ORIGINAL PAPER

Chitosan/Hydrophilic Plasticizer-Based Films: Preparation,Physicochemical and Antimicrobial Properties

Jesus R. Rodrıguez-Nunez • Tomas J. Madera-Santana •

Dalia I. Sanchez-Machado • Jaime Lopez-Cervantes •

Herlinda Soto Valdez

Published online: 8 November 2013

� Springer Science+Business Media New York 2013

Abstract The addition of plasticizers to biopolymer films

is a good method for improving their physicochemical

properties. The aim of this study was to evaluate the effect

of chitosan (CHI) blended with two hydrophilic plasticizers

glycerol (GLY) and sorbitol (SOR), at two concentrations

(20 and 40 wt%) on their mechanical, thermal, barrier,

structural, morphological and antimicrobial properties. The

chitosan was prepared through the alkaline deacetylation of

chitin obtained from fermented lactic from shrimp heads.

The obtained chitosan had a degree of deacetylation (DA)

of 84 ± 2.7 and a molecular weight of 136 kDa, which

indicated that a good film had formed. The films composed

of CHI and GLY (20 wt%) exhibited the best mechanical

properties compared to the neat chitosan film. The per-

centage of elongation at break increase to over 700 % in

the films that contained 40 % GLY, and these films also

exhibited the highest values for the water vapor transmis-

sion rate (WVTR) of 79.6 ± 1.9 g m2 h-1 and a yellow

color (bo = 17.9 ± 2.0) compared to the neat chitosan

films (bo = 8.8 ± 0.8). For the structural properties, the

Fourier transform infrared spectroscopy (FTIR) and X-ray

diffraction analyses revealed an interaction in the acetam-

ide group and changes in the crystallinity of plasticized

films. The scanning electron micrographs revealed that all

formulations of the chitosan films were smooth, and that

they did not contain aggregations, pores or microphase

separation. The thermal analysis using differential scanning

calorimetry (DSC) revealed a glass transition temperature

(Tg) of 130 �C for neat chitosan film, but the addition of

SOR or GLY elicited a decrease in the temperature of the

peak (120 �C). In addition, the antimicrobial activity of the

chitosan films was evaluated against Listeria monocytog-

enes, and reached a reduction of 2 log after 24 h. The

plasticizer concentration of 20 % GLY is sufficient for

obtaining flexible chitosan films with good mechanical

properties, and it could serve as an alternative as a pack-

aging material to reduce environmental problems associ-

ated with synthetic packaging films.

Keywords Chitosan � Sorbitol � Glycerol �Listeria monocytogenes � Antimicrobial properties

Introduction

The growing environmental concerns regarding non-bio-

degradable petrochemical-based plastic packaging materi-

als have increased interest in the use of biodegradable

alternatives from renewable sources. In recent years, there

has been remarkable development in edible films and

coatings using biopolymers [1]. These materials are thin

films prepared from edible materials such as cellulose,

chitosan, alginate and starch that act as a barrier to the

external elements (such as moisture, oil and vapor), thereby

protecting the products and extending their shelf life [2].

Chitosan is a natural polymer derived through the

deacetylation of chitin, which is the second most abundant

biopolymer after cellulose and has a unique nature for a

cationic polysaccharide. Chitosan has been demonstrated to

be nontoxic, biodegradable and biocompatible, and it is

J. R. Rodrıguez-Nunez � D. I. Sanchez-Machado �J. Lopez-Cervantes

Departamento de Biotecnologıa y Ciencias Alimentarias,

Instituto Tecnologico de Sonora, 5 de Febrero 818 Sur,

85000 Ciudad Obregon, Sonora, Mexico

T. J. Madera-Santana (&) � H. Soto Valdez

Centro de Investigacion en Alimentacion y Desarrollo,

A.C. CTAOV, A.P. 1735, 83304 Hermosillo, Sonora, Mexico

e-mail: tmadera.santana@gmail.com

123

J Polym Environ (2014) 22:41–51

DOI 10.1007/s10924-013-0621-z

insoluble in water, but soluble in acidic solvents such as

dilute hydrochloric, formic and acetic acids. In acidic

solutions, the amine groups on the chitosan molecule are

protonated to NH3? and thus acquire a positive charge [3].

Because of the film forming properties of chitosan, it has

been reported to be a potential material for producing food

packaging, particularly as edible films or coatings [4].

Chitosan films are generally clear and transparent, and they

do not possess any pores. In addition, Suyatma et al. [5]

reported that the production of chitosan from shrimp shells,

which are wastes from the seafood industry, is economi-

cally feasible.

The properties of chitosan films depend on the mor-

phology of the chitosan, which is affected by its molecular

weight, degree of N-acetylation, solvent evaporation and

the free amine regenerating mechanism. Additionally,

chitosan films have intrinsic antimicrobial activity and

inhibit the growth of a wide variety of bacteria (Esche-

richia coli, Salmonella typhimurium, Staphylococcus aur-

eus and Listeria monocytogenes) and fungi (Botrytis

cinerea, Fusarium oxysporum and Piricularia oryzae) [6,

7]. Due to this property, chitosan has been successfully

used to maintain the quality of post-harvest fruit and veg-

etables [8]. One of the reasons for the antimicrobial char-

acter of chitosan is its positively charged amino group

which interacts with the negatively charged microbial cell

membrane, leading to the leakage of proteinaceous and

other intracellular constituents of the microorganism [9].

Although chitosan has good film-forming ability, the

chitosan film is brittle. The brittleness of the films is

primarily determined by the strength of polymer–polymer

interactions, which can be controlled through the polymer

chemistry and the addition of a plasticizer. Plasticizers are

generally small molecules, such as polyols including sor-

bitol, glycerol and polyethylene glycol (PEG), which

intersperse and intercalate between the polymer chains,

thereby disrupting hydrogen bonding and spreading the

chains apart to increase the flexibility, water vapor

transmission rate and the gas permeability of the films.

Glycerol (GLY) and sorbitol (SOR) are considered to be

good plasticizers for use in edible films [5]. However, the

films plasticized with GLY are sensitive to water, such as

environments with high relative humidity, and the

migration of GLY out of the film was observed. Fur-

thermore, SOR can crystallize when the films are stored

under low and intermediate relative humidity conditions

[10].

The aim of the present study was to evaluate the effect

of two hydrophilic plasticizers, specifically glycerol (GLY)

and sorbitol (SOR), on the mechanical, optical, thermal,

structural, morphological and antimicrobial properties of

chitosan films, where the chitosan was prepared from chitin

obtained from lactic fermentation.

Materials and Methods

The chitosan powder was prepared in our laboratory fol-

lowing the procedure described below. Glycerol (GLY)

(Lot. 1891) and sorbitol (SOR) (Lot. 4072C7) were pur-

chased from REASOLTM Iztapalapa, Mexico.

Preparation of Chitin and Chitosan from Shrimp Waste

Shrimp waste (cephalothorax and exoskeleton) obtained

from a local shrimp processing plant in South Sonora,

Mexico was used as the raw material from which the chitin

was obtained. The chitin was obtained at pilot scale levels

by lactic fermentation from shrimp waste, which was based

on the procedure of Bueno-Solano et al. [11]. Chitosan

with a low molecular weight was prepared according to the

method of Weska et al. [12] with some modifications.

During the first step, the chitin was immersed in a 4.5 %

(w/v) NaOH solution at 65 �C for 4 h to remove the pro-

teins. Then, the solid precipitate was immersed in a 3.6 %

(w/w) HCl solution at room temperature for 4 h to remove

the remaining minerals in the chitin. The final step con-

sisted of an alkaline deacetylation with 45 % (w/v) NaOH

solution at 120 �C for 2 h, which was followed by

expensive washes with water and drying at 40 �C for 12 h.

Before use, the chitosan was milled to obtain a fine powder

with a 180 lm particle size.

Physicochemical Characterization of Chitosan

To determine the moisture content, the samples were dried

overnight in an electric oven at 60 �C. To determine the

ash content, dried ground samples were heated for 5 h in an

electric oven at 525 �C. The nitrogen content was deter-

mined using the micro-Kjeldahl method [13]. The degree

of deacetylation in the chitosan was measured by UV

spectrophotometry using a dual standard based on a pro-

cedure reported by Liu et al. [14], with some modifications.

The shear viscosity of the chitosan was measured at room

temperature using a digital viscometer (Brookfield, Mid-

dleboro, MA, USA) with a cylindrical spindle (LV #1) at

30 rpm.

The viscosity molecular weight (Mw) was investigated

using an Ubbelohde viscometer at 25 ± 1 �C. Five chito-

san concentrations of 0.0014, 0.0012, 0.001, 0.0008 and

0.0006 g ml-1 were dissolved in 0.3 M CH3COOH/0.2 M

CH3COONa solution, and filtered through 0.45 lm mem-

branes. The viscosity Mw was calculated based on the Mark

Houwink relationship (Eq. 1).

g ¼ K MaV ð1Þ

where [g] is the intrinsic viscosity and K and a are con-

stants whose values depend on the nature of the polymer

42 J Polym Environ (2014) 22:41–51

123

and solvent as well as the temperature. MV is the average

viscosity molecular weight. In this paper the values for

K = 0.074 mL g-1 and a = 0.76 were used [15].

Preparation of the Plasticized Chitosan Films

Plasticized chitosan films were prepared according to the

method mentioned by Suyatma et al. [5] with some mod-

ifications. A solution with a concentration of 1 % neat

chitosan or chitosan/plasticizer was prepared by adding

4.2 g of the chitosan powder to 420 mL of a 1 % acetic

acid solution, which was then stirred for 12 h. The com-

positions of the chitosan/plasticizers blends were 100/0,

80/20 and 60/40 by weight. The films were cast onto a

polypropylene plate and dried at 60 �C for 12 h.

Characterization of the Films

The thickness of the films were measured using a

micrometer (Mitutoyo, Japan) with a precision of 0.01 mm

each of the chitosan films and plasticized films were

measured in ten different zones, and the average value was

calculated.

Optical Properties

The transparencies of the film were determined using the

procedure described by Shiku et al. [16] with some modi-

fications. Samples of the films were cut into a rectangle and

placed on the internal side of a spectrophotometer cell. The

absorbance spectrum (200–800 nm) of each sample was

recorded using a UV–visible spectrophotometer (Varian,

Cary 50 Bio, USA). The transparencies of the films were

determined by measuring the absorbance at 600 nm (A600).

The transparency at the unit light path length was calcu-

lated using Eq. (2), which was derived by Han and Floros

[17].

Transparency ¼ A600

Tð2Þ

where A600 is the absorbance at 600 nm and T is the

thickness of the film (mm). The measurement was repeated

three times for each of the films and the average value was

reported.

The film color was determined using a Minolta color-

imeter CR 300 (Osaka, Japan) calibrated with a standard

(Y = 93.2, x = 0.3133, y = 0.3192) based on the method

reported by Garcıa et al. [18]. The CIELab scale was used,

and the lightness (L) and the chromaticity parameters a*

(red-green) and b* (yellow-blue) were measured. Mea-

surements were performed by placing the films sample

over the standard. The samples were analyzed in triplicate,

and six measurements were recorded for each sample. The

color differences (DE) were calculated using Eq. (3).

DE ¼ ðDL�Þ2 þ ðDa�Þ2 þ ðDb�Þ2 ð3Þ

where DL* = L* - Lo, Da = a* - ao, and Db* = b* - bo,

with L*, a*, b* representing the color parameter values of

the standard and Lo, ao, and bo, representing the color

parameters of the sample.

Degree of Swelling, Solubility and WVTR of Chitosan

Films

The degree of swelling and solubility of the films were

determined using the method reported by Bourtoom [19]

with some modification. Specifically, the dried films were

immersed in distilled water at room temperature. After

obtaining equilibrium (1 h), the moisture on the surface of

the films was removed, and the weight of the films was

measured. The percentage of degree of the swelling (% DS)

of the films was calculated using Eq. (4).

%DS ¼ we � wo

wo

� 100 ð4Þ

where we is the weight of the films at absorbing equilibrium

and wo is the initial dry weight of the films. The swelled

chitosan films were dried again for 24 h at 60 �C and their

percentages of solubility in water (% SW) were calculated

using Eq. (5).

% SW ¼ wo � wd

wo

� 100 ð5Þ

where wo is the initial dry weight and wd is the dry weight

of the chitosan films after the drying process. The % DS

and % SW tests for each formulation performed in

triplicate.

The gravimetric modified cup method based on ASTM

E96 [20] was used to determine the WVTR and perme-

ability of the films. Specifically, each sample of the films

with an area of 2.85 9 10-4 m2 was stored at 25 �C in a

desiccator (with dry silica gel). To maintain a relative

humidity (RH) gradient across the films, distilled and

deionized water was place inside the cell. The RH outside

the cell was always lower than that inside the cell, and the

water transport was determined from the weight loss of the

permeation cell. After steady-state conditions were reached

(8 h), changes in the weight of the cell were recorded to the

nearest 0.0001 g as a function of time, and the gain weight

(GW) g/m2 was obtained when it was divided by the cell

area. The water vapor transmission rate (g H2O/m2/h) was

calculated from the slope of each line through a linear

regression (r2 [ 0.98) using Eq. (6).

WVTR ¼ w

t � A ð6Þ

J Polym Environ (2014) 22:41–51 43

123

where t is the time in hours, and A is the permeation area

(1.257 9 10-3 m2). The measurements for each film were

performed in triplicate.

Mechanical Characterization

The mechanical properties of the chitosan films with

plasticizers were determined using a texturometer (Texture

Technologies Corp, New York, USA) at 25 �C. The sample

dimensions were 10 mm in width and 60 mm length. The

employed gauge length and crosshead speed were 30 mm

and 10 mm min-1, respectively. The measured mechanical

parameters were the tensile strength, elongation at break

(%), elastic modulus and the tear strength. The tensile test

procedure was based on the ASTM D882 standard method

[21]. A single-tear test was performed to determine the tear

propagation force based on the ASTM D1938 standard

method [22]. The average of six replicate measurements

was determined for each formulation, and the specimens

consisted of strips that were 60 mm long by 10 mm wide

with a longitudinal slit that was 30 mm long that was

produced using a sharp razor blade.

Thermal Analysis

The thermal characterization of the films was performed

using a Perkin Elmer Diamond Pyris differential scanning

calorimeter (DSC). A total of 10 mg of each sample was

loaded into an aluminum pan and sealed with pressure. An

empty pan was used as a reference; both pans were heated

from 25 to 200 �C at a heat rate of 10 �C min-1. Then, the

sample was cooled to the initial temperature (25 �C). The

measured thermal parameters were the heat of fusion

(DHm) and the melting temperature (Tm). The heat of

fusion values were calculated from the area heat and are

given in units of J/g.

Structural Characterization

The FTIR spectra of the chitosan films were obtained using

an infrared spectrometer with Fourier transformation

(Nicolet 670 spectrometer). The measurement range was

4,000–650 cm-1, with a 4 cm-1 resolution, a

0.475 cm-1 s-1 scan speed and 100 scans. The attenuated

total reflectance (ATR) technique was applied an Avatar

multibounce HATR accessory with ZnSe crystal at 45�.

X-ray diffraction (XRD) patterns of the chitosan films

were recorded using a Siemens D-5000 difractometer

(CuKa wavelength of 1.5418 A) operated at 35 kV and

25 mA. Chitosan films were scanned in a zero background

rotator (15 rpm) and Si samples holder to avoid back-

ground interference at room temperature. Measurements

were recorded over the range of 5�–60� (2h) with a step-

time of 8 s and step-size of 0.02� (2h).

Morphological Analysis

The surface morphology of the chitosan films was char-

acterized using a Phillips XL 30 ESEM scanning electron

microscope (SEM). Before examination, the films were

mounted onto a cylindrical stub with a 10 mm diameter

cylindrical using double–sided adhesive tape. The surface

morphology was observed in over 5 9 5 mm samples fixed

to the stub.

Antimicrobial Activity Test

Listeria monocytogenes ATCC 7644 was obtained from

MediMark Europe (Florida, USA) and the media tryptone

soy broth (TSB), tryptone soy agar (TSA), and Mueller–

Hinton broth (MHB) were obtained from BD Bioxon

(Atizapan de Zaragoza, Estado de Mexico, Mexico). For

experimental use, the stock cultures were maintained by

regular subculture on agar tryptone soy agar slant at 4 �C

and transferred monthly. A loop of bacteria was added to

10 mL of TSB and incubated at 37 �C for 12 h. A 100 lL

aliquot from the culture was again transferred to TSB and

grown at 37 �C to the mid-exponential phase of growth.

The inoculums began with approximately 105–106

CFU mL-1 in the test tubes. These CFU counts were

determined to be accurate based on an inoculation of

0.1 mL of culture having an absorbance value of 0.2

measured by UV/vis spectroscopy for its optical density at

600 nm [23].

To assess the effectiveness of the antimicrobial prop-

erties of the chitosan films, the bacterial growth was

evaluated based on the macro-dilution method described by

the National Committee of Clinical Laboratory Standards

[24] with some modifications. For this assay, chitosan films

with 40 % GLY were introduced in test tubes containing

10 mL of sterile Mueller–Hinton broth (MHB) at a pH of

6.2. After 15 min, the tubes were inoculated with

105 CFU mL-1 of L. monocytogenes in the mid-exponen-

tial growth phase and incubated at 37 �C for 24 h. Then, a

0.1 mL aliquot of MHB from each tube was subcultivated

on TSA plates. Finally, the plates were read by the plate

count method after 24 h at 37 �C. These results were

expressed as CFU/mL and compared with a control sample

did not contain the film.

Statistical Analysis

A completely randomized design was used as an experi-

mental design, where film-forming solutions containing

different concentrations of GLY or SOR were applied as

44 J Polym Environ (2014) 22:41–51

123

treatments. Chitosan films without plasticizers were used as

the control. All of the data are presented as the

means ± standard deviations. The statistical significance

of the differences between the means (p \ 0.05) was esti-

mated using an ANOVA test with the Statgraphic plus 4.0

software package.

Results and Discussion

Physicochemical Characterization of Chitosan

Table 1 presents the results from the physicochemical

characterization of the chitosan prepared from chitin

extracted by lactic fermentation of shrimp wastes. The ash

and moisture values are similar to those reported by

Ohtakara et al. [25] with percentages of 0.08 and 4.75 %,

respectively. However, these values are lower than the 6.78

and 6.84 % reported by Marmol et al. [26]; the low ash

content is due to the demineralization process. Further-

more, according to Martınez-Camacho et al. [15], a high-

quality chitosan sample must contain\1 % of residual ash.

The nitrogen content in the obtained chitosan was similar

to the value of 6.89 % reported by Rabea et al. [6]. Yen

et al. [27] reported a nitrogen content of 6.2 % in purified

chitin and values between 8.8 and 9.5 % for chitosan.

Furthermore, by eliminating the proteins during the puri-

fication process, a more precise nitrogen content value in

the chitosan molecule can be determined.

An important factor for the molecular structure of

chitosan is the preparation processes. The preparation has

an influence on certain parameters, such as the degree of

deacetylation, acetyl group distribution, and molecular

weight. The degree of deacetylation is similar to the values

reported by Yen et al. [27], which ranged from 75 to 85 %.

In addition, Weska et al. [12] reported that chitosan with a

90 % level of deacetylation results in a higher quality

chitosan. The chitosan obtained in this work presented a

low intrinsic viscosity, which indicates a low degree of

polymerization (Table 1) and is consistent with the low

molecular weight (136 kDa). This result is similar to those

reported by Martınez-Camacho et al. [15], who obtained

low molecular weight chitosan (100 and 263 kDa, respec-

tively). The low molecular weight of the obtained chitosan

may be related to the biological treatment administered to

the chitin/or depolymerization during the removal of pro-

teins and minerals in the silage as well as in the purification

procedures and subsequent deacetylation. Similarly, the

viscosity parameter is affected by the molecular weight of

the polymer and the degree of deacetylation. According to

Bough et al. [28] when the deacetylation process is applied

at high temperatures (140–150 �C), the chitosan viscosity

will range from 311 to 511 cps. However, Lertsutthiwong

et al. [29] reported viscosity values ranging from 106 to

6,370 cps, which were obtained through different chitin

treatments. The reported values in the present investigation

agree with the previous report.

Color and Transparency of the Films

The effects of the concentration of GLY and SOR on the

color and transparency of the films are shown in Table 2.

The addition of GLY and SOR as a plasticizer into the

chitosan films exhibited have shown in some significant

effects (p \ 0.05) on the a* (redness/greenness) and b*

values of the surface of the films. In contrast, the L* values

of the films did not exhibit a significant difference

(p [ 0.05) when GLY and SOR were added. In terms of

the redness (a* value) the chitosan films with 20 and 40 %

SOR exhibited the largest difference, where an increase

from -0.97 ± 0.29 to 4.00 ± 0.59 (indicator of the ten-

dency towards redness) compared to the other films. In the

case of yellowness (b* value), the chitosan films with 40 %

SOR plasticizer exhibited more yellow color than the other

chitosan films. This result is due to the presence of SOR,

which modifies the color of the chitosan films from trans-

parent to a yellow tone. Figure 1 shows neat chitosan film

and the chitosan film with different concentrations of GLY

and SOR. All of the examined chitosan films were visually

colorless, transparent, homogeneous and uniform. The

average thickness of the films was 0.035 ± 0.06 mm. The

measurement of transparency using an instrumental tech-

nique indicated that this property is affected by plasticizers,

and even with the naked eye, this effect can be observed

across the films in Fig. 1. Chitosan films formulated with

GLY exhibited a significant decrease in their transparency

values. Similar results have been reported by Rivero et al.

[4] in bilayer films of gelatin and chitosan plasticized with

GLY. In contrast, the transparency of the chitosan films

with SOR was significantly increased (p \ 0.05) with

increasing concentration of the plasticizer. These results

Table 1 Physicochemical characterization of chitosan

Parameters (%) Average value

Moisture (%) 5.6 ± 0.054

Ash (%) 0.2 ± 0.020

Nitrogen (%) 6.2 ± 0.11

DA (%) 84 ± 2.7

Viscosity (cps) 152 ± 0.30

[g] (mL g-1) 2.76 ± 0.51

Mv (kDa) 136

Means values of n = 3 ± SD

DA deacetylation degree; [g] intrinsic viscosity; Mv average vis-

cosity molecular weight

J Polym Environ (2014) 22:41–51 45

123

are important for potential applications for the films, spe-

cifically if the film will be used as a surface coating for

food or for improving product appearance.

Swelling, Solubility and WVTR of the Chitosan

FilmsThe surface morphology of the

The degree of swelling and solubility of the chitosan films

are shown in Table 3. The percentage of the degree of

swelling and solubility in water of neat chitosan films and

films with 20 % SOR were not able to be determined

because the samples were completely solubilized. The

percentage of the degree of swelling (% DS) of the chitosan

films using GLY exhibited higher swelling values than the

films with 40 % SOR. This result is associated with the

hydrophilic character of SOR because it is less hydrophilic

than GLY. With regard to the percentage of solubility in

water (% SW), the neat chitosan films had high solubility

(easy dissolution), and thus the solubility was not detected;

however, when GLY was added to the films, the % SW was

measured. A similar behavior was only observed in the

films with 40 % SOR. Assuming that neat chitosan and

chitosan with 20 % SOR have a 100 % SW, a reduction of

the % SW of the plasticized films could be attributed to the

formation of hydrogen bonds between the chitosan and the

plasticizer, which reduced its capacity to absorb water. In

all cases, the chitosan films became rubbery when they

were dipped in water, but they did not maintain their

integrity because the soluble plasticized part of the film

disrupts the structure. Nevertheless, the solubility of the

film can be tailored by controlling the concentration of the

plasticizer, which allows a wide range of possible appli-

cations. In some food packaging applications insoluble

films are required insolubility to enhance product integrity

and water resistance.

The WVTR values of the chitosan films as a function of

the plasticizer content are also shown in Table 3. The

addition of GLY in the chitosan films increases the WVTR

values, which is contrary to the behavior that was observed

with the use of SOR as a plasticizer. The maximum WVTR

value occurred at a GLY content of 40 %; in contrast, at a

SOR content of 40 %, this value was minimal. It was

observed that the chitosan films plasticized with SOR

exhibited a slight reduction in the WVTR values compared

to those plasticized with GLY. This result might be due to

the SOR having a ring molecular conformation which may

sterically hinder its insertion between the chains of chito-

san, whereas GLY has a short linear chain, which are

inserted and positioned within the three-dimensional

polymer network. The neat chitosan film had a WVTR

value that was similar to the value reported by Xu et al.

[30]. Note that a low WVTR opens a wide range of

applications for these films in packaging, especially in a

considerably humid environment.

Mechanical Properties

The mechanical properties of the neat chitosan films and

those plasticized with GLY and SOR are shown in Table 4.

It was observed that the concentrations of the plasticizers,

GLY and SOR, significantly affected the tensile parameters

Table 2 Color and

transparency value of chitosan

films

Values are given of

n = 5 ± SD. Different letters in

the same column indicate a

significant difference (p \ 0.05)

Film samples L* a* b* Transparency

Neat chitosan 92.77 ± 0.64a -1.26 ± 0.21a 8.88 ± 0.86a 1.77 ± 0.13a

CHI ? 20 % GLY 92.43 ± 0.99a -1.79 ± 0.26b 11.44 ± 1.22b 0.41 ± 0.27b

CHI ? 40 % GLY 92.41 ± 0.47a -1.25 ± 0.21a 10.80 ± 1.06b 0.30 ± 0.06b

CHI ? 20 % SOR 92.41 ± 0.89a -0.97 ± 0.29a 8.54 ± 1.30a,b 2.70 ± 0.89c

CHI ? 40 % SOR 92.08 ± 0.27a -4.00 ± 0.59c 17.90 ± 2.01c 3.13 ± 0.46c

Fig. 1 Photograph of the chitosan films showing that shows their

transparency with different concentrations of plasticizer

Table 3 Swelling, solubility and WVTR of chitosan films

Film samples % DS % SW WVTR (g/m2/h)

Neat chitosan ND ND 57 ± 0.7a

CHI ? GLY 20 % 519.3 ± 80a 46.5 ± 0.3a 59 ± 1.6a

CHI ? GLY 40 % 447.4 ± 17b 65.6 ± 1.1b 79.6 ± 1.9b

CHI ? SOR 20 % ND ND 53.5 ± 0.4c

CHI ? SOR 40 % 382.1 ± 68b,c 86.3 ± 3.0c 52.9 ± 0.8c

Mean values of n = 3 ± SD. Different letters in the same column

indicate a significant difference (p \ 0.05)

46 J Polym Environ (2014) 22:41–51

123

(tensile strength, elongation at break, and tensile modulus)

of the films. The neat chitosan films had a higher tensile

strength and tensile modulus and a low elongation at

breakage. However, when GLY was added, the films

became more flexible. Chitosan films with a 20 % content

of GLY exhibited plasticization characteristics, such as a

decrease in the tensile strength and tensile modulus as well

as an increase in the elongation at breakage values. This

result is due to GLY penetrating through the biopolymer

matrix and interfering with the chitosan chains, thereby

decreasing the intermolecular attraction and increasing the

chain mobility of this biopolymer, which resulted in the

films being more flexible [2]. However, when the GLY

content was increased to 40 %, the tensile strength and

elongation at breakage remained constant, and the elastic

modulus exhibited a significant increase (p [ 0.05). At this

concentration of GLY, the chitosan films with GLY do not

exhibit the conventional effect of common plasticizers.

This behavior is well known as the antiplasticization effect,

and it has been observed in polysaccharides plasticized

with water, GLY, and other components. This effect is

attributed to the disruption of the carbohydrate–carbohy-

drate hydrogen bond, which facilitates rearrangements of

the carbohydrate chains and consequently alters the struc-

ture of the matrix. From our results, the limit of GLY

concentration in the chitosan films is 20 wt%; at higher

GLY concentrations, the interactions of the chitosan chains

are increased, and the hole volume caused by the GLY

cannot be accounted for by a simple ‘‘hole filling’’. How-

ever, the increased concentration of SOR as plasticizer

produced a decrease in the tensile strength from 72.2 to

40.4 MPa when the concentration of SOR increased from

20 to 40 %, while and the elastic modulus also decreased

from 3,441.3 to 2,204.8 MPa, respectively (Table 4). The

elongation at breakage of the chitosan films with SOR as a

plasticizer was lower than that of the GLY plasticized

films; this result is due to the ring structure of SOR, which

hinders the interaction between chitosan molecules [31].

The tear strength of the plasticized chitosan films with

20 and 40 % GLY were the lowest and highest values,

respectively. The chitosan films with SOR as plasticizers

increased the tear strength without a significant difference

(p \ 0.05). The behavior of the tear strength as a function

of the concentration of plasticizers is in agreement with the

tensile strength from the mechanical tests. The plasticizing

effect was remarkably different in chitosan films with 40 %

GLY. The reduction of the tear strength observed in some

formulations could be due to crack propagation, which is

enhanced by the brittle nature of chitosan.

Thermal Analysis

Figure 2 shows thermograms of the films produced with

neat chitosan and films with GLY and SOR as plasticizers

obtained from the first DSC run. The neat chitosan films

present a bimodal thermogram; the first peak is at 110 �C

and second peak is observed at 130 �C. According to the

literature, the first peak results from water evaporation. The

enthalpies for this endothermic peak represent the energy

required to vaporize the water present in the films. Fur-

thermore, Rueda et al. [32] reported that the endothermic

area of the first DSC run, for starch and chitosan was

correlated with the water content of the sample. The second

apparent peak is related to the glass transition temperature

(Tg) of chitosan. Our results agree with Fernandez Cervera

et al. [33], who observed that the Tg of chitosan determined

using DSC measurements was 130 to 139 �C, which is

Table 4 Tensile strength,

percent of elongation at break,

elastic modulus and tear

strength of chitosan films

Mean values of n = 6 ± SD.

Different letters in the same

column indicate a significant

difference (p \ 0.05)

Film samples Tensile

strength (MPa)

Elongation

at break (%)

Elastic modulus

(MPa)

Tear

strength (N)

Neat chitosan 196.7 ± 23.3a 5.34 ± 0.9a 7,697.3 ± 110a 0.26 ± 0.02a

CHI ? GLY 20 % 22.2 ± 3.2b 41.98 ± 7.5b 153.9 ± 45b 0.09 ± 0.02b

CHI ? GLY 40 % 245.2 ± 19.1c 43.69 ± 5.2b 2,488.9 ± 354c 0.41 ± 0.02c

CHI ? SOR 20 % 74.2 ± 8.6d 2.70 ± 0.22a 3,441.3 ± 381d 0.32 ± 0.03d

CHI ? SOR 40 % 40.3 ± 4.1e 1.81 ± 0.3a 2,204.8 ± 471c 0.21 ± 0.12e

Fig. 2 DSC thermograms of neat chitosan films (a), with 20 % GLY

(b), 40 % GLY (c), 20 % SOR (d) and 40 % SOR (e)

J Polym Environ (2014) 22:41–51 47

123

similar to the results of the present study. The addition of

SOR produced a decrease in the temperature of both peaks,

as was observed in the neat chitosan. In contrast, when

GLY was added only one broad small peak was observed

in both thermograms at approximately 120 �C. The

behavior of the thermogram’s of both plasticizers may be

used to compare the efficiency of the plasticizers. The

presence of only one peak indicates a miscible system. The

occurrence of two endothermic peaks at 20 % SOR and a

broad peak indicate that this system is not miscible and that

SOR has a plasticizer effect. This behavior is well-known

for films based on biopolymers.

Structural Properties

The effect of the plasticizers in the chitosan films was

evaluated by FTIR analysis. Fig. 3 shows comparative

FTIR spectra of the neat chitosan film and the film with

plasticizers. The spectrum of the neat chitosan films

exhibited a broad absorption band in region between 3,600

and 3,000 cm-1, which was attributed to hydrogen-bonded

hydroxyl groups (O–H) and to asymmetric/symmetric

stretching of the N–H bonds in the amino group. Bands at

2,910, 2,867, 1,420 and 1,260 cm-1, belong to the asym-

metric CH2 vibrations of the carbohydrate ring, and the

band at 1,330 cm-1 is characteristic of –OH, –NH2 and

–CO groups and measures the extent of N-acetylation.

Other peaks situated at wavenumbers of 1,730 cm-1, cor-

respond to the ester linkage, and that at 896 cm-1 corre-

sponds to the C–O–C of the pyranose ring [4]. The bands

obtained between 1,580 and 1,550 cm-1 are attributed to

N–H bonds of the N-acetyl group (amide II). As observed

in Fig. 3, the addition of GLY and SOR did not modify the

shape of the spectra obtained for chitosan films; however,

some absorption intensities were slightly affected by the

presence of the GLY and SOR plasticizers. The major

changes in the plasticized chitosan films can be observed in

two peaks in the 1,660–1,550 cm-1 region, which are the

peaks are associated with the –NH groups and are in

agreement with the amide I and amide II bands. In addition,

the strong water band at 1,400 cm-1, which is associated

with the in plane bending to the –OH is discernible in the

plasticized films. These observations supported the

assumption that there could be a modification in the

arrangement in the films due to the interactions of the GLY

and SOR plasticizers with the hydroxyl and amino groups

in the chitosan matrix. The results from the FTIR technique

analyses exhibited a similar trend to those observed for all

of the studied properties. Furthermore, these findings sup-

ported that the values obtained with the addition of GLY

and SOR were intermediate between those obtained with

the incorporation of a plasticizer.

X-ray diffraction patterns of the neat and plasticized

chitosan films are shown in Fig. 4. The diffraction peaks at

11� and 20� from the neat chitosan and plasticized films are

typical finger print for chitosan and are attributed to the

crystal lattice arrangements of the films. The X-ray dif-

fraction patterns of the chitosan films plasticized with GLY

exhibit a diffraction peak in the region from 2h = 10� to

25� with the highest peak intensity at 2h = 20.2�. Changes

in the diffraction pattern (peak intensities) indicate that the

incorporation of GLY into the chitosan might have affected

the packaging of the molecular chains and changed its

crystallinity. In contrast, the chitosan films with 20 % did

not present a different shape than the neat chitosan films.

However, the films with 40 % SOR exhibited a significant

decrease in the intensity of the diffraction peak. This result

Fig. 3 FTIR spectra of neat chitosan films (a), with 20 % GLY (b),

40 % GLY (c), 20 % SOR (d) and 40 % SOR (e)

Fig. 4 XRD patterns of neat chitosan films (a), with 20 % GLY (b),

40 % GLY (c), 20 % SOR (d) and 40 % SOR (e)

48 J Polym Environ (2014) 22:41–51

123

suggests that SOR produces a lack of crystalline and semi-

crystalline regions during the production of the film [33].

Morphological Characterization

The SEM micrographs of the neat chitosan films and the

plasticized films are shown in Fig. 5. It was observed that

all of the chitosan films were smooth with a complete

absence of aggregation, pores and microphase separation.

These results indicate that the chitosan and plasticizers

(GLY and SOR) were well mixed at the microstructure

level. Our results are in agreement with Ziani et al. [2] and

Rivero et al. [4], who reported that the incorporation of

plasticizers into chitosan produces changes in the bio-

polymer structure and improves the optical properties of

the films (transparency). It has been reported that GLY can

enter the interior of the polysaccharide chain, where it

disrupts the inter- and intramolecular hydrogen bonds and

makes the polymer plastic, thereby forming a continuous

phase of a plasticized film.

Fig. 5 Scanning electron micrographs of neat chitosan films (a), with 20 % GLY (b), with 40 % GLY (c), with 20 % SOR (d) and with 40 %

SOR (e). The scale is 50 lm for the samples

J Polym Environ (2014) 22:41–51 49

123

Susceptibility Tests

L. monocytogenes is commonly used as a bacterial indi-

cator of contamination in meat products [9]; therefore, the

chitosan film with the best mechanical properties (chitosan

with 20 % GLY) was selected, and its effects on the growth

of this bacterial species were evaluated (Table 5). The

results indicate a bacterial reduction by at least 2 log10

units from the initial inoculum size with 20 mg of chitosan

with 20 % GLY film and did not show a significant dif-

ference at higher concentrations (p \ 0.05). Additionally,

it was observed that lower doses do not achieve a loga-

rithmic reduction. These results are similar to those

reported by Bento et al. [34] who assayed the chitosan

obtained from the fungi Mucor rouxii against L. monocyt-

ogenes and achieved a reduction of 2 log10 with a con-

centration of 5 mg mL-1.

Previous studies have shown that chitosan has a high

capacity for the reduction of L. monocytogenes. The

antimicrobial effect of chitosan films against L. mono-

cytogenes at 37 �C has been tested, and it was observed

that L. monocytogenes growth was completely inhibited

[35]. Furthermore, emulsions with 0.58 % acetic acid and

0.1 % chitosan reduced the initial inoculum of L. mon-

ocytogenes from a level of 107 to 1 CFU mL-1 after

24 h. Several authors have reported that there are close

relationships between the molecular weight and degree of

deacetylation of chitosan with the its antimicrobial

activity [6, 36]. Accordingly, Devlieghere et al. [36]

reported that chitosan with a low degree of polymeriza-

tion (45 KDa) and a high degree of deacetylation (94 %)

exhibited good antimicrobial activity against gram-nega-

tive bacteria, whereas gram-positive bacteria are reported

to have a variable susceptibility to chitosan. The origin

and characteristics of chitosan can explain the variations

in the obtained results.

Conclusions

The chitosan used in this work was prepared from chitin

extracted by the lactic fermentation of shrimp wastes, and

the obtained chitosan has a low molecular weight. Neat

chitosan films and films plasticized with SOR and GLY were

prepared using the solution casting method. The prepared

films were colorless, transparent, homogeneous and uni-

form. However, the WVTR values were increased when

GLY was added to the chitosan films. Furthermore, the

chitosan films plasticized with 20 % GLY exhibited plasti-

cization characteristics, but when the content was increased,

the antiplasticization effect was observed. The structural

properties of the neat and plasticized chitosan films revealed

good compatibility between chitosan and both plasticizers.

The primary interaction with chitosan and the plasticizers

was through hydrogen bonds. Furthermore, the plasticized

chitosan films exhibit antimicrobial activity, which makes

the development of active packaging possible.

Acknowledgments This research was supported by funds from

FONCICYT C002-2008-1/ALA-127 249 and by AGRINOS AS Bio

Tech Company. Mr. Rodrıguez-Nunez is grateful to CONACYT

(213712). The authors wish to thank to D. Aguilar-Trevino and D.

Huerta-Quitanilla for their technical assistance with the XRD and

SEM analyses, respectively.

References

1. Hashem HM, Razavi SH, Mousavi AMS, Shaidi YS, Ghorbani

HA (2008) Improving antibacterial activity of edible films based

on chitosan by incorporation thyme and clove essential oil and

EDTA. J Appl Sci 8:2895–2900

2. Ziani K, Oseas J, Coma V, Mate JI (2008) Effect of the presence

of glycerol and Tween 20 on the chemical properties of films base

on chitosan with different degree of deacetylation. LWT Food Sci

Technol 41:2159–2165

3. Ye M, Neetoo H, Chen H (2008) Control of Listeria monocyt-

ogenes on ham steaks by antimicrobials incorporated into chito-

san-coated plastic films. Food Microbiol 25:260–268

4. Rivero S, Garcıa MA, Pinotti A (2009) Composite and bi-layer

films based on gelatin and chitosan. J Food Eng 90:531–539

5. Suyatma E, Tighzert L, Copinet A (2005) Effect of hydrophilic

plasticizers on mechanical, thermal, and surface properties of

chitosan films. J Agric Food Sci Chem 53:3950–3967

6. Rabea EI, Badawy ET, Stevens CV, Smagghe G, Steurbaut W

(2003) Chitosan as antimicrobial agent: applications and mode of

action. Biomacromolecules 4:1457–1465

7. Holappa J, Hjalmarsdottir M, Masson M, Runarsson O, Asplund

T, Soininen P, Nevalainen T, Jarvinen T (2006) Antimicrobial

activity of chitosan N-bentainates. Carbohydr Polym 65:114–118

8. CisseM Montet D, Loiseau G, Ducamp-Collin MN (2012)

Influence of the concentrations of chitosan and glycerol on edible

film properties showed by response surface methodology.

J Polym Environ. doi:10.1007/s10924-012-0437-2

9. Pranoto Y, Rakshit SK, Salokhe VM (2005) Enhancing antimi-

crobial activity of chitosan films by incorporating garlic oil,

potassium sorbate and nisin. Food Sci Technol 38:859–865

Table 5 Logarithmic reduction of L. monocytogenes by chitosan

films with 20 % GLY after 24 h at different concentrations

Films mass (mg) Listeria monocytogenes

Log count (CFU/ml)

0 6.93 ± 0.01a

5 6.38 ± 0.01a

10 6.39 ± 0.02a

15 6.39 ± 0.77a

20 4.92 ± 0.02b

25 4.91 ± 0.20b

Means values of n = 3 ± SD. The moisture content of the films was

98.77 ± 0.02 %. Means in each column with different superscript

letter are significantly different (p \ 0.05)

50 J Polym Environ (2014) 22:41–51

123

10. Ray D, Roy P, Sengupta S, Segupta SP, Mohanty AK, Misra M

(2009) A study of physicomechanical and morphological prop-

erties of starch/poly(vinyl alcohol) based films. J Polym Environ

17(1):56–63

11. Bueno-Solano C, Lopez-Cervantes J, Campas-Baypoli ON,

Lauterio-Garcıa R, Adan Bante NP, Sanchez-Machado DI (2009)

Chemical and biological characteristics of protein hydrolysates

from fermented shrimp by-products. Food Chem 112:671–675

12. Weska RF, Moura JM, Batista LM, Rizzi J, Pinto LAA (2007)

Optimization of deacetylation in the production of chitosan from

shrimp wastes: use of response surface methodology. J Food Eng

80:749–753

13. AOAC (1984) Protein nitrogen content of milk. In: Williams S

(ed) Official methods of analysis of the Association of Official

Analytical Chemistry (AOAC), 14th edn. AOAC International,

Gaithersburg, MD

14. Liu D, Wei Y, Yao P, Jiang L (2006) Determination of degree of

acetylation of chitosan by UV spectrophotometry using dual

standards. Carbohydr Res 341:782–785

15. Martınez-Camacho AP, Cortez-Rocha MO, Ezquerra-Brauer JM,

Graciano-Verdugo AZ, Rodriguez-Felix F, Castillo-Ortega MM,

Yepiz-Gomez MS, Plascencia-Jatomea M (2010) Chitosan com-

posite films: thermal, structural, mechanical and antifungal

properties. Carbohydr Polym 82:305–315

16. Shiku Y, Yuca Hamaguchi P, Tanaka M (2003) Effect of pH on

the preparation of edible films based on fish myofibrillar proteins.

Fish Sci 69(5):1026–1032

17. Han JH, Floros JD (1997) Casting antimicrobial packaging films

and measuring their physical properties and antimicrobial activ-

ity. J Plast Films Sheet 13:287–298

18. Garcıa MA, Pinnotti A, Martino MA, Zaritzky NE (2004)

Characterization of composite hydrocolloid films. Carbohydr

Polym 56:339–345

19. Bourtoom T (2008) Plasticizer effect on the properties of bio-

degradable blend film from rice starch-chitosan. Songklanakarin J

Sci Technol 30:149–165

20. ASTM (2010) Standard test methods for water vapor transmis-

sion of materials. E96/E96M-10. Annual book of ASTM stan-

dards. American Society for Testing and Materials, Philadelphia,

PA

21. ASTM (2002) Standard test method for tensile properties of thin

plastics sheeting. D882-02. Annual book of ASTM standards.

American Society for Testing and Materials, Philadelphia, PA

22. ASTM (2008) Standard test method for tear-propagation resis-

tance (trouser tear) of plastic film and thin sheeting by a single-

tear method. D1938-08. Annual book of ASTM standards.

American Society for Testing and Materials, Philadelphia, PA

23. Fernandez-Saiz P, Ocio MJ, Lagaron JM (2010) Antibacterial

chitosan-based blends with ethylene–vinyl alcohol copolymer.

Carbohydr Polym 80:874–884

24. National Committee for Clinical Laboratory Standards (2001)

Performance standards for antimicrobial susceptibility testing—

eleventh informational supplement. NCCLS, Wayne, PA

25. Othakara A, Izume M, Mitsutomi M (1998) Action of microbi-

ological chitinases on chitosan with different degrees of deacet-

ylation. Agric Biol Chem 521:3181–3182

26. Marmol Z, Gutierrez E, Paez G, Ferrer J, Rincon M (2004) De-

sacetilacion termoalcalina de la quitina de conchas de camaron.

Multiciencias 4:1–10

27. Yen MJ, Yang JA, Mauc JL (2009) Physicochemical character-

ization of chitin and chitosan from crab shells. Carbohydr Polym

75:15–21

28. Bough W, Salter W, Perkin B (1978) Influence of manufacturing

variables on the characteristic an effectiveness of chitosan

product. I. chemical composition, viscosity and molecular-weight

distribution of chitosan product. Biotechnol Bioeng

20:1931–1943

29. Lertsutthiwong P, Rojsitthisak P, Nimmannit P (2009) Prepara-

tion of turmeric oil-loaded chitosan-alginate biopolymeric nano-

capsules. Mater Sci Eng C 29:856–860

30. Xu YX, Kimb KM, Hanna Y, Nag D (2005) Chitosan–starch

composite film: preparation and characterization. Ind Crops Prod

21(2):185–192

31. Yang L, Paulson AT (2000) Effect of lipids on the mechanical

and moisture barriers properties of edible gellan films. Food Res

Int 55:571–578

32. Rueda DR, Secall T, Bayer RK (1999) Differences in the inter-

action of water with starch and chitosan films as revealed by

infrared spectroscopy and differential scanning calorimetry.

Carbohydr Polym 40:49–56

33. Fernandez Cervera M, Heinamak J, Rasanen M, Maunu SL,

Karjalainen M, Nieto Acosta OM, Iraizoz Colarte A, Yliruusi J

(2004) Solid-state characterization of chitosans derived from

lobster chitin. Carbohydr Polym 58:401–408

34. Bento RA, Stamford TLM, Campos-Takaki GMS, Thayza CM,

Souza EL (2009) Potential of chitosan from Mucor rouxxi

UCP064 as alternative natural compound to inhibit Listeria

monocytogenes. Braz J Microbiol 40(3):583–589

35. Coma V (2008) Bioactive packaging technologies for extendedshelf life of meat-based products. Meat Sci 78:90–103

36. Devlieghere F, Vermeulen A, Debevere J (2004) Chitosan: anti-

microbial activity, interactions with food components and

applicability as a coating on fruit and vegetables. Food Microbiol

21:703–714

J Polym Environ (2014) 22:41–51 51

123