A kind of preparation method of chitosan quaternary ammonium salt

Abstract

The present invention relates to a preparation method of chitosan quaternary ammonium salt, comprising the following steps: step 1, making a carboxyl-containing small molecule quaternary ammonium salt; step 2, dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution with a mass percentage concentration of 1%-5%; step 3, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the above-mentioned small molecule quaternary ammonium salt to obtain a small molecule quaternary ammonium salt solution; step 4, adding the small molecule quaternary ammonium salt solution to the chitosan solution for reaction; step 5, after the reaction is completed, transferring the product to an ultrafiltration device, adding water to ultrafiltration to remove the small molecule quaternary ammonium salt that is not involved in the reaction, and concentrating and drying to obtain the chitosan quaternary ammonium salt. The chitosan quaternary ammonium salt antibacterial agent synthesized by the preparation method of the present invention has good water solubility, high inhibition and antibacterial activity, can inhibit biofilm formation, and has a certain removal effect on mature bacterial biofilms.

Description

Technical Field

[0001] The invention belongs to the field of preparation and application of chitosan antibacterial agents, and particularly relates to a method for preparing chitosan quaternary ammonium salt and its application in inhibiting bacteria and bacterial biofilm. Background Art

[0002] Biofilm is a self-protection method for bacteria to adhere to the surface of objects in order to adapt to the natural environment during their growth and reproduction. It is the main form of bacteria in nature. After the formation of biofilm, it can not only adapt to changes in the external environment, but also block the attack of drugs like a shield, evade the effects of drugs, and develop drug resistance. At present, among the common antimicrobial agents, organic small molecule antimicrobial agents have the characteristics of rapid sterilization, but are prone to drug resistance.

[0003] Chitosan has a broad spectrum of antibacterial properties, but the antibacterial properties of pure chitosan are not significant, and its solubility is limited. The research on the antibacterial properties of modified chitosan has made significant progress. By quaternizing chitosan, not only the solubility of chitosan can be improved, but also the antibacterial properties of chitosan can be improved.

[0004] In view of this, the inventor of this case conducted in-depth research on the above-mentioned issues, which led to the emergence of this case. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a chitosan quaternary ammonium salt which is simple to operate, has convenient raw material sources, is easily soluble in water, and has the functions of antibacterial and inhibiting biofilm formation and removing biofilm.

[0006] In order to achieve the above object, the present invention adopts such technical solution:

[0007] A method for preparing a chitosan quaternary ammonium salt comprises the following steps:

[0008] Step 1, preparing a small molecule quaternary ammonium salt containing a carboxyl group;

[0009] Step 2, dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution with a mass percentage concentration of 1%-5%;

[0010] Step 3, dissolving the above-mentioned small molecule quaternary ammonium salt in water or a mixed solution of water / ethanol, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide until they are completely dissolved to obtain a small molecule quaternary ammonium salt solution;

[0011] Step 4, adding the small molecule quaternary ammonium salt solution into the chitosan solution for reaction, the reaction temperature is 20-50° C., and the reaction time is 12-48 hours;

[0012] Step 5, after the reaction is completed, the product is transferred to an ultrafiltration device, water is added to ultrafilter to remove the small molecule quaternary ammonium salt that does not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

[0013] As a preferred embodiment of the present invention, step 1 is to dissolve substance A in an organic solvent, wherein the mass percentage concentration of substance A is 0.5-20%, add substance B, react at 50-140° C. for 4-48 hours, and after the reaction is completed, filter, wash and dry to obtain a small molecule quaternary ammonium salt.

[0014] Wherein, substance A is p-chloromethylbenzoic acid, p-bromomethylbenzoic acid, chloroacetic acid, chloropropionic acid or bromoacetic acid; substance B is N,N-dimethyldodecylamine, trimethylamine, N,N-dimethylbutylamine, N,N-dimethyloctylamine, N,N-dimethyltetradecylamine, N-methylmorpholine, N-methylpiperidine or pyridine;

[0015] As a preferred embodiment of the present invention, the organic solvent is one or more of ethyl acetate, tetrahydrofuran, acetone, petroleum ether and acetonitrile.

[0016] As a preferred embodiment of the present invention, the substance A is p-chloromethylbenzoic acid, and the substance B is N,N-dimethyldodecylamine, and the molar ratio of the substance A and B is 1:1-2.

[0017] As a preferred embodiment of the present invention, in step 2, the mass percentage concentration of the acetic acid aqueous solution is 1% to 5%.

[0018] As a preferred embodiment of the present invention, the amount of the small molecule quaternary ammonium salt is 1-3 times the molar amount of chitosan.

[0019] As a preferred embodiment of the present invention, in step 3, the mass percentage concentration of the small molecule quaternary ammonium salt is 1%-10%.

[0020] As a preferred embodiment of the present invention, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.

[0021] As a preferred embodiment of the present invention, in step 3, the molar ratio of the small molecule quaternary ammonium salt to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-3.

[0022] As another preferred embodiment of the present invention, the carboxyl-containing small molecule quaternary ammonium salt in step 1 is isonicotinic acid quaternary ammonium salt.

[0023] The technical scheme of the present invention is adopted to first synthesize a carboxyl-terminated small molecule, and then under the action of EDC and NHS, the small molecule is grafted onto a chitosan molecular chain, and ultrafiltration and concentration are performed to obtain chitosan quaternary ammonium salt. The chitosan quaternary ammonium salt antibacterial agent synthesized by the preparation method of the present invention has good water solubility, high inhibitory and antibacterial activity, can inhibit the formation of biofilm, and has a certain removal effect on mature bacterial biofilm. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is N-(4-carboxybenzyl)-N,N-dimethyldodecylammonium chloride in Example 1 1 HNMR.

[0025] Figure 2 is N-(4-carboxybenzyl)-N,N-dimethyldodecylammonium chloride in Example 1 13 CNMR.

[0026] Figure 3 For the chitosan quaternary ammonium salt in Example 1 1 H NMR.

[0027] Figure 4 is N-(carboxymethyl)-N,N-dimethyldodecyl ammonium chloride in Example 2 1 H NMR.

[0028] Figure 5 is N-(4-carboxymethyl)-N,N-dimethyldodecyl ammonium chloride in Example 2 13 C NMR.

[0029] Figure 6 For the chitosan quaternary ammonium salt in Example 2 1 H NMR.

[0030] Figure 7 is N-(4-carboxybenzyl)-N-methylpiperidinium chloride in Example 3 1 H NMR.

[0031] Figure 8 is N-(4-carboxybenzyl)-N-methylpiperidinium chloride in Example 3 13 C NMR.

[0032] Fig. 9 For the chitosan quaternary ammonium salt in Example 3 1 H NMR.

[0033] Fig.10 isonicotinic acid quaternary ammonium salt in Example 4 1 H NMR.

[0034] Fig.11isonicotinic acid quaternary ammonium salt in Example 4 13 C NMR.

[0035] Fig.12 For the chitosan quaternary ammonium salt in Example 4 1 H NMR.

[0036] Fig.13 isonicotinic acid quaternary ammonium salt in Example 5 1 H NMR.

[0037] Fig.14 isonicotinic acid quaternary ammonium salt in Example 5 13 C NMR.

[0038] Fig.15 For the chitosan quaternary ammonium salt in Example 5 1 H NMR. DETAILED DESCRIPTION

[0039] Reference Figures 1 to 15 In order to further explain the technical solution of the present invention, it is described in detail below in conjunction with embodiments.

[0040] Example 1

[0041] Step 1, weigh 1.70 kg of p-chloromethylbenzoic acid and dissolve it in 20 kg of ethyl acetate, and put it into a reactor, add 2.2 kg of N, N-dimethyldodecylamine under stirring, raise the temperature to 85 ° C, reflux and stir for 8 hours, a large amount of white solid precipitates during the reaction, after the reaction is completed, filter, wash the filter cake with ethyl acetate several times, and dry to obtain N-(4-carboxybenzyl)-N, N-dimethyldodecylamine chloride.

[0042] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 100 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution, wherein the mass percentage concentration of chitosan is 1.6%; chitosan can be directly purchased from the market.

[0043] Step 3, weigh 3.87kg N-(4-carboxybenzyl)-N,N-dimethyldodecyl ammonium chloride and dissolve it in 50kg ethanol water with a volume fraction of 50%, add 1.92kg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.15kg N-hydroxysuccinimide, and obtain a small molecule quaternary ammonium salt solution after complete dissolution, wherein the mass percentage concentration of N-(4-carboxybenzyl)-N,N-dimethyldodecyl ammonium chloride in the solution is 7.7%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.

[0044] Step 4: slowly drop the small molecule quaternary ammonium salt solution into the chitosan acetic acid aqueous solution in the reaction kettle, and react at room temperature for 24 hours after the addition is completed. In this embodiment, the molar ratio of N-(4-carboxybenzyl)-N,N-dimethyldodecylammonium chloride to the chitosan added in step 2 can be 1:1.

[0045] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added to ultrafilter to remove N-(4-carboxybenzyl)-N,N-dimethyldodecylammonium chloride and carboxyl activator (EDC and NHS) that do not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

[0046] Example 2

[0047] Step 1, weigh 0.95kg of p-chloroacetic acid and dissolve it in 20kg of ethyl acetate, and put it into a reactor, add 2.3kg of N,N-dimethyldodecylamine under stirring, raise the temperature to 85°C, reflux and stir for 8h, a large amount of white solid precipitates during the reaction, and filter after the reaction. The filter cake is washed with ethyl acetate for several times and dried to obtain N-(carboxymethyl)-N,N-dimethyldodecylamine chloride.

[0048] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 50 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution. At this time, the mass percentage concentration of chitosan is 3.2%.

[0049] Step 3, weigh 3.70kg N-(carboxymethyl)-N,N-dimethyldodecyl ammonium chloride and dissolve it in 100kg water, add 2.30kg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.3kg N-hydroxysuccinimide, and obtain a small molecule quaternary ammonium salt solution after complete dissolution, wherein the mass percentage concentration of N-(carboxymethyl)-N,N-dimethyldodecyl ammonium chloride in the solution is 3.7%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.

[0050] Step 4: slowly drop the small molecule quaternary ammonium salt solution into the chitosan acetic acid aqueous solution in the reaction kettle, and react at 30° C. for 24 hours after the addition is completed. In this embodiment, the molar ratio of N-(carboxymethyl)-N,N-dimethyldodecylammonium chloride to the chitosan added in step 2 is 1.2:1.

[0051] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added to ultrafiltration to remove N-(carboxymethyl)-N,N-dimethyldodecyl ammonium chloride and carboxyl activator that do not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

[0052] Example 3

[0053] Step 1, weigh 1.70kg of p-chloromethylbenzoic acid and dissolve it in 20kg of ethyl acetate, and put it into a reactor, add 1.0kg of N-methylpiperidine under stirring, raise the temperature to 85°C, reflux and stir for 10h, a large amount of white solid precipitates during the reaction, and filter after the reaction. The filter cake is washed with ethyl acetate several times and dried to obtain N-(4-carboxybenzyl)-N-methylpiperidinium chloride.

[0054] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 100 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution, at which the mass percentage concentration of chitosan is 1.6%.

[0055] Step 3, weigh 2.70kg N-(4-carboxybenzyl)-N-methylpiperidinium chloride and dissolve it in 50kg ethanol water with a volume fraction of 50%, add 1.92kg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.15kg N-hydroxysuccinimide, and obtain a small molecule quaternary ammonium salt solution after complete dissolution, wherein the mass percentage concentration of N-(4-carboxybenzyl)-N-methylpiperidinium chloride in the solution is 7.7%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.

[0056] Step 4: slowly dropwise add the small molecule quaternary ammonium salt solution to the chitosan acetic acid aqueous solution in the reaction kettle, and react at room temperature for 24 hours after the addition is completed. In this embodiment, the molar ratio of N-(4-carboxybenzyl)-N-methylpiperidinium chloride to the chitosan added in step 2 is 1:1.

[0057] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added to ultrafiltration to remove N-(4-carboxybenzyl)-N-methylpiperidinium chloride and carboxyl activator that do not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

[0058] Example 4

[0059] Step 1, weighing 1.79 kg of tert-butyl isonicotinate and adding it to a reactor, adding 2.25 kg of chlorododecane under stirring, raising the temperature to 130 ° C, reflux stirring for 36 hours, washing with ethyl acetate for several times after the reaction is completed, and drying to obtain tert-butyl isonicotinate quaternary ammonium salt; tert-butyl isonicotinate quaternary ammonium salt is dissolved in formic acid, stirred at room temperature for 24 hours, rotary evaporation to remove formic acid, washing with ethyl acetate, and drying to obtain quaternary ammonium salt of isonicotinate.

[0060] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 50 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution. At this time, the mass percentage concentration of chitosan is 3.2%.

[0061] Step 3, weigh 3.84kg of isonicotinoic acid quaternary ammonium salt and dissolve it in 50kg of ethanol water with a volume fraction of 50%, add 1.92kg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.15kg of N-hydroxysuccinimide, and obtain a small molecule quaternary ammonium salt solution after complete dissolution, wherein the mass percentage concentration of isonicotinoic acid quaternary ammonium salt in the solution is 3.84%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.

[0062] Step 4: slowly dropwise add the small molecule quaternary ammonium salt solution to the chitosan acetic acid aqueous solution in the reactor, and react at room temperature for 24 hours after the addition is completed. In this embodiment, the molar ratio of isonicotinic acid quaternary ammonium salt to the chitosan added in step 2 is 1:1.

[0063] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added to ultrafiltration to remove the isonicotinic acid quaternary ammonium salt and carboxyl activator that have not participated in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

[0064] Example 5

[0065] Step 1, weigh 1.5 kg of isonicotinic acid and dissolve it in 30 kg of N, N-dimethylformamide, add 9.0 kg of 1,3-propane sultone under stirring, raise the temperature to 60 ° C, reflux and stir for 24 hours, filter after the reaction is completed, wash the filter cake with N, N-dimethylformamide for several times, wash with ethyl acetate, and dry to obtain isonicotinic acid quaternary ammonium salt.

[0066] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 50 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution. At this time, the mass percentage concentration of chitosan is 3.2%.

[0067] Step 3, weigh 2.45kg of isonicotinate quaternary ammonium salt and dissolve it in 50kg of water, add 2.30kg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.3kg of N-hydroxysuccinimide, and obtain an isonicotinate quaternary ammonium salt solution after complete dissolution, wherein the mass percentage concentration of isonicotinate quaternary ammonium salt in the solution is 2.45%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.

[0068] Step 4: slowly dropwise add the isonicotinic acid quaternary ammonium salt solution into the chitosan acetic acid aqueous solution in the reaction kettle, and react at 25° C. for 24 hours after the addition is completed. In this embodiment, the molar ratio of isonicotinic acid quaternary ammonium salt to the chitosan added in step 2 is 1.2:1.

[0069] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added to ultrafiltration to remove the isonicotinic acid quaternary ammonium salt and carboxyl activator that have not participated in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

[0070] 2. Antibacterial performance test

[0071] The antibacterial properties of the chitosan quaternary ammonium salt in the examples were tested by the plate count method, and the antibacterial rate was calculated by formula 1. The prepared chitosan quaternary ammonium salt had good antibacterial activity, and its antibacterial activity was better than that of chitosan.

[0072]

[0073] Table 1 Antibacterial effect of different concentrations of chitosan quaternary ammonium salt on Staphylococcus aureus

[0074] Group 2.5mg / mL 1.0mg / mL 0.5mg / mL Chitosan raw materials 61.69％ 46.39％ 23.58％ Example 1 100％ 100％ 100％ Example 2 100％ 100％ 100％ Example 3 100％ 100％ 100％ Example 4 100％ 100％ 100％ Example 5 100％ 100％ 100％

[0075] Table 2 Antibacterial effect of different concentrations of chitosan quaternary ammonium salt on Escherichia coli

[0076] Group 2.5mg / mL 1.0mg / mL 0.5mg / mL Chitosan raw materials 45.37％ 21.33％ 10.39％ Example 1 100％ 100％ 100％ Example 2 100％ 98.18％ 93.66％ Example 3 98.14％ 96.98％ 76.80％ Example 4 100％ 98.62％ 96.03％ Example 5 100％ 98.18％ 93.66％

[0077] The minimum inhibitory concentration (MIC) and the minimum biofilm inhibition concentration (MBC) of the chitosan quaternary ammonium salt in the example were determined by turbidimetry, and the minimum bactericidal concentration (MBC) and the minimum biofilm killing concentration (MBBC) of the chitosan quaternary ammonium salt in the example were determined by combining the plate count method.

[0078] Add 100 μL of diluted bacterial solution and 100 μL of chitosan solution diluted by half to a 96-well plate, and then make a control group with only chitosan quaternary ammonium salt solution but no bacterial solution and a control group with only bacterial solution but no chitosan quaternary ammonium salt solution, respectively. Make three parallel groups for each concentration of chitosan quaternary ammonium salt, and observe the results after culturing in a 37°C incubator for 18 hours. Take the lowest concentration of chitosan quaternary ammonium salt corresponding to the clarified wells as MIC, take 100 μL of the mixture of bacterial solution and sample solution in the clarified wells for plating, and record the lowest concentration corresponding to less than 5 colonies per dish as MBC.

[0079] Add diluted bacterial solution to a 96-well plate and place it in a 37°C incubator for 36-48 hours to form a biofilm on the plate. Then discard the floating bacteria, rinse with sterile saline or PBS, dry and fix, add 100 μL of diluted chitosan quaternary ammonium salt sample solution and 100 μL of culture medium to the 96-well plate, use sterile water as the control group, place it in a 37°C incubator for 18 hours and observe the results. The minimum concentration of chitosan quaternary ammonium salt corresponding to the clarified well is taken as MBIC, and 100 μL of the mixture of the bacterial solution and the sample solution in the clarified well is taken for plating. The minimum chitosan quaternary ammonium salt concentration corresponding to less than 5 colonies per dish is recorded as MBBC. The minimum inhibitory concentration and minimum bactericidal concentration data show that the anti-floating bacteria activity and anti-film bacteria activity of the modified chitosan are improved.

[0080] Table 3 Changes in drug resistance of Staphylococcus aureus before and after BF formation

[0081]

[0082] Table 4 Changes in drug resistance of Escherichia coli before and after BF formation

[0083]

[0084]

[0085] The crystal violet staining method can quickly and easily measure the intervention effect of drugs on biofilms. The OD value under the action of different drugs can reflect the inhibitory effect of chitosan quaternary ammonium salt on biofilm formation.

[0086] 100 μL of diluted bacterial solution and 100 μL of chitosan quaternary ammonium salt solution diluted to a certain concentration (4MIC, 2MIC, MIC) were added to a 96-well plate, with water as the control group, and the final concentration of chitosan quaternary ammonium salt was 2MIC, MIC and 1 / 2MIC. Three groups of drugs were made in parallel for each concentration, and cultured in a 37°C incubator for 36-48 hours, then the floating bacteria and chitosan quaternary ammonium salt solution were discarded, rinsed with sterile saline, dried and fixed, 0.1% crystal violet solution was added for staining for 15 minutes, the crystal violet solution was discarded and rinsed with sterile saline for 3 times, and dried with sterile air. 95% ethanol solution was added, and the absorbance was measured at 595nm with an enzyme marker after 5 minutes, and the biofilm formation inhibition rate was calculated by formula 2.

[0087]

[0088] Table 5 Inhibitory effect of chitosan quaternary ammonium salt on biofilm formation of Staphylococcus aureus

[0089] Group 1 / 2MIC MIC 2MIC Chitosan raw materials 30.78％ 56.03％ 74.53％ Example 1 76.96％ 82.34％ 92.90％ Example 2 66.89％ 81.37％ 92.79％ Example 3 50.18％ 91.61％ 93.67％ Example 4 70.30％ 80.81％ 92.55％ Example 5 59.67％ 83.52％ 91.91％

[0090] Table 6 Inhibitory effect of chitosan quaternary ammonium salt on Escherichia coli biofilm formation

[0091] Group 1 / 2MIC MIC 2MIC Chitosan raw materials 28.43％ 43.89％ 60.55％ Example 1 76.43％ 86.94％ 89.76％ Example 2 55.55％ 86.59％ 89.99％ Example 3 76.47％ 85.25％ 90.55％ Example 4 56.84％ 73.25％ 80.86％ Example 5 52.23％ 70.46％ 76.16％

[0092] Add 200 μL of diluted bacterial solution to a 96-well plate, then place it in a 37°C constant temperature incubator for 36-48 hours, discard the bacterial solution, rinse with sterile saline, dry and fix, then add 200 μL of chitosan quaternary ammonium salt solution, and use sterile water as a control group. Keep the temperature in a 37°C incubator for 1 hour, discard the chitosan solution, rinse with sterile saline, stain with crystal violet, dry, add 95% ethanol solution, and measure the absorbance at 595 nm with an enzyme reader after 5 minutes. Calculate the clearance rate of the biofilm using Formula 3.

[0093]

[0094] Table 7 Effect of chitosan quaternary ammonium salt on the removal of Staphylococcus aureus biofilm

[0095] Group 2.5mg / mL 5.0mg / mL Chitosan raw materials 41.47％ 67.47％ Example 1 92.93％ 93.98％ Example 2 93.07％ 94.28％ Example 3 85.68％ 92.88％ Example 4 92.57％ 93.91％ Example 5 73.30％ 78.25％

[0096] Table 8 The removal effect of chitosan quaternary ammonium salt on Escherichia coli biofilm

[0097]

[0098]

[0099] The above results show that the chitosan quaternary ammonium salt prepared by this method is effective against Staphylococcus aureus and Escherichia coli, can also inhibit the biofilm formation of Staphylococcus aureus and Escherichia coli, and has a certain clearing effect on mature biofilms, and the effects are better than those of unmodified chitosan raw materials.

[0100] The product form of the present invention is not limited to the embodiments of this case. Any appropriate changes or modifications made by anyone with similar ideas should be deemed to be within the patent scope of the present invention.

Claims

1. A method for preparing a chitosan quaternary ammonium salt, characterized in that: The steps include: Step 1, preparing a small molecule quaternary ammonium salt containing a carboxyl group; Step 2, dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution with a mass percentage concentration of 1%-5%; Step 3, dissolving the above-mentioned small molecule quaternary ammonium salt in water or a mixed solution of water / ethanol, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide until they are completely dissolved to obtain a small molecule quaternary ammonium salt solution; Step 4, adding the small molecule quaternary ammonium salt solution into the chitosan solution for reaction, the reaction temperature is 20-50° C., and the reaction time is 12-48 hours; Step 5, after the reaction is completed, the product is transferred to an ultrafiltration device, water is added to ultrafilter to remove the small molecule quaternary ammonium salt that does not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary ammonium salt.

2. The method for preparing a chitosan quaternary ammonium salt according to claim 1, wherein: Step 1 is to dissolve substance A in an organic solvent, wherein the mass percentage concentration of substance A is 0.5-20%, add substance B, react at 50-140° C. for 4-48 hours, and after the reaction is completed, obtain a small molecule quaternary ammonium salt by suction filtration, washing and drying. Among them, substance A is p-chloromethylbenzoic acid, p-bromomethylbenzoic acid, chloroacetic acid, chloropropionic acid or bromoacetic acid; substance B is N,N-dimethyldodecylamine, trimethylamine, N,N-dimethylbutylamine, N,N-dimethyloctylamine, N,N-dimethyltetradecamine, N-methylmorpholine, N-methylpiperidine or pyridine.

3. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 2, characterized in that, The organic solvent is one or more of ethyl acetate, tetrahydrofuran, acetone, petroleum ether and acetonitrile.

4. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 3, characterized in that, The substance A is p-chloromethylbenzoic acid, and the substance B is N,N-dimethyldodecylamine, and the molar ratio of the substance A and B is 1:1-2.

5. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 4, characterized in that, In step 2, the mass percentage concentration of the acetic acid aqueous solution is 1% to 5%.

6. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 4, characterized in that, The amount of the small molecule quaternary ammonium salt is 1-3 times the molar amount of chitosan.

7. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 6, characterized in that: In step 3, the mass percentage concentration of the small molecule quaternary ammonium salt is 1%-10%.

8. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 7, characterized in that, The molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:

1.

9. A method for preparing a chitosan quaternary ammonium salt as claimed in claim 8, characterized in that: In step 3, the molar ratio of the small molecule quaternary ammonium salt to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-3.

10. The method for preparing a chitosan quaternary ammonium salt according to claim 1, wherein: The small molecule quaternary ammonium salt containing a carboxyl group in step 1 is isonicotinic acid quaternary ammonium salt.

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