JP2015093857A - Water-insoluble silk protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a silk protein which can impart transparency and flexibility without weakening samples by water-insolubilizing a fibroin film or fibroin nanofiber.SOLUTION: The silk protein related to the invention has flexibility and transparency as well as water-insolubility. It is obtained by immersing a silk protein film obtained by a dry solidification of an organic solvent comprising a silk protein solution or silk protein, or silk protein nanofiber by electro-spinning the organic solvent comprising the silk protein solution or silk protein with a glycerol/alcohol mixed solution, subsequently taking it out from the glycerol/alcohol mixed solution, and drying in a standard state.

Description

  The present invention relates to a silk protein subjected to water insolubilization and softening treatment.

  Silkworm-derived silk protein (also sometimes referred to as fibroin or simply silk) is a highly safe material for living organisms, and has been used as a surgical suture to be implanted in living organisms in Japan. It is also possible to use fibroin as an enzyme or pharmaceutical immobilization material. A pure fibroin aqueous solution can be obtained by dissolving fibroin fiber, which is produced by scouring silkworm-like or silk-like fibers made by silkworms, and removing sericin in a concentrated aqueous solution of neutral salt, and replacing it with cellulose dialysis membrane. . When this fibroin aqueous solution is spread on the surface of the polymer film and dried and solidified, a transparent fibroin film can be produced. When an organic solvent in which the fibroin film is dissolved is electrospun, fibroin nanofibers can be produced.

  Fibroin fiber can be dissolved in a heated neutral salt solution such as calcium chloride, calcium nitrate, lithium bromide, lithium thiocyanate. That is, a fibroin aqueous solution prepared by dissolving fibroin fibers in a heated concentrated neutral salt aqueous solution is placed in a cellulose dialysis membrane, both ends are sewn with thread, and replaced with tap water or pure water for 4-5 days. By removing ions based on neutral salts, a pure fibroin aqueous solution can be obtained. For example, by spreading this fibroin aqueous solution on the substrate surface of a polyethylene membrane, air drying and evaporating the moisture, a transparent fibroin membrane can be produced. After adding alcohol to the fibroin aqueous solution and freeze-drying, fibroin sponge Can be manufactured. In addition, as described below, fibroin nanofibers (also referred to as silk nanofibers) can be produced by electrospinning fibroin sponge, fibroin membrane or fibroin fiber dissolved in trifluoroacetic acid (TFA).

Since fibroin membranes, fibroin sponges, or fibroin nanofibers produced by the method described above are soluble in water, the use of these fibroin is limited. In order to increase the utility value as various industrial materials, it is necessary to treat the fibroin material into a water-insolubilized sample that does not dissolve in water.
In order to make the fibroin material insoluble in water, a method is adopted in which the fibroin material is dipped in an organic solvent such as alcohol for a predetermined time and then lightly dried at room temperature. According to this method, cohesion between fibroin molecules increases with alcohol treatment, and as a result, the fibroin material becomes insoluble in water. However, in the alcohol treatment, when the alcohol enters the fibroin material and the cohesion between the fibroin molecules increases and the fibroin crystallizes, the fibroin material becomes stiff and the flexibility of the sample is lost, as described in Experimental Example 3 described later. However, since there is a problem that the sample film is cut only by being pulled a little, it is a problem in applying fibroin for various uses. In addition, it is a fibroin film | membrane and a fibroin nanofiber that the extent by which a raw material becomes brittle by alcohol treatment is especially remarkable.

  Fibroin nanofibers can be produced by electrospinning. First, the principle of electrospinning will be explained. An anode electrode is attached to a storage tank containing a fibroin solution of a predetermined concentration, and a cathode plate (collector) is installed at a certain distance from the anode electrode. A high voltage is applied between the cathode and the anode to generate an electric attractive force between the electrodes. When this electric attractive force is equal to or greater than the surface tension of the polymer solution, a polymer jet in a mist state is jetted from the nozzle of the spinneret toward the cathode plate by electrostatic force, and nanofibers are laminated on the cathode plate. As a result, a fine fibroin nanofiber having a fiber diameter on the order of nano to micrometer can be produced. The specific surface area of nanofibers is extremely large, and thus fibroin nanofibers that can be produced can be used in a wide range of fields such as regenerative medicine engineering, wound care and other healthcare fields, biotechnology fields, and energy fields.

  Fibroin nanofibers immediately after electrospinning are highly soluble in water enough to absorb water in the air, so it is necessary to insolubilize the nanofibers for a wide range of applications. It is essential. The simplest and most effective conventional method for insolubilizing silk in water is to immerse the sample in an ethanol or methanol solution, which is a poor solvent for silk, and lift the sample from the alcohol solution and dry at room temperature. As described in Experimental Example 3, since the sample becomes brittle when the silk nanofibers are soaked in methanol or ethanol because they are insolubilized by immersion in an alcohol solution, this is the most important point to consider when applying silk. Met.

As another technique for insolubilizing the fibroin membrane with water, a technique for insolubilizing the sample membrane with water by adding glycerin to a fibroin aqueous solution has been disclosed (Non-patent Document 1). Fibroin fiber is dissolved in a heated neutral salt solution and dialyzed into a fibroin solution prepared by directly adding a predetermined amount of a glycerin / ethanol mixed solution. A technique for producing a silk film rich in a degree is disclosed (Non-Patent Document 2). In addition, in order to modify the water absorption of natural proteins, the change in the hygroscopicity of the sample film prepared by immersing the peanut protein film in an aqueous solution of glycerin, polyethylene glycol, or polypropylene glycol with different pH acts as a plasticizer. It has been studied (Non-Patent Document 4). Furthermore, it was clarified that the fibroin membrane becomes flexible by measuring the strength and elongation of a silk fibroin membrane obtained by adding a predetermined amount of glycerin to a fibroin aqueous solution and solidifying it by drying (Non-patent Document 5).

  The conventional technology for water-insolubilizing silk films is a common treatment method in that a water-insoluble silk film is produced by directly adding a predetermined amount of glycerin to a silk aqueous solution and then drying and solidifying the sample (non-patented). Literature 1-5). According to the conventional method, if the amount of glycerin added to the silk aqueous solution is small, there is no effect of water insolubilization. If the amount of glycerin is large, the sample becomes water insoluble, but excess glycerin adheres to the inside and outside of the sample. It was. Furthermore, when glycerin is added directly to the silk aqueous solution, the interaction between silk and glycerin is strong, so that the silk is rapidly solidified at the molecular level, or the silk is rapidly denatured. There was a problem of being inferior. It was necessary to add an appropriate amount of glycerin that would produce the maximum effect in making the amount of glycerin added to the silk minimum and water insolubilizing the silk.

Shenzhou Lu, Xiaoquin Wang, Qiang Lu, Xiaohi Zhang, Jonathan A. Kluge, Neha Uppal, Fiorenzo Omenetto, and David Kaplan, Insoluble and flexible silk films containing glycerol, Biomacromolecules, 2010, 11, 143-150. Mariana F. Silva, Mariana A. de Moraes, Grinia M. Nogueira, AndreaC.D. Rodas, Olga Z. Higa, Marisa M. Beppu, Glycerin and Ethanol asaddtiives on silk fibroin films: Insoluble and Malleable Films, Journal of Applied Plymer Science , 2013, DOI: 10.1002 / APP.38139. A. Jangchuld and M.S.Chinnan, Properties of Peanut Protein Film: Sorption Isotherm and Plasticizer Effect, Lebensm.-Wiss.u-Technol., 32, 89-94 (1999) Tsutsumi Kazuyo, Ogawa Akane, Fukuda Tsubasa, Morita Hiroshi, Elucidation of Factors Insoluble in Fibroin Membrane, Journal of Japan Food Industry Association, Vol. 11, (2), 105-112 (2010) Shinichiro Hiraide, Softening of silk fibroin film, Nissan Zoshi, 66 (2), 138-140 (1997)

According to the above-mentioned technical disclosure, all samples are fibroin derived from rabbits, and in order to produce a water-insoluble fibroin membrane, a predetermined amount of glycerin is directly added to an aqueous fibroin solution, and then, for example, the surface is coated on the substrate membrane surface. The method of expanding and drying and solidifying is adopted.
That is, as described above, when glycerin is added to the fibroin aqueous solution, the glycerin has a strong interaction with the fibroin molecule and coagulates the fibroin molecule, or rapidly forms a hydrogen bond between the fibroin molecules. Since the fibroin molecule rapidly transitions to the β structure, the type of fibroin film that dissolves in water becomes insoluble in water.

  When glycerin is added directly to an aqueous fibroin solution by a conventional method, if the amount of glycerin added is too small, the degree of fibroin modification is not sufficient, and the desired water insolubilization effect is not exhibited, or the amount of glycerin added is too large. Fibroin molecules coagulate in an aqueous solution of fibroin and a fibroin membrane with the desired characteristics cannot be produced.If too much glycerin is added, the fibroin membrane becomes water-insoluble, but the highly viscous glycerin becomes excessive and the fibroin membrane Glycerin is contained inside and on the surface, which is inconvenient for application.

  The present invention has been made to solve these problems, and is obtained by electrospinning a fibroin membrane obtained by first spreading a fibroin aqueous solution on a substrate membrane and drying and solidifying it, or fibroin prepared using silk as a raw material. The desired water-insolubilized fibroin material can be manufactured by immersing fibroin nanofibers in a glycerin / alcohol mixed solution, and the sample is fragile by making fibroin insoluble in water while using the minimum amount of glycerin. The object is to provide a silk protein that can be imparted with flexibility.

  The present invention has been completed by discovering a method different from the conventional method, that is, a fibroin membrane or a fibroin nanofiber is first manufactured, and then a new method for immersing these fibroin samples in a glycerin / alcohol mixed solution is discovered. It is. That is, an aqueous solution of rabbit or wild silk fibroin or a fibroin membrane obtained by spreading and solidifying an organic solvent obtained by dissolving rabbit or wild silk fibroin fiber / fibroin sponge on the surface of the substrate membrane can be used as a sample. In addition, a certain amount of glycerin / alcohol mixed solution of a certain concentration is applied to a rabbit or wild silk fibroin nanofiber produced by electrospinning an organic solvent prepared by dissolving wild silk fibroin aqueous solution or rabbit or wild silk fibroin fiber / fibroin sponge. It is characterized in that the silk membrane or silk nanofiber is water-insolubilized by dipping, removing a sample from the glycerin / alcohol mixed solution, and lightly drying at room temperature.

In the present invention, in addition to glycerin / alcohol, in addition to glycerin / alcohol, a desired effect can be obtained by using a mixed solution of polyethylene glycol / alcohol or polypropylene glycol / alcohol, but preferably a glycerin / alcohol mixed solution is used. Easy to use.
If desired, the glycerin component adhering to the inside or the surface of the sample by dipping in the glycerin / alcohol mixed solution can be completely removed by dipping the material in alcohol for a predetermined time.

The silk protein according to the present invention is characterized by having water insolubility and flexibility and transparency.
Flexibility refers to whether the sample shows flexibility when it is folded by hand, or the sample is cracked or broken at the folding site, or whether the sample is sufficiently flexible and bends well. Visually evaluated.
Transparency is a visual observation of the transparency of the sample. When the sample is brought into contact with the printed matter printed with the Mincho font written in 10.5 points, the characters appear transparent through the sample. The evaluation was based on the difference in whether the characters were not visible because the sample was not transparent.
The silk protein is characterized by using silk derived from rabbit or wild silk, and the silk protein is in the form of membrane or nanofiber.

Silk proteins that can be used in the present invention include silkworm silk thread obtained from Bombyx mori larvae and silk fiber products of rabbit silkworm, larvae of silkworm, tengu, erimo, muga, and Shinju moths belonging to wild silkworms Both wild silk and silk fiber products obtained from can be used. It may also be a silk protein accumulated in the silk gland within the larvae of a rabbit or wild silkworm.
Since glue-like sericin is attached around the silk thread of a rabbit or wild silkworm, it is preferable to produce an aqueous fibroin solution after removing sericin as follows. First, after fibroin fibers formed by removing sericin by scouring with a heated alkaline aqueous solution are dissolved in a neutral salt aqueous solution, a fibroin aqueous solution can be produced by a dialysis treatment using a cellulose dialysis membrane to replace water. Alternatively, water-soluble fibroin in the silk gland extracted from the silkworm body of rabbit or wild silkworm can be used in the same manner.

  Fibroin membranes are prepared by dissolving fibroin fibers or fiber products in heated neutral salt solutions such as calcium chloride, calcium nitrate, lithium bromide, and lithium thiocyanate and then dialyzing them with a cellulose dialysis membrane. Thereafter, it can be produced by spreading on a polyethylene substrate film and solidifying it by drying.

In the neutral salt compound, the conditions for dissolving silk thread will be described with reference to lithium bromide having high silk solubility. In order to dissolve fibroin fiber using lithium bromide, the lithium bromide concentration may be about 8.0 to 9.8M, and the dissolution temperature may be about 45 to 70 ° C. The dissolution temperature is preferably 60 ° C. or lower. If the melting temperature is excessively high, the molecular weight of the silk protein decreases, and there is a risk that the high molecular weight of the material is lost. If the molecular weight is excessively reduced, the moldability of silk becomes poor.
When dissolving fibroin fiber with neutral salt solution, if the temperature of the neutral salt solution becomes higher than necessary, there is a concern that the molecular weight of fibroin will decrease and the high molecular weight of the material may be lost. The temperature must not be higher than necessary or the dissolution time should not be too long. In order to dissolve fibroin fibers efficiently, lithium salts excellent in solubility of fibroin fibers are preferably used among neutral salts, and lithium bromide is particularly preferable. If lithium bromide is 8M or more, preferably 8.5M or more, the fibroin fiber can be completely dissolved by treatment at 55 ° C. for about 15 minutes.

  Fibroin fibers are dissolved in these heated concentrated neutral salt aqueous solutions, put into cellulose dialysis membranes, both ends are sewn with thread, and placed in room temperature tap water or pure water for 4-5 days to replace the aqueous solution. A pure fibroin aqueous solution is obtained by completely removing ions based on the neutral salt. When this aqueous solution of fibroin is spread on the surface of a substrate such as a polyethylene membrane and blown and dried as necessary, water is evaporated on the sample and a transparent fibroin membrane can be prepared. A fibroin sponge can be prepared by freeze-drying the aqueous fibroin solution.

Add an organic acid such as hydrochloric acid, acetic acid or formic acid to the aqueous fibroin solution to adjust the pH of the aqueous fibroin solution to 2.5, add ethanol to coagulate the fibroin and leave it at 5 ° C for 1 week to freeze the fibroin aqueous solution. When dried, a fibroin sponge is formed.
Fibroin nanofibers can be produced by electrospinning a fibroin TFA solution obtained by dissolving a fibroin film or silk sponge with trifluoroacetic acid (hereinafter TFA).

Since the fibroin membrane or the fibroin nanofiber immediately after being manufactured by the above method is easily dissolved in water so as to absorb and dissolve the humidity of the sample environment, the water insolubilization treatment is performed as follows.
That is, it is immersed for a predetermined time in a mixed solution of glycerin / alcohol, polyethylene glycol / alcohol, or polypropylene glycol / alcohol having different composition ratios (hereinafter simply referred to as composition ratios) that can be expressed by volume ratio of fibroin membrane or fibroin nanofibers. Thus, the fibroin membrane or fibroin nanofiber can be insolubilized in water, and thus the sample membrane or sample nanofiber can be given flexibility and transparency.

A specific method for water-insolubilizing silk protein will be described with reference to an example of a glycerin / alcohol mixed solution that can be used to insolubilize fibroin.
If it is a rabbit or silkworm fibroin membrane, it is immersed in a glycerin / alcohol mixed solution with a composition ratio of 75/25 to 100/0 for 2 to 10 minutes, preferably 3 to 8 minutes. If the sample is left in the state for 20 minutes or more and the crystallinity of the sample is increased, the water insolubility of the sample is reliably improved.
In the case of savage or savage fibroin nanofiber, after immersion treatment in a glycerin / alcohol mixed solution having a composition ratio of 50/50 to 10/90 for 2 to 10 minutes, preferably 3 to 8 minutes, the mixture is taken out from the mixture at room temperature. By leaving the sample in the standard state for 20 minutes or more and increasing the crystallinity of the sample, the water insolubility of the sample is reliably improved.

  When fibroin nanofibers are immersed in a glycerin / alcohol mixed solution, if the alcohol concentration is too low, the fibroin nanofibers can be quickly immersed in a glycerin / alcohol mixed solution, for example, a 100/0 glycerin / alcohol mixed solution. It swells and becomes a muddy state, and the sample cannot be pulled up from the glycerin / alcohol mixed solution.

  When a fibroin membrane produced from rabbit fibroin or nanofibers that can be produced by electrospinning is immersed in a glycerin / alcohol mixed solution for water insolubilization treatment, if the glycerin concentration is high, glycerin tends to adhere excessively to the sample surface. In order to apply the water-insolubilized fibroin, it is preferable to remove the glycerin contained in the sample by immersing the sample in the alcohol solution for a short time after the water insolubilization treatment soaking in the glycerin / alcohol mixed solution. As a method for removing glycerin contained in the sample, a method of immersing in a mixed solution of alcohol / water having a composition ratio of 40/60 to 100/0 for 2 to 10 minutes is preferable. More preferably, from an economic point of view, a mixed solution of alcohol / water having a composition ratio of 40/20 to 100/0 is used, and the immersion time is 3 to 8 minutes.

According to the present invention, it becomes possible to insolubilize a sample by immersing a fibroin membrane or fibroin nanofibers in a glycerin / alcohol, polyethylene glycol / alcohol, or polypropylene glycol / alcohol mixed solution. Can be granted.
When treated with a glycerin / alcohol mixed solution, the sample contains a trace amount of glycerin, which cannot be avoided. However, in order to apply fibroin as an industrial material, it is necessary to remove all glycerin from the sample as desired. is there. For this purpose, a water-insolubilized sample obtained by immersing the sample in a glycerin / alcohol mixed solution for 2 to 10 minutes must be immersed in the alcohol solution for 2 to 10 minutes as a post-treatment step, and thus the glycerin contained in the sample Can be completely removed.

  The sample immersed in water and insolubilized with a glycerin / alcohol mixed solution according to the present invention is flexible, unlike a sample in which water is insolubilized with a known alcohol, and is not fragile even when bent, and has good transparency. There is a feature that there is. Even if fibroin is implanted in the body, it does not become an antigen and does not react with antigen-antibody, and is a biocompatible material that is a biocompatible material that decomposes when subjected to the action of enzymes in the body. Therefore, the water-insoluble fibroin material of the present invention can also be used as an organ adhesion separation membrane during surgery. Furthermore, on the fibroin surface, useful cells are easy to attach and proliferate and have biodegradability, so that it can be widely applied as a leading material in the field of regenerative medicine as a material for producing a cell sheet.

According to the present invention, fibroin membrane (silk protein membrane) and fibroin nanofiber (silk protein nanofiber) are immersed in a mixed solution of glycerin / alcohol, polyethylene glycol / alcohol, or polypropylene glycol / alcohol to insolubilize the sample. Transparency and flexibility can be imparted.
Since fibroin is a body-compatible material and has biochemical properties such as being a biodegradable material, the silk protein of the present invention can also be used as an organ adhesion separation membrane at the time of surgery. It can be widely applied as a leading material in the field of regenerative medicine as a material for making sheets.

It is a graph which shows the viscoelasticity measurement (DMA) result of the fibroin nanofiber which carried out the water insolubilization process and was produced by electrospinning a rabbit fibroin aqueous solution. It is a SEM image of rabbit fibroin nanofibers prepared from rabbit silk aqueous solutions with sample concentrations of 22.4, 27.3, and 47.1 wt%. It is the SEM image (a) of the nanofiber before being immersed in glycerin, and the SEM image (b) of the nanofiber after being immersed in glycerin for 5 minutes and subjected to a drying treatment. a: fibroin nanofiber immediately after electrospinning, b: sample in which a is immersed in glycerin, c: sample in which a is immersed in a glycerol / ethanol mixed solution (composition ratio 50/50), d: a is immersed in ethanol It is the photograph of the processed sample. a: FTIR spectra for fibroin nanofibers immediately after electrospinning, b: a soaked with a mixed solution of glycerin / ethanol (composition ratio 50/50), and c: a soaked b with ethanol. a Fibroin nanofibers immediately after electrospinning are immersed in a glycerin / ethanol mixed solution (composition ratio 90/10) for 5 minutes, b Fibroin nanofibers immediately after electrospinning are mixed with a glycerin / ethanol mixed solution (composition ratio 50/50) 5 5 minutes immersion treatment with fibroin nanofibers just after electrospinning with glycerin / ethanol mixed solution (composition ratio 10/90), d 5 minute immersion treatment of fibroin nanofibers immediately after electrospinning with glycerin, e electrospinning It is a FTIR spectrum about the fibroin nanofiber immediately after. a Fibroin nanofiber immediately after electrospinning, b Fibroin nanofiber immediately after electrospinning is immersed in a glycerin / ethanol mixed solution (composition ratio 90/10), then immersed in ethanol for 5 minutes, c Fibroin nanofiber immediately after electrospinning After dipping with glycerin / ethanol mixed solution (composition ratio 50/50), dipping with ethanol for 5 minutes, d After dipping the fibroin nanofiber immediately after electrospinning with glycerin / ethanol mixed solution (composition ratio 10/90) , FTIR spectrum for e-glycerin, treated with ethanol for 5 minutes.

Hereinafter, although an example explains an example of the present invention in detail, the present invention is not limited to these examples.
The chemicals and measurement methods used in the experimental examples and the sample preparation methods used will be described.

Reagents used:
The reagents used are all manufactured by Wako Pure Chemical Industries, Ltd., and the Lot numbers are as follows.
・ Polyethylene glycol (PEG) Lot WEH1268 Molecular weight 200,000 ・ Glycerin Lot CDE1348
・ Ethyl alcohol Lot AWQ4906
・ Sodium carbonate Lot PDR6423
・ Calcium chloride Lot WEE4700

Mechanical properties:
The strength and elongation of the fibroin nanofiber were stretched until the sample was cut, and evaluated by the sample strength and elongation at the time of cutting. A universal material testing machine (RTC1250A) manufactured by Orientec Co., Ltd. was used as a measuring device. The measurement conditions are a sample size of 1 cm × 2 cm, a film thickness of 45 to 72 μm, and a pulling speed of 1 mm / min.

Fourier transform infrared absorption spectrum:
The infrared absorption spectrum of the silk fibroin nanofiber was observed using an FTIR (Fourier transform infrared absorption spectrum) measuring device manufactured by PerkinElmer. The measurement wave number is 2000 to 400 cm ⁻¹ and the number of repeated measurements is 20 times.

Viscoelasticity measurement (DVA):
The loss elastic modulus (E "), which is the viscoelastic property of the sample, was measured by DMA. With DMA, the temperature dependence of glass transition temperature and elastic modulus by temperature dispersion measurement can be measured. E" corresponds to molecular motion. The characteristics are reflected. The heating rate was 10 ° C./min.

(1) Rabbit fibroin aqueous solution preparation method The fibroin fiber formed by treating the sericin adhering to the surface of the silkworm with a boiled alkaline aqueous solution and removing the sericin (this is called scouring treatment) at 80 ° C. It was immersed in a heated 60 wt% calcium chloride aqueous solution for 1 hour to completely dissolve the fibroin fiber so that the concentration of the aqueous fibroin solution was 20 wt / v%. After dissolution, the solution was placed in a cellulose dialysis membrane and desalted by replacing with distilled water for 3 days to prepare a pure silk solution with a concentration of 6.8 wt%.

(2) Concentration of Rabbit Fibroin Aqueous Solution The above-mentioned 6.8 wt% silk aqueous solution was too thin as shown in Experimental Example 1 and could not be electrospun. Therefore, the concentration was increased as follows. Place 30 mL of 6.8 wt% silk aqueous solution in cellulose dialysis membrane, and place cellulose dialysis membrane containing silk aqueous solution in 100 mL 50 wt% polyethylene glycol (average molecular weight 20,000, 20000 ± 5000) aqueous solution and replace with polyethylene glycol aqueous solution. As a result, the aqueous fibroin solution was concentrated.

(3) Fibroin membrane Rabbit fibroin fiber is prepared by immersing rabbit silk fibers in 0.11% sodium carbonate aqueous solution 50 times its weight and removing silk sericin covering the circumference of silk yarn for 1 hour at 98 ° C. did. Dissolve this rabbit fibroin fiber in lithium thiocyanate aqueous solution, put this aqueous solution into cellulose dialysis membrane, wrap both ends with sewing thread and put it in room temperature tap water for 2 days to completely remove lithium ions, pure rabbit fibroin An aqueous solution was prepared. The aqueous rabbit fibroin aqueous solution thus prepared was spread on a polyethylene membrane and dried by blowing air to prepare a transparent rabbit fibroin membrane.

(4) Fibroin sponge Rabbit fibroin fiber was boiled in 0.07% sodium carbonate solution for 1 hour to remove silk sericin, and 6.5 g of the resulting fibroin fiber was dissolved in an aqueous lithium bromide solution and distilled at 5 ° C. Was dialyzed for 4 days to remove lithium ions and bromide ions to obtain an aqueous fibroin solution having a concentration of 1.8 wt%. When 20 mL of 99% methanol is added to 300 mL of this 1.8 wt% fibroin aqueous solution and allowed to stand at room temperature, the fibroin aqueous solution gels and precipitates. This was put into a freeze-drying apparatus and dried under reduced pressure to produce a fibroin sponge.

(5) Production of Rabbit Fibroin Nanofiber Fibroin nanofiber can be produced by electrospinning using an aqueous fibroin solution. Fibroin nanofibers can be produced by electrospinning fibroin TFA prepared by dissolving fibroin sponge in TFA.

(6) Silkworm fibroin membrane and silkworm fibroin sponge The silkworm silk with completely different chemical structure from the above rabbit silk thread is immersed in 0.1% sodium peroxide aqueous solution 50 times the weight of silk thread and treated at 98 ° C for 1 hour. Then, silk sericin and tannin covering the periphery of the silk thread were removed, and silkworm fibroin fiber was prepared. A cocoon fibroin fiber from which sericin and tannin have been removed in advance is completely dissolved in an aqueous lithium cyanate solution at 60 ° C. to produce an cocoon fibroin aqueous solution. This aqueous solution is placed in a dialysis membrane made of cellulose, and both ends are tied together with sewing threads. It was replaced with tap water for 4 days to completely remove lithium ions, and a pure sputum fibroin aqueous solution was prepared. The soot fibroin aqueous solution thus prepared was spread on a polyethylene membrane and dried by blowing to produce a soot fibroin membrane. Moreover, this cocoon fibroin aqueous solution was lyophilized | freeze-dried and the cocoon fibroin sponge was manufactured.柞 蚕 Fibroin nanofibers can be produced by dissolving fibroin in TFA and electrospinning it.

(7) Electrospinning Spinning Fibroin nanofibers using an electrospinning apparatus manufactured by Kato Tech Co., Ltd. using a rabbit fibroin sponge prepared from the rabbit rabbit fibroin aqueous solution described above and a rabbit trifluoroacetic acid (THA) solution dissolved Manufactured. A Terumon bevel needle (22G × 1 1/2 ″ (0.70 × 38 mm) manufactured by Terumo Corporation was used as a nozzle for the spinneret.
As a polymer storage tank, a lock type / spiral type 5 mL top plastic syringe manufactured by Top Co., Ltd. was used.
In addition, when there is no description of electrospinning conditions in an experiment example, the following conditions were employ | adopted. The spinning voltage was 20 kv, the spinning speed was 0.5 mm / min, and the spinning distance was 18 cm.

(8) Average fiber diameter of nanofibers Fibroin nanofibers produced by electrospinning are sputtered with gold ions using an ion coater device to give a gold coating having a thickness of 300 angstroms. 10 types of nanofiber images with different field of view are photographed with a scanning electron microscope at a magnification of 50,000 times, and the images are printed with a printer. Ten fiber diameters were measured for each visual field based on the scale described in the image.

(Experimental Example 1) Nanofiber using Rabbit Fibroin Aqueous Solution Rabbit yarn is scoured to remove sericin, and the fibroin fiber prepared at a sample concentration of 20 wt / v% is placed in a calcium chloride aqueous solution. Heat the sample for 1 hour to completely dissolve the sample. Subsequently, this was put into a cellulose dialysis membrane (fraction 14,000) and dialyzed against pure water for 3 days to prepare an aqueous fibroin solution. In order to adjust the concentration of this fibroin aqueous solution, it was placed in a cellulose dialysis membrane and then replaced with a 50 wt% PEG aqueous solution (molecular weight 20,000 ± 5,000). By changing the time of substitution with PEG, the aqueous fibroin solution can be concentrated.
The concentration of the fibroin aqueous solution was measured as follows. The mass (g) of a certain amount of silk aqueous solution is measured, completely dried at 105 ° C. to completely evaporate water, and the mass (g) of the dried solidified product is measured. The concentration (wt%) of the sample was calculated by calculating the mass (g) of the dried solidified product / the mass (g) of the silk aqueous solution.
The conditions for electrospinning are as follows. The applied voltage was 20 kV, the spinning distance was 18 cm, the spinning speed was 20 μl / min, the temperature was 30 ° C., and the relative humidity was 10 RH%.
Table 1 shows the results of studying whether or not fibroin nanofibers can be produced by electrospinning an aqueous fibroin solution adjusted to a concentration of 6.8 to 43.1 wt%.

In Table 1, the state at the time of electrospinning (also referred to as ES) during the production of fibroin nanofibers was evaluated in the following two categories.
X: At the time of electrospinning, the sample jet is not ejected from the spinning port, and nothing is attached to the collector.
○: During electrospinning, the sample jet was ejected well from the spinneret, and fibroin nanofibers with a fine fiber diameter were laminated on the collector, and the ES state was good.

  From Table 1, the following was confirmed. When the fibroin aqueous solution concentration was 14.5 wt% or less, fibroin nanofibers could not be produced even by electrospinning. Fibroin nanofibers could be produced by electrospinning when the concentration of fibroin aqueous solution was over 22.4 wt%. The fiber diameter of the obtained fibroin nanofiber was 800 to 900 nm. The preferred concentration of the fibroin aqueous solution for producing the fibroin nanofibers by electrospinning the silk aqueous solution was 22.4-43.1%.

(Experimental example 2) Dissolving rabbit fibroin membrane in TFA and electrospinning Fibroin membrane or fibroin sponge made from rabbit silk thread dissolved in TFA at room temperature and electrospinning 10wt% fibroin TFA solution to fine-diameter fibroin nanofibers Could be manufactured. The fiber diameter of the obtained fibroin nanofiber was 300 to 400 nm. Next, the rabbit fibroin membrane was electrospun using 8 wt% or 12 wt% fibroin TFA dissolved in TFA, and the spinning state was observed. In each case, fibroin nanofibers with a fine diameter could be produced.
When electrospun 10% by weight of 柞 蚕 fibroin membrane prepared from 柞 蚕 fibroin fiber or wtfibroin sponge dissolved in TFA, 10 柞 蚕% 柞 蚕 fibroin nanofibers were produced. The fiber diameter of the obtained fibroin nanofiber was 200-250 nm.

(Experimental Example 3) Water Insolubilization Treatment of Fibroin Nanofibers Produced from Rabbit Fibroin Aqueous Solution When a rabbit's fibroin aqueous solution described in Experimental Example 1 was electrospun, the fibroin nanofibers immediately after spinning were dissolved in water. In order to develop a wide range of applications of nanofibers, it is essential to insolubilize fibroin nanofibers in water. Therefore, silk nanofibers were immersed in a mixed solution of glycerin / ethanol having different concentrations at room temperature for 5 minutes, and then a sample was taken out from the mixed solution system of glycerin / ethanol and lightly dried at room temperature for 20 minutes. The fibroin nanofiber thus lightly dried was immersed in 100% (0/100) ethanol for 5 minutes to remove glycerin contained in the sample. The characteristics and flexibility of the surface of fibroin nanofibers before (after Gly removal) and after (after Gly removal) removal of glycerin by ethanol treatment were examined by visual observation and tentacle observation, respectively. The composition ratio of the glycerin / ethanol mixed solution means the volume ratio of glycerin and ethanol. The examination results are shown in Table 2.

In Table 2, “Before Gly removal” and “After Gly removal” mean that fibroin nanofibers were insolubilized with glycerin / ethanol mixed solution, and then before treatment to remove glycerin with ethanol (before Gly removal) and after (After removing Gly).
The items “Sample characteristics” and “Flexibility” in Table 2 were evaluated in the following categories.
Sample characteristics:
+: Sample surface is sticky ±: Sample surface is slightly sticky
-: The sample surface is not sticky.
Flexibility:
+: A sample of 1 cm x 2 cm is folded in half and bent along the fold line so that the sample is folded without breaking and has sufficient flexibility
-: A 1 cm x 2 cm sample is folded in half, and along the fold line, the sample becomes brittle and partially breaks.

  Silk fibroin nanofibers obtained by electrospinning fibroin TFA in which the silkworm silk fibroin membrane and the silkworm silk fibroin sponge produced by the method described in Experimental Example 2 are dissolved in TFA are immersed in a mixed solution of glycerin / ethanol and water insolubilized. It was. When the characteristics and flexibility of the sample were evaluated by the same method as described above, the same result as that of the water insolubilized sample of fibroin nanofibers produced from the rabbit fibroin aqueous solution was obtained.

As shown in Table 2, when water insolubilization treatment of rabbit fibroin nanofibers with a glycerin / ethanol mixed solution, when the composition ratio becomes 50/50 or more and the glycerin concentration increases, glycerin adheres to the surface of the silk nanofibers, However, when the composition ratio was 50/50 or less, the surface of the silk nanofiber was not sticky due to glycerin and adhered to glycerin.
The flexibility of the fibroin nanofibers was not changed before and after the ethanol treatment for removing glycerin, and the flexibility was maintained after the treatment as before the treatment.
Since the characteristic FTIR spectrum of glycerin appears at 1030 cm ⁻¹ as described in Experimental Example 16, the composition ratio of the glycerin / ethanol mixed solution is 50/50 or more, that is, fibroin nano treated with the mixed solution with increased glycerin concentration. A peak of 1030 cm ^-1 appears in the FTIR spectrum of the fiber, and it can be confirmed that glycerin is attached to the sample. It was confirmed that the glycerin contained in the sample could be completely removed by simply immersing the glycerin-containing sample in alcohol for 2 to 10 minutes, and the sticky feeling of the sample surface was eliminated.
Even when the sample was immersed in a 25/75 or 0/100 glycerol / ethanol mixed solution having a high ethanol concentration, the flexibility of the sample was maintained.
In addition, when fibroin nanofibers are treated with 100% (0/100) ethanol, water insolubilization occurs, but the thickness of the sample increases, making it brittle and brittle. Fibroin is treated with water by immersing with a glycerin / ethanol mixed solution according to the present invention. You can claim inventive step to insolubilize.

(Experimental example 4) Water insolubilization treatment of rabbit and silkworm fibroin membranes using PEG: Changes in sample properties with or without ethanol treatment after PEG / ethanol treatment rabbit silk fibroin aqueous solution or silkworm fibroin aqueous solution and rabbit as described in Experimental Example 1 An aqueous fibroin solution was spread on the surface of the polyethylene membrane and evaporated to dryness to produce a fibroin membrane of rabbit or silkworm.
Polyethylene glycol (PEG) with a molecular weight of 200,000 is dissolved in water to prepare a 5 wt% PEG aqueous solution, ethanol is added thereto, and the concentration ratio of PEG and ethanol is 100/0, 75/25, 50/50, A 25/75, 0/100 PEG / ethanol mixed solution was prepared, and the fibroin membranes of rabbits and rabbits were immersed for 5 minutes. Thereafter, the sample was taken out from the PEG / ethanol mixed solution and lightly dried at room temperature for 20 minutes. If the fibroin membranes of rabbits and rabbits are immersed in a mixed solution of PEG / ethanol (100/0), the sample membrane swells, and when the sample is removed from the mixed solution of PEG / ethanol (100/0), the form of the sample is lost. I have. However, the morphology of the sample was not lost by removing the fibroin membranes of rabbit and rabbit from the PEG / ethanol mixed solution of composition ratio 75/25, 50/50, 25/75.

FT FTIR spectrum of 柞 蚕 fibroin membrane that has been insolubilized by immersing the 柞 蚕 fibroin membrane in PEG / ethanol (0/100) shows a peak at 1655 cm ^-1 as in the case of water insolubilized sample. However, no change was observed in the molecular form of the sample film. When immersed in a mixed solution of PEG / ethanol (75/25, 50/50, 25/75) for 5 minutes, a peak is observed at 1630 cm ^-1 in the sample, and the molecular form of the fibroin film changes from the random coil form to the beta form. And the sample membrane became insoluble in water. The molecular form of the sample even if the fibroin membrane soaked in PEG / ethanol (75/25, 50/50, 25/75) is soaked in ethanol to remove the PEG contained in the sample. Remained transformed into β-form.
In addition, even though the rabbit fibroin membrane was soaked in PEG / ethanol (0/100), the molecular morphology of the sample did not change, but PEG / ethanol (75/25, 50/50, 25/75) mixed When immersed in the solution for 5 minutes, the molecular form of the sample changed from the random coil form to β-type, and the sample film became insoluble in water. The rabbit fibroin membrane soaked in PEG / ethanol (75/25, 50/50, 25/75) was further treated with ethanol to remove the PEG contained in the sample. It remained changing.

(Experimental example 5) Manufacture of a cocoon fibroin nanofiber, and water insolubilization processing The silkworm silk thread which is a field cocoon was scoured, the cocoon fibroin sponge was manufactured, it melt | dissolved in TFA, and electrospinning produced the fibroin nanofiber.
The soot fibroin nanofibers immediately after being produced by electrospinning are extremely soluble in water.す る In order to insolubilize fibroin nanofibers in water, after immersing in glycerin / ethanol mixed solution of different concentration for 5 minutes, remove the sample from glycerin / ethanol mixed solution and lightly dry at room temperature for 20 minutes. Fibroin nano before (after Gly removal) and after (after Gly removal) removal of glycerin after removing glycerin contained in the sample by immersing the wild silk fibroin nanofibers thus prepared in 100% (0/100) ethanol for 5 minutes The characteristics and flexibility of the fiber were examined visually and with tentacles. Table 3 shows the obtained results. The meanings of notations such as + and − in the items of sample characteristics and flexibility are the same as those in Experimental Example 3.

As shown in Table 3, in order to insolubilize the fibroin nanofibers, when immersed in a glycerin / ethanol mixed solution having a different composition ratio, the surface of the fibroin nanofibers does not become sticky even if the glycerin concentration is high. This was in contrast to the stickiness on the fibroin nanofiber surface.
In addition, the flexibility of sputum fibroin nanofibers was not changed before and after the treatment to remove glycerin contained in the sample by ethanol treatment after water insolubilization treatment, similar to the rabbit fibroin nanofiber shown in Experimental Example 3. Even after the treatment, the same flexibility as before the treatment was maintained.

(Experimental example 6) Swelling state of sample during glycerin / ethanol aqueous solution treatment Rabbit fibroin nanofibers (BSF nf) prepared in Experimental example 1 were insolubilized in water, so the samples were mixed in glycerin / ethanol mixed solutions with different composition ratios. Was immersed for 5 minutes, and when the sample was pulled up, it was investigated whether the sample shape collapsed by evaluating the swelling state of the sample. The same experiment was performed on rabbit fibroin membrane (BSF membrane) and rabbit fibroin fiber (BSF fiber) as target samples. In addition, the same experiment was carried out for soot fibroin nanofiber (TSF nf), soot fibroin membrane (TSF membrane), and soot fibroin fiber (TSF fiber).
Table 4 shows the evaluation results of the degree of swelling of various samples.

The swelling state in Table 4 was evaluated in the following two categories.
Swelling state:
-: The sample swelled significantly in the glycerin / ethanol mixed solution, and the shape of the sample was broken when the sample was taken out from the glycerin / ethanol mixed solution.
+; The sample was removed from the glycerin / ethanol mixed solution without swelling in the glycerin / ethanol mixed solution.

In the present invention, the process of putting the sample into a glycerin / ethanol mixed solution having a different composition ratio in order to insolubilize the sample is an important aspect of the invention. To efficiently produce a water-insolubilized sample, glycerin / ethanol It is important to control the composition ratio of the mixed solution. When silk nanofibers were placed in a glycerin solution with a composition ratio of 100/0, glycerin entered the sample and the sample swelled significantly, and the sample became muddy, making it impossible to pull up the sample from the glycerin solution.
From Table 4, it was found that the fibroin fibers of rabbits and silkworms did not collapse at all even when the glycerin concentration was different.
Rabbit and silkworm fibroin membranes were swollen by the treatment with a glycerin / ethanol mixed solution having a composition ratio of 100/0, and the sample shape collapsed.
Rabbits and silkworm fibroin nanofibers were crushed with 75/25 to 100/0 glycerin / ethanol treatment, so the water insolubilization treatment of rabbits and silkworm fibroin nanofibers was glycerin with a composition ratio of 50/50 to 90/10. / Ethanol mixed solution for at least 2 minutes, preferably 3-8 minutes.
The water insolubilization treatment of the rabbit and silkworm fibroin membrane may be performed by immersing with a glycerin / ethanol mixed solution having a composition ratio of 75/25 to 100/0 for at least 2 minutes, preferably 3 to 8 minutes.

(Experimental Example 7) Mechanical Properties of Rabbit Fibroin Nanofibers Fibroin nanofibers prepared by electrospinning 22.4 wt% fibroin aqueous solution described in Experimental Example 1 were immersed in glycerin / ethanol mixed solutions of different concentrations for 5 minutes. Water insolubilization treatment was performed. A sample was taken from the glycerin / ethanol mixed solution and lightly dried at room temperature for 20 minutes. Then, in order to remove glycerin adhering to the sample surface, it was immersed in a 100% concentration ethanol solution for 5 minutes. The strength and elongation of fibroin nanofibers dried at room temperature were measured. The strength and elongation of silk nanofiber (As spun) immediately after electrospinning were also measured. The results obtained are shown in Table 5.

As shown in Table 5, the silk nanofiber immediately after electrospinning exhibited an excellent elongation characteristic of 25.8% and strength of 0.1 MPa, but the strength of the sample was small.
On the other hand, the strength of rabbit fibroin nanofibers, which were insolubilized in water using a glycerin / ethanol mixed solution and then removed the glycerin on the sample surface by ethanol treatment, increased in all samples compared to As-spun. .
The sample machine was prepared by immersing a silkworm silk fibroin nanofiber produced by electrosping using TFA as a solvent in the method described in Experimental Example 2, and then drying the sample for 20 minutes at room temperature. As a result, the same tendency as the measurement result of the fibroin nanofiber produced by electrospinning the fibroin aqueous solution described in Experimental Example 7 was observed.
The elongation of rabbit fibroin nanofibers was higher than that of As-spun after treatment with a glycerin / ethanol mixed solution with a glycerin composition ratio of 10/90 or more. When the composition ratio of glycerin was 10/90, the elongation of the As-spun sample increased specifically to 76%. It should be noted that when As-spun nanofibers were immersed in glycerin with a composition ratio of 100/0, the viscosity of glycerin was too high, and the nanofibers became muddy and the sample was broken and could not be measured. It was not described in 5.

(Experimental example 8) DMA measurement of rabbit fibroin nanofiber
The viscoelastic properties of the samples were measured by DMA. Here, the loss elastic modulus (E ″) of fibroin nanofibers was measured. The measurement temperature range was 20 to 240 ° C. With DMA, the temperature dependence of glass transition temperature and elastic modulus by temperature dispersion measurement can be measured. E "reflects the properties corresponding to molecular motion.
Fibroin nanofibers produced by electrospinning the 22.4 wt% rabbit rabbit fibroin aqueous solution of Experimental Example 1 were immersed in glycerin / ethanol mixed solutions having different concentrations for 5 minutes to insolubilize in water. Thereafter, a sample was taken out from the glycerin / ethanol mixed solution, and viscoelasticity measurement (DMA) was performed on fibroin nanofibers which were lightly dried at room temperature for 20 minutes. The obtained results are shown in FIG.

It can be seen from FIG. Immediately after electrospinning, fibroin nanofiber (a) fibroin molecules begin to move above 140 ° C, and the molecular motion is highest at 180 ° C. In sample (b), in which sample (a) is immersed in a glycerin / ethanol mixed solution (composition ratio 50/50) and water insolubilized, molecular motion begins to occur at 120 ° C. or higher, and the maximum motion is 210. ° C. In the sample (c) obtained by immersing (b) in ethanol and removing the glycerin contained in the sample, molecular motion occurs at 80 ° C. and 140 ° C., and the maximum motion is 209 ° C.
The temperature at which the molecular motion of sample (b) or sample (c) reaches the highest temperature of 30 ° C or higher is higher than that of fibroin nanofiber (a) immediately after electrospinning. It suggests that crystallization is performed by immersion treatment, and that thermal molecular motion occurs at a high temperature accompanying crystallization, that is, the structure of fibroin is stabilized.

(Experimental Example 9) SEM Observation of Rabbit Fibroin Nanofibers SEM images of rabbit fibroin nanofibers immediately after electrospinning of a rabbit silk solution having concentrations of 22.4, 27.3, and 47.1 wt% are shown in FIG. This rabbit fibroin nanofiber is a silk nanofiber immediately after electrospinning the rabbit silk solution described in Experimental Example 1.
Figures 2 (a), (b), and (c) are SEM images of rabbit fibroin nanofibers prepared from rabbit silk solutions with sample concentrations of 22.4, 27.3, and 47.1 wt%, respectively. The average fiber diameters were 800, 1000, and 1800 nm, respectively, as is clear in Experimental Example 10. From the SEM image of FIG. 2, the cross-sectional shape of rabbit fibroin nanofibers was not circular but often had a flat “ximen” shape.

(Experimental example 10) Relationship between fiber diameter of rabbit fibroin nanofiber and sample concentration Immediately after electrospinning a silk solution of rabbit with different concentrations, that is, an SEM image of fibroin nanofiber not subjected to glycerol / ethanol treatment or ethanol treatment at all. Based on this, the fiber diameter of the fibroin nanofiber was measured. The results obtained are shown in Table 6.

In Table 6, “NT” means that electrospinning was impossible and SEM measurement was not performed.
From Table 6, the fiber diameter of the fibroin nanofiber is 800 nm when the silk aqueous solution concentration is 22.4 wt%. The fiber diameter increases as the silk aqueous solution concentration increases, and when the silk aqueous solution concentration is 43.1 wt%, It became a scale order.
On the other hand, the fiber length of silkworm silk fibroin nanofibers obtained by dissolving the silk nanofiber and rabbit silk fibroin membrane described in Experimental Example 2 in TFA and electrospinning was 200 to 250 nm.

Experimental Example 11 SEM Image of Rabbit Fibroin Nanofiber Treated with Glycerin Nanofibers obtained by electrospinning a rabbit silk solution with a sample concentration of 43.1 wt% were immersed in 100% glycerin for 10 minutes. After completion of the immersion, the glycerin attached to the sample was carefully wiped off with a Kimwipe. Thereafter, the sample was allowed to stand at room temperature for 24 hours and dried, but the sample was wet and a trace amount of glycerin remained in the sample.
FIG. 3 (a) is an SEM image of the nanofiber before being immersed in glycerin, and FIG. 3 (b) is an SEM image of the nanofiber after being immersed in glycerin for 5 minutes and dried.
The fiber diameter of the rabbit fibroin nanofibers did not change much before and after the immersion treatment with glycerin, but there was a tendency for a very thin film of glycerin to adhere to the surface of the silk nanofibers after the immersion treatment.

(Experimental example 12) Sample form accompanying glycerol or methanol treatment
Immediately after the electrospinning of the 22.4wt% rabbit fibroin aqueous solution, the fibroin nanofibers were immersed in 100/0 glycerin, glycerin / ethanol mixed solution (composition ratio 50/50), and 0/100 ethanol for 5 minutes. Thereafter, the sample was pulled up from each solution and lightly dried at room temperature. The state of the fibroin nanofiber after drying was photographed. The obtained result is shown in FIG.
As can be seen from FIG. When fibroin nanofiber (a) is treated with 100/0 glycerin (b), the sample size (size) does not change, but becomes slightly transparent. In the glycerin / methanol mixed solution treatment (c), the sample size does not change, but the transparency of the fibroin nanofibers increases. When the 0/100 methanol treatment (d) was performed, the size of the fibroin nanofibers contracted significantly and the original shape could not be maintained, and the sample became brittle and opaque.

(Experimental Example 13) Transparency of Rabbit Fibroin Nanofiber Treated with Glycerin / Ethanol Mixture Solution or Methanol
Immediately after electrospinning 22.4wt% rabbit rabbit fibroin aqueous solution (immediately after ES), treatment with 100% glycerin (Gly), glycerin / ethanol mixed solution (Gly / E (50/50)), 100% ethanol treatment The transparency of the sample subjected to (E) for 5 minutes was visually observed. Table 7 shows the observation results.

In Table 7, the transparency of the sample was evaluated in the following three categories.
+: The characters on the print paper that are transparent and printed are visible.
++: The characters on the printed paper can be seen with extremely high transparency.
-: Characters on the print paper with low transparency are not visible.

As shown in Table 7, fibroin nanofibers obtained by electrospinning a 22.4 wt% silk aqueous solution and 0/100 ethanol-treated fibroin nanofibers were both opaque, but glycerin or glycerin / ethanol ( The transparency of fibroin nanofibers soaked with 50/50) mixed solution increased.
The transparency and visual observation of the samples by various treatments described in Table 7 are as follows.
The sample treated with 100% ethanol (E) was opaque and the sample became hard and brittle and bent during the drying process.
The fibroin nanofibers whose transparency was improved by treatment with 100/0 glycerin (G) and Gly / E (50/50) were immersed in 0/100 ethanol. As is apparent from the measurement results of the FTIR spectrum described in Experimental Example 15 below, glycerin contained in the sample was removed by immersion treatment in 0/100 ethanol, but the sample remained in an opaque state.

(Experimental example 14) Water-insoluble rabbit fibroin nanofibers treated with a glycerin / ethanol mixed solution Rabbit fibroin nanofibers immediately after electrospinning a silk solution with sample concentrations of 22.4, 27.3, and 47.1 wt% After being immersed in a mixed solution of ethanol (composition ratio: 90/10, 70/30, 50/50, 30/70, 10/90, 0/100) for 5 minutes, then treated with 0/100 ethanol The solubility of rabbit silk fibroin nanofibers in water was investigated.
Of the samples subjected to the above treatment, fibroin nanofibers obtained by immersing a sample immersed in a mixed solution of glycerin / ethanol (composition ratio 0/100) with ethanol were immersed in water at room temperature for 24 hours. As a result, the sample did not become water-insoluble when treated with 100/0 glycerin.
On the other hand, a sample in which fibroin nanofibers are immersed in a mixed solution of glycerin / ethanol (composition ratio: 90/10, 70/30, 50/50, 30/70, 10/90) is immersed in water at room temperature for 24 hours. Even if it was made to do, the weight loss of a sample did not occur at all, and it was confirmed that the sample became water-insoluble. Even if water-insolubilized fibroin nanofibers are dipped in a mixed solution of glycerin / ethanol with a composition ratio of 10/90 to 90/10, the sample size does not change and the sample can be given flexibility. , Transparency increased. These effects were most effective when the glycerol concentration was 10 / 90-90 / 10.
In addition, after the silk nanofiber used in Experimental Example 3 was immersed in a mixed solution of glycerin / ethanol having different concentrations for 2 minutes and then immersed in water for 24 hours in the same manner as in Example 14, a decrease in the sample weight was observed. It was confirmed that the sample was insoluble in water.

(Experimental example 15) Glycerin adhering to sample by treatment with glycerin / ethanol mixed solution Fibroin nanofiber produced by electrospinning 22.4 wt% silk aqueous solution described in Experimental example 1, and this fibroin nanofiber immersed in 100/0 glycerin In order to clarify whether glycerin adheres to fibroin nanofibers that have been subjected to a treatment to be immersed in a 0/100 ethanol solution for 5 minutes, FTIR spectra were measured. FIG. 5 is the obtained FTIR spectrum.
In the sample (b) in which the fibroin nanofiber (a) immediately after electrospinning was dipped in a glycerin / ethanol mixed solution (composition ratio 50/50), a clear peak appeared at a wave number of 1030 cm ^-1 , which was experimental. It is a peak due to glycerin as discussed in FIG. On the other hand, no peak at 1030 cm ⁻¹ is observed in the fibroin nanofiber (a) immediately after electrospinning and the sample (c) obtained by treating the nanofiber (a) with ethanol. This means that when the sample (a) is treated with a glycerin / ethanol mixed solution, the glycerin adheres to the sample (a) or the sample contains glycerin, but the sample (b) is immersed in the ethanol solution for 5 minutes. Sample (c) suggests that glycerin has been removed. It was confirmed that all the ethanol contained in the sample was removed even when the sample (b) was immersed in an ethanol solution for 2 minutes.

According to FIG. 5, the fibroin nanofiber (a) immediately after electrospinning produced an amide I band at 1645 cm ⁻¹ and an amide II band at 1535 cm ⁻¹ . The fibroin nanofiber (c) treated with ethanol after water insolubilization treatment showed an amide I band at 1622 cm ⁻¹ and an amide II band at 1516 cm ⁻¹ . On the other hand, for the fibroin nanofiber (b) treated with the glycerin / ethanol mixed solution, the amide I band appeared at 1622 cm ^-1 , the amide II band at 1516 cm ^-1 and a peak attributed to glycerin contained in the sample appeared at 1037 cm ^-1 .
Fibroin nanofibers produced by electrospinning are in the form of a random coil, but it can be seen that the molecular structure of fibroin nanofibers treated with ethanol or mixed with glycerin / ethanol after water insolubilization treatment has a β structure, It was experimentally confirmed that fibroin nanofibers crystallized during this treatment.

(Experimental example 16) FTIR spectrum by glycerol / ethanol treatment
Fibroin nanofibers produced by electrospinning 22.4 wt% of silkworm silk solution are immersed in glycerin / ethanol mixed solution (composition ratio: 90/10, 50/50, 10/90) for 5 minutes, and mixed with glycerin / ethanol. After pulling up from the solution, it was dried at room temperature for one day. FIG. 6 shows a: fibroin nanofibers obtained by electrospinning immersed in a glycerin / ethanol mixed solution (composition ratio 90/10), b: immersed in a glycerin / ethanol mixed solution (composition ratio 50/50) Treated: c: soaked with glycerin / ethanol mixed solution (composition ratio 10/90), d: soaked fibroin nanofibers obtained by electrospinning with 100% glycerin, e: immediately after electrospinning The result of having measured the FTIR spectrum about each sample of fibroin nanofiber of is shown.

From FIG. 6, the spectrum of the sample (a, b, c) obtained by treating the fibroin nanofiber (e) produced by electrospinning with a mixed solution of glycerin / ethanol having different composition ratios shows fibroin nanofibers in the control group. An absorption peak that is not observed in the FTIR spectrum of (e) appears at 1030 cm ⁻¹ . Since the peak to 1030 cm ^-1 in the FTIR spectra of fibroin nanofibers (e) was immersed for 5 minutes in glycerol solution sample (d) has appeared quite clear, the peak of 1030 cm ^-1 conclusion shall belong to glycerin it can. Therefore, it is suggested that all the samples (a, b, c) treated with the glycerin / ethanol mixed solution contain glycerin, and whether the sample contains glycerin is 1030 cm ^{−1 by} FTIR spectrum measurement. It can be evaluated by the presence or absence of a peak.

(Experimental example 17) FTIR spectrum by glycerol / ethanol treatment
Fibroin nanofibers (a) produced by electrospinning 22.4wt% of rabbit silk solution are immersed in glycerin / ethanol mixed solutions (composition ratio: 90/10, 50/50, 10/90) with different concentrations. The FTIR spectrum was measured for a sample that had been subjected to water insolubilization and further immersed in ethanol for 5 minutes. FIG. 7 shows the FTIR spectrum. In FIG. 7, a: fibroin nanofiber immediately after electrospinning, b: fibroin nanofiber obtained by electrospinning is immersed in a glycerin / ethanol mixed solution (composition ratio 90/10), and further immersed in ethanol for 5 minutes. C: Immersion treatment with glycerin / ethanol mixed solution (composition ratio 50/50), further immersion treatment with ethanol for 5 minutes, d: Immersion treatment with glycerin / ethanol mixture solution (composition ratio 10/90) Further, the sample was immersed in ethanol for 5 minutes, and e: a fibroin nanofiber obtained by electrospinning was immersed in 100/0 glycerin for 5 minutes.

According to FIG. 7, a peak of 1030 cm ⁻¹ appears very clearly in the FTIR spectrum (e) of glycerin. A sample of silk nanofibers immediately after electrospinning treated with water insolubilization using a mixed solution of glycerin / ethanol, followed by immersion in ethanol (b, c, d) is the same as sample (a). No glycerin absorption peak is observed. When water insolubilization treatment was performed using a glycerin / ethanol mixed solution, the sample adhered or impregnated with glycerin, but it was proved that glycerin could be completely removed from the sample by immersion treatment with ethanol for 5 minutes.

Claims (8)

  Silk protein with water insolubility and flexibility and transparency   2. The silk protein according to claim 1, wherein the silk protein is derived from a rabbit or a wild boar.   The silk protein according to claim 1 or 2, wherein the silk protein is in the form of a membrane or a nanofiber. Silk protein membrane formed by drying and solidifying an organic solvent containing silk protein aqueous solution or silk protein, or silk protein nanofiber obtained by electrospinning an organic solvent containing silk protein aqueous solution or silk protein,
2. The silk protein according to claim 1, wherein the silk protein is soaked with a glycerin / alcohol mixed solution, then taken out from the glycerin / alcohol mixed solution and dried in a standard state.   The silk protein film or silk protein nanofiber is taken out from the glycerin / alcohol mixed solution, dried in a standard state, and then immersed in an alcohol solution to remove glycerin contained in the material. 4. The silk protein according to 4.   The silk protein nanofiber is immersed in a glycerin / alcohol mixed solution having a composition ratio of 50/50 to 90/10 for 2 to 10 minutes, and then taken out from the glycerin / alcohol mixed solution and dried in a standard state. The silk protein according to claim 4 or 5.   The silk protein film is soaked in a glycerin / alcohol mixed solution having a composition ratio of 75/25 to 100/0 for 2 to 10 minutes, and then taken out from the glycerin / alcohol mixed solution and dried in a standard state. The silk protein according to claim 4 or 5. Silk protein membrane formed by drying and solidifying an organic solvent containing silk protein aqueous solution or silk protein, or silk protein nanofiber obtained by electrospinning an organic solvent containing silk protein aqueous solution or silk protein,
2. The silk protein according to claim 1, wherein the silk protein is soaked in a polyethylene glycol / alcohol mixed solution or a polypropylene glycol / alcohol mixed solution, then taken out from the mixed solution and dried in a standard state.