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Journal of Food Engineering 116 (2013) 889–899

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Journal of Food Engineering
journal homepage: www.elsevier.com/locate/jfoodeng

Characterization and antimicrobial analysis of chitosan-based films
I. Leceta a, P. Guerrero a, I. Ibarburu b, M.T. Dueñas 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 Sebastián, Spain
University of the Basque Country (UPV/EHU), Department of Applied Chemistry, Faculty of Chemistry, Donostia-San Sebastián, 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 films
Antimicrobial activity
Maillard reaction

a b s t r a c t
Chitosan-based films 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,
films were flexible and transparent, regardless of chitosan molecular weight, glycerol content, and temperature. Regarding antimicrobial activity, chitosan film forming solutions showed antimicrobial behaviour against Escherichia coli and Lactobacillus plantarum. It was also observed that the bacteriostatic
property of chitosan-based films against bacteria employed in this study was notably affected by temperature. Moreover, temperature produced significant variation in the functional properties of chitosanbased films, 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 films, possibly due to Maillard reaction when heat treatment was used.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
The world market for plastic films is dominated by petroleumderived polymers in a wide range of industrial applications and, in
particular, in food packaging. In any field, 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 fit 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 Ó 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 films is one of the most promising ways, being
chitosan one of the most perspective active films (Aider, 2010;
Sánchez-González 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; Martínez-Camacho et al., 2010; Sebti
et al., 2005) has received considerable attention in recent years.
However, research has been mostly confined 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).
Chitosan’s bactericidal efficacy depends on various factors that
can be classified into four categories: microbial factors related
microorganism species; intrinsic factor of chitosan, including
molecular weight and concentration; physical state, solution or
film; 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
film. Nevertheless, pure chitosan films 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,

2. Colour values of films were measured using a portable colorimeter (CR-400 Minolta Chroma Meter).39... Golden Gate (Specac). the gram-positive bacterium. Two representative bacteria.4.2. 2012). After this time. 2. relative to a highly polished surface of black glass standard with a value near to 100. 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. a = 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. After that. 2. Measurements were made in triplicate.8. b were  measured.0 ± 0. L. Three thickness measurements were taken on each specimen at five different positions. All films were stored for 48 h in a controlled environment chamber (ACS SU700 V) at 25 °C and 50% relative humidity before testing. where h is the angle of incidence of the X-ray beam on the sample.001 mm with a hand-held digimatic micrometer (QuantuMike Mitutoyo). there are no many previous reports on the effect of high temperatures on the physicochemical and antimicrobial properties of films based on chitosans of different molecular weights and plasticized with different contents of polyols. according to ASTM D523 (ASTM. a (greenness) to +a (redness) and b (blueness) to +b (yellowness). final pH (pH = 4. . 2005).03 and b = 1. glycerol was added. Although many studies dealing with chitosan films have been performed. Escherichia coli. Stirring was continued for 30 min until total homogenization of the mixture. and considering standard light source D65 and standard observer 2° colour parameters. Light absorption 2. and Lactobacillus plantarum. 1999). solutions were filtered and 25 mL of the solution were poured into 90 mm diameter Petri dishes. being in the range of 60–80 lm in all cases. and five replicates were made per formulation. The gloss was measured at a 60° incidence angle. functional properties of films depend strongly on temperature (Rivero et al. 2011.9. Colour measurements 2.1. Film thickness 2. Acetic acid was used to fix the solution pH and glycerol as plasticizer. Thermogravimetric analysis (TGA) Thermal stability of films was analysed by TGA. 2..5. Non-isothermal degradation measurements were performed in a Mettler Toledo TGA SDTA 851. Film preparation Chitosan films were prepared by casting. using a UV-Jasco spectrophotometer (Model V-630). Fourier transform infrared (FTIR) spectroscopy 2.6. Gloss Film thickness was measured to the nearest 0.11..1) was measured with a digital pH-meter (Crison pH-meter BASIC 20+). Films prepared with 0%.5° to 50°. Results were expressed as gloss units. Experimental 2. Five replicates were made per formulation. A total of 32 scans were performed at 4 cm1 resolution. Park et al. Film specimens were placed on a white plate. the gram-negative bacterium.10. Furthermore. Among many possible plasticizers. TSM was calculated in relation to the dry mass and it was expressed as the percentage of the film dry matter solubilized. mechanical properties and. dataphysics instruments) was used to measure the contact angle of water in air on the The light-barrier properties of films were determined by measuring their light absorption at wavelengths ranging from 200 nm to 800 nm. specimens were dried in an air-circulating oven at 105 °C for 24 h.7. With these values.77. The gloss of the films was measured with a Minolta gloss meter (MultiGloss 268 Plus). Total colour difference (DE) was calculated as: DE ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi   ðLstandard  Lsample Þ2 þ ðastandard  asample Þ2 þ ðbstandard  bsample Þ2 Values were expressed as the means of ten measurements on different areas of each film. and the CIELAB colour scale was used to measure colour: L = 0 (black) to L = 100 (white). the aim of this work is focused on analyzing the effect of temperature on functional properties and antimicrobial activity of chitosan-based films for food packaging applications. 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). in general. / Journal of Food Engineering 116 (2013) 889–899 which noticeably affects the mechanical properties of the film (Osés et al. 2009. and were used as received. a. The diffraction data were collected from 2h values from 2. X-ray diffraction (XRD) XRD studies were performed with a diffraction unit (PANalytical Xpert PRO) operating at 40 kV and 40 mA. Same film was used to make RT and HT measurements in non-destructive tests. Suyatma et al. The measurements were recorded between 4000 and 700 cm1. Leceta et al. Total soluble matter (TSM) Total soluble matter was measured by immersion in 50 mL of distilled water.3. After 15 min under continuous stirring. Both of them were food grade reactants. Thus. Standard values for the white calibration plate were L = 97. glycerol is commonly used to plasticize biodegradable films and can essentially improve mechanical properties of chitosan-based films (Kerch and Korkhov. 2002. which are common spoilage bacteria for food contamination. 2.. 2. 2. 2010). The flasks were stored in the environmental chamber at 25 °C for 24 h. Thakhiew et al. have been tested.890 I. A 1% by weight chitosan solution was prepared in a 1% acetic acid solution. Finally. purchased from Panreac (Spain).54060 Å) source. Contact angle Fourier transform infrared spectra of chitosan/glycerol films were carried out on a Nicolet Nexus FTIR spectrometer using ATR A contact angle meter (model Oca20. The radiation was generated from a Cu Ka (k = 1. Chrome (C ab ) and hue angle (hab ) were calculated from: C ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  ða Þ2 þ ðb Þ2   h ¼ arctg b a The change of colour was evaluated by comparing total colour differences between films.

WVP of three specimens for each sample was calculated and reported. As it is shown in Fig.00 ± 0. All the results are the mean of three replicate assays.00a 18. The absorption peak at 1405 cm1 corresponds to the carboxylate groups associated with the antimicrobial activity of the biopolymer (Lagaron et al. On one hand. TSM values decreased significantly (P < 0. Agar plates were spread with 0. the antimicrobial effect of chitosan films and film forming solutions in the bacterial growth was also evaluated by the liquid culture medium assay on 96-well polypropylene microplates.05. referred as melanoidins.. L. Total soluble matter An electromechanical testing system (MTS Insight 10) was used to determine mechanical properties. 2. E. such as Maillard reaction. .. 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. which fixes temperature and relative humidity conditions as 38 °C and 90%.57a 20.05) for higher glycerol contents. However.00 ± 0. Ten replicates were made per formulation.60 ± 0. as it is shown in Table 1. 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 CECT748.00a 84. as it was also reported by other authors (Guerrero et al. 2007). Antimicrobial activity 3. The band that appears at 1378 cm1 is assigned to the acetamide groups. plantarum strains. indicating a change in the chemical structure of the film. suggesting that there is certain interaction between chitosan and glycerol chains.. In the case of film forming solutions. The liquid culture assay was conducted in 200 lL of both broths with Taking into account solubility results. coli.40 ± 1. which subsequently cyclises to produce the Amadori compound and insoluble polymeric compounds. 1.00 ± 0. Each assay was performed in duplicate. FTIR analysis was carried out in order to study the interactions between functional groups in chitosan-based films. WVP was calculated as: WVP ¼ 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 film.98b LMw 0 15 30 100. The same procedure was applied for the films dried in an air-circulating oven at 105 °C for 24 h. The contact angle was measured in a conditioned room by recording contact angle values. The sample film was cut into a circle of 7. provided by J.M. The absorption peak at 1050 cm1 is assigned to CAO G WVTR ¼ tA where G is the change in weight (g). a–c Chitosan Glycerol (%) TSM (%) RT HT HMw 0 15 30 100. and a Gram-positive bacterium.891 I. TSM values decreased drastically (P < 0. 2012). plantarum CECT748 and at 37 °C for E.39 ± 0. provided by the Spanish type culture collection. Two methods were used to assess the antimicrobial activity.1 mL of inoculum containing approximately 105–106 CFU/mL of bacteria. agar diffusion method adapted by Pereda et al. Measurements were performed in an water vapour transmission rate tester (PERME™ W3/0120. After incubation for 24 h at 37 °C to E. (2011) was performed. Each well was inoculated with 2% (v/v) of an overnight bacteria culture. to NAH bending (amide II) at 1558 cm1. A film sample (20 mm  80 mm) was put on a movable sample stage and levelled horizontally. 2. Cell growth was followed by reading the medium turbidity at 620 nm by a microplate reader (Labsystems iEMS Reader MF). plantarum strain and on LB (Lysogeny Broth) agar for E.84a 15.66c 21.13. then a drop of about 3 lL of distilled water was placed on the surface of the film using a microsyringe.12. Labthink). Leceta et al. 2000). Moreover. The setup was subjected to a temperature and relative humidity of water vapour transmission rate (WVTR) was calculated as: 2. Yasir et al.2. The total area was used to evaluate the antimicrobial potential of films. 2.32b 11. On the other hand. Five replicates were tested for each composition. Statistical analysis 3. 2012. This reaction produces a Schiff base.05). Water vapour permeability (WVP) The data were subjected to one-way analysis of variant (ANOVA) by means of an SPSS computer program (SPSS Statistic 19.70c 24. coli 0517H for 24 h in both cases. which indicate that chitosan is not totally desacetylated. 1993). t is the time (h). Rodriguez (Veterinary Faculty.01b 14. as it is shown in Table 1. coli and at 28 °C to L.1. 30 lL of solution were poured into agar wells and the same procedure described above was applied.40 cm diameter and the test area was 33 cm2.05) when 30% glycerol was added. / Journal of Food Engineering 116 (2013) 889–899 surface of chitosan/glycerol films. indicating a good degree of interaction between the two components of the film and the formation of a more compact network.30 ± 1. Cell cultures were incubated at 28 °C for L.14 ± 0.15. As could be appreciated in Fig. and to CAN stretching (amide III) at 1320 cm1. the plates were optically examined for width of inhibition in the contact area.05) different thought the Tukey’s multiple range test. 2. Image analyses were carried out using SCA20 software.52 ± 0. film pieces (sterilized with UV light) or 60 lL of film forming solutions.0). 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 films dried at room temperature (RT) and heat treated (HT). when films were heat-treated. WVP of films was determined according to ASTM E96-00 (ASTM.98 ± 0. Tensile strength (TS) and elongation at break (EB) were determined according to ASTM D1708-93 (ASTM. Results and discussion 3.00a 100.14. Post hoc multiple comparision were determined by the Tukey’s test with the level of significance set at P < 0. 2007). Madrid). respectively. the main absorption peaks of chitosan are attributed to C@O stretching (amide I) at 1650 cm1. FTIR spectroscopy The antimicrobial activity of chitosan-based films and chitosan film forming solutions was tested against the growth of two typical food spoilage bacteria: a Gram-negative bacterium. are formed (Leceta et al. coli 0517H. It is worth noting that the films prepared without glycerol and dried at room temperature were totally soluble. TSM values were lower (P < 0. and A is the test area of the test area (m2)..62c Two values followed by the same letter in the same column are not significant (P > 0.71a 12.35 ± 4.47 ± 1. This behaviour could mean that temperature promoted chemical reactions.

2009. Fernandez-Saiz et al. On one hand. The typical absorption bands of glycerol are located in the region from 800 cm1 up to 1150 cm1. Ziani et al. stretching and the broad band above 3000 cm1 corresponds to OAH and NAH bonds (Brugnerotto et al. comparing the relative intensity of the bands at 1405 cm1 and 1378 cm1. 925 cm1. and 995 cm1 are assigned to the vibration of CAC skeleton. 1. and the one at 1117 cm1 is related to the stretching of CAO in C2.892 I. 2. Variations in the FTIR spectra produced by the effect of temperature can be observed in Fig. which is in agreement with the decrease of solubility observed for heattreated films.. regardless of the molecular weight of chitosan or glycerol content. Leceta et al. 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. / Journal of Food Engineering 116 (2013) 889–899 OH HO O O O OH H O H2O O O HO N + CH C O CHOH H2O O O OH NH 2 CHOH OH O NH CH2 C O H2O R R R Schiff Base Amadori Compound Fig. Maillard reaction between carbonyl ends and the amine groups in chitosan. the band at 1045 cm1 is associated to the stretching of CAO linkage in C1 and C3. (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. 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 films dried at room temperature. 3.. Infrared spectra for (a) high molecular weight (HMw) and (b) low molecular weight (LMw) chitosan-based films dried at room temperature (RT) and heat treated (HT). The peaks at 850 cm1. the difference in the relative intensity of those two bands became smaller as the glycerol content increased for both HMw and LMw chitosan-based films dried in the oven.. and correspond to the vibrations of CAC and CAO linkages. On the other hand. it can be observed that the intensity . However. 2001. 2008).

and a shoulder around 175 °C. there was a small weight loss at temperatures below 100 °C. 3. / Journal of Food Engineering 116 (2013) 889–899 (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. 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 films. it is also worth noting that the absorption peaks at 1045 cm1 and 1117 cm1. 2009). 1973). which is 170 °C (Perry and Chilton. suggesting interactions between hydroxyl groups of chitosan and glycerol by hydrogen bonding. Thermo-gravimetric analysis (TGA) 3. 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. joined to become a single peak when glycerol content was increased. This peak showed about 15% loss weight for the films prepared with 30% glycerol and the temperature was higher than the one estimated for pure glycerol. but when 15% glycerol was added. In the case of the films heat-treated. The X-ray diffraction patterns of chitosan- . 3. Regarding thermo-gravimetric analysis for the films with 30% glycerol. The decrease of the relative intensity of the band at 1405 cm1. the peak related to glycerol disappeared. In the case of HMw films. attributed to the biocide character of chitosan. the relative intensity of those bands showed the same tendency observed at room temperature. for the films heat-treated was also observed by the antimicrobial tests. Fig. as shown below. Nevertheless. it must be noted that there was a new peak located around 225 °C that was related to the evaporation of glycerol. thus X-ray analysis was carried out. the relative intensity of the bands became similar for the films without glycerol and was higher for the band at 1378 cm1 when glycerol was added.3. films containing 0% and 30% glycerol. associated to CAO stretching. as it was previously shown by the decrease of TSM values.893 I. which can be attributed to the loss of moisture. the relative intensity became similar. indicating a change in the structure due to the Maillard reaction. which may correspond to the loss of adsorbed and bound water (Lewandowska. For the films without glycerol. For LMw chitosan films without glycerol. and the relative intensity of the band at 1378 cm1 became higher for the films prepared with 30% glycerol. Leceta et al.4. of the band at 1405 cm1 was always higher than the one at 1378 cm1 for the films dried at room temperature. 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. as shown by FTIR results. It is worth noting that there was no significant change between chitosan-based films dried at room temperature or heat-trated. Magnification of infrared spectra from 1800 to 800 cm1 for (a) high molecular weight (HMw) and b) low molecular weight (LMw) chitosan-based films dried at room temperature (RT) and heat treated (HT). the relative intensity of those two bands changed for the films heat-treated. These two facts could indicate a good degree of interaction between chitosan and glycerol. Finally.

05) happened in all films. As observed in Fig. . 5. 5. Total colour difference between chitosan films dried at room temperature or heat treated was more significant for LMw films and increased (P < 0. 4. 0 10 20 30 40 50 2θ Fig. Regarding the effect of temperature. but it can be observed that chrome values increased with the addition of glycerol. the chitosan-based films dried at room temperature were in a semicrystalline state with two main diffraction peaks at 2h around 10° and 20°. Thermogravimetric analysis for (a) high molecular weight (HMw) and (b) low molecular weight (LMw) chitosan-based films with 0% and 30% glycerol. being this effect more remarkable for LMw chitosan-based films. so becoming an important quality factor to be taken into account. based films are shown in Fig. Chitosan is a partially crystalline polysaccharide due to its regular chain and has two reflection fall at 2h = 10° and 20° (Luo et al. dried at room temperature (RT) and heat treated (HT). Leceta 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.. / Journal of Food Engineering 116 (2013) 889–899 (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 0 100 200 300 Temperature (ºC) 400 500 600 700 800 Temperature (ºC) Fig. whereas the peak of chitosan at 10° disappeared in the films heat-treated. dried at room temperature (RT) and heat treated (HT). Films were homogeneous and transparent. Moreover.894 I. indicating that chitosan structure was influenced by the effect of temperature. This could suggest that there was an interaction between these two components that lead to new crystalline structures. 5. 2013).. 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.5.. X-ray diffractograms of high molecular weight (HMw) chitosan-based films with 0% and 30% glycerol. 2012).05) with the addition of glycerol. the intensity of the peak at 20° was enhanced in the films prepared with glycerol. The reflection fall at 10° was assigned to crystal forms I and the strongest reflection at 20° to crystal forms II (Wu et al. RT 0HMw HT 0HMw RT 30HMw HT 30HMw 3. Colour values of all the films prepared in this study are shown in Table 2. significant total colour change (P < 0.

25 ± 0.12 ± 2. gloss values were reduced by the addition of plasticizer (P > 0.64 ± 1.61a 31.99 ± 0.22 ± 0. 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. UV–vis spectra for a) high molecular weight (HMw) and b) low molecular weight (LMw) chitosan-based films dried at room temperature (RT) and heat treated (HT). 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. However.94 ± 0.20c 94.18e 8.65 ± 1. Gloss is also an optical phenomenon related to the appearance of the surface. 1996).40 ± 0.21 ± 0.38 ± 7. increasing the roughness of the film surface.05) different thought the Tukey’s multiple range test.29 ± 0.45f 23.82 ± 0.6.16c 97.23cd LMw 0 15 30 4. 6 shows the UV–vis absorption spectra from 200 to 800 nm for all the films.08abc 17.15 ± 0.15a 8. Moreover.14a 3.66 ± 0.18c 98.40d 9.70 ± 0.67 ± 0.17b 5.05).22c 1.33 ± 0.50 ± 0.51b 6.19 ± 0. it can be said that the films prepared in this work were slightly glossy.13e 15.52 ± 0.36 ± 0. As it can be seen. Leceta et al. It is noteworthy that films had excellent barrier properties to light in the UV region.01 ± 0.77 ± 0. the spectra for chitosan-based films dried at room temperature were similar.20d 4.95 ± 0.42c 30.54bc 22. Light absorption Fig.39d 6.24a 3.36 ± 0. 3.51 ± 0. . 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.50a 95.98 ± 0. 6.89 ± 0. which is favoured at high temperatures.17e 17.31d 11.53 ± 0.22c 10.90 ± 0.75b 2.57 ± 1. being the preventive effect slightly better for LMw chitosan-based films.63 ± 1.41f 100.38b 100.05b 3.30b 4.65 ± 0.37a 97.73d 20.44f 24. changes in these two aspects resulted in changes in gloss.46 ± 0.08cd 99. 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. gloss values were lower than 40 for all the compositions.15c 97.28b 28. there was no significant difference in the UV–vis absorption spectra for HMw chitosan-based films dried at room temperature or heat-treated. As gloss is related to surface composition and morphology.10 ± 0.81 ± 0.97a 28. generated discontinuities in the chitosan matrix.65 ± 0. Chitosan Glycerol (%) Colour measurements Gloss 60° (Gloss units) RT HT C ab a–f  C ab hab  hab DE  RT HT HMw 0 15 30 3.81 ± 1. there was a sharp increase in absorbance in the range of 200–400 nm for higher glycerol contents.81 ± 0.13d 100. regardless of glycerol amount. Table 2 shows the values obtained at 60° incidence angle.05) and showed a slight decrease due to the effect of temperature (P > 0. As it is shown. Moreover.73 ± 0.895 I. It represents the capacity of the surface to reflect directed light and it is considered to be the proportion of incident light that is reflected at the specular reflectance angle with respect to the normal plane of the surface. when films were heat-treated.90d Two values followed by the same letter in the same column are not significant (P > 0.24c 36.18a 99.68 ± 0.04 ± 1. / Journal of Food Engineering 116 (2013) 889–899 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 films dried at room temperature (RT) and heat treated (HT).01e 96.87cd 12.

85 ± 3.05) and elongation at break decreased (P < 0. small glycerol molecules could get into chitosan chains more easily when chitosan molecular weight was lower. so the final state of the water drop on the film surface can be taken as an indication of surface wettability (Guerrero et al.05) when glycerol content increased due to the plasticization effect.83 ± 2.96a 36.05) different through the Tukey’s multiple range test.11c 31. This fact could hinder the proximity between amino and carbonyl groups to react through Maillard reaction.59 ± 0.47 ± 2.41b 10.8.72e LMw 0 15 30 55.87 ± 3.58 ± 0.05) for LMw chitosan-based films plasticized with glycerol.14a 9. 2003). However.00a 8. . which produces chromophores that absorb the light at wavelength below 400 nm. There was no significant difference (P > 0.51 ± 1. The addition of glycerol did not show a remarkable change in water vapour permeability and chitosan-based films showed adequate barrier properties against water vapour. 7.47 ± 0. 2013). many authors reported that the influence of molecular weight of chitosan on WVP was not significant (Park et al.82 ± 3.38c 37.65b 8.75c 68. Hsieh et al.38a 27. Leceta et al.08b 32. / Journal of Food Engineering 116 (2013) 889–899 160 RT HMw HT HMw RT LMw HT LMw 140 Contact angle (º) 120 a b a a a b b b c cd 100 d d 80 60 40 20 0 0% GLY 15% GLY 30% GLY Fig. which is a crucial property for the films intended to be used as food packaging (Ahvenainen. As it can be observed. 2005). heat-treatment caused a slight decrease in contact angle values. When chitosan molecular weight increased.23 ± 2. However.40 ± 0. Comparison of mechanical properties of chitosan-based films is difficult since there are a great dispersion in deacetylation degrees.93cd 4. This could be due to the formation of a more compact network induced by the Maillard reaction.20b 17. Hwang et al. are more static owing to its higher length. Chitosan a–f Glycerol (%) RT HT TS (MPa) EB (%) TS (MPa) EB (%) HMw 0 15 30 61. Table 3 Water vapour permeability (WVP) average values and standard deviations of high molecular weight (HMw) and low molecular weight (LMw) chitosan-based films dried at room temperature (RT) and heat treated (HT). However. 3.05) different thought the Tukey’s multiple range test. contact angles were investigated for the films prepared with different contents of glycerol and dried at room temperature or heat-treated.60 ± 5.05) different thought the Tukey’s multiple range test. indicating changes in the conformation of molecules.7.76 ± 0.41 ± 3..12 ± 1.46e Two values followed by the same letter in the same column are not significant (P > 0. regardless of glycerol content. 2007.96f 20.21ab 9.58 ± 0. 7. Regarding molecular weight.70b 30. 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 films dried at room temperature (RT) and heat treated (HT).49a 11. Average contact angle values and standard deviation of high molecular weight (HMw) and low molecular weight (LMw) chitosan-based films dried at room temperature (RT) and heat treated (HT).91ab 9.89 ± 5.38 ± 0. being higher when hydrophilicity is lower. as shown by previous results.12 ± 0.05) and elongation at break increased (P < 0. 2003.46 ± 0.67 ± 2.14 ± 0. which indicated that total colour difference was higher for LMw chitosan films. tensile strength decreased (P < 0. Mechanical properties The mechanical behaviour of chitosan-based films prepared in this work is shown in Table 4.37 ± 2.01 ± 2.78 ± 0.25 ± 1. the values were similar for all the films. 2002. elongation at break decreased (P < 0.23c 37.43a 43.05 ± 0.1 ± 0. and the exposure of hydrophilic groups toward the surface. These results were in good agreement with the colour values shown above.58e 43.50 ± 0.2 ± 0. On the other hand.48ab 10. providing exceptional barrier properties to UV light.21 ± 0.55bc 23.34 ± 3. tensile strength increased (P < 0.77b 15. water contact angles of chitosan-based films with 0% and 15% glycerol were around 105°. LMw chitosan films dried at room temperature exhibited slightly higher WVP than HMw chitosan films.05) between contact angle values for the films dried at room temperature or heat-treated.55ab a–b Two values followed by the same letter in the same column are not significant (P > 0. a-dColumns with the same letter are not significantly (P > 0. However.16d 53. which would facilitate the diffusion of water vapour through the film. As it can be seen.65a 38.00f 17.93 ± 4. the concentration of glycerol notably affected mechanical properties. The WVP values are shown in Table 3. both tensile strength and elongation at break increased (P < 0.. molecular weights. water vapour permeability provides information about water vapour transmission through the film..00 ± 2.04b 8. On the other hand. when films were heat-treated. WVP.. Chen and Zhao. 2011). contact angle values decreased with the addition of 30% plasticizer owing to the hydrophilic character of glycerol..05) by the effect of temperature for HMw chitosan-based films. However.56ab 7. To understand the effect of temperature on film wettability.07 ± 1. In all cases. As it is shown in Fig. in agreement with the results shown by other authors (De Britto and Assis.05).79d 4. 2012). Barrier properties Water contact angle values are good indicators of the degree of hydrophilicity of films.50b 10.98 ± 7. 3.54ab LMw 0 15 30 9. 1013 (g cm1 s1 Pa1) Chitosan Glycerol (%) RT HT HMw 0 15 30 8.49a 9.21a 8.896 I. concentrations of chitosan and plasticizer as well as film preparation and test conditions (Silva-Weiss et al. This fact could favour Maillard reaction. This fact could be due to shorter length of the chains.

. wettability. as could be observed in Fig. Agar diffusion method results for chitosan-based films (a) dried at room temperature and (b) heat-treated. However. 2013). which has been related to the antimicrobial character of chitosan in the literature (Lagaron et al. resistance against UV light... / Journal of Food Engineering 116 (2013) 889–899 897 Fig. Khan et al. as it can be seen in Fig. 8.2 1. 2013. The absence of inhibitory character could be explained by the limitation of the diffusion of chitosan in agar medium. indicating that there was no inhibitory effect. samples used (solutions or films). TGA and XRD results. 2007).I. These changes were explained by chemical interactions through Maillard reaction. Control 0HMw 15HMw 30HMw 0LMw 15LMw 30LMw Fig. These results were in good agreement with FTIR results. Antimicrobial activity Antimicrobial activity of chitosan-films was evaluated by the agar diffusion method. remarkable variations were observed in some functional properties such as colour.. which was promoted by temperature. 10. Results showed that there was no inhibition zone on agar solid medium. 9. However. indicating that chitosan-based films can exhibit bacteriostatic effect against E. there was bacterial growth under the films heat-treated. 24 and 48 h of bacteria inoculation.2 e e 1. FTIR. suggesting changes in the structure of films due to the effect of temperature. Average absorbance values and standard deviations at 620 nm for chitosanbased films dried at room temperature after 0.4 d de 1.4 weight. Valenzuela et al.. In addition.05). glycerol content. 2012.. there was no bacterial growth under the film for the films dried at room temperature.8 e e e bc e d 1. As the previous results suggest that agar diffusion method is not an appropriate method to characterize the antimicrobial properties of chitosan. giving rise to the reduction of the bacteriostatic property.coli 0h 24 h 48 h 0. It was also worth noting that films dried at room temperature shrunk as time went by. which were reduced when films were heat-treated. 9. which must be taken into account not only for fabrication purposes . 8. irrespective of chitosan molecular 4. On the other hand. there was no significant decrease (P > 0.2 Absorbance 620 nm 0h 24 h 48 h Lactobacillus Plantarum 2. which indicated that there was a reduction of the absorption band at 1405 cm1. as well as in agar. suggesting that temperature caused a structural change in films. Marroquin et al.6 c 1. absorbance values were strongly reduced (P < 0. This could be owing to the limitation of the biocide’s diffusion in the case of films in the liquid culture medium. as it has been reported by other authors (Coma et al. diffusion in liquid culture medium was also used.0 bc 1. pure chitosan films are higher than the values obtained by other authors with chitosan of similar deacetylation degree and by similar preparation conditions (Hosseini et al.0 a 0. Conclusions The results of this work showed that chitosan film forming solutions presented antibacterial properties. 2013. 2.0 1. regarding chitosan-based films. or drying conditions employed to prepare the films. in the case of film forming solutions. as shown above.05) different through the Tukey’s multiple range test. plantarum.05) in the absorbance at 620 nm. 2002). coli and L.9.6 d cd 1. 3.6 a a a a E. Leceta et al.8 a 0.8 bc bc c bc bc bc bc b 1.4 e e e e 1.8 a a 0. a–eColumns with the same letter are not significantly (P > 0.6 a a d a a a a 0. whereas chitosan-based films dried at room temperature only showed bacteriostatic properties.0 d 0.4 Absorbance 620 nm 2. showing an important inhibition of bacteria growth and good antimicrobial activity. as shown by TSM. As can be appreciated in Fig. These results show that temperature is a critical factor affecting physicochemical and antibacterial properties of chitosan-based films. and mechanical properties due to the effect of temperature.

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