9. The application of an amino acid-modified quaternized chitosan in the preparation of antibacterial products, characterized in that, The amino acid-modified quaternized chitosan includes the amino acid-modified quaternized chitosan as described in any one of claims 1 to 3, or the amino acid-modified quaternized chitosan prepared by the preparation method of the amino acid-modified quaternized chitosan as described in any one of claims 4 to 8.

10. The application of the amino acid-modified quaternized chitosan as described in claim 9 in the preparation of antibacterial products, characterized in that, The antibacterial product includes at least one of antibacterial liquid, antibacterial coating, antibacterial film, and antibacterial composite material.

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