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Journal of Food Engineering 116 (2013) 889899

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Journal of Food Engineering


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

Characterization and antimicrobial analysis of chitosan-based lms


I. Leceta a, P. Guerrero a, I. Ibarburu b, M.T. Dueas b, K. de la Caba a,
a
b

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,

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which noticeably affects the mechanical properties of the lm (Oss


et al., 2009; Park et al., 2002; Suyatma et al., 2005). Among many
possible plasticizers, glycerol is commonly used to plasticize biodegradable lms and can essentially improve mechanical properties of
chitosan-based lms (Kerch and Korkhov, 2011; Thakhiew et al.,
2010). Furthermore, mechanical properties and, in general, functional properties of lms depend strongly on temperature (Rivero
et al., 2012). Although many studies dealing with chitosan lms have
been performed, there are no many previous reports on the effect of
high temperatures on the physicochemical and antimicrobial properties of lms based on chitosans of different molecular weights and
plasticized with different contents of polyols. Thus, the aim of this
work is focused on analyzing the effect of temperature on functional
properties and antimicrobial activity of chitosan-based lms for
food packaging applications. Two representative bacteria, Escherichia coli, the gram-negative bacterium, and Lactobacillus plantarum, the gram-positive bacterium, which are common spoilage
bacteria for food contamination, have been tested.

Golden Gate (Specac). A total of 32 scans were performed at


4 cm1 resolution. The measurements were recorded between
4000 and 700 cm1.
2.6. Thermogravimetric analysis (TGA)
Thermal stability of lms was analysed by TGA. Non-isothermal
degradation measurements were performed in a Mettler Toledo
TGA SDTA 851. Tests were running from room temperature up to
800 C at a heating rate of 10 C/min under nitrogen atmosphere
(10 mL/min) to avoid thermo-oxidative reactions.
2.7. X-ray diffraction (XRD)
XRD studies were performed with a diffraction unit (PANalytical Xpert PRO) operating at 40 kV and 40 mA. The radiation was
generated from a Cu Ka (k = 1.54060 ) source. The diffraction data
were collected from 2h values from 2.5 to 50, where h is the angle
of incidence of the X-ray beam on the sample.

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

The change of colour was evaluated by comparing total colour


differences between lms. Total colour difference (DE) was calculated as:

DE

q


Lstandard  Lsample 2 astandard  asample 2 bstandard  bsample 2

Values were expressed as the means of ten measurements on


different areas of each lm, and ve replicates were made per
formulation.

2.3. Film thickness

2.9. Gloss

Film thickness was measured to the nearest 0.001 mm with a


hand-held digimatic micrometer (QuantuMike Mitutoyo). Three
thickness measurements were taken on each specimen at ve different positions, being in the range of 6080 lm in all cases.

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.

2.4. Total soluble matter (TSM)


Total soluble matter was measured by immersion in 50 mL of
distilled water. The asks were stored in the environmental chamber at 25 C for 24 h. After this time, specimens were dried in an
air-circulating oven at 105 C for 24 h. TSM was calculated in relation to the dry mass and it was expressed as the percentage of the
lm dry matter solubilized. Measurements were made in triplicate.

2.10. Light absorption

2.5. Fourier transform infrared (FTIR) spectroscopy

2.11. Contact angle

Fourier transform infrared spectra of chitosan/glycerol lms


were carried out on a Nicolet Nexus FTIR spectrometer using ATR

A contact angle meter (model Oca20, dataphysics instruments)


was used to measure the contact angle of water in air on the

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).

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I. Leceta et al. / Journal of Food Engineering 116 (2013) 889899

surface of chitosan/glycerol lms. A lm sample (20 mm  80 mm)


was put on a movable sample stage and levelled horizontally; then
a drop of about 3 lL of distilled water was placed on the surface of
the lm using a microsyringe. The contact angle was measured in a
conditioned room by recording contact angle values. Image analyses were carried out using SCA20 software. Ten replicates were
made per formulation.

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.

2.12. Water vapour permeability (WVP)

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.

WVP of lms was determined according to ASTM E96-00


(ASTM, 2000), which xes temperature and relative humidity conditions as 38 C and 90%, respectively. Measurements were performed in an water vapour transmission rate tester (PERME
W3/0120, Labthink). The sample lm was cut into a circle of
7.40 cm diameter and the test area was 33 cm2. The setup was subjected to a temperature and relative humidity of water vapour
transmission rate (WVTR) was calculated as:

2.15. Statistical analysis

3. Results and discussion


3.1. Total soluble matter

An electromechanical testing system (MTS Insight 10) was used


to determine mechanical properties. Tensile strength (TS) and elongation at break (EB) were determined according to ASTM D1708-93
(ASTM, 1993). Five replicates were tested for each composition.

It is worth noting that the lms prepared without glycerol and


dried at room temperature were totally soluble, as it is shown in
Table 1. TSM values were lower (P < 0.05) for higher glycerol contents, suggesting that there is certain interaction between chitosan
and glycerol chains. Moreover, TSM values decreased drastically
(P < 0.05) when 30% glycerol was added, indicating a good degree
of interaction between the two components of the lm and the formation of a more compact network. However, when lms were
heat-treated, TSM values decreased signicantly (P < 0.05), as it is
shown in Table 1, indicating a change in the chemical structure
of the lm. This behaviour could mean that temperature promoted
chemical reactions, such as Maillard reaction, as it was also reported by other authors (Guerrero et al., 2012). As could be appreciated in Fig. 1, the early stage of the Maillard reaction involves the
formation of conjugates between the carbonyl group of the carbohydrate ends with the amine group in chitosan. This reaction produces a Schiff base, which subsequently cyclises to produce the
Amadori compound and insoluble polymeric compounds, referred
as melanoidins, are formed (Leceta et al., 2012; Yasir et al., 2007).

2.14. Antimicrobial activity

3.2. FTIR spectroscopy

The antimicrobial activity of chitosan-based lms and chitosan


lm forming solutions was tested against the growth of two typical
food spoilage bacteria: a Gram-negative bacterium, E. coli 0517H,
provided by J.M. Rodriguez (Veterinary Faculty, Madrid), and a
Gram-positive bacterium, L. plantarum CECT748, provided by the
Spanish type culture collection.
Two methods were used to assess the antimicrobial activity. On
one hand, agar diffusion method adapted by Pereda et al. (2011)
was performed. Agar plates were spread with 0.1 mL of inoculum
containing approximately 105106 CFU/mL of bacteria. Films (sterilized with UV light) were cut into a disc shape of 15 mm diameter
and then placed on the surface of MRS (Man Rogosa and Sharpe)
agar for L. plantarum strain and on LB (Lysogeny Broth) agar for
E. coli. After incubation for 24 h at 37 C to E. coli and at 28 C to
L. plantarum strains, the plates were optically examined for width
of inhibition in the contact area. The same procedure was applied
for the lms dried in an air-circulating oven at 105 C for 24 h. The
total area was used to evaluate the antimicrobial potential of lms.
In the case of lm forming solutions, 30 lL of solution were poured
into agar wells and the same procedure described above was applied. Each assay was performed in duplicate.
On the other hand, the antimicrobial effect of chitosan lms and
lm forming solutions in the bacterial growth was also evaluated
by the liquid culture medium assay on 96-well polypropylene
microplates. Cell cultures were incubated at 28 C for L. plantarum
CECT748 and at 37 C for E. coli 0517H for 24 h in both cases. The
liquid culture assay was conducted in 200 lL of both broths with

Taking into account solubility results, FTIR analysis was carried


out in order to study the interactions between functional groups in
chitosan-based lms. As it is shown in Fig. 2, the main absorption
peaks of chitosan are attributed to C@O stretching (amide I) at
1650 cm1, to NAH bending (amide II) at 1558 cm1, and to CAN
stretching (amide III) at 1320 cm1. The absorption peak at
1405 cm1 corresponds to the carboxylate groups associated with
the antimicrobial activity of the biopolymer (Lagaron et al.,
2007). The band that appears at 1378 cm1 is assigned to the acetamide groups, which indicate that chitosan is not totally desacetylated. The absorption peak at 1050 cm1 is assigned to CAO

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.

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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

Wave Number (cm )

4000

3600

3200

2800

2400

2000

1600

1200

800

-1

Wave Number (cm )

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).

stretching and the broad band above 3000 cm1 corresponds to


OAH and NAH bonds (Brugnerotto et al., 2001; Fernandez-Saiz
et al., 2009; Ziani et al., 2008). The typical absorption bands of glycerol are located in the region from 800 cm1 up to 1150 cm1, and
correspond to the vibrations of CAC and CAO linkages. The peaks
at 850 cm1, 925 cm1, and 995 cm1 are assigned to the vibration
of CAC skeleton, the band at 1045 cm1 is associated to the
stretching of CAO linkage in C1 and C3, and the one at
1117 cm1 is related to the stretching of CAO in C2.
Variations in the FTIR spectra produced by the effect of temperature can be observed in Fig. 3. On one hand, the intensity of the
band at 1650 cm1 (amide I) was always lower than the intensity

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

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I. Leceta et al. / Journal of Food Engineering 116 (2013) 889899

(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

Wave Number (cm )

1800

1600

1400

1200

1000

800

-1

Wave Number (cm )

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.

3.3. Thermo-gravimetric analysis (TGA)


3.4. X-ray diffraction (XRD)
Thermogravimetric analysis was also carried out in order to
study the changes promoted by the effect of temperature and glycerol content on the interactions between polar groups. Fig. 4 shows
the weight loss as a function of temperature for chitosan-based

The changes showed above require information about the


molecular conformation of chitosan-based lms, thus X-ray analysis was carried out. The X-ray diffraction patterns of chitosan-

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(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).

based lms are shown in Fig. 5. Chitosan is a partially crystalline


polysaccharide due to its regular chain and has two reection fall
at 2h = 10 and 20 (Luo et al., 2012). The reection fall at 10
was assigned to crystal forms I and the strongest reection at 20
to crystal forms II (Wu et al., 2013). These two phases correspond
to crystalline (less hydrated and harder) zones dispersed in an
amorphous (more hydrated and softer) zone and describe the
development of crystallinity in chitosan matrices due to the formation of hydrogen bonds between chains (Pastor et al., 2013).
As observed in Fig. 5, the chitosan-based lms dried at room
temperature were in a semicrystalline state with two main diffraction peaks at 2h around 10 and 20, whereas the peak of chitosan
at 10 disappeared in the lms heat-treated, indicating that chitosan structure was inuenced by the effect of temperature. Moreover, the intensity of the peak at 20 was enhanced in the lms
prepared with glycerol. This could suggest that there was an interaction between these two components that lead to new crystalline
structures.

RT 0HMw
HT 0HMw

RT 30HMw
HT 30HMw

3.5. Optical properties


Optical properties are essential in packaging applications since
they affect the appearance of the product and make consumers accept or reject the product packaged, so becoming an important
quality factor to be taken into account. Colour values of all the lms
prepared in this study are shown in Table 2. Films were homogeneous and transparent, but it can be observed that chrome values
increased with the addition of glycerol, being this effect more
remarkable for LMw chitosan-based lms. Regarding the effect of
temperature, signicant total colour change (P < 0.05) happened
in all lms. Total colour difference between chitosan lms dried
at room temperature or heat treated was more signicant for
LMw lms and increased (P < 0.05) with the addition of glycerol.

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).

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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

Gloss 60 (Gloss units)

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

I. Leceta et al. / Journal of Food Engineering 116 (2013) 889899

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

as an indication of surface wettability (Guerrero et al., 2011). To


understand the effect of temperature on lm wettability, contact
angles were investigated for the lms prepared with different contents of glycerol and dried at room temperature or heat-treated. As
it is shown in Fig. 7, water contact angles of chitosan-based lms
with 0% and 15% glycerol were around 105, in agreement with
the results shown by other authors (De Britto and Assis, 2007;
Hsieh et al., 2005). There was no signicant difference (P > 0.05)
between contact angle values for the lms dried at room temperature or heat-treated. However, contact angle values decreased with
the addition of 30% plasticizer owing to the hydrophilic character
of glycerol. In all cases, heat-treatment caused a slight decrease
in contact angle values, indicating changes in the conformation
of molecules, as shown by previous results, and the exposure of
hydrophilic groups toward the surface.
On the other hand, water vapour permeability provides information about water vapour transmission through the lm, which is a
crucial property for the lms intended to be used as food packaging
(Ahvenainen, 2003). The WVP values are shown in Table 3. As it can
be observed, the values were similar for all the lms. The addition of
glycerol did not show a remarkable change in water vapour permeability and chitosan-based lms showed adequate barrier properties against water vapour. Regarding molecular weight, many
authors reported that the inuence of molecular weight of chitosan
on WVP was not signicant (Park et al., 2002; Hwang et al., 2003;
Chen and Zhao, 2012). However, LMw chitosan lms dried at room
temperature exhibited slightly higher WVP than HMw chitosan
lms. This fact could be due to shorter length of the chains, which
would facilitate the diffusion of water vapour through the lm.

3.8. Mechanical properties


The mechanical behaviour of chitosan-based lms prepared in
this work is shown in Table 4. As it can be seen, the concentration
of glycerol notably affected mechanical properties; tensile strength
decreased (P < 0.05) and elongation at break increased (P < 0.05)
when glycerol content increased due to the plasticization effect.
When chitosan molecular weight increased, tensile strength increased (P < 0.05) and elongation at break decreased (P < 0.05). On
the other hand, when lms were heat-treated, both tensile strength
and elongation at break increased (P < 0.05) by the effect of temperature for HMw chitosan-based lms, regardless of glycerol content.
However, elongation at break decreased (P < 0.05) for LMw chitosan-based lms plasticized with glycerol. This could be due to the
formation of a more compact network induced by the Maillard
reaction.
Comparison of mechanical properties of chitosan-based lms is
difcult since there are a great dispersion in deacetylation degrees,
molecular weights, concentrations of chitosan and plasticizer as
well as lm preparation and test conditions (Silva-Weiss et al.,
2013). However, both TS and EB values obtained in this work for

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.

I. Leceta et al. / Journal of Food Engineering 116 (2013) 889899

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.

Control 0HMw 15HMw 30HMw 0LMw 15LMw 30LMw


Fig. 9. Average absorbance values and standard deviations at 620 nm for chitosanbased lms dried at room temperature after 0, 24 and 48 h of bacteria
inoculation. aeColumns with the same letter are not signicantly (P > 0.05)
different through the Tukeys multiple range test.

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

I. Leceta et al. / Journal of Food Engineering 116 (2013) 889899

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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