Chitosan is a natural polysaccharide that has received great attention in a
variety of applications because of their biodegradability and biocompatibility
(Ravi Kumar et al., 2004; Muzzarelli
and Muzzarelli, 2005). Because of its excellent film-forming property, chitosan
can be used effectively as a film-forming material to use for food packaging
(Park and Zhao, 2004; Rhim et al.,
2006), wound dressing (Yang et al., 2008)
and drug delivery applications (Senel et al., 2000;
Shu et al., 2001). However, the broader applications
of chitosan films were limited due to its film brittleness. Blending is a simple
and convenient method to improve the film flexibility. Various plasticizers
such as glycerol, sorbitol, erythritol, polyethylene glycol and fatty acids
have been blended with chitosan (Rhim et al., 2006;
Kittur et al., 2001; Jeon
et al., 2002; Cervera et al., 2004;
Srinivasa et al., 2007; Ziani
et al., 2008). Different types and amounts of plasticizers used may
affect the flexibility of the chitosan films. However, the other plasticizers
for improving chitosan film flexibility are still interesting.
Although, the preparation of chitosan films by dissolving chitosan in lactic
acid solution before film casting has been reported (Hoagland
and Parris, 1996) but there are no reports in the systematic results on
the effect of lactic acid with different concentrations and configurations on
characteristics of chitosan films.
The objectives of this research were to prepare lower and higher molecular weights chitosan films with two dissolving agents (L- and DL-lactic acids) and to determine the intermolecular interactions between chitosan film matrices and these acids. Mechanical properties of chitosan films were measured to compare plasticizing effect with acetic acid. The morphology, transparency and water vapor sensitivity of the films were also investigated.
MATERIALS AND METHODS
This research was conducted on November 2008-May 2009 at Mahasarakham University, Mahasarakham, Thailand.
Chitosans (90% deacetylation) with molecular weights of 100 and 740 kDa
were purchased from Sea fresh Chitosan Lab Co., Ltd., (Thailand). L- and DL-lactic
acids (90%, Fluka, Switzerland) and acetic acid (99.7%, Lab Scan, Ireland) were
used without further purification.
Lactic acid can protonate the chitosan molecules by changing -NH2
of the chitosan molecule to -NH3+ in order to make the
chitosan more soluble in water. The 1% (w/v) chitosan solutions were prepared
by using lactic acid aqueous solution with different concentration as a solvent
to obtain chitosan/lactic acid ratios of 1/1, 1/1.5 and 1/2 (w/w), respectively.
Twenty milliliter of chitosan solution was poured onto glass Petri dish and
dried at 40°C for 2 days before characterization. The film with chitosan/acetic
acid ratio of 1/2 (w/w) was also prepared as control by using acetic acid aqueous
solution as a solvent. All formulations of chitosan films were shown in Table
Fourier transform infrared (FTIR) spectroscopy was used to characterize
functional groups of the films. The FT-IR spectra were obtained by FTIR spectroscopy
using a Perkin-Elmer Spectrum GX FTIR spectrometer with air as the reference.
The resolution of 4 cm-1 and 32 scans were chosen.
Tensile strength and percentage of elongation at break of the films were measured
by tensile tester using a Charra TA-XT2I Texture Analyzer Machine. The films
were tested at the 40 mm of gauge length with speed of 10 mm min-1
and 10 N load cell. The tensile strength and elongation at break are calculated
from Eq. 1 and 2, respectively. The tensile
strength and percentage of elongation at break are obtained from average values
of at least three independent samples.
|| Formulations, transparency and moisture uptakes of chitosan
|aSD were less than 5%
Morphology of film surface and cross-section were determined by Scanning Electron Microscopy (SEM) using a JEOL JSM-6460LV SEM. The film cross-section was obtained after cutting film with paper-scissors. Before SEM measurement, the films were sputter coated with gold for enhancing the surface conductivity.
Film transparency was measured by UV-Vis spectroscopy using a Perkin-Elmer
Lambda 25 UV-Visible spectrophotometer at 660 nm as previously described by
Rhim et al. (2006).
Moisture uptake of the films was determined by the method as earlier described
by Khamhan et al. (2008). The typical procedure
was explained as follows. The sample films with 20x20 mm in size were dried
in vacuum at room temperature for a week. After weighing, they were kept in
a desiccator with 90±5% Relative Humidity (RH) maintained with a saturated
sodium chloride solution at 30±2°C. The sample films were weighed
again after kept in the desiccator for a week. The percentage of moisture uptake
was calculated from Eq. 3. The moisture uptakes are the average
of three different measurements.
where, Mi and Mf are the initial and final weights (g) of the films before and after moisture uptake, respectively.
The data were expressed as means and SD. Statistical analysis was performed
using a one-way analysis of variance (one-way ANOVA).
The FTIR spectra of chitosan films with M.W. of 100 and 740 kDa prepared
by using acetic acid solution as a solvent are shown in Fig. 1a
(film No. 1) and 2a (film No. 5), respectively that showed
free amino absorption bands at 1547 and 1551 cm-1, respectively and
polysaccharide structure broad bands in the range of 1200-1000 cm-1.
The absorption bands at 1652-1648 and 1315-1310 cm-1 were assigned
to amide I and II of residue chitin fractions, respectively indicated that the
chitosan used in present study was not fully deacetylated.
The FTIR spectra of chitosan films prepared by using lactic acid solution showed
the absorption bands of the both chitosan and lactic acid characteristics as
shown in Fig. 1b-d, 2b-d and 3b-d.
The carbonyl bands of lactic acids were existed in the range of 1718-1729 cm-1.
The position of amide I and II bands of the residue chitin were also slightly
shifted when lactic acid solution was used as a solvent instead of acetic acid
The tensile strength and elongation at break of chitosan films increased
with the M.W. of chitosan as shown in Fig. 4a and b. The chitosan
films prepared by using lactic acid solution as a solvent showed more flexible
than by using acetic acid solution. The tensile strengths at break of chitosan
films decreased and the elongations increased as the lactic acid ratio increased
for the all chitosan films prepared by using lactic acid solutions (Fig.
The tensile strengths at break of chitosan films slightly increased and elongations slightly decreased when the DL-lactic acid solution was used instead of L-lactic acid solution as a solvent to prepare the chitosan films.
||FTIR spectra of chitosan films with molecular weights of 100
kDa obtained from (a) 2.0% acetic acid (film No. 1), (b) 1.0% L-lactic acid
(film no. 2), (c) 1.5% L-lactic acid (film No. 3) and (d) 2.0% L-lactic
acid (film No. 4)
||FTIR spectra of chitosan films with molecular weights of 740
kDa obtained from (a) 2.0% acetic acid (film No. 5), (b) 1.0% L-lactic acid
(film No. 6), (c) 1.5% L-lactic acid (film No. 7) and (d) 2.0% L-lactic
acid (film No. 8)
All chitosan film thicknesses determined from several SEM images were in
the range of 30-40 μm and slightly increased with the lactic acid ratio.
The all chitosan films showed homogeneous and continuous phase as example of
which is shown in Fig. 5 for the chitosan film with 1/2 (w/w)
chitosan/L-lactic acid ratio (film No. 8). The phase separation did not found.
The all chitosan films were transparent and slight yellowish. The transmittance
(%) at λmax 660 nm (T660%) was used for studying
the film transparency. The T660 (%) values of chitosan films are
shown in Table 1. It was found that the T660 (%)
values of all chitosan films were similar.
||FTIR spectra of chitosan films with molecular weights of 740
kDa obtained from (a) 2.0% acetic acid (film No. 5), (b) 1.0% DL-lactic
acid (film No. 9), (c) 1.5% DL-lactic acid (film No. 10) and (d) 2.0% DL-lactic
acid (film No. 11)
||(a) Tensile strength and (b) elongation at break of chitosan
films of various film No. in Table 1
Moisture Uptakes of the Films
The moisture uptakes of chitosan films were measured instead of water uptake
(immersion in water) because of the partial dissolution of chitosan. The moisture
(%) uptakes were calculated from Eq. 3 and also shown in Table
1. The moisture uptakes of chitosan films were increased as the lactic acid
|| SEM micrograph of film No. 8
As would be expected, the intensities of carbonyl bands in the FTIR spectra
(Fig. 1-3) increased as the increasing lactic
acid ratio. It was found that the free amino bands of chitosan were shifted
to higher wave number when the lactic acid was used instead of acetic acid.
The existence of intermolecular bonds between free amino groups of chitosan
and carbonyl groups of polylactide in the chitosan/polylactide blends has been
determined from their FTIR spectra as previously reported by Chen
et al. (2005). Thus, the shifting of free amino bands of chitosan
in our research could be explained by the fact that the intermolecular hydrogen
bonds between free amino groups of chitosan and carbonyl groups of lactic acid
had occurred. The intermolecular interactions between chitosan film matrix and
lactic acid were also confirmed by shifting of the carbonyl bands of lactic
acid to higher wave number. In addition, the interactions between residue chitin
and lactic acid were detected by shifting of their amide I and II bands.
The carbonyl bands of lactic acid in low M.W. Chitosan films (Fig. 1a-d) shifted to higher wave number than the high M.W. films (Fig. 2a-d) for the same chitosan/lactic acid ratio suggested that the shorter chitosan molecules can interact with lactic acid more than another one. In addition, the carbonyl bands of L-lactic acid in the films (Fig. 2) shifted to higher wave number than the DL-lactic acid in the films (Fig. 3a-d) for the same chitosan/lactic acid ratio. The results indicated that the lactic acid in L-form can interact with chitosan better than the DL-form.
All high M.W. Chitosan films showed higher tensile strength and elongation
at break than the lower M.W. films for all chitosan/acid ratios proposed that
an entanglement network forming of high M.W. chitosan molecules during film
formation. The L-lactic acid solution has been used to dissolve chitosan for
film preparation as previously reported by Hoagland and
Parris (1996). However, they did not compare the mechanical properties of
chitosan films prepared by using different lactic acid configurations (L- and
DL-forms) and ratios. In this study, we add more results of mechanical properties
on chitosan film analysis data.
The results of mechanical properties indicated that the both lactic acids act as better plasticizers for the chitosan films than the acetic acid. It is important to note that the chitosan films changed from rigid to highly flexible films when the lactic acid blend ratio was increased for the both chitosan molecular weights (100 and 740 kDa) and lactic acid configurations (L- and DL-forms). In addition, the L-lactic acid showed greater plasticization effect than the DL-lactic acid for the all chitosan/lactic acid ratios. This may be indicated that the D-lactic acid contained in DL-lactic acid should be worse than the L-lactic acid for chitosan film plasticization.
From the morphological results, the adding lactic acids did not effect to
film morphology suggested that the film properties were consistent throughout
the film matrices.
The T660 (%) values of all chitosan films did not different suggested
that their film transparencies were similar. The results showed that the lactic
acid loading did not effect to chitosan film transparency. This can be explained
that the lactic acids are well homogeneous blended through the chitosan film
matrix. These clear chitosan films can be used as edible invisible films for
Moisture Uptakes of the Films
The moisture uptakes of chitosan films were decreased when the methoxy poly
(ethylene glycol)-b-poly(ε-caprolactone) contained hydrophobic character
was nano-composited (Khamhan et al., 2008). In
our study, increasing of moisture uptakes of chitosan films with the lactic
acid ratio may be due to the lactic acid had higher moisture adsorption than
the acetic acid. Then, lactic acids enhanced wetability of the chitosan films.
The L- and DL-lactic acid solutions can be used as the solvents instead of acetic acid solution to prepare the chitosan films. The intermolecular interactions between chitosan film matrix and lactic acids were determined from the FTIR analysis. The carbonyl groups of lactic acids can interact with the free amino and amide groups of chitosan and residue chitin, respectively. The both L- and DL-lactic acids showed potential for using as good plasticizers to improve film flexibility (increasing elongations at break of the films) of the both low and high chitosan molecular weights. The plasticization effect of L-lactic acid showed greater than the DL-lactic acid. Then, it may be expected that the lactic acid in L- and DL-forms can act as better plasticizers than in D-form. The using lactic acid instead of acetic acid for chitosan film preparation did not effect to film transparency but increasing the moisture uptakes.
These highly flexible edible chitosan films may possibly have potential for use as the invisible food coating and the drug delivery applications. The film flexibility can be adjusted by varying the chitosan/lactic acid ratio and lactic acid configuration.
The authors gratefully acknowledge the Research Development and Support Unit, Mahasarakham University and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand for financial supports.