CN113666995A - Method for quickly and efficiently preparing native tussah silk fibroin powder - Google Patents
Method for quickly and efficiently preparing native tussah silk fibroin powder Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 10
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 10
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- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims abstract description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 4
- 235000011089 carbon dioxide Nutrition 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
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- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 7
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- ZJZXSOKJEJFHCP-UHFFFAOYSA-M lithium;thiocyanate Chemical compound [Li+].[S-]C#N ZJZXSOKJEJFHCP-UHFFFAOYSA-M 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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Abstract
The invention discloses a method for quickly and efficiently preparing native tussah silk fibroin powder. Boiling the washed tussah cocoon shell in sodium carbonate solution, repeatedly boiling, removing sericin, cleaning and drying; and (2) putting the dried tussah silk fibroin fibers into a crushing tank, putting the rotary beads, covering the cover tightly, then immersing the tank into liquid nitrogen, a refrigerator or dry ice for freezing, fixing the fully frozen crushing tank on a stainless steel adapter, starting a freezing crusher for crushing, and sieving to prepare the tussah silk fibroin powder with micron-sized particle size. The method has the characteristics of rapidness, high efficiency, no pollution and the like, can retain the biological function and the nano-scale natural conformation structure of the tussah silk fibroin, overcomes various problems brought by the existing method for processing the tussah silk fibroin powder, and has wide application.
Description
Technical Field
The invention relates to a preparation method of a silk fibroin protein product, in particular to a method for quickly and efficiently preparing native tussah silk fibroin protein powder, belonging to the field of natural polymers.
Background
The tussah silk cocoon mainly comprises silk fibroin and sericin, and the sericin can be removed in the degumming process to obtain fibrous tussah silk fibroin fiber. Tussah silk fibroin is a typical natural protein polymer, and has excellent biocompatibility, degradability and low immunogenicity. The repetitive structure fragment of the primary sequence of the tussah silk fiber is similar to the primary sequence of the spider silk traction silk, is rich in alanine and beta-folded structures, and has a multi-layer structure from micro to nano, so that the tussah silk fiber is endowed with super-strong mechanical strength, such as high modulus, tensile strength and the like. Meanwhile, the tussah silk fibroin contains more essential amino acids required by a human body and highly repeated RGD (Arg-Gly-Asp) tripeptide, and the RGD tripeptide has important physiological functions and can promote the adhesion and growth of cells, so that the tussah silk fibroin has potential application values in the field of biological medicine, such as the application of a tussah protein porous scaffold in cell culture, tissue repair, drug carriers and the like.
At present, the annual output of tussah raw material cocoons in China is more than 8.0 ten thousand tons, the tussah silk yield accounts for 3/4 of the total world output, the tussah silk protein resource is very rich, and the production of the tussah silk fibroin powder by utilizing the tussah cocoons is an effective way for improving the economic benefit and the functional utilization of the tussah silk fibroin powder. For a long time, due to the limitation of extraction and processing modes of tussah silk fibroin, the main application direction of tussah silk is still concentrated in the textile industry, and the maximum utilization value of the tussah silk is not exerted. In recent years, although silkworm silk fibroin powder, as a new protein source, plays an important role in the fields of cosmetics, biomedicines, food preservation and environmental protection, there are few reports on the application of tussah silk fibroin having better biological properties than silkworm silk fibroin in the field of biomedicines. The reason is that the fiber form of the tussah silk fiber is not easy to damage and break due to the existence of spider silk-like amino acid fragments, high beta-folding structures and multi-level structures, so that subsequent processing and material remanufacturing are hindered, and the processing mode of the domestic silkworm fibroin protein powder is difficult to succeed if the processing mode is simply applied.
The current methods for preparing the tussah silk fibroin powder mainly comprise physical, chemical and biological methods. The chemical treatment method generally comprises treating degummed tussah fiber with acid (hydrochloric acid), alkali (sodium hydroxide), neutral salt (such as lithium thiocyanate) at high temperature, hydrolyzing, dialyzing, purifying, and spray drying. The method not only destroys the natural primary structure and RGD sequence of tussah silk fibroin, reduces the denaturation and biocompatibility of tussah silk peptide chain, loses biological functionality, but also has the problems of long processing time, complex operation process, high cost, environmental pollution and the like. For example, in a Chinese patent with the application date of 2013, 3 and 20 and the publication number of CN102977381A, tussah silk fibroin microspheres and a preparation method thereof are disclosed, wherein tussah silk fibroin microspheres (powder particles) with controllable particle sizes are prepared by dissolving tussah fibers in a LiSCN solvent and adjusting the pH value of the solution to 3-6. The residue of the toxic solvent LiSCN used in the above preparation method can lose the activity of protein particles and reduce biocompatibility, and also can bring a heavy burden to the environment. The biological treatment method mainly comprises an enzymolysis method, wherein the tussah silk fibroin fiber is subjected to enzymolysis by using active protease and then subjected to spray drying treatment. However, the method requires strict temperature and pH conditions to prevent enzyme inactivation, and simultaneously needs the auxiliary action of chemicals in the enzymolysis process, the preparation process is slow, the efficiency and the yield are both low, and the further application of the tussah silk fibroin powder prepared by the enzymolysis method is limited. The method can overcome some defects of chemical and biological methods for preparing silk fibroin powder by physical methods such as high temperature and high pressure method without using any chemical. However, the high-temperature and high-pressure method has harsh preparation conditions (about 200 ℃, 1.96MPa), high requirements on equipment and high energy consumption, and needs to be combined with other various physical means (such as a high-pressure homogenizer) to prepare the tussah silk fibroin powder (the particle size is often larger than 100 micrometers), and in the process, a carbonization product is easily generated in a high-temperature environment, so that the primary structure and the peptide chain structure of the tussah silk fiber are damaged.
In summary, since the tussah silk fiber has superior mechanical strength and complex structural composition, the preparation of tussah silk fibroin powder becomes a problem to be solved urgently in research and industry, and a method capable of retaining the native structure and functional polypeptide structure of tussah silk fibroin and preparing the native tussah silk fibroin powder rapidly, efficiently and in a pollution-free manner does not exist so far, so that the yield of the tussah silk fibroin powder is limited, and the industrial added value is reduced.
Disclosure of Invention
In order to overcome the defects of environmental unfriendliness, complicated preparation method and damage to the natural structure of silk in the prior art for preparing the tussah silk fibroin powder, the invention provides a method for quickly and efficiently preparing the native tussah silk fibroin powder. The method of the invention is specially aimed at the treatment of tussah, obtains the native tussah silk fibroin powder with excellent biological performance by a specific crushing mode, has the characteristics of rapidness, high efficiency, no pollution and the like, can retain the biological function and the nano-scale natural conformation structure (native structure) of the tussah silk fibroin, and overcomes a plurality of problems brought by the existing method for processing the tussah silk fibroin powder.
In order to achieve the purpose, the technical scheme provided by the invention adopts the following specific steps:
(1) putting the cleaned tussah cocoon shells into a sodium carbonate solution with the mass fraction of 0.75% to be boiled for 0.5h, taking out the tussah cocoon shells, repeatedly boiling again, removing sericin, cleaning, and putting into an oven to be dried to obtain dried tussah silk fibroin fibers;
(2) and carrying out specific crushing treatment on the dried tussah silk fibroin fibers to obtain micron-sized tussah silk fibroin powder.
The processing raw materials in the step (1) not only comprise tussah silk fibroin fibers, but also can be used for silk types such as tussah cocoon shells, tussah cocoon leftovers and the like, but are not limited to the above.
The step (2) is specifically as follows:
and (2) putting the dried tussah silk fibroin fibers into a rotary cover type stainless steel crushing tank, putting a stainless steel rotary bead, covering the cover tightly, then immersing the tank into liquid nitrogen, a low-temperature refrigerator or dry ice for freezing, fixing the fully frozen crushing tank on a stainless steel adapter, starting a low-temperature freezing crusher for crushing, and sieving to obtain the tussah silk fibroin powder with the micron-sized particle size.
The frequency of the freezing crusher is set to be 30-60Hz, and the time is 10-120 s.
The invention aims at the tussah silk fibroin fiber and adopts a specific crushing mode of matching a crushing tank and a rotating bead to process, thereby realizing micron-scale crushing without generating nano-scale damage.
The freezing temperature in the step (2) is lower than-30 ℃, and the preferable temperature range is-80 ℃ to-196 ℃.
And (3) sieving the tussah silk fibroin powder in the step (2) by different meshes, and screening out micron-sized tussah silk fibroin powder with different particle sizes and uniform particles.
The tussah silk fibroin powder obtained in the step (3) contains an undamaged RGD polypeptide sequence.
The RGD polypeptide sequence contained in the tussah silk fibroin powder obtained by the invention is not damaged, and the early adhesion and growth of mammalian cells can be promoted.
The tussah silk fibroin fiber is plasticized and embrittled by freezing the tussah silk fibroin fiber at low temperature, and the natural tussah silk fibroin fiber is directly crushed into tussah silk fibroin powder completely in a physical and mechanical mode. The fibrous silk can be processed into micron-sized powder within the shortest 10s, and the prepared tussah silk fibroin powder has no toxic or side effect on mammalian cells and can remarkably promote the early adhesion of the cells.
Specifically, in a high-frequency oscillation mode, the stainless steel rotating beads in the crushing tank do circular arc radial oscillation, the frozen tussah fibers can be impacted at a high speed, and the silk fibers can be quickly damaged by the generated huge friction force, low-temperature freezing force and impact force, so that micron-sized powder is obtained. Due to the low-temperature freezing process, the tussah silk fibroin fiber can be protected from being carbonized under the high-frequency action, and the structural integrity of protein molecules is kept, so that the obtained tussah silk fibroin powder has a nano fibril form, an integral beta-folding conformation and an RGD polypeptide structure.
The method has the advantages of simple and convenient operation, low cost and no environmental pollution, and the prepared tussah silk fibroin powder has wide application prospect in the fields of tissue engineering, chemical engineering, biological medicine, food, environmental protection and the like.
Compared with the prior art, the invention has the following outstanding characteristics:
(1) the invention does not need any other chemical product to treat the tussah silk fibroin fiber, and can prepare the natural tussah silk fibroin fiber into the tussah silk fibroin powder only by a simple operation mode.
(2) The method is processed at low temperature, so that the degradation of tussah silk fibroin molecules due to pressure and heating can be effectively prevented, and the nano-scale structure of tussah silk fibroin is more completely reserved.
(3) Compared with the prior art for preparing the tussah fibroin powder, the method has the advantages of high biological safety, simple and convenient operation, high efficiency and no pollution to the environment.
(4) The powder prepared by the invention keeps the natural structure of the fibroin and has good biocompatibility, and has wide application prospect in the fields of food, chemical industry, medicine, biological materials and the like.
Drawings
Fig. 1 is a schematic diagram of tussah silk fibroin powder obtained in example 1.
Fig. 2 is a scanning electron microscope image of tussah silk fibroin powder obtained in example 1.
Fig. 3 is a scanning electron microscope image of tussah silk fibroin powder obtained in example 2.
FIG. 4 is the attached drawing of cell adhesion of the fibroblasts on the tussah silk fibroin powder/silk fibroin membrane in example 2.
FIG. 5 is a graph showing the cell adhesion number of fibroblasts on the tussah silk fibroin powder/silk fibroin membrane in example 2.
FIG. 6 is a schematic diagram of a silkworm silk powder obtained in example 4.
FIG. 7 is a scanning electron micrograph of Bombyx mori silk powder obtained in example 4.
Detailed Description
The present invention is further illustrated by the following examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
The examples of the invention are as follows:
example 1
The method for quickly and efficiently preparing the native tussah silk fibroin powder in the embodiment sequentially comprises the following steps:
(1) and (3) putting the cleaned tussah cocoon shells into 0.75% sodium carbonate solution, boiling for 0.5h, repeating the step once, removing sericin, cleaning, and drying in a 50 ℃ oven for later use.
(2) And (3) putting 1.0g of tussah silk fiber degummed in the step (1) into a crushing tank, putting 1 stainless steel rotating bead, and tightly covering a cover. And immersing the crushing tank filled with the sample into liquid nitrogen for precooling for 2 minutes, and quickly fixing the crushing tank on a stainless steel adapter of a crusher after taking out. Setting the frequency at 30Hz and the time at 30s to obtain tussah silk fibroin powder.
(3) And (3) transferring the tussah silk fibroin powder obtained in the step (2) into a standard sieve with 100 meshes, and sieving and separating to obtain the tussah silk fibroin powder with the particle size of 50-100 micrometers (as shown in figures 1 and 2).
Example 2
(1) And (3) putting the cleaned tussah cocoon shells into 0.75% sodium carbonate solution, boiling for 0.5h, repeating the step once, removing sericin, cleaning, and drying in a 50 ℃ oven for later use.
(2) And (3) taking 0.2g of tussah silk fiber degummed in the step (1), putting into a crushing tank, putting 1 stainless steel rotating bead, and tightly covering with a cover.
And immersing the crushing tank filled with the sample into liquid nitrogen for precooling for 10 minutes, and quickly fixing the crushing tank on a stainless steel adapter of a crusher after taking out. Setting frequency at 60Hz and time at 60s to obtain tussah silk fibroin powder (shown in FIG. 3).
(3) And (3) transferring the tussah silk fibroin powder obtained in the step (2) into a standard sieve with 100 meshes, and sieving and separating to obtain the tussah silk fibroin powder with the particle size of 20-50 microns.
(4) And (3) taking the tussah silk fibroin powder in the step (3), fully dispersing the tussah silk fibroin powder in the silk fibroin solution according to the mass ratio of the tussah silk fibroin powder to the silk fibroin solution with the concentration of 5% being 1: 99 and 5: 95, and naturally airing for 24 hours at normal temperature to obtain the blending film of the tussah silk fibroin powder and the silk fibroin solution.
(5) Inoculating the blend membrane in the step (4) with L929 fibroblasts, culturing for three hours in an incubator, and observing the early adhesion condition of the cells on different blend membranes. The number of cell adhesion on pure silk fibroin membranes (SF) is low; when the tussah silk fibroin powder in the blended film accounts for 1 wt% (SFT1), the adhesion quantity is higher than that of the pure silk fibroin film; when the tussah silk fibroin powder in the blend membrane is 5 wt% (SFT5), the cell adhesion number is significantly higher than that of SFT1 membrane (as shown in fig. 4 and 5, the arrows in fig. 4 indicate cells). Therefore, the tussah silk fibroin powder prepared by the method can effectively promote early adhesion of cells on the surface of the material.
The product of the invention can effectively prevent the degradation of tussah silk fibroin molecules due to pressure and heating, and completely reserve the protein structure and the molecular structure of the tussah silk fibroin in a nanometer scale.
Example 3
(1) Weighing 0.5g of cut tussah cocoon shell, putting into a crushing tank, putting into stainless steel rotary beads, and tightly covering.
(2) And (2) immersing the crushing tank filled with the sample in the step (1) into a refrigerator with the temperature of-80 ℃ for precooling for 30 minutes, and quickly fixing the crushing tank on a stainless steel adapter of a crusher after being taken out. Setting the frequency at 60Hz and the time at 60s, and preparing the tussah cocoon powder.
(3) And (3) transferring the tussah powder obtained in the step (2) into a standard sieve with 100 meshes, and sieving and separating to obtain the tussah cocoon powder with the particle size of about 20-100 microns.
Example 4
(1) Weighing 1.0g of the silkworm silk fiber, filling into a crushing tank, putting 1 stainless steel rotary bead, and tightly covering a cover.
(2) And (3) immersing the crushing tank filled with the sample into dry ice for precooling for 1 hour, and quickly fixing the crushing tank on a stainless steel adapter of a crusher after taking out. The frequency was set at 30Hz and the time was set at 120s, and the silk powder was prepared.
(3) And (3) transferring the silk powder obtained in the step (2) into a standard sieve with a size of 100 meshes, and sieving and separating to obtain the silkworm silk powder with the particle size of about 100 microns (as shown in figures 6 and 7).
Claims (6)
1. A method for quickly and efficiently preparing native tussah silk fibroin powder is characterized by comprising the following steps:
(1) putting the washed tussah cocoon shells into a sodium carbonate solution with the mass fraction of 0.75% for boiling, taking out the tussah cocoon shells, repeating the boiling again, removing sericin, washing, and putting into an oven for drying to obtain dried tussah fibroin fibers;
(2) and carrying out specific crushing treatment on the dried tussah silk fibroin fibers to obtain micron-sized tussah silk fibroin powder.
2. The method for rapidly and efficiently preparing the native tussah silk fibroin powder as claimed in claim 1, is characterized in that: the step (2) is specifically as follows: and (2) putting the dried tussah silk fibroin fibers into a crushing tank, putting the rotary beads, covering the cover tightly, then immersing the tank into liquid nitrogen, a refrigerator or dry ice for freezing, fixing the fully frozen crushing tank on a stainless steel adapter, starting a freezing crusher for crushing, and sieving to prepare the tussah silk fibroin powder with micron-sized particle size.
3. The method for rapidly and efficiently preparing the native tussah silk fibroin powder as claimed in claim 2, is characterized in that: the frequency of the freezing crusher is set to be 30-60Hz, and the time is 10-120 s.
4. The method for rapidly and efficiently preparing the native tussah silk fibroin powder as claimed in claim 2, is characterized in that: the freezing temperature in the step (2) should be lower than-30 ℃.
5. The method for rapidly and efficiently preparing the native tussah silk fibroin powder as claimed in claim 2, is characterized in that: and (3) sieving the tussah silk fibroin powder in the step (2) by different meshes, and screening out micron-sized tussah silk fibroin powder with different particle sizes and uniform particles.
6. The method for rapidly and efficiently preparing the native tussah silk fibroin powder as claimed in claim 2, is characterized in that: the tussah silk fibroin powder obtained in the step (3) contains an undamaged RGD polypeptide sequence.
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