Viscosity changing agent

Abstract

The objective of the present invention is, by providing an aqueous solution tendigng to deviate significantly from the temperature dependence of the viscosity of an ordinary fibrous protein aqueous solution in a specific range of temperature, to provide a simple means for obtaining a desired viscosity at a desired temperature.　More specifically, provided is a viscosity changing agent containing an acidic pH buffering agent to be added to a fibrous protein aqueous solution for changing the viscosity. The viscosity changing agent is configured so that the aqueous solution of the fibrous protein exhibits a pH of less than 5 and a concentration of the acidic pH buffering agent of 5.1 mM or more, and the viscosity is changed in a temperature range of 5°C to 55° C.

Description

Viscosity switching agent

[0001] The present invention relates to a viscosity switching agent, a viscosity-modifiable aqueous fibrous protein solution, and a method for changing the viscosity of an aqueous fibrous protein solution.

[0002] The viscosity of a liquid changes depending on temperature. For example, the viscosity of an aqueous solution of fibrous protein such as fibroin decreases as the temperature rises, similarly to other polymer solutions. This is because an increase in temperature increases the thermal momentum of molecules in the solution, which results in an increase in the fluidity of the solution, that is, a decrease in the viscosity of the solution.

[0003] The viscosity itself of an aqueous fibrous protein solution can be appropriately changed depending on factors such as the concentration of the fibrous protein that is the solute. However, as described above, the temperature dependence tendency of the viscosity of an aqueous fibrous protein solution is constant in accordance with the thermal motion of the constituent molecules, and it is not easy to reverse the overall tendency. Therefore, generally, once the viscosity at low temperature is determined by the concentration of the solute and other factors, the viscosity in a higher temperature range will inevitably become a viscosity that decreases in accordance with the amount of temperature increase.

[0004] On the other hand, as a method for changing the viscosity by deviating from the above temperature dependence in a higher temperature range, post-addition of a solute or a thickener has been known (Non-Patent Document 1).

[0005] Agostinacchio, F., et al. Bioactive materials vol. 35 122-134. 25 Jan. 2024

[0006] Generally, in order to efficiently process, transport, and deliver solutions, it is preferable that the solution has a viscosity suitable for its intended purpose at the processing temperature. However, as mentioned above, fibrous protein aqueous solutions have the constraint that their viscosity cannot be significantly altered due to its temperature dependence. For example, fibrous protein aqueous solutions are usually stored at low temperatures such as 4°C, but when processed at room temperature, the viscosity drops to about half, forcing processing under completely different viscosity conditions than during storage. As a result, additional effort and costs were incurred, such as changing the processing temperature or adjusting equipment to match the viscosity determined by the temperature, in order to achieve the desired viscosity.

[0007] Furthermore, as mentioned above, when viscosity is increased by adding additional solutes or thickeners, the viscosity increases regardless of temperature. As a result, the impact is not limited to the equipment or conditions for a portion of the process, but often necessitates a review of the entire equipment and flow.

[0008] Furthermore, when a protein is the solute, irreversible cross-linking, precipitation, and / or denaturation of that protein irreversibly alters the viscosity of the aqueous solution. Therefore, it is not possible to significantly change the temperature to achieve large viscosity changes, and there are stricter constraints on temperature-based changes in solution viscosity.

[0009] Therefore, the object of the present invention is to provide a simple means for obtaining a desired viscosity at a desired temperature by providing an aqueous solution that exhibits a tendency to deviate significantly from the temperature dependence of the viscosity of a normal fibrous protein aqueous solution in a specific temperature range.

[0010] To solve the above problems, the present inventors conducted intensive research and found that by including an acidic pH buffer at a specific concentration and setting the pH to a specific level, the temperature dependence of the viscosity of the fibrous protein aqueous solution changes, and that the temperature dependence of the viscosity reverses within a specific temperature range. As a result, an aqueous solution is provided that exhibits a tendency to reversibly reverse the temperature dependence of the viscosity of a normal fibrous protein aqueous solution within a specific temperature range, making it possible to utilize a wide range of viscosity changes over a broad temperature range, and providing a simple means to obtain a desired viscosity at a desired temperature.

[0011] The present invention is based on the aforementioned novel findings and provides the following: [1] A viscosity converter comprising an acidic pH buffer for adding to an aqueous solution of fibrous protein to convert its viscosity, wherein the addition causes the aqueous solution of fibrous protein to exhibit a pH of less than 5 and a concentration of the acidic pH buffer of 5.1 mM or more, and the viscosity is converted in a temperature range of 5°C to 55°C. [2] The viscosity converter according to [1], wherein the acidic pH buffer has a pKa value of less than 6. [3] The viscosity converter according to [2], wherein the acidic pH buffer comprises one or more salts selected from the group consisting of citrate, formate, phosphate, phthalate, acetate, glycine salt, maleate, and malate, and / or imidazole, pyridine. [4] The viscosity converter according to any one of [1] to [3], further comprising a metal salt, wherein the addition causes the aqueous solution of fibrous protein to exhibit a concentration of the metal salt of 5.5 mM or more. [5] The viscosity converter according to [4], wherein the metal salt contains an anion having hydration properties equal to or greater than chloride ions in the Hofmeister series. [6] The viscosity converter according to [4] or [5], wherein the metal ion in the metal salt is a cosmotropic ion. [7] The viscosity converter according to any one of [1] to [6], wherein the fibrous protein contains fibroin protein. [8] A viscosity-variable fibrous protein aqueous solution comprising the viscosity converter according to any one of [1] to [7]. [9] The viscosity-variable fibrous protein aqueous solution according to [8], having a maximum viscosity in the temperature range of 20°C to 40°C.

[10] A method for changing the viscosity of a fibrous protein aqueous solution, comprising a temperature adjustment step of adjusting the temperature of the viscosity-variable fibrous protein aqueous solution according to [8] or [9] to 5°C.

[0012] The viscosity converter of the present invention can convert the temperature dependence of the viscosity of fibrous proteins and reverse the temperature dependence in a certain temperature range.

[0013] According to the present invention, the viscosity of the fibrous protein aqueous solution can be reversibly changed simply by changing the temperature.

[0014] This figure shows the temperature dependence of the viscosity of the fibroin protein aqueous solution in Example 1. Figure 1A shows the temperature dependence of the viscosity of a control aqueous solution without salt and acidic pH buffer (dashed line) and a mixture containing salt and acidic pH buffer (solid line). Figure 1B shows a schematic diagram of the calculation method for the minimum value V1, minimum temperature T1, maximum value V2, and maximum temperature T2 of viscosity in the temperature range where the temperature dependence of viscosity reverses. In Figure 1B, the diagonal dashed line indicates an approximate straight line of the viscosity change curve. This figure shows the temperature dependence of the viscosity of the fibroin protein aqueous solution in Example 1. Figure 2A shows the viscosity measurement results at 15°C and 26°C in each temperature change cycle. Figure 2B shows the viscosity measurement results in the 4th and 5th cycles of each temperature change cycle. In Figure 2A, the white circles indicate the measurement results for each cycle and each temperature. In Figure 2B, the white circles indicate the measurement results for the 4th cycle, and the black circles indicate the measurement results for the 5th cycle. This figure shows the temperature dependence of the viscosity of the fibroin protein aqueous solution under each condition in Example 2. Figure 3A shows the results for conditions A to D, and Figure 3B shows the results for conditions A and E to G. This figure shows the temperature dependence of the viscosity of the fibroin protein aqueous solution for each condition (condition A and conditions H to J) in Example 2. This figure shows the temperature dependence of the viscosity of the fibroin protein aqueous solution for each condition (condition A and conditions H to J) in Example 3. Figure 5A shows the results for conditions with and without magnesium sulfate, and Figure 5B shows the results for conditions with salts other than magnesium sulfate. This figure shows the temperature dependence of the viscosity of the fibroin protein aqueous solution for each pH condition in Example 4.

[0015] 1. Viscosity Converter 1-1. Overview The first aspect of the present invention is a viscosity converter. The viscosity converter of the present invention contains an acidic pH buffering agent as an active ingredient and is added to an aqueous solution of fibrous protein to convert the viscosity of the aqueous solution of fibrous protein in a temperature range of 5°C to 55°C. The viscosity converter of the present invention can be an active ingredient in a viscosity-variable aqueous solution of fibrous protein.

[0016] 1-2. Definitions The terms used herein are defined below. "Viscosity" is a measure of the degree of stickiness of a substance. In particular, viscosity as used herein refers to the fluid resistance of a liquid. More specifically, viscosity as used herein refers to the viscosity of a liquid. Methods for measuring viscosity can be known and are not particularly limited, but examples include methods using a capillary viscometer, rotary viscometer, falling ball viscometer, rolling ball viscometer, vibrating viscometer, rheometer, Zahn cup method, flow cup method, etc. For example, a method using a vibrating viscometer can be preferably used.

[0017] "Liquid state" or "liquid" refers to a state in which colloidal particles are dispersed in a dispersion medium and have fluidity, or a state that has been achieved in such a state. In this specification, the liquid state broadly encompasses states that have fluidity, but does not include gel states that result from irreversible gelation. For example, this includes colloids consisting of solutes that can be gelled by raising the temperature, which have become fluid in a dispersion medium. "Colloid" refers to a state in which molecules or ions aggregate to form fine particles and are dispersed in a dispersion medium. The fine particles that form a colloid are called "colloidal particles".

[0018] A "gel" refers to a substance in which colloidal particles self-organize in a dispersion medium, lose their fluidity, and solidify, becoming a solid.

[0019] A "gel state" refers to a state in which colloidal particles self-assemble in a dispersion medium, losing fluidity and solidifying. Generally, this includes a state in which a liquid solidifies due to cooling, and in the case of an aqueous solution of fibrous protein, a state in which all or part of the fibrous protein is cross-linked (chemically and / or physically), precipitated, or denatured, resulting in a loss of fluidity. In this specification, the gel state specifically refers to a gel state resulting from irreversible gelation.

[0020] "Gellation" refers to a phase transition phenomenon from a liquid state to a gel state. In this specification, gelation specifically refers to irreversible gelation.

[0021] "Gelation temperature" refers to the temperature at which a phase transition occurs from a liquid state to a gel state. In this specification, gelation temperature specifically refers to the temperature at which an irreversible phase transition occurs from a liquid state to a gel state.

[0022] In this specification, "converting" viscosity refers to changing the viscosity tendency of a liquid. More specifically, it refers to changing the temperature dependence tendency of viscosity.

[0023] In this specification, "change" of viscosity refers to a reversible alteration of the viscosity of a liquid. However, irreversible gelation is not included in the definition of viscosity change.

[0024] In this specification, "multiple" means, for example, 2 to 1000, 2 to 800, 2 to 600, 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 150, 2 to 100, 2 to 80, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0025] 1-3. Composition The viscosity converter of the present invention contains an acidic pH buffering agent as an essential component. This will be explained in detail below.

[0026] <Acid pH Buffers> In this specification, "acid pH buffer" refers to a pH buffer having a buffer range in the pH range of less than 7. "pH buffer" refers to a drug that exhibits an action that counteracts pH changes (buffering action), and "buffer range" refers to the pH range in which each pH buffer exhibits a buffering action. Typically, a mixture of a weak acid and a salt containing the weak acid as an anion, or an aqueous solution using such a mixture as a solute, is used.

[0027] The acidic pH buffer included in the viscosity converter of the present invention is not particularly limited, as long as it is a pH buffer that includes the pH of the viscosity converter within its buffer range. For example, an acidic pH buffer that includes the pH described later within its buffer range can be used. For example, if the buffer has multiple buffer ranges, at least one of them should include the pH of the viscosity converter.

[0028] As an acidic pH buffer, for example, a pH buffer having a pKa value of less than 6 can be used. The pKa value in this case is not particularly limited, but can be, for example, less than 6, 5.5 or less, 5.2 or less, 5.1 or less, 5 or less, 4.8 or less, 4.7 or less, 4.5 or less, 4.1 or less, 4 or less, 3.9 or less, 3.8 or less, 3.7 or less, 3.6 or less, 3.5 or less, 3.4 or less, 3.3 or less, 3.2 or less, etc. The lower limit of pKa is not particularly limited, but for example, it can be 0.5 or higher, 1 or higher, 1.5 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, 3.5 or higher, 3.6 or higher, 3.7 or higher, 3.8 or higher, 3.9 or higher, 4 or higher, 4.5 or higher, 4.6 or higher, 4.7 or higher, etc. Specifically, for example, the ranges could be 0.5 or more and less than 6, 1 or more and less than 6, 1.5 or more and less than 6, 2 or more and less than 6, 2.5 or more and less than 6, 3 or more and less than 6, 3.5 or more and less than 6, 4 or more and less than 6, 4.5 or more and less than 6, 2 or more and 5 or less, 2.5 or more and 5 or less, 3 or more and 5 or less, 3.5 or more and 5 or less, 4 or more and 5 or less, 4.5 or more and 5 or less, 2 or more and 4.5 or less, 2.5 or more and 4.5 or less, 3 or more and 4.5 or less, 3.5 or more and 4.5 or less, 2.5 or more and 4 or less, 3 or more and 4 or less, 2.5 or more and 3.5 or less, 3 or more and 3.5 or less, etc. For example, if the buffer has multiple pKas, it is sufficient that at least one of them falls within these ranges.

[0029] The specific types of salts included in the acidic pH buffer are not particularly limited. Acidic pH buffers that satisfy these conditions are known in the art, and any of them may be used. For example, acidic pH buffers containing one or more salts selected from the group consisting of citrate (a mixture of citrate and citric acid, etc.), formate (a mixture of formate (e.g., ammonium formate) and formic acid, etc.), phosphate (a mixture of phosphate and phosphoric acid, etc.), phthalate (a mixture of phthalate and phthalic acid, etc.), acetate (a mixture of acetate and acetic acid, etc.), glycine salt (glycine-hydrochloride buffer, etc.), maleate (maleate buffer, etc.), and malate (a mixture of malate and malic acid, etc.), as well as acidic pH buffers containing imidazole, pyridine (pyridine-formate buffer, etc.), etc., can be used.

[0030] The type of cation included in the acidic pH buffer is not particularly limited. For example, the cations described later can be used as metal ions. Typically, sodium salts can be used.

[0031] The acidic pH buffering agent of the present invention can be a combination of several compounds that satisfy the above-mentioned conditions.

[0032] The concentration of the acidic pH buffering agent contained in the viscosity converter of the present invention is not particularly limited. For example, the final concentration when added to an aqueous solution of fibrous protein can be 5.1 mM or higher, 5.5 mM or higher, 6 mM or higher, 7 mM or higher, 8 mM or higher, 9 mM or higher, 10 mM or higher, 15 mM or higher, 20 mM or higher, 21 mM or higher, 22 mM or higher, 23 mM or higher, 24 mM or higher, 25 mM or higher, 30 mM or higher, 35 mM or higher, 40 mM or higher, 50 mM or higher, 75 mM or higher, 100 mM or higher, 125 mM or higher, 150 mM or higher, 200 mM or higher, 250 mM or higher, etc. Furthermore, there is no particular upper limit to this final concentration, but for example, it can be 1000mM or less, 900mM or less, 800mM or less, 700mM or less, 600mM or less, 500mM or less, 400mM or less, 300mM or less, 250mM or less, 240mM or less, 230mM or less, 225mM or less, 220mM or less, 210mM ​​or less, 200mM or less, 190mM or less, 180mM or less, 170mM or less, 160mM or less, 150mM or less, 120mM or less, 100mM or less, etc. More specifically, for example, the final concentration could be 5.1mM to 1000mM, 5.5mM to 1000mM, 6mM to 800mM, 8mM to 500mM, 9mM to 400mM, 10mM to 300mM, 20mM to 300mM, 20mM to 260mM, 25mM to 260mM, 30mM to 260mM, 50mM to 260mM, 100mM to 260mM, 120mM to 260mM, 120mM to 250mM The range can be less than or equal to mM, 120 mM to 230 mM, 120 mM to 200 mM, 120 mM to 180 mM, 140 mM to 260 mM, 140 mM to 250 mM, 140 mM to 230 mM, 140 mM to 200 mM, 140 mM to 180 mM, 140 mM to 170 mM, 150 mM to 260 mM, 150 mM to 250 mM, 150 mM to 230 mM, 150 mM to 200 mM, etc.

[0033] <pH> The viscosity converter of this embodiment is configured such that the pH of the fibrous protein aqueous solution of this embodiment is less than 5 when added. The pH of the viscosity converter itself is not particularly limited.

[0034] The specific pH when added to the aqueous solution of fibrous protein according to this embodiment is not particularly limited, but for example it can be less than 5, 4.9 or less, 4.8 or less, 4.7 or less, 4.6 or less, 4.5 or less, 4.4 or less, 4.3 or less, 4.2 or less, 4.1 or less, 4 or less, 3.5 or less, 3 or less, 2.9 or less, 2.8 or less, 2.5 or less, 2 or less, etc. The lower limit of pH is not particularly limited as long as it is within the range in which the target fibrous protein does not denature. For example it can be 0.5 or more, 1 or more, 1.5 or more, 2 or more, 2.5 or more, 2.7 or more, 2.8 or more, 2.9 or more, 3 or more, etc. Specifically, for example it can be 0.5 or more and less than 5, 1 or more and less than 5, 1.5 or more and less than 5, 2 or more and less than 5, 2.5 or more and less than 5, 2 or more and 4.5 or less, 2 or more and 4 or less, 2 or more and 3.5 or less, 2 or more and 3 or less, 2.5 or more and 3 or less, etc.

[0035] pH adjustment can be performed using any pH adjusting agent. Any acid and / or base can be used as the pH adjusting agent. Specific examples of pH adjusting agents include inorganic acids such as hydrochloric acid and phosphoric acid, organic acids such as formic acid, acetic acid, lactic acid, gluconic acid, malic acid, and citric acid, alkali metal hydroxides such as sodium hydroxide, ammonia salts, carbonates such as sodium bicarbonate, or combinations thereof. For example, basic salts containing metal ions that constitute the conjugate acid of an acidic pH buffer and / or metal salts described later may also be used.

[0036] <Metal Salts> The viscosity converter of this embodiment may further contain metal salts. "Metal salt" means a salt containing a metal ion as a cation, and in this specification, a metal salt includes any salt containing a metal ion. If the cation and / or anion is divalent or greater, it may contain one or more hydrogen ions and / or hydroxide ions, or it may not contain any of them.

[0037] The type of metal ion is not particularly limited. For example, it may be a monovalent, divalent, trivalent, or combination thereof. For example, metal ions of the Hofmeister series can be suitably used. More specifically, for example, metal ions in the Hofmeister series that have a salting-out effect greater than or equal to that of potassium ions can be used. Typical examples of metal ions in the Hofmeister series, from those with the weakest salting-out effect, include cesium ions, rubidium ions, potassium ions, sodium ions, lithium ions, barium ions, calcium ions, magnesium ions, and aluminum ions. In addition, for example, cosmotropic ions can be used as metal ions.

[0038] A "cosmotropic ion" is an ion with a positive Jones-Dole B coefficient. Among the metal ions in the Hofmeister series mentioned above, the ions from sodium onward are considered cosmotropic ions. The values ​​of the Jones-Dole B coefficients for each cation are known in the art and can be measured according to known methods as needed.

[0039] While there are no specific limitations on the Jones-Dole B coefficient values ​​for metal ions, metal ions with values ​​of -0.05 or higher, -0.045 or higher, -0.04 or higher, -0.035 or higher, -0.03 or higher, -0.02 or higher, -0.01 or higher, -0.009 or higher, -0.008 or higher, -0.007 or higher, -0.005 or higher, -0.001 or higher, 0 or higher, 0.01 or higher, 0.02 or higher, 0.05 or higher, 0.06 or higher, 0.07 or higher, 0.08 or higher, 0.086 or higher, 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.25 or higher, 0.28 or higher, 0.29 or higher, 0.3 or higher, 0.35 or higher, 0.37 or higher, 0.38 or higher, and 0.385 or higher can be used.

[0040] Specific metal ions include, for example, one or more of potassium ions, sodium ions, lithium ions, barium ions, calcium ions, magnesium ions, and aluminum ions, and in particular, one or more of potassium ions, sodium ions, and magnesium ions can be suitably used.

[0041] The metal ions used may be one type or a combination of multiple types. When using multiple types of metal ions, a salt with a common anion may be used, or a combination of salts with multiple types of anions may be used.

[0042] The type of anion contained in the metal ion is not particularly limited. For example, it may be a monovalent, divalent, trivalent, or combination thereof anion. For example, anions of the Hofmeister series can be suitably used. More specifically, for example, metal ions that have a hydration effect (salting-out effect) of chloride ions or greater in the Hofmeister series can be used. Typical anions of the Hofmeister series, from those with weaker salting-out effects, include thiocyanate ions, perchlorate ions, iodide ions, nitrate ions, bromide ions, chloride ions, fluoride ions, bicarbonate ions, acetate ions, formate ions, citrate ions, thiosulfate ions, phosphate ions, monohydrogen phosphate ions, dihydrogen phosphate ions, sulfate ions, carbonate ions, tartrate ions, etc. Also, for example, cosmotropic ions can be used as anions.

[0043] Among the Hofmeister series anions mentioned above, ions that exhibit a salting-out effect greater than or equal to that of fluoride ions in the Hofmeister series are considered cosmotropic ions. The Jones-Dole B coefficient values ​​for each anion are known in the art and can be measured according to known methods as needed.

[0044] There are no particular limitations on the specific value of the Jones-Dole B coefficient of anions, and for example, anions having a Jones-Dole B coefficient of -0.05 or higher, -0.04 or higher, -0.032 or higher, -0.03 or higher, -0.02 or higher, -0.01 or higher, -0.009 or higher, -0.008 or higher, -0.007 or higher, -0.005 or higher, -0.001 or higher, 0 or higher, 0.01 or higher, 0.02 or higher, 0.05 or higher, 0.06 or higher, 0.08 or higher, 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.25 or higher, 0.3 or higher, 0.35 or higher, 0.4 or higher, 0.45 or higher, 0.5 or higher, 0.55 or higher, 0.57 or higher, 0.58 or higher, or 0.59 or higher can be used.

[0045] Specific examples of suitable anions include one or more selected from the group consisting of chloride ions, fluoride ions, bromide ions, hydrogen carbonate ions, acetate ions, citrate ions, thiosulfate ions, phosphate ions, monohydrogen phosphate ions, dihydrogen phosphate ions, sulfate ions, and carbonate ions; for example, one or more selected from the group consisting of chloride ions, fluoride ions, acetate ions, citrate ions, dihydrogen phosphate ions, and sulfate ions; and particularly preferably one or more selected from the group consisting of chloride ions, acetate ions, citrate ions, dihydrogen phosphate ions, sulfate ions, and potassium ions. One or more of these can be suitably used.

[0046] One type of anion may be used alone, or a plurality of types of anions may be used in combination. When a plurality of types of anions are used, salts thereof with a common metal ion may be used, or salts with a plurality of types of metal ions may be used in combination.

[0047] There are no particular limitations on the specific metal salt contained in the viscosity converting agent of the present embodiment. For example, the viscosity converting agent can contain any metal salt constituted by a combination of the above-mentioned metal ions and anions. Specifically, for example, one or more selected from the group consisting of sodium citrate, magnesium sulfate, magnesium chloride, and potassium dihydrogen phosphate can be suitably used.

[0048] When the viscosity modifier of the present invention contains a metal salt, the concentration of the metal salt is not particularly limited. For example, the final concentration of the metal salt when added to an aqueous fibrous protein solution can be the same as the concentration described above for the acidic pH buffer. Specifically, for example, the final concentration of the metal salt can be 5.1 mM or more.

[0049] When the viscosity modifier of the present invention contains a metal salt, the ratio of the concentration of metal ions to the concentration of the acidic pH buffer is not particularly limited. For example, the molar concentration ratio of metal ions to the acidic pH buffer can be 10:1 to 1:10, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2, 1.5:1 to 1:1.5, 1.2:1 to 1:1.2, 1.1:1 to 1:1.1, 1:1, or the like.

[0050] <Fibrous Protein> The viscosity modifier of the present embodiment modifies the viscosity of an aqueous fibrous protein solution. The fibrous protein that is the target of the viscosity modifier of the present embodiment will be described in detail in the second aspect. Specific fibrous proteins are not particularly limited, and for example, fibroin proteins derived from silk of various silk-producing organisms (such as bagworm silk) or artificial fibroin proteins can be used as the target.

[0051] 1-4. Effects According to the viscosity modifier of the present embodiment, the viscosity of an aqueous fibrous protein solution can be modified over a partial or entire temperature range of 5°C to 55°C. The temperature range in which viscosity can be modified is not particularly limited as long as it falls within 5°C to 55°C. For example, the viscosity can be modified in the temperature range described in the section on properties of the viscosity-variable aqueous fibrous protein solution according to the second aspect. In the viscosity-modified aqueous fibrous protein solution, the temperature dependence of the viscosity in the liquid state changes, and as described below, the viscosity reaches a maximum at a low temperature of about 20°C to 40°C. This modification causes inversion of temperature dependence in a specific temperature range, an increase in the amount of viscosity change accompanying temperature change, and the like, as described below.

[0052] Since this viscosity modification is not based on irreversible changes in the structure of the fibrous protein, the original temperature dependence can be restored by removing the viscosity modifier of the present embodiment (including dilution of the concentration and change of pH).

[0053] 2. Viscosity-Variable Fibrous Protein Aqueous Solution 2-1. Overview A second aspect of the present invention is a viscosity-variable fibrous protein aqueous solution. The viscosity-variable fibrous protein aqueous solution of the present invention contains a viscosity converter and a fibrous protein as essential components. According to the composition of the present invention, the viscosity can be changed by adjusting the temperature.

[0054] 2-2. Composition The components of the viscosity-variable fibrous protein aqueous solution of the present invention will now be described. The viscosity-variable fibrous protein aqueous solution of the present invention contains a viscosity converter and fibrous protein as essential components, and additives as optional components. Each component will be described in detail below.

[0055] An "aqueous solution" refers to a liquid mixture composed of two or more substances, with water as the primary solvent. The solute dissolved in the aqueous solution may be a solid, liquid, or gas. Examples of solutes include electrolytes and water-soluble substances. Liquid solutes may also include organic solvents such as lower alcohols.

[0056] 2-2-1. Viscosity Converter The viscosity-variable fibrous protein aqueous solution of the present invention contains the viscosity converter described in the first embodiment as an essential active ingredient. Furthermore, it may contain one or more types of viscosity converters as needed.

[0057] The composition of the viscosity converter is described in detail in the first embodiment, so a specific explanation will be omitted here.

[0058] The concentration of the viscosity converter is not particularly limited. For example, the viscosity converter can be added so that the acidic pH buffer, metal ions, anions, etc. reach the final concentrations described in the first embodiment, and the pH reaches the pH described in the first embodiment. Specifically, the fibrous protein aqueous solution of this embodiment has a pH of less than 5 and a concentration of acidic pH buffer of 5.1 mM or more. Furthermore, if a metal salt is further included, the concentration of the metal salt can be, for example, 5.1 mM or more.

[0059] The fibrous protein aqueous solution of this embodiment may contain multiple types of viscosity converters as described in the first embodiment. In this case, the mixing ratio and concentration are not particularly limited, but the sum of their final concentrations will be equal to the final concentration described in the first embodiment, and the final pH will be equal to the pH described in the first embodiment.

[0060] 2-2-2. Fibrous Proteins The aqueous solution of fibrous proteins in this embodiment contains fibrous proteins. In this specification, "fibrous proteins" means proteins that have a fibrous morphology.

[0061] In the context of proteins, "insoluble" means having a three-dimensional structure that does not dissolve in water or aqueous solutions. However, the three-dimensional structure of an insoluble protein can be dissolved through solubilization treatment, converting it back into a water-soluble protein (solubilized) before it formed its three-dimensional structure.

[0062] Specific examples of fibrous proteins include fibroin (including spirulin), keratin, collagen, elastin, myosin, laminin, fibrin, or their artificial proteins. For example, fibroin protein or its artificial protein can be suitably used.

[0063] "Fibroin protein" refers to the silk protein that makes up silk threads derived from insects such as silkworms and bagworms. When simply referred to as fibroin protein, it includes both wild-type and mutant varieties. In this specification, when "fibroin protein" is used, it refers to fibroin H-chain protein unless otherwise specified.

[0064] Generally, natural fibroin heavy chain proteins (FibH) consist of an N-terminal region, a central region, and a C-terminal region as their basic components, in that order from the N-terminus.

[0065] The "N-terminal region" refers to the region in the amino acid sequence that constitutes natural FibH that is located on the N-terminal side of the central region described below, does not contain repeating units, and consists of 70 to 250 amino acid residues.

[0066] Furthermore, the "central region" is the main region that exhibits the physical properties of natural FibH. This region is responsible for the fibrous properties of FibH and is composed of multiple repeating units consisting of the same and / or different amino acid sequences linked together. A "repeating unit" is the main constituent unit of the central region, and one repeating unit can contain multiple glycine (G) and alanine (A) molecules. Therefore, more than 70% of the amino acid sequence of the central region is composed of glycine and alanine.

[0067] Furthermore, the "C-terminal region" refers to the region in the amino acid sequence that constitutes natural FibH that is located on the C-terminal side of the central region, does not contain repeating units, and consists of 30 to 60 amino acid residues.

[0068] The fibroin protein may be natural fibroin protein or artificial fibroin protein.

[0069] In this specification, "natural fibroin protein" refers to fibroin protein that exists in nature. Since natural fibroin protein is a protein encoded by the wild-type genes of various organisms, it is treated as synonymous with "wild-type fibroin protein." Specific examples of natural fibroin protein include natural fibroin H-chain protein, etc.

[0070] "Fibroin H-chain protein" (often abbreviated as "FibH" in this specification) is the main protein constituting the fibroin complex (silk fibroin elementary unit; SFEU complex), which is the fibrous protein component of silk thread. Generally, fibroin H-chain protein refers to the high molecular weight fibrous protein that makes up silk thread derived from insects such as silkworms and bagworms. However, in this specification, spidoin protein, which is a high molecular weight fibrous protein that makes up silk thread, particularly the dragline thread, derived from organisms of the order Araneae or Acari, is also included in the definition of fibroin H-chain protein in the following description. Fibroin H-chain protein usually has an amino acid sequence in which clusters of glycine residues (G) and alanine residues (A) are repeated. In this specification, fibroin H-chain protein may be natural fibroin H-chain protein or artificial fibroin H-chain protein.

[0071] In this specification, "natural fibroin H-chain protein" (natural FibH) refers to FibH that exists in nature. Natural FibH is defined as a protein whose full-length amino acid sequence is identical to that of naturally occurring FibH. Regardless of the collection process, any single molecule of FibH whose composition is identical to that of naturally occurring FibH is included in the definition of natural FibH. Examples include FibH directly collected from bagworms, silkworms, or spiders, or FibH obtained through the expression of the FibH gene. Since natural FibH is a protein encoded by the wild-type FibH gene of various organisms, it is often referred to as "wild-type FibH" in this specification.

[0072] In this specification, "one molecule" means one molecule of FibH, that is, FibH consisting of one peptide chain, unless otherwise specified.

[0073] In this specification, "artificial fibroin protein" refers to fibroin proteins that do not exist in nature, and typically includes mutant fibroin proteins.

[0074] In this specification, "artificial fibroin H-chain protein" (artificial FibH) refers to FibH that does not exist in nature. It is a fibroin H-chain protein in which all or part of natural FibH has been artificially modified using genetic engineering or other technologies.

[0075] In this specification, "mutant fibroin H-chain protein" (often referred to as "mutant FibH" in this specification) is FibH composed of an amino acid sequence different from that of wild-type FibH, and is synonymous with artificial FibH in this specification. Whether or not the FibH has the same physical properties as wild-type FibH is irrelevant. Examples of mutant FibH include mutant FibH obtained by introducing the addition, deletion, and / or substitution of one or more amino acids into the amino acid sequence of FibH, and chimeric FibH (hybrid FibH) obtained by fusing the amino acid sequences of FibH from two or more different insect species.

[0076] In this specification, "wild-type fibroin protein" refers to naturally occurring fibroin protein encoded by the wild-type silk protein gene of various organisms.

[0077] In this specification, "mutant fibroin protein" refers to a silk protein in which all or part of a wild-type fibroin protein has been artificially modified by genetic engineering or other means. It consists of an amino acid sequence different from that of wild-type fibroin protein and, in principle, is a fibroin protein that does not exist in nature. However, it is assumed that mutant fibroin proteins include the N-terminal region, the central region, and the C-terminal region, in order from the N-terminus, which are the basic components of wild-type fibroin protein. Examples of mutant silk proteins include fibroin proteins in which one or more amino acids have been added, deleted, and / or substituted into the amino acid sequence of a fibroin protein, and chimeric fibroin proteins (hybrid fibroin proteins) in which the amino acid sequences of fibroin proteins from two or more different insects have been fused. Mutant fibroin proteins may have the same physical properties as wild-type fibroin proteins or they may have different physical properties.

[0078] The amino acid sequence of the mutant fibroin protein is not particularly limited, but examples include proteins consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence of the natural fibroin protein, or proteins consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the wild-type amino acid sequence.

[0079] In this specification, "amino acid identity" refers to the percentage of identical amino acids in one polypeptide relative to the total number of amino acids in the other polypeptide, after the amino acid sequences of two polypeptides have been aligned and, if necessary, gaps have been introduced into either amino acid sequence to maximize the degree of amino acid agreement between the two polypeptides. This percentage of amino acid identity can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al, J. Mol. Biol., 215, 403-410, 1990).

[0080] The species from which fibroin proteins are derived in this specification are not particularly limited. For example, species belonging to the silkworms may be included.

[0081] In this specification, "silky organisms" refers to a general term for organisms that have silk glands and are capable of spinning or secreting silk. Examples include silkworms and organisms belonging to the order Araneae.

[0082] In this specification, "silkworm" refers to a general term for insects that possess silk glands and are capable of spinning silk. Specifically, it refers to species from the orders Lepidoptera, Hymenoptera, Neuroptera, Trichoptera, etc., that are primarily capable of spinning silk during the larval stage for nesting, cocooning, or migration. Silkworms are defined as insects that are capable of spinning silk, and include those that spin silk at any stage of development, such as larvae and adults. For example, in the order Lepidoptera, this includes species belonging to families such as Bombycidae, Saturniidae, Psychidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Archtiidae, and Noctuidae, which are capable of spinning large amounts of silk. Specifically, for example, silkworms (B. mori) and mulberry silkworms (B. mandarina) belonging to the genus Bombyx, the pearl silkworm (S. cynthia) and the Eri silkworm (S. cynthia ricini) belonging to the genus Samia, the Japanese oak silkworm (A. yamamai) and the Japanese silkworm (A. pernyi) belonging to the genus Antheraea, and the small Japanese oak silkworm (S. yamamai) belonging to the genus Saturnia. This includes species belonging to the genera Acanthopsyche, Anatolopsyche, Bacotia, Bambalina, Canephora, Chalioides, Dahlica, Diplodoma, Eumeta, Eumasia, Kozhantshikovia, Mahasena, Nipponopsyche, Paranarychia, Proutia, Psyche, Pteroma, Siederia, Striglocyrbasia, Taleporia, Theriodopteryx, and Trigonodoma, which are all members of the Psychidae family. In particular, silkworms, which are the larvae of the silkworm moth, and bagworms, which are the larvae of moths belonging to the Psychidae family, are suitable as silkworms. Specific examples of bagworm moths include the giant bagworm moth (Eumeta japonica), the small bagworm moth (Eumeta minuscula), and the moss bagworm moth (Nipponopsyche fuscescens).

[0083] Examples of organisms belonging to the order Araneae include species belonging to the families Araneidae, Nephilidae, Tetragnathidae, Theridiidae, and Linyphiidae. Specific examples include the Araneus genus species Araneus ventricosus, Araneus uyemurai, Araneus diadematus, and Araneus maccacus; the Argiope genus Araneus amoena; the Caerostris genus Caerostris darwini; and the Nephila genus Nephila species Nephila clavata, Nephila clavipes, and Nephila pilipes.

[0084] In this specification, fibrous proteins such as those derived from silkworm silk, spider silk, and bagworm silk can be suitably used.

[0085] In this specification, "bagworm silk" refers to silk derived from bagworms.

[0086] In this specification, "silk" refers to protein-based threads derived from insects and spiders, which are spun by their larvae and adults for purposes such as nesting, movement, anchoring, cocooning, and prey capture. The term "silk" in this specification includes monofilaments, spun fibers, and aggregated fibers.

[0087] In this specification, "spun silk fibers" refer to silk threads in their spun state in the case of bagworms and silkworms, and threads in their secreted state in the case of spiders. The spinned silk fibers of bagworms and silkworms are composed of difilaments, which are pairs of two single fibers. This form is based on the fact that during spinning, two single fibers extruded from the silk thread sacs located on the left and right sides of the bagworm or silkworm, through the spinning opening, are bound together by a sericin-like adhesive substance.

[0088] In this specification, "single fiber" refers to the smallest unit of fiber components, a filament, and is also called a monofilament. Single fibers are mainly composed of fibroin-like proteins that make up silk threads. When a spun fiber is composed of multiple single fibers, for example, adhesive substances can be removed by scouring the spun fiber to obtain single fibers.

[0089] In this specification, "composite fiber" refers to a fiber composed of multiple fiber bundles, also known as a multifilament. This is so-called raw silk, and in principle is composed of multiple single fibers, but in this specification, it also includes cases where it is composed of multiple single fibers and spinning fibers, or multiple spinning fibers. Composite fibers in this specification also include mixed fibers made by mixing multiple types of silk yarn (for example, multiple types of bagworm silk; multiple types of silkworm silk; multiple types of spider silk; silk composed of two or more of bagworm silk, silkworm silk, and spider silk). Composite fibers in this specification include not only twisted yarn fibers but also untwisted yarn fibers.

[0090] Silk threads can include scaffolding silks and nesting silks, but in this specification, silk includes both. "Scaffolding silks" are silk threads spun or secreted by insects or spiders for movement, and they function as scaffolding to prevent them from falling from branches, leaves, etc., during movement. "Nesting silks," on the other hand, are silk threads that make up nests, and are spun or secreted to bind leaf fragments or twig fragments together, or to create the nest or the inner wall of the nest, which is the living area.

[0091] Specific examples of fibroin proteins include α-helix-β-turn / sheet coexisting fibroin H-chain proteins such as the bagworm fibroin H-chain protein.

[0092] In this specification, "α-βt / s coexisting FibH (α-helix-β-turn / sheet coexisting fibroin H-chain protein)" refers to a fibroin H-chain protein characterized by containing one or more α-helix-forming sequences and β-turn-forming sequences in a single FibH molecule. Due to this characteristic, α-βt / s coexisting FibH has the property of allowing α-helix and β-turn to coexist in stable molecular forms within the peptide chain in aqueous solution, and allowing α-helix and β-sheet structures to coexist in stable molecular forms within the peptide chain in fibrous protein coagulation molded products.

[0093] An "α-helix" is a secondary structure in polypeptides that forms a right-handed helical structure. The pitch per helix is ​​0.54 nm, and hydrogen bonds are formed between the imino group of the first amino acid residue and the carbonyl group of the fourth amino acid residue in a peptide chain of four consecutive amino acid residues, with 3.6 amino acid residues per turn.

[0094] In this specification, "β-turn" refers to a secondary structure in polypeptides in which a single molecular chain is bent and forms a sheet structure through intramolecular hydrogen bonding.

[0095] A "β-sheet structure" is a secondary structure in polypeptides in which at least two to three parallel β-chains (β-strands) are bonded laterally by intermolecular hydrogen bonds, forming a pleated sheet structure that is twisted as a whole. Substances that have a β-sheet structure within their molecule may be insoluble in water.

[0096] In this specification, "fibrous protein molded article" means a molded article containing a fibrous protein such as fibroin protein as its main component. It may also be composed solely of fibrous protein. Examples include thin film molded articles, fine particle molded articles, or fiber molded articles. In this specification, "fibrous protein molded article" means a molded article of regenerated fibrous protein unless otherwise specified. Furthermore, when "fibrous protein molded article" is simply referred to in this specification, it shall include both fibrous protein coagulation molded articles and fibrous protein immobilization molded articles, which will be described later.

[0097] In this specification, "regenerated fibrous protein" refers to a solid fibrous protein obtained by coagulating a liquid fibrous protein that was dissolved in an aqueous solution. In this specification, this mainly refers to regenerated fibroin H-chain protein obtained from an aqueous solution of fibroin H-chain protein.

[0098] In this specification, "regenerated fibrous protein molded product" refers to a fibrous protein coagulation molded product or a fibrous protein insolubilized molded product obtained by molding regenerated fibrous protein to have a certain firmness and a specific shape. Examples include thin film molded products, fine particle molded products, or fiber molded products.

[0099] In this specification, "fibrous protein coagulation molded product" refers to a regenerated fibrous protein molded product that is not insolubilized and is obtained by coagulating an aqueous solution of fibrous protein, for example, by dehydration. For example, this includes fibrous protein coagulation molded products made of fibrous proteins that contain one or more α-helix-forming sequences and β-turn-forming sequences in their molecules, i.e., α-βs coexisting fibrous protein coagulation molded products (α-helix-β-sheet coexisting coagulation molded products), etc.

[0100] In this specification, "fibrous protein immobilized molded article" refers to a regenerated fibrous protein molded article that has been immobilized, obtained by coagulating an aqueous solution of fibrous protein, for example, by dehydration. A fibrous protein immobilized molded article usually refers to a β-sheet fibrous protein molded article that has become insoluble in water because the immobilization treatment induces most of the α-helices or β-turns of the fibrous protein to crystallize into a β-sheet structure. In this specification, a fibrous protein molded article obtained by immobilizing a coagulated fibrous protein molded article also falls under the category of fibrous protein immobilized molded article.

[0101] In this specification, "α-helix-forming sequence" refers to an amino acid sequence that, in whole or in part, forms an α-helix in an aqueous solution or a fibrous protein coagulation molded product. For example, an amino acid sequence represented by (A)n (where n is an integer ≥ 6), specifically the amino acid sequence shown in Sequence ID No. 1, is an example. Another example is an amino acid sequence represented by (GGX)n (where X is an alanine residue (A) or a tyrosine residue (Y), and n is an integer ≥ 1), specifically the amino acid sequence shown in Sequence ID No. 2. The helix formed by (GGX)n corresponds to a "31-helix," which has a helix pitch slightly longer than that of an α-helix, but in this specification, the "31-helix" is also included as a structure of the α-helix.

[0102] In this specification, "β-turn forming sequence" refers to an amino acid sequence that, in whole or in part, forms a β-turn in an aqueous solution, and in whole or in part, forms a β-sheet structure in a fibrous protein coagulation molded product. Thus, this β-turn forming sequence can form different secondary structures in an aqueous solution and in a fibrous protein coagulation molded product. Examples of amino acid sequences exhibiting such characteristics include the amino acid sequence represented by (GX)n (where X is an alanine residue (A) or a serine residue (S), and n is an integer ≥ 3), specifically the amino acid sequence shown in Sequence ID No. 3. Also, the amino acid sequence represented by (GAGAGX)n (where X is a serine residue or a tyrosine residue, and n is an integer ≥ 1), specifically the amino acid sequence shown in Sequence ID No. 4. However, exceptionally, in the wild-type silkworm fibroin H chain protein, (GX)n and (GAGAGX)n do not form a β-sheet structure in fibrous protein coagulation molded products, but instead form a water-soluble crystalline structure consisting of aggregates of β-turns called Silk-I type.

[0103] α-βt / s coexisting FibH is defined as having at least one α-helix-forming sequence and one β-turn-forming sequence in its amino acid sequence, regardless of their position or number. For example, in the amino acid sequence of α-βt / s coexisting FibH, the α-helix-forming sequences and β-turn-forming sequences may be arranged in separate, consecutive clusters, or they may be arranged in alternating consecutive sequences. Furthermore, the α-helix-forming sequences and β-turn-forming sequences may be randomly distributed within the amino acid sequence of α-βt / s coexisting FibH.

[0104] In natural FibH, the α-helix-forming sequence and the β-turn-forming sequence may be contained within the repeating unit of the central region, or they may be contained outside of the repeating unit, such as the N-terminal region and / or the C-terminal region.

[0105] An example of artificial FibH having a structure in which α-βt / s coexistence is mutant bagworm FibH.

[0106] In this specification, "mutant bagworm FibH (mutant bagworm fibroin H-chain protein)" refers to a mutant FibH based on bagworm FibH, which is a mutant FibH obtained by introducing amino acid mutations into the amino acid sequence of wild-type bagworm FibH, and / or a chimeric FibH obtained by fusing the amino acid sequences of two or more different insects, including bagworms, and which has the structure of α-βt / s coexisting FibH.

[0107] Mutant bagworm FibH contains one or more repeat units in its amino acid sequence. Examples of specific amino acid sequences that constitute one repeat unit in bagworm FibH include the amino acid sequences shown in SEQ ID NOs. 5-13, amino acid sequences in which one or more amino acids are added, deleted, or substituted in the amino acid sequences shown in SEQ ID NOs. 5-13, or amino acid sequences that have 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequences shown in SEQ ID NOs. 5-13 are amino acid sequences of repeat units unique to wild-type bagworm FibH of the giant bagworm moth, which has the α-βt / s coexisting FibH configuration.

[0108] Specific examples of mutant bagworm FibH include the amino acid sequence shown in SEQ ID NO: 14, which contains four repeat units consisting of the amino acid sequences shown in SEQ ID NOs: 5 to 13; the amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 14; or the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 14. The amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence consisting of a part of the wild-type bagworm FibH of the giant bagworm moth.

[0109] An example of a specific base sequence that encodes the amino acid sequence shown in Sequence ID No. 14 is the base sequence shown in Sequence ID No. 15.

[0110] Furthermore, specific examples of mutant or artificial fibroin proteins include artificial FibH having the α-βt / s coexisting FibH structure.

[0111] As mutant or artificial fibroin proteins, chimeric (hybrid) fibroin proteins can be used, which are a combination of fibroin protein from one species and fibroin protein from another species. For example, chimeric FibH can be used, which is a combination of FibH from a bagworm and FibH from another insect. Specific examples of such chimeric FibH include, for instance, chimeric FibH of bagworm FibH and silkworm FibH. A concrete example of such a chimeric FibH is the chimeric FibH of the bagworm moth (Philadelphia japonica) and silkworm FibH, which consists of the amino acid sequence shown in Sequence ID No. 22. In this chimeric FibH, positions 1-153 and 466-524 are amino acid sequences derived from silkworm FibH, while positions 156-463 contain amino acid sequences derived from bagworm FibH of the bagworm moth. In the case of chimeric FibH, it may possess the physical properties of the FibH of each of the species from which it is derived. Furthermore, FibH may consist of an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 22. An example of a specific nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 22 is the nucleotide sequence shown in SEQ ID NO: 23.

[0112] Another example of a mutant bagworm FibH is a terminal mutant bagworm FibH, which contains a mutation in either the N-terminal region, the C-terminal region, or both of the wild-type bagworm FibH.

[0113] The aforementioned mutation, if it is a deletion, may involve the complete deletion of either the N-terminal region or the C-terminal region, or both, or a partial deletion. Here, "partial" refers to one or two or more consecutive or discontinuous amino acids from the amino acid sequence constituting a specific amino acid region (here, the N-terminal region and / or C-terminal region), and less than the total number of amino acids. Preferably, this corresponds to the number of amino acids that may cause loss of function in the terminal region. For example, this could include 5, 8, 10, 12, 15, 18, or 20 or more consecutive or discontinuous amino acids. In the terminal mutant bagworm FibH, either the N-terminal region or the C-terminal region, or both, may be deleted.

[0114] In addition to deletions, mutations may also include the addition or substitution of one or more amino acids. The amino acids to be added or substituted are not particularly limited, but amino acids that may lose the function of their terminal region are preferred.

[0115] Specific examples of terminal region mutant bagworm FibH include the amino acid sequences shown in SEQ ID NOs: 16-18. SEQ ID NO: 16 is the amino acid sequence of terminal region mutant bagworm FibHΔC, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with the entire C-terminal region deleted. SEQ ID NO: 17 is the amino acid sequence of terminal region mutant bagworm FibHΔN, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with the entire N-terminal region deleted. SEQ ID NO: 18 is the amino acid sequence of terminal region mutant bagworm FibHΔN / C, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with both the N-terminal and C-terminal regions completely deleted.

[0116] Examples of specific base sequences of genes encoding terminal region mutant bagworm FibH include the terminal region mutant bagworm FibHΔC gene shown in Sequence ID No. 19, the terminal region mutant bagworm FibHΔN gene shown in Sequence ID No. 20, and the mutant bagworm FibHΔN / C gene shown in Sequence ID No. 21.

[0117] The fibrous proteins described herein may be produced by their source organism, synthesized by other means, or a combination of both. For example, methods other than those used by the source organism include chemical synthesis and synthesis by organisms other than the source organism (including cells and microorganisms).

[0118] In this specification, "microorganism" refers to a single-celled organism, encompassing both eukaryotic single-celled organisms (such as yeast) and prokaryotes. Typically, prokaryotes are included. The type of microorganism is not limited; any microorganism commonly used in the field of genetic engineering is acceptable. For example, yeast and Escherichia coli are suitable. Unless otherwise specified, in this specification, "microorganism" refers to a transformed organism (genetically modified microorganism) that has been made capable of expressing the target protein through genetic engineering.

[0119] The fibrous protein aqueous solution of this embodiment may additionally contain components of organic fibers other than fibrous proteins or silk threads, or components of inorganic fibers, etc. Examples of organic fibers include plant-based natural fibers such as cotton and hemp, which are mainly composed of cellulose; animal fibers such as wool and other animal hair fibers; and synthetic fibers such as aramid, polyamide (including nylon), polyester, polyethylene, acrylic, and rayon. Examples of inorganic fibers include carbon fibers, glass fibers, metal fibers (stainless steel, titanium, copper, aluminum, nickel, iron, tungsten, molybdenum, etc.), and amorphous fibers (ceramic fibers, rock wool, etc.).

[0120] The concentration of fibrous protein is not particularly limited. For example, the concentration can be 0.1% or more, 1% or more, 2% or more, 2.5% or more, 2.6% or more, 2.7% or more, 2.8% or more, 2.9% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.1% or more, 5.2% or more, 5.3% or more, 5.5% or more, 5.6% or more, 5.7% or more, etc., in terms of the mass percentage of fibrous protein relative to the mass of the aqueous solution (hereinafter the same applies in this specification). Furthermore, there is no particular upper limit, but for example, concentrations of 50% or less, 40% or less, 30% or less, 25% or less, 22% or less, 20% or less, 15% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5.8% or less, 5.7% or less, 5.6% or less, 5.5% or less, 5.3% or less, 5.2% or less, 5.1% or less, 5% or less, 4.5% or less, 4% or less, 3.9% or less, 3.7% or less, 3.5% or less, 3.4% or less, 3.3% or less, 3.2% or less, 3.1% or less, 3% or less, etc. Specific concentration ranges include, for example, 0.1% to 50%, 2.5% to 50%, 1% to 30%, 2.5% to 30%, 2% to 25%, 2.5% to 25%, 2.5% to 20%, 2.5% to 10%, 2.5% to 9%, 2.7% to 9%, 2.5% to 7%, 2.7% to 7%, and so on. Examples include 5% to 6%, 2.7% to 6%, 2.8% to 6%, 2.8% to 5.7%, 2.8% to 5.5%, 2.8% to 5.3%, 3% to 6%, 2.8% to 5.7%, 3% to 5.5%, 3% to 5.3%, 2.5% to 5%, 2.5% to 4%, 2.5% to 3.5%, etc.

[0121] If the aqueous solution of fibrous proteins contains multiple types of fibrous proteins, it can be configured so that the total concentration of those fibrous proteins falls within the above-mentioned range. Furthermore, if it contains a specific protein as a fibrous protein, such as fibroin protein derived from silk threads, for example, bagworm silk, and additionally contains other fiber-derived components, it can be configured so that the concentration of that specific fibrous protein falls within the above-mentioned range.

[0122] 2-2-3. Additives The aqueous solution of fibrous protein according to this embodiment may contain any solvent and additives as long as they do not impair the function of the protein.

[0123] Any solvent commonly used in the art can be used as the solvent. Examples include water or aqueous solutions, or organic solvents. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other auxiliary agents, phosphate buffers, sodium acetate buffers, etc. Examples of auxiliary agents include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Examples of organic solvents include methanol, ethanol, or propanol.

[0124] Any additive commonly used in this art can be used as an additive. Examples include emulsifiers, solubilizers, suspending agents, diluents, dispersants, surfactants, stabilizers, bulking agents, humectants, moisturizers, wetting agents, adsorbents, flavoring and deodorizing agents, coloring agents, preservatives, antiseptics, antioxidants, buffering agents, isotonic agents, and the like.

[0125] For example, emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0126] 2-3. Properties The fibrous protein aqueous solution of this embodiment exhibits a viscosity conversion and a change in the temperature dependence of its viscosity compared to a fibrous protein aqueous solution that does not contain a viscosity converter.

[0127] Specifically, for example, the viscosity has a maximum value in the temperature range in which it exhibits a liquid state. The temperature at which viscosity exhibits a maximum value (maximum temperature) is not particularly limited, but the fibrous protein aqueous solution of this embodiment has maximum temperatures at, for example, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C or higher, 25°C or higher, 26°C or higher, 27°C or higher, 29°C or higher, 29.5°C or higher, etc. Also, for example, the maximum temperature is 50°C or lower, 45°C or lower, 44°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, etc. More specifically, the fibrous protein aqueous solution of this embodiment has maximum temperatures in temperature ranges such as 20°C to 50°C, 20°C to 45°C, 20°C to 40°C, 24°C to 45°C, 24°C to 42°C, 24°C to 41°C, 24°C to 40°C, 24°C to 39°C, 24°C to 38°C, 24°C to 37°C, 25°C to 40°C, 27°C to 39°C, 29°C to 38°C, and 24°C to 37°C.

[0128] The specific value of the maximum viscosity at the maximum temperature varies depending on the concentration of fibrous protein in the aqueous solution of fibrous protein, and is not particularly limited. For example, the maximum value calculated as the coordinates of the intersection of straight lines that approximate the decrease and increase portions of viscosity measured with a vibrating viscometer such as the VM-10A (Sekonic Corporation) is, for example, 10 mPa·s or more, 11 mPa·s or more, 12 mPa·s or more, 13 mPa·s or more, 14 mPa·s or more, 15 mPa·s or more, 16 mPa·s or more, 17 mPa·s or more, 18 mPa·s or more, 19 mPa·s or more, 20 mPa·s or more, 21 mPa·s or more, 22 mPa·s or more, 23 mPa·s or more, 24 mPa·s or more, 25 mPa·s or more, 26 mPa·s or more, 27 mPa·s or more, 28 mPa·s or more, etc. Furthermore, maximum values ​​include, for example, 65 mPa·s or less, 61 mPa·s or less, 60 mPa·s or less, 55 mPa·s or less, 50 mPa·s or less, 45 mPa·s or less, 40 mPa·s or less, 35 mPa·s or less, 34 mPa·s or less, 33 mPa·s or less, 32 mPa·s or less, 31.5 mPa·s or less, 31 mPa·s or less, 30 mPa·s or less, 29 mPa·s or less, 28.5 mPa·s or less, etc. Specifically, the maximum viscosity values ​​of the fibrous protein aqueous solution in this embodiment are, for example, 10 mPa·s to 65 mPa·s, 12 mPa·s to 61 mPa·s, 13 mPa·s to 60 mPa·s, 10 mPa·s to 55 mPa·s, 13 mPa·s to 55 mPa·s, 10 mPa·s to 35 mPa·s, 13 mPa·s to 34 mPa·s, 15 mPa·s to 32 mPa·s, 18 mPa·s to 35 mPa·s, 18 mPa·s to 33 mPa·s, 18 mPa·s to 31.5 mPa·s, 18 mPa·s to 30 mPa·s, 18 mPa·s to 29 mPa·s, etc.

[0129] The fibrous protein aqueous solution of this embodiment may have a minimum viscosity at a temperature below the maximum temperature. The temperature at which viscosity exhibits a minimum value (minimum temperature) is not particularly limited, but the fibrous protein aqueous solution of this embodiment has minimum temperatures at, for example, 5°C or higher, 6°C or higher, 7°C or higher, 8°C or higher, 9°C or higher, 10°C or higher, 11°C or higher, 11.5°C or higher, 12°C or higher, 13°C or higher, 14°C or higher, 15°C or higher, etc. The minimum temperature is also, for example, 30°C or lower, 25°C or lower, 24°C or lower, 23°C or lower, 22°C or lower, 21°C or lower, 20°C or lower, 19°C or lower, 18°C ​​or lower, etc. More specifically, the fibrous protein aqueous solution of this embodiment has minimum temperatures in temperature ranges such as 5°C to 30°C, 5°C to 25°C, 5°C to 23°C, 6°C to 23°C, 7°C to 23°C, 5°C to 22°C, 5°C to 22°C, 6°C to 22°C, 7°C to 22°C, 5°C to 21°C, 5°C to 21°C, 6°C to 21°C, 7°C to 21°C, 10°C to 21°C, 11°C to 20°C, and 14°C to 19°C.

[0130] The specific value of the minimum viscosity at the lowest temperature varies depending on the concentration of fibrous protein in the aqueous solution of fibrous protein, and is not particularly limited. For example, the minimum value calculated as the coordinates of the intersection of straight lines that approximate the decrease and increase portions of viscosity measured with a vibrating viscometer such as the VM-10A (Sekonic Corporation) is, for example, 10 mPa·s or higher, 11 mPa·s or higher, 12 mPa·s or higher, 13 mPa·s or higher, 14 mPa·s or higher, 15 mPa·s or higher, 16 mPa·s or higher, 17 mPa·s or higher, 18 mPa·s or higher, 19 mPa·s or higher, 20 mPa·s or higher, etc. Furthermore, local minimums include, for example, values ​​below 55 mPa·s, below 54 mPa·s, below 53 mPa·s, below 50 mPa·s, below 45 mPa·s, below 40 mPa·s, below 35 mPa·s, below 30 mPa·s, below 25 mPa·s, below 24 mPa·s, below 23 mPa·s, below 22.5 mPa·s, below 22 mPa·s, below 21 mPa·s, below 20 mPa·s, below 19 mPa·s, below 18.5 mPa·s, below 18 mPa·s, below 17 mPa·s, below 16 mPa·s, below 15 mPa·s, etc. Specifically, the minimum viscosity values ​​of the fibrous protein aqueous solution in this embodiment are, for example, 10 mPa·s to 55 mPa·s, 11 mPa·s to 54 mPa·s, 12 mPa·s to 54 mPa·s, 10 mPa·s to 50 mPa·s, 11 mPa·s to 40 mPa·s, 12 mPa·s to 30 mPa·s, 10 mPa·s to 25 mPa·s, and 11 mPa. These ranges include s to 25 mPa·s, 12 mPa·s to 25 mPa·s, 13 mPa·s to 25 mPa·s, 10 mPa·s to 23 mPa·s, 10 mPa·s to 22.5 mPa·s, 11 mPa·s to 22.5 mPa·s, 12 mPa·s to 22.5 mPa·s, 13 mPa·s to 22.5 mPa·s, 10 mPa·s to 22 mPa·s, etc.

[0131] The difference between the minimum and maximum temperatures in the fibrous protein aqueous solution of this embodiment is not particularly limited, but the fibrous protein aqueous solution of this embodiment has minimum and maximum temperatures that differ by, for example, 5°C or more, 7°C or more, 9°C or more, 10°C or more, 10.5°C or more, 11°C or more, 13°C or more, 15°C or more, 15.5°C or more, 16°C or more, 17°C or more, and 18°C ​​or more. In addition, the difference between the minimum and maximum temperatures is, for example, 30°C or less, 27°C or less, 26°C or less, 25°C or less, 21°C or less, 20°C or less, 19°C or less, 18°C ​​or less, 17°C or less, etc. Specifically, the difference between the minimum and maximum temperatures in the fibrous protein aqueous solution of this embodiment is, for example, 5°C to 30°C, 5°C to 27°C, 5°C to 26°C, 10°C to 30°C, 10°C to 27°C, 10°C to 26°C, 13°C to 26°C, 13°C to 25°C, 15°C to 25°C, 15°C to 21°C, 15.5°C to 21°C, 15.5°C to 20°C, etc.

[0132] The specific values ​​of the difference between viscosity extrema (the difference between the maximum and minimum values) are not particularly limited. For example, the difference between extrema calculated as the coordinates of the intersection of straight lines that approximate the decreasing and increasing portions of viscosity measured with a vibrating viscometer such as the VM-10A (Sekonic Corporation) can be, for example, 0.1 mPa·s or more, 0.5 mPa·s or more, 0.8 mPa·s or more, 0.9 mPa·s or more, 1 mPa·s or more, 1.5 mPa·s or more, 1.6 mPa·s or more, 1.7 mPa·s or more, 2 The values ​​of mPa·s and above include 2.2 mPa·s and above, 2.4 mPa·s and above, 2.5 mPa·s and above, 3 mPa·s and above, 3.5 mPa·s and above, 4 mPa·s and above, 4.2 mPa·s and above, 4.4 mPa·s and above, 4.5 mPa·s and above, 5 mPa·s and above, 6 mPa·s and above, 6.5 mPa·s and above, 7 mPa·s and above, 7.5 mPa·s and above, 8 mPa·s and above, 8.5 mPa·s and above, etc. Furthermore, the difference in the extreme values ​​of viscosity can be, for example, 20 mPa·s or less, 15 mPa·s or less, 13 mPa·s or less, 12 mPa·s or less, 11 mPa·s or less, 10.5 mPa·s or less, 10.3 mPa·s or less, 10.1 mPa·s or less, 10 mPa·s or less, etc. Specifically, the minimum viscosity of the fibrous protein aqueous solution in this embodiment may be, for example, 0.1 mPa·s to 20 mPa·s, 0.5 mPa·s to 15 mPa·s, 0.8 mPa·s to 13 mPa·s, 1 mPa·s to 13 mPa·s, 1.5 mPa·s to 11 mPa·s, 2 mPa·s to 11 mPa·s, 2.4 mPa·s to 11 mPa·s, 4 mPa·s to 10.5 mPa·s, 4.4 mPa·s to 10.5 mPa·s, etc.

[0133] The method for determining the maximum (minimum) value and maximum temperature (minimum temperature) is not particularly limited. For example, approximation of viscosity temperature change data by piecewise lines, curves, or partial curves can be used. In the case of piecewise line approximation, for example, the decreasing and increasing portions of viscosity can be approximated by straight lines, and the extreme value and / or the temperature that gives the extreme value can be determined based on the coordinates of the intersection points of these lines. In the case of curve approximation, for example, the viscosity temperature change data can be approximated by exponential function curves, logarithmic function curves, higher-order function curves, or combinations thereof, and the extreme value and / or the temperature that gives it can be determined based on the coordinates of the extreme value of that function. In the case of partial curve approximation, for example, only the data around the extreme value can be considered, and the extreme value and / or the temperature that gives it can be determined based on the coordinates of the maximum value obtained by approximating with a function curve having an extreme value, such as a quadratic function curve.

[0134] The methods for determining the extreme values ​​and the temperatures that give them may differ from one another. For example, the maximum temperature may be determined based on an approximation, and the measured viscosity value of the data closest to the calculated maximum temperature may be taken as the maximum value.

[0135] Any method known in the art can be used as an approximation, and is not particularly limited. Examples include regression analysis, least squares method, and maximum likelihood method.

[0136] The fibrous protein aqueous solution in this embodiment has a maximum viscosity in the temperature range where it is in a liquid state. Therefore, in some temperature ranges (below the maximum temperature, especially in the temperature range between the minimum temperature and the maximum temperature), the temperature dependence of the viscosity is reversed from the usual. Specifically, in some temperature ranges (below the maximum temperature), temperature and viscosity show a negative correlation.

[0137] Furthermore, in some cases, the temperature dependence of viscosity is greater than usual (for example, in the case of a fibrous protein aqueous solution of similar concentration without a viscosity converter) in the temperature range above the maximum temperature. The extent of this increase in temperature dependence is not particularly limited.

[0138] Furthermore, in some cases, the upper limit temperature for viscosity change (for example, a change of 1 mPa·s or more for a temperature change of 5°C) in the fibrous protein aqueous solution of this embodiment is higher. The specific upper limit temperature is not particularly limited, but for example, it falls within the range of 40°C or higher, 45°C or higher, 46°C or higher, 47°C or higher, 50°C or higher, 52°C or higher, 53°C or higher, 55°C or higher, and 56°C or higher. Also, for example, compared to the usual case (for example, a fibrous protein aqueous solution of similar concentration that does not contain a viscosity converter), the upper limit temperature for viscosity change (for example, a change of 1 mPa·s or more for a temperature change of 5°C) may change by 5°C or higher, 6°C or higher, 7°C or higher, 8°C or higher, 9°C or higher, 10°C or higher, 11°C or higher, 15°C or higher, 20°C or higher, and 25°C or higher.

[0139] Preferably, in the aqueous fibrous protein solution of this embodiment, the viscosity in the temperature range below the minimum temperature and / or above the upper limit temperature of viscosity change (where gelation occurs) (i.e., outside the temperature range where temperature conversion occurs) is equivalent to that of the aqueous fibrous protein solution without the viscosity converter described in the first embodiment (i.e., there is no significant difference).

[0140] By utilizing the above-mentioned properties of the fibrous protein aqueous solution according to this embodiment, the viscosity can be reversibly changed solely by temperature changes. The specific method for doing so is as described in the third embodiment.

[0141] 3. Method for Changing the Viscosity of a Fibrous Protein Aqueous Solution 3-1. Overview The third aspect of the present invention is a method for changing the viscosity of a fibrous protein aqueous solution. The method of this aspect includes a temperature control step as an essential step and a preparation step as an optional step. According to the method of this aspect, the viscosity of a fibrous protein aqueous solution can be reversibly changed.

[0142] 3-2. Process 3-2-1. Preparation Process The "Preparation Process" is an optional process and is the process of preparing an aqueous solution of viscosity-variable fibrous protein.

[0143] For viscosity-variable fibrous protein aqueous solutions, the description in the second embodiment applies.

[0144] The preparation method used in this process can be any method used to prepare an aqueous solution containing multiple components. For example, it may be prepared by adding the fibrous protein and viscosity converter to the solvent simultaneously or separately, or by adding the solvent to the fibrous protein and / or viscosity converter and then adding the remaining components.

[0145] Fibrous proteins can be solubilized as needed. The method of solubilization is not particularly limited. For example, it can be done by adding a solubilizing agent.

[0146] Any solubilizing agent known in the art may be used, and the specific solubilizing agent used is not particularly limited. Examples of usable solubilizing agents include urea, guanidine salts, nitrates, acyl sarcosine salts, lithium salts, sodium iodide, perchlorates, or combinations thereof.

[0147] The temperature used during preparation is not particularly limited, as long as it is a temperature at which the fibrous protein aqueous solution can be brought into a liquid state by temperature control. For example, it may be within the temperature range used in the temperature control process, or it may be outside that range. The specific temperature is not particularly limited, but for example, it may be any of the following: freezing temperature (below 0°C), refrigeration temperature (0°C to 10°C), low temperature (10°C to 20°C), cool place (1°C to 15°C), normal temperature (15°C to 25°C), room temperature (1°C to 30°C), lukewarm temperature (30°C to 40°C), or any temperature between 40°C and 100°C.

[0148] In addition to addition, any additional processing can be performed. The specific additional processing is not limited, but for example, mixing, stirring, dilution, concentration, and / or pH adjustment can be performed after addition. If the concentration or pH changes due to the additional processing, the resulting concentration or pH should remain within the ranges described in the first and second embodiments.

[0149] 3-2-2. Temperature Control Process The "temperature control process" is a process of adjusting the temperature of the viscosity-variable fibrous protein aqueous solution to 5°C to 55°C. If the preparation process is performed, this process can be performed simultaneously with or after the preparation process.

[0150] Any method known in the art can be used for temperature control, and is not particularly limited. Typically, temperature control can be achieved by combining heating and cooling methods.

[0151] For example, the heating method may be based on either direct heating (resistance heating, induction heating, etc.) or indirect heating (radiant heating, conduction heating, convection heating, etc.). The heat source is not particularly limited. For example, heating by combustion of gas or oil, electrical resistance, electromagnetic waves, lasers, electron beams, electrical discharges, etc., heating based on heat pumps, heating based on chemical reactions such as specific oxidation reactions or hydration reactions, or a combination thereof can be used.

[0152] Furthermore, the cooling method is not particularly limited, but for example, it may be used for convection cooling (natural convection cooling, forced convection cooling, etc.), radiative cooling, liquid cooling, evaporative cooling, thermoelectric cooling, phase change cooling, cooling based on a heat pump, cooling based on a chemical reaction such as a specific dehydration reaction or decarboxylation reaction, or a combination thereof.

[0153] In this process, the temperature of the fibrous protein aqueous solution is adjusted to 5°C to 55°C. The temperature range can be any range from 5°C to 55°C and is not particularly limited. For example, it can be a temperature range above the minimum temperature described above in the second embodiment and below the upper limit of the temperature at which viscosity changes. The specific lower limit of the temperature range can be, for example, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 11.5°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, etc. The specific upper limit of the temperature range can be, for example, 55°C, 53°C, 52°C, 50°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41°C, 40°C, etc. Specific temperature ranges can include, for example, 5°C to 55°C, 6°C to 55°C, 7°C to 55°C, 9°C to 55°C, 10°C to 55°C, 11°C to 55°C, 12°C to 55°C, 13°C to 55°C, 14°C to 55°C, 15°C to 55°C, 16°C to 55°C, 18°C ​​to 55°C, 20°C to 55°C, 5°C to 53°C, 10°C to 53°C, 15°C to 53°C, 20°C to 53°C, 5°C to 52°C, 10°C to 52°C, 15°C to 52°C, 20°C to 52°C, 5°C to 51°C, 10°C to 51°C, 15°C to 51°C, 20°C to 51°C, 5°C to 50°C, 10°C to 50°C, 15°C to 50°C, 20°C to 50°C, etc.

[0154] Within this temperature range, a fibrous protein aqueous solution can be thickened or thinned by performing specific temperature changes. The thickening and thinning steps for each method are described below. In this process, either the thickening or thinning step may be performed individually, or both may be combined. Furthermore, the thickening and / or thinning step may be combined with temperature changes in other temperature ranges (e.g., below the minimum temperature). Each step can be performed once or more times. Additionally, by performing the thickening step in this process, the method of this embodiment can be implemented as a reversible thickening method. Similarly, by performing the thinning step in this process, the method of this embodiment can be implemented as a reversible thinning method.

[0155] Even if temperature changes occur in temperature ranges other than those described later, changes within the temperature range described later are included in this process and each step.

[0156] <Thickening Step> This step involves increasing the viscosity of the fibrous protein aqueous solution by changing its temperature.

[0157] Examples of temperature changes in this case include (i) heating from a temperature above the minimum temperature but below the maximum temperature towards the maximum temperature, and (ii) cooling from a temperature above the maximum temperature towards the maximum temperature.

[0158] In case (i), the starting temperature is not particularly limited, as long as it is above the minimum temperature and below the maximum temperature described in the second embodiment. Specifically, for example, the starting temperature can be in the range of 5°C to 35°C, 7°C to 35°C, 9°C to 35°C, 10°C to 35°C, 11°C to 35°C, 13°C to 35°C, 15°C to 35°C, 17°C to 35°C, 5°C to 30°C, 10°C to 30°C, 11°C to 30°C, 13°C to 30°C, 15°C to 30°C, 17°C to 30°C, 5°C to 25°C, 10°C to 25°C, 11°C to 25°C, 13°C to 25°C, 15°C to 25°C, 17°C to 25°C, 5°C to 20°C, 10°C to 20°C, 11°C to 20°C, 13°C to 20°C, 15°C to 20°C, or 17°C to 20°C.

[0159] In case (i), the final temperature is not particularly limited, and should be higher than the starting temperature and below the maximum temperature. Specifically, for example, the final temperature can be any temperature in the range of 10°C to 45°C, 15°C to 45°C, 20°C to 45°C, 25°C to 45°C, 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, 25°C to 40°C, 20°C to 37°C, 24°C to 37°C, 25°C to 37°C, 10°C to 35°C, 15°C to 35°C, 20°C to 35°C, 25°C to 35°C, 10°C to 30°C, 15°C to 30°C, 20°C to 30°C, or 25°C to 30°C, and is higher than the starting temperature.

[0160] Specific temperature changes in case (i) include, for example, a change from a starting temperature of 5°C to 35°C to a final temperature of 10°C to 45°C, from a starting temperature of 10°C to 35°C to a final temperature of 20°C to 45°C, from a starting temperature of 5°C to 30°C to a final temperature of 20°C to 45°C, from a starting temperature of 10°C to 30°C to a final temperature of 20°C to 45°C, from a starting temperature of 5°C to 20°C to a final temperature of 20°C to 45°C, from a starting temperature of 10°C to 20°C to a final temperature of 25°C to 45°C, from a starting temperature of 5°C to 30°C to a final temperature of 25°C to 45°C, and from a starting temperature of 10°C to 20°C to a final temperature of 25°C to 45°C. In all of these cases, the final temperature is higher than the starting temperature.

[0161] In case (ii), the starting temperature is not particularly limited, as long as it is higher than the maximum temperature described in the second embodiment and below the upper limit of the temperature at which viscosity changes. Specifically, for example, temperatures within the ranges of 20°C to 56°C, 24°C to 56°C, 25°C to 56°C, 29°C to 56°C, 30°C to 56°C, 35°C to 56°C, 37°C to 56°C, 20°C to 53°C, 24°C to 53°C, 25°C to 53°C, 29°C to 53°C, 30°C to 53°C, 35°C to 53°C, 37°C to 53°C, 20°C to 50°C, 24°C to 50°C, 25°C to 50°C, 29°C to 50°C, 30°C to 50°C, 35°C to 50°C, 37°C to 50°C, 20°C to 45°C, 24°C to 45°C, 25°C to 45°C, 29°C to 45°C, 30°C to 45°C, 35°C to 45°C, and 37°C to 45°C can be used as the defrosting temperature.

[0162] In case (ii), the final temperature is not particularly limited and can be any temperature in the range of 20°C to 50°C, 24°C to 50°C, 25°C to 50°C, 29°C to 50°C, 30°C to 50°C, 35°C to 50°C, 37°C to 50°C, 20°C to 45°C, 24°C to 45°C, 25°C to 45°C, 29°C to 45°C, 30°C to 45°C, 35°C to 45°C, 37°C to 45°C, 20°C to 40°C, 24°C to 40°C, 25°C to 40°C, 29°C to 40°C, 30°C to 40°C, 35°C to 40°C, or 37°C to 40°C, that is lower than the starting temperature.

[0163] Specific temperature changes in case (ii) include, for example, a change from an initial temperature of 20°C to 56°C to a final temperature of 20°C to 50°C, from an initial temperature of 24°C to 56°C to a final temperature of 20°C to 45°C, from an initial temperature of 29°C to 56°C to a final temperature of 20°C to 45°C, from an initial temperature of 24°C to 56°C to a final temperature of 20°C to 40°C, from an initial temperature of 24°C to 56°C to a final temperature of 20°C to 40°C, from an initial temperature of 24°C to 50°C to a final temperature of 20°C to 45°C, from an initial temperature of 24°C to 50°C to a final temperature of 20°C to 40°C, and from an initial temperature of 29°C to 50°C to a final temperature of 20°C to 40°C. In all of these cases, the final temperature is lower than the initial temperature.

[0164] <Thickness Reduction Step> This step involves reducing the viscosity of the fibrous protein aqueous solution by changing its temperature.

[0165] Examples of temperature changes in this case include (i) cooling from a temperature above the minimum temperature but below the maximum temperature towards the minimum temperature, and (ii) heating from a temperature above the maximum temperature but below the upper limit of the temperature at which viscosity changes.

[0166] In case (i), the starting temperature is not particularly limited, and should be higher than the minimum temperature described in the second embodiment and less than or equal to the maximum temperature.

[0167] In case (i), the final temperature is not limited to any temperature above the minimum temperature and below the starting temperature. Specifically, for example, the starting temperature and final temperature can be selected from the temperature ranges exemplified as the starting temperature and final temperature in case (i) of the thickening step.

[0168] Specific temperature changes in case (i) include, for example, a change from a starting temperature of 10°C to 45°C to a final temperature of 5°C to 35°C, from a starting temperature of 20°C to 45°C to a final temperature of 10°C to 35°C, from a starting temperature of 20°C to 45°C to a final temperature of 5°C to 30°C, from a starting temperature of 20°C to 45°C to a final temperature of 10°C to 30°C, from a starting temperature of 20°C to 45°C to a final temperature of 5°C to 20°C, from a starting temperature of 20°C to 45°C to a final temperature of 10°C to 20°C, from a starting temperature of 25°C to 45°C to a final temperature of 5°C to 30°C, from a starting temperature of 25°C to 45°C to a final temperature of 10°C to 20°C, and from a starting temperature of 25°C to 45°C to a final temperature of 10°C to 20°C. In all of these cases, the final temperature is lower than the starting temperature.

[0169] In the case of (ii), the starting temperature is not particularly limited, as long as it is above the maximum temperature described in the second embodiment and below the upper limit of the temperature at which viscosity changes.

[0170] In case (ii), the final temperature is not particularly limited, as long as it is higher than the starting temperature and below the upper limit of the temperature at which viscosity changes. Specifically, for example, the starting temperature and final temperature can be selected from the temperature ranges exemplified as the starting temperature and final temperature in case (ii) of the thickening step.

[0171] Specific temperature changes in case (ii) include, for example, a change from an initial temperature of 20°C to 50°C to a final temperature of 20°C to 56°C, from an initial temperature of 20°C to 45°C to a final temperature of 24°C to 56°C, from an initial temperature of 20°C to 45°C to a final temperature of 29°C to 56°C, from an initial temperature of 20°C to 40°C to a final temperature of 24°C to 56°C, from an initial temperature of 20°C to 40°C to a final temperature of 29°C to 56°C, from an initial temperature of 20°C to 45°C to a final temperature of 24°C to 50°C, from an initial temperature of 20°C to 40°C to a final temperature of 29°C to 50°C, and from an initial temperature of 20°C to 40°C to a final temperature of 29°C to 50°C. In all of these cases, the final temperature is higher than the initial temperature.

[0172] 3-3. Effects According to the method of this embodiment, the viscosity of an aqueous solution of fibrous protein can be easily and reversibly changed by temperature change alone and adjusted to a desired viscosity.

[0173] <Example 1. Change in viscosity of fibroin protein aqueous solution upon addition of salt> (Objective) To investigate how the viscosity of fibroin protein aqueous solution changes upon addition of salt.

[0174] (Methods) (1) Preparation of Regenerated Fibroin Protein Aqueous Solution First, a fibroin protein aqueous solution derived from bagworm silk was prepared. To remove components other than fibroin protein from the silk of bagworms (Larva of the giant bagworm moth: larvae collected in Tsukuba City, Ibaraki Prefecture), the silk was scouring with an aqueous sodium carbonate solution. Subsequently, after dissolving in an aqueous lithium thiocyanate solution, the obtained fibroin protein aqueous solution was sealed in a cellulose dialysis tube and desalted by dialysis with pure water to obtain a regenerated fibroin protein aqueous solution of approximately 0.3% by mass (the same applies below). The concentration of the regenerated fibroin protein aqueous solution was calculated by measuring the dry weight.

[0175] (2) Preparation of salt-added regenerated fibroin protein aqueous solutions Magnesium sulfate and pH3 citrate buffer (containing trisodium citrate) were added to the obtained regenerated fibroin protein aqueous solutions to final concentrations of 0.5 mM to 250 mM, respectively, and mixed to prepare a regenerated fibroin protein mixture of approximately 3% to 6%. An approximately 3% regenerated fibroin protein aqueous solution without the addition of magnesium sulfate aqueous solution and pH3 citrate buffer was used as a control. The pH of the buffer was measured using a benchtop pH meter LAQUA F-72 (HORIBA).

[0176] (3) Measurement of viscosity temperature dependence The viscosity of the obtained solution was measured at 0.5°C intervals over a temperature range from around 2°C to around 60°C where gelation begins, and its temperature dependence was evaluated. A laboratory vibrating viscometer VM-10A (Sekonic Corporation) was used for viscosity measurement. Temperature control was performed using a digital hot plate stirrer (Corning Inc.; model number: PC-420D), and heating was performed so that the water temperature rose at a rate of approximately 1°C / min. Water temperature was measured using a digital controller TTM-004W (Toho Electronics; model number: TTM-004W-PA).

[0177] (4) For the reversibility evaluation mixture, the viscosity was measured while the temperature was changed over 5 cycles, with one cycle being heating from 15°C to 26°C. After each cycle, the mixture was cooled to 6°C at a rate of approximately 1°C / min of water temperature decrease. The temperature change and viscosity measurement were performed in the same manner as in (3), except that viscosity measurements were taken every 1°C.

[0178] (Results) The results are shown in Figures 1 and 2. When no salt was added, the viscosity of the fibroin protein aqueous solution decreased as the temperature increased below the gelation temperature (Figure 1A, dashed line). On the other hand, the viscosity of the mixture reached a maximum value at around 30°C, and a transient increase in viscosity was observed (Figure 1A, solid line).

[0179] Hereafter, the temperature at which the viscosity takes its minimum value V1 will be called the minimum temperature T1, and the temperature at which the viscosity takes its maximum value V2 will be called the maximum temperature T2 (Figure 1B). Here, each value was calculated based on the intersection of the approximate straight lines in the viscosity decrease and increase ranges. In this mixture, the viscosity took its minimum value V1 = 18.1 (mPa·s) at T1 = 11.9 (°C), and its maximum value V2 = 28.2 (mPa·s) at T2 = 29.9 (°C).

[0180] As a result, the trend of viscosity temperature dependence reversed between approximately 12°C, where viscosity is at its minimum, and approximately 30°C, with viscosity increasing as the temperature rises.

[0181] When the viscosity of the same mixture was measured while repeatedly changing the temperature, the viscosity near the minimum and maximum remained almost constant no matter how many times the experiment was repeated, as shown in Figure 2A. Furthermore, as illustrated in the 4th and 5th cycles, the viscosity change with temperature between them was also almost constant (Figure 2B).

[0182] <Example 2. Effects of Salt Concentration and Buffer Concentration on Viscosity Conversion> (Objective) To investigate the effects of salt concentration and buffer concentration on viscosity conversion.

[0183] (Method) The procedure was the same as in Example 1, except that the mixed solution (or aqueous solution) of each condition was prepared under the conditions shown in Table 1.

[0184]

[0185] (Results) The results are shown in Table 2 and Figures 3 and 4.

[0186]

[0187] As shown in Table 2 and Figure 3, under conditions A to D, where the concentrations of magnesium sulfate and pH3 citrate buffer were between 0 mM and 5.0 mM, the viscosity conversion observed in Example 1 was not seen (Figure 3A). On the other hand, under conditions E to G, where the concentrations of magnesium sulfate and pH3 citrate buffer were 25.0 mM or higher, the viscosity conversion observed in Example 1 was seen (Figure 3B). A similar trend was observed when the fibroin protein concentration (dope concentration) was changed; no viscosity conversion was observed under low conditions of magnesium sulfate and pH3 citrate buffer concentrations of 0.8 mM (condition H) or 3.0 mM (condition I), while viscosity conversion was observed under high condition J at 9.0 mM (Figure 4).

[0188] Furthermore, it was found that the extreme values ​​and their temperatures changed depending on the salt concentration and buffer concentration. Generally, as the concentration increased, the extreme temperature tended to shift to lower temperatures, and the viscosity at the extreme values ​​tended to increase.

[0189] These results suggest that viscosity conversion occurs at least when the salt concentration and buffer concentration are 9.0 mM or higher.

[0190] <Example 3. Effects of the presence or absence of buffer solution and the type of salt on viscosity conversion> (Objective) To investigate the effects of the presence or absence of buffer solution and the type of salt on viscosity conversion.

[0191] (Method) The procedure was the same as in Example 1, except that the mixed solution (or aqueous solution) of each condition was prepared under the conditions shown in Table 3.

[0192]

[0193] (Results) The results are shown in Table 4 and Figure 5.

[0194]

[0195] Under conditions A, F-, and K, which did not contain a buffer, no viscosity conversion was observed. However, viscosity conversion was observed under conditions A+, F, K+, and L, which did contain a buffer (Figure 5), suggesting that the presence of a buffer is essential for viscosity conversion. Furthermore, viscosity conversion was also observed under condition A+, which contained only a buffer and no additional salts (Figure 5), indicating that the presence of additional metal ions is not necessarily required. In addition, since citric acid is an anion with a high salting-out effect in the Hofmeister series, it is suggested that an anion with a relatively high salting-out effect is necessary for viscosity conversion.

[0196] Furthermore, regarding the type of salt, viscosity conversion was observed under conditions where chloride was used as the magnesium salt (condition K+), similar to the conditions where sulfate was used (condition F) (Figure 5B). Moreover, viscosity conversion was also observed under conditions where potassium phosphate, which has different metal ions and anions, was used (condition L) (Figure 5B), suggesting that viscosity conversion occurs regardless of the specific type of ion, as long as an ion with a certain level of salting-out effect is included.

[0197] <Example 4. Effect of pH on viscosity conversion> (Objective) To investigate the effect of pH on viscosity conversion.

[0198] (Method) The procedure was the same as in Example 1, except that the mixtures of each condition were prepared under the conditions shown in Table 5. pH adjustment was performed by changing the ratio of citric acid to citrate.

[0199]

[0200] (Results) The results are shown in Table 6 and Figure 6.

[0201]

[0202] Viscosity conversion was observed under conditions where the pH was 4 or less (Conditions A+ and A+(4)), while no viscosity conversion was observed under conditions where the pH was 5 or more (Conditions A+(5) and A+(6)). This indicates that a certain level of acidity is necessary for viscosity conversion. Furthermore, since there was no significant difference in the temperature dependence of viscosity between conditions A+ and A+(4), it is suggested that a certain level of acidity is sufficient for a certain viscosity conversion to occur, regardless of the specific pH value. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A viscosity converter comprising an acidic pH buffering agent for adding to an aqueous solution of fibrous protein to convert its viscosity, wherein the addition causes the aqueous solution of fibrous protein to exhibit a pH of less than 5 and a concentration of the acidic pH buffering agent of 5.1 mM or higher, and the viscosity is converted within a temperature range of 5°C to 55°C.

2. The viscosity converter according to claim 1, wherein the acidic pH buffer has a pKa value of less than 6.

3. The viscosity converter according to claim 2, wherein the acidic pH buffering agent comprises one or more salts selected from the group consisting of citrate, formate, phosphate, phthalate, acetate, glycine salt, maleate, and malate, and / or imidazole, pyridine.

4. The viscosity converter according to any one of claims 1 to 3, further comprising a metal salt, wherein the addition causes the aqueous solution of the fibrous protein to exhibit a metal salt concentration of 5.5 mM or more.

5. The viscosity converter according to claim 4, wherein the metal salt contains an anion having hydration properties equal to or greater than that of a chloride ion in the Hofmeister series.

6. The viscosity converter according to claim 4 or 5, wherein the metal ion in the metal salt is a cosmotropic ion.

7. The viscosity converter according to any one of claims 1 to 6, wherein the fibrous protein comprises fibroin protein.

8. A viscosity-variable fibrous protein aqueous solution comprising the viscosity converter according to any one of claims 1 to 7.

9. The viscosity-variable fibrous protein aqueous solution according to claim 8, having a maximum viscosity in the temperature range of 20°C to 40°C.

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