Natural polymers have been wildly used in biotechnological and biomedical applications.
These were due to their unique properties including nontoxicity, biocompatibility
and biodegradability. Unfortunately, natural homopolymer is inadequate to meet
the diversity of demands for biomaterials by itself (Kweon
et al., 2001). Silk is a kind of natural polymer which produced by
the family Bombycidae (domestic silk; Bombyx mori) and Saturniidae (wild
silk; Antheraea pernyi, Philosamia ricini, etc.) of the order
Lepidoptera (Dash et al., 2007). Silk is one of
the most valuable materials for applications such as the textile and biomedical
devices (Taddei et al., 2006). Generally, silk
fiber consists of two main proteins; fibroin and sericin. Silk Fibroin (SF)
is a typical fibrous protein and shows excellent both physical and chemical
properties. SF has been used in various fields such as cosmetics, food additives
and medical materials (Min et al., 2004). In
addition, SF can be prepared and used in various forms including gel, powder,
fiber or membrane, depending on application (Park et
The efforts at adjusting or preparing the biomaterial forms to their required
functions are the aims in last decades. The silk powder is a kind of material
form and now produced commercially as an additive in cosmetics and functional
foods. Moreover, potential application of silk powder including surface coating,
fibre treatment, fillers in films, ink, wound care, enzyme immobilization, composite
scaffold for cell growth and drug delivery have also been reported by Rajkhowa
et al. (2009). Making available novel versatile biomaterials addressing
a broad range of biomedical needs is a crucial technological challenge (Taddei
et al., 2006). Recently, much attention has been studied on B.
mori either in producing process or application forms (Park
et al., 2004). Contrast few studies have been carried out on the
wild silk SF, especially Eri (P. ricini) silk.
Silk Fibroin (SF) powder can be prepared either by a solution route or by a
mechanical method. With the solution route, SF is firstly dissolved in a concentrated
salts followed by removal of the salts and then produced regenerated SF powder
(Yoshimizu and Asakura, 1990; Jin
and Young, 2001). On the other hand, the mechanical method avoids lengthy,
costly and environmentally sensitive production process (Rajkhowa
et al., 2009). It has been reported that dissolution of the SF fiber
of wild silk is hardly work. In addition, dissolution of SF is often required
for non textiles applications (Kweon et al., 2000).
Therefore, the aims of this study are to prepare and characterize SF powder of Eri (P. ricini) silk. The conformation structure and thermal properties of the silk were investigated by using Fourier Transform Infrared (FT-IR) and TA-Instrument TG SDT Q600 thermogravimetric analyzer, respectively.
MATERIALS AND METHODS
This study was constructed for 5 months from October 1, 2008 to March 5, 2009. The preparation of SF powder and characterization were performed at the Central Instrument, Faculty of Science, Mahasarakham University.
Materials: The Eri (P. ricini) silk cocoons were kindly supplied from Silk Innovation Center (SIC) Mahasarakham University, Thailand. The cocoons were degummed twice using 0.5% Na2CO3 (w/v) and thoroughly rinsed 2 times in warm distilled water. They were then air-dried at room temperature.
Dissolution of silk fibroin: The Eri SF was dissolved with 9 M Ca (NO3)2
solution followed by Tao et al. (2007). Briefly,
dried Eri SF was mixed with dissolving solution of 1 g SF to 10 mL of 9 M Ca(NO3)2
solution. The solution was firstly warmed to about 90°C on the hot plate,
then gradually added SF into the solution and magnetic stirred at 100-105°C
until SF completely dissolved (approximately 45 min). The SF hydrolysate was
filtrated and then dialyzed in cellulose tube against distilled water for 3
days at room temperature.
SF powder preparation: There are many reports about SF powder preparation
such as spray drying (Rajkhowa et al., 2009).
However, lyophilization or freeze-drying method is a simple and popular method
(Yao et al., 2004). Fifty milliliters of SF
solution was freeze-dried about 2 days. The obtained SF powder was then grinned
by using mortar and pestle to obtain fine powder. The SF powder was returned
to dehydrate in vacuum oven before storing in desiccators. They were then observed
their appearances and also measured particle sizes by sieving method.
Investigation of SF powder: The samples were prepared using KBr disc in the ratio of 9:1 (KBr : sample) and then analyzed with FT-IR (Perkin Elmer-Spectrum Gx, USA) in the spectral range of 4000-400 cm-1 at 4 cm-1 spectral resolution and 32 scans. A TA-Instrument TG SDT Q600 thermogravimetric analyzer was used to determine the thermal behavior. The analysis condition was 50-1000°C for heating sample at 20°C min-1 rates under nitrogen atmosphere. Difference of silk structure between Eri and B. mori was compared.
The Eri SF powder could be prepared. Generally, the powder appeared light white color like cocoon and luster.
|| Absorption bands of FT-IR spectra of the SF in different
||FTIR spectra of the different forms of SF, (A) B. mori
SF powder, (B) Eri SF fiber and (C) Eri SF powder
SF powder was arranged in the micrometer of their sizes under 200 micrometers pore sieved.
FT-IR spectra: Table 1 showed the absorption bands of silk cocoon and SF powder. The absorption bands of Eri silk cocoon appear at 1655 cm-1 with shoulder peak at 1702 cm-1 (amide I), 1559 cm-1 (amide II), 1235 cm-1 (amide III) and 695 cm-1 (amide IV). Contrast FT-IR spectrum of the SF powder showed intense absorption bands at 1643 cm-1 (amide I), 1542 cm-1 (amide II), 1235 cm-1 (amide III) and 695 cm-1 (amide IV). The FT-IR result of B. mori powder appeared at similar absorption bands of Eri SF powder, except at amide IV (Fig. 1).
Thermogravimetry: At low temperature, Eri silk has decomposed weight
higher than that of B. mori silk. However, both SF fiber and powder of
B. mori silk started decomposition at lower temperature than Eri silk
about 300°C which completely decomposed at 360°C (Fig.
2). The Eri silk underwent of three thermal decomposition stages which are
200 to 300°C, 300 to 350°C and complete decomposition at approximately
350 to 400°C. The detail of those decomposition peaks were clearly evidenced
by differential thermogravimetric (DTG) curves (Fig. 3). The
maximum decomposition temperatures of Eri SF fiber were 235, 350 and 400°C,
while Eri SF powder was 235, 350 and 390°C.
|| TG curves of SF in different forms
|| DTG curves of the SF in different forms
|| DSC thermograms of the SF in different forms
Differential scanning calorimetry: With DSC thermograms, the peak at below 100°C distributions to the dehydration of the SF appeared in all of samples. The Eri silk showed multiple endothermic peaks at approximately 310, 380 and 450°C. Contrast the B. mori silk occurred strong single step at 310 and 340°C for SF powder and SF fiber, respectively (Fig. 4).
Eri SF solution could be achieved by dissolving with 9 M Ca(NO3)2.
The SF solution was then used to construct SF powder and subject to investigate
properties. In general, silk protein composed of many amino acid residues. They
were linked with peptide bonds. The secondary structures of silk protein are
indicated by the amide groups of the amino acid components (Kweon
et al., 2000; Hino et al., 2003). The
amide I (1700-1600 cm-1), amide II (1600-1500 cm-1), amide
III (1300-1200 cm-1) and amide IV (700-600 cm-1) bands
were measured. From FT-IR results, the lower wave number bands of amide I and
amide II of the Eri SF powder were assigned to β-sheet structures (Kweon
et al., 2001). The result indicating that the main structures of
Eri SF powder were co-existed of α-helix and β-sheet structures with
predominantly β-sheet form (Tao et al., 2007).
It is suggested that the step of SF powder preparation could be induced structural
transition of the amino acid components of the SF. The result may suggest that
the dehydration of moisture from the silk could be affected on the silk structure.
However, FT-IR result of the Eri and B. mori SF powder were differed. Thermal
properties of domesticated silk, B. mori took place in a single step;
contrast from wild silk, P. ricini which underwent at least two steps
(Kweon et al., 2000). This might be expected that
structure and amino acid between B. mori and Eri SF were differed. In
addition, decomposition temperature of Eri silk is similar to other wild silk,
especially A. pernyi. However, they are different in details such as
the endothermic peaks at about 310 and 380°C of Eri silk which were differed
from A. pernyi (Kweon et al., 2000). The
differences of some properties between wild silk should be affected by the unique
strain and genetic information. These mean that amino acid compositions and
bond formation between them were main factors on the silk characteristics. It
was also observed that the maximum decomposition temperatures of Eri SF powder
and SF fiber were higher than that of B. mori SF powder or SF fiber.
The result indicated that different characteristics of the silk were influenced
by the components, form or silk strains.
The Eri SF powder could be achieved by the present study. The obtained SF powder
was light white color, luster and arranged in micrometer sizes. With FT-IR spectra,
the SF powder co-existed of α-helix and β-sheet structures and differed
from the Eri SF fiber and B. mori SF powder. Those of TG, DTG and DSC
indicated that the thermal behavior could be affected by silk form and strain.
We thank Central Instrument staff, for guidance in FT-IR investigation. We thank Division of Support and Development, Mahasarakham University for financial support. Finally, we also thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand to support this study.