JP7580092B2 - Method for producing silk masses derived from bagworm silk spun onto a substrate surface - Google Patents

Description

The present invention relates to a method for producing a mass of silk threads by spinning scaffold silk threads onto the surface of a substrate using bagworms.

The threads that make up insect cocoons and the hair of mammals have been used as animal fibers for clothing and other purposes since ancient times. In particular, silk threads derived from silkworms, the larvae of the silkworm moth (Bombyx mori) (often referred to as "silkworm silk threads" in this specification) are highly valued as a high-quality natural material even today, due to their excellent moisture absorption and release properties, moisture retention, and heat retention, as well as their unique luster and smooth texture.

However, there are animal fibers in nature that have properties comparable to or even superior to those of silkworm silk. In recent years, there has been much research and development being done to discover and utilize such animal fibers with superior properties as new natural materials.

One of the silks that has attracted attention is that derived from spiders (often referred to as "spider silk" in this specification). Spider silk has flexibility, stretchability, and high elasticity that is 5 to 6 times that of polystyrene, and is expected to be used as a medical material such as surgical sutures, and as a special material for disaster prevention ropes and protective clothing (Non-Patent Documents 1 and 2). However, spider silk has problems such as the difficulty of mass-rearing spiders and extracting a large amount of silk from spiders, making it impossible to mass-produce, and the production costs are high. Attempts have been made to solve this problem by using recombinant DNA technology to make hosts such as silkworms and E. coli produce spider silk (Patent Document 1 and Non-Patent Document 2). However, since the silkworms and E. coli that produce spider silk are recombinant, they can only be reared and cultivated in facilities equipped with the required equipment, which poses a problem of heavy maintenance and management burden. In addition, the liquid spider silk protein expressed in E. coli needs to be converted into fiber, which increases the number of steps. Furthermore, at the current stage, the spider silk spun by genetically modified silkworms is only a few percent mixed in with the silkworm silk, and there is also the problem that it is not possible to obtain 100% spider silk that utilizes 100% of the properties of spider silk.

By the way, there is an insect called a basket worm (also known as a "bag worm"). Bagworms are a general term for the larvae of moths belonging to the order Lepidoptera and family Psychidae. They are known to live in their nests throughout their entire larval stage, usually hiding in spindle-shaped or cylindrical nests made of pieces of leaves or branches entangled with silk, as shown in Figure 1A, and even moving the nest when feeding.

The silk thread spun by these bagworms (often referred to as "bagworm silk thread" in this specification) has been attracting attention in recent years as a new natural animal fiber material with superior properties to silkworm silk thread and spider silk thread. For example, the elastic modulus of bagworm silk thread from the brown bagworm moth (Eumeta minuscula) is 3.5 times that of silkworm silk thread and 2.5 times that of spider silk thread from the golden orb spider (Nephila clavata), and it boasts extremely high strength (Non-Patent Documents 1 and 2). In addition, not only does it have the same or greater luster and gloss as silkworm silk thread, but the cross-sectional area of a single fiber is only about 1/7 that of silkworm silk thread, making it possible to produce thinner, lighter fabrics with a finer grain and a smoother feel than silkworm silk thread.

Bagworms also have advantages over silkworms and spiders in terms of rearing. Like silkworms, bagworms are herbivorous, so unlike carnivorous spiders, food is easy to obtain and can be supplied steadily. In addition, even though they are both herbivorous, they have advantages over silkworms. For example, silkworms feed only on fresh leaves of species belonging to the genus Morus (e.g., M. bombycis, M. alba, and M. ihou), so the rearing area and rearing period depend on the source of mulberry leaves and the period when the leaves open. On the other hand, bagworms are polyphagous and have low specificity for food leaves, so many species can feed on the leaves of various tree species. Therefore, food leaves are easy to obtain and they can be reared in any area. Also, depending on the species, evergreen tree leaves can be used as food leaves, so unlike deciduous mulberry trees, food leaves can be supplied throughout the year. Furthermore, bagworms are smaller than silkworms, so they require less space than silkworms and can be easily raised in large numbers. This helps keep breeding costs down.

Furthermore, bagworms are superior to silkworms in terms of productivity. For example, silkworms only spin large amounts of silk when spinning their cocoons, and all larvae spin their cocoons at the same time. This causes their silk-harvesting periods to overlap, resulting in a concentrated work period. However, bagworms spin silk repeatedly throughout their larval stage, when building nests and when moving. This means that their work periods can be dispersed by artificially adjusting the silk-harvesting periods.

As described above, bagworm silk has properties that surpass those of silkworm silk and spider silk, and it also has many advantages in terms of production, making it a promising new natural material.

However, there are some problems with putting bagworm silk to practical use. One of these problems is related to the characteristics of bagworm nests. The surface of a bagworm nest is always covered with impurities such as pieces of leaves and branches. This is due to the bagworm's habit of incorporating surrounding twigs and leaves into the nest for camouflage during the process of building and expanding the nest. In order to commercialize bagworm silk, these impurities must be completely removed. Conventionally, these impurities have been removed from nests by hand, or by soaking the nests in warm water for a long time to soften them and release the impurities. However, the removal of these impurities requires a huge amount of work. In addition, existing technology is unable to completely remove the impurities, and there is a problem that only low-quality products can be obtained, such as small pieces of leaves being mixed in the final product or bagworm silk being dyed light brown with pigments derived from the impurities. Although it is possible to use base or acid to remove the pigment, this can result in a significant decrease in the quality of the bagworm silk, including a loss of strength.

In addition to the nest silk that constitutes the nest, bagworm silk also contains silk called scaffold silk. As shown in Figure 1B, this scaffold silk is a silk that is spun by bagworms as a foothold to prevent them from falling when they move. The inventors' research results have revealed that this scaffold silk is stronger than nest silk and has excellent mechanical properties. Furthermore, unlike nest silk, scaffold silk does not contain impurities such as pieces of leaves or branches. Therefore, if scaffold silk can be collected and used, it may be possible to put it to practical use as bagworm silk.

However, there are problems. First, the scaffold thread is usually spun in a zigzag pattern in the direction of travel, as shown in Figure 2A. Bagworm silk is spun in a mixture of fiber components and a glue-like protein that covers its surface, and the scaffold silk is fixed to the substrate surface by the glue-like protein (arrowhead in large circle in Figure 2A) at the zigzag turning point. This fixation is very strong, so a strong tension is required to mechanically peel the scaffold silk from the substrate. In addition, in many cases, the scaffold silk is torn and fragmented around the fixed part by this operation. Furthermore, it is generally difficult to control the movement of bagworms, and they may move back and forth to the same place multiple times. As a result, as shown in Figure 2B, the silk threads spun in a zigzag pattern overlap and become intricately entangled, making it even more difficult to recover them from the substrate without damage. For this reason, scaffold silk threads spun on a substrate have not been effectively used as natural fiber materials until now.

WO2012/165477

Shigeyoshi Osaki, 2002, Journal of the Society of Fiber Science and Technology (Fibers and Industry), 58: 74-78 Kuwana Y, et al., 2014, PLoS One, DOI: 10.1371/journal.pone. 0105325

The object of the present invention is to develop and provide a method for peeling bagworm scaffold silk threads, which have been difficult to recover and could not be used in the past, from the substrate with a weak force without damaging the threads, and recovering and utilizing them as thread masses.

In order to solve the above problems, the inventors conducted extensive research and have succeeded in peeling the bagworm silk threads from the substrate without damaging them in the form of a lump, using only 15% or less of the force required for a negative control in which no such wetting liquid is used, by spraying or applying a wetting liquid such as ethanol, an aqueous solution, or an organic solvent onto the bagworm silk threads spun onto a substrate. Based on the above method, the present invention provides the following:
(1) A method for producing a mass of bagworm silk thread, comprising a wetting liquid application step of applying a wetting liquid to bagworm silk thread spun onto the surface of a substrate, and a separation step of separating the substrate and the bagworm silk thread, wherein the wetting liquid is a pure substance or mixture that is in a liquid state at least at 20°C or higher and lower than 30°C under atmospheric pressure and does not damage, denature, or dissolve the fibroin protein, which is the fiber component of bagworm silk thread.
(2) The method described in (1), which includes a spinning process of placing a bagworm on the substrate surface prior to the wetting liquid application process and causing the bagworm to spin a spinning thread.
(3) The method described in (2), further comprising a bagworm recovery process of recovering the bagworms together with their nests after the spinning process and before the wetting liquid application process.
(4) A method according to any one of (1) to (3), comprising a washing step of washing the separated bagworm silk.
(5) A method according to any one of (1) to (4), comprising a refining step of refining the separated bagworm silk.
(6) The method according to any one of (1) to (5), wherein the wetting liquid is a pure substance or a mixture having a melting point below 20°C and a boiling point between 30°C and 300°C.
(7) The method according to any one of (1) to (6), wherein the wetting liquid is an aqueous solution or an organic solvent.
(8) A method for harvesting bagworm silk threads spun onto the surface of a substrate, comprising a wetting liquid application step of applying a wetting liquid to the bagworm silk threads spun onto the surface of the substrate, and a separation step of separating the substrate and the bagworm silk threads, wherein the wetting liquid is a pure substance or mixture that is in a liquid state at least at 20°C or higher and lower than 30°C under atmospheric pressure and does not damage, denature, or dissolve the fibroin protein, which is the fibrous component of bagworm silk threads.
(9) The method according to (8), wherein the wetting liquid is an aqueous solution or an organic solvent.
(10) Bagworm silk obtained using the method for producing silk clumps described in any one of (1) to (7), or the method for harvesting silk described in (8) or (9).
(11) A nonwoven fabric made of bagworm silk thread obtained using the method for producing thread clumps described in any one of (1) to (7).
This specification includes the disclosure of Japanese Patent Application No. 2018-158762, which is the priority basis of this application.

The method for producing bagworm silk lumps of the present invention makes it possible to recover bagworm scaffolding threads spun onto the surface of a substrate with weak force and without fragmentation, which was previously difficult to recover.

A: An external view of a giant bagworm nest (Psacroilia maxima). B: A diagram showing the spinning behavior of a Psacroilia bagworm as it moves. You can see how the bagworm moves forward while spinning scaffold silk threads (arrowheads) and how it hooks its claws around the spun bagworm silk threads (scaffold silk threads) (thin arrows). A: A schematic diagram showing the state of bagworm silk threads (scaffold silk threads) spun by a bagworm as it moves across the surface of a substrate. As shown in the figure, the bagworm's scaffold silk threads are zigzag like a ladder. In the figure, the black arrow indicates the direction in which the bagworm moves while spinning. The figure in the large circle is an enlarged view of the small circle, and the arrowhead in the large circle represents a glue-like protein. The scaffold silk threads are fixed to the substrate at the turning point of the zigzag section, which corresponds to the horizontal boards of the ladder. B: A diagram showing the state of bagworm silk threads as the giant bagworm spun them onto a plastic plate. It can be seen that the zigzag-spun bagworm silk threads are intricately entangled. FIG. 1 is a diagram showing the process flow of the method for producing lump of bagworm silk of the present invention. 1 shows the results of the peel tension evaluation test carried out in Example 1. In the figure, (A) shows the test results for the negative control where no wetting liquid was applied, (B) shows the test results when water was applied, and (C) shows the test results when ethanol (99.5%) was applied.

1. Method for producing a lump of bagworm silk thread 1-1. Concept The first aspect of the present invention is a method for producing a lump of bagworm silk thread. The production method of the present invention is a method for obtaining a lump of the desired bagworm silk thread by applying a wetting liquid to the bagworm silk thread spun onto the surface of a substrate, and then separating the substrate and the bagworm silk thread. According to the method of the present invention, scaffold silk thread, which has excellent physical properties but has not been used in the past because it has been difficult to recover it without causing physical damage, can be efficiently recovered without causing damage.

1-2. Definitions of Terms The following terms frequently used in this specification are defined as follows.
As mentioned above, "bagbugs" refers to the general term for the larvae of moths belonging to the family Psychidae in the order Lepidoptera. Moths of the family Psychidae are distributed all over the world, and all larvae (bagbugs) live in nests made of natural materials such as leaves and branches spun with silk threads that they spin themselves throughout the entire larval stage. In addition, all species have the habit of moving while spinning scaffolding silk threads in the direction of travel to prevent them from falling. Therefore, the bagbugs used in this specification are larvae of moths belonging to the family Psychidae, and as long as they have the above-mentioned habit, there are no restrictions on the type, age, or sex. For example, the Psychidae family includes genera such as Acanthopsyche, Anatolopsyche, Bacotia, Bambalina, Canephora, Chalioides, Dahlica, Diplodoma, Eumeta, Eumasia, Kozhantshikovia, Mahasena, Nipponopsyche, Paranarychia, Proutia, Psyche, Pteroma, Siederia, Striglocyrbasia, Taleporia, Theriodopteryx, and Trigonodoma, and the bagworm used in this specification may be a species belonging to any of the genera. In addition, the larvae may be of any age from the first age to the final age. However, in order to obtain a large amount of bagworm silk in terms of mass, a large bagworm is preferable. For example, if the same species, the final age larvae are preferable, and if male and female, the larger female is preferable. In addition, the larger the species within the Psychidae family, the more preferable it is. For example, the large species Eumeta japonica and Eumeta minuscula are suitable species for use in the present invention.

The bagworms used in the production method of the present invention are preferably, but not limited to, bagworms that retain their nests. "Retaining a nest" refers to a state in which the bagworm carries the nest with it. As mentioned above, bagworms live with their nests, and when feeding or moving, as shown in Figure 1B, only a portion of their body is exposed outside the nest, and in principle, their entire body is not exposed from the nest throughout the entire larval stage. In general, when a bagworm is artificially separated from its nest and its entire body is exposed to the outside world, the naked bagworm will minimize movement and quickly begin to reconstruct the nest in order to protect and keep its body warm. Therefore, the reason why bagworms that retain their nests are preferable is that they do not prioritize the spinning of nest silk threads, but rather actively spin scaffold silk threads, which is the purpose of the present invention.

In this specification, "silk thread" refers to thread derived from insects, a protein thread spun by insect larvae and adults for the purposes of nesting, moving, anchoring, spinning cocoons, capturing food, etc. In this specification, when simply written as "silk thread", it means a broad and general silk thread without specifying the name of the insect from which it originates, and when referring to silk thread derived from a specific insect, the name of the organism from which it originates is placed before the word "silk thread", such as silkworm silk thread or bagworm silk thread.

In this specification, "bagworm silk" refers to silk thread spun by bagworms. Bagworm silk in this specification includes single fibers, spun fibers, and aggregate fibers. A "single fiber" is the smallest unit filament that constitutes fiber components, and is also called a monofilament. A single fiber is mainly composed of fibroin protein. In the natural state, bagworm silk and silkworm silk are spun in the form of a difilament in which two single fibers are bonded by the adhesive substance sericin protein. This difilament is called a "spun fiber." Bagworm nests and silkworm cocoons are composed of spun fibers. In addition, a fiber bundle in which multiple spun fibers are combined is called an "aggregated fiber (multifilament)." In general, raw silk corresponds to this aggregate fiber. Furthermore, silk thread from which raw silk has been treated with soap, lye, and alkaline chemicals such as sodium carbonate and urea, and enzymes to remove the sericin protein, is called degummed silk.

There are two types of bagworm silk: scaffold silk and nest silk. As mentioned above, "scaffold silk" is silk that bagworms spin to move around, and functions as a scaffold (foothold) to prevent them from falling off branches and leaves. On the other hand, "nest silk" is silk that bagworms spin to form nests, and is used to bind leaf and branch pieces together and to create a comfortable environment for the inner walls of the nest, which is their habitat. For this purpose, the bagworm silk of this invention is scaffold silk. Therefore, unless otherwise specified, in this specification, when the term "bagworm silk" is used, it refers to scaffold silk.

In this specification, the term "thread mass" refers to an assembly of threads composed only of bagworm scaffold silk threads. Although a bagworm nest is an assembly of bagworm silk threads, it does not fall under the thread mass of the present invention because it usually contains impurities such as twigs and leaves and is composed of nest silk threads. Although not limited, the thread mass in this specification corresponds to an assembly of scaffold silk threads produced through some artificial process that does not contain impurities. For example, a bagworm is placed on a substrate and allowed to spin threads. The state of the thread mass is not limited, but for example, one or more bagworm silk threads may be intricately entangled to form a sheet like a nonwoven fabric, or it may be in a state where it is gathered together and ready to be spun.

"Wetting liquid" refers to a pure substance or mixture that is in a liquid state at least at 20°C or higher but lower than 30°C under atmospheric pressure and that does not damage, denature, or dissolve the fibroin protein, which is the fiber component of bagworm silk. Therefore, pure substances that are in a state other than liquid within the above temperature range, and mixtures such as strongly acidic or strongly basic solvents that denature proteins even in a liquid state, or solutions containing proteases, etc., are not suitable as wetting liquids for the present invention.

"Pure substance" refers to a chemical substance that has certain properties, including a simple substance consisting of a single element and a compound consisting of multiple elements. In the present invention, the pure substance is usually, but not limited to, a compound.

"Mixture" refers to a substance that is made up of multiple pure substances. For example, a solution.

The wetting liquid may be composed of any pure substance or mixture as long as it satisfies the above conditions. Examples include, but are not limited to, compounds that have a melting point (MP) below 20°C and a boiling point (BP) between 30°C and 300°C at room temperature (25°C) and normal pressure (100 kPa). Many compounds with such properties have the property of being a "solvent" that can dissolve other compounds as solutes.

Specific examples of wetting liquids that are compounds are listed below, but the wetting liquids in this specification are not limited to the following.

The wetting liquid may be a liquid consisting of polar molecules (polar solvent). For example, protic polar solvents such as water (MP: 0°C; BP: 100°C), methanol (MP: -96°C; BP: 64.7°C), ethanol (MP: -117°C; BP: 78.3°C), 1-propanol (MP: -127°C; BP: 97.2°C), 1-butanol (MP: -90°C; BP: 118°C), glycerin (MP: 17.8°C; BP: 290°C), formic acid (MP: 8.3°C; BP: 100.8°C), acetic acid (MP: 15°C; BP: 118°C) and butyric acid (MP: -7.9°C; BP: 164°C); and aprotic polar solvents such as DMSO (MP: 18.5°C; BP: 189°C), acetonitrile (MP: -48°C; BP: 81.6°C), acetone (MP: -94°C; BP: 56°C), dimethylformamide (MP: -61°C; BP: 153°C), dimethylsulfoxide (MP: 19°C; BP: 189°C), tetrahydrofuran (MP: -108°C; BP: 66°C), and 1,1,1,3,3,3-hexafluoro-2-propanol (MP: -3.3°C; BP: 58.2°C). Other examples include ionic liquids. The numbers in parentheses for each compound are the melting point (MP) and boiling point (BP) at room temperature and pressure (hereinafter the same applies in this specification).

The wetting liquid may also be a non-polar liquid (non-polar liquid: non-polar solvent) made of non-polar molecules. Examples include oils and many organic solvents (low polarity organic solvents), with some exceptions. Oils are compounds that are in a liquid state at room temperature and pressure, and include, for example, fatty acids represented by the general formula R-COOH (where R is an alkyl group with 4≦C≦8). Specific examples of non-polar liquids include fats such as valeric acid (valeric acid: MP: -34.5°C; BP: 186°C), caproic acid (hexanoic acid: MP: -3°C; BP: 205°C), enanthic acid (heptylic acid, heptanoic acid: MP: -7.5°C; BP: 223°C), caprylic acid (octanoic acid: MP: 16.7°C; BP: 239.7°C), pelargonic acid (nonanoic acid: MP: 11°C; BP: 247°C), palmitoleic acid (hexadecenoic acid: MP: -0.1°C; BP: 230°C), linoleic acid (octadecadienoic acid: MP: -5°C; BP: 229°C), linolenic acid (octadecatrienoic acid: MP: -11°C; BP: 278°C), and arachidonic acid (icosatetraenoic acid: MP: -49°C; BP: 169°C). These include acid, hexane (MP: -95.3°C; BP: 68.7°C), toluene (MP: -95°C; BP: 110.6°C), chloroform (MP: -63.5°C; BP: 61°C), dichloromethane (MP: -95°C; BP: 39.8°C), 1,2-dichloroethane (MP: -35.7°C; BP: 83.4°C), trichloroethylene (MP: -86.4°C; BP: 87°C), acetone (MP: -94°C; BP: 56°C), diethyl ether (MP: -116°C; BP: 34.6°C), xylene (MP: -25°C; BP: 137°C), carbon tetrachloride (MP: -23°C; BP: 76.7°C), methyl acetate (MP: -98°C; BP: 57°C), and ethyl acetate (MP: -84°C; BP: 77°C).

On the other hand, when the wetting liquid in this specification is a mixture, a specific example thereof is a solution. The solution is not limited to a solution consisting of a polar or non-polar liquid in which one or more different solutes are dissolved, a colloidal solution or sol in which a colloid is dispersed in a liquid in which the colloid is a dispersion medium, two or more different liquids (for example, a mixed liquid consisting of different polar liquids), or a combination thereof. For example, a polar solution is an aqueous solution in which a solute is dissolved in water, a mixed solution of solvents such as ethanol and water, etc. In the case of an aqueous solution, the solute is not limited. For example, salt, sugar, surfactant, etc. are included, but are not limited to these. The salt is not limited to a salt with high solubility, but is preferably a salt with high solubility. For example, sodium chloride salt, potassium chloride salt, sodium carbonate salt, sodium bicarbonate salt, etc. are included. In the case of a solution, the concentration of the solute is not particularly limited. For example, it may be solubility. In the case of a mixed solution, there is also no particular limit. For example, a mixture of ethanol and water may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99.5% ethanol.

As mentioned above, the type of pure substance or mixture that constitutes the wetting liquid is not important, but in consideration of ease of handling (including waste liquid treatment, etc.), safety, and purchase cost, water (including warm water and hot water), an aqueous solution, ethanol, or a mixture thereof is preferred, with an aqueous solution and ethanol being particularly preferred.

In this specification, the term "substrate" refers to a base for harvesting bagworm silk threads. By placing and moving a bagworm on the surface of this substrate, the bagworm silk threads are spun onto the surface.

The material constituting the substrate is not limited as long as the bagworm silk can be fixed to its surface by the paste-like protein. Examples include glass (including enamel), metal, synthetic resin (including thermoplastic resin, thermosetting resin, and synthetic rubber), ceramics, or paper, plant pieces (including wood chips), and animal pieces (including bone chips, shells, and sponges). However, substrates that dissolve themselves in the wetting liquid used or react with the wetting liquid in a redox reaction or other reaction do not fit the purpose of the present invention, which is to recover the bagworm silk spun onto the substrate. Therefore, it is desirable that the material constituting the substrate is insoluble and non-reactive with the wetting liquid used in the present invention. "Insoluble in wetting liquid" refers to the property of not dissolving in the wetting liquid used in the present invention. Also, "non-reactive with wetting liquid" refers to the property of not causing a chemical reaction with the wetting liquid used in the present invention. Therefore, the material of the substrate of the present invention can change depending on the type of wetting liquid used. For example, if the wetting liquid is water or an aqueous solution, synthetic resins such as polyethylene, polypropylene, polystyrene, vinyl acetate, cellulose acetate, acrylic resin, and polycarbonate can be used, but if the wetting liquid is a low-polarity organic solvent, polystyrene, vinyl acetate, cellulose acetate, acrylic resin, and polycarbonate, which are soluble in the wetting liquid, cannot be used. Glass, ceramics, polypropylene, and the like, which are easy to obtain, relatively inexpensive, and have low reactivity, are suitable materials for the substrate.

The thickness of the substrate used in this process is not limited. It may be appropriately determined taking into consideration the manufacturing cost, rigidity, ease of processing in the subsequent process, etc. of the substrate. For example, the average thickness of the substrate is preferably 0.5 mm or more, 0.6 mm or more, 0.7 mm or more, 0.8 mm or more, 0.9 mm or more, 1.0 mm or more, 1.2 mm or more, or 1.5 mm or more, and may be 3.0 mm or less, 2.8 mm or less, 2.5 mm or less, 2.2 mm or less, or 2.0 mm or less. When the substrate is composed of a thin film with an average thickness of less than 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.5 mm, the substrate itself does not have the rigidity to maintain a constant shape, so the substrate may be placed on a suitable support.

In this specification, the term "support" refers to a member on whose surface a substrate is placed, which can impart rigidity and/or shape to the substrate. The support is an optional component used in the method of the present invention, and can be used as needed. There are no particular limitations on the material of the support, so long as it has sufficient rigidity to maintain a certain shape. Examples of the material include glass, metal, plastic, synthetic rubber, ceramics, or paper, plant fragments, and animal fragments.

The shape and size of the substrate used in this process are not limited. The shape may be, for example, a flat sheet or plate shape, or a three-dimensional shape, but a flat shape is preferred considering the ease of separation of the bagworm silk from the substrate. The surface condition of the substrate is also not limited, but a smooth surface that is easier to separate is preferred over a rough surface that has a strong adhesion to the bagworm silk. The size of the substrate can be adjusted as needed, but considering that the scaffold silk is bagworm silk that is spun as the bagworm moves, the lower limit is preferably equal to or greater than the size of the bagworm, that is, the body length of the bagworm. For example, the long axis or long diameter can be 1 cm or more, 2 cm or more, 3 cm or more, 4 cm or more, or 5 cm or more. On the other hand, there is no upper limit to the size of the substrate, but when the long axis or long diameter is 10 cm or more, 15 cm or more, 20 cm or more, 25 cm or more, or 30 cm or more, it is preferable to have multiple bagworms spun the silk.

1-3. Method The process flow of this embodiment is shown in Figure 3. As shown in this figure, the method of this embodiment includes the essential steps of a wetting liquid application step (S0103) and a separation step (S0104), and the selection steps of a spinning step (S0101), a bagworm recovery step (S0102), a washing step (S0105), and a scouring step (S0106). Each step will be explained below in the order of the flow.

The "thread-spinning process" (S0101) is a process in which a bagworm is placed on the surface of a substrate and the bagworm to be spun is placed together with the substrate, and is an optional process in the present invention. This process is carried out before the wetting liquid application process described below.

"Placing bagworms on the substrate surface" means positioning both the bagworms and the substrate surface so that they can come into contact with each other. For example, the bagworms may be placed directly on the substrate, or may be positioned so that the bagworms can reach the substrate by moving. An example of the latter is placing bagworms on the bottom of a wide-mouthed plastic container without a lid, and then covering the container with the substrate. Bagworms prefer high positions, so they move along the sides of the wide-mouthed plastic container and reach the bottom of the substrate, which corresponds to the container ceiling, and then spin scaffold silk threads while moving there. There are no particular limitations on the position of the substrate from which the bagworm silk threads are spun. As mentioned above, it may be placed on the container ceiling or on the wall. In this way, if the substrate is placed somewhere other than the bottom, even if the bagworms defecate, the feces will fall to the bottom, and the spinning surface will not be contaminated with feces, which is convenient.

The type and number of bagworms placed does not matter. For example, one or more bagworms may be placed at a time on each substrate from which bagworm silk is spun. The type and age of the bagworms placed also does not matter. When placing multiple bagworms, each individual may be of the same species and age, or bagworms of different species and ages may be mixed.

The duration of this process is not particularly limited. The duration depends on the type and age of the bagworms and the number of individuals used, but in any case, it is usually sufficient to continue the process until the required amount of silk is spun onto the substrate. As an example, when a single final-stage bagworm is used to spun silk onto a circular substrate with a diameter of 9 cm, it can be spun for 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Since the bagworm's scaffold silk is spun as it moves, as mentioned above, the amount of silk obtained is generally proportional to the distance moved by the bagworm. Therefore, the time required for the silk-spinning process is shorter when multiple bagworms are spun than when a single bagworm is spun. Also, since the bagworms continue to spun silk without feeding, they often stop spun silk during this process. In such cases, the bagworm can be replaced with a new one and the silk-spinning process can be continued.

In order to increase the amount of silk spun by the bagworms per unit time, it is preferable that the temperature and humidity during this process are constant or change little. The temperature is preferably around 20°C, for example, in the range of 15°C to 25°C or 18°C to 22°C, and the humidity is preferably around 50%, for example, in the range of 40% to 65% or 45% to 60%. There are no particular restrictions on the light and dark periods during this process, and it may be a light period only, but it may also be possible to add cyclical light and dark periods. For example, in a 24-hour period, the light period may be 6 to 18 hours, 7 to 17 hours, 8 to 16 hours, 9 to 15 hours, 10 to 14 hours, 11 to 13 hours, or 12 hours, with the rest being a dark period.

The "bagworm recovery process" (S0102) is a process for recovering the bagworms used in the spinning process together with their nests, and is an optional process in the present invention. The purpose of this process is to separate and recover the bagworms that are no longer needed after the spinning process from the substrate.

After the spinning process, the base material is mixed with the spun scaffold silk threads and the bagworms that spun them. However, in principle, bagworms are not needed for the next wetting liquid application process. Furthermore, if the liquid used in the wetting liquid application process is applied to bagworms, the bagworm silk threads may be undesirably stained by the bagworms' body fluids or extracts from dead leaves, etc. used in the nests. Therefore, although this process is optional, it is preferable to carry it out after the spinning process.

The method of recovering the bagworms from the substrate is not limited. Any method of separating the bagworms from the substrate can be used. For example, the bagworms in contact with the substrate can be peeled off together with the nest. However, for the purpose of the invention, a method that can reduce damage to the bagworm silk thread as much as possible is preferable. For example, the bagworms can be induced to spontaneously leave the substrate. A specific example of this method is a method in which the substrate that was previously placed at the ceiling of the container is inverted upside down to the bottom, utilizing the aforementioned property of the bagworms moving to high places. After moving the bagworms to the side of the container, the substrate can be recovered. Another method is to heat the substrate. The bagworms spontaneously leave the substrate to escape from the high temperature, so the substrate can be recovered after the movement. The heating temperature may be any temperature that is above room temperature, does not damage the bagworm silk thread, and does not melt the substrate. For example, the temperature may be 30°C or higher, 33°C or higher, 35°C or higher, 38°C or higher, 40°C or higher, 42°C or higher, 45°C or higher, 48°C or higher, or 50°C or higher, and 80°C or lower, 75°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, or 55°C or lower.
Furthermore, the collected bagworms can be reused in the production method of the present invention after feeding.

The "wetting liquid application step" (S0103) is a step of applying a wetting liquid to the bagworm silk threads spun onto the substrate surface, and is the most important and essential step in the present invention.

There are no particular limitations on the method of applying the wetting liquid. Any method can be used as long as it sufficiently wets the bagworm silk threads on the substrate surface with the wetting liquid. Examples include a method of spraying, jetting, or applying the wetting liquid to the substrate surface on which the bagworm silk threads have been spun, or a method of immersing the substrate on which the bagworm silk threads have been spun in the wetting liquid.

After applying the wetting liquid, it is preferable to hold it for a specified time. This is to ensure that the wetting liquid has time to fully penetrate between the substrate and the bagworm silk thread. There are no particular limitations on the "specified time" mentioned here. It depends on the amount of wetting liquid applied and the application method, but it is usually 1 second to 1 hour, 1 minute to 40 minutes, 2 minutes to 30 minutes, 3 minutes to 20 minutes, 4 minutes to 15 minutes, or 5 minutes to 10 minutes after application.

There are no particular limitations on the temperature of the wetting liquid used in this process, so long as it does not damage, denature, or dissolve the bagworm silk. It is usually in the room temperature range; for example, if the melting point of the wetting liquid is less than 1°C and the boiling point is higher than 35°C, it may be 1°C to 35°C, 5°C to 32°C, 10°C to 30°C, 12°C to 27°C, 15°C to 25°C, or 18°C to 20°C. However, since the higher the temperature of the wetting liquid, the more reactive it is in general, a higher wetting liquid temperature is also preferable in this process. For example, if the wetting liquid is an aqueous solution, the temperature is preferably 35°C or higher, 38°C or higher, 40°C or higher, 42°C or higher, 45°C or higher, 48°C or higher, 50°C or higher, 52°C or higher, 55°C or higher, 58°C or higher, 60°C or higher, 62°C or higher, 65°C or higher, 68°C or higher, 70°C or higher, 72°C or higher, 75°C or higher, 78°C or higher, 80°C or higher, 82°C or higher, 85°C or higher, 88°C or higher, 90°C or higher, 92°C or higher, 95°C or higher, or 98°C or higher under atmospheric pressure. The wetting liquid may be heated before and/or during this step.

1-3-4. Separation process The "separation process" (S0104) is a process for separating the substrate from the bagworm silk thread spun onto its surface after the wetting liquid application process, and is an essential process in the present invention. There is no particular limitation on the method for separating the substrate from the bagworm silk thread. Because the bonding strength between the substrate and the bagworm silk thread is reduced by the wetting liquid application process, the two can be separated with a relatively weak tension. For example, the method includes a method of gripping the end of the bagworm silk thread and peeling it off from the substrate, a method of high-pressure injection of air or liquid onto the bonding surface between the substrate and the bagworm silk thread to separate them, a method of fixing the substrate and then suctioning and peeling the bagworm silk thread, or a method of immersing the substrate in a liquid, shaking the substrate or stirring the liquid to cause the liquid to flow, and separating the two by the liquid pressure generated at that time. When the liquid is used in this process, although there is no limitation, the liquid is preferably the wetting liquid used in the wetting liquid application process. In particular, water is suitable. This process makes it possible to obtain bagworm scaffolding silk threads spun onto the surface of a substrate, which was difficult to recover without damaging using conventional methods.

The "washing step" (S0105) is a step for washing the bagworm silk separated in the separation step. This step is optional and may be performed as needed.

The wetting liquid used in the wetting liquid application process remains on the surface of the bagworm silk obtained after the separation process. If the wetting liquid used is a solution or a low-polarity organic solvent, it may dry and adhere to the bagworm silk surface and cause the bagworm silk to deteriorate or discolor over time. Therefore, it is preferable to completely remove the wetting liquid used in this process by washing. In addition, if any parts of feces or the like are attached to the bagworm silk, they can also be removed at the same time in this process.

The cleaning liquid used in this process is not limited. If the wetting liquid used is a polar liquid, an aqueous solution, or a colloidal solution, a suitable liquid for the cleaning liquid is water (including warm water). If the wetting liquid used is a non-polar liquid such as a low-polarity organic solvent, another highly volatile solvent that has a high affinity with the low-polarity organic solvent is suitable for the cleaning liquid. As an example, if toluene or benzene is used in the wetting liquid application process, other xylene or ethanol can be used as the cleaning liquid.

There are no limitations on the cleaning method, so long as it is a method that can remove the wetting liquid used in the wetting liquid application process from the bagworm silk thread. The bagworm silk thread may be sprayed with the cleaning liquid or immersed in the cleaning liquid.

There is no limit to the number of washings. It can be done once or multiple times. In this specification, "multiple times" means, for example, 2 to 20 times, 2 to 15 times, 2 to 10 times, 2 to 7 times, 2 to 5 times, 2 to 4 times, or 2 to 3 times. In general, it is preferable to wash multiple times. When washing multiple times, the washing liquid used each time may be the same or different. The washing method may also be the same or different. After washing, it may be left to dry naturally, or the washing liquid may be separated and removed by centrifugation using a dehydrator or the like.

The "refining process" (S0106) is a process for refining the lump of bagworm scaffold silk produced in this process. This process is optional and may be performed as needed.

"Refining" refers to the process of removing the sericin-like adhesive substance (glue-like protein) from bagworm silk to obtain fibroin fibers.

There are no particular limitations on the scouring method, so long as it can remove adhesive substances without reducing the strength of the fiber components of the bagworm silk thread. For example, the scouring method for silkworm silk thread can be applied. In the scouring method for silkworm silk thread, a 0.01 mol/L to 0.1 mol/L, 0.03 to 0.08 mol/L, or 0.04 to 0.06 mol/L sodium carbonate solution is used as the scouring solution, and the same can be used in the scouring step of this method. The obtained bagworm silk thread can be boiled in the sodium carbonate solution, which is the scouring solution, for 1 second to 1 hour, 5 seconds to 30 minutes, 10 seconds to 15 minutes, 20 seconds to 10 minutes, or 30 seconds to 5 minutes.

When a refining solution such as a sodium carbonate solution is used as the wetting solution used in the wetting solution application process, the same refining solution is used in the subsequent refining process, which is convenient as it does not require a washing process before the refining process. After treatment in this process, the material can be washed in the same manner as in the washing process.

After the refining process, the harvested bagworm silk thread may be dried. There are no particular limitations on the drying method, so long as it can reduce the amount of wetting liquid, cleaning solution, or refining solution remaining in the bagworm silk thread without denaturing or altering the scaffold silk thread. Examples include a natural drying method (including drying in the sun) in which the wetting liquid, cleaning solution, or refining solution is evaporated by exposing the silk thread to the outside air, an air drying method in which hot or cold air is applied using a blower or the like, a dehumidification drying method in which the silk thread is placed in an enclosed space together with a dehumidifier for a certain period of time, a heat drying method in which the wetting liquid, cleaning solution, or refining solution is evaporated by heating, a reduced pressure drying method in which the silk thread is deaerated and evaporated using a vacuum pump or the like in a container, or a combination of these methods.

2. Bagworm silk thread and nonwoven fabric made therefrom 2-1. Overview The second aspect of the present invention is a scaffold silk thread made of bagworm silk thread, and a nonwoven fabric made therefrom. The bagworm silk thread and nonwoven fabric of the present invention are obtained using the thread lump production method of the first aspect.

2-2. Structure When the thread mass obtained by the method of the first aspect is spun on the surface of a substrate without controlling the movement of the bagworm, it often takes the form of a nonwoven fabric in which the bagworm silk threads are overlapped lengthwise and widthwise on the surface of the substrate. Therefore, the bagworm silk threads peeled off from the surface of the substrate by the thread mass production method of the first aspect can be used as a nonwoven fabric as is. Furthermore, the scaffold silk threads obtained by the method of the first aspect can be further made into a nonwoven fabric by an existing nonwoven fabric manufacturing method. Existing nonwoven fabric manufacturing methods that can be used include, but are not limited to, the spunlace method and the needle punch method.

On the other hand, if a cord is produced from the bagworm silk thread on the surface of the substrate and then peeled off, or if the cord is pulled out from the peeled nonwoven fabric and reeled, long bagworm silk thread can be obtained.

Example 1
(the purpose)
A peel tension evaluation test was conducted to verify that the silk mass production method of the present invention makes it easy to harvest and recover bagworm silk (scaffold silk) spun onto a substrate, which was difficult to harvest using conventional methods.

(material)
The bagworms used were the final stage larvae of the giant bagworm moth (Giant bagworm) collected from a fruit orchard in Tsukuba City, Ibaraki Prefecture. The substrate was an acrylic board of about 30 ^cm2 .

(method)
The bagworms used in the examples were fed a sufficient amount of food leaves until the day before the spinning process was carried out.
The bagworms were spun onto the surface of the acrylic substrate by placing them on a vertically standing acrylic plate and allowing them to climb up the wall of the plate.

After the bagworm silk was sufficiently spun onto the surface of the acrylic plate, a few drops of water (pure water), 30%, 50% and 70% ethanol aqueous solutions, and 99.5% ethanol were dropped onto the evaluation area using a syringe. The negative control was bagworm silk without the wetting liquid (sample without the wetting liquid).
Here, the peeling tension evaluation test used in this embodiment will be described. As shown in FIG. 2A, the scaffold silk of the bagworm has a ladder-like zigzag shape. This scaffold silk is fixed to the surface of the substrate at the turning point (referred to as the "fixed point") of the zigzag part (referred to as the "zigzag block" in this specification) corresponding to the horizontal board of the ladder shown by the dashed oval in FIG. 2A. When peeling off the scaffold silk, the tension peaks at the fixed point of each zigzag block because it requires the greatest force, but after peeling, the tension relaxes to the initial value of zero because it is not fixed to the substrate until the next fixed point. Similarly, at the next fixed point, a large tension is required for peeling, but immediately returns to the initial value after peeling. When peeling off the scaffold silk, this cycle is repeated between each zigzag block. In the peel tension evaluation test, one end of the bagworm scaffold silk thread spun onto the surface of the substrate is fixed to the load cell of a tensile testing machine, and the bagworm silk thread is then pulled away at a constant speed.The change in tension at this time is then continuously recorded, and the adhesion at the fixed point is released, and the process in which the zigzag scaffold thread is gradually unraveled into a straight line can be measured as the relationship between the peel length (pulling distance) and tension.

After applying each wetting liquid, and confirming that the bagworm silk thread was sufficiently wet, one end of the bagworm silk thread on each acrylic plate was pulled out and fixed to the load cell of a tensile tester. The fixed bagworm silk thread was peeled off at a constant speed (100 μm/sec), and the change in tension was continuously recorded by the load cell. A tensile tester (Shimadzu compact tabletop testing machine EZ Test) was used for the measurements.

In this example, the sum of all peak areas per zigzag block obtained in the peel test was divided by the peel length to calculate the peel energy per unit peel length. In order to minimize the effect on adhesive strength due to individual differences in the bagworms used in the spinning process, the results when each wetting liquid was used were normalized by the peel energy of the negative control (sample not treated with wetting liquid) scaffold silk spun by the same individual bagworm, and evaluated and compared as a percentage (%) when the peel energy of the negative control was taken as 100.

(result)
The results are shown in FIG.

In Figure 4, the horizontal axis represents the peel length (mm) and the vertical axis represents the peel tension (N). As shown in Figure 4 (A), the negative control required a relatively strong peel tension of 0.003 to 0.004 N at each fixed point per zigzag block. On the other hand, when water was applied, the peel tension was about 0.001 N, approximately 1/4 of that of the negative control, as shown in Figure 4 (B). Furthermore, when ethanol (99.5%) was applied, the peel tension was 0.0001 to 0.0002 N, approximately 1/20 to 1/40 of that of the negative control, as shown in Figure 4 (C).

As shown in Table 1, when water was applied to the substrate and bagworm silk, the peel energy was reduced to approximately 30% of the negative control. When ethanol (99.5%) was applied, the peel energy was reduced to 6% of the negative control, demonstrating a significant effect greater than that of water. This effect of ethanol was also maintained across various concentrations in the case of an ethanol aqueous solution, which is a mixed solution of ethanol and water, and it was confirmed that all concentrations were more effective than water.

Example 2
(the purpose)
The fact that the yarn lump production method of the present invention can be used with wetting liquids other than water and ethanol is verified by a peel tension evaluation test.

(Method)
The basic procedure and operation were the same as in Example 1. Here, the wetting liquids used were methanol (MeOH), a polar monohydric alcohol; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), a polar halogen-containing organic solvent; dimethyl sulfoxide (DMSO), a polar sulfur-containing organic solvent; carbon tetrachloride, a low-polarity halogen-containing organic solvent; glycerin, a polar trihydric alcohol; and a 0.05M sodium chloride aqueous solution (NaCl aq), and 0.05M sodium carbonate solution (Na₂CO₃aq) was used.

(result)
The results are shown in Table 2.

As shown in Table 2, the peel energy of each of the wetting liquids was reduced to 15% or less of the negative control by the application treatment. In addition, the peel energy of each of the liquids was confirmed to be highly effective, being less than half that of water.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (8)

A bagworm silk thread obtained by a silk harvesting method including a wetting liquid application step of applying a wetting liquid to the bagworm silk thread spun onto the surface of a substrate, and a separation step of separating the substrate from the bagworm silk thread,
The wetting liquid is a pure substance or mixture that is in a liquid state at least at 20°C or higher and lower than 30°C under atmospheric pressure, and does not damage, denature, or dissolve the fibroin protein, which is the fiber component of bagworm silk. The bagworm silk thread according to claim 1, which includes a spinning process in which bagworms are placed on the surface of the substrate prior to the wetting liquid application process and the bagworms are caused to spin threads. The bagworm silk thread according to claim 2, further comprising a bagworm recovery process for recovering the bagworms together with their nests after the spinning process and before the wetting liquid application process. The bagworm silk thread according to any one of claims 1 to 3, which includes a washing step for washing the separated bagworm silk thread. The bagworm silk thread according to any one of claims 1 to 4, which includes a refining step of refining the separated bagworm silk thread. Bagworm silk according to any one of claims 1 to 5, wherein the wetting liquid is a pure substance or mixture having a melting point below 20°C and a boiling point between 30°C and 300°C. The bagworm silk thread according to any one of claims 1 to 6, wherein the wetting liquid is an aqueous solution or an organic solvent. A nonwoven fabric made from bagworm silk thread according to any one of claims 1 to 7.

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