J Therm Anal Calorim (2015) 122:1069–1076
DOI 10.1007/s10973-015-4736-4
Comparative studies of regenerated water-based Mori, Thai, Eri,
Muga and Tussah silk fibroin films
Fang Wang1,2 • Nathan Wolf1 • Eva-Marie Rocks1 • Trinh Vuong3
Xiao Hu1,4,5
Received: 7 November 2014 / Accepted: 30 April 2015 / Published online: 20 May 2015
 Akadémiai Kiadó, Budapest, Hungary 2015
Abstract Silk materials from different silkworm species
can be utilized in various medical and material applica-
tions. In this study, five types of silk proteins: Indian An-
theraea mylitta (Tussah), Philosamia ricini (Eri),
Antheraea assamensis (Muga), Thailand and Chinese
Bombyx mori mulberry (Thai, Mori) were successfully re-
generated in the aqueous solution and were fabricated into
films. Differential scanning calorimetry (DSC) and tem-
perature-modulated DSC were used to identify glass tran-
sition temperatures (Tg), heat capacity increments at Tg and
degradation temperatures of these silk films. Besides this,
the bound water contents and the thermal degradation
mechanisms of different silk systems were quantified using
thermogravimetric analysis. In addition, Fourier transform
infrared spectrometer was also utilized to investigate the
structural differences of these silk films. Compared with
the mulberry silk films, wild silk films showed higher
thermal stabilities, and variable degradation and structural
profiles. These comparative studies offered a new pathway
Fang Wang, Nathan Wolf, Eva-Marie Rocks and Trinh Vuong have
contributed equally to this manuscript.
& Xiao Hu
hu@rowan.edu
1
Department of Physics and Astronomy, Rowan University,
Glassboro, NJ 08028, USA
2
Center of Analysis and Testing, Nanjing Normal University,
Nanjing 210023, China
3
Department of Chemistry and Biochemistry, Rowan
University, Glassboro, NJ 08028, USA
4
Department of Biomedical and Translational Sciences,
Rowan University, Glassboro, NJ 08028, USA
5
Department of Biomedical Sciences, Cooper Medical School
of Rowan University, Camden, NJ 08103, USA
•
to understand the tunable properties of silk-based bioma-
terials and would provide important insights for the de-
velopment of novel silk-based functional materials with
controllable physical and biochemical properties in the
future.
Keywords DSC  TG  FTIR  Silk fibroin  Glass
transition temperatures  Stabilities
Introduction
Silk is a protein polymer spun by insects and other or-
ganisms for natural purposes [1, 2]. For 5000 years, silk
has been used as a textile; a practice originating in Asia.
However, in recent years, there has been increasing interest
in using silk as a biomedical material thanks to its unique
physical and chemical properties since the human body can
better process the introduction of this protein material into
its internal environment. Silk is made up of smaller units
called fibroin fibers. These fibers have the same repeating
amino acid sequences, and this greatly affects the structure
of the protein chains, which in turn affects the material
properties of the silk. These properties of silk have led to a
significant increase in their study for biomedical applica-
tions [3–5]. Some applications of silk as a biomaterial in-
clude, but are not limited to, building silk scaffolding for
tissue regeneration, in vitro release of small drugs using
silk particles, and fabricating silk fibers as suture bioma-
terials [3, 6–9]. However, current studies often focus on
one type of silk materials, and the processing method to
regenerate various silk solutions is very different. There-
fore, there is a need for a comparative study of different
species of silk samples using the same approach. In this
study, we successfully regenerated five different types of
123
1070
silk samples using a same processing method and have
their material properties, especially the thermal properties,
tested and compared in order to determine their usefulness
in biomedical applications.
The five types of silk being tested are Mori, Thai, Eri,
Muga and Tussah (Fig. 1). The Mori silk comes from the
Bombyx Mori silkworm, which is the domesticated silk-
worm that has been used to produce silk for textile pur-
poses for millennia [1]. The silk is white, and the cocoon is
spun into a hard egg-like shape. Thai silk (yellow) also
comes from domesticated silkworms; however, these
worms are raised in Thailand, so the different climate and
environment affects the cocoon differently than those
raised elsewhere. The other three silks come from wild
silkworms. Tussah silk is produced by the Antheraea
mylitta silkworm, and the cocoon is dark tan or brown in
color and fairly smooth, similar to paper. The structure of
the cocoon is also significantly harder than the other co-
coons, and they are the largest in this study [10]. Eri silk
comes from the Philosamia ricini silkworm. The silk is
white in color, and the cocoons have a rough fibrous out-
side [5, 11]. These cocoons are also less stiff than the
others. Finally, Muga silk comes from the Antheraea as-
samensis species of silkworm and is light tan to gold/yel-
low in color. The cocoon has many loose fibers on the
outside and is also fairly flexible [12].
In order to test the material properties of silks, first
different silks were made into thin films from solutions.
Then, the film samples are compared using several thermal
Mori Thai Eri Muga Tussah
Silkworm Cocoons
Regenerated Solutions
Silk Fibroin Films
Fig. 1 Preparation of variable silkworm cocoons into silk fibroin
films. The solutions of Mori, Thai, Eri, Muga and Tussah silks were
cast onto the PMMA substrates to form films
123
F. Wang et al.
analysis methods, including temperature-modulated varia-
tion of differential scanning calorimetry techniques
(TMDSC), as well as thermogravimetric analysis (TG)
techniques. These methods compared and determined the
phase changes (such as glass transitions), crystallizations,
degradations and water–protein interactions of different
silk materials when it is heated through a broad tem-
perature range. Additionally, Fourier transform infrared
spectrometry (FTIR) was used to determine the structures
of various silk polymers. This comparative knowledge is
significantly important for silk-based biomaterial studies
that are currently conducted on different silk proteins,
where their properties are useful for variable biomedical
applications, but the differences are not well discussed
previously.
Experimental
Materials and preparation
Cocoons of Antheraea mylitta (Tussah), Philosamia ricini
(Eri) and Antheraea assamensis (Muga) were obtained
from India, while B. mori silkworm cocoons were obtained
from China and Thailand, individually. Degumming is one
of the most important procedures in the production of silk-
based materials. Through degumming, the protein layer on
the silk fibroin fiber, silk sericin (or called silk gum), can be
removed completely, which improves the color, sheen,
purity and texture of silk fibers [13–17]. In order to max-
imize the silk cocoons molecular stability during the
degumming process, 0.02 M sodium bicarbonate
(NaHCO3) solution (which is a weaker alkalinity than
sodium carbonate) was applied [13]. Silk cocoons were
first boiled for 2 h in an aqueous solution of 0.02
M NaHCO3 and rinsed thoroughly with water to remove the
glue-like sericin [18–24]. The final degummed silk fibers
were air dried overnight, then put into a vacuum oven at
room temperature for 1 day to remove moisture on their
surfaces. Then, they were dissolved into a melted calcium
nitrate hydrate (Ca(NO3)2xH2O) (Sigma-Aldrich, USA)
solution at 90 C for 12 h at a concentration of 10 mass%.
The silk solution was then dialyzed against pH = 7–8 Milli-
Q water for at least 2 days using dialysis cassettes (Pierce
Snake Skin MWCO 3500; Thermo Fisher Scientific,
Waltham, MA). After centrifugation and filtration to remove
insoluble residues, a 6 mass% silk fibroin aqueous
solution was obtained from all kinds of silk samples. Finally,
the products were cast onto polydimethylsiloxane
(PDMS) substrates to form different regenerated films.
Figure 1 shows the preparation processes of variable
silkworm cocoons to become their final water-based silk
fibroin films.
Comparative studies of regenerated water-based Mori, Thai, Eri, Muga and Tussah silk fibroin…
Differential scanning calorimetry
Samples with masses of about 6 mg were encapsulated in
Al pans and heated in a TA Instruments Q100 DSC with a
dry nitrogen gas flow of 50 mL min-1 and equipped with a
refrigerated cooling system (RCS). The instrument was
calibrated for empty cell baseline, with indium for heat
flow and temperature. Aluminum and sapphire reference
standards were used for calibration of the heat capacity
through a three-run method described previously [13].
Standard mode DSC measurements were taken from
-25 C (248 K) to 400 C (673 K) at a heating rate of
2 K min-1, while temperature-modulated differential
scanning calorimetry (TMDSC) measurements were taken
at a heating rate of at 2 K min-1 with a modulation period
of 60 s and temperature amplitude of 0.318 K. The Lis-
sajous figures of modulated heat flow versus modulated
temperature were also plotted to check the establishment of
steady state.
The ‘‘reversing heat capacity’’ in TMDSC can be cal-
culated within the temperature range of the modulation
from [21–24]:
1071
was continuously purged by nitrogen gas to eliminate the
spectral contributions of atmospheric water vapor. For each
measurement, 128 scans were co-added with a resolution of
4 cm-1 and the wavenumbers that included in the test
ranged from 400 to 4000 cm-1.
Results and discussion
DSC analysis
The thermal properties of different regenerated silk fibroin
films were first examined by standard DSC and tem-
perature-modulated DSC (TMDSC). Figure 2 shows the
standard DSC scans of various silk fibroin film samples:
Mori, Thai, Eri, Muga and Tussah. During the heating
scans, all samples first showed a bound water evaporation
peak (Tw) around 60–160 C (333–433 K) [28, 29]. The
bound water peak of Thai film occurred at 96 C (369 K),
which is the lowest Tw temperature among all samples,
while the Tw of Tussah film has the highest water peak
temperature found in all silk samples, which is close to
  131 C (404 K). For Tw of five silk film samples, the trend
mCp þ Cs Cr  DCcell  ¼ K 0 ðAHF Þ=A ð1Þ
where Cp is the specific heat capacity of a sample of mass,
m; Cs is the heat capacity of the sample pan; Cr is the heat
capacity of the empty reference pan; and DCcell is the cell
asymmetry correction which can be determined by running
a series of empty pans with various mass differences versus
the reference pan [25–28]. On the right-hand side of
Eq. (1), AHF is the heat flow amplitude, A is the sample
0
temperature modulation amplitude and K is a calibration
constant to assure the same heat transfer is maintained at
the same experimental conditions.
Thermal gravimetric analysis
was found in the order of Thai \ Mori \ Mu-
ga \ Eri \ Tussah (Fig. 2). This result indicates that do-
mesticated silk films (Thai, Mori) were more easily to be
dehydrated than wild silks films (Muga, Eri and Tussah).
This advantage could help them exchange water molecules
easily in an ordinary atmosphere. While in the wild envi-
ronment, wild silk materials with better water inhibiting or
Tussah Td
Tw
Muga

Thermal gravimetric analysis (TG) through a simultaneous Eri Tw

Td
Heat Flow/a.u.

DSC and TG instrument (SDT, TA Instruments Q600) was

used to measure changes in mass of regenerated silk film Thai Tw
samples with increasing temperature. Thermal gravimetric Td
Tw Td
curves were obtained under nitrogen atmosphere with a gas Mori
Exo

flow of 50 mL min-1. The instrument tested the samples at

a range of temperatures from ambient temperature to Tw Td
500 C (773 K) at a heating rate of 2 K min-1.
Endo

0.2 W g–1

Fourier transform infrared spectrometry –50 0 50 100 150 200 250 300 350 400
Temperature/°C
Samples were performed on a Bruker Tensor 27 Fourier
transform infrared spectrometer (FTIR), equipped with a Fig. 2 Standard DSC scans of different silk fibrin film samples. The
samples were heated at 2 K min-1 from -25 C (248 K) to 275 C
deuterated triglycine sulfate detector and a multiple re- (548 K, Thai and Mori films) or 400 C (673 K, Tussah, Muga and
flection, horizontal MIRacle ATR attachment [using a Ge Eri films) with temperature regions related to bound water evapora-
crystal, from Pike Tech. (Madison, WI)]. The instrument tions (Tw) and sample degradations (Td)

123
1072
reservation ability are more favorable for the surviving of
silkworms in the cocoons.
The phase changes shown in Fig. 2 include the glass
transition and the degradation regions of various silk films.
During the glass transition, the uncrystallized silk macro-
molecules transfer into a liquid-like phase from their ori-
ginal rigid solid phase. And the midpoint temperature of
this transition is chosen as Tg in this study [25–28]. The
degradation region, on the other hand, indicates the silk
molecule backbones have been destroyed after being ex-
posed to heat, and cannot return to their original compo-
sition. After the water removal, the glass transition (Tg)
regions of all silk films can be found in their temperature-
modulated DSC (TMDSC) curves (Fig. 3). Then, all sam-
ples thermally degraded around Td, while a melting peak
was not shown (Fig. 2). The melting temperature of Mori
fibers has been found close to 340 C (613 K), previously
by a fast-scanning DSC at a heating rate of 2000 K s-1
[28]. Therefore, the 2 K min-1 DSC scans in this study
could not reach the samples’ melting points before the
thermal degradations. And we will discuss the melting
behaviors of wild silk films by a separate study in the
future. Table 1 lists the Td temperatures of all regenerated
silk samples. It is clearly shown that the degradation peak
temperatures (Td) of Thai and Mori films were 245.44 C
(518.44 K), 244.33 C (517.33 K), respectively, which
were significantly lower than those of wild silk films (see
Fig. 2). In addition, the Tussah film sample showed a big
exothermic peak before its degradation. This peak might be
related to the thermal crystallization or self-assembly of
Tg=188°C
Reversing Heat Capacity/J g–1°C–1
Tg=168°C
Tg=176°C
Tg=149°C
Tg=175°C
0.5 J g–1°C–1
0 50 100 150 200 250
Temperature/°C
Fig. 3 Reversing heat capacities of the silk fibrin film samples (Mori,
Thai, Eri, Muga and Tussah) from -25 C (248 K) to 250 C (523 K)
(glass transition region), which were measured by TMDSC with a
2 K min-1 heating rate, a modulation period of 60 s and a
temperature amplitude of 0.318 K
123
F. Wang et al.
Tussah silk proteins in this temperature region. The results
also indicated that wild silk protein films are more ther-
mally stable than domesticated silk protein films, which is
consistent with our previous studies on the wild and do-
mesticated silk cocoons and fibers [13].
To fully understand the glass transitions of different silk
samples, the temperature-modulated DSC (TMDSC) was
used to measure the reversing thermal properties of silk
film materials. Figure 3 shows the reversing heat capacity
curves of different silk samples from -10 C (263 K) to
275 C (548 K), which were measured with a 2 K min-1
heating rate, and a modulation amplitude of 0.318 K per
60 s. All samples showed clear heat capacity increments
(DCp) during their glass transition regions, indicating oc-
currence of the conformational change from solid to vis-
cous liquid stage [24–28]. Generally, all silk samples
showed similar slopes of heat capacity baselines before the
glass transition regions. It is also found that the baselines of
all silk films were slightly drifted in the low temperature
range (0–100 C), which is due to the loss of bound water
in the silk films [20–23]. Pyda et al. [23] pointed out that
the heat capacity of solid silk-bound water system can be
estimated from a sum of linear combinations of the vi-
brational Cp of dry silk and glassy water below the glass
transition. After the evaporation of bound water molecules
above 100 C, the baselines in Fig. 3 dropped back to the
vibrational Cp of pure silks and then their glass transitions
appeared. This phenomenon can be also found in other silk
blending systems, such as the silk–tropoelastin materials
studied previously [22]. Table 1 lists the glass transition
temperatures (Tg) of various silk samples and their heat
capacity increments (DCp) during the glass transition. It
shows that the Tussah, Eri and Mori samples have higher
Tussah glass transition temperatures than those of Muga and Thai
samples, and with a Tg trend of Thai \ Mu-
Muga
Eri ga \ Mori \ Eri \ Tussah. It needs to be noted that the Tg
Thai of Thai film is the lowest in these five samples, while the
Tussah film is the highest. In contrast, the heat capacity
Mori
increment (DCp) at Tg of Thai film is the highest and that of
Tussah sample is the lowest among all silk samples. This
could be explained that the Thai film is the softest and
weakest silk sample, whereas Tussah film is the hardest silk
material found in the wild environments. The heat capacity
increment (DCp) is directly proportional to the average
chain mobility of proteins, reflecting the number of freely
rotating bonds capable of changing the chain conformation.
300 For the Mori film sample, the DCp increment at Tg was
0.475 J kg-1 K-1, which is consistent with our previous
results [30, 31]. The Thai film showed a similar DCp in-
crement at Tg since it also belongs to the B. mori species.
Comparing the heat capacity increment (DCp) of these five
fibroin films (Table 1), a DCp trend of Tussah \ Mu-
ga \ Eri \ Mori \ Thai was found. Since all samples
Comparative studies of regenerated water-based Mori, Thai, Eri, Muga and Tussah silk fibroin… 1073

Table 1 Thermal analysis data of different silk fibroin film samples (measured by DSC and TG)
Silk sample Tg/C DCp at Tg/J g-1 K-1 Degradation Water content/% Degradation middle Remaining
Td/C temperature Tdm/C mass at 450 C/%

Thai 149.0 0.564 245.44 7.26 288.46 47.23

Mori 175.0 0.475 244.33 9.70 289.65 29.31
Muga 168.0 0.370 330.32 8.57 351.42 43.80
Eri 176.0 0.509 329.02 5.54 352.61 62.27
Tussah 188.0 0.282 336.62 10.28 321.72 50.84
All numbers have an error bar within ±5 %, the first three columns (Tg, DCp at Tg and degradation Td) were determined by DSC analysis and the
rest were determined by TG analysis

were uncrystallized after forming the films (proved by

(a)
FTIR below), this implied that the uncrystallized Thai 100
proteins can easily change their chain conformation when
90

Percent Mass Remaining/%

they were heated, while the average chain mobility of
uncrystallized Tussah protein chains is the lowest among 80
the five silk film samples. In general, the average chain
70
mobility of domesticated (Mori and Thai) silk films were
higher than those of the wild silks, which may indicate the 60 Eri
wild silks could contain other noncrystalline ordered Tussah
50
structures such as alpha-helix, in addition to the random Thai
40 Muga
coil noncrystalline structures [1, 25, 29]. Therefore, this
may be the reason that domesticated silks are less thermally 30 Mori
stable than wild silks in nature, which has been proved in
0 100 200 300 400
the standard DSC study.
Temperature/°C

TG analysis
(b)
Muga
To identify the water content and the thermal degradation
0.6
process of regenerated silk fibroin films, thermogravimetric
Eri
Deriv. Mass/%°C–1

analysis was used on different silk samples [10, 32–37]. Tussah

Figure 4a shows the mass percentage change of Mori, Thai, Mori
0.4
Eri, Muga and Tussah films during a heating from room
temperature to 500 C (773 K), while Fig. 4b shows the Thai
first derivatives of their mass percentage curves in Fig. 4a, 0.2 Tw
which reveals the middle degradation temperatures (Tdm)
and the degradation rates of different silk samples. Td
During the initial heating from room temperature to 0.0
150 C (423 K), bound water molecules were removed 0 100 200 300 400
from all silk samples as we demonstrated in the DSC study. Temperature/°C
Table 1 lists the water content (%) of various silk samples
measured by TG. It shows that all samples contained bound Fig. 4 a Percent mass remaining of different silk fibrin film samples
measured by thermogravimetric analysis during heating from room
water around 5.5–10.3 %, with a trend of
temperature to 500 C (773 K) at 10 K min-1. b The first derivative of
Eri \ Thai \ Muga \ Mori \ Tussah. It is interesting to the percent mass remaining in a, which reveals the degradation rates of
see that the water content in domesticated silk films (Thai silk film samples and demonstrates temperature regions related to
and Mori) was not the largest in these five samples bound water evaporations (Tw) and the sample degradations (Td)
although they can be easily dehydrated which was indi-
cated by the Tw in the standard DSC curves. Meanwhile, demonstrated by the first derivative curves in Fig. 4b.
the initial decompositions of the proteins started around These slight mass decreases may be related to the de-
175 C (448 K) and their mass changing rates were clearly compositions of protein side chains. After these small

123
1074 F. Wang et al.

peaks in Fig. 4b, the major thermal degradation process of (a)

1651 1541 1331
silk proteins occurred due to the decomposition of mole- 1051
1404 1265
cular backbones started around 250 C (523 K). Figure 4b
also showed that some wild silk samples (Muga and Eri)
had a very quick mass loss during their major degradation

Absorbance/a.u.
stages, and the Tdm of all wild silks (Tussah, Muga and Eri) Tussah
were higher than those of domesticated silks (Thai and
Mori), with a trend of Thai \ Mori \ Tussah \ Mu- Muga
ga \ Eri. The degradation middle temperatures of different Eri
silk films were listed in Table 1. It proved again that the Thai

wild silk films are stronger than the domesticated samples 1641 1511 1011
1409 1336 1234 Mori
as we predicted in the DSC study. Above 380 C (653 K), 1451 1170
1375 0.2
the degradation rates of all samples slowed down (Fig. 4b) A-I A-II A-III
and no peak appeared in the first derivative. Therefore, we 1800 1600 1400 1200 1000
listed the remaining mass (%) of all samples at 450 C
Wavenumber/cm–1
(723 K) in Table 1. For the Mori silk samples, the re-
(b)
maining mass was 29.31 % at 450 C (723 K), which is the
lowest content among all film samples, suggesting that the 3292 V(N–H)
thermal stability of Mori film in high temperature regions is
0.1
weaker than those of others. In contrast, the Eri silk films
showed the strongest thermal stability, and about 62 % of Absorbance/a.u. Tussah
the sample mass was left at 450 C (723 K).
Muga
FTIR analysis
Eri
FTIR analysis was performed to understand the structural
Thai
differences between the five regenerated silk films. Figure 5
shows the FTIR absorbance spectra of different silk samples Amide N Mori
2848
(Mori, Thai, Eri, Muga and Tussah), for the wavenumber
regions (a) 900–1800 cm-1 and (b) 2500–3750 cm-1. 3750 3500 3250 3000 2750 2500
Generally, the IR spectral region between 1700 and Wavenumber/cm–1
1500 cm-1 is assigned to the peptide backbone absorptions
of Amide I (A-I: 1700–1600 cm-1) and Amide II (A-II: Fig. 5 FTIR absorbance spectra of different regenerated silk fibroin
films (Mori, Thai, Eri, Muga and Tussah) for the regions mentioned
1600–1500 cm-1) regions. And the Amide III (A-III) region above, a 900–1800 cm-1 and b 2500–3750 cm-1
ranges from 1350 to 1200 cm-1 [38–41]. The Amide I region
mainly comes from the C=O stretching vibrations ([80 %) analyzing the N–H stretching vibration of silk materials
of the protein backbones [38] and is the most commonly used [38, 39]. The Amide N region is a part of the Fermi resonance
region for the quantitative analysis of different protein sec- doublet connected with its second component Amide N0 ,
ondary structures. In addition, the out-of-phase combination which absorbs weakly between 3100 and 3030 cm-1
of the C–N stretching and the N–H in-plane bending vibra- [38, 39].
tions form the Amide II bands [38, 39]. And then, the mi- All five silk films showed the main characteristics of
croenvironment of protein side chain groups can easily affect protein structures in the FTIR spectra (Fig. 5a). However,
the Amide II (A-II) region [34, 39]. The region of absorbance differences were also observed in some regions.
1490–1460 cm-1 comes from a larger C–N stretching vi- First, although all samples had a strong peak in A-I region,
bration mode converted by N-deuteration [38, 39]. The fin- their peak positions are different. The peak shifted gradually
gerprint region [38, 39] is located within 1340–1000 cm-1 from 1641 cm-1 (random coil structure) to 1643, 1648, 1650
with a low absorbance intensity. The peak near 1405 cm-1 and 1651 cm-1 (alpha-helix structure), for Mori, Thai, Eri,
corresponds to the major deformation of CH2 and CH3 Muga and Tussah silk, respectively. In addition, Mori silk
groups and the minor COO- symmetric stretching bands film in A-II region showed a strong peak at 1514 cm-1 along
[38, 39]. The peaks ranging from 1350 to 1200 cm-1 are with a shoulder around 1541 cm-1. While the Tussah silk
associated with NH bending vibration modes of Amide III demonstrated a contrary pattern, the peak at 1541 cm-1 was
band [38, 39]. In this study, the Amide N region obvious, but the shoulder at 1514 cm-1 was fuzzy. Other silk
(3310–3270 cm-1) was also investigated (Fig. 5b) when samples, such as Thai, Eri and Muga films displayed both

123
Comparative studies of regenerated water-based Mori, Thai, Eri, Muga and Tussah silk fibroin…
these two peaks simultaneously in the A-II region. More-
over, for the A-III and IR fingerprinting regions, Thai silk
sample showed multiple peaks at 1451, 1409, 1375, 1336,
1234 and 1170 cm-1, as well as at 1051 and 1011 cm-1,
which were similar to the peaks found in the Mori film. Their
similar IR features indicated that these two kinds of silks did
come from genetically similar silkworm species, which both
ate mulberry leaves although they grew in different areas.
For the Muga and Eri samples, their peak positions, heights
and shapes were almost identical in the region of
1477–1421 cm-1, while there were a few differences around
the 1051 cm-1 region. The biggest difference came from the
Tussah silk: It had a large and broad peak from 1487 to
1187 cm-1 with many small peaks (1404, 1375, 1265 cm-1)
on it. These unique structures may be directly related to the
unique thermal properties of Tussah silk found in the DSC
study (Fig. 2), where a broad exothermic peak was found
just before its degradation region. It is known that protein is
consisted of a series of amino acids, while each amino acid
has at least three freely rotating bonds in the backbone [23].
The unique and strong absorbance spectra of Tussah silk in
this region indicated that the Tussah silk molecules are more
active than other silk molecules regarding to their CH2, CH3
groups (1405 cm-1) and NH bending vibrations
(1350–1200 cm-1). Therefore, with the increase of tem-
perature, large-scale conformational changes such as NH
bending can easily occur in the mobile regions of the Tussah
silk molecular chains, which helped Tussah silk molecules
thermally crystallized or self-assembled at a high tem-
perature, and showed a big nonisothermal exothermic peak
between 250 and 350 C in its standard DSC curve. In ad-
dition, both Tussah and Muga samples had a similar large
peak at 1041 cm-1 which represented the major deformation
of enhanced CH2 and CH3 groups. Since they both belong to
a moth genus Antheraea, this might be one of the indicators
to demonstrate their structural similarity in IR spectra. Fi-
nally, all samples had a large peak around 3292 cm-1 in the
Amide N region (Fig. 5b), which represented the N–H
stretching vibration of silk proteins. Mulberry-based Thai
and Mori films also showed a sharp peak at 2848 cm-1,
while other wild silk films did not.
Conclusions
Advanced thermal analysis methods were utilized to
comparatively study five different regenerated water-based
silk fibroin film systems—Indian Antheraea mylitta (Tus-
sah), Philosamia ricini (Eri), Antheraea assamensis (Mu-
ga), Thailand and Chinese mulberry (Bombyx mori).
Standard DSC was used to detect the bound water re-
movals, the phase transitions and the multiple-stage ther-
mal degradations of silk films. And their precise heat
1075
capacities and heat capacity increments (DCp) during the
glass transition regions were also measured by the tem-
perature-modulated DSC (TMDSC). Comparative thermal
gravimetric (TG) analyses were further used to confirm the
results and predictions obtained from DSC and TMDSC.
Results indicated that wild silks (Tussar, Muga and Eri)
were significantly different from the domesticated silks
(Thai and Mori), in terms of their thermal stability,
degradation rate and temperature, bound water sustaining
ability, as well as their molecular mobility during the glass
transitions. These findings are consistent with the previous
studies of silk materials reported by other groups [38, 39].
As evidenced by FTIR, all samples had the characteristics
of protein structures in general, while silkworms ate the
same foods or had the similar genes can show similar
structural features of their silks in some regions of IR
spectra. These features could be further utilized as indi-
cators in medical and material tests of different silk ma-
terials [5, 19]. The results also demonstrate that a
combinational method with both advanced thermal analysis
and spectra analysis is a powerful tool to comparatively
study protein-based biomaterials, which would be useful
broadly for other biopolymer systems in the future. Further
studies will focus on investigating their mechanical prop-
erty differences as well as their unique applications for
tissue regeneration [41, 42].
Acknowledgements This study was supported by the Rowan
University Start-up Grants, Rowan University 2013–2014 Seed Fund-
ing Program (10110-60930-7460-12), NSF-MRI Program (DMR-
1338014) and New Jersey Space Grant Consortium. Fang Wang also
thanks Nanjing Normal University Scholarship for the support through
Overseas Studies Foundation of China (2013–2014).
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