Silk non-woven fabric and manufacturing method thereof

Abstract

The present invention relates to a silk nonwoven fabric and a method for manufacturing the same. The method for manufacturing a silk nonwoven fabric according to the present invention allows for the production of a silk nonwoven fabric with desired physical properties by extracting silk fibers from a specific layer of a silkworm cocoon. The method for manufacturing a silk nonwoven fabric according to the present invention comprises the steps of: preparing a silkworm cocoon comprising a plurality of layers; immersing the silkworm cocoon in a solution to produce a swollen silkworm cocoon; winding a silk fiber from one of the plurality of layers of the swollen silkworm cocoon to produce a silk web; and heat-pressing the silk web after wet treatment.

Description

Technology Field

[0001] The present invention relates to a silk nonwoven fabric and a method for manufacturing the same. More specifically, it relates to a silk nonwoven fabric manufactured using distinct characteristics according to the layers constituting the silkworm cocoon and a method for manufacturing the same. Background Technology

[0003] Silk is a naturally occurring biomaterial composed of fibroin and sericin. It exhibits excellent biocompatibility, biodegradability, blood compatibility, mechanical properties, and cellular compatibility, and rarely causes inflammatory reactions in the human body. Due to these biocompatible characteristics, silk has been the subject of intensive research over the past few decades in various biomedical and cosmetic applications, including wound dressings, membranes for bone-induced regeneration, drug delivery, nerve conduits, and facial mask packs.

[0004] In addition, silk has been used in various forms of recycled silk, such as electrospun webs, films, and gels, but recently, nonwoven fabrics made from natural silk are attracting the attention of researchers due to their excellent mechanical properties, the ability to produce them simply in various sizes, and the ease of mass production.

[0006] Meanwhile, the silkworm cocoon is composed of several layers with different structures and characteristics. The silk fibers contained in each layer of the silkworm cocoon may differ in fiber diameter, sericin content, etc. Because the structural characteristics and composition of silk fibers differ depending on the layer of the silkworm cocoon, there may also be differences in the biomedical performance that is expressed.

[0007] In biomedical and cosmetic applications, there is a need for silk materials with diverse and excellent performance. Therefore, studies have previously been conducted to investigate the effects of various conditions, such as pressing temperature, pressing time, winding bath temperature, air-laid method, silkworm breed, amount of rayon added, and sericin content, on the structure and properties of silk nonwoven fabrics. However, the effects of the various layers of the silkworm cocoon on silk nonwoven fabrics have not yet been investigated. Accordingly, the present invention aims to produce a silk nonwoven fabric using silk fibers contained in the various layers of the silkworm cocoon. Prior art literature

[0009] Korean Patent Publication No. 10-1869342, "Silk Nonwoven Fabric and Method of Manufacturing the Same" The problem to be solved

[0010] One objective of the present invention is to manufacture a silk nonwoven fabric capable of exhibiting target properties depending on the application.

[0011] Another objective of the present invention is to manufacture a silk nonwoven fabric having various mechanical properties and cell viability. means of solving the problem

[0013] A method for manufacturing a silk nonwoven fabric according to the present invention comprises the steps of: preparing a silkworm cocoon comprising a plurality of layers; immersing the silkworm cocoon in a solution to produce a swollen silkworm cocoon; winding a silk fiber from one of the plurality of layers of the swollen silkworm cocoon to produce a silk web; and heat-pressing the silk web after wet treatment.

[0014] In one embodiment, the plurality of layers may consist of five layers.

[0015] In one embodiment, silk fibers can be selectively wound from one of the plurality of layers to control the mechanical properties and cell viability of the silk nonwoven fabric.

[0016] In one embodiment, the diameter of the silk fibers in the plurality of layers may differ from layer to layer.

[0017] In one embodiment, the composition of the silk fibers in the plurality of layers may differ from layer to layer.

[0018] In one embodiment, the step of winding silk fibers from one of the plurality of layers of the swollen silkworm cocoon to produce a silk web may further include the step of producing a plurality of silk webs by repeating the step of producing the silk web for each of the plurality of layers, and the step of arranging the plurality of silk webs in a laminated structure.

[0019] In one embodiment, at least two of the plurality of silk webs are placed at the top and bottom of the laminated structure to control the cell viability of the silk nonwoven fabric.

[0020] In one embodiment, the wetting treatment can be performed for 1 to 30 minutes.

[0021] In one embodiment, the heat pressing can be performed at 100°C to 270°C.

[0022] In one embodiment, the heat pressing can be performed for 5 seconds to 500 seconds.

[0023] The silk nonwoven fabric according to the present invention is manufactured according to the method for manufacturing a silk nonwoven fabric according to the present invention.

[0024] In one embodiment, the silk nonwoven fabric may have one of the plurality of layers selectively wound to control mechanical properties and cell viability.

[0025] In one embodiment, the silk nonwoven fabric may have at least two of the plurality of silk webs positioned at the top and bottom of the laminated structure to control cell viability.

[0026] In one embodiment, the silk nonwoven fabric may have a breaking strength of 60 MPa to 91 MPa.

[0027] In one embodiment, the silk nonwoven fabric may have a cell viability of 150% to 184%. Effects of the invention

[0029] According to one embodiment of the present invention, a silk nonwoven fabric prepared by selectively extracting silk fibers from a silkworm cocoon layer can exhibit desired mechanical properties and cell viability.

[0030] According to one embodiment of the present invention, silk fibers are selectively extracted from a silkworm cocoon layer to produce a silk web, and a silk nonwoven fabric produced by laminating the silk web can exhibit desired mechanical properties and cell viability. Brief explanation of the drawing

[0032] Figure 1 is a schematic for manufacturing five types of silk webs and silk nonwoven fabrics using silk fibers obtained from different silkworm cocoon layers. Figure 2 is a schematic diagram showing the manufacturing process of a silk web and a silk nonwoven fabric. Figure 3 is an FE-SEM image of a silk web and a silk nonwoven fabric made from silk fibers of various silkworm cocoon layers. FIG. 4a is a graph showing the thickness of silk webs prepared using silk fibers from different silkworm cocoon layers according to Comparative Examples 1 to 5. FIG. 4b is a graph showing the thickness of silk nonwoven fabrics manufactured using silk fibers of different cocoon layers according to Examples 1 to 5. Figure 5 summarizes the amino acid composition of silk nonwoven fabrics prepared from silk fibers of various silk cocoon layers according to Examples 1 to 5. FIG. 6 is a graph showing the yellowness index of silk webs and silk nonwoven fabrics made from silk fibers of various cocoon layers according to Comparative Examples 1 to 5 and Examples 1 to 5. Figure 7a is the ATR-FTIR spectrum of a silk web prepared using silk fibers of various cocoon layers according to Comparative Examples 1 to 5. Figure 7b is the ATR-FTIR spectrum of silk nonwoven fabrics prepared using silk fibers of various cocoon layers according to Examples 1 to 5. FIG. 7c is a graph showing the ratio of molecular structures of silk webs prepared using silk fibers from various silkworm cocoon layers according to Comparative Examples 1 to 5. FIG. 7d is a graph showing the molecular structure ratio of silk nonwoven fabrics prepared using silk fibers of various cocoon layers according to Examples 1 to 5. Figure 8a shows the XRD analysis results of silk webs prepared using silk fibers from different silkworm cocoon layers according to Comparative Examples 1 to 5. Figure 8b shows the XRD analysis results of silk nonwoven fabrics prepared using silk fibers from different silkworm cocoon layers according to Examples 1 to 5. FIG. 9 is an XRD pattern of a silk web and a silk nonwoven fabric made from silk fibers of various silk cocoon layers according to Comparative Examples 1 to 5 and Examples 1 to 5. Figure 10a shows the results of measuring the moisture retention rate of silk webs prepared from silk fibers of different silkworm cocoon layers according to Comparative Examples 1 to 5. FIG. 10b is the result of measuring the contact angle of silk nonwoven fabrics made from silk fibers of different silkworm cocoon layers according to Examples 1 to 5. FIG. 11a is a DSC thermal analysis of silk webs prepared using silk fibers from different silkworm cocoon layers according to Comparative Examples 1 to 6. FIG. 11b is a DSC thermal analysis of silk nonwoven fabrics prepared using silk fibers of different cocoon layers according to Examples 1 to 6. FIG. 12a is a stress-strain curve of a silk nonwoven fabric made from silk fibers of various cocoon layers according to Examples 1 to 5. FIG. 12b shows the breaking strength of silk nonwoven fabrics made from silk fibers of various silkworm cocoon layers according to Examples 1 to 5. FIG. 12c is a graph of the elongation at break of silk nonwoven fabrics made from silk fibers of various silkworm cocoon layers according to Examples 1 to 5. FIG. 12d is a graph of the work of rupture of silk nonwoven fabrics made from silk fibers of various silkworm cocoon layers according to Examples 1 to 5. FIG. 12e is a graph of the initial Young's modulus of silk nonwoven fabrics made from silk fibers of various cocoon layers according to Examples 1 to 5. Figure 13 is a graph showing the cell viability of silk nonwoven fabrics made from silk fibers of different silkworm cocoon layers according to Examples 1 to 5. Figure 14 is a fluorescence image of the cell viability analysis of silk nonwoven fabrics prepared using silk fibers of various silk cocoon layers according to Examples 1 to 5. Specific details for implementing the invention

[0033] The terms used herein are for describing the embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used herein, "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components, steps, actions, and / or elements to the mentioned components, steps, actions, and / or elements.

[0034] As used herein, terms such as “examples,” “examples,” “aspects,” “examples,” etc., are not to be interpreted as implying that any described aspect or design is superior or more advantageous than other aspects or designs.

[0035] The terms used in the following description have been selected as common and universal in the relevant technical field, but other terms may exist depending on technological development and / or changes, conventions, preferences of the skilled technician, etc. Therefore, the terms used in the following description should not be understood as limiting the technical concept, but as illustrative terms to explain the embodiments.

[0036] In addition, in specific cases, there are terms arbitrarily selected by the applicant, and in such cases, their detailed meanings will be described in the relevant explanatory section. Therefore, the terms used in the description below must be understood not merely as their names, but based on their meanings and the content throughout the specification.

[0037] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0038] Meanwhile, in describing the present invention, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the invention, such detailed description will be omitted. Furthermore, the terminology used in this specification is used to appropriately express embodiments of the present invention, and such terminology may vary depending on the intent of the user or operator, or the conventions of the field to which the invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification.

[0040] Silkworm cocoons are natural biopolymer materials that possess diverse and complex structures and forms. Silkworm cocoons consist of a multilayer structure, and each layer can have different morphological structures and mechanical properties.

[0041] In addition, silk filaments can be extracted from silkworm cocoons, and silk filaments have a structure in which sericin wraps around two strands of fibroin.

[0043] The method for manufacturing a silk nonwoven fabric according to the present invention comprises the steps of: preparing a silkworm cocoon comprising a plurality of layers; immersing the silkworm cocoon in a solution to produce a swollen silkworm cocoon; winding a silk fiber from one of the plurality of layers of the swollen silkworm cocoon to produce a silk web; and heat-pressing the silk web after wet treatment.

[0044] The method for manufacturing a silk nonwoven fabric according to the present invention is characterized by producing a silk nonwoven fabric using only the silk fibers contained in a single layer of a silkworm cocoon. In this case, compared to a silk nonwoven fabric produced from multiple layers without distinguishing the layers and a silkworm cocoon layer separated in its shell form, mechanical properties such as high breaking strength and breaking elongation may be superior.

[0046] According to one embodiment, the plurality of layers may consist of five layers. In this specification, the outermost layer (outermost layer) that the silkworm cocoon is in contact with the external environment is named Layer 1, the outer layer (outer layer) is named Layer 2, the middle layer (middle layer) is named Layer 3, the inner layer (inner layer) is named Layer 4, and the innermost layer (innermost layer) is named Layer 5.

[0048] In the step of preparing swollen silkworm cocoons by immersing them in a solution, purified water may preferably be used as the solution. Swollen silkworm cocoons can facilitate the separation of the layers of the silkworm cocoon compared to unswollen silkworm cocoons, and can facilitate the extraction of silk fibers from the layers of the silkworm cocoon.

[0050] The step of manufacturing a silk web by winding silk fibers from one layer among the plurality of layers of the swollen silkworm cocoon can be more specifically divided into a process of extracting silk fibers from the silkworm cocoon layers and a process of winding, that is, reeling, the extracted silk fibers. In the step of manufacturing the silk web, since the characteristics of the silk fibers contained in each layer differ for each silkworm cocoon layer, the silk fibers are not wound indiscriminately from two or more layers in order to selectively utilize the desired characteristics.

[0052] According to one embodiment, the mechanical properties and cell viability of the silk nonwoven fabric can be controlled by selectively winding silk fibers from one of the multiple layers. That is, the silkworm cocoon contains different silk fibers in each layer, and these can exhibit different chemical compositions and various physical properties.

[0054] According to one embodiment, the diameter of the silk fibers in the plurality of layers constituting the silkworm cocoon may differ from layer to layer. The silk fibers constituting the silkworm cocoon layers may have the largest diameter in the outermost layer and decrease in diameter as they go toward the inner layers.

[0055] As a result, when manufacturing a silk nonwoven fabric using silk fibers of the same length, if the outer layer is used to manufacture the silk nonwoven fabric, a thick nonwoven fabric with a thickness of approximately 0.25 mm can be obtained, and if the inner layer is used to manufacture the silk nonwoven fabric, a thin nonwoven fabric with a thickness of approximately 0.15 mm can be obtained. Therefore, different silkworm cocoon layers can be selected and used depending on the thickness of the nonwoven fabric to be obtained.

[0057] According to one embodiment, the composition of the silk fibers in the plurality of layers constituting the silkworm cocoon may differ from layer to layer. That is, the sericin content and fibroin content of the silk fibers in each layer may differ. As the sericin content increases toward the outer layer of the silkworm cocoon and the diameter of the silk fibers increases, the breaking strength and breaking elongation are higher, allowing for the production of a silk nonwoven fabric that is hard and has high mechanical strength. The fibroin content may increase toward the inner layer of the silkworm cocoon.

[0058] In this regard, the amino acid composition contained in the silkworm cocoon may differ depending on the layer. Sericin, one of the components of silk fibers in the silkworm cocoon, contains a large amount of serine. In this case, the serine content may increase from the inner layer to the outer layer of the silkworm cocoon. Accordingly, the content of sericin, which contains a large amount of sericin, may also increase from the inner layer to the outer layer.

[0060] Regarding the yellowness index of the silkworm cocoon layer by layer, the yellowness index of the silk nonwoven fabric formed from it may increase as one moves from the inner layer to the outer layer. This is because the sericin content of the silkworm cocoon increases as one moves from the inner layer to the outer layer. Sericin has lower thermal stability than fibroin, so decomposition occurs even at lower temperatures, and amino acids such as serine, which are abundant in sericin, contain many hydroxyl groups (OH groups). Therefore, the more sericin and hydroxyl groups a layer contains, such as layers 1 to 3 of the silkworm cocoon, the more likely the silk nonwoven fabric is to yellow. Silk nonwoven fabrics manufactured using silk fibers from layers 4 and 5 of the silkworm cocoon may exhibit relatively less yellowing.

[0062] Regarding the layer-by-layer crystallinity of silkworm cocoons, the β-sheet crystals of sericin located on the surface of the silk nonwoven fabric can be destroyed during the heat pressing process associated with the formation of the silk nonwoven fabric. Consequently, as the β-sheet crystals on the surface of the silk nonwoven fabric decrease, the proportion of random coils may relatively increase. In terms of the layers of the silkworm cocoon, there may be a tendency for the proportion of β-sheets to increase and the proportion of random coils to decrease from the outer layer to the inner layer. As one moves toward the inner layer, the silk fibers contain more fibroin and less sericin. Fibroin exhibits greater crystallinity compared to sericin because it not only contains a large number of β-sheet crystals but is also not destroyed by heat pressing.

[0064] Regarding water retention and hydrophilicity by layer of the silkworm cocoon, water retention and hydrophilicity may increase from the inner layer to the outer layer. This is because the sericin content increases and the fibroin content decreases from the inner layer to the outer layer of the silkworm cocoon; sericin has stronger hydrophilicity and lower crystallinity than fibroin, exhibiting a higher affinity for water. Therefore, silk nonwoven fabrics manufactured using the first and second layers of the silkworm cocoon have superior water retention and hydrophilicity compared to the fourth and fifth layers, making them highly suitable for use in the fields of cosmetics and medical materials.

[0066] Regarding the mechanical properties of silkworm cocoon layers, mechanical strengths such as breaking strength, breaking elongation, and breaking work of the silk nonwoven fabric may increase as one moves from the inner layer to the outer layer. As one moves from the inner layer to the outer layer, sericin increases and highly crystalline fibroin decreases, and the adhesive effect of sericin acts more strongly than the high crystallinity of fibroin in terms of mechanical properties. Therefore, silk nonwoven fabrics produced from the first and second layers may exhibit superior mechanical strength compared to silk nonwoven fabrics produced from the fourth and fifth layers.

[0068] Regarding cell viability by layer of the silkworm cocoon, silk nonwoven fabrics prepared from layers 2 to 5, preferably layers 2 and 3, based on a cell culture time of 48 hours, can show significantly improved cell viability compared to a control group that does not use nonwoven fabric. This is because fibroin is present in a higher cell viability than sericin, and the fibroin content increases towards the inner layers. Therefore, silk nonwoven fabrics prepared using layers 2 to 5 of the silkworm cocoon have superior cell viability compared to layer 1, making them highly useful in biomedical and cosmetic applications. However, since all silk nonwoven fabrics prepared from all layers of the silkworm cocoon have a cell viability of 80% or higher, they do not exhibit cytotoxicity and can all be used in biomedical and cosmetic applications.

[0070] Various methods for manufacturing nonwoven fabrics can be conceived to utilize the diverse characteristics of silkworm cocoons in their layers as natural nonwoven fabrics. Generally, methods include carefully peeling off the layers of the silkworm cocoon to use the separated layers in their shell form, or extracting silk fibers from the silkworm cocoon to form a silk nonwoven fabric. The present invention aims to provide a method for manufacturing nonwoven fabric that can exhibit synergistic effects while utilizing the layer-by-layer characteristics of the silkworm cocoon as described above. Accordingly, in addition to the above methods, we intend to compare the effects of a method in which silk fibers are extracted by separating the silkworm cocoon into layers and the extracted silk fibers from a single layer are manufactured into a nonwoven fabric for use; a method in which silk fibers extracted from each layer are formed into a web, these webs are laminated to have desired physical properties, and then heat-pressed to form a silk nonwoven fabric with a laminated structure; and a method in which silk fibers are extracted from the silkworm cocoon as a whole without separating the layers and then formed into a silk nonwoven fabric.

[0072] According to one embodiment, the step of manufacturing a silk web by winding silk fibers from one of the plurality of layers of the swollen silkworm cocoon may further include the step of manufacturing a plurality of silk webs by repeating the step of manufacturing a silk web for each of the plurality of layers, and the step of arranging the plurality of silk webs in a laminated structure. That is, after individually manufacturing multiple silk webs by winding silk fibers layer by layer, multiple different silk webs can be laminated in various order. Subsequently, the silk webs in the laminated structure can be subjected to the same wet treatment and heat-pressing treatment to form a single nonwoven fabric having an internal laminated structure. The silk nonwoven fabric manufactured in this manner is referred to as a "composite silk nonwoven fabric" in the following examples.

[0073] The important point here is that when manufacturing a silk web, silk fibers are not extracted and wound from multiple layers at once, but rather only one silk web is produced from one layer and one silk web is produced from another layer, and then these separate silk webs are stacked so that they overlap at the top and bottom.

[0075] According to one embodiment, the cell viability of the silk nonwoven fabric can be controlled by placing at least two of a plurality of silk webs at the top and bottom of the laminated structure. The order of lamination during the lamination of silk webs may affect the physical properties of the silk nonwoven fabric. The mechanical properties and cell viability of the silk nonwoven fabric having a laminated structure can be controlled to suit the application of the silk nonwoven fabric.

[0077] In the step of wetting and heat-pressing the silk web, the wetting process is a process of supplying moisture by methods such as immersing the silk web in water or spraying water onto the silk web. Due to the wetting treatment, a swelling phenomenon occurs in which the sericin within the silk web swells. Through a series of wetting and heat-pressing treatments, adjacent silk fibers adhere to each other due to the binding effect of sericin, thereby forming a silk nonwoven fabric.

[0078] Preferably, a water spraying method can be used as the wetting treatment, and the water spraying method has the advantage of not applying external force to the nonwoven fabric. When water is sprayed, depending on one embodiment, the wetting treatment may be carried out for 1 minute to 30 minutes. If the wetting treatment is less than 1 minute, there is insufficient time for the sericin to swell, and if the wetting treatment exceeds 30 minutes, time may remain even after the sericin is completely swollen, resulting in wasted process time.

[0079] A silk web is a form in which silk fibers are uniformly arranged, and since the silk fibers in the silk web are not bonded, it is difficult to use it as a fabric exhibiting excellent mechanical properties. Therefore, a silk nonwoven fabric with improved mechanical properties is manufactured by bonding and intertwining the silk fibers through a silk web bonding process, such as a heat pressing process.

[0081] According to one embodiment, the heat pressing process can be performed at 100°C to 270°C. Preferably, it can be performed at 150°C to 220°C. If the temperature of the heat pressing process is below 100°C, the sericin acting as a binder is not sufficiently deformed by heat, making it difficult to fully express the sericin's adhesive ability to the silk fibers, and additional costs may be incurred as the time required for the heat pressing process increases due to the lower process temperature. On the other hand, if the temperature of the heat pressing process exceeds 270°C, the molecular chains of the silk fibers begin to decompose, forming a silk nonwoven fabric with degraded mechanical properties, or severe yellowing may occur, causing the silk nonwoven fabric to turn brown or black.

[0083] According to one embodiment, the heat pressing process may be performed for 5 to 500 seconds. The temperature at which the heat pressing is performed is based on 200°C. Preferably, the heat pressing may be performed for 10 to 300 seconds. Most preferably, the heat pressing may be performed for 90 seconds. If the silk web is pressed for too long at a high temperature, severe yellowing may occur in the formed silk nonwoven fabric, the porosity may decrease, and mechanical properties may deteriorate. In addition, there is a problem that additional process costs are incurred if the heat pressing is performed for too long.

[0085] The silk nonwoven fabric according to the present invention is manufactured according to the method for manufacturing a silk nonwoven fabric according to the present invention.

[0086] According to one embodiment, in the silk nonwoven fabric, one of the plurality of layers may be selectively wound to control mechanical properties and cell viability. A composite silk nonwoven fabric having a laminated structure may exhibit better mechanical properties and cell viability than a silk nonwoven fabric made from a single silkworm cocoon layer. In this case, the mechanical properties and cell viability may exhibit a synergistic effect greater than simply the sum of the characteristics expected from the selected silkworm cocoon layer in the composite silk nonwoven fabric having a laminated structure.

[0087] In particular, a silk nonwoven fabric with a laminated structure can exhibit a breaking strength stronger than the average breaking strength of the laminated silkworm cocoon layers, because this can be determined by the layer with the strongest breaking strength among the silkworm cocoon layers forming the laminated structure. Furthermore, this effect can occur simply by the presence of the layer with the strongest breaking strength, regardless of the lamination position of that layer.

[0088] According to one embodiment, the silk nonwoven fabric may have a breaking strength of 60 MPa to 91 MPa.

[0090] According to one embodiment, in the silk nonwoven fabric, at least two of the plurality of silk webs are positioned at the top and bottom of the laminated structure, so that cell viability can be controlled. That is, in the silk nonwoven fabric of the laminated structure, cell viability can be controlled to be superior to the average value because it represents the cell viability of the outermost layer rather than the average cell viability of the entire layer constituting the laminated structure.

[0091] According to one embodiment, the silk nonwoven fabric may have a cell viability of 150% to 184%.

[0093] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples.

[0095] Figure 1 is a schematic for manufacturing five types of silk webs and silk nonwoven fabrics using silk fibers obtained from different silkworm cocoon layers.

[0097] [Preparation Example 1] Silkworm cocoon

[0098] The Bombyx mori silkworm cocoons were provided by the Gangwon-do Agricultural Products Seedling Center (Chuncheon, South Korea). To minimize variations in cocoon weight and fiber content, cocoons weighing 0.7–0.8g were selected and used.

[0100] [Comparative Example 1] Silk web (1st layer)

[0101] The silkworm cocoons of Preparation Example 1 were swollen by immersing them in a distilled water bath at 85°C for 60 minutes. Afterward, the silkworm cocoons were transferred to a reeling bath at 70°C. Subsequently, one layer of silk fibers from the silkworm cocoons was wound onto a winder of a nonwoven fabric manufacturing device (SNWFM-1, Dong-A Machinery, Namyangju, South Korea) for 4.8 minutes. At this time, to create a cross angle of 30° between the silk fibers, the reeling speed and lateral speed were fixed at 66.5 cm / s and 19 cm / s, respectively. After winding one layer of silkworm cocoons from 50 silkworm cocoons, the silk fibers were cut and separated from the bobbin of the reeler to produce a silk web.

[0103] [Comparative Example 2] Silk web (2 layers)

[0104] A silk web was manufactured in the same manner as in Comparative Example 1, except that two layers of silkworm cocoons were used instead of one layer in Comparative Example 1 for winding.

[0106] [Comparative Example 3] Silk web (3 layers)

[0107] A silk web was manufactured in the same manner as in Comparative Example 1, except that three layers of silkworm cocoons were used instead of one layer in Comparative Example 1.

[0109] [Comparative Example 4] Silk web (4 layers)

[0110] A silk web was manufactured in the same manner as in Comparative Example 1, except that four layers of silkworm cocoons were used instead of one layer in Comparative Example 1.

[0112] [Comparative Example 5] Silk web (5 layers)

[0113] A silk web was manufactured in the same manner as in Comparative Example 1, except that five layers of silkworm cocoons were used instead of one layer in Comparative Example 1.

[0115] [Example 1] Silk nonwoven fabric (1 layer)

[0116] The silk web of Comparative Example 1 was moistened by spraying distilled water with a spray bottle for 10 minutes, and then compressed twice at 200°C for 10 seconds using a heat press (HK 2008-1-5, Gwangju, Korea). At this time, to prevent the silk web from adhering to the hot pressing plate, polyester nonwoven fabrics were placed above and below the silk web, respectively. After the heat pressing was completed, the polyester nonwoven fabrics were removed to produce a silk nonwoven fabric.

[0117] Figure 2 is a schematic diagram showing the manufacturing process of a silk web and a silk nonwoven fabric.

[0119] [Example 2] Silk nonwoven fabric (2 layers)

[0120] A silk nonwoven fabric was prepared in the same manner as in Example 1, except that Comparative Example 2 was used instead of Comparative Example 1 in Example 1.

[0122] [Example 3] Silk nonwoven fabric (3 layers)

[0123] A silk nonwoven fabric was prepared in the same manner as in Example 3, except that Comparative Example 3 was used instead of Comparative Example 1 in Example 1.

[0125] [Example 4] Silk nonwoven fabric (4 layers)

[0126] A silk nonwoven fabric was prepared in the same manner as in Example 1, except that Comparative Example 4 was used instead of Comparative Example 1 in Example 1.

[0128] [Example 5] Silk nonwoven fabric (5 layers)

[0129] A silk nonwoven fabric was prepared in the same manner as in Example 1, except that Comparative Example 5 was used instead of Comparative Example 1 in Example 1.

[0131] [Comparative Example 6] Silk nonwoven fabric (entire layer)

[0132] A silk web was prepared in the same manner as in Comparative Example 1, except that the entire layer of the silkworm cocoon was used instead of the single layer of the silkworm cocoon in Comparative Example 1 and the silk nonwoven fabric was prepared in the same manner as in Example 1.

[0134] [Comparative Example 7] Silkworm cocoon layer (1st layer)

[0135] The first layer of the shell was separated from the silkworm cocoon of Preparation Example 1.

[0137] [Comparative Example 8] Silkworm cocoon layer (2 layers)

[0138] The second layer of the shell was separated from the silkworm cocoon of Preparation Example 1.

[0140] [Comparative Example 9] Silkworm cocoon layer (3 layers)

[0141] The three layers of shell were separated from the silkworm cocoon of Preparation Example 1.

[0143] [Comparative Example 10] Silkworm cocoon layer (4 layers)

[0144] The four layers of shell were separated from the silkworm cocoon of Preparation Example 1.

[0146] [Comparative Example 11] Silkworm cocoon layer (5 layers)

[0147] The 5th layer of shell was separated from the silkworm cocoon of Preparation Example 1.

[0149] [Example 6] Composite nonwoven fabric (5-1-5-1-5 layers)

[0150] A silk nonwoven fabric was manufactured in the same manner as in Example 1, except that instead of Comparative Example 1 in Example 1, a silk web laminated in the order of Comparative Example 5, Comparative Example 1, Comparative Example 5, Comparative Example 1, Comparative Example 5 from bottom to top was used.

[0152] [Example 7] Composite nonwoven fabric (5-3-5-3-5 layers)

[0153] A silk nonwoven fabric was manufactured in the same manner as in Example 1, except that instead of Comparative Example 1 in Example 1, a silk web laminated in the order of Comparative Example 5, Comparative Example 3, Comparative Example 5, Comparative Example 3, Comparative Example 5 from bottom to top was used.

[0155] [Example 8] Composite nonwoven fabric (4-2-4-2-4 layers)

[0156] A silk nonwoven fabric was manufactured in the same manner as in Example 1, except that instead of Comparative Example 1 in Example 1, a silk web laminated in the order of Comparative Example 4, Comparative Example 2, Comparative Example 4, Comparative Example 2, Comparative Example 4 from bottom to top was used.

[0158] [Example 9] Composite nonwoven fabric (2-4-2-4-2 layers)

[0159] A silk nonwoven fabric was manufactured in the same manner as in Example 1, except that instead of Comparative Example 1 in Example 1, a silk web laminated in the order of Comparative Example 2, Comparative Example 4, Comparative Example 2, Comparative Example 4, Comparative Example 2 from bottom to top was used.

[0161] [Example 10] Composite nonwoven fabric (1-3-1-3-1 layer)

[0162] A silk nonwoven fabric was manufactured in the same manner as in Example 1, except that instead of Comparative Example 1 in Example 1, a silk web laminated in the order of Comparative Example 1, Comparative Example 3, Comparative Example 1, Comparative Example 3, Comparative Example 1 from bottom to top was used.

[0164] All comparative examples and embodiments were summarized as shown in Table 1 below.

[0165] form silkworm cocoon layer Comparative Example 1 Web 1st floor Comparative Example 2 Web 2nd floor Comparative Example 3 Web 3rd floor Comparative Example 4 Web 4th floor Comparative Example 5 Web 5th floor Example 1 non-woven fabric 1st floor Example 2 non-woven fabric 2nd floor Example 3 non-woven fabric 3rd floor Example 4 non-woven fabric 4th floor Example 5 non-woven fabric 5th floor Comparative Example 6 non-woven fabric 1st to 5th floors Comparative Example 7 shell 1st floor Comparative Example 8 shell 2nd floor Comparative Example 9 shell 3rd floor Comparative Example 10 shell 4th floor Comparative Example 11 shell 5th floor Example 6 Composite nonwoven fabric 5-1-5-1-5th floor Example 7 Composite nonwoven fabric 5-3-5-3-5th floor Example 8 Composite nonwoven fabric 4-2-4-2-4th floor Example 9 Composite nonwoven fabric 2-4-2-4-2nd floor Example 10 Composite nonwoven fabric 1-3-1-3-1st floor Example 11 Composite nonwoven fabric 1-5-1-5-1st floor

[0167] [Experimental Example 1] Form of silk fiber

[0168] The morphology of the silk web and silk nonwoven fabric was analyzed using a field emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Tokyo, Japan) equipped with a secondary electron detector. Prior to observation, the comparative example and the example were coated with Pt-Pd, and the acceleration voltage was set to 5.0 kV. The mean and standard deviation of the silk fiber diameter of the silk web were calculated by measuring 50 silk filaments using an image analysis program (DIMIS-PRO 2.0, Siwon Optical Technology, Anyang, South Korea).

[0169] Figure 3 shows FE-SEM images of a silk web and a silk nonwoven fabric made from silk fibers of various silkworm cocoon layers, with a magnification bar of 100 μm. Since the silk web is a fabric made by winding silk fibers extracted from silkworm cocoons with an electric winder, the silk fibers are arranged in a regular pattern. In contrast, the silk fibers of the silk nonwoven fabric became uneven after wet and hot pressing treatments. The bending and unevenness of the silk fibers in the silk nonwoven fabric is caused by the rapid removal of moisture during wet and hot pressing treatments. Referring to Table 1, it can be seen that the silk fibers of the silk web become thinner as one moves from the first layer (outermost layer) to the fifth layer (innermost layer) of the silkworm cocoon. The diameter of the silk fibers in the silk web (layer 1) was 33±4.7μm, in the silk web (layer 2) was 32±3.4μm, in the silk web (layer 3) was 31±4.6μm, in the silk web (layer 4) was 28±3.0μm, and in the silk web (layer 5) was 25±2.9μm. Through this, it can be confirmed that the diameter of the silk fibers of the silkworm cocoon decreases from the outer layer to the inner layer.

[0170] At this time, there was no significant difference between the diameter of the silk fibers constituting the silk web and the diameter of the silk fibers constituting the cocoon layer.

[0172] [Experimental Example 2] Thickness of silk web and silk nonwoven fabric

[0173] The thickness of the silk web and silk nonwoven fabric was measured at 21 points using a thickness gauge (MDC-25PXT, Mitutoyo, Kawasaki, Japan). Then, the average and standard deviation of the thickness were calculated based on the measurements.

[0174] FIG. 4a is a graph showing the thickness of silk webs prepared using silk fibers from different silkworm cocoon layers according to Comparative Examples 1 to 5. Referring to FIG. 4a, the thickness of Comparative Example 1 (1 layer) is 0.40 mm, and the thickness of Comparative Example 5 (5 layers) is 0.23 mm, and the thickness of the silk web decreased from the 1st layer to the 5th layer.

[0175] FIG. 4b is a graph showing the thickness of silk nonwoven fabrics manufactured using silk fibers from different cocoon layers according to Examples 1 to 5. Referring to FIG. 4b, the thickness of Example 1 (1 layer) is 0.25 mm, and the thickness of Example 5 (5 layers) is 0.15 mm, indicating that the thickness of the silk nonwoven fabric decreased from the 1st layer to the 5th layer. Considering a constant winding time during the manufacture of the silk web and silk nonwoven fabric for all comparative examples and examples, the tendency for the thickness of the silk web and silk nonwoven fabric to decrease from the 1st layer to the 5th layer is due to the decrease in the diameter of the silk fibers. In this case, the identical winding time implies that the length of the silk fibers is identical. The decrease in the thickness of the silk nonwoven fabric compared to the silk web is because the silk web is compressed due to the heat pressing treatment.

[0176] Figure 4c is a graph to confirm the linear correlation between the diameter of the silk fiber and the thickness of the silk web. R of the graph 2 The value was 0.99. Referring to Fig. 4c, it can be seen that the diameter of the silk fiber is proportional to the thickness of the silk web, and the diameter of the silk fiber increases from the 5th layer (inner layer) to the 1st layer (outer layer).

[0178] [Experimental Example 3] Sericin Content

[0179] The amino acid composition of the silk nonwoven fabric was analyzed using an automatic amino acid analyzer (L-8900, Hitachi, Tokyo, Japan). Each example was hydrolyzed with 6N HCl and then passed through an ion exchange column to separate the amino acids. When the amino acids separated from the column reacted with ninhydrin at a high temperature (135°C) to form a colored compound, the absorbance of the compound was measured at wavelengths of 570 nm and 440 nm.

[0180] Figure 5 summarizes the analysis of the amino acid composition of silk nonwoven fabrics prepared from silk fibers of various silk cocoon layers according to Examples 1 to 5. Referring to Figure 5, as the layers progressed from 1 to 5, the glycine content increased from 35.92% to 40.30%, and the alanine content increased from 23.78% to 27.83%. The serine content decreased from 14.19% to 11.36%, and the aspartic acid content decreased from 5.55% to 3.55%. Sericin, one of the components constituting the silk cocoon fibers, contains a large amount of serine. Therefore, as the serine content decreased as the layers progressed from 1 to 5, the sericin content decreased as the layers progressed from 1 to 5.

[0182] [Experimental Example 4] Yellowness index

[0183] To quantitatively determine the color change of the silk web and nonwoven fabric, the yellowness index was measured. The colors of the silk web and silk nonwoven fabric were investigated using the CIE 1931 color space. The CIE tristimulus (XYZ) values ​​were based on the CIE standard light source D65 using the specular component exclusion mode of a colorimeter (Konica Minolta, CR-300 Chromameter, Osaka, Japan). The yellowness index was calculated using Equation 1 below.

[0184] [Mathematical Formula 1]

[0185]

[0187] Figure 6 is a graph showing the yellowness index of silk webs and silk nonwoven fabrics prepared from silk fibers of various silkworm cocoon layers according to Comparative Examples 1–5 and Examples 1–5. Referring to Figure 6, the yellowness index of the silk nonwoven fabric is generally higher than that of the silk web. This result is due to the yellowing of the silk caused by heat treatment during the process of manufacturing the nonwoven fabric by hot pressing at high temperatures. It is known that the yellowing of silk occurs due to the loss of amino acids containing hydroxyl groups within the silk. Furthermore, both the silk web and the silk nonwoven fabric showed a tendency for the yellowness index to decrease from layer 1 to layer 5. Considering that the sericin content of the silk decreases from layer 1 to layer 5, sericin contributed more to the yellowness of the silk than fibroin. Similarly, the difference in yellowness index between the silk nonwoven fabric and the silk web was greater in layers 1, 2, and 3 than in layers 4 and 5 of the silkworm cocoon. This is because the sericin content decreases from the first floor to the fifth floor.

[0188] Sericin is thermally weaker than fibroin and has a lower decomposition temperature. Because it has a high content of amino acids containing hydroxyl groups, similar to serine, it has a greater effect on the yellowing of silk nonwoven fabrics than fibroin.

[0190] [Experimental Example 5] Degree of Crystallinity (ATR-FTIR Analysis)

[0191] The crystallinity of silk affects mechanical properties and moisture retention. The molecular morphology of silk webs and nonwoven silk fabrics was investigated using FTIR (Nicolet 380, Thermo Fisher Scientific, Waltham, MA, USA) via the ATR method (Smart iTR ZnSe). The scan range, number of scans, and resolution were 4000–650 cm⁻¹, respectively. -1 , 32 cm -1 , 8cm -1The ratio of β-sheet and random coil forms was in the amide I band (1600–1700 cm⁻¹) using the Fourier self-deconvolution fitting method of Origin 8.0 software to investigate the differences between silk webs and silk nonwovens made from silk fibers of various silk cocoon layers. -1 It was determined by deconvolution of ).

[0192] FIG. 7a shows the ATR-FTIR spectra of silk webs prepared using silk fibers from various cocoon layers according to Comparative Examples 1 to 5. (a) Layer 1, (b) Layer 2, (c) Layer 3, (d) Layer 4, and (e) Layer 5. Referring to FIG. 7a, the silk web is 1620 cm⁻¹. -1 It exhibits an IR absorption peak, which is due to the β-sheet crystal.

[0193] FIG. 7b shows the ATR-FTIR spectra of silk nonwoven fabrics prepared using silk fibers of various cocoon layers according to Examples 1 to 5. (a) Layer 1, (b) Layer 2, (c) Layer 3, (d) Layer 4, and (e) Layer 5. Referring to FIG. 7b, for the nonwoven silk, 1620 cm⁻¹ -1 IR peak and 1643 cm⁻¹ -1 Shoulder peaks (shoulders) appear, which are due to β-sheet crystals and random coil structures, respectively. This means that random coil shapes appear more frequently in silk nonwoven fabrics due to the destruction of β-sheet crystals caused by high-temperature hot pressing treatment.

[0195] To quantitatively investigate structural changes on the surface of silk webs and silk nonwoven fabrics manufactured using silk fibers from various silkworm cocoon layers, ATR-FTIR spectra capable of measuring the structure of the material surface were obtained, and then deconvolution was performed on them to calculate the molecular conformation ratio of silk.

[0196] Figure 7c is a graph showing the molecular structure ratio of silk webs prepared using silk fibers from various cocoon layers according to Comparative Examples 1 to 5. Figure 7d is a graph showing the molecular structure ratio of silk nonwoven fabrics prepared using silk fibers from various cocoon layers according to Examples 1 to 5. Referring to Figures 7c and 7d, for the silk web and silk nonwoven fabric, the proportion of β-sheets increased from layer 1 to layer 5, and the proportion of random coils decreased from layer 1 to layer 5. As the proportion of β-sheet crystals increased from layer 1 to layer 5, it can be confirmed that the materials contain more highly crystalline fibroin as they progress from layer 1 to layer 5. Furthermore, as sericin decreases from layer 1 to layer 5, β-sheet crystals also decrease; this implies that fibroin contains a larger amount of β-sheet crystals than sericin, and that fibroin is more crystallized than sericin. In Fig. 7c, the silk web shows a β-sheet crystal content of 33.6–42.8% from layer 1 to layer 5, and in Fig. 7d, the silk nonwoven fabric has a β-sheet crystal content of 27.5–34.9% from layer 1 to layer 5, so it can be confirmed that the β-sheet crystal content of the silk web is higher than that of the silk nonwoven fabric. Referring to Fig. 7d, the β-sheet crystal content of the silk nonwoven fabric made from silk fibers in layer 1 was 27.55±1.28%, layer 2 was 31.34±2.01%, layer 3 was 32.64±2.00%, layer 4 was 33.53±0.93%, and layer 5 was 34.92±2.39%. In addition, the random coil content of the silk nonwoven fabric made from silk fibers in layer 1 was 33.98±1.26%, layer 2 was 32.04±1.17%, layer 3 was 31.54±1.87%, layer 4 was 31.45±1.20%, and layer 5 was 31.42±1.97%. As confirmed by the ATR-FTIR spectrum, the β-sheet crystals of sericin, which are mainly located on the surface of the nonwoven fabric, are destroyed due to the high-temperature hot pressing treatment.

[0198] [Experimental Example 6] Degree of Crystallinity (XRD Analysis)

[0199] Unlike ATR-FTIR technology, which reflects sericin from the silk surface, X-ray diffraction (XRD) analysis can detect the entire sample by penetrating X-rays into it. Therefore, XRD analysis can be used as a complementary tool to investigate the microstructure of silk. The crystal structures of silk webs and silk nonwovens are Cu K α It was determined using a wide-angle X-ray scattering system (D8 Discover, Bruker, Karlsruhe, Germany) that uses radiation. The irradiation conditions were set to 50 kV, 1000 μA, and the measurement time was 600 seconds.

[0200] Figure 8a shows the XRD analysis results of silk webs prepared using silk fibers of different cocoon layers according to Comparative Examples 1 to 5. Figure 8b shows the XRD analysis results of silk nonwoven fabrics prepared using silk fibers of different cocoon layers according to Examples 1 to 5. Regardless of the cocoon layer, all silk webs and silk nonwoven fabrics have XRD peaks at 2θ of 8.8°, 20.7°, and 24.8° due to the presence of β-sheet crystals. Referring to Figures 8a and 8b, the XRD peak of the silk nonwoven fabric at 20.7° has a higher intensity than the XRD peak of the silk web, which means that the crystallinity is higher in the silk nonwoven fabric than in the silk web.

[0201] The result of XRD analysis showing that the crystallinity of the silk nonwoven fabric is higher than that of the silk web contradicts the FTIR measurement result of Experimental Example 5, which is a result of different detection methods. ATR-FTIR measurement focuses on the surface of the sample analyzed (i.e., corresponding to sericin in silk), whereas XRD measurement detects the entire sample analyzed (i.e., corresponding to fibroin and sericin in silk). Therefore, the ATR-FTIR result indicated that the proportion of β-sheet crystals in the silk nonwoven fabric was lower than that of the silk web due to the destruction of sericin crystals during the hot pressing treatment. On the other hand, since the crystallization of silk fibroin is induced by heat during the hot pressing treatment to overcome the destruction of sericin crystals, the overall degree of crystallinity of the silk nonwoven fabric was found to be higher than that of the silk web.

[0202] In addition, referring to Figures 8a and 8b, the XRD peaks of the silk web and silk nonwoven fabric of the 5th layer of the silkworm cocoon at 24.8° were more distinct than the XRD peaks of the silk web and silk nonwoven fabric of the 1st layer. This indicates that the crystallinity of the silk web and silk nonwoven fabric increases as you move toward the 5th layer. This is because the fibroin content of the 5th layer is higher than that of the 1st layer, and the crystallinity of fibroin is higher than that of sericin.

[0204] Figure 9 shows the XRD patterns of silk webs and silk nonwoven fabrics prepared from silk fibers of various silk cocoon layers according to Comparative Examples 1 to 5 and Examples 1 to 5. Referring to Figure 9, four XRD spots are observed at 2θ of 20.7° and 24.8° in the silk web, which is due to the straight alignment of silk fibers with an intersection angle of 30°. In the case of the silk nonwoven fabric, as confirmed in Figure 9, the XRD spots appear as blunt arc shapes because the straight alignment of the silk web bends during wetting and heat pressing treatments.

[0206] [Experimental Example 7] Water retention

[0207] The water absorption capacity of a material is one of the important performance characteristics in biomedical and cosmetic applications. To measure the moisture retention rate, a silk web was stored for 24 hours under standard conditions (20°C and 65% relative humidity). The moisture retention rate of the silk web was calculated using Equation 3 below. The dry weight of the silk web was determined using a moisture balancer (XM60, Precisa Gravimetrics, Dietikon, Switzerland).

[0208] [Mathematical Formula 3]

[0209]

[0211] Figure 10a shows the results of measuring the moisture retention rate of silk webs made from silk fibers of different silkworm cocoon layers according to Comparative Examples 1 to 5. The moisture retention rate of the silk web made from silk fibers of the first silkworm cocoon layer was measured to be 10.62±0.39%, the second layer 10.05±0.35%, the third layer 10.04±0.28%, the fourth layer 10.03±0.05%, and the fifth layer 9.75±0.18%, indicating that the moisture retention rate decreased as the layer increased to the fifth layer.

[0213] [Experimental Example 8] Contact angle

[0214] Hydrophilicity of materials is one of the important performance characteristics in biomedical and cosmetic applications. The water contact angle was measured using the drop-and-go method with a contact angle meter (Dino-Lite, AM703MZT, Seoul, South Korea). The nonwoven fabrics of Examples 1–5 were 2x2 cm 2 The nonwoven fabric was cut into pieces, and 10 μL of distilled water was dropped onto each piece at room temperature. After 30 seconds, an image of the dropped distilled water was taken, and the contact angle was calculated from this.

[0215] FIG. 10b shows the results of measuring the contact angle of silk nonwoven fabrics made from silk fibers of different silkworm cocoon layers according to Examples 1 to 5. Regarding the contact angle of the silk nonwoven fabrics by layer, the first layer was measured to be 95.4±4.8°, the second layer to be 99.1±4.2°, the third layer to be 101.3±4.5°, the fourth layer to be 102.2±3.6°, and the fifth layer to be 104.4±4.6°.

[0216] The contact angle of the silk nonwoven fabric increased from layer 1 to layer 5. This result implies that the hydrophilicity of the silk web and silk nonwoven fabric decreases from layer 1 to layer 5. The change in hydrophilicity is related to changes in the composition of the silkworm cocoon layers. Specifically, the fibroin content increases and the sericin content decreases from layer 1 to layer 5 of the silkworm cocoon. Since sericin has higher hydrophilicity and lower crystallinity than fibroin, it can access water more easily, resulting in a higher moisture retention rate and a lower contact angle.

[0218] [Experimental Example 9] Thermal properties

[0219] Differential scanning calorimetry (DSC) was performed to investigate the thermal behavior of silk webs and silk nonwoven fabrics. DSC analysis was conducted using the thermal analyzer Q10 (DS25, TA Instrument, New Castle, DE, USA) at a scan rate of 10°C / min in the range of 60–270°C. The analysis was performed under a nitrogen stream of 50 mL / min.

[0220] Figure 11a shows the DSC measurement results of silk webs prepared using silk fibers from different cocoon layers according to Comparative Examples 1 to 6. Figure 11b shows the DSC measurement results of silk nonwoven fabrics prepared using silk fibers from different cocoon layers according to Examples 1 to 6. In the figure, (a) represents layer 1, (b) layer 2, (c) layer 3, (d) layer 4, and (e) layer 5. Referring to Figure 11a, the silk web exhibited a small, broad endothermic peak at approximately 220°C and a large peak at approximately 320°C. These two peaks are generated by the thermal decomposition of sericin and fibroin, respectively. Comparing Figure 11b with Figure 11a, it can be confirmed that in the case of the silk nonwoven fabric, the endothermic peak corresponding to sericin has shifted to 210°C. This result is due to the partial destruction of sericin crystals by the hot pressing treatment.

[0222] In silk webs and silk nonwoven fabrics, the cocoon layer does not significantly affect the thermal decomposition temperature of sericin and fibroin. Referring to Figures 11a and 11b, the 210°C endothermic peak of the silk nonwoven fabric and the 220°C endothermic peak of the silk web decreased from layer 1 to layer 5. This is because the sericin content of sericin decreases from layer 1 to layer 5.

[0224] [Experimental Example 10] Mechanical properties

[0225] The mechanical properties of the silk nonwoven fabric were evaluated using a universal testing machine (OTT-003, Oriental TM, Ansan, South Korea) with a load of 200 kgf and an elongation of 10 mm / min. The gauge length was 30 mm. The silk nonwoven fabrics of Examples 1 to 5 were 50 x 10 mm 2 The fabrics were cut into pieces and pre-conditioned at 20°C and 65% relative humidity. Seven nonwoven fabrics were tested for each condition, and the mean and standard deviation of the measurement results were calculated from five results after removing the maximum and minimum values. Strength was obtained as a corrected value regardless of the thickness of the nonwoven fabric, and elongation is a physical property independent of the thickness of the nonwoven fabric.

[0226] FIG. 12a is a stress-strain curve of a silk nonwoven fabric made from silk fibers of various cocoon layers according to Examples 1 to 5.

[0227] FIG. 12b shows the breaking strength of silk nonwoven fabrics made from silk fibers of various cocoon layers according to Examples 1 to 5. The breaking strength was measured as 90 MPa for layer 1, 86 ± 4 MPa for layer 2, 82 ± 11 MPa for layer 3, 70 ± 9 MPa for layer 4, and 63 ± 6 MPa for layer 5.

[0228] FIG. 12c is a graph of the elongation at break of silk nonwoven fabrics made from silk fibers of various cocoon layers according to Examples 1 to 5. The elongation at break was measured as 18.9±1.3% for layer 1, 16.5±1.2% for layer 2, 15.6±0.4% for layer 3, 13.4±0.5% for layer 4, and 11.1±0.3% for layer 5.

[0229] FIG. 12d is a graph of the work of rupture of silk nonwoven fabrics made from silk fibers of various cocoon layers according to Examples 1 to 5. The work of rupture was measured as 13.7±2.1MPa for layer 1, 11.4±0.7MPa for layer 2, 10.2±0.9MPa for layer 3, 7.3±0.6MPa for layer 4, and 5.2±0.5MPa for layer 5.

[0230] FIG. 12e is a graph of the initial Young's modulus of silk nonwoven fabrics made from silk fibers of various cocoon layers according to Examples 1 to 5. The initial Young's modulus was measured as 1560.7±209.3MPa for layer 1, 1500.9±126.3MPa for layer 2, 1538.5±283.4MPa for layer 3, 1034.5±335.3MPa for layer 4, and 1028.4±67.9MPa for layer 5.

[0231] Referring to FIGS. 12a to 12e, the breaking strength, elongation at break, and work at break decreased as the number of layers of the silk nonwoven fabric increased. Since the silk nonwoven fabric has a higher crystallinity fibroin as the number of layers increases from 1 to 5, the mechanical properties of the silk nonwoven fabric are inversely proportional to crystallinity. This is a result of the binding effect of sericin, which overwhelms the effect of crystalline fibroin. In other words, the silk nonwoven fabric is manufactured by the binding force of sericin, and since the amount of sericin increases as the number of layers of the silkworm cocoon increases from 5 to 1, a silk nonwoven fabric with improved mechanical properties can be obtained as the number of layers increases. In summary, through Experimental Example 10, it can be confirmed that the binding effect of sericin is stronger than the effect of fibroin in improving the mechanical properties of the silk nonwoven fabric.

[0233] Table 2 shows the results of measuring the fracture strength and fracture elongation of Comparative Examples 6 to 11 and Examples 1 to 10.

[0234] Breaking strength (MPa) Breaking Faith (%) Comparative Example 6 51 10.2 Comparative Example 7 45 9.2 Comparative Example 8 44 8.1 Comparative Example 9 41 7.4 Comparative Example 10 35 6.6 Comparative Example 11 32 5.4 Example 1 90 18.9 Example 2 86 16.5 Example 3 82 15.6 Example 4 70 13.4 Example 5 63 11.1 Example 6 90 14.1 Example 7 81 13.2 Example 8 86 14.0 Example 9 87 15.8 Example 10 91 17.0 Example 11 90 15.9

[0235] Referring to Table 2, as the structure progressed from Comparative Example 7 (silkworm cocoon layer, 1 layer) to Comparative Example 10 (silkworm cocoon layer, 5 layers), the breaking strength decreased from 45 MPa to 32 MPa, and the breaking elongation also decreased from 9.2% to 5.4%. When the silkworm cocoons were separated by layer and the silkworm cocoon layers were used as is, the breaking strength and breaking elongation were generally lower than when fibers were extracted from the entire structure without distinguishing the layers and made into a nonwoven fabric (Comparative Example 6).

[0236] Examples 1 to 5, in which nonwoven fabrics were manufactured from each layer of a silkworm cocoon, exhibited higher breaking strength and breaking elongation than Comparative Example 6 (nonwoven fabric, entire layer) or Comparative Examples 7 to 11, in which the silkworm cocoon layers were used as they were. Regarding breaking strength and breaking elongation, Example 1 (nonwoven fabric, 1 layer) showed the highest values ​​of 90 MPa and 18.1%. As the process moved toward Example 5 (nonwoven fabric, 5 layers), both breaking strength and breaking elongation decreased, showing values ​​of 65 MPa and 11.5%, respectively.

[0237] As in Comparative Example 6, the nonwoven fabric made using silk fibers extracted from the entire layer of a silkworm cocoon has silk fibers arranged in a disorderly manner in layers 1 to 5, and as a result, it breaks in parts with weak mechanical strength. However, as in Examples 1 to 5, the nonwoven fabric made using silk fibers extracted from specific layers exhibited higher mechanical strength due to the effect of uniformity, as the characteristics of the silk fibers are uniform.

[0238] In addition, if silk fibers from the entire layer of the silkworm cocoon are used, there are differences in the amount of sericin contained, and the distribution of sericin in the manufactured nonwoven fabric may vary, and the distribution of silk fiber diameters is not uniform, which becomes a factor that lowers mechanical strength. On the other hand, in the case of a nonwoven fabric composed of silk fibers with uniform characteristics, the mechanical strength is relatively higher because the silk fibers are arranged uniformly throughout.

[0239] In addition, both the cocoon layer and the nonwoven fabric show results where breaking strength and elongation at break increase as they approach the first layer, which is because the chemical composition and fiber diameter of the silk fibers differ depending on the cocoon layer. As it approaches the first layer, the sericin content is high and the fiber diameter is large, whereas as it approaches the fifth layer, the sericin content decreases and the fiber diameter also decreases. Since the nonwoven fabric is implemented with a stiffer structure as the diameter of the silk fibers constituting the silk nonwoven fabric increases, the breaking strength and elongation at break tend to increase in the case of the cocoon layer and the nonwoven fabric as they approach the first layer.

[0241] Meanwhile, when a nonwoven fabric was manufactured by combining a single layer of silk fibers, it exhibited various characteristics in terms of breaking strength and breaking elongation compared to when the nonwoven fabric was manufactured by combining them. The breaking strength of Example 6 (composite nonwoven fabric, 5-1-5-1-5 layers) was similar to the breaking strength of Example 1 (nonwoven fabric, 1 layer), which was 90 MPa, and the breaking elongation was 14.1%, which was similar to the average value of Example 1 (nonwoven fabric, 1 layer) and Example 5 (nonwoven fabric, 5 layers). A similar trend was observed in Examples 7 to 10, where the breaking strength of the nonwoven fabric was measured to be similar to the layer with the best breaking strength among the silkworm cocoon layers used, and the breaking elongation was measured to be similar to the average value of the layers used. In addition, the breaking strength was measured to be the best value when a single layer of silk fibers was included in the composite nonwoven fabric, as in Examples 6, 10, and 11.

[0242] From this, it can be confirmed that when manufacturing a laminated nonwoven fabric using silk fibers extracted from various cocoon layers, a synergistic effect exceeding the average value in mechanical properties is observed compared to the case where only silk fibers extracted from a single cocoon layer are used. More specifically, regarding breaking strength, if high-strength silk fibers are included regardless of the position of the cocoon layers forming the laminated structure, these silk fibers can act as pillars and contribute to the overall strength of the composite nonwoven fabric. Regarding elongation at break, it appears that no separate synergistic effect is observed, as it approaches the average elongation at break of the silk fibers forming the laminated structure.

[0244] [Experimental Example 11] Cell viability

[0245] Cell viability serves as an important characteristic of materials in biomedical and cosmetic applications. To evaluate cell viability, L929 cells were grown in RPMI1640 medium (Gibco, Billings, MT, USA) supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) antibiotic-antifungal solution. L929 cells were cultured at 37°C in a humidified 5% CO2 atmosphere. Subculture was performed twice a week once 80% confluence was observed.

[0246] Figure 13 is a graph showing the cell viability of silk nonwoven fabrics prepared from silk fibers of different silkworm cocoon layers according to Examples 1 to 5. ** indicates p < 0.01, and *** indicates p < 0.001. Referring to Figure 13, no significant difference was observed between the control group (which did not use nonwoven fabric) and the silk nonwoven fabrics after a culture time of 24 hours (h). After a culture time of 48 hours (h), the silk nonwoven fabric prepared from a single layer of silk fibers showed a cell viability similar to the control group, whereas the silk fibers from layers 2 to 5, particularly layers 2 and 3, showed a significant difference from the control group. The cell viability was measured as 164±6.6% for layer 1, 183±3.9% for layer 2, 187±5.0% for layer 3, 179±14.5% for layer 4, and 177±6.5% for layer 5.

[0248] Table 3 shows the results of measuring cell viability relative to TCP for Comparative Examples 6–11 and Examples 1–10.

[0249] Cell viability relative to TCP (based on 48 hours, %) Comparative Example 6 123 Comparative Example 7 112 Comparative Example 8 117 Comparative Example 9 119 Comparative Example 10 120 Comparative Example 11 121 Example 1 163 Example 2 181 Example 3 184 Example 4 180 Example 5 180 Example 6 183 Example 7 182 Example 8 180 Example 9 181 Example 10 163 Example 11 164

[0250] Referring to Table 3, the cell viability increased from 112% to 121% as it progressed from Comparative Example 7 (silkworm cocoon layer, 1 layer) to Comparative Example 10 (silkworm cocoon layer, 5 layers). When the silkworm cocoons were separated by layer and the silkworm cocoon layers were used as is, the overall cell viability was lower than when fibers were extracted from the entire layer without distinguishing the layers and made into a nonwoven fabric (Comparative Example 6).

[0251] Examples 1 to 5, in which nonwoven fabrics were prepared from each layer of a silkworm cocoon, showed higher cell viability than Comparative Example 6 (nonwoven fabric, entire layer) or Comparative Examples 7 to 11, in which the silkworm cocoon layers were used as they were. In Example 1 (nonwoven fabric, 1 layer), the cell viability was 163%, but in Example (nonwoven fabric, 2 layers), it increased to 181%, and up to Example 5 (nonwoven fabric, 5 layers), a similarly high cell viability was observed.

[0252] Both the silkworm cocoon layer and the nonwoven fabric showed results indicating that cell viability increases as the layer approaches the fifth layer. This is because the chemical composition and fiber diameter of the silk fibers vary by cocoon layer. While the sericin content is higher and the fiber diameter is larger as one approaches the first layer, the sericin content and fiber diameter decrease as one approaches the fifth layer. Regarding cell viability, superior characteristics are observed when fibroin is present rather than sericin; therefore, superior cell viability was observed as one moved toward the fifth layer, which has a lower sericin content. Since fibroin is insoluble while sericin is hydrophilic and partially leaches into the cell culture medium, fibroin exhibits superior cell viability compared to sericin.

[0254] Meanwhile, when a nonwoven fabric was manufactured by combining a single layer of silk fibers, it exhibited various characteristics in cell viability compared to when the nonwoven fabric was manufactured by combining them. The cell viability of Example 6 (composite nonwoven fabric, 5-1-5-1-5 layers) was measured to be 183%, similar to Example 5 (nonwoven fabric, 5 layers). A similar trend was observed in Examples 7 to 10, where the cell viability of the nonwoven fabric was measured to be similar to that of the outermost layer in the laminated structure of the composite nonwoven fabric. That is, in the case of the composite nonwoven fabric, Example 10, in which the 1st layer with the lowest cell viability was laminated on the outside, showed a cell viability of 163–164%, similar to Example 1, while Examples 6 to 9 showed a cell viability of over 180%, similar to the examples laminated on the outermost layer.

[0255] From this, it can be confirmed that when a laminated nonwoven fabric is manufactured using silk fibers extracted from various silkworm cocoon layers, a synergistic effect exceeding the average value in cell viability is observed compared to when only silk fibers extracted from a single silkworm cocoon layer are used. In the case of cell viability, since the outer (topmost and bottommost) layers corresponding to the surface of the silk nonwoven fabric come into contact with the cells, the characteristics of the silkworm cocoon layers laminated on the outside determine the cell viability.

[0257] [Experimental Example 12] Cytotoxicity

[0258] In vitro cytotoxicity tests were performed using the extraction method according to ISO 10993-5. Prior to extraction, each nonwoven fabric was sterilized with ethylene oxide gas. Extraction was performed on the nonwoven fabric (6x3 cm 2 This was performed by immersing ) in 6 mL of RPMI1640 culture medium and gently shaking at 37°C for 24 hours. The ratio of the sample surface area to the extraction vehicle volume was 3 cm² 2 It was / mL. The cytotoxicity of the sample against L929 cells was determined by applying the CCK-8 assay (Cell Counting Kit 8, Dojindo, Japan) in vitro. L929 cells were seeded into a 96-well plate at 1 x 10⁴ cells / well and cultured for 24 hours under a 5% CO₂ atmosphere at 37°C. Subsequently, the culture medium was replaced with 100 μL / well nonwoven extract. After 24 and 48 hours of incubation, the extract was discarded for CCK assay, and 100 μL of 10% (v / v) CCK-8 solution was added to each well. After incubation for 1 hour, the absorbance was measured at 450 nm. Subsequently, the cell viability of sericin was calculated using Equation 3 below.

[0259] [Mathematical Formula 3]

[0260]

[0261] Cytotoxicity was evaluated by performing fluorescent staining using the viability / apoptosis viability / cytotoxicity kit (L3224, Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. L929 cells were seeded into 24-well plates at a density of 3 x 10⁴ cells / well and cultured for 24 hours at 37°C under a 5% CO₂ atmosphere. Subsequently, the culture medium was replaced with 300 μL / well nonwoven extract. After culturing for 24 and 48 hours, the extract was discarded, and 300 μL of staining solution was added to each well. After culturing for 45 minutes, the stain was removed, and the cells were observed using a fluorescence inverted microscope (IX83, Olympus, Tokyo, Japan).

[0263] Figure 14 is a fluorescence image of the cell viability analysis of silk nonwoven fabrics prepared using silk fibers from various silk cocoon layers according to Examples 1 to 5. The white bar represents 200 μm. The cell image of the silk nonwoven fabric in Figure 14 supports the results of the CCK (Cell Counting Kit) test in Figure 13. Referring to Figure 14, more viable cells were observed as the culture period increased to 48 hours. Regardless of the cocoon layer used, all silk nonwoven fabrics showed a similar number of live cells as the control group without nonwoven fabric, suggesting that all silk nonwoven fabrics prepared with silk fibers from layer 1 to layer 5 possessed cell compatibility. Considering that the silk nonwoven fabric is composed of natural silk fibers, the excellent cell viability of the silk nonwoven fabric is consistent with the high cell viability of the silk fibers.

[0265] In this invention, five silk webs and five non-woven silk fabrics were produced by winding silk fibers obtained from five different silkworm cocoon layers, and their structural characteristics and physical properties were analyzed. As a result, the diameter of the silk fibers and the thickness of the silk non-woven fabric decreased from layer 1 to layer 5. The hydrophilicity and crystallinity of the silk webs and silk non-woven fabrics increased from layer 1 to layer 5. The silk non-woven fabric exhibited better mechanical properties as it moved from layer 5 to layer 1. Furthermore, since all silk non-woven fabrics from layer 1 to layer 5 possessed excellent cell viability, there is high potential for their application in the fields of cosmetics and biomedical science.

[0267] Meanwhile, the embodiments of the present invention disclosed in this specification and drawings are merely specific examples provided to aid understanding and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that other variations based on the technical concept of the present invention are possible in addition to the embodiments disclosed herein.

Claims

Claim 1 A method for manufacturing a silk nonwoven fabric comprising: a step of preparing a silkworm cocoon having a plurality of layers; a step of immersing the silkworm cocoon in a solution to produce a swollen silkworm cocoon; a step of winding a silk fiber from one of the plurality of layers of the swollen silkworm cocoon to produce a silk web; and a step of heat-pressing the silk web after wet treatment, wherein at least one of the diameter and composition of the silk fibers differs in each of the plurality of layers. Claim 2 A method for manufacturing a silk nonwoven fabric according to claim 1, characterized in that the plurality of layers consists of five layers. Claim 3 A method for manufacturing a silk nonwoven fabric according to claim 1, characterized by selectively winding silk fibers from one of the plurality of layers to control the mechanical properties and cell viability of the silk nonwoven fabric. Claim 4 delete Claim 5 delete Claim 6 A method for manufacturing a silk nonwoven fabric according to claim 1, wherein the step of winding a silk fiber from one of the plurality of layers of the swollen silkworm cocoon to manufacture a silk web further comprises the step of manufacturing a plurality of silk webs by repeating the step of manufacturing the silk web for each of the plurality of layers and the step of arranging the plurality of silk webs in a laminated structure. Claim 7 A method for manufacturing a silk nonwoven fabric according to claim 6, characterized in that at least two of the plurality of silk webs are placed at the top and bottom of the laminated structure to control the cell viability of the silk nonwoven fabric. Claim 8 A method for manufacturing a silk nonwoven fabric according to claim 1, characterized in that the wetting treatment is performed for 1 minute to 30 minutes. Claim 9 A method for manufacturing a silk nonwoven fabric according to claim 1, characterized in that the heat pressing is performed at 100℃ to 270℃. Claim 10 A method for manufacturing a silk nonwoven fabric according to claim 1, characterized in that the heat pressing is performed for 5 seconds to 500 seconds. Claim 11 Silk nonwoven fabric manufactured in accordance with Paragraph 1 or Paragraph 6. Claim 12 In claim 11, the silk nonwoven fabric is characterized in that one of the plurality of layers is selectively wound to control mechanical properties and cell viability. Claim 13 In claim 11, the silk nonwoven fabric is characterized in that at least two of the plurality of silk webs are positioned at the top and bottom of the laminated structure to control cell viability. Claim 14 In claim 11, the silk nonwoven fabric is characterized by having a breaking strength of 60 MPa to 91 MPa. Claim 15 In claim 11, the silk nonwoven fabric is characterized by having a cell viability of 150% to 184%.

Images