Amphiphilic chitosan derivative with efficient antibacterial activity as well as preparation and application of amphiphilic chitosan derivative

Abstract

The invention discloses an amphiphilic chitosan derivative with efficient antibacterial activity and a preparation method and application thereof, and the preparation method comprises the following specific steps: dissolving chitosan in acetic acid, adding benzaldehyde, and reacting at 60-100 DEG C for 6-24 hours to obtain N-benzylidene chitosan (CSBA); the method comprises the following steps: reacting CSBA with halogenated hydrocarbon with different molar ratios at 50-80 DEG C for 12-36 hours to obtain O-alkyl-N-benzylidene chitosan (TCSBA); stirring the TCSBA in a hydrochloric acid / ethanol solution overnight to obtain O-alkyl chitosan; and adding the O-alkyl chitosan into 2, 3-epoxypropyl trimethyl ammonium chloride, and reacting at 60-100 DEG C for 6-48 hours to obtain a final product. The derivative has a broad-spectrum antibacterial property, has a killing effect on various bacteria such as staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter lwoffii, inhibits the formation of biological membranes of the pseudomonas aeruginosa and the acinetobacter lwoffii, and has high compatibility with metal materials. The method can be applied to the fields of petroleum and natural gas industrial pipeline protection, marine ship fuel oil tank antibiosis and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial materials, and in particular relates to an amphiphilic chitosan derivative with high-efficiency antibacterial activity, a preparation method thereof, and an application in the antibacterial field. Background Art

[0002] The importance of antimicrobial and anti-corrosion technologies is becoming increasingly prominent in modern industry and daily life. In the consumer chemical sector, products ranging from lotions and creams to shampoos are rich in moisture and nutrients, creating an ideal habitat for microorganisms. Once microbial growth occurs, not only can products deteriorate, resulting in odor, discoloration, and delamination, reducing the user experience, but they can also produce harmful metabolites, such as bacterial toxins, posing a health threat to consumers. Critical infrastructure, such as industrial lubrication systems and oil and gas pipelines, has long faced the dual challenges of mechanical corrosion and microbial attack. In industrial lubrication systems, microbial growth alters the physical and chemical properties of lubricants, reducing viscosity, weakening antioxidant properties, and accelerating equipment wear. Microbial corrosion in oil and gas pipelines, resulting in thinning of pipe walls, can easily lead to leaks, resulting in resource waste, environmental pollution, and, in severe cases, even catastrophic consequences such as explosions. Bacterial contamination of marine fuel tanks and food processing and packaging machinery is also a significant concern. In the fuel tanks of ocean vessels, the growth and reproduction of bacteria will produce acidic substances, corroding the tanks and creating the risk of fuel leakage; in the field of food processing and packaging machinery, bacterial contamination may cause foodborne diseases and affect consumer health.

[0003] Traditional antimicrobial agents and preservatives suffer from toxicity, drug resistance, and environmental concerns, making them unable to meet increasingly stringent safety and performance requirements across multiple sectors. Therefore, the development of novel, highly effective, safe, and environmentally friendly antimicrobial formulations is an urgent industry need. Industrial antimicrobial agents often face residual risks, contact toxicity, and material incompatibility. Naturally derived chitosan exhibits moderate antimicrobial activity, but its water insolubility limits its application. Quaternization is currently an effective strategy to enhance the water solubility and antimicrobial activity of chitosan. Current research focuses on optimizing its antimicrobial properties by manipulating substitution sites and groups, and progress has been made in areas such as food preservation, medical dressings, and composite antimicrobial materials. However, quaternized chitosan, which relies solely on cationic charge, often performs poorly against mature biofilms in complex environments, and is particularly difficult to effectively remove biofilm contamination from hydrophobic surfaces. Therefore, developing novel derivatives of quaternized chitosan with both high antimicrobial and antibiofilm activity through specific functional modifications holds significant research value and promise for addressing microbial contamination in practical applications. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In response to the shortcomings of existing product technology, the present invention provides an amphiphilic chitosan derivative with high antibacterial activity, its preparation method and implementation application, to solve the problems of strong drug resistance of mature biofilms, low stripping efficiency and material incompatibility in the above-mentioned complex environment. The prepared antibacterial agent has high antibacterial activity, effectively strips the biofilm formed on the surface of the material, and has good compatibility with metal materials, thereby improving the technical problem of applicability of product applications.

[0006] (2) Technical solution

[0007] An amphiphilic chitosan derivative, characterized in that: the amphiphilic chitosan derivative is O-alkyl-N-hydroxypropyltrimethylammonium chitosan, and the specific structure is shown in Formula 1:

[0008]

[0009] The R is one or more of H, C1-C20 alkyl or aromatic hydrocarbon.

[0010] The method for preparing the amphiphilic chitosan derivative is characterized by:

[0011] The following steps are involved:

[0012] a: Chitosan and benzaldehyde are reacted at 60-100°C for 6-24 hours, and the pH is adjusted to 7.0-7.2 with one or more of potassium hydroxide, sodium hydroxide, sodium hydride, sodium carbonate, potassium carbonate, or triethylamine. The mixture is filtered, Soxhlet extracted for 12-48 hours, and vacuum dried overnight to obtain product 1: N-benzylidene chitosan (CSBA);

[0013] b: Product 1 reacts with halogenated hydrocarbon at 50-80°C for 12-36h, then precipitates with 3-5 volumes of ethanol, washes with acetone 3-5 times, and filters to obtain product 2: O-alkyl-N-benzylidene chitosan (TCSBA);

[0014] c: Product 2 is stirred in a 0.1-0.5 mol / L hydrochloric acid / ethanol mixed solution for 12-48 hours, neutralized with an alkaline solution of potassium hydroxide, sodium hydroxide, sodium hydride, sodium carbonate, potassium carbonate or triethylamine to pH: 7.0-7.2, added with 3-5 volumes of ethanol for precipitation, filtered and washed with acetone 3-5 times to obtain product 3: O-alkyl-chitosan.

[0015] d: Product 3 and 2,3-epoxypropyltrimethylammonium chloride are added in a molar ratio of glucosamine to 2,3-epoxypropyltrimethylammonium chloride of 1:5-1:20, and the reaction is carried out at 60-100° C. for 6-48 hours to obtain the final product O-alkyl-N-hydroxypropyltrimethylammonium chitosan.

[0016] The preparation method of the amphiphilic chitosan derivative is characterized in that: in the step a, chitosan is dissolved in a 1%-3% w / v acetic acid aqueous solution, and benzaldehyde is added in a ratio of glucosamine:benzaldehyde of 1:10, stirred at 60-100°C for 6-24 hours, the pH is adjusted to 7.0-7.4 with a 3 mol / L alkaline solution, the filter residue is filtered, washed with anhydrous ethanol 3-5 times, placed in a Soxhlet extractor, extracted with anhydrous ethanol as the extraction solvent, extracted for 12-48 hours, and then vacuum dried to obtain product 1: N-benzylidene chitosan (CSBA).

[0017] The preparation method of the amphiphilic chitosan derivative is characterized in that: the preparation method of the product 2 in step b is as follows: N-benzylidene chitosan is dispersed in a mixed solvent of isopropanol and potassium hydroxide, stirred at room temperature for 2-4 hours, and added according to a molar ratio of glucosamine:N-benzylidene chitosan of 1:5-1:20. The reaction is carried out at 50-80°C for 12-36 hours. After cooling to room temperature, ethanol is added to precipitate the filter residue, washed with acetone, and vacuum dried at 40-60°C to obtain product 2: O-alkyl-N-benzylidene chitosan (RCSBA). The preparation method of the amphiphilic chitosan derivative is characterized in that the halogenated hydrocarbon used in the preparation is an alkane or aromatic hydrocarbon with a chain length of C3-C20, and is one or more of n-propyl chloride, sec-butyl chloride, 1-n-pentane chloride, n-hexane chloride, n-heptane chloride, 1-octane chloride, 1-nonane chloride, 1-decane chloride, 1-undecane chloride, dodecane chloride, 1-tridecane chloride, tetradecane chloride, 1-pentadecane bromide, hexadecane chloride, 1-heptadecane chloride, octadecane chloride, 1-nonadecane bromide, 1-eicosane chloride, 1-eicosane bromide, isopropyl bromide, n-butyl bromide, 1-pentane bromide, 1-heptane bromide, 1-octane bromide, n-nonane bromide, decane bromide, undecane bromide, dodecane bromide, 1-fluorododecane, bromobenzene, p-chlorotoluene, benzyl chloride, and p-chlorobenzyl bromide.

[0018] The preparation method of the amphiphilic chitosan derivative is characterized in that: the preparation method of step d is to add product 3 and 2,3-epoxypropyltrimethylammonium chloride in a molar ratio of glucosamine:2,3-epoxypropyltrimethylammonium chloride of 1:5-1:20, react in an aqueous reaction system at 60-100°C for 6-48 hours, precipitate the product with ethanol, dissolve the filter residue with water, place it in a 14000Da molecular weight dialysis bag for dialysis, and then freeze-dry to obtain the final product O-alkyl-N-hydroxypropyltrimethylammonium chitosan (RTAC). The use of the amphiphilic chitosan derivative in a broad-spectrum antibacterial agent is characterized in that the amphiphilic chitosan derivative has a killing effect on bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter lwoffi, and can be used as a broad-spectrum antibacterial agent.

[0019] The use of the amphiphilic chitosan derivative in an anti-biofilm preparation is characterized in that the amphiphilic chitosan derivative can also be used as an anti-biofilm preparation, has the effect of inhibiting the biofilm formation and adhesion of bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter lwoffi, and can be used as an anti-biofilm preparation.

[0020] The application of the amphiphilic chitosan derivative in daily chemical products, food processing and packaging machinery, industrial lubrication systems and mechanical corrosion protection, oil and gas industry pipeline protection, and marine ship fuel tank antibacterial agent is characterized in that the addition amount of the amphiphilic chitosan derivative is different, wherein the addition amount of the daily chemical product is 0.05-10%, the addition amount of the antibacterial and antibacterial coating of food processing and packaging machinery is 0.05-15%, and the addition amount of the industrial lubrication system and mechanical corrosion protection, oil and gas industry pipeline protection, and marine ship fuel tank antibacterial agent is 0.1-30%.

[0021] The amphiphilic chitosan derivatives are used as antimicrobial agents in industrial anti-biofilm preparations. The addition amount of the amphiphilic chitosan derivatives as antimicrobial agents is different. The addition amount for industrial anti-biofilm is 0.2-30%, and the addition amount for food processing and packaging machinery antimicrobial and coating materials is 0.1-20%.

[0022] A series of O-alkane-N-quaternized chitosan derivatives were obtained by selectively grafting long-chain alkanes or aromatic hydrocarbons and quaternary ammonium salt branches on the 6-hydroxyl end and 2-amino end of chitosan. The general structure is shown in Formula 1:

[0023]

[0024] The R is H, one or more of C1-C20 alkanes or aromatic hydrocarbons.

[0025] A method for preparing an amphiphilic chitosan derivative with high antibacterial activity comprises the following steps:

[0026] a: Chitosan and benzaldehyde are reacted at 60-100°C for 6-24 hours to obtain product 1: N-benzylidene chitosan (CSBA). After acid-base neutralization, filtration, Soxhlet extraction for 24 hours, and vacuum drying, product 1 (CSBA) is obtained.

[0027] b: N-benzylidene chitosan reacts with halogenated hydrocarbon at 50-80°C for 12-36h, precipitates with ethanol, washes with acetone three times, and filters to obtain product 2: O-alkyl-N-benzylidene chitosan;

[0028] c: Product 2 is stirred in a 0.1-0.5 mol / L hydrochloric acid / ethanol solution for 12-48 hours. After acid-base neutralization, it is precipitated with ethanol, filtered, and washed with acetone three times to obtain product 3: O-alkyl-chitosan.

[0029] d: Product 3 and 2,3-epoxypropyltrimethylammonium chloride are added in a molar ratio of glucosamine to 2,3-epoxypropyltrimethylammonium chloride of 1:5-1:20, and the mixture is reacted at 60-100° C. for 6-48 hours to obtain the final product.

[0030] Preferably, in the preparation step b, the 3 mol / L alkaline solution is one or more of potassium hydroxide, sodium hydroxide, sodium hydride, sodium carbonate, potassium carbonate, and triethylamine.

[0031] Preferably, in the preparation step b, N-benzylidene chitosan is dispersed in isopropanol and stirred for 0.5-1 h, and then an alkaline solution is added and stirred at room temperature for 1-1.5 h.

[0032] Preferably, in the preparation step b, the halogenated hydrocarbon can be one or more of fluoroalkanes, chloroalkanes, bromoalkanes and halogenated aromatic hydrocarbons with different chain lengths.

[0033] Preferably, in the preparation step c, the preparation method is to place product 2 in a 0.1-0.5 mol / L hydrochloric acid / ethanol solution, stir for 12-48 hours, neutralize with acid and alkali, precipitate with ethanol, filter and wash with acetone three times to obtain product 3: O-alkyl-chitosan.

[0034] Preferably, in the preparation method of step d, after O-alkyl-chitosan is dispersed in pure water for 0.5-2h, 2,3-epoxypropyltrimethylammonium chloride is added and the glucosamine is added at a molar ratio of 1:5-1:20, and the reaction is carried out at 60-100°C for 6-48h. The product is precipitated with ethanol, the filter residue is dissolved with water, dialyzed in a 14000Da molecular weight dialysis bag, and freeze-dried to obtain the final product (RTAC).

[0035] Preferably, the O-alkyl-N-hydroxypropyltrimethylammonium chitosan derivative has a killing effect on bacteria such as Staphylococcus aureus (abbreviated as S.aureus), Escherichia coli (abbreviated as E.coli), Pseudomonas aeruginosa (abbreviated as P.aeruginosa) and Acinetobacter lwoffii (abbreviated as A.lwoffiii) under in vitro culture conditions and minimum bactericidal concentration (MBC) conditions.

[0036] Preferably, the O-alkyl-N-hydroxypropyl trimethyl ammonium chitosan derivatives have an anti-metallic effect on the single colony concentration of P. aeruginosa and A. lwoffiii at 1×10 7 Under the conditions of CFU / mL, it inhibits the growth of biofilm and improves the tensile strength of metal materials.

[0037] Preferably, the amount of O-alkyl-N-hydroxypropyltrimethylammonium chitosan derivative added varies according to different fields, among which the amount added in daily chemical products is 0.05-10%, the amount added in antibacterial and antibacterial coatings for food processing and packaging machinery is 0.05-15%, and the amount added in industrial lubrication systems and mechanical corrosion protection, oil and gas industry pipeline protection, and marine ship fuel tank antibacterial is 0.1-30%.

[0038] Preferably, the daily chemical products are cleaning and hygiene cosmetics, skin care cosmetics, beauty cosmetics, hair care cosmetics, etc., and the antibacterial coating includes water-based coatings, powder coatings, etc.

[0039] Beneficial effects of the present invention

[0040] Compared with the prior art, the present invention provides a series of amphiphilic chitosan derivatives and their preparation and application technologies, which have the following beneficial effects:

[0041] 1. The amphiphilic chitosan derivative prepared by the present invention has excellent antibacterial activity, killing Staphylococcus aureus, Escherichia coli, Acinetobacter lwoffii, and Pseudomonas aeruginosa. The bactericidal activity is strong even at relatively low concentrations, achieving a highly efficient bactericidal process through a synergistic effect of penetration-affinity-rupture-destruction. It has great potential as an antibacterial agent for use in daily chemical products, materials, industrial pipelines, and fuel tank antibacterial protection.

[0042] 2. The amphiphilic chitosan derivative prepared by the present invention has both water-soluble and oil-soluble properties, and exhibits strong antibacterial effects in oil, water, and at the oil-water interface. It has good compatibility with a variety of daily chemical products and has no effect on the properties of metal materials, significantly improving the practicality of the present invention.

[0043] 3. The quaternary amphiphilic chitosan derivative prepared by the present invention has a destructive effect on biological membranes. The hydrophobic end branches of the derivatives are inserted into the interior of the biological membrane and adsorbed on the cell membrane surface through charge, thereby improving the characteristics of the present invention for use in special environments. At the same time, the method of the present invention is simple, the experimental materials are easily available, and it is convenient for large-scale preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the infrared spectrum of chitosan and its derivatives of the present invention;

[0045] Figure 2 is the nuclear magnetic hydrogen spectrum of chitosan and its derivatives of the present invention;

[0046] Figure 3 This is a graph showing the water contact angles of the amphiphilic chitosan derivatives grafted with alkanes of different lengths according to the present invention;

[0047] Figure 4 The MIC and MBC test results of the derivatives of the present invention against different bacterial species are shown in FIG.

[0048] Figure 5 The inhibitory effect of TTAC on the growth curves of different bacterial species under the minimum inhibitory concentration condition, including a) A.lwoffiii, b) P.aeruginosa, c) E.coli, d) S.aureus.

[0049] Figure 6 Scanning electron microscopy images of different bacterial morphologies after TTAC treatment;

[0050] Figure 7 Figure 2 shows the in vitro anti-biofilm activity assay of TTAC; a) 3D fluorescence images of biofilms after TTAC treatment for 0 h and 20 h; b) and c) single-layer scanning fluorescence images and fluorescence quantitative analysis images.

[0051] Figure 8The effect of TTAC on the tensile properties of 7050 aluminum alloy includes a) test group diagram, b) room temperature tensile curve, and c) maximum load and tensile strength.

[0052] Figure 9 The figure shows the flow chart for the preparation of an amphiphilic chitosan derivative with high antibacterial activity. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0054] Example 1: Preparation of an amphiphilic chitosan derivative high-efficiency antibacterial agent

[0055] a: Chitosan and benzaldehyde: Chitosan was dissolved in a 1% w / v aqueous acetic acid solution and added with benzaldehyde at a molar ratio of glucosamine to benzaldehyde of 1:10. The mixture was reacted at 60°C for 24 hours. The pH was adjusted to 7.0-7.4 (pH 7 in this embodiment) with a 3 mol / L potassium hydroxide alkaline solution. The residue was filtered and washed three times with anhydrous ethanol. The residue was then extracted in a Soxhlet extractor with anhydrous ethanol for 24 hours and then dried in vacuo to obtain N-benzylidene chitosan (CSBA).

[0056] b: N-benzylidene chitosan was dispersed in a mixed solvent of isopropanol and potassium hydroxide, stirred at room temperature for 2 h, and n-propyl chloride was added at a molar ratio of glucosamine to N-benzylidene chitosan of 1:8. The mixture was reacted at 50°C for 36 h. After cooling to room temperature, ethanol was added for precipitation. The precipitate was washed with acetone three times, filtered, and dried in vacuo at 40°C to obtain the product: O-propyl-N-benzylidene chitosan.

[0057] c: O-propyl-N-benzylidene chitosan was stirred in 1 mol / L hydrochloric acid / ethanol solution for 12 h, neutralized with 3 mol / L potassium hydroxide to pH 7.0, precipitated with ethanol, filtered and washed with acetone three times to obtain the product: O-propyl-chitosan.

[0058] d: O-alkyl-chitosan and 2,3-epoxypropyltrimethylammonium chloride were added in a molar ratio of 1:5 of glucosamine:2,3-epoxypropyltrimethylammonium chloride in an aqueous reaction system at 80°C for 24 hours. The product was precipitated with ethanol, and the residue was dissolved in water, dialyzed in a 14,000 Da molecular weight dialysis bag, and freeze-dried to obtain the final product, O-propyl-N-hydroxypropyltrimethyl chitosan derivative (PTAC).

[0059] Example 2: Preparation of an amphiphilic chitosan derivative high-efficiency antibacterial agent

[0060] a: Chitosan and benzaldehyde: Chitosan was dissolved in a 1% w / v aqueous acetic acid solution and added with benzaldehyde at a molar ratio of glucosamine to benzaldehyde of 1:10. The mixture was reacted at 70°C for 18 hours. The pH was adjusted to 7.0-7.4 (pH 7 in this embodiment) with a 3 mol / L sodium carbonate alkaline solution. The residue was filtered and washed three times with anhydrous ethanol. The residue was then extracted in a Soxhlet extractor with anhydrous ethanol for 24 hours and then dried in vacuo to obtain N-benzylidene chitosan.

[0061] b: N-benzylidene chitosan was dispersed in a mixed solvent of isopropanol and potassium hydroxide, stirred at room temperature for 2 h, N-benzylidene chitosan and n-hexyl chloride were added at a molar ratio of glucosamine: N-benzylidene chitosan of 1:10, reacted at 60°C for 36 h, precipitated with ethanol, washed with acetone three times, filtered, and dried in vacuo at 40°C to obtain the product: O-hexyl-N-benzylidene chitosan;

[0062] c: O-hexyl-N-benzylidene chitosan was stirred in 1 mol / L hydrochloric acid / ethanol solution for 18 h, neutralized with 3 mol / L sodium carbonate to pH 7.0, precipitated with ethanol, filtered and washed with acetone three times to obtain the product: O-hexyl-chitosan.

[0063] d: O-hexyl-chitosan and 2,3-epoxypropyltrimethylammonium chloride were added in a molar ratio of 1:15 of glucosamine:2,3-epoxypropyltrimethylammonium chloride in an aqueous reaction system at 80°C for 24 hours. The product was precipitated with ethanol, and the residue was dissolved in water, dialyzed in a 14,000 Da molecular weight dialysis bag, and freeze-dried to obtain the final product, O-hexyl-N-hydroxypropyltrimethyl chitosan derivative (HTAC).

[0064] Example 3: Preparation of an amphiphilic chitosan derivative as a highly effective antibacterial agent

[0065] a: Chitosan and benzaldehyde. Chitosan was dissolved in a 1% w / v aqueous acetic acid solution and added with benzaldehyde at a molar ratio of glucosamine to benzaldehyde of 1:10. The mixture was reacted at 80°C for 12 hours. The pH was adjusted to 7.0-7.4 (pH 7 in this embodiment) with a 3 mol / L sodium hydroxide alkaline solution. The residue was filtered and washed three times with anhydrous ethanol. The residue was then extracted in a Soxhlet extractor with anhydrous ethanol for 24 hours, and then dried in vacuo to obtain product 1: N-benzylidene chitosan.

[0066] b: N-benzylidene chitosan was dispersed in a mixed solvent of isopropanol and potassium hydroxide, stirred at room temperature for 2 h, and added with chlorotetradecane at a molar ratio of glucosamine: N-benzylidene chitosan of 1:8. The mixture was reacted at 70°C for 36 h. After cooling to room temperature, ethanol was added for precipitation, and the mixture was washed with acetone three times. After filtration, the mixture was dried in vacuo at 40°C to obtain product 2: O-tetradecyl-N-benzylidene chitosan.

[0067] c: O-tetradecyl-N-benzylidene chitosan was stirred in a 2 mol / L hydrochloric acid / ethanol solution for 24 h, neutralized with 3 mol / L sodium hydroxide to pH 7.0, precipitated with ethanol, filtered and washed with acetone three times to obtain O-tetradecyl-chitosan.

[0068] d: O-tetradecyl-chitosan and 2,3-epoxypropyltrimethylammonium chloride were added in a molar ratio of 1:13 of glucosamine:2,3-epoxypropyltrimethylammonium chloride in an aqueous reaction system at 80°C for 24 hours. The product was precipitated with ethanol, and the residue was dissolved in water, dialyzed in a 14,000 Da molecular weight dialysis bag, and freeze-dried to obtain the final product, O-tetradecyl-N-hydroxypropyltrimethyl chitosan derivative (TTAC).

[0069] Example 4: Preparation of an amphiphilic chitosan derivative as a highly effective antibacterial agent

[0070] a: Chitosan and benzaldehyde. Chitosan was dissolved in a 1% w / v aqueous acetic acid solution and added with benzaldehyde at a molar ratio of glucosamine to benzaldehyde of 1:10. The mixture was reacted at 80°C for 12 hours. The pH was adjusted to 7.0-7.4 (pH 7 in this embodiment) with a 3 mol / L potassium carbonate alkaline solution. The residue was filtered and washed three times with anhydrous ethanol. The residue was then extracted in a Soxhlet extractor with anhydrous ethanol for 24 hours, and then dried under vacuum to obtain product 1: N-benzylidene chitosan.

[0071] b: N-benzylidene chitosan was dispersed in a mixed solvent of isopropanol and potassium hydroxide, stirred at room temperature for 2 h, N-benzylidene chitosan and chlorooctadecane were added at a molar ratio of glucosamine: N-benzylidene chitosan of 1:8, reacted at 80°C for 36 h, cooled to room temperature, precipitated with ethanol, washed with acetone three times, filtered, and dried in vacuo at 40°C to obtain product 2: O-octadecyl-N-benzylidene chitosan;

[0072] c: O-octadecyl-N-benzylidene chitosan was stirred in 2 mol / L hydrochloric acid / ethanol solution for 24 h, neutralized with 3 mol / L potassium carbonate to pH 7.0, precipitated with ethanol, filtered and washed with acetone three times to obtain O-octadecyl-chitosan.

[0073] d: O-octadecyl-chitosan and 2,3-epoxypropyltrimethylammonium chloride were added in a molar ratio of 1:20 of glucosamine:2,3-epoxypropyltrimethylammonium chloride in an aqueous reaction system at 80°C for 36 hours. The product was precipitated with ethanol, and the residue was dissolved in water, dialyzed in a 14,000 Da molecular weight dialysis bag, and freeze-dried to obtain the final product, O-octadecyl-N-hydroxypropyltrimethyl chitosan derivative (OTAC).

[0074] Example 5, Infrared Spectroscopic Structural Characterization of Amphiphilic Chitosan Derivatives

[0075] Methods: The structural characteristics of the samples were determined using a Nicolet IS 50 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA) at 25°C with a scanning wavenumber range of 4000–500 cm -1 The test sample was mixed with KBr and pressed into a pellet, and the spectral resolution was 4 cm -1 .

[0076] Results: The amphiphilic chitosan derivatives prepared in Examples 1, 2, 3, and 4 showed Fourier infrared spectra. Figure 1 As shown, compared with the CS infrared spectrum, the 1597 cm -1 The characteristic NH bending vibration peak of primary amine disappears. CSBA at 1644 cm -1 and 1581cm -1 The new peaks appearing at 757cm are attributed to the symmetric and asymmetric stretching vibrations of ν(C=N) in the Schiff base structure. -1 and 636cm -1 The characteristic peak at 2927cm corresponds to the in-plane bending vibration of the aromatic ring CH. -1 and 2868cm -1 The (alkyl CH) shows enhanced stretching vibrations at 1091 cm-1 compared to chitosan. -1 The C6-OH characteristic peak at 1493 cm is significantly weakened, indicating that the alkyl grafting occurs at the hydroxyl site. -1 The absorption peak at is trimethylammonium (-N + (CH3)3) Deformation vibration.

[0077] Example 6, Structural Characterization of Amphiphilic Chitosan Derivatives by Proton NMR Spectroscopy

[0078] Method: The samples were measured at 25℃ using a Bruker AVIII 600 NMR spectrometer. 1 H NMR spectroscopy. Approximately 10 mg of the test sample was dissolved in 0.6 mL of a D2O / CD3COOD mixed solvent, filtered through a 0.22 μm filter membrane, and injected into a NMR tube for analysis.

[0079] Results: The structures of the derivatives prepared in Examples 1, 2, 3, and 4 were further characterized by H NMR spectroscopy. Figure 2 The characteristic peaks of CS appear at δ = 2.10 [N-acetyl], δ = 3.14 [H2], δ = 3.76-3.99 [H3-H6], and δ = 4.64 [H1]. Characteristic signals of the aromatic ring appear in the CSBA derivative: a multiplet at δ = 7.58-8.76 (Ar-H) and a significant downfield shift signal at δ = 9.79 (imine proton - N = CH-Ar).

[0080] The characteristic peaks of the alkyl chain are δ = 0.95 [-CH3], δ = 1.65 [-CH2-] and δ = 1.22 [-O-CH2-]. The characteristic peaks of quaternary ammonium modification appear at δ = 2.61, δ = 2.80 [-CH2-], δ = 3.28 [-N + (CH3)3] and [-CH-OH] at δ=4.37, and the structural formula of the amphiphilic chitosan derivatives of Examples 1-4 was determined.

[0081] In summary, the structures of each derivative met the expected requirements.

[0082] Example 7, Determination of the Degree of Substitution of Alkanization and Quaternization in the Amphiphilic Chitosan Derivatives Prepared in Examples 1, 2, 3, and 4

[0083] Results: According to Figure 2 According to the H-NMR spectrum, the deacetylation degree of chitosan (CS) raw material was calculated to be 93% according to formula 1-3. The alkyl substitution degree (DA) and quaternization substitution degree (DQ) are shown in Table 1.

[0084]

[0085] Table 1 Degree of substitution of chitosan and its derivatives

[0086]

[0087]

[0088] Example 8, Hydrophilic / Hydrophobic Properties-Water Contact Angle Measurement

[0089] Method: The contact angle of the chitosan double-grafted derivative was measured using an optical contact angle meter. The freeze-dried powder was pressed into a tablet and placed on a transparent stage. 5 μL of water was added to the PLEO test tube by an automatic sampler. The droplet image was captured after standing for 30 seconds at room temperature (25 ± 1 ° C). The results are shown in the figure. Figure 3 shown.

[0090] Results: The water contact angles of quaternary ammonium salt derivatives of chitosan grafted with alkane of different lengths were measured. The water contact angle of PTAC obtained in Example 1 was θ=65.78°, the water contact angle of HTAC obtained in Example 2 was θ=68.66°, the water contact angle of TTAC obtained in Example 3 was θ=75.84°, and the water contact angle of OTAC obtained in Example 4 was θ=82.97°. It can be seen that as the length of the substituted alkane chain increases, the hydrophobicity increases.

[0091] Example 9, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Determination

[0092] Method: Take the sterilized LB medium and dilute the A.lwoffiii, P.aeruginosa, E.coli and S.aureus bacterial solutions to a concentration of (1-5)×10 6 CFU / mL. Inoculate 50 μL of bacterial solution into each well of a 96-well plate. Add 50 μL of the derivative at final concentrations of 1024 mg / L, 512 mg / L, 256 mg / L, 128 mg / L, 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L, 2 mg / L, and 1 mg / L. After incubation at 37°C for 20 hours, the concentration at which no turbidity is observed by naked eye is the minimum inhibitory concentration (MIC). Inoculate the liquid from different wells onto LB solid medium. After incubation at 37°C for 24 hours, the concentration at which no colonies appear is the minimum bactericidal concentration (MBC).

[0093] Results: The minimum bactericidal concentration and minimum inhibitory concentration of the amphiphilic chitosan derivatives in Examples 1-4 against the four bacteria are as follows: Figure 4 As shown in Table 2, TTAC had an MIC of 32 mg / L and an MBC of 64 mg / L against P. aeruginosa. Compared to PTAC and HTAC, OTAC exhibited superior antibacterial activity. The preparation process for OTAC is more demanding than that for TTAC, and its degree of substitution is lower. Therefore, TTAC was used in subsequent experiments.

[0094] Table 2 MIC and MBC of chitosan derivatives against four bacteria

[0095]

[0096]

[0097] Example 10, Antibacterial Activity of Chitosan Derivatives against Different Bacteria

[0098] Methods: A. lwoffiii, P. aeruginosa, E. coli and S. aureus were cultured for 24 hours and then centrifuged at 4000 rpm for 10 minutes. The supernatant was removed and the bacterial solution was diluted with PBS to a concentration of 0.5 using a McFarland turbidimetric tube. The bacterial concentration was about 1.5-2.5×10 8 CFU / mL. Add LB liquid medium and adjust the bacterial concentration to 1.5-2.5×10 5 CFU / mL. 50 μL of bacterial solution and 50 μL of sample (normal saline for the blank group, 8 mg / L for A. lwoffii, 32 mg / L for P. aeruginosa, and 64 mg / L for E. coli and S. aureus) were added to a 96-well plate. The plate was incubated at 37°C in a microplate reader and the absorbance at 600 nm was monitored continuously for 15 hours.

[0099] Results: As Figure 5 As shown in the figure, under normal conditions, the number of bacteria (turbidity) increases with time. After being treated with the amphiphilic chitosan derivatives in Examples 1-4 at the MIC concentration, the four bacteria all showed a significant growth inhibition effect, and the OD 600nm The growth curve was measured and it was found that the growth period of each bacteria was prolonged and its logarithmic growth phase was inhibited within 15 hours. Figure 5 a showed that the amphiphilic diffractoids had broad-spectrum antibacterial activity against different types of bacteria.

[0100] Example 11, Effect of TTAC of Example 3 on Bacterial Morphology

[0101] Method: The concentration of A. lwoffiii, P. aeruginosa, E. coli and S. aureus was adjusted to 1×10 8 CFU / mL, PBS was added as a blank control group, and samples of different concentrations for different bacteria were added, specifically A.lwoffiii: 8 mg / L, P.aeruginosa: 32 mg / L, E.coli and S.aureus: 64 mg / L concentrations of TTAC were added as sample groups, and then the bacteria were cultured at 37 ° C for 20 hours. After cultivation, the culture medium was discarded, the samples were washed three times with PBS, and fixed in 5% glutaraldehyde for 2 hours. Subsequently, ethanol gradient dehydration was used. After the samples were critical point dried with carbon dioxide, gold sprayed, and observed under a field emission scanning electron microscope.

[0102] Results: As Figure 6As shown in the figure, scanning microscopy was used to observe changes in the microstructure of each bacterial cell before and after co-culture with TTAC. The results show that in the blank control group, all bacteria displayed a rod-shaped or spherical morphology with an intact, plump surface. However, after 20 hours of TTAC treatment, the microstructure of all four bacterial cells showed significant disruption, manifesting as typical bacteriolysis phenomena, such as disruption of cell wall / membrane integrity and leakage of intracellular substances. In addition, E. coli cells also exhibited a concave morphology.

[0103] Example 12, Inhibitory Effect of TTAC on Different Bacterial Biofilms

[0104] Methods: The concentration of A. lwoffiii, P. aeruginosa, E. coli and S. aureus bacterial suspensions in the logarithmic growth phase was adjusted to 1×10 8 CFU / mL, added to a 24-well plate and cultured statically for 48 hours (37°C). Subsequently, samples of different concentrations for different bacteria were added, specifically A.lwoffiii: 8 mg / L, P.aeruginosa: 32 mg / L, E.coli and S.aureus: 64 mg / L TTAC concentrations, and continued to be cultured for 20 hours. After discarding the supernatant, it was gently rinsed three times with PBS. SYTO-9 (2 μM) / PI (5 μg / mL) mixed dye was used for staining in the dark for 15 minutes. A laser confocal microscope (CLSM, Zeiss LSM 900, Germany, 40x objective lens) was used to collect Z-axis tomographic images (1 μm) to reconstruct the 3D biofilm structure, and the fluorescence intensity was quantitatively analyzed by ImageJ software.

[0105] Results: As Figure 7 As shown in Figure 3, TTAC treatment resulted in decreased biofilm fluorescence intensity and thickness compared to the control group. Figure 7 a) Shows collapse of the EPS matrix and loss of structural integrity, with a significant change in the live / dead cell ratio ( Figure 7 b), indicating that TTAC has an anti-biofilm effect. Fluorescence quantitative analysis showed that at TTAC concentrations of 8 mg / L for A. lwoffiii, 32 mg / L for P. aeruginosa, and 64 mg / L for E. coli and S. aureus, TTAC killed 23.8%, 19.5%, 14.9%, and 19.9% ​​of the bacteria in biofilms of A. lwoffiii, P. aeruginosa, E. coli, and S. aureus, respectively ( Figure 7 c) The results confirmed that TTAC has a dual mechanism of action: hydrophobic interaction destabilizes EPS, while the quaternary ammonium cations act as an affinity bactericidal agent against biofilm bacteria. Therefore, TTAC exhibited excellent in vitro anti-biofilm activity against all four bacterial strains, suggesting potential application in the field of anti-biofilm materials.

[0106] Example 13, Effect of TTAC on Tensile Properties of Aluminum Alloy Materials

[0107] Method: A. lwoffiii and P. aeruginosa are bacteria that have been reported to have significant effects on the performance of metal materials in actual industrial applications. Therefore, these two bacteria were selected for the evaluation of their effects on tensile properties. Figure 8 The microenvironment system was constructed as shown in a: the test piece was placed in 24 mL of 1 / 4 concentration Bushnell-Haas medium (A. lwoffiii and P. aeruginosa bacterial solution concentration 1×10 7 CFU / mL) and 24mL oil in a culture tube. All materials were sterilized before use. After the system was statically cultured at 28°C for 2 weeks, 10% TTAC was added to the TTAC group and an equal amount of PBS was added to the control group. In this experiment, a PWS-100 dynamic and static universal testing machine was used for tensile testing. The test parameters were set as follows: maximum tensile load 100kN, measurement speed range 0.005-500mm / min, displacement resolution 0.025μm, maximum stroke 1200mm, and constant tensile speed 1mm / min. Three parallel samples were set in each group to ensure data reliability.

[0108] Results: Compared with the sterile blank group ( Figure 8 b) The tensile strength of the biofilm-contaminated specimens decreased by 7.8% (A. lwoffiii) and 9.9% (P. aeruginosa), respectively, and the maximum load decreased from 11.0 kN to 10.1 kN and 9.9 kN. After antimicrobial treatment, the mechanical properties of the materials significantly improved compared to those of the biofilm-contaminated group, with tensile strength increasing by 2.7% and 3.7% compared to the contaminated group, reaching maximum loads of 10.7 kN and 10.6 kN, respectively. These results demonstrate that the addition of TTAC has a high affinity for metal materials, significantly counteracting the effects of microbial influences on their tensile properties.

[0109] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An amphiphilic chitosan derivative, characterized in that: The amphiphilic chitosan derivative is O-alkyl-N-hydroxypropyltrimethylammonium chitosan, and its specific structure is shown in Formula 1: The R is one or more of H, C1-C20 alkyl or aromatic groups.

2. A method for preparing the amphiphilic chitosan derivative according to claim 1, characterized in that: The following steps are involved: a: Chitosan and benzaldehyde are reacted at 60-100°C for 6-24 hours, and the pH is adjusted to 7.0-7.2 with one or more of potassium hydroxide, sodium hydroxide, sodium hydride, sodium carbonate, potassium carbonate, or triethylamine. The mixture is filtered, Soxhlet extracted for 12-48 hours, and vacuum dried overnight to obtain product 1: N-benzylidene chitosan (CSBA); b: Product 1 reacts with halogenated hydrocarbon at 50-80°C for 12-36h, then precipitates with 3-5 volumes of ethanol, washes with acetone 3-5 times, and filters to obtain product 2: O-alkyl-N-benzylidene chitosan (TCSBA); c: Product 2 is stirred in a 0.1-0.5 mol / L hydrochloric acid / ethanol mixed solution for 12-48 hours, neutralized with an alkaline solution of potassium hydroxide, sodium hydroxide, sodium hydride, sodium carbonate, potassium carbonate or triethylamine to pH: 7.0-7.2, added with 3-5 volumes of ethanol for precipitation, filtered and washed with acetone 3-5 times to obtain product 3: O-alkyl-chitosan. d: Product 3 and 2,3-epoxypropyltrimethylammonium chloride are added in a molar ratio of glucosamine to 2,3-epoxypropyltrimethylammonium chloride of 1:5-1:20, and the reaction is carried out at 60-100° C. for 6-48 hours to obtain the final product O-alkyl-N-hydroxypropyltrimethylammonium chitosan.

3. The method for preparing the amphiphilic chitosan derivative according to claim 2, wherein: In the step a, chitosan is dissolved in a 1%-3% w / v acetic acid aqueous solution, and benzaldehyde is added in a ratio of glucosamine to benzaldehyde of 1:

10. The mixture is stirred at 60-100° C. for 6-24 hours, and the pH is adjusted to 7.0-7.4 with a 3 mol / L alkaline solution. The residue is filtered, washed with anhydrous ethanol 3-5 times, and then placed in a Soxhlet extractor with anhydrous ethanol as the extraction solvent. After extraction for 12-48 hours, the mixture is vacuum-dried to obtain product 1: N-benzylidene chitosan (CSBA).

4. The method for preparing the amphiphilic chitosan derivative according to claim 2, wherein: The preparation method of the product 2 in the step b is as follows: N-benzylidene chitosan is dispersed in a mixed solvent of isopropanol and potassium hydroxide, stirred at room temperature for 2-4 hours, added in a ratio of glucosamine:N-benzylidene chitosan of 1:5-1:20, reacted at 50-80° C. for 12-36 hours, cooled to room temperature, added ethanol to precipitate the filter residue, washed with acetone, and vacuum dried at 40-60° C. to obtain product 2: O-alkyl-N-benzylidene chitosan (RCSBA).

5. The method for preparing the amphiphilic chitosan derivative according to claim 2, wherein the halogenated hydrocarbon used in the preparation is one or more of n-propyl chloride, sec-butyl chloride, 1-n-chloropentane, n-hexane chloride, n-heptane chloride, 1-chlorooctane, 1-chlorononane, 1-chlorodecane, 1-chloroundecane, chlorododecane, 1-chlorotridecane, chlorotetradecane, 1-bromopentadecane, chlorohexadecane, 1-bromoheptadecane, chlorooctadecane, 1-bromononadecane, 1-chloroeicosane, 1-bromoeicosane, isopropyl bromide, n-butyl bromide, 1-bromopentane, 1-bromoheptane, 1-n-bromooctane, n-nonane bromide, n-decane bromide, bromoundecane, bromododecane, 1-fluorododecane, bromobenzene, p-chlorotoluene, benzyl chloride, and p-chlorobenzyl bromide.

6. The method for preparing the amphiphilic chitosan derivative according to claim 2, wherein: The preparation method of step d is as follows: product 3 and 2,3-epoxypropyltrimethylammonium chloride are added in a molar ratio of glucosamine to 2,3-epoxypropyltrimethylammonium chloride of 1:5-1:20, reacted at 60-100° C. for 6-48 hours in an aqueous reaction system, the product is precipitated with ethanol, the filter residue is dissolved with water, dialyzed with a 14,000 Da molecular weight dialysis bag, and freeze-dried to obtain the final product, O-alkyl-N-hydroxypropyltrimethylammonium chitosan (RTAC).

7. Use of the amphiphilic chitosan derivative according to claim 1 in a broad-spectrum antimicrobial agent, characterized in that: The amphiphilic chitosan derivative has a killing effect on bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter lwoffi, and can be used as a broad-spectrum antibacterial agent.

8. Use of the amphiphilic chitosan derivative according to claim 1 in an anti-biofilm preparation, characterized in that: Amphiphilic chitosan derivatives can also be used as anti-biofilm agents, and can inhibit the biofilm formation and adhesion of bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter lwoffi, and can be used as anti-biofilm agents.

9. Use of the amphiphilic chitosan derivative according to claim 1 in daily chemical products, food processing and packaging machinery, industrial lubrication systems and machinery corrosion protection, oil and gas industry pipeline protection, and marine fuel tank antimicrobial agent, characterized in that: The addition amount of the amphiphilic chitosan derivative is different, among which the addition amount of daily chemical products is 0.05-10%, the addition amount of food processing and packaging machinery antibacterial and antibacterial coating is 0.05-15%, and the addition amount of industrial lubrication system and machinery anticorrosion, oil and gas industry pipeline protection, and marine ship fuel tank antibacterial is 0.1-30%.

10. Use of the amphiphilic chitosan derivative according to claim 1 as an antimicrobial agent in industrial anti-biofilm applications, characterized in that: The addition amount of the amphiphilic chitosan derivative as an anti-biofilm preparation is different. The addition amount for industrial anti-biofilm is 0.2-30%, and the addition amount for food processing and packaging machinery antibacterial and coating materials is 0.1-20%.

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