CN101962812B - Method for preparing antibacterial nanofibre composite membrane by utilizing electrostatic spinning and application thereof - Google Patents
Method for preparing antibacterial nanofibre composite membrane by utilizing electrostatic spinning and application thereof Download PDFInfo
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- CN101962812B CN101962812B CN2010102863110A CN201010286311A CN101962812B CN 101962812 B CN101962812 B CN 101962812B CN 2010102863110 A CN2010102863110 A CN 2010102863110A CN 201010286311 A CN201010286311 A CN 201010286311A CN 101962812 B CN101962812 B CN 101962812B
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Abstract
The invention belongs to the fields of nano material preparation technique and biological material, in particular to a method for preparing an antibacterial EGCG (Epigallocatechin Gallate)_Cu(II)/PVA (Polyvinyl Alcohol) nanofibre composite membrane by utilizing electrostatic spinning and an application thereof. The method of the invention comprises the following steps of weighing copper sulfate and EGCG according to a ratio; dissolving in distilled water; regulating the pH value to prepare an EGCG_Cu (II) complex; mixing the EGCG_Cu (II) complex and PVA according to a ratio; and generating the EGCG_Cu (II) /PVA nanofibre composite membrane by utilizing an electrostatic spinning technique. The invention has the advantage that the sterilizing rate on escherichia coli can reach 99%. The nanofibre composite membrane has the advantages of good toughness and biocompatibility, and wide application prospects in the field of the biological materials.
Description
Technical field
The invention belongs to nano material preparation technology and technical field of biological material, be specifically related to method and application thereof that a kind of electrostatic spinning prepares antibiotic EGCG_Cu (II)/PVA nano-fiber composite film.
Background technology
Catechin is the main component that is rich in polyhydric phenolic substance-Tea Polyphenols in the tealeaves, the EGCG(Epigallo-catechin gallate (EGCG)) be the maximum catechin of content in the tealeaves, have antibacterial and bacteriostasis.EGCG makes it when meeting with metal ion owing to have the special construction of a plurality of phenolic hydroxyl groups and have stronger soda acid buffer capacity, very easily with metal ion absorption and complexing takes place, and forms complex compound.People such as Japan scholar Kimura find that the bactericidal effect of Tea Polyphenols obviously increases in the presence of copper ion.
Electrostatic spinning is a kind of nano material preparation technology with broad prospect of application, and its equipment price is cheap, preparation technology is simple, can prepare the nanofiber silk with very big volumetric surface area, therefore is subjected to paying close attention to widely.There has been report to utilize electrostatic spinning technique, up to the present do not appeared in the newspapers about the blending of component in polymer and the tealeaves prepares nano fibrous membrane with polymer and metallic particles, protein and some inorganic material blending.
Above-mentioned research is not furtherd investigate active ingredient in the Tea Polyphenols and complex reaction, and the complex compound of catechin and copper ion formation simultaneously is unstable in solution, redox reaction easily takes place, thereby influence its application at biomedical sector.
Summary of the invention
The object of the invention is to provide a kind of electrostatic spinning to prepare the method and the application thereof of antibiotic EGCG_Cu (II)/PVA nano-fiber composite film.
The electrostatic spinning that the present invention proposes prepares the method for antibiotic EGCG_Cu (II)/PVA nano-fiber composite film, and concrete steps are as follows:
(1) takes by weighing copper sulphate and EGCG respectively, use dissolved in distilled water, under abundant stirring condition, copper-bath is added drop-wise in the EGCG solution control EGCG and Cu
2Mol ratio be 1:1-1:4, under 22-30 ℃ of temperature, be 5.0-8.0 with the pH value of alkaline solution regulator solution;
(2) take by weighing the PVA(polyvinyl alcohol) be dissolved in the distilled water, add step (1) gained solution, control PVA concentration is 5-12%(w/v, g/ml), the concentration of EGCG is 0.05 * 10
-3-4 * 10
-3Mol/l, CuSO
4The concentration of solution is 0.1 * 10
-3-10 * 10
-3Mol/l; The room temperature lower magnetic force stirs 1-4h, obtains spinning solution;
(3) step (2) gained spinning solution is sucked the 20ml syringe, load onto diameter 0.6-1.2mm syringe needle, the adjustment syringe needle is 5-20cm to the distance of receiver, regulation voltage is at 10-20kv, the spinning solution flow velocity is controlled at 0.5-6ml/h, spinning 2h, 78-82 ℃ of following vacuumize 2-8h promptly obtains required product.
Among the present invention, described alkaline solution is 10%Na
2CO
3Or 1mM NaOH.
The electrostatic spinning that the present invention proposes prepares the application of antibiotic EGCG_Cu (II)/PVA nano-fiber composite film as the bactericidal material, and concrete grammar is:
(1) yeast culture, centrifugal and collection somatic cells are used the phosphate buffer re-suspended cell, again nano-fiber composite film are put into reactant liquor, and 28-37 ℃, shaking speed 100-250 r/min, reaction 2-8h;
(2) centrifugal collection thalline is observed.
Concrete, used Escherichia coli among the present invention
Esehedchia coliBe DH5 α.
Concrete, described method is as follows:
(1) slant culture: get routine and be applicable to
E.coliSlant medium, the inoculation, 37 ℃ of slant culture 6-12h are as the inclined-plane.
(2) yeast culture: get routine and be applicable to
E.coliFluid nutrient medium, be inoculated into the fluid nutrient medium from the inclined-plane, 37 ℃ of shaking speed 100-250 r/min cultivate 4-12h, collect thalline.
The invention has the advantages that:
The present invention is to EGCG and Cu
2+The complex reaction condition optimize.
The present invention adopts polymer and EGCG_Cu (II) compound co spun technology to prepare nano fibrous membrane, is one of electrostatic spinning technique new application.
The polymer that the present invention adopts has highly-water-soluble, advantages such as low toxicity and good biocompatibility, and it is natural extract that institute adds medicine, and this composite membrane finally can be degraded, and environmental pollution is little, is a kind of technology of environmental protection.
Technology of the present invention is simple, and mild condition is easy and simple to handle, and is with low cost, and product can reuse, and is suitable for large-scale industrial production.
The nano-fiber composite film of the present invention's preparation has toughness and biocompatibility preferably, in technical field of biological material comparatively wide application prospect is arranged.
Description of drawings
Abosrption spectrogram when Fig. 1 is 1:1 for EGCG and Cu (II) mol ratio.
Abosrption spectrogram when Fig. 2 is 1:2 for EGCG and Cu (II) mol ratio.
Abosrption spectrogram when Fig. 3 is 1:3 for EGCG and Cu (II) mol ratio.
Fig. 4 is 7.4 o'clock abosrption spectrograms for the pH of solution.
Fig. 5 is product E GCG_Cu (II) nano composite membrane figure.
Fig. 6 is 8%PVA+0.5mM EGCG_Cu (II) nano composite membrane sem photograph.
Fig. 7 is 8%PVA+0.05mM EGCG_Cu (II) nano composite membrane sem photograph.
Fig. 8 and Fig. 9 are for adopting EGCG_Cu (II) the nano composite membrane transmission electron microscope picture of 1 mM EGCG.
Figure 10 is for adopting EGCG_Cu (II) nano composite membrane and the EGCG fungistatic effect figure of 0.5 mM EGCG.
Figure 11 is blank figure before EGCG_Cu (II) nano composite membrane is handled.
Figure 12 is that bacteria suspension was coated with Butut after EGCG_Cu (II) nano composite membrane was handled.
The specific embodiment
The invention is further illustrated by the following examples.
Embodiment 1:
(1) gets 5 * 10 of 3ml
-3Mol/l EGCG solution adds 5 μ l, 0.01 mol/l CuSO with the micropipette rifle again in cuvette
4Solution detects with UV-Visible all band spectrophotometer all band, and the result as shown in Figure 1.
(2) take by weighing the PVA(polyvinyl alcohol) be dissolved in the distilled water, be heated to 95 ℃ it is fully dissolved, add the solution in the step (1) again, the PVA final concentration is 8%(w/v, g/ml), it is standby that the room temperature lower magnetic force stirs 2h.Spinning solution is sucked the 20ml syringe, load onto diameter 0.6mm syringe needle, the adjustment syringe needle is 6cm to the distance of receiver, regulation voltage is at 12kv, and the spinning solution flow velocity is controlled at 2ml/h, spinning 2h, 80 ℃ of following vacuumize 6h obtain EGCG_Cu (II) nano composite membrane.
Embodiment 2:
(1) gets 3ml 5 * 10
-3Mol/l EGCG solution adds 10 μ l, 0.01 mol/l CuSO with the micropipette rifle again in cuvette
4Solution detects with UV-Visible all band spectrophotometer all band, and the result as shown in Figure 2.
(2) take by weighing the PVA(polyvinyl alcohol) be dissolved in the distilled water, be heated to 95 ℃ it is fully dissolved, add the solution in the step (1) again, the PVA final concentration is 8%(w/v, g/ml), it is standby that the room temperature lower magnetic force stirs 2h.Spinning solution is sucked the 20ml syringe, load onto diameter 0.6mm syringe needle, the adjustment syringe needle is 6cm to the distance of receiver, regulation voltage is at 12kv, and the spinning solution flow velocity is controlled at 2ml/h, spinning 2h, 80 ℃ of following vacuumize 6h obtain EGCG_Cu (II) nano composite membrane.
Embodiment 3:
(1) gets 3ml 5 * 10
-3Mol/l EGCG solution adds 15 μ l, 0.01 mol/l CuSO with the micropipette rifle again in cuvette
4Solution detects with UV-Visible all band spectrophotometer all band, and the result as shown in Figure 3.
(2 is specific as follows: take by weighing the PVA(polyvinyl alcohol) is dissolved in the distilled water, is heated to 95 ℃ it is fully dissolved, and adds the solution in the step (1) again, and the PVA final concentration is 8%(w/v, and g/ml), it is standby that the room temperature lower magnetic force stirs 2h.Spinning solution is sucked the 20ml syringe, load onto diameter 0.6mm syringe needle, the adjustment syringe needle is 6cm to the distance of receiver, regulation voltage is at 12kv, and the spinning solution flow velocity is controlled at 2ml/h, spinning 2h, 80 ℃ of following vacuumize 6h obtain EGCG_Cu (II) nano composite membrane.
Embodiment 4:
Get 0.15ml 1 * 10 with the micropipette rifle
-3Mol/l EGCG solution is in cuvette, and adding distil water drips 0.05 mol/l NaOH solution to 3ml, fully stirs, and transfers pH to 7.4, detects with UV-Visible all band spectrophotometer all band, and the result as shown in Figure 4.
(2) take by weighing the PVA(polyvinyl alcohol) be dissolved in the distilled water, be heated to 95 ℃ it is fully dissolved, add the solution in the step (1) again, the PVA final concentration is 8%(w/v, g/ml), it is standby that the room temperature lower magnetic force stirs 2h.Spinning solution is sucked the 20ml syringe, load onto diameter 0.6mm syringe needle, the adjustment syringe needle is 6cm to the distance of receiver, regulation voltage is at 12kv, and the spinning solution flow velocity is controlled at 2ml/h, spinning 2h, 80 ℃ of following vacuumize 6h obtain EGCG_Cu (II) nano composite membrane.
Embodiment 5:
Prepare EGCG_Cu (II) complex solution according to the method among embodiment 2 and the embodiment 4.
Take by weighing 0.8 g PVA(polyvinyl alcohol) be dissolved in the 10 ml distilled water, PVA concentration is 8%(w/v, g/ml), it is standby that the room temperature lower magnetic force stirs 2h.Spinning solution is sucked the 20ml syringe, load onto diameter 0.6mm syringe needle, the adjustment syringe needle is 6cm to the distance of receiver, regulation voltage is at 12kv, the spinning solution flow velocity is controlled at 2ml/h, spinning 2h, 80 ℃ of following vacuumize 6h, obtain EGCG_Cu (II) nano composite membrane, diameter is seen Fig. 5-9 at 100-500nm().Wherein Fig. 6-7 is a sem photograph, and Fig. 8-9 is a transmission electron microscope picture.
Embodiment 6:
Prepare EGCG_Cu (II) complex solution according to the method among embodiment 2 and the embodiment 4.
Prepare EGCG_Cu (II) nano composite membrane according to the method among the embodiment 5.
Cultivate 16～20h's from 37 ℃
E.coliGet a ring in the flat board, be transferred in the 250ml triangular flask that 50ml LB culture medium is housed, be cultured to OD600=0.4 ~ 0.5 in 37 ℃ of 200 r/min; 4 ℃ of 4000 centrifugal 10 min of r/min collects thalline; Making cell concentration with sterilized water is 10
6 ~ 7Bacteria suspension.
About 15ml in the culture dish that the 0.5ml bacteria suspension is arranged is dripped in the culture medium impouring of melting and be cooled to about 50 ℃, shake up rapidly to make and contain the bacterium flat board; Maybe splash into the 0.5ml bacteria suspension after waiting to solidify in the culture medium impouring sterilization culture dish that melts, spreading rod is coated with even the bacterium flat board that contains with bacteria suspension with sterilizing.
The EGCG_Cu of gained (II) nano composite membrane is cut into the disk that diameter is 9mm, is placed on sterilization 2h under the uviol lamp, is affixed on above-mentioned containing on the bacterium flat board, is inverted and cultivates 24h, observes the inhibition zone (see figure 10).
Embodiment 7:
Prepare EGCG_Cu (II) complex solution according to the method among embodiment 2 and the embodiment 4.
Prepare EGCG_Cu (II) nano composite membrane according to the method among the embodiment 5.
Under 37 ℃
E.coliDull and stereotyped 16 ~ the 20h that cultivates, be transferred in the test tube that 2ml LB culture medium is housed, be cultured to OD600=1.0 in 37 ℃ of 200 r/min, 4 ℃ of 4000 centrifugal 10 min of r/min, collect thalline, resuspended with pbs, EGCG_Cu (II) nano composite membrane with embodiment 2 gained adds in the above-mentioned resuspended liquid again, EGCG_Cu (II) final concentration is EGCG_Cu (II) nano composite membrane of 0.5mM, 37 ℃ of 200 r/min cultivates 24h, is coated with flat board with decimal dilution method, and colony counting method is counted bacterium colony, detect fungistatic effect (seeing Figure 11-12), bacteriostasis rate is up to 99%.
Claims (4)
1. an electrostatic spinning prepares the method for antibacterial nano fiber composite membrane, it is characterized in that concrete steps are as follows:
(1) takes by weighing copper sulphate and EGCG respectively, use dissolved in distilled water, under abundant stirring condition, copper-bath is added drop-wise in the EGCG solution control EGCG and Cu
2+Mol ratio be 1:1-1:4, under 22-30 ℃ of temperature, be 5.0-8.0 with the pH value of alkaline solution regulator solution;
(2) weighing polyvinyl alcohol is dissolved in the distilled water, adds step (1) gained solution, and the control polyvinyl alcohol concentration is 5-12%, and the concentration of EGCG is 0.05 * 10
-3-4 * 10
-3Mol/l, CuSO
4The concentration of solution is 0.1 * 10
-3-10 * 10
-3Mol/l; The room temperature lower magnetic force stirs 1-4h, obtains spinning solution;
(3) step (2) gained spinning solution is sucked the 20ml syringe, load onto diameter 0.6-1.2mm syringe needle, the adjustment syringe needle is 5-20cm to the distance of receiver, regulation voltage is at 10-20kv, the spinning solution flow velocity is controlled at 0.5-6ml/h, spinning 2h, 78-82 ℃ of following vacuumize 2-8h promptly obtains required product.
2. electrostatic spinning according to claim 1 prepares the method for antibacterial nano fiber composite membrane, it is characterized in that described alkaline solution is 10%Na
2CO
3Or 1mM NaOH.
3. an electrostatic spinning as claimed in claim 1 prepared antimicrobial nano composite membrane of method for preparing the antibacterial nano fiber composite membrane is as the application of bactericidal material, and concrete grammar is:
(1) yeast culture, centrifugal and collection somatic cells are used the phosphate buffer re-suspended cell, again nano-fiber composite film are put into reactant liquor, and 28-37 ℃, shaking speed 100-250 r/min, reaction 2-8h;
(2) centrifugal collection thalline is observed.
4. the prepared antibacterial nano fiber composite membrane of the method that electrostatic spinning according to claim 3 prepares the antibacterial nano fiber composite membrane is characterized in that as the application of bactericidal material this antibacterial nano fiber composite membrane is used for Escherichia coli
Esehedchia coliConcrete grammar is as follows:
(1) slant culture: get routine and be applicable to
E.coliSlant medium, the inoculation, 37 ℃ of slant culture 6-12h are as the inclined-plane;
(2) yeast culture: get routine and be applicable to
E.coliFluid nutrient medium, be inoculated into the fluid nutrient medium from the inclined-plane, 37 ℃ of shaking speed 100-250 r/min cultivate 4-12h, collect thalline.
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| CN102206623A (en) * | 2011-04-20 | 2011-10-05 | 同济大学 | Method for preparing nano cellulase membrane through electrostatic spinning and application of nano cellulase membrane |
| CN103757727B (en) * | 2013-12-30 | 2016-03-02 | 江苏大学 | A kind of nano-fiber material fresh-keeping for pork |
| CN103898625B (en) * | 2013-12-30 | 2016-04-27 | 江苏大学 | A kind of catechin-Cu iIthe preparation method of/polyvinylpyrrolidone composite fibre |
| CN105963762B (en) * | 2015-11-19 | 2019-04-09 | 华南理工大学 | A kind of nontoxic dressing of wide spectrum of water dispersible abandonment and preparation method thereof |
| CN106757464B (en) * | 2016-11-15 | 2019-03-08 | 天津捷盛东辉保鲜科技有限公司 | Food oxydating resistance cellulose acetate nanofiber preservative film |
| CN106319757B (en) * | 2016-11-15 | 2019-05-07 | 天津捷盛东辉保鲜科技有限公司 | The anti-oxidant nano fibrous membrane of electrospinning polyvinyl alcohol based tannic acid |
| CN113106635B (en) * | 2021-03-15 | 2023-01-10 | 广东金发科技有限公司 | Electrostatic spinning nanofiber non-woven fabric and preparation method and application thereof |
| CN113123017B (en) * | 2021-04-19 | 2022-06-21 | 天津工业大学 | Photodynamic filtering antibacterial composite membrane and preparation method and application thereof |
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| CN1709887A (en) * | 2005-07-13 | 2005-12-21 | 东北林业大学 | Method for preparing high-purity tea polypenols |
| CN101358382A (en) * | 2008-08-26 | 2009-02-04 | 东华大学 | Antibacterial nano fiber material and preparation method thereof |
| CN101638830A (en) * | 2009-08-25 | 2010-02-03 | 江南大学 | Method for preparing nanofibre membrane |
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| JP2008007464A (en) * | 2006-06-29 | 2008-01-17 | Bio Verde:Kk | Antifungal composition of external preparation |
| EP1958611A1 (en) * | 2006-12-20 | 2008-08-20 | DSMIP Assets B.V. | Oral composition containing EGCG and lycopene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1709887A (en) * | 2005-07-13 | 2005-12-21 | 东北林业大学 | Method for preparing high-purity tea polypenols |
| CN101358382A (en) * | 2008-08-26 | 2009-02-04 | 东华大学 | Antibacterial nano fiber material and preparation method thereof |
| CN101638830A (en) * | 2009-08-25 | 2010-02-03 | 江南大学 | Method for preparing nanofibre membrane |
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