International Journal of Biological Macromolecules 105 (2017) 1412–1420

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International Journal of Biological Macromolecules

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

Enhancement of extracellular polymeric substances (EPS) production

in Spirulina (Arthrospira sp.) by two-step cultivation process and
partial characterization of their polysaccharidic moiety
Imene Chentir a,∗ , Marwa Hamdi b , Amel Doumandji a , Abelkader HadjSadok c ,
Hatem Ben Ouada d , Moncef Nasri b , Mourad Jridi b
a
Plants Production and Biotechnology Laboratory, Nature and Life Science Faculty, Biotechnology Department, Blida 1 University, BP 270, 09000, Blida,
Algeria
b
Enzyme Engineering and Microbiology Laboratory, Sfax-University, National School of Engineering of Sfax (ENIS), BP 1173, Sfax, 3038, Tunisia
c
Functional Analysis of Chemical Processes Laboratory, Process Engineering Department, Blida 1 University, BP 270, 09000, Blida, Algeria
d
Marine Biodiversity and Biotechnology Laboratory, National Institute of Marine Sciences and Technology, BP 59, 5000, Monastir, Tunisia

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

Article history: The interactive effects of light intensity and NaCl concentration were investigated for Spirulina two-step
Received 30 May 2017 cultivation process using Full Factorial Design. In the experiment interval, light intensity had no effect
Received in revised form 27 June 2017 while the NaCl concentration had significant effect on the enhancement of extracellular polymeric sub-
Accepted 3 July 2017
stances (EPS) production. Interestingly, results revealed a significant negative interaction between light
Available online 5 July 2017
and NaCl concentration indicating that high NaCl concentration (40 gL−1 ) and low light intensity (10 ␮mol
photons m−2 s−1 ) enhanced the EPS production. Under these conditions, EPS production reached a max-
Keywords:
imum of 1.02 g g−1 of biomass (dry weight), which is 1.67-folds greater than EPS content under optimal
Arthrospira sp.
EPS production
growth conditions (10 ␮mol photons m−2 s−1 , 1 g L−1 , 30 ◦ C). Desalting and deproteinezation steps of EPS
Two-step culture were efficient to obtain polysaccharides (PS) with high carbohydrate (67.3 ± 1.1%), low soluble proteins
Rheological behavior (5.14 ± 0.32%), ash (5.85 ± 0.71%) and sulfate (2.42 ± 0.12%) contents. Rheological studies of PS at different
concentrations (1%, 2.5% and 5%) revealed that the viscosity of the solution increased with the increase
of PS concentration. In addition, PS exhibited a non Newtonian shear-thinning nature, a predominant
gel-like behavior and a good resistance to consecutive heating-cooling cycles. The adopted process could
be, then, a promising and economic strategy to enhance EPS production and extract polysaccharides with
interesting rheological properties.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction numerous industries for their proprieties as thickening or gelling

Exopolysaccharides are produced by several microorganisms
and show an extensive variety of biochemical complex struc-
tures, engaging different monosaccharides generally between 9
and 12 with the presence of several non sugars substituents such
as acyl groups, proteins, sulfate groups, or uronic acids, which
may be attached on their backbones [1,2]. This complexity lim-
its the structural investigations, however, exopolysaccharides have
attracted much attention because of their proven biological activi-
ties, such as antibacterial, anticoagulant, anti-oxidative, anticancer
and anti-inflammatory activities [1,3,4]. Moreover, they are used in
∗ Corresponding author.
E-mail address: chentir.imene@hotmail.com (I. Chentir).
agents [5].
Autotrophic microorganisms including microalgae and
cyanobacteria are unicellular photosynthetic microorganisms,
which convert captured light energy into biomass [6]. The world
market of microalgae and cyanobacteria can be considered as very
emergent with annual world industrial production estimated at
10,000–20,000 tons [7], leading to the decrease of the biomass
production and extracts cost, and thereby allowing possible
exploitation of their metabolites [1,8].
Among the most investigated autotrophic microorganisms, Spri-
ulina (Arthrospira sp.) is the first one that has attracted industrial
attention since a long time because of its relative fast growth,
large-scale and commercial cultivation potential as well as higher
photosynthetic efficiency compared with high plants [9]. Besides
its high protein content (50–70% of dry weight matter (DW)), Spir-

http://dx.doi.org/10.1016/j.ijbiomac.2017.07.009
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 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420
ulina releases a large amount of extracellular polymeric substances
(EPS) in its extracellular environment [10,11]. These released EPS
are polymers of 81–98 kDa [12], constituted of proteins, sulfated
substitutes (0.5–22%), uronic acids (14–40%) and polysaccharide
moiety, commonly composed of galactose, glucose, xylose, fruc-
tose, rhamnose, arabinose and mannose [10,13,14].
Nevertheless, EPS are rarely considered as high-value added
molecules by industrial and scientific community but they are
much considered as by-products of other intracellular metabolites
such as proteins, pigments or lipids. Consequently, few investiga-
tions have been carried out on studying culture parameters in order
to optimize the EPS production. However, downstream processes
for extraction, fractionation and characterization of released EPS
and their moieties by autotrophic microorganisms, especially Spir-
ulina, are frequently not adjusted for the treatment of these low
soluble polymers, which are, often, produced in media with high
salts contents [15].
Indeed, several important large-scale factors of culture process,
such as light intensity, temperature, salt and nutrient have been
modulated in combined manner to enhance the cellular growth
[16].
It is important to note that robust algal growth and high metabo-
lites production are usually jointly exclusive and reciprocally high
metabolites content produced under stress conditions are usu-
ally correlated with lower biomass productivity [17]. Grounded on
these considerations, a two-step culture strategy was adopted in
this study, with a first optimal-growth biomass production step
(Step I) followed by EPS induction step under stress conditions (step
II).
Hence, the aim of the present work is, first, to study the effect of
combination of two crucial large-scale factors i.e. light intensity and
NaCl concentration on Spirulina’s EPS production using an exper-
imental design methodology, to contribute therefore to specific
culture and downstream processes proposal of EPS polysaccharides
moiety, and finally to achieve the physico-chemical characteriza-
tion for the assessment of their possible industrial application.
2. Materials and methods
2.1. Strain
Arthrospira sp. (Spirulina) strain was isolated from pound
(23◦ 06 11 , N5◦ 49 01 E) in Tamanrasset area (Algeria). The col-
lected water samples were treated according to the standard
microbiological protocols [18,19], then the purified strain was iden-
tified according to the keys and description established by Komèrak
et al. [20].
Preliminary in lab experiments were achieved in different media
such as Zarrouk, BG-11, and Paoletti liquid media, under dif-
ferent temperatures and light intensity regimes, to define the
optimal growth conditions of this cyanobacterium strain. The
highest growth was obtained in Zarrouk liquid medium (pH
9.0) containing (g L−1 ): NaNO3 2.50, K2 HPO4 0.50, NaHCO3 10.0,
NaCl 1.00, MgSO4 ·7H2 O 0.2, CaCl2 ·2H2 O 0.02, FeSO4 ·7H2 O 0.01,
1 mL trace metal solution (g L−1 : H3 BO3 2.86, MnCl2 ·4H2 O 1.81,
Na2 MoO4 ·2H2 O 0.39, CuSO4 ·5H2 O 0.079, Co(NO3 )2 ·6H2 O 0.049,
and ZnSO4 · 7H2 O 0.222) [21], under 80 ␮mol photons m−2 s−1 of
light intensity and at 30 ± 1 ◦ C.
2.2. Cultivation steps
2.2.1. Step I: pre-cultures
Pre-cultures, under optimal growth conditions (80 ␮mol pho-
tons m−2 s−1 and at 30 ± 1 ◦ C) with inoculum size of 0.08 g L−1 , were
carried out in triplicate in 5 L reactors containing 3 L of Zarrouk
1413
medium, until exponential growth phase was reached. Each reac-
tor was equipped with a device for aseptic removal of samples and
stirred continuously with air at a constant flow rate (0.1 v/v/min).
The growth phase control was achieved daily, based on the deter-
mination of biomass concentration (n = 3) according to Costa et al.
[22]. The specific growth rate (max , d−1 ) was obtained by referring
to the following formula:
 
lnx2 − lnx1
max =
t2 − t1
where X1 and X2 are biomass concentrations at exponential phase,
t1 and t2 are the times corresponding to the beginning and the end
of exponential phase, respectively.
2.2.2. Step II: experimental stressed cultures
Biomass from pre-cultures, harvested at the end of the expo-
nential growth phase by filtration (20 ␮m nylon mesh), was used
as an inoculum for the stress experiments. Each 250 mL reactor,
containing 100 mL of the appropriate Zarrouk medium, where NaCl
concentrations varied respectively from 1 to 40 g L−1 , was equally
inoculated with 1 mg mL−1 DW of pre-culture biomass. Further,
each reactor, was exposed to fluorescent lamps (Phyto-Claude halo-
gen lamps 400 W) to give constant and continuous light intensities
of 10, 65 or 120 ␮mol photons m−2 s−1 . All stressed cultures were
incubated at 30 ± 1 ◦ C in a thermostat chamber.
After three days of treatment, all experimental cultures were
harvested by filtration (20 ␮m nylon mesh) and the total recovered
filtrates of each experiment were used for EPS content determina-
tion.
2.3. EPS production determination
EPS content was determined according to the method described
by Lewin [23]. Briefly, after three days of treatment, the total recov-
ered filtrates from each experimental culture (stressed and control
cultures conducted under optimal growth conditions) were used
for EPS content determination by evaporation of water in open cru-
cibles in a forced-air oven for 24 h at 105 ◦ C. The obtained dried
matter in each crucible was incinerated in a muffle furnace at
550 ± 5 ◦ C until white ashes remain. The change in weight of each
crucible containing EPS free-water and free-ashes, was considered
as the EPS content, and expressed in g per g of biomass DW.
2.4. Experimental design
The two independent factors light intensity (10, 65, 120 ␮mol
photons m−2 s−1 ) and NaCl concentrations (1, 20.5, 40 g L−1 ) were
examined at three levels, chosen according to preliminary tests.
A full factorial design was applied to the factors using MODDE
6.0 software for experimental design and optimization (Umetrics
AB, Sweden) to evaluate the effect of each factor. A total of 12
experiments (9 points of the factorial design and 3 center points,
indicating the medians of the studied factor interval, to determine
the experimental errors) were carried out (Table 1). The selected
response variable was the EPS production (g per g of biomass
DW). The model parameters were estimated by multiple linear
regressions (MLR) and the model fit accuracy was evaluated by the
explained variation (R2 ) and the validity of model (lack of fit). A
Box-Cox mathematical transformation [24] was applied when the
response was varied by more than a magnitude of ten in the experi-
mental domain. The statistical effect of each factor was determined
by the analysis of variance (ANOVA) for a confidence level of 90%.
The insignificant terms were removed, the model was refitted and
the significant linear, quadratic and interactive terms were used to
assess the effect of each factor on the response optimization trend.
1414 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420

Table 1
Experimental EPS production according to different factor combinations of light 500 mL culture media
intensity (␮mol photon m−2 s −1 ) and NaCl concentration in step I and step II of the
two-phase cultivation process.
UF concentration 5 kDa
Factors Response

Run Light NaCl EPS Retentate

(10 ␮mol photons m−2 s−1 ) (g L−1 ) (g g−1 )

Step I 80 1 0.62 ± 0.08

Washing 5 times with ultrapure water (v/5v)
Step II
1 10 1 0.152
2 65 1 0.192 Desalted EPS
3 120 1 0.454
4 10 20.5 0.387
Enzymatic hydrolysis (Purafect 1%, 50 °C, pH 10.0)
5 65 20.5 0.313
6 120 20.5 0.326
7 10 40 0.98 Centrifugation 8000 rpm 30 min at 4 °C
8 65 40 0.97
9 120 40 0.465
10a 65 20.5 0.38 Supernatant precipitation with EtOH (v/3v)
11a 65 20.5 0.336
12a 65 20.5 0.468
a
Values correspond to the central points of the experiment design matrix.
Pellet: Enriched fraction with polysaccharides (PS)

2.5. Polysaccharidic moiety (PS) extraction Lyophilisation of PS

EPS extraction was performed from Arthrospira sp. culture (in Fig. 1. Diagram of PS extraction.
10 L reactor, containing 5 L of experimental medium) following the
experimented two-phase culture process and under conditions of
thermal profile of EPS and PS samples was analyzed in a tempera-
NaCl concentration and light intensity allowing the highest EPS
ture range from 0 to 100 ◦ C. All tests were run at a heating rate scan
production level.
of 5 ◦ C/min. An empty capsule was used as reference. Nitrogen was
The culture was then filtered through Whatman filter paper No.
used as the purge gas at a flow rate of 50 mL/min. The glass transi-
2 (Whatman International Ltd., Maidston, UK) to separate the cells
tion temperature (Tg) for each sample was then determined from
from the culture medium. Then, the resulting culture filtrate, which
the mid-point of the second heating cycle using TA Universal Anal-
contained culture medium and the soluble EPS, was concentrated
ysis 2000 software (version 4.5 A, TA Instrument) and the enthalpy
(20 times) using a tangential ultrafiltration system (TFF Millipore,
change (H) was measured by the integration of the area under
USA) through Millipore membranes of 5 kDa cut-off. The desalting
the curve using the Pyris StepScan DSC software.
step was optimized by washing the obtained concentrate 8 times
with ultrapure water (v/5 v), and the salt content was evaluated
2.6.3. EPS and PS infrared spectroscopy analysis
using conductivity measurements (conductivity meterHI 8633).
The spectroscopic measurements were performed using
Subsequently, the desalted EPS were deproteinized according
approximately 1 mg of PS and EPS mixed with 100 mg of dried
to the method adopted by Abdelhedi et al. [25]. Briefly, EPS were
KBr. The Fourier Transform Infrared (FTIR) spectra between 500
suspended in water (20%, w/v). The pH of the solution was adjusted
® and 4000 cm−1 were recorded using a Nicolet FTIR spectrometer
to 10.0 and 1% of an alkaline protease (Purafect ) was added for the
equipped with an attenuated total reflection (ATR) accessory. The
deproteinization process. The reaction was performed during 24 h
background noise was corrected using the data for pure KBr.
at 50 ◦ C. After cooling, the mixture was centrifuged at 8000 rpm
for 30 min at 4 ◦ C and the supernatant was precipitated with abso-
2.6.4. Rheological analysis
lute ethanol (96%) (v/3 v) over night at 4 ◦ C. The formed precipitate
Prior to analysis, the freeze-dried PS was dissolved in 0.1 M NaCl
was recovered by centrifugation at 8000 rpm for 30 min at 4 ◦ C and
at pH 6.5 achieving concentrations of 1%, 2.5% and 5% (w/v). Samples
the obtained pellet (PS) was re-dissolved in distilled water and
were subjected to a controlled shear rate ramp from 10−3 to 103 s−1
lyophilized (Fig. 1).
in a plate geometry (25 mm diameter, 0.5 mm gap) of a rheome-
ter equipment (Physica MCR 302, Anton Paar, GmbH,Germany)
2.6. EPS and PS physico-chemical characterization
at a selected temperature of 20 ◦ C. Temperature modulation was
achieved with a Viscotherm VT 2 circulating bath and controlled
2.6.1. EPS and PS composition
with a Peltier system (Anton Paar) to an accuracy of 0.1 ◦ C. The data
Different parameters were used to characterize the obtained
were analyzed with proprietary software (Rheoplus, Anton Paar).
EPS and PS. The efficiency of desalting process was evaluated by
In order to explore the rheological behavior of PS in aqueous media,
conductivity determination and consolidated by total ash content
three types of analysis were performed:
measurement for both EPS and PS [26]. Protein content of both EPS
and PS was estimated according to Lowry et al. [27]. Total carbohy-
a) The flow behavior: where curves were fitted according to rhe-
drates were determined by the phenol-sulfuric acid method [28].
ological Carreau model [30] for overall shear rate curve of 10−3
Sulfate content was determined according to the classical method
to 103 s−1 model is presented by the following equation:
of barium chloride–gelatin as described by Lloyd et al. [29].

2.6.2. Differential scanning calorimetric analysis (DSC)  − ∞ 1

Thermal proprieties of EPS and PS powders were studied using app − ∞
=  2
p
Differential Scanning Calorimeter (Mettler Toledo Star). The sam- 1 − (/
˙ ˙ c )
ples were accurately weighed into aluminum pans and sealed. The
 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420
The flow behavior is appreciated by the four parameters ␩app ,
. Scaled and centered coefficient (USC. Coeff.), R2 and lack of fit of the EPS production
␩∞ , c and P where: ␩app : Apparent viscosity; ␩∞ : Infinite-shear
. .
viscosity; P: Carreau Model exponent; c : Critical shear rate;  :
Shear rate;
b) Linear and dynamic viscoelasticity proprieties: which were
investigated in linear regime under a sinusoidal shear of fre-
quency f = 1 Hz, and where the variation of the conservation
modulus (G ) and the loss modulus (G ) as a function of the strain
(y) (0.1–1000%) were measured at 20 ◦ C.
c) Thermo-viscoelasticity proprieties: where the ramp tempera-
ture effect was carried out by varying the temperature by heating
from 20 to 80 ◦ C and then cooling from 80 ◦ C to 2 ◦ C under con-
stant shear (f = 1, y = 1%).
Distillated water was added around the analytic plate disposi-
tive to avoid evaporation of water solution when heating.
2.7. Statistical analysis
Data were expressed as means ± SD (standard deviation) and
analyzed using SPSS ver. 19.0 for Windows professional edition
(SPSS Inc., Chicago,USA). A one-way analysis of variance (ANOVA)
was then applied to estimate the significance of variation at a con-
fidence level of 95%.
3. Results and discussion
3.1. Growth kinetic
The biomass concentration curve of Spirulina in batch culture,
under optimal growth conditions (Zarrouk medium, 80 ␮mol pho-
tons m−2 s−1 , 30 ◦ C) over 5 days of culture, is indicated in Fig. 2.
Results showed that the exponential growth reached its maximum
after 4 day of culture, where the maximum biomass density and
growth rate were about 0.67 ± 0.03 g L−1 and max of 0.41 ± 0.003
d−1 , respectively, while the EPS production (at day 4) was estimated
at 0.62 ± 0.08 g g−1 DW (Table 1). This result is not in congru-
ence with that found by Trabelsi et al. [31], who recorded an
EPS production of 0.433 g g−1 DW under autotrophic culture of
inlab-acclimated Arthrospira platensis strain. The variation in EPS
production amount could be related to the culture conditions.
Indeed, the difference observed between Arthrospira platensis and
Arthrospira sp. in EPS production is due to the extreme environ-
ment (desert) from where Arthrospira sp. was isolated, which would
leads this strain to possess efficient mechanisms for producing EPS
to preserve cells from desiccation and therefore from death.
3.2. EPS production modelization
The variation of the Spirulina EPS production under induced
experimental stress conditions, with respect to different combi-
nations of the experimental factors, is shown in Table 1. Results
revealed a large variation of the EPS production from 0.15 to
0.98 g g−1 DW.
To examine the interactive effects between the studied fac-
tors and their influence on EPS production, a modelization was
performed using response surface methodology (RSM). Results
illustrated in Table 2 indicates the statistical model parameters
of the obtained EPS model and the resulted unscaled coefficients
(Coeff.USC). The EPS response exhibited a satisfactory coefficient of
determination (R2 = 0.92) and non-significant lack of fit (p > 0.05),
resulting in sufficient model to accurately fit the experimental data
after computing power transformation (E−0.25 ) as suggested by
Box-Cox analysis.
1415
Table 2
model (Confidence interval of 90%).
R2 Lack of fit
0.92 0.26
SC. Coeff. p-value
Constant 0.777653 4.98E-10
Light −0.000973 0.954511
NaCl 0.117659 0.0001856a
NaCl × NaCl 0.0424055 0.111279
Light × NaCl −0.091409 0.0026852a
a
significant effect.
The effects of linear, quadratic and interactive terms of NaCl
concentration and light intensity on the EPS production are shown
in Fig. 3. Results revealed that light intensity had a non-significant
linear effect on EPS production in the experimental interval. Nev-
ertheless, the NaCl concentration displayed a positive linear effect,
while its quadratic term is not significant. Interestingly, results
indicated a significant negative interaction between light and NaCl,
revealing that the maximum EPS production required high NaCl
concentrations and low light intensity into the experimental stud-
ied intervals.
Several studies demonstrated that enhancement in EPS produc-
tion in cyanobacteria microorganisms was obtained at high light
intensities [11], while another study [16] specified that the light
was not the determining factor for EPS maximization in batch cul-
ture but was significantly interacting with temperature. In addition,
the obtained highe amounts of EPS under these high light inten-
sities could reflect only a greater biomass production, as cellular
productivities are generally not mentioned.
In agreement with these results, the EPS production increased
concomitantly with increasing NaCl concentration in different
cyanobacteria microorganisms and particularly in Spirulina platen-
sis [32]. The same data have been observed for various microalgae
strains such as Aphanocapsa halophyta [33], Microcoleus vaginatus
[34], Cyanothece [35] or Synechocystis [36]. Indeed, under phys-
iological conditions, the enhancement of EPS amount could be
suggested as a way to avoid the oxidative stress in cells and to pro-
tect membranes from desiccation [37]. In homology with bacteria,
the presence of EPS layer around the cell may reduce the diffusion of
ions from media through the cell surface and therefore preventing
cell death [38].
3.3. Maximized factor combinations for biomass composition and
EPS production
The factor combination conditions that maximize the EPS
production were evaluated by the established equations of the
obtained model. Low light intensity of 10 ␮mol photons m−2 s−1
combined with high concentration of NaCl (39 g L−1 ) triggered an
EPS maximum amount of 1.02 g g−1 biomass DW. Based on the
highest EPS content obtained under the stressed conditions (step
II), the EPS maximization ratio was 1.67-folds greater than that
obtained at the step I of cultivation (optimal growth conditions).
3.4. Desalting optimization and chemical composition of both EPS
and PS
Results displayed in Fig. 4 indicated the conductivity values of
the concentrated culture medium through washing times. Data
showed that five washing steps were efficient to bring conductivity
of the concentrated culture medium from 104.63 ± 1.01 mS cm−1
to 0.514 ± 0.02 mS cm−1 . The total ash content consolidated this
1416 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420

Fig. 2. Growth curve of Arthrospira sp. under optimal growth conditions. Error bars represent the standard deviation (n = 3).

Fig. 3. The values of process factor effects (computed as twice the MLR coefficients) on EPS production. LI (Light Intensity) and NaCl concentration. The standard error at 90%
confidence interval is shown as error bars.

Table 3
Chemical composition and thermal proprieties of concentrated culture media, EPS and polysaccharidic moiety (PS).

Concentratedculture media EPS(After desalting) PS(After deproteinization)

Moisture% ND 14.8 ± 1.39a 12.85± 0.78a

Ash%* 90.1 ± 1.11a 5.08 ± 0.66b 5.85 ± 0.71b
Total Sugar%* 2.1 ± 0.052a 60.56 ± 1.16b 67.3 ± 1.1c
Soluble proteins%* ND 11.12 ± 0.65a 5.14 ± 0.32b
Sulfate group%* ND 1.84 ± 0.02a 2.42 ± 0.12b
Tg (◦ C) ND 55.6 50.1
Hg (J/g) ND 60.7 51.35

ND: Not determined. (a,b,c) Different letters in the same line mean significant differences between concentrated culture media, EPS and PS moiety.
*
Results based on the wet weight of freeze-dried powder.
 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420 1417

Fig. 4. Desalting dynamic of concentrated culture medium according to number of washing cycles.a, b and c: different letters in the different column mean significant
difference between number of washing cycles of concentration media.

result and decreased from 90.1 ± 1.11% in the concentrated culture

medium to 5.08 ± 0.66% in the EPS after 5 washing cycles (Table 3).
The chemical composition of the concentrated culture medium,
EPS and PS is shown in Table 3. The sugar content was esti-
mated at 2.1 ± 0.052% in the concentrated culture medium, and
Transmittance

increased after the desalting steps to reach 60.56 ± 1.16% for EPS
and 67.3 ± 1.1% for PS after the EPS deproteinization. The depro-
teinization step allowed a decrease of the soluble protein content
in EPS by 2.16-folds, and reached a final content of 5.14 ± 0.32% in
PS.
The present findings are in discordance with those obtained by
Trabelsi et al. [10], where the composition of the extracted EPS from
Arthrospira platensis,cultivated in one step batch autotrophic cul-
ture process over 25 days, was mostly dominated by protein moiety
(55% DW) while carbohydrates amount was at 13% (DW). Accord-
ing to the available literature, protein moiety in cyanobacterial EPS
are ranged from 0.6 to 50.3% (DW) and depended on the species
and culture conditions [13,39]. Indeed, high NaCl concentration
may have an indirect effect on EPS composition. Increasing NaCl
concentration in culture medium could reduce the availability of
nutrients like nitrogen, which contributes to the C/N ratio increase
and thus promoting the incorporation of carbon into exopolysac-
charidic fraction [13]. However, even after repeated purification
steps, most authors are agreed in considering that proteins are not
reducible below a certain usual proportion ranged from 1 to 3% DW
of EPS, thereby, pointing out that it must be a true component of
the studied polymer [13,39].
3.5. DSC and infrared spectroscopy analysis of EPS and PS
The calorimetric proprieties of EPS and PS were evaluated and
data are presented in Table 3. Results indicated an endothermic
peak associated with the helix-coil transition characterized the dif-
ferential scanning calorimetric thermograms of PS and EPS. The
transition temperature (Tg) and enthalpy (H) of EPS and PS pow-
ders are reported in Table 3. PS had a Tg value of 50.1 ◦ C, which is
lower than that of EPS (55.6 ◦ C). Suyatma et al. [40] reported that Tg
is an important criterion for polymer uses in food or pharmaceuti-
cal application. In addition, both samples were wholly amorphous,
as they did not exhibit any melting event in the temperature region
EPS
PS
600 1100 1600 2100 2600 3100 3600
Wavenumbers (cm-1)
Fig. 5. Infrared spectra of EPS and PS.
that would, otherwise, be expected based on the heat stability
characteristics of the crude polysaccharide (PS). In addition, the
transition enthalpy associated with this peak is related to the rela-
tive interaction between components in the samples.
Furthermore, obtained results indicated that deproteinization
decreases transition enthalpy (51.35 J/g). The higher enthalpy value
for EPS (60.7 J/g), indicates that it had higher renaturation level than
the deproteinized polysaccharide (Table 3).
The presence of polysaccharide in both polymers was likewise
confirmed by FTIR analysis [41]. As shown in Fig. 5, for the two sam-
ples, absorption bands were observed in the region of 3200 cm−1
and 2900 cm−1 , which could be attributed to the stretching of O H
and the C H, respectively [42], typical for alkyl and hydroxyl func-
tionality of a carbohydrate. The most important peak for both PS
and EPS at 1250 cm−1 corresponds to the stretching vibration of
the ester sulfate groups (S O) [27]. Moreover, the presence of
pyranose units, C O C group and C O S bindings were consis-
tent with absorption band at 1050 cm−1 , 950 cm−1 and 780 cm−1 ,
respectively, and further evidenced sulfate groups as substituent
[29,42].
1418 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420
Fig. 6. (a) Fitted apparent viscosity variation of PS solution (1%, 2.5%, 5%) as a func-
tion of shear rate (s−1 ), T = 20 ◦ C; (b): PS solution G and G [Pa] variation as a function
of strain (%), f = 1 Hz,T = 20 ◦ C; (c): PS solution G and G temperature dependence
f = 1 Hz, y = 1%.
3.6. Rheological characterization results
3.6.1. Viscosity: shear-thinning behavior
Fig. 6a shows the flow curves of PS solutions at various concen-
trations (5%, 2.5% and 1% (w/v)). Under shear rate variation from
10−3 to 103 s−1 , the flow behavior is monitored by the four parame-
.
ters ␩app , ␩∞ ,  and P, supported by the constitutive Carreau model,
which gives a good fit with ␩app values (R2 = 0.99). After adjust-
ment, the values of the four characteristic parameters of the three
PS solutions were determined and grouped in Table 4A.
Table 4A
Carreau model parameters values as function of PS concentration.
.
␩app (Pa.s) ␩∞ (Pa.s) c (s−1 ) P
1% 0.007 15.8 0.002 0.282
2.5% 0.011 36.5 0.045 0.360
5% 0.029 335.6 0.059 0.384
The curves of apparent viscosity variation ␩ (Pa.s), as function
of the shear rate (s−1 ), indicated that all PS solutions (1%, 2.5% and
5%) exhibited a rheological behavior of structural type, which is
composed of three regions:
(i) The first Newtonian region characterized by a relative con-
stant apparent viscosity (␩app ) at the zero shear rate indicated a
non-solid behavior, suggesting that the inter-macromolecular
bonds are not enough to form a rigid network responsible for
the gelling behavior. All over the experiment, the PS samples
showed an unlimited viscosity that reaches 102 Pa.s, particu-
larly in 5% PS.
.
(ii) Beyond a certain shear value associated with critical shear, c ,
the apparent viscosity ␩ (Pa.s) of the PS solutions decreased,
generating thereby an intermediate rheo-fluidifiant behavior.
This behavior was characterized by the exponent (P) of Car-
reau model, which measures the degree of the Newtonian
anomaly or the gap in Newtonian behavior. When P = 0, the
fluid is Newtonian. In this intermediate region, PS macro-
molecules undergo shear forces resulting in partial destruction
and deployment.
(iii) Thereafter, the following region is characterized by a high
shear rate in which the apparent viscosity ␩ (Pa.s) seems to
be stabilized and was associated with a viscosity at the infinite
shear rate, ␩∞ . It is the second Newtonian region.
Moreover as shown in Table 4A, ␩app and ␩∞ increased with
the concentration of the PS solutions increasing, which denote the
structural character of the colloidal system and its sensitivity to
the increase when PS concentration increases. The increase of ␩app
.
showed that in the absence of shear ( = 0), the intermolecular
interactions were more reinforced. The increase in ␩∞ explained
the increase in flow resistance, under extreme flow, due to the
increase in the number of macromolecules. However, the value
of this parameter is considerably smaller than ␩app values, as in
infinite shear, the macromolecules are completely dispersed and
aligned in the direction of the shear and hence their resistance
.
to flow is reduced. Furthermore, the critical shear rate, c (s−1 ),
was similarly concentration-dependent, explaining the fact that the
molecular bonds become more cohesive, requiring more intense
shears to be destructed. Finally, the Carreau model exponent (P),
last parameter characteristic of the flow, was similar in the differ-
ent samples and ranged from 0.28 to 0.38 when PS concentration
increased from 1% to 5% (Table 4A). These P values,showed that the
relative drop in apparent viscosity under the effect of the flow (the
Newtonian anomaly), was not dependent to the concentration.
All these observations of shearing-thinning behavior lead to
conclude that the extracellular polysaccharide (PS) of Arthrospira
sp. displayed a non-Newtonian behavior and had an extremely
strong pseudoplastic characteristic, which is appeared at the
different studied concentrations and may be due to their poly-
electrolytic propriety. The flow results are in agreement with
those found for EPS from Arthrospira platensis [14], different bac-
terial extracellular polysaccharides such as Pseudomonas stutzeri
AS22 [43], Rhizobium radiobacter [44,45] and for polysaccharides
extracted from Lepidium sativum [46],where the shear-thinning
was directly proportional to the polymer concentration.
 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420
Grounded on this result, the assessed rheological propriety
of Arthrospira sp. extracellular polysaccharides suggests several
industrial applications as a thinking agent or viscosity-reducer in
some applications, which are involved in food processes at differ-
ent shear rates like mixing, pouring or pumping [47]. In addition,
this rheological behavior may affect the flavor release, food texture,
mouth feeling and suspension ability [45].
3.6.2. Viscoelastic proprieties: gel-like behavior
This test describes the variation of the two rheological moduli:
the storage modulus (G ) and the loss modulus (G ). The G values
provide information about the energy stored in the material during
deformation stress, while G describes its viscous character. With
increasing strain, two different regions were distinguished, namely
linear viscoelastic range (LVE) where G and G are almost constant,
and non-linear region (NVE) in which G and G start to drop.
The viscoelastic proprieties were evaluated and data are pre-
sented in Fig. 6a. Results demonstrated the presence of the linear
viscoelastic range (LVE), in which the two moduli were constant
and the storage modulus G was greater than the loss modulus G for
the different concentrations of PS solutions (G > G ). These results
indicated that the three solutions exhibited a viscoelastic gel-like
behavior, which is consistent with the determined liquid behavior
in continuous regime.
In addition, results indicated that PS gel-like behavior increased
as the PS concentration increased from 1% to 5% and thereby,
increasing the strength of the polymer to be more rigid.
Moreover, interestingly, at high PS concentration, the two mod-
uli get more spaced-paralleled to each other. Indeed an increase in
PS concentration leaded to rigidify the polymer’s network through
enhanced molecular contact, and thus making it more resistant to
deformation and flow [48]. In addition, in oscillation, the probe
time, which is the inverse of the frequency, is relatively small com-
pared to the relaxation time of the fluid, indicating that Arthrospira
sp. PS behaves more like solid, as shown by the elastic character.
This behavior is in accordance with those found for other hydro-
colloids such as Lepidium perfoliatum seed gum [49] and Auricularia
auricular-judae polysaccharide [50].
Beyond LVE, G begun to decrease and intercepted the curve
G at the gel point (G = G ). This point marked the transition from
viscoelastic solid to viscoelastic liquid behavior. However, the tran-
sition point was different in the tasted samples and depended on
the PS concentration. At the highest PS concentration (5%), this
transition point is reached under a strain (y) value to above 100%,
whereas it is reached at a much lower strain (<1%) for 1% PS.
Indeed, the hydrophilic nature of Arthrospira sp. PS allows water
absorption through hydrogen bonds belt between water molecules
and polymer carboxyl groups. Thus, at the lowest PS concentra-
tion (1%), the higher sensitivity of PS solution to the imposed strain
could be probably related to the lower density of junction zones
in the polymer network, having a weak nature leading to its easy
break-down by the strain action. Conversely, by increasing PS con-
centration, more hydrogen bonds are formed between the free PS
carboxylic groups and water molecules. Hence, the polymer net-
work gets much stronger and more elastic propriety because of
the higher crosslinking density [51]. Nevertheless, the PS rheolog-
ical behavior could be equally due to the electrostatic repulsions
caused by the charged groups on the polymer’s backbone, leading
to a highly extended structure. Consequently, these molecules may
be aligned and associated via hydrogen bonding to form a weakly
structured material [52].
On the other hand, the transition point is associated with a char-
acteristic parameter called “threshold stress”, ␶0 (Pa) (Table 4B).
When the applied stress is less than ␶0 , the solution behaves like
a viscoelastic gel, while high ␶0 (Pa) values enhanced the behavior
switch to a viscoelastic liquid.
1419
Table 4B
G ,G and ␶0 threshold strain values as function of PS concentration.
G (Pa) G (Pa) ␶0 (Pa)
1% 0.7 0.51 0.02
2.5% 6.2 4.34 6.10
5% 91.5 40.9 69.19
The values of G , G and ␶0 for the three concentrations of the PS
solution are in Table 4B. It is clearly shown that the three param-
eters register a large increase, varying by decade, displayed the
concentration varies linearly. In agreement with the study of Miao
et al. [53], this behavior indicates the potential use of PS as a tex-
turizing agent, affecting both very low consistency systems, such
as milky or liquid products, or pasts with high consistency and
thickness.
3.6.3. Thermo-viscoelastic proprieties: thermo-evaluation of PS
gel-like behavior
Fig. 6c shows the dependence of G and G at different PS concen-
trations (1%, 2.5% and 5%) through a temperature ramp test during
heating and cooling step.
For the investigated concentrations, the storage modulus G
always remains higher than the loss modulus G , even though
the solutions have been subjected to temperatures up to 80 ◦ C,
suggesting that the increase in the Brownian motion of the macro-
molecules (thermal agitation), due to temperature, did not affect
the PS structure, and thereby preserving the viscoelastic gel char-
acter of the PS. Furthermore, the two moduli G and G decreased
during heating, while increased during subsequent cooling. This
variation appears to be more significant for PS concentration of
1%, which recorded a gain of one decade after returning to ambi-
ent temperature (G heating(20 ◦ C) = 0.5 Pa and G cooling(20 ◦ C) = 2 Pa).
This observation may suggest that the macromolecules under-
went, after cooling, a new reorganization or aggregation of the
PS chains, generating a more compact structure particularly for
low PS concentrations [54]. This PS behavior is in accordance with
that observed in bacterial exopolysaccharides (EPSA22) [43]. How-
ever, other studies reported that the temperature treatment did
not affected the polysaccharide structure extracted from Rhizobium
radiobacter CAS [44], Pseudomonas oleovoranse [55] and Lepidium
sativum [45].
4. Conclusion
The present work demonstrated that two steps culture of
Arthropira sp. modify significantly and in a dynamic manner the EPS
production, and showed that the application of combined multiple
stress factors gave an appreciate EPS maximization ratio The opti-
mized combination between light and NaCl for EPS enhancement is
energy economic and could be easily adopted by industry in a short
time (3 days) according to the adopted two-step cultivation pro-
cess, which avoids biomass productivity restriction. Under these
conditions, Arthrospira sp. produced extracellular polysaccharides
with low protein moiety. The isolated polysaccharide (PS) has good
calorimetric proprieties with amorphous character. The rheologi-
cal studies suggested that PS displayed a pseudoplastic behavior,
exhibited a typical shear-thinning nature, gel-like behavior, and can
resist to thermal cycle. The outcomes of this study demonstrate the
potential use of PS in several food and pharmaceutical applications.
Acknowledgment
The authors thank Youcef Krichen and Ali Aouabed for permit-
ting access to their laboratory.
1420 I. Chentir et al. / International Journal of Biological Macromolecules 105 (2017) 1412–1420
References
[1] M.P.F.J. Raposo, R.M.S.C. Morais, A.M.M.B. Morais, Bioactivity and applications
of sulphated polysaccharides from marine microalgae, Mar. Drug 11 (2013)
233–252.
[2] F. Donot, A. Fontana, J.C. Baccou, S. Schorr-Galindo, Microbial
exopolysaccharides: main examples of synthesis, excretion, genetics and
extraction, Carbohydr. Polym. 87 (2012) 951–962.
[3] H. Majdoub, M.B. Mansour, F. Chaubet, M.S. Roudesli, R.M. Maaroufi,
Anticoagulant activity of a sulfated polysaccharide from the green alga
Arthrospira platensis, Biochem. Biophys. Acta. 1790 (2009) 1377–1381.
[4] R. Challouf, L. Trabelsi, R. Ben Dhieb, O. El Abed, A. Yahia, K. Ghozzi, J. Ben
Ammar, H. Omran, H. Ben Ouada, Evaluation of cytotoxicity and biological
activities in extracellular polysaccharides released by cyanobacterium
Arthrospira platensis, Braz. Arch. Biol.Technol. 54 (2011) 831–838.
[5] F. Freitas, V.D. Alves, M.A.M. Reis, Advances in bacterial exopolysaccharides:
from production to biotechnological applications, Trends Biotech. 29 (2011)
388–398.
[6] P.G. Stephenson, C.M. Moore, M.J. Terry, M.V. Zubkov, T.S. Bibby, Improving
photosynthesis for algal biofuels: toward a green revolution, Trend.
Biotechnol. 29 (2011) 615–623.
[7] A.C. Guedes, H.M. Amaro, F.X. Malcata, Microalgae as source of high-value
compounds a brief review of recent works, Biotechnol. Prog. 27 (2011)
597–613.
[8] O. Pignolet, S. Jubeau, C. Vacca-Garcia, P. Michaud, Highly valuable
microalgae: biochemical and topological aspects, J. Ind. Microbiol. Biotechnol.
40 (2013) 781–796.
[9] G. Markou, I. Chatzipavlidis, D. Georgakakis, Carbohydrates production and
bio-flocculation characteristics in cultures of Arthrospira (Spirulina) platensis:
Improvements through phosphorus limitation process, BioEnergy Res. 5
(2012) 915–925.
[10] L. Trabelsi, N.H. M’sakni, H.B. Ouada, H. Bacha, S. Roudelsi, Partial
characterization of extracellular polysaccharides produced by
cyanobacterium Arthrospira platensis, Biotechnol. Bioprocess Eng. 14 (2009)
27–31.
[11] C. Delattre, G. Pierre, C. Laroche, P.H. Michaud, Production, extraction and
characterization of microalgal and cyanobacterial exopolysaccharides,
Biotechnol. Adv. (2016), http://dx.doi.org/10.1016/j.biotechadv.2016.08.001.
[12] R. De Philippis, M. Vincenzini, exocellular polysaccharides from cyanobactéria
and their possible applications, Fems. Micro. Rev. 22 (1998) 151–175.
[13] A. Otero, M. Vincenzini, Extracellular polysaccharide synthesis by Nostoc
strains as affected by N source and light intensity, J. Biotechnol. 102 (2003)
43–152.
[14] R. Filali Mouhim, J.F. Cornet, T. Fontaine, B. Fournet, G. Dubertret, Production,
isolation and preliminary characterization of the exopolysaccharide of the
cyanobacterium Spirulina platensis, Biotech. Lett. 15 (1993) 567–572.
[15] H. Li, Z. Li, S. Xiong, H. Zhang, N. Li, S. Zhou, Y. Liu, Z. Huang, Pilot-scale
isolation of bioactive extracellular polymeric substances from cell-free media
of mass microalgal cultures using tangential-flow ultrafiltration, Process
Biochem. 46 (2011) 1104–1109 (L.).
[16] Trabelsi, H.B. Ouada, H. Bacha, M. Ghoul, Combined effect of temperature and
light intensity on growth and extracellular polymeric substance production by
the cyanobacterium Arthrospira platensis, J. Appl. Phycol. 21 (2009) 405–412.
[17] C. Adams, V. Godfrey, B. Wahlen, L. Seefeldt, B. Bugbee, Understanding
precision nitrogen stress to optimize the growth and lipid content trade off in
oleaginous green microalgae, Biores. Technol. 131 (2013) 88–194.
[18] R.Y. Stanier, R. Kunisawa, M. Mandel, G. Cohen-Bazire, Purification and
properties of unicellular blue-green algae (order Chroococcales), Bacterio.
Rev. 35 (1971) 171–205.
[19] R. Rippka, J. Deruelles, J.B. Waterbury, M. Herdman, R.Y. Stanier, Generic
assignments, strain histories and properties of pure cultures of cyanobacteria,
J. Gen. Microbiol. 111 (1979) 1–6.
[20] J. Komárek, H. Kling, J. Komárková, Filamentous cyanobacteria, in: John D.
Wehr, Robert G. Sheath (Eds.), Freshwater Algae of North America: Ecology
and Classification, New York, 1998, pp. 117–196.
[21] C. Zarrouk, Contribution to the Study of a Cyanophyceae Influence of Various
Physical and Chemical Factors on Growth and Phototsynthesis of Spirulina
Maxima (Setch. And Gardner) Geithner. Ph.D Thesis, University of Paris,
France, 1966.
[22] J.A.V. Costa, L.M. Colla, P.F. Duarte Filho, K. Kabke, A, Weber Modeling of
Spirulina platensis growth in fresh water using response surface methodology,
W. J. Microbiol. Biotechnol. 18 (2002) 603–607.
[23] R.A. Lewin, Extracellular polysaccharides of green algae, Can. J. Microbiol. 2
(1956) 665–672.
[24] M.R. Peltier, C.J. Wilcox, D.C. Sharp, Technical note: application of the Box-Cox
data transformation to animal science experiments, J. Anim. Sci. 76 (1998)
847–849.
[25] O. Abdelhedi, R. Nasri, N. Souissi, M. Nasri, M. Jridi, Sulfated polysaccharides
from common smooth hound: extraction and assessment of anti-ACE,
antioxidant and antibacterial activities, Carbohydr. Polym. 152 (2016)
605–614.
[26] AOAC, Official Methods of Analysis, 17th ed., Associationof Official Analytical
Chemists, Washington, DC, 2000.
[27] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with
the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
[28] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric
method for determination of sugars and related substances, Anal. Chem. 28
(1956) 350–356.
[29] A.G. Lloyd, K.S. Dodgson, R.B. Price, F.A. Rose, Infrared studies onsulfate esters.
I. Polysaccharide sulfates, Bioch. Bioph. Acta. 46 (1961) 108–115.
[30] C.W. Macosko, Rheology Principles, Measur & Applic, Willey Edition, 1994, pp.
86.
[31] L. Trabelsi, H. Ben Ouada, F. Zili, N. Mazhoud, J. Ammar, Evaluation of
Arthrospira platensis extracellular polymeric substances production in
photoautotrophic, heterotrophic and mixotrophic conditions, Folia. Microbiol.
58 (2013) 39–45.
[32] M.C. Lee, Y.C. Chen, T.C. Peng, Two-stage culture method for optimized
polysaccharide production in Spirulina platensis, J. Sci. Food. Agric. 92 (2012)
1562–1569.
[33] H. Sudo, J.G. Burgess, H. Takemasa, N. Nakamura, T. Matsunaga, Sulfated
exopolysaccharide production by the halophilic cyanobacterium Aphanocapsa
halophytia, Curr. Microbiol. 30 (1995) 219–222.
[34] C. Hu, Y. Liu, B.S. Paulsen, D. Petersen, D. Klaveness, Extracellular
carbohydrate polymers from five desert soil algae with different cohesion in
the stabilization of fine sand grain, Carbohyd. Polym. 54 (2003) 33–42.
[35] C. Su, C. Zhenming, L. Weidong, Optimization of medium and cultivation
condition for enhanced exopolysaccharide yield by marine Cyanothece sp.
113, Chin. J. Oceanol. Limnol. 25 (2007) 411–417.
[36] S. Ozturk, B. Aslim, Modification of exopolysaccharide composition and
production by three cyanobacterial isolates under salt stress, Environ. Sci.
Pollut. Res. 17 (2010) 595–602.
[37] L.Z. Chen, D.H. Li, L.R. Song, C.X. Hu, G.H. Wang, Y.D. Liu, Effects of salt stress
on carbohydrate metabolism in desert soil alga Microcoleus vaginatus Gom, J.
Integr. Plant Biol 48 (2006) 914–919.
[38] A.S. Kumar, K. Mody, B. Jha, Bacterial exopolysaccharides −a perception, J.
Basic Microbiol. 47 (2007) 103–117.
[39] R. De Philippis, M.C. Margheri, E. Pelosi, S. Ventura, Exopolysaccharide
production by a unicellular cyanobacteriumisolated froma hypersaline
habitat, J. Appl. Phycol 5 (1993) 387–394.
[40] N.E. Suyatma, A. Copinet, L. Tighzert, V. Coma, Mechanical and barrier
properties of biodegradable films made from chitosan and poly (lactic acid)
blends, J. Polym. Environ. 12 (2004) 1–6.
[41] C. Rochas, M. Lahaye, W. Yaphe, Sulfate content of carrageenan and
agardetermined by infrared spectroscopy, Botanica Marina 29 (1986)
335–340.
[42] D. Santhiya, S. Subramanian, K.A. Natarajan, Surface chemical studies on
sphalerite and galena using extracellular polysaccharides isolated from
bacillus polymyxa, J. Coll. Interface Sci. 256 (2002) 237–248.
[43] H. Maalej, N. Hmidet, C. Boisset, E. Bayma, A. Heyraud, M. Nasri, Rheological
and emulsifying properties of a gel-like exopolysaccharide produced by
Pseudomonas stutzeri AS22, Food. Hydrocoll. 52 (2016) 634–647.
[44] F. Zhou, Z. Wu, C. Chen, J. Han, L. Ai, B. Guo, Exopolysaccharides produced by
Rhizobium radiobacter S10 in whey and their rheological properties, Food.
Hydrocoll. 36 (2014) 362–368.
[45] P. Andhare, C. Delattre, G. Pierre, P. Michaud, H. Pathak, Characterization and
rheological behaviour analysis of the succinoglycan produced by Rhizobium
radiobacter strain CAS from curd sample, Food Hydrocoll. (2016), http://dx.
doi.org/10.1016/j.biotechadv.2016.08.001.
[46] S. Naji, S.M.A. Razavi, H. Karazhiyan, Effect of thermal treatment on functional
properties of cress seed (Lepidium sativum) and xanthan gums: a comparative
study, Food. Hydrocoll. 28 (2012) 75–81.
[47] S.E. Velasco, J. Areizaga, A. Irastorza, M.T. Duenas, A. Santamaria, M.E. Munoz,
Chemical and rheological properties of the beta-glucan produced by
Pediococcus parvulus 2.6, J. Agric. Food Chem. 57 (2009) 1827–1834.
[48] D. Saha, S. Bhattacharya, Hydrocolloids as thickening and gelling agents in
food: a critical review, J. Food. Sci. Technol 47 (2010) 587–597.
[49] M. Hesarinejad, A. Koocheki, S. Razavi, Dynamic rheological properties of
lepidium perfoliatum seed gum: effect of concentration, temperature and
heating/cooling rate, Food Hydrocoll. 35 (2014) 583–589.
[50] H. Bao, S. You, L. Cao, R. Zhou, Q. Wang, S.W. Cui, Chemical and rheological
properties of polysaccharides from fruit body of Auricularia auricular-judae,
Food. Hydrocoll. (2016), http://dx.doi.org/10.1016/j.foodhyd.2015.12.031.
[51] M. Gottlieb, C.W. Macosko, G.S. Benjamin, K.O. Meyers, E.W. Merrill,
Equilibrium modulus of model poly(dimethylsiloxane) networks,
Macromolecules 14 (1981) 1039–1046.
[52] W. Rochefort, S. Middleman, Rheology of xanthan gum: salt, temperature, and
strain effects in oscillatory and steady shear experiments, J. Rheol. 31 (1987)
337.
[53] M. Miao, C. Huang, X. Jia, S.W. Cui, B. Jiang, T. Zhang, Physicochemical
characteristics of a high molecular weight bioengineered ␣-D-glucan from
Leuconostoccitreum SK24. 002, Food Hydrocoll. 50 (2015) 37–43.
[54] F. Sun, Q. Huang, J. Wu, Rheological behaviors of an exopolysaccharide from
fermentation medium of a Cordyceps sinensis fungus (Cs-HK1), Carbohydr.
Polym. 114 (2014) 506–513.
[55] F. Freitas, V.D. Alves, M. Carvalheira, N. Costa, R. Oliveira, M.A.M. Reis,
Emulsifying behaviour and rheological properties of the extracellular
polysaccharide produced by Pseudomonas oleovorans grown on glycerol
byproduct, Carbohydr. Polym. 78 (2009) 549–556.