CN113981610A

CN113981610A - Antibacterial keratin nanofiber membrane and preparation method thereof

Info

Publication number: CN113981610A
Application number: CN202110116952.XA
Authority: CN
Inventors: 杨世玉; 曹风采; 张东; 王晓龙
Original assignee: Zhejiang Zhili New Material Co ltd
Current assignee: Zhejiang Zhili New Material Co ltd
Priority date: 2021-01-28
Filing date: 2021-01-28
Publication date: 2022-01-28

Abstract

The invention relates to a preparation method of a functional nanofiber membrane, in particular to an antibacterial keratin nanofiber membrane and a preparation method thereof, and belongs to the technical field of functional nanofiber materials. The method takes waste hairiness as a raw material, removes soluble impurities and grease through a pretreatment process, and extracts high molecular weight keratin through a metal salt method; then mixing and dissolving keratin, a spinning aid and an antibacterial agent in formic acid to obtain a spinning solution; and finally, preparing the antibacterial keratin nano fiber membrane by using an electrostatic spinning technology. The material can be used as wound dressing, bracket material and filling material, and can be widely applied to the fields of medical treatment, health care and the like.

Description

Antibacterial keratin nanofiber membrane and preparation method thereof

Technical Field

The invention relates to a preparation method of a functional nanofiber membrane, in particular to an antibacterial keratin nanofiber membrane and a preparation method thereof, and belongs to the technical field of functional nanofiber materials.

Technical Field

Keratin is a filamentation protein insoluble in water and most organic solvents, and is the main component constituting the keratin layer and epidermal appendages (hair, nails, feathers, horns, claws) of animal epidermis. The keratin waste is used as an agricultural byproduct, and the yield is very rich. According to statistics, the feather, the pig hair, the waste wool and the human hair which are produced in China every year are as high as millions of tons, and the biomass resource is huge in number. In addition, the high disulfide crosslinking in the keratin molecular structure gives it high toughness and high modulus, with good mechanical properties and stability compared to other proteinaceous materials. However, keratin resources are not fully utilized at present, and most of them are incinerated and landfilled except that a small part of them are hydrolyzed into protein powder by high-pressure steaming as an animal feed additive with low nutritional value and low added value. This not only causes serious resource waste, but also brings about huge environmental pollution.

As a natural biological polymer, the keratin has the characteristics of good biocompatibility, biodegradability, low immunogenicity and the like, meets the requirements of medical materials to a certain extent, has good application prospect in the aspect of medicine, and can be used for wound dressing, stent materials, filling materials and the like. However, good medical materials must have a large specific surface area, high porosity and pore morphology to provide a good environment for cell proliferation, tissue growth, transport of oxygen and nutrients and excretion of metabolites. In addition, the medical material also needs to have an antibacterial function, so that bacterial breeding and virus infection are avoided.

As a classical nanofiber preparation method, the electrostatic spinning is simple to operate, low in cost and capable of realizing large-scale production, and basically meets the structural requirements of medical materials. However, the electrostatic spinning of pure keratin as a raw material to produce nanofibers is not ideal. On one hand, the pure keratin has poor spinnability, and a nozzle is easy to block during spinning; on the other hand, the pure keratin nanofiber membrane has the advantages of high brittleness, poor mechanical properties and no antibacterial function, and is difficult to meet the requirements of medical and health materials. Therefore, it is urgently needed to add chemical additives such as spinning aids and functional finishing agents into the keratin spinning solution to prepare functional nanofiber membranes which are applied to the field of medical health. The keratin functional nanofiber membrane can be applied to medical treatment, and can also make up the current situation of shortage of high-end medical products in the current market.

Disclosure of Invention

The invention provides an antibacterial keratin nanofiber membrane, which not only has good mechanical flexibility, but also has antibacterial functionality.

The invention also provides a preparation method of the antibacterial keratin nanofiber membrane.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of an antibacterial keratin nanofiber membrane comprises the following steps:

(1) pretreatment of waste hairiness:

respectively cleaning the waste hairiness in clear water and NaOH solution to remove soluble impurities and grease, and drying for later use

(2) Keratin extraction:

extracting keratin by metal salt method.

Putting the dried hairiness in the step (1) into a reducing agent NaHSO₃Adjusting the pH value to a proper value in a metal salt LiBr/surfactant SDS dissolving system, and dissolving at a certain temperature for a plurality of times to obtain a keratin solution;

dialyzing and centrifuging the keratin solution, and removing small-molecular polypeptide protein and residual reagent to obtain macromolecular keratin stock solution;

freezing the macromolecular keratin stock solution at low temperature, freeze-drying, and grinding to obtain keratin powder.

(3) Preparing a spinning solution:

and (3) mixing the keratin powder obtained in the step (2) with a certain amount of spinning aid and antibacterial agent, dissolving in formic acid solution, and fully stirring at a certain temperature to obtain the spinning solution.

(4) And (4) performing electrostatic spinning on the spinning solution obtained in the step (3) to obtain the keratin nano fiber membrane.

Preferably, the spinning aid in step (3) may be a hydrophilic spinning aid or a hydrophobic spinning aid, wherein the hydrophilic spinning aid is polyethylene oxide (PEO), polyvinyl alcohol (PVA), or the like; the hydrophobic spinning aid is Polycaprolactone (PCL), Polyurethane (PU), polyamide-6 (PA-6) and the like.

Preferably, the antibacterial agent in step (3) may be an inorganic antibacterial agent, an organic antibacterial agent or a natural antibacterial agent, wherein the inorganic antibacterial agent may be nano silver particles, nano copper particles, titanium dioxide (TiO)₂) One or more of zinc oxide (ZnO), montmorillonite, etc.; the organic antibacterial agent can be halamine compound, quaternary ammonium salt, biguanide, imidazole micromolecule antibacterial agent, and high-molecule organic antibacterial agent prepared from micromolecule antibacterial monomer by copolymerization, homopolymerization and grafting methods. The natural antibacterial agent can be chitosan, Chinese herbal medicine, tea leaf, and antibacterial component extracted from animal and plant with antibacterial function.

Preferably, in the step (3), the weight ratio of the keratin powder to the formic acid is 0.08-0.15:1, the weight ratio of the keratin powder to the spinning aid is 1:0.05-0.3, the weight ratio of the keratin powder to the antibacterial agent is 1:0.05-0.2, the stirring temperature is 50-80 ℃, and the stirring time is 24-48 h.

Preferably, in the step (4), when the electrostatic spinning is carried out, the spinning voltage is 12-18kV, the flow rate is 0.5-1.5mL/h, and the distance between the spinneret and the receiving plate is 10-20 cm.

Preferably, the waste feather in step (1) may be keratin-rich biomass such as wool, bird and poultry feathers, pig hair, cow hair and human hair.

Preferably, the concentration of NaOH in the step (1) is 5-10g/L, the cleaning temperature is 60-80 ℃, and the drying process is drying for 24h at 80 ℃.

Preferably, the reducing agent NaHSO in the dissolving system in the step (2)₃The concentration of the sodium ion-exchange resin is 0.5-1.5mol/L, the concentration of the metal salt LiBr is 0.1-0.25mol/L, the concentration of the surfactant SDS is 0.02-0.1mol/L, the pH value of a dissolving solution is 12-14, the dissolving temperature is 85-100 ℃, and the dissolving time is 4-6 h.

Preferably, when the keratin solution is dialyzed in the step (2), the cut-off molecular weight of the dialysis bag is 8000-14000, and the dialysis time is 72 h; the freezing temperature is-25 ℃ and the time is 24 hours; the freeze drying temperature is-50 deg.C, pressure is 40Pa, and time is 24 h.

An antibacterial nanofiber membrane taking keratin as a raw material is prepared by the preparation method. The material has good flexibility, high mechanical strength and antibacterial function, and can be applied to various fields such as textile and clothing, medical treatment and health care and the like.

The method takes waste hairiness as a raw material, removes soluble impurities and grease through a pretreatment process, and extracts high molecular weight keratin through a metal salt method; then mixing and dissolving keratin, a spinning aid and an antibacterial agent in formic acid to obtain a spinning solution; and finally, preparing the antibacterial keratin nano fiber membrane by using an electrostatic spinning technology. The material can be used as wound dressing, bracket material and filling material, and can be widely applied to the fields of medical treatment, health care and the like.

Compared with the prior art, the method has the following characteristics;

(1) the preparation method is simple, the reaction conditions are easy to control and realize, and the large-scale production can be realized;

(2) by adding the spinning aid into the spinning solution, the spinnability of the keratin is improved, the three-dimensional network structure is formed by crosslinking the keratin and the spinning aid, the defect that the keratin nanofiber membrane is fragile and brittle is overcome, and the mechanical strength of the keratin nanofiber membrane is improved.

(3) The antibacterial agent is added into the spinning solution for blending, so that the keratin nanofiber membrane can be directly endowed with antibacterial functionality, the working procedure of performing functional finishing at the later stage is reduced, and the process flow is shortened.

(4) Compared with the traditional acid-base method for extracting keratin, the metal salt method can be used for obtaining the keratin solution with higher concentration, and a large amount of alkaline waste water and waste acid steam can not be generated, so that the harm to the human health and the surrounding environment is avoided.

Description of the drawings:

FIG. 1 is a Scanning Electron Microscope (SEM) photograph of a pure keratin nanofiber membrane prepared in example 1;

FIG. 2 is an SEM photograph of the keratin/PEO composite nanofiber membrane prepared in example 2;

FIG. 3 is an SEM photograph of the keratin/PEO/nanosilver composite antibacterial nanofiber membrane prepared in example 3;

FIG. 4 is an SEM photograph of the keratin/PVA composite nanofiber membrane prepared in example 4;

FIG. 5 is a graph showing the bacteriostatic activity of the nanofiber membranes prepared in examples 4 to 6 against Staphylococcus aureus and Escherichia coli.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the invention, all parts and percentages are weight units, and all equipment, raw materials and the like can be purchased from the market or are commonly used in the industry, if not specified.

Example 1

A preparation method of a keratin nanofiber membrane comprises the following specific steps:

(1) pretreatment of waste hairiness:

weighing 20g of waste wool, and washing with clear water to remove soluble impurities; then putting the wool into 5g/L NaOH solution, cleaning the wool for 1h at 80 ℃ to remove grease on the wool, taking the wool out, and washing the wool with clean water; finally, the wool was dried in an oven at 80 ℃ for 24 h.

(2) Keratin extraction:

weighing 0.5mol of NaHSO₃0.1mol of LiBr and 0.02mol of SDS are dissolved in 500mL of deionized water to prepare a hair feather dissolving solution, then 10g of the dried wool obtained in the step (1) is placed in the dissolving solution, the pH value is adjusted to 12, and the dried wool is dissolved for 5 hours at 90 ℃ to obtain a keratin solution.

The keratin solution is poured into a dialysis bag (molecular weight cut-off: 8000-.

Freezing the pure keratin stock solution at-25 deg.C for 24 hr, freeze-drying at-50 deg.C under 40Pa for 24 hr to obtain dried keratin, and fully grinding to obtain keratin powder.

(3) Preparing a spinning solution:

0.8g of keratin powder was dissolved in 10g of formic acid and stirred at 50 ℃ for 24 hours to obtain a spinning solution.

(4) And (3) carrying out electrostatic spinning on the spinning solution to obtain the pure keratin nanofiber membrane. Wherein the spinning voltage is 15kV, the flow rate is 1.0mL/h, and the distance between the spinneret and the receiving plate is 15 cm.

SEM photographs of pure keratin nanofiber membranes are shown in fig. 1.

Example 2

(1) pretreatment of waste hairiness:

(2) Keratin extraction:

(3) Preparing a spinning solution:

0.8g of keratin powder and 0.08g of PEO were dissolved in 10g of formic acid and stirred at 50 ℃ for 24 hours to obtain a spinning solution.

(4) And (3) carrying out electrostatic spinning on the spinning solution to obtain the keratin/PEO composite nanofiber membrane. Wherein the spinning voltage is 15kV, the flow rate is 1.0mL/h, and the distance between the spinneret and the receiving plate is 15 cm.

An SEM photograph of the keratin/PEO composite nanofiber membrane is shown in fig. 2.

Example 3

A preparation method of an antibacterial keratin nanofiber membrane comprises the following specific steps:

(1) pretreatment of waste hairiness:

(2) Keratin extraction:

(3) Preparing a spinning solution:

0.8g of keratin powder, 0.08g of PEO and 0.08g of nano-silver particles were dissolved in 10g of formic acid and stirred at 50 ℃ for 24 hours to obtain a spinning solution.

(4) And (3) carrying out electrostatic spinning on the spinning solution to obtain the keratin/PEO/nano-silver composite antibacterial nanofiber membrane. Wherein the spinning voltage is 15kV, the flow rate is 1.0mL/h, and the distance between the spinneret and the receiving plate is 15 cm.

The SEM photograph of the keratin/PEO/nano silver composite antibacterial nanofiber membrane is shown in fig. 3.

Example 4

(1) pretreatment of waste hairiness:

(2) Keratin extraction:

(3) Preparing a spinning solution:

0.8g of keratin powder and 0.1g of PVA were dissolved in 10g of formic acid and stirred at 50 ℃ for 24 hours to obtain a spinning solution.

(4) And (4) carrying out electrostatic spinning on the spinning solution to obtain the keratin/PVA composite nanofiber membrane. Wherein the spinning voltage is 16kV, the flow rate is 0.8mL/h, and the distance between the spinneret and the receiving plate is 12 cm.

An SEM photograph of the keratin/PVA composite nanofiber membrane is shown in fig. 4.

Example 5

(1) pretreatment of waste hairiness:

weighing 20g of waste duck feather, and washing with clear water to remove soluble impurities; then putting the duck feather into 5g/L NaOH solution, washing the duck feather for 1h at 80 ℃ to remove grease on the duck feather, taking out the duck feather, and washing the duck feather with clear water; and finally, drying the duck feather in an oven at 80 ℃ for 24 h.

(2) Keratin extraction:

weighing 0.5mol of NaHSO₃Dissolving 0.1mol of LiBr and 0.02mol of SDS in 500mL of deionized water to prepare a hair feather solution, then placing 10g of the dried duck hair in the step (1) in the solution, adjusting the pH to 12, and dissolving at 90 ℃ for 5 hours to obtain a keratin solution.

(3) Preparing a spinning solution:

0.8g of keratin powder, 0.1g of PVA and 0.08g of nano-silver particles were dissolved in 10g of formic acid and stirred at 50 ℃ for 24 hours to obtain a spinning solution.

(4) And (3) carrying out electrostatic spinning on the spinning solution to obtain the keratin/PVA/nano-silver composite antibacterial nanofiber membrane. Wherein the spinning voltage is 16kV, the flow rate is 0.8mL/h, and the distance between the spinneret and the receiving plate is 12 cm.

Example 6

(1) pretreatment of waste hairiness:

weighing 20g of waste pig hair, and washing with clear water to remove soluble impurities; then putting the pig hair into 5g/L NaOH solution, washing the pig hair for 1h at 80 ℃ to remove grease on the pig hair, taking the pig hair out, and washing the pig hair with clear water; and finally, drying the pig hair in an oven at 80 ℃ for 24 hours.

(2) Keratin extraction:

weighing 0.5mol of NaHSO₃0.1mol of LiBr and 0.02mol of SDS are dissolved in 500mL of deionized water to prepare a hairiness solution, and then 10g of the solution obtained in the step (1) is addedPlacing the dried pig hair in the dissolving solution, adjusting pH to 12, and dissolving at 90 deg.C for 5 hr to obtain keratin solution.

(3) Preparing a spinning solution:

0.8g of keratin powder, 0.1g of PVA and 0.08g of TiO were mixed₂Dissolved in 10g of formic acid and stirred at 50 ℃ for 24 hours to give a spinning solution.

(4) Carrying out electrostatic spinning on the spinning solution to obtain keratin/PVA/TiO₂And (3) compounding an antibacterial nanofiber membrane. Wherein the spinning voltage is 16kV, the flow rate is 0.8mL/h, and the distance between the spinneret and the receiving plate is 12 cm.

The bacteriostatic properties of the nanofiber membranes prepared in examples 4-6 against staphylococcus aureus and escherichia coli are shown in fig. 5.

A Scanning Electron Microscope (SEM) photograph of the pure keratin nanofiber membrane prepared in example 1 is shown in fig. 1. Pure keratin has poor spinnability, and the nano fiber prepared by electrostatic spinning has high brittleness, easy fracture and poor mechanical property. In addition, in the spinning process, a large number of spinning bead knots are generated, the fiber thickness is not uniform, the shape is not good, and continuous nano fibers cannot be obtained.

An SEM photograph of the keratin/PEO composite nanofiber membrane prepared in example 2 is shown in fig. 2. Compared with a pure keratin nanofiber membrane, after the spinning aid PEO is added, the spinnability of keratin is obviously improved, the fiber diameter is uniform, and spinning bead knots disappear. This shows that by adding a spinning aid to blend with keratin, the spinnability of keratin is obviously improved, and the fiber integrity is improved.

An SEM photograph of the keratin/PEO/nano silver composite antibacterial nanofiber membrane prepared in example 3 is shown in fig. 3. Compared with keratin/PEO composite nanofiber membranes, the addition of the antibacterial agent nano silver has no obvious influence on the spinnability of keratin. The fiber surface is loaded with a large amount of nano silver particles, the particles are uniformly dispersed, and the agglomeration phenomenon is avoided.

An SEM photograph of the keratin/PVA composite nanofiber membrane prepared in example 4 is shown in fig. 4. Compared with a pure keratin nanofiber membrane, PVA is selected as a spinning aid, so that the spinnability of keratin can be obviously improved, and the fiber form is complete. In addition, compared with keratin/PEO composite nanofiber membranes, when the spinning aid is changed from PEO to PVA, cross-linking between fibers is more likely to occur, a three-dimensional interconnected network structure is formed, and the fiber surface is rougher. These results show that the addition of different types of spinning aids can improve the spinnability of keratin, but the effect on the fiber morphology is greatly different, and the proper spinning aid can be selected according to different requirements on the nanofiber morphology finally.

The bacteriostatic properties of the nanofiber membranes prepared in examples 4-6 against staphylococcus aureus and escherichia coli are shown in fig. 5. When no antibacterial agent is added, the keratin/PVA composite nanofiber membrane has the bacteriostasis rate of about 1 percent on staphylococcus aureus and escherichia coli, and almost has no antibacterial effect. Adding antibacterial agent nano silver or TiO₂During the process, the antibacterial effect of the composite keratin nanofiber membrane is greatly improved, and the antibacterial rate is basically maintained at about 95%. The bacteriostatic effect of the same antibacterial agent on different bacteria is different, but the difference is not great. Similarly, the antibacterial effect of different antibacterial agents is slightly different for the same bacteria, wherein the antibacterial effect of the nano-silver is better than that of the TiO₂。

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Claims

1. A preparation method of an antibacterial keratin nanofiber membrane is characterized by comprising the following steps:

(1) pretreatment of waste hairiness:

(2) Keratin extraction:

extracting keratin by metal salt method.

(3) Preparing a spinning solution:

2. The method of claim 1, wherein: the spinning aid in the step (3) can be a hydrophilic spinning aid or a hydrophobic spinning aid, wherein the hydrophilic spinning aid is polyethylene oxide (PEO), polyvinyl alcohol (PVA) and the like; the hydrophobic spinning aid is Polycaprolactone (PCL), Polyurethane (PU), polyamide-6 (PA-6) and the like.

3. The method of claim 1, wherein: the antibacterial agent in step (3) can be inorganic antibacterial agent, organic antibacterial agent or natural antibacterial agent, wherein the inorganic antibacterial agent can be nano silver particles, nano copper particles, titanium dioxide (TiO)₂) One or more of zinc oxide (ZnO), montmorillonite, etc.; the organic antibacterial agent can be small molecule antibacterial agent such as halamine compound, quaternary ammonium salt, biguanide, imidazole, and high molecule organic antibacterial monomer prepared by copolymerization, homopolymerization and graftingAn antibacterial agent. The natural antibacterial agent can be chitosan, Chinese herbal medicine, tea leaf, and antibacterial component extracted from animal and plant with antibacterial function.

4. The method of claim 1, wherein: in the step (3), the weight ratio of the keratin powder to the formic acid is 0.08-0.15:1, the weight ratio of the keratin powder to the spinning aid is 1:0.05-0.3, the weight ratio of the keratin powder to the antibacterial agent is 1:0.05-0.2, the stirring temperature is 50-80 ℃, and the stirring time is 24-48 hours.

5. The method of claim 1, wherein: and (4) when electrostatic spinning is carried out in the step (4), the spinning voltage is 12-18kV, the flow rate is 0.5-1.5mL/h, and the distance between a spinning nozzle and a receiving plate is 10-20 cm.

6. The method of claim 1, wherein: the waste feather in the step (1) can be keratin-rich biomass such as wool, bird and poultry feather, pig hair, cattle hair, human hair and the like.

7. The method of claim 1, wherein: the concentration of NaOH in the step (1) is 5-10g/L, the cleaning temperature is 60-80 ℃, and the drying process is drying for 24h at 80 ℃.

8. The method of claim 1, wherein: reducing agent NaHSO in dissolution system in step (2)₃The concentration of the sodium ion-exchange resin is 0.5-1.5mol/L, the concentration of the metal salt LiBr is 0.1-0.25mol/L, the concentration of the surfactant SDS is 0.02-0.1mol/L, the pH value of a dissolving solution is 12-14, the dissolving temperature is 85-100 ℃, and the dissolving time is 4-6 h.

9. The method of claim 1, wherein: when the keratin solution is dialyzed in the step (2), the cut-off molecular weight of the dialysis bag is 8000-14000, and the dialysis time is 72 h; the freezing temperature is-25 ℃ and the time is 24 hours; the freeze drying temperature is-50 deg.C, pressure is 40Pa, and time is 24 h.

10. An antibacterial keratin nanofiber membrane using waste hairiness as a raw material, which is manufactured by the manufacturing method of claim 1.