CN112064145A - Preparation method of poly-gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber - Google Patents
Preparation method of poly-gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber Download PDFInfo
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Abstract
The invention belongs to the technical field of fiber material preparation and application, and particularly relates to a preparation method of poly-gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber. The method is realized by the following steps: firstly, dripping poly gamma-glutamic acid solution into chitosan spinning solution to prepare nano particles; then mixing poly-gamma-glutamic acid/chitosan and nano copper powder, adding water for dissolving, adding glutaraldehyde for crosslinking, uniformly mixing, standing for defoaming, and preparing a composite spinning solution; and (3) performing electrostatic spinning on the composite spinning solution, and performing vacuum drying to obtain the poly-gamma-glutamic acid/chitosan/nano-copper composite fiber. The composite fiber prepared by the invention overcomes the environmental and toxicity problems caused by inorganic copper through crosslinking and complexing, and the prepared composite fiber has a high-efficiency antibacterial function, so that the product does not need antibacterial after-finishing, the production cost is reduced, and the composite fiber is biodegradable and has no pollution to the environment.
Description
Technical Field
The invention relates to the technical field of fiber material preparation and application, in particular to a preparation method of poly-gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber.
Background
As a natural neutral polymer material, chitosan has the characteristics of inherent antibacterial property, wide sources, high yield and the like, and in recent decades, chitosan-based materials are the hot points of research in the antibacterial field, and have excellent biocompatibility, antibacterial property and capability of promoting oral healing, so that the chitosan-based materials become one of the most ideal antibacterial materials. However, chitosan is soluble and exerts antibacterial action only under acidic conditions, which greatly restricts the application of chitosan-based antibacterial materials under physiological conditions. To address this problem, two strategies are generally available. One is to chemically modify chitosan to enhance its antimicrobial properties, or to enhance its water solubility. The chitosan-based derivatives mainly comprise quaternized chitosan, succinylated chitosan, carboxyalkylated chitosan, sulfonated and sulfobenzoyl chitosan, amino acid grafted chitosan and the like. Another strategy is to combine chitosan with other nanoparticles with antibacterial properties by using the complexation of amino groups on chitosan.
Poly gamma-glutamic acid, called gamma-PGA for short, is a non-ribosomal polypeptide formed by linking L-and D-glutamic acid monomers through gamma-glutamyl bonds under the catalytic action of enzymes, and a molecular chain has a large number of side chain carboxyl groups which can form hydrogen bonds in the molecule or among the molecules. Has biodegradability, easy modification, water retention property, solubilization property and slow release property.
Copper is a natural metal element and is one of essential elements of human bodies, but the use of inorganic copper often causes environmental pollution and is also easy to cause poisoning, so that the application of copper is limited.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method of poly-gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber, the composite fiber converts inorganic copper into organic copper, and simultaneously improves the antibacterial and bactericidal effects of chitosan, and the compound obtained by crosslinking poly-gamma-glutamic acid and chitosan is easy to dissolve in water, has slow release performance, low toxicity and better antibacterial activity. And has good biocompatibility and dispersibility.
In order to achieve the purpose, the invention relates to the following specific technical scheme:
the invention provides a preparation method of poly-gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber, which comprises the following steps:
(1) dripping poly gamma-glutamic acid solution into the chitosan spinning solution to prepare nano particles;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method;
(3) mixing poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, adding water for dissolving, adding glutaraldehyde for crosslinking, uniformly mixing, standing for defoaming, and preparing a composite spinning solution;
(4) and (4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare the poly-gamma-glutamic acid/chitosan/nano-copper composite fiber.
The poly-gamma-glutamic acid used by the invention is obtained by fermenting and extracting bacillus licheniformis (bacillus licheniformis), and the fermentation and extraction method comprises the following steps:
weighing a basic culture medium, dissolving to obtain a basic culture medium solution, weighing each component of an optimized culture medium, adding into the basic culture medium solution, fixing the volume, and adjusting the pH value to 7.5 by solid NaOH;
b, subpackaging the culture medium into conical flasks (50 ml/flask), sterilizing at the temperature of 150 ℃ by 100-;
c, centrifuging the fermentation liquor, adding 2-7 times of ethanol into supernatant to precipitate for 10-24h, centrifuging, precipitating the obtained supernatant with 2-6 times of ethanol again, centrifuging, and drying the precipitate to constant weight to obtain a constant weight sample;
d, dissolving the constant weight sample in distilled water, and dialyzing and purifying to obtain a purified sample;
e, dissolving the purified sample in distilled water, and spray drying to obtain poly gamma-glutamic acid powder.
Further, in the step (1), the mass fraction of chitosan in the chitosan spinning solution is 3-8%, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 8 multiplied by 105～15×105g/mol, degree of deacetylation greater than 90%.
Further, in the step (1), the nanoparticle preparation method comprises: dissolving gamma-PGA in ultrapure water at room temperature and a stirring speed of 600 r/min, respectively dripping 10mL and 2 g/L gamma-PGA into 50mLCS acetic acid buffer solution at a speed of 6 mL/h by using a micro-injection pump, carrying out ultrasonic treatment at 598W for 10min, continuing stirring for 12 h, and dialyzing for 1-3h to remove unbound small molecular polymers.
In the preparation method provided by the invention, the specific preparation method of the nano-copper comprises the following steps: adding copper chloride into a CTAB aqueous solution of 150mL and 0.1 mol/L, wherein the concentration of the copper chloride is 0.01 mol/L, and adjusting the pH value of the solution to 10 by using ammonia water, and recording the solution as A solution; adding hydrazine hydrate into 150ml of 0.1 mol/L CTAB aqueous solution to respectively prepare solutions with the hydrazine hydrate concentrations of 0.8, 0.4, 0.2 and 0.1 mol/L, and recording the solution as a B solution; stirring at high speed at 30 ℃, dropwise adding the solution B into the solution A, continuing to react for 2.5 hours after dropwise adding to obtain a red brown nano-copper hydrosol, and then standing, precipitating, centrifuging, washing and drying the product.
Further, in the step (3), the composite spinning solution is prepared from the following raw materials in parts by weight: 5-15 parts of poly gamma-glutamic acid/chitosan, 3-5 parts of nano copper, 2 parts of glutaraldehyde and 78-90 parts of water.
Further, in the step (4), the electrostatic spinning parameters are as follows: the injection speed is 0.05-0.2 mm/min; the spinning temperature is 25-30 ℃; the receiving distance is 10-20 cm; the positive pressure is 8-25 kV; the negative pressure is-3 to-0.2 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
In the preparation process of poly-gamma-glutamic acid, in the step (a), the volume ratio of the optimized culture medium to the basal culture medium is 1: 4.
the basic culture medium used by the invention comprises the following raw materials: 10g/L of tryptone and 5 g/L, NaCl 10g/L of yeast extract; the optimized culture medium comprises the following raw materials: NaCl 10-15 g/L, alpha-ketoglutaric acid 1.0-2.5 g/L, Mn (II) 0.02-0.1 g/L, L-glutamine 0.2-1.0 g/L, and glycerin 5-15 g/L.
In the process of preparing poly-gamma-glutamic acid, the working parameters of spray drying are as follows: the maximum water evaporation capacity is 50kg/h, the inlet temperature is 200 ℃, the outlet temperature is 85 ℃, and the centrifugal spray head mechanically rotates at the rotating speed of 18000 r/min.
The invention has the beneficial effects that:
1. the poly-gamma-glutamic acid and chitosan used in the invention are colorless, nontoxic, tasteless and easily degradable microbial fermentation extracts, the carboxyl of gamma-PGA and the amino of CS have high coordination coefficient and stable structure, and the loading rate of nano-copper can be increased.
2. The chitosan and the nano-copper have a synergistic effect in combined antibiosis, and the finally prepared composite fiber has better antibacterial performance, antistatic performance and flame retardance and strong antibacterial durability.
3. The composite fiber prepared by the invention overcomes the environmental and toxicity problems caused by inorganic copper through crosslinking and complexing, and the prepared composite fiber has a high-efficiency antibacterial function, so that the product does not need antibacterial after-finishing, the production cost is reduced, and the composite fiber is biodegradable and has no pollution to the environment.
4. Compared with the antibacterial agent added into the fabric, the antibacterial fiber prepared by the invention has a lasting antibacterial effect, can ensure the natural style of the fabric, is good in comfort, omits the after-finishing process, and is simple to operate and convenient to use.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but it should not be construed that the scope of the present invention is limited to the following examples. Various substitutions and alterations can be made by those skilled in the art and by conventional means without departing from the spirit of the method of the invention described above.
Example 1
(1) The poly-gamma-glutamic acid solution was dropped into the chitosan spinning solution, and nanoparticles were prepared using electrostatic interaction between carboxyl groups of poly-gamma-glutamic acid (gamma-PGA) and amino groups of Chitosan (CS) in a ratio of the amounts of the carboxyl groups to the amino groups. The mass fraction of chitosan in the chitosan spinning solution is 3 percent, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 8 multiplied by 105g/mol, degree of deacetylation greater than 90%. Dropwise adding 10mL and 2 g/L of gamma-PGA into CS acetic acid buffer (50 mL, pH = 6) by using a micro-syringe pump at the rate of 6 mL/h under stirring at room temperature (600 r/min), carrying out ultrasonic treatment at 598W for 10min, continuing stirring for 12 h, and dialyzing for 3h to remove unbound small-molecule polymer;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method. A CTAB aqueous solution was prepared in a fixed amount of 0.1 mol/L, copper chloride was added to 150mL of the CTAB aqueous solution to make the copper chloride concentration 0.01 mol/L, and the pH of the solution was adjusted with ammonia water to finally obtain a blue solution with pH = 10, which was referred to as a solution a. Different amounts of hydrazine hydrate are added into 150ml of CTAB aqueous solution to prepare solutions with hydrazine hydrate concentration of 0.4 mol/L, which are recorded as B solutions. Dropwise adding the solution B into a three-neck flask containing the solution A at a certain speed under the conditions of high-speed stirring and a temperature of 30 ℃. After the end of the dropwise addition, the reaction was continued for 2.5 h. The reaction product is red brown nano copper hydrosol. Finally, carrying out post-treatment such as standing precipitation, centrifugation, washing, drying and the like on the product;
(3) dissolving poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, crosslinking through glutaraldehyde, uniformly mixing, standing and defoaming to prepare the composite spinning solution. In the composite spinning solution, 14 parts of poly-gamma-glutamic acid/chitosan, 5 parts of nano-copper, 2 parts of glutaraldehyde and 81 parts of water by weight;
(4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare poly-gamma-glutamic acid/chitosan/nano-copper composite fibers; the electrostatic spinning parameter injection speed is 0.2 mm/min; the spinning temperature is 28 ℃; the receiving distance is 15 cm; the positive voltage is 20 kV; the negative pressure is-2 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
Example 2
(1) The poly-gamma-glutamic acid solution was dropped into the chitosan spinning solution, and nanoparticles were prepared using electrostatic interaction between carboxyl groups of poly-gamma-glutamic acid (gamma-PGA) and amino groups of Chitosan (CS) in a ratio of the amounts of the carboxyl groups to the amino groups. The mass fraction of chitosan in the chitosan spinning solution is 4%, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 10 multiplied by 105g/mol, degree of deacetylation greater than 90%. 10mL and 2 g/L of gamma-PGA were added dropwise to CS acetate buffer (50 mL, pH = 5) at a rate of 6 mL/h with a micro syringe pump under stirring at room temperature (600 r/min), sonicated at 598W for 10min, stirred for 12 h, and dialyzed for 3h to remove unbound small molecule polymer.
(2) Copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method. A CTAB aqueous solution was prepared in a given amount of 0.1 mol/L, copper chloride was added to 150mL of the CTAB aqueous solution so that the copper chloride concentration was 0.01 mol/L, and the pH of the solution was adjusted with ammonia water to finally obtain a blue solution having a pH = 10, which was referred to as a solution a. Different amounts of hydrazine hydrate are added into 150ml of CTAB aqueous solution to prepare solutions with hydrazine hydrate concentration of 0.2 mol/L, which are recorded as B solutions. Dropwise adding the solution B into a three-neck flask containing the solution A at a certain speed under the conditions of high-speed stirring and a temperature of 30 ℃. After the end of the dropwise addition, the reaction was continued for 2.5 h. The reaction product is red brown nano copper hydrosol. Finally, carrying out post-treatment such as standing precipitation, centrifugation, washing, drying and the like on the product;
(3) dissolving poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, crosslinking through glutaraldehyde, uniformly mixing, standing and defoaming to prepare the composite spinning solution. In the composite spinning solution, 13 parts by weight of poly-gamma-glutamic acid/water-soluble chitosan, 3 parts by weight of nano-copper, 2 parts by weight of glutaraldehyde and 84 parts by weight of water are added;
(4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare poly-gamma-glutamic acid/chitosan/nano-copper composite fibers; the electrostatic spinning parameter injection speed is 0.05 mm/min; the spinning temperature is 25 ℃; the receiving distance is 5 cm; the positive pressure is 8 kV; the negative pressure is-0.5 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
Example 3
(1) The poly-gamma-glutamic acid solution was dropped into the chitosan spinning solution, and nanoparticles were prepared using electrostatic interaction between carboxyl groups of poly-gamma-glutamic acid (gamma-PGA) and amino groups of Chitosan (CS) in a ratio of the amounts of the carboxyl groups to the amino groups. The mass fraction of chitosan in the chitosan spinning solution is 5%, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 12 multiplied by 105g/mol, degree of deacetylation greater than 90%. Dropwise adding 10mL and 2 g/L of gamma-PGA into CS acetic acid buffer (50 mL, pH = 3) by using a micro-syringe pump at the rate of 6 mL/h under stirring at room temperature (600 r/min), carrying out ultrasonic treatment at 598W for 10min, continuing stirring for 12 h, and dialyzing for 3h to remove unbound small-molecule polymer;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method. A CTAB aqueous solution was prepared in a given amount of 0.1 mol/L, copper chloride was added to 150mL of the CTAB aqueous solution so that the copper chloride concentration was 0.01 mol/L, and the pH of the solution was adjusted with ammonia water to finally obtain a blue solution having a pH = 10, which was referred to as a solution a. To 150ml CTAB aqueous solution, different amounts of hydrazine hydrate were added to prepare solutions with hydrazine hydrate concentration of 0.1 mol/L, respectively, which are referred to as B solutions. Dropwise adding the solution B into a three-neck flask containing the solution A at a certain speed under the conditions of a temperature of 30 ℃ and high-speed stirring. After the end of the dropwise addition, the reaction was continued for 2.5 h. The reaction product is red brown nano copper hydrosol. Finally, carrying out post-treatment such as standing precipitation, centrifugation, washing, drying and the like on the product;
(3) dissolving poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, crosslinking through glutaraldehyde, uniformly mixing, standing and defoaming to prepare the composite spinning solution. In the composite spinning solution, 12 parts by weight of poly-gamma-glutamic acid/water-soluble chitosan, 10 parts by weight of nano-copper, 2 parts by weight of glutaraldehyde and 78 parts by weight of water are added;
(4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare poly-gamma-glutamic acid/chitosan/nano-copper composite fibers; the electrostatic spinning parameter injection speed is 0.3 mm/min; the spinning temperature is 35 ℃; the receiving distance is 25 cm; the positive voltage is 27 kV; the negative pressure is-5 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
Example 4
(1) The poly-gamma-glutamic acid solution was dropped into the chitosan spinning solution, and nanoparticles were prepared using electrostatic interaction between carboxyl groups of poly-gamma-glutamic acid (gamma-PGA) and amino groups of Chitosan (CS) in a ratio of the amounts of the carboxyl groups to the amino groups. The mass fraction of chitosan in the chitosan spinning solution is 6 percent, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 13 multiplied by 105 g/mol, degree of deacetylation greater than 90%. Dropwise adding 10mL and 2 g/L of gamma-PGA into CS acetic acid buffer (50 mL, pH = 2.5) by using a micro-syringe pump at the rate of 6 mL/h under stirring at room temperature (600 r/min), carrying out ultrasonic treatment at 598W for 10min, continuing stirring for 12 h, and dialyzing for 3h to remove unbound small-molecule polymer;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method. A CTAB aqueous solution was prepared in a given amount of 0.1 mol/L, copper chloride was added to 150mL of the CTAB aqueous solution so that the copper chloride concentration was 0.01 mol/L, and the pH of the solution was adjusted with ammonia water to finally obtain a blue solution having a pH = 10, which was referred to as a solution a. Different amounts of hydrazine hydrate are added into 150ml of CTAB aqueous solution to prepare solutions with the hydrazine hydrate concentration of 0.8 mol/L, which are recorded as B solutions. Dropwise adding the solution B into a three-neck flask containing the solution A at a certain speed under the conditions of a temperature of 30 ℃ and high-speed stirring. After the end of the dropwise addition, the reaction was continued for 2.5 h. The reaction product is red brown nano copper hydrosol. Finally, carrying out post-treatment such as standing precipitation, centrifugation, washing, drying and the like on the product;
(3) dissolving poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, crosslinking through glutaraldehyde, uniformly mixing, standing and defoaming to prepare the composite spinning solution. In the composite spinning solution, 14 parts by weight of poly-gamma-glutamic acid/water-soluble chitosan, 7 parts by weight of nano-copper, 2 parts by weight of glutaraldehyde and 79 parts by weight of water are added;
(4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare poly-gamma-glutamic acid/chitosan/nano-copper composite fibers; the electrostatic spinning parameter injection speed is 0.05 mm/min; the spinning temperature is 25 ℃; the receiving distance is 10 cm; the positive pressure is 8 kV; the negative pressure is 0.2 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
Example 5
(1) The poly-gamma-glutamic acid solution was dropped into the chitosan spinning solution, and nanoparticles were prepared using electrostatic interaction between carboxyl groups of poly-gamma-glutamic acid (gamma-PGA) and amino groups of Chitosan (CS) in a ratio of the amounts of the carboxyl groups to the amino groups. The mass fraction of chitosan in the chitosan spinning solution is 7 percent, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 14 multiplied by 105g/mol, degree of deacetylation greater than 90%. Dropwise adding 10mL and 2 g/L of gamma-PGA into CS acetic acid buffer (50 mL, pH = 2.5) by using a micro-syringe pump at the rate of 6 mL/h under stirring at room temperature (600 r/min), carrying out ultrasonic treatment at 598W for 10min, continuing stirring for 12 h, and dialyzing for 3h to remove unbound small-molecule polymer;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method. A CTAB aqueous solution was prepared in a given amount of 0.1 mol/L, copper chloride was added to 150mL of the CTAB aqueous solution so that the copper chloride concentration was 0.01 mol/L, and the pH of the solution was adjusted with ammonia water to finally obtain a blue solution having a pH = 10, which was referred to as a solution a. Different amounts of hydrazine hydrate are added into 150ml of CTAB aqueous solution to prepare solutions with the hydrazine hydrate concentration of 0.8 mol/L, which are recorded as B solutions. Dropwise adding the solution B into a three-neck flask containing the solution A at a certain speed under the conditions of a temperature of 30 ℃ and high-speed stirring. After the end of the dropwise addition, the reaction was continued for 2.5 h. The reaction product is red brown nano copper hydrosol. Finally, carrying out post-treatment such as standing precipitation, centrifugation, washing, drying and the like on the product;
(3) dissolving poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, crosslinking through glutaraldehyde, uniformly mixing, standing and defoaming to prepare the composite spinning solution. In the composite spinning solution, 14 parts by weight of poly-gamma-glutamic acid/water-soluble chitosan, 2 parts by weight of nano-copper, 2 parts by weight of glutaraldehyde and 90 parts by weight of water are added;
(4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare poly-gamma-glutamic acid/chitosan/nano-copper composite fibers; the electrostatic spinning parameter injection speed is 0.1 mm/min; the spinning temperature is 28 ℃; the receiving distance is 17 cm; the positive voltage is 10 kV; the negative pressure is-1 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
Example 6
(1) The poly-gamma-glutamic acid solution was dropped into the chitosan spinning solution, and nanoparticles were prepared using electrostatic interaction between carboxyl groups of poly-gamma-glutamic acid (gamma-PGA) and amino groups of Chitosan (CS) in a ratio of the amounts of the carboxyl groups to the amino groups. The mass fraction of chitosan in the chitosan spinning solution is 8 percent, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 15 multiplied by 105g/mol, degree of deacetylation greater than 90%. Dropwise adding 10mL and 2 g/L of gamma-PGA into CS acetic acid buffer (50 mL, pH = 2.5) by using a micro-syringe pump at the rate of 6 mL/h under stirring at room temperature (600 r/min), carrying out ultrasonic treatment at 598W for 10min, continuing stirring for 12 h, and dialyzing for 3h to remove unbound small-molecule polymer;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method. A CTAB aqueous solution was prepared in a given amount of 0.1 mol/L, copper chloride was added to 150mL of the CTAB aqueous solution so that the copper chloride concentration was 0.01 mol/L, and the pH of the solution was adjusted with ammonia water to finally obtain a blue solution having a pH = 10, which was referred to as a solution a. Different amounts of hydrazine hydrate are added into 150ml of CTAB aqueous solution to prepare solutions with the hydrazine hydrate concentration of 0.8 mol/L, which are recorded as B solutions. Dropwise adding the solution B into a three-neck flask containing the solution A at a certain speed under the conditions of a temperature of 30 ℃ and high-speed stirring. After the end of the dropwise addition, the reaction was continued for 2.5 h. The reaction product is red brown nano copper hydrosol. Finally, carrying out post-treatment such as standing precipitation, centrifugation, washing, drying and the like on the product;
(3) dissolving poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, crosslinking through glutaraldehyde, uniformly mixing, standing and defoaming to prepare the composite spinning solution. In the composite spinning solution, 14 parts by weight of poly-gamma-glutamic acid/water-soluble chitosan, 4 parts by weight of nano-copper, 2 parts by weight of glutaraldehyde and 82 parts by weight of water are added;
(4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare poly-gamma-glutamic acid/chitosan/nano-copper composite fibers; the electrostatic spinning parameter injection speed is 0.15 mm/min; the spinning temperature is 27 ℃; the receiving distance is 16 cm; the positive voltage is 14 kV; the negative pressure is-1.5 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
Comparative example 1
Composite fibers were prepared using only poly gamma-glutamic acid/chitosan, and other preparation conditions were identical to those of example 6.
In this comparative example, the diameter of the prepared fiber was enlarged to 98nm and the surface area was reduced, and the continuity of the prepared fiber was inferior to that of the example.
Comparative example 2
The composite fiber was prepared using only chitosan/nano-copper, and other preparation conditions were the same as in example 6.
In this comparative example, the diameter of the prepared fiber was enlarged to 93nm and the surface area was reduced, and the continuity of the prepared fiber was inferior to that of the example.
And (3) performance testing:
(1) 4 groups of nano-copper powder with different particle sizes are prepared by changing the concentration of the reducing agent, and the characterization finds that the particle size of the nano-copper powder is increased along with the reduction of the concentration of hydrazine hydrate. The minimum inhibitory concentration of the nano-copper is tested by a broth dilution shaking culture method. The data obtained are shown in Table 1.
Table 1: detection of minimum inhibitory concentration of nano-copper with different particle sizes by using escherichia coli as test strain
(2) γ -PGA contains abundant free carboxyl groups, CS contains a large number of free amino groups, and strong electrostatic attraction exists between the amino groups and the carboxyl groups to combine γ -PGA and CS together to form particles of different sizes, specific particle size, Potential and Dispersion Index (PDI). When the ratio of the amount of the carboxyl group to the amino group is 1: 2 and 2: 1, respectively, the particle size of the nanoparticles is kept around 200 nm, the dispersion index is small, and the dispersibility is good. The reason for this is that the amino group and the carboxyl group stabilize their internal structures by electrostatic attraction, and the excessive amino group or carboxyl group is distributed outside the nanoparticles to repel each other, resulting in good dispersibility. When the mole fraction of amino groups in the nanoparticle is higher than the corresponding value of carboxyl groups, CS is coated on the surface of γ -PGA, so that the surface of the nanoparticle is positively charged, while the excessive amount of carboxyl groups causes the surface of the nanoparticle to be negatively charged.
(3) Escherichia coli and staphylococcus aureus are used as test strains, an antibacterial ability test is carried out according to ISO 22196-2007 and QB/T2591-2003 standards, an Oxford cup method is used for detecting the composite fiber prepared by the method, the size of a 24-hour antibacterial ring of the test fiber is detected, the total number of colonies before and after the test is determined according to GB 4789.2-2010 standard, the colony culture condition is 37 +/-1 ℃, the relative humidity is more than 90%, the obtained test data is statistically processed by SPSS13.0 software, and P < 0.05 shows that the size of the 24-hour antibacterial ring is obtained.
The data obtained are shown in Table 2.
Table 2: bacteriostatic effect (bacteriostasis circle diameter)
(4) Cutting a 2cm dried fiber sample, weighing the fiber sample to obtain a mass Md, immersing the fiber sample in deionized water for 24h to fully swell the fiber sample, quickly absorbing the surface moisture of the fiber sample by using filter paper, weighing the sample to obtain a mass of Mw., measuring for 3 times in parallel, and taking an average value. The degree of swelling (Sd) can be calculated according to the formula:
in the formula: md is the dry weight (g) of the sample and Mw is the wet weight (g) of the sample.
The result shows that the swelling degree of the antibacterial fiber is greatly increased along with the introduction of the poly-gamma-glutamic acid, so that the stability of the antibacterial fiber is greatly improved, and an important basis is provided for the application and popularization of the antibacterial fiber.
Table 3: measurement result of swelling degree
As can be seen from the above embodiments, the technical solution provided by the present invention has the following advantages:
(1) the poly-gamma-glutamic acid/chitosan/nano-copper composite fiber prepared by the invention has obvious bacteriostatic effect.
(2) The poly-gamma-glutamic acid/chitosan/nano-copper composite fiber prepared by the invention has simple preparation method and lower cost of raw materials.
(3) The poly-gamma-glutamic acid/chitosan/nano-copper prepared by the invention can be applied to a plurality of fields.
Claims (8)
1. A preparation method of poly gamma-glutamic acid/chitosan/nano-copper antibacterial composite fiber is characterized by comprising the following steps:
(1) dripping poly gamma-glutamic acid solution into the chitosan spinning solution to prepare nano particles;
(2) copper chloride is used as a copper source, hydrazine hydrate is used as a reducing agent, Cetyl Trimethyl Ammonium Bromide (CTAB) is used as a stabilizing agent, ammonia water is used as a complexing agent, and the nano copper powder is synthesized by a liquid phase reduction method;
(3) mixing poly-gamma-glutamic acid/chitosan and nano-copper powder according to a certain mass ratio, adding water for dissolving, adding glutaraldehyde for crosslinking, uniformly mixing, standing for defoaming, and preparing a composite spinning solution;
(4) and (4) performing electrostatic spinning on the composite spinning solution prepared in the step (3), and performing vacuum drying to prepare the poly-gamma-glutamic acid/chitosan/nano-copper composite fiber.
2. The method according to claim 1, wherein the poly-gamma-glutamic acid is obtained by fermentation and extraction of Bacillus licheniformis (Bacillus licheniformis), and the fermentation and extraction method comprises the following steps:
weighing a basic culture medium, dissolving to obtain a basic culture medium solution, weighing each component of an optimized culture medium, adding into the basic culture medium solution, fixing the volume, and adjusting the pH value to 7.5 by solid NaOH;
b, subpackaging the culture medium into conical flasks (50 ml/flask), sterilizing at the temperature of 150 ℃ by 100-;
c, centrifuging the fermentation liquor, adding 2-7 times of ethanol into supernatant to precipitate for 10-24h, centrifuging, precipitating the obtained supernatant with 2-6 times of ethanol again, centrifuging, and drying the precipitate to constant weight to obtain a constant weight sample;
d, dissolving the constant weight sample in distilled water, and dialyzing and purifying to obtain a purified sample;
e, dissolving the purified sample in distilled water, and spray drying to obtain poly gamma-glutamic acid powder.
3. The preparation method according to claim 1, characterized in that in the step (1), the mass fraction of chitosan in the chitosan spinning solution is 3-8%, and the solvent is acetic acid solution; the weight average molecular weight of the chitosan is 8 multiplied by 105～15×105g/mol, degree of deacetylation greater than 90%.
4. The method according to claim 1 or 3, wherein in the step (1), the nanoparticle is prepared by: dissolving gamma-PGA with ultrapure water at the room temperature and the stirring speed of 600 r/min, respectively dripping 10mL of gamma-PGA of 2 g/L into 50mLCS acetic acid buffer solution by using a micro-injection pump according to the speed of 6 mL/h, carrying out 598W ultrasonic treatment for 10min, continuing stirring for 12 h, and dialyzing for 1-3h to remove the unbound small molecular polymer.
5. The preparation method according to claim 1, wherein in the step (2), the specific preparation method of the nano-copper is as follows: adding copper chloride into a CTAB aqueous solution of 150mL and 0.1 mol/L, wherein the concentration of the copper chloride is 0.01 mol/L, and adjusting the pH value of the solution to 10 by using ammonia water, and recording the solution as A solution; adding hydrazine hydrate into 150ml of 0.1 mol/L CTAB aqueous solution to respectively prepare solutions with the hydrazine hydrate concentrations of 0.8, 0.4, 0.2 and 0.1 mol/L, and recording the solution as a B solution; stirring at high speed at 30 ℃, dropwise adding the solution B into the solution A, continuing to react for 2.5 hours after dropwise adding to obtain a red brown nano-copper hydrosol, and then standing, precipitating, centrifuging, washing and drying the product.
6. The preparation method according to any one of claims 1 to 5, wherein in the step (3), the composite spinning solution is composed of the following raw materials in parts by weight: 5-15 parts of poly gamma-glutamic acid/chitosan, 3-5 parts of nano copper, 2 parts of glutaraldehyde and 78-90 parts of water.
7. The production method according to any one of claims 1 to 6, wherein in the step (4), the electrostatic spinning parameters: the injection speed is 0.05-0.2 mm/min; the spinning temperature is 25-30 ℃; the receiving distance is 10-20 cm; the positive pressure is 8-25 kV; the negative pressure is-3 to-0.2 kV; the diameter of the finally prepared nano fiber is between 60 and 70 nm.
8. The method according to claim 2, wherein in the step (a), the volume ratio of the optimized culture medium to the basal culture medium is 1: 4.
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