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[Note]Enhanced Mechanical Property of Silk Sericin Beads Prepared fromEthanol-precipitated Sericin

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Int. J. Indust. Entomol. Vol. 15, No. 2, 2007, pp. 171~174

[Note]

Enhanced Mechanical Property of Silk Sericin Beads Prepared from Ethanol-precipitated Sericin

HanJin Oh, Ji Young Lee, Young-Kyu Lee1 and Ki Hoon Lee*

Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, 151 - 921, Seoul, Korea

1National Instrument Center of Environmental Management, Seoul National University, 151 - 921, Seoul, Korea (Received 11 September 2007; Accepted 30 November 2007)

Sericin beads were prepared from ethanol-precipi- tated sericin. The addition of ethanol into hot-water extracted sericin solution induced precipitation of seri- cin and thereby some sericin could be fractionated.

The ethanol-precipitated sericin (EpSS) had narrower molecular weight distribution than original sericin.

The EpSS had mainly random coil structure with small portion of β-sheet structure. With the EpSS, spherical beads could be prepared at lower concen- tration than with original sericin due to higher vis- cosity. The EpSS beads had better compressive strength than the original sericin beads and had rub- ber-like property. Our results suggest that EpSS is more compatible in the polymeric field, since it has better mechanical strength than original sericin.

Key words: Silk, Sericin, Bead, Biopolymer, Ethanol pre- cipitation

Introduction

Sericin derived from silkworm is a useful bioresource as it can be easily separated from silkworm cocoon. Usually, it can be obtained from degumming process using distilled water at high temperature (Lee, 2004). However, degra- dation occurs during the degumming process and it has been reported that the main cleavage site is next to the aspartic acid (Teramoto et. al., 2006). The final molecular weight of hot-water extracted sericin is usually in the range of 30-110 kDa, which is far low than the original

molecular weight in the silkworm gland (150-400 kDa) (Takasu et. al., 2002a).

There were many attempts to reuse the sericin in the polymeric field, but none of these results got final success due to their inherent properties. Films made from sericin were brittle and susceptible to water. Recently, we have successfully prepared sericin in a bead form by using LiCl/DMSO solvent system (Oh et. al., 2007). The con- centration of sericin solution could be increased up to 30

% (w/v). However, their mechanical strength was not suf- ficient to use in general applications.

There are many factors that affect the mechanical strength of polymeric materials. Molecular weight of a polymer is one of these factors, and a polymer of high molecular weight shows better mechanical strength than that of low one. The molecular weight distribution is also an important factor. In general, polymers do not have a definite molecular weight; instead they use “average”

molecular weight. Therefore, it is a mixture of various molecular weights, and their properties are also an aver- age of each molecular weight. Usually a polymer with narrow molecular weight has better mechanical strength than that with wider one.

In this paper, we added ethanol into sericin solution and recovered the precipitates thereafter. The ethanol-precip- itated sericin had narrower molecular weight distribution and has better mechanical properties.

Materials and Methods

Preparation of ethanol-precipitated silk sericin (EpSS) Sericin solution was obtained from silkworm cocoons by boiling them in the distilled water at 120oC for 1 hour.

After quenching the solution to room temperature, ethanol was added in ratio of 25, 50, and 75% (v/v). It was stirred for 1 hour and the precipitated sericin was recovered after

International Journal of

Industrial Entomology

*To whom the correspondence addressed

Department of Biosystems and Biomaterials Science and Engi- neering, Seoul National University San 56-1, Shillim, Kwanak, Seoul 151-921, Korea. Tel: +82-2-880-4625; FAX: +82-2-873- 2285; E-mail: prolee@snu.ac.kr

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172 HanJin Oh et al. centrifugation at 10,000 g for 30 mins. Finally, it was

freeze dried for 3 days.

Preparation of beads with EpSS

The dope solution of EpSS was prepared by dissolving a given amount of EpSS in 10ml of 1M LiCl/DMSO solu- tion for 2 hours at 50oC. The dope solution was dripped into ethanol bath, and the height of needle from the sur- face of ethanol was carefully controlled in order to obtain well-shaped beads. The SS beads were kept in the coag- ulant for additional 1 hour and washed at least 10 times with the same coagulant. Finally, it was washed 3 times with distilled water.

Characterization of EpSS

The secondary structure of EpSS was observed with FT- IR spectrometer (MIDAC, USA). The molecular weights of EpSS were observed by SDS-PAGE. The compression curves of a single EpSS bead were measured using Mim- imat (Rheometric Scientific, USA), after applying 0.1 N to EpSS bead. The pictures of solidified EpSS were taken with a digital camera (Olympus, Japan).

Results and Discussion

Takasu et. al. (2002a) adopted ethanol to isolate three main sericin components from silkworm cocoon. The addition of ethanol into sericin solution induced aggre- gation of sericin molecules. In general, the solubility of protein is determined by the dielectric constant of the sol- vent. The addition of ethanol changes the dielectric con- stant of the system, and therefore, sericin became insoluble. While they added ethanol into non-degraded sericin solution obtained from LiSCN extraction method, we used sericin solution from hot-water extraction method, which is more economically valuable method to extract sericin from cocoon. The precipitated sericin was collected by centrifugation and further experiments were conducted.

The molecular weights EpSS were shown in Fig. 1. The molecular weight of hot-water extracted sericin (HS) was in the range of 17-140 kDa. As the amount of added eth- anol increased, the molecular weight distribution was get- ting narrower. At present, we are not sure why the molecular weight distribution of EpSS is narrower than HS. One assumption could be made on the fact that sericin is composed of various molecules (Hu et. al., 1992). Each molecule will have different amino acid composition, and thereby, they will have different solubility in different sol- vent system. In the case of EpSS75, there are 75% of eth- anol and 25% of water in the solvent system. Therefore, it

could be thought that sericin molecules in the range of 36- 72 kDa are insoluble in this solvent system while others are remained soluble.

The secondary structure of HS and EpSS did not much differ (Fig. 2). The amide I peak in between 1700 and 1600 cm−1 indicates random coil structure in all samples.

However, there is a shoulder at 1630 cm−1 on amide I peak in EpSSs, indicating the existence of β-sheet structure. The amide V peak also shows an increase at 690 cm−1, which is due to the β-sheet structure of EpSS. The addition of eth- anol induces structural transition of sericin from random coil to β-sheet structure. It has been reported that silk pro-

Fig. 1.SDS-PAGE of hot-water extracted sericin (HS) and eth- anol-precipitated sericin (EpSS25, EpSS50 and EpSS75).

EpSS25:EpSS from ethanol 25%;EpSS50:EpSS from etha- nol 50%;EpSS75:EpSS from ethanol 75%.

Fig. 2.FT-IR spectra of hot-water extracted sericin (HS) and ethanol-precipitated sericin (EpSS50 and EpSS 75). EpSS50:

EpSS from ethanol 50%;EpSS75:EpSS from ethanol 75%.

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Silk Sericin Beads Prepared from Ethanol-precipitated Sericin 173

tein can be made insoluble by treating it with alcohols (Oh et. al., 2007). Therefore, the addition of ethanol not only ch- anges the dielectric constant in the solvent system but also makes sericin insoluble in water by structural transition.

Fig. 3 shows the digital images of beads prepared from EpSS. Previously, we have reported that a spherical bead could be formed when the concentration of sericin solu- tion is above 20% in the case of HS (Oh et. al., 2007). As shown in Fig. 3a, the shape of solidified sericin from 15%

HS solution has a disk shape rather than a sphere. How- ever, a spherical bead was formed at the concentration of 15% in case of EpSS. This is due to the differences in vis- cosity between HS and EpSS solutions. The concentration of sericin solution has significant effect on the shape of solidified sericin, since a solution of higher concentration has higher viscosity. EpSS solution exhibited higher vis- cosity than HS solution at the same concentration. The relationship between the molecular weight distribution and the viscosity is not yet clearly understood.

The mechanical properties of sericin beads were also compared (Fig. 4). The EpSS beads showed better com- pressive strength than HS beads. As mentioned before, the mechanical property could be affected by the molecular

weight distribution. If a polymer has a wide molecular weight distribution, then the molecules of low molecular weight can act as a defect in the polymer sample. It is interesting that the EpSS beads did not show any sign of rupture. In the case of HS beads, there is a sudden decrease in compressive stress due to the rupture of bead surface. However, in the EpSS bead, the compressive stress increases exponentially as the compressive strain increases. This kind of behavior could be found in rubbery materials.

Our results suggest that the EpSS is more adaptable in the polymeric field than the HS. Even though there were significant efforts to utilize sericin in the polymeric field, materials prepared from sericin alone had insufficient strength and were even brittle. Several strategies were adopted to solve problems through crosslinking or blend- ing with synthetic polymers (Zhang, 2002). However, these methods are not appropriate because these are not truly biodegradable. Another method that might increase the mechanical strength of sericin is using salt like urea for extraction of sericin of higher molecular weight (Takasu et. al., 2002b). But the salts should be removed by time- and cost-consuming dialysis procedure. The method that we present in this study is quite simple and efficient.

We use only distilled water for sericin extraction and sim- ple addition of ethanol can precipitate sericin that has bet- ter mechanical strength. However, more detailed studies are needed in the future to identify the precipitated sericin.

Acknowledgement

This work was supported by NICEM Grant.

Fig. 3.Images of hot-water extracted sericin (HS) beads (a) and ethanol-precipitated sericin (EpSS) beads (b). The concen- tration of each dope solution was equal (15%).

Fig. 4.Compressive stress and strain curve of of hot-water extracted sericin (HS) beads and ethanol-precipitated sericin (EpSS75) beads.

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174 HanJin Oh et al.

References

Hu, W. J., T. Shimada and M. Kobayashi (1992) Developmen- tal changes in electrophoretic patterns of sericin in the silkgland of Bombyx mori. J. Seric. Sci. Jpn. 61, 236-240.

Lee, K. H. (2004) Silk sericin retards the crystallization of silk fibroin. Macromol. Rapid Commun.25, 1792-1796.

Oh, H., J. Y. Lee, A. Kim, C. S. Ki, J. W. Kim, Y. H. Park and K. H. Lee (2007) Preparation of silk sericin beads using LiCl/DMSO solvent and their potential as a drug carrier for oral administration. Fiber. Polym. 8, 470-476.

Takasu, Y., H. Yamada and K. Tsubouchi (2002a) Isolation of

three main sericin components from cocoon of the silkworm, Bombyx mori. Biosci. Biotechnol. Biochem. 66, 2715-2718.

Takasu, Y., H. Yamada and K. Tsubouchi (2002b) Extraction and chromatographic analysis of cocoon sericin of the silk- worm, Bombyx mori. J. Insect Biotech. Sericology71, 151- Teramoto, H., A. Kakazu and T. Asakura (2006) Native struc-156.

ture and degradation pattern of silk sericin studied by 13C NMR spectroscopy. Macromolecules39, 6-8.

Zhang Y.-Q. (2002) Applications of natural silk protein sericin in biomaterials. Biotechnol. Adv.20, 91-100.

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