Chitin extraction and chitosan production

from cell wall of two mushroom species

(Lactarius vellereus and Phyllophora ribis)
Cite as: AIP Conference Proceedings 1809, 020012 (2017); https://doi.org/10.1063/1.4975427
Published Online: 24 February 2017

S. Erdogan, M. Kaya, and I. Akata

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AIP Conference Proceedings 1809, 020012 (2017); https://doi.org/10.1063/1.4975427 1809, 020012

© 2017 Author(s).
 Chitin Extraction and Chitosan Production
from Cell Wall of Two Mushroom Species
(Lactarius Vellereus and Phyllophora Ribis)
S. Erdogan1,*, M. Kaya2 and I. Akata3
1
Laborant and Veterinary Programme, Keşan Vocational College, Trakya University, 22800
Keşan, Edirne, Turkey
2
Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray
University, 68100, Aksaray, Turkey
3
Department of Biology, Science Faculty, Ankara University, 06100, Tandoğan, Ankara, Turkey
*
Corresponding author: serdogantrakya@gmail.com

Abstract. Chitin is an important polysaccharide found as supporting material in the cell wall of
mushrooms. In this study, chitin and chitosan were obtained from the cell wall of two different
mushroom species using chemical method and physicochemically characterized. The dry weight
chitin contents of the mushroom species were determined as 11.4% for Lactarius vellereus and
7.9% for Phyllophora ribis. Chitosan yields of the chitins isolated from L. vellereus and P. ribis
were 73.1% and 75.3%, respectively. While, the maximum degradation temperatures of L.
vellereus and P. ribis chitins were found to be 354°C and 275°C by thermogravimetric analysis
(TGA), the maximum degradation temperature of the chitosans obtained from these chitins were
recorded as 262°C and 229°C, respectively. The crystalline index values of L. vellereus and P.
ribis chitins were calculated as 64% and 49%, respectively according to the X-ray diffraction
analysis (XRD) results. The scanning electron microscopy (SEM) indicated that there were no
nanofiber or nanopores on the surface of the chitins and chitosans obtained from these two
mushroom species. The results of this study revealed that L. vellereus and P. ribis had higher chitin
contents than some other insects and mushroom species recorded in the literature and these species
may be used as a potential chitin sources.
Keywords: Biopolymer; Isolation; Characterization; Crystallinity.
PACS: 61.10.Nz, 68.37.Hk, 68.60.Dv, 78.40.Me, 82.80.-d, 87.15.-v.

INTRODUCTION
Mushrooms have been consumed for centuries as a valuable food due to their high
protein content and good taste [1, 2]. Besides, some mushroom species have been used
for medical purposes [3, 4]. Therefore, researches on mushrooms have mostly focused
on their nutritional value, antimicrobial properties, metabolites, and some other issues
[5-9]. There are limited number of studies on the properties of chitin and chitosan which
are important components of the cell wall of mushrooms.
Chitin, which is an N-acetylglucosamine polymer, is a structural component found
in the exoskeleton of invertebrates such as crustacean and insects, in the skeleton of
sponges and in the inner skeleton of squid and cuttlefish. Chitin also exist within the
structure of cell wall of mushrooms as well as invertebrates and it gives shape to cell
wall and determine its rigidity [10]. Chitosan is a chitin derivative and also it is naturally
found in the cell wall of species belonging to Mucoraceae [11]. Chitin and chitosan are

Proceedings of the 6th International Advances in Applied Physics and Materials Science Congress & Exhibition
AIP Conf. Proc. 1809, 020012-1–020012-10; doi: 10.1063/1.4975427
Published by AIP Publishing. 978-0-7354-1477-8/$30.00

020012-1
used in many application area including textile, cosmetics, food, biomedical,
pharmaceutical, waste water treatment and agriculture thanks to their biocompatible,
biodegradable and non-toxic nature [12-14]. Crustacean organisms have been mostly
used as a chitin source up until now. However, it is known that chitins and chitosans
from different sources exhibited different physicochemical properties and these
different properties expanded the application area of these biomaterials [15]. Thus, in
this study, chitin and chitosan were obtained from two different mushroom species and
their physicochemical properties compared with each other.
The genus Lactarius, which is characteristic for producing milky latex when
damaged, have about 400 species. The genus includes mushrooms, which some are rich
in nutritional value and some are valuable due to their antimicrobial and antioxidant
activities [2]. Some species of the genus Lactarius are cultivated. Lactarius vellerius
used in this study is a common mushroom and used as a food by local people in Turkey
[16]. It is also known that this species has antioxidant and antimicrobial activity [5, 16].
Other species, Phylophora ribis is a wild mushroom. Considering the easily cultivation
of mushroom species, it can be said that mushrooms have a potential as an alternative
chitin source. Therefore, this study will contribute to expand the using area of mushroom
chitin and chitosan by determining the physicochemical properties of chitin and chitosan
from L. vellereus and P. ribis.
This study aims a) to extract chitin from two mushroom species and to product
chitosan from these two chitins, b) to determine some physicochemical properties of
these chitins and chitosans and c) to compare these two mushroom species in terms of
their chitin contents.

MATERIALS and METHODS

MATERIAL
Two mushroom species (L. vellereus, P. ribis) were used in this study. Mushrooms
were collected from the Işık Mountain-Kızılcahamam (Ankara) in 2015. Samples of
these mushrooms have been stored at the herbarium in the department of Biology in
Ankara University. Fruit bodies of mushroom samples were washed and dried in an
owen at 50 °C for 10 days. Samples were ground into powder in a mixer before chitin
extraction.

CHITIN ISOLATION
Twenty gram milled mushroom samples were weighted for the chitin extraction. The
mushroom samples were treated with 250 ml 2M HCl solution to remove the minerals.
The demineralization step took 15 h at 60°C. By the end of this time, the samples were
filtered through a filter paper with the pore size of 2 μm by rinsing with distilled water.
Then, the filtrates were treated with 2M NaOH solution at 85°C for removal of protein
residues in the structure. After NaOH treatment of 24 hours, samples washed with
distilled water until a neutral pH. This step was followed by a decolorization process
using a mixture of chloroform, methanol and distilled water in the ratio of 1:2:4.

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Bleaching the samples took one hour. Finally, the samples were rinsed again with
distilled water and kept in an oven at 50°C until drying.

CHITOSAN PRODUCTION
The chitin samples isolated in previous step were refluxed with 60% NaOH at 130°C
for 4 hours for chitosan output. After this process, the samples were filtered and washed
with distilled water until reaching a neutral pH. The obtained chitosans were kept in the
oven at 50°C for one day to dry.

SCANNING ELECTRON MICROSCOPY (SEM)

Scanning electron microscopy was used to examine the surface morphologies of the
chitins isolated from two mushroom species and the chitosans derived from these
chitins. Examination was carried out on samples coated with gold using an EVO LS 10
ZEISS scanning electron microscope.

THERMOGRAVIMETRIC ANALYSIS (TGA)

A thermogravimetric analysis was performed on the chitin and chitosan samples
obtained from the two mushroom species by EXSTAR S11 7300 thermogravimetric
analyser. The samples were heated at temperature intervals (10°C per min.) ranging
from 25 to 650°C.

X-RAY DIFFRACTION (XRD)

A Rigaku D max 2000 system X-ray diffractometer was used to perform X-Ray
Diffraction analysis on the chitin and chitosan samples obtained from mushroom species
L. vellereus and P. ribis. XRD patterns of the samples were recorded at 40 kV, 30 mA,
and 2θ with a scan angle from 5° to 45°. The crystalline index value (CrI) was calculated
according to the formula [17] that I110 is the maximum intensity at 2θ 20° and Iam is
the intensity of amorphous diffraction at 2θ 16°.
CrI110 = [(I110− Iam)/I110] × 100 (1)

RESULTS and DISCUSSION

CHITIN CONTENT
The dry weight chitin content of L. vellereus was found to be 11.4% while the dry
weight chitin content of P. ribis was determined as 7.9%. Chitosan yields of these chitins
were also recorded as 73.1% for L. vellereus and 75.3% for P. ribis. In literature studies,
it was reported that the dry weight chitin contents of crustaceans vary between 13% -

020012-3
42% [18]. Similarly, it was determined that dry weight chitin contents of some insect
species varied between 2.59% and 36% [17, 19-23]. While, the chitin contents of L.
vellereus and P. ribis are lower than crustaceans, they are higher than that of some insect
species. The chitin contents of fungi are also considerably different among species as in
crustacean and insects. Synowiecki and Al-Khateeb [18] observed that the dry weight
chitin contents of mycelium of micro fungi species from different phylums varied
between 2.3-42.0%. In another study, it was observed that the crude chitin content of
shiitake stipes (Lentinula edodes) varied between 25.08 and 36.72% depending on the
isolation method [24]. Vetter [10] was investigated the chitin contents of some
cultivated mushroom species belonging to Basidiomycota phylum. The author reported
that the chitin contents of Agaricus bisporus, Pleurotus ostreatus and L. edodes were
6.67%, 3.78% and 8.07% for the pileus and 7.31%, 2.8% and 6.55% for the stipes,
respectively. Dry weight chitin content of a medicinal fungus Fomitopsis pinicola was
found to be 30.11 % and the chitosan yield of this chitin was 71.75 % [25]. Finally, the
dry weight chitin content of a lichen species Xanthoria parietina consisting of fungi and
algae was determined as 4.23% by Kaya et al. [26]. The chitin content of the two
mushroom species in this study are higher than the mushroom species A. bisporus, P.
ostreatus, L. edodes and lichen X. parietina.
As is seen, there are significant differences between mushroom species in terms of
chitin contents. Ifuku et al. [4] stated that there are branched β-1,3-glucan components
embedded between chitin nanofibres in the cell wall of mushrooms, unlike crustaceans.
These differences in the chitin contents may depend on the existence of β-1,3-glucan
components that couldn’t effectively remove from the mushroom chitin as well as
factors such as mushroom species, extraction method and age of thallus [4, 27, 28].
Although, P. ribis has lower chitin content when compared with L. vellereus, both
mushroom species had higher chitin contents than some other insects and mushroom
species recorded in the literature and these species may be used as a potential chitin
sources. In addition, easily cultivation of some species increases the significance of
mushrooms as a chitin source [29].

SEM
The surface morphologies of the chitins and chitosans from the two mushroom
species (L. vellereus and P. ribis) were examined with SEM analysis. The analysis
results indicated that there were no nanofibres or nanopores on the surfaces of the chitins
and chitosans obtained from L. vellereus and P. ribis (Fig. 1). In a previous study, it was
reported that lichen species X. parietina has a smooth surface [26]. A similar study
revealed that shitake stipes chitosans exhibited a firm structure without nanopores and
nanofibres [30]. On the contrary, Ifuku et al. [4] obtained chitins which has uniform
nanofibre structure from five mushroom species (P. eryngii, A. bisporus, L. edodes,
Grifola frondosa and Hypsizygus marmoreus) by grinding the chitins in acidic
conditions. Kaya et al. [25] revealed that the surface morphologies of chitin and chitosan
from F. pinicola consist of nanofibres 10–15 nm in size and nanopores 15–25 nm in size.
While, the surface morphologies of chitins and chitosans from L. vellereus and P. ribis
were similar to that of lichen X. parietina, they were different from the surface

020012-4
morphologies of mushroom species F. pinicola, P. eryngii, A. bisporus, L. edodes, G.
frondosa and H. marmoreus.

Figure 1. SEM images of chitins and chitosans from two mushroom species: a) Chitin from L.
vellereus; b) Chitin from P. ribis; c) Chitosan from L.vellereus; d) chitosan from P. ribis.

The surface morphologies of the chitins and chitosans of these two mushroom species
exhibited smooth and firm structure differently from the fibrillar and porous chitins in
crustacean and insect [23, 31]. The smooth surface morphologies observed in this study
indicated that β-1,3-glucan components couldn’t effectively remove from the chitin
structure by chemical method.
Surface morphology is one of the important factors which determine the using area
of chitin and chitosan. Muzzarelli [32] emphasizes that nanofibres of fungal origin can
be used in anti-tumor applications and immune-modulating activity. Fibrillar chitin,
which will be obtained from these two mushroom species by removing β-1,3-glucan
components with a suitable extraction method, can be used for these purposes.

TGA
The TGA analyses results indicated that the mass loss of chitins and chitosans from
two mushroom species occurred in two steps (Fig. 2). While the chitin from L. vellereus
lost 6% of its mass in the first step, it lost 68% of it in the second step. The mass loss of
P. ribis chitin was 2% in the first step and 15% in the second step. The mass loss in the

020012-5
first step proceed from evaporation of water in the chitin structure and the mass loss in
the second step results from degradation of carbohydrate [21]. Similarly, mass loss of
chitins from crustacean, insect, lichen and some mushroom species also occurred in two
steps [21, 23, 26, 33].
While, L. vellereus chitosan lost 4% of its mass in the first step due to the evaporation
of water in it, P. ribis chitosan lost 5% of its mass. In the second step, the mass losses
of L. vellereus and P. ribis chitosans were 38% and 54%, respectively owing to the
degradation of carbohydrate structure. On the contrary, Mesa Ospina et al. [34] reported
that the mass loss of the mushroom Ganoderma lucidium chitosan was in three steps.
The authors explained that the first mass loss was due to evaporation of water in the
chitin and the mass losses in the last two steps were due to the degradation of sugar and
secondly degradation of organic material.
The maximum degradation temperatures (DTGmax) of chitins from L. vellereus and
P. ribis was measured as 354 and 275 °C, respectively. The DTGmax value determined
between 300 - 400 oC for α-chitin [35]. The DTGmax values were reported around 350
°C for crustaceans, around 380°C for insects, 341 oC for F. pinicola and 342 oC for X.
parietina [22, 25, 26, 31, 33]. A similar result was recorded for potato beetle
(Leptinotarsa decemlineata) larvae (DTGmax: 307 °C) by Kaya et al. [36]. Considering
these results, it is seen that the thermal stabilities of the chitins from these two mushroom
species were lower than the chitins of crustacean, insect, lichen and mushroom F.
pinicola. It was reported in previous studies that the thermal stability of chitosan is lower
than that of chitin and DTGmax value of chitosan is around 300 °C [31, 37]. In this study,
DTGmax values of chitosans obtained from two mushroom species were found to be
262°C for L. vellereus and 229 °C for P. ribis. The DTGmax values of chitosans were
reported as 300°C for silkworm chrysalides [21], 290 °C for potato beetle [36], 265 °C
for F. pinicola [25] and 273.2 °C for G. lucidium [34].
The thermal stabilities of chitin and chitosan of L. vellereus were found to be higher
than those of P. ribis. However, the thermal stabilities of chitins and chitosans from
these two mushroom species are lower than those of crustacean and insects. Kaya et al.
[25] asserted that the low DTGmax value of mushroom chitin and chitosan stems from
the glucan residues couldn’t remove from the chitin structure.

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Figure 2. TGA results of chitins and chitosans from two mushroom species: a) Chitin from L. vellereus;
b) Chitosan from L. vellereus; c) Chitin from P. ribis; d) Chitosan from P. ribis.

XRD
Crystallinities of the chitins and chitosans from L. vellereus and P. ribis were
determined by x-ray diffraction analysis and two sharp peaks at 9.20 and 19.64° and
four weak peaks at 12.20, 21.50, 23.40 and 26.68° were observed for L. vellereus chitin
(Fig. 3). Similarly, a total of six peaks were recorded for P. ribis chitin at 9.38, 19.60,
14.20, 21.90, 23.50 and 26.80° (Fig. 3). Of these, 9.38° and 19.60° were the peaks with
higher intensity while other peaks with lower intensity. It was reported that crustacean
chitins has two sharp peaks around 9° and 19° [33]. These peaks are typical crystal
patterns of α-chitin. Similar diffraction peaks were observed for the insect, mushroom
and lichen chitins in previous studies [4, 22, 23, 25, 26, 36].
Two sharp peaks were recorded for L. vellereus chitosan at 12.05° and 20.01° and
for P. ribis chitosan at 12.20° and 20.30° (Fig. 3). In a study conducted on crustacean
chitosan, two sharp peaks at around 10° and 20° were observed [38]. Similarly, these
two sharp peaks at 10° and 20° were recorded for the chitosans from mushroom species
F. pinicola and G. lucidum [25, 34]. Mesa Ospina et al. [34] reported that these peaks
are characteristic peaks of the pure chitosan. This indicated that the purity of L. vellereus
and P. ribis chitosans are low.
Crystalline index (CrI) values of the chitins isolated from L. vellereus and P. ribis
were calculated as 64% and 49%, respectively. In literature, the CrI values was reported

020012-7
around 67.8% and 64.1% for crab and shrimp chitins [39]. This value was found to be
89.05% for H. parallela chitin, 89.7% for cicada sloughs chitin, 76% for adult potato
beetle chitin and 72% for larval potato beetle chitin [17, 22, 36]. Ifuku et al. [4] reported
that the CrI values of chitin nanofibers isolated from five mushroom species varied
between 47,6% and 88,5%. In other studies, while the CrI value of lichen chitin was
found to be 70.1%, the CrI value of F. pinicola chitin was determined as 52 % [25, 26].
In this study, the CrI value calculated for the L. vellereus chitin was higher than the CrI
value of P. ribis chitin. However, the CrI values of the chitins obtained from these two
mushroom species are lower than the values given in literature. Ifuku et al. [4] remarked
that low crystalline index values indicate the excess amount of amorphous glucan in the
chitin structure. This revealed that the purities of mushroom chitins isolated from L.
vellereus and P .ribis were lower.

Figure 3. X-ray diffraction peaks of chitins and chitosans from L. vellereus and P. ribis: a) Chitin from
L. vellereus; b) Chitosan from L. vellereus; c) Chitin from P. ribis; d) Chitosan from P. ribis.

CONCLUSION
In this study, chitin and chitosan were obtained from two different mushroom species
and their physicochemical properties were determined with SEM, TGA and XRD
analyses. While the dry weight chitin content of L. vellereus was determined as 11.4%,

020012-8
the dry weigh chitin content of P. ribis was found to be 7.9%. It was observed that the
thermal stabilities of the chitin and chitosan from L. vellereus (DTG max: 354°C for chitin,
262°C for chitosan) were higher than those of P. ribis (DTG max: 275°C for chitin, 229
°C for chitosan). In addition, crystalline index value of L. vellereus chitin (64%) was
also higher than that of P. ribis (49%). SEM analysis revealed that chitin and chitosan
of both mushroom species had a smooth surface morphology without porous and fibers.
Considering that L. vellereus and P. ribis had higher dry weight chitin content in
comparison to some insect and mushroom species, it is suggested that these two
mushroom species can be used as an alternative chitin source. In addition, it can be
suggested that chitin and chitosan from L. vellereus can be used as antimicrobial and
antioxidant agents in medical, pharmaceutics and cosmetics industry due to
antimicrobial and antioxidant properties of this mushroom species. Also, as reported in
a previous study [28], chitin-glucan complex extracted from these mushroom can be
used as food additives and effective enterosorbents in food sector.

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