International Journal of Biological Macromolecules 75 (2015) 7–12

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Description of a new surface morphology for chitin extracted from

wings of cockroach (Periplaneta americana)
Murat Kaya a,b,∗ , Talat Baran b,c
a
Faculty of Science and Letters, Department of Biotechnology and Molecular Biology, Aksaray University, 68100 Aksaray, Turkey
b
Science and Technology Application and Research Center, Aksaray University, 68100 Aksaray, Turkey
c
Department of Chemistry, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: In this study a new morphology of chitin, which could find wide applications in the fields of medicine,
Received 30 October 2014 pharmacy, agriculture, food and textiles, has been described. The chitin was isolated from the wings of
Received in revised form 3 December 2014 Periplaneta americana employing a conventional method. Considering chitin isolation studies conducted
Accepted 8 January 2015
previously, chitin has three surface morphologies, which are (1) hard and rough surface without pores or
Available online 15 January 2015
nanofibers, (2) surface solely composed of nanofibers and (3) surfaces with both pores and nanofibers. In
this study, the surface of the chitin, examined with environmental scanning electron microscopy (ESEM),
Keywords:
only has oval nanopores (230–510 nm) without nanofibers, and this is different from the above mentioned
Chitin
New morphology
surface morphologies. The nanopores are not distributed on the chitin surface randomly. Typically, there
Nanoporous is a pore in the center that is surrounded by six or seven other pores in an ordered manner. Structures
ESEM similar to cell walls exist between the pores. Chitin with the new surface morphology was characterized
Insect using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction
Thermal properties (XRD) and elemental analysis.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction description of a new surface morphology of chitin that has a wide

The molecular weight, the degree of acetylation or deacety-
lation of the chitin and its derivatives together with the surface
morphology are very important aspects of chitin when considering
the effective use of the biomaterial [1–3]. Chitin and chitosan can
be used in a lot of areas thanks to their non-toxicity, biocompa-
bility and biodegradable properties. These biopolymers have been
widely used in controlled drug release capsule production and
weight loss pills that have a high oil retention capacity [4,5]. They
are also used in the food and textile industries due to their antimi-
crobial and antioxidant effects [5–7]; while, additionally they are
used in agriculture, waste water treatment and veterinary and
human medicine [6]. For more applications of chitin and its deriva-
tives, there are a range of review studies [8–12]. Therefore, the
∗ Corresponding author at: Faculty of Science and Letters, Department of Biotech-
nology and Molecular Biology, Aksaray University, 68100 Aksaray, Turkey.
Tel.: +90 382 288 2184; fax: +90 382 288 2125.
E-mail addresses: muratkaya3806@yahoo.com (M. Kaya),
talatbaran 66@hotmail.com (T. Baran).
range of applications is closely related to many branches of science.
Scientists have focused on chitin isolation and characterization
from crayfish, crabs and shrimp because of their commercial pro-
duction [13]. The use of chitin and its derivatives is increasing
worldwide, both in overall consumption levels and areas of appli-
cation. Due to this reason, new chitin sources are being sought.
Recently, some organisms such as insects, anthozoan, fungi and
sponges have been researched as alternative chitin sources [13–23].
In the present study, the wings and other body parts of Periplaneta
americana were screened for their ability to be an alternative chitin
source.
Periplaneta americana is an insect species that is distributed
worldwide. The adults have a body size of 35–40 mm, and they
show excessive growth, especially when it is hot, humid and there
is abundant food. People are familiar with this insect because they
live in the trash, kitchens, bathrooms, near radiators, sewers and
drainage channels. Therefore, they are described as a big problem in
terms of human health; they cause asthma and intestinal diseases
in humans [24,25]. In addition, the body of this insect has been
found to carry pathogenic bacteria such as Klebsiella, Pseudomonas,
Escherichia coli, Staphylococcus, Enterobacter, Streptococcus, Serra-
tia, Bacillus and Proteus [25]. They can achieve huge numbers with

http://dx.doi.org/10.1016/j.ijbiomac.2015.01.015
0141-8130/© 2015 Elsevier B.V. All rights reserved.
8 M. Kaya, T. Baran / International Journal of Biological Macromolecules 75 (2015) 7–12
extreme reproduction and development in moist and warm areas
[26].
In this study, a new surface morphology of chitin, from the wings
of an insect (P. americana), was recorded and defined. The surface
morphologies of the chitins isolated from the wings of an insect
and other body part were examined comparatively using ESEM. In
addition, the new chitin morphology was characterized by Fourier
transform infrared spectroscopy (FTIR), thermogravimetric analy-
sis (TG), X-ray diffraction (XRD) and elemental analysis.
2. Materials and methods
2.1. Sample collection
Individuals belonging to P. americana were collected by hand-
picking in Kirikan (Hatay, Turkey) on October 14, 2013. After
collection, the samples were taken to the laboratory in a 20%
ethanol solution. The samples were washed with distilled water for
the removal of possible residues, and then the wings were removed
with the help of forceps. The separated wings and other parts of
the insect were incubated for 2 days to dry in an oven at 60 ◦ C. The
pictures of P. americana and its wings were shown in Fig. 1.
Fig. 1. Periplaneta americana: (a) habitus, (b) wings.
2.2. Chitin extraction
The dried wings and other parts were pulverized separately,
in a mortar, into a powdered form. Then a demineralization step
was performed in which 10 g of each material was weighted and
refluxed in a 4 M HCl solution for 2 h at 75 ◦ C. Subsequently, the
mixture was filtered with a 1 ␮m filter paper and rinsed with dis-
tilled water to a neutral pH. To remove the protein, the filtrate was
refluxed in a 4 M NaOH solution for 20 h at 150 ◦ C. Then the mixture
was filtered and washed with distilled water to a neutral pH. After-
wards, the extract was mixed with a magnetic heating stirrer in a
solution of water, methanol and chloroform (in the ratio of 4:2:1)
for 4 h at 30 ◦ C. Then the extract was filtered and washed again with
distilled water to a neutral pH. Finally, the material was dried in an
oven at 60 ◦ C for 2 days. The chitin contents of the dried wings and
other parts of the insects were calculated.
2.3. ESEM
The dried wings and other parts of P. americana were coated with
gold using a “Sputter Coater” (Cressingto Auto 108) at Selçuk Uni-
versity. Then the surfaces of the samples were examined employing
a QUANTA- FEG 250 ESEM (at 15,000×, 40,000×, 80,000× and
100,000× magnifications) at the Science and Technology Applica-
tion and Research Center (Aksaray University).
2.4. Fourier transform infrared analysis (FTIR)
Analysis of chitin isolated from the wings of P. americana was
conducted using a Perkin–Elmer FTIR at 4000–625 cm−1 .
2.5. Thermogravimetric analysis (TG)
TGA analysis of extracted chitin from the wings of P. americana
was done using an EXSTAR S11 7300 from 25 to 650 ◦ C at a heating
rate of 10 ◦ C min−1 .
2.6. X-ray diffraction (XRD)
XRD peaks of chitin extracted from the wings of P. americana
were taken at 40 kV, 30 mA and 2 with a scan angle from 5◦ to 45◦
using a Rigaku D max 2000 system in Harran University (HUMEL).
The crystalline index (CrI) values were calculated with the formula
given below:
 (I − Iam )

110
CrI110 = × 100 (1)
I110
where I110 is the maximum intensity at 2 = ∼ 19.58◦ and Iam is
the intensity of amorphous diffraction at 2 ∼
= 12.88◦ .
2.7. Elemental analysis
A Thermo Flash 2000 was employed to determine the N, H and C
contents of the chitin structure isolated from the wings of P. amer-
icana. Then degree of acetylation (DA) of the chitin was calculated
using the following formula [27]:
 
(C/N − 5.14)
DA = × 100 (2)
1.72
3. Results and discussion
3.1. ESEM
ESEM analyses conducted on chitin have revealed that it has
three main surface morphologies: (1) a hard and rough surface
 M. Kaya, T. Baran / International Journal of Biological Macromolecules 75 (2015) 7–12 9

Fig. 2. ESEM pictures of chitin isolated from the wings of Periplaneta americana (magnifications: (a) 15,000×, (b) 40,000×, (c) 80,000× and (d) 100,000×).

without pores or nanofibers, with chitin isolated from certain fungi
and Daphnia magna eggs having this type of surface morphology
[28,29]. (2) A surface solely composed of nanofibers, with this type
of morphology having been observed in samples isolated from fungi
[30,31] as well as Antarctic krill, certain aquatic insect species and
aquatic Asellus aquaticus (Cructacea) [23,32]. (3) A surface with
both pores and nanofibers, which is the most common morphol-
ogy [33–35]. In this study, a new morphology was defined: pores
but no nanofibers. The shape of the pores is oval and their diam-
eter ranges from 230 to 510 nm. Previous studies reported that
the diameters of pores on chitin isolated from shrimp, Gammarus
and silkworm chrysalides ranged from 150 to 250 nm [33–35]. The
pores on the chitin that are defined in this study are larger than the
ones reported in the literature. This new morphology has cell wall-
like structures between the pores. These pores are hollow and not
very deep (Fig. 2). The distance between two pores is in the range of
125–340 nm. Fig. 2 shows this new chitin morphology at 15,000×,
40,000×, 80,000× and 100,000× magnifications. Another impor-
tant point is that more than 50% of the chitin surface is composed
of pores (Fig. 2). This highly porous surface could have significant
biomaterial applications. The pores are regular and positioned on
the surface orderly (Fig. 3). It is evident from Fig. 3 that there is
a pore in the center that is surrounded by six (Fig. 3a) or seven
(Fig. 3b) other pores in an ordered manner. Chitin with this mag-
nificent design may find important applications in various fields.
Fig. 4 depicts a comparison of the new type morphology with pre-
viously known types. Fig. 4a represents a hard and rough surface
without pores or nanofibers [29]. Fig. 4b shows the surface mor-
phology solely composed of nanofibers [23]. A surface with both
pores and nanofibers can be seen Fig. 4c [35]. The surface of chitin
extracted from other parts of P. americana (not the wings) has both
pores and nanofibers (Fig. 5), which is an example of type 3.

Fig. 3. ESEM pictures of chitin extracted from the wings of Periplaneta americana ((a) one pore in the middle and six pores around and (b) one pore in the middle and seven
pores around).
10 M. Kaya, T. Baran / International Journal of Biological Macromolecules 75 (2015) 7–12

Fig. 4. SEM pictures of chitin types isolated from various organisms: (a) hard and rough surface without pores or nanofibers (isolated from Daphnia magna resting eggs), (b)
surface solely composed of nanofibers (isolated from Asellus aquaticus) and (c) surface with both pores and nanofibers (isolated from Gammarus).

3.2. FTIR
Chitin has three crystalline forms: alpha, beta and gamma, but
there is little information regarding the gamma form [36]. The most
important feature of FTIR spectra is that they can be assessed to
distinguish the alpha form from the beta via whether the Amide
I band is split or not. In the alpha form, the Amide I band splits
into two bands at about 1650 and 1620 cm−1 [32]. In the beta form,
only one Amide I band is observed at 1656 cm−1 . Beta chitins are
found in organisms such as squid pens [36]. It is known that alpha
chitin is found in the Arthropoda [19,27]. In the FTIR spectra of the
chitin extracted in this study, the Amide I band is split at 1654
and 1621 cm−1 (Fig. 6), which indicates that this chitin from P.
americana is in the alpha form.
The other FTIR spectrum bands were observed as the follow-
ing; 3435 cm−1 (O–H stretching), 3259 cm−1 (asymmetric N–H
stretching), 3108 cm−1 (symmetric N–H stretching), 2924 cm−1
(asymmetric C–H stretching), 2857 cm−1 (symmetric C–H stretch-
ing), 1420 cm−1 (CH2 ending and CH3 deformation), 1376 cm−1
(CH bend, CH3 symmetric deformation), 1308 cm−1 (CH2 wagging),
1154 cm−1 (C–O–C asymmetric stretching), 1114 cm−1 (asymmet-
ric bridge oxygen stretching), 1114 cm−1 (asymmetric in-phase
ring stretching mode), 1068 m−1 (C–O–C asymmetric stretch in
phase ring), 1012 cm−1 (C–O asymmetric stretch in phase ring),
952 cm−1 (CH3 wagging), 895 cm−1 (CH ring stretching) [15].
The FTIR bands of commercial chitin were observed to be as fol-
lows; 3438, 3260, 3103, 2932, 2865, 1654, 1623, 1555, 1428, 1376,
1310, 1155, 1115, 1068, 1016, 952, 896 cm−1 . High similarity was
recorded between the commercial and the chitin extracted from
wings of P. americana.
3.3. TG
In the literature, characterization studies for chitin extracted
from crab, shrimp and insects using TG analysis have revealed that
there are two mass loss steps: one at around 100 ◦ C and the other
at 350–390 ◦ C [36]. In the present work we obtained similar results
to the previous studies (Fig. 7). The first mass loss step represents
the evaporation of absorbed water molecules; the second one cor-
responds with the decomposition of the chitin polymer. In the first

Fig. 5. ESEM pictures of chitin isolated from the insect’s (Periplaneta americana) body without wings (magnifications: (a) 30,000× and (b) 40,000×).
 M. Kaya, T. Baran / International Journal of Biological Macromolecules 75 (2015) 7–12 11

Fig. 8. XRD peaks of chitin extracted from the wings of Periplaneta americana.

organisms can vary in the range of 54–90% [13,37]. This wide range
can be attributed to differences in chitin source and purity of the
isolated chitin.

Fig. 6. FTIR bands of chitin isolated from the wings of Periplaneta americana. 3.5. Elemental analysis

We determined the C, N and H contents of the chitin from the

step the mass loss is 5% and in the second step it is 76%. The DTGmax
wings of P. americana to be 45.74%, 6.69% and 6.59% respectively.
where chitin monomers decompose has been determined to be
The N percentage value of completely acetylated chitin is known
389 ◦ C. It has been reported in the literature that the DTGmax values
to be 6.89 [17]. The N percentage values for chitin from different
for chitin from crab, shrimp and Antarctic krill were 365–380 ◦ C.
organisms are as follows: shrimp, 4.85%; bumblebee, 5.92%; crab,
Another chitin characterization study by Sajomsang and Gonil [19]
5.3%; Holotrichia parallela (beetle), 6.45%; another shrimp, 6.24%;
reported that the DTGmax for chitin from cicada sloughs (insect)
and cicada sloughs, 5.92% [13,17,19,38]. It is known that the N con-
was 379 ◦ C. The DTGmax value that we determined in this work is
tent of chitin from fungi is low (2.96%) due to the glucan residues
very similar to the values reported in literature.
in chitin [31]. The N content determined in this study approached
the value of completely acetylated chitin. This indicates that chitin
3.4. XRD
obtained in this study has higher purity than that reported in the
literature. The degree of acetylation of chitin cannot exceed 100%
X-ray diffraction analysis of chitin shows two strong peaks and
[19], and the degree of acetylation of the chitin we isolated was cal-
four faint peaks [19,31,32,36]. We observed two strong peaks (9.14
culated employing the formula given in the materials and methods
and 19.58) and four faint peaks (12.88, 20.98, 23.12 and 26.8)
section; it was found to be 98.67%. Some workers reported that the
(Fig. 8). The crystallinity index (CrI) has been calculated to be 86.7%.
degrees of acetylation of chitin from shrimp, insect and crap were
Liu et al. [13] reported that the CrI for chitin from Holotrichia
112–237%, 101–132% and 104% respectively [13,17,19,38]. These
parallela (insect) was 89%. The CrI values of chitin from different
values are higher than 100%, which indicates that the isolated chitin
contains some impurities, like protein and inorganic materials.

3.6. Chitin content of wings and insect body (without wings)

It has been determined that the dry weight of the wings of the P.
americana was 18% chitin, while the chitin content of the other parts
of the organism was 13%. The dry weights in other organisms are
Holotrichia parallela contains 15% chitin [13], Bombyx mori 15–20%
[37] and Cicada sloughs 36% [19]. Our chitin content result is similar
to those of H. parallela and B. mori.

4. Conclusion

Periplaneta americana is a cosmopolitan species that can be cul-

tured easily. Twenty individuals of this species were obtained from
Hacettepe University (Ankara) and cultured in our animal biodiver-
sity laboratory. The temperature and humidity of the laboratory is
conditioned to be 30 ◦ C. Many newborns and eggs have been gen-
Fig. 7. TG thermograms and DTG curves of chitin from the wings of Periplaneta
erated in only 2 months; this species multiples in hot and humid
americana. conditions [26]. Since this species is an invader and can be easily
12 M. Kaya, T. Baran / International Journal of Biological Macromolecules 75 (2015) 7–12
cultured, this organism could be considered as an alternative chitin
source to crab, shrimp, crayfish and prawn.
Chitin is found in the body structures of more than one million
invertebrate organisms including representatives of fungi, diatoms,
protists, anthozoans, nematods, sponges and mostly arthropods.
Considering the chitin isolation and characterization studies con-
ducted so far, around 50 different species have been investigated.
In this study, the isolation and characterization of chitin was con-
ducted using only the wings of an organism and not the whole
body for the first time, and a new surface morphology for chitin was
defined. The study showed that the physical characteristics of chitin
can be different depending on the body part of the same organ-
ism. The chitin isolation and characterization procedure that we
described here can be applied to various organisms. Furthermore,
it is possible that this chitin isolation and characterization process
can be assessed regarding commercial chitin sources, such as crab,
shrimp and crayfish. The number of pores on the chitin’s surface is
an important parameter in metal sorption studies [9]. Chitin with
this new morphology may be utilized to remove heavy metal ions.
In addition, chitin with pores can be used in tissue engineering
applications [39].
In the most common morphology (referred to as type 3)
nanofibers and pores are randomly disturbed on the chitin surface
[33–35]. In the new morphology that we defined here, the distri-
bution pattern of the pores on the chitin has a magnificent design,
and this design is depicted in this study for the first time.
In conclusion, (1) chitin from the wing of P. americana was iso-
lated and characterized using FTIR, TG, XRD and elemental analysis,
(2) the isolated chitin is in the alpha form and (3) very similar to
the commercial chitin from shrimp, crab and crayfish. The feature
that distinguishes this chitin from the others is its surface morphol-
ogy. We believe that chitin with this new morphology could be a
promising material for further studies and a product that is desired
in a wide range of applications.
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