Study on the Structure and Properties of Silk Fibers Obtained from Factory All-Age Artificial Diets

The traditional production mode of the sericulture industry is no longer suitable for the development requirements of modern agriculture; to facilitate the sustainable development of the sericulture industry, factory all-age artificial diet feeding came into being. Understanding the structural characteristics and properties of silk fibers obtained from factory all-age artificial diet feeding is an important prerequisite for application in the fields of textiles, clothing, biomedicine, and others. However, there have been no reports so far. In this paper, by feeding silkworms with factory all-age artificial diets (AD group) and mulberry leaves (ML group), silk fibers were obtained via two different feeding methods. The structure, mechanical properties, hygroscopic properties, and degradation properties were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Structurally, no new functional groups appeared in the AD group. Compared with the ML group, the structure of the two groups was similar, and there was no significant difference in mechanical properties and moisture absorption. The structure of degummed silk fibers is dominated by crystalline regions, but α-chymotrypsin hydrolyzes the amorphous regions of silk proteins, so that after 28 d of degradation, the weight loss of both is very small. This provides further justification for the feasibility of factory all-age artificial diets for silkworms.


Introduction
Silk is an important biological protein polymer in which liquid silk proteins undergo a conformational transition from the spinning tubes of the silkworm to form solid fibers [1].Natural silk fibers consist of two main protein components, silk fibroin (72-81%) and silk sericin (19-28%), forming a core-shell structure [2].Silk fibroin acts as the core structure of a semicrystalline material to provide mechanical properties, while silk sericin acts as a globular protein that binds the two fibroin fibers together [3].In recent years, due to the excellent mechanical properties [4], stable biocompatibility [5], tunable biodegradability [6], low immunogenicity, and low toxicity of silk proteins [7], the application of silk has no longer been limited to the textile industry and has been further extended to biomedical fields.Since sericin could cause inflammatory reactions in the body, silk fibroin and silk sericin are usually separated [8].Degummed silk fibers have received increasing attention as a biomaterial, especially in wound-healing materials [9,10] and bone tissue engineering [11][12][13][14].
The surface morphology of degummed silk fibers obtained from the two feeding methods is shown in Figure 1.It is obvious that the surface of silk fibroin fibers was smooth after steam degumming treatment.Silk sericin, which was easily soluble in hot water, dissolved under the action of steam heat and humidity.After washing, the silk fibroin, which was originally adhered to by the sericin, had no adhesive force and separated.Therefore, both of them were in a dispersed state.However, the silk fibroin fibers obtained by mulberry leaf (ML group) feeding showed slight fibrillation on the fibroin fiber surface, and fine longitudinal streaks of fibrillar structures began to appear on some of the silk fibroin fibers.This may have been due to the low sericin content in the silk from the mulberry leaf feeding group, which made it faster to remove sericin in the degumming process, and then, it would have acted on the silk fibroin fiber to form split fibers.Moreover, the scanning electron microscopy (SEM) results also showed that the fineness of the degummed silk fibers in the ML group was larger than that in the AD group.The average size of silk fibers was 3.18dtex in the ML group and 2.97dtex in the AD group.Qu et al. [27] suggested that this was caused by the difference in silk protein content per unit length.

Effect of Factory All-Age Artificial Diet Feeding on the Structure of Silk Fibers
Analyzing the structure of the silk fibroin fibers is useful for understanding its properties.The conformational differences in protein chains and the structural changes in aggregates between mulberry leaf and artificial feed silk fibroin fibers were investigated by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD).As shown in Figure 2a, FTIR can reflect the structure of and changes in molecules, can be used to analyze the structure and conformation of silk proteins, and is often used to observe many complex amide bands of silk proteins [28], which are mainly divided into three main vibration bands, in the range of 1600 to 1700 cm −1 for amide I (C=O stretching), 1500 to1580 cm −1 for amide II (in-plane N-H bending and C-N stretching), and 1200 to1300 cm −1 for amide III (N-H bending and C-N stretching) [29].The frequency splitting observed for the amide I and II bands was mostly due to the vibrational interactions among peptide groups in the chain and through hydrogen bonds [30].Among them, the peak at 1694 cm −1 was the β-turn conformation in the amide I band [31], and the peaks at 1620 cm −1 , 1516 cm −1 , and 1227 cm −1 were the stretches of the vibration absorption peaks of amide I-III bands in the β-sheet structure, respectively, which was consistent with the literature [32].There was no significant shift in the characteristic peaks of the silk fibers obtained from factory all-age artificial diet feeding, and no new peaks appeared, indicating that the main chemical structure of the two groups of silk was the same.
The amide I band of silk fibroin fibers was further deconvoluted and integrated with peaks, and the fitting results of the peaks are shown in Figure 2b-d.The content of the βsheet structure of silk fibroin fibers after factory all-age artificial diets was slightly lower than that of the mulberry leaf group.This may be due to the fact that the intake of artificial feed hinders the transformation of α-helix and random coil into β-sheet, resulting in a decrease in β-sheet structures.
The changes in the crystalline structure of the silk fibers obtained by artificial feed being given to silkworms are shown in Figure 3.According to the positions of the two groups of silk diffraction peaks, the silk crystal diffraction peaks were detected at about 9.3, 20.6, and 24.5°.Moreover, the shapes and positions of the two sets of X-ray diffraction curves were almost identical.A distinct peak at 20.6° and two minor peaks at around 9.3 and 24.5° in the XRD spectrum were attributed to the predominant β-sheet crystalline structure of silk fibroin fibers [33,34].MDI Jade was used to fit the peaks and calculate their crystallinity.The crystallinity of ML and AD was 46.16% and 45.27%, respectively.The crystal structure of the two groups was still dominated by a β-sheet structure, which was consistent with the above FTIR analysis conclusion.

Effect of Factory All-Age Artificial Diet Feeding on the Structure of Silk Fibers
Analyzing the structure of the silk fibroin fibers is useful for understanding its properties.The conformational differences in protein chains and the structural changes in aggregates between mulberry leaf and artificial feed silk fibroin fibers were investigated by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD).As shown in Figure 2a, FTIR can reflect the structure of and changes in molecules, can be used to analyze the structure and conformation of silk proteins, and is often used to observe many complex amide bands of silk proteins [28], which are mainly divided into three main vibration bands, in the range of 1600 to 1700 cm −1 for amide I (C=O stretching), 1500 to1580 cm −1 for amide II (in-plane N-H bending and C-N stretching), and 1200 to1300 cm −1 for amide III (N-H bending and C-N stretching) [29].The frequency splitting observed for the amide I and II bands was mostly due to the vibrational interactions among peptide groups in the chain and through hydrogen bonds [30].Among them, the peak at 1694 cm −1 was the β-turn conformation in the amide I band [31], and the peaks at 1620 cm −1 , 1516 cm −1 , and 1227 cm −1 were the stretches of the vibration absorption peaks of amide I-III bands in the β-sheet structure, respectively, which was consistent with the literature [32].There was no significant shift in the characteristic peaks of the silk fibers obtained from factory all-age artificial diet feeding, and no new peaks appeared, indicating that the main chemical structure of the two groups of silk was the same.
The amide I band of silk fibroin fibers was further deconvoluted and integrated with peaks, and the fitting results of the peaks are shown in Figure 2b-d.The content of the β-sheet structure of silk fibroin fibers after factory all-age artificial diets was slightly lower than that of the mulberry leaf group.This may be due to the fact that the intake of artificial feed hinders the transformation of α-helix and random coil into β-sheet, resulting in a decrease in β-sheet structures.
The changes in the crystalline structure of the silk fibers obtained by artificial feed being given to silkworms are shown in Figure 3.According to the positions of the two groups of silk diffraction peaks, the silk crystal diffraction peaks were detected at about 9.3, 20.6, and 24.5 • .Moreover, the shapes and positions of the two sets of X-ray diffraction curves were almost identical.A distinct peak at 20.6 • and two minor peaks at around 9.3 and 24.5 • in the XRD spectrum were attributed to the predominant β-sheet crystalline structure of silk fibroin fibers [33,34].MDI Jade was used to fit the peaks and calculate their crystallinity.The crystallinity of ML and AD was 46.16% and 45.27%, respectively.The crystal structure of the two groups was still dominated by a β-sheet structure, which was consistent with the above FTIR analysis conclusion.

Effect of Factory All-Age Artificial Diet Feeding on the Amino Acid Composition of Silk Fibers
Silk protein contains 18 kinds of amino acids.The type and content of amino acids often affect the structure of silk, but also, to a certain extent, they determine the basic properties of silk.In this experiment, acid hydrolysis was used to determine the content of various amino acids in the two groups of filaments, and tryptophan was destroyed in this process, so Table 1 presents the results of the determination of only 17 amino acids.As seen in the table, there was no significant difference in the amino acid composition of fibroin between feeding methods.Silk proteins had high glycine, alanine, and serine

Effect of Factory All-Age Artificial Diet Feeding on the Amino Acid Composition of Silk Fibers
Silk protein contains 18 kinds of amino acids.The type and content of amino acids often affect the structure of silk, but also, to a certain extent, they determine the basic properties of silk.In this experiment, acid hydrolysis was used to determine the content of various amino acids in the two groups of filaments, and tryptophan was destroyed in this process, so Table 1 presents the results of the determination of only 17 amino acids.As seen in the table, there was no significant difference in the amino acid composition of fibroin between feeding methods.Silk proteins had high glycine, alanine, and serine

Effect of Factory All-Age Artificial Diet Feeding on the Amino Acid Composition of Silk Fibers
Silk protein contains 18 kinds of amino acids.The type and content of amino acids often affect the structure of silk, but also, to a certain extent, they determine the basic properties of silk.In this experiment, acid hydrolysis was used to determine the content of various amino acids in the two groups of filaments, and tryptophan was destroyed in this process, so Table 1 presents the results of the determination of only 17 amino acids.As seen in the table, there was no significant difference in the amino acid composition of fibroin between feeding methods.Silk proteins had high glycine, alanine, and serine contents, accounting for about 75% of the total amino acid composition.In the crystalline region, the highly repetitive hexapeptide sequence GAGAGS (amino acids Gly-Ala-Gly-Ala-Gly-Ser) is considered to be the building block of the β-sheet microcrystalline region in silk proteins [35].In the previous literature, it was reported that glycine and alanine are predominantly located in the crystalline region and that changes in the ratio of these two amino acids influence the crystal structure of silk proteins [36].The calculated results showed that the ratio was slightly lower in the ML group, and fibroin proteins were more likely to form crystal structures.Another important factor affecting the crystallization process is the ratio between amino acid residues in the small and large side chains.The large side chain amino acid has a high molecular weight and is difficult to fold, which prevents the close arrangement of the peptide chains when the ratio is high, resulting in a coiled molecular conformation [37,38].Tyrosine and phenylalanine are large sidearm amino acids, mainly located in the amorphous region, with a share of 11.37% and 11.22% in the ML and AD groups, respectively.Furthermore, amino acids can be categorized into hydrophilic and hydrophobic amino acids.Hydrophilic amino acids are amino acids that are hydrophilic in water, such as aspartic acid, threonine, serine, glutamic acid, tyrosine, lysine, etc., also known as polar amino acids.The percentage of polar amino acids in the ML and AD groups was 30.82% and 30.30%, respectively.The common hydrophobic amino acids mainly included seven amino acids such as glycine, alanine, and isoleucine, and the proportion of them in the ML and AD groups was 67.45% and 67.96%, respectively.

Effect of Factory All-Age Artificial Diet Feeding on the Mechanical Properties of Silk Fibers
It is well known that silk is a semi-crystalline biopolymer material, with crystalline regions formed by a β-sheet embedded in an amorphous protein matrix formed by a random coil.The strength of the silk fiber depends mainly on the crystalline zone, while the amorphous zone accounts for its extensibility [39].The test results of the breaking strength and elongation at break of silk before and after degumming are shown in Figure 4.In degummed silk fibers without the support of the outer layer of silk glue, the cocoon filaments were scattered between the monofilament.At the same time, the covalent bonds between the fibers were weakened, so the breaking strength and elongation at break of the silk fibroin fibers decreased after degumming.Surprisingly, the strength of the silk fibers in the AD group was not much different from that of the ML group, due to their amino acid content and crystalline structure essentially being the same.The extensibility was 14.35% and 13.88% in the ML and AD groups, respectively.This was due to the higher proportion of large side chain amino acids in the ML group.However, there was no significant difference overall.It should be noted that there are inconsistencies in the results of silk fiber strength obtained by the factory all-age artificial diet feeding test on the market, which may be due to the results of different feed varieties.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 14 the silk fibroin fibers decreased after degumming.Surprisingly, the strength of the silk fibers in the AD group was not much different from that of the ML group, due to their amino acid content and crystalline structure essentially being the same.The extensibility was 14.35% and 13.88% in the ML and AD groups, respectively.This was due to the higher proportion of large side chain amino acids in the ML group.However, there was no significant difference overall.It should be noted that there are inconsistencies in the results of silk fiber strength obtained by the factory all-age artificial diet feeding test on the market, which may be due to the results of different feed varieties.

Effect of Factory All-Age Artificial Diet Feeding on Hygroscopic Properties of Silk Fibers
In addition to the temperature and humidity of external conditions, the factors affecting the hygroscopicity and moisture regain of fiber are also related to its internal factors such as hydrophilic group, crystallinity, specific surface area, and so on.The test results of moisture absorption and moisture regain of silk fibroin fibers are shown in Figure 5.It can be seen from Figure 5a that the moisture absorption curves of the two groups of silk fibroin fibers were the same in each period.In the first 20 min, the moisture absorption first increased rapidly, and then, the upward trend slowed down and tended to be saturated after 60 min.The reason for the hygroscopic phenomenon is the presence of amorphous regions and hydrophilic amino acids in the internal structure of silk fibroin fibers.Hydrophilic functional groups such as amino groups and carboxyl groups in hydrophilic amino acids can form hydrogen bonds with water molecules, which enhances their ability to interact with water.However, according to "2.3.Effect of Factory All-Age Artificial Diet Feeding on the Amino Acid Composition of Silk Fibers", it can be found that the proportion of hydrophilic amino acids is smaller, the proportion of hydrophobic amino acids is greater, and the non-polar amino acids are the main components of silk fibroin fibers.Moreover, there is a crystalline region where the peptide chains are arranged neatly and tightly, and it is difficult for water molecules to enter, so the moisture absorption rate is less than 9%.Moisture regain also reflects the moisture absorption capacity of the fibers to a certain extent.As can be seen from Figure 5b, the AD group also had the same moisture regain rate as the ML group.

Effect of Factory All-Age Artificial Diet Feeding on Hygroscopic Properties of Silk Fibers
In addition to the temperature and humidity of external conditions, the factors affecting the hygroscopicity and moisture regain of fiber are also related to its internal factors such as hydrophilic group, crystallinity, specific surface area, and so on.The test results of moisture absorption and moisture regain of silk fibroin fibers are shown in Figure 5.It can be seen from Figure 5a that the moisture absorption curves of the two groups of silk fibroin fibers were the same in each period.In the first 20 min, the moisture absorption first increased rapidly, and then, the upward trend slowed down and tended to be saturated after 60 min.The reason for the hygroscopic phenomenon is the presence of amorphous regions and hydrophilic amino acids in the internal structure of silk fibroin fibers.Hydrophilic functional groups such as amino groups and carboxyl groups in hydrophilic amino acids can form hydrogen bonds with water molecules, which enhances their ability to interact with water.However, according to "Section 2.3.Effect of Factory All-Age Artificial Diet Feeding on the Amino Acid Composition of Silk Fibers", it can be found that the proportion of hydrophilic amino acids is smaller, the proportion of hydrophobic amino acids is greater, and the non-polar amino acids are the main components of silk fibroin fibers.Moreover, there is a crystalline region where the peptide chains are arranged neatly and tightly, and it is difficult for water molecules to enter, so the moisture absorption rate is less than 9%.Moisture regain also reflects the moisture absorption capacity of the fibers to a certain extent.As can be seen from Figure 5b, the AD group also had the same moisture regain rate as the ML group.

Effect of Factory All-Age Artificial Diet Feeding on Thermal Stability of Silk Fibers
The thermal properties of silk fibroin fibers in ML and AD were studied to further understand the effect of feed feeding.The thermogravimetric (TG) and differential thermogravimetric (DTG) curves of two sets of silk fibers from room temperature to 600 °C were obtained, and the results are shown in Figure 6.The thermal degradation character-

Effect of Factory All-Age Artificial Diet Feeding on Thermal Stability of Silk Fibers
The thermal properties of silk fibroin fibers in ML and AD were studied to further understand the effect of feed feeding.The thermogravimetric (TG) and differential thermogravimetric (DTG) curves of two sets of silk fibers from room temperature to 600 • C were obtained, and the results are shown in Figure 6.The thermal degradation characteristics of the two groups of silk fibroin samples were almost identical, which meant that different feeding methods did not cause significant changes in the thermal degradation process.An initial weight drop was observed at about 100 • C, and the main factor of mass loss was the evaporation of moisture inside the silk fibers.From 280 • C, the temperature continued to rise, which intensified the vibration of the internal groups of the silk fiber, the carboxyl and amino groups were gradually destroyed and decomposed into gas overflow, resulting in a sharp drop in the percentage of weight residue.The DTG curve reflects the change in the rate of mass loss with temperature.The figure showed that the two groups of samples had a large thermal degradation peak near 320 • C, the weight loss was mainly caused by the breakage of peptide bonds and side groups, and the silk fiber was violently decomposed [35].A small degradation peak appeared at around 555 • C, and the degradation of silk fibers was slowed down.According to the test results, the thermal stability of silk fibers did not change significantly after artificial feed feeding.

Effect of Factory All-Age Artificial Diet Feeding on Thermal Stability of Silk Fibers
The thermal properties of silk fibroin fibers in ML and AD were studied to further understand the effect of feed feeding.The thermogravimetric (TG) and differential thermogravimetric (DTG) curves of two sets of silk fibers from room temperature to 600 °C were obtained, and the results are shown in Figure 6.The thermal degradation characteristics of the two groups of silk fibroin samples were almost identical, which meant that different feeding methods did not cause significant changes in the thermal degradation process.An initial weight drop was observed at about 100 °C, and the main factor of mass loss was the evaporation of moisture inside the silk fibers.From 280 °C, the temperature continued to rise, which intensified the vibration of the internal groups of the silk fiber, the carboxyl and amino groups were gradually destroyed and decomposed into gas overflow, resulting in a sharp drop in the percentage of weight residue.The DTG curve reflects the change in the rate of mass loss with temperature.The figure showed that the two groups of samples had a large thermal degradation peak near 320 °C, the weight loss was mainly caused by the breakage of peptide bonds and side groups, and the silk fiber was violently decomposed [35].A small degradation peak appeared at around 555 °C, and the degradation of silk fibers was slowed down.According to the test results, the thermal stability of silk fibers did not change significantly after artificial feed feeding.

Effect of Factory All-Age Artificial Diet Feeding on In Vitro Degradation of Silk Fibers
In recent years, with the development of tissue engineering and the great potential of silk protein-based biomaterials in biomedical applications, it has become particularly important to explore the enzymatic degradation properties of silk protein materials.To facilitate later purification and application, the enzyme hydrolysis method with efficient

Effect of Factory All-Age Artificial Diet Feeding on In Vitro Degradation of Silk Fibers
In recent years, with the development of tissue engineering and the great potential of silk protein-based biomaterials in biomedical applications, it has become particularly important to explore the enzymatic degradation properties of silk protein materials.To facilitate later purification and application, the enzyme hydrolysis method with efficient and mild action was used in this case.Some in vitro studies on the degradation behavior of two groups of filamentous protein fibers were carried out using α-chymotrypsin.As can be seen in Figure 7, the residual mass of degummed silk fibers treated with PBS buffer and chymotrypsin solution decreased gradually with the increase in treatment time.A comparison of silk fibers prepared on the basis of different feeding methods showed that AD and ML had the same degree of reduction in silk residual mass.The rate of degradation was analyzed to be closely related to the structure and molecular weight [40,41] of filamentous fibrous proteins.This implies that aa non-dense structure and low molecular weight have a better ability to increase the rate of degradation of silk fibers.Soluble silk fiber has been reported to be degraded by α-chymotrypsin [42].However, the ML and AD groups still had more than 97% mass residue after 28d of enzymatic hydrolysis.This shows that αchymotrypsin has no serious effect on the degradation of silk fibers, which is in agreement with descriptions in the literature [43].In fact, the rate of degradation is dependent on the concentration of the protease used, with larger concentrations leading to faster degradation.To increase the in vitro degradation of silk fibers, the concentration of the enzyme solution can be increased or other types of proteases can be selected.fiber has been reported to be degraded by α-chymotrypsin [42].However, the ML and AD groups still had more than 97% mass residue after 28d of enzymatic hydrolysis.This shows that α-chymotrypsin has no serious effect on the degradation of silk fibers, which is in agreement with descriptions in the literature [43].In fact, the rate of degradation is dependent on the concentration of the protease used, with larger concentrations leading to faster degradation.To increase the in vitro degradation of silk fibers, the concentration of the enzyme solution can be increased or other types of proteases can be selected.The surface of the silk fibers was damaged to varying degrees after exposure to αchymotrypsin solution for 14 and 28 d.The results are shown in Figure 8.A closer look at Figure 8a,b shows that after 14 d of degradation, the surface of both groups of silk fibers showed fine traces of corrosion.The fibers of the ML group showed splitting, while the surfaces of the fibers of the AD group were relatively smooth.As the time of enzymatic degradation increased, the fiber surface roughening became more severe.As shown in Figure 8c,d, a large number of solubilized cracked particles appeared on the surface of the silk fibers.The AD group had its most severe erosion after 28 d.This was in general agreement with the results of the above analysis of the degradation rate of silk fibers after treatment with α-chymotrypsin solution.The surface of the silk fibers was damaged to varying degrees after exposure to αchymotrypsin solution for 14 and 28 d.The results are shown in Figure 8.A closer look at Figure 8a,b shows that after 14 d of degradation, the surface of both groups of silk fibers showed fine traces of corrosion.The fibers of the ML group showed splitting, while the surfaces of the fibers of the AD group were relatively smooth.As the time of enzymatic degradation increased, the fiber surface roughening became more severe.As shown in Figure 8c,d, a large number of solubilized cracked particles appeared on the surface of the silk fibers.The AD group had its most severe erosion after 28 d.This was in general agreement with the results of the above analysis of the degradation rate of silk fibers after treatment with α-chymotrypsin solution.

Materials
The raw materials for this experiment were silkworm cocoons with two feeding methods, one of which was fed on mulberry leaves from Haian, Jiangsu Province, and the other was fed on a factory all-age artificial diet from the Zhejiang Babei group.The α-

Materials
The raw materials for this experiment were silkworm cocoons with two feeding methods, one of which was fed on mulberry leaves from Haian, Jiangsu Province, and the other was fed on a factory all-age artificial diet from the Zhejiang Babei group.The α-chymotrypsin for the in vitro degradation tests was purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd., and the phosphate-buffered saline solution (PBS) was provided by Yida Technology Co., Ltd (Quanzhou, China).Deionized water was used throughout the process.

Preparation of Silk Fibroin Fibers
The preparation process of silk fibroin fibers is shown in Figure 9.The steam degumming method was used for the acquisition of silk fiber, which was used as the material in this study.First of all, silkworm cocoons were torn apart into three layers to facilitate complete degumming.The deionized water-soaked cocoon shells were then subjected to degumming in a modified pressure cooker (XFS-280CB, Xinfeng Medical Apparatus, Zhejiang, China, a pressure meter was equipped on the cover lib) at 125 • C for 90 min [44].Finally, the degummed cocoon shells were ultrasonically cleaned and then rinsed in warm water and dried to obtain the silk fibroin sample.

Morphological Observation
The surface morphology of the silk fibroin specimens was scanned and observed using an Ultra55 scanning electron microscope (Carl Zeiss, Oberkochen, Germany) after gold palladium coating at an accelerating voltage of 3 kV with a magnification of 1000 times.

Fourier Transform Infrared Spectroscopy Test
The Attenuating Total Reflection Fourier transform infrared (ATR-FTIR) spectra of degummed silk fibroin fibers were obtained using a Nicolet 5700 spectrometer (Thermo Nicolet, Shanghai, China) within a wavelength range of 4000-400 cm −1 with a spectral resolution of 4 cm −1 .

X-ray Diffraction Test
The crystalline structure was tested using an X'TRA X-ray diffractometer with an Xray tube that produces monochromatic Cu-Kα radiation.When scanning, the SF fibers were cut into powders, placed on the sample stage, and scanned from 5° to 50° at a scan-

Morphological Observation
The surface morphology of the silk fibroin specimens was scanned and observed using an Ultra55 scanning electron microscope (Carl Zeiss, Oberkochen, Germany) after gold palladium coating at an accelerating voltage of 3 kV with a magnification of 1000 times.

Fourier Transform Infrared Spectroscopy Test
The Attenuating Total Reflection Fourier transform infrared (ATR-FTIR) spectra of degummed silk fibroin fibers were obtained using a Nicolet 5700 spectrometer (Thermo Nicolet, Shanghai, China) within a wavelength range of 4000-400 cm −1 with a spectral resolution of 4 cm −1 .

X-ray Diffraction Test
The crystalline structure was tested using an X'TRA X-ray diffractometer with an X-ray tube that produces monochromatic Cu-Kα radiation.When scanning, the SF fibers were cut into powders, placed on the sample stage, and scanned from 5 • to 50 • at a scanning speed of 2( • )/min under the conditions of 40 kV and 40 mA.

Amino Acid Composition Test
The amino acid composition of the hydrolysates was measured with a Hitachi L8900 Amino Analyzer (Hitachi Company, Tokyo, Japan).The two kinds of silk fibroin fiber samples were hydrolyzed in 10 mL of 6 mol/L HCL in a vacuum atmosphere at 110 • C for 24 h and then filtered.Subsequently, each sample was deacidified and dried on a rotary evaporator at 45 • C and filtered again by adding buffer solution.Three samples of SF fibers from each group were tested to obtain the average values.

Mechanical Property Test
The breaking strength and elongation at break of the degummed silk fibers were determined by the YG004 electronic strength machine (Zhongxian Testing Equipment, Changzhou, China) with a gauge length of 25 mm at a stretching speed of 100 mm/min.All specimens were subjected to experiments at (20 ± 2) • C and (65 ± 4) % relative humidity (RH).Thirty samples of degummed silk fibers were tested in each group, and the results were averaged.

Hygroscopic Test
The moisture absorption rate of silk fibers indicates the percentage of water absorbed compared to its moisture-free weight.The dried samples were weighed and quickly transferred to a desiccator containing a saturated copper sulphate solution, where they were subjected to hygroscopic experiments at a temperature of (20 ± 2) • C and a relative humidity range of (65 ± 4)%.In the moisture regain test, the sample was subjected to standard conditions for 12 h.The mass of each set of specimens was recorded and calculated according to the following formulas: Moisture absorption rate (%) = m 2 − m 0 m 0 ×100, Moisture regain rate (%) = where m 2 is the mass after moisture absorption, m 1 is the initial mass under standard conditions, and m 0 is the mass of drying to constant weight.

Thermal Stability Test
Thermogravimetric studies of the silk fibroin powders were conducted by a thermogravimetric analyzer (TGA550, TA, New Castle, DE, USA).The thermograms were obtained under a nitrogen atmosphere (60 mL/min) at a uniform heating rate of 10 • C/min in the temperature range of 50-650 • C.

In vitro Enzymatic Degradability Test
Centrifuge tubes were first sterilized in an autoclave for 20 min.Each sample was then individually placed in a 50 mL centrifuge tube and sterilized by UV irradiation for 30 min.After complete sterilization, 1 mg/mL of α-chymotrypsin and 30 mL of PBS buffer were added to the centrifuge tubes in an ultra-clean bench.Finally, they were placed in a constant-temperature shaker at 37 • C for degradation, and the buffer was replaced with

Figure 1 .
Figure 1.SEM images of silk fibers with two different feeding methods.(a) ML group, (b) AD group.

Figure 2 .
Figure 2. (a) ATR-FTIR spectra of silk fibers with two different feeding methods, (b) secondary structure content, (c) deconvolution details of amide I band of ML group, (d) deconvolution details of amide I band of AD group.Black line, original spectra; orange dash line, simulated spectra from the total peaks.

Figure 3 .
Figure 3. (a) XRD of silk fibers with two different feeding methods, (b) degree of crystallinity.

Figure 2 . 14 Figure 2 .
Figure 2. (a) ATR-FTIR spectra of silk fibers with two different feeding methods, (b) secondary structure content, (c) deconvolution details of amide I band of ML group, (d) deconvolution details of amide I band of AD group.Black line, original spectra; orange dash line, simulated spectra from the total peaks.

Figure 3 .
Figure 3. (a) XRD of silk fibers with two different feeding methods, (b) degree of crystallinity.

Figure 3 .
Figure 3. (a) XRD of silk fibers with two different feeding methods, (b) degree of crystallinity.

Figure 4 .
Figure 4. Mechanical properties of silk fibers with two different feeding methods.

Figure 4 .
Figure 4. Mechanical properties of silk fibers with two different feeding methods.

Figure 5 .
Figure 5. Hygroscopic properties of silk fibers with two different feeding methods.(a) Moisture absorption, (b) moisture regain.

Figure 5 .
Figure 5. Hygroscopic properties of silk fibers with two different feeding methods.(a) Moisture absorption, (b) moisture regain.

Figure 6 .
Figure 6.TG and DTG curves of silk fibers with two different feeding methods.(a) TG, (b) DTG.

Figure 6 .
Figure 6.TG and DTG curves of silk fibers with two different feeding methods.(a) TG, (b) DTG.

Figure 7 .
Figure 7. Enzymatic and PBS degradation curves of silk fibers with two different feeding methods.

Figure 7 .
Figure 7. Enzymatic and PBS degradation curves of silk fibers with two different feeding methods.

14 Figure 8 .
Figure 8. SEM image of silk fiber degradation process.(a) ML group after 14 d, (b) AD group after 14 d, (c) ML group after 28 d, (d) AD group after 28 d.

Figure 8 .
Figure 8. SEM image of silk fiber degradation process.(a) ML group after 14 d, (b) AD group after 14 d, (c) ML group after 28 d, (d) AD group after 28 d.

Table 1 .
Differences in amino acid composition of silk fibers with two different feeding methods.