JP7683933B2 - Method for preparing cocoons for reeling - Google Patents

Description

The present invention relates to a method for preparing cocoons for reeling, the cocoons prepared thereby, and a method for producing raw silk.

The use of biological materials has attracted more attention as genetic manipulation and genomic information have become easier to use, and is currently expanding. Research and development into these areas is not only being actively carried out in the development of pharmaceuticals using cells, but also in the field of materials.

Silk thread, which is one of the biological materials, is an ideal material for the development of new materials from the viewpoints of its strength, texture, manufacturing method, processing method, etc. In fact, a great deal of research is being conducted on imparting new functions to silk thread (Patent Document 1 and Non-Patent Document 1).

Conventional silk manufacturing methods include several steps involving heating, such as drying, boiling cocoons, and degumming. In recent years, attempts have been made to add various proteins to silk threads using recombinant gene technology in order to impart new functions to the threads. However, problems have arisen in that the added proteins are thermally denatured during the heating process, causing them to lose their function. There is an increasing need to develop new methods that do not inactivate the added proteins.

Cocoon boiling is a process in which cocoons are boiled to make it easier to pull out the thread (cord). Conventionally, this process required boiling the cocoons in hot water. The inventors have developed a method in which a basic solution is used to treat cocoons at a low temperature of around 60°C, as a method that can be carried out at a relatively low temperature that does not denature proteins (Patent Document 2). This method has the problem that it is not suitable for proteins that are easily denatured in a basic environment, as the cocoons are exposed to a basic environment for a relatively long period of time. Therefore, there has been a demand for the development of a method that can be carried out at a lower temperature without inactivating the added functional protein.

JP 2020-70293 A JP 2010-95833 A

K. Kojima, et al., Biosci. Biotechnol. Biochem. 71, 2943 (2007)

The objective of the present invention is to provide a new cocoon boiling method that does not require high-temperature heat treatment of silk threads and does not require a basic solution.

The present inventors have conducted extensive research to solve the above problems. As a result, they have found that by contacting the cocoons with an organic solvent or organic acid, it is possible to prepare cocoons that can be reeled without denaturing the proteins added to the silk thread at a lower temperature than previously developed methods, particularly at room temperature. Furthermore, they have found that when this method is applied to silk thread containing a functional region, the activity of the functional region is maintained. They have also found that, as a result of treatment with an organic solvent or organic acid significantly promoting the penetration of water, the same effect can be obtained in a shorter time than before, even when boiling normal cocoons at conventional temperatures. The present invention is based on the above novel findings and provides the following.

(1) A method for preparing cocoons for reeling, comprising a contacting step of contacting the cocoons with an organic solvent and/or an organic acid, and an infiltration step of infiltrating water into the cocoon shell and cocoon cavity after the contact.
(2) The preparation method according to (1), further comprising a removal step of removing the organic solvent and/or organic acid before the penetration step.
(3) The preparation method according to (1) or (2), wherein the infiltration step is carried out by a dipping method.
(4) A preparation method described in (1) or (2), wherein the infiltration step includes a step of reducing the pressure inside the cocoon cavity.
(5) The preparation method described in (4), wherein the infiltration step is carried out by a method selected from the group consisting of a low-temperature reduced pressure method, a boiled cocoon cooling method, and a contact steaming method.
(6) The preparation method according to any one of (1) to (5), wherein the organic solvent comprises one or more organic solvents selected from the group consisting of lower alcohols, acetone, N,N-dimethylformamide, acetonitrile, and ethyl acetate.
(7) The preparation method according to any one of (1) to (6), wherein the organic acid comprises acetic acid and/or propionic acid.
(8) A cocoon for reeling prepared by the preparation method described in any one of (1) to (7).
(9) A method for producing raw silk, comprising: a stringing step of stringing a cocoon for reeling obtained by a preparation method described in any one of (1) to (7); and a reeling step of reeling silk based on the thread obtained in the stringing step.

According to the present invention, it is possible to prepare cocoons for reeling at a temperature at which proteins do not denature. According to the present invention, it is possible to prepare cocoons for reeling in a short time. Furthermore, according to the manufacturing method of the present invention, it is possible to produce raw silk from the prepared cocoons for reeling.

1 shows the amount of water absorbed by cocoons when treated with an organic solvent using aqueous ethanol solutions of different concentrations, in which the error bars indicate standard deviation. 1 is a graph showing the amount of water absorbed by cocoons when treated with different organic solvents, in which error bars indicate standard deviation. This is a diagram showing the appearance of cocoons containing the fluorescent protein Azami Green as a functional region when they were subjected to organic solvent treatment. In the figure, the upper part shows the appearance in bright field, and the lower part shows the appearance observed by fluorescence. The order of arrangement of the cocoons is the same in the upper and lower parts. The dashed line in the lower part indicates the position of the control cocoon. In the figure, "-" indicates that Azami Green or ethanol treatment is not included, and "+" indicates that Azami Green or ethanol treatment is included. This is a diagram showing the appearance of cocoons containing the fluorescent protein Azami Green as a functional region when subjected to normal cocoon boiling treatment. In the figure, the upper part shows the appearance in bright field, and the lower part shows the appearance observed by fluorescence. The order of arrangement of the cocoons is the same in the upper and lower parts, with the left side showing cocoons that were not boiled and the right side showing cocoons that were boiled. The dashed line in the lower part indicates the position of the cocoons that were boiled. In the figure, "-" indicates that the cocoon did not undergo boiling treatment, and "+" indicates that the cocoon did undergo either Azami Green or boiling treatment. This is a diagram showing the appearance of cocoons containing the fluorescent protein Azami Green as a functional region when they were subjected to organic solvent treatment using acetic acid. In the figure, the upper part shows the appearance in bright field, and the lower part shows the appearance observed by fluorescence. The order of arrangement of the cocoons is the same in the upper and lower parts, with the left side showing cocoons that were not treated with organic solvent, and the right side showing cocoons that were treated with organic solvent. The dashed line in the lower part indicates the position of the cocoon that was subjected to organic solvent treatment. In the figure, "-" indicates that no organic solvent treatment was included, and "+" indicates that either Azami Green or organic solvent treatment was included.

1. Method for preparing cocoons for reeling 1-1. Overview The first aspect of the present invention is a method for preparing cocoons for reeling. The method of this aspect includes a contact step and a penetration step as essential steps. According to the method of this aspect, cocoons for reeling can be prepared at a lower temperature or in a shorter time.

1-2. Definition "Reeling" refers to the process of producing raw silk by pulling silk threads from the ends of corded cocoons.
In the present specification, the term "cocoon" refers to a structure consisting of a cocoon layer and an adult, a pupa, an exuvia, and/or a larva. The term "cocoon" in the present specification includes cocoons produced by any silkworm, as long as the cocoon layer is composed of silk threads.

In this specification, "reeling cocoons" refers to cocoons that have been processed so that the thread can be easily pulled out using a cord. Normally, reeling cocoons have a structure in which the cocoon cavity is filled with water and the cocoon itself cannot be pulled up during reeling.

In this specification, the term "cocoon cavity" refers to the space within the cocoon, i.e., the pupal chamber. For example, in the case of a silkworm cocoon, it means the space that houses the silkworm larvae, pupae, or adults.

As used herein, the term "cocoon layer" refers to the wall of a cocoon that is made up of silk threads.
"Reeling" refers to the process of searching for and pulling out the ends of the threads from the cocoons. This is usually done with a reeling broom or the like, but in this specification, reeling includes any process in which the ends of the threads are pulled out from the reeling cocoons, regardless of the means.

As used herein, the term "thread end" refers to the end of a silk thread that has been released from a cocoon.
As used herein, "silk thread" refers to thread derived from silkworms, and refers to protein thread produced by larvae or adults of silkworms for the purposes of nesting, moving, fixing, spinning cocoons, capturing food, etc. Silk thread as used herein includes threads derived from any silkworm.

As used herein, "silkworm" refers to a general term for insects that have silk glands and can spin silk threads. It usually refers to species that can spin silk threads during the larval stage for nest-building, cocoon-making, or movement.

In this specification, "raw silk" refers to silk thread that has been reeled from a cocoon and still has adhesive substances such as sericin remaining. In this specification, raw silk includes not only silk that has been simply reeled, but also silk that has been doubled and/or twisted.

In this specification, "organic solvent" refers to an organic compound that is in liquid form at room temperature and pressure.

In this specification, "lower alcohol" refers to an organic compound having 4 or less carbon atoms and a hydroxyl group, and its derivatives. In this specification, lower alcohol includes both straight-chain lower alcohols with unbranched carbon chains and branched-chain lower alcohols with branched carbon chains.

In this specification, "organic acid" refers to an organic compound that can donate a proton to another compound. In this specification, organic acid refers mainly to an acidic organic solvent that is liquid at room temperature and normal pressure.

"Contact" means direct physical contact of one substance with another substance.
"Permeation" refers to the movement of a liquid present outside a substance or space through the surface of the substance or through the structure surrounding the space to the inside. For example, "permeating into the cocoon layer" refers to the liquid outside the cocoon soaking into the cocoon layer, and "permeating into the cocoon cavity" refers to the liquid outside the cocoon passing through the cocoon layer and moving into the cocoon cavity.

"Removal" refers to the loss of at least a portion of a specific substance contained in a structure from the structure. In this specification, removal includes both active removal of a specific substance and passive removal, such as by placing the specific substance in a situation in which it may be lost.

As used herein, the term "immersion" refers to immersing a part or the whole of a target substance in a liquid.
In this specification, the term "reduced pressure" refers to reducing the atmospheric pressure or vapor pressure. In this specification, the term "reduced pressure" refers to both a direct reduction in atmospheric pressure or vapor pressure and an indirect reduction thereof by a temperature change or the like.

1-3. Configuration The method of the present embodiment includes a contacting step and a permeation step as essential steps, and includes a drying step, a removing step, and a washing step as optional steps.

1-3-1. Drying step The "drying step" is an optional step in which the cocoons and/or pupae in the cocoon cavity are dried to prepare dried cocoons.

This step is usually carried out for the purpose of killing the pupae in the cocoon cavity and/or for long-term storage. Therefore, the necessity of this step can be determined taking into consideration the conditions such as the cocoons to be used and the necessity of storage. For example, this step does not need to be carried out when using cocoons that do not contain pupae in the cocoon cavity or when they are not to be stored.

The drying method used in this step is not particularly limited, but may be, for example, a method used for drying raw silkworm cocoons. Specific examples include air heat drying such as hot air drying, natural drying such as drying in the sun, drying by infrared rays, reduced pressure drying in which degassing and evaporation is performed using a vacuum pump or the like in a container, freeze drying in which water is evaporated while still frozen, air drying using hot or cold air, dehumidification drying using a dehumidifier or the like, drying by electromagnetic waves, or a combination thereof. All of these methods can be performed manually or automatically using a machine or the like.

The cocoons can be subjected to additional processing during drying. For example, if natural drying is performed, the cocoons can be frozen or refrigerated before or after the drying process.

The "contact step" is an essential step in which the cocoons are contacted with an organic solvent and/or an organic acid (hereinafter, in this specification, often collectively referred to as "organic solvent, etc."). When a drying step is performed, this step can be performed thereafter.

＜Cocoon＞
The cocoons used in this step are not particularly limited. For example, the insect from which they are derived, their composition and type can be arbitrarily selected depending on the purpose.

The silkworm from which the cocoons are derived is not particularly limited, but is preferably a species belonging to the order Lepidoptera that can spin a large amount of silk. For example, species belonging to the families Bombycidae, Saturniidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Archtiidae, Noctuidae, etc. are preferred as silkworms in this specification. Species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically Bombyx mori (domestic silkworm; hereinafter, in this specification, both larvae and adults of silkworms are collectively referred to as "silkworms"), Bombyx mandarina, Samia cynthia (including Samia cynthia ricini and hybrids of Samia cynthia and Samia ricini), Antheraea yamamai, Antheraea pernyi (tussah silkworm), Saturnia japonica, and Actias gnoma are particularly preferred. Genetically modified versions of these and mutants produced by genome editing or the like can also be used. The cocoons used in this method may be derived from a single individual, may be derived from multiple individuals, or may be a mixture of those derived from multiple species.

When using cocoons derived from genetically modified organisms, the cocoons may be made of genetically modified silk thread.

In this specification, "genetically modified silk thread" refers to silk thread in which genes of silk thread components have been genetically modified. Genetically modified silk thread includes not only silk threads in which the sericin and fibroin that compose the silk thread have been genetically modified, but also silk threads whose properties have been changed by genetic recombination, such as silk threads whose internal structure has been changed by genetic recombination. Genetically modified silk threads can include, for example, functional regions.

As used herein, the term "functional region" refers to a protein region that is introduced into silk thread by genetic recombination to impart a desired function. In addition, in this specification, silk thread into which a functional region has been introduced is referred to as "functional silk thread" or the like. "Functional" refers to the functional region exhibiting activity.

The type of functional region is not particularly limited, and includes any protein or part thereof. Examples include binding proteins such as enzymes, antibodies, receptors, and ligands, neuropeptides, hormones, fluorescent proteins, luminescent proteins, and parts thereof.

For example, fluorescent proteins include GFP, DsRed, TurboRFP and their modifications, as well as various fluorescent proteins with different origins, such as thistle green.

The number and types of functional regions are not particularly limited. The silk thread used in the present invention can contain, for example, one or more functional regions of one type, or multiple functional regions of multiple types. When multiple functional regions are contained in one molecule, their arrangement is also not particularly limited. For example, one or more types of functional regions can be introduced linked to each other at the same position, or distributed at different positions.

The functional domain to be used can be appropriately selected depending on its properties. For example, among fluorescent proteins, Azami Green has a higher quantum yield than GFP, and stronger fluorescence can be obtained from the same intensity of excitation light, but Azami Green, especially in the monomer form, has properties such as being inferior to GFP in terms of pH stability (Karasawa et al., J. Biol. Chem. 278(36): 34167-71 (2003)).

The functional region used in the method of the present invention may be a protein or a portion thereof that is susceptible to thermal denaturation and/or denaturation under basic conditions.

The origin of the functional region is not particularly limited. For example, it may be derived from an endogenous gene of the silkworm, or it may be derived from an exogenous gene.

As used herein, "exogenous gene" refers to any gene introduced from outside. As used herein, exogenous genes include both natural genes and artificial genes. In the case of exogenous genes, there are no particular limitations on the organism from which they are derived. There are no particular limitations on the type of exogenous gene. For example, the exogenous gene may be a gene not derived from silk thread, a gene derived from silk thread, or a combination thereof.

The method for introducing the gene is not particularly limited. It can be introduced using gene recombination techniques known in the art.

There are no particular limitations on the treatment that is applied in advance to the cocoons used in this process. Specifically, for example, the cocoons produced by the silkworms can be used as is, or can be treated as desired prior to this process. Specifically, for example, both live cocoons and dried cocoons obtained by drying live cocoons can be used.

There are no particular limitations on the treatment that can be applied to the silk thread prior to this method, but if the aim is to avoid high-temperature heat treatment, it is preferable that this process does not include treatment at high temperatures (e.g., 80°C or higher).

<Organic solvents, etc.>
The temperature of the organic solvent is not particularly limited. For example, it can be a temperature equal to or higher than the melting point or equal to or lower than the boiling point of the organic solvent used. Specifically, for example, it is -120°C or higher, -100°C or higher, -90°C or higher, -80°C or higher, -50°C or higher, -30°C or higher, -20°C or higher, -10°C or higher, -5°C or higher, -2°C or higher, -1°C or higher, 0°C or higher, 3°C or higher, 5°C or higher, 10°C or higher, 12°C or higher, 15°C or higher, 17°C or higher, 18°C or higher, 19°C or higher, 20°C or higher, 23°C or higher, 24°C or higher, or 25°C or higher. Specific upper limits of the temperature are, for example, 100°C or less, 90°C or less, 80°C or less, 79°C or less, 75°C or less, 70°C or less, 65°C or less, 60°C or less, 55°C or less, 50°C or less, 45°C or less, 40°C or less, 37°C or less, 35°C or less, 33°C or less, 31°C or less, or 30°C or less. The temperature does not need to be kept constant during this step as long as it is generally within the above temperature range. If necessary, the temperature can be adjusted using freezing means, refrigeration means, warming means, warming means and/or heating means known in the art.

There are no particular limitations on the type of organic solvent. Either water-soluble or water-insoluble organic solvents can be used.

In this specification, "water-soluble" refers to the property of having a solubility in water at one atmosphere and 20°C that is greater than a certain level. The solubility of the water-soluble organic solvent is not particularly limited, but is, for example, greater than 8000 mg/L. Specifically, for example, it is 8500 mg/L or more, 9000 mg/L or more, 9500 mg/L or more, 10000 mg/L or more, 15000 mg/L or more, 20000 mg/L or more, 25000 mg/L or more, 30000 mg/L or more, 50000 mg/L or more, 100000 mg/L or more, 250000 mg/L or more, 500000 mg/L or more, or 1000000 mg/L. "Water-insoluble" refers to the property of not being water-soluble.

Examples of water-soluble organic solvents include, but are not limited to, lower alcohols, acetone, N,N-dimethylformamide, acetonitrile, carboxylic acid derivatives such as ethyl acetate and formamide, polyhydric alcohols such as ethylene glycol and glycerin, other alcohols such as diethylene glycol, alcohol derivatives such as alcohol ethers and alcohol ether esters, sulfoxides such as dimethyl sulfoxide, polyethers, and lactones.

Non-water-soluble organic solvents include, but are not limited to, chloroform, isoamyl alcohol (also called isopentyl alcohol), and aromatic compounds such as xylene, benzene, hexane, and toluene.

As the organic solvent, for example, a low surface tension solvent may be used. A low surface tension solvent refers to an organic solvent having a surface tension lower than that of pure water. Since the surface tension of pure water is 72.75 mN/m at 20°C, the surface tension of a low surface tension solvent may be less than 72.75 mN/m (20°C). Specifically, for example, solvents having a surface tension at 20°C of less than 72.75 mN/m, less than 70 mN/m, less than 65 mN/m, less than 60 mN/m, less than 55 mN/m, less than 50 mN/m, less than 45 mN/m, less than 40 mN/m, or less than 38 mN/m may be used.

Organic acids include carboxylic acids including aliphatic carboxylic acids such as formic acid, acetic acid, and propionic acid, and aromatic carboxylic acids such as benzoic acid and salicylic acid, sulfonic acids such as methanesulfonic acid, and acidic alcohols such as phenols and enols.

The organic solvent, etc., does not have to be a pure substance. For example, a mixture with an inorganic compound such as water and/or other organic solvent, etc., can be used. In that case, the concentration of the organic solvent, etc. is not particularly limited. For example, the concentration of the organic solvent, etc., is 20% by volume or more, 30% by volume or more, 40% by volume or more, 45% by volume or more, 50% by volume or more, 55% by volume or more, 59% by volume or more, 60% by volume or more, 61% by volume or more, 64% by volume or more, 65% by volume or more, 66% by volume or more, 69% by volume or more, 70% by volume or more, 71% by volume or more, 74% by volume or more, 75% by volume or more, 76% by volume or more, 79% by volume or more, 80% by volume or more, 81% by volume or more, 90% by volume or more, or 99.5% by volume or more, based on the total volume of the mixture. The concentration may be within the above range during this process and does not need to be kept constant. Also, for example, organic solvent, etc., or other components may be added during this process, or the solution may be replaced.

In this process, it is sufficient that at least a portion of the organic solvent, etc., is liquid. It is not necessary for the entire organic solvent, etc., to be maintained in liquid form during this process. For example, a portion of the organic solvent, etc., may be vaporized or solidified during this process. For example, if the pure substance is in a form other than liquid, but can become liquid when mixed with another substance, it may be preferable to use it as a mixture.

The organic solvents used in the present invention may contain any other components as necessary. Specific examples include buffers, pH adjusters, surfactants, chelating agents, etc.

The amount of the organic solvent or other liquid is not particularly limited. For example, when contact is performed by immersion, the bath ratio, which indicates the total volume of the liquid when the weight of the cocoon layer is taken as 1, is usually 1:0.5 or more, 1:1 or more, 1:1.5 or more, 1:2 or more, 1:5 or more, or 1:10 or more.

The conditions of the organic solvent, such as temperature, can be appropriately selected depending on the purpose and the properties of the silk threads that make up the cocoons. For example, the temperature of the organic solvent may be determined by the physical properties, such as the boiling point and melting point, of the organic solvent used. Conditions may also be selected depending on the purpose. For example, if the purpose is not to perform a heat treatment, an organic solvent at a temperature of less than 80°C, less than 60°C, room temperature (15°C or higher and less than 40°C), or below room temperature can be used, and if the purpose is to shorten the time, an organic solvent at a higher temperature can be used.

The organic solvent and the like used in this process may be prepared before this process or may be prepared simultaneously with this process. Specifically, for example, components may be added and/or updated during this process.

<Contact>
There is no particular limitation on the method as long as the solid (cocoons) and the liquid (organic solvent, etc.) can be directly contacted with each other. In this step, since both components are brought into contact with each other, a method suitable for contacting a solid with a liquid is preferable. Specifically, for example, the cocoons can be contacted by immersing them in an organic solvent, spraying, spraying, or applying the organic solvent to the cocoons, or a combination thereof.

The contact can be performed by a known method used in boiling or scouring silkworm cocoons. Specific methods include manual methods, such as hanging the cocoons directly from something or placing them in a bag etc. and immersing them in liquid, or spraying liquid using a machine. The hanging method, bagging method, and spraying methods can be performed in accordance with the methods used in scouring, such as the hanging method, bagging method, and spray scouring method. The "hanging method" is a method in which the fabric or silk thread is hung from a pole or rod placed above or inside a pot and immersed in liquid in the pot, and can be divided into the pole method, stick method, etc. depending on the method used. The "bag method" is a method in which the cocoons or silk thread are placed in a bag such as a cotton bag and immersed in liquid. The "spray scouring method" is a method in which liquid is sprayed on the silk thread by a machine.

When using a method that uses immersion, it is not necessary for the entire cocoon to be under the liquid surface at once; it is sufficient that the entire cocoon comes into contact with the organic solvent, etc. as a result, for example, by changing the position, mixing, rotating, etc. Even when using a method other than immersion, it is preferable to perform an operation such that the entire cocoon can come into contact with the organic solvent, etc., by changing the position or rotating the cocoon, etc.

The contact time is not particularly limited as long as the organic solvent etc. can penetrate into the cocoon layer. For example, it can be determined in relation to the temperature and properties of the organic solvent etc. In general, the lower the temperature, the longer the time, and the higher the temperature, the shorter the time. Specifically, for example, the contact time is 1 second or more, 3 seconds or more, 5 seconds or more, 6 seconds or more, 9 seconds or more, 10 seconds or more, 11 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, or 30 seconds or more. Note that in this process, the organic solvent etc. can penetrate into the cocoon cavity.

The contact can be carried out multiple times. In this case, the composition of the organic solvent, etc., the contact time, temperature, etc. may be changed for each contact, or the same organic solvent used in the previous contact may be used as is.

The "removal step" is an optional step of removing the organic solvent and/or the organic acid from the cocoon shell. This step can be performed after the contact step.

Whether or not this step is necessary can be determined taking into consideration the conditions such as the type and concentration of the organic solvent, etc., used in the contact step. This step is usually carried out when a water-insoluble organic solvent, etc. is used in the contact step. Note that even if an organic solvent, etc., containing a water-insoluble organic solvent, etc. is used in the contact step, this step may not be necessary depending on the type, for example, or if the organic solvent, etc., contains a water-soluble organic solvent, etc., or a surfactant.

The removal method used in this process is not particularly limited. For example, it can be performed by drying, dropping, centrifugation, suction, air blowing, or a combination thereof.

When drying, the method is not particularly limited, but for example, the above-mentioned method can be used for the drying process such as reducing pressure.

When dripping, any method can be used as long as it allows the organic solvent, etc. to fall off due to gravity, and there are no particular limitations on the method. For example, the cocoons can be left to stand to allow the organic solvent, etc. to fall off. At this time, the cocoons may be given stimulation such as rotation or vibration.

When centrifuging, there are no particular limitations on the method as long as it is a method that uses centrifugal force to remove the organic solvent, etc. For example, it can be performed manually or automatically using a centrifuge, etc.

When aspirating, the method is not particularly limited. For example, it can be done manually by breathing or using a syringe, or by using an aspirator, etc.

When blowing air, there are no particular limitations on the method, as long as it is a method that uses wind pressure to remove the organic solvent, etc. For example, it can be done manually by breathing or using a fan, or by using a blower, etc.

This process can be carried out multiple times. In that case, the method and conditions used may be the same each time or may be different each time.

1-3-4. Washing step The "washing step" is an optional step in which the silk thread is brought into contact with water, an aqueous solution, or another organic solvent, etc., to wash the cocoons. This step can be carried out after the contacting step. Furthermore, when a removing step is carried out, this step can be carried out simultaneously with or after the removing step. In particular, when the method includes a plurality of steps, this step can be carried out between each of the steps. For example, when a drying step is carried out, this step can be carried out between the drying step and the contacting step and/or after the contacting step, and when the contacting step is carried out multiple times, this step can be carried out between each contact.

The cleaning solution used in this process is not particularly limited, but examples include surfactants, water-soluble or volatile organic solvents such as toluene, benzene, and ethanol, and water.

This process can be carried out multiple times. In that case, the composition and temperature of the liquid used may be the same each time or may be different each time.

The "permeation step" is an essential step in which water or an aqueous solution is permeated into the cocoon shell and the cocoon cavity after contact. This step can be carried out simultaneously with or after the contact step, and if a removal step is carried out, simultaneously with or after the removal step.

The composition of the aqueous solution used in this step is not particularly limited. For example, the aqueous solution may contain any other components as necessary. Specific examples include alkaline agents such as sodium carbonate, sodium bicarbonate, and calcium carbonate, acid solutions such as citric acid, acetic acid, and propionic acid, metal ions, buffers, pH adjusters, surfactants, and chelating agents. Water for boiling silkworm cocoons may also be used.

The temperature of the water and aqueous solution used in this process is not particularly limited. For example, water at the temperatures exemplified for the organic solvent, etc. (including cold water (less than 15°C), room temperature water (15°C or more and less than 40°C), warm water (40°C or more and less than 80°C), and hot water (80°C or more)) can be used. The temperature of the hot water is not particularly limited, but is, for example, 80°C or more, 85°C or more, 90°C or more, 93°C or more, or 95°C or more.

The method used in this step is not particularly limited. For example, a method used in normal boiling of silkworm cocoons can be used. For example, this step can be carried out by the immersion method. In this method, the cocoons are immersed in water for a certain period of time. The time is not particularly limited. For example, the immersion can be carried out for 30 seconds or more, 1 minute or more, 1 minute 30 seconds or more, 2 minutes or more, 5 minutes or more, 6 minutes or more, 8 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, or 90 minutes or more. Other conditions are similar to those described in detail for the immersion in the contact step.

This process may include a step of reducing the pressure inside the cocoon cavity. In this step, the pressure inside the cocoon cavity is reduced. The reduction in pressure in this step is performed for the purpose of assisting the penetration of water into the cocoon cavity, so there are no particular limitations on the method used or the degree of reduction in pressure as long as this purpose is achieved.

Specific examples of the method include a reduced pressure method, a cooling method, and a steam method.
The decompression method is a method of reducing the air pressure in the space containing the cocoons while they are immersed in water or an aqueous solution. In this method, the pressure is usually reduced during immersion, and the reduced pressure is maintained for a certain period of time before the pressure is restored to atmospheric pressure. In particular, the decompression method using cold water, room temperature water or hot water is called the low-temperature decompression method, and the decompression method using hot water is called the high-temperature decompression method.

There are no particular limitations on the means for changing the air pressure. Examples include a method of degassing and/or supplying air to the space (such as inside a container or a room) that contains the cocoons, or a method of changing the volume of the space (particularly inside the container). A commercially available pressure reducer may also be used.

The degree of decompression is not particularly limited. Usually, the pressure is reduced to a low vacuum (approximately 10 ⁵ Pa to 10 ² Pa). For example, the gauge pressure, which indicates the strength of the decompression, may be −50 mmHg or less, specifically, −750 mmHg to −50 mmHg, −700 mmHg to −100 mmHg, −650 mmHg to −200 mmHg, or −600 mmHg to −300 mmHg.

The time for which the reduced pressure state is maintained is not particularly limited. For example, it can be maintained for the time exemplified in the immersion method.

After maintaining the reduced pressure, the pressure is restored to normal pressure (for example, one atmosphere). In this case, it is preferable to restore the pressure slowly, but this is not limited to this. Specifically, the pressure is restored over, for example, 1 second or more, 2 seconds or more, 5 seconds or more, 10 seconds or more, 30 seconds or more, 40 seconds or more, 1 minute or more, or 2 minutes or more.

The cooling method involves varying the temperature of the water used for immersion. In this method, a first immersion in water of a relatively high temperature is followed by a second immersion in water of a relatively low temperature.

The temperature difference between the first and second soaking is not particularly limited, but can be changed by, for example, 10°C or more, 15°C or more, 20°C or more, 25°C or more, 30°C or more, or 35°C or more. The temperature of each soaking is not particularly limited, but can be appropriately selected, for example, from the water temperatures exemplified in this process. In this specification, a method using hot water for the first soaking is called the cocoon boiling and cooling method, and a method using water at a temperature lower than warm water for the first soaking is called the low-temperature cooling method. For example, as the cocoon boiling and cooling method, hot water can be used for the first soaking and warm water can be used for the second soaking. The temperature of the hot water used in the cocoon boiling and cooling method can be, for example, 90°C or more or 95°C or more.

The time for which the first and second immersions are performed is not particularly limited. For example, the immersions can be performed for the times exemplified in the immersion method, but according to the method of the present invention, the same effect can be obtained in a shorter time than usual. Therefore, it is sufficient to perform each immersion for, for example, 5 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute 30 seconds or less. The times for the first and second immersions may be the same or different from each other. For example, the first immersion can be performed longer than the second immersion.

The steaming method is a method in which high-temperature steam is applied to the cocoons instead of the first immersion in the cooling method. The temperature of the steam may be, for example, the same as the temperature of the hot water used in the cooling method.

Other conditions such as temperature difference are the same as for the cooling method.
This step may be carried out using a known machine such as a cocoon cooker.

The method and conditions used can be appropriately selected taking into consideration the purpose, the properties of the silk threads that make up the cocoons, the facilities, etc. For example, if the purpose is to avoid heat treatment, the low-temperature reduced pressure method or low-temperature cooling method can be used, and if the purpose is to shorten the cocoon boiling time, the high-temperature reduced pressure method, cocoon boiling and cooling method, or contact steaming method can be used.

This process can be carried out multiple times. In that case, the same method and conditions can be used, or the method and conditions can be changed each time.

The method and conditions used in the method of the present invention may be determined, for example, in relation to the stringing efficiency (the proportion of cocoons that can be stringed), the unwinding rate (the number of cocoons per cut), or the amount of water absorption. For example, they may be determined so that the stringing efficiency is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. Also, for example, they may be determined so that the unwinding rate is 80% or more.

The amount of water absorbed per cocoon varies depending on the cocoon used, but it is usually 1 g or more, 1.1 g or more, 1.2 g or more, 1.3 g or more, or 1.4 g or more, or, for example, 1.05 times or more, 1.06 times or more, 1.075 times or more, 1.1 times or more, 1.15 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, or 1.6 times or more compared to when the same infiltration process is carried out using cocoons that have not been treated with an organic solvent, etc.

1-4. Effects The method of this embodiment makes it possible to prepare cocoons for reeling at low temperatures or in a short time. These cocoons for reeling can be untied and reeled as is. In addition, it is possible to obtain cocoons for reeling that retain the activity of functional regions that are easily denatured by heat and/or under basic conditions. Furthermore, according to the method of this embodiment, cocoons can be boiled in a shorter time using normal cocoon boiling equipment as is. Therefore, the method of the present invention can be used for various purposes, such as reducing the load on silk threads, shortening the time, or reducing silk reeling costs.

Although the method of this embodiment can produce a cord and a cocoon ready for reeling, for example, a cocoon prepared without carrying out the infiltration step of this method can be stored or provided as a cocoon for infiltration before reeling. In this case, the infiltration step can be carried out on the stored or provided cocoon for infiltration before reeling to prepare a cocoon for reeling, which can then be used for reeling.

2. Method for producing raw silk 2-1. Overview The second aspect of the present invention is a method for producing raw silk. The method of this aspect includes a stringing step and a reeling step as essential steps. According to the method of this aspect, raw silk can be produced from the reeling cocoons prepared in the first aspect.

2-2. Configuration The method of this embodiment includes a stringing step and a reeling step as essential steps.

The "stringing step" is an essential step in which stringing is performed on the reeling cocoons obtained in the first embodiment.

As the reeling cocoon has been described in detail in the first embodiment, a detailed description thereof will be omitted here.
The method and conditions used in this step are not particularly limited. For example, a method normally used for stringing silkworm cocoons can be used. For example, the surface of the cocoon in the water in which the cocoon is soaked during reeling (hereinafter, in this specification, often referred to as "water for reeling") may be rubbed with a broom or the like. As long as a large amount of water is not lost within the cocoon cavity, it is not necessary to perform this step while the cocoon for reeling is soaked in water. In addition, the method for rubbing the cocoon surface is not particularly limited as long as it allows the thread end to be pulled out.

The temperature and composition of the reeling water are not particularly limited, but for example, the reeling water used in normal reeling of silkworm cocoons may be used, or one exemplified in the section on permeation in the first embodiment may be used. For example, when this method is carried out consecutively with the method described in the first embodiment, the water used for permeation may be used as is for reeling water, or the water used in this step may be replaced after this step or its composition may be changed before being used in the reeling step.

The "thread reeling process" is an essential process in which thread is reeled from the thread obtained in the stringing process. This process can be carried out simultaneously with or after the stringing process.

Any one of the threads pulled out in the stringing process can be selected for reeling. For example, similar to the stringing process used in the normal silkworm cocoon reeling method, the threads can be further pulled out until only one thread remains.

There are no particular limitations on the method used in this process. For example, a method that is normally used for reeling silkworm cocoons can be used. Also, since the main purpose of this process is to produce raw silk from the cocoons, any method that can reel thread from the cocoons can be used in this process.

This process may be carried out manually by hand or counter-reeling, or may be carried out by controlling the fineness using a counter-reeling machine or multi-row reeling machine to produce fixed-size yarn, or an automatic reeling machine to produce fixed-size yarn.

If necessary, it is possible to address defects in the yarn, such as the joining of the yarn, or to control the fineness of the yarn, such as the doubling and/or twisting. These methods are well known in the art and are not particularly limited.

This method may be carried out as a series of steps using an automatic reeling machine that can perform stringing and reeling continuously.

Any product can be manufactured from the raw silk obtained by the method of this embodiment and/or the degummed silk obtained by degumming the raw silk. Specific examples of such products include nonwoven fabrics, fillers, surgical sutures, woven fabrics, knitted fabrics, and strings for musical instruments.

In this case, any other fiber material may be included. Although not particularly limited, specific examples include natural fibers including other animal fibers such as wool and plant fibers such as cotton, semi-synthetic fibers such as acetate, regenerated fibers such as rayon, synthetic fibers such as polyester, and inorganic fibers such as glass fibers.

The use of the product is not particularly limited. For example, if the reeling cocoons used in the method of this embodiment are prepared at low temperatures, even if the silk contains a functional region that is easily thermally denatured, the method of this embodiment can produce raw silk that retains its activity. In that case, the product may be used for an application in which the functional region can exert its function.

By carrying out the method of the first embodiment at a low temperature and then scouring using a scouring method that can be carried out at a low temperature after the method of the present embodiment, it is possible to obtain a degummed yarn that contains a functional region that can function, even if the silk thread contains a functional region that is easily thermally denatured. Any known method can be used as the scouring method in this case.

Example 1. Improvement of retrieval efficiency by organic solvent treatment
(the purpose)
The effect of treatment with organic solvents on the efficiency of stringing cocoons is investigated.

(method)
As samples, dried cocoons of Gunma 200 silkworm moths were used.
The organic solvent treatment was carried out by immersing the cocoons in the organic solvent of the following composition for 30 seconds.

As the organic solvent, aqueous ethanol solutions with ethanol concentrations of 20, 40, 60, 80, and 99.5% by volume were used. As a control, water containing no ethanol (0% by volume aqueous ethanol solution) was used.

After the organic solvent treatment, the cocoon was immersed in water at room temperature and left to stand for 8 minutes under reduced pressure (gauge pressure: -600 mmHg) to allow water to permeate into the cocoon cavity.
After restoring the pressure to atmospheric pressure over a period of 2 minutes, the surface of the cocoon was rubbed with a cord broom to pull out the threads.

The threading efficiency was calculated as the percentage of cocoons from which the correct thread could be pulled out out of the 10 cocoons used in each condition.

(result)
The results are shown in Table 1.

Table 1 shows the retrieval efficiency when each organic solvent was used. The retrieval efficiency increased depending on the ethanol concentration. In the 0% ethanol control, the retrieval efficiency was 20%, whereas in the 40% ethanol aqueous solution, the retrieval efficiency was 60%, in the 60% ethanol aqueous solution, the 80% ethanol aqueous solution, the 80% ethanol aqueous solution, and the 99.5% ethanol aqueous solution, the retrieval efficiency was excellent, being 100%.

When the cocoons were observed, in the control cocoon, there were patches of water-permeated (semi-transparent) and non-permeated (white) areas. In contrast, in the cocoons treated with organic solvent, the cocoon layer was translucent throughout, indicating that water had been uniformly permeated.

Furthermore, when reeling was performed from the thread slits pulled out by the cord, it was possible to reel more than 450 meters of silk thread from each of the corded cocoons. According to the selection criteria for water-reeling strains (Dainippon Sanshikai Silk News No. 41 p. 2-3 (2011)), all of them were deemed capable of reeling.

The physical properties of raw silk produced by reeling raw cocoons of Ariake, a four-way hybrid of Japan and China, using the same process as above were compared with those of raw silk produced by boiling dried cocoons of the same variety in the usual way. As a result, no significant difference was found in the physical properties of the two, with the former having a strength of 4.25±0.10g/d and the latter having a strength of 4.29±0.05g/d.

From the above, it was found that organic solvent treatment makes it possible to prepare cocoons that can be corded and reeled without heating, without affecting the physical properties of raw silk.

Example 2. Relationship between water penetration into cocoon shell and organic solvent concentration
(the purpose)
We investigate the effect of organic solvent treatment on water penetration into the cocoon layer and the relationship between organic solvent concentration and penetration.

(method)
The sample and organic solvent treatment were basically the same as in Example 1.
The amount of water absorbed was calculated for each cocoon by subtracting the weight of the cocoon before the organic solvent treatment from the weight of the cocoon after the water in the cocoon cavity was removed after the pressure was restored. Here, the water in the cocoon cavity was removed by pulling the cocoon out of the water after the pressure was restored and reducing the pressure again.
Experiments were carried out using 10 cocoons for each concentration condition.

(result)
The results are shown in Figure 1.
Figure 1 is a graph showing the amount of water absorbed by cocoons (cocoon shells) when organic solvent treatment was performed using ethanol aqueous solutions of different concentrations. The amount of water absorbed by the cocoon shells changed depending on the ethanol concentration, and the amount of water absorbed increased significantly when 60% ethanol aqueous solution was used compared to the 0% ethanol control. Furthermore, the amount of water absorbed increased further at concentrations of 80% or more by volume, and no significant difference was observed between the amounts of water absorbed at 80% and 99.5% by volume.

From the above, it was found that the water absorption of the cocoon layer increases with organic solvent treatment, and that this increase is concentration-dependent.

Example 3: Relationship between water penetration into cocoon shell and types of organic solvents, etc.
(the purpose)
The amount of water absorbed by the cocoon layer is measured by changing the type of organic solvent used, and the relationship between the type of organic solvent and penetration is examined.

(method)
The sample and the treatment with the organic solvent were basically the same as in Example 2.
As the organic solvent, 99.5 to 99.8% by volume of an aqueous solution of ethanol, an aqueous solution of methanol, and an aqueous solution of 2-propanol were used.
Ten cocoons were used in each experiment.

(result)
The results are shown in Figure 2.
Figure 2 is a graph showing the amount of water absorbed by the cocoon shell when treated with different organic solvents. Regardless of which organic solvent was used, good water absorption of 1.4 g/cocoon was observed. Furthermore, there was no significant difference in the amount of water absorbed depending on the type of solvent.

In addition, in a simple cocoon layer penetration test using cocoon pieces, organic solvents (ethyl acetate, acetone, N,N-dimethylformamide, and acetonitrile) and organic acids (acetic acid and propionic acid) were found to penetrate the cocoon layer well and were replaced by water.

When chloroform and isopentyl alcohol were used as organic solvents, the organic solvents penetrated the cocoon layer well, but replacement with water was difficult, and they had to be removed by washing with a water-soluble organic solvent (ethanol, etc.).

This shows that any organic solvent can promote water penetration into the cocoon shell, regardless of its type.

Example 4. Effect of exposure to organic solvents on functional regions in silk threads.
(the purpose)
The cocoons containing the functional regions are exposed to organic solvents to examine the effect of organic solvents on the functional regions.

(method)
As samples, we used dried cocoons obtained from silkworms that spin silk containing green fluorescent protein as a functional region in the fibroin H chain, and as controls, we used dried cocoons of a normal variety (variety: Japan 137 × China 146) that does not contain the functional region.

Silkworms expressing fibroin fused with GFP or Azami Green (background strains: GFP: Gunma x 200; Azami Green: Nihon no. 604 x Chu no. 511) were used as green fluorescent proteins.

An 80% by volume aqueous solution of ethanol was used as the organic solvent.
The cocoons were exposed to the organic solvent by immersing them in an 80% ethanol solution for 30 seconds, then leaving them to dry naturally for a day after immersion. This allowed the cocoons to be exposed to the ethanol until the ethanol had completely evaporated from the cocoon shell and the cocoon cavity.

In addition, some cocoons containing Azami Green as a functional region were boiled in the usual way without being treated with an organic solvent. The boiling process was carried out in the usual way using a VP type vacuum boiling machine (Harada Co., Ltd.).

Control cocoons, cocoons that were not exposed to the above-mentioned exposure treatment, and treated cocoons were observed under bright field.
We also observed the fluorescence of the cocoons in the dark using a blue LED as excitation light.

(result)
The results are shown in Figures 3 and 4.
3 is a diagram showing the appearance of cocoons containing Azami Green as a functional region when they were subjected to organic solvent treatment. The intensity of fluorescence observed did not change regardless of whether or not they were exposed to ethanol.

On the other hand, as shown in Figure 4, when normal cocoon boiling treatment was performed (Figure 4, right column), the fluorescence of Azami Green was lost compared to when no cocoon boiling treatment was performed (Figure 4, left column).

Similarly, no effect of organic solvents was observed when cocoons containing GFP as the functional region were used.

From the above, it was found that even when using cocoons containing functional regions whose activity may be lost by normal cocoon boiling, it is possible to prepare cocoons that can be reeled without losing their activity.

Example 5. Effect of organic acid treatment on functional regions and improvement of binding efficiency
(the purpose)
We investigate the effect of treatment with organic acids on the functional regions in silk threads and on the efficiency of stringing cocoons.

(method)
The samples used were dried cocoons obtained from silkworms that spin silk containing Azami Green as a functional region in the fibroin H chain, and dried cocoons of a normal variety (variety: Gunma 200) that does not contain the functional region.

As the organic acid, a 99.7% by volume aqueous solution of acetic acid was used.
For the cocoons containing Azami Green, the treatment was carried out in the same manner as in Example 4, except that a 99.7% by volume aqueous solution of acetic acid was used as the organic solvent, and the effect of the acetic acid treatment on the cocoons was examined.

For normal varieties of cocoons (10 cocoons), the process was carried out in the same manner as in Example 1, except that a 99.7% by volume aqueous solution of acetic acid was used as the organic solvent, and the stringing efficiency was measured.

(result)
The results are shown in Figure 5.
5 is a diagram showing the appearance of cocoons containing Azami Green as a functional region when the cocoons were subjected to organic acid treatment using acetic acid. The intensity of the fluorescence observed did not change regardless of whether or not the cocoons were exposed to acetic acid.

Even when organic acid treatment was performed using acetic acid, an excellent retrieval efficiency of 80% was observed.

From the above, it was found that even when an organic acid such as acetic acid is used as the organic solvent, it is possible to prepare cocoons that can be reeled without impairing the activity of the functional regions.

Example 6. Shortening of cocoon boiling time using organic solvent treatment
(the purpose)
The effects of organic solvent treatment on the cocoon boiling time, reeling efficiency, and physical properties of the reeled raw silk are investigated when the cocoon boiling and cooling method is performed after the organic solvent treatment.

(method)
Dried cocoons of size 137 (Japan) and 146 (China) were used as samples.
The organic solvent treatment was carried out by immersing the cocoons in an 80% by volume aqueous solution of ethanol for 30 seconds.

The organic solvent-treated cocoons were immersed in hot water heated to 95°C for approximately 90 seconds, then transferred to warm water at 60°C and immersed for approximately 60 seconds to carry out the cocoon boiling and cooling method.

As a control group, cocoons were used that were not treated with organic solvents and were boiled in the usual manner using a VP type vacuum boiling machine (Harada Co., Ltd.).

The cocoons that had been boiled under each condition were then stringed using a string broom and reeled into silk.
The reeling rate was calculated by a reeling test based on the old cocoon inspection method (Cocoon Inspection Station Management Council, 1984).
The physical properties of raw silk obtained by reeling were measured.

(result)
The results are shown in Table 2.

Table 2 shows the reeling efficiency from cocoons that were boiled in a shortened time using organic solvent treatment and those that were boiled normally, and the physical properties of the raw silk produced by reeling. In the group that used organic solvent treatment, although the boiling time was significantly shorter (approximately 3 minutes compared to the usual approximately 15 minutes), there was no significant difference in the reeling efficiency, such as the unraveling rate and cocoon thread length, compared to those of normally boiled cocoons. Similarly, there was no difference in the physical properties of the reeled raw silk, such as the strength and elongation, depending on the boiling method.

This shows that organic solvent treatment makes it possible to boil cocoons in a significantly shorter time without compromising reeling efficiency or the physical properties of raw silk.

Claims (9)

A method for preparing a cocoon for reeling, comprising the steps of:
A contacting step of contacting the cocoons with an organic solvent and/or an organic acid having a concentration of 45% by volume or more ; and
The method includes a permeation step of permeating water or an aqueous solution into the cocoon shell and the cocoon cavity after the contact step , and
The temperature of the organic solvent and/or organic acid is 65° C. or less;
The temperature of the water or aqueous solution is 65 ° C. or less ;
Does not include high temperature heat treatment above 80℃.
The preparation method. The preparation method according to claim 1, further comprising a removal step of removing the organic solvent and/or organic acid before the penetration step. The preparation method according to claim 1 or 2, wherein the infiltration step is carried out by a dipping method. The preparation method according to claim 1 or 2, wherein the infiltration step includes a step of reducing the pressure inside the cocoon cavity. The preparation method according to claim 4, wherein the infiltration step is carried out by a low-temperature decompression method and/or a low-temperature cooling method. The preparation method according to any one of claims 1 to 5, wherein the organic solvent comprises one or more organic solvents selected from the group consisting of lower alcohols, acetone, N,N-dimethylformamide, acetonitrile, and ethyl acetate. The preparation method according to any one of claims 1 to 6, wherein the organic acid comprises acetic acid and/or propionic acid. A cocoon for reeling prepared by the preparation method according to any one of claims 1 to 7. A method for producing raw silk, comprising the steps of:
The manufacturing method includes a stringing step of stringing the cocoons for reeling obtained by the preparation method according to any one of claims 1 to 7 , and a reeling step of reeling silk based on the threads obtained in the stringing step.

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