Silk fibroin molded body and molding mixture

Abstract

Disclosed are a molded body and a method for manufacturing the same, in which the molded body comprises silk fibroin having high impact strength and flexural performance, the molded body is capable of maintaining the impact strength and flexural performance over a long period of time, the molded body comprises 80 parts by weight or more of silk fibroin, and the molded body is a molded body which is capable of maintaining the impact strength and flexural performance over a long period of time. The silk fibroin comprises fibrous silk fibroin and non-fibrous silk fibroin.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 579,884, filed August 31, 2023, in the U.S. Patent and Trademark Office, the disclosure of which is incorporated herein in its entirety. BACKGROUND 1. TECHNICAL FIELD

[0004] The present disclosure relates to a molded body formed from fibrous fibroin and non-fibrous fibroin.

[0005] 2. DESCRIPTION OF RELATED ART

[0006] Currently, efforts are being made to process fibroin, which is a protein having high biocompatibility, into a product. Fibroin is one of the main proteins that make up a cocoon. For example, JP7096549B2 discusses a method for mixing raw silk into a molded body to improve strength when a molded body is obtained from a powder derived from silk.

[0007] However, since raw silk contains a large amount of highly water-soluble sericin, it is difficult for conventional methods to maintain the strength of the molded body over a long period of time, particularly when the method discussed in JP7096549B2 is utilized. SUMMARY

[0008] The present disclosure overcomes the shortcomings of conventional systems by providing a molded body that can maintain high strength over an extended period of time, which includes 80 parts by weight or more of fibroin, wherein the fibroin is composed of fibrous fibroin and non-fibrous fibroin, thereby allowing the molded body to maintain its strength over a long period of time.

[0009] Accordingly, one aspect of the present disclosure provides a molded body including at least 80 parts by weight of fibroin, wherein the fibroin includes fibrous fibroin and non-fibrous fibroin.

[0010] Another aspect of the present disclosure provides a mixture for molding including fibrous fibroin and fibroin in powder form, wherein the solid component of the mixture contains 80 parts by weight or more of fibroin.

[0011] Still another aspect of the present disclosure provides a method for manufacturing a molded body, the method including: obtaining a mixture including fibrous fibroin and fibroin in powder form, wherein the solid component of the mixture contains 80 parts by weight or more of fibroin; and compressing the mixture such that the fibrous fibroin is combined with the fibroin in powder form.

[0012] These and other embodiments, objects, features, and advantages of this disclosure will become clear when read in conjunction with the accompanying drawings and the provided claims in the following detailed description of exemplary embodiments of this disclosure. Attached Figure Description

[0013] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate various embodiments, objects, features and advantages of this disclosure.

[0014] Figure 1A and Figure 1B A table summarizing various embodiments of this disclosure is provided; and

[0015] Figure 2 It is a perspective view of the molded body according to this disclosure. Detailed Implementation

[0016] This disclosure has several embodiments, and details known to those skilled in the art rely on patents, patent applications, and other references. Therefore, when patents, patent applications, or other references are cited or paraphrased herein, it should be understood that the entirety of the patent, patent application, and non-patent documents are incorporated by reference for all purposes and for the propositions set forth herein.

[0017] <Molding Mixture>

[0018] This disclosure provides a molding mixture comprising 80 parts by weight or more of silk fibroin in its solid component. The silk fibroin included in the mixture comprises fibrous silk fibroin and powdered silk fibroin. Preferably, the powdered silk fibroin is amorphous. Examples of manufacturing using this molding mixture will be described below.

[0019] The molding mixture includes a solid component, which is a substantially solid component excluding solvents (such as water). For example, when heated to 80 degrees Celsius at atmospheric pressure, the component can be substantially solid when the weight loss rate is 0.1% or less per day. For example, in conventional molding methods, solvents (such as water) can be used in the molding mixture. However, the solid component in this specification is a substantially solid component excluding solvents. Various methods can be considered to determine the total amount of solvent-free solid component. For example, when heated to 80 degrees Celsius at atmospheric pressure, a weight loss rate can be determined to be 0.1% or less per day of total solid component content.

[0020] The solid component of the molding mixture disclosed herein contains fibroin derived from silkworm cocoons, spider silk, or wasp silk produced by wasp larvae, and is a bio-based polymer composed of amino acids (such as glycine and alanine) or more. From the viewpoint of biocompatibility and biodegradability, fibroin is considered superior. In the case of natural silkworm cocoons (raw silk), typically 25 to 30 parts by weight are sericin and 75 to 80 parts by weight are fibroin. On the other hand, the molding mixture of this disclosure contains 80 parts by weight or more of fibroin. Therefore, for example, fibroin with a sericin content reduced in raw silk through a degumming process is used.

[0021] When using silkworm cocoons as raw material, it is permissible to use only degummed cocoons as the molding mixture. Sericin is removed from the degummed cocoons, ensuring that the amount of fibroin is 80 parts by weight or more. It is also permissible to prepare the molding mixture by mixing raw silk containing sericin with degummed cocoons, thereby ensuring that the amount of fibroin is 80 parts by weight or more. Furthermore, the purity of fibroin can be improved by methods such as sericin dialysis utilizing the difference in water solubility between fibroin and sericin. Additionally, silkworm cocoons from which the concentration of fibroin in raw silk can be artificially increased by using silkworms or wasps whose DNA has been modified.

[0022] As described above, sericin is highly water-soluble and can dissolve in water after molding. Therefore, the sericin content of the molding mixture disclosed herein is preferably 10 parts by weight or less, more preferably 3 parts by weight or less. To simplify the degumming process, 0.1 parts by weight or more of sericin may be included. For example, when using a general degumming process, approximately 2 parts by weight of sericin are retained.

[0023] The weight of sericin contained in the molding mixture can be verified by analyzing the completely dissolved mixture using liquid chromatography-mass spectrometry (LC-MS) / MS, as described in Int. J. Mol. Sci. 2023, 24(1), 259, or by infrared absorption spectroscopy, etc.

[0024] When using silkworm cocoons, the sericin content can be determined by comparing the weight before and after degumming. Degummed cocoons typically contain 2 parts by weight of sericin and 98 parts by weight of fibroin. The sericin weight in the cocoon can be determined by adding the weight of the sericin lost during the degumming process.

[0025] Furthermore, the molding mixture of this disclosure may contain less than 20 parts by weight of components other than silk fibroin. However, from the viewpoint of the durability of the molded body, the components other than silk fibroin preferably have low solubility in water. Additionally, if the molded body is to be used as a medical material, the components other than silk fibroin are preferably highly biocompatible. For example, silicone or hydrogel compounds can be used as other components, such as those used in contact lenses, carbon fiber cloth with proven biocompatibility, glass fiber cloth, combinations thereof, etc., and the molded body includes at least one component selected from carbon fiber and glass fiber.

[0026] 1-A. Fibrous silk fibroin

[0027] The fibrous fibroin disclosed herein is a filamentous fibroin or a processed form, wherein the fibroin forms crystals in the radial direction by hydrogen bonds and extends in a direction perpendicular to the radial direction.

[0028] The average thickness (diameter) of fibrous silk fibroin is greater than 1 micrometer and less than 200 micrometers. If it is less than 1 micrometer, the strength of the molded body after molding may be insufficient. In addition, if it is greater than 200 micrometers, the miscibility with powder may deteriorate.

[0029] From the viewpoint of the mechanical strength of the molded body, the fibrous fibroin comprises fibrous fibroin with a length of 0.2 mm or more, and in one embodiment, preferably comprises fibrous fibroin with a length of 1.0 mm or more. For silkworms, since the length of cocoon silk obtainable from a single individual exceeds 1 kilometer, it is preferable to appropriately cut the cocoon to mix it with powdered fibroin. Preferably, the cut fibrous fibroin comprises fibroin with a length of 50 mm or less, and in one embodiment, it preferably comprises fibroin with a length of 30 mm or less.

[0030] The average thickness and average length of fibrous fibroin can be calculated from the area and perimeter by taking images of 100 or more fibers using a camera or scanner and analyzing the images. Specifically, the area is obtained by multiplying X and Y based on the thickness X and fiber length Y, and the perimeter is twice the sum of X and Y. After determining X and Y for each fibrous fibroin, the average thickness of each X of the fibrous fibroin is obtained, and the average length Y of each fiber of the fibrous fibroin is also obtained.

[0031] The proportion of fibrous fibroin in the mixture is preferably 10 parts by weight or more. If it is less than this, the strength of the molded article obtained after molding may be reduced. Preferably, the proportion of fibrous fibroin to silk fibroin is 50 parts by weight or more. On the other hand, when there is too much fibrous fibroin, it is difficult for the fibrous fibroin to bind together during molding because there is less powdery fibroin, as described herein. As a result, the strength of the molded article is reduced. Therefore, the proportion of fibrous fibroin to silk fibroin in the mixture is preferably 85 parts by weight or less.

[0032] Filamentous fibroin includes, for example, cocoon, degummed cocoon, and spun fibroin. Processed products may include: cut filamentous fibroin products, such as mesh products processed like woven fabrics or webs, or cut products thereof. That is, the fibrous fibroin of this disclosure may be one-dimensional long filaments, two-dimensional sheets (i.e., sheet-like) processed like woven fabrics, or cut forms of these.

[0033] The molecular weight of fibrous fibroin is preferably 130 kDa or higher. If it is less than 130 kDa, the fibers become brittle, and the mechanical strength of the formed body may decrease. Typically, the molecular weight in silkworm cocoons is said to be 300 kDa or higher, and this molecular weight is reduced by boiling. Therefore, the molecular weight of fibroin can be controlled by the boiling time. In this disclosure, when used after cleaning to remove dust or other contaminants that may adhere to the cocoon, the molecular weight of fibrous fibroin is limited to below 300 kDa. The molecular weight can be confirmed by using the fibroin after desalting dissolved fibroin in an aqueous solution such as lithium bromide (LiBr).

[0034] For the fibrous silk fibroin of this disclosure, a silk fibroin with a crystallinity of 50% or more is preferred. Typically, the crystallinity of silkworm cocoons is 50% or more, unless dissolved at the molecular level. For the crystallinity in this disclosure, the method described in Nature Materials 19, 102-108 (2020) (hereinafter referred to as Non-Patent Document 2) is referenced and the following method is used.

[0035] <Methods for measuring crystallinity>

[0036] From 1580 cm -1 Up to 1720 cm -1 Within the range of 1 cm -1 Measure the infrared absorption spectrum of the molded body surface. Calculate the 1580 cm⁻¹ wavelength of the obtained spectrum. -1 The value at 1720 cm -1The value at a given point is a linear function, which is used as a baseline and subtracted from the measured value. The resulting spectrum is considered to be the sum of four spectra derived from random coil, β-sheet I, β-sheet II, and β-turn, and is referred to as the effective spectrum.

[0037] The spectrum derived from random curl can be approximated as having a value at 1645 cm⁻¹ -1 and 1655 cm -1 The sum of Gaussian functions of the variable peak centers between them, and the spectra derived from β-fold I, β-fold II and β-turn can be approximated by having 1620, 1698 and 1685 cm⁻¹, respectively. -1 The sum of Gaussian functions with fixed peak centers. Among these four Gaussian functions, the sum of the peak intensities and deviations of these Gaussian functions is determined as variables to obtain a synthetic spectrum with nine variables.

[0038] At 1580 cm -1 Up to 1720 cm -1 Within the range, at every 1 cm -1 The absolute value of the difference between the effective spectrum and the synthesized spectrum is determined, and the sum is calculated as the spectral error. The spectral error relative to the integral value of the effective spectrum is set as the error rate, and the nine variables of the synthesized spectrum are fitted to reduce the error rate. When the fitted error rate becomes 3% or less, the error rate is considered to be the convergent error rate, and the four spectra constituting the synthesized spectrum are considered to be the actual spectra of each component. Then, relative to 1580 cm⁻¹... -1 Up to 1720 cm -1 The integral value of the effective spectrum within the range is calculated from the sum of the integral values ​​of the three spectra of β-sheet I, β-sheet II, and β-turn, which is used as the crystallinity of this disclosure. Since the crystallinity (the sum of β-sheet I, β-sheet II, and β-turn) in the case of silk fibroin, according to Non-Patent Document 2, is less than 20% in the dry state before thermoforming and more than 50% after thermoforming, if the crystallinity in this disclosure exceeds 50%, the molded body is determined to have the desired strength. However, the index of crystallinity can vary depending on the required strength of the molded body.

[0039] Fitting can be performed using software provided with the infrared absorption spectroscopy measurement device, graphical analysis software, solver plugins in Microsoft Excel, etc.

[0040] 1-B. Powdered silk fibroin

[0041] In this disclosure, powdered silk fibroin is used as a non-fibrous silk fibroin. Powdered silk fibroin is silk fibroin that has been turned into powder. For the diameter of the powdered silk fibroin, a diameter of 200 micrometers or less is preferred. If it is greater than 200 micrometers, powdered silk fibroin is not preferred because it becomes difficult to mix with fibrous silk fibroin. The diameter can be determined by a device for measuring powder size.

[0042] Powdered silk fibroin is amorphous. As mentioned above, silk fibroin is in a crystalline state and has poor reactivity, while amorphous silk fibroin melts during molding. Therefore, when a mixture of amorphous powdered silk fibroin and fibrous silk fibroin is thermoformed, the melting process binds the amorphous powdered silk fibroin to the fibrous silk fibroin, thereby improving the strength of the molded article. Alternatively, low-pressure curing during the molding process provides similar strength. See also Figure 1A and 1B The figure summarizes the physical properties and provides a table summarizing the various embodiments of this disclosure.

[0043] Amorphous silk fibroin can be obtained by the method described in Non-Patent Document 2. That is, it can be manufactured by dissolving degummed silk fibroin in an aqueous solution and then removing water from the aqueous solution. Since dehydration by heating can accelerate crystallization and impair meltability during molding, methods such as freeze-drying, which enables dehydration while cooling, and spray drying, which requires very little time to heat the aqueous solution, can be used.

[0044] When using a freeze-drying method, there is no particular limitation on the freezing temperature, as long as the aqueous solution is frozen. However, since the freezing point of the aqueous solution containing the solute is lower than that of water, it is preferably below -10°C, and more preferably below -20°C. When drying under reduced pressure after freezing, the temperature is preferably above -20°C, and more preferably above -15°C, because the lower the drying temperature, the longer the dehydration time. In a fully frozen sample, the pressure is preferably below 600 Pa, which is the pressure at the triple point of water. However, if the frozen sample partially liquefies due to uneven temperature distribution, the aqueous solution will foam, so below 100 Pa is preferred, and below 50 Pa is more preferred. For the dried material after moisture removal, it is preferable to release the vacuum after the temperature reaches above 10°C. Below this value, condensation may occur inside the sample, and the dried material may adsorb water, leading to gelation of the dried silk fibroin material, etc.

[0045] When using spray drying, the concentration of silk fibroin in the aqueous solution can be pre-adjusted to promote spraying. Preferably, the heat exposure time of the silk fibroin is minimized by adjusting the air pressure and flow rate of the spray. If the silk fibroin is heated and crystallized due to the equipment being heated during spraying, spraying can also be performed while cooling the periphery of the equipment and the sample collection area using a cooling device or similar means.

[0046] To prepare powder, it can be ground using a jet mill, hammer mill, ball mill, needle mill, etc., after freeze-drying, or it can be obtained by spray drying.

[0047] In this disclosure, amorphous refers to silk fibroin with a crystallinity of less than 20%. The crystallinity is calculated using the same method described in Section 1-A.

[0048] 1-C. A mixture of fibrous silk fibroin and powdery silk fibroin

[0049] Fibrous silk fibroin and non-fibrous (powdered or powdered) silk fibroin are mixed and stirred in a chamber. From a biocompatibility point of view, the amount of impurities (e.g., sericin) contained in fibrous and powdered silk fibroin is preferably 20 parts by weight or less, more preferably 10 parts by weight or less. Generally, since cocoons that have been degummed to improve the purity of silk fibroin contain about 2 parts by weight of sericin, they may contain more than 2 parts by weight of impurities (sericin).

[0050] Preferably, mixing and stirring are performed using methods that do not easily generate heat. In this disclosure, for example, cyclone separators utilizing airflow, propeller agitation, vibration stirring, etc., can be used.

[0051] This process yields a mixture in which amorphous, non-fibrous (powdered / powdered form) silk fibroin is attached to crystalline fibrous silk fibroin.

[0052] Molding method

[0053] The molding temperature of the molding mixture disclosed herein is preferably above 80°C, so as to melt the powdered silk fibroin and bind the fibrous silk fibroin together. Furthermore, since silk fibroin thermally decomposes above 200°C, the molding temperature is preferably below 180°C.

[0054] The molding pressure is preferably 300 MPa or less, more preferably 100 MPa or less. When using a pressure of 300 MPa, the capital investment cost may be high. In addition, in order to melt the powdered silk fibroin and bind it with the fibrous silk fibroin, it is preferable to mold it at a pressure of 30 MPa or more.

[0055] After the powdered silk fibroin has crystallized during the molding process, it is preferable to select an appropriate molding time so that the crystallization rate becomes more than 50%.

[0056] Various molding methods can be used, including injection molding, extrusion molding, compression molding, etc., but are not limited to these.

[0057] Molded body

[0058] The molded article disclosed herein contains 80 parts by weight or more of silk fibroin. In this case, the silk fibroin includes fibrous silk fibroin and non-fibrous silk fibroin. The fibrous silk fibroin is bound to the non-fibrous silk fibroin.

[0059] The molded articles disclosed herein may include components other than silk fibroin, but preferably, any component other than silk fibroin has high biocompatibility and low water solubility. If the component other than silk fibroin is sericin, the sericin is preferably 10 parts by weight or less in order to reduce degradation to the maximum extent that may occur during long-term use, as described herein. Preferably, the amount of sericin is 3 parts by weight or less. In addition, since it is difficult to use silk fibroin from which sericin can be completely removed, the sericin can be 0.1 parts by weight or more. The amount of sericin in the molded article can be confirmed by LC-MS / MS or infrared absorption spectroscopy, as described herein.

[0060] Since fibrous silk fibroin is crystalline and does not melt during the molding process, it exists in the molded body in a fibrous state. Therefore, the preferred conditions for the length and thickness of the fibrous silk fibroin are the same as those described herein regarding the molding mixture.

[0061] In the mixture, the proportion of fibrous fibroin in the silk fibroin is preferably 10 parts by weight or more. If the proportion is less than this, the strength of the molded body obtained after molding may be insufficient. Preferably, the proportion of fibrous fibroin in the silk fibroin is 50 parts by weight or more. If there is too much fibrous fibroin, it is difficult for the fibrous fibroin to bind together during molding because there is less non-fibrous fibroin. Since this reduces the strength of the molded body, the proportion of fibrous fibroin to silk fibroin in the mixture is preferably 85 parts by weight or less. Non-fibrous fibroin is tightly integrated silk fibroin, which is different from the fibrous fibroin in the molded body. It is equivalent to amorphous powdered silk fibroin, which is prepared by the molding mixture and is molded and integrated. Since the powdered silk fibroin adheres to the fibrous fibroin in the molding mixture, the non-fibrous fibroin in the molded body adheres to the fibrous fibroin, and the fibrous fibroin is bound by the non-fibrous fibroin.

[0062] To achieve high strength in the molded body, it is crucial that the silk fibroin crystallizes throughout the entire molded body. When in a mixture prior to molding, fibrous silk fibroin is crystalline, while powdered silk fibroin is amorphous. Therefore, in terms of strength, the crystallization of the powdered silk fibroin that melts during molding is important. Since non-fibrous silk fibroin adheres to the fibrous silk fibroin, the crystallinity of the non-fibrous silk fibroin can be determined by measuring the crystallinity of the molded body surface.

[0063] The crystallinity of the molded body surface is preferably 50% or more, which indicates a crystalline state of non-fibrous silk fibroin. If it is less than this, the molded body may not have sufficiently high strength. The method for calculating the crystallinity, as described herein, can be the same as that described in the section on molding mixtures.

[0064] Verification of the mixing of fibrous fibroin in a molded body can be observed in optical micrographs of a cross-section of a bent molded body. For example, the vertical orientation (i.e., upright) of the tangled fibrous fibroin can be observed, but is not limited to a specific orientation. That is, the molded body that fractures after use in an impact strength test or a flexural modulus stress test can be magnified and observed.

[0065] The amount of fibrous fibroin in a fibroin mixture can be quantified proportionally during the mixing stage, but it can also be determined through analysis of the molded body itself. For example, non-fibrous fibroin has a high density and is pale yellow, while fibrous fibroin has a low density and is white. Therefore, the ratio of yellow to white areas in the cross-section of the molded body can distinguish between non-fibrous and fibrous fibroin. Furthermore, the low density of fibrous fibroin can be used to calculate its proportion based on fluorescence staining properties or gas adsorption properties.

[0066] In the molded body of this disclosure, fibrous silk fibroin and non-fibrous silk fibroin are bonded together. That is, the gaps between the fibrous silk fibroin are filled with dense non-fibrous silk fibroin, and the fibrous and non-fibrous silk fibroin are substantially integrated without any gaps. Since the cross-sectional view of the fibrous silk fibroin in the molded part is substantially circular, i.e. elliptical, these cross-sectional views can be observed by scanning electron microscopy to confirm whether the elliptical integration occurs without any gaps between the ellipses.

[0067] Figure 2 This is a perspective view of the molded body according to this disclosure. For example... Figure 2 As shown, the molded body 1 is provided with embedded fibrous silk protein.

[0068] The mechanical properties of the molded parts were confirmed using the Charpy impact strength test according to ISO 179-1 and the flexural properties test according to ISO 178. The preferred impact strength was 5 kJ / m. 2 The maximum bending stress is 50 MPa or more, and the impact strength is more preferably 12 kJ / m. 2 The maximum bending stress is 70 MPa or higher. Specifically, the maximum bending stress of the molded body after immersion in a 0.02M sodium carbonate aqueous solution at 60 degrees Celsius for 60 minutes is 50 MPa or higher. Furthermore, the impact strength is 50 kJ / m². 2 Below that, and the maximum bending stress is below 200 MPa.

[0069] When the molded body is not large enough to be measured by impact strength and bending tests, a larger sample is used for strength testing based on the strength uniformity throughout the sample.

[0070] The impact strength of 100 parts by weight can be evaluated by manufacturing a molded article using an additional material to which 0, 25, 50 or 75 parts by weight of the silk fibroin mixture of this disclosure are added, and by strength measurement of said additional material.

[0071] Regarding the aforementioned impact strength and flexural properties, it is preferable that these properties are maintained even after immersion in a 0.02 M sodium carbonate aqueous solution heated to 60°C for 60 minutes. Although proteins are generally considered to be weakly alkaline, if they maintain their strength under the above conditions, they can be said to be strong enough to withstand long-term use.

[0072] <Preparation Method of Fiber Molded Body>

[0073] Example 1

[0074] Step 1. Preparation of fibrous silk fibroin

[0075] After washing the silkworm cocoons with water, a 0.02 mol / L sodium carbonate aqueous solution was heated to 95°C and degummed for 15 minutes to convert them into degummed silk fibroin.

[0076] To determine the molecular weight, degummed silk fibroin was dissolved in a 9.3 mol / L LiBr aqueous solution by stirring at 60°C for 4 hours. Desalting was performed using cellulose tubes 30 / 32 (fractionated molecular weight 12,000 to 14,000) manufactured by Sekisui Chemical Co., Ltd. After dilution with pure water, the aqueous solution was visually inspected to confirm it was homogeneous and free of precipitate. Molecular weight was determined using SDS-PAGE. Details of the measurement method are as disclosed in LS Wray et al., J Biomed Mater Res B Appl Biomater. 2011 Oct.; 99 (1): 89-101. The molecular weight was determined to be 225 kDa.

[0077] The degummed silk fibroin before dissolution was finely ground into fibrous silk fibroin using a shear mill (model SM300, manufactured by Verder Scientific) with a sieve having 8 mm square holes. The fiber diameter was determined to be 13.1 μm when verified using a scanning electron microscope (model JSM-F100, manufactured by JEOL Ltd.). Furthermore, the average length of 100 fibers was determined to be 10.3 mm when dispersed on a scanner with a resolution of 10 μm. Additionally, the infrared absorption spectrum of the fibers was measured using an ATR method on a Frontier FT-IR / NIR spectrometer (manufactured by PerkinElmer), and the crystallinity was determined to be 64.9%, confirming that the fibers are crystalline.

[0078] Step 2. Preparation of silk fibroin powder

[0079] After washing the silkworm cocoons with water, they were boiled in a 0.02 mol / L sodium carbonate aqueous solution for 30 minutes to degummify. The degummed silk fibroin was then dissolved in a 9.3 mol / L LiBr aqueous solution by stirring at 60°C for 4 hours. Desalination was performed using a cellulose tube 30 / 32 (fractionated molecular weight 12,000 to 14,000) manufactured by Sekisui Chemical Co., Ltd. Furthermore, after dilution with pure water, the aqueous solution was visually confirmed to be homogeneous and free of precipitate. This aqueous solution was used as the silk fibroin aqueous solution. The molecular weight of this aqueous solution was determined to be 85 kDa.

[0080] After spreading the silk fibroin aqueous solution onto a tray to a thickness of 5 mm, the tray was placed on a rack in a freeze dryer (model FD-550P, manufactured by Tokyo Electric Machine) and cooled to -30°C. After 3 hours, and after visual confirmation of freezing, decompression was initiated at -6°C and 30 Pa to begin dehydration. After 24 hours, the sample temperature was raised to 15°C, the decompression was released, and the sheet-like dried body was removed.

[0081] The sample was then pulverized by grinding it for 30 seconds in a grinding device (Crash Milser IFM-C 20G, manufactured by Iwatani Industries).

[0082] When the infrared absorption spectrum of the powder was measured using the Frontier FT-IR / NIR spectrometer via the ATR method, the crystallinity was determined to be 10%, thus confirming that the sample was an amorphous form of powdered silk fibroin.

[0083] Step 3. Preparation of the molding mixture

[0084] Ten parts by weight of the fibrous silk fibroin obtained in step 1 and 90 parts by weight of the powdered silk fibroin obtained in step 2 are mixed in a bag and stirred by a strong vibration bench vibrator manufactured by Azwan for about 1 minute to obtain a molding mixture.

[0085] Step 4. Molding process

[0086] 4.8g of the molding mixture was filled into the bottom mold, which had a hole machined in it with a length of 10mm, a width of 85mm, and a depth of 50mm. After installing the top mold, it was moved to a 20t hand press and compressed to achieve a molding pressure of 35 MPa at room temperature. After releasing the load, the mold was temporarily removed. The hand press was heated to 140°C, and the mold was moved back to the hand press and heated and compressed to achieve a molding pressure of 100 MPa. After 10 minutes, the load was released and the mold was air-cooled. When the temperature cooled to 40°C, the mold was disassembled and the molded body was removed. The crystallinity of the molded body surface was measured to be 65.5%. This process was repeated four times, resulting in four molded bodies for physical property evaluation.

[0087] Step 5. Mechanical Performance Evaluation Process

[0088] A grooving machine (model 189-PN, manufactured by Yasuda Seiki Co., Ltd.) was used on the first molded body to groove to a depth of 2 mm. The molded body was then placed in a Charpy impact testing machine (model 258-D, manufactured by Yasuda Seiki Co., Ltd.), and the impact test determined the impact strength to be 6.1 kJ / m. 2 When the fractured surface of the sample was examined using a scanning electron microscope, it was confirmed that non-fibrous fibroin was densely packed between fibrous fibroin.

[0089] The second molded body was also measured using a general-purpose material testing machine (model 5582, manufactured by Instron Japan Co., Ltd.) at a pressing speed of 2 mm / min. The maximum bending stress was 91 MPa.

[0090] Step 6. Mechanical Performance Deterioration Evaluation Process

[0091] The third and fourth molded bodies manufactured in step 4 were immersed in a 0.02 M sodium carbonate aqueous solution heated to 60°C for 60 minutes, and their mechanical properties were evaluated using the same method as in step 5. The evaluation results are shown below. Figure 1A middle.

[0092] Example 2

[0093] In Example 2, silk fibroin was mixed in step 3, so that the fibrous silk fibroin obtained in step 1 was 33 parts by weight and the powdered silk fibroin obtained in step 2 was 67 parts by weight, and then stirred by vibration. Steps 4, 5, and 6 were performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1A middle.

[0094] Example 3

[0095] In Example 3, silk fibroin was mixed in step 3, making 50 parts by weight of the fibrous silk fibroin obtained in step 1 and 50 parts by weight of the powdered silk fibroin obtained in step 2, and then stirred by vibration. Steps 4, 5, and 6 were performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1A middle.

[0096] Example 4

[0097] In step 1 of Example 4, the same procedures were performed except that the molecular weight of the degummed silk fibroin was set to 130 kDa by extending the boiling time of the silkworm cocoons. Step 2 was performed in the same manner as in Example 1. In step 3, the silk fibroin was mixed so that the fibrous silk fibroin obtained in step 1 was 75 parts by weight and the powdered silk fibroin obtained in step 2 was 25 parts by weight, and then stirred by vibration. Steps 4, 5, and 6 were performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1A middle.

[0098] Example 5

[0099] In step 1 of Example 5, the same procedures were performed except that the molecular weight of the degummed silk fibroin was set to 273 kDa by shortening the boiling time of the silkworm cocoons. Step 2 was performed in the same manner as in Example 1. In step 3, the silk fibroin was mixed so that 20 parts by weight of the fibrous silk fibroin obtained in step 1 and 80 parts by weight of the powdered silk fibroin obtained in step 2 were mixed, and then stirred by vibration. Steps 4, 5, and 6 were performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1A middle.

[0100] Example 6

[0101] Following steps 1 and 2 of Example 5, in step 3 of Example 6, silk fibroin is mixed such that the fibrous silk fibroin obtained in step 1 is 5 parts by weight and the powdered silk fibroin obtained in step 2 is 95 parts by weight, and then stirred by vibration. Steps 4, 5, and 6 are performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1B middle.

[0102] Example 7

[0103] In step 1 of Example 6, the degummed silk fibroin was pulverized using a shear mill (model SM300, manufactured by Verder Scientific) equipped with a sieve having 4 mm square holes. When the fiber diameter and average length were measured using the measurement methods described in Example 1, they were determined to be 12.9 μm and 6.6 mm, respectively. Furthermore, when the infrared absorption spectrum of the fiber was measured and the crystallinity was measured, it was determined to be 59.1%, confirming that the fiber was crystalline.

[0104] Step 2 and subsequent steps were performed in the same manner as in Example 6. The crystallinity and mechanical properties of the resulting molded article are shown in... Figure 1B middle.

[0105] Example 8

[0106] In step 1 of Example 6, the degummed silk fibroin was pulverized using a shear mill (model SM300, manufactured by Verder Scientific) with a sieve having 1 mm trapezoidal apertures. When the fiber diameter and average length were measured using the measurement methods described in Example 1, they were determined to be 13.3 μm and 1.3 mm, respectively. Furthermore, when the infrared absorption spectrum of the fiber was measured, it was determined to be 61.9%, and when the crystallinity was measured, it was confirmed that the fiber was crystalline. Steps 2 and subsequent steps were performed in the same manner as in Example 6. The crystallinity and mechanical properties of the resulting molded article are shown in… Figure 1B middle.

[0107] Example 9

[0108] In step 1 of Example 3, the degummed silk fibroin was formed into fibrous silk fibroin without cutting. When the diameter of the fibers was measured using the measurement method described in Example 1, they were each determined to be 12.9 μm. When the infrared absorption spectrum of the fibers was measured, it was determined to be 63.4%, and when the crystallinity was measured, it was confirmed to be crystalline. Steps 2 and subsequent steps were performed in the same manner as in Example 3. The crystallinity and mechanical properties of the resulting molded articles are shown in… Figure 1B middle.

[0109] Comparative Example 1

[0110] Step 2 was performed in the same manner without performing step 1 of Example 1. In step 3, only powdered silk fibroin was used and prepared into a molding mixture. Steps 4, 5, and 6 were performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1B middle.

[0111] Comparative Example 2

[0112] Without performing step 1 of Example 1, in step 2, the boiling time of the silkworm cocoons was shortened, and after preparing a 100 kDa silk fibroin aqueous solution, it was freeze-dried and ground in the same manner to produce powdered silk fibroin. Step 3 and subsequent steps were performed in the same manner as in Comparative Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1B middle.

[0113] Comparative Example 3

[0114] Without performing step 1 of Example 1, in step 2, after washing and drying the silkworm cocoons in water, they were pulverized using a shear mill (model SM300, manufactured by Verder Scientific) equipped with a 0.25 mm trapezoidal mesh screen, without degumming. When measuring the sericin content and crystallinity, they were determined to be 28 parts by weight and 59% by weight, respectively, and used as powdered silk fibroin. Then, in step 3, only powdered silk fibroin was used as the molding mixture. Steps 4, 5, and 6 were performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in… Figure 1B middle.

[0115] Example 10

[0116] In step 1 of Example 1, the silkworm cocoons were washed with water without degumming. The sericin content was measured to be 26.5%. The cocoons were then finely ground using a shear mill (model SM300, manufactured by Verder Scientific) with a sieve having 8mm square holes, producing fibrous silk fibroin. The crystallinity was measured to be 65.0%.

[0117] Subsequently, after preparing a 3% by weight aqueous solution of silk fibroin in step 2 of Example 1, spray drying was performed. The crystallinity of the resulting powder was measured and determined to be 12%.

[0118] In step 3, silk fibroin is placed in a bag, making 30 parts by weight of the fibrous silk fibroin obtained in step 1 and 70 parts by weight of the powdered silk fibroin obtained in step 2, and then vibrated and stirred for 1 minute to prepare a molding mixture. The sericin content of this molding mixture is calculated to be 8 parts by weight. Steps 4, 5, and 6 are performed in the same manner as in Example 1. The crystallinity and mechanical properties of the resulting molded articles are shown in... Figure 1B middle.

[0119] Unless otherwise stated, implementation of this disclosure may employ conventional techniques and descriptions within the art of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and immunology.

[0120] Referring to the specification, specific details are set forth to provide a thorough understanding of the disclosed examples. In other instances, well-known methods, processes, components, and circuits have not been described in detail to avoid unnecessarily lengthening this disclosure.

[0121] It should be understood that if an element or part is referred to herein as "on another element or part," "against," "connected to," or "linked to" another element or part, it may be directly on, against, connected to, or linked to that other element or part, or an intermediary element or part may be present. In contrast, if an element is referred to herein as "directly on another element or part," "directly connected to," or "directly linked to" another element or part, there is no intermediary element or part. When used, the term "and / or" includes any and all combinations of one or more of the associated listed items (if so provided).

[0122] For ease of description, spatial relative terms such as “under,” “beneath,” “below,” “lower,” “above,” “upper,” “near,” and “farer” may be used herein to describe the relationship of one element or feature to other elements or features as shown in the figures. However, it should be understood that, in addition to the orientations depicted in the figures, these spatial relative terms are intended to cover different orientations of the device in use or operation. For example, if the device in the figures is flipped, an element described as “below” or “beneath” other elements or features will be oriented “above” other elements or features. Therefore, spatial relative terms such as “below” can cover both above and below orientations. The device may be oriented in other ways (rotated 90 degrees or in other orientations), and the spatial relative descriptors used herein should be understood accordingly. Similarly, where applicable, the relative spatial terms “near” and “farer” may also be interchangeable.

[0123] As used herein, the term “about” means, for example, within 10%, within 5%, or less. In some embodiments, the term “about” may mean within the measurement error.

[0124] The terms first, second, third, etc., may be used herein to describe various elements, components, regions, parts, and / or sections. It should be understood that these elements, components, regions, parts, and / or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, part, or section from another. Therefore, without departing from the teachings herein, the first element, component, region, part, or section discussed below may be referred to as the second element, component, region, part, or section.

[0125] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise specified herein or the context clearly contradicts it, the use of the terms “a” and “an,” as well as “the,” and similar pronouns in the context of describing this disclosure (especially in the context of the following claims) should be interpreted as covering both the singular and plural. Unless otherwise specified, the terms “comprising,” “having,” “including,” “containing,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Specifically, these terms, when used in this specification, indicate the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof not expressly stated. Unless otherwise specified herein, the description of numerical ranges herein is intended only as a shorthand method for individually referring to each independent value falling within the range, and each independent value is incorporated into the specification as if individually stated herein. For example, if ranges 10-15 are disclosed, then 11, 12, 13, and 14 are also disclosed. Unless otherwise specified herein or otherwise obviously contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate this disclosure and does not constitute a limitation on the scope of this disclosure. None of the language in the specification should be construed as indicating that any unclaimed element is essential to the implementation of this disclosure.

[0126] It should be understood that the methods and components of this disclosure can be incorporated in various embodiments, of which only some are disclosed herein. Variations of those embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will employ such variations where appropriate, and that this disclosure will be practiced in ways other than those specifically described herein. Therefore, this disclosure includes all variations and equivalents of the subject matter recited in the appended claims, as permitted by applicable law. Furthermore, unless otherwise specified herein or otherwise obviously contradicted by the context, any combination of the foregoing elements in all possible variations is covered by this disclosure.

Claims

1. A molded body comprising: At least 80 parts by weight of silk fibroin, in, The silk fibroin includes fibrous silk fibroin and non-fibrous silk fibroin.

2. The molded body according to claim 1, wherein, The non-fibrous fibroin is mixed with the fibrous fibroin to form the molded body.

3. The molded article according to claim 1, wherein, During mixing, the fibrous fibroin adheres to the non-fibrous fibroin.

4. The molded article according to claim 1, wherein, The average diameter of the fibrous fibroin is greater than 1 μm and less than 200 μm.

5. The molded article according to claim 1, wherein, The fibrous fibroin includes fibrous fibroin with a length of 0.1 mm or more and 50 mm or less.

6. The molded article according to claim 1, wherein, The molded body contains at least 10 parts by weight of the fibrous silk protein.

7. The molded article according to claim 1, wherein, The molecular weight of the fibrous fibroin is above 130 kDa.

8. The molded article according to claim 1 further comprises sericin. in, The content of sericin in the molded body is more than 0.1 parts by weight and less than 10 parts by weight.

9. The molded article according to claim 1, wherein, The fibrous fibroin includes sheet-like fibrous fibroin.

10. The molded article according to claim 1 further comprises at least one component selected from carbon fiber and glass fiber.

11. The molded article according to claim 1, wherein, The crystallization rate of the silk fibroin is over 50%.

12. The molded article according to claim 1, wherein, The impact strength of the molded body is 5 kJ / m. 2 The maximum bending stress of the molded body is 50 MPa or more.

13. The molded article according to claim 1, wherein, The maximum bending stress of the molded body after immersion in a 0.02 M sodium carbonate aqueous solution at 60 degrees Celsius for 60 minutes is above 50 MPa.

14. A molding mixture comprising: Fiber fibroin; and Silk fibroin in powder form, in, The solid component of the mixture contains more than 80 parts by weight of silk fibroin.

15. The mixture according to claim 14, wherein, The crystallization rate of the silk fibroin in powder form is less than 20%.

16. A method for manufacturing a molded article, the method comprising: A mixture comprising fibrous silk fibroin and powdered silk fibroin is obtained, wherein the solid component of the mixture contains more than 80 parts by weight of silk fibroin, and The mixture is compressed so that the fibrous silk fibroin binds to the powdered silk fibroin.

17. The method of claim 16, wherein, The fibrous fibroin and the powdered fibroin are produced from silkworm cocoons.

18. The method of claim 16, wherein, The powdered silk fibroin is produced through the following method: Degumming silkworm cocoons yields silk fibroin. Dissolve the silk fibroin in an aqueous solution; and The silk fibroin in powder form is obtained by dehydrating the aqueous solution containing degummed silkworm cocoons.

19. The method of claim 18, wherein, The dehydration is performed by at least one of freeze drying and spray drying.

20. The method of claim 15, wherein, Temperatures above 80 degrees Celsius and below 180 degrees Celsius The compression is performed at a pressure between 30 MPa and 300 MPa. The mixture is compressed and heated.

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