1856 International Journal of Food Science and Technology 2011, 46, 18561862

Original article
Preparation of biopolymer film from chitosan modified with lipid
fraction

Vanderlei C. Souza, Micheli L. Monte & Luiz A. A. Pinto*

Unit Operation Laboratory, School of Chemistry and Food, Federal University of Rio Grande (FURG), PO Box 474, Zip 96201-900, Rio Grande,
RS, Brazil
(Received 25 December 2010; Accepted in revised form 15 May 2011)

Summary Films formed from polysaccharides, as chitosan, present a high permeability in water vapour. In order to
increase resistance to water vapour for chitosan-based lms, dierent lipid fractions were incorporated into a
lmogenic matrix: sh and vegetable oils, stearic and oleic acids. The chitosan showed a molecular weight of
150 kDa and a deacetylation degree of 86 1%. Results showed that incorporation of dierent lipid
fractions decreased the water vapour permeability (WVP) (1.31.8 g mm m)2 day)1 kPa)1) as compared
with pure chitosan lm (3.8 g mm m)2 day)1 kPa)1). A higher reduction in WVP (65%) was found with the
addition of rened sh oil to the continuous matrix of the lms than with the addition of rened rice oil, oleic
or stearic acid (5060%). However, pure chitosan lms showed better tensile strength (TS = 33 MPa) and
elongation percentage (E = 18%) than lipids fractionchitosan lms (719 MPa and 713%, respectively).
Keywords Biopolymer lms, chitosan, fatty acid, sh oil, lipids, vegetable oil.

In order to improve the water barrier properties, the

Introduction
Owing to concerns about limited natural resources and
environmental impacts caused by the use of synthetic
polymers and rigid packaging, there is a great interest in
developing biodegradable lms that act as packing
material and or component (Arvanitoyannis, 1999;
Ferreira et al., 2009). Two major promising applications
of such lms are the replacement of short-life plastic in
food packaging and use as edible food lms (Adebiyi
et al., 2008). Films formed from polysaccharides have
good mechanical properties; however, the fact that they
are highly permeable to water vapour owing to their
strong hydrophilic character limits their potential appli-
cations, because an eective control of moisture transfer
is desirable for most foods (Arvanitoyannis et al., 1998;
Liu et al., 2006; Fernandez-Saiz et al., 2009).
Chitosan is an amine polysaccharide type of carbo-
hydrate with antimicrobial, nontoxic and biocompati-
bility properties, which when combined with its cationic
character and lm-forming capacity makes it one of the
biopolymers with the greatest potential in obtaining
food packaging, especially as edible lms (Arvanitoyan-
nis et al., 1997; No et al., 2007).
*Correspondent: Fax: +55 53 32338745;
e-mail: dqmpinto@furg.br
main lipid fractions incorporated into the base of
biopolymer lms are fatty acids (Srinivasa et al., 2007;
Fabra et al., 2008; Vargas et al., 2009), vegetable oils
(Bourtoom & Chinnan, 2009), hydrogenated oils (The
et al., 2009) and waxes (Hambleton et al., 2009). For
example, Bourtoom & Chinnan (2009) added lipids to
chitosanstarch composite lms, decreasing the water
vapour permeability (WVP) when palm oil and marga-
rine were used. The eect of adding some vegetable oils,
such as oregano essential, has also been investigated
(Chi et al., 2006). However, little information exists
about the impact of sh oil incorporation on biopoly-
mer lms properties.
Waxes have been the most widely used lipids in
polysaccharide lms. Studies show that they are more
eective than saturated fatty acids in improving the
water vapour barrier property, and this is because of its
high hydrophobicity (Fabra et al., 2008; Hambleton
et al., 2009). Owing to the hydrophobic nature of the
sh oil, it could be combined with polysaccharides;
furthermore, use of sh oil as the dispersed phase could
aggregate value to the lm, mainly due to the presence
of eicosapentaenoic (C20:5 EPA x-3) and docosahexa-
enoic (C22:6 DHA x-3) acids (Crexi et al., 2010).
The aim of this study was to increase resistance to
water vapour of chitosan-based lms, incorporating
dierent lipid fractions into the lmogenic matrix: sh

doi:10.1111/j.1365-2621.2011.02692.x
 2011 The Authors. International Journal of Food Science and Technology  2011 Institute of Food Science and Technology

oil (rened from carp viscera), vegetable oil (rened
from rice), saturated fatty acid (stearic acid) and
unsaturated fatty acid (oleic acid). Moreover, mechan-
ical properties (tensile strength and elongation percent-
age at break point) of the lms were evaluated.
Material and methods
Production of chitosan
Chitin was obtained through the steps of demineralisa-
tion, deproteinisation and deodorisation of shrimp
(Farfantepenaeus brasiliensis) waste. Chitin was dried
in a tray drier until commercial moisture content
(< 10%). Chitosan was obtained from chitin according
to the methodology of Weska et al. (2007) through
alkaline deacetylation (NaOH 0.42 kg L)1, at 130 C
for 90 min). Chitosan was puried and dried in spouted
bed according to the methodology of Halal et al. (2011).
The viscosity average molecular weight of chitosan
was determined by viscosimetric method (model Schott
Gerate, GMBH-D65719; Cannon-Fenske, Mainz, Ger-
many). The intrinsic viscosity was obtained by Huggins
equation and converted into molecular weight by Mark
HouwinkSakurada equation, eqn 1, where k = 1.81
10)3 mL g)1 and a = 0.93.
g KMav
)1
where g is intrinsic viscosity (mL g ) and Mv is viscosity
average molecular weight (Da) (Weska et al., 2007).
Deacetylation degree was veried through Fourier
transform infrared spectroscopy, FT-IR (Prestige 21,
210045, Kyoto, Japan) analysis. Chitosan powder was
prepared with potassium bromide (WF Cient ca, Pelo-
tas, Brazil) (Sakkayawong et al., 2007). Deacetylation
degree was determined according to eqn 2 (Cervera
et al., 2004).
%DD 87:8b3ACO =AOH c
where %DD is deacetylation degree (%); AC=O is
absorbance of C=O group; and A-OH is absorbance of -
OH group.
Obtainment of lipid fractions
Crude sh oil was obtained from carp (Cyprinus carpio)
viscera by shmeal process according to the methodol-
ogy proposed by Crexi et al. (2009). Crude oil was
subjected to chemical renement, comprising degum-
ming, neutralisation, washing, dehumidication, bleach-
ing, winterisation and deodorisation steps (Crexi et al.,
2010). Fatty acids (stearic and oleic acids) were provided
by the WF Cient ca, and commercial rice oil purchased
from a local market.
Physicochemical characterisation of the rened oils
was carried out. Thiobarbituric acid value (TBA) was
 2011 The Authors
International Journal of Food Science and Technology  2011 Institute of Food Science and Technology
Preparation of biopolymer film V. C. Souza et al. 1857
found according to Vyncke (1970), using spectrophoto-
metric method (model Q-108 DRM; Quimis, Sao Paulo,
Brazil), and calculated from a standard curve obtained
by reacting known amounts of 1,1,3,3 tetramethoxy-
propane with TBA. Free fatty acid (FFA) content was
found according to the methods of American Oil
Chemists Society (AOCS, 1980). FFA method (Ca 5a-
40) was used, based on titration of the oil with a sodium
hydroxide solution (phenolphthalein as an indicator),
suitably diluted with an ethyl alcoholethyl ether
mixture.
Through the preparation of methyl esters, it was
possible to determine the fatty acid prole of rened oil
in accordance with Metcalfe & Schimtz (1966). Fatty
acid methyl esters were identied using gas chromatog-
raphy (model Varian-3400 CX, Minneapolis, MN,
USA).
Film preparation
Film-forming dispersions (FFDs) were obtained by
dissolution, at room temperature, of chitosan powder
(1% w v) in an aqueous solution of glacial acetic acid
(1% v w). When required, Tween 80 at 5% (w w) with
respect to the amount of chitosan and lipid fraction (sh
oil, vegetable oil, oleic acid or stearic acid) was added
1 (chitosan lipid ratio of 4:1). FFD were emulsied
(model 1100-01; Dremel Stylus, Rancine, WI, USA) at
10 000 rpm for 4 min. Subsequently, pH (MB10 MB-
10P; Marte, Sao Paulo, Brazil) was adjusted to 5.0 with
NaOH solution (1 m) (Vargas et al., 2009). Before the
incorporation of lipid fraction, this was dissolved
through its heating by a hot plate (752A; Fisaton, Sao
Paulo Brasil). The dispersions were ltered through a
paper lter under vacuum. Thickness was controlled
through the volume of the lmogenic solution on petri
dishes. All the lms were prepared with 100 mL of
lmogenic solution per petri dish. The lms were
2
obtained by evaporating the solvent in an oven (model
Q-314M; Quimis) with air circulation at 25 C for about
48 h. Later, the lms were removed from dishes and
placed in desiccators at 25 C and 55% relative humid-
ity for at least 48 h prior to analysis. The experiments
were carried out in replicate.
Film characterisation
After the storage period, the lm thickness was mea-
sured using a digital calliper (model VTC; Stainless
Hardened, Sao Paulo, Brazil) with 0.0100 0.0005 mm
resolution. The thickness was set as the arithmetic mean
of twelve random measurements over the area of the
lm.
Water vapour permeability of lms was determined
gravimetrically at 25 C, using the ASTM standard
method E96-95 (ASTM, 1995). Samples of each lm in
International Journal of Food Science and Technology 2011
1858 Preparation of biopolymer film V. C. Souza et al.

the form of discs (diameter = 70 mm) were xed with
paran cell permeation of aluminium, containing
anhydrous calcium chloride. These cells were placed in
desiccators at 25 C and 75% relative humidity. By
increasing the mass of anhydrous calcium chloride
(measured in intervals of 24 h for 7 days), it was
possible to determine the water vapour transferred
through the lm according to eqn 3.
wma: L
WVP
t A:DP
where wma is the weight of absorbed moisture (g); t is the
time duration of the test (days); L is the average lm
thickness (mm); A is the area of the exposed lm surface
(m2); and DP is the partial vapour pressure difference
across the lm (Pa).
Tensile strength (TS) and elongation percentage (E) at
break point were measured uniaxially by stretching the
specimen in one direction using a Texture Analyzer
(model TA.XP; Stable Microsystems SMD, Godalming,
UK) according to the ASTM D-882 standard (ASTM,
2001), with a 50 N load cell. Sample lms were cut into
25-mm-wide and 100-mm-long strips. The initial grip
separation and cross-head speed were set to 50 mm and
50 mm min)1, respectively.
The lms were analysed using FT-IR spectrophotom-
etry (Prestige 21), in a spectral range from 4000 to
400 cm)1. Film samples were examined for surface
characteristics using a scanning electron microscope
(SEM) (model JSM-5800 LV, Jeol, Tokyo, Japan)
operated at 10 kV. Five samples were mounted on a
bronze stub and sputter-coated (Sputter coater SPI-
Module, Santa Clara, CA, USA) with a layer of gold
prior to imaging.
groups linked in chitosan structure (Baskar & Kumar,
2009) were observed. Deacetylation degree obtained
from FT-IR analysis was 86 1%.
Refined oil characterisation
Thiobarbituric acid and FFA values of carp rened oil
(TBA = 5.9 0.2 mg malonaldehyde kg)1; FFA =
0.09 0.01%) and rice oil (TBA = 0.14 0.02 mg
3 malonaldehyde kg)1; FFA = 0.22 0.01%) are in
accordance with the standards required for the accept-
ability of rened oils for human consumption (TBA =
78 mg malonaldehyde kg)1 and FFA = 1.83.5%
(Sathivel et al., 2003; Boran et al., 2006). The rened
oil thus obtained oer quality and oxidative stability
and thereby can be used as edible components.
The fatty acid proles and lipid classes of rened oils
are shown in Table 1.
According to Table 1, the major fatty acids present in
rened carp (Cyprinus carpio) oil are palmitic acid
(C16:0), palmitoleic acid (C16:1), oleic acid (C18:1 x-9),
linoleic acid (C18:2 x-6) and linolenic acid (C18:3 x-3),
which constitute approximately 68% of the total fatty
acids. Furthermore, the rened oil showed 69% of
unsaturated and polyunsaturated fatty acids (MU-
FA + PUFA), of which 28% correspond to x-3 and
x-6. These proportions are in agreement with those
found by Crexi et al. (2010). The majority of fatty acids
present in rice oil are palmitic acid (20.2%), stearic acid
(1.2%), oleic acid (41.9%) and linoleic acid (28.6%).
Table 1 Fatty acid (%) proles and lipid classes (% of total fatty acids)
of the carp and rice rened oils

Fatty acids and lipid classes Refined carp oil Refined rice oil
Statistical analysis
C16:0 15.47 0.02 20.16 0.01
One-way analysis of variance and Tukeys multiple C18:0 3.09 0.02 1.18 0.02
comparison tests were used to statistically determine C16:1 x-7 7.59 0.04 0.22 0.02
signicant dierences (P 0.05) among averages, using C18:1 x-9 cis 26.52 0.03 41.39 0.02
the software Statistic 6.0 (Statsoft, Tulsa, OK, USA). C18:1 x-9 trans 0.33 0.01 0.46 0.01
C18:2 x-6 trans 9,12 0.44 0.01 0.60 0.03
C18:2 x-6 cis 9.32 0.04 28.00 0.02
Results and discussion C18:3 x-6 0.39 0.02 0.27 0.01
C18:3 x-3 7.61 0.04 1.24 0.03
Chitosan powder characterisation C20:5 x-3 (EPA) 3.99 0.03
C22:6 x-3 (DHA) 1.30 0.01 0.10 0.01
P
Chitosan showed viscosity average molecular weight of ni 5.24 0.19 1.08 0.24
P
150 5 kDa (eqn 1). The FT-IR spectrum was anal- P
SFA 25.25 0.15 23.98 0.08
ysed (gure not shown): a strong band at 1556 cm)1 P
MUFA 41.81 0.14 43.80 0.11
showed a typical chitosan amino group (-NH2) (Ziani PUFA 27.69 0.20 31.11 0.18
et al., 2008). At 1640 cm)1, an axial deformation of x-3 14.29 0.10 1.48 0.08
x-6 13.48 0.13 28.99 0.09
C=O (amide band I) (Mart nez-Camacho et al., 2010)
was observed. Weak bands at 1020 and 1080 cm)1 were Mean value standard error (in replicate). EPA, eicosapentaenoic acid;
relative to C-N links and at 2933 cm)1 to primary amine DHA, docosahexaenoic acid;
P
ni, sum of unidentified fatty acids;
P
P
stretching. These peaks are related to functional chito- SFA, sum of unsaturated fatty acids; MUFA, sum of monounsaturated
san amino group. In addition, at 3470 cm)1, hydroxyl
P
fatty acids; PUFA, sum of polyunsaturated fatty acids.

International Journal of Food Science and Technology 2011  2011 The Authors
International Journal of Food Science and Technology  2011 Institute of Food Science and Technology

The proportion of linolenic acid (1.2%) in rice oil is
lower when compared with that of sh oil (7.6%)
(Table 1). The metabolic conversion of linolenic acid to
EPA and DHA is not ecient, and for this reason, it is
considered essential and must be supplied in the diet
(Horrocks & Yeo, 1999).
Film characterisation
All the lms showed an average thickness of
0.030 0.001 mm and were characterised in terms of
WVP and mechanical (tensile strength and elongation
percentage at break point) properties (Table 2).
Table 2 shows signicant dierences (P 0.05) be-
tween the permeability and mechanical properties of
lms through Tukeys mean comparison test.
The incorporation of dierent lipid fractions
decreased the WVP [WVP, (eqn 3)] as compared with
pure chitosan lm (Table 2). The results showed that the
addition of rened sh oil or oleic acid resulted in a
higher reduction in WVP than the addition of rened
rice oil or stearic acid. For the lm containing fatty
acids, WVP decreased with an increase in the unsatu-
ration degree of lipids.
The low transfer of water vapour between the polymer
chains of the composite (chitosanrened oil) lms may
be due to low interaction of highly polar molecules such
as hydroxyl fatty alcohols and carboxyl groups of fatty
acids present in the oil (Table 1). In this sense, the
obtained results indicate that the predominance of long-
chain lipid molecules, especially EPA and DHA, may
have been responsible for the higher resistance to water
vapour diusion of the chitosansh oil composite lms,
where EPA + DHA contents in sh and rice oils were
5.3% and 0.1%, respectively (Table 1). Hence, mole-
cules of the polar portion may have had little inuence
on the matrix of long-chain hydrocarbons, and the
Table 2 Tensile strength and elongation percentage at break point and
water WVP
WVP Tensile
Biopolymer (g mm m)2 strength
films* day)1 kPa)1) (MPa)
CH 3.80 0.03a 33.0
CH-SA 1.80 0.01b 19.0
CH-OA 1.41 0.03c 16.0
CH-RRO 1.53 0.02d 14
CH-RFO 1.32 0.05e 7.1
Mean values standard error (in replicate). Different superscripts letters
in the same column indicate significant differences (P 0.05) between
the properties of the biopolymer films.
*CH, pure chitosan; CH-SA, chitosanstearic acid; CH-OA, chitosanoleic
acid; CH-RRO, chitosanrefined rice oil; CH-RFO, chitosanrefined fish oil
films; WVP, water vapour permeability.
 2011 The Authors
International Journal of Food Science and Technology  2011 Institute of Food Science and Technology
Preparation of biopolymer film V. C. Souza et al. 1859
water migrated preferentially through the continuous
matrix (hydrophilic) and the dispersed lipid phase,
increasing the tortuosity in the polymer chains and
decreasing the diusion of water vapour (Shellhammer
& Rhim, 2005).
Oleic acid has a certain degree of mobility because of
its double bond, which might result in reducing WVP
properties (Fabra et al., 2008; Monedero et al., 2009).
Despite the high amount of oleic acid present in rened
rice oil (Table 1), the lms containing pure oleic acid
(CH-OA) had lower values of WVP than those that
contained rice oil (CH-RRO). The results of CH-RRO
lms could be due to the development of particular
interactions of oleic acid with chitosan matrices, which
did not increase the hydrophobicity of the matrix when
the rened rice oil was incorporated.
Elongation percentage (E) at break point and tensile
strength (TS) values of pure chitosan (CH) lm
(Table 2) are in the range found by the literature,
around 40 MPa (Srinivasa et al., 2007) and 20% (Var-
gas et al., 2009) for TS and E, respectively. The addition
of the dierent lipid fractions decreased TS and E
values, and similar results were found by Srinivasa et al.
(2007) and Vargas et al. (2009), who added saturated
fatty acids (palmitic and stearic acids) and unsaturated
fatty acids (oleic acid) to a chitosan matrix, respectively,
and by Bourtoom & Chinnan (2009) through the
incorporation of oleic acid, palm oil or margarine to a
chitosanstarch matrix. The lower tensile strength of
composite lms can be attributed to a reduction in
proximity and interaction between the polymers chains,
caused by the surfactant addition. According to Bour-
toom & Chinnan (2009), the lower elongation is the
consequence of a reduction in interaction between the
lipid molecules (nonpolar) and carbohydrates (polar).
Films obtained with rened rice oil showed higher
elongation compared with those obtained with fatty
acids or rened sh oil. The high amount of oleic acid
in the oils (Table 1), especially the rened rice oil,
caused a plasticising eect of the lms (Monedero et al.,
2009). If plasticisation does occur, the tensile strength
should decrease and at the same time the elongation
Elongation increases. However, it was not the case when the
at break
elongation value of the chitosan-only lm is compared
point (%)
with that of the composites. It may be a case of phase
0.4a 18.0 0.6a separation caused by incompatibility. This can be
0.4b 7.0 0.4b deduced from imaging techniques such as scanning
0.5c 12.0 0.7c electron microscopy, where surface microstructure was
1d 13.0 0.1d observed for lms. According to the classication
0.5e 8.5 0.1e established by Krochta & De Mulder-Johnston (1997)
for biopolymer lms, the mechanical attributes
obtained by the composite chitosan and lipid lms
developed in this study can be considered as poor,
compared with synthetic lms.
FT-IR spectroscopy was used to examine the interac-
tions between chitosan and rened sh oil. The infrared
International Journal of Food Science and Technology 2011
1860 Preparation of biopolymer film V. C. Souza et al.

(a)

(b)

Figure 1 Infrared spectra of lm: (a) pure

chitosan lm (CH); (b) chitosanrened sh
oil composite lm (CH-FRO).

(a) (b)

(c) (d)

(e)

Figure 2 Scanning electron microscope

micrographs of lms. (a) CH, pure chitosan;
(b) CH-OA, chitosanoleic acid; (c) CH-
RFO, chitosanrened sh oil; (d) CH-RRO,
chitosanrened rice oil; (e) CH-SA, chito-
sanstearic acid lm.

International Journal of Food Science and Technology 2011  2011 The Authors
International Journal of Food Science and Technology  2011 Institute of Food Science and Technology

spectra of pure chitosan and chitosanrened sh oil
composite lm are presented in Fig. 1.
The IR spectra of pure chitosan lm showed peaks at
3292 cm)1 of OH stretching, which overlaps the NH
stretching in the same region. The peaks at 2877, 2927
and 2981 cm)1 are related to the symmetric and asym-
metric axial deformation of CH (CH3 and CH2). The
band at 1560 cm)1 was the NH bending (amide II). A
small peak near 1415 cm)1 was because of C=O
stretching (amide I), and a peak at 1707 cm)1 suggested
the presence of a carbonyl group in the lm (Xu et al.,
2005). In the spectrum of chitosanrened sh oil
composite lm, the amino peak of chitosan has not
moved with the addition of oil. Unlike the work of Xu
et al. (2005) that prepared composite lms, this result
indicated that interactions were not present between the
hydroxyl groups of fatty acids present in oil and the
amino groups of chitosan. Peaks at 15351251 cm)1
refer to the predominance of unsaturation present in the
rened oil. The peaks at 2924 and 2852 cm)1 correspond
to CH2 and CH, respectively.
Scanning electron microscope micrographs of CH lm
(Fig. 2a) show increased surface irregularity in the lm
with the incorporation of lipids. However, CH-OA
(Fig. 2b) and rened sh oil (CH-RFO) (Fig. 2c)
appeared to be well incorporated and embedded in the
chitosan matrix, resulting in a relatively smooth and
continuous surface, whereas the lms with CH-RRO
(Fig. 2d) and stearic acid (CH-SA) (Fig. 2e) had a highly
irregular surface, suggesting a phase separation because
of incompatibility. The dierences in surface structure
may have contributed to some extent to the dierences
in properties (mechanical and WVP) of the two lms.
Aggregation was probably more dicult for formulation
with rened rice oil or stearic acid because of the phase
separation or higher surface tension of liquid droplets.
This separation could explain the higher WVP (as
compared with pure chitosan and rened sh oil lms)
of the lms because of preferential paths for water
vapour diusion and the low TS (as compared with pure
chitosan lm) for structure discontinuities (Table 2).
Conclusions
Film-forming dispersions were obtained by dissolution
at room temperature (chitosan lipid ratio of 4:1), and it
was emulsied at 10 000 rpm for 4 min with pH
adjusted to 5.0. Results showed that the incorporation
of dierent lipid fractions (sh oil, vegetable oil,
saturated fatty acid and unsaturated fatty acid)
decreased the WVP (from 50% to 65%) as compared
with pure chitosan lm. Addition of rened sh oil to
the continuous matrix of the lms resulted in a higher
reduction in WVP (WVP) than the addition of rened
rice oil, oleic acid or stearic acid. However, the lms
with dierent lipid fractions showed lower mechanical
 2011 The Authors
International Journal of Food Science and Technology  2011 Institute of Food Science and Technology
Preparation of biopolymer film V. C. Souza et al. 1861
properties, tensile strength and elongation percentage at
break point (719 MPa and 713%, respectively), than
the pure chitosan lm (33 MPa and 18%). These results
suggest that the resulting lms mainly of chitosansh
oil may be classied as packaging material for foods.
Acknowledgments
The authors thank CAPES (Brazilian Agency for
Improvement of Graduate Personnel) and CNPq (Na-
tional Council of Science and Technological Develop-
ment) for the nancial support.
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