J. Biomedical Science and Engineering, 2011, 4, 397-402
doi:10.4236/jbise.2011.45050 Published Online May 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online May 2011 in SciRes. http://www.scirp.org/journal/JBiSE
The effects of different sterilization methods on silk fibroin
Yahong Zhao, Xiaoli Yan, Fei Ding, Yumin Yang, Xiaosong Gu
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, China.
Received 10 March 2011; revised 17 March 2011; accepted 17 April 2011.
ABSTRACT
The aim of this study was to investigate the changes
in the molecular structure and physiological activities
of silk fibroin induced by three different sterilization
methods (steam, gamma radiation and ethylene oxide)
with different dose or time period of sterilization by
means of Fourier transform infrared (FT-IR) spec-
troscopy, X-ray diffraction, mechanical properties
and assessment of molecular weight. The results
showed that the steam sterilization darkened the
color of silk fibroin and obviously affected the me-
chanical property; gamma irradiation slightly de-
graded the molecular weight of silk fibroin and the
speed of degradation increased with increasing irra-
diation dose; and ethylene oxide almost had no in-
fluence on silk fibroin expect for some slight hydroly-
sis on molecular weight. Because ethylene oxide ster-
ilization had the smallest influence on the quality of
silk fibroin with compared to other sterilization
methods, it could be used as an efficient method to
make fibroin more suitable for the development of
functional foods and cosmetics.
Keywords: Silk Fibroin; Sterilization; Degradation;
Mechanical Properties
1. INTRODUCTION
Silk, popularly known in the textile industry for its luster
and mechanical properties, is produced by cultured
silkworms [1]. The discovery of silk production by the
silkworm Bombyx mori can be traced back to a myste-
rious and romantic legend from ancient China [2]. Natu-
ral silk has long been used as fabric materials in textile
industry and also as surgical sutures in medical field [3],
but only recently has it been found rapidly increasing
applications in biomedical fields including the genera-
tion of tissue engineered bones, skins and cartilages
[4,5].
Native silkworm silk protein from Bombyx mori con-
sists of a core structural fibroin protein surrounded by
sericin, which is a family of glue-like proteins. A highly
repeated hydrophobic and crystallizable sequence has
been described for the primary structure of fibroin heavy
chain: Gly-Ala-Gly-Ala-Gly-X (X stands for Ser or Tyr).
Sericin is a more hydrophilic protein, whose primary
structure is richer in polar residues, but some of its frac-
tions are not completely water-soluble due to β-sheet
portion [6,7].
Silk is considered as one of the most promising natu-
ral polymers for a packaging application because of its
impressive mechanical properties [8], in addition to en-
vironmental stability, biocompatibility [7], controlled
proteolytic biodegradability, morphologic flexibility and
the ability for amino acid side change modification to
immobilize growth factors [9,10]. It will probably be
used to an even greater extent in the near future.
Tissue engineering is a growing research area where
the goal is to augment, replace, or restore complex hu-
man tissue functions [11]. Crude biomaterials normally
carry a great number of bacteria and fungi. Current prac-
tices of harvesting, handling, storage and production
may cause additional contamination and microbial
growth. The determination of these microorganisms may
indicate the quality of production and harvesting prac-
tices. Methods for decontamination of crude biomate-
rials mostly turn to the sterilization [12]. In order to
make tissue engineering a commercially successful con-
cept, the manufactured materials must be able to with-
stand processing and sterilization.
Most of the commonly used sterilization methods for
silk include steam, ethylene oxide and gamma ray radia-
tion [13,14]. The different forms of sterilization may
attack the materials of polymers by the same mechanism
resulting in hydrolysis, oxidation, chain scission and
depolymerization [15]. Some studies have shown that
gamma radiation can modify the chemical, physical and
mechanical properties of silk fibroin. However few
studies have discussed the modification of silk fibroin
properties induced by steam or ethylene oxide steriliza-
tion, which will restrict people from selecting the most
adequate sterilization method for silk fibroin and its
products.
People often assume that existing sterilization tech-

Y. H. Zhao et al. / J. Biomedical Science and Engineering 4 (2011) 397-402 398
nologies will be appropriate for silk and, for most in-
stances; the more popular methods are adequate for the
silk material [1]. But for selecting the best sterilization
method, we not only focus on the efficacy of a steriliza-
tion process in terms of effect of killing microorganism
and the nature of the residuals formed, but also take into
account the effect of sterilization processes on the prop-
erties of the silk material, which is often ignored. Obvi-
ously, the effect of sterilization processes on the proper-
ties of the silk material should be as minor as possible.
Before the sterilization method is endorsed for silk, its
effects on the properties and end performance of silk
should be well investigated [13].
The goal of this work was to investigate the changes
in the color, molecular weight and structure of silk pro-
duced by three different sterilization methods (steam,
gamma radiation and ethylene oxide) with different dose
or time period of sterilization by means of FT-IR (Fou-
rier Transform Infrared Spectroscopy) spectroscopy, X-
ray diffraction, mechanical properties and assessment of
molecular weight.
2. EXPERIMENTAL METHODS AND
MATERIALS
2.1. Materials
Raw silk fibers (Bombyx mori cocoons) were bought
from Xinyuan sericulture company, Hai’an, Jiangsu,
China. All other reagents used in the study were of ana-
lytical grade.
2.2. Preparation of Silk Fibroin
Raw silk fibers’ sericin coating was removed via de-
gumming process of boiling in 0.5% (w/w) aqueous
Na2CO3 solution for 30 min at 100˚C for three times [7].
2.3. Steam Sterilization
Steam sterilization is a relatively simple process, and it
kills microorganisms by destroying metabolic and struc-
tural components essential to their replication. In this
study the silk fibroin samples were exposed to saturated
steam at 121˚C for 30 min at a pressure of 115 kPa [16].
2.4. Gamma Irradiation
In this study, the gamma irradiation sample was carried
out in a Nantong Meikeer irradiation company (Jiangsu,
China). Radiation sterilization utilizes ionizing radiation
from a cobalt-60 (60Co) isotope source with sterilization
achieved when highly reactive free radicals induce
breaks in the DNA double helix of microorganisms,
preventing replication [17]. The most commonly vali-
dated dose used to sterilize medical materials is 25 kGy.
In this study, samples were exposed to 10 and 20 kGy
gamma irradiation using a 60Co source, respectively.
2.5. Ethylene Oxide Sterilization [18]
The ethylene oxide sterilization process utilizes ethyl-
ene oxide which has bactericidal, sporicidal and viru-
cidal effects resulting from alkylation of sulphydryl,
amino, carboxyl, phenolic and hydroxyl groups in nu-
cleic acids causing cell injury or death [11]. In this
study, samples were exposed to ethylene oxide, the
concentration of which was 800 g/m3, at 50˚C for 0.5 h,
1 h, 2 h, 4 h, 8 h respectively and aeration for 10 days.
After aeration, no ethylene oxide residue was tested in
silk fibroin samples [13].
2.6. FT-IR Analysis
FT-IR spectra were obtained with Nexus model 870
Fourier Transform IR Spectrophotometer. Dry silk fib-
roin was ground with KBr powder and compressed into
discs for FT-IR examination and analyzed with a model
Nexus 870 Fourier transform infrared spectrophotometer
(Nicolet Instruments Co, Madison, WI, USA). FT-IR
was used to study changes in the chemical structure of
the silk fibroin samples after different sterilization pro-
cedures, which were steam heated for 30 min, gamma
irradiated with 10, 20 kGy, or exposed to ethylene oxide
for 8 h.
2.7. X-Ray Di ff ra c ti on Analysis
X-ray diffraction patterns in the 2  range of 4˚ to 60˚
were obtained for the samples of unsterilized and steril-
ized at room temperature using a scan rate of 10˚ (2
 )/min
at 40 kV and 40 mA (x’TRA, Switzerland ARL). The
samples of sterilized silk fibroins were steam heated 30
min, gamma irradiated with 10, 20 kGy, or exposed to
ethylene oxide for 8 h.
2.8. Mechanical Analysis
The tensile strength of silk fibroin sterilized with differ-
ent methods was measured on a J-100 N strength-testing
machine with or without the sample being soaked in
phosphate buffered solution (pH 7.4) at 37˚C. The silk
fibroins processed with steam sterilization were post-
dried, and the samples sterilized by other methods were
treated in the same way. The strand of silk fibroin with
the same thickness was pre-measured. The sample length
was 20 mm. The tensile speed and gauge length were set
as 10 mm/min and 60 mm, respectively. The crosshead
speed was maintained at 200 mm/min. Every sample
was tested for 6 times.
2.9. Assessment of Molecular Weight
(SDS-PAGE) (Polyacrylamide Gel
Electrophoresis)
Samples were dissolved in a tertiary solvent system of
CaCl2/H2O/EtOH solution (mole ratio 1 : 8 : 2) at 80˚C
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Y. H. Zhao et al. / J. Biomedical Science and Engineering 4 (2011) 397-402 399
for 30 min and then dialyzed against milli-Q water in
dialysis tubing (molecular cutoff 12 kDa) at room tem-
perature. Milli-Q water was changed every 6 hours. Af-
ter 72 h of dialysis, the samples were removed from the
cassettes and stored at 4˚C [7]. Samples were reduced
and run on a NuPage 4% - 12% Bis-Tris gel in electro-
phoresis buffer. Prestained Protein (NEB #P7702) was
run as the molecular weight marker (6 - 175 kDa) on all
gels [19]. Gels were stained with the Commassie®
G-250 Stain (Bio-Rad, Catalog #161-0786). Digital im-
ages of the gel were captured with a GS-800 Calibrated
Densitometer (Bio-Rad).
3. RESULTS AND DISCUSSION
3.1. Visual Inspectio n
Visual inspection of all samples was performed before
and after sterilization. A color change from white to yel-
low in silk samples was observed after exposure to
steam, and the intensity of color change increased with
increasing heating time of sterilization. The yellow color
darkening of the steam-sterilized silk may result from
the Maillard reaction [20] between NH2 and OH groups.
However, no obvious color change was found in silk
samples sterilized by using ethylene oxide or gamma
irradiation.
3.2. FT-IR Analysis
Fig.1 showed the FT-IR spectra of silk fibroin (a) and
silk sterilized using steam (b), ethylene oxide (c), and
gamma irradiation (d), respectively. They were showed
absorption bands at 3307.0 cm–1 (ν N-H), 1647.5 cm–1
(amide Ι), 1516.1 cm–1 (amide II), 1231.9 cm–1 (amide
ш). And it could also be found that there were absorption
bands at 1516.1 cm–1 and 1067.4 cm–1, which were re-
lated to β-sheet conformation. From Figure 1, we can
see that the peak shape at amide Ι region change a little
by steam sterilization (b). This result seems that steam
sterilization process results in increasing beta-sheet con-
formation. And ethylene oxide (c) and gamma irradiation
(d) sterilization process did not modify the silk fibroin’s
composition and did not change the crystal conformation
of silk fibroin either.
3.3. X-Ray Diff ra ct io n
X-ray diffraction curves of silk fibroin and silk fibroin
exposed to different sterilizations were shown in Figure
2. They were almost the same and all showed a very
broad peak at 2
 = 20˚ and a little peak at 2  = 24˚,
which was a typical characteristic diffraction pattern of
silk fibroin. It belonged to β-crystal (silk II). The results
suggested that the unsterilized silk fibroin and silk fib-
roin exposed to different sterilizations all had β-sheet
crystal structures and the sterilization process did not
Figure 1. FT-IR spectra of unsterilized silk fibroin (a) and silk
fibroin sterilized using steam (b), gamma irradiation (c), and
ethylene oxide (d).
Figure 2. X-ray diffraction curves of unsterilized silk fibroin (a)
and silk fibroin sterilized using steam (b), gamma irradiation
(c), and ethylene oxide (d).
change the conformation of silk fibroin, which con-
firmed the results of FT-IR.
3.4. Mechanical Analysis
The mechanical measurement results indicated that the
maximum fracture strength of silk fibroin unsterilized,
and sterilized using steam, gamma irradiation and ethyl-
ene oxide were 4.3 ± 0.2 N, 2.9 ± 0.3 N, 4.2 ± 0.2 N and
4.0 ± 0.3 N, respectively, under dry conditions. In addi-
tion, statistical analysis in Figure 3 revealed that there
was no significant difference between gamma irradiation
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Figure 3. The average percent of maximum fracture strength
of silk fibroin unsterilized (a), and sterilized using steam (b),
gamma irradiation (c), and ethylene oxide (d) (n = 6). *p < 0.05
versus the unsterilized group.
and ethylene oxide sterilization for both groups were
compared to unsterilized group. But there was marked
difference between silk fibroins sterilized using steam
and other silk fibroin samples.
3.5. SDS-PAGE
In order to understand the distribution range of silk fib-
roin peptides processed with different sterilization
methods, the relative molecular mass of the protein was
measured by SDS-PAGE with 12% gel. As shown in
Figure 4, both large and small molecular weight of silk
fibroins was affected with sterilization. Compared with
unsterilized silk fibroin, the intensity of the 25 kDa band
for samples exposured to gamma irradiation and ethyl-
ene oxide decreased, suggesting that the 25 kDa chain
was cleaved and degraded to smaller peptides. In addi-
tion, the intensity of light chain decreased from 10 to 20
kGY. It was likely that the peptides within this smear
decreased in size with increased dose of irradiation.
Corresponding bands were found in all samples at
large molecular weight, which was likely due to minor
degradation of the heavy chain during sterilization proc-
essing and the difficulty in breaking the tight β-sheet
structure of crystalline region during processing sterili-
zation. But from the pattern, the intensity of all steriliza-
tion bands especially that of gamma irradiation and eth-
ylene oxide weakened. These results indicated that both
the intermediary regions of the fibroin as well as regions
within the heavy chain (possibly those between the
crystalline regions) were degraded by sterilization.
3.6. Steam Sterilization
It has been reported that the high temperature, humidity
and pressure used during steam sterilization can lead to
Figure 4. SDS-PAGE spectra of unsterilized silk fibroin (1)
and silk fibroin sterilized using steam (2), gamma irradiation10,
20 kGY (3,4), ethylene oxide (5) and Marker (6).
hydrolysis, softening and degradation of biomedical
polymers [16]. It was investigated that the high tem-
perature and moist environment created by the autoclave
sterilization process was found to change the color of
silk fibroin and the color of silk fibroin darkened with
increasing time period of sterilization. However, the
chemical structure of silk fibroin sustained during steam
sterilization as shown in FT-IR and X-ray spectrum, and
the SDS-PAGE pattern of silk fibroin upon steam ster-
ilization showed a slight decrease in molecular weight.
The result seems that steam sterilization process results
in increasing beta-sheet conformation. The mechanical
property changed a lot with contrast to other silk fibroin
samples. This was possibly due to little degradation of
silk fibroin occurred in the process of steam sterilization.
Thus, there are some limitations for steam sterilization
concerning batch process scale-up.
3.7. Gamma Irradiation Sterilization
Gamma irradiation, as one of the most commonly em-
ployed sterilization agent, is a rapid and highly effective
sterilization method for medical devices and materials,
but it has been reported that this form of sterilization
results in a range of physical changes including embrit-
tlement, degradation, odor generation, stiffening, sof-
tening, enhancement or reduction of chemical resistance
[11,21]. In this study, FT-IR spectra suggest that this
sterilization process does not change chemical properties
and structure or crystallinity and network structure of
silk fibroin and this is consistent with the observed
X-ray diffraction patterns. It has been found that the
gamma irradiation decreases the molecular mass of silk
fibroin, which implies that gamma irradiation breaks
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acetylamino group, and results in significant degradation
in the noncrystalline region of silk fibroin. This is be-
cause the radiation induces the difference in the amino
acid composition, especially the aromatic series amino
acid such as tyrosine and phenylalanine.
3.8. Ethylene Oxide Sterilization
Ethylene oxide is one of the most widely used commer-
cial sterilization processes in the medical device and care
industries. However, the alkylating reactions, which this
process utilizes to achieve sterilization, have also been
reported to react with the functional groups of some
biomedical materials [17]. Although the side chain such
as carboxy group, amino group, hydrosulfide group and
hydroxy group on the molecular weight are reactive and
provide a mechanism for side group attachment using a
variety of mild reaction conditions [17]. FT-IR spectra
and X-ray spectrum showed that ethylene oxide used in
sterilization process does not affect the chemical proper-
ties and structure of silk fibroin. From the SDS-PAGE
spectrum, the molecular mass decreased a little. There-
fore, ethylene oxide sterilization can be considered to be
the most adequate sterilizing agent for silk fibroin, de-
spite ethylene oxide sterilization associated with residues,
toxicity, flammability, and environmental risks.
4. CONCLUSIONS
In summary, the experiment results indicated that silk
fibroins were weakened by thermal methods of steriliza-
tion and steam sterilization also darkened the color of
silk fibroin. Silk fibroin can be processed with gamma
irradiation without significant loss of tensile strength but
the molecular weight decreased with the increase of ir-
radiation dose.
Except for some degradation on molecular weight,
silk fibroin sterilized with ethylene oxide does not
change its morphology or β-sheet structure, and its im-
pact on mechanical property can be ignored. Therefore
the ethylene oxide sterilization method can be consid-
ered as the most adequate sterilization method for silk
fibroin.
5. ACKNOWLEDGEMENTS
The financial supports of the Hi-Tech Research and Development
Program of China (863 Program, Grant No. 2006AA02A128), the Nature
Science Foundation of China (Grant No. 30770585), Basic Research
Program of Jiangsu Education Department (Grant no. 07KJA31025)
and Program for New Century Excellent Talents in University (NCET-
07-0466) are gratefully acknowledged.
REFERENCES
[1] Veparia, C. and Kaplan, D.L. (2008) Silk as a biomaterial.
Progress in Polymer Science, 32, 991-1007.
doi:10.1016/j.progpolymsci.2007.05.013
[2] Yang, Y.M., Ding, F., Wu, J., Hu, W., Liu, W., Liu, J. and
Gu, X.S. (2007) Development and evaluation of silk fib-
roin-based nerve grafts used for peripheral nerve regen-
eration. Biomaterials, 28, 5526-5535.
doi:10.1016/j.biomaterials.2007.09.001
[3] Postlethwait, R.W. (1970) Long-term comparative study
of nonabsorbable sutures. Ann Surg, 171, 892-897.
doi:10.1097/00000658-197006010-00010
[4] Altman, G.H., Horan, R.L., Lu, H.H., Moreau, J., Martin,
I., Richmond, J.C. and Kaplan, D.L. (2002) Silk matrix
for tissue engineered anterior cruciate ligaments. Bioma-
terials, 23, 4131-4141.
doi:10.1016/S0142-9612(02)00156-4
[5] Altman, G .H., Diaz, F., Jakuba, C., Calabro, T., Horan,
R.L., Chen, J.S., Lu, H., Richmond, J. and Kaplan, D.L.
(2003) Silk-based biomaterials. Biomaterials, 24,
401-416. doi:10.1016/S0142-9612(02)00353-8
[6] Yang, Y.M., Chen, X.M., Ding, F., Zhang, P.Y., Liu, J.
and Gu, X.S. (2007) Biocompatibility evaluation of silk
fibroin with peripheral nerve tissues and cells in vitro.
Biomaterials, 28, 1643-1652.
[7] Chen, X.M., Yang, Y.M., Wu, J., Zhao, Y.H., Ding, F. and
Gu, X.S. (2007) Biocompatibility studies of silk fib-
roin-based artificial nerve grafts in vitro and in vivo.
Progress in Natural Science, 17, 1029-1034.
[8] Minoura, N., Aiba, S.I., Gotoh, Y., Tsukada, M. and Imai,
Y. (1995) Attachment and growth of cultured fibroblast
cells on silk protein matrices. Journal of Biomeical Ma-
terials Research, 29, 1215-1221.
doi:10.1002/jbm.820291008
[9] Kim, U.J., Park, J., Kim, H.J., Wada, M. and Kaplan, D.L.
(2005) Three-dimensional aqueous-derived biomaterial
scaffolds from silk fibroin. Biomaterials, 26, 2775-2785.
doi:10.1002/jbm.820291008
[10] Meinel, L., Hofmann, S., Karageorgiou, V., Kirker-Head,
C., McCool, J., Gronowicz, G., Zichner, L., Langer, R.,
Vunjak-Novakovia, G. and Kaplan, D.L. (2005) The in-
flammatory responses to silk films in vitro and in vivo.
Biomaterials, 26, 147-55.
doi:10.1016/j.biomaterials.2004.02.047
[11] Odelius, K., Plikk, P. and Albertsson, A.C. (2008) The
influence of composition of porous copolyester scaffolds
on reactions induced by irradiation sterilization. Bioma-
terials, 29, 129-140.
doi:10.1016/j.biomaterials.2007.08.046
[12] Kamakuraa, H., Hiranob, T., Itoc, H. and Sunagac, H.
(2002) Studies on the sterilization methods for the crude
drugs. Possibility of EB machine for decontamination of
crude drugs and influence on the components of crude
drugs by irradiation. Radiation Physics and Chemistry,
63, 685-689. doi:10.1016/S0969-806X(01)00659-4
[13] Yang, Y.M., Zhao, Y.H., Liu, X.H., Ding, F. and Gu, X.S.
(2007) The effect of different sterilization procedures on
chitosan. Journal of Applied Polymer Science, 104,
1968-1972. doi:10.1002/app.25906
[14] Kawano, Y. and Logarezzi, A.J.M. (1995) X-ray induced
degradation of regenerated cellulose membrane films.
Polymer Degradation and Stability, 50, 125-130.
doi:10.1016/0141-3910(95)00145-C
[15] Booth, A.F. (1999) Sterilization of Medical Devices,
Interpharm Press, Illinois, USA.
C
opyright © 2011 SciRes. JBiSE

Y. H. Zhao et al. / J. Biomedical Science and Engineering 4 (2011) 397-402 402
JBiSE
[16] Najafpour, G.D. (2007) Sterilisation. Biochemical Engi-
neering and Biotechnology, Elsevier, Netherlands,
342-350. doi:10.1016/B978-044452845-2/50015-X
[17] Kim, J.K., Jo, C., Park, H.J. and Byun, M.W. (2008)
Effect of gamma irradiation on the physicochemical
properties of a starch-based film. Food Hydrocolloids, 22,
248-254. doi:10.1016/B978-044452845-2/50015-X
[18] Mendes, G.C.C., Brandão, T.R.S. and Silva, C.L.M.
(2007) Ethylene oxide sterilization. American Journal of
Infection Control, 35, 574-581.
doi:10.1016/j.ajic.2006.10.014
[19] Horan, R.L., Antle, K., Collette, A.L., Wang, Y.Z., Huang,
J., Moreau, J.E., Volloch, V., Kaplan, D.L. and Altman,
G.H. (2005) In vitro degradation of silk fibroin. Biomate-
rials, 26, 3385-3393.
doi:10.1016/j.biomaterials.2004.09.020
[20] Zheng, W.H. and Xu, X. (2005) Research progress on
Maillard reaction. Progress in Chemistry, 17, 122-129.
[21] Alariqi, S.A.S., Kumar, A.P., Rao, B.S.M. and Sing, R.P.
(2007) Stabilization of
 -sterilized biomedical polyolefins
by synergistic mixtures of oligomeric stabilizers. Part II:
Polypropylene matrix. Polymer Degradation Stability, 92,
299-309. doi:10.1016/j.polymdegradstab.2006.10.010