WO2025053734A1 - Bioplastic and manufacturing method therefor - Google Patents

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

Bioplastic and its manufacturing method

The present invention relates to bioplastic and a method for producing the same.

Medical bioplastic materials used in living organisms must satisfy not only antibacterial properties and biocompatibility, but also mechanical properties such as formability, tensile strength, elastic modulus, and toughness depending on the product to which they are applied. In particular, in the case of materials used in orthodontics, if a material does not exhibit stress relaxation behavior, the patient may experience severe pain or discomfort due to orthodontic force exceeding the patient's pain threshold, so it is desirable to use bioplastic materials exhibiting stress relaxation behavior. In addition, orthodontic devices must be worn continuously for approximately 20 hours a day, excluding meals and washing, and must be repeatedly reused for several months. Therefore, dental plastics are exposed to repeated and significant loads that occur during essential oral activities, and furthermore, they must exhibit viscoelasticity to accurately transmit orthodontic force to the teeth as the tooth position is adjusted in submillimeter units (0.6 mm or less). Therefore, dental bioplastic materials must be flexible, tough, and viscoelastic to enable 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.

Meanwhile, with the development of bioplastics, the United States generates an average of 0.5 kg of medical plastic waste per day and a total of 6 million tons per year. Of this medical plastic waste, 91% is inappropriately infiltrated into nature through landfill or incineration, worsening the environment. In particular, in orthodontic treatment using orthodontic devices, since the positions of teeth are precisely adjusted with a series of orthodontic devices, patients must change to a new type of orthodontic device every 1-2 weeks, inevitably generating plastic waste every week. For example, it was reported that a single institution collected 11,483 pieces of plastic waste related to orthodontic devices weighing 117.8 kg in just one year. In other words, a huge amount of dental plastic waste is generated in dental treatment, so bioplastics must exhibit excellent biodegradation properties so that they can be disposed of without worsening the environment.

In response to these demands, recent studies have reported bioplastics with excellent biodegradability properties using materials such as cellulose, wood, food, silk fibroin, and pollen, but the effect of biodegradation of bioplastics on the destruction of microorganisms in landfills has been rarely discussed. On the other hand, since dental bioplastics are used in the oral cavity, which is very rich in microorganisms, microorganisms that easily grow on bioplastics can affect 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 should have high microbial resistance, be compostable without leaving toxic residues in landfills, and affect the microbiological characteristics.

Meanwhile, silk is the second most abundant natural material after cellulose. Generally, silk refers to a fibrous protein released from a silkworm cocoon, and specifically, cocoon thread released from a silkworm cocoon is largely composed of two proteins: silk sericin and silk fibroin. Specifically, it contains about 75% fibroin protein, about 25% sericin protein, and about 3% minerals and carbohydrates. In order to expand the practicality of silk, it goes through a degumming process to remove impurities and sericin using alkali, soap, enzymes, bleach, etc., and separates pure fibroin protein.

Silk fibroin material has excellent toughness among known protein materials and is utilized in various material industries. Specifically, it is used as a scaffold for sutures and cell therapy. The above-mentioned properties can be explained by examining the protein secondary structure of silk fibroin. In particular, the more beta-sheet crystals there are with strong internal force, the better it can withstand the applied external force and exhibit high tensile strength. However, as the beta-sheet crystal content increases, the amorphous content decreases, which lowers flexibility and easily breaks, exhibiting poor stretchability.

Therefore, research and development of 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 are necessary.

[Prior art literature]

[Patent Document]

Patent Document 1. Korean Publication Patent No. 10-2023-0174848

The present invention has been made to solve the problems described above, and the purpose of the present invention is to provide a bioplastic including 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.

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

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

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 including denatured silk fibroin.

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.

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.

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.

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

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

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

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.

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.

Another aspect of the present invention provides a method for re-molding a bioplastic, comprising: a step of preparing a bioplastic solution by dissolving the bioplastic and an acidic solvent; a step of injecting the bioplastic solution into a urethane oligomer casting mold; and a step of drying the bioplastic solution to prepare a recycled bioplastic.

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 excellent maximum tensile strength and toughness as well as 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, so that it can be easily used in an in vivo environment where fluid always exists, and can be widely utilized in the biomedical industry.

The effects of the present invention are not limited to the effects mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

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

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.

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.

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

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

Figure 2e shows the glass transition temperature (T _g ) of BECOM (Templated) manufactured in Example 1 and natural silk fibroin (Native) manufactured in Comparative Example 2.

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

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

Figure 3a is a schematic diagram illustrating a process of performing orthodontic treatment using BECOM and PETG manufactured in Example 1 as an orthodontic device.

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.

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

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

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.

Figure 3f shows the tooth displacement profile of the right maxilla after correction of teeth using the orthodontic appliance using BECOM of Example 1.

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

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

Figure 4b schematically illustrates a three-step process for orthodontic correction 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.

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

Figure 4d shows a frontal 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 appliance manufactured in Example 1.

Figure 4e shows a plan view image of teeth in which malocclusion is progressing due to non-orthodontic treatment with an orthodontic appliance.

Figure 5a is a schematic diagram illustrating a recycling procedure based on disentanglement of BECOM manufactured in Example 1.

Figure 5b shows the chemical structures of the antibacterial group (R _3A ) and dye tracer (Rhodamin B, R _3B ) of the urethane oligomer casting mold used in Example 1.

Figure 5c shows photographic images of BECOM prepared in Example 1 and silk fibroin extracted in Example 2 according to the substitution ratio (R _3A /(R _3A + R _3B )) of the antimicrobial group and the dye tracer of the urethane oligomer casting mold used in Example 1.

FIG. 5d shows ¹³ C nuclear magnetic resonance (NMR) spectra of BECOM prepared in Example 1, silk fibroin extracted in Example 2, and natural silk fibroin prepared in Comparative Example 2.

Figure 5e shows photographic images of the urethane oligomer casting mold (1 mL) extracted by recycling BECOM of Example 1 having a substitution ratio of 50 mmol% of the antimicrobial group and dye tracer of the urethane oligomer casting mold in Example 2 after the first and fifth extraction processes.

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.

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

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.

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

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

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.

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.

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

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 on BECOM and PETG in Example 1. In Figure 6f, PC1 and PC2 represent the first and second coordinates, respectively.

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

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

Figure 7a is a schematic diagram 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.

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).

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

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

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

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.

Figure 7g shows the principal coordinate analysis (PCoA) analysis 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.

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

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

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.

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

Figure 11 shows (a) the normalized strain versus time profile of BECOM manufactured in Example 1 when subjected to a creep stress of 1.0 kPa 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.

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 under tooth resistances of 0.1-0.5 mm of BECOM and PETG manufactured in Example 1.

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.

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

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.

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.

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.

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

Figure 19 shows (a) ¹ H nuclear magnetic resonance (NMR; Avance III HD 300, Bruker) spectra and (b) photograph images (1 mL per vial) of the urethane oligomer casting mold extracted by recycling BECOM having a substitution ratio of 50 mmol% of the antimicrobial group and dye tracer of the urethane oligomer casting mold in Example 2 according to the extraction cycle.

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.

Figure 21 shows stress-strain curves (n=3) when a uniform tension is applied at a displacement rate of 5.0 mm min ^-1 to beam-shaped specimens of 4 cm ² using (a) BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4, 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.

Figure 22 is a graph showing the reaction distance of fibroblast confluent monolayer when treated with (a) negative control (NC; high-density polyethylene film), positive control (PC; 0.1% ZDEC polyurethane film), and BECOM prepared in Example 1 (n=3), (b) cell viability after co-culturing the 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) relative expression levels of inflammatory biomarkers by BECOM prepared in Example 1 and the control (Mock).

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

Figure 24 shows the photographic images and the thickness over time of BECOM manufactured in Example 1 immersed in an enzyme medium containing 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) according to immersion time.

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.

Figure 26 shows (a) observation and (b) Chao1 alpha diversity metric of compost soil obtained after 8 weeks of composting of BECOM prepared in Example 1 and EPSF prepared in Example 2.

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

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

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.

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

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).

The advantages and features of the present invention, and the methods for achieving them, will become clear 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, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person having ordinary skill in the art to which the present invention belongs of the scope of the invention, and the present invention is defined only by the scope of the claims.

In describing the present invention, if it is judged that a specific description of a related known technology may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. When "includes," "has," "consists of," etc. are used in this specification, other parts may be added unless "only" is used. In addition, the terms "includes" or "has" etc. are intended to specify the presence of a feature, number, step, component, or combination thereof described in the specification, and should not be understood as excluding 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 singular, it includes a case where the plural is included unless otherwise explicitly stated. In the following specification, units used without special mention are based on weight, and for example, a unit of % or ratio means weight% or a weight ratio.

Additionally, the numerical range used in this specification may include a lower limit and an upper limit 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 a numerical range defined in different shapes. Unless otherwise specifically defined in the specification of the present invention, values outside the numerical range that may occur due to experimental error or rounding of values are also included in the defined numerical range.

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

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.

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 analysis conditions or the analyst.

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

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 a rock, and silkworm silk from the cocoon of the silkworm (Bombyx mori). Considering high productivity, it is more common to use silk fibroin protein obtained by extraction from the cocoon of the silkworm (Bombyx mori). In addition, according to a preferred embodiment, the natural silk fibroin may be derived from the silkworm moth.

Silk fibroin protein is a form in which a crystal structure is added to an irregular matrix, and the crystal structure is formed by stacking β-sheets in a layered form through hydrogen bonding. The representative base sequence of silk fibroin extracted from silkworm cocoons is GAGAGS at about 53% and GAGAGY at about 18%. A large portion is occupied by hydrophobic amino acids such as glycine (G) and alanine (A).

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

The above-mentioned modified silk fibroin can exhibit an entangled structure.

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

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

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.

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 can mean dimensionless %.

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, and the like, 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, and the like.

According to one embodiment of the present invention, the bioplastic may have a peak intensity (I _PU ) in a region of 1590 ±10 cm ^-1 to a peak intensity (I _BIO ) in a region of 1650 ±10 cm ^-1 of a spectrum according to FT-IR spectroscopy of 0 to 0.8:1, or 0 to 0.5:1, or 0 to 0.2:1. The I _PU is a peak intensity indicating polyurethane, and I _BIO is a peak intensity indicating the bioplastic according to one embodiment. In other words, the bioplastic according to one embodiment may not substantially contain polyurethane therein.

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

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

The above bio-based carbon content is an indicator used to define bioplastics, and means the content of carbon derived from biomass-based resources, and can be calculated from ^{the 14} C radiocarbon content measured according to ASTM D 6866-22 Method B and the modern carbon content.

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, It exhibits an effective peak at q= 0.9 to 1.0 nm ^-1 , and the intensity of the effective peak can also significantly increase compared to natural silk fibroin.

Bioplastics that exhibit a valid 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.

According to one embodiment of the present invention, as shown in FIG. 9b, the bioplastic according to one embodiment exhibits an a-axis stacking peak at 14.9 nm ^-1 and a c-axis stacking peak at 17.6 nm ^-1 as a result of wide-angle X-ray scattering (WAXS) analysis, and a 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.

Bioplastics satisfying the above-described range of I _a /I _c values exhibit excellent flexibility and toughness, so that no damage may occur even after repeated twisting and bending.

According to one embodiment of the present invention, the bioplastic may include an antibacterial urethane oligomer. The antibacterial urethane oligomer may be mixed inside the bioplastic, or the antibacterial urethane oligomer may not be mixed inside the bioplastic, but may be released in a small amount from the antibacterial urethane oligomer casting mold during the process in which the casted liquid bioplastic self-assembles into a solid and present in a small amount in the bioplastic. The content of the antibacterial urethane oligomer may be less than 20%, preferably less than 17%, and more preferably 7 to 15%, with respect to the total 100% of the bioplastic, but is not limited thereto. Accordingly, the surface of the bioplastic may exhibit antibacterial properties.

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

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

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 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 weight 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, or the like can be used.

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.

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.

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. The molecular weight may be controlled as the polyol is excessively added, 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.

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, more specifically 1,000 to 3,000 g/mol.

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

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 Equation 1 of greater than 1.5, preferably greater than 2.0.

[Formula 1] X ₁ /X ₂

(In the above equation 1, X ₁ is the beta sheet content of the bioplastic calculated through Raman spectroscopy, and X ₂ is the beta sheet content of natural silk fibroin.)

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 exceeds 1.5, preferably exceeds 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 being modified. In addition, when the above formula 1 exceeds 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 exceeds 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.

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 manufactured through a simple manufacturing method, and can exhibit significantly improved beta-sheet content and high crystallinity, and can also implement excellent mechanical properties such as toughness, tensile strength, and elastic modulus.

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

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.

It was confirmed that when the glass transition temperature of the bioplastic satisfies the above range, the entangled structure of silk fibroin within the bioplastic is formed densely.

The above glass transition temperature can be measured using a differential scanning calorimeter (DSC) at a heating rate of 5 to 20 ℃ min ^-1 .

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.

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.

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/㎥.

The above ultimate stress, elastic modulus (E), and toughness (T) may be measured from a specimen in the form of a beam with a thickness of 400 μm and a size of 4 cm ² .

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 improves both tensile strength and ductility, which were previously in conflict with each other, thereby overcoming the limitations between the two properties, and has the advantage of having excellent maximum tensile strength and toughness as well as antibacterial properties and exhibiting excellent stress relaxation behavior.

According to one embodiment of the present invention, the bioplastic may have a maximum strain (ε _max ) 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 of 40 to 200%, preferably 60 to 100%.

According to one embodiment of the present invention, the bioplastic may have a stress relaxation rate expressed by Equation 2 below of 25% or more, preferably 50% or more.

[Formula 2] (F ₀ -F _30m )/F ₀ x 100

(In the above equation 2, when a stress relaxation test is performed on a 500 ㎛ thick bioplastic under a strain condition of 2 to 3%, F ₀ is the initial stress and F _30m is the stress after 30 minutes.)

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.

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 composting is completed.

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

[Formula 3] (CM ₀ -CM ₈ )/CM ₀ X 100

(In the above equation 3, CM ₀ means the weight of bioplastic before composting, and CM ₈ means the weight of bioplastic after 8 weeks of composting.)

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

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, Pseudomonas sps, Enterobacter sps , Klebsiella sps , B. licheniformis , and B. paralicheniformis .

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.

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

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

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.

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.

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%.

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

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 .

Bioplastics that exhibit antibacterial effects against the above 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 when used as orthodontic devices.

The present invention can provide a biomedical molded article comprising the above-described bioplastic. The molded article can 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 article, 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 preferable because spinning is difficult.

In addition, the bioplastic does not cause stickiness between materials because internal hydrogen bonds are maintained due to the high crystallinity even when exposed to water. Accordingly, 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.

The present invention can provide an orthodontic device including the bioplastic described above. The orthodontic device can be manufactured with a structure of a commonly used or known orthodontic device (aligner) or orthodontic retainer. For example, it can be a mouth piece type structure of registered patent 10-1476715 or a structure such as Hawley, Wraparound, Begg, VFR vacuum formed, etc., but is not limited thereto.

As shown in the above Fig. 3a, in a 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 exhibit resistance to 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 and at the same time withstand the resistance of the teeth to enable successful treatment. In the case of the above-described bioplastic, not only can it sufficiently withstand the resistance of the teeth, but also exhibit excellent viscoelastic properties, making it suitable for use as an orthodontic device.

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

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

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

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

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 mixing a polyol and an antimicrobial isocyanate compound to manufacture a polymerizable composition; and (A-2) a step of casting the polymerizable composition into a mold and then reacting it.

The above isocyanate compound means a compound containing an isocyanate group, and may preferably be a cyclic diisocyanate having two or more isocyanate groups.

The above antimicrobial isocyanate compound can be produced by substituting an antimicrobial compound for an isocyanate compound, for example, a reactive compound containing a quaternary ammonium salt can be reacted with an isocyanate compound to ultimately produce an antimicrobial isocyanate compound containing a quaternary ammonium salt, but is not limited thereto.

Specifically, the above 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.

Specific examples of the polyol compounds are the same as those described above and are therefore omitted.

The above polyol and antimicrobial isocyanate compound can be mixed, the polymerizable composition is stirred with a mixer, and then cast into a mold of a specific shape and reacted. The above reaction can be applied without significant limitation as long as it is a common and known condition for urethane reaction, and can be reacted for 5 hours, specifically 10 hours or more at a temperature of 30° C. or higher, specifically 50° C. or higher. Furthermore, a drying step can be further performed, and can be dried for 1 hour or more at a temperature of 70° C. or higher, but is not limited thereto.

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.

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

The above 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 is preferably a sodium carbonate solution, but is not limited thereto.

The acidic solvent may be a C _1-7 organic acid. For example, it may be, but is not limited to, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, fumaric acid, malic acid, citric acid or a mixed solvent thereof.

The above cosmotropic salt is known to contribute to the stabilization of water molecules in an aqueous environment, and also to contribute to the stabilization of intramolecular interactions in macromolecules such as proteins. The above cosmotropic salt may be a combination of an alkali metal ion or an alkaline earth metal ion; and one or more ions selected from the group consisting of sulfate (SO ₄ ^2- ), phosphate (HPO ₄ ^2- ), acetate (CH ₃ COO ^- ), hydroxide (OH ^- ), chloride (Cl ^- ), bromide (Br ^- ), and formate (HCOO ^- ). Preferably, the cosmotropic salt may be one or more or two or more ions selected from calcium chloride, lithium bromide, potassium chloride, sodium chloride, and sodium acetate, and may be calcium chloride, but is not limited thereto.

According to one embodiment of the present invention, the step (A) is performed by preparing the natural silk fibroin solution described above 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 vary.

(B) A step of drying the natural silk fibroin solution to produce a bioplastic containing denatured silk fibroin.

The above step (B) is a step of drying the natural silk fibroin solution to produce a bioplastic containing denatured silk fibroin.

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 significantly improved beta-sheet content than the natural silk fibroin protein, thereby producing a bioplastic including denatured silk fibroin having a high crystallinity.

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

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

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 longer, 30 minutes or longer, or 1 hour or longer, and by including the step, a bioplastic having better physical properties such as tensile strength can be manufactured.

According to one embodiment of the present invention, the storage solution may include a polyol, specifically a C _1-20 alkyl polyol or a C _1-7 alkyl polyol. As an 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.

The bioplastic according to one embodiment of the present invention has the advantage of being easily recyclable. As a method for recycling, silk fibroin can be extracted from the bioplastic and recycled, or the entangled structure can be unraveled and then manufactured (reshaped) into a bioplastic using a urethane oligomer casting mold and recycled.

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.

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 released, 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.

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 contain a cosmotropic salt like the above-described natural silk fibroin solution.

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, and at this time, the silk fibroin in the bioplastic and the urethane oligomer casting mold may be separated. The recovery may be performed multiple times until the urethane oligomer casting mold is completely separated.

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

The above yield can be calculated by the following equation 4.

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

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).

The present invention provides a method for re-molding a bioplastic, comprising: a step of preparing a bioplastic solution by dissolving the bioplastic and an acidic solvent; a step of injecting the bioplastic solution into a urethane oligomer casting mold; and a step of drying the bioplastic solution to prepare a recycled bioplastic.

In the method for re-molding 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 manufacturing the above-described bioplastic.

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

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

Manufacturing Example 1. Manufacturing of natural silk fibroin

Mori silkworm (silkworm moth) cocoons were degummed by soaking them in a sodium carbonate solution (0.02 M Na ₂ CO ₃ ) for 1 hour. Afterwards, they were washed and dried at room temperature to obtain degummed natural silk fibroin.

Manufacturing Example 2. Manufacturing of urethane oligomer casting mold (template)

Propylene carbonate (Sigma Aldrich, USA) was added 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) and reacted at 80 °C for more than 1 hour to prepare an antimicrobial diisocyanate compound.

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. Next, 4 mL of the polymerizable composition was placed 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 chemical structure of the antibacterial group (R _3A ) in the urethane oligomer is shown in Fig. 5b.

Example 1. Manufacturing of bioplastic (BECOM)

The refined silk fibroin of Manufacturing Example 1 was added to a solution containing 0.3 wt% calcium chloride (CaCl ₂ ) in formic acid to a concentration of 0.1 g mL ^-1 and dissolved at 60 ° C. for 1 hour to obtain a natural silk fibroin solution.

The above natural silk fibroin solution was injected into the urethane oligomer casting mold of the above-mentioned 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.

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.

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

Figure 5a is a schematic diagram illustrating a recycling procedure based on disentanglement of BECOM manufactured in Example 1.

Figure 5b shows the chemical structures of the antibacterial group (R _3A ) and the dye tracer (Rhodamin B, R _3B ) of the template used in Example 1.

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

First, in order to visualize the process of recovering silk fibroin, the antibacterial group (R _3A ) of the urethane oligomer casting mold used in Example 1 was partially substituted with a pink dye tracer (Rhodamin B, R _3B ), as shown in FIG. 5b. Then, 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. Then, 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 residual silk fibroin was dispersed in 10 mL formic acid for 10 minutes, treated with 10 mL 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 added in sequence and centrifuged again. Finally, the solution was centrifuged three times to remove the residual solvent at 80 °C to obtain silk fibroin, and the yield (weight of recovered natural silk fibroin/initial bioplastic weight 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).

Example 3. Recycling of bioplastics 2 (BECOM-R1)

As shown in the above Fig. 5a, in order to manufacture a 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 the bioplastic was manufactured again.

In the above Example 2, 0.5 g of BECOM manufactured 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 formic acid was obtained, and this was named BECOM-R1.

Example 4. Recycling of bioplastics 2 (BECOM-R2)

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

The above natural silk fibroin solution was injected into the urethane oligomer casting mold of the above-mentioned 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.

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).

Comparative Example 1

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

Comparative Example 2. Native silk fibroin

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, thereby obtaining native silk fibroin according to Comparative Example 2.

Experimental Example 1. Behavior Evaluation after Exposure to Water

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 placed over each other, the two 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 the two 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.

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

Measuring bio-based carbon content

BECOM manufactured in Example 1 and native silk fibroin manufactured in Comparative Example 2 were each prepared in an amount of 2.0 g. The Korea Apparel Testing & Research Institute measured ^{the 14} C radiocarbon content and modern carbon content using an accelerator mass spectrometer in accordance with ASTM D 6866-22 Method B, and the bio-based carbon content was calculated based on the measured content. The results are shown in Fig. 2a.

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.

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 exceeds the indicator of a high-quality bioplastic of 80% indicated by the horizontal dotted line, and it can be confirmed 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.

Structural analysis of silk fibroin

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

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.

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

As shown in the above FIGS. 2b and 2c, the urethane oligomer casting mold (template) used in the above Example 1 contains an antibacterial group including two ether groups (entanglement-inducing groups) and one quaternary ammonium salt (FIG. 2c), and when the silk fibroin denatured in an acid in the above Example 1 is injected into the urethane oligomer casting mold and regenerated through acid removal (template regeneration method), the two ether groups can be 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 a loose entanglement expressed in yellow.

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.

More specifically, X-ray scattering experiments were performed using beamline 9A of the Pohang Light Source (PLS) in Korea, with a synchrotron X-ray beam wavelength of 0.626 ㅕ (X-ray energy = 19.8 keV) and a typical beam size of 0.8 × 0.8 ^mm2 , a 2D Mar CCD detector (SX 165, Rayonix LLC, USA) to acquire the scattered beam intensity, and SAXS measurements at a sample-to-detector distance of 2.5 m and an exposure time in the range of 10-30 s.

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

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.

As shown in the above Fig. 2d, in the Kratky plot, Example 1 and Comparative Example 2 exhibit effective peaks at q=0.958 nm ^-1 and q=0.187 nm ^-1 , respectively, indicating that the effective peak position of Example 1 shifts 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 in Comparative Example 2 (I( q ), 1.54×), confirming that a densely entangled structure is formed in BECOM through the template regeneration method and that the protein is compressed to have a more solid β-sheet stacking.

As shown in the above Fig. 9a, in the case of Example 1, it was confirmed that the peak showing the maximum intensity in the 1663 cm ^-1 region corresponding to the β-sheet, the half width of the peak was 32.5 cm ^-1 , and the intensity of the peak in the 1663 cm ^-1 region was about 4 times higher than that of Comparative Example 2. In addition, as a result of calculating the β-sheet content using the Raman signal at 1665±10 cm ^-1 corresponding to the β-sheet structure in the Raman spectrum, 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 the Comparative Example and exhibited more solid β-sheet stacking.

In addition, as shown in the above Fig. 9b, the WAXS analysis results show that the intensity ratio of the a-axis stacking peak appearing at 14.9 nm ^-1 to the intensity of the c-axis stacking peak appearing at 17.6 nm ^-1 (I _a /I _c ) is 1.01 and 0.791 for Example 1 and Comparative Example 2, respectively, confirming that the BECOM of Example 1 manufactured by template regeneration exhibits more intense a-axis stacking and exhibits a silk II structure.

Glass transition temperature

1-2 mg of BECOM samples manufactured in Example 1 and native silk fibroin samples manufactured in Comparative Example 2 were used (PerkinElmer, Korea) to purge the chamber with N ₂ gas and measure the glass transition temperature (T _g ) under the conditions of a temperature range of 25 to 300 ℃ and a heating rate of 5 ℃ min ^-1 , 10 ℃ min ^-1 , and 20 ℃ min ^-1 . The results are shown in Fig. 2e.

Figure 2e shows the glass transition temperature (T _g ) of BECOM (Templated) manufactured in Example 1 and natural silk fibroin (Native) manufactured in Comparative Example 2.

As shown in the above Fig. 2e, the natural silk fibroin manufactured in Comparative Example 2 exhibited a glass transition temperature (200.5 ℃/173.5 ℃/161.6 ℃) similar to that of the reported silk fibroin, but the BECOM manufactured in Example 1 exhibited a glass transition temperature of 257.6 ℃, 261 ℃, and 277.5 ℃, which were significantly increased compared to that of natural silk fibroin. Considering that it was previously reported that when the chain mobility of a semi-flexible polymer decreases by about 12.5%, the T _g increases by about 100 ℃, it can be confirmed that the chain mobility of BECOM is reduced due to the densely entangled structure.

Experimental Example 2. Evaluation of Mechanical Properties

To measure the ultimate stress, strain (λ), toughness (T), and elastic modulus (E), a tensile test was performed using a UTM (Model 3366, Instron, USA) according to ASTM D882 by applying a uniaxial tension of 5.0 mm min ^-1 until the sample was broken on 400 μm thick and 4 cm ² beam-shaped specimens. In addition, a cyclic bending test was performed by bending the specimens at a rate of 5.0 mm min ^-1 at 1%, 3%, and 5% strains and then allowing them to recover, which was repeated 100 times per strain point. In addition, a cyclic torsion test (exposed to 600 deformation cycles for each sample) was performed by twisting the specimens at 10° min ^-1 at 10°, 30°, and 60° angles, 100 times per step.

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

As shown in the above Fig. 10, the BECOM manufactured in the above Example 1, when manufactured into a specimen having a thickness of 400 μm and a beam shape of 4 cm ² , exhibited an ultimate stress of 17.6 MPa, an elastic modulus of 645.1 MPa, and a toughness of 1.57 GJ m ^-3 . In particular, it can be seen that the BECOM in the above Example 1 has higher toughness than the high-performance hierarchical poly(vinyl alcohol) hydrogel and lignocellulosic bioplastic, which are high-toughness polymers, which showed toughness values of 210 MJ m ^-3 and 2.8 MJ m ^-3 , respectively, which are less than MJ m ^-3 (expressed by a horizontal dotted line).

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

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

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 through this, 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.

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

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

Experimental Example 3. Performance Evaluation of Orthodontic Device

Figure 3a is a schematic diagram illustrating a process of performing orthodontic treatment using BECOM and PETG manufactured in Example 1 as an orthodontic device.

As shown in the above Fig. 3a, in order to confirm whether the BECOM manufactured in the above 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.

Creep recovery

The BECOM and PETG manufactured in the above Example 1 were prepared in the form of a 1.0 cm ² film with a thickness of 1.0 mm, and 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.

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.

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. In Figure 5a, permanent recovery indicates plastic deformation.

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 can be delivered quickly 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 user-friendly orthodontic force of an appropriate size.

Stress relaxation test

To investigate the durability under tooth resistance conditions, the BECOM and PETG manufactured in Example 1 were prepared in the form of a 4.0 cm ² beam with a thickness of 500 μm, and then a pre-displacement (0.1-0.5 mm) was applied at a rate of 5.0 mm min ^-1 (initial strain of 0.2 mm, 0.4 mm, and 0.5 mm for a 2 cm long sample) (strain = 1%, 2%, and 2.5%), and the corresponding relaxation profiles were monitored for 30 min, and the results are shown in Fig. 3c and Fig. 12. At this time, tooth resistance for tooth movement on a scale of less than millimeters was reflected by pre-displacements lower than 0.5 mm, and the stress relaxation rate (stress reduction rate, %) was calculated as shown in Equation 2 below.

[Formula 2] Stress relaxation rate = [(F ₀ -F ₃₀ )/F ₀ ]x100

(In the above equation 2, when a stress relaxation test is performed on a 500 μm sample under the first strain (ε) condition, F ₀ means the initial stress, and F ₃₀ means the stress after 30 minutes.)

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

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 under tooth resistances of 0.1-0.5 mm of BECOM and PETG manufactured in Example 1.

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 stress relaxation rates of 29%, 54%, and 62% according to Equation 2 described above when the first strain was 1%, 2%, and 2.5%, respectively. In addition, BECOM of Example 1 relaxed stresses of 2.39 J min m ^-3 and 43.2 J min m ^-3 when 0.1 mm and 0.5 mm tooth resistances were applied, respectively, confirming that it can disperse concentrated stress depending on the tooth resistance level, thereby ensuring stable tooth movement and durability at the same time.

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 used in an actual 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, thereby improving the limitations of the conventional technology.

Experimental Example 4. In silico finite element analysis

Analysis conditions

In order to verify the orthodontic performance of the BECOM manufactured in the above Example 1, a simulation was performed to correct a human maxilla using BECOM and PETG as orthodontic devices, and the results were analyzed in silico by finite element analysis. The conditions, process, and results for the simulation are shown in Figs. 3d-g.

Figure 3d is a schematic diagram of 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.

First, as shown in the above 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. A 0.2 mm midline diastema was formed. Then, 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 the shape of the orthodontic appliance was created by merging them, which was then obtained through a subtractive Boolean operation. Then. A symmetrical arch pattern was assumed and a finite element model of the teeth and the orthodontic appliance was constructed only on the right side, aiming at eliminating the 0.2 mm gap (spur) between the upper incisors and moving the two central incisors by 0.1 mm toward the center. The material properties were assumed to be homogeneous, isotropic, and linear elastic material constitutive models, and the 3D model was meshed using the finite element method with the properties shown in Table 1 using modified tetrahedral second-order elements (C3D10M).

Model Elastic odulus Poisson's Ratio Node number Element number Teeth 18600 MPa 0.31 20463 69329 Periodontal ligament 0.68 MPa 0.48 41380 64716 PETG 668 MPa 0.45 7879 15279 BECOM 645.1 MPa 0.40 7879 15279

A series of iterative adjustments were made within the simulation to allow for gradual and controlled movements of the teeth, taking into account the natural flexibility of the periodontal ligament, and to investigate the initial stages of tooth movement. Initially, the orthodontic appliance was fitted accurately and closely to the teeth, and the tooth sockets of the central incisors were slightly adjusted. The initial tooth movements were calculated by considering each tooth as a rigid and immovable object, to increase precision, and the tooth sockets were then adjusted based on the observed initial movements. These adjustments involved slight changes in position and angle, and were mainly applied to the outer surface of the periodontal ligament. In addition, this method allowed for continuous updating and improvement of the forces applied to the teeth, which improved 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).

Analysis Results

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 the above Example 1 was calculated, and the final three-dimensional displacement and stress magnitude of the central incisor (U1) and the lateral incisor (U2) of the BECOM and PETG manufactured in the above Example 1 were compared, and the tooth displacement profiles are shown in FIGS. 3e, 3f, and 13.

Figure 3e shows the distribution of orthodontic 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 orthodontic force between BECOM and PETG manufactured in Example 1 is observed is expressed as a red circle.

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

Figure 3f shows the tooth displacement profile of the right maxilla after correction of teeth using the orthodontic appliance using BECOM of Example 1.

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

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.

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.

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.

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

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

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

Experimental Example 5. In vivo orthodontic performance

In order to confirm the orthodontic performance of the orthodontic appliance 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.

After establishing a midline expansion model that requires reduction of the space between the mandibular incisors, a repetitive midline gap closure model was designed to simulate orthodontic treatment using transparent aligners, which is typically performed in stages. In each repetition, interdental reduction was meticulously performed and evaluated three times in total, each targeting a 2.5 mm spur closure.

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 1/3 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. 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.

To objectively measure tooth movement, intraoral anatomical pick-up impressions of the mandibular incisor region were recorded using a lightweight and medium-sized impression material, polyvinyl siloxane (I-Sil™ Bite, Spident, Incheon, Korea), and the rabbits were routinely examined for general and periodontal health. All procedures were performed under intravenous sedation (Zoletil). Each repetition did not last longer than 30 min 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.

The transmittance according to the thickness of the orthodontic device using BECOM manufactured in the above 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 2.5 mm using this are shown in Figs. 4a to 4e.

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.

As shown in the above Figure 15, the orthodontic device using 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%.

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

Figure 4b schematically illustrates a three-step process for orthodontic correction 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.

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

FIG. 4d shows a frontal 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 appliance 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 appliance manufactured in Example 1.

Fig. 4e is a plan view image of teeth in which malocclusion progresses due to non-orthodontic treatment with an orthodontic appliance. In Fig. 4e, θ _day denotes the incisal edge angle on a specific day after interproximal reduction surgery, and Δθ denotes undesirable internal rotation expressed as θ ₀ - θ _day .

As shown in FIGS. 4a to 4e, in order to secure a tooth movement site, a midline diastema, i.e., an incisor gap, was created through interproximal reduction surgery (FIG. 4a), and then 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). As a result, the teeth were sufficiently moved, and in particular, the blue portion of the tooth movement indicator showed a clear re-approximation of the incisal mesial edge (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 θ ₀ = 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 positions of teeth, prevent unwanted tooth movement, and ultimately enable satisfactory orthodontic treatment.

Experimental Example 6. Recycling Evaluation-Silk Fibroin Extraction

Pure silk fibroin extraction process and results

The procedure and recycling yield (yield of silk fibroin) of the BECOM manufactured in Example 1 in the above Example 2 are shown in FIGS. 5A and 16, and in order to visually confirm the process as shown in FIG. 5B, the antibacterial group (R _3A ) of the urethane oligomer casting mold used in Example 1 was partially substituted with a pink dye tracer (Rhodamin B, R _3B ).

Fig. 5a is a schematic diagram showing a recycling procedure based on disentanglement of BECOM manufactured in Example 1. The symbols l and s shown in Fig. 5a represent liquid phase and solid phase, respectively.

Figure 5b shows the chemical structures of the antibacterial group (R _3A ) and dye tracer (Rhodamin B, R _3B ) of the urethane oligomer casting mold used in Example 1.

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.

As shown in the above FIGS. 5A and 16, the BECOM manufactured in Example 1 exhibited an entangled structure, which was expected to be untangled and thus have recyclable properties. In fact, when the BECOM manufactured in Example 1 was treated with formic acid in Example 2, the entangled structure was untangled based on hydrogen bonds, thereby generating a liquid BECOM containing a freely movable urethane oligomer casting mold (template) and denatured silk fibroin strands. Then, 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).

Meanwhile, the photographic images of BECOM manufactured in Example 1 and silk fibroin extracted in Example 2 according to the substitution ratio (R _3A /(R _3A + R _3B )) of the antimicrobial group and the dye tracer of the urethane oligomer casting mold used in Example 1 are shown in FIG. 5c and FIG. 17.

Figure 5c shows photographic images of BECOM prepared in Example 1 and silk fibroin extracted in Example 2 according to the substitution ratio (R _3A /(R _3A + R _3B )) of the antimicrobial group and the dye tracer of the urethane oligomer casting mold used in Example 1.

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

As shown in the above FIG. 5c and FIG. 17, as the ratio (substitution rate) of the antibacterial group (R _3A ) of the urethane oligomer casting mold being replaced with a pink dye (R _3B ) increased up to a maximum of 10 mmol%, BECOM exhibited a more intense pink color, but the extracted silk fibroin exhibited a yellow color regardless of the substitution rate, indicating that the template was completely removed.

FIG. 5d shows ¹³ C nuclear magnetic resonance (NMR) spectra of BECOM prepared in Example 1, silk fibroin extracted in Example 2, and natural silk fibroin prepared in Comparative Example 2.

As shown in the above FIG. 5d, the BECOM of the above 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 the above 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 the above Example 2 was very pure and did not contain any residual urethane oligomer casting mold.

In order to analyze the characteristics of the urethane oligomer casting mold obtained in the process of extracting silk fibroin from the 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 purged with helium at a flow rate of 1 mL min ^-1 in the chamber using a single-shot pyrolysis gas chromatography-mass spectrometer (8890 GC-5977B MSD, Agilent) equipped with a 30 m long UA-5 column, and after an initial stabilization stage at an injection temperature of 40 °C for 5 min, the temperature was increased to 320 °C at a heating rate of 10 °C min ^-1 and held for 10 min to obtain the analysis results, and based on this, the extraction amount of the urethane oligomer casting mold was calculated using the following Equation 5, and the process and results are shown in FIGS. 18, 5e, and 19.

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

(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.)

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

Figure 5e shows photographic images of the urethane oligomer casting mold (1 mL) extracted by recycling BECOM of Example 1 having a substitution ratio of 50 mmol% of the antimicrobial group and dye tracer of the urethane oligomer casting mold in Example 2 after the first and fifth extraction processes.

Figure 19 shows (a) ¹ H nuclear magnetic resonance (NMR; Avance III HD 300, Bruker) spectra and (b) photograph images (1 mL per vial) of the urethane oligomer casting mold extracted by recycling BECOM having a substitution ratio of 50 mmol% of the antimicrobial group and dye tracer of the urethane oligomer casting mold in Example 2 according to the extraction cycle.

As shown in the above FIGS. 18, 5e, and 19, 76.8% of the urethane oligomer casting mold was extracted in the first extraction step in Example 2 (FIG. 18), and the broad peak in the range of 7.1 to 7.4 ppm in the ¹ H NMR spectrum derived from Rhodamine B disappeared after three extractions (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).

Evaluation of inflammatory biomarker expression

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.

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.

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.

Experimental Example 7. Recycling Evaluation-Reforming

In order to verify whether the used bioplastic can be recycled by re-molding 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 re-entanglement, thereby recycling BECOM-R1 (Example 3), and BECOM-R2 (Example 4), in which silk fibroin was extracted from the BECOM manufactured in Example 1 and then recycled using a template regeneration method, were analyzed for the morphology, FT-IR spectrum analysis, and ultimate stress, and the results are shown in FIGS. 5g, 20, and 5h.

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

As shown in the above Fig. 5g, all of the above Examples 1 (BECOM), 3 (BECOM-R1), and 4 (BECOM-R2) showed similar morphologies, confirming that BECOM can be recycled by inducing re-entanglement 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 the template regeneration method again (Example 4, BECOM-R2).

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).

As shown in the above Figure 20, all of the above Examples 1 (BECOM), 3 (BECOM-R1) and 4 (ECOM-R2) showed β-sheet-related Fourier transform infrared amide I spectra including a strong signal at 1620 cm ^-1 in the amide I region corresponding to the β-sheet structure of silk fibroin, indicating that the tightly entangled structure was reconstructed in the above Examples 3 and 4.

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 the results compared with conventional plastics are shown in FIG. 5h and FIG. 21.

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.

Figure 21 shows stress-strain curves (n=3) when a uniform tension is applied at a displacement rate of 5.0 mm min ^-1 to beam-shaped specimens of 4 cm ² using (a) BECOM manufactured in Example 1, BECOM-R1 manufactured in Example 3, and BECOM-R2 manufactured in Example 4, 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.

As shown in the above 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 BECOM of Example 1. On the other hand, BECOM-R2 of Example 4 exhibited an ultimate stress that was reduced by 35%, but considering that the ultimate stress of the biodegradable starch plastic is ~10 MPa, it was confirmed that it exhibited an ultimate stress of 11.5 MPa, which is equivalent to that of the biodegradable starch plastic. Through this, it was confirmed that the excellent mechanical properties of BECOM can be maintained even after the recycling process of BECOM is completed.

8. Evaluation of microbial resistance of BECOM in experiments

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

As shown in the above Fig. 6a, oral tissues contain numerous microorganisms, and when correcting teeth using an orthodontic appliance, the orthodontic appliance is applied to the oral cavity for a long period of time, and after 6 hours, the outer surface shows a strong affinity for plaque formation. In addition, since the inner area between the orthodontic appliance and the teeth is relatively more anaerobic than the outer surface, an environment favorable for biofilm formation, such as accumulation of thick extracellular polysaccharides, can easily be created. Therefore, gingival tissues and orthodontic appliances are vulnerable to biofilm growth during orthodontic treatment. Therefore, in order to evaluate the microbial resistance of the BECOM manufactured in Example 1, the microbial resistance was confirmed using human saliva samples.

In vitro microbial resistance

Saliva sample collection was conducted in strict accordance 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 Number 2-2019-0049), and the recruitment of participants was performed fairly without considering gender or sex 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 h. After that, 1.5 mL volume was dispensed into the BECOM and PETG prepared in Example 1 and incubated at a constant temperature of 37 °C for 48 h, and fresh medium was replenished at intervals of 8 h, 16 h, and 24 h during the incubation period to maintain the integrity and relevance of the experiment, and the surface biofilm was collected using a sterile loop for 16s rRNA sequencing.

As described above, after treating the human saliva medium with the 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. The culture without BECOM and PETG was used as the control group (Mock). At this time, the CFU value was expressed on a log scale, and every increase corresponded to an increase of 10 ² in CFU. For example, with regard to S. mutans attachment, BECOM and PETG showed average CFU of 56 and 3.2 × 10 ⁶ , respectively.

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

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

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.

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.

As shown in the above Fig. 6c, PETG showed a similar proliferation 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 .

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.

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

Cell viability assessment

Concentrated quaternary ammonium compounds have concerns about toxicity despite their excellent antibacterial performance, so the BECOM of Example 1 was used to measure cell viability 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 to culture at 37°C, 95% humidity, 5% CO ₂ , and after confluence, the 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 then diluted in culture medium, and the growth medium was used as a control (Mock).

1 × 10 ⁴ cells were plated in a 96-well plate (SPL Life Sciences Co., Pocheon-si, Gyeonggi-do, South Korea) and maintained at 37 ℃, 95% humidity, and 5% CO ₂ for 24 h. After the incubation time, 100 μL of each material was extracted into the 96-well plate cultured with cells. 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 h, and then 100 μL of isopropanol (Duksan Pure Chemical Co., Ansan-si, South Korea) was added to each well. The optical density (OD) was measured at 57 nm using a microplate reader (Epoch, BioTek, Winooski, VT, USA) for cell viability analysis. The cell viability (Cell viability, %) was calculated using the following Equation 6:

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

In addition, to perform pro-inflammatory response induction using qPCR analysis, 1 × 10 ⁵ RAW 264.7 cells were cultured in 6-well plates (SPL Life Science Co.) for 24 h, the existing medium was discarded and replaced with various sample extractions, and 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 SYBR® Premix Ex TaqTM kit (Takara Bio Co.). The relative messenger RNA levels of the target genes were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase.

Figure 22 is a graph showing the reaction distance of fibroblast confluent monolayer when treated with (a) negative control (NC; high-density polyethylene film), positive control (PC; 0.1% ZDEC polyurethane film), and BECOM prepared in Example 1 (n=3), (b) cell viability after co-culturing the 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) relative expression levels of inflammatory biomarkers by BECOM prepared in Example 1 and the control (Mock).

The above Fig. 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 Fig. 22b indicates 70% cell viability, which is the cytotoxicity criterion according to the ISO 10993-5:2009 standard. The control group in Fig. 22c indicates an experimental group without EPSF.

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

Analysis of human salivary biofilm microbial communities

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

More specifically, DNA sequencing was performed after DNA extraction, we prepared the sequenced samples following the Illumina 16S rRNA sequencing library protocol, and assessed DNA quality and quantity using PicoGreen and VICTOR Nivo. 16S V3-V4 primers were used to amplify the 16S rRNA gene, and an additional limited cycle amplification step was performed to include the multiplexing index 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 in the background of amplicon sequencing. Quality checks were performed on the raw reads, except when low-score reads (<25) were performed, and then the paired-end sequence data were merged and the primers were trimmed. Unique 16S rRNA reads were separated using a 97% similarity threshold and taxonomic assignments were performed based on the EzBioCloud 16S rRNA database.

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

As shown in the above Fig. 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.

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 on BECOM and PETG in Example 1. In Figure 6f, PC1 and PC2 represent the first and second coordinates, respectively.

As shown in the above Fig. 6f, there was a difference in beta diversity between the microbial community of BECOM and PETG of the above Example 1, and specifically, the BECOM group of the above 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) because the variation accounted for 64.71%. In other words, it can be confirmed that substantially different microbial communities grew in BECOM and PETG.

Figure 6g shows a significantly (*p<0.05) revealed 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, and the specific correlation analysis results comparing the occurrence patterns of different genera with BECOM and PETG are shown in Table 2 below.

Taxa Correlation T-statistic P -value ^a FDR PETG BECOM Abiotrophia 0.94153 5.5891 0.0050276 0.033182 1.652E-04 9.304E-03 Granulocytes 0.89932 4.1131 0.014695 0.048493 3.922E-02 3.861E-01 AF366267_g 0.71346 2.0364 0.11139 0.22975 0.000E+00 1.844E-03 Panthea 0.67093 1.8096 0.14461 0.26513 8.258E-05 6.193E-03 Serratia 0.58106 1.4279 0.2265 0.36453 6.331E-04 1.617E-01 Neisseria 0.57786 1.4161 0.22969 0.36453 0.000E+00 5.230E-04 Eikenella 0.56467 1.3684 0.24302 0.36453 4.129E-04 3.716E-03 Rothia 0.38784 0.84155 0.44741 0.58056 3.303E-04 1.514E-03 Saccharimonas 0.38002 0.82169 0.45741 0.58056 5.505E-05 2.753E-04 Porphyromonas 0.25802 0.53412 0.62156 0.71877 2.202E-04 4.129E-04 Atopobium 0.063319 0.12689 0.90515 0.90515 1.376E-04 1.376E-04 Weissella -0.06624 -0.13277 0.90078 0.90515 2.477E-04 1.927E-04 Parvimonas -0.16664 -0.338 0.75236 0.80089 1.374E-02 7.762E-03 Fusobacterium -0.21504 -0.44038 0.68241 0.75066 3.854E-04 1.927E-04 Actinomyces -0.25083 -0.51822 0.63165 0.71877 3.854E-04 1.927E-04 Prevotella -0.27664 -0.57576 0.59562 0.71877 7.157E-04 3.854E-04 Peptostreptococcus -0.47169 -1.0699 0.34494 0.47429 6.496E-03 5.505E-05 Streptococcus -0.50947 -1.1841 0.30191 0.43318 6.807E-01 4.134E-01 Oribacterium -0.567 -1.3767 0.24064 0.36453 7.047E-03 0.000E+00 Megasphaera -0.67946 -1.8521 0.13765 0.26513 3.110E-03 0.000E+00 Leptotrichia -0.77913 -2.4858 0.067788 0.14913 8.258E-04 1.101E-04 Dialister -0.82781 -2.9511 0.041922 0.098817 2.587E-03 0.000E+00 Gemella -0.85427 -3.2867 0.030308 0.076935 3.083E-03 3.028E-04 Haemophilus -0.85527 -3.3011 0.029904 0.076935 2.863E-03 7.432E-04 Lactobacillus -0.88573 -3.8161 0.018841 0.056524 6.372E-02 0.000E+00 Campylobacter -0.90066 -4.1454 0.014314 0.048493 3.248E-03 2.753E-05 Alloscardovia -0.91471 -4.527 0.010602 0.043732 5.230E-04 0.000E+00 Lachnoanaerobaculum -0.92242 -4.777 0.0087951 0.041462 4.266E-03 8.258E-05 Moryella -0.93279 -5.1762 0.0066238 0.036431 2.532E-02 0.000E+00 Staphylococcus -0.94507 -5.7828 0.0044424 0.033182 1.354E-02 3.276E-03 Enterococcus -0.9773 -9.2261 0.000767 0.008437 6.193E-03 3.303E-04 Veillonella -0.98407 -11.07 0.0003788 0.00625 1.144E-01 1.129E-03 Bulleidia -0.98616 -11.896 0.000286 0.00625 5.312E-03 8.258E-05

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

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

Taxonomic unit ^a log2FC ^b lfcSE P-values FDR Veillonella_atypica -8.1412 0.75531 4.34E-27 2.61E-25 Lactobacillus_fermentum -11.335 1.2905 1.59E-18 4.76E-17 Abiotrophia_defectiva 8.3326 0.96772 7.27E-18 1.45E-16 Streptococcus_salivarius_group -5.1207 0.60528 2.67E-17 4.01E-16 Stomatobaculum_longum -9.8701 1.3082 4.52E-14 5.43E-13 Veillonella_uc -5.9381 0.82473 6.02E-13 6.02E-12 Serratia_ureilytica 8.9542 1.2793 2.57E-12 2.20E-11 Serratia_marcescens_group 10.183 1.5006 1.15E-11 8.66E-11 Streptococcus_anginosus_group 4.3332 0.65642 4.08E-11 2.72E-10 Granulicatella_adiacens_group 4.883 0.81327 1.92E-09 1.15E-08 Enterococcus_faecalis -4.4909 0.79728 1.77E-08 9.67E-08 Pantoea_agglomerans_group 8.6794 1.5609 2.69E-08 1.35E-07 Moryella_uc -7.2319 1.3049 2.99E-08 1.38E-07 Granulicatella_elegans 8.8031 1.594 3.34E-08 1.43E-07 Eikenella_corrodens 5.1284 0.99902 2.84E-07 1.09E-06 Enterobacteriaceae_group 1.8405 0.35883 2.91E-07 1.09E-06 Solobacterium_moorei -4.998 0.9796 3.36E-07 1.19E-06 Veillonella_parvula_group -3.8777 0.76397 3.86E-07 1.29E-06 Lachnoanaerobaculum_saburreum_group -6.8494 1.3762 6.45E-07 2.04E-06 FWNZ_s 1.8514 0.39149 2.25E-06 6.76E-06 Dialister_pneumosintes -6.6126 1.4189 3.15E-06 9.01E-06 Campylobacter_concisus_group -4.8133 1.1002 1.21E-05 3.31E-05 Serratia_uc 8.6984 2.0662 2.55E-05 6.66E-05 AF366267_s 7.0167 1.7022 3.75E-05 9.38E-05 Megasphaera_micronuciformis -7.2851 1.8164 6.05E-05 0.000145 Granulicatella_uc 3.8112 0.95804 6.94E-05 0.00016 Abiotrophia_uc 6.916 1.8721 0.0002205 0.00049 Campylobacter_showae_group -5.5295 1.5022 0.0002324 0.000494 Oribacterium_asaccharolyticum -7.2149 1.9638 0.0002388 0.000494 Bulleidia_uc -5.0492 1.3866 0.0002712 0.000542 Streptococcus_gordonii_group -2.6357 0.74466 0.000401 0.000776 Veillonella_rogosae -2.4715 0.7079 0.0004807 0.000901 Streptococcus_sinensis_group 2.9841 0.88415 0.0007377 0.001341 JH815185_s_group -3.8395 1.2203 0.0016532 0.002917 Pantoea_uc 4.5063 1.452 0.0019121 0.003278 Gemella_haemolysans_group -2.1074 0.69198 0.0023231 0.003872 Peptostreptococcus_stomatis_group -5.8129 1.9819 0.0033575 0.005445 Klebsiella_uc 1.1767 0.43734 0.0071346 0.011265 Streptococcus_constellatus_group 2.6392 0.99248 0.0078327 0.01205

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

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

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

Experimental Example 9. Composting

Composting Methods

Figure 7a is a schematic diagram 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.

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

The above standard compost was prepared by mixing 40 wt% of sawdust, 30 wt% of rabbit feed, 10 wt% of mature compost, 10 wt% of corn starch, 5% sucrose, 4% corn seed oil, and 1% urea, and a polypropylene composting reactor having two holes with a diameter of 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 circulation oven at 60 °C 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.

[Formula 3] (CM ₀ -CM ₈ )/CM ₀ X 100

(In the above equation 3, CM ₀ means the weight of bioplastic before composting, and CM ₈ means the weight of bioplastic after 8 weeks of composting.)

Composting Results

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).

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

As shown in FIGS. 7b and 7c, the initial 4.0 cm ² film-shaped single BECOM and EPSF samples at week 0 were degraded and turned black, distorted, and fragmented after 8 weeks (FIG. 7b), while 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 week 4, indicating that the area of residual BECOM was reduced by 20% due to fragmentation (FIG. 7c).

Bacillus species in the soil produce proteases during the aerobic metabolic process, and these proteases induce effective protein degradation of silk fibroin, thereby causing rapid composting of silk fibroin. Therefore, in order to confirm whether the BECOM manufactured in Example 1 is sufficiently decomposed by the silk fibroin-decomposing enzyme, 1 x 1 cm ² of the BECOM (n=4) manufactured in Example 1 was immersed in 4 mL of an enzyme medium containing matrix metallopeptidase 9, protease XIV (1.0 U mL ^-1 , n=4), and collagenase IA, known as silk fibroin-decomposing enzymes, and cultured at 37°C for up to 72 hours, and the photographic images over time are shown in FIG. 24.

Figure 24 shows the photographic images and the thickness over time of BECOM manufactured in Example 1 immersed in an enzyme medium containing 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) according to immersion time.

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

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

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

Soil Microbial Analysis after Composting

BECOM composting can create an environment that is helpful for 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 composting weeks 0, 4, and 8 of BECOM manufactured in Example 1 and EPSF manufactured in Example 2, and DNA of each compost was extracted. 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. After that, 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 maintained at 4 °C.

For library preparation, the protocol provided by ONT was used as described below. For the barcoding process, carefully selected 16S barcodes were selected from 96-well plates, carefully isolated and thawed, and the foil surface was pierced using a sterile pipette tip. 10 μL volume of mixed barcodes was then added to the corresponding PCR tubes containing previously amplified DNA, and 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, centrifuged to form a magnetic pellet, and 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 into a sterile DNA LoBind tube. In the quantification and pooling step, 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 to 50–100 ng for ~1500 base pair (bp) 16S amplicons. Finally, 1 sL of RAP was added to the pooled DNA, mixed thoroughly, and incubated for 5 min.

Sequencing was performed and supervised using MinKNOW software (version 5. 2. 13), which handles various tasks like data acquisition, real-time analysis, run feedback, and local base calling. Sequencing data were basecalled in real-time using Guppy version 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 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.

Considering that the EPSF manufactured in the above Example 2 is a high-purity silk fibroin and that silk fibroin participates fairly 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 and the compost microbial community were monitored for microbiological characteristics, such as similarity with the EPSF control group, at 8 weeks through 16S rRNA gene sequence analysis, thereby proving the composting action without side effects.

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

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 (EPSF).

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 matured according to the experimental schedule through the improvement in microbial diversity and species richness (FIG. 7e), and the Chao1 richness and observed species index confirmed that the microbial species richness increased significantly (FIG. 25).

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.

Figure 26 shows (a) observation and (b) Chao1 alpha diversity metric of compost soil obtained after 8 weeks of composting of BECOM prepared in Example 1 and EPSF prepared in Example 2.

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.

Figure 7g shows a principal coordinate analysis (PCoA) analysis plot of the compost soil obtained after 8 weeks of composting of the standard compost at week 0, the BECOM prepared in Example 1, and the EPSF prepared in Example 2, where 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 of the BECOM prepared in Example 1 and the EPSF prepared in Example 2 are shown in Table 4 below.

Taxon Average dissimilarity ^a Contribution % Cumulative % Mean abundance BECOM ESPF Parageobacillus genomosp 1.714 6.078 6.078 7280.00 256.00 Steroidobacter denitrificans 1.287 4.563 10.64 881.00 4810.00 Bacillus velezensis 1.271 4.507 15.15 13900.00 9050.00 Amphibacillus xylanus 1.044 3.703 18.85 8600.00 6380.00 Bacillus cereus 0.9436 3.345 22.2 11300.00 7550.00 Bacillus thuringiensis 0.7782 2.759 24.95 10200.00 7370.00 Ureibacillus thermophilus 0.7012 2.486 27.44 4300.00 1480.00 Symbiobacterium thermophilum 0.6153 2.181 29.62 681.00 2830.00 Ureibacillus thermosphaericus 0.4779 1.694 31.32 2500.00 842.00 Caloranaerobacter azorensis 0.4442 1.575 32.89 1940.00 2470.00 Candidatus Filomicrobium marinum 0.4338 1.538 34.43 684.00 1990.00 Bacillus subtilis 0.3925 1.392 35.82 5320.00 5010.00 Salicibibacter halophilus 0.2974 1.054 36.87 3380.00 2500.00 Herbinix luporum 0.297 1.053 37.93 399.00 1270.00 Lentibacillus sp. CBA3610 0.2921 1.036 38.96 3690.00 2550.00

As shown in the above Fig. 7g and Table 4, the compost soils of BECOM prepared in Example 1 and EPSF prepared in Example 2 showed a remarkable overlap with the ^R2 value of 0.201 (F value = 0.75), and the groups 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 demonstrated that the composting of BECOM could be included in the natural life cycle of soil microorganisms.

Microorganisms that thrive in compost can dissolve nutrients combined with compost, mediate enzymatic reactions to ultimately help plant growth, and exhibit the effects of compost maturation, phosphorus dissolution, and nitrogen fixation through microbial metabolism. Accordingly, the microbial characteristics of the compost soil of BECOM manufactured in Example 1 and EPSF manufactured in Example 2 were analyzed.

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

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

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

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

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.

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

As shown in FIGS. 27, 28, 29, and 30 above, in the compost soil obtained after 8 weeks of composting of BECOM of Example 1 and EPSF of Example 2, Serratia sps , which contributes to phosphate dissolution, was observed in large quantities (FIG. 27), and the dominance of nitrifying bacteria, Enterobacter sps and Klebsiella sps, was confirmed (FIG. 28). In addition, Pseudomonas sps , 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 ratio was low (FIG. 29). In addition, B. licheniformis and B. paralicheniformis , which have a positive effect on improving microbial cooperation that enhances microbial metabolism, were not detected in the initial compost, but showed sufficient amounts for both BECOM- and EPSF-based composts (FIG. 30). Through this, it was confirmed that the beneficial microbial species increased in BECOM and EPSF-based composts, which could improve the compost soil condition and provide significant microbiological benefits.

The results of the above Experimental Example 7 demonstrated 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. Accordingly, when its lifespan has ended, it can be disposed of through an environmentally friendly and practical composting method without incineration, thereby becoming a sustainable technology. In addition, the used composting protocol ISO 20200-17 was confirmed to be an efficient method for preparing microbiologically superior compost using BECOM.

According to recent agro-ecosystem studies, among the characteristics of compost, pH, carbon-to-nitrogen (C/N) ratio, and abundance of B. subtilis can greatly determine the productivity of crops. 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 being 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.

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 level, confirming that BECOM can potentially contribute to agricultural production systems as an excellent compost resource.

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.

Taxa Absolute count Relative abundance (%) BECOM ESPF BECOM ESPF Firmicutes 151065.00 131292.00 85.3% 74.1% Proteobacteria 14445.50 30649.67 8.2% 17.3% Actinobacteria 9147.50 11346.33 5.2% 6.4% Planctomycetes 301.50 645.00 0.2% 0.4% Armatimonadetes 297.50 464.67 0.2% 0.3% Acidobacteria 225.00 463.00 0.1% 0.3% Chloroflexi 219.50 336.67 0.1% 0.2% Atribacterota 231.00 315.00 0.1% 0.2% Fusobacteria 225.50 267.33 0.1% 0.2% Spirochaetes 214.00 242.33 0.1% 0.1% Bacteroidetes 170.50 238.00 0.1% 0.1% Tenericutes 116.00 120.33 0.1% 0.1% Synergistic 101.50 118.33 0.1% 0.1% Gemmatimonadetes 63.50 110.33 0.0% 0.1% Chrysiogenetes 70.50 97.33 0.0% 0.1% Deferribacteres 75.00 72.33 0.0% 0.0% Deinococcus-Thermus 50.50 83.67 0.0% 0.0% Thermodesulfobacteria 33.50 79.00 0.0% 0.0% Fibrobacteres 33.50 73.00 0.0% 0.0% Coprothermobacterota 23.00 27.67 0.0% 0.0% Aquatic 6.50 34.00 0.0% 0.0% Cyanobacteria 17.50 22.67 0.0% 0.0% Verrucomicrobia 12.50 25.00 0.0% 0.0% Thermotogae 12.50 17.33 0.0% 0.0% Nitrospirae 9.50 13.33 0.0% 0.0% Chlorobi 6.00 14.00 0.0% 0.0% Kiritimatiellaeota 7.50 6.33 0.0% 0.0% Chlamydiae 1.50 5.67 0.0% 0.0% Calditrichaeota 3.50 4.00 0.0% 0.0% Elusimicrobia 3.50 3.33 0.0% 0.0% Ignavibacteriae 1.50 3.67 0.0% 0.0% Caldiserica 1.50 1.67 0.0% 0.0%

As shown in Table 5 above, the dominant phyla order of BECOM of Example 1 and EPSF of Example 2 were identical to each other as Firmicutes , Proteobacteria and Actinobacteria after 8 weeks of composting, and the relative abundances of Firmicutes and Actinobacteria showed great similarity. Meanwhile, the relative abundances of Proteobacteria were observed to be 8.2% and 17.3% in BECOM of Example 1 and EPSF of Example 2, respectively. In particular, since Proteobacteria play an important role in composting by promoting the early mesophilic stage and assisting plant farming, it was confirmed that EPSF could 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-infection by contaminated medical plastic waste should be considered, and the preferential growth of pathogens in the compost should be considered to prevent the possibility of secondary cross-infection caused by contaminated landfills. Accordingly, the presence of E. coli, a common pathogen commonly found in compost due to the presence of animal organic matter, was analyzed to what extent it was present in the compost soil obtained after composting BECOM manufactured in Example 1 and EPSF manufactured in Example 2 for 8 weeks, and the results are shown in Fig. 31.

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).

As shown in the above Fig. 31, in the initial 0-week compost, E. coli was prevalent and accounted 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 microbiologically 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.

Above, the embodiments of the present invention have been described, but those skilled 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 (28)

A bioplastic comprising denatured silk fibroin having an increased content of β-sheets by being denatured from natural silk fibroin, wherein the denatured silk fibroin has an entangled structure. In the first paragraph, a bioplastic comprising 80% or more of the modified silk fibroin based on 100% of the entire bioplastic. In the first paragraph, the bioplastic has a peak exhibiting maximum intensity in the 1665±10 cm ^-1 region of the spectrum according to Raman spectroscopy, and the half-width of the peak is 10 to 80 cm ^-1 . In the first paragraph, the bioplastic is a bioplastic having a bio-based carbon content of 80% or more. In the first paragraph, the bioplastic is a bioplastic that exhibits an effective peak at q= 0.5 to 1.3 nm ^-1 in a Kratky plot. In the first paragraph, the bioplastic exhibits an a-axis stacking peak at 14.9 nm ^-1 and a c-axis stacking peak at 17.6 nm ^-1 as a result of wide-angle X-ray scattering (WAXS) analysis, and has a ratio of the a-axis stacking peak intensity to the c-axis stacking peak intensity (I _a /I _c ) of 0.85 to 1.7. A bioplastic further comprising an antibacterial urethane oligomer in claim 1. In claim 7, the urethane oligomer is a bioplastic manufactured by reacting a polyol and an antibacterial diisocyanate. A bioplastic in claim 8, wherein the antibacterial isocyanate compound is a heterocyclic diisocyanate compound containing a quaternary ammonium salt. In claim 8, the antibacterial urethane oligomer is a bioplastic having a hydroxyl group at the terminal. In the first paragraph, the bioplastic is a bioplastic in which the ratio of the beta sheet content of modified silk fibroin to the beta sheet content of natural silk fibroin expressed by the following formula 1 exceeds 1.5. [Formula 1] X ₁ /X ₂ (In the above equation 1, X ₁ is the beta sheet content of the bioplastic calculated through Raman spectroscopy, and X ₂ is the beta sheet content of natural silk fibroin.) In the first paragraph, the bioplastic is a bioplastic having an ultimate stress of 10 MPa or more. 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. In the first paragraph, the bioplastic is a bioplastic having a toughness (T) of 300 MJ/㎥ or more as measured according to ASTM D882. 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] (F ₀ -F _30m )/F ₀ (In the above equation 2, when a stress relaxation test is performed on a 500 ㎛ thick bioplastic under a strain condition of 2 to 3%, F ₀ is the initial stress and F _30m is the stress after 30 minutes.) In the first paragraph, the bioplastic is a bioplastic having a composting rate of 60% or more, expressed by Equation 3 below, when composted according to ISO 20200-17. [Formula 3] (CM ₀ -CM ₈ )/CM ₀ X 100 (In the above equation 3, CM ₀ means the weight of bioplastic before composting, and CM ₈ means the weight of bioplastic after 8 weeks of composting.) In claim 1, 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 . A biomedical molded article comprising a bioplastic according to any one of claims 1 to 17. An orthodontic device comprising a bioplastic according to any one of claims 1 to 17. (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: a step of drying the natural silk fibroin solution to produce a bioplastic containing denatured silk fibroin. In Article 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 manufacturing a bioplastic, comprising a step of casting the polymerizable composition into a mold and then reacting it. In Article 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 a cosmotropic salt are dissolved in an acidic solvent. In claim 20, the cosmotropic salt is a method for producing a bioplastic, which is a combination of an alkali metal ion or an alkaline earth metal ion; and one or more ions selected from the group consisting of sulfate (SO ₄ ^2- ), phosphate (HPO ₄ ^2- ), acetate (CH ₃ COO ^- ), hydroxide (OH ^- ), chloride (Cl ^{- )} , bromide (Br - ), and formate (HCOO ^- ). In Article 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 step (B) to produce denatured silk fibroin. In Article 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. In claim 25, a method for producing a bioplastic comprising the storage solution containing C _1-7 alkyl polyol. 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. A step for producing a bioplastic solution by dissolving the bioplastic and an 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: a step of drying the bioplastic solution to produce recycled bioplastic.

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