Bioplastic and manufacturing method therefor

Abstract

The present invention relates to a bioplastic and a manufacturing method therefor. The bioplastic according to the present invention can retrain antibacterial properties and biocompatibility and exhibit excellent stress relaxation behavior.

Description

Bioplastics and their manufacturing methods

[0001] The present invention relates to bioplastics and a method for producing the same.

[0002] Medical bioplastic materials used in living organisms must not only exhibit antibacterial properties and biocompatibility, but also satisfy mechanical properties such as formability, tensile strength, elastic modulus, and toughness, depending on the application. In particular, materials used in orthodontic treatment lacking stress relaxation behavior can cause patients to experience significant pain or discomfort due to the application of orthodontic force exceeding their pain threshold. Therefore, bioplastic materials exhibiting stress relaxation behavior are preferable. Furthermore, orthodontic appliances must be worn continuously for approximately 20 hours a day, excluding eating and cleaning, and must be reused repeatedly for several months. Therefore, dental plastics are exposed to the repetitive and significant loads encountered during essential oral activities. Furthermore, they must exhibit viscoelastic properties to accurately transmit orthodontic force to the teeth as tooth positions are adjusted to submillimeter levels (less than 0.6 mm). Therefore, dental bioplastic materials must be flexible, tough, and viscoelastic to ensure satisfactory dental treatment. However, bioplastics and biodegradable petrochemical plastics used in conventional orthodontic devices lack flexibility and viscoelasticity, which limits the successful orthodontic treatment effect.

[0003] Meanwhile, with the development of bioplastics, the United States generates an average of 0.5 kg of medical plastic waste per day, totaling 6 million tons annually. Of this waste, 91% is inappropriately released into the environment through landfills or incineration, degrading the environment. In particular, orthodontic treatment using orthodontic appliances involves precise tooth positioning with a series of appliances. Therefore, patients must replace their appliances with new ones every one to two weeks, inevitably generating plastic waste every week. For example, a single institution once reported collecting 11,483 pieces of orthodontic appliance-related plastic waste, weighing 117.8 kg, in just one year. Therefore, given the enormous amount of dental plastic waste generated in dental practice, bioplastics must exhibit excellent biodegradability so that they can be disposed of without harming the environment.

[0004] In response to this demand, recent studies have reported bioplastics with excellent biodegradability using materials such as cellulose, wood, food, silk fibroin, and pollen. However, the impact of bioplastic biodegradation on landfill microbial destruction has been rarely discussed. Furthermore, because dental bioplastics are used in the oral cavity, which is rich in microorganisms, microorganisms that readily grow on bioplastics can influence the characteristics of soil microbial communities when contaminated bioplastics are buried. For example, oral microorganisms can spread antibiotic resistance to soil microorganisms. Therefore, dental bioplastics must have high microbial resistance, be compostable without leaving toxic residues in landfills, and impact microbiological characteristics.

[0005] Meanwhile, silk is the second most abundant natural material after cellulose. Generally, silk refers to a fibrous protein released from a silkworm cocoon. Specifically, cocoon threads released from a silkworm cocoon are largely composed of two proteins: silk sericin and silk fibroin. Specifically, it contains approximately 75% fibroin protein, approximately 25% sericin protein, and approximately 3% minerals and carbohydrates. To expand the practicality of silk, it goes through a degumming process that removes impurities and sericin using alkali, soap, enzymes, and bleach, and separates pure fibroin protein.

[0006] Silk fibroin, a known protein material, boasts exceptional toughness and is utilized in various material industries. Specifically, it is used as a suture material and as a scaffold for cell therapy. These properties can be explained by examining the secondary protein structure of silk fibroin. Specifically, the higher the content of beta-sheet crystals, which possess strong internal forces, the better it can withstand external forces, resulting in high tensile strength. However, as the content of beta-sheet crystals increases, the amorphous content decreases, resulting in reduced flexibility, increased breakage, and poor stretchability.

[0007] Therefore, there is a need for research and development on bioplastic materials that have excellent tensile strength due to high beta-sheet crystal content, while also exhibiting excellent ductility, excellent stress relaxation behavior, and antibacterial and biocompatibility properties.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Patent Document 1. Korean Patent Publication No. 10-2023-0174848

[0011] The present invention has been devised to solve the above-mentioned problems, and the purpose of the present invention is to provide a bioplastic comprising modified silk fibroin having an increased content of β-sheets by being modified from natural silk fibroin, and wherein the modified silk fibroin has an entangled structure.

[0012] In addition, the present invention aims to provide a biomedical molded product comprising the bioplastic.

[0013] In addition, the present invention aims to provide an orthodontic device comprising the bioplastic.

[0014] In addition, the present invention aims to provide a method for producing a bioplastic, comprising the steps of (A) injecting a natural silk fibroin solution into a urethane oligomer casting mold; and (B) drying the natural silk fibroin solution to produce a bioplastic containing modified silk fibroin.

[0015] In addition, the present invention aims to provide a method for recovering silk fibroin from a bioplastic, comprising the steps of: immersing the bioplastic in an acidic solvent; and recovering silk fibroin from the acidic solvent in which the bioplastic is immersed.

[0016] In addition, the present invention aims to provide a method for remolding a bioplastic, comprising the steps of: preparing a bioplastic solution by dissolving the bioplastic and an acidic solvent; injecting the bioplastic solution into a urethane oligomer casting mold; and drying the bioplastic solution to prepare a recycled bioplastic.

[0017] One aspect of the present invention provides a bioplastic comprising modified silk fibroin having an increased content of β-sheets by being modified from natural silk fibroin, wherein the modified silk fibroin has an entangled structure.

[0018] Another aspect of the present invention provides a biomedical molded article comprising the bioplastic.

[0019] Another aspect of the present invention provides an orthodontic device comprising the bioplastic.

[0020] Another aspect of the present invention provides an orthodontic device comprising the bioplastic.

[0021] Another aspect of the present invention provides a method for producing a bioplastic, comprising the steps of: (A) injecting a natural silk fibroin solution into a urethane oligomer casting mold; and (B) drying the natural silk fibroin solution to produce a bioplastic including modified silk fibroin.

[0022] Another aspect of the present invention provides a method for recovering silk fibroin from a bioplastic, comprising the steps of: immersing the bioplastic in an acidic solvent; and recovering silk fibroin from the acidic solvent in which the bioplastic is immersed.

[0023] Another aspect of the present invention provides a method for remolding a bioplastic, comprising the steps of: preparing a bioplastic solution by dissolving the bioplastic and an acidic solvent; injecting the bioplastic solution into a urethane oligomer casting mold; and drying the bioplastic solution to prepare a recycled bioplastic.

[0024] The bioplastic according to the present invention comprises modified silk fibroin having significantly improved toughness, tensile strength, and elastic modulus compared to conventional silk fibroin, thereby exhibiting not only excellent maximum tensile strength and toughness, but also antibacterial properties and superior stress relaxation behavior. Furthermore, the bioplastic of the present invention exhibits non-sticky properties because hydrogen bonds are not broken even when exposed to water, making it easily usable in the body environment where fluids are always present, and can be widely utilized in the biomedical industry.

[0025] The effects of the present invention are not limited to those mentioned above. It should be understood that the effects of the present invention encompass all effects inferred from the following description.

[0026] FIG. 1 is a schematic diagram showing the design and advantages of a bioplastic (BECOM) according to one embodiment of the present invention.

[0027] Figure 2a shows a graph of the bio-based carbon content of BECOM manufactured in Example 1 of the present invention and native silk fibroin manufactured in Comparative Example 2.

[0028] Figure 2b is a schematic diagram schematically showing the manufacturing process and morphology of BECOM manufactured in Example 1 and native silk fibroin manufactured in Comparative Example 2 in terms of the degree of entanglement.

[0029] Figure 2c shows the chemical structure of the biomass-derived silk fibroin and urethane oligomer casting mold (template) used in Example 1.

[0030] Figure 2d shows the Kratky graph of BECOM manufactured by template regeneration in Example 1 and native silk fibroin (native renaturation) manufactured in Comparative Example 2.

[0031] Figure 2e shows the glass transition temperature (T) of BECOM (Templated) manufactured in Example 1 and natural silk fibroin (Native) manufactured in Comparative Example 2. g ) is shown.

[0032] Figure 2f shows the ultimate stress and elastic modulus of BECOM, petrochemical plastic, degradable petrochemical plastic, and bio-based bioplastic manufactured in Example 1.

[0033] Figure 2g shows the cyclic bending and torsion profiles of BECOM manufactured in Example 1.

[0034] Figure 3a is a schematic diagram schematically showing a process of performing orthodontic treatment using BECOM and PETG manufactured in Example 1 as orthodontic devices.

[0035] Figure 3b shows the results of creep-recovery response evaluation at initial activation conditions in the range of 0.3-1.0 kPa of BECOM and PETG manufactured in Example 1.

[0036] Figure 3c shows the results of a stress relaxation experiment of BECOM and PETG manufactured in Example 1.

[0037] Figure 3d is a schematic diagram showing the maxillary morphology for in silico simulation of human maxillary orthodontic surgery.

[0038] Figure 3e shows the distribution of corrective force according to the y-axis position of the corrective device using BECOM and PETG manufactured in Example 1.

[0039] Figure 3f shows the tooth displacement profile of the right maxilla after correcting the teeth using the orthodontic device using BECOM of Example 1.

[0040] Figure 3g shows the maximum residual force after orthodontic treatment using the BECOM and PETG orthodontic devices manufactured in Example 1.

[0041] Figure 4a shows photographic images of rabbit incisors at the initial stage (pre-treatment), after the occurrence of an incisal gap (midline diastema), and after orthodontic treatment using an orthodontic appliance using BECOM manufactured in Example 1 (BECOM orthodontics).

[0042] Figure 4b schematically illustrates a three-step process for orthodontic treatment of a rabbit's front teeth using an orthodontic device using BECOM manufactured in Example 1. In Figure 4b, PT indicates the post-treatment state.

[0043] Figure 4c schematically illustrates the process by which undesirable tooth movement and malocclusion occur in a control group that did not undergo orthodontic treatment with an orthodontic device.

[0044] Figure 4d shows a front view of a three-dimensional scanned tooth movement profile of a rabbit's front teeth after orthodontic treatment of the rabbit's front teeth using the BECOM orthodontic device manufactured in Example 1.

[0045] Figure 4e shows a plan view image of teeth in which malocclusion progresses due to non-orthodontic treatment with an orthodontic device.

[0046] Figure 5a is a schematic diagram schematically showing a recycling procedure based on disentanglement of BECOM manufactured in Example 1.

[0047] Figure 5b shows the antibacterial group (R) of the urethane oligomer casting mold used in Example 1. 3A ) and dye tracers (Rhodamin B, R 3B ) shows the chemical structure.

[0048] Figure 5c shows the substitution ratio (R) of the antibacterial group and dye tracer of the urethane oligomer casting mold used in Example 1. 3A / (R 3A + R 3B )) shows photographic images of BECOM manufactured in Example 1 and silk fibroin extracted in Example 2.

[0049] Figure 5d shows the BECOM prepared in Example 1, the silk fibroin extracted in Example 2, and the natural silk fibroin prepared in Comparative Example 2. 13 C shows the nuclear magnetic resonance (NMR) spectrum.

[0050] Figure 5e shows photographic images of the urethane oligomer casting mold (1 mL) extracted by recycling BECOM of Example 1, in which the substitution ratio of the antibacterial group and the dye tracer of the urethane oligomer casting mold in Example 2 was 50 mmol%, after the first and fifth extraction processes.

[0051] Figure 5f is a graph showing the relative expression intensity of inflammatory biomarkers (n=3) between silk fibroin (EPSF) extracted in Example 2 and the control group.

[0052] Figure 5g shows photographic images of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4, each having a thickness of 200 μm.

[0053] Figure 5h shows the ultimate stress graphs (n=3) of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4.

[0054] Figure 6a is a schematic diagram showing the phenomenon of microbial biofilm growth on orthodontic appliances and gingival tissue during orthodontic treatment.

[0055] Figure 6b shows a graph of the attachment behavior (n=3) of Streptococcus mutans and Staphylococcus aureus of the experimental and control groups treated with BECOM and PETG of Example 1 in vitro on human saliva medium.

[0056] Figure 6c shows a graph of the growth behavior (n=3) of Streptococcus mutans and Staphylococcus aureus in the experimental and control groups treated with BECOM and PETG of Example 1 in vitro in human saliva medium.

[0057] Figure 6d shows scanning electron microscope images (scale bar = 5.0 μm) of S. mutans and S. aureus in the experimental group treated with BECOM and PETG of Example 1 in vitro in human saliva medium.

[0058] Figure 6e shows alpha diversity metrics at the amplicon sequence variant level of human saliva biofilms formed on BECOM and PETG in Example 1.

[0059] Figure 6f shows a principal component (PC) analysis plot of amplicon sequence variants based on the Bray-Curtis dissimilarity method of human saliva biofilms formed from BECOM and PETG in Example 1. In Figure 6f, PC1 and PC2 represent the first and second coordinates, respectively.

[0060] Figure 6g shows a significantly (*p<0.05) correlation pattern for the occurrence of different taxa at the genus level in human saliva biofilms formed on BECOM and PETG in Example 1.

[0061] Figure 6h is a graph showing the number of three pathogens (LactoBacillusfermentum, Veillonella atypica, and Enterococcus faecalis) in human saliva biofilms formed on BECOM and PETG in Example 1.

[0062] Figure 7a is a schematic diagram schematically illustrating composting content based on the International Organization for Standardization (ISO) 20200-17 for BECOM prepared in Example 1 and EPSF prepared in Example 2 (total 4.6 g per sample) under thermophilic and aerobic conditions.

[0063] Figure 7b shows photographic images of BECOM manufactured in Example 1 and EPSF manufactured in Example 2 before composting (week 0) and after 8 weeks (week 8).

[0064] Figure 7c shows the initial (1st and 4th week) composting photographic images of BECOM manufactured in Example 1.

[0065] Figure 7d shows the composting rate at the 8th week of composting of BECOM manufactured in Example 1 and EPSF manufactured in Example 2.

[0066] Figure 7e shows the ACE alpha diversity matrix (metrics) of the compost soil obtained over 8 weeks of the control (EPSF) (EPSF).

[0067] Figure 7f shows the results of comparing the ACE richness of composted soil obtained after 8 weeks of composting of BECOM manufactured in Example 1 and EPSF manufactured in Example 2.

[0068] Figure 7g shows a principal coordinate analysis (PCoA) plot of the compost soil obtained after 8 weeks of composting with standard compost at week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2.

[0069] Figure 7h shows the relative abundance of the 30 most abundant species of compost soil obtained according to the composting time of BECOM manufactured in Example 1 and EPSF manufactured in Example 2.

[0070] Figure 8 shows a photographic image of a urethane oligomer casting mold manufactured in Manufacturing Example 2 of the present invention and BECOM manufactured in Example 1.

[0071] Figure 9 shows (a) the Raman spectrum and (b) the wide-angle X-ray scattering (WAXS) analysis results of BECOM (Templated renaturation) manufactured in Example 1 and natural silk fibroin (native renaturation) manufactured in Comparative Example 2.

[0072] Figure 10 shows the BECOM (4 cm) manufactured in Example 1. 2 (a) 5.0 mm min of beam shape -1 This is a graph showing (a) stress-strain curves and (b) ultimate stress, elastic modulus, and toughness when uniform tension is applied at a tensile and displacement rate.

[0073] Figure 11 shows (a) the normalized strain versus time profile of BECOM manufactured in Example 1 when a creep stress of 1.0 kPa was applied for 2 minutes, and (b) the permanent strains induced at creep stresses of 0.1 kPa, 0.3 kPa, 0.5 kPa, 1.0 kPa, and 2.0 kPa of BECOM and PETG manufactured in Example 1.

[0074] Figure 12 shows (a) the stress relaxation pattern of BECOM manufactured in Example 1 when the pre-displacements are 0.4 mm and (b) the relaxed energy determined by the upper region of the stress versus time curve of BECOM manufactured in Example 1 and PETG under tooth resistance of 0.1-0.5 mm.

[0075] Figure 13 shows the displacement of a human maxillary central incisor (U1) measured using in silico finite element analysis according to the thickness (300 μm, 500 μm, 750 μm) of the orthodontic appliance using BECOM manufactured in Example 1.

[0076] Figure 14 shows the residual force distribution after tooth position correction of the orthodontic device using BECOM and PETG manufactured in Example 1.

[0077] Figure 15 shows the transmittance according to the thickness ranging from 50 μm to 400 μm of the orthodontic device using BECOM of Example 1.

[0078] Figure 16 shows (a) the weight (n=4) of pure silk fibroin (EPSF) extracted by recycling BECOM of Example 1 and BECOM of Example 1 in Example 2, and (b) the average mass yield of pure silk fibroin (EPSF) extracted by recycling BECOM of Example 1.

[0079] Figure 17 shows photographic images of silk fibroin extracted in Example 2 according to the molar ratio of the antibacterial group and dye tracer of the urethane oligomer casting mold used in the production of BECOM in Example 1.

[0080] Figure 18 shows the extraction amount profile of the urethane oligomer casting mold according to the extraction cycle in Example 2.

[0081] Figure 19 shows the extraction cycle of the urethane oligomer casting mold extracted by recycling BECOM with a substitution ratio of 50 mmol% of the antibacterial group and dye tracer of the urethane oligomer casting mold in Example 2 (a). 1 (a) H nuclear magnetic resonance (NMR; Avance III HD 300, Bruker) spectrum) and (b) photographic image (1 mL per vial).

[0082] Figure 20 shows the FT / IR spectra of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4.

[0083] Figure 21 shows (a) 4 cm using BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4. 2 5.0 mm·min of beam-shaped specimen -1 When uniform tension was applied at a displacement rate of , stress versus strain curves (n=3) and (b) ultimate stress and elastic modulus of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4, petrochemical plastics, degradable petrochemical plastics, and bio-based bioplastics are shown.

[0084] Figure 22 shows (a) the reaction distance of fibroblast confluent monolayer when treated with negative control (NC; high-density polyethylene film), positive control (PC; 0.1% ZDEC polyurethane film) and BECOM prepared in Example 1 (n=3), (b) a graph showing cell viability after co-culturing BECOM of Example 1 and EPSF of Example 2 with human gingival fibroblasts (HGF-1), mouse fibroblasts (L929) and mouse macrophages (Raw264.7) (n=3), and (c) the relative expression level of inflammatory biomarkers by BECOM prepared in Example 1 and the control (Mock).

[0085] Figure 23 is a graph showing the relative abundance at the species level within the microbial community formed in the human saliva biofilm from BECOM and PETG of Example 1.

[0086] Figure 24 shows the activity of BECOM prepared in Example 1 against metallopeptidase 9 (MMP-9; 1 nM activity, n=4), protease XIV (1.0 U mL-1 , n=4) and collagenase IA (1.0 U mL -1 , n= 4) after immersion in the enzyme medium, the photographic images according to the immersion time and the thickness according to time are shown.

[0087] Figure 25 shows (a) the observation and (b) the Chao1 alpha diversity metric of the compost soil obtained at 0, 4, and 8 weeks of the control group.

[0088] Figure 26 shows (a) observations and (b) Chao1 alpha diversity metrics of composted soil obtained after 8 weeks of composting of BECOM prepared in Example 1 and EPSF prepared in Example 2.

[0089] Figure 27 is a graph showing the relative abundance of Serratia species in composted soil obtained after composting standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2 for 8 weeks.

[0090] Figure 28 is a graph showing the relative abundance of (a) Enterobacter species and (b) Klebsiella species in composted soil obtained after composting standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2 for 8 weeks.

[0091] Figure 29 is a graph showing the relative abundance of Pseudomonas species in composted soil obtained after composting standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2 for 8 weeks.

[0092] Figure 30 is a graph showing the relative abundance of B. licheniformis and B. paralicheniformis in composted soil obtained after 8 weeks of composting with standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2.

[0093] Figure 31 is a graph showing the abundance of E. coli in composted soil obtained after composting BECOM manufactured in Example 1 and EPSF manufactured in Example 2 for 8 weeks (p-value = 2.45E-12, FDR value = 5.32E-10).

[0094] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined solely by the scope of the claims.

[0095] In describing the present invention, if a detailed description of a related known technology is judged to unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. When "comprises," "has," "consists of," etc. are used in this specification, other parts may be added unless "only" is used. In addition, terms such as "comprises" or "has" are intended to specify the presence of a feature, number, step, component, or combination thereof described in the specification, and should not be understood to exclude the possibility of the presence or addition of one or more other features, numbers, steps, components, or combinations thereof. In addition, when a component is expressed in the singular, it includes the case of including the plural unless specifically stated otherwise. In the following specification, unless specifically stated otherwise, units used are based on weight, and for example, units of % or ratio mean weight% or weight ratio.

[0096] Additionally, the numerical ranges used herein may include lower and upper limits and all values ​​within that range, increments logically derived from the shape and width of the defined range, all doubly defined values, and all possible combinations of upper and lower limits of numerical ranges defined in different shapes. Unless otherwise specifically defined in the present specification, values ​​outside the numerical range that may arise due to experimental error or rounding of values ​​are also included in the defined numerical range.

[0097] Additionally, the term “oligomer” as used herein may mean a polymer having a weight average molecular weight of 100 to 10,000 g / mol.

[0098] In addition, the term "full width at half maximum (FWHM)" used in this specification is a term indicating the width of a function, and means the difference between two independent variable values ​​that is half the maximum value of the function.

[0099] Additionally, the term "significant or effective peak" as used herein means a peak that is repeatedly detected in substantially the same pattern without being significantly affected by the analysis conditions or the person performing the analysis.

[0100]

[0101] The bioplastic according to the present invention comprises modified silk fibroin, and the modified silk fibroin can be produced from natural silk fibroin.

[0102] The above natural silk fibroin may be extracted from one or more of silkworm silk obtained from the cocoons of silkworms such as the common silkworm that feeds on oak leaves, spider silk, which is a spider web protein secreted by spiders, seaweed silk secreted when seaweed adheres to rocks, and silkworm silk from the cocoon of Bombyx mori. Considering high productivity, it is more common to use silk fibroin protein obtained by extraction from the cocoon of Bombyx mori. In addition, according to a preferred embodiment, the above natural silk fibroin may be derived from the silkworm moth.

[0103] Silk fibroin protein is a protein with a crystalline structure added to an amorphous matrix. This crystal structure is formed by layered β-sheets formed by hydrogen bonds. The representative base sequence of silk fibroin extracted from silkworm cocoons is GAGAGS, accounting for approximately 53%, and GAGAGY, approximately 18%. A significant portion of this sequence is comprised of hydrophobic amino acids such as glycine (G) and alanine (A).

[0104] The present invention provides a bioplastic comprising modified silk fibroin having an increased content of β-sheets by being modified from natural silk fibroin.

[0105] The above-mentioned modified silk fibroin can exhibit an intertwined structure.

[0106] According to one embodiment of the present invention, the natural silk fibroin is denatured and exhibits an entangled structure, so that the content of β-sheets can increase.

[0107] According to one embodiment of the present invention, the bioplastic may contain 80% or more, preferably 83% or more, and more preferably 85 to 93% of the modified silk fibroin based on 100% of the total. The bioplastic may substantially not contain other synthetic polymers such as polyurethane, polyacrylate, and polyolefin other than the modified silk fibroin.

[0108] If the modified silk fibroin is included in less than 80% of the entire 100% of the above bioplastic, the mechanical properties may deteriorate, and if it is included in more than 93%, the content of the antibacterial urethane oligomer may be relatively low, so that the antibacterial properties may not be sufficiently exhibited.

[0109] In the present invention, the content of modified silk fibroin and antibacterial urethane oligomer is a value obtained through carbon isotope analysis results, and the unit of this content may mean dimensionless %.

[0110] According to one embodiment of the present invention, the bioplastic may further comprise a commonly known biocompatible polymer. The biocompatible polymer may be a protein, a polysaccharide, etc., and non-limiting examples thereof include collagen, hyaluronic acid, alginic acid, chitin, keratin, cellulose, polydioxanone, polycaprolactone, poly(b-hydroxybutyrate), poly(hydroxyvalerate), polyglycolic acid, polylactic acid, etc.

[0111] According to one embodiment of the present invention, the bioplastic has a spectrum of 1590 ±10 cm according to FT-IR spectroscopy. -1 Intensity of peaks in the region (I PU ) 1650 ±10 cm -1 Intensity of peaks in the region (I BIO ) may be 0 to 0.8:1, or 0 to 0.5:1, or 0 to 0.2:1. The above I PUis the intensity of the peak, which means polyurethane, and I BIO is the peak intensity of a bioplastic according to one embodiment. In other words, in the case of a bioplastic according to one embodiment, it may substantially not contain polyurethane therein.

[0112] According to one embodiment of the present invention, as shown in FIG. 9a, the bioplastic according to one embodiment has a spectrum of 1665±10 cm according to Raman spectroscopy. -1 It has a peak showing the maximum intensity in the region, and the half width of the peak is 10 to 80 cm. -1 , specifically 15 to 60 cm -1 , more specifically 20 to 50 cm -1 , or 30 to 42 cm -1 It can be 1665±10 cm above. -1 The peak in the region may represent a β-sheet formed by modification from natural silk fibroin.

[0113] According to one embodiment of the present invention, the bioplastic may have a bio-based carbon content of 80% or more, preferably 83% or more, more preferably 85% or more, and most preferably 85% or more.

[0114] The above bio-based carbon content is an indicator used to define bioplastics, and refers to the content of carbon derived from biomass-based resources, and is measured according to ASTM D 6866-22 Method B. 14 C may be calculated from radiocarbon content and modern carbon content.

[0115] According to one embodiment of the present invention, as shown in FIG. 2d, the bioplastic according to one embodiment shifts the position of the effective peak appearing in the Kratky plot to a higher q value than natural silk fibroin, q = 0.5 to 1.3 nm. -1 , Specifically, q = 0.7 to 1.1 nm -1 , More specifically q = 0.9 to 1.0 nm -1 It exhibits a valid peak, and the intensity of the valid peak can also be significantly increased compared to natural silk fibroin.

[0116] Bioplastics that exhibit a significant peak in the Kratky plot at the above-described q value have a structure in which silk fibroin is densely intertwined inside and exhibit a rigid β-sheet stacking structure, so that even when deformation is applied from the outside, they can quickly recover to their original shape and effectively disperse the applied stress.

[0117] According to one embodiment of the present invention, as shown in FIG. 9b, the bioplastic according to one embodiment has a wide-angle X-ray scattering (WAXS) analysis result of 14.9 nm. -1 It exhibits an a-axis stacking peak at 17.6 nm. -1 Indicates the c-axis stacking peak, and the ratio of the a-axis stacking peak intensity to the c-axis stacking peak intensity (I a / I c ) may be 0.85 to 1.7, preferably 0.87 to 1.5, and more preferably 0.9 to 1.2.

[0118] I mentioned above a / I c Bioplastics that satisfy the range of values ​​exhibit excellent flexibility and toughness, so that no damage may occur even after repeated twisting and bending.

[0119] According to one embodiment of the present invention, the bioplastic may include an antimicrobial urethane oligomer. The antimicrobial urethane oligomer may be mixed within the bioplastic, or the antimicrobial urethane oligomer may not be mixed within the bioplastic, but may be released in small amounts from the antimicrobial urethane oligomer casting mold during the process in which the casted liquid bioplastic self-assembles into a solid, and thus exists in the bioplastic in small amounts. The content of the antimicrobial urethane oligomer may be less than 20%, preferably less than 17%, and more preferably 7 to 15%, based on 100% of the total bioplastic, but is not limited thereto. Accordingly, the surface of the bioplastic may exhibit antimicrobial properties.

[0120] If the content of the antibacterial urethane oligomer is 20% or more based on the total 100% of the bioplastic, the mechanical properties may be deteriorated, which may be undesirable, and if it is less than 7% by weight, the antibacterial properties may not be sufficiently exhibited.

[0121] According to one embodiment of the present invention, the antibacterial urethane oligomer may be prepared by reacting a polyol with an antibacterial isocyanate compound.

[0122] The above polyol may be a polyol having a weight average molecular weight of 50 to 2,000 g / mol, specifically 100 to 1,000 g / mol, more specifically 150 to 700 g / mol, and specifically may be a polyether polyol. Non-limiting examples of the above polyether polyol include a polyether polyol obtained by addition polymerizing an alkylene oxide such as ethylene oxide, propylene oxide, or butylene oxide using water, a low molecular polyol (propylene glycol, ethylene glycol, glycerin, trimethylolpropane, pentaerythritol, etc.), bisphenols (bisphenol A, etc.), dihydroxybenzene (catechol, resorcin, hydroquinone, etc.) as an initiator. Specifically, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, etc. can be used.

[0123] In particular, when the polyol is a polyether polyol, the ether group of the polyether polyol can bind to the amine of the peptide backbone of the silk fibroin to induce the initial entanglement of the silk fibroin.

[0124] According to one embodiment of the present invention, the antimicrobial isocyanate compound may be used without significant limitation as long as it is a diisocyanate compound containing an antimicrobial compound, and specifically, may be a heterocyclic diisocyanate compound containing a quaternary ammonium salt. The heterocyclic diisocyanate may be a compound derived from a trimer (HMDI trimer) in which hexamethylene diisocyanate is bonded to isocyanurate, but is not limited thereto.

[0125] According to one embodiment of the present invention, the polyol and the antimicrobial isocyanate compound may be included in a molar ratio of 1.1 to 10:1, specifically a molar ratio of 1.3 to 5:1, and more specifically a molar ratio of 1.5 to 3:1. As the polyol is added in excess, the molecular weight may be controlled, and further, the antimicrobial urethane oligomer may have a hydroxyl group at the terminal, and the antimicrobial urethane oligomer has an advantage in that the molecular weight decreases when the terminal is a hydroxyl group.

[0126] According to one embodiment of the present invention, the antibacterial urethane oligomer may have a weight average molecular weight of 200 to 10,000 g / mol, specifically 500 to 5,000 g / mol, and more specifically 1,000 to 3,000 g / mol.

[0127] The bioplastic according to the present invention can realize a remarkable improvement in mechanical properties and excellent stress relaxation behavior compared to the prior art by manufacturing a bioplastic including modified silk fibroin having an improved beta-sheet content by inducing modification of the natural silk fibroin using the above-described urethane oligomer as a casting mold.

[0128] According to one embodiment of the present invention, the bioplastic may have a ratio of the beta sheet content of the modified silk fibroin to the beta sheet content of the natural silk fibroin expressed by the following formula 1 of greater than 1.5, preferably greater than 2.0.

[0129] [Formula 1] X1 / X2

[0130] (In the above equation 1, X1 is the beta sheet content of the bioplastic calculated through Raman spectroscopy, and X2 is the beta sheet content of natural silk fibroin.)

[0131] A bioplastic having a ratio of the beta-sheet content of the modified silk fibroin to the beta-sheet content of the natural silk fibroin expressed by the above formula 1 of more than 1.5, preferably more than 2.0, exhibits a high beta-sheet content by having the modified silk fibroin, and can implement significantly improved mechanical properties compared to the natural silk fibroin before modification. In addition, when the above formula 1 is more than 1.5, it means that the beta-sheet content of the modified silk fibroin has increased by 1.5 times or more compared to the beta-sheet content of the natural silk fibroin, and when it is more than 2.0, it may mean that the beta-sheet content has increased by 2.0 times or more, and further, it may increase by 2.2 times or more, or 2.2 to 10 times, or 2.5 times or more, or 2.5 to 5 times.

[0132] According to one embodiment of the present invention, the modified silk fibroin may have a beta-sheet content of 20 to 80%, preferably 30% or more, more preferably 40% or more, and even more preferably 45 to 80%, as calculated through Raman spectroscopy. The bioplastic according to the present invention is produced through a simple production method, and can exhibit significantly improved beta-sheet content and high crystallinity, and can also realize excellent mechanical properties such as toughness, tensile strength, and elastic modulus.

[0133] According to one embodiment of the present invention, the bioplastic is a transparent, hard, rigid plastic, and exhibits excellent mechanical properties such as toughness, tensile strength, toughness, and elastic modulus, and at the same time exhibits excellent stress relaxation behavior, so that when used in an orthodontic device, it can exhibit excellent orthodontic power.

[0134] According to one embodiment of the present invention, the bioplastic may have a glass transition temperature of 220°C or higher, 220°C to 320°C, 240°C to 300°C, or 255°C to 300°C.

[0135] It was confirmed that when the glass transition temperature of the bioplastic satisfies the above range, a dense and well-entangled structure of silk fibroin within the bioplastic is formed.

[0136] The above glass transition temperature was measured using differential scanning calorimetry (DSC) at 5 to 20 ℃ min. -1 It may be measured by the heating rate.

[0137] According to one embodiment of the present invention, the bioplastic may have an ultimate stress of 10 MPa or more, more preferably 12 MPa or more, and even more preferably 15 to 20 MPa.

[0138] According to one embodiment of the present invention, the bioplastic may have an elastic modulus (E) measured according to ASTM D882 of 500 MPa or more, preferably 550 MPa or more, and more preferably 600 to 700 MPa.

[0139] According to one embodiment of the present invention, the bioplastic may have a toughness (T) measured according to ASTM D882 of 300 MJ / ㎥ or more, preferably 400 MJ / ㎥ or more, more preferably 500 MJ / ㎥ or more, still more preferably 800 to 5,000 MJ / ㎥, and most preferably 1,000 to 3,000 MJ / ㎥.

[0140] The ultimate stress, elastic modulus (E) and toughness (T) are 400 μm and 4 cm thick, respectively. 2 It may be measured from a beam-shaped specimen.

[0141] A bioplastic according to one embodiment of the present invention satisfies all of the above-described ultimate stress, tensile strength, and elastic modulus, and at the same time, overcomes the limitations between the two properties by improving both tensile strength and ductility, which were previously in conflict, and has the advantage of exhibiting excellent maximum tensile strength and toughness as well as antibacterial properties and excellent stress relaxation behavior.

[0142] According to one embodiment of the present invention, the bioplastic has a maximum strain (ε) to failure measured according to ASTM D882 at a width of 0.5 cm, a length of 2 cm, and a thickness of 400 μm. max ) can be 40 to 200%, preferably 60 to 100%.

[0143] According to one embodiment of the present invention, the bioplastic may have a stress relaxation rate expressed by the following equation 2 of 25% or more, preferably 50% or more.

[0144] [Formula 2] (F0-F 30m ) / F0x 100

[0145] (In the above equation 2, when a stress relaxation test is performed on a bioplastic with a thickness of 500 ㎛ under a strain condition of 2 to 3%, F0 is the initial stress, and F 30m is the stress after 30 minutes.)

[0146] Bioplastics that satisfy the stress relaxation rate expressed by the above formula 2 of 25% or more, preferably 50% or more, can exhibit excellent stress relaxation behavior, and in particular, when applied to an internal orthodontic device, have the advantage of minimizing pain and discomfort in patients while exhibiting excellent orthodontic power.

[0147] According to one embodiment of the present invention, the bioplastic has the advantage of being compostable and can be disposed of in an environmentally friendly manner by composting without using a method that causes environmental pollution, such as incineration, after the bioplastic is completed.

[0148] According to a preferred embodiment of the present invention, the bioplastic may have a composting rate expressed by Equation 3 below of 60% or more, preferably 65% ​​or more, and more preferably 70% or more when composted according to ISO 20200-17.

[0149] [Formula 3] (CM0-CM8) / CM0X 100

[0150] (In the above equation 3, CM0 means the weight of bioplastic before composting, and CM8 means the weight of bioplastic after 8 weeks of composting.)

[0151] According to one embodiment of the present invention, the bioplastic can increase beneficial microorganisms in the soil compared to the initial soil when composted.

[0152] The above soil-beneficial microorganisms may refer to microorganisms that dissolve nutrients combined in compost, mediate enzymatic reactions to ultimately help plant growth, and exhibit the effects of compost maturation, phosphorus dissolution, and nitrogen fixation through microbial metabolism, and specifically, may refer to one or more species selected from the group consisting of Serratia sps, nitrifying bacteria, Pseudomonassps, Enterobacter sps, Klebsiella sps, B. licheniformis, and B. paralicheniformis.

[0153] According to a preferred embodiment of the present invention, the bioplastic can increase Bacillus subtilis in soil by 3 times, preferably 4 times, more preferably 5 times, even more preferably 6 times, and most preferably 7 times or more compared to the initial level when composted for 8 weeks or more according to ISO 20200-17.

[0154] Bioplastic according to one embodiment of the present invention can improve crop productivity by increasing beneficial microorganisms in the soil as described above.

[0155] According to a preferred embodiment of the present invention, the bioplastic can increase the pH of soil compared to the initial pH when composted according to ISO 20200-17. More specifically, when the bioplastic is composted for 8 weeks or more, the pH of soil can be 7 or higher, preferably 7.5 or higher, and even more preferably 8 or higher.

[0156] According to a preferred embodiment of the present invention, the bioplastic can reduce the C / N ratio in soil compared to the initial level when composted according to ISO 20200-17. More specifically, when the bioplastic is composted for 8 weeks or more, the C / N ratio in soil can be 13.7%, preferably 13.5% or less.

[0157] The bioplastic according to one embodiment of the present invention has the advantage of being able to provide soil that can significantly increase crop productivity by increasing the pH of standard compost and reducing the C / N ratio as described above.

[0158] According to a preferred embodiment of the present invention, the bioplastic can reduce the abundance of E. coli in soil when composted according to ISO 20200-17. More specifically, when the bioplastic is composted for 8 weeks or more, the abundance of E. coli in soil can be less than 5%, preferably less than 1%.

[0159] According to one embodiment of the present invention, the bioplastic can significantly reduce the possibility of cross-contamination during movement and transportation of the bioplastic by reducing the abundance of E. coli in the soil after composting as described above.

[0160] According to one embodiment of the present invention, the bioplastic can exhibit an antibacterial effect against one or more oral disease or periodontal disease causing bacteria selected from the group consisting of Streptococcus mutans and Staphylococcus aureus.

[0161] Bioplastics that exhibit antibacterial effects against the above-mentioned oral disease or periodontal disease-causing bacteria significantly inhibit the growth of microbial communities in saliva, and thus can prevent microbial growth during the orthodontic treatment period even when used as orthodontic devices.

[0162]

[0163] The present invention can provide a biomedical molded article comprising the bioplastic described above. The molded article may be in one or more forms selected from the group consisting of a film, a sheet, a mesh, a mat, a non-woven mat, a scaffold, a tube, a block, an implant, a foam, a needle, and a freeze-dried product, but is not limited thereto. However, considering the characteristics of the manufacturing method of the bioplastic according to one embodiment, a fiber form is not preferred due to the difficulty in spinning.

[0164] In addition, the bioplastic maintains internal hydrogen bonds due to its high crystallinity even when exposed to water, thus preventing stickiness between materials. Therefore, the molded product does not deteriorate in shape or usability even when inserted into the body, making it suitable for use as a biomedical material.

[0165]

[0166] The present invention can provide an orthodontic device comprising the aforementioned bioplastic. The orthodontic device can be manufactured in the structure of a conventionally used or known orthodontic device (aligner) or orthodontic retainer. For example, it can be a mouthpiece-type structure as described in Patent No. 10-1476715, or a Hawley, Wraparound, Begg, VFR vacuum-formed structure, etc., but is not limited thereto.

[0167] As shown in the above Fig. 3a, in the general process of orthodontic treatment using plastic as an orthodontic device, a series of orthodontic devices are designed to correct the position and alignment of teeth, and these aligning devices are stretched and then restored to their original shape to exert orthodontic force. At this time, the teeth may resist the orthodontic force, which may cause distortion or damage to the orthodontic device. Therefore, the orthodontic device must be able to transmit sufficient recoverable orthodontic force to the teeth while withstanding the resistance of the teeth to ensure successful treatment. The bioplastic described above not only sufficiently withstands the resistance of the teeth, but also exhibits excellent viscoelastic properties, making it suitable for use as an orthodontic device.

[0168]

[0169] Hereinafter, a method for manufacturing a bioplastic according to one embodiment of the present invention will be described in more detail.

[0170] The present invention can provide a method for producing a bioplastic, comprising the steps of: (A) injecting a natural silk fibroin solution into a urethane oligomer casting mold; (B) drying the natural silk fibroin solution to produce a bioplastic containing modified silk fibroin; and (C) detaching the bioplastic from the mold.

[0171] (A) Step of injecting a natural silk fibroin solution into a urethane oligomer casting mold

[0172] The above step (A) is a step of injecting a natural silk fibroin solution into a urethane oligomer casting mold.

[0173] According to one embodiment of the present invention, the urethane oligomer casting mold may be manufactured through a step including: (A-1) a step of preparing a polymerizable composition by mixing a polyol and an antimicrobial isocyanate compound; and (A-2) a step of casting the polymerizable composition into a mold and then reacting it.

[0174] The above isocyanate compound refers to a compound containing an isocyanate group, and may preferably be a cyclic diisocyanate having two or more isocyanate groups.

[0175] The above antibacterial isocyanate compound can be prepared by substituting an antibacterial compound for an isocyanate compound, and for example, a reactive compound containing a quaternary ammonium salt can be reacted with an isocyanate compound to ultimately prepare an antibacterial isocyanate compound containing a quaternary ammonium salt, but is not limited thereto.

[0176] Specifically, the isocyanate compound may be a heterocyclic triisocyanate compound, and more specifically, the heterocyclic triisocyanate may be a trimer (HMDI trimer) in which hexamethylene diisocyanate is bonded to isocyanurate, but is not limited thereto. In addition, the reactive compound containing the quaternary ammonium salt may include a functional group such as amine or hydroxy at the terminal, and the functional group and the isocyanate of the above-described isocyanate compound may react to produce an antibacterial isocyanate compound containing a quaternary ammonium salt.

[0177] Examples of specific compounds of the above polyols are the same as those described above and are therefore omitted.

[0178] The above polyol and antimicrobial isocyanate compound may be mixed, the polymerizable composition may be stirred with a mixer, and then cast into a mold of a specific shape and reacted. The reaction may be applied without significant limitations under the usual and known conditions for urethane reaction, and may be reacted at a temperature of 30°C or higher, specifically 50°C or higher, for 5 hours, specifically 10 hours or longer. Furthermore, a drying step may be further performed, and drying may be performed at a temperature of 70°C or higher for 1 hour or longer, but is not limited thereto.

[0179] According to one embodiment of the present invention, in the step (A), the natural silk fibroin solution may be a natural silk fibroin protein and a kosmotropic salt dissolved in an acidic solvent. Specifically, the natural silk fibroin protein may be contained in an amount of 0.1 to 50% (w / v), specifically 0.5 to 30% (w / v), with respect to the acidic solvent, and the kosmotropic salt may be contained in an amount of 0.01 to 20% (w / v), specifically 0.1 to 10% (w / v) with respect to the acidic solvent.

[0180] The above refined natural silk fibroin protein may be obtained by soaking silkworms in a refining solution.

[0181] The refining solution may be an alkaline aqueous solution, an acidic aqueous solution, an enzyme aqueous solution, an amine aqueous solution, or a mixed solvent thereof, and may preferably be a sodium carbonate solution, but is not limited thereto.

[0182] The above acidic solvent is C 1-7It may be an organic acid. Examples thereof include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, fumaric acid, malic acid, citric acid, or a mixture thereof.

[0183] The above kosmotropic salt is known to contribute to the stabilization of water molecules in an aqueous environment and also to the stabilization of intramolecular interactions in macromolecules such as proteins. The above kosmotropic salt is an alkali metal ion or an alkaline earth metal ion; and sulfate (SO4 2- ), phosphate (HPO4 2- ), acetate (CH3COO - ), hydroxide (OH - ), chloride (Cl - ), bromide (Br - ) and formate (HCOO - ) and the like. Preferably, the cosmotropic salt may be one or more ions selected from the group consisting of calcium chloride, lithium bromide, potassium chloride, sodium chloride, and sodium acetate, and may be preferably calcium chloride, but is not limited thereto.

[0184] According to one embodiment of the present invention, the step (A) is performed by preparing the above-described natural silk fibroin solution and injecting it into a urethane oligomer casting mold. Depending on the shape to be prepared, the shape of the casting mold may vary, and accordingly, the amount of the solution to be injected may also vary.

[0185] (B) A step of drying the natural silk fibroin solution to produce a bioplastic containing modified silk fibroin.

[0186] The above step (B) is a step of manufacturing a bioplastic containing modified silk fibroin by drying the natural silk fibroin solution.

[0187] In the step (B), the drying is performed at room temperature, preferably in a vacuum, for 12 hours or more, preferably 24 hours or more, and during the drying, the natural silk fibroin protein contained in the natural silk fibroin solution may be crystallized to produce denatured silk fibroin. Specifically, the denatured silk fibroin may exhibit a structure in which silk fibroin is entangled with each other due to the influence of the urethane oligomer casting mold described above during the crystallization process, and as a result, may have a beta-sheet content significantly improved compared to the natural silk fibroin protein, thereby producing a bioplastic including denatured silk fibroin having a high crystallinity.

[0188] The above step (B) may include a step of detaching the manufactured bioplastic from the mold.

[0189] (C) Step of immersing the above bioplastic in a storage medium

[0190] According to one embodiment of the present invention, after step (B), a step (C) of immersing the bioplastic in a storage medium may be further performed. The immersion may be performed for 10 minutes or more, 30 minutes or more, or 1 hour or more, and by including this step, a bioplastic having better physical properties such as superior tensile strength can be manufactured.

[0191] According to one embodiment of the present invention, the storage solution comprises a polyol, specifically C 1-20 Alkyl polyol or C 1-7It may include an alkyl polyol. For example, the storage solution may include at least one selected from, but not limited to, ethylene glycol, diethylene glycol, triethylene glycol, 1,3-propanediol, cyclohexanedimethanol, 4-butanediol, glycerol, pentaerythritol, sorbitol, and trimethylolpropane.

[0192]

[0193] The bioplastic according to one embodiment of the present invention has the advantage of being easily recyclable. Methods for this recycling include extracting and recycling silk fibroin from the bioplastic, or re-forming it into a bioplastic using a urethane oligomer casting mold after untangling its entangled structure.

[0194]

[0195] The present invention can provide a method for recovering silk fibroin from a bioplastic, comprising the steps of: immersing the bioplastic in an acidic solvent; and recovering silk fibroin from the acidic solvent in which the bioplastic is immersed.

[0196] According to one embodiment of the present invention, the bioplastic has a structure in which silk fibroin is entangled therein. If only this entangled structure is unraveled, it is easy to separate and recover silk fibroin, and thus has the advantage of being able to recover silk fibroin from a used bioplastic.

[0197] The above acidic solvent may have the same content as the acidic solvent used in the above-described natural silk fibroin solution, and may further include a cosmotropic salt like the above-described natural silk fibroin solution.

[0198] The step of recovering the above natural silk fibroin may be performed by centrifuging or filtering the acidic solvent in which the bioplastic is immersed, wherein the silk fibroin and the urethane oligomer casting mold within the bioplastic may be separated. The recovery may be performed multiple times until the urethane oligomer casting mold is completely separated.

[0199] According to one embodiment of the present invention, the yield of the recovered silk fibroin may be 65% or more, preferably 67% or more, and more preferably 70% or more.

[0200] The above yield may be calculated by the following equation 4.

[0201] [Formula 4] (Weight of recovered natural silk fibroin / Initial weight of bioplastic) x 100

[0202] According to one embodiment of the present invention, the recovered silk fibroin can reduce the expression of one or more inflammatory biomarkers selected from the group consisting of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

[0203]

[0204] The present invention provides a method for remolding a bioplastic, comprising the steps of: preparing a bioplastic solution by dissolving the bioplastic and an acidic solvent; injecting the bioplastic solution into a urethane oligomer casting mold; and drying the bioplastic solution to prepare a recycled bioplastic.

[0205] In the method for re-forming the above bioplastic, the bioplastic solvent may further include a cosmotropic salt, and the acidic solvent, urethane oligomer casting mold, and drying may be the same as the method for producing the above-described bioplastic.

[0206] The recycled bioplastic manufactured by the above method of re-forming the bioplastic can exhibit mechanical properties equivalent to those of the bioplastic before recycling.

[0207] Hereinafter, the present invention will be described in more detail through examples, etc., but the scope and content of the present invention cannot be interpreted as being reduced or limited by the examples, etc. below.

[0208]

[0209] Manufacturing Example 1. Manufacturing of natural silk fibroin

[0210] Mori silkworm (silkworm moth) cocoons were degummed by soaking them in a sodium carbonate solution (0.02 M Na2CO3) for 1 hour. Afterwards, they were washed and dried at room temperature to obtain degummed natural silk fibroin.

[0211] Manufacturing Example 2. Manufacturing of a urethane oligomer casting mold (template)

[0212] 0.425 M diisocyanate isocyanurate trimer (hexamethylene diisocyanate isocyanurate trimer, 504.6 g / mol, BLD Pharm, China) and 0.425 M aminooxoethyl dimethyl dodecananium chloride (N-(2-Amino-2-oxoethyl)-N,N-dimethyldodecan-1-aminium chloride, 306.9 g / mol, Angenechemical, Hong Kong) were added to propylene carbonate (Sigma Aldrich, USA) and reacted at 80°C for more than 1 hour to prepare an antibacterial diisocyanate compound.

[0213] Next, 0.85 M polyethylene glycol (Mw: 200 g / mol) was added to the solution containing the antibacterial diisocyanate compound to prepare a polymerizable composition. At this time, the molar ratio of the polyethylene glycol to the antibacterial diisocyanate compound was added so as to satisfy 2:1. Then, 4 mL of the polymerizable composition was put into a polystyrene mold (Ø60 mm) and baked in an oven at 80°C for 7 days to finally prepare a urethane oligomer casting mold. The weight average molecular weight of the prepared urethane oligomer was measured to be 1211.5 g / mol, and the antibacterial group (R) in the urethane oligomer 3A ) is shown in Figure 5b.

[0214] Example 1. Manufacturing of bioplastic (BECOM)

[0215] 0.1 g mL of the refined silk fibroin of the above manufacturing example 1 was added to a solution containing 0.3 wt% of calcium chloride (CaCl2) in formic acid. -1 The solution was added to the concentration and dissolved at 60°C for 1 hour to obtain a natural silk fibroin solution.

[0216] The above natural silk fibroin solution was injected into the urethane oligomer casting mold of Manufacturing Example 2, and templated renaturation was performed by evaporating formic acid for 3 days. Then, the solidified denatured silk fibroin was separated from the mold by treating with an ethanol-deionized water co-solvent (7:3 vol%) for 1 hour.

[0217] Next, the modified silk fibroin was immersed in a glycerol-deionized water co-solvent (5:5 wt%) for 1 hour and then dried for 2 days to obtain a bioplastic (beneficially compostable medical bioplastic, BECOM). The content ratio of the modified silk fibroin and the urethane oligomer casting mold in the obtained bioplastic was 87.4:12.6.

[0218] Example 2. Recycling of bioplastics 1-Recovery of silk fibroin (EPSF)

[0219] Figure 5a is a schematic diagram schematically showing a recycling procedure based on disentanglement of BECOM manufactured in Example 1.

[0220] Figure 5b shows the antibacterial group (R) of the template used in Example 1. 3A ) and dye tracers (Rhodamin B, R 3B ) shows the chemical structure.

[0221] As shown in the above Figure 5a, in order to obtain silk fibroin from the BECOM manufactured in Example 1, the entangled structure within the BECOM was unraveled using the following method to obtain silk fibroin.

[0222] First, in order to visualize the process of recovering silk fibroin, as shown in Fig. 5b, the antibacterial group (R) of the urethane oligomer casting mold used in Example 1 3A ) is a dye tracer that shows pink color (Rhodamin B, R 3B) was partially substituted. Next, 0.5 g of BECOM prepared in Example 1 was immersed in 15 mL of formic acid at 60°C for 3 hours to disentangle the entangled structure. Subsequently, 30 mL of ethanol was added and mixed, and centrifuged at 7000 rpm at 25°C for 10 minutes to selectively precipitate silk fibroin, and then the supernatant containing the urethane oligomer casting mold was recovered. Next, the remaining silk fibroin was dispersed in 10 mL of formic acid for 10 minutes, treated with 10 mL of ethanol, and centrifuged in the same manner to separate the urethane oligomer casting mold solution, after which 10 mL of formic acid and 20 mL of ethanol were sequentially added and centrifuged again. Finally, the solution was centrifuged three times to remove the residual solvent at 80°C, and silk fibroin was obtained. The yield (weight of recovered natural silk fibroin / weight of initial bioplastic x 100) expressed as a percentage of the weight of the extracted silk fibroin with respect to the initial bioplastic weight was 72.6%. The obtained silk fibroin was pure and no residual urethane oligomer casting mold was detected, so it was named extracted pure silk fibroin (EPSF).

[0223] Example 3. Recycling of Bioplastics 2 (BECOM-R1)

[0224] As shown in the above Figure 5a, in order to manufacture bioplastic by recycling the BECOM manufactured in the above Example 1, the entangled structure within the BECOM was unraveled using the following method, and then it was manufactured into bioplastic again.

[0225] In the above Example 2, 0.5 g of BECOM prepared in the above Example 1 was immersed in 15 mL of formic acid at 60°C for 3 hours, and the formic acid was removed from the solution (pink solution in Fig. 5a) in which the entangled structure was disentangled. At this time, BECOM in which silk fibroin re-formed an entangled structure upon removal of the formic acid was obtained, and this was designated BECOM-R1.

[0226] Example 4. Recycling of Bioplastics 2 (BECOM-R2)

[0227] Pure silk fibroin (EPSF) extracted in Example 2 was added to a formic acid solution at 0.1 g mL -1 The solution was added to the concentration and dissolved at 60°C for 3 hours to obtain a natural silk fibroin solution.

[0228] The above natural silk fibroin solution was injected into the urethane oligomer casting mold of Manufacturing Example 2, and templated renaturation was performed by evaporating formic acid for 3 days. Then, the solidified denatured silk fibroin was separated from the mold by treating with an ethanol-deionized water co-solvent (7:3 vol%) for 1 hour.

[0229] Next, the modified silk fibroin was immersed in a glycerol-deionized water co-solvent (5:5 wt%) for 1 hour and then dried for 2 days to obtain recycled bioplastic (BECOM-R2).

[0230] Comparative Example 1

[0231] The refined natural silk fibroin of the above Manufacturing Example 1 was manufactured into a sample having a size of 40 x 5.0 x 0.4 ㎣ to obtain natural silk fibroin according to Comparative Example 1.

[0232] Comparative Example 2. Native silk fibroin

[0233] The natural silk fibroin solution of Example 1 was cast into a polystyrene mold (Ø60 mm), dried at room temperature for 24 hours, and the specimen was detached from the casting substrate to undergo native renaturation to obtain native silk fibroin according to Comparative Example 2.

[0234]

[0235] Experimental Example 1. Behavior Evaluation after Exposure to Water

[0236] The BECOM manufactured in Example 1 and the natural silk fibroin manufactured in Comparative Example 2 were immersed in water and the changes were observed. As a result, in the case of Comparative Example 2, when immersed in water and then overlapped, they became sticky and were not easily separated, whereas in the case of Example 1, the hydrogen bonds were not broken even when exposed to water and they were easily separated. Through this, it was confirmed that the bioplastic according to one embodiment of the present invention having a high beta-sheet content exhibits a non-sticky property because the hydrogen bonds are not broken even when exposed to water, so that it can be easily used in an environment in the body where fluids such as blood always exist, and that it can be utilized in the medical industry, such as biomedical materials that can be inserted into the body, by utilizing these characteristics.

[0237] Experimental Example 2. Evaluation of physical properties, shape, and structure

[0238] Measuring bio-based carbon content

[0239] BECOM manufactured in Example 1 and native silk fibroin manufactured in Comparative Example 2 were each prepared in an amount of 2.0 g, and analyzed using an accelerator mass spectrometer at the Korea Apparel Testing & Research Institute according to ASTM D 6866-22 Method B. 14 C Radiocarbon content and modern carbon content were measured, and bio-based carbon content was calculated based on this, and the results are shown in Fig. 2a.

[0240] Figure 2a shows a graph of the bio-based carbon content of BECOM manufactured in Example 1 of the present invention and native silk fibroin manufactured in Comparative Example 2.

[0241] As shown in the above Fig. 2a, the natural silk fibroin, which is a pure biomass resource of the above Comparative Example 2, had a bio-based carbon content of 97%, and the BECOM manufactured in the above Example 1 had a bio-based carbon content of 87%, which significantly exceeded the 80% indicator of a high-quality bioplastic indicated by the horizontal dotted line, confirming that it can be used as a high-quality bioplastic. In addition, the above Example 1 contained 87.4% of silk fibroin and 12.6% of antibacterial urethane oligomer.

[0242] Structural analysis of silk fibroin

[0243] In order to analyze the form of BECOM manufactured in Example 1, a schematic diagram schematically showing the manufacturing process and form of BECOM manufactured in Example 1 and native silk fibroin manufactured in Comparative Example 2 is shown in FIG. 2b, and the chemical structure of silk fibroin and urethane oligomer casting mold (template) used in manufacturing the same is shown in FIG. 2c.

[0244] Figure 2b is a schematic diagram schematically showing the manufacturing process and morphology of BECOM manufactured in Example 1 and native silk fibroin manufactured in Comparative Example 2 in terms of the degree of entanglement.

[0245] Figure 2c shows the chemical structure of the biomass-derived silk fibroin and urethane oligomer casting mold (template) used in Example 1.

[0246] As shown in the above FIGS. 2b and 2c, the urethane oligomer casting mold (template) used in Example 1 includes two ether groups (entanglement-inducing groups) and an antibacterial group including one quaternary ammonium salt (FIG. 2c). In Example 1, when the silk fibroin denatured in acid was injected into the urethane oligomer casting mold and regenerated through acid removal (template regeneration method), the two ether groups were attached to the peptide backbone of the silk fibroin to induce entanglement, thereby exhibiting a densely entangled structure expressed in red. On the other hand, in Comparative Example 2, it was naturally regenerated without the urethane oligomer casting mold, exhibiting loose entanglement expressed in yellow.

[0247] In order to confirm the formation of a densely entangled structure in the BECOM manufactured through the above template regeneration method, the BECOM of Example 1 and the natural silk fibroin of Comparative Example 2 were subjected to small-angle X-ray scattering (SAXS) and Raman spectrum analysis, and the results are shown in FIG. 2d and FIG. 9.

[0248] More specifically, using beamline 9A of the Pohang Light Source (PLS) in Korea, the synchrotron X-ray beam has a wavelength of 0.626 ㅕ (X-ray energy = 19.8 keV) and a typical beam size of 0.8 x 0.8 mm. 2 The X-ray scattering experiment was performed by setting the detector to , acquiring the scattered beam intensity using a 2D Mar CCD detector (SX 165, Rayonix LLC, USA), and performing SAXS measurements at a sample-to-detector distance of 2.5 m and an exposure time in the range of 10-30 s.

[0249] Figure 2d shows the Kratky graph of BECOM manufactured by template regeneration in Example 1 and native silk fibroin (native renaturation) manufactured in Comparative Example 2.

[0250] Figure 9 shows (a) the Raman spectrum and (b) the wide-angle X-ray scattering (WAXS) analysis results of BECOM (Templated renaturation) manufactured in Example 1 and natural silk fibroin (native renaturation) manufactured in Comparative Example 2.

[0251] As shown in the above Figure 2d, in the Kratky plot, Example 1 and Comparative Example 2 each have q=0.958 nm -1 and q=0.187 nm -1 By showing the effective peak in , it can be seen that the effective peak position of Example 1 moves to a higher q (scattering vector) value than that of Comparative Example 2. In addition, the intensity of the effective peak is correlated with the degree of protein compression and entanglement, and the intensity of the effective peak in Example 1 is higher than that of Comparative Example 2 (I(q), 1.54×), confirming that a densely entangled structure is formed in BECOM through the template regeneration method and the protein is compressed to have a more solid β-sheet stacking.

[0252] As shown in the above Figure 9a, in the case of Example 1, 1663 cm corresponding to the beta sheet -1 It has a peak showing the maximum intensity in the region, and the half width of the peak is 32.5 cm. -1 and 1663 cm -1 It was confirmed that the intensity of the peak in the region was about 4 times higher than that of Comparative Example 2. In addition, in the Raman spectrum, 1665±10 cm corresponding to the β-sheet structure -1 As a result of calculating the β-sheet content using the Raman signal, the β-sheet content was 51.8%, 18.0%, and 28.7% in Example 1, Comparative Example 1, and Comparative Example 2, respectively, confirming that Example 1 had at least 1.80 times more β-sheet crystals than Comparative Example and exhibited more robust β-sheet stacking.

[0253] In addition, as shown in the above Fig. 9b, the WAXS analysis results showed that 17.6 nm -1 14.9 nm for the intensity of the c-axis stacking peak appearing in -1 The intensity ratio of the a-axis stacking peaks appearing in (I a / I c ) It can be confirmed that the BECOM of Example 1 manufactured by template regeneration exhibits more intense a-axis stacking and exhibits a silk II structure, with Example 1 and Comparative Example 2 having values ​​of 1.01 and 0.791, respectively.

[0254] glass transition temperature

[0255] 1-2 mg of BECOM prepared in Example 1 and native silk fibroin prepared in Comparative Example 2 were placed in a chamber purged with N2 gas using PerkinElmer, Korea, at a temperature range of 25 to 300°C and a temperature of 5°C min. -1 , 10 ℃ min -1 and 20 ℃ min -1 Glass transition temperature (T) under heating rate conditions g ) was measured and the results are shown in Fig. 2e.

[0256] Figure 2e shows the glass transition temperature (T) of BECOM (Templated) manufactured in Example 1 and natural silk fibroin (Native) manufactured in Comparative Example 2. g ) is shown.

[0257] As shown in the above Fig. 2e, the natural silk fibroin prepared in Comparative Example 2 showed a glass transition temperature (200.5 ℃ / 173.5 ℃ / 161.6 ℃) similar to that of the reported silk fibroin, but the BECOM prepared in Example 1 showed a glass transition temperature of 257.6 ℃, 261 ℃, and 277.5 ℃, which was significantly increased compared to the natural silk fibroin. When the chain mobility of the semi-flexible polymer decreased by about 12.5%, T gConsidering that it has been previously reported that the temperature increases by approximately 100 ℃, it can be confirmed that the chain mobility is reduced due to the tightly entangled structure of BECOM.

[0258] Experimental Example 2. Mechanical Properties Evaluation

[0259] To measure ultimate stress, strain (λ), toughness (T), and elastic modulus (E), UTM (Model 3366, Instron, USA) was used according to ASTM D882 at thicknesses of 400 μm and 4 cm. 2 5.0 mm min for beam-shaped specimens -1 Tensile tests were performed by applying uniaxial tension until the sample was destroyed, at strains of 1%, 3% and 5%, with a strain rate of 5.0 mm min. -1 Repeated bending experiments were performed by bending the specimen at a speed of 10° min and then recovering it, repeating 100 times per strain point. -1 Repeated torsion experiments (exposure to 600 deformation cycles per sample) were performed with 100 repetitions per step at 10°, 30° and 60° speeds.

[0260] Figure 10 shows the BECOM (thickness 400 μm and 4 cm) manufactured in Example 1. 2 (a) 5.0 mm min of beam shape -1 This is a graph showing the stress-strain curve and (b) ultimate stress, elastic modulus, and toughness when uniform tension is applied at a tensile and displacement rate of . In Fig. 10, n=3, and is expressed as the mean ± standard deviation (SD).

[0261] As shown in the above Figure 10, the BECOM manufactured in Example 1 has a thickness of 400 μm and 4 cm. 2When manufactured as beam-shaped specimens, the ultimate stress was 17.6 MPa, the elastic modulus was 645.1 MPa, and the elongation was 1.57 GJ m -3 In particular, the toughness values ​​of high-performance hierarchical poly(vinyl alcohol) hydrogel and lignocellulose bioplastic, which are high-toughness polymers, were 210 MJ m, respectively. -3 and 2.8 MJ m -3 By MJm -3 Compared to the values ​​shown below (expressed by the horizontal dotted line), it can be seen that Example 1 has higher toughness.

[0262] Meanwhile, thickness 400 μm and 4 cm 2 The ultimate stress, elastic modulus (E), and toughness (T) of BECOM of Example 1 manufactured as a beam-shaped specimen were compared with those of conventional plastics, and the results are shown in Fig. 2f.

[0263] Figure 2f shows the ultimate stress and elastic modulus of BECOM, petrochemical plastic, degradable petrochemical plastic, and bio-based bioplastic manufactured in Example 1.

[0264] As shown in the above Fig. 2f, the ultimate stress and elastic modulus of BECOM of Example 1 had mechanical properties equivalent to those of bio-based bioplastic (PLA) and biodegradable petrochemical plastic (PCL), and it was confirmed that the bioplastic according to one embodiment of the present invention exhibited excellent maximum tensile strength, elastic modulus, and toughness due to the increased beta sheet content.

[0265] Figure 2g shows the cyclic bending and torsion profiles of BECOM manufactured in Example 1.

[0266] As shown in the above Figure 2g, it can be confirmed that Example 1 exhibits excellent toughness and flexibility simultaneously by adapting to repeated deformation without breakage when repeated cycles of bending and twisting are applied.

[0267] Experimental Example 3. Performance Evaluation of Orthodontic Devices

[0268] Figure 3a is a schematic diagram schematically showing a process of performing orthodontic treatment using BECOM and PETG manufactured in Example 1 as orthodontic devices.

[0269] As shown in the above Fig. 3a, in order to confirm whether the BECOM manufactured in Example 1 can exhibit suitable performance as an orthodontic device, an orthodontic device was manufactured, and polyethylene terephthalate (PETG; Scheu-Dental Duran Thermoforming Foils, Duran), which is widely used as a conventional dental plastic, was used as a control group.

[0270] Creep recovery

[0271] BECOM and PETG manufactured in the above Example 1 were 1.0 cm thick and 1.0 mm thick. 2 It was prepared in the form of a film, compressed using a rheometer (MCR 302, Anton Paar, Austria) and a cylindrical probe (Ø8.0 mm) until the force reached 5.0 N, then a creep stress in the range of 0.1 to 1.0 kPa was applied for 2 minutes, and the subsequent recovery behavior was monitored for 2 minutes after the stress was released to 0 kPa, and the results are shown in Fig. 3b and Fig. 11.

[0272] Figure 3b shows the results of creep-recovery response evaluation at initial activation conditions in the range of 0.3-1.0 kPa of BECOM and PETG manufactured in Example 1.

[0273] Figure 11 shows (a) the normalized strain versus time profile of BECOM manufactured in Example 1 when a creep stress of 1.0 kPa was applied for 2 minutes, and (b) the permanent strains induced at creep stresses of 0.1 kPa, 0.3 kPa, 0.5 kPa, 1.0 kPa, and 2.0 kPa for BECOM and PETG manufactured in Example 1. In Figure 5a, permanent recovery indicates plastic deformation.

[0274] As shown in the above Figs. 3b and 11, unlike PETG which only exhibits strong elastic recovery, BECOM of Example 1 exhibited both elastic and viscoelastic recovery, and showed a higher recovery rate than PETG based on the increased viscoelasticity, confirming that BECOM can exert orthodontic force more effectively than PETG. Furthermore, PETG showed a pattern dominated by elastic recovery, confirming that strong orthodontic force was quickly delivered at once, which may cause excessive stress on the periodontal ligaments and cause pain in the patient, whereas BECOM of Example 1 showed a slower recovery response in the measured viscoelastic region, confirming that it can continuously and smoothly deliver a user-friendly orthodontic force of an appropriate size.

[0275] Stress relaxation test

[0276] In order to investigate the durability under tooth resistance conditions, BECOM and PETG manufactured in Example 1 were made into a 500 μm, 4.0 cm thick 2 After preparation in beam form, pre-displacement (0.1-0.5 mm) was applied at 5.0 mm min -1The strain was applied at a rate of (initial strain of 0.2 mm, 0.4 mm, 0.5 mm on a 2 cm long sample) (strain = 1%, 2%, and 2.5%) and the corresponding relaxation profiles were monitored for 30 minutes, and the results are shown in Fig. 3c and Fig. 12. At this time, tooth resistance to sub-millimeter tooth movement was reflected by pre-displacements lower than 0.5 mm, and the stress relaxation rate (stress reduction rate, %) was calculated as in Equation 2 below.

[0277] [Formula 2] Stress relaxation rate = [(F0-F 30 ) / F0]x100

[0278] (In the above equation 2, when a stress relaxation test was performed on a 500 μm sample under the first strain (ε) condition, F0 means the initial stress, and F 30 (means stress after 30 minutes)

[0279] Figure 3c shows the results of a stress relaxation experiment of BECOM and PETG manufactured in Example 1.

[0280] Figure 12 shows (a) the stress relaxation pattern of BECOM manufactured in Example 1 when the pre-displacements are 0.4 mm and (b) the relaxed energy determined by the upper region of the stress versus time curve of BECOM manufactured in Example 1 and PETG under tooth resistance of 0.1-0.5 mm.

[0281] As shown in the above Fig. 3c and Fig. 12, PETG with low viscoelasticity hardly relaxed the applied stress with a relaxation rate of less than 8%, but BECOM of Example 1 showed excellent stress relief characteristics with a stress relaxation rate of 29%, 54%, and 62% according to the above-described Equation 2 when the first strain was 1%, 2%, and 2.5%, respectively. In addition, BECOM of Example 1 showed a stress relaxation rate of 2.39 J min m when 0.1 mm and 0.5 mm tooth resistances were applied, respectively. -3 and 43.2 J min m -3 It was confirmed that the stress can be relieved and the concentrated stress can be distributed according to the tooth resistance level, thereby ensuring stable tooth movement and durability at the same time.

[0282] In summary of the above creep recovery and stress relaxation evaluations, it was confirmed that the BECOM of Example 1 exhibits excellent viscoelastic properties with stress relaxation behavior like human tissues with viscoelasticity because it exhibits a densely entangled structure in which the biopolymer network of the extracellular matrix and the cytoskeleton is entangled. In addition, Comparative Example 3, which is actually used in an orthodontic device, exhibits elastic properties with insufficient stress relaxation behavior, but Example 1 can effectively suppress patient pain or discomfort and significantly improve orthodontic force compared to Comparative Example 3, and thus it was confirmed that it can improve the limitations of the conventional technology.

[0283] Experimental Example 4. In silico finite element analysis

[0284] Analysis conditions

[0285] In order to verify the orthodontic performance of the BECOM manufactured in Example 1 above, a simulation was performed to correct a human maxilla using BECOM and PETG as orthodontic devices, and the results were subjected to in silico finite element analysis. The conditions, process, and results for the simulation are shown in Fig. 3dg.

[0286] Figure 3d is a schematic diagram illustrating the maxillary morphology for in silico simulation of human maxillary orthodontic surgery. In Figure 3d, U1 and U2 represent the central and lateral maxillary incisors, respectively, and the x-axis, y-axis, and z-axis designate coordinates in the transverse, anteroposterior, and vertical orientations.

[0287] First, as shown in Fig. 3d, a detailed 3D model of each tooth was created from a laser-scanned Nissin Dental Products (Kyoto, Japan, Model-i21D-400G) image of an adult with normal occlusion using 3D modeling software, and periodontal ligament (uniform thickness of 0.1 mm) was added to the root of each tooth, followed by a 0.2 mm midline diastema. Next, an orthodontic appliance using BECOM or PETG of Example 1 with a uniform thickness of 350 μm was positioned at the target position with the 3D model of the tooth crown, and these were merged to create the shape of the orthodontic appliance, which was then obtained through a subtractive Boolean operation. Then. A symmetrical arch pattern was assumed, with the goal of eliminating the 0.2 mm gap (spur) between the upper front teeth and moving the two central incisors 0.1 mm mediolaterally. Finite element models of the teeth and orthodontic appliance were constructed only on the right side. Material properties were assumed to be homogeneous, isotropic, and linear elastic, and the 3D model was meshed using the finite element method using modified tetrahedral second-order elements (C3D10M) with the properties shown in Table 1 below.

[0288] ModelElastic odulusPoisson's RatioNode numberElement numberTeeth18600 MPa0.312046369329Periodontal ligament0.68 MPa0.484138064716PETG668 MPa0.45787915279BECOM645.1 MPa0.40787915279

[0289] A series of iterative adjustments within the simulation enabled gradual and controlled tooth movements, taking into account the natural flexibility of the periodontal ligament to investigate the initial stages of tooth movement. Initially, the orthodontic appliance was fitted precisely and closely to the teeth, and the tooth sockets of the central incisors were slightly adjusted. The initial tooth movements were calculated with greater precision by treating each tooth as a rigid, immovable object. Afterwards, the tooth sockets were adjusted based on the observed initial movements. These adjustments involved slight changes in position and angle and were applied primarily to the outer surface of the periodontal ligament. This method also allowed for continuous updating and refinement of the forces applied to the teeth, thereby enhancing the accuracy of the simulation. The entire simulation process was automated using APDL (Ansys Parametric Design Language) in ANSYS (version 11.0, Ansys Inc., Canonsburg, PA, USA).

[0290] Analysis results

[0291] The magnitude of the initial stress acting on various points of the periodontal ligament as a function of the thickness of the orthodontic appliance using BECOM and PETG manufactured in Example 1 was calculated, and the final three-dimensional displacement and stress magnitude of the central incisor (U1) and lateral incisor (U2) of BECOM and PETG manufactured in Example 1 were compared, and the tooth displacement profiles are shown in FIGS. 3e, 3f, and 13.

[0292] Figure 3e shows the distribution of corrective force according to the y-axis position of the orthodontic device using BECOM and PETG manufactured in Example 1. In Figure 3e, the outline of U1 is expressed as a black line, and the part where a significant difference in corrective force between BECOM and PETG manufactured in Example 1 was observed is expressed as a red circle.

[0293] As shown in FIG. 3e, the BECOM of Example 1 generated force magnitudes 250-300% higher than those induced by PETG at the tip (y = 0.0 mm) and anterior (y = -3.2 mm), and unlike PETG, which showed a force pattern that tended to be concentrated toward the incisal side, the BECOM of Example 1 showed forces distributed along the anteroposterior tooth surface (y-axis).

[0294] Figure 3f shows the tooth displacement profile of the right maxilla after correcting the teeth using the orthodontic device using BECOM of Example 1.

[0295] As shown in the above Fig. 3f, the tooth position was moved three-dimensionally (3D) after orthodontic treatment using the BECOM orthodontic device of Example 1 compared to the original tooth position expressed as a black mesh, and in particular, U1 showed a maximum displacement of 0.07 mm within the range of clinical standards.

[0296] Figure 13 shows the displacement of a human maxillary central incisor (U1) measured using in silico finite element analysis according to the thickness (300 μm, 500 μm, 750 μm) of the orthodontic appliance using BECOM manufactured in Example 1.

[0297] As shown in the above Figure 13, the orthodontic performance could be controlled by adjusting the thickness of the BECOM, and as the BECOM orthodontic device became thicker, the three-dimensional tooth movement increased, and in particular, when a BECOM with a thickness of 750 μm was used, the transverse movement (x-axis) was improved by 1.60 times.

[0298] In addition, the residual force and its distribution within the teeth after orthodontic treatment using the BECOM and PETG orthodontic devices manufactured in Example 1 are shown in FIG. 3g and FIG. 14.

[0299] Figure 3g shows the maximum residual force after orthodontic treatment using the BECOM and PETG orthodontic devices manufactured in Example 1.

[0300] Figure 14 shows the residual force distribution after tooth position correction of the orthodontic device using BECOM and PETG manufactured in Example 1.

[0301] As shown in the above Fig. 3g and Fig. 14, the residual force accumulated by tooth resistance was analyzed, and the BECOM manufactured in Example 1 sufficiently alleviated the resistance force, showing a residual force 55.7% lower than that of PETG, confirming that the BECOM of Example 1 exhibits highly desirable mechanical characteristics that ensure successful orthodontic treatment.

[0302] Experimental Example 5. In vivo orthodontic performance

[0303] In order to confirm the orthodontic performance of the orthodontic device using the BECOM manufactured in Example 1 in vivo, rabbit front teeth were selected as the orthodontic target, and after receiving approval from the Animal Care and Use Committee of Yonsei Medical Center in Seoul (Approval No. 2022-0190), three male New Zealand white rabbits weighing 3 ± 0.2 kg were individually housed in a controlled laboratory environment with unrestricted access to water and food, and an adaptation period of at least 1 week was allowed before the start of the study.

[0304] After establishing a midline expansion model that required reduction of the space between the mandibular incisors, a repetitive midline gap closure model was designed to simulate orthodontic treatment typically performed in stages using clear aligners. Each iteration involved meticulous interproximal reduction, and three iterations were evaluated, each aiming to achieve a 2.5 mm interproximal closure.

[0305] The BECOM manufactured in Example 1 was fabricated into a band-shaped orthodontic device with a length of 5-5.5 mm, a width of 6.8-7.2 mm, and a thickness of 50-400 μm that could cover the labial aspect of the lower incisors of rabbits and extend from the incisal edge to the junction of the middle and gingival third of the teeth. Two of the three rabbits underwent active BECOM orthodontic space closure, and the third rabbit did not undergo orthodontic treatment using BECOM and served as a control group. In the control group, evaluations were performed every 48 hours to evaluate tooth movement while closely monitoring physiological changes when there was no active orthodontic tooth movement.

[0306] To objectively measure tooth movement, intraoral anatomical pickup impressions of the mandibular incisor region were recorded using a lightweight and medium-weight impression material, polyvinyl siloxane (I-Sil™ Bite, Spident, Incheon, Korea). The rabbits were regularly examined for general health and periodontal health. All procedures were performed under intravenous sedation (Zoletil). Each repetition lasted no longer than 30 minutes per animal. To accurately assess changes in tooth movement, dental calculus (Mounting Stone, Whip Mix Corp) was poured onto the impression records and scanned using a tabletop scanner (3Shape E3 scanner, Copenhagen, Denmark). Morphometric analysis of tooth movement was performed using best-fit superimposition (Geomagic Control X, 3D Systems, SC, USA), and scans of the mandibular incisors after interproximal reduction served as reference for comparison purposes.

[0307] The transmittance according to the thickness of the orthodontic device using BECOM manufactured in Example 1 is shown in Fig. 15, and the results of clinical orthodontic treatment performed by gradually moving the rabbit's front teeth up to a maximum of 2.5 mm using this are shown in Figs. 4a to 4e.

[0308] Figure 15 shows the transmittance according to the thickness ranging from 50 μm to 400 μm of the orthodontic device using BECOM of Example 1.

[0309] As shown in the above Figure 15, the orthodontic device using the BECOM of Example 1 used in the in vivo study showed a maximum transmittance of 70%, and the thinner it was, the higher the transmittance was, up to a maximum of 82%.

[0310] Figure 4a shows photographic images of rabbit incisors at the initial stage (pre-treatment), after the occurrence of an incisal gap (midline diastema), and after orthodontic treatment using an orthodontic appliance using BECOM manufactured in Example 1 (BECOM orthodontics).

[0311] Figure 4b schematically illustrates a three-step process for orthodontic treatment of a rabbit's front teeth using an orthodontic device using BECOM manufactured in Example 1. In Figure 4b, PT indicates the post-treatment state.

[0312] Figure 4c schematically illustrates the process by which undesirable tooth movement and malocclusion occur in a control group that did not undergo orthodontic treatment with an orthodontic device.

[0313] FIG. 4d shows a front view of a 3D scanned tooth movement profile of a rabbit's front teeth after orthodontic treatment using the BECOM orthodontic device manufactured in Example 1. This is the result of morphometric analysis based on multi-axis 3D scans after orthodontic treatment was performed for 72 hours using the BECOM orthodontic device manufactured in Example 1.

[0314] Figure 4e shows a plan view image of teeth with malocclusion that has progressed due to non-orthodontic treatment with an orthodontic device. In Figure 4e, θ dayrefers to the incisal edge angle on a specific date after interproximal reduction surgery, and Δθ is θ0- θ day This represents an undesirable internal rotation expressed as .

[0315] As shown in the above FIGS. 4a to 4e, after a midline diastema, i.e., an incisor gap, was created through interproximal reduction surgery to secure a tooth movement site (FIG. 4a), the orthodontic device using the BECOM manufactured in Example 1 was used to gradually correct the rabbit incisor position to a total displacement of 2.5 mm through three correction steps (FIG. 4b), and as a result, the teeth were sufficiently moved, and in particular, it was confirmed that the re-approximation of the incisal mesial edge was distinct in the blue portion of the tooth movement indicator (FIG. 4d). On the other hand, in the control group that did not use the orthodontic appliance using the BECOM manufactured in Example 1, acute malocclusion with internal rotation was observed as expected (Fig. 4c), and in particular, the normal incisal edge with θ0 = 143° was significantly distorted to Δθ = 8.0°. Through this, it was confirmed that the BECOM manufactured in Example 1 can be used as an orthodontic appliance to finely adjust the position of teeth, prevent unwanted tooth movement, and consequently enable satisfactory orthodontic treatment.

[0316] Experimental Example 6. Recycling Evaluation - Silk Fibroin Extraction

[0317] Pure silk fibroin extraction process and results

[0318] In the above Example 2, the procedure and recycling yield (yield of silk fibroin) of the BECOM manufactured in the above Example 1 are shown in FIGS. 5a and 16, and the antibacterial group (R) of the urethane oligomer casting mold used in Example 1 was used to visually confirm the process as shown in FIG. 5b. 3A ) is a dye tracer that shows pink color (Rhodamin B, R 3B ) was partially replaced.

[0319] Figure 5a is a schematic diagram schematically illustrating a recycling procedure based on disentanglement of BECOM manufactured in Example 1. The symbols l and s shown in Figure 5a represent the liquid phase and the solid phase, respectively.

[0320] Figure 5b shows the antibacterial group (R) of the urethane oligomer casting mold used in Example 1. 3A ) and dye tracers (Rhodamin B, R 3B ) shows the chemical structure.

[0321] Figure 16 shows (a) the weight (n=4) of pure silk fibroin (EPSF) extracted by recycling BECOM of Example 1 and BECOM of Example 1 in Example 2, and (b) the average mass yield of pure silk fibroin (EPSF) extracted by recycling BECOM of Example 1.

[0322] As shown in FIG. 5a and FIG. 16, the BECOM manufactured in Example 1 exhibited an entangled structure, which was expected to be unraveled and thus have recyclable properties. In fact, in Example 2, when the BECOM manufactured in Example 1 was treated with formic acid, the entangled structure was unraveled based on hydrogen bonds, resulting in the generation of a liquid BECOM containing a freely movable urethane oligomer casting mold (template) and denatured silk fibroin strands. Subsequently, when ethanol was added, the silk fibroin precipitated, and the urethane oligomer casting mold and silk fibroin were separated. After this process was repeated five times, the urethane oligomer casting mold and silk fibroin were extracted in a liquid phase and a solid phase, respectively. The extraction yield of silk fibroin in Example 2 was 72.6% on a mass basis (FIG. 16).

[0323] Meanwhile, the substitution ratio (R) of the antibacterial group and dye tracer of the urethane oligomer casting mold used in Example 1 3A / (R 3A + R 3B )) Photographic images of BECOM manufactured in Example 1 and silk fibroin extracted in Example 2 are shown in FIG. 5c and FIG. 17.

[0324] Figure 5c shows the substitution ratio (R) of the antibacterial group and dye tracer of the urethane oligomer casting mold used in Example 1. 3A / (R 3A + R 3B )) shows photographic images of BECOM manufactured in Example 1 and silk fibroin extracted in Example 2.

[0325] Figure 17 shows photographic images of silk fibroin extracted in Example 2 according to the molar ratio of the antibacterial group and dye tracer of the urethane oligomer casting mold used in the production of BECOM in Example 1.

[0326] As shown in the above Figure 5c and Figure 17, the antibacterial group (R) of the urethane oligomer casting mold 3A ) with pink dye (R 3B ) increased to a maximum of 10 mmol%, BECOM showed a more intense pink color, but the extracted silk fibroin showed a yellow color regardless of the substitution rate, indicating that the template was completely removed.

[0327] Figure 5d shows the BECOM prepared in Example 1, the silk fibroin extracted in Example 2, and the natural silk fibroin prepared in Comparative Example 2. 13 C shows the nuclear magnetic resonance (NMR) spectrum.

[0328] As shown in the above Fig. 5d, the BECOM of Example 1 showed both a peak at 29.2 ppm related to the aliphatic chain of the antibacterial group of the urethane oligomer casting mold and a peak at 70.9 ppm related to the ether group that induced the entanglement, indicating the presence of the urethane oligomer casting mold. On the other hand, the silk fibroin extracted in Example 2 did not show any signals related to the urethane oligomer casting mold, and only showed the Gly, Ala, and Ser signals of the silk fibroin. Through this, it was confirmed that the silk fibroin extracted in Example 2 was very pure and did not contain any residual urethane oligomer casting mold.

[0329] In order to analyze the characteristics of the urethane oligomer casting mold obtained in the process of extracting silk fibroin from BECOM of Example 1 in the above Example 2, 0.320 mg of the urethane oligomer casting mold extracted after each extraction cycle in Example 2 was introduced into the chamber at 1 mL min using a single-shot pyrolysis gas chromatography-mass spectrometer (8890 GC-5977B MSD, Agilent) equipped with a UA-5 column of 30 m length. -1Purged with helium at a flow rate of , and after an initial stabilization stage of 5 min at an injection temperature of 40°C, the temperature was increased by 10°C min -1 The heating rate was increased to 320 ℃ and stopped for 10 minutes to obtain the analysis results, and based on this, the extraction amount of the urethane oligomer casting mold was calculated as in the following equation 5, and the process and results are shown in Figs. 18, 5e, and 19.

[0330] [Formula 5] (Peak area related to urethane oligomer casting mold) / (Solvent peak area + Peak area related to urethane oligomer casting mold)

[0331] (In the above formula 5, the peaks related to the urethane oligomer casting mold are peaks corresponding to methane, ethylene, propene, acetylene, 1,3-butadiene, and benzene as a result of gas chromatography analysis, and the solvent peaks are peaks corresponding to formic acid and ethanol as a result of gas chromatography analysis.)

[0332] Figure 18 shows the extraction amount profile of the urethane oligomer casting mold according to the extraction cycle in Example 2.

[0333] Figure 5e shows photographic images of the urethane oligomer casting mold (1 mL) extracted by recycling BECOM of Example 1, in which the substitution ratio of the antibacterial group and the dye tracer of the urethane oligomer casting mold in Example 2 was 50 mmol%, after the first and fifth extraction processes.

[0334] Figure 19 shows the extraction cycle of the urethane oligomer casting mold extracted by recycling BECOM with a substitution ratio of 50 mmol% of the antibacterial group and dye tracer of the urethane oligomer casting mold in Example 2 (a). 1 (a) H nuclear magnetic resonance (NMR; Avance III HD 300, Bruker) spectrum) and (b) photographic image (1 mL per vial).

[0335] As shown in the above Figures 18, 5e, and 19, 76.8% of the urethane oligomer casting mold was extracted in the first extraction step in Example 2 (Figure 18), and derived from Rhodamine B. 1 The broad peak in the range of 7.1 to 7.4 ppm in the H NMR spectrum disappeared after the third extraction (Fig. 19a), and the pink color corresponding to the urethane oligomer casting mold was hardly observed after the third extraction, indicating that the urethane oligomer casting mold was completely removed (Fig. 19b, Fig. 5e).

[0336] Assessment of inflammatory biomarker expression

[0337] Macrophages were treated with the EPSF extracted in Example 2 above, and the expression levels of immune response and inflammatory biomarkers (tumor necrosis factor-α (TNF-α); interleukin-1β (IL-1β); interleukin-6 (IL-6), and nitric oxide synthase 2 (NOS2)) were compared with those of the control group (Mock (n=3)) that was not treated with EPSF, and the results are shown in Fig. 5f.

[0338] Figure 5f is a graph showing the relative expression intensity of inflammatory biomarkers (n=3) between silk fibroin (EPSF) extracted in Example 2 and the control group.

[0339] As shown in the above Fig. 5f, macrophages treated with EPSF extracted in Example 2 showed decreased expression of TNF-α, IL-1β, and IL-6 compared to the control group, and showed a level of NOS2 expression equivalent to the control group. Since this has been reported to also appear in natural silk fibroin, it can be confirmed that EPSF shows excellent cytocompatibility like natural silk fibroin.

[0340] Experimental Example 7. Recycling Evaluation - Reshaping

[0341] In order to verify whether the used bioplastic can be recycled by reshaping it back into the initial bioplastic, the BECOM manufactured in Example 1 was immersed in an acidic solution to disentangle the entangled structure, and then the acidic solution was removed to induce reentanglement, thereby recycling BECOM-R1 (Example 3), and BECOM-R2 (Example 4), which was prepared by extracting silk fibroin from the BECOM manufactured in Example 1 and recycling it using a template regeneration method, were analyzed for morphology, FT-IR spectrum analysis, and ultimate stress, and the results are shown in FIGS. 5g, 20, and 5h.

[0342] Figure 5g shows photographic images of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4, each having a thickness of 200 μm.

[0343] As shown in the above Fig. 5g, the above Examples 1 (BECOM), 3 (BECOM-R1), and 4 (BECOM-R2) all exhibited similar morphologies, confirming that BECOM could be recycled by inducing reentanglement of the BECOM manufactured in Example 1 through an acidic solution immersion and removal process (Example 3, BECOM-R1), or by extracting silk fibroin from the BECOM manufactured in Example 1 and then performing a template regeneration method again (Example 4, BECOM-R2).

[0344] Figure 20 shows the FT / IR spectra of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4. The FT / IR spectra of Figure 20 were obtained by repeating scanning 32 times per sample using a Fourier transform infrared spectrometer (FT / IR-4700, JACSO, Japan).

[0345] As shown in the above Figure 20, in all of the above Examples 1 (BECOM), 3 (BECOM-R1) and 4 (ECOM-R2), the amide I region corresponding to the β-sheet structure of silk fibroin was 1620 cm -1 The β-sheet-related Fourier transform infrared amide I spectrum containing strong signals was shown, indicating that the tightly entangled structure was reconstructed in Examples 3 and 4.

[0346] Meanwhile, in order to confirm whether the mechanical properties of the bioplastic of the present invention are maintained even after recycling, the results of measuring the stress versus strain curve, ultimate stress, maximum tensile strength, and elastic modulus, and comparing them with conventional plastics are shown in Fig. 5h and Fig. 21.

[0347] Figure 5h shows the ultimate stress graphs (n=3) of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4.

[0348] Figure 21 shows (a) 4 cm using BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4. 2 5.0 mm·min of beam-shaped specimen -1 When uniform tension was applied at a displacement rate of , stress versus strain curves (n=3) and (b) ultimate stress and elastic modulus of BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4, petrochemical plastics, degradable petrochemical plastics, and bio-based bioplastics are shown.

[0349] As shown in FIG. 5h and FIG. 21, the ultimate stresses of Example 1 (BECOM), Example 3 (BECOM-R1), and Example 4 (BECOM-R2) were 17.6 MPa, 17.4 MPa, and 11.5 MPa, respectively, and the elastic moduli were 645.1 MPa, 413.2 MPa, and 292.8 MPa, respectively. That is, BECOM-R1 of Example 3 exhibited an ultimate stress very similar to that of BECOM of Example 1. On the other hand, BECOM-R2 of Example 4 exhibited an ultimate stress of 11.5 MPa, which is equivalent to the ultimate stress of a biodegradable starch plastic, although the ultimate stress was reduced by 35%, considering that the ultimate stress of the biodegradable starch plastic is ~10 MPa. Through this, it was confirmed that the excellent mechanical properties of BECOM can be maintained even after the recycling process of BECOM is completed.

[0350] Experiment 8. Evaluation of microbial resistance of BECOM

[0351] Figure 6a is a schematic diagram showing the phenomenon of microbial biofilm growth on orthodontic appliances and gingival tissue during orthodontic treatment.

[0352] As shown in Fig. 6a, oral tissues contain numerous microorganisms. When orthodontic appliances are used to correct teeth, the external surface exhibits a strong affinity for plaque formation after 6 hours of long-term oral application of the appliance. In addition, the internal area between the appliance and the teeth is relatively more anaerobic than the external surface, which can easily create an environment favorable for biofilm formation, such as the accumulation of thick extracellular polysaccharides. Therefore, gingival tissue and orthodontic appliances during orthodontic treatment are vulnerable to biofilm growth. Therefore, to evaluate the microbial resistance of the BECOM manufactured in Example 1, human saliva samples were used to confirm the microbial resistance.

[0353] In vitro microbial resistance

[0354] Saliva sample collection was conducted in strict compliance with ethical guidelines, including compliance with the 64th World Medical Association Declaration of Helsinki, and the entire procedure was strictly approved by the Institutional Review Board of Yonsei University Dental Hospital, Republic of Korea (Approval No. 2-2019-0049). Participant recruitment was conducted fairly without considering sex or gender as a variable in the study. Saliva samples collected from six healthy donors were mixed in equal volumes and diluted with sterile glycerol to achieve a 30% concentration and then stored at -80 °C. To promote the culture of the biofilm model, a special medium known as McBain's medium was prepared, and human saliva samples were cultured in McBain's medium for 24 hours. Afterwards, 1.5 mL of volume was dispensed onto the BECOM and PETG prepared in Example 1 and cultured at a constant temperature of 37°C for 48 hours, and fresh medium was replenished at intervals of 8, 16, and 24 hours during the culture period to maintain the integrity and relevance of the experiment, and the surface biofilm was collected using a sterile loop for 16s rRNA sequencing.

[0355] As described above, after treating the human saliva medium with BECOM and PETG of Example 1, the behavioral characteristics against Streptococcus mutans and Staphylococcus aureus were evaluated by the degree of colony formation (unit = CFU), and the results are shown in Fig. 6 bc, and the culture without BECOM and PETG was used as the control group (Mock). At this time, the CFU values ​​were expressed on a log scale, and all increases were 10 of CFU. 2 corresponded to an increase, for example, BECOM and PETG were 56 and 3.2 × 10, respectively, in relation to S. mutans attachment. 6 The average CFU corresponding to .

[0356] Figure 6b shows a graph of the attachment behavior (n=3) of Streptococcus mutans and Staphylococcus aureus of the experimental and control groups treated with BECOM and PETG of Example 1 in vitro on human saliva medium.

[0357] As shown in the above Fig. 6b, the BECOM of Example 1 showed almost no attachment behavior of S. aureus, confirming that the BECOM could block the formation of the initial pellicle layer.

[0358] In addition, to consider secondary pathogenic growth, S. mutans and S. aureus were cultured in liquid medium with BECOM or PETG prepared in Example 1 for 72 hours, and then the growth behavior was evaluated, and the results are shown in Fig. 6c.

[0359] Figure 6c shows a graph of the growth behavior (n=3) of Streptococcus mutans and Staphylococcus aureus in the experimental and control groups treated with BECOM and PETG of Example 1 in vitro in human saliva medium.

[0360] As shown in the above Figure 6c, PETG showed a similar growth pattern to the control group (Mock), but BECOM of Example 1 significantly inhibited microbial growth, and in particular, completely inhibited the growth of S. aureus.

[0361] Figure 6d shows scanning electron microscope images (scale bar = 5.0 μm) of S. mutans and S. aureus in the experimental group treated with BECOM and PETG of Example 1 in vitro in human saliva medium.

[0362] As shown in the above Fig. 6d, the biofilm formed on PETG based on S. mutans and S. aureus had a more organized form, but it was confirmed that the BECOM of Example 1 exhibited excellent sterilization performance with minimal biofilm growth and asymmetrically torn cocci.

[0363] Cell viability assessment

[0364] Concentrated quaternary ammonium compounds have concerns about toxicity despite their excellent antibacterial performance, so the cell viability of BECOM of Example 1 was measured in L-929, RAW264.7, and human gingival fibroblast (HGF-1) cell lines. Dulbecco's Modified Eagle Medium media (DMEM; HyClone Co., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin and streptomycin (P / S; Gibco; Thermo Fisher Scientific) was used for culturing at 37°C, 95% humidity, 5% CO2, and after confluence, adherent cells were detached with 0.05% trypsin / EDTA (Gibco, Thermo Fisher Scientific), centrifuged at 1300 rpm for 3 minutes, and resuspended in growth medium. CEB and EPSF were extracted from DMEM containing 10% FBS and 1% P / S at an extraction ratio of 25 mg / mL at 37°C for 72 h. The supernatant was collected, sterile filtered (0.2 μm syringe filter), and diluted in culture medium. Growth medium served as a control (Mock).

[0365] 1×10 4Cells were plated in 96-well plates (SPL Life Sciences Co., Pocheon-si, Gyeonggi-do, Korea) and maintained at 37°C, 95% humidity, and 5% CO2 for 24 h. After the incubation period, 100 μL of each substance was extracted from the 96-well plates in which the cells were cultured. After 24 h of incubation, 50 μL of MTT solution (1 mg mL -1 ) was added, and the 96-well plate was placed in the incubator for 2 more hours, and then 100 μL of isopropanol (Duksan Pure Chemical Co., Ansan-si, Republic of Korea) was added to each well. Optical density (OD) was measured at 57 nm using a microplate reader (Epoch, BioTek, Winooski, VT, USA) for cell viability analysis, and cell viability (Cell viability, %) was calculated using the following equation 6.

[0366] [Formula 6] Cell viability (%) = (OD of experimental group) 570 ) / (OD of control group (Mock) 570 ) × 100

[0367] Additionally, to induce pro-inflammatory response using qPCR analysis, 1×10 5 RAW 264.7 cells were cultured in 6-well plates (SPL Life Science Co.) for 24 h, and the existing medium was discarded and replaced with various sample extractions. After 48 h, total RNA was extracted (QIAzol lysis reagent; Qiagen, Hilden, Germany) and complementary deoxyribonucleic acid (DNA) was synthesized (PrimeScript™ RT reagent kit; Takara Bio Co., Tokyo, Japan). qPCR was then performed on a real-time PCR system using the SYBR® Premix Ex Taq™ kit (Takara Bio Co.). The relative messenger RNA levels of the target genes were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase.

[0368] Figure 22 shows (a) the reaction distance of fibroblast confluent monolayer when treated with negative control (NC; high-density polyethylene film), positive control (PC; 0.1% ZDEC polyurethane film) and BECOM prepared in Example 1 (n=3), (b) a graph showing cell viability after co-culturing BECOM of Example 1 and EPSF of Example 2 with human gingival fibroblasts (HGF-1), mouse fibroblasts (L929) and mouse macrophages (Raw264.7) (n=3), and (c) the relative expression level of inflammatory biomarkers by BECOM prepared in Example 1 and the control (Mock).

[0369] The above Figure 22a is the result obtained by performing an agar diffusion test (ISO 10993-5:2009) according to the International Organization for Standardization (ISO) 10993-5:2009 standard at a Good Laboratory Practice approved institution (Seoul National University Hospital, Seoul, Korea). The dotted line in the above Figure 22b indicates the 70% cell viability, which is the cytotoxicity criterion according to the ISO 10993-5:2009 standard. The control group in Figure 22c refers to an experimental group without EPSF.

[0370] As shown in the above Figure 22, the reactivity of the BECOM manufactured in the above Example 1 was grade 1 (Figure 22a), and it showed high cell viability in the viability test for human gingival fibroblasts and macrophages (Figure 22b), and it did not induce an allergic reaction in vivo, and it was confirmed that it showed excellent biocompatibility by reducing the expression of inflammatory biomarkers (TNF-α, IL-1β, IL-6, and NOS2) in macrophages (Figure 22c).

[0371] Analysis of human salivary biofilm microbial communities

[0372] Human saliva, rich in glycoproteins, can accelerate pathogenic plaque accumulation on orthodontic devices by promoting the attachment of various microorganisms and the subsequent formation of complex biofilms. Contaminated orthodontic devices with plaque accumulation can induce oral diseases such as gingivitis and dental caries during orthodontic treatment. Therefore, 16S ribosomal ribonucleic acid (rRNA) amplicon sequencing was used to obtain microbiome information of human salivary biofilms formed in Example 1 (BECOM) or PETG.

[0373] More specifically, DNA sequencing was performed after DNA extraction. We prepared the sequenced samples according to the Illumina 16S rRNA sequencing library protocol and assessed DNA quality and quantity using PicoGreen and VICTOR Nivo. The 16S V3-V4 primers were used to amplify the 16S rRNA gene, and an additional limited-cycle amplification step was performed to incorporate multiplexing indices and Illumina sequencing adapters. The resulting products were then normalized, pooled, and size-checked before sequencing on the MiSeq™ platform. The Illumina Miseq Sequencing System (Illumina, USA) was used for amplicon sequencing. Quality checks were performed on raw reads, except for cases where low-score reads (<25) were performed, followed by merging of paired-end sequence data and primer trimming. Unique 16S rRNA reads were separated using a 97% similarity threshold, and taxonomic assignments were performed based on the EzBioCloud 16S rRNA database.

[0374] Figure 6e shows alpha diversity metrics at the amplicon sequence variant level of human saliva biofilms formed on BECOM and PETG in Example 1.

[0375] As shown in the above Figure 6e, the BECOM of Example 1 had a low observed species index, which resulted in a decrease in species diversity within the microbial community of BECOM, and both the Chao1 and ACE levels decreased, which also resulted in a decrease in total diversity, confirming that BECOM has a high resistance to microbial community growth.

[0376] Figure 6f shows a principal component (PC) analysis plot of amplicon sequence variants based on the Bray-Curtis dissimilarity method of human saliva biofilms formed from BECOM and PETG in Example 1. In Figure 6f, PC1 and PC2 represent the first and second coordinates, respectively.

[0377] As shown in the above Fig. 6f, there was a difference in beta diversity between the microbial community of BECOM and PETG of Example 1, and specifically, the BECOM group of Example 1 was closely clustered and was distinguished from PETG in the second principal component. In particular, BECOM showed a significant difference in the first principal component with an ANOSIM R-value of 0.9259 (p=0.101) accounting for 64.71% of the variation. In other words, it can be confirmed that microbial communities that were substantially quite different grew in BECOM and PETG.

[0378] Figure 6g shows a significantly (*p<0.05) correlation pattern for the occurrence of different taxa at the genus level in human saliva biofilms formed on BECOM and PETG in Example 1. In Figure 6g, the pink bars represent species that are relatively more dominant in PETG than in BECOM, and the green bars represent species that are dominant in BECOM. The specific correlation analysis results comparing the occurrence patterns of different genera in BECOM and PETG are shown in Table 2 below.

[0379] TaxaCorrelationT-statisticP-value aFDRPETGBECOMAbiotrophia0.941535.58910.00502760.0331821.652E-049.304E-03Granulicatella0.899324.11310.0146950.0484933.922E-023.861E-01AF366267_g0.713462.03640.111390.229750.000E+001.844E-03Pantoea0.670931.80960.144610.265138.258E-056.193E-03Serratia0.581061.42790.22650.364536.331E-041.617E-01Neisseria0.577861.41610.229690.364530.000E+005.230E-04Eikenella0.564671.36840.243020.364534.129E-043.716E-03Rothia0.387840.841550.447410.580563.303E-041.514E-03Saccharimonas0.380020.821690.457410.580565.505E-052.753E-04Porphyromonas0.258020.534120.621560.718772.202E-044.129E-04Atopobium0.0633190.126890.905150.905151.376E-041.376E-04Weissella-0.06624-0.132770.900780.905152.477E-041.927E-04Parvimonas-0.16664-0.3380.752360.800891.374E-027.762E-03Fusobacterium-0.21504-0.440380.682410.750663.854E-041.927E-04Actinomyces-0.25083-0.518220.631650.718773.854E-041.927E-04Prevotella-0.27664-0.575760.595620.718777.157E-043.854E-04Peptostreptococcus-0.47169-1.06990.344940.474296.496E-035.505E-05Streptococcus-0.50947-1.18410.301910.433186.807E-014.134E-01Oribacterium-0.567-1.37670.240640.364537.047E-030.000E+00Megasphaera-0.67946-1.85210.137650.265133.110E-030.000E+00Leptotrichia-0.77913-2.48580.0677880.149138.258E-041.101E-04Dialister-0.82781-2.95110.0419220.0988172.587E-030.000E+00Gemella-0.85427-3.28670.0303080.0769353.083E-033.028E-04Haemophilus-0.85527-3.30110.0299040.0769352.863E-037.432E-04Lactobacillus-0.88573-3.81610.0188410.0565246.372E-020.000E+00Campylobacter-0.90066-4.14540.0143140.0484933.248E-032.753E-05Alloscardovia-0.91471-4.5270.0106020.0437325.230E-040.000E+00Lachnoanaerobaculum-0.92242-4.7770.00879510.0414624.266E-038.258E-05Moryella-0.93279-5.17620.00662380.0364312.532E-020.000E+00Staphylococcus-0.94507-5.78280.00444240.0331821.354E-023.276E-03Enterococcus-0.9773-9.22610.0007670.0084376.193E-033.303E-04Veillonella-0.98407-11.070.00037880.006251.144E-011.129E-03Bulleidia-0.98616-11.8960.0002860.006255.312E-038.258E-05.

[0380] As shown in the above Figure 6g and Table 2, the taxonomic distribution within the microbial community of PETG had a positive correlation with the commonly observed pathogenic genera Bulleida, Veillonella, Enterococcus, Staphylococcus, LactoBacillus, and Gemella, but a significant negative correlation was observed with the commensal Granulicatella.

[0381] Figure 6h is a graph showing the number of three pathogens (LactoBacillusfermentum, Veillonella atypica, and Enterococcus faecalis) in human saliva biofilms formed from BECOM and PETG of Example 1. Specific results of species-level taxonomic units analyzed using DESeq2 in the human saliva biofilm formed from BECOM of Example 1 are shown in Table 3 below.

[0382] Taxonomic unit a log2FC blfcSEP-valuesFDRVeillonella_atypica-8.14120.755314.34E-272.61E-25Lactobacillus_fermentum-11.3351.29051.59E-184.76E-17Abiotrophia_defectiva8.33260.967727.27E-181.45E-16Streptococcus_salivarius_group-5.12070.605282.67E-174.01E-16Stomatobaculum_longu m-9.87011.30824.52E-145.43E-13Veillonella_uc-5.93810.824736.02E-136.02E-12Serratia_ureilytica8.95421.27932.57E-122.20E-11Ser ratio_marcescens_group10.1831.50061.15E-118.66E-11Streptococcus_anginosus_group4.33320.656424.08E-112.72E-10Granulicatella_a diacens_group4.8830.813271.92E-091.15E-08Enterococcus_faecalis-4.49090.797281.77E-089.67E-08Pantoea_agglomerans_group8.67941 .56092.69E-081.35E-07Moryella_uc-7.23191.30492.99E-081.38E-07Granulicatella_elegans8.80311.5943.34E-081.43E-07Eikenella_corr odens5.12840.999022.84E-071.09E-06Enterobacteriaceae_group1.84050.358832.91E-071.09E-06Solobacterium_moorei-4.9980.97963.36E-071.19E-06Veillonella_parvula_group-3.87770.763973.86E-071.29E-06Lachnoanaerobaculum_saburreum_group-6.84941.37626.45E-072.04E-06FWNZ_s1.85140.391492.25E-066.76E-06Dialister_pneumosintes-6.61261.41893.15E-069.01E-06Campylobacter_concisus_group-4.81331.10021.21E-053.31E-05Serratia_uc8.69842.06622.55E-056.66E-05AF366267_s7.01671.70223.75E-059.38E-05Megasphaera_micronuciformis-7.28511.81646.05E-050.000145Granulicatella_uc3.81120.958046.94E-050.00016Abiotrophia_uc6.9161.87210.00022050.00049Campylobacter_showae_group-5.52951.50220.00023240.000494Oribacterium_asaccharolyticum-7.21491.96380.00023880.000494Bulleidia_uc-5.04921.38660.00027120.000542Streptococcus_gordonii_group-2.63570.744660.0004010.000776Veillonella_rogosae-2.47150.70790.00048070.000901Streptococcus_sinensis_group2.98410.884150.00073770.001341JH815185_s_group-3.83951.22030.00165320.002917Pantoea_uc4.50631.4520.00191210.003278Gemella_haemolysans_group-2.10740.691980.00232310.003872Peptostreptococcus_stomatis_group-5.81291.98190.00335750.005445Klebsiella_uc1.17670.437340.00713460.011265Streptococcus_constellatus_group2.63920.992480.00783270.01205.

[0383] As shown in the above Figure 6h and Table 3, the BECOM manufactured in Example 1 was free of the anaerobic cariogenic pathogen L. fermentum and the major biofilm-bridging species V. atypica and E. faecalis, confirming that the BECOM had a significantly reduced preference for pathogenic species. This confirmed that the BECOM of Example 1 could maintain a cleaner orthodontic device even during an orthodontic treatment period lasting several months.

[0384]

[0385] Figure 23 is a graph showing the relative abundance at the species level within the microbial community formed in the human saliva biofilm from BECOM and PETG of Example 1.

[0386] As shown in the above Figure 23, it can be confirmed that the BECOM and PETG manufactured in Example 1 exhibit distinct variations in members of the Streptococccaceae family, including S. parasanguinis, S. salivarius, and S. anginosus.

[0387] Experimental Example 9. Composting

[0388] Composting Methods

[0389] Figure 7a is a schematic diagram schematically illustrating composting content based on the International Organization for Standardization (ISO) 20200-17 for BECOM prepared in Example 1 and EPSF prepared in Example 2 (total 4.6 g per sample) under thermophilic and aerobic conditions.

[0390] As shown in the above Figure 7a, in order to confirm whether the BECOM manufactured in Example 1 can be disposed of in an environmentally friendly manner, Example 1 (BECOM) was made into a 4.0 cm particle having a dry weight of 4.6 g. 2After preparing the film-shaped samples, they were randomly divided into 2-3 groups and 3 batches per sample were used for thermophilic and aerobic composting experiments for 8 weeks using a standard compost soil with Bacillus as the dominant species defined in ISO 20200-17 in compliance with ISO 20200-17. In addition, in order to compare the natural composting characteristics of silk fibroin observed in the ecosystem, 4.55 g of EPSF prepared in Example 2 was used as a control group in a 4.0 cm 2 It was prepared and used in film form.

[0391] The above standard compost was prepared by mixing 40 wt% sawdust, 30 wt% rabbit feed, 10 wt% mature compost, 10 wt% corn starch, 5% sucrose, 4% corn seed oil, and 1% urea, and a polypropylene composting reactor with two holes of diameter Ø60 mm to allow gas exchange with the external environment was used. The initial mass ratio of BECOM or EPSF fibroin to wet compost was set to 2%, and then thermophilic incubation was performed in an air circulating oven at 60 ℃ by mixing the composting materials and periodically adding water to ensure sufficient aeration and moisture content. Then, the compost obtained at the 8th week was sieved, and the dry mass of BECOM and EPSF was measured to determine the composting rate according to Equation 3 below.

[0392] [Formula 3] (CM0-CM8) / CM0X 100

[0393] (In the above equation 3, CM0 means the weight of bioplastic before composting, and CM8 means the weight of bioplastic after 8 weeks of composting.)

[0394] Composting results

[0395] Figure 7b shows photographic images of BECOM manufactured in Example 1 and EPSF manufactured in Example 2 before composting (week 0) and after 8 weeks (week 8).

[0396] Figure 7c shows the initial (1st and 4th week) composting photographic images of BECOM manufactured in Example 1.

[0397] As shown in the above Figures 7b and 7c, the initial 4.0 cm of the 0th week 2 The single BECOM and EPSF samples in film form decomposed, turned black, distorted and fragmented after 8 weeks (Fig. 7b), and the BECOM prepared in Example 1 was slightly deformed from the first week of composting, and was shattered during composting and fragmented as indicated by the red arrows, and significant fragmentation was observed after the 4th week, indicating that the area of ​​the remaining BECOM was reduced by 20% due to fragmentation (Fig. 7c).

[0398] Bacillus species in the soil produce proteases during aerobic metabolism, and these proteases induce effective protein degradation of silk fibroin, resulting in rapid composting of silk fibroin. To confirm whether the BECOM prepared in Example 1 is sufficiently decomposed by the silk fibroin-decomposing enzyme, 1 x 1 cm of BECOM (n=4) prepared in Example 1 2 Matrix metallopeptidase 9, known as silk fibroin-degrading enzyme, and protease XIV (1.0 U mL) -1 , n=4) and collagenase IA were immersed in 4 mL of enzyme medium and cultured at 37°C for up to 72 hours, and the photographic images over time are shown in Figure 24.

[0399] Figure 24 shows the activity of BECOM prepared in Example 1 against metallopeptidase 9 (MMP-9; 1 nM activity, n=4), protease XIV (1.0 U mL -1 , n=4) and collagenase IA (1.0 U mL -1 , n= 4) after immersion in the enzyme medium, the photographic images according to the immersion time and the thickness according to time are shown.

[0400] As shown in the above Figure 24, after 24 hours of soaking the BECOM manufactured in Example 1 in protease XIV, the medium showed a slightly cloudy color, and it was found that protein degradation began after 24 hours due to the protease reaction of silk fibroin, and within 48 hours, the concentrated protease XIV medium (1.0 U mL -1 ) was completely decomposed, but was not well decomposed by collagenase. Through this, it was confirmed that the bioplastic according to one embodiment of the present invention can have excellent composting properties because it is well decomposed by protease secreted by B. subtilis, a beneficial bacterium present in actual compost, and it does not dissolve well in collagenase, so it shows high stability in saliva (spit) in the oral cavity.

[0401] Figure 7d shows the composting rate at the 8th week of composting of BECOM manufactured in Example 1 and EPSF manufactured in Example 2.

[0402] As shown in the above Figure 7d, the BECOM manufactured in Example 1 showed an excellent composting rate of 72.1% in the 8th week through protease reaction, and the EPSF of Example 2 achieved a complete composting rate of 96.7%, confirming that complete composting can be achieved within a few weeks after 8 weeks.

[0403] Soil microbial analysis after composting

[0404] BECOM composting can create an environment that is conducive to the development and growth of various microorganisms, and the compost soil condition is closely connected to the ecosystem. Therefore, in order to analyze the microbial status in the composted soil, well-mixed composts were obtained at weeks 0, 4, and 8 of composting of BECOM prepared in Example 1 and EPSF prepared in Example 2, and DNA was extracted from each compost. More specifically, in the preparation step, a mixture consisting of 5 μL of nuclease-free water, 10 μL of 10 ng input DNA, and 25 μL of LongAmp Hot Start Taq 2X Master Mix was strictly prepared in a 0.2 mL thin-walled PCR tube, and the tube was briefly centrifuged for homogenization, and PCR amplification was performed in a thermocycler under carefully optimized conditions. Afterwards, initial denaturation was performed at 95 °C for 1 min, followed by 25 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 30 s, and extension at 65 °C for 2 min, and the cycles were terminated with a final extension at 65 °C for 5 min, after which the sample was kept at 4 °C.

[0405] For library preparation, the protocol provided by ONT was used as described below. For the barcoding process, carefully selected 16S barcodes were selected from a 96-well plate, carefully isolated, thawed, and pierced through the foil surface using a sterile pipette tip. A 10 μL volume of mixed barcodes was then added to the corresponding PCR tube containing the previously amplified DNA. In the subsequent purification step, each sample was treated with 30 μL of resuspended AMPure XP beads. The samples were incubated on a rotating mixer at room temperature for 5 minutes and centrifuged to form a magnetic pellet. The clear supernatant was discarded, and the bead pellet was washed twice with 70% ethanol for magnetic separation. After magnetic separation, 10 μL of the clear eluate was collected in a sterile DNA LoBind tube. In the quantification and pooling steps, each eluted sample was quantified using a Qubit fluorometer, and then barcoded libraries were pooled to obtain concentrations in the range of 50–100 fmol, which were correlated with 50–100 ng for the ~1500 base pair (bp) 16S amplicon. Finally, 1 sL of RAP was added to the pooled DNA, mixed thoroughly, and incubated for 5 min.

[0406] Sequencing was performed and supervised using MinKNOW software (version 5.2.13), which handles various tasks such as data acquisition, real-time analysis, run feedback, and local base calling. Sequencing data were basecalled in real time using a version of Guppy integrated into MinKNOW software (version 3.3.2) with the help of MinIT device (version 19.05.2), and all FASTQ files were concatenated and used in a bioinformatics pipeline. MinION sequencing statistics (analyzed using adapters) were cleaned using Porechop (version 0.2.4) (read: minimum read length=100 bp, >80% ensure, minimum Q-score of five using filtlong (version 0.2.1)). The average sequence length of all samples was 1574 bases, and taxonomic classification of microbial 16S rRNA reads was performed with Kraken2 (version 2.0.8b) using the kraken2-microbe PlusPF-16 database.

[0407] Considering that the EPSF manufactured in the above Example 2 is a high-purity silk fibroin and that silk fibroin participates equally in the natural ecosystem, it can be assumed that the composting of EPSF will not affect the condition of the compost soil. Accordingly, the BECOM manufactured in the above Example 1 was monitored for microbiological characteristics, such as similarity with the EPSF control group, at 8 weeks through 16S rRNA gene sequence analysis of the compost microbial community, and also proved the composting action without side effects.

[0408] Figure 7e shows the ACE alpha diversity matrix (metrics) of the compost soil obtained over 8 weeks of the control (EPSF).

[0409] Figure 25 shows (a) observations and (b) Chao1 alpha diversity metrics of compost soil obtained at 0, 4, and 8 weeks of the control group (EPSF).

[0410] As shown in FIG. 7e and FIG. 25, the change in microbial diversity over time expressed by the ACE alpha diversity estimator confirmed that the compost had matured according to the experimental schedule, indicating an improvement in microbial diversity and species richness (FIG. 7e), and the Chao1 richness and observed species index confirmed that the microbial species richness had increased significantly (FIG. 25).

[0411] Figure 7f shows the results of comparing the ACE abundance of composted soil obtained after 8 weeks of composting of BECOM manufactured in Example 1 and EPSF manufactured in Example 2.

[0412] Figure 26 shows (a) observations and (b) Chao1 alpha diversity metrics of composted soil obtained after 8 weeks of composting of BECOM prepared in Example 1 and EPSF prepared in Example 2.

[0413] As shown in the above FIG. 7f and FIG. 26, it can be seen that the BECOM manufactured in the above Example 1 and the EPSF manufactured in the above Example 2 exhibit similar characteristics with respect to microbial diversity at the end of composting.

[0414] Figure 7g shows a principal coordinate analysis (PCoA) plot of the compost soil obtained after 8 weeks of composting with the standard compost at week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2, wherein the amplicon sequence variants in Figure 7g were measured using the Bray-Curtis dissimilarity method. In addition, the specific analysis results of the species-level similarity ratio of the compost soil obtained after 8 weeks of composting with the BECOM prepared in Example 1 and EPSF prepared in Example 2 are shown in Table 4 below.

[0415] TaxonAverage dissimilarity aContribution %Cumulative %Mean abundanceBECOMESPFParageobacillus genomosp1.7146.0786.0787280.00256.00Steroidobacter denitrificans1.2874.56310.64881.004810.00Bacillus velezensis1.2714.50715.1513900.009050.00Amphibacillus xylanus1.0443.70318.858600.006380.00Bacillus cereus0.94363.34522.211300.007550.00Bacillus thuringiensis0.77822.75924.9510200.007370.00Ureibacillus thermophilus0.70122.48627.444300.001480.00Symbiobacterium thermophilum0.61532.18129.62681.002830.00Ureibacillus thermosphaericus0.47791.69431.322500.00842.00Caloranaerobacter azorensis0.44421.57532.891940.002470.00CandidatusFilomicrobium marinum0.43381.53834.43684.001990.00Bacillus subtilis0.39251.39235.825320.005010.00Salicibibacter halophilus0.29741.05436.873380.002500.00Herbinix luporum0.2971.05337.93399.001270.00Lentibacillus sp. CBA36100.29211.03638.963690.002550.00

[0416] As shown in the above Figure 7g and Table 4, the compost soil of BECOM manufactured in Example 1 and EPSF manufactured in Example 2 had R 2A notable overlap was observed with a value of 0.201 (F value = 0.75), and the groups of composted soils of BECOM prepared in Example 1 and EPSF prepared in Example 2 were closely clustered with the first principal component, accounting for 69.98%, and further analysis of taxon-level similarity percentage (SIMPER) showed a low average dissimilarity of 28.21%. This impressive microbial community similarity between BECOM and EPSF decomposed composts indicated that composting of BECOM could be included in the natural life cycle of soil microorganisms.

[0417] Microorganisms that thrive in compost dissolve nutrients bound to the compost, mediate enzymatic reactions, ultimately aiding plant growth. Through microbial metabolism, they can contribute to compost maturation, phosphorus dissolution, and nitrogen fixation. Therefore, the microbial characteristics of the compost soils produced with BECOM in Example 1 and EPSF in Example 2 were analyzed.

[0418] Figure 7h shows the relative abundance of the 30 most abundant species of compost soil obtained according to the composting time of the control group (Compost), BECOM prepared in Example 1, and EPSF prepared in Example 2, and the microbial community structure of at least two samples was analyzed.

[0419] In particular, B. subtilis, an essential microorganism of compost soil that promotes compost maturation and improves compost quality, had an initial log-scaled abundance of 0.48, but the final log-scaled abundance significantly increased to 3.73 in the compost soil composted with BECOM of Example 1, which was very close to the value (3.70) of the compost soil composted with ESPF prepared in Example 2.

[0420] Figure 27 is a graph showing the relative abundance of Serratia species in composted soil obtained after composting standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2 for 8 weeks.

[0421] Figure 28 is a graph showing the relative abundance of (a) Enterobacter species and (b) Klebsiella species in composted soil obtained after composting standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2 for 8 weeks.

[0422] Figure 29 is a graph showing the relative abundance of Pseudomonas species in composted soil obtained after composting standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2 for 8 weeks.

[0423] Figure 30 is a graph showing the relative abundance of B. licheniformis and B. paralicheniformis in composted soil obtained after 8 weeks of composting with standard compost of week 0, BECOM prepared in Example 1, and EPSF prepared in Example 2.

[0424] As shown in FIGS. 27, 28, 29, and 30, Serratia sps, which contributes to phosphate dissolution, was observed in large quantities in the compost soil obtained after 8 weeks of composting of BECOM of Example 1 and EPSF of Example 2 (FIG. 27), and the dominance of nitrifying bacteria, Enterobacter sps, and Klebsiella sps was confirmed (FIG. 28). In addition, Pseudomonassps, which is known to assist heterotrophic nitrification and denitrification together with Serratia sps and increase available phosphorus content, was observed to increase in all samples after composting, although the relative proportion was low (FIG. 29). In addition, B. licheniformis and B. paralicheniformis, which have a positive effect on improving microbial cooperation to enhance microbial metabolism, were not detected in the initial compost, but showed sufficient amounts in both BECOM- and EPSF-based composts (FIG. 30). Through this, it was confirmed that the beneficial microbial species increased in BECOM and EPSF-based compost, which could improve the compost soil condition and provide significant microbiological benefits.

[0425] The results of the above Experimental Example 7 demonstrate that the BECOM manufactured in the above Example 1 is not only compostable but also exhibits microbiological benefits to the soil, so that it can be disposed of in an environmentally friendly manner through the composting process. Therefore, when its lifespan ends, it can be disposed of through an environmentally friendly and practical composting method without incineration, making it a sustainable technology. The composting protocol ISO 20200-17 used was confirmed to be an efficient method for preparing microbiologically superior compost using BECOM.

[0426] According to recent agroecosystem research, among the characteristics of compost, pH, carbon-to-nitrogen (C / N) ratio, and abundance of B. subtilis can greatly determine crop productivity. Specifically, it has been reported that the closer the pH is to alkaline, the more active the compost soil can be, and the lower the C / N ratio, the more mature the compost can be. Based on this, inoculation of B. subtilis is performed in the agricultural field to improve crop growth. From this point of view, in order to evaluate the characteristics of the compost soil composted with the BECOM manufactured in Example 1 and the EPSF manufactured in Example 2, the pH, C / N ratio, and B. subtilis of the compost soil obtained 8 weeks after composting with the BECOM manufactured in Example 1 and the EPSF manufactured in Example 2 were compared with the initial ones.

[0427] As a result, the pH of the initial compost soil was 6.4 and the C / N ratio was 23.2%, but 8 weeks after composting with BECOM of Example 1 and EPSF of Example 2, the pH increased to 8.2 and the C / N ratio significantly decreased to 13.8% and 13.5%, respectively. Furthermore, the abundance of B. subtilis increased approximately 7.77 times after BECOM composting compared to the initial period, confirming that BECOM can potentially contribute to agricultural production systems as an excellent compost resource.

[0428] The microorganisms in the compost soil obtained 8 weeks after composting the BECOM manufactured in Example 1 and the EPSF manufactured in Example 2 were analyzed at the phylum level, and the number and relative ratio are shown in Table 5 below.

[0429] TaxaAbsolute countRelative abundance (%)BECOMESPFBECOMESPFFirmicutes151065.00131292.0085.3%74.1%Proteobacteria14445.5030649.678.2%17.3%Actinobacteria9147.5011346.335.2%6.4%Planctomycetes301.50645.000.2%0.4%Armatimonadetes297.50464.670.2%0.3%Acidobacteria225.00463.000.1%0.3%Chloroflexi219.50336.670.1%0.2%Atribacterota231.00315.000.1%0.2%Fusobacteria225.50267.330.1%0.2%Spirochaetes214.00242.330.1%0.1%Bacteroidetes170.50238.000.1%0.1%Tenericutes116.00120.330.1%0.1%Synergistetes101.50118.330.1%0.1%Gemmatimonadetes63.50110.330.0%0.1%Chrysiogenetes70.5097.330.0%0.1%Deferribacteres75.0072.330.0%0.0%Deinococcus-Thermus50.5083.670.0%0.0%Thermodesulfobacteria33.5079.000.0%0.0%Fibrobacteres33.5073.000.0%0.0%Coprothermobacterota23.0027.670.0%0.0%Aquificae6.5034.000.0%0.0%Cyanobacteria17.5022.670.0%0.0%Verrucomicrobia12.5025.000.0%0.0%Thermotogae12.5017.330.0%0.0%Nitrospirae9.5013.330.0%0.0%Chlorobi6.0014.000.0%0.0%Kiritimatiellaeota7.506.330.0%0.0%Chlamydiae1.505.670.0%0.0%Calditrichaeota3.504.000.0%0.0%Elusimicrobia3.503.330.0%0.0%Ignavibacteriae1.503.670.0%0.0%Caldiserica1.501.670.0%0.0%.

[0430] As shown in Table 5 above, the dominant phyla order of BECOM of Example 1 and EPSF of Example 2 were identical, Firmicutes, Proteobacteria, and Actinobacteria, after 8 weeks of composting, and the relative abundances of Firmicutes and Actinobacteria showed great similarity. On the other hand, the relative abundance of Proteobacteria was observed to be 8.2% and 17.3% in BECOM of Example 1 and EPSF of Example 2, respectively. In particular, Proteobacteria play an important role in composting by promoting the early mesophilic stage and assisting plant farming, so EPSF may be a better choice than BECOM if the goal is to quickly prepare better compost during the same composting period. Medical plastic waste contaminated with microorganisms can cause public cross-infection, and cases of cross-infection with Mycobacterium tuberculosis, human immunodeficiency virus, and hepatitis B have been reported. Therefore, cross-contamination by contaminated medical plastic waste must be considered, and the preferential growth of pathogens in compost must be considered to prevent the possibility of secondary cross-contamination caused by contaminated landfills. Accordingly, the presence of Escherichia coli, a common pathogen commonly found in compost due to the presence of animal organic matter, was analyzed in the compost soil obtained after composting BECOM produced in Example 1 and EPSF produced in Example 2 for 8 weeks, and the results are shown in Figure 31.

[0431] Figure 31 is a graph showing the abundance of E. coli in composted soil obtained after composting BECOM manufactured in Example 1 and EPSF manufactured in Example 2 for 8 weeks (p-value = 2.45E-12, FDR value = 5.32E-10).

[0432] As shown in the above Figure 31, E. coli was prevalent in the initial 0-week compost, accounting for 46% of the relative abundance, but as composting progressed, both BECOM and EPSF showed remarkable resistance, and the relative abundance significantly decreased to 0.32% and 0.54%, respectively. It was confirmed that BECOM is microbially clean after dental treatment due to its excellent antibacterial performance, and thus concerns about cross-infection can be alleviated during post-processing (pickup, transportation, etc.) of used BECOM.

[0433] Above, embodiments of the present invention have been described, but those of ordinary skill in the art will be able to modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., within the scope that does not depart from the spirit of the present invention described in the claims, and this will also be considered to be included within the scope of the rights of the present invention.

Claims

1. A bioplastic comprising modified silk fibroin having an increased content of β-sheets by being modified from natural silk fibroin, wherein the modified silk fibroin has an intertwined structure.

2. A bioplastic comprising 80% or more of the modified silk fibroin based on 100% of the entire bioplastic in the first paragraph.

3. In the first paragraph, the bioplastic has a spectrum of 1665±10 cm according to Raman spectroscopy. -1 It has a peak showing the maximum intensity in the region, and the half width of the peak is 10 to 80 cm. -1 In bioplastics.

4. In the first paragraph, the bioplastic is a bioplastic having a bio-based carbon content of 80% or more.

5. In the first paragraph, the bioplastic has a Kratky plot of q = 0.5 to 1.3 nm. -1 Bioplastics exhibiting effective peaks in .

6. In the first paragraph, the bioplastic has a wide-angle X-ray scattering (WAXS) analysis result of 14.9 nm. -1 It exhibits an a-axis stacking peak at 17.6 nm. -1 Indicates the c-axis stacking peak, and the ratio of the a-axis stacking peak intensity to the c-axis stacking peak intensity (I a / I c ) bioplastics with a molecular weight of 0.85 to 1.

7.

7. A bioplastic further comprising an antibacterial urethane oligomer according to claim 1.

8. In the 7th paragraph, the urethane oligomer is a bioplastic manufactured by reacting a polyol and an antibacterial diisocyanate.

9. A bioplastic according to claim 8, wherein the antibacterial isocyanate compound is a heterocyclic diisocyanate compound containing a quaternary ammonium salt.

10. In the 8th paragraph, the antibacterial urethane oligomer is a bioplastic having a hydroxyl group at the terminal.

11. In the first paragraph, the bioplastic is a bioplastic in which the ratio of the beta sheet content of the modified silk fibroin to the beta sheet content of the natural silk fibroin expressed by the following formula 1 is greater than 1.

5. [Formula 1] X1 / X2 (In the above equation 1, X1 is the beta sheet content of the bioplastic calculated through Raman spectroscopy, and X2 is the beta sheet content of natural silk fibroin.) 12. In the first paragraph, the bioplastic is a bioplastic having an ultimate stress of 10 MPa or more.

13. In the first paragraph, the bioplastic is a bioplastic having an elastic modulus (E) of 500 MPa or more as measured according to ASTM D882.

14. In the first paragraph, the bioplastic is a bioplastic having a toughness (T) of 300 MJ / ㎥ or more as measured according to ASTM D882.

15. In the first paragraph, the bioplastic is a bioplastic having a stress relaxation rate of 0.2 or more, as expressed by the following equation 2. [Formula 2] (F0-F 30m ) / F0 (In the above equation 2, when a stress relaxation test is performed on a bioplastic with a thickness of 500 ㎛ under a strain condition of 2 to 3%, F0 is the initial stress, and F 30m is the stress after 30 minutes.) 16. In the first paragraph, the bioplastic is a bioplastic having a composting rate of 60% or more, as expressed by the following formula 3, when composted according to ISO 20200-17. [Formula 3] (CM0-CM8) / CM0X 100 (In the above equation 3, CM0 means the weight of bioplastic before composting, and CM8 means the weight of bioplastic after 8 weeks of composting.) 17. In the first paragraph, the bioplastic is a bioplastic that exhibits an antibacterial effect against at least one oral disease or periodontal disease-causing bacteria selected from the group consisting of Streptococcus mutans and Staphylococcus aureus.

18. A biomedical molded product comprising a bioplastic according to any one of claims 1 to 17.

19. An orthodontic device comprising a bioplastic according to any one of claims 1 to 17. 20.(A) A step of injecting a natural silk fibroin solution into a urethane oligomer casting mold; and (B) A method for producing a bioplastic, comprising the step of drying the natural silk fibroin solution to produce a bioplastic containing denatured silk fibroin.

21. In paragraph 20, The above urethane oligomer casting mold (A-1) a step of preparing a polymerizable composition by mixing a polyol and an antibacterial isocyanate compound; and (A-2) A method for producing a bioplastic, comprising a step of casting the above polymerizable composition into a mold and then reacting it.

22. In paragraph 20, In the above step (A), The above natural silk fibroin solution is a method for producing a bioplastic in which natural silk fibroin protein and cosmotropic salt are dissolved in an acidic solvent.

23. In the 20th paragraph, the cosmotropic salt comprises an alkali metal ion or an alkaline earth metal ion; and sulfate (SO4 2- ), phosphate (HPO4 2- ), acetate (CH3COO - ), hydroxide (OH - ), chloride (Cl - ), bromide (Br - ) and formate (HCOO - ) is selected from the group consisting of one or more ions; a method for producing a bioplastic which is a combination of:

24. In paragraph 20, A method for producing a bioplastic, wherein the natural silk fibroin protein contained in the natural silk fibroin solution is crystallized during drying in the above step (B) to produce denatured silk fibroin.

25. In paragraph 20, After step (C) above, A method for producing a bioplastic, further comprising the step of immersing a mold containing the bioplastic in a storage solution.

26. In paragraph 25, the storage solution is C 1-7 A method for producing a bioplastic containing alkyl polyol.

27. A step of immersing the bioplastic of any one of claims 1 to 17 in an acidic solvent; and A method for recovering silk fibroin from a bioplastic, comprising the step of recovering silk fibroin from an acidic solvent in which the bioplastic is immersed.

28. A step of preparing a bioplastic solution by dissolving the bioplastic and acidic solvent of any one of claims 1 to 17; A step of injecting the above bioplastic solution into a urethane oligomer casting mold; and A method for reshaping a bioplastic, comprising the step of drying the bioplastic solution to produce recycled bioplastic.

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