JP2009280715A - Amorphous fibroin film and method for manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel amorphous fibroin film mainly having both flexibility and sufficient strength. <P>SOLUTION: The amorphous fibroin film is formed from a fibroin separated from a liquid silk contained in a silk grand of a silkworm as a main component raw material. The film is constituted of a crystal part and an amorphous part, crystallinity in the film is less than 10% and a crystal structure of the crystal part contained in the film is a silk I type structure to be an existence state in the liquid silk. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an amorphous fibroin film and a method for producing the same. More specifically, the present invention relates to a method for producing an amorphous fibroin film without destroying the supramolecular structure of fibroin, which is an existing form in the silk gland.

  The silk thread made by cocoons is composed of two proteins, fibroin and sericin. Silk fibroin obtained by removing sericin in silk thread by high-temperature water washing or the like is known to dissolve in a high-concentration aqueous solution of calcium chloride (see, for example, Patent Document 1), and thus obtained. A regenerated silk fibroin film is produced from the obtained silk fibroin solution.

  In addition, since the silk fibroin film increases its strength by being crystallized, its use in contact lenses, artificial skin, wigs, sweating clothing, and the like has been studied (see, for example, Patent Documents 2 to 4). .

  As a method for crystallizing a silk fibroin film, for example, (I) a method for ethanol treatment (for example, see Non-Patent Document 1) and (II) a method for heat treatment (for example, see Non-Patent Document 2) are known. ing.

  However, in the method (I), since crystallization proceeds rapidly, it is impossible to adjust the crystallinity of the film. Further, the film is distorted by local crystallization, and it is difficult to supply a large amount of film having homogeneous mechanical properties.

  The method (II) has a disadvantage that the decomposition of the silk fibroin and the deterioration of the film proceed simultaneously because the film needs to be heated to the glass transition point (about 175 ° C.).

By the way, the crystallized silk fibroin film loses flexibility and has the disadvantage of being hard and brittle. As a method of eliminating such drawbacks, (i) a method of obtaining flexibility by adding ethanol or water to the film, and (ii) film formation and crystallization after adding glycerin to the silk fibroin solution, the flexibility is obtained. A method for obtaining the information is known (for example, see Non-Patent Document 3).
JP 7-173192 A JP-A 63-246169 Japanese Unexamined Patent Publication No. 1-1118545 JP-A-11-70160 Satoshi Magoshi, “Transition mechanism of silk fibroin”, Polymer papers, Vol.31, 765-770, 1974 J. Magoshi and Y. Magoshi, "Physical properties and structure of silk", J. Polym. Sci., Vol. 15, 1675-1682, 1977 Shinichiro Hiraide, “Flexibility of silk fibroin film”, Japanese Journal of Yarn Science, Vol. 66, 138-140, 1997 Jikusen et al., "Flexibility of laminated silk sheets", Nihon Sericultural Journal, Vol. 65, 81-85, 1996

  However, the film obtained by the above method (i) easily volatilizes the added ethanol and water, and instead loses flexibility and becomes brittle, and it is difficult to ensure flexibility for a long time. It was.

  The film obtained by the above method (ii) is difficult to crystallize the silk fibroin film by 20% or more because glycerin inhibits the crystallization of silk fibroin (see, for example, Non-Patent Document 4). Sufficient strength cannot be obtained.

  In addition, glycerin added to the silk fibroin solution is phase-separated into droplets and scatters light, so that the film produced from such a solution becomes cloudy and loses its transparency.

Furthermore, when the amount of glycerin added is increased to 10% or more, in addition to the sheet-like Silk II type crystal (β type crystal), a Silk I type crystal (α type crystal) is generated (for example, see Non-Patent Document 4). It is believed that the mechanical properties of silk fibroin films made from this solution are reduced.

  An object of the present invention is mainly to provide a novel amorphous fibroin film having both flexibility and sufficient strength.

  Another object of the present invention is to provide a novel method for producing such an amorphous fibroin film.

  The invention according to claim 1 is based on fibroin separated from liquid silk contained in silkworm gland of silkworm as a main ingredient, the film is composed of a crystalline part and an amorphous part, and the crystallinity in the film is 10%. A non-crystalline fibroin film characterized in that the crystal structure of the crystal part contained in the film is a Silk I-type structure which is an existing form in liquid silk.

  In the invention according to claim 2, in the silk gland extracted from the body of the cocoon, the middle silk gland cut out from the silk gland is immersed in pure water maintained at a temperature of 10 ° C. or lower and immersed in pure water. A process of obtaining silk in the gland by removing silk gland cells from the middle silk gland, and immersing the liquid silk in pure water maintained at a temperature of 10 ° C. or lower, maintaining this soaked state, soaking the liquid silk Discarding the purified water, supplying pure water maintained at a temperature of 10 ° C. or lower again, and performing elution and separation of water-soluble components of sericin from liquid silk by performing pure water exchange at regular intervals, A step of separating the poorly water-soluble sericin component from the liquid silk and removing the separated precipitate, and a transparent fibroin by dissolving the liquid silk from which the sericin has been separated in pure water maintained at a temperature of 10 ° C. or lower. A step of preparing an aqueous solution, Put the aqueous solution in a film-forming container with a smooth bottom, spread the aqueous solution on the bottom of the container, hold the forming container with the developed aqueous solution in a dry atmosphere at 0 to 10 ° C., and evaporate and remove the moisture in the developed aqueous solution. And a step of forming a film on the bottom of the container by drying and solidifying, and a method for producing an amorphous fibroin film.

  The amorphous fibroin film of the present invention is mainly advantageous in that it has both flexibility and sufficient strength and exhibits extremely excellent mechanical properties.

  Next, the best mode for carrying out the present invention will be described.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor did not use a silk raw material melted with a neutral salt or the like as a film raw material, but separated directly from the silk gland extracted from the body of the cocoon. Neutral salt obtained by conventional methods by using liquid silk fibroin as a film raw material, suppressing the crystallization of crystalline H chain while preserving the supramolecular structure of fibroin during film formation, and making it amorphous As a result, the present inventors have found that the mechanical properties can be drastically improved as compared with a film produced by using the material, and that the above-mentioned problems can be solved, thereby completing the present invention.

  That is, the non-crystalline fibroin film of the present invention comprises fibroin separated from liquid silk contained in silkworm gland of silkworm as a main component raw material, and the film is composed of a crystalline part and an amorphous part, and is crystallized in the film. The degree is less than 10%, and the crystal structure of the crystal part contained in the film is a Silk I type structure which is an existing form in liquid silk. An amorphous fibroin film having such characteristics is mainly advantageous in that it has both flexibility and sufficient strength and exhibits extremely excellent mechanical properties.

  Here, the supramolecular structure is not a strong bond such as a covalent bond, but a weak intermolecular interaction (eg, hydrophobic interaction, hydrogen bond, coordination). The structure is formed by bonding, electrostatic interaction, etc.) and expresses the function as a whole supramolecule. For example, a DNA double helix structure, an enzyme and the like can be mentioned. In the case of silk fibroin, an intermolecular interaction occurs between the fibroin H chain and P25 to form a supramolecular structure.

  The crystallinity is determined by the following method. Several layers of the produced fibroin film are laminated to form a laminate, X-rays (radiation source: CuKα ray) are incident on the film surface of this laminate, and the diffraction intensity distribution curve is in the range of 2θ = 5 to 30 °. taking measurement. Then, after the intensity curve is separated into a peak of a crystalline portion and an amorphous portion within a range of 2θ = 15 to 30 ° by the peak separation method, the crystallinity is obtained from the area ratio.

  In addition, the method for producing an amorphous fibroin film of the present invention includes immersing the middle silk gland cut out from the silk gland in pure water maintained at a temperature of 10 ° C. or less among the silk glands extracted from the body of the cocoon, A process of obtaining liquid silk in the gland by removing silk gland cells from the middle silk gland while immersed in pure water, and immersing the liquid silk in pure water maintained at a temperature of 10 ° C. or less. The water-soluble component of sericin from liquid silk is maintained by throwing away pure water soaked in liquid silk and supplying pure water maintained at a temperature of 10 ° C. or lower again. And separating the poorly water-soluble components of sericin from the liquid silk, removing the precipitate that has been separated and precipitated, and pure water in which the liquid silk from which the sericin has been separated is maintained at a temperature of 10 ° C. or less Transparent fibroin dissolved in A step of preparing a solution; and a fibroin aqueous solution is placed in a film forming container having a smooth bottom, the aqueous solution is developed at the bottom of the container, and the forming container in which the aqueous solution is developed is maintained in a dry atmosphere of 0 to 10 ° C. And a step of forming a film on the bottom of the container by evaporating and removing moisture therein.

  When obtaining the liquid silk in the gland, when elution and separation of the water-soluble component of sericin from the liquid silk, when preparing the fibroin aqueous solution from the liquid silk from which sericin has been separated, the temperature of pure water used is 10 ° C. or less, respectively. Use pure water maintained at a temperature, preferably 0-5 ° C. The reason why the temperature of pure water is specified is that when water having a temperature exceeding 10 ° C. is used, there is a problem that fibroin existing in the liquid silk is crystallized.

  Moreover, the reason why the drying atmosphere is set to 0 to 10 ° C. when drying and solidifying is that a film having excellent mechanical performance can be obtained within the above range. Within the above range, if the temperature of the drying atmosphere is low, the drying processing time will be longer. Therefore, the drying processing time may be shortened by reducing the drying atmosphere. If the upper limit of 10 ° C. is exceeded, crystallization occurs during drying and solidification, resulting in a film having a high degree of crystallinity and a film having poor mechanical performance. If it is less than 0 degreeC which is a lower limit, dry solidification does not advance and it is inferior to production efficiency.

  By passing through the above steps, a novel amorphous fibroin film having both flexibility and sufficient strength can be obtained.

  When the fibroin aqueous solution is stored for a certain period of time before forming the fibroin film using the prepared aqueous fibroin solution, it is kept at a low temperature of about 0 to 4 ° C. and stored in order to prevent freezing and crystallization of fibroin. It is preferable to set the liquid for about 12 to 24 hours for long-term storage without adding vibration, shaking, or stirring to the liquid.

  Next, embodiments of the present invention will be described in detail.

<Example 1>
First, a 5 year old cocoon was prepared, and a tear was made in the back of the heel with tweezers to naturally expose a part of the silk gland from the inside of the cocoon. In the silk gland, the part of the middle silk gland was separated with scissors and removed without applying as much force as possible to the exposed silk gland. The reason for applying no force when extracting the silk gland is that if force is applied to the silk gland, fibroin crystallization occurs. Subsequently, after removing the extracted middle silk gland in ultrapure water for several minutes, the silk gland cells in the gland are carefully removed from the middle silk gland in a state of being immersed in ultrapure water. Obtained. The reason why the middle silk gland was immersed in ultrapure water was maintained because the silk gland cells were swollen and easily removed from the liquid silk. In addition, although the operation so far used the ultrapure water maintained at about 4 degreeC, if the state of water is maintained, it has confirmed that it may be 10 degrees C or less. When the water temperature exceeds 10 ° C., there is a problem that fibroin crystallization occurs.

  Next, sericin was separated from the liquid silk. Specifically, gel-like liquid silk is immersed in ultrapure water maintained at a temperature of 4 ° C. or less at a bath ratio of about 1: 100, and this soaked state is maintained, and the water-soluble component of sericin is fibroin. The water-soluble component of sericin was eluted and separated from the liquid silk by exchanging ultrapure water at regular intervals while eluting the water-soluble component of sericin by utilizing its high water solubility.

  Specifically, liquid silk is immersed in ultrapure water for a certain period of time, the soaked ultrapure water is discarded, and ultrapure water maintained at the same temperature is again supplied at the same bath ratio. It was. In addition, if vibration, shaking, or stirring is applied to the liquid silk during the elution separation, the liquid silk will crystallize. Therefore, the liquid that is being treated with a gentle flow due to a gentle seesaw motion once per second. Elution was carried out by adding to In this example, the elution separation process was performed for about 4 hours, but it was confirmed that almost all sericin can be eluted and separated from the liquid silk if it is at least 30 minutes or more, preferably about 3 to 4 hours. . Moreover, although the ultrapure water maintained at 4 degrees C or less was used in the elution separation, if the state of water is maintained, it has confirmed that it may be 10 degrees C or less. When the water temperature exceeds 10 ° C., there is a problem that fibroin crystallization occurs. The liquid silk forms a fourth layer (the layer closest to fibroin) of the sericin surrounding fibroin in a layered form. The poorly water-soluble component of this sericin At this time, since it gradually separated from fibroin and precipitated in the liquid, the precipitate was removed with tweezers etc. at appropriate times.

  Next, the liquid silk from which sericin was separated was dissolved in ultrapure water maintained at a temperature of 4 ° C. or less to prepare a 1 to 2 mass% transparent fibroin aqueous solution.

  Further, an aqueous solution of fibroin is put in a styrene petri dish and the aqueous solution is developed in a petri dish. The petri dish in which the aqueous solution is developed is kept in a desiccator in which the inside is kept at 4 ° C. The transparent fibroin film was obtained by evaporating and removing the water in the developed aqueous solution by evaporating and drying and solidifying. The obtained fibroin film had a thickness of about 10 μm.

<Evaluation test 1>
Several sheets of the obtained fibroin film are prepared, these films are laminated to form a laminate, X-rays (radiation source: CuKα ray) are incident on the film surface of the laminate, and the diffraction intensity distribution curve is 2θ = 5-30. Measured in the range of °. The intensity curve was separated into peaks of a crystalline part and an amorphous part within a range of 2θ = 15 to 30 ° by the peak separation method, and the crystallinity was measured from the area ratio. FIG. 1 shows an intensity curve.

  As is clear from FIG. 1, a small peak showing the characteristics of the Silk I type crystal structure was observed at 2θ = 19.8 °. This is presumed to be a result of supporting that the supramolecular structure existing only in the liquid silk is maintained in the formed film without being destroyed.

  When the crystallinity was evaluated by the peak separation method in the range of 2θ = 15 to 30 °, the crystallinity was 8.7%.

<Evaluation Test 2>
A tape having a width of 5 mm was cut out from the obtained fibroin film, and a high elongation test was performed at room temperature under conditions of a gauge length of 25 mm and a pulling speed of 40 mm / min. As a result, it was confirmed that the film had both high flexibility and sufficient strength, with a breaking stress of 41.9 MPa and a breaking elongation of 7.4%.

  The mechanical properties of commercially available wafer films, polyhydroxybutane (PHB) films, and commercially available biodegradable films were compared by the same measurement method. The results are shown in FIG. In addition, the area | region represented by the rectangle in FIG. 2, and is displaying (A) inside shows the dynamic characteristic of NOVON.

  As is apparent from FIG. 2, it was found that the mechanical properties of the fibroin film of the present invention are extremely excellent.

  In addition, although the density | concentration of the fibroin aqueous solution prepared in a present Example is 1-2 mass%, if the density | concentration of a fibroin aqueous solution is the range of 0.5-5 mass%, the fibroin film which has the effect of this invention will be obtained. Make sure you can.

  The non-crystalline fibroin film of the present invention can be applied to wound dressing for medical use and special fiber for clothing.

The figure which shows the diffraction intensity distribution curve of the evaluation test 1. FIG. The figure which shows the mechanical characteristic result of the evaluation test 2. FIG.

Claims (2)

Using fibroin separated from liquid silk contained in silkworm gland of silkworm as the main ingredient,
The film is composed of a crystalline part and an amorphous part, and the crystallinity in the film is less than 10%,
An amorphous fibroin film, wherein the crystal structure of a crystal part contained in the film is a Silk I type structure which is a form existing in the liquid silk. Among the silk glands extracted from the body of the cocoon, the middle silk gland cut out from the silk gland is immersed in pure water maintained at a temperature of 10 ° C. or less, and the silk gland cells from the middle silk gland are immersed in the pure water. Removing liquid to obtain liquid silk in the gland;
The liquid silk is soaked in pure water maintained at a temperature of 10 ° C. or lower, the soaked state is maintained, the pure water soaked in the liquid silk is discarded, and pure water maintained at a temperature of 10 ° C. or lower again. The water-soluble component of sericin is eluted and separated from the liquid silk by separating pure water at regular intervals, and the poorly water-soluble component of sericin is separated from the liquid silk. Removing the precipitated precipitate,
Dissolving the liquid silk from which the sericin has been separated in pure water maintained at a temperature of 10 ° C. or lower to prepare a transparent fibroin aqueous solution;
The fibroin aqueous solution is placed in a film forming container having a smooth bottom, and the aqueous solution is developed on the bottom of the container. The forming container in which the aqueous solution is developed is maintained in a dry atmosphere of 0 to 10 ° C. And a step of forming a film on the bottom of the container by evaporating and removing moisture to dry and solidify the film.