Carbon fiber fused direct writing silk fibroin composite for microbial culture and preparation method thereof

Abstract

The application discloses a carbon fiber melt direct writing silk fibroin composite material for microbial culture and a preparation method thereof, and belongs to the field of microbial culture. The carbon fiber melt direct writing silk fibroin composite material is prepared by taking silk fibroin, diatom earth powder and polymer powder as direct writing raw materials, physically blending the direct writing raw materials, and spraying and extruding the direct writing raw materials on carbon fibers soaked in a hyaluronic acid solution. A strong specific surface area is provided on the carbon fiber base to obtain more attachment sites. The silk fibroin and the polymer are biocompatible and biodegradable, provide nutrients for microorganisms, promote the synthesis of microbial bodies and the growth of microorganisms, and have good biological affinity. In addition, the diatom earth can provide minerals for the growth of microorganisms, which is beneficial to the growth and reproduction of microorganisms. After the carbon fibers are soaked in the hyaluronic acid solution and then covered with silk fibroin and other substances by direct writing, the slow-release effect of the hyaluronic acid can be achieved, so that the service life of the microbial carrier is prolonged.

Description

Technical Field

[0001] This invention relates to the field of microbial culture, and in particular to a carbon fiber melt-written silk fibroin composite material for microbial culture and its preparation method. Background Technology

[0002] Silk is a continuous long fiber formed from the solidified silk fluid secreted by mature silkworms during cocoon spinning. It is one of the earliest animal fibers utilized by humans and a product of ancient Chinese civilization. Silk fibroin possesses excellent biocompatibility, is non-toxic, non-polluting, non-irritating, and biodegradable. In previous research and practice, silk fibroin has been mainly used in cosmetics, medical aesthetics, controlled drug release, wound repair, and tissue engineering. Silk fibroin can be used to make surgical sutures; relying on electrospinning technology, it can be made into a sheet-like material, currently contributing value in wound dressings, hernia repair patches, skin regeneration, and heart valve patches. In recent years, many researchers have combined osteoblastic seed cells and silk fibroin to prepare silk fibroin materials with bone repair capabilities. The silk fibroin in these materials can provide the necessary space and environment for cell growth, adhesion, and differentiation. The development of silk fibroin-based bone repair materials has become one of the hot topics in bone tissue engineering research. Currently, various forms of silk fibroin biomaterials, including silk fibroin membranes, silk fibroin nanofibers, silk fibroin hydrogels, and silk fibroin porous scaffolds, have been widely used in the repair of bone loss and injury. However, dried silk fibroin films are brittle and fragile. Studies have shown that blending silk fibroin with polymers such as polyethylene glycol, polycaprolactone, and polyvinyl alcohol can improve the mechanical properties of silk fibroin materials.

[0003] Traditional microbial culture uses petri dishes for planar culture. However, the culture medium in the petri dish does not have a large specific surface area, so the microorganisms are restricted to growing on the surface of the culture medium, and the culture effect needs to be improved. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides a carbon fiber melt-written silk fibroin composite material for microbial culture and its preparation method. This invention utilizes melt-written technology to physically blend silk fibroin, diatomaceous earth, and polymers, improving the brittleness and fragility of silk fibroin films. Using melt-written technology, silk fibroin is directly written onto carbon fibers to prepare micro / nano functional fibers, constructing a network structure to provide a high specific surface area and porosity. The excellent mechanical properties and stable physicochemical properties of carbon fibers enable the silk fibroin composite material to be used stably and easily collected. The silk fibroin and polymers in this invention have good biocompatibility and are biodegradable, providing nutrients for microorganisms and promoting cell synthesis and microbial growth. The presence of diatomaceous earth provides minerals for microbial growth, which is beneficial for microbial growth and reproduction. Implanting carbon fibers with hyaluronic acid solution before directly writing and coating them with silk fibroin and other substances achieves a sustained-release effect of hyaluronic acid, extending the service life of the microbial carrier. Carbon fiber can exert its excellent mechanical properties in aquatic environments, providing a stable carrier for microbial attachment. The micro-nano network structure obtained by melt-writing and the bundle morphology of composite materials have a larger specific surface area and higher porosity, thus providing more attachment sites to achieve the enrichment and cultivation of microorganisms.

[0005] The technical solution of the present invention is as follows:

[0006] A method for preparing carbon fiber melt-written silk fibroin composite material for microbial culture includes the following steps:

[0007] (1) Mix silk fibroin, diatomaceous earth powder and polymer powder evenly, and add them to the feed cylinder as direct writing raw material;

[0008] (2) The blend in the feed cylinder is heated and melted into a molten state at high temperature, and the air pressure generated by the air pump is used as a power source to extrude the molten mixture from the spinning nozzle;

[0009] (3) The carbon fiber soaked in hyaluronic acid solution is placed on a collection plate, and the spinning of step (2) is sprayed onto the carbon fiber using melt direct writing technology. After cooling and drying, carbon fiber melt direct writing silk fibroin composite material is prepared.

[0010] In one embodiment of the present invention, the mass ratio of silk fibroin, diatomaceous earth powder and polymer powder is 1:5 to 20:10 to 20, preferably 1:5:15.

[0011] In one embodiment of the present invention, the polymer powder is any one or more of polycaprolactone, ethylene-vinyl acetate, and polyvinyl chloride.

[0012] In one embodiment of the present invention, the polymer powder is preferably polycaprolactone or ethylene-vinyl acetate, both of which have low melting temperatures and are less likely to damage the structure and properties of biological materials.

[0013] In one embodiment of the present invention, in step (3), the concentration of the hyaluronic acid solution is 20-50 g / L.

[0014] In one embodiment of the present invention, the molecular weight of the hyaluronic acid is 10-20 kDa.

[0015] In one embodiment of the present invention, the carbon fiber is immersed in the hyaluronic acid solution for 10 to 30 minutes.

[0016] In one embodiment of the present invention, the high temperature in step (2) is 80-200°C.

[0017] In one embodiment of the present invention, in step (2), the nozzle is subjected to a voltage of 2.5-3.5KV, the distance from the nozzle to the collecting plate is 1-6mm, and the collecting plate moves at a speed of 30-36mm·s. －1 The nozzle inner diameter is 0.2-0.5mm.

[0018] The second objective of this invention is to provide a carbon fiber melt-written silk fibroin composite material prepared by the above-described preparation method.

[0019] In one embodiment of the present invention, the fiber diameter of the carbon fiber melt-written silk fibroin composite material is 1-20 μm, and the porosity of the mesh material is 80-90%.

[0020] A third objective of this invention is to provide a microbial culture apparatus comprising the aforementioned carbon fiber melt-written silk fibroin composite material.

[0021] In one embodiment of the present invention, the carbon fiber melt-written silk fibroin composite material is used as a carrier for microbial culture.

[0022] A fourth objective of this invention is to provide a method for culturing microorganisms, wherein the method uses the aforementioned carbon fiber melt-written silk fibroin composite material for microbial culture as a carrier, or uses the aforementioned microbial culture apparatus for culturing.

[0023] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0024] 1. The carbon fiber melt-written silk fibroin composite material provided by this invention is prepared by melt-written technology, which can precisely control the functional fiber material. The resulting fiber has high uniformity and uniform size, with fiber diameter ranging from nanometers to tens of micrometers. After the fibers overlap and cross-link on the carbon fiber substrate, they form a network structure, providing a strong specific surface area to obtain larger attachment sites, which is conducive to achieving efficient microbial culture.

[0025] 2. The functional fiber mesh material provided by this invention uses carbon fiber as a substrate. Carbon fiber has excellent mechanical properties, providing a solid carrier for the growth and cultivation of microorganisms, while also improving the brittleness and fragility of single silk fibroin. Silk fibroin and polymers have good biocompatibility and are biodegradable, providing nutrients for microorganisms and promoting cell synthesis and growth. In addition, the presence of diatomaceous earth can provide minerals for microbial growth, which is beneficial to the growth and reproduction of microorganisms. After impregnating the carbon fiber with hyaluronic acid solution and then directly writing and covering it with silk fibroin and other substances, a slow-release effect of hyaluronic acid can be achieved, thereby extending the service life of the microbial carrier.

[0026] 3. Traditional microbial culture uses petri dishes for planar culture. The culture medium in the petri dish does not have a large specific surface area, and the microorganisms are restricted to growing on the surface of the culture medium. This invention provides a strong specific surface area and porosity. Compared with ordinary culture materials and methods, this invention can achieve exponential growth of microorganisms.

[0027] 4. The composite material provided by this invention uses carbon fiber as a substrate. The stable structure of carbon fiber makes it easy to handle, and it is easy to recover and obtain microorganisms after enriching and culturing them in an aquatic environment. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the use of the composite material of the present invention.

[0029] Figure 2 These are images showing the microbial culture effects of Examples 1-3 and Comparative Examples 1-4 of the present invention. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0031] Example 1:

[0032] The method for preparing carbon fiber melt-written silk fibroin composite material for microbial culture provided by this invention is as follows:

[0033] (1) Immerse the carbon fiber in a hyaluronic acid solution with a concentration of 40 g / L and a molecular weight of 15-20 kDa for 30 min, and set aside.

[0034] (2) Select silk fibroin, diatomaceous earth powder and polycaprolactone (PCL) powder in a mass ratio of 1:5:15 and mix them physically. Shake well and add to the feeding cylinder.

[0035] (3) Set the nozzle temperature of the melt direct writing spinneret to 80℃, heat the blend in the feed cylinder to melt it into a molten state at high temperature, and use the air pressure generated by the air pump as a power source to extrude the molten mixture from the spinning nozzle; place the carbon fiber impregnated with hyaluronic acid obtained in step (1) (wherein the mass ratio of carbon fiber to silk fibroin is 50:1) on the collecting plate in advance, set the nozzle loading voltage to 2.5KV, and the collecting plate movement speed to 30mm·s. －1 The nozzle has an inner diameter of 0.2 mm and a distance of 2 mm from the nozzle to the collection plate. By using melt direct writing technology to uniformly spray the spinning onto the carbon fiber, carbon fiber melt direct writing silk fibroin composite material can be prepared.

[0036] After measurement, the fiber diameter of the final composite material was 5-10 μm, and the porosity of the mesh material was 84.2%.

[0037] Example 2:

[0038] The method for preparing carbon fiber melt-written silk fibroin composite material for microbial culture provided by this invention is as follows:

[0039] (1) Immerse the carbon fiber in a hyaluronic acid solution with a concentration of 40 g / L and a molecular weight of 15-20 kDa for 30 min, and set aside.

[0040] (2) Select silk fibroin, diatomaceous earth powder and ethylene-vinyl acetate powder in a mass ratio of 1:10:20 for physical mixing, shake well and add to the feeding cylinder.

[0041] (3) Set the nozzle temperature of the melt direct writing spinneret to 120℃, heat the blend in the feed cylinder to melt it into a molten state at high temperature, and use the air pressure generated by the air pump as a power source to extrude the molten mixture from the spinning nozzle; place the carbon fiber impregnated with hyaluronic acid obtained in step (1) (wherein the mass ratio of carbon fiber to silk fibroin is 60:1) on the collecting plate in advance, set the nozzle loading voltage to 3.0KV, and the collecting plate movement speed to 35mm·s. －1 With a nozzle inner diameter of 0.2 mm and a distance of 5 mm from the nozzle to the collection plate, carbon fiber melt-written silk fibroin composite material can be prepared by uniformly spraying the spun fibers onto the carbon fiber using melt-writing technology.

[0042] Measurements showed that the fiber diameter of the composite material was 5–10 μm, and the porosity of the mesh material was 87.2%.

[0043] Example 3:

[0044] The method for preparing carbon fiber melt-written silk fibroin composite material for microbial culture provided by this invention is as follows:

[0045] (1) Immerse the carbon fiber in a hyaluronic acid solution with a concentration of 40 g / L and a molecular weight of 15-20 kDa for 30 min, and set aside.

[0046] (2) Select silk fibroin, diatomaceous earth powder and polyvinyl chloride powder in a mass ratio of 1:5:15 for physical mixing, shake well and add to the feeding cylinder.

[0047] (3) Set the nozzle temperature of the melt direct writing spinneret to 200℃, heat the blend in the feed cylinder to melt it into a molten state at high temperature, and use the air pressure generated by the air pump as a power source to extrude the molten mixture from the spinning nozzle; place the carbon fiber impregnated with hyaluronic acid obtained in step (1) (wherein the mass ratio of carbon fiber to silk fibroin is 50:1) on the collecting plate in advance, set the nozzle loading voltage to 2.5KV, and the collecting plate movement speed to 30mm·s. －1 The nozzle has an inner diameter of 0.2 mm and a distance of 2 mm from the nozzle to the collection plate. By using melt direct writing technology to uniformly spray the spinning onto the carbon fiber, carbon fiber melt direct writing silk fibroin composite material can be prepared.

[0048] Comparative Example 1:

[0049] Comparative Example 1 did not utilize the melt-write method or use carbon fiber as a substrate. Instead, it directly physical blended the same mass of silk fibroin, diatomaceous earth, and polymer particles as in Example 1 at a mass ratio of 1:1 to 10, and then used them as raw materials for microbial culture.

[0050] Comparative Example 2

[0051] The addition of silk fibroin in step (2) of Example 1 was omitted, and the rest of the preparation methods and parameters were the same as in Example 1, and the composite material was prepared.

[0052] Comparative Example 3

[0053] The addition of diatomaceous earth powder in step (2) of Example 1 was omitted, and the rest of the preparation methods and parameters were the same as in Example 1, and the composite material was prepared.

[0054] Comparative Example 4

[0055] Step (1) of Example 1 is omitted. The remaining preparation methods and parameters are the same as in Example 1, and the composite material is prepared.

[0056] 50g of carbon fiber melt-written silk fibroin composite materials prepared in Examples 1-3 and Comparative Examples 1-4 of this invention were unpacked into bundles and placed in 250mL Erlenmeyer flasks. Sludge taken from the secondary sedimentation tank of a wastewater treatment plant was inoculated into the Erlenmeyer flasks to achieve a sludge concentration of 6000mg / L. KNO3 was added to adjust the NO3 concentration. - The nitrogen concentration was increased to 30 mg / L, the total volume was 200 mL, and the mixture was incubated at 25 °C for 7 days.

[0057] After cultivation, the amount of major bacterial phyla in the sludge was measured, and the results are shown in [the table below]. Figure 2 As can be seen, the carbon fiber melt-written silk fibroin composite materials prepared in Examples 1-3 can all provide a good growth environment for microbial growth and culture. Silk fibroin and polymers have good biocompatibility and are biodegradable, providing nutrients for microorganisms, promoting cell synthesis and microbial growth, and exhibiting good biocompatibility. The presence of diatomaceous earth provides minerals for microbial growth, which is beneficial to microbial growth and reproduction. Implanting carbon fibers with hyaluronic acid solution before direct writing and coating with silk fibroin and other substances can achieve a sustained-release effect of hyaluronic acid, thereby extending the service life of the microbial carrier.

[0058] The mixture in Comparative Example 1, due to the presence of unbundled carbon fibers, could not provide a strong specific surface area to obtain larger attachment sites, which was not conducive to achieving efficient microbial culture.

[0059] Comparative Example 2 lacked silk fibroin, resulting in a lack of protein nutrients during microbial culture, which affected the long-term growth of microorganisms.

[0060] Comparative Example 3 lacked diatomaceous earth, which led to a lack of minerals in the microbial culture process, thus preventing the microorganisms from reproducing and growing effectively.

[0061] In Comparative Example 4, the lack of hyaluronic acid did not significantly affect the microbial culture during short-term microbial culture.

[0062] When the microbial culture time of Example 1 and Comparative Example 4 was extended to 14 days, the number of microorganisms cultured in the composite material of Comparative Example 4 was significantly less than that cultured in the composite material of Example 1 (a difference of 10^2 times). This shows that impregnating carbon fibers with hyaluronic acid solution and then directly writing and covering them with silk fibroin and other substances can achieve a sustained-release effect of hyaluronic acid, thereby extending the service life of the microbial carrier.

[0063] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, and for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims

1. A method for preparing carbon fiber melt-written silk fibroin composite material for microbial culture, characterized in that, Includes the following steps: (1) Mix silk fibroin, diatomaceous earth powder and polymer powder evenly, and add them to the feed cylinder as direct writing raw material; The blend in the feed cylinder is heated and melted into a molten state at high temperature, and the air pressure generated by the air pump is used as a power source to extrude the molten mixture from the spinning nozzle. Carbon fibers soaked in hyaluronic acid solution were placed on a collection plate, and the spinning process of step (2) was sprayed onto the carbon fibers using melt direct writing technology. After cooling and drying, carbon fiber melt direct writing silk fibroin composite material was prepared.

2. The preparation method according to claim 1, characterized in that, The mass ratio of silk fibroin, diatomaceous earth powder, and polymer powder is 1:5-20:10-20.

3. The preparation method according to claim 1, characterized in that, The mass ratio of silk fibroin, diatomaceous earth powder, and polymer powder is 1:5:

15.

4. The preparation method according to claim 1, characterized in that, The polymer powder is any one or more of polycaprolactone, ethylene-vinyl acetate, and polyvinyl chloride.

5. The preparation method according to claim 4, characterized in that, The polymer powder is polycaprolactone or ethylene-vinyl acetate.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In step (3), the concentration of the hyaluronic acid solution is 20-50 g / L.

7. The preparation method according to claim 6, characterized in that, The molecular weight of the hyaluronic acid is 10-20 kDa.

8. The preparation method according to claim 1, characterized in that, The high temperature mentioned in step (2) is 80-200℃; the nozzle loading voltage is 2.5-3.5KV; the distance from the nozzle to the collecting plate is 1-6mm; and the collecting plate movement speed is 30-36mm·s. -1 The nozzle inner diameter is 0.2-0.5mm.

9. The carbon fiber melt-written silk fibroin composite material prepared by the preparation method according to any one of claims 1 to 8.

10. A microbial culture apparatus comprising the carbon fiber melt-written silk fibroin composite material of claim 9.

11. A method for culturing microorganisms, characterized in that, The method uses the carbon fiber melt-written silk fibroin composite material for microbial culture as described in claim 9 as a carrier, or the microbial culture device as described in claim 10 for cultivation.

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