Preparation method of antibacterial and antioxidant phenolic chitosan biquaternary ammonium salt

Abstract

The preparation method comprises the following steps: firstly, introducing a quaternary ammonium salt group to an amino group of chitosan to improve the antibacterial property and water solubility of the chitosan, then further introducing a pyridine group to provide a basis for introducing a phenolic group and biquaternary ammonium salt, and finally, carrying out quaternization reaction to obtain the antibacterial and antioxidant phenolic chitosan biquaternary ammonium salt. Quaternary ammonium salt is introduced for the second time, and the phenol-based chitosan biquaternary ammonium salt is obtained. The preparation method of the antibacterial and antioxidant phenolic chitosan biquaternary ammonium salt is simple and convenient to operate and wide in raw material source, and the prepared phenolic chitosan biquaternary ammonium salt is soluble in water and has good antibacterial and antioxidant properties.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of chitosan quaternary ammonium salt, and in particular to a method for preparing antibacterial and antioxidant phenol-based chitosan diquaternary ammonium salt. Background Art

[0002] Chitosan is obtained by deacetylation of chitin. It is abundant in nature, second only to cellulose in content. It has good biocompatibility and degradability and is widely used in medicine, food packaging, cosmetics, agriculture, daily chemicals and other fields.

[0003] Chitosan has been shown to have certain antibacterial properties against both Gram-negative and Gram-positive bacteria, but its poor solubility limits its application. Chitosan contains a large number of active groups such as amino and hydroxyl groups on the molecular chain, which can be used to perform various chemical modifications on chitosan, such as alkylation, quaternization, carboxylation, ring-opening and acylation, to obtain water-soluble or organic-soluble chitosan derivatives. Such modified chitosan derivatives can be used in the fields of cosmetics, food packaging and biomedicine. Summary of the invention

[0004] The invention aims to provide a method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt, which is simple to operate and has a wide source of raw materials. The prepared phenolic chitosan diquaternary ammonium salt is soluble in water and has good antibacterial and antioxidant properties.

[0005] In order to achieve the above object, the solution of the present invention is: A method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt comprises the following steps: Step 1, first dissolving chitosan in an acetic acid aqueous solution or a hydrochloric acid aqueous solution to obtain a chitosan aqueous solution; Step 2, then dissolving the quaternary ammonium agent A in deionized water to obtain an aqueous solution of the quaternary ammonium agent A; Step 3, then slowly adding the aqueous solution of quaternizing agent A to the chitosan aqueous solution, carrying out quaternization reaction at a constant temperature of 70-100° C. for 12-36 h, and ultrafiltration, concentration, and freeze-drying of the reaction product to obtain chitosan quaternary ammonium salt; Step 4, the dried chitosan quaternary ammonium salt is then crushed and fully dispersed in an aprotic solvent, the temperature of the reaction system is lowered to 0-5°C, nicotinyl chloride is slowly added, and then a catalyst is added, and the reaction is carried out at 20-100°C for 12-36 hours. After the reaction is completed, the reaction product is poured into ice water, and pyridyl chitosan quaternary ammonium salt is obtained after ultrafiltration, concentration, and freeze-drying; Step 5, then the dried pyridyl chitosan quaternary ammonium salt is crushed and fully dispersed in a non-protonic solvent, a quaternizing agent B is added, the temperature of the reaction system is increased to 70-100° C., a secondary quaternization reaction is carried out, and the reaction time is 6-24 h. After the reaction is completed, a filter cake is obtained by suction filtration, and finally the filter cake is washed with anhydrous ethanol for multiple times, and the phenolic chitosan diquaternary ammonium salt is obtained after drying; In step 2, the quaternizing agent A is selected from 2,3-epoxypropyl-trimethylammonium chloride, sodium 3-chloro-2-hydroxypropanesulfonate, 1,3-propane sultone, 1,4-propane sultone, cyanamide or dicyandiamide; In step 4 and step 5, the aprotic solvent is selected from N -Methylpyrrolidone, N , N - dimethylformamide, dimethyl sulfoxide or tetrahydrofuran; In step 4, the catalyst is an acid-binding agent for promoting an acylation reaction or an acid for promoting an esterification reaction; In step 5, the quaternizing agent B is selected from 3,4-dihydroxy-2'-chloroacetophenone, 4-(2-bromoethyl)benzene-1,2-diol, 4-(2-chloroethyl)benzene-1,2-diol, m-hydroxybenzyl chloride, 4-hydroxybenzyl chloride, o-hydroxybenzyl chloride, 2-(chloromethyl)-6-methylphenol or 3-(chloromethyl)-2-methylphenol.

[0006] In step 1, the mass ratio of chitosan to acetic acid aqueous solution is 1:50-100, or the mass ratio of chitosan to hydrochloric acid aqueous solution is 1:50-100.

[0007] In step 2, the mass concentration of the aqueous solution of the quaternizing agent A is 10% to 50%.

[0008] In step 3, the molar ratio of chitosan to the quaternary ammonium agent A in the chitosan aqueous solution is 1:1-10.

[0009] In step 4, the mass ratio of the chitosan quaternary ammonium salt to the aprotic solvent is 1: 8-25, and the molar ratio of the chitosan quaternary ammonium salt, nicotinoyl chloride, and the catalyst is 1: 1-5: 0.001-0.03.

[0010] In step 5, the mass ratio of the pyridyl chitosan quaternary ammonium salt to the aprotic solvent is 1: 8-25, and the molar ratio of the pyridyl chitosan quaternary ammonium salt to the quaternizing agent B is 1: 1-5.

[0011] In step 4, the acid binding agent is selected from N , N -dimethylformamide, N , N- diisopropylethylamine, pyridine, trimethylamine, isopropylamine or triethylamine, and the acid is sulfuric acid or p-toluenesulfonic acid.

[0012] In step 4, the nicotinoyl chloride is replaced by isonicotinoyl chloride, isonicotinic acid or nicotinic acid.

[0013] After adopting the above technical scheme, the preparation method of the antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt of the present invention first introduces a quaternary ammonium salt group on the amino group of chitosan to improve the antibacterial property and water solubility of chitosan, then further introduces a pyridine group in step 4 to provide a basis for introducing a phenolic group and a diquaternary ammonium salt, and finally introduces a quaternary ammonium salt for a second time through a quaternization reaction in step 5 to further improve the positive charge density of the pyridyl chitosan quaternary ammonium salt, which has a gain effect on improving the antibacterial activity of the pyridyl chitosan quaternary ammonium salt. In addition, the quaternizing agent B selected in step 5 also contains a phenolic group, so that the prepared chitosan derivative also has excellent antioxidant activity.

[0014] Therefore, the phenolic chitosan diquaternary ammonium salt synthesized by the preparation method of the present invention has good water solubility, high antibacterial and antimicrobial activities, and excellent antioxidant activity, and has broad application prospects in the fields of cosmetics, food packaging, and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the synthetic route of the preparation method of an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt in Example 1. DETAILED DESCRIPTION

[0016] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0017] 1. Preparation of phenolic chitosan diquaternary ammonium salt Example 1 A method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt, wherein the synthetic route is as follows: Figure 1 As shown, the following steps are included: Step 1, first dissolving 1.61 kg of chitosan in 150 kg of 2% by volume acetic acid aqueous solution to obtain a chitosan aqueous solution; Step 2, then dissolving 6.07 kg of 2,3-epoxypropyl-trimethylammonium chloride in 15 kg of deionized water to obtain an aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride; Step 3, then slowly adding the aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride to the chitosan aqueous solution, carrying out quaternization reaction at a constant temperature of 80°C, reflux stirring for 24 hours, and transferring to an ultrafiltration device after the reaction is completed, removing unreacted raw materials by ultrafiltration, and obtaining chitosan quaternary ammonium salt after concentration and freeze drying; Step 4: Then, 1.56 kg of the dried chitosan quaternary ammonium salt was crushed and dispersed in 20 kg N -methyl pyrrolidone, after fully swelling, lower the temperature of the reaction system to 0°C, slowly add 1.77 kg of nicotinyl chloride, and then add 10 mL N , N -dimethylformamide, react at 25 ° C for 24 h, after the reaction is completed, pour the reaction product into ice water, ultrafiltration, concentration, freeze drying to obtain pyridyl chitosan quaternary ammonium salt, in this step, chitosan quaternary ammonium salt, nicotinyl chloride, N , N - the molar ratio of dimethylformamide is 1: 2.5: 0.027; Step 5: Then, the dried 2.27 kg pyridyl chitosan quaternary ammonium salt was crushed and fully dispersed in 20 kg N -methyl pyrrolidone, after fully swelling, add 0.93 kg 3,4-dihydroxy-2'-chloroacetophenone (the molar ratio of pyridyl chitosan quaternary ammonium salt to 3,4-dihydroxy-2'-chloroacetophenone is 1:1), increase the temperature of the reaction system to 80°C, carry out secondary quaternization reaction, reflux and stir for 24 h, after the reaction is completed, filter and obtain filter cake, finally wash the filter cake with anhydrous ethanol for several times, and obtain phenolic chitosan diquaternary ammonium salt after drying, the structure of which is as follows:

[0018] Example 2 A method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt comprises the following steps: Step 1, first dissolving 1.61 kg of chitosan in 100 kg of 2% by volume acetic acid aqueous solution to obtain a chitosan aqueous solution; Step 2, then dissolving 1.22 kg of 1,3-propane sultone in 2.5 kg of deionized water to obtain an aqueous solution of 1,3-propane sultone; Step 3, then slowly adding the aqueous solution of 1,3-propane sultone into the chitosan aqueous solution, carrying out quaternization reaction at a constant temperature of 70° C., reflux stirring for 18 h, transferring to an ultrafiltration device after the reaction is completed, removing unreacted raw materials by ultrafiltration, and obtaining chitosan quaternary ammonium salt after concentration and freeze drying; Step 4: 2.82 kg of dried chitosan quaternary ammonium salt was crushed and dispersed in 50 kg of dimethyl sulfoxide. After sufficient swelling, the temperature of the reaction system was lowered to 0°C, 4.24 kg of nicotinyl chloride was slowly added, and then 10 mL of triethylamine was added. The reaction was carried out at 40°C for 12 h. After the reaction was completed, the reaction product was poured into ice water, and pyridyl chitosan quaternary ammonium salt was obtained after ultrafiltration, concentration, and freeze-drying. In this step, the molar ratio of chitosan quaternary ammonium salt, nicotinyl chloride, and triethylamine was 1:3:0.01; Step 5: Then, the dried 3.87 kg pyridyl chitosan quaternary ammonium salt was crushed and fully dispersed in 80 kg dimethyl sulfoxide. After fully swelling, 1.43 kg 4-hydroxybenzyl chloride was added (the molar ratio of pyridyl chitosan quaternary ammonium salt to 4-hydroxybenzyl chloride was 1:1), the temperature of the reaction system was raised to 90°C, a secondary quaternization reaction was carried out, and reflux stirring was performed for 12 h. After the reaction was completed, a filter cake was obtained by suction filtration. Finally, the filter cake was washed with anhydrous ethanol for several times, and phenolic chitosan diquaternary ammonium salt was obtained after drying. The structure is as follows:

[0019] Example 3 A method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt comprises the following steps: Step 1, first dissolving 1.61 kg of chitosan in 100 kg of a 2% by volume hydrochloric acid aqueous solution to obtain a chitosan aqueous solution; Step 2, then dissolving 2.52 kg of dicyandiamide in 20 kg of deionized water to obtain an aqueous solution of dicyandiamide; Step 3, then slowly adding the aqueous solution of dicyandiamide to the chitosan aqueous solution, carrying out quaternization reaction at a constant temperature of 95° C., reflux stirring for 12 h, transferring to an ultrafiltration device after the reaction is completed, removing unreacted raw materials by ultrafiltration, and obtaining chitosan quaternary ammonium salt after concentration and freeze drying; Step 4: Then, the dried 2.45 kg chitosan quaternary ammonium salt was crushed and dispersed in 20 kg N -methyl pyrrolidone, after fully swelling, lower the temperature of the reaction system to 0°C, slowly add 3.69 kg of isonicotinic acid, then add 2 mL of concentrated sulfuric acid, react at 95°C for 12 h, after the reaction is completed, pour the reaction product into ice water, ultrafilter, concentrate, and freeze-dry to obtain pyridyl chitosan quaternary ammonium salt; in this step, the molar ratio of chitosan quaternary ammonium salt, isonicotinic acid, and concentrated sulfuric acid is 1: 3: 0.003; Step 5: Then, 3.5 kg of the dried pyridyl chitosan quaternary ammonium salt was crushed and fully dispersed in 20 kg N-methyl pyrrolidone, after fully swelling, add 7.83 kg 2-(chloromethyl)-6-methylphenol (the molar ratio of pyridyl chitosan quaternary ammonium salt to 2-(chloromethyl)-6-methylphenol is 1:5), raise the temperature of the reaction system to 85°C, carry out secondary quaternization reaction, reflux and stir for 18 h, after the reaction is completed, filter to obtain a filter cake, finally wash the filter cake with anhydrous ethanol for several times, and after drying, obtain phenolic chitosan diquaternary ammonium salt, the structure of which is as follows:

[0020] 2. Antibacterial property test of phenolic chitosan diquaternary ammonium salt The antibacterial properties of chitosan, the chitosan quaternary ammonium salt obtained in Example 1, the pyridyl chitosan quaternary ammonium salt and the phenolic chitosan quaternary ammonium salt prepared in each example were tested by plate counting method: Specifically, chitosan, chitosan quaternary ammonium salt obtained in Example 1, pyridyl chitosan quaternary ammonium salt and phenolic chitosan quaternary ammonium salt prepared in each embodiment are first dissolved in deionized water to obtain a corresponding solution with a concentration of 1.0 mg / mL, which is sterilized for standby use. The test bacteria are activated and diluted to a certain concentration, and then 0.5 mL of the diluted bacterial solution is fully mixed with the sterilized chitosan and chitosan derivative solutions, at which time the concentration of chitosan or chitosan derivatives in each solution is 0.5 mg / mL, and then incubated in a 37°C constant temperature shaker for 10 min, and then an appropriate amount is evenly coated on LB agar medium, placed in a 37°C incubator and cultured for 18 to 24 h, deionized water is used as a blank control group, and the antibacterial rate of chitosan, chitosan quaternary ammonium salt, pyridyl chitosan quaternary ammonium salt and phenolic chitosan quaternary ammonium salt prepared in each embodiment is tested, and the antibacterial rate is calculated using the following formula, and the results are shown in Table 1.

[0021]

[0022] Table 1 Antibacterial rate of chitosan, chitosan quaternary ammonium salt, pyridyl chitosan quaternary ammonium salt, and phenolic chitosan quaternary ammonium salt against Escherichia coli and Staphylococcus aureus

[0023] The results in Table 1 show that the antibacterial rates of the unmodified chitosan sample against Escherichia coli and Staphylococcus aureus are 10.4% and 20.5%, respectively; the antibacterial rates of the quaternary ammonium salt of chitosan after quaternization against Escherichia coli and Staphylococcus aureus are 30.2% and 96.7%, respectively, indicating that the introduction of quaternary ammonium salt can improve the antibacterial properties of chitosan, and further introducing a pyridine group on the hydroxyl group of the quaternary ammonium salt of chitosan has a certain positive effect on improving the antibacterial properties of chitosan, but the improvement effect is not obvious. The antibacterial rates of the phenolic chitosan diquaternary ammonium salt prepared in Example 1 against Escherichia coli and Staphylococcus aureus are 85.1% and 100%, respectively, and the antibacterial rates of the phenolic chitosan diquaternary ammonium salt prepared in each example are significantly improved compared with the antibacterial rates of chitosan and chitosan quaternary ammonium salt, which is mainly attributed to the fact that the phenolic chitosan diquaternary ammonium salt contains two quaternary ammonium salt groups, which increases the positive charge density. In addition, the phenolic group also has a certain positive effect on improving the antibacterial properties of chitosan.

[0024] 3. Antioxidant performance test of phenolic chitosan diquaternary ammonium salt First, DPPH was dissolved in anhydrous ethanol to obtain a 180 μmol / L DPPH-ethanol solution; then chitosan, the chitosan derivatives prepared in each step of Example 1, and the phenolic chitosan quaternary ammonium salt obtained in each example were dissolved in deionized water to obtain a corresponding chitosan solution with a concentration of 1.0 mg / mL, and then 0.5 mL of DPPH-ethanol solution was mixed with 0.5 mL of chitosan solution, and after mixing evenly, the final concentration of the sample was 0.5 mg / mL. Finally, the mixture was incubated at room temperature in the dark for 20 min, and its absorption value at 520 nm was tested. The mixed solution of 0.5 mL of deionized water and 0.5 mL of DPPH-ethanol solution was used as the blank group, and the mixed solution of 0.5 mL of anhydrous ethanol and 0.5 mL of DPPH-ethanol solution was used as the control group. The DPPH free radical scavenging rate was calculated according to the following formula, and the results are shown in Table 2.

[0025]

[0026] Table 2 DPPH radical scavenging ability of chitosan, chitosan quaternary ammonium salt, pyridyl chitosan quaternary ammonium salt and phenolic chitosan diquaternary ammonium salt

[0027] The results in Table 2 show that chitosan, chitosan quaternary ammonium salt, and pyridyl chitosan quaternary ammonium salt have certain antioxidant properties, but their antioxidant properties are not obvious. The scavenging rates of 0.5 mg / mL chitosan, chitosan quaternary ammonium salt, and pyridyl chitosan quaternary ammonium salt on DPPH free radicals are 14.9%, 15.5%, and 19.3%, respectively. The introduction of pyridyl can improve the antioxidant properties of chitosan, but the performance is not obvious. Phenol has a strong antioxidant capacity. Therefore, the antioxidant properties of the prepared phenolic chitosan diquaternary ammonium salt are significantly improved compared with pyridyl chitosan quaternary ammonium salt. The scavenging rate of 0.5 mg / mL phenolic chitosan diquaternary ammonium salt on DPPH free radicals can reach about 90%.

[0028] The above results show that the antibacterial activity of the phenolic chitosan diquaternary ammonium salt prepared by the present invention is significantly improved compared with chitosan, and the antibacterial performance is also better than that of a single chitosan quaternary ammonium salt. Since the phenolic group has a strong antioxidant capacity, the introduction of the phenolic group significantly improves the antioxidant performance of the chitosan quaternary ammonium salt.

[0029] The phenolic chitosan diquaternary ammonium salt prepared by the invention has broad application prospects in the fields of cosmetics, food packaging, biomedicine and the like.

[0030] The above embodiments and drawings do not limit the product form and style of the present invention. Any appropriate changes or modifications made by ordinary technicians in the relevant technical field should be deemed to be within the patent scope of the present invention.

Claims

1. A method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt, characterized in that: The following steps are involved: Step 1, first dissolving chitosan in an acetic acid aqueous solution or a hydrochloric acid aqueous solution to obtain a chitosan aqueous solution; Step 2, then dissolving the quaternary ammonium agent A in deionized water to obtain an aqueous solution of the quaternary ammonium agent A; Step 3, then slowly adding the aqueous solution of quaternizing agent A to the chitosan aqueous solution, carrying out quaternization reaction at a constant temperature of 70-100° C. for 12-36 h, and ultrafiltration, concentration, and freeze-drying of the reaction product to obtain chitosan quaternary ammonium salt; Step 4, the dried chitosan quaternary ammonium salt is then crushed and fully dispersed in an aprotic solvent, the temperature of the reaction system is lowered to 0-5°C, nicotinyl chloride is slowly added, and then a catalyst is added, and the reaction is carried out at 20-100°C for 12-36 hours. After the reaction is completed, the reaction product is poured into ice water, and pyridyl chitosan quaternary ammonium salt is obtained after ultrafiltration, concentration, and freeze-drying; Step 5, then the dried pyridyl chitosan quaternary ammonium salt is crushed and fully dispersed in a non-protonic solvent, a quaternizing agent B is added, the temperature of the reaction system is increased to 70-100° C., a secondary quaternization reaction is carried out, and the reaction time is 6-24 h. After the reaction is completed, a filter cake is obtained by suction filtration, and finally the filter cake is washed with anhydrous ethanol for multiple times, and the phenolic chitosan diquaternary ammonium salt is obtained after drying; In step 2, the quaternizing agent A is selected from 2,3-epoxypropyl-trimethylammonium chloride, sodium 3-chloro-2-hydroxypropanesulfonate, 1,3-propane sultone, 1,4-propane sultone, cyanamide or dicyandiamide; In step 4 and step 5, the aprotic solvent is N -Methylpyrrolidone, N , N - dimethylformamide, dimethyl sulfoxide or tetrahydrofuran; In step 4, the catalyst is an acid-binding agent for promoting an acylation reaction or an acid for promoting an esterification reaction; In step 5, the quaternizing agent B is selected from 3,4-dihydroxy-2'-chloroacetophenone, 4-(2-bromoethyl)benzene-1,2-diol, 4-(2-chloroethyl)benzene-1,2-diol, m-hydroxybenzyl chloride, 4-hydroxybenzyl chloride, o-hydroxybenzyl chloride, 2-(chloromethyl)-6-methylphenol or 3-(chloromethyl)-2-methylphenol.

2. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 1, the mass ratio of chitosan to acetic acid aqueous solution is 1:50-100, or the mass ratio of chitosan to hydrochloric acid aqueous solution is 1:50-100.

3. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 2, the mass concentration of the aqueous solution of the quaternizing agent A is 10% to 50%.

4. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 3, the molar ratio of chitosan to the quaternary ammonium agent A in the chitosan aqueous solution is 1:1-10.

5. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 4, the mass ratio of the chitosan quaternary ammonium salt to the aprotic solvent is 1:8-25, and the molar ratio of the chitosan quaternary ammonium salt, nicotinoyl chloride, and the catalyst is 1:1-5:0.001-0.

03.

6. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 5, the mass ratio of the pyridyl chitosan quaternary ammonium salt to the aprotic solvent is 1: 8-25, and the molar ratio of the pyridyl chitosan quaternary ammonium salt to the quaternizing agent B is 1: 1-5.

7. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 4, the acid binding agent is selected from N , N -dimethylformamide, N , N - diisopropylethylamine, pyridine, trimethylamine, isopropylamine or triethylamine, and the acid is sulfuric acid or p-toluenesulfonic acid.

8. The method for preparing an antibacterial and antioxidant phenolic chitosan diquaternary ammonium salt according to claim 1, characterized in that: In step 4, the nicotinoyl chloride is replaced by isonicotinoyl chloride, isonicotinic acid or nicotinic acid.

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