CN103300071A - Method for preparing nanofiber composite membrane for fixing lysozyme-rectorie - Google Patents
Method for preparing nanofiber composite membrane for fixing lysozyme-rectorie Download PDFInfo
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- CN103300071A CN103300071A CN2013102460772A CN201310246077A CN103300071A CN 103300071 A CN103300071 A CN 103300071A CN 2013102460772 A CN2013102460772 A CN 2013102460772A CN 201310246077 A CN201310246077 A CN 201310246077A CN 103300071 A CN103300071 A CN 103300071A
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Abstract
The invention relates to a method for preparing a modified nanofiber composite membrane for fixing enzyme-phyllosilicate by using an electric injection method. The method for preparing a lysozyme-rectorie/cellulose nano composite membrane material comprises the following steps of: firstly, preparing a cellulose acetate nanofiber membrane by utilizing an electrospinning technology; and fixing a mixed solution of lysozyme and rectorite on the surface of the nanofiber membrane by utilizing the electric injection technology, thereby producing the lysozyme-rectorie/cellulose nano composite membrane material. The mass ratio of lysozyme to rectorite in the mixed solution of lysozyme to rectorite is 2.5 to 10:1. The prepared lysozyme-rectorie/cellulose nano composite membrane material can be used as an antimicrobial material, and is widely used in fields of agriculture, sanitation, food, bio-medical materials and the like.
Description
Technical field
The present invention relates to a kind of preparation method of nano-fiber composite film of fixedly lysozyme-rectorite, belong to packaging material for food and bio-medical material technical field.
Background technology
A kind of natural anti-infective material with bactericidal action of lysozyme; antibiotic, antiviral, hemostasis, swelling and pain relieving are arranged and accelerate the effect such as organized renewing function; but because the lysozyme suitability for industrialized production often can run into poor stability; be subject to the external environment impact; easy inactivation; be difficult to the problems such as recycling, limited it in the extensive use of bio-medical material, medicine controlled releasing and daily-use chemical industry and field of food, and the immobilization of lysozyme can effectively improve these problems.
The lysozyme fixing means of bibliographical information is mainly layer by layer self assembly, microcapsules technology at present.The patent No. be " 201010610000 " patent report graphene oxide/lysozyme antibiotic nano composite film and preparation method thereof, but the preparation of graphene oxide is time-consuming, productive rate is low, is unsuitable for large-scale production.
Plant cellulose is renewable resources the abundantest on the earth, plays the support structure effect in plant, has good mechanical property.By electrostatic spinning technique and electric jet technology lysozyme is fixed on nano composite membrane made on the cellulose nano-fibrous membrane and has not only kept the good fungistatic effect of lysozyme itself, but also increased its stability and had reusing, and lysozyme free of losses in the immobilization process is for the biological safety developing material of natural degradable provides a new effective way.
Rectorite, a kind of have special construction, comparatively rare layer aluminosilicate clay mineral, alternately piled up regularly under special natural condition by class mica elementary layer and class montmorillonite elementary layer, but not the simple combination of the two, especially its banded microstructure is rather rare, and has the structure sheaf separation property, and itself is without fungistatic effect, but have the assistance bacteriostasis, but and the slowly-releasing of regulation protein and enzyme.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of nano-fiber composite film of fixedly lysozyme-rectorite.
The present invention utilizes electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, lysozyme-rectorite is stirred at phosphate buffer solution it is uniformly dispersed, and then utilizes electric jet technology that mixed solution is fixed on vacuum drying nano fibrous membrane.
By the method, lysozyme stability is significantly improved and can be repeated and recycle, and have controlled sustained release performance.The cellulose acetate nano fibrous membrane plays the support structure effect in the nano-fiber composite film of preparation, provides material good mechanical property.Lysozyme-rectorite is fixed on the surface of nano fibrous membrane by the method for EFI, because cellulose acetate is electronegative, the compound positively charged of lysozyme-rectorite so that both also have electrostatic adsorption in fixation procedure, thereby has higher envelop rate.
The concrete technical scheme that the present invention adopts is:
(1) cellulose acetate is dissolved, be made into the solution that contains cellulose acetate 16wt%, utilize electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, temperature and relative moisture are respectively 25 ℃ and 50% in the electrostatic spinning process, then vacuum drying cellulose acetate nano fibrous membrane;
(2) lysozyme and rectorite being dissolved in pH is in 6.24 the phosphate buffer solution, preparation obtains containing the mixing suspension of 2.5 ~ 10wt% lysozyme and 1wt% rectorite, stirring is uniformly dispersed it, then utilize the EFI method that mixing suspension is solidificated on the prepared cellulose acetate nano fibrous membrane of step (1), the temperature of EFI process and relative moisture are respectively 25 ℃ and 50%, are fixed the nano-fiber composite film of lysozyme-rectorite.
In the said process, the voltage of electrostatic spinning is 16kV in the step (1), and flow velocity is 1ml/h, and the duration is 24h.
Distance between metal needle and fiber receiver is 15cm.
The electric jet technology concrete operation method is the syringe of lysozyme-rectorite mixed solution being put into 10ml in the step (2), the cellulose acetate nano fibrous membrane is fixed on the metallic plate, be spaced apart 12cm between syringe metal needle and the metallic plate, and between adds the high-tension electricity of 12kV, the syringe flow velocity is 0.25ml/h, and the duration is 4h.
Utilize the nano-fiber composite film of the fixedly lysozyme-rectorite of preparation to carry out bacteriostatic experiment and slowly-releasing experiment, the result shows that this nano composite material all has certain fungistatic effect to representing bacterium staphylococcus aureus and Escherichia coli, fungistatic effect to staphylococcus aureus is better, and rectorite has obvious assistance bacteriostasis; The envelop rate of this nano fibrous membrane is very high, and rectorite has controlled effect to its slowly-releasing.Therefore, the lysozyme-rectorite nano composite material of the present invention's preparation is a kind of good anti-biotic material.And the method can make lysozyme percent of loss in immobilization process almost nil.Preparation method of the present invention is simple, and cost of material is low, whole simple operation of process, and the gained composite nano materials has good bacteriostasis property, higher envelop rate and controlled sustained release performance.
Description of drawings
Fig. 1 is the pattern of lysozyme-rectorite of the present invention/cellulose nano compound film.A is the cellulose acetate nano fibrous membrane, b is the 5wt% lysozyme/cellulose nano compound film of embodiment 2 preparation, c is the 5wt% lysozyme-1wt% rectorite/cellulose nano compound film of embodiment 3 preparation, and d is the 10wt% lysozyme-1wt% rectorite/cellulose nano compound film of embodiment 4 preparations.
Fig. 2 is nano compound film bacteriostasis property result of study of the present invention, and what bacteriostatic experiment adopted is the inhibition zone method.Wherein a is the cellulose acetate nano fibrous membrane, b is the 2.5wt% lysozyme/cellulose nano compound film of embodiment 1 preparation, c is the 5wt% lysozyme/cellulose nano compound film of embodiment 2 preparation, d is the 5wt% lysozyme-1wt% rectorite/cellulose nano compound film of embodiment 3 preparation, and e is the 10wt% lysozyme-1wt% rectorite/cellulose nano compound film of embodiment 4 preparations.
Fig. 3 is inhibition zone method experiment experimental results picture, the a-e in the 1-5 difference corresponding diagram 2 among the figure on the culture dish.
Embodiment
Below by specific embodiment technical scheme of the present invention is described further, its purpose is to help better to understand content of the present invention, but the protection domain that these specific embodiments do not limit the present invention in any way.
Embodiment 1
Utilize electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, be about to cellulose acetate be dissolved in contain acetone and
N, NIn the mixed solvent of dimethylacetylamide (volume ratio of the two is 2:1), gained 16%(w/w) cellulose acetate solution carries out electrostatic spinning, and the voltage of electrostatic spinning is 16kV, and flow velocity is 1ml/h, and the duration is 24h.Lysozyme being dissolved in stir in the phosphate buffer solution (pH=6.24) of 100mM is uniformly dispersed it, then utilize electric jet technology that the solution of gained 2.5wt% lysozyme is solidificated on vacuum drying nano fibrous membrane, the technical conditions of EFI process are that voltage is 12kV, flow velocity is 0.25ml/h, and the duration is 4h.The temperature of EFI and electrostatic spinning process is 25 ℃, and relative moisture is 50%.Lysozyme on the composite material nanometer tunica fibrosa that makes is almost lossless, and namely the lysozyme on the film is 25mg, and enzyme is lived and is 50.925Ucm
-2, staphylococcus aureus and colibacillary antibacterial circle diameter are respectively 1.125cm and 1.000cm.
Embodiment 2
Utilize electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, be about to cellulose acetate be dissolved in contain acetone and
N, NIn the mixed solvent of dimethylacetylamide (volume ratio of the two is 2:1), gained 16%(w/w) cellulose acetate solution carries out electrostatic spinning, and the voltage of electrostatic spinning is 16kV, and flow velocity is 1ml/h, and the duration is 24h.Lysozyme being dissolved in stir in the phosphate buffer solution (pH=6.24) of 100mM is uniformly dispersed it, then utilize electric jet technology that the solution of gained 5wt% lysozyme is solidificated on vacuum drying nano fibrous membrane, the technical conditions of EFI process are that voltage is 12kV, flow velocity is 0.25ml/h, and the duration is 4h.The temperature of EFI and electrostatic spinning process is 25 ℃, and relative moisture is 50%.Lysozyme on the composite material nanometer tunica fibrosa that makes is almost lossless, and namely the lysozyme on the film is 50mg, and enzyme is lived and is 152.778Ucm
-2, staphylococcus aureus and colibacillary antibacterial circle diameter are respectively 1.125cm and 1.267cm.
Embodiment 3
Utilize electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, be about to cellulose acetate be dissolved in contain acetone and
N, NIn the mixed solvent of dimethylacetylamide (volume ratio of the two is 2:1), gained 16%(w/w) cellulose acetate solution carries out electrostatic spinning, and the voltage of electrostatic spinning is 16kV, and flow velocity is 1ml/h, and the duration is 24h.Lysozyme and rectorite being dissolved in stir in the phosphate buffer solution (pH=6.24) of 100mM is uniformly dispersed it, then utilize electric jet technology that the mixing suspension that gained contains the rectorite of the lysozyme of 5wt% and 1wt% is solidificated on vacuum drying nano fibrous membrane, the technical conditions of EFI process are that voltage is 12kV, flow velocity is 0.25ml/h, and the duration is 4h.The temperature of EFI and electrostatic spinning process is 25 ℃, and relative moisture is 50%.Lysozyme on the composite material nanometer tunica fibrosa that makes is almost lossless, and namely the lysozyme on the film is 50mg, and rectorite is 10mg, and enzyme is lived and is 349.075Ucm
-2, staphylococcus aureus and colibacillary antibacterial circle diameter are respectively 1.550cm and 1.375cm.
Embodiment 4
Utilize electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, be about to cellulose acetate be dissolved in contain acetone and
N, NIn the mixed solvent of dimethylacetylamide (volume ratio of the two is 2:1), gained 16%(w/w) cellulose acetate solution carries out electrostatic spinning, and the voltage of electrostatic spinning is 16kV, and flow velocity is 1ml/h, and the duration is 24h.Lysozyme with 10% and 1% rectorite are dissolved in stirring in the phosphate buffer solution (pH=6.24) of 100mM it are uniformly dispersed, then utilize electric jet technology that the mixing suspension that gained contains the rectorite of 10% lysozyme and 1% is solidificated on vacuum drying nano fibrous membrane, the technical conditions of EFI process are that voltage is 12kV, flow velocity is 0.25ml/h, and the duration is 4h.The temperature of EFI and electrostatic spinning process is 25 ℃, and relative moisture is 50%.Lysozyme on the composite material nanometer tunica fibrosa that makes is almost lossless, and namely the lysozyme on the film is 100mg, and rectorite is 10mg, and enzyme is lived and is 396.297Ucm
-2, staphylococcus aureus and colibacillary antibacterial circle diameter are respectively 1.950cm and 1.625cm.
Claims (7)
1. the fixing preparation method of the nano-fiber composite film of lysozyme-rectorite is characterized in that, comprises the steps:
(1) cellulose acetate is dissolved, be made into the solution that contains cellulose acetate 5-20wt%, utilize electrostatic spinning technique to prepare the cellulose acetate nano fibrous membrane, temperature and relative moisture are respectively 25 ℃ and 50% in the electrostatic spinning process, then vacuum drying cellulose acetate nano fibrous membrane;
(2) lysozyme and rectorite being dissolved in pH is in 6.24 the phosphate buffer solution, preparation obtains containing the mixing suspension of 2.5 ~ 10wt% lysozyme and 1wt% rectorite, stirring is uniformly dispersed it, then utilize the EFI method that mixing suspension is solidificated on the prepared cellulose acetate nano fibrous membrane of step (1), the temperature of EFI process and relative moisture are respectively 25 ℃ and 50%, are fixed the nano-fiber composite film of lysozyme-rectorite.
2. preparation method according to claim 1 is characterized in that, the voltage of electrostatic spinning is 5-25kV in the step (1), and flow velocity is 0.1-2ml/h, and the duration is 1-96h.
3. preparation method according to claim 1 is characterized in that, in step (1) electrostatic spinning, the distance between metal needle and fiber receiver is 1-50cm.
4. preparation method according to claim 1 is characterized in that, the voltage of EFI process is 12kV in the step (2), and flow velocity is 0.25ml/h, and the duration is 1-48h.
5. preparation method according to claim 1 is characterized in that, in step (2) the EFI process, the distance between metal needle and metallic plate is 5-20cm.
6. preparation method according to claim 1 is characterized in that, in the step (1), with cellulose acetate be dissolved in acetone and
N, NBe made into the solution that contains cellulose acetate 5-20wt% in the dimethylacetylamide mixed solvent that 2:1 forms by volume.
7. the nano-fiber composite film of the fixedly lysozyme-rectorite of each described method preparation of claim 1-5.
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Cited By (6)
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| CN103536958A (en) * | 2013-09-29 | 2014-01-29 | 武汉大学 | Modified cellulose nanofiber membrane based on layer-by-layer self-assembly of lysozyme and silk protein based as well as preparation and application thereof |
| CN105925013A (en) * | 2016-04-28 | 2016-09-07 | 长兴凯鸿新型墙体材料有限公司 | Weatherproof heavy calcium carbonate preparation method |
| CN105925012A (en) * | 2016-04-28 | 2016-09-07 | 长兴凯鸿新型墙体材料有限公司 | Preparation method of modified ultrafine heavy calcium carbonate |
| CN106282153A (en) * | 2016-08-31 | 2017-01-04 | 武汉大学 | Sandwich micro nanometer fiber composite membrane of loading microorganisms and its preparation method and application |
| CN106581786A (en) * | 2016-11-22 | 2017-04-26 | 华南理工大学 | Functional patch and preparation method and applications thereof |
| CN113980921A (en) * | 2021-11-05 | 2022-01-28 | 江苏奕农生物股份有限公司 | Method for immobilizing laccase by using ionic liquid modified composite material |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103536958A (en) * | 2013-09-29 | 2014-01-29 | 武汉大学 | Modified cellulose nanofiber membrane based on layer-by-layer self-assembly of lysozyme and silk protein based as well as preparation and application thereof |
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| CN105925013A (en) * | 2016-04-28 | 2016-09-07 | 长兴凯鸿新型墙体材料有限公司 | Weatherproof heavy calcium carbonate preparation method |
| CN105925012A (en) * | 2016-04-28 | 2016-09-07 | 长兴凯鸿新型墙体材料有限公司 | Preparation method of modified ultrafine heavy calcium carbonate |
| CN105925012B (en) * | 2016-04-28 | 2018-03-09 | 长兴凯鸿新型墙体材料有限公司 | A kind of preparation method of modified superfine powdered whiting |
| CN105925013B (en) * | 2016-04-28 | 2018-04-13 | 长兴凯鸿新型墙体材料有限公司 | A kind of preparation method of weatherability powdered whiting |
| CN106282153A (en) * | 2016-08-31 | 2017-01-04 | 武汉大学 | Sandwich micro nanometer fiber composite membrane of loading microorganisms and its preparation method and application |
| CN106282153B (en) * | 2016-08-31 | 2019-08-02 | 武汉大学 | Sandwich micro nanometer fiber composite membrane of loading microorganisms and its preparation method and application |
| CN106581786A (en) * | 2016-11-22 | 2017-04-26 | 华南理工大学 | Functional patch and preparation method and applications thereof |
| CN113980921A (en) * | 2021-11-05 | 2022-01-28 | 江苏奕农生物股份有限公司 | Method for immobilizing laccase by using ionic liquid modified composite material |
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