Solidified silk fibroin, aqueous silk fibroin solution and production method therefor, and gel-state fixing material

Abstract

Provided are: solidified silk fibroin including silk fibroin having a specific proportion of molecular weights; an aqueous silk fibroin solution and a production method therefor; and a gel-state fixing material. The silk fibroin included in the solidified silk fibroin, the aqueous silk fibroin solution, and the gel-state fixing material has a weight-average molecular weight, as determined by gel filtration chromatography, of 55-150 kDa.

Description

Solidified silk fibroin, aqueous silk fibroin solution and its manufacturing method, and gel-like fixative Related Applications

[0001] This application claims priority from Japanese Patent Application No. 2024-055040, filed on March 28, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a solidified silk fibroin containing silk fibroin, an aqueous silk fibroin solution containing the silk fibroin, a method for producing the same, and a gel-like fixing material.

[0003] Silk materials generally have low solubility, and this low solubility is not a problem when added to solid materials such as soap, but when dissolving silk materials in water, the solubility must be taken into consideration. To make them soluble in water, silk materials are decomposed to reduce their molecular weight.

[0004] For example, Patent Document 1 (Japanese Patent No. 6019506) discloses a method for producing an aqueous solution of high molecular weight silk fibroin, in which the weight-average molecular weight of the silk fibroin contained in the aqueous solution of high molecular weight silk fibroin is in the range of 10,000 to 50,000 as measured by gel permeation chromatography (GFC).

[0005] On the other hand, Patent Document 2 (JP 2016-517443 A) discloses a low-molecular-weight silk fibroin composition containing a population of silk fibroin fragments having a certain range of molecular weights, wherein 15% or less of the total number of the silk fibroin fragments in the population have a molecular weight of more than 200 kDa, and at least 50% of the total number of the silk fibroin fragments in the population have a molecular weight within a specified range, the specified range being between a lower limit of about 3.5 kDa or more and an upper limit of about 120 kDa or less.

[0006] Patent Document 1 proposes a method for producing a high-molecular-weight silk fibroin aqueous solution with excellent storage stability, but the weight-average molecular weight of the silk fibroin used is at most 50,000 (i.e., 50 kDa), which is insufficient for the degree of molecular weight increase. Therefore, the effect of utilizing the molecular structure of silk fibroin to improve the physical properties of the liquid is insufficient. On the other hand, Patent Document 2 proposes a method for reducing the molecular weight of silk material, but the method is characterized by requiring a long refining time, which limits the degree of molecular weight reduction.

[0007] Therefore, the object of the present invention is to provide a solidified silk fibroin product that has a medium molecular weight, can be stored in a solidified state, has excellent solubility in water, and can have improved physical properties in liquids.

[0008] Another object of the present invention is to provide an aqueous silk fibroin solution containing medium-molecular-weight silk fibroin and having excellent storage stability.

[0009] Another object of the present invention is to provide a gel-like fixing material that contains medium-molecular-weight silk fibroin and is useful for fixing a material to be fixed.

[0010] As a result of extensive research to achieve the above-mentioned objective, the inventors of the present invention discovered that when silk fibroin has a medium molecular weight, not only can it be stored for a long period of time as a solidified silk fibroin, but the solidified product also has good solubility in water and can have improved physical properties in liquids, which led to the completion of the present invention.

[0011] That is, the present invention can be configured in the following aspects. [Aspect 1] A solidified silk fibroin product containing silk fibroin, wherein the weight-average molecular weight of the silk fibroin measured by gel filtration chromatography is 55 to 150 kDa (preferably 60 to 140 kDa, more preferably 70 to 130 kDa). [Aspect 2] The solidified silk fibroin product according to Aspect 1, wherein the proportion of fragments having a molecular weight exceeding 100 kDa in the molecular weight distribution measured by polyacrylamide gel electrophoresis is 15% by weight or less (preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less) and / or the proportion of fragments having a molecular weight of 25 to 100 kDa is 15 to 40% by weight (preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight). [Aspect 3] The solidified silk fibroin according to Aspect 1 or 2, wherein the peak top molecular weight is smaller than the weight average molecular weight. [Aspect 4] The solidified silk fibroin according to any one of Aspects 1 to 3, wherein the solidified silk fibroin is used for cosmetics, foods, or experimental materials. [Aspect 5] The solidified silk fibroin according to any one of Aspects 1 to 4, wherein the moisture absorption after standing for 3 hours under high temperature and humidity conditions of 37°C and 100% RH is 25% or less. [Aspect 6] The solidified silk fibroin according to any one of Aspects 1 to 5, wherein the density is 20 x 10 －３ g / cm ３A solidified silk fibroin as described above. [Aspect 7] The solidified silk fibroin according to any one of Aspects 1 to 6, which is in the form of a powder or a freeze-dried product. [Aspect 8] A method for producing the solidified silk fibroin according to any one of Aspects 1 to 7, which comprises an alkali treatment step of dissolving refined silk fibroin in a solvent and reacting the resulting solution with an alkali treatment agent, and a step of solidifying an aqueous solution containing the alkali-treated silk fibroin. [Aspect 9] The method for producing a solidified silk fibroin according to Aspect 8, which is performed at 40°C or lower (e.g., 5 to 40°C, preferably 10 to 40°C, more preferably 15 to 35°C). [Aspect 10] The method for producing a solidified silk fibroin according to Aspect 8 or 9, wherein the solidifying step comprises obtaining a freeze-dried silk fibroin product from an aqueous solution containing the silk fibroin. [Aspect 11] The method for producing a solidified silk fibroin according to Aspect 10, wherein the solidifying step further comprises a step of pulverizing the freeze-dried silk fibroin. [Aspect 12] A silk fibroin aqueous solution containing silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel filtration chromatography, and containing at least water as a solvent. [Aspect 13] The silk fibroin aqueous solution according to Aspect 12, wherein the proportion of fragments having a molecular weight of more than 100 kDa in the molecular weight distribution as measured by polyacrylamide gel electrophoresis is 15% by weight or less (preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less) and / or the proportion of fragments having a molecular weight of 25 to 100 kDa is 15 to 40% by weight (preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight). [Embodiment 14] The silk fibroin aqueous solution according to embodiment 12 or 13, which is used for oral or transdermal administration.[Aspect 15] A method for producing an aqueous silk fibroin solution, comprising dissolving the solidified silk fibroin according to any one of Aspects 1 to 7 in a solvent containing at least water. [Aspect 16] A gel-like fixing material for fixing an object to be fixed, comprising silk fibroin having a weight-average molecular weight of 55 to 150 kDa (preferably 60 to 140 kDa, more preferably 70 to 130 kDa) as measured by gel filtration chromatography, and comprising a silk fibroin gel produced from an aqueous solution containing at least water as a solvent. [Aspect 17] The gel-like fixative according to Aspect 16, wherein the proportion of fragments with a molecular weight of more than 100 kDa in the molecular weight distribution measured by polyacrylamide gel electrophoresis in the silk fibroin is 15% by weight or less (preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less), and / or the proportion of fragments with a molecular weight of 25 to 100 kDa is 15 to 40% by weight (preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight). [Aspect 18] The gel-like fixative according to Aspect 16 or 17, wherein the object to be fixed is a biological sample or a microorganism.

[0012] In this specification, a "solidified material" refers to a substance that does not easily deform when subjected to external forces, and is distinguished from semi-solidified materials and gels that are easily deformed when subjected to external forces. In addition, in this specification, a gel-like fixing material refers to a semi-solid substance that does not have fluidity even when subjected to external forces or heat, from the perspective of fixing a material to be fixed therein.

[0013] The solidified silk fibroin material and the silk fibroin gel-like fixing material may be distinguished, for example, by their moisture contents. The solidified silk fibroin material may have a moisture content of 50% or less, and the silk fibroin gel-like fixing material may have a moisture content of 80% or more.

[0014] The moisture content of solidified silk fibroin can be calculated from the following formula, where WB is the weight of the target after being left to stand at room temperature (37°C) and 100% RH for 12 hours, and WC is the constant weight of the target after drying at 50°C for a predetermined time: Moisture content (%) = (WB - WC) / WB x 100 Similarly, the moisture content of the silk fibroin gel-like fixing material can be calculated from the above formula, where WB is the weight of the target before drying, and WC is the constant weight of the target after drying at 50°C for a predetermined time.

[0015] In addition, in this specification, the term "aqueous solution" refers to a solution that contains at least water as a solvent, and in which no precipitates are visible to the naked eye in an aqueous solution at or below the limit solubility of silk fibroin.

[0016] In an aqueous solution, the proportion of water in the solvent may be, for example, 50 wt % or more, preferably 70 wt % or more, more preferably 90 wt % or more, and even more preferably 95 wt % or more. There is no particular upper limit, and the solvent may contain only water (100 wt %). Note that a solution containing only water as a solvent is called an aqueous solution.

[0017] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms, including "at least one," unless the content clearly dictates otherwise. As used herein, the terms "and / or," "at least one," and "one or more" include any and all combinations of the associated listed items.

[0018] It should be noted that any combination of at least two elements disclosed in the claims and / or the specification and / or the drawings is included in the present invention, and in particular any combination of two or more of the claims set forth in the claims is included in the present invention.

[0019] The silk fibroin constituting the solidified silk fibroin of the present invention has a medium molecular weight, which not only allows for long-term storage in the form of a solidified product (e.g., powder, freeze-dried product), but also has good solubility in water and an effect of improving the physical properties of liquids (e.g., thickening). Furthermore, the silk fibroin aqueous solution of the present invention has a medium molecular weight, which not only improves the physical properties of aqueous solutions but also prevents gelation in the aqueous solution state, allowing for long-term storage (e.g., about one to one and a half years). On the other hand, the silk fibroin aqueous solution of the present invention can also be gelled by actively carrying out a predetermined gelation treatment, and therefore, in the gelled state, it can also be used as a fixative for a desired object to be fixed (e.g., a biological sample).

[0020] Photographs showing the state of silk fibroin powder (100 mg) of Example 1 before and 5 minutes after adding water (1 mL). Photographs showing the state of silk fibroin powder (100 mg) of Comparative Example 1 before and 5 minutes after adding water (1 mL). Photographs showing the state of silk fibroin powder (100 mg) of Comparative Example 2 before and 5 minutes after adding water (1 mL). Photographs showing the state of silk fibroin powder (100 mg) of Comparative Example 2 before and 5 minutes after adding water (1 mL). Photographs showing the state of cells in the silk fibroin gel fixative of Example 4 before and after vibrating the container by shaking the observation stage (left) and after vibrating the container (right). Photographs showing the state of cells in the silk fibroin gel fixative obtained in Example 4 at 0 hours (left) and 76 hours (right) after the start of culture. Arrows indicate flattened cells, and arrowheads indicate cells undergoing cell division.

[0021] (Solidified Silk Fibroin) The solidified silk fibroin contains medium-molecular-weight silk fibroin. More specifically, the medium-molecular-weight silk fibroin has a weight-average molecular weight (Mw) of 55 to 150 kDa as measured by gel permeation chromatography (GFC).

[0022] The weight-average molecular weight of the silk fibroin may be preferably 60 to 140 kDa, more preferably 70 to 130 kDa. Specifically, the weight-average molecular weight is a value measured by GFC according to the method described in the Examples below.

[0023] Furthermore, the peak top molecular weight (Mp) of silk fibroin measured by GFC is preferably smaller than the weight average molecular weight, and the difference between the peak top molecular weight and the weight average molecular weight may be, for example, 1 to 70 kDa, preferably 10 to 60 kDa, and more preferably 20 to 50 kDa. The peak top molecular weight refers to the molecular weight corresponding to the peak top position of the differential molecular weight distribution curve detected by GFC, and is specifically a value measured by the method described in the Examples below.

[0024] Preferably, the medium-molecular-weight silk fibroin of the present invention has a specific weight-average molecular weight, and the molecular weight distribution includes many low-molecular-weight fragments. In the silk fibroin, the proportion of fragments with a molecular weight exceeding 100 kDa in the molecular weight distribution measured by polyacrylamide gel electrophoresis (SDS-PAGE) may be, for example, 15% by weight or less, preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less. The lower limit is not particularly limited, and may be 0% by weight.

[0025] Furthermore, in the silk fibroin, the proportion of fragments having a molecular weight of 25 to 100 kDa as measured by polyacrylamide gel electrophoresis (SDS-PAGE) may be, for example, 15 to 40% by weight, preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight. The "fragment proportion" refers to the weight ratio of fragments present in a specific range of molecular weight to the weight of all fragments. More specifically, the molecular weight distribution is a value measured by SDS-PAGE using the method described in the Examples below.

[0026] Furthermore, the number average molecular weight (Mn) of the silk fibroin measured by GFC may be 80 kDa or less (e.g., 1 to 80 kDa), preferably 50 kDa or less, more preferably 40 kDa or less, and even more preferably 30 kDa or less. The lower limit of the number average molecular weight is not particularly limited, but may be, for example, 1 kDa or more. Specifically, the number average molecular weight is a value measured by GFC using the method described in the Examples below.

[0027] The polydispersity index (PDI) of silk fibroin may be 1 to 10, preferably 1.5 to 8, more preferably 2 to 6, and even more preferably 3 to 5. The polydispersity index (PDI) is a value (Mw / Mn) obtained by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn).

[0028] Silk fibroin is generally known as a type of fibrous protein, with molecules arranged in a regular pattern and composed of a crystalline portion containing mainly glycine, alanine, and serine, and an amorphous portion containing tyrosine and the like. The raw material for silk fibroin is not particularly limited as long as it has this structure, and silk fibroin can be obtained, for example, from the silk raw material described below. Furthermore, silk fibroin does not need to be chemically modified, but may be chemically modified to the extent that the effects of the present invention are not impaired. In this specification, when the term "silk fibroin" is simply mentioned, the definition also includes silk fibroin that has been chemically modified to the extent that the effects of the present invention are not impaired.

[0029] The solidified silk fibroin is not particularly limited as long as it has excellent solubility in water and can improve the physical properties of an aqueous solution. From the viewpoint of easily adjusting the concentration of silk fibroin in the aqueous silk fibroin solution, a powdery solidified product or a three-dimensional solidified product (e.g., approximately rectangular parallelepiped, approximately cubic, approximately cylindrical, approximately spherical, etc.) that is easy to measure is preferred. From the viewpoint of measurement, the solidified silk fibroin may not preferably contain a silk fibroin film.

[0030] The solidified product may be, for example, a freeze-dried product, a crushed freeze-dried product, a spray-dried product, or the like, since it is derived from an aqueous silk fibroin solution.

[0031] The hygroscopicity of the solidified silk fibroin may be 50% or less, preferably 40% or less, after being left for 3 hours under high-temperature and high-humidity conditions, for example, 37°C and 100% RH. The lower limit of the hygroscopicity is not particularly limited, but may be, for example, 5% or more. Furthermore, from the viewpoint of maintaining solubility in water even when absorbing moisture under high-temperature and high-humidity conditions, the hygroscopicity of the solidified silk fibroin may be 25% or less. Here, the hygroscopicity of the solidified silk fibroin is specifically a value measured in the examples described later.

[0032] When the solidified silk fibroin is a three-dimensional solidified material, for example, the density is 7×10 －３ g / cm ３ or more, preferably 15 x 10 －３ g / cm ３ The upper limit of the density is not particularly limited, but may be, for example, 130 g / cm ３ In order to maintain solubility in water even when absorbing moisture under high temperature and humidity, the density of the solidified silk fibroin may be 25×10 or less. －３ g / cm ３ The density of the solidified silk fibroin material is specifically a value measured in the examples described below.

[0033] The solubility of the solidified silk fibroin in water at 20±5°C according to JIS K 8001 (2017) may be, for example, 1 mL or more but less than 10 mL, as the amount of water required to dissolve 1 g of solidified silk fibroin. Alternatively, when a certain amount of solidified silk fibroin is placed in water and vigorously shaken for 30 seconds every 5 minutes at 20±5°C, the amount of silk fibroin that dissolves within 30 minutes is preferably as high as possible, and may be, for example, 50 mg / mL or more, preferably 110 mg / mL or more, and more preferably 120 mg / mL or more. The upper limit is not particularly limited, but may be 150 mg / mL or less.

[0034] The solidified silk fibroin may be stored at low temperatures or in a specified environment (temperature of 15 to 30°C), and may be used in foods, cosmetics, experiment-related materials, etc. Experiment-related materials may be materials used in connection with experiments conducted on a lab scale, such as various types of experimental culture media.

[0035] (Method for manufacturing solidified silk fibroin) The method for manufacturing solidified silk fibroin includes an alkali treatment step of dissolving refined silk fibroin in a solvent and reacting it with an alkali treatment agent, and a step of solidifying the aqueous solution containing the alkali-treated silk fibroin.

[0036] As the silk fibroin raw material, cocoons and raw silk produced by insects (such as Lepidoptera insects as domesticated silkworms, wild silkworms, and wild silkworms, and other silkworms producing insects such as hornets and honeybees, and other Hymenoptera insects such as wasps and honeybees) or spiders can be used, and there are no particular limitations on the silk raw material as long as it contains fibroin and sericin. Silk fibroin from which sericin has been removed can be obtained by scouring the silk raw material. Silk fibroin can also be obtained from silk glands. Scouring can be performed by known methods, including, for example, a method of removing sericin by swelling it using an alkaline scouring agent such as sodium carbonate, sodium silicate, or sodium phosphate; a method of removing sericin by decomposing it using a sericin-degrading enzyme; and a method of removing sericin by decaying it. From the viewpoint of ease of setting conditions, scouring using an alkaline scouring agent is preferred. The main purpose of degumming is to remove sericin, and from the viewpoint of suppressing the decomposition of silk fibroin, the degumming time using an alkaline degumming agent varies depending on the type of silk raw material and alkaline degumming agent, but may be 5 to 60 minutes, preferably 10 to 50 minutes, and more preferably 15 to 45 minutes.

[0037] Next, the refined silk fibroin can be subjected to an alkali treatment to hydrolyze the silk fibroin and improve its water solubility. From the viewpoint of molecular weight control, the hydrolysis of silk fibroin is preferably carried out in an alkali treatment step. The alkali treatment can be carried out by dissolving silk fibroin in a solvent and reacting it with an alkali treatment agent (e.g., an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, or ammonia). For example, silk fibroin may be dissolved in a neutral salt solution containing a neutral salt such as lithium bromide or calcium chloride. The alkali treatment can be carried out by dissolving silk fibroin in a mixed solution containing a neutral salt and an alkali treatment agent, or by dissolving silk fibroin in a neutral salt solution and then adding an alkali treatment agent to carry out the alkali treatment.

[0038] By performing the alkali treatment under relatively mild conditions, the weight-average molecular weight can be adjusted to a predetermined range. The temperature of the alkali treatment may be 40°C or lower (e.g., 5 to 40°C), preferably 10 to 40°C, and more preferably 15 to 35°C. The alkali treatment time may be longer than 3 hours, preferably 4 hours or more, more preferably 10 hours or more, and even more preferably 15 hours or more. The upper limit of the alkali treatment time is not particularly limited, but may be, for example, 48 hours or less. In this specification, the alkali treatment time refers to the time during which the silk fibroin is in contact with the alkali treatment agent. For example, when a mixed solution containing a neutral salt and an alkali treatment agent is used, the time refers to the time from the addition of the mixed solution to the silk fibroin (or from the silk fibroin to the mixed solution). When the alkali treatment agent is added after dissolving the silk fibroin in a neutral salt solution, the time refers to the time from the addition of the alkali treatment agent.

[0039] After the alkali treatment, the neutral salt and the alkali treatment agent may be removed by a known method such as dialysis or ultrafiltration. Furthermore, if necessary, it is preferable to purify or sterilize the silk fibroin before the freeze-drying treatment in order to remove impurities other than the neutral salt and the alkali treatment agent. Regarding the sterilization, known methods such as autoclave sterilization and filter sterilization can be used, but autoclave sterilization is preferred.

[0040] Next, a step of solidifying the silk fibroin aqueous solution (preferably the purified and / or sterilized silk fibroin aqueous solution) is carried out. In the solidification step, the obtained silk fibroin aqueous solution can be solidified by freeze-drying, spray-drying, or the like.

[0041] For example, freeze-drying is a drying method in which an aqueous silk fibroin solution is frozen at a temperature below its eutectic point and water is removed by sublimation. Freeze-drying can be performed using a known freeze-drying apparatus, and may be performed in multiple stages, for example. Alternatively, pre-freezing may be performed before using the freeze-drying apparatus. Pre-freezing may involve freezing the aqueous silk fibroin solution at a temperature of, for example, about −80 to −30°C. The solution may then be freeze-dried in the freeze-drying apparatus at a trap cooling temperature of, for example, −100 to −60°C, preferably −90 to −70°C, to obtain a freeze-dried product. The freeze-dried product may be powdered to a desired size, or may be used as is without being powdered.

[0042] When the freeze-dried product is powdered, a known method can be used, and for example, the powdering treatment may be carried out using a grinder, a mill mixer, a ball mill, a hammer mill, a roller mill, or the like.

[0043] Spray drying is a method of atomizing an aqueous silk fibroin solution using compressed air. The method of powdering by spray drying is not particularly limited, and a known spray drying device can be used. The spray drying conditions are, for example, preferably set at an inlet temperature of 80°C to 160°C and an outlet temperature of 60°C to 120°C, and more preferably set at an inlet temperature of 100°C to 140°C and an outlet temperature of 70°C to 90°C.

[0044] The powdered silk fibroin powder may be classified as needed, and may be further enclosed in a sterilization pack or the like.

[0045] (Silk Fibroin Aqueous Solution) The silk fibroin aqueous solution contains silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel filtration chromatography (GFC) and at least water as a solvent. Furthermore, the silk fibroin may have, either alone or in combination, various properties described in the description of the solidified silk fibroin, such as the weight-average molecular weight (Mw), peak-top molecular weight (Mp), number-average molecular weight (Mn), polydispersity (Mw / Mn), and molecular weight distribution as measured by SDS-PAGE. The silk fibroin aqueous solution does not gel and has excellent storage stability unless subjected to an active gelation treatment, as described below.

[0046] The concentration of silk fibroin in the aqueous solution may be adjusted depending on the application. For example, the concentration of silk fibroin in the aqueous silk fibroin solution may be 1 to 10% (w / v), preferably 2 to 8% (w / v), and more preferably 3 to 6% (w / v).

[0047] The silk fibroin aqueous solution may be used as an oral aqueous solution or a transdermal aqueous solution. The oral aqueous solution may be used as a drinkable preparation, and may contain, in addition to the silk fibroin aqueous solution, various active ingredients (such as various cosmetic ingredients, hair growth ingredients, and blood circulation promoting ingredients) and additives (described later) depending on the desired use, such as a nutritional tonic or cosmetic application.

[0048] The transdermal aqueous solution may be used as a cosmetic. For example, the silk fibroin aqueous solution may be used as an aqueous cosmetic, or may be used as an oil-based cosmetic by combining the silk fibroin aqueous solution with an oily base. The aqueous cosmetic may be a cosmetic containing 50 wt% or more, preferably 60 wt% or more, of water. The oil-based cosmetic may be a cosmetic containing 50 wt% or more, preferably 60 wt% or more, of an oily base. Furthermore, the cosmetic may contain various active ingredients (various beauty ingredients, hair growth ingredients, blood circulation promoting ingredients, etc.), additives as described below, etc., depending on the intended use.

[0049] As the oily base, known or conventional oils can be used, for example, natural oils such as coconut oil, palm oil, squalane, olive oil, castor oil, almond oil, jojoba oil, orange oil, camellia oil, argan oil, bergamot oil, lavender oil, rosemary oil, rosehip oil, tea tree oil, shea oil, macadamia nut oil, hazelnut oil, kukui nut oil, meadowfoam oil, persic oil, peppermint oil, lanolin, etc.; mineral oils such as mineral oil, petrolatum, paraffin, liquid paraffin, etc.; synthetic oils such as silicone oil, etc. These oily bases may be used alone or in combination of two or more.

[0050] Examples of cosmetics include liquid cosmetics (e.g., lotion, emulsion, serum, cosmetic liquid (e.g., all-in-one cosmetic liquid), emulsified foundation, oil-based foundation, two-layer foundation, nail enamel, shampoo, rinse, conditioner, treatment, tonic, hair growth agent, perm, hair manicure, liquid soap, various oils (e.g., oil cleanser, moisturizing skin oil, hair oil), foam, mist, etc.), and semi-solid cosmetics (e.g., cream, gel, grease, coating agent, ointment, etc.). Liquid cosmetics may be used as a sheet impregnated with the liquid cosmetic. Of these, moisturizing liquid cosmetics, various oils (preferably hair oil), cream, etc. are particularly preferred. For example, silk fibroin is preferred for moisturizing hair oil because it can moisturize the hair while protecting it from heat damage caused by dryers, hair irons, etc.

[0051] When the liquid cosmetic contains water and oil, it may be an emulsion cosmetic such as an oil-in-water (O / W) type, water-in-oil (W / O) type, W / O / W type, or O / W / O type.

[0052] The additives can be appropriately selected depending on the application, and examples thereof include surfactants, antioxidants, antiseptics, disinfectants, preservatives, pH adjusters, ultraviolet absorbers, chelating agents, refreshing agents, stabilizers, fragrances, colorants, etc. These additives may be used alone or in combination of two or more.

[0053] (Gel-like Fixing Material) A silk fibroin aqueous solution may be gelled by actively subjecting it to a gelling treatment and used as a gel-like fixing material. For example, the gel-like fixing material is a gel-like fixing material for fixing an object to be fixed, and contains silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel permeation chromatography (GFC).

[0054] Furthermore, the silk fibroin may have various properties described in the above-described solidified silk fibroin, such as the weight-average molecular weight (Mw) measured by GFC, the molecular weight distribution measured by SDS-PAGE, the peak-top molecular weight (Mp), the number-average molecular weight (Mn), and the polydispersity (Mw / Mn), either alone or in combination of two or more of them.

[0055] When obtaining a gel-like fixing material, the silk fibroin aqueous solution before the gelation treatment may contain other materials (e.g., other solvents, buffer solutions, etc.) depending on the type of object to be fixed, as long as they do not interfere with gelation.

[0056] For example, when a gel-like fixative is used, there are no particular limitations as long as the desired fixation target (e.g., a biological sample, a microorganism, etc.) can be fixed with the gel-like fixative, and the fixation target can be fixed according to its physical properties.

[0057] Biological samples include, for example, cells, tissues, blood (including, for example, whole blood, plasma, umbilical cord blood, platelets, and serum), milk products (e.g., milk), amniotic fluid, sputum, urine, saliva, mucus, semen, cerebrospinal fluid, bronchial aspirate, sweat, nasal secretions, vaginal fluid, liquefied feces, synovial fluid, lymphatic fluid, tears, tracheal aspirate, or fractions thereof (e.g., proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins), or combinations thereof.

[0058] The biological sample may be derived from either a plant or an animal. The animal may be a human or a non-human animal. Examples of non-human animals include non-human mammals, such as apes, other primates, mice, rats, hamsters, guinea pigs, horses, cows, pigs, sheep, goats, dogs, cats, and rabbits.

[0059] Furthermore, microorganisms are a general term for all minute living organisms, and in this specification include viruses, bacteria, fungi (yeast, mold, mushrooms, etc.), microalgae, protozoa (amoeba, paramecium, etc.), etc.

[0060] For example, when the object to be fixed is not easily affected by the gelation treatment (for example, the object to be fixed is an organic material or an inorganic material that is a non-biological sample), the method for producing a gel-like fixative may include a dispersion step in which a predetermined amount of the desired object to be fixed is dispersed in an aqueous silk fibroin solution to obtain a dispersion of the aqueous silk fibroin solution and the object to be fixed, and a gelation step in which a gelation treatment is performed on the dispersion.

[0061] Furthermore, when the object to be fixed is susceptible to the gelation treatment (for example, the object to be fixed is a biological sample, a soft sample such as a microorganism), the method for producing a gel-like fixative may include, for example, a gelation induction step for inducing gelation in an aqueous silk fibroin solution, a mixing step for mixing the desired object to be fixed directly or as a suspension with the aqueous silk fibroin solution that has been subjected to the gelation induction treatment to obtain a dispersion, and a gelation completion step for leaving the dispersion to complete gelation.

[0062] With gel-type fixatives, the object to be fixed is first dispersed in a highly fluid liquid, and then a gelation process is performed to fix the object by forming a network of entangled silk fibroin molecules. This allows the object to be fixed in a state where it is uniformly dispersed in the silk fibroin gel.

[0063] Gelation treatment can be carried out using various known methods. Furthermore, the gelation treatment may comprise a gelation-inducing step and a gelation-completion step. For example, the gelation-inducing step is carried out on a silk fibroin aqueous solution, and examples include a step of adding an acidic substance such as citric acid to adjust the pH to an acidic condition, for example, pH 3 to 6, preferably pH 3 to 4; a step of adding an organic solvent such as ethanol (for example, the amount of organic solvent added is 1 volume or less per volume of the silk fibroin aqueous solution); vortexing; ultrasonic treatment; and spraying with carbon dioxide gas. These gelation-inducing steps may be carried out alone or in combination. Alternatively, for example, when the object to be fixed is a soft sample, vortexing and ultrasonic treatment, as described below, are preferred over gelation treatments under acidic conditions or gelation treatments involving the addition of an organic solvent, in order to reduce damage to the soft sample.

[0064] The vortex treatment conditions can be appropriately set depending on the silk fibroin concentration of the silk fibroin aqueous solution, and may be, for example, a rotation speed of 1,000 to 5,000 rpm, preferably 2,000 to 4,000 rpm, and a vortex treatment time of 1 to 20 minutes, preferably 2 to 11 minutes.

[0065] The ultrasonic treatment conditions can be appropriately set depending on the silk fibroin concentration of the silk fibroin aqueous solution, and the amplitude (Ampl) may be, for example, 10 to 40%, preferably 15 to 30%. The ultrasonic treatment time can be appropriately set depending on the amplitude, and may be, for example, 10 to 60 seconds, preferably 15 to 50 seconds.

[0066] In the gelation completion step, the liquid to be treated that has been treated in the gelation induction step may be allowed to stand at a predetermined temperature, for example, 15 to 40°C, preferably 35 to 40°C, for 100 minutes or less (e.g., 2 to 90 minutes), preferably 80 minutes or less (e.g., 3 to 70 minutes), and more preferably 60 minutes or less (e.g., 4 to 50 minutes), to complete the gelation treatment. By shortening the time until gelation is completed, it is possible to prevent the substances to be fixed that are dispersed in the liquid from settling.

[0067] The gel-like fixative is not particularly limited as long as it can uniformly disperse and fix the object to be fixed. For example, from the viewpoint of preventing the object to be fixed from settling at the bottom of the liquid to be treated, the concentration of silk fibroin in the liquid to be treated may be, for example, 4 to 15% (w / v), preferably 5 to 9% (w / v).

[0068] The gel-like fixative can fix an object to be fixed in a desired container. For example, when the object to be fixed is a biological sample or a microorganism, the object can be fixed by the gel-like fixative preferably in a container such as a multi-well culture plate or a culture dish.

[0069] In addition to the object to be fixed, the gel-like fixative may further contain the additives described above in the silk fibroin aqueous solution, various biologically active substances, etc. Biologically active substances can be selected appropriately depending on the type of object to be fixed, and include, for example, various therapeutic agents, organic molecules, and inorganic materials. Therapeutic agents are not particularly limited, but include, for example, anticancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anticonvulsants, hormones, muscle relaxants, antispasmodics, ophthalmological agents, prostaglandins, antidepressants, antipsychotic substances, nutritional factors, osteoinductive proteins, growth factors, peptide drugs, and vaccines.

[0070] The present invention will be described in more detail below with reference to examples, but the present invention is not limited by these examples. In the following examples and comparative examples, various physical properties were measured by the following methods.

[0071] [Molecular Weight by GFC] The molecular weight of silk fibroin was measured using an aqueous silk fibroin solution adjusted to a concentration of 1% (w / v) as the measurement sample. This measurement sample was used for measurement by gel filtration chromatography (GFC) using the following measurement equipment and conditions. The weight-average molecular weight (Mw), number-average molecular weight (Mn), polydispersity index (PDI), and peak top molecular weight (Mp) of silk fibroin were calculated using molecular weight markers included in the low molecular weight (LMW) gel filtration calibration kits and the high molecular weight (HMW) gel filtration calibration kits (both manufactured by Cytiva) as standards. The measured values ​​are the average of three or more measurements. However, in Comparative Example 1, evaluation was based on a single measurement due to the availability of samples. Apparatus: GFC apparatus "AKTA go" manufactured by Cytiva Separation column: "HiLoad 16 / 600 Superdex 200pg" manufactured by Cytiva Mobile phase: 20 mM sodium phosphate buffer containing 500 mM sodium chloride (pH: 7.4) Flow rate: 1 mL / min Temperature: Room temperature

[0072] [Molecular weight distribution by SDS-PAGE] Silk fibroin solution adjusted to 0.5% (w / v) by dilution with 6 M urea aqueous solution was used as the measurement sample. Sample buffer (4x Laemmli Sample Buffer, BioRad) and 2-mercaptoethanol were mixed at a volume ratio of 9:1 to prepare a reducing agent-containing sample buffer. The measurement sample and the reducing agent-containing sample buffer were mixed at a volume ratio of 3:1 and heated at 98°C for 5 minutes. After cooling to room temperature, the mixture was loaded onto a polyacrylamide gel (EHR-R520L, Atto) at 10 μL / lane and electrophoresed at a constant current (30 mA) using an electrophoresis apparatus (AE-8155 Mypower II500, Atto). The electrophoresis buffer used was electrophoresis buffer solution (30329-61, manufactured by Nacalai Tesque) diluted 10-fold with deionized water, and Precision Plus Protein Two-Color Standard (manufactured by BioRad) was used as a molecular weight marker. After electrophoresis, the polyacrylamide gel was immersed in gel staining solution (EzStain AQua, manufactured by Atto) in a steel square case (1-4698-12, manufactured by AS ONE Corporation) and shaken at room temperature for 2 hours for staining. The gel staining solution was then removed, and deionized water was added. A washing procedure was then performed in which the gel was shaken overnight at room temperature. The deionized water was exchanged five times during the washing procedure. After washing, the polyacrylamide gel was imaged using a gel imaging device (WSE-5400 Printgraph Classic, Atto) equipped with a white light source (Flatview, Atto) (exposure time: 1 msec, magnification: ×1.8). The resulting image data was analyzed using ImageJ software (National Institute of Health). The color tone was set to 8 bits, and background subtraction was performed using the Subtract Background command (Rolling ball radius: 90 pixels). Then, the Gels command was used to obtain the change in brightness relative to the molecular weight in each lane. Based on the brightness peak of the molecular weight marker lane, the amount of silk fibroin distributed between the two molecular weight markers was calculated as an area. The weight ratio of silk fibroin fragments within a certain molecular weight range to the total silk fibroin fragments was calculated by dividing the area by the total area of ​​each lane.

[0073] [Storage stability test for powder] The storage stability test for powder is an accelerated test, in which the powder packed in an aluminum bag is tested under conditions of 40°C ± 2°C / 75% RH ± 5% at the start, for 2 months (guaranteed for 1 year), and for 3 months (guaranteed for 1.5 years), and the color, pH, and moisture content of the powder after storage can be evaluated according to the following criteria.

[0074] (Color tone) A: No change in color tone B: Slight yellowing C: Significant yellowing

[0075] (pH) A: The pH is in the range of 0 or more and less than 0.5 relative to the pH measured at the start. B: The pH is in the range of 0.5 or more and less than 0.8 relative to the pH measured at the start. C: The pH is in the range of 0.8 or more and less than 1.1 relative to the pH measured at the start.

[0076] (Moisture content) A: The moisture content has increased by 0 to less than 5% by weight compared to the moisture content measured at the start. B: The moisture content has increased by 5 to less than 10% by weight compared to the moisture content measured at the start. C: The moisture content has increased by 10% by weight or more compared to the moisture content measured at the start.

[0077] [Storage test in aqueous solution] Silk fibroin powder was dissolved to obtain 1% and 3% aqueous silk fibroin solutions. After storage at 5°C, the solutions were evaluated for color, pH, and gelation at the start, 2 months, 4 months, and 6 months, according to the following criteria.

[0078] (Color tone) A: No change in color tone B: Slight yellowing C: Significant yellowing

[0079] (Gelling) A: The aqueous solution did not gel. B: The aqueous solution gelled.

[0080] [Improvement of physical properties] Five evaluators dropped a drop of the silk fibroin aqueous solution onto the back of their hand, and evaluated the improvement of physical properties relative to water by spreading the drop over the back of their hand according to the following criteria. The following criteria are relative evaluations of two types of silk fibroin aqueous solutions (Example 2 and Comparative Example 1), and the evaluators washed their hands before dropping each drop. A: The feel is relatively sticky. B: The feel is relatively smooth.

[0081] [Solubility in Water] According to JIS K 8001 (2017), silk fibroin powder (100 mg) was placed in deionized water and vigorously shaken for 30 seconds every 5 minutes at 20-22°C. The degree of solubility of the silk fibroin powder in water was determined from the volume of deionized water required for dissolution within 30 minutes.

[0082] [Density of freeze-dried product] 1 mL of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w / v) silk fibroin aqueous solution was placed in a 2 mL screw-cap tube (manufactured by WATSON) with a known weight (W1 (mg)), and freeze-dried using the method described below. Immediately after freeze-drying, the cap of the tube was closed, and the weight (W2 (mg)) of the tube and the freeze-dried silk fibroin product was measured. The tube containing the freeze-dried product was placed next to a tube containing water with a known volume, and the volume (V (cm ３ The density of the freeze-dried product was calculated using the following formula: Density (×10 －３ g / cm ３ )=(W2-W1) / V

[0083] [Hygroscopicity (%)] The lyophilized product was left to stand under high temperature and humidity conditions of 37°C and 100% RH for 3 hours, after which its hygroscopicity was measured. When the mass before standing under high temperature and humidity conditions was Wa and the mass after standing for a predetermined time was Wb, the hygroscopicity was calculated using the following formula: Hygroscopicity (%) = (Wb - Wa) / Wa × 100

[0084] Example 1 Silkworm cocoons were cut into small pieces and degummed in a boiling 0.02 M aqueous solution of sodium carbonate for 30 minutes to obtain silk fibroin.

[0085] 3 g of the refined silk fibroin was immersed in a mixed aqueous solution (50 mL) containing 9 M lithium bromide and 0.1 M sodium hydroxide (NaOH) and left at room temperature for 1 to 6 hours. Next, this mixed aqueous solution was stirred at room temperature for 17 hours to dissolve the silk fibroin in the mixed aqueous solution and to perform an alkali treatment, thereby obtaining a silk fibroin aqueous solution.

[0086] The obtained silk fibroin aqueous solution was dialyzed against deionized water using a dialysis membrane (molecular weight cutoff: 6-8 kJ). Each dialysis session was performed at room temperature for 6-12 hours, and this was repeated six times (bath ratio: 160). This removed the lithium bromide and sodium hydroxide contained in the silk fibroin aqueous solution.

[0087] After removing the lithium bromide and sodium hydroxide, the silk fibroin aqueous solution was concentrated by air-drying at room temperature until the volume of the silk fibroin aqueous solution was reduced to 1 / 5 to 1 / 3.

[0088] The concentrated silk fibroin aqueous solution was subjected to autoclave sterilization using an autoclave device (Tomy Seiko Co., Ltd., "LBS-245"). Autoclave sterilization was performed twice at 121°C for 20 minutes. All subsequent treatments were performed under sterile conditions.

[0089] The autoclave-sterilized silk fibroin aqueous solution was centrifuged (40,000×g, 20° C., 30 minutes) using a centrifugal separator (Beckman Coulter, "Avanti 20I") to remove precipitated insoluble matter.

[0090] After removing the insoluble matter, the silk fibroin aqueous solution was left overnight in a deep freezer (VT-78HC, manufactured by Nippon Freezer Co., Ltd.) and pre-frozen at -80°C. The solution was then freeze-dried (trap cooling temperature: -90°C) for three days using a freeze dryer (FDS-1000, manufactured by Tokyo Rikakikai Co., Ltd.) to obtain a solidified silk fibroin. The solidified product was then crushed in a mortar and pestle to obtain a powder.

[0091] Table 1 shows various physical properties of silk fibroin powder.

[0092] Figure 1 shows the state of silk fibroin powder (100 mg) placed in a glass tube (left) and the state of the tube after adding deionized water (1 mL) and shaking vigorously for 30 seconds after 5 minutes (right). As shown in Figure 1, the silk fibroin powder dissolved in water, and no visible precipitate was observed.

[0093] [Example 2] The silk fibroin powder obtained in Example 1 was redissolved in water at room temperature. The obtained silk fibroin aqueous solution exhibited the various physical properties shown in Table 2.

[0094] [Example 3] The silk fibroin powder obtained in Example 1 was redissolved in water at room temperature to obtain a 4% (w / v) silk fibroin aqueous solution. 500 μL of this solution was placed in a 1.5 mL tube (Eppendorf) and subjected to ultrasonic irradiation (Ampl: 20%, 30 seconds) using an ultrasonic homogenizer (SONICS & MATERIALS "VCX-750"). 300 μL of this solution was transferred to a 2 mL tube (Eppendorf), and the time until gelation was complete was measured using the tilt method.

[0095] Example 4 The silk fibroin powder obtained in Example 1 was redissolved in water at room temperature to obtain a 5% (w / v) silk fibroin aqueous solution. 500 μL was placed in a 1.5 mL tube (Eppendorf) and subjected to ultrasonic irradiation (Ampl: 20%, 30 seconds) using an ultrasonic homogenizer (SONICS & MATERIALS "VCX-750"). 270 μL was then transferred to a new 1.5 mL tube and mixed with 30 μL of 10x concentrated phosphate-buffered saline (Wako Pure Chemical Industries, Ltd.). 285 μL of this mixture was transferred to a new 1.5 mL tube and mixed with 15 μL of a 1.7 × 107 cell / mL suspension of mouse embryonic fibroblasts (NIH / 3T3, provided by the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition). 200 μL of this cell-suspended silk fibroin aqueous solution was spread into one well of a 24-well tissue culture plate (manufactured by Iwaki Co., Ltd.) and placed in a cell culture incubator (temperature: 37°C, CO2 concentration: 5%, humidity: 100%, manufactured by Panasonic Co., Ltd.) After 5 minutes, the cell suspension did not flow even when the plate was tilted, confirming gelation of the cell-suspended silk fibroin aqueous solution.

[0096] Furthermore, cells in the silk fibroin gel fixative were observed using a phase-contrast microscope (BioStudio-mini, equipped with a 4x objective lens, manufactured by Nikon Corporation). As a result, as shown in Figure 4, the relative positions of the cells hardly changed before and after the application of vibration by shaking the observation stage, confirming the immobilization of the cells in the gel fixative.

[0097] One mL of Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco) was added to the gel-like fixative containing the immobilized cells, and the cells were cultured in a cell culture incubator for 0 and 76 hours. The cells in the gel-like fixative were then observed using a phase-contrast microscope (IX-70, equipped with a 10x objective lens, Olympus). As shown in Figure 5, only spherical cells were observed at 0 hours of culture, whereas flattened cells and cells undergoing cell division were observed at 76 hours of culture, indicating that the cells were successfully immobilized in the gel-like fixative without damage.

[0098] Comparative Example 1 Commercially available silk fibroin powder (product name: "Water-soluble Fibroin") was redissolved in water at room temperature. Figure 2 shows the state of silk fibroin powder (100 mg) placed in a glass tube (left) and the state of the tube after adding deionized water (1 mL) and shaking vigorously for 30 seconds after 5 minutes (right). As shown in Figure 2, the silk fibroin powder dissolved in water, and no visible precipitates were observed. This silk fibroin powder was also redissolved in water at room temperature to form a silk fibroin aqueous solution (4% (w / v)), which was subjected to a gelation treatment in the same manner as in Example 3, and the time until gelation was complete was measured.

[0099] Comparative Example 2 Silk fibroin powder was prepared in the same manner as in Example 1, except that a 9M lithium bromide aqueous solution was used as the solution for dissolving the refined silk fibroin, as in Patent Document 2. Table 1 shows the physical properties of the silk fibroin powder. Figure 3 shows the state of silk fibroin powder (100 mg) placed in a glass tube (left) and the state after adding deionized water (1 mL) to the tube and shaking vigorously for 30 seconds after 5 minutes (right). In Comparative Example 2, the silk fibroin aqueous solution was powdered. As shown in Figure 3, the silk fibroin powder was barely soluble in water, and precipitates were visually observed. Note that because precipitates were observed in the silk fibroin powder obtained in Comparative Example 2, experiments using aqueous solutions were not performed.

[0100]

[0101]

[0102]

[0103] As shown in Table 1, the powder obtained in Example 1 had a weight-average molecular weight in the range of 55 to 150 kDa, indicating that silk fibroin could be made into a medium molecular weight. Example 1 and Comparative Example 1 had good solubility in water, but Comparative Example 1 showed insufficient improvement in physical properties. Furthermore, Comparative Example 2 failed to achieve sufficient molecular weight reduction, resulting in poor solubility in water.

[0104] As shown in Table 2, the silk fibroin powder obtained in Example 1 had good storage stability, and even when an accelerated storage test was performed, there was little change in color, pH, and water content after 6 months (guaranteed period) from the start of storage. Furthermore, the silk fibroin aqueous solutions of 1% (w / v) and 3% (w / v) obtained in Example 2 also showed good storage stability, and there was no gelation after 6 months of storage, and there was little change in color and pH.

[0105] Furthermore, as shown in Table 3, the silk fibroin of Comparative Example 1 did not have a medium molecular weight compared to the silk fibroin of Example 3, and therefore required four times as long to complete gelation as that of Example 3. Furthermore, in Example 4, gelation was completed in five minutes.

[0106] Example 5 Silk fibroin was obtained by shredding domesticated silkworm cocoons and degumming them in a boiling 0.02 M aqueous sodium carbonate solution for 30 minutes. 3 g of the degummed silk fibroin was immersed in a mixed aqueous solution (50 mL) containing 9 M lithium bromide and 0.1 M sodium hydroxide (NaOH) and allowed to stand at room temperature for 1 to 6 hours. The mixed aqueous solution was then stirred at room temperature for 17 hours, thereby dissolving the silk fibroin in the mixed aqueous solution and subjecting it to an alkali treatment, yielding a silk fibroin aqueous solution.

[0107] The obtained silk fibroin aqueous solution was dialyzed against deionized water using a dialysis membrane (molecular weight cutoff: 6-8 kJ). Each dialysis session was performed at room temperature for 6-12 hours, and this was repeated six times (bath ratio: 160). This removed the lithium bromide and sodium hydroxide contained in the silk fibroin aqueous solution.

[0108] After removing the lithium bromide and sodium hydroxide, the silk fibroin aqueous solution was concentrated by air-drying at room temperature until the volume of the silk fibroin aqueous solution was reduced to 1 / 5 to 1 / 3.

[0109] The concentrated silk fibroin aqueous solution was subjected to autoclave sterilization using an autoclave device (Tomy Seiko Co., Ltd., "LBS-245"). Autoclave sterilization was performed twice at 121°C for 20 minutes. All subsequent treatments were performed under sterile conditions.

[0110] The autoclave-sterilized silk fibroin aqueous solution was centrifuged (40,000×g, 20° C., 30 minutes) using a centrifugal separator (Beckman Coulter, "Avanti 20I") to remove precipitated insoluble matter.

[0111] The molecular weight of the obtained silk fibroin was Mw: 88 kDa, Mn: 26 kDa, PDI: 3.0, and Mp: 36 kDa.

[0112] Next, the concentration of the silk fibroin aqueous solution was adjusted as shown in Table 4, and the obtained silk fibroin aqueous solution was left to stand overnight in a deep freezer (VT-78HC, manufactured by Nippon Freezer Co., Ltd.) and pre-frozen at -80°C. It was then freeze-dried (trap cooling temperature: -90°C) for 3 days using a freeze dryer (FDS-1000, manufactured by Tokyo Rikakikai Co., Ltd.). Table 4 shows the density of the obtained freeze-dried product, the hygroscopicity of the freeze-dried product in a high-temperature, high-humidity environment, and the solubility in water after storage in a high-temperature, high-humidity environment.

[0113]

[0114] As shown in Table 4, the freeze-dried products obtained by changing the concentration of the silk aqueous solution had different hygroscopicity and density. When the hygroscopicity of the freeze-dried product was low and / or the density of the freeze-dried product was high, the freeze-dried product was able to maintain its solubility in water even after being left under high temperature and high humidity conditions for 3 hours.

[0115] The solidified silk fibroin of the present invention has excellent storage stability and solubility in water, and can improve the physical properties of liquids. Furthermore, the obtained aqueous silk fibroin solution can be stored in a stable state for a long period of time without gelation unless actively subjected to a gelation treatment, and can therefore be used, for example, as cosmetics or drinks. Furthermore, when the aqueous silk fibroin solution is actively gelled, the obtained gel-like fixative can easily fix an object to be fixed.

[0116] While the preferred embodiments of the present invention have been described above, those skilled in the art will readily recognize various changes and modifications within the scope of the present invention, which are obvious from the description of the present invention. Therefore, such changes and modifications are to be interpreted as falling within the scope of the invention as defined by the claims.

Claims

1. A solidified silk fibroin containing silk fibroin, wherein the weight-average molecular weight of the silk fibroin measured by gel filtration chromatography is 55 to 150 kDa.

2. A solidified silk fibroin according to claim 1, wherein the proportion of fragments with a molecular weight exceeding 100 kDa is 15% by weight or less, and / or the proportion of fragments with a molecular weight of 25 to 100 kDa is 15 to 40% by weight, in the molecular weight distribution measured by polyacrylamide gel electrophoresis.

3. A solidified silk fibroin according to claim 1, wherein the peak top molecular weight is smaller than the weight average molecular weight.

4. The solidified silk fibroin according to claim 1, which is used for cosmetics, food or experimental materials.

5. A solidified silk fibroin according to claim 1, which has a moisture absorption of 25% or less after being left to stand for 3 hours under high temperature and humidity conditions of 37°C and 100% RH.

6. The solidified silk fibroin according to claim 1, having a density of 20 x 10 －３ g / cm ３ This is the solidified silk fibroin.

7. The solidified silk fibroin according to claim 1, which is a powder or freeze-dried product.

8. A method for producing a solidified silk fibroin according to any one of claims 1 to 7, comprising an alkali treatment step of dissolving refined silk fibroin in a solvent and reacting it with an alkali treatment agent, and a step of solidifying an aqueous solution containing the alkali-treated silk fibroin.

9. The method for producing solidified silk fibroin according to claim 8, wherein the alkali treatment step is carried out at a temperature of 40°C or less.

10. A method for producing a solidified silk fibroin product according to claim 8, wherein the solidification step comprises a step of obtaining a freeze-dried silk fibroin product from an aqueous solution containing the silk fibroin.

11. A method for producing solidified silk fibroin according to claim 10, wherein the solidifying step further comprises a step of pulverizing the freeze-dried silk fibroin.

12. A silk fibroin aqueous solution containing silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel filtration chromatography, and containing at least water as a solvent.

13. The silk fibroin aqueous solution according to claim 12, wherein the proportion of fragments with a molecular weight exceeding 100 kDa in the molecular weight distribution of the silk fibroin measured by polyacrylamide gel electrophoresis is 15% by weight or less, and / or the proportion of fragments with a molecular weight of 25 to 100 kDa is 15 to 40% by weight.

14. The silk fibroin aqueous solution according to claim 12 or 13, which is used orally or transdermally.

15. A method for producing an aqueous silk fibroin solution, comprising dissolving the silk fibroin powder according to any one of claims 1 to 7 in a solvent containing at least water.

16. A gel-like fixative for immobilizing an object to be fixed, the gel-like fixative containing silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel filtration chromatography, and containing a silk fibroin gel prepared from an aqueous solution containing at least water as a solvent.

17. A gel-like fixative according to claim 16, wherein the silk fibroin has a molecular weight distribution measured by polyacrylamide gel electrophoresis in which the proportion of fragments with a molecular weight exceeding 100 kDa is 15% by weight or less, and / or the proportion of fragments with a molecular weight of 25 to 100 kDa is 15 to 40% by weight.

18. The gel-type fixative according to claim 16 or 17, wherein the object to be fixed is a biological sample or a microorganism.

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