CN100360036C - Preparation method of chitosan/metal copper composite antibacterial agent - Google Patents

Abstract

The invention discloses a method for preparing a chitosan/metal copper composite antibacterial agent. In the method, after the raw material chitosan is purified, it coordinates with the transition metal ion copper, precipitates with acetone-ethanol mixed solvent, washes and dries to obtain the chitosan/metallic antibacterial agent. Metal copper composite antimicrobial agent. The organic/inorganic composite antibacterial agent prepared by this method has excellent broad-spectrum antibacterial properties, and the antibacterial activity against various pathogenic bacteria is greatly improved. The novel high-efficiency antibacterial agent can be used in the fields of food, textile industry, antibacterial of daily necessities, wood preservation and the like, and the preparation method is simple, easy, pollution-free and has broad application prospects.

Description

Preparation method of chitosan/metal copper composite antibacterial agent

technical field

The invention relates to a method for preparing a novel antibacterial agent by using a metal copper complex of chitosan, which is a natural renewable resource, and belongs to the field of chemical biology of renewable resources.

Background technique

Chitin is a natural polysaccharide extracted from the shells of crustaceans such as shrimps and crabs and the cell walls of fungi. It is also the second largest biomass resource in nature after cellulose. Chitosan is a deacetylated derivative of chitin, which has good biocompatibility, biodegradability, natural non-toxicity and various biological activities, especially spectrum antibacterial properties, and because there are many amino groups in its molecular chain , Hydroxyl and other active groups have high reactivity and are very easy to undergo complexation reactions with transition metal ions. In recent years, chitosan has received widespread attention as a natural antibacterial agent. However, compared with traditional antibacterial agents, chitosan's antibacterial activity is low, and it is not enough to be widely used in food, medicine, and textile industries. . Therefore, improving the antibacterial activity of chitosan through modification, blending and other methods is very important for the application of chitosan as an antibacterial agent. At present, the ways to improve the antibacterial activity of chitosan mainly focus on the modification of chitosan such as derivatization, including carboxymethylation, sulfation, and quaternization of chitosan. Although the antibacterial activity of chitosan can be improved to a certain extent through the derivatization treatment of chitosan, the effect is not obvious, and it still cannot meet its application requirements, and the derivatization process of chitosan is complicated, and the reaction conditions are harsh conducive to large-scale production. Metal ion is a traditional antibacterial agent with strong antibacterial activity, but its biological toxicity cannot meet the requirements of the current development of green non-toxic antibacterial agents.

Contents of the invention

Aiming at the problems existing in the above-mentioned prior art, the present invention provides a kind of method for preparing chitosan/metal copper composite antibacterial agent, and this method adopts the coordination reaction of raw material chitosan and transition metal ion copper to prepare novel high-efficiency broad-spectrum Antibacterial agents. The new chitosan/metal copper composite antibacterial agent prepared by this method has significantly enhanced antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has antibacterial activity against Staphylococcus aureus and Escherichia coli. It is 8 times that of chitosan alone, and its antibacterial activity against Staphylococcus epidermidis and Pseudomonas aeruginosa is 4 times that of chitosan alone.

The technical scheme provided by the invention is to prepare chitosan/metal copper composite antibacterial agent according to the following steps:

(1) Dissolve an appropriate amount of chitosan in a dilute acetic acid solution with a concentration of 0.005-0.03g/ml to form a chitosan dilute acetic acid solution with a concentration of 0.005-0.02g/ml, and then add chitosan under magnetic stirring. Divalent copper ions with an amino molar ratio of 0.25:1-4:1, then adjust the pH value of the solution to 4.5-6.0 with a sodium hydroxide solution with a concentration of 0.05-1mol/L, and continue stirring for 3-4 hours to obtain a shell Glycan Metal Copper Complex Solution

(2) The obtained chitosan metal copper complex solution is added in a sufficient amount of acetone and ethanol mixed solvent to obtain a blue precipitate, then separate the precipitate and the solution system by decompression suction filtration, and the obtained chitosan The metal copper complex precipitate is washed with a sufficient amount of absolute ethanol, and the detergent is removed by vacuum filtration again, and washed 2-3 times according to the above method, and the obtained chitosan metal copper complex precipitate is vacuum-dried, ground and ready to use. A chitosan/copper complex powder is obtained.

(3) The obtained chitosan/copper complex powder is dissolved in 0.05-2mol/l dilute hydrochloric acid to make a solution with a concentration of 1000 μg/ml-31.3 μg/ml, and the sodium hydroxide with a concentration of 0.05-1mol/l Solution Adjust the pH value of the solution to 4.5-6.0, and finally sterilize the solution in an autoclave at 121-135° C. for 20-40 minutes to obtain the chitosan/copper composite antibacterial agent.

Wherein, the chitosan used is preferably purified by conventional methods, and the molecular weight of chitosan is in the range of 26,000-788,000, and the degree of deacetylation is in the range of 85%-95%; The range is 4.6-6.0; the divalent copper ion comes from copper sulfate pentahydrate, and the volume ratio of acetone:ethanol in the mixed solvent of acetone and ethanol is 3:1-1:3.

Since the chitosan/metal copper composite antibacterial agent is the product of the coordination reaction between chitosan and metal ions with antibacterial activity, it combines the antibacterial properties of chitosan and metal copper, showing much stronger antibacterial properties than chitosan. At the same time, the toxicity of metal copper antibacterial agents is greatly reduced, and the source of raw materials is abundant, the preparation method is simple, and it is easy to produce on a large scale, so it can be used as a new type of antibacterial bactericide, additive, antibacterial drug, etc. Wood preservation and other fields have broad application prospects.

Detailed ways

The method for preparing the chitosan/metal copper composite antibacterial agent provided by the invention will be described in detail below through specific examples.

Example 1: Weigh 1g of purified chitosan with a molecular weight of 388,000 and a degree of deacetylation of 95%, add 100ml of chitosan with a concentration of 1g/ml in dilute acetic acid solution, stir for 1-2h until dissolved, and add 1.55 gCuSO ₄ ·5H ₂ O powder, adjust the pH value of the solution to 6.0 with 0.5mol/l NaOH solution, and keep stirring for 3-4h until coordination equilibrium. Stirring was stopped, and 200 ml of acetone:ethanol mixed solvent with a volume ratio of 1:1 was added to the solution to obtain a blue mass precipitate. The precipitate was separated by suction filtration under reduced pressure, the precipitate was washed with 100 ml of absolute ethanol, and the detergent and unreacted ions eluted were removed by suction filtration under reduced pressure again. Repeat washing twice with 100ml of deionized water, filter the precipitate, and dry in vacuum to obtain the chitosan/copper complex. Weigh 0.1g chitosan/copper complex solubilizer 10ml concentration is 0.3g/ml dilute hydrochloric acid solution, place in an autoclave, sterilize at 121°C for 25 minutes to obtain chitosan/copper composite antibacterial agent , can be directly used in antibacterial experiments.

Example 2: Take 1g of purified chitosan with a molecular weight of 184,000 and a degree of deacetylation of 93%, add 100ml of chitosan with a concentration of 0.02g/ml to dilute acetic acid solution, stir for 1-2h until dissolved, and add under magnetic stirring 1.55g CuSO ₄ ·5H ₂ O powder, adjust the pH value to 4.5 with 0.05mol/l NaOH solution, and keep stirring for 3-4h until coordination equilibrium. Stirring was stopped, and 200 ml of acetone:ethanol mixed solution with a volume ratio of 1:1 was added to the solution to obtain a blue mass precipitate. The precipitate was separated by suction filtration under reduced pressure, the precipitate was washed with 100 ml of absolute ethanol, and the detergent and unreacted ions eluted were removed by suction filtration under reduced pressure again. Repeat washing twice with 100ml of deionized water, filter the precipitate, and dry in vacuum to obtain the chitosan/copper complex. Weigh 0.1g of chitosan/copper complex and dissolve it in 10ml of dilute hydrochloric acid solution with a concentration of 0.3g/ml, place it in an autoclave, and sterilize it at 130°C for 25 minutes to obtain the chitosan/copper composite antibacterial agent , can be directly used in antibacterial experiments.

Embodiment 3: Take by weighing 1g purified chitosan samples with a molecular weight of 26,000 and a degree of deacetylation of 93%, add 100ml of a concentration of 1g/ml dilute acetic acid solution, stir for 1-2h to dissolve, and add under magnetic stirring 1.55g CuSO ₄ ·5H ₂ O powder, adjust the pH value to 6.0 with 1mol/l NaOH solution, and keep stirring for 3-4h until coordination equilibrium. Stirring was stopped, and 200 ml of acetone:ethanol mixed solution with a volume ratio of 1:1 was added to the solution to obtain a blue mass precipitate. The precipitate was separated by suction filtration under reduced pressure, the precipitate was washed with 100 ml of absolute ethanol, and the detergent and unreacted ions eluted were removed by suction filtration under reduced pressure again. Repeat washing twice with 100ml of deionized water, filter the precipitate, and dry in vacuum to obtain the chitosan/copper complex. Weigh 0.1g of chitosan/copper complex and dissolve it in 10ml of dilute hydrochloric acid solution with a concentration of 0.3g/ml, place it in an autoclave, and sterilize it at 121°C for 25 minutes to obtain chitosan/copper composite antibacterial can be directly used in antibacterial experiments.

The following table is the component analysis of the chitosan/metal copper composite antibacterial agent that embodiment 1 makes.

Molecular formula C content H content N content metal content Cu(C₆H₆O₄N)₁₇·SO₄· 4H₂O 23.93(23.8) 5.47(5.2) 4.49 (4.6) 12.32(12.6)

The following table is the test result of comparing the minimum inhibitory concentration (unit: μg/ml) of the chitosan/metal copper composite antibacterial agent prepared in Examples 1, 2, and 3 with chitosan respectively.

Microorganisms Comparative example Instance 1 Example 2 Example 3 Staphylococcus aureus Staphylococcus epidermidis Escherichia coli Pseudomonas aeruginosa Candida albicans Yeast   2505005001000＞1000＞1000  31.362.5125125＞1000＞1000  31.362.5125500＞1000＞1000 62.5125500＞1000＞1000500

The test results show that the chitosan/metal copper composite antibacterial agent is significantly better than chitosan in the antibacterial properties of gram-positive bacteria, gram-negative bacteria and fungi.

Claims (6)

1, the preparation method of chitosan/metal copper composite antibacterial agent is characterized in that preparation steps are as follows: (1) Dissolve an appropriate amount of chitosan in a dilute acetic acid solution with a concentration of 0.005-0.03g/ml to form a chitosan dilute acetic acid solution with a concentration of 0.005-0.02g/ml, and then add chitosan under magnetic stirring. Divalent copper ions with amino molar ratio of 4:1-0.25:1, then adjust the pH value of the solution to 4.5-6.0 with a sodium hydroxide solution with a concentration of 0.05-1mol/l, and continue stirring for 3-4 hours to obtain chitosan Sugar metal copper complex solution; (2) The obtained chitosan metal copper complex solution is added in a sufficient amount of acetone and ethanol mixed solvent to obtain a blue precipitate, and then the precipitate and solution system are separated by vacuum filtration, and the obtained chitosan The sugar metal copper complex precipitate was washed with a sufficient amount of absolute ethanol, and the detergent was removed by vacuum filtration again, and washed 2-3 times according to the above method, and the obtained chitosan metal copper complex precipitate was vacuum-dried and ground Promptly obtain chitosan/copper complex powder; (3) Dissolve the obtained chitosan/copper complex powder in 0.05-2mol/l dilute hydrochloric acid to make a solution with a concentration of 1000μg/ml-31.3μg/ml, and oxidize it with a concentration of 0.05-1mol/l The sodium solution adjusts the pH value of the solution to 4.5-6.0, and finally sterilizes the solution in an autoclave at 121-135° C. for 20-40 minutes to obtain the chitosan/copper composite antibacterial agent. 2. The preparation method of chitosan/metal copper composite antibacterial agent according to claim 1, characterized in that: the chitosan used is chitosan purified by conventional methods. 3. The preparation method of chitosan/metal copper composite antibacterial agent according to claim 1, characterized in that: the chitosan used has a molecular weight ranging from 26,000 to 788,000. 4. The preparation method of chitosan/metal copper composite antibacterial agent according to claim 1, characterized in that the deacetylation degree of the chitosan used is in the range of 85%-95%. 5. The preparation method of chitosan/metal copper composite antibacterial agent according to claim 1, characterized in that: divalent copper ions come from hydrated copper sulfate, copper chloride, copper nitrate or copper acetate. 6. The preparation method of chitosan/metal copper composite antibacterial agent according to claim 1, characterized in that: the volume ratio of acetone:ethanol in the mixed solvent of acetone and ethanol is 3:1-1:3.