Beruflich Dokumente
Kultur Dokumente
BIOMAT Research Group, University of the Basque Country (UPV/EHU), Department of Chemical and Environmental Engineering, Polytechnic School, Donostia-San Sebastin, Spain
University of the Basque Country (UPV/EHU), Department of Applied Chemistry, Faculty of Chemistry, Donostia-San Sebastin, Spain
a r t i c l e
i n f o
Article history:
Received 15 November 2012
Received in revised form 22 January 2013
Accepted 27 January 2013
Available online 8 February 2013
Keywords:
Chitosan
Biodegradable lms
Antimicrobial activity
Maillard reaction
a b s t r a c t
Chitosan-based lms for food packaging applications were prepared by casting and dried at room temperature or heat-treated in order to study functional properties and antimicrobial activity. In all cases,
lms were exible and transparent, regardless of chitosan molecular weight, glycerol content, and temperature. Regarding antimicrobial activity, chitosan lm forming solutions showed antimicrobial behaviour against Escherichia coli and Lactobacillus plantarum. It was also observed that the bacteriostatic
property of chitosan-based lms against bacteria employed in this study was notably affected by temperature. Moreover, temperature produced signicant variation in the functional properties of chitosanbased lms, such as colour, wettability, resistance against UV light and mechanical properties. In good
agreement with this behaviour, total soluble matter (TSM), fourier transform infrared (FTIR) spectroscopy, thermo-gravimetric analysis (TGA) and X-ray diffraction (XRD) results suggested a change in the
chemical structure of chitosan lms, possibly due to Maillard reaction when heat treatment was used.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
The world market for plastic lms is dominated by petroleumderived polymers in a wide range of industrial applications and, in
particular, in food packaging. In any eld, manufacturers must satisfy economical, social, legal, and environmental concerns, so the
balance between good properties and sustainability must be taken
into account, and material selection becomes critical to face environmental demands and the need for sustainable products (Leceta
et al., 2013). Nowadays, there is an increasing interest in biopolymers, natural and biodegradable polymers, as an alternative to
commodity polymers in terms of raw material supply and waste
product generation. In this context, chitin is the second most abundant biopolymer after cellulose and chitosan is the biopolymer obtained from deacetylation of chitin, which is the major structural
component of the exoskeleton of invertebrates and the cell walls
of fungi (Knorr, 1991; Pillai et al., 2009; Rinaudo, 2006). In addition,
since most biopolymers are either biodegradable or compostable, it
can also be argued that chitosan could t with the cradle-to-cradle
concept, which means that on disposal it could become food for the
next generation of materials (McDonough and Braungart, 2002).
On the other hand, although the use of conventional packaging
materials such as commodity polymers and their derivatives is
effective for food preservation, the environmental problems that
their disposal create contribute to increase the interest of manufactures in the food industry and scientists specialized in food
engineering in new polymers with antimicrobial activity. The
Corresponding author.
E-mail address: koro.delacaba@ehu.es (K. de la Caba).
0260-8774/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2013.01.022
growing consumer demand for foods without chemical preservatives has focused efforts in natural antimicrobials and the use of
active bio-based lms is one of the most promising ways, being
chitosan one of the most perspective active lms (Aider, 2010;
Snchez-Gonzlez et al., 2011). The antimicrobial activity of
chitosan against different groups of microorganisms such as bacteria (Bulwan et al., 2012; Doulabi et al., 2013; Fernandez-Saiz et al.,
2008; Kristo et al., 2008) and fungi (Assis and De Britto, 2011;
Avila-Sosa et al., 2012; Martnez-Camacho et al., 2010; Sebti
et al., 2005) has received considerable attention in recent years.
However, research has been mostly conned to biomedical applications (Kong et al., 2008). Chitosan has inherent antimicrobial
activity owing to the fact that long positively charged chitosan
molecules interact with negatively charged bacteria causing
disruption on the cell (Coma et al., 2003; Zivanovic et al., 2005).
Chitosans bactericidal efcacy depends on various factors that
can be classied into four categories: microbial factors related
microorganism species; intrinsic factor of chitosan, including
molecular weight and concentration; physical state, solution or
lm; and environmental conditions like pH and temperature (Kong
et al., 2010).
Owing to its renewable, biodegradable, and antimicrobial character, chitosan is a potential material for its use as food packaging
lm. Nevertheless, pure chitosan lms are fragile and need plasticizers to reduce frictional forces between the polymer chains, as
hydrogen bonds or ionic forces, thus improving mechanical properties (Olabarrieta et al., 2001; Srinivasa et al., 2007; Suyatma
et al., 2005). Plasticizers act as internal lubricant weakening intra
and intermolecular interactions and increasing the mobility of
biopolymeric chains. As a consequence, free volume increases,
890
2. Experimental
2.8. Colour measurements
2.1. Materials
Low molecular weight (LMw) chitosan (batch MKBB9037) and
high molecular weight (HMw) chitosan (batch MKBC0059) with a
degree of deacetylation higher than 75% were provided by
Sigma-Aldrich (Spain). Acetic acid was used to x the solution
pH and glycerol as plasticizer. Both of them were food grade reactants, purchased from Panreac (Spain), and were used as received.
2.2. Film preparation
Chitosan lms were prepared by casting. A 1% by weight chitosan solution was prepared in a 1% acetic acid solution. After 15 min
under continuous stirring, glycerol was added. Stirring was continued for 30 min until total homogenization of the mixture. After
that, nal pH (pH = 4.0 0.1) was measured with a digital pH-meter (Crison pH-meter BASIC 20+). Finally, solutions were ltered
and 25 mL of the solution were poured into 90 mm diameter Petri
dishes. Films prepared with 0%, 15% and 30% wt glycerol were allowed to dry at room temperature (RT) or heat-treated (HT) in an
air-circulating oven at 105 C for 24 h. Same lm was used to make
RT and HT measurements in non-destructive tests. All lms were
stored for 48 h in a controlled environment chamber (ACS
SU700 V) at 25 C and 50% relative humidity before testing.
Colour values of lms were measured using a portable colorimeter (CR-400 Minolta Chroma Meter). Film specimens were placed
on a white plate, and the CIELAB colour scale was used to measure
colour: L = 0 (black) to L = 100 (white), a (greenness) to +a
(redness) and b (blueness) to +b (yellowness). Standard values
for the white calibration plate were L = 97.39, a = 0.03 and
b = 1.77. With these values, and considering standard light source
D65 and standard observer 2 colour parameters, L, a, b were
measured. Chrome (C ab ) and hue angle (hab ) were calculated from:
C
q
a 2 b 2
h arctg
b
a
DE
q
Lstandard Lsample 2 astandard asample 2 bstandard bsample 2
2.9. Gloss
The gloss of the lms was measured with a Minolta gloss meter
(MultiGloss 268 Plus). The gloss was measured at a 60 incidence
angle, according to ASTM D523 (ASTM, 1999). Five replicates were
made per formulation. Results were expressed as gloss units, relative to a highly polished surface of black glass standard with a value near to 100.
The light-barrier properties of lms were determined by measuring their light absorption at wavelengths ranging from
200 nm to 800 nm, using a UV-Jasco spectrophotometer (Model
V-630).
891
lm pieces (sterilized with UV light) or 60 lL of lm forming solutions. Each well was inoculated with 2% (v/v) of an overnight bacteria culture. Cell growth was followed by reading the medium
turbidity at 620 nm by a microplate reader (Labsystems iEMS
Reader MF). All the results are the mean of three replicate assays.
The data were subjected to one-way analysis of variant (ANOVA) by means of an SPSS computer program (SPSS Statistic 19.0).
Post hoc multiple comparision were determined by the Tukeys
test with the level of signicance set at P < 0.05.
G
WVTR
tA
where G is the change in weight (g), t is the time (h), and A is the
test area of the test area (m2). WVP was calculated as:
WVP
WVRT T
DP
where T is the thickness of the test specimen (mm) and DP is the partial pressure difference of the water vapour across the lm. WVP of
three specimens for each sample was calculated and reported.
2.13. Mechanical properties
Table 1
Total soluble matter (TSM) average values and standard deviations of high molecular
weight (HMw) and low molecular weight (LMw) chitosan-based lms dried at room
temperature (RT) and heat treated (HT).
ac
Chitosan
Glycerol (%)
TSM (%)
RT
HT
HMw
0
15
30
100.00 0.00a
100.00 0.00a
18.98 0.32b
11.39 0.71a
12.47 1.84a
15.52 0.98b
LMw
0
15
30
100.00 0.00a
84.35 4.70c
24.30 1.01b
14.40 1.57a
20.60 0.66c
21.14 0.62c
Two values followed by the same letter in the same column are not signicant
(P > 0.05) different thought the Tukeys multiple range test.
892
OH
HO
O
O
OH
H2O
O
O
HO
CH
C O
CHOH
H2O
O
O
OH
NH 2
CHOH
OH
O
NH
CH2
C O
H2O
R
R
Schiff Base
Amadori Compound
Fig. 1. Maillard reaction between carbonyl ends and the amine groups in chitosan.
(b)
Transmittance
Transmittance
(a)
RT 0HMw
HT 0HMw
Transmittance
Transmittance
RT 0LMw
H T 0LM w
RT 15LMw
HT 15LMw
Transmitance
Transmittance
RT 15HMw
HT 15HMw
RT 30LMw
HT 30LMw
RT 30HMw
HT 30HMw
4000
3600
3200
2800
2400
2000
1600
1200
800
-1
4000
3600
3200
2800
2400
2000
1600
1200
800
-1
Fig. 2. Infrared spectra for (a) high molecular weight (HMw) and (b) low molecular weight (LMw) chitosan-based lms dried at room temperature (RT) and heat treated (HT).
of the band at 1558 cm1 (amide II) for the lms dried at room
temperature, regardless of the molecular weight of chitosan or
glycerol content. However, the difference in the relative intensity
of those two bands became smaller as the glycerol content increased for both HMw and LMw chitosan-based lms dried in
the oven. These results could indicate that Maillard reaction between carbonyl and amine groups in chitosan was promoted by
temperature and favoured at higher contents of glycerol, which is
in agreement with the decrease of solubility observed for heattreated lms.
On the other hand, comparing the relative intensity of the bands
at 1405 cm1 and 1378 cm1, it can be observed that the intensity
893
(b)
Transmittance
Transmittance
(a)
RT 0HMw
HT 0HMw
Transmittance
Transmittance
RT 0LMw
HT 0LMw
RT 15HMw
HT 15HMw
Transmittance
Transmittance
RT 15LMw
HT 15LMw
RT 30HMw
HT 30HMw
1800
1600
RT 30LMw
HT 30LMw
1400
1200
1000
800
-1
1800
1600
1400
1200
1000
800
-1
Fig. 3. Magnication of infrared spectra from 1800 to 800 cm1 for (a) high molecular weight (HMw) and b) low molecular weight (LMw) chitosan-based lms dried at room
temperature (RT) and heat treated (HT).
of the band at 1405 cm1 was always higher than the one at
1378 cm1 for the lms dried at room temperature. Nevertheless,
the relative intensity of those two bands changed for the lms
heat-treated. For LMw chitosan lms without glycerol, the relative
intensity of those bands showed the same tendency observed at
room temperature, but when 15% glycerol was added, the relative
intensity became similar, and the relative intensity of the band at
1378 cm1 became higher for the lms prepared with 30% glycerol.
In the case of HMw lms, the relative intensity of the bands became similar for the lms without glycerol and was higher for
the band at 1378 cm1 when glycerol was added. The decrease of
the relative intensity of the band at 1405 cm1, attributed to the
biocide character of chitosan, for the lms heat-treated was also
observed by the antimicrobial tests, as shown below.
Finally, it is also worth noting that the absorption peaks at
1045 cm1 and 1117 cm1, associated to CAO stretching, joined
to become a single peak when glycerol content was increased, suggesting interactions between hydroxyl groups of chitosan and glycerol by hydrogen bonding.
lms containing 0% and 30% glycerol. For the lms without glycerol, there was a small weight loss at temperatures below 100 C,
which can be attributed to the loss of moisture, and a shoulder
around 175 C, which may correspond to the loss of adsorbed
and bound water (Lewandowska, 2009). The main stage of weight
loss due to the degradation of chitosan started up to 290 C and
showed about 21% loss weight for all the samples. It is worth noting that there was no signicant change between chitosan-based
lms dried at room temperature or heat-trated.
Regarding thermo-gravimetric analysis for the lms with 30%
glycerol, it must be noted that there was a new peak located
around 225 C that was related to the evaporation of glycerol. This
peak showed about 15% loss weight for the lms prepared with
30% glycerol and the temperature was higher than the one estimated for pure glycerol, which is 170 C (Perry and Chilton,
1973). These two facts could indicate a good degree of interaction
between chitosan and glycerol, as shown by FTIR results. In the
case of the lms heat-treated, the peak related to glycerol disappeared, indicating a change in the structure due to the Maillard
reaction, as it was previously shown by the decrease of TSM values.
894
(b)
DTGA (%/min)
DTGA (%/min)
(a)
RT 0LMw
HT 0LMw
DTGA (%/min)
DTGA (%/min)
RT 0HMw
HT 0HMw
RT 30HMw
HT 30HMw
0
100
200
300
400
500
600
700
RT 30LMw
HT 30LMw
800
100
200
300
Temperature (C)
400
500
600
700
800
Temperature (C)
Fig. 4. Thermogravimetric analysis for (a) high molecular weight (HMw) and (b) low molecular weight (LMw) chitosan-based lms with 0% and 30% glycerol, dried at room
temperature (RT) and heat treated (HT).
RT 0HMw
HT 0HMw
RT 30HMw
HT 30HMw
10
20
30
40
50
2
Fig. 5. X-ray diffractograms of high molecular weight (HMw) chitosan-based lms
with 0% and 30% glycerol, dried at room temperature (RT) and heat treated (HT).
895
Table 2
Colour (C ab and hab ) and gloss average values and standard deviations of high molecular weight (HMw) and low molecular weight (LMw) chitosan-based lms dried at room
temperature (RT) and heat treated (HT).
Chitosan
Glycerol (%)
Colour measurements
RT
HT
C ab
af
C ab
hab
hab
DE
RT
HT
HMw
0
15
30
3.21 0.24a
3.19 0.05b
3.94 0.20c
94.67 0.50a
95.46 0.37a
97.70 0.30b
4.81 0.15a
8.57 1.51b
6.15 0.16c
97.73 0.18a
99.53 0.38b
100.01 0.22c
1.68 0.14a
3.10 0.75b
2.33 0.24c
36.22 0.61a
31.64 1.28b
28.95 0.42c
30.38 7.97a
28.04 1.54bc
22.12 2.23cd
LMw
0
15
30
4.77 0.20d
4.98 0.22c
10.81 1.01e
96.89 0.15c
97.65 0.18c
98.36 0.40d
9.99 0.31d
11.65 0.17e
17.82 0.41f
100.40 0.13d
100.36 0.08cd
99.52 0.17b
5.29 0.39d
6.81 0.18e
8.90 0.44f
24.50 0.73d
20.63 1.13e
15.25 0.45f
23.65 1.08abc
17.66 0.87cd
12.51 0.90d
Two values followed by the same letter in the same column are not signicant (P > 0.05) different thought the Tukeys multiple range test.
This fact could indicate that Maillard reaction was more pronounced for these systems due to the fact that there was less stearic
hindrance when the molecular weight of chitosan was lower.
Gloss is also an optical phenomenon related to the appearance
of the surface. It represents the capacity of the surface to reect directed light and it is considered to be the proportion of incident
light that is reected at the specular reectance angle with respect
to the normal plane of the surface. Table 2 shows the values obtained at 60 incidence angle. As it is shown, gloss values were lower than 40 for all the compositions. Since a surface with a gloss of
70 or greater at an incidence angle of 60 is generally considered a
high gloss surface (Kible-Boeckler, 1996), it can be said that the
lms prepared in this work were slightly glossy. Moreover, gloss
values were reduced by the addition of plasticizer (P > 0.05) and
showed a slight decrease due to the effect of temperature
(P > 0.05). As gloss is related to surface composition and morphology, changes in these two aspects resulted in changes in gloss, so
the decrease in gloss values could be due to the fact that the
(a)
addition of glycerol or the compound formed during Maillard reaction, which is favoured at high temperatures, generated discontinuities in the chitosan matrix, increasing the roughness of the
lm surface.
3.6. Light absorption
Fig. 6 shows the UVvis absorption spectra from 200 to 800 nm
for all the lms. As it can be seen, the spectra for chitosan-based
lms dried at room temperature were similar, regardless of glycerol amount. It is noteworthy that lms had excellent barrier properties to light in the UV region, being the preventive effect slightly
better for LMw chitosan-based lms. Moreover, when lms were
heat-treated, there was a sharp increase in absorbance in the range
of 200400 nm for higher glycerol contents. However, there was no
signicant difference in the UVvis absorption spectra for HMw
chitosan-based lms dried at room temperature or heat-treated.
This difference could be due to the fact that HMw chitosan chains
(b)
RT 0HMw
RT 0LMw
HT 0LMw
Absorbance
Absorbance
HT 0HMw
RT 15LMw
HT 15HMw
HT 15LMw
Absorbance
Absorbance
RT 15HMw
RT 30LMw
HT 30HMw
HT 30 LMw
200
Absorbance
Absorbance
RT 30HMw
300
400
500
600
Wavelenght (nm)
700
800
200
300
400
500
600
700
800
Wavelenght (nm)
Fig. 6. UVvis spectra for a) high molecular weight (HMw) and b) low molecular weight (LMw) chitosan-based lms dried at room temperature (RT) and heat treated (HT).
896
160
RT HMw
HT HMw
RT LMw
HT LMw
140
Contact angle ()
120
a
b
cd
100
80
60
40
20
0
0% GLY
15% GLY
30% GLY
Fig. 7. Average contact angle values and standard deviation of high molecular
weight (HMw) and low molecular weight (LMw) chitosan-based lms dried at room
temperature (RT) and heat treated (HT). a-dColumns with the same letter are not
signicantly (P > 0.05) different through the Tukeys multiple range test.
Table 3
Water vapour permeability (WVP) average values and standard deviations of high
molecular weight (HMw) and low molecular weight (LMw) chitosan-based lms
dried at room temperature (RT) and heat treated (HT).
WVP. 1013 (g cm1 s1 Pa1)
Chitosan
Glycerol (%)
RT
HT
HMw
0
15
30
8.07 1.00a
8.38 0.21a
8.76 0.56ab
7.78 0.49a
9.40 0.21ab
9.47 0.54ab
LMw
0
15
30
9.21 0.48ab
10.1 0.41b
10.2 0.04b
8.46 0.14a
9.05 0.91ab
9.12 0.55ab
ab
Two values followed by the same letter in the same column are not signicant
(P > 0.05) different thought the Tukeys multiple range test.
are more static owing to its higher length. This fact could hinder
the proximity between amino and carbonyl groups to react
through Maillard reaction. However, small glycerol molecules
could get into chitosan chains more easily when chitosan molecular weight was lower. This fact could favour Maillard reaction,
which produces chromophores that absorb the light at wavelength
below 400 nm, providing exceptional barrier properties to UV light.
These results were in good agreement with the colour values
shown above, which indicated that total colour difference was
higher for LMw chitosan lms.
3.7. Barrier properties
Water contact angle values are good indicators of the degree of
hydrophilicity of lms, being higher when hydrophilicity is lower,
so the nal state of the water drop on the lm surface can be taken
Table 4
Tensile strength (TS) elongation at break (EB) average values and standard deviations of high molecular weight (HMw) and low molecular weight (LMw) chitosan-based lms
dried at room temperature (RT) and heat treated (HT).
Chitosan
af
Glycerol (%)
RT
HT
TS (MPa)
EB (%)
TS (MPa)
EB (%)
HMw
0
15
30
61.82 3.43a
43.41 3.11c
31.89 5.93cd
4.59 0.49a
11.14 0.70b
30.51 1.75c
68.47 2.58e
43.60 5.23c
37.93 4.77b
15.37 2.00f
17.00 2.96f
20.58 0.72e
LMw
0
15
30
55.83 2.96a
36.85 3.55bc
23.87 3.79d
4.58 0.38a
27.34 3.38c
37.67 2.16d
53.98 7.65a
38.25 1.08b
32.01 2.65b
8.50 0.50b
10.12 1.20b
17.23 2.46e
Two values followed by the same letter in the same column are not signicant (P > 0.05) different thought the Tukeys multiple range test.
897
Fig. 8. Agar diffusion method results for chitosan-based lms (a) dried at room temperature and (b) heat-treated.
2.2
Absorbance 620 nm
0h
24 h
48 h
Lactobacillus Plantarum
2.0
bc
1.8
bc
bc
bc
bc
bc
bc
1.6
1.4
d
de
1.2
e e
1.0
a
0.8
0.6
E.coli
0h
24 h
48 h
0.4
Absorbance 620 nm
2.0
1.8
e
bc
1.4
1.6
d
cd
1.2
1.0
0.8
a
0.6
a
a
0.4
weight, glycerol content, samples used (solutions or lms), or drying conditions employed to prepare the lms. The absence of inhibitory character could be explained by the limitation of the diffusion
of chitosan in agar medium, as it has been reported by other
authors (Coma et al., 2002). However, as it can be seen in Fig. 8,
there was no bacterial growth under the lm for the lms dried
at room temperature, indicating that chitosan-based lms can exhibit bacteriostatic effect against E. coli and L. plantarum. It was
also worth noting that lms dried at room temperature shrunk
as time went by. On the other hand, there was bacterial growth under the lms heat-treated, suggesting that temperature caused a
structural change in lms, giving rise to the reduction of the bacteriostatic property. These results were in good agreement with FTIR
results, which indicated that there was a reduction of the absorption band at 1405 cm1, which has been related to the antimicrobial character of chitosan in the literature (Lagaron et al., 2007).
As the previous results suggest that agar diffusion method is not
an appropriate method to characterize the antimicrobial properties
of chitosan, diffusion in liquid culture medium was also used. As
can be appreciated in Fig. 9, regarding chitosan-based lms, there
was no signicant decrease (P > 0.05) in the absorbance at 620 nm,
indicating that there was no inhibitory effect. This could be owing
to the limitation of the biocides diffusion in the case of lms in the
liquid culture medium, as well as in agar, as shown above. However, in the case of lm forming solutions, as could be observed
in Fig. 10, absorbance values were strongly reduced (P < 0.05),
showing an important inhibition of bacteria growth and good antimicrobial activity.
pure chitosan lms are higher than the values obtained by other
authors with chitosan of similar deacetylation degree and by similar preparation conditions (Hosseini et al., 2013; Khan et al., 2012;
Marroquin et al., 2013; Valenzuela et al., 2013).
3.9. Antimicrobial activity
Antimicrobial activity of chitosan-lms was evaluated by the
agar diffusion method. Results showed that there was no inhibition
zone on agar solid medium, irrespective of chitosan molecular
4. Conclusions
The results of this work showed that chitosan lm forming solutions presented antibacterial properties, whereas chitosan-based
lms dried at room temperature only showed bacteriostatic properties, which were reduced when lms were heat-treated, suggesting changes in the structure of lms due to the effect of
temperature. These changes were explained by chemical interactions through Maillard reaction, which was promoted by temperature, as shown by TSM, FTIR, TGA and XRD results. In addition,
remarkable variations were observed in some functional properties
such as colour, wettability, resistance against UV light, and
mechanical properties due to the effect of temperature. These results show that temperature is a critical factor affecting physicochemical and antibacterial properties of chitosan-based lms,
which must be taken into account not only for fabrication purposes
898
2.0
Lactobacillus plantarum
1.8
0h
24 h
48 h
Absorbance 620 nm
1.6
1.4
k
1.2
ijk
ij
1.0
jk
i
h
g
0.8
0.6
f
a
ae
e
d
0.4
fh
a
d
0.2
E. coli
0h
24 h
48 h
Absorbance 620 nm
1.2
c
b
1.0
0.8
0.6
0.4
a
d
d d
0.2
d
d d
e e
d
e e
e
d
0.0
Control 0HMw 15HMw 30HMw 0LMw 15LMw 30LMw
Fig. 10. Average absorbance values and standard deviation at 620 nm for chitosan
lm forming solutions after 0, 24 and 48 h of bacteria inoculation. akColumns with
the same letter are not signicantly (P > 0.05) different through the Tukeys
multiple range test.
but also for xing storage conditions. Moreover, the effect of temperature must also be taken into account when designing active
packaging lms to prevent microorganisms growth.
Acknowledgments
The authors thank MICINN (Project MAT2009-07735) and Plan
E as well as Basque Government (Project S-PE12UN002) and
UPV/EHU (Project PES11/35) for their nancial support. Itsaso Leceta thanks Basque Government (Fellowship BFI-2010-82). Thanks
also SGIker service from the University of the Basque Country.
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