Polymer Degradation and Stability 96 (2011) 2088e2097

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Polymer Degradation and Stability

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

How the biodegradability of wheat gluten-based agromaterial can be modulated

by adding nanoclays
Anne Chevillard a, *, Hlne Angellier-Coussy a, Bernard Cuq b, Valrie Guillard a, Guy Csar c,
Nathalie Gontard a, Emmanuelle Gastaldi a
a
b
c

UMR IATE, Universit Montpellier II, CC023, pl. E Bataillon, 34095 Montpellier Cedex, France
UMR IATE, Montpellier SupAgro, Bat 37, 2 place Viala, 34060 Montpellier, France
SERPBIO, Laboratoire LIMATB-L2PIC, Universit de Bretagne Sud, rue Saint Maud, 56321 Lorient Cedex, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 9 August 2011
Received in revised form
23 September 2011
Accepted 28 September 2011
Available online 6 October 2011

The objective of this work was to investigate the inuence of clay nanoparticles on the biodegradability
of wheat gluten-based materials through a better understanding of multi-scale relationships between
biodegradability, water transfer properties and structure of wheat gluten/clay materials. Wheat gluten/
clay (nano)composites materials were prepared via bi-vis extrusion by using an unmodied sodium
montmorillonite (MMT) and an organically modied MMT. Respirometric experiments showed that the
rate of biodegradation of wheat gluten-based materials could be slowed down by adding unmodied
MMT (HPS) without affecting the nal biodegradation level whereas the presence of an organically
modied MMT (C30B) did not signicantly inuence the biodegradation pattern. Based on the evaluation
of the water sensitivity and a multi-scale characterization of material structure, three hypotheses have
been proposed to account for the underlying mechanisms. The molecular/macromolecular afnity
between the clay layers and the wheat gluten matrix, i.e. the ability of both components to establish
interactions appeared as the key parameter governing the nanostructure, the water sensitivity and, as
a result, the overall biodegradation process.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Biodegradation
Wheat gluten
Montmorillonite
Nanocomposite
Water sensitivity

1. Introduction
The huge development of conventional plastics made from
petroleum-based synthetic polymers unable to degrade in landll
or compost-like environment had led to serious environmental
issues. In response to this increasing awareness pushed by
governments and societies, the use of polymers stemming from
renewable and sustainable resources to develop bioplastics
constitutes an innovative and promising alternative. Among biosourced polymers, proteins such as wheat gluten are natural heteropolymers constituted by different amino acids which offer
a large spectrum of chemical functionalities and thus, various
polymer network structures [1]. Wheat gluten is a by-product of
the wheat starch industry available at a reasonable price (around

* Corresponding author. Tel.: 33 467 144 235; fax: 33 467 144 990.
E-mail addresses: anne.chevillard@univ-montp2.fr, anne.chevillard@gmail.com
(A. Chevillard), helene.coussy@univ-montp2.fr (H. Angellier-Coussy), cuq@
supagro.inra.fr (B. Cuq), guillard@univ-montp2.fr (V. Guillard), cesar.guy@
neuf.fr (G. Csar), gontard@univ-montp2.fr (N. Gontard), egastald@univmontp2.fr (E. Gastaldi).
0141-3910/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymdegradstab.2011.09.024

1.3 V/kg) and displaying functional properties interesting for

packaging or agricultural applications [2]. Wheat gluten is mainly
constituted of two main storage proteins that are gliadins (monomeric proteins with molecular weight ranging from 15 to 85 kDa)
and glutenins (macropolymer with molecular weight ranging from
150 to more than 103 kDa). Gluten proteins can undergo disulphide
interchange upon heating, which leads to the formation of a threedimensional macromolecular network [3]. Owing to good thermoplastic properties, wheat gluten can be processed by extrusion
at temperature as low as 60
C in the presence of hydrophilic
plasticizers [4].
Domenek et al. [5] have demonstrated the high biodegradability
and non ecotoxicity of wheat gluten-based materials. Even if
covalent cross-linking induced by thermal treatments allowed to
signicantly improve water resistance and mechanical properties
of wheat gluten-based materials [6,7], it was shown that the
biodegradability was not affected when evaluated in a liquid
medium (modied Sturm test) [5]. Nevertheless, under composting
conditions, Zhang et al. [8] have recently reported that the biodegradability of wheat gluten-based materials can be affected by
chemical modication.

A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

Besides the application of thermal and chemical treatments, the

creation of a nanocomposite structure through the introduction of
layered silicates constitutes another promising route to modulate
properties of wheat gluten materials [7,9e12]. Wheat gluten-based
nanocomposites are commonly prepared using either a solvent
(casting) or a thermomolding process but not yet by extrusion. It
has been shown that the introduction of unmodied montmorillonite led to a signicant decrease in water sensitivity, water vapor
permeability [7,9,10] and liquid water diffusivity [11], together with
an increase in rigidity and resistance [7,9] of wheat gluten-based
materials.
Although many studies have focused on the effect of layered
silicates on some of functional properties (especially mechanical,
thermal and transfer properties) of wheat gluten-based materials,
new insights are still required to understand how the biodegradability can be modulated through the introduction of nanoclays.
Some literature is already available concerning the biodegradability
of some bio-sourced polymers and their related nanocomposites
(gelatin [13], casein [14], methyl cellulose [15], polylactic acid
[16e18] or polyhydroxybutyrate valerate [19]). However, the effects
resulting from nanoclays addition on the rate, the level and the
underlying mechanisms of biodegradability are not yet clearly
elucidated and often contradictory. This state of the art can be
explained by (i) a wide range of raw materials (clays and polymer
matrices) differing in chemical and physical features, (ii) a large
number of structures achieved at the nanometric scale, (iii) a large
variety of methods used to evaluate biodegradability, and (iv) the
fact that biodegradation data are not always normalized by the total
carbon content, making difcult the comparison between studies.
Moreover, most of the assumptions proposed to account for the
biodegradability behavior are often not sufciently supported by
other complementary experimental approaches.
The objective of this work was to investigate the inuence of
nanoclays on the biodegradability of wheat gluten-based materials
by focusing on a better understanding of multi-scale relationships
between biodegradability, water transfer properties and structure
of resulting materials. For this purpose, wheat gluten/clay (nano)
composites were prepared via bi-vis extrusion, a process largely
used at the industrial scale, by using two types of montmorillonites
(MMT): an unmodied sodium MMT and organically modied
MMT. Biodegradation patterns were evaluated through respirometric experiments and water sensitivity was assessed by soaking
experiments. The multi-scale structural characterization involved
complementary technical approaches enabling to evaluate the
cross-linking degree of the matrix, the glass transition temperature
of materials, the nano-scale structure i.e. the level of dispersion of
nanoclays within the matrix, and to nish with, the macroscale
structure.

2089

in C30B) on nanoclays are given in Chevillard et al. [20]. Chemicals,

unless specied separately, were purchased from Sigma Aldrich in
per analytical quality.
2.2. Preparation of wheat gluten-based materials
Extrusion was performed using a co-rotating twin screw
extruder (Coperion, ZSK25, Stuttgart, Germany) connected to
a computer interface and controller unit (Brabender, O.H.G, Duisburg, Germany). The barrel consisted of twelve zones, each zone
being equipped with an independent temperature control and a die
head constituted of two 5-mm diameter holes. The total length of
the screw was 42D. The rst and second heating zones were
constantly set at 40
C and the other heating zones at 60
C. The
screw speed was set at 150 rpm. Wheat gluten and nanoclays
powders were fed using two distinct weight feeders (Brabender,
Duisburg, Germany) leading to a cumulate powder feed rate of
4.5 kg h1. The ratio nanoclays/wheat gluten was adjusted to have
a nal inorganic ller content corresponding to 5 wt%. Water was
fed with a weight pump (Movacolor, WL Sneek, Netherlands) at
a ow rate of 2 kg h1. Immediately after extrusion, extrudates were
cooled and air-dried in ambient conditions during approximately
30 min before being cut using a Scheer SGS 50E pelletizer (Scheer
Reduction Engineering GmbH, Stuttgart, Germany). Granulates
were allowed to dry in ambient room conditions until constant
weight. Water content of nal granulates was 9 wt% (measured
after drying 24 h at 105
C). They were characterized by a height of
2.3  0.2 mm, a diameter of 5.1  0.4 mm, and a weight of
53  2 mg. Samples were packed in polyethylene hermetic bags and
stored in dark room at 4
C until experiments.
2.3. Biodegradation tests
Respirometric tests were conducted in aerobic conditions to
evaluate the biodegradability of wheat gluten-based materials.
Method was adapted from the US standard ASTM D5988-96, which
is a Standard Test Method for Determining Aerobic Biodegradation
in Soil of Plastic Materials. The released CO2 being proportional to
the percentage of biodegraded substrate, CO2 evolution measures
ultimate degradation (i.e. mineralization) in which a substance is
broken down to its nal products. Beforehand, materials were
ground with a domestic blender to obtain particles around 1e2 mm
and carbon contents were measured with an elementary analyser
(ThermoQuest NA 2500). Biodegradation tests were carried out in
cylindrical hermetic glass vessels (1000 mL capacity) containing
three small open polypropylene asks (60 mL capacity) (Fig. 1). The

2. Materials and methods

2.1. Materials
Commercial vital wheat gluten was kindly supplied by Syral
(Belgium) under the reference AMYGLUTEN 110. Its moisture and
protein content was approximately 10% and 80%, respectively. Two
types of nanoclays were used as received in this study: an
unmodied sodium montmorillonite provided by Laviosa (Italy)
under the reference HPS and an organically modied montmorillonite carrying a methyl, tallow, bis-2-hydroxyethyl quaternary
alkylammonium salt, supplied by Southern Clay under the reference Cloisite30B (C30B). CEC (cation exchange capacity) values are
129 meq 100 g1 for HPS and around 93 meq 100 g1 for C30B.
Further information (interlayer distance, organic content, interlayer
cation organic cation saturation 3D models organic cations present

Fig. 1. Schematic representation of the glass vessel used for respirometric test for
biodegradation evaluation.

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A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

rst ask contained 25 g of dry soil (previously passed through

a 2 mm sieve) mixed with ground material samples whose weight
corresponded to 50 mg of carbon. The water content of soil samples
was adjusted to reach 80% of the soil water retention capacity. Soil
characteristics were as follows: pH 6.8 (H2O), 2.3 wt% of organic
matter, 16.85 wt% of clay, 26.85 wt% of lime and 56.3 wt% of sand.
The second ask contained 30 mL NaOH solution (0.1 M) to trap the
CO2 produced by microorganisms. The third ask contained
distilled water, in order to maintain the relative humidity at 100%
inside the vessel. The glass vessels were hermetically closed and
incubated in the dark at 28  1
C. At selected time, glass vessels
were open to ensure the back titration of the excess of NaOH which
has not reacted with CO2. Before titrating the residual NaOH with
HCl solution (0.1 M) in the presence of thymophthaleine 0.10% in
ethanol 95
, 5 mL of barium chloride solution (20% in water) was
added in each ask to precipitate carbonate ions. The asks containing soil were weighted and appropriate amount of water was
added (if necessary) in order to keep constant the water content
(initially xed at 80% of the soil water retention capacity). During
this procedure, the glass vessels were left open during 2 min in
order to air the vessel. At each time interval, a new polypropylene
ask containing 30 mL NaOH 0.1 M was placed in each glass vessel
before being closed and put in the dark at 28
C until the next
measurement. Biodegradation experiments included a control and
a blank. For the control, cellulose was chosen as reference because
of its well-known degradation characteristics. For the blank, the
test was conducted without addition of a carbon source to measure
both the CO2 produced by the soil carbon substrate and the CO2
present in the air of the glass vessel. All biodegradation tests were
measured in triplicate.
Results were calculated by subtracting the CO2 production of the
blank. The theoretical maximum CO2 potential (CO2 (max) (mg))
produced by total oxidation of the material is calculated from CO2
max C  44.01/12.01, where C is the amount of carbon of the
sample introduced in the soil for the test (mg). The percentage of
degradation Deg is calculated by using equation (1):

Deg

CO2 material  CO2 blank

CO2 max

(1)

2.4. Liquid water sensitivity

2.4.1. Maximum water uptake, swelling and dry matter losses
at equilibrium
Liquid water uptake was determined gravimetrically for all
materials in triplicate at 20
C. Four days before experiment,
materials were stored in a desiccator upon silicagel (relative
humidity close to 0%). After drying, granulate samples (3 granulates
per sample) were weighted using a four-digit balance (m0) and
their dimensions (thickness and diameter) were measured with
a digital caliper to calculate the volume of the samples (V0). Then,
samples were immersed in 50 mL distilled water (stirred solution)
and removed at specic time t in order to be weighted again (mt)
after having carefully removed the excess of water using tissue
paper. The water uptake at time t (WUt) was calculated using
equation (2).

WUt

mt  m0
m0

(2)

When the equilibrium was reached, i.e. when materials absorbed no more water, samples were weighed, granulate dimensions
(thickness and diameter) were measured with a digital caliper to
calculate the volume of the samples at equilibrium (Veq). Then they
were stored again 4 days in a desiccator upon silicagel before being
weighed (meq dried). The maximum water uptake (WUeq) was

calculated at this specic time using equation (2) while the swelling
of the materials (SWeq) as well as the dry mass losses (DMLeq) were
calculated using equations (3) and (4), respectively.

SWeq

Veq  V0
$100
V0

DMLeq

(3)

m0  meq dried
m0

(4)

2.4.2. Liquid water diffusivity

Identication of liquid water diffusivity was done from water
uptake kinetics data (equation (2)). The solution of Ficks second
law for diffusion from a nite cylinder of diameter 2r and height 2l
immersed in a stirred solution of innite volume was obtained by
the superposition of the analytical solution for an innite cylinder
of diameter 2r (equation (5)) and that for an innite slab of thickness 2l (equation (7)).

jr 1 



4
2
a
$exp

D
$
$t
app
n
r 2 $a2n
n1
N
X

(5)

where the an are the positive roots of:

J0 r an 0

(6)

where J0 (x) is the Bessel function of the rst kind of order zero.
Roots of equation (6) are tabulated in tables of Bessel functions [21].

jz 1 

N
8 X

p2 n 0 2n 12

$exp 

2n 12 $p2
2l2

!
Dapp $t

(7)

In equations (5) and (7), jr and jz , are the quantities of water

which have entered in the theoretical innite cylinder and innite
plane sheet at time t to the corresponding quantity after innite
time and D is the effective liquid water diffusivity (supposed
constant) in a stirred solution (considering no external resistance to
transport). The hypothesis of a negligible external mass transfer
coefcient, which is the case for a well-stirred solution or a Biot
number greater than 100 [22] was rst tentatively validated. To do
this, the external mass transfer coefcient (k) was evaluated using
the methodology extensively detailed in Mascheroni et al. [23]. and
the Biot number was calculated. The external mass transfer
coefcient, k, was found varying between 1.33  105 and
1.68  105 m s1 for all the liquid water sorption kinetics and the
corresponding Biot numbers ranged from 513 to 536. It is obvious
from these results that the external mass transfer resistance at the
interface material/water could be neglected (Bi > 100) conrming
the effectiveness of the stirring. In this approach, the swelling of the
materials was neglected. Therefore, the diffusivities identied were
considered as apparent diffusivity values called (Dapp). As demonstrated in Carslaw and Jaeger [24], solution of Ficks second law for
nite cylinder can be written down as products of the solutions
obtained for simple geometries, i.e. innite cylinder of 0 < r < a
and innite slab of l < x < l. This product solution is given by
equation (8). This equation has been successfully used by different
authors for modeling mass transfer in nite cylinder geometry
[25,26]. The quantity of water entering in the nite cylinder at time
t jt , is then calculated as follows:

jt

WUt
jr $jz
WUN

(8)

2.4.3. Identication procedure

Apparent diffusivity parameters were identied from the
experimental curve by minimizing the root mean square deviations

A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

between simulated and experimental results using the Levq

^  y2 =N  P
enbergeMarquardt procedure [27] RMSE y
^
where y and y represent experimental and predicted values
respectively, N is the number of experimental measurements and p
is the number of estimated model parameters. If RMSE tends
toward 0, that means that the calculated concentrations are very
close to the experimental ones and the model is thus able to
represent experimental data. All analytical solutions were programmed using MATLAB software (The Mathworks Inc., Natick,
MA, USA) and the LevenbergeMarquardt procedure was used via
a dedicated routine lsqnonlin developed in MATLAB.
2.5. Gluten network cross-linking degree
The degree of network cross-linking of wheat gluten-based
materials was evaluated through the determination of the sodium
dodecyl sulfate (SDS)-insoluble protein fraction (Fi) according to
Domenek et al. [28]. Ground samples (160 mg) were stirred for
80 min at 60
C into 20 mL 0.1 M sodium phosphate buffer (pH 6.9)
containing 1% SDS and subsequently centrifuged at 18,000 rpm for
30 min. The supernatant contained the SDS-soluble protein fraction
(Fs). The SDS-insoluble protein fraction (Fi) was extracted with 5 mL
of SDS-phosphate buffer containing 20 mmol L1 dithioerythriol
(DTE) under stirring for 60 min at 60
C, then tip sonicated for 3 min
and nally centrifugated 30 min at 18,000 rpm. 500 mL of the
resulting supernatant was mixed with 500 mL of SDS-phosphate
buffer containing 40 mmol L1 iodoacetamide (IAM), (a
sulfhydryl-reactive alkylating agent) used to block reduced cysteine
residues. Both Fs and Fi extracts were submitted to size-exclusion
HPLC [28]. Fi was expressed in percent as the ratio of the SDSinsoluble protein fraction on the total protein fraction (Fi Fs).
2.6. Differential scanning calorimetry (DSC)
Differential scanning calorimetry was used to measure the glass
transition temperature (Tg) of wheat gluten-based materials.
Ground samples (around 12 mg) were placed in open aluminum
pans (Tzero pan, TA Instruments New Castle, USA) and stored at
aw 0.753 over a saturated salt solution of NaCl. After equilibration, pans were immediately and hermetically sealed. Measurements were done with a thermo-modulated calorimeter (Q200
modulated DSC, TA Instruments, New Castle, USA). Each sample
was heated from 40
C to 130
C at a heating rate of 3
C min1.
The period and the amplitude of modulation were respectively
100 s and 0.796
C. The glasserubber transition was characterized
by three different temperatures in the DSC traces (heat ow
curves), i.e. Tg onset, corresponding to the onset of the specic
heat increment ascribed to glass transition, in other words, the
temperature at which some polymer chains start to undergo the
transition; Tgi the temperature at the inexion point, corresponding
to the temperature at which the differential heat ow is maximum;
and Tg offset corresponding to the offset of the glasserubber transition. Tg values were measured in triplicate.
2.7. Wide angle X-ray scattering analysis
Ground material samples and pristine nanoclays were characterized by wide angle X-ray scattering (WAXS) at room temperature
and relative humidity. Experiments were performed using
a PHILIPS XPert MPD diffractometer (diffractometer qe2q) with
a Xcelerator detector and a nickel lter operating at 40 kV and
20 mA with a Cu-Ka radiation (l 1.5418 ). The spectra were
recorded between 2
and 25
by using a scan speed of
0.035368
s1 and a step size of 0.0334226
.

2091

2.8. Microscopic observations

2.8.1. Scanning electron microscopy (SEM)
The morphology of the wheat gluten-based extrudate sections
was observed after platinum coating (5 nm thickness) using a eld
emission scanning electron microscope (FESEM S-4500, Hitachi,
Japan) at magnication 30 and an acceleration voltage of 2000V.
SEM observations were coupled with image analysis (Image J software) to measure the diameter of the holes observed on extrudate
sections. For each formulation, three samples were analyzed, corresponding to a total number of holes ranging between 260 and 700
depending on the material. The average number of holes (nb holes),
the median diameter (d50) and the cumulated hole area (Sholes) were
calculated for each granulate. The cumulated hole area was expressed
as a percentage of the total observed area.
2.8.2. Transmission electron microscopy (TEM)
Materials (about 1 mm3) were initially xed in glutaraldehyde
2.5% (v/v), dehydrated in an ethanol gradient, then impregnated in
propylene oxide and nally embedded in epoxy resin epon-812
substitute, (Electron Microscopy Science, England). After 3 days of
incubation at 60
C, ultra-thin sections of 70 nm were cut with an
ultramicrotome diamond and mounted on 100 mesh grids covered
by a colodion lm. Samples were examined with a Jeol JEM-1200EX
II TEM (Jeol ltd., Tokyo, Japan) using magnications from 10 to
100 K.
3. Results and discussion
Layered silicates (montmorillonites) were introduced within the
wheat gluten matrix with the aim to modulate the biodegradability
of materials. The approach consisted in selecting two different
montmorillonites: (i) a hydrophilic MMT (HPS) and (ii) an organically modied MMT containing a quaternary alkylammonium with
two hydroxy-ethanol groups as interlayer cation (C30B). Indeed,
since unmodied MMT is hydrophilic and negatively charged, it
was supposed to be naturally compatible with the hydrophilic and
positively charged wheat gluten matrix and thus, appeared suitable
for nanocomposite preparation. However, the very low charged
groups frequency of gluten proteins combined with a rather high
frequency in non-polar side chain [29] dropped hints that the
compatibility between protein and clay might be improved by
using an organically modied MMT (OMMT). The interest of using
an organically modied MMT such as C30B is the presence on its
surfactant of apolar (tallow chain and methyl group) and polar
(hydroxyl groups) groups, which confer both hydrophobicity and
hydrophilicity. Moreover, since the degree of penetration of the
polymer into the clay gallery was expected to be enhanced by
a greater interlayer distance, C30B (which displays a d001 of 18.3
instead of 12.7 for HPS) could favor the formation of an intercalated/exfoliated structure.
3.1. Biodegradation pattern of wheat gluten-based materials under
indoor soil conditions
Respirometric tests were used to evaluate the biodegradability
of the wheat gluten-based materials. CO2 evolution provides an
indicator of the ultimate biodegradability ascribed to mineralization of the test samples. The experimental degradation data were
modeled with the Hill equation [5,30]:

tn
Deg Degmax $ n
k t n

(9)

where Deg [%] is the percentage of degradation at time t [days],

Degmax [%] the percentage of degradation at innite time, k [days]

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A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

into the matrix and enzymes to cleave the protein chains [32]. Once
the size of polymer fragments is small enough, they are transported
into the cell where they are ultimately mineralized. The products of
the mineralization process are gasses (CO2, CH4, N2, H2), water,
minerals, and biomass [31].
As compared to pure cellulose (i.e. the control), wheat glutenbased materials biodegraded very rapidly since the maximum
degradation rate was observed after only 4 days (Fig. 2b) as indicated by Timerate max values, and it took less than 6 days to reach
50% of Dmax as indicated by k values (Table 1). The high biodegradability of proteins, and especially wheat gluten, has already
been reported [5,30]. It worth noting that experiments have been
conducted using ground materials (1e2 mm) in accordance with
normative tests. Indeed, even if our objective was only to investigate the inuence of the material formulation, we could remind
that sample size and shape, and notably its specic surface area, are
very important factors in determining biodegradation pattern.
The biodegradability of the materials was not signicantly
inuenced by the presence of organically modied MMT (C30B)
(Fig. 2, Table 1). As already reported in literature, the effect of
organically modied montmorillonites on biodegradation is often
attributed to a catalytic effect involving the hydroxyl groups located
at the edges of the clay layers [18,33e36]. It is worth noting that
investigated polymers are generally water resistant polymers
notably poly(lactic acid) (PLA) [16e18,33e35,37,38] making difcult the comparison with protein based materials. In the case of
these hydrophobic polymers, the use of OMMT usually favors the
layers dispersion/exfoliation within the matrix and results in
a higher exchange surface for catalytic effect. It has been notably
reported that C30B enabled enhancement of PLA biodegradability
under compost conditions [16,37]. In the present study, the lack of
evidence of such a potential catalytic effect could be due to a low
level of dispersion/exfoliation of C30B within the wheat gluten
matrix due to a probable low compatibility between these two
components.
On the contrary, the unmodied MMT (HPS) signicantly
decreased the biodegradation kinetic of wheat gluten-based
materials without changing the nal biodegradation (Fig. 2,
Table 1). Both the time required to reach the maximum degradation
rate (Timerate max) as well as the time to attain 50% of Dmax (k) were
doubled. In addition, the maximum degradation rate was twice
lower (4.5% instead of 9.4% degradation day1). These results
highlighted that the biodegradation phenomenon of wheat glutenbased materials was clearly slowed down in the presence of
unmodied MMT, as already observed for methyl cellulose lms
[15] or gelatin materials [13]. Three hypotheses can be proposed to
explain the decrease of biodegradability in the presence of MMT:

Fig. 2. Kinetic of biodegradation (a) and biodegradation rate (b) of neat wheat
gluten-based materials (WG B), and wheat gluten-based materials lled with HPS
(WG-HPS 6) and C30B (WG-C30B >) in the respirometric test. Symbols are experimental data points. Solid and dot lines correspond to the degradation curves calculated
with the Hill equation of wheat gluten-based materials and cellulose, respectively.
Error bars represent standard deviation.

the time for which Deg Degmax and n the curve radius of the
sigmoid function. The degradation kinetic and the biodegradation
rate of wheat gluten-based materials are presented in Fig. 2 and Hill
parameters are reported in Table 1.
The biodegradation curves of all wheat gluten-based materials
displayed a sigmoidal shape, which is characteristic of biodegradation measurements (Fig. 2). Indeed, two key steps occur in the
microbial polymer degradation process: rst, depolymerization or
chain cleavage, and second, mineralization. The rst step normally
occurs outside the organism thanks to extracellular enzymes [31].
As a result, a lag phase can sometimes be observed on sigmoid
curves, corresponding to the time needed for water to penetrate

(i) a reduced water adsorption capacity of the materials in the

presence of such llers [13,15],

Table 1
Hill parameters (Degmax, k, n) and related biodegradation indicators (Timerate max, Degrate max) of wheat gluten-based materials (WG), and wheat gluten-based materials lled
with HPS (WG-HPS) and C30B (WG-C30B) in respirometric test.
Sample

Control
(cellulose)
WG
WG-HPS
WG-C30B

Hill parametersa
2

Timerate
[days]

max

Degrate maxc
[% day1]

Degmax [%]

k [days]

100

20.6 (0.6)

1.5 (0.1)

0.97

3.0

87 (1.6)
87 (1.9)
92 (3.0)

5.8 (0.2)
12.6 (0.5)
6.3 (0.4)

1.9 (0.2)
2.1 (0.1)
1.7 (0.2)

0.98
0.99
0.95

4
8
3

9.4
4.5
8.9

Values in parentheses represent the condent intervals.

a
Degmax: percentage of degradation at innite time; k: constant of the Hill equation representing the time for which Deg Degmax; n: constant of the Hill equation
representing the curve radius of the sigmoid function.
b
Timerate max: time to reach the maximum biodegradation rate.
c
Degrate max: maximum degradation rate.

A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

(ii) the presence of a tortuous path induced by the nanodispersion

of layered silicates resulting in a slower diffusion of penetrants
(water, enzymes, microorganisms) [15,19,39,40],
(iii) the establishment of specic interactions between layered
silicates and the matrix leading to a lower availability for the
matrix to be biodegraded [15,19,39].
3.2. Water sensitivity of wheat gluten-based materials
Because changes in polymer biodegradability are often explained
by modication of water sensitivity, soaking experiments have been
carried out to verify the rst assumption. This approach was conducted to allow evaluating both equilibrium parameters (notably
water adsorption capacity) and kinetic parameters (diffusivity) with
the objective to evaluate the contribution of each phenomenon on the
overall biodegradation pattern.
Fig. 3 gives an example of liquid water sorption kinetic in wheat
gluten material at 20
C. The mathematical model t very well the
data from 0 to 0.5 days then slightly overestimated the water
uptake between 0.5 and 1.5 days. This discrepancy could be related
to the swelling of the material (not taken into account in the
modeling) that modies the apparent water diffusion rate. Nevertheless, in spite of its simplications, the mathematical model used
here is satisfying to predict the water uptake at equilibrium.
Equilibrium parameters and apparent diffusivity values
(Table 2) of wheat gluten-based materials were in agreement with
values previously reported in literature [6] which reected a high
sensitivity to liquid water. This has been already highlighted in
previous works [6,7,9,41] and is generally explained by the hydrophilic nature of the wheat gluten proteins (high content of polar
amino acid) [29].
The presence of organically modied MMT (C30B) did not
impact the water sensitivity, since equilibrium parameters were
not signicantly different from values obtained for the neat matrix
(Table 2). On the contrary, the presence of unmodied MMT (HPS)
signicantly increased water resistance leading to a reduction of
the liquid water uptake of 25% while concomitantly, the dry mass
losses and swelling values were reduced of respectively 28% and
53% (Table 2). This improvement occurred in spite of the high water
retention capacity of unmodied MMT, known to absorb water up
to 30 times their weight [42]. These results were consistent with
Tunc et al. [9] who also reported a decrease in water uptake of
wheat gluten nanocomposite lms while increasing the ller
content. Such a reduction in water sensitivity in presence of
unmodied MMT could be explained either by the establishment of

Fig. 3. Kinetic of water uptake of wheat gluten-based materials. Symbols are experimental data points. Solid lines correspond to the model.

2093

Table 2
Liquid water sensitivity of wheat gluten-based materials: equilibrium parameters
(water uptake (WUeq), dry matter losses (DMLeq), swelling (SWeq) and kinetic
parameter (water diffusivity (D)).
Sample

WUeq
[g g1]

DMLeq
[g g1]

SWeq
[%]

D
[1010 m2 s1]

RMSE
[g g1]

WG
WG-HPS
WG-C30B

1.36 (0.01)
1.03 (0.01)
1.34 (0.02)

0.21 (0.02)
0.15 (0.03)
0.25 (0.06)

142 (15)
67 (8)
134 (21)

0.61 (0.09)
0.76 (0.09)
0.61 (0.09)

0.07
0.07
0.07

RMSE: roots mean square error. Values in parentheses represent the condent
intervals.

hydrogen bonds between HPS and protein hydrophilic sites that

would become less available for interactions with water molecules,
or by a potential exfoliated structure giving rise to a tortuous
pathway able to restrict water penetration.
Nevertheless, diffusivity values remained unchanged whatever
the presence and the type of nanoclays (HPS or C30B). In spite of
a better water resistance in the presence of HPS, the layered silicates did not slow down the kinetic of water penetration through
the material. Thus, the second hypothesis mentioned above should
be invalidated: even if a nanocomposite structure leading to the
creation of a tortuous pathway was supposed to be obtained in the
presence of HPS, it would not be effective enough to decrease the
rate of water penetration.
These results evidenced a direct relationship between biodegradability and water sensitivity parameters at equilibrium, but not
with kinetic parameters. Indeed, it was demonstrated that the
introduction of nanoclays able to reduce material water adsorption
were also assumed to decrease biodegradation rate. Indeed, given
that water is required for enzymatic hydrolysis reaction occurring
in the rst stage of the biodegradation process and also for the
transport of solutes and microorganisms, a signicant decrease in
water content would be expected to slow down the biodegradation
process. At this stage, further investigation was required to deepen
our understanding of multi-scale relationships between biodegradability, water transfer properties and structure of resulting
materials.
3.3. Multi-scale structure characterization of wheat gluten-based
materials: from the macromolecular to the macroscopic level
This multi-scale approach aimed at exploring the structure of
the materials from the macromolecular to the macroscopic level
through the characterization of the network structure, the potential
establishment of specic interactions between nanoclays and
wheat gluten, the level of dispersion of nanoclays within the matrix
and to nish, the presence of macroscopic defects in the materials.
Such an approach would enable to settle on the three hypotheses
proposed to explain changes in biodegradation pattern induced by
the addition of nanoclays.
The macromolecular structure of wheat gluten-based materials
has been investigated through the evaluation of the degree of
covalent cross-linking between protein chains, as revealed by the
fraction of SDS-insoluble proteins (Fi) [28,43]. As shown in Fig. 4,
the extrusion process led to an increased Fi value of the wheat
gluten-based material as compared to the raw powder (from 9%
to 29%). During extrusion, a considerable amount of thermomechanical energy is conferred to the material, resulting in
a reversible change in protein conformation followed by aggregation reactions involving disulphide interchanges and leading to
new intermolecular disulphide cross-links [28]. Among the two
main protein fractions constituting wheat gluten, glutenins start to
cross-link above 60e70
C, whereas for gliadins, the reactive zone

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A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

Fig. 4. Fi values (%) of wheat gluten raw powder, wheat gluten-based materials (WG)
and wheat gluten-based materials lled with HPS (WG-HPS) and C30B (WG-C30B).

is clearly evidenced around 90
C. In the present study, the processing conditions (combining heating and shearing treatments)
would be sufcient to reduce the activation energy of gluten crosslinking and enable new intermolecular bonds [44]. Indeed, even if
the barrel temperature was set at 60
C, the temperature at the core
of the product was supposed to be shifted toward higher temperatures, as already observed upon wheat gluten extrusion [4]. In
close conditions of setting temperature and screw speed (60
C and
100 rpm), these authors reported a huge increase of the temperature (reaching 101
C) in the converging section of the die. It could
be noted that the Fi increase (from 9 to 29%) appeared consistent
with the high reactivity of gluten proteins upon heating and
shearing [45,46] even if it was quite moderate as compared to the Fi
values that might be reachable when applying a more drastic
heating treatment. For example, Domenek et al. [5] obtained Fi
values up to 80% for wheat gluten materials thermo-pressed during
35 min at 120
C and 150 bar.
The introduction of unmodied MMT (HPS) led to a slightly
higher Fi value (33%) as compared to the neat wheat gluten matrix
(29%), whereas the introduction of organically modied MMT
(C30B) did not inuence this parameter (Fig. 4). This implied that
the presence of unmodied MMT might favor protein cross-linking
via disulphide bonds, contrary to the use of organically modied
MMT. An increased value of Fi is known to reect biochemical
changes induced by temperature suggesting the occurrence of
heat-activated reactions during the extrusion process. In the presence of unmodied MMT (HPS), it could be supposed that an
increase in thermo-mechanical energy (due to an increased
viscosity) might occur at the core of the product in the extruder
converging section even if the barrel temperature was maintained
at 60
C. An increase in Fi value was also reported by Angellier et al.
[7] when applying a thermo-mechanical process to a wheat gluten
matrix in the presence of unmodied MMT. This was ascribed to
a concomitant temperature increase of the material during the
extrusion process due to intense shearing effects. The increase in Fi
value could also result from an anti-plasticizing effect of unmodied MMT (HPS). The hydrophilic character of HPS might favor the
establishment of hydrogen bonds with the plasticizer (water), this
latter becoming less available for the plasticization of the wheat
gluten matrix. A decrease in plasticizer content has already been
found to increase Fi values [7]. We could thus conclude that the
macromolecular structure of wheat gluten-based materials, which
is directly related to the rheology of the melt upon extrusion
process, was affected by the nature of the reinforcing ller, and
more precisely, by the interactions that might be established
between the different constituents.

DSC measurements have been carried out to show how the

presence of nanoclays can affect the glass transition temperature of
wheat gluten-based materials. This method would enable to
evidence the potential establishment of interactions between
nanoclays and proteins which were expected to affect the polymer
segmental motion at the interface. In the case of wheat glutenbased materials reinforced with unmodied MMT (HPS), a signicant shift of Tg values toward higher temperatures (around 4.5
C
higher) was noted, indicating that the presence of HPS strongly
restricted the protein chain mobility (Fig. 5). This result was
consistent with the increase in Fi values and can also be ascribed to
the establishment of strong interactions between unmodied MMT
and the wheat gluten matrix. The fact that the amplitude of the
glasserubber transition was not signicantly affected by the presence of HPS suggested that only one type of interaction would be
involved. No change in Tg values was observed for materials lled
with organically modied MMT (C30B), indicating that the mobility
of protein chains was not inuenced by the introduction of such
MMT. It can thus be deduced that no specic interaction was
formed between the two components in that case.
Since Fi values and DSC measurements highlighted that the
afnity between nanoclays and the gluten matrix strongly depended on the nature of the clay, it could be assumed that different
levels of clay dispersion would be achieved according to the type of
clay used. Based on results presented above, a low dispersion level
was expected in the presence of organically modied MMT (C30B)
due to its low afnity for the wheat gluten matrix. On the opposite,
a good dispersion/exfoliation of clays was supposed to be obtained
in the case of unmodied MMT (HPS) owing to its good afnity for
wheat gluten. To go further in this investigation, characterization of
the nanostructure by TEM and WAXS appeared essential to
conclude about all the hypotheses proposed above.
The nano-scale structure of wheat gluten-based materials was
evaluated using WAXS analysis (Fig. 6) combined with TEM
observations (Fig. 7). The neat wheat gluten-based matrix displayed a typical amorphous structure characterized by two very
broad peaks centered around 2q 8
and 2q 20
on WAXS
patterns (Fig. 6). In the presence of organically modied MMT
(C30B), a microcomposite structure was achieved, as demonstrated
by both WAXS (Fig. 6) and TEM analyses (Fig. 7). TEM pictures were
characterized by the presence of huge agglomerates of clays
(micrometer sized) and very little dispersed particles. Nevertheless,
the peak around 2q 4.8
characteristic of the pristine C30B
(d001 18.3 ) was slightly shifted toward lower angles (2q 4.60

Fig. 5. Glass transition parameters (Tg onset, Tgi and Tg offset) of wheat gluten-based
materials (WG) and wheat gluten-based materials lled with HPS (WG-HPS) and
C30B (WG-C30B).

A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

Fig. 6. Wide angle X-ray diffractograms of pristine unmodied MMT (HPS) and
organically modied MMT (C30B), wheat gluten-based materials (WG) and wheat
gluten-based materials lled with HPS (WG-HPS) and C30B (WG-C30B).

corresponding to d001 19.2 ), conrming that organically

modied MMT were not unable to well disperse within the wheat
gluten matrix until exfoliation, even if C30B nanoclays were slightly
intercalated by partial penetration of the protein chain in the

Fig. 7. TEM pictures of wheat gluten-based materials lled with HPS (a) and C30B (b).

2095

interlayer (Fig. 6). Although C30B displayed a higher interlayer

distance and a certain hydrophilicity brought by hydroxyl groups, it
seemed that its hydrophobic character was preponderant, leading
to a poor compatibility with the wheat gluten matrix. It should be
noted that our ndings were different from those of Zhang et al.
[10] who concluded that a nanocomposite structure was achieved
for wheat gluten-C30B material prepared by casting. Nevertheless,
the presence of the d001 peak of the C30B on their XRD pattern of
the corresponding nanocomposite suggested that the nanoclays
would be not really exfoliated.
Based on WAXS diffractograms (Fig. 6), the introduction of
unmodied MMT (HPS) resulted in the disappearance of the
diffraction peak around 2q 7
, corresponding to the basal interlayer spacing value of HPS (d001 12.7 ). The presence of the group
of peaks (at around 20
) ascribed to the crystallographic planes of
the MMT on the diffraction pattern of wheat gluten material containing HPS, demonstrated that the WAXS analysis was sufciently
sensitive to detect the presence of MMT (5 wt%) in the nanocomposite. These results were supported by TEM observations
(Fig. 7) since almost all nanoclays appeared well dispersed. This
demonstrated that such materials displayed a well intercalatedexfoliated nanocomposite structure in favor of the creation of
a tortuous pathway for penetrants.
However, even if it has been shown that the water adsorption
capacity was reduced in the presence of HPS, the water diffusivity
was not affected in spite of a well-exfoliated nanocomposite
structure achieved in the presence of HPS. Thus, the tortuosity
effect, which is often mentioned in literature but rarely supported
by structural characterization combined with water diffusivity
measurements, seemed ineffective to reduce water diffusion in the
present study.
In addition, it is worth noting that the characteristic times for
water sorption and biodegradation kinetics were not in the
same order of magnitude in our experimental conditions. The
time required to reach 50% of maximum water uptake (WUeq)
was around 3 h, whereas the time to reach 50% of maximum
degradation (Degmax) was up to 5 days. Nevertheless, in soil
conditions, the temperature is usually lower (<10
C) and the
water is less available; thus the water uptake kinetic is expected
to be slower. Moreover, once the sine qua non water activity
condition would be reached for microbial growth, it would still
require at least 24e48 h of incubation to reach the growing
stage of soil microora. Thus, in soil conditions, the kinetic of
water uptake could be considered as a limiting factor and
consequently, the tortuosity effect might be a key parameter for
limiting biodegradation.
Finally, the analysis of the micro/macroscopic structure of the
wheat gluten materials revealed that granulates were porous as
reected by the presence of holes on SEM pictures (Fig. 8). The
porosity was evaluated by image analysis of SEM scans (Fig. 8). For
unlled wheat gluten-based materials, around 90 holes per granulate were counted, with a median hole diameter around 36 mm. As
shown in Fig. 8, the granulate porosity was affected by the addition
of nanoclays. The presence of organically modied MMT (C30B) led
to a two-fold increase of the number of holes and a decrease of their
size, also by a factor of 2. In the case of unmodied MMT (HPS),
a more contrasted macrostructure was observed: holes were 2.7
fold more numerous and 1.5 fold bigger with a larger diameter
distribution. As a result, these latter materials displayed a relatively
important total hole area (4.6% of the total area as compared to 1.1%
for the neat matrix). Whatever the formulation, these holes could
be ascribed to combined phenomena including air incorporation
during the extrusion process and/or a rapid water evaporation
in the die, resulting in material expansion. In the presence of
hydrophilic MMT (HPS), this latter phenomenon was probably

2096

A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

Fig. 8. SEM pictures of wheat gluten-based materials (WG) and wheat gluten-based materials lled with HPS (WG-HPS) and C30B (WG-C30B) and corresponding hole area
distribution.

emphasized by the supposed temperature increase at the core of

the product.
4. Conclusion
The rate of biodegradation of wheat gluten-based materials has
been reduced by adding unmodied MMT (HPS) without affecting
the nal biodegradation level whereas the presence of an organically modied MMT (C30B) did not signicantly inuence the
biodegradation pattern. Three hypotheses have been proposed to
explain how the presence of MMT could slow down biodegradation
patterns of wheat gluten-based materials: (i) a reduced water
adsorption capacity of the materials in the presence of such llers,
(ii) the establishment of interactions between MMT and the matrix,
resulting in a lower availability for the matrix to be biodegraded,
and/or (iii) the presence of a tortuous path induced by the nanodispersion of layered silicates leading to a slower diffusion of
penetrants.
In the case of organically modied MMT (C30B), no change
in biodegradation pattern was observed based on the three

hypotheses proposed above (no change in water sensitivity, poor

compatibility between C30B and wheat gluten and no tortuous
pathway due to a bad dispersion of nanoclays). On the contrary,
the presence on unmodied MMT (HPS) led to a signicant
reduction in biodegradation rate which is fully consistent with
the two rst hypotheses (decrease of liquid water adsorption and
good afnity between HPS and wheat gluten). Concerning the
third hypothesis, a good dispersion/exfoliation of nanoclays was
achieved, but the resulting tortuosity effect appeared not effective to signicantly reduce the liquid water diffusion even if it
might be sufcient to limit diffusion of other penetrants like
enzymes and microorganisms.
To conclude, biodegradation pattern of wheat gluten-based
materials can be modulated by incorporating nanoclays at
ller content as low as 5 wt%. Among the three hypotheses
proposed to explain the underlying mechanisms, all of
them were validated by the results obtained. The molecular/
macromolecular compatibility between the clay layers and the
wheat gluten matrix, i.e. the ability of both components to
establish interactions, appeared as the key parameter governing

A. Chevillard et al. / Polymer Degradation and Stability 96 (2011) 2088e2097

the nanostructure, the liquid water sensitivity and, as a result,

the biodegradation process.
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