Chitosan derivative antibacterial liquid and its preparation method and application

Abstract

The present invention belongs to the technical field of new functional polymer materials, and discloses a chitosan derivative antibacterial liquid and its preparation method and application. The preparation method is as follows: under an atmosphere of protective gas, chitosan is swelled and then sulfonated to obtain sulfonated chitosan; in water, the sulfonated chitosan is subjected to a quaternization reaction with 2,3-epoxypropyltrimethylammonium chloride to introduce a quaternary ammonium salt group on the sulfonated chitosan to obtain a chitosan derivative; the chitosan derivative is dispersed in water and sterilized by radiation to obtain the chitosan derivative antibacterial liquid. The preparation process of the present invention is green and pollution-free, which is conducive to industrial production, and can promote the use of chitosan as a biological antibacterial liquid; and the chitosan derivative antibacterial liquid prepared by the present invention not only has a wide pH value application range, but also shows high selectivity for the antibacterial property of different bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of new functional polymer materials, and in particular to a chitosan derivative antibacterial liquid, a preparation method thereof and applications thereof. Background Art

[0002] In recent years, with increasing attention paid to environmental awareness and ecological sustainability, the use of safe and environmentally friendly biological extracts as natural antimicrobial agents has gradually become widespread. This trend reflects the urgent need to replace traditional synthetic antimicrobial agents to reduce their adverse impact on the environment and minimize potential harm to humans.

[0003] Chitosan, the second largest bioresource in nature after cellulose, is a deacetylated product of chitin. It is non-toxic, antibacterial, renewable, biodegradable, and biocompatible, making it widely used in biomedicine, agriculture, and the food industry. However, due to the high crystallinity induced by hydrogen bonds and acetylamino groups, chitosan has poor solubility at neutral pH and a high viscosity even in dilute acid solutions.

[0004] In order to solve the above technical problems, those skilled in the art have attempted to improve the water solubility of chitosan in a wider pH range by introducing permanent cationic charges - quaternary ammonium salt groups. And the common methods for introducing quaternary ammonium salt functional groups into chitosan currently include using iodomethane to prepare N,N,N-trimethyl chitosan (TMC), or using 2,3-epoxypropyltrimethylammonium chloride (GTMAC) or 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTMAC) to synthesize N-2-hydroxypropyl-3-trimethylammonium chloride chitosan (HTCC). However, the preparation of TMC requires strong alkaline conditions, and the iodomethane used has high biological toxicity and high cost. Formaldehyde and sodium borohydride are sometimes used in the formation process of TMC, which are also toxic.

[0005] Therefore, obtaining and providing a low-viscosity chitosan antibacterial liquid that can be directly dissolved in water and has antibacterial properties and a preparation method thereof have important social and economic value. To this end, the present invention provides a chitosan derivative antibacterial liquid and a preparation method and application thereof. Summary of the Invention

[0006] In order to address the deficiencies in the above-mentioned prior art, the present invention provides a chitosan derivative antibacterial liquid, a preparation method thereof, and an application thereof. The present invention introduces sulfonate groups and quaternary ammonium salt groups into chitosan to achieve a modification treatment of chitosan by the sulfonate groups and the quaternary ammonium salt groups, thereby improving the water solubility and antibacterial properties of chitosan. The sulfonating agent and the quaternary ammonium salt group introducing agent used in the present invention are low in cost, and the preparation process is green and pollution-free, which is conducive to industrial production, thereby promoting the use of chitosan as a biological antibacterial liquid.

[0007] The chitosan derivative antibacterial liquid, preparation method and application thereof of the present invention are achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a method for preparing a chitosan derivative antibacterial solution, comprising the following steps:

[0009] Step 1: Under a protective gas atmosphere, chitosan with a deacetylation degree of ≥97% is swelled in anhydrous ethanol, and then 1,3-propane sultone is added to sulfonate the chitosan to introduce sulfonate groups into the chitosan molecules to obtain sulfonated chitosan.

[0010] Step 2: Using 2,3-epoxypropyltrimethylammonium chloride as a quaternary ammonium group introducing agent, the 2,3-epoxypropyltrimethylammonium chloride and the sulfonated chitosan are subjected to a quaternization reaction in water to introduce quaternary ammonium groups into the sulfonated chitosan to obtain a chitosan derivative.

[0011] Step 3: Dispersing the chitosan derivative in water and sterilizing it by radiation to obtain the chitosan derivative antibacterial solution.

[0012] Preferably, the mass ratio of the sulfonated chitosan to the 2,3-epoxypropyltrimethylammonium chloride is 8:15-30.

[0013] Preferably, when preparing the sulfonated chitosan, the usage ratio of the chitosan, 1,3-propane sultone and anhydrous ethanol is 30 g: 4.5-18.2 g: 500-1000 mL.

[0014] Preferably, the temperature of the sulfonation treatment is 45-55° C., and the reaction time is 5-7 days.

[0015] Preferably, the reaction temperature of the quaternization reaction is 75-80°C.

[0016] Preferably, the temperature of the swelling treatment is 90-95° C., and the swelling time is 20-30 hours.

[0017] Preferably, the mass fraction of the chitosan derivative in the chitosan derivative antibacterial solution is 0.5% to 2%.

[0018] Preferably, during the radiation sterilization process, the radiation dose is 10 to 30 kGy.

[0019] The second object of the present invention is to provide a chitosan derivative antibacterial liquid prepared by the above preparation method.

[0020] The third object of the present invention is to provide a use of the chitosan derivative antibacterial solution in the preparation of antibacterial materials.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention introduces sulfonate groups and quaternary ammonium salt groups into chitosan, thereby achieving modification treatment on chitosan through the sulfonate groups and the quaternary ammonium salt groups, thereby achieving improvement in the water solubility and antibacterial performance of chitosan. The sulfonating agent and the quaternary ammonium salt group introduction agent used in the invention have low costs, and the preparation process is green and pollution-free, which is conducive to industrial production, and can further promote the use of chitosan as a biological antibacterial liquid.

[0023] The chitosan derivatives prepared in the present invention (structural formula shown in Formula I) not only have a wide pH range in water but also exhibit high selectivity against different bacteria. For example, the chitosan derivative antibacterial liquid prepared in the present invention was tested for its antibacterial properties against Escherichia coli and Staphylococcus aureus in accordance with the Health Industry Standard of the People's Republic of China (WS / T650-2019). The antibacterial rate against Staphylococcus aureus reached 99.4% after a 2-minute exposure time. However, even when the exposure time was extended to 60 minutes, the inhibition rate against Escherichia coli was only 45.9%. This shows that the antibacterial selectivity of chitosan derivatives against different bacteria can be adjusted by introducing different groups. Further research is underway to further investigate other properties of the chitosan derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a macroscopic photograph of the chitosan derivative antibacterial liquid prepared in the present invention. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be described clearly and completely below.

[0026] As described in background technology, due to the poor water solubility of chitosan, cause chitosan to be difficult to use as antibacterial liquid, seriously limit the further application and development of chitosan material. Although prior art proposes to improve the water solubility of chitosan by the mode of introducing quaternary ammonium salt functional group, there is cost height to the method for improving the water solubility of chitosan at present, and has the defect of toxicity, still can't affect chitosan in the production and use of actual antibacterial liquid. For this reason, the invention provides a kind of chitosan derivative antibacterial liquid, the present invention attempts by introducing sulfonate radical group and quaternary ammonium salt group on chitosan, to realize the modification treatment to chitosan by sulfonate radical group and quaternary ammonium salt group jointly, to realize the improvement of chitosan water solubility, viscosity and antibacterial property, and the cost of sulfonating agent and quaternary ammonium salt group group introducing agent adopted by the present invention is not high, and preparation process is green and pollution-free, is conducive to industrialized production, and then can promote chitosan to use as biological antibacterial liquid, solve the technical barrier existing in above-mentioned prior art. But the present invention finds in the process of inquiry, if first introduce quaternary ammonium salt group, then introduce sulfonate radical group, need to all carry out a series of dialysis, freeze drying etc. a series of operations to the product obtained by twice introducing, process operation is complicated and time consuming, is unfavorable for suitability for industrialized production.Simultaneously the present invention is by first introducing sulfonate radical group on chitosan molecule, first first introduce sulfonate radical group on chitosan molecule, reaction solution can directly filter, wash, dry and obtain sulfonated chitosan, to realize the sulfonated modification to chitosan.Reintroduce quaternary ammonium salt group, to realize the quaternary modification to chitosan, to realize jointly realizing the modification treatment to chitosan by sulfonate radical group and quaternary ammonium salt group, method of the present invention is on the basis of simplifying treatment step, also improves the introduction efficiency of sulfonate radical group simultaneously, makes to realize more efficiently jointly realizing the modification treatment to chitosan of sulfonate radical group and quaternary ammonium salt group, to further improve water-soluble, viscosity and the antibacterial property of chitosan, makes chitosan can be used as antibacterial liquid.

[0027] The chitosan derivative antibacterial liquid of the present invention is prepared by the following steps:

[0028] Step 1, preparation of sulfonated chitosan:

[0029] Under a protective gas atmosphere, chitosan with a deacetylation degree of ≥97% is swelled in anhydrous ethanol, and then 1,3-propane sultone is added to sulfonate the chitosan to introduce sulfonate groups into the chitosan molecules to obtain sulfonated chitosan.

[0030] It should be noted that, in order to ensure the safety of the reaction, the present invention is carried out under a protective gas atmosphere during the process of introducing the sulfonate group, and the protective gas used in the present invention is nitrogen or an inert gas, wherein the inert gas can be argon or helium.

[0031] In order to facilitate the introduction of sulfonate groups into chitosan, the present invention uses anhydrous ethanol as a swelling treatment solvent. The chitosan is first fully swollen with anhydrous ethanol, thereby facilitating the entry of the sulfonate groups during subsequent treatment. Therefore, in a preferred embodiment of the present invention, the chitosan is swollen at a temperature of 90 to 95° C. for 20 to 30 hours under reflux stirring in an atmosphere of nitrogen or an inert gas, so that the chitosan is fully swollen in the anhydrous ethanol, thereby facilitating full contact between 1,3-propane sultone and the chitosan, thereby improving the efficiency of introducing sulfonate groups into the chitosan.

[0032] The present invention takes into account that 1,3-propanesultone is an active ester and easily attacked by nucleophilic reagents, and in particular, no catalyst is required when reacting with amino groups, and the reaction can be carried out at a relatively low temperature. High temperature may cause other side reactions, and the temperature of the system after the swelling treatment is as high as 90-95°C. In order to avoid the occurrence of other side reactions and improve the efficiency of introducing sulfonate groups on chitosan by 1,3-propanesultone, the system is first cooled to 45-55°C, and then 1,3-propanesultone is added to carry out a sulfonation reaction, so as to ensure that the added 1,3-propanesultone undergoes a nucleophilic ring-opening reaction with part of the amino groups on the chitosan as much as possible, thereby introducing sulfonate groups and improving the water solubility of the chitosan through the sulfonate groups.

[0033] In order to ensure that some amino groups on chitosan undergo nucleophilic ring-opening reaction with 1,3-propane sultone and introduce sulfonate groups, in a preferred embodiment of the present invention, the reaction time of the sulfonation treatment after adding 1,3-propane sultone is 5 to 7 days.

[0034] In order to ensure that quaternary ammonium groups can be further introduced into sulfonated chitosan during subsequent treatment, the present invention should ensure that after the added 1,3-propane sultone reacts with the amino groups on the chitosan, unreacted amino groups still exist on the chitosan, so the amount of 1,3-propane sultone added should not be too much; at the same time, it was found during the exploration process that when too little 1,3-propane sultone was added, the sulfonate group and the quaternary ammonium group could not be effectively coordinated to achieve the modification of the chitosan, and thus the antibacterial effect of the chitosan could not be effectively improved. Therefore, in a preferred embodiment of the present invention, the amount ratio of the chitosan to the 1,3-propane sultone and anhydrous ethanol is 30g:4.5-18.2g:500-1000mL.

[0035] Since the above preparation is carried out in a solution state, in order to improve the subsequent introduction effect of quaternary ammonium salt groups, the product obtained by the above sulfonation reaction is first filtered to remove excess solvent, and then the filter residue obtained after filtration is washed with anhydrous ethanol at least three times to remove the residual solvent and impurities on the surface of the filter residue, and then vacuum dried to remove the residual washing solvent to obtain pure solid sulfonated chitosan.

[0036] Step 2: Using 2,3-epoxypropyltrimethylammonium chloride as a quaternary ammonium group introducing agent, the 2,3-epoxypropyltrimethylammonium chloride and the sulfonated chitosan are subjected to a quaternization reaction in water to introduce quaternary ammonium groups into the sulfonated chitosan to obtain a chitosan derivative.

[0037] It should be noted that in order to better allow 2,3-epoxypropyltrimethylammonium chloride and sulfonated chitosan to undergo quaternization reaction in water, in a preferred embodiment of the present invention, sulfonated chitosan and 2,3-epoxypropyltrimethylammonium chloride are first prepared into solutions, and then mixed to undergo quaternization reaction, so as to improve the effect of introducing quaternary ammonium salt groups on the sulfonated chitosan, thereby improving the modification of the water solubility and antibacterial properties of the sulfonated chitosan by the quaternary ammonium salt groups, and further reducing the viscosity of the material.

[0038] 1) Preparation of sulfonated chitosan solution:

[0039] The sulfonated chitosan is dispersed in water, and the mixture is stirred and heated to 75-80° C. to obtain solution A.

[0040] It should be noted that the present invention takes into account that when the temperature is too low, it is not conducive to the rapid dissolution of sulfonated chitosan, and when the temperature is too high, it will cause the hydrolysis of 2,3-epoxypropyltrimethylammonium chloride and the occurrence of other side reactions. Therefore, the optimal reaction temperature of sulfonated chitosan and 2,3-epoxypropyltrimethylammonium chloride is 80°C, which should not exceed 80°C. Heating at the same time is more conducive to the dissolution of sulfonated chitosan in water. Therefore, the present invention disperses sulfonated chitosan in water while stirring and dissolving it, so that the sulfonated chitosan is quickly dissolved and evenly dispersed in water, and at the same time prepares for the subsequent reaction after adding 2,3-epoxypropyltrimethylammonium chloride. Among them, in order to improve the dissolution efficiency of sulfonated chitosan, in a preferred embodiment of the present invention, the stirring rate during stirring and heating is 300-350r / min.

[0041] 2) Prepare 2,3-epoxypropyltrimethylammonium chloride solution:

[0042] 2,3-Epoxypropyltrimethylammonium chloride is used as a quaternary ammonium salt and is dispersed in water to obtain a 2,3-epoxypropyltrimethylammonium chloride solution, namely, solution B.

[0043] It should be noted that, in order to facilitate the further introduction of quaternary ammonium salt groups on sulfonated chitosan, the present invention preferably introduces them in the form of a solution, and first prepares the quaternary ammonium salt into a solution, that is, 2,3-epoxypropyltrimethylammonium chloride is dispersed in water and mixed to form a 2,3-epoxypropyltrimethylammonium chloride solution with uniform components for standby use.

[0044] 3) Introducing quaternary ammonium salt groups:

[0045] The solution B is added to the solution A to allow the 2,3-epoxypropyltrimethylammonium chloride to undergo a quaternization reaction with the sulfonated chitosan, thereby introducing a quaternary ammonium salt group into the sulfonated chitosan to obtain a chitosan derivative.

[0046] It should be noted that, in order to uniformly introduce quaternary ammonium groups into the sulfonated chitosan molecules, the introduced quaternary ammonium salt can further improve the water solubility and antibacterial properties of the sulfonated chitosan while reducing the viscosity of the sulfonated chitosan, so that the chitosan derivative finally obtained can be prepared with water to form an antibacterial solution with uniform components. Therefore, in a preferred embodiment of the present invention, solution B is divided into 2 to 4 parts and then added to solution A in batches. The time interval between the addition of two adjacent batches is 4 to 6 hours, and the reaction time after the addition is completed is 24 to 30 hours, so that the remaining amino groups in the sulfonated chitosan fully react with 2,3-epoxypropyltrimethylammonium chloride to undergo nucleophilic ring-opening reaction, thereby achieving the uniform introduction of quaternary ammonium groups into the sulfonated chitosan molecules, obtaining chitosan derivatives having both sulfonate groups and quaternary ammonium groups, so as to improve the water solubility, antibacterial properties and viscosity of chitosan through the sulfonate groups and quaternary ammonium groups. The chemical structure of the chitosan derivative having a sulfonate group and a quaternary ammonium salt group is shown in Formula I, wherein, in Formula I, the molar fraction of the quaternary ammonium salt group introduced into the chitosan molecule, y is the molar fraction of the chitosan monomer that does not participate in the reaction, z is the molar fraction of the sulfonate group introduced into the chitosan molecule, and x+y+z=1:

[0047]

[0048] In another preferred embodiment of the present invention, the mass ratio of the sulfonated chitosan in the solution A to the 2,3-epoxypropyltrimethylammonium chloride in the solution B is 8:15-30.

[0049] The present invention takes into account that in addition to the chitosan derivative, residual 2,3-epoxypropyltrimethylammonium chloride and its hydrolysis products may still exist in the reaction solution obtained after the quaternization reaction. Therefore, in order to improve the purity of the chitosan derivative and thus improve the antibacterial effect of the antibacterial solution prepared from the chitosan derivative, in a preferred embodiment of the present invention, the reaction solution obtained after the quaternization reaction is cooled to room temperature, the cooled reaction solution is dialyzed and then freeze-dried to obtain a pure solid chitosan derivative.

[0050] In order to ensure that the residual 2,3-epoxypropyltrimethylammonium chloride and its hydrolysis products can be removed by dialysis, in a preferred embodiment of the present invention, the dialysis bag used for dialysis has a molecular weight cutoff of 1000 to 3000 Da, and the deionized water is replaced every 8 hours until the "exudate" is identified as free of Cl negative ions by a dilute silver nitrate solution, and then the "retained solution" is freeze-dried to obtain a solid chitosan derivative.

[0051] The present invention takes into account the factors that foaming is easy to occur during reduced pressure distillation on a rotary evaporator, and the retained liquid is easy to rush into the buffer bottle and cause sample loss. Therefore, the freeze-drying method is adopted to obtain the target product of the chitosan derivative.

[0052] Step 5, prepare antibacterial solution:

[0053] The chitosan derivative is dispersed in water to obtain an aqueous solution of the chitosan derivative; and the aqueous solution of the chitosan derivative is sterilized by radiation to obtain the chitosan derivative antibacterial solution.

[0054] It should be noted that, in consideration of the antibacterial effect and preparation cost, when dispersing the chitosan derivative in water, the content of the chitosan derivative should not be too much or too little, so that the mass fraction of the chitosan derivative in the obtained chitosan derivative antibacterial solution is preferably 0.5% to 2%.

[0055] The present invention mainly sterilizes the aqueous solution of chitosan derivatives by radiation, and the radiation sterilization can also degrade the chitosan main chain to further reduce the viscosity of the target product antibacterial solution. Therefore, in a preferred embodiment of the present invention, during radiation sterilization, the absorbed dose of the aqueous solvent of the chitosan derivative is 10 to 30 kGy.

[0056] Example 1

[0057] This embodiment provides a chitosan derivative antibacterial liquid, which is prepared by the following steps:

[0058] Step 1, preparation of sulfonated chitosan:

[0059] 30.2 g of chitosan with a deacetylation degree of ≥97% was dispersed in a reaction bottle containing 500 mL of anhydrous ethanol. The reaction bottle was placed in an oil bath at 95° C. and refluxed under nitrogen protection. The reaction bottle was swelled for 24 hours under magnetic stirring. After the swelling was completed, the reaction solution obtained after the swelling treatment was cooled to 50° C., 9.0 g of 1,3-propane sultone was added, and a sulfonation reaction was carried out for 5 days to introduce sulfonate groups on the chitosan molecules. The sulfonated product was filtered to obtain a solid product. The solid product was washed three times with anhydrous ethanol and then dried in a vacuum drying oven at 50° C. for 12 hours to obtain solid sulfonated chitosan.

[0060] Step 2, prepare sulfonated chitosan solution:

[0061] 8.0 g of the sulfonated chitosan prepared in step 1 above was dispersed in 180 mL of deionized water, and heated to 80° C. with stirring at a stirring rate of 320 r / min to obtain solution A.

[0062] Step 3, prepare 2,3-epoxypropyltrimethylammonium chloride solution:

[0063] 22.0 g of 2,3-epoxypropyltrimethylammonium chloride was dispersed in 44 mL of deionized water and magnetically stirred at room temperature for 1 min to obtain a 2,3-epoxypropyltrimethylammonium chloride solution, namely, solution B.

[0064] Step 4, introduction of quaternary ammonium salt groups:

[0065] 1) After the solution B obtained in step 3 was divided into three equal parts, the solution B was added dropwise to the solution A in three portions while stirring the solution A at a stirring rate of 320 r / min, and the time interval between two adjacent additions was 4 h. After the addition was completed, the reaction was continued for 24 h. After the reaction was completed, the mixture was cooled to room temperature to obtain a reaction solution.

[0066] 2) The reaction solution obtained above was dialyzed through a dialysis bag with a molecular weight cut-off of 1000 Da to remove unreacted 2,3-epoxypropyltrimethylammonium chloride and its hydrolysis products. Deionized water was replaced every 8 hours until the "exudate" was free of Cl by silver nitrate dilute solution. - ions, and then the "retained solution" is freeze-dried to obtain a slightly yellow porous solid, that is, a chitosan derivative.

[0067] Step 5, prepare antibacterial solution:

[0068] 1 g of the chitosan derivative obtained in step 4 was dispersed in 99 mL of deionized water, and sterilized by radiation with an absorbed dose of 20 kGy to obtain a chitosan derivative antibacterial solution with a chitosan derivative mass fraction of 1%.

[0069] Example 2

[0070] The difference between this embodiment and embodiment 1 is that:

[0071] In this embodiment, the mass fraction of the chitosan derivative in the chitosan derivative antibacterial solution is 0.5%.

[0072] Example 3

[0073] This embodiment provides a chitosan derivative antibacterial liquid, which is prepared by the following steps:

[0074] Step 1, preparation of sulfonated chitosan:

[0075] 30.1 g of chitosan with a deacetylation degree of ≥97% was dispersed in a reaction flask containing 500 mL of anhydrous ethanol. The reaction flask was refluxed in an oil bath at 93° C. under nitrogen protection and refluxed. The mixture was swelled for 26 hours under magnetic stirring. After the swelling was completed, the reaction solution obtained after the swelling treatment was cooled to 50° C., 13.0 g of 1,3-propane sultone was added, and a sulfonation reaction was carried out for 6 days to introduce sulfonate groups into the chitosan molecules. The sulfonated product was filtered to obtain a solid product. The solid product was washed four times with anhydrous ethanol and then dried in a vacuum drying oven at 50° C. for 12 hours to obtain solid sulfonated chitosan.

[0076] Step 2, prepare sulfonated chitosan solution:

[0077] 8.0 g of the sulfonated chitosan prepared in step 1 above was dispersed in 200 mL of deionized water, and the mixture was heated to 80° C. with stirring at a stirring rate of 310 r / min to obtain solution A.

[0078] Step 3, prepare 2,3-epoxypropyltrimethylammonium chloride solution:

[0079] 18.0 g of 2,3-epoxypropyltrimethylammonium chloride was dispersed in 36 mL of deionized water and stirred magnetically at room temperature for 1 min to obtain a 2,3-epoxypropyltrimethylammonium chloride solution, namely, solution B.

[0080] Step 4, introduction of quaternary ammonium salt groups:

[0081] 1) After the solution B obtained in step 3 was divided into three equal parts, the solution B was added dropwise to the solution A in three portions while stirring the solution A at a stirring rate of 310 r / min, and the time interval between two adjacent additions was 5 h. After the addition was completed, the reaction was continued for 25 h. After the reaction was completed, the mixture was cooled to room temperature to obtain a reaction solution.

[0082] 2) The reaction solution obtained above was dialyzed through a dialysis bag with a molecular weight cut-off of 2000Da to remove unreacted 2,3-epoxypropyltrimethylammonium chloride and its hydrolysis products. Deionized water was replaced every 8 hours until the "exudate" was free of Cl by silver nitrate dilute solution. - ions, and then the "retained solution" is freeze-dried to obtain a slightly yellow porous solid, that is, a chitosan derivative.

[0083] Step 5, prepare antibacterial solution:

[0084] 1.5 g of the chitosan derivative obtained in step 4 was dispersed in 98.5 mL of deionized water, and sterilized by radiation with an absorbed dose of 20 kGy to obtain the chitosan derivative antibacterial solution.

[0085] Example 4

[0086] This embodiment provides a chitosan derivative antibacterial liquid, which is prepared by the following steps:

[0087] Step 1, preparation of sulfonated chitosan:

[0088] 30.2 g of chitosan with a deacetylation degree of ≥97% was dispersed in a reaction flask containing 500 mL of anhydrous ethanol. The reaction flask was refluxed in an oil bath at 93° C. under nitrogen protection and refluxed. The mixture was swelled for 28 h under magnetic stirring. After the swelling was completed, the reaction solution obtained after the swelling treatment was cooled to 50° C., 18.0 g of 1,3-propane sultone was added, and a sulfonation reaction was carried out for 7 days to introduce sulfonate groups into the chitosan molecules. The sulfonated product was filtered to obtain a solid product. The solid product was washed with anhydrous ethanol 5 times and then dried in a vacuum drying oven at 50° C. for 16 h to obtain solid sulfonated chitosan.

[0089] Step 2, prepare sulfonated chitosan solution:

[0090] 8.0 g of the sulfonated chitosan prepared in step 1 above was dispersed in 220 mL of deionized water, and the mixture was stirred and heated to 80° C. at a stirring rate of 330 r / min to obtain solution A.

[0091] Step 3, prepare 2,3-epoxypropyltrimethylammonium chloride solution:

[0092] 16.0 g of 2,3-epoxypropyltrimethylammonium chloride was dispersed in 32 mL of deionized water and magnetically stirred at room temperature for 3 min to obtain a 2,3-epoxypropyltrimethylammonium chloride solution, namely, solution B.

[0093] Step 4, introduction of quaternary ammonium salt groups:

[0094] 1) After the solution B obtained in step 3 was divided into three equal parts, the solution B was added dropwise to the solution A in three portions while stirring the solution A at a stirring rate of 320 r / min, and the time interval between two adjacent additions was 6 h. After the dropwise addition was completed, the reaction was continued for 28 h. After the reaction was completed, the mixture was cooled to room temperature to obtain a reaction solution.

[0095] 2) The reaction solution obtained above was dialyzed through a dialysis bag with a molecular weight cut-off of 1000 Da to remove unreacted 2,3-epoxypropyltrimethylammonium chloride and its hydrolysis products. Deionized water was replaced every 8 hours until the "exudate" was free of Cl by silver nitrate dilute solution. - ions, and then the "retained solution" is freeze-dried to obtain a slightly yellow porous solid, that is, a chitosan derivative.

[0096] Step 5, prepare antibacterial solution:

[0097] 2 g of the chitosan derivative obtained in step 4 was dispersed in 98 mL of deionized water, and sterilized by radiation with an absorbed dose of 20 kGy to obtain the chitosan derivative antibacterial solution.

[0098] Comparative Example 1

[0099] In order to demonstrate the modification effects of sulfonation and quaternization on chitosan, this comparative example attempted to directly prepare an antibacterial liquid from the chitosan subjected to sulfonation and quaternization for comparison. However, during the actual experiment, it was found that since the chitosan was not modified, it could not be fully dissolved in water, and thus no antibacterial liquid could be obtained.

[0100] Experimental part

[0101] (1) Macromorphology analysis

[0102] The present invention takes the chitosan derivative antibacterial liquid prepared in Example 1 as an example, and records its physical photos as follows: Figure 1 As shown, it can be seen that the chitosan derivative antibacterial liquid prepared in Example 1 is a light yellow liquid, and the liquid is clear and transparent, indicating that the preparation method of the present invention can successfully improve the water solubility of chitosan, and successfully prepare an antibacterial liquid with good solubility and uniform components.

[0103] (2) Antibacterial properties

[0104] The present invention takes the chitosan derivative antibacterial liquid prepared in Example 1 and Comparative Example 2 as examples, and tests the antibacterial properties thereof.

[0105] Test method:

[0106] Take the fresh slant culture of the test bacteria for 24 hours, wash it with PBS, and dilute it with PBS to about 5.0×10 5 ~4.5×106 Prepare a suspension of Staphylococcus aureus (or Escherichia coli) at a concentration of 5.0 CFU / mL. Add 5.0 mL of the antibacterial solution to be tested to a sterile test tube. Place in a 20°C ± 1°C water bath for 5 minutes. Then add 0.1 mL of the test bacterial suspension, mix quickly, and start the timer immediately.

[0107] When the interaction time between the test bacteria and the antibacterial solution sample is 2 min, 5 min, and 10 min, 1.0 mL of the test bacteria and sample mixture is respectively aspirated to inoculate two plates, and the culture medium is poured.

[0108] If the bacterial count is too high to count, make a 10-fold serial dilution with PBS. Select an appropriate dilution and inoculate 1.0 mL onto two plates for viable bacterial count. Simultaneously, perform a parallel experiment using PBS instead of the sample as a positive control.

[0109] The number of colonies recovered in the positive control was 1.0×10 4 ~9.0×10 4 CFU / mL.

[0110] PBS and culture medium from the same batch were used as negative controls.

[0111] All test samples and control samples were cultured at 36°C ± 1°C, and the bacterial propagules were cultured for 48 hours to observe the final results; the test was repeated three times, and the inhibition rates at action times of 2 minutes, 5 minutes, and 10 minutes were calculated according to Formula II.

[0112] The calculation formula of the antibacterial rate is shown in Formula II:

[0113]

[0114] Where:

[0115] X is the inhibition rate, %;

[0116] A0 is the amount of bacteria recovered in the positive control group, CFU / mL;

[0117] A1 is the amount of bacteria recovered in the test group, CFU / mL.

[0118] The results showed that the antibacterial rates of Example 1 against Staphylococcus aureus were 99.4%, 99.5%, and 99.99% when the exposure time was 2 minutes, 5 minutes, and 10 minutes, respectively; and the antibacterial rates of Example 2 against Staphylococcus aureus were 98.0%, 98.9%, and 99.3% when the exposure time was 5 minutes, 10 minutes, and 20 minutes, respectively. It can be seen that the antibacterial solution obtained by the present invention not only has good water solubility but also has excellent antibacterial effect against Staphylococcus aureus.

[0119] The test results also show that the antibacterial liquid of Example 1 has an inhibition rate of 33.9%, 41.9%, and 45.9% against Escherichia coli for 2 minutes, 10 minutes, and 60 minutes, respectively. This shows that the antibacterial liquid obtained by the present invention not only has different antibacterial properties against different strains, but also indicates that the present invention can regulate the antibacterial selectivity of chitosan derivatives against different bacteria by introducing different groups. Other properties are under further investigation.

[0120] Obviously, the above embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A chitosan derivative antibacterial liquid for preparing a selective antibacterial antibacterial material, characterized in that: The preparation method of the chitosan derivative antibacterial liquid comprises the following steps: Under a protective gas atmosphere, chitosan with a deacetylation degree of ≥97% is swelled in anhydrous ethanol, and then 1,3-propane sultone is added to sulfonate the chitosan to introduce a sulfonate group into the chitosan molecule to obtain sulfonated chitosan; Using 2,3-epoxypropyltrimethylammonium chloride as a quaternary ammonium group introducing agent, the 2,3-epoxypropyltrimethylammonium chloride and the sulfonated chitosan are subjected to a quaternization reaction in water to introduce a quaternary ammonium group into the sulfonated chitosan to obtain a chitosan derivative; The chitosan derivative is dispersed in water to obtain an aqueous solution of the chitosan derivative; the aqueous solution of the chitosan derivative is sterilized by radiation, and the viscosity of the aqueous solution of the chitosan derivative is reduced while sterilizing the aqueous solution, thereby obtaining the chitosan derivative antibacterial solution; during the radiation sterilization process, the radiation dose is 10 to 30 kGy; The mass ratio of the sulfonated chitosan to the 2,3-epoxypropyltrimethylammonium chloride is 8:15-30; When preparing the sulfonated chitosan, the chitosan, 1,3-propane sultone and anhydrous ethanol are used in a ratio of 30 g: 4.5-18.2 g: 500-1000 mL; The selective antibacterial property means that the antibacterial material has an antibacterial rate of 99.4% against Staphylococcus aureus and an antibacterial rate of 45.9% against Escherichia coli.

2. The use according to claim 1, characterized in that The temperature of the sulfonation treatment is 45-55° C., and the reaction time is 5-7 days.

3. The use according to claim 1, characterized in that The reaction temperature of the quaternization reaction is 75-80°C.

4. The use according to claim 1, wherein The temperature of the swelling treatment is 90-95° C., and the swelling time is 20-30 hours.

5. The use according to claim 1, characterized in that The mass fraction of the chitosan derivative in the chitosan derivative antibacterial liquid is 0.5% to 2%.

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