LWT - Food Science and Technology 44 (2011) 1133e1138

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LWT - Food Science and Technology

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Evaluation of antimicrobial and physical properties of edible lm based on

carboxymethyl cellulose containing potassium sorbate on some mycotoxigenic
Aspergillus species in fresh pistachios
Sara Sayanjali*, Babak Ghanbarzadeh, Shiva Ghiassifar
Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, P.O.Box 51666-16471, Tabriz, Iran

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 1 December 2009
Received in revised form
14 December 2010
Accepted 16 December 2010

Active packaging is a relatively novel concept of packaging that changes the conditions of the packaged
food to extend its shelf-life and improve its safety. In this study, antimicrobial effects of carboxymethyl
cellulose based-edible lm containing potassium sorbate as an antimicrobial agent were studied against
Aspergillus avus (PTCC-5004), Aspergillus parasiticus (PTCC-5286) and A. parasiticus (PTCC-5018) by using
agar diffusion assay. Results showed suitable inhibition effects against A. parasiticus (PTCC-5286) and A.
avus (PTCC-5004) in comparison with A. parasiticus (PTCC-5018). Pistachios were coated with this edible
antimicrobial lm containing three concentrations of sorbate (1, 0.5 and 0.25 g/100 mL lm solution); all
concentrations showed no growth of molds. Tensile strength values of lms with potassium sorbate,
decreased when compared to control, and lms exibility, was 28.82 percent for 3 g/100 mL sorbate, while
higher concentration of sorbate, decreased the exibility. The water vapor permeability values (WVP) of
lms were determined to be 1.18 (g mm/m2 day kPa) for lms plasticized with glycerol, without sorbate
while WVP values for the lms containing 1 and 2 g/100 mL sorbate increased to 3.77 and 15.5 (g mm/m2
day kPa), respectively. The observed glass transition temperature (TG) depression for these polymer blends
was related to the plasticizer content (water, polyethylene, and glycerol), especially water.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Edible antimicrobial coating
Potassium sorbate
Physical properties
Carboxymethyl cellulose lm

1. Introduction
The traditional concept of a packaging is to preserve the quality of
the product with a minimal product/packaging interaction,
however, in recent years, a wide variety of packages have been
employed for interaction with food to provide desirable effects
(Silveira et al., 2007). Active packaging technology is a relatively new
concept that is aimed for extending the product shelf-life, maintaining its nutritional and sensory quality, as well as providing
microbial safety (Cha & Chinnan, 2004). The ability of edible lm or
coating as a type of active packaging to carry some food additives
such as antioxidants, antimicrobials, colorants, avors, fortied
nutrients and spices are being studied (Han, 2001; Pena & Torres,
1991). In term of microbial properties, direct surface application of
antibacterial substances has limited benets, because the active
substances are neutralized on contact or diffused rapidly into the
bulk of food (Siragusa & Dickson, 1992; Torres, Motoki, & Karel,
1985). Antimicrobial substances incorporated into packaging
* Corresponding author. Tel./fax: 98 2166937177.
E-mail addresses: sara.sayanjali@gmail.com (S. Sayanjali), ghanbarzadeh@
tabrizu.ac.ir (B. Ghanbarzadeh), shghiassifar@yahoo.com (S. Ghiassifar).
0023-6438/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2010.12.017

materials can control microbial contamination by reducing the

growth rate and maximum growth population and/or extending the
lag-phase of the target microorganism or by inactivating microorganisms by contact (Quintavalla & Vicini, 2002). Numerous
researchers have studied the applied potential of active materials to
use in food packaging industry. Pranoto, Salokhe, and Rakshit (2005)
reported that an antibacterial alginate edible lm incorporated with
garlic oil has good potential in many food applications. Plant
essential oils have ability to protect food against pathogenic bacteria
contaminating apple juice (Friedman, Henika, Levin, & Mandrell,
2004) and other foods (Burt, 2004; Seydim & Sarikus, 2006). For
example Rojas-Grau, Avena-Bustillos et al (2007) reported that
antimicrobial activity of oregano essential oil and carvacrol in alginateeapple puree edible lms and lm forming solutions against
Escherichia coli O157:H7 was signicantly greater than the activity of
lemongrass oil, citral, cinnamon oil, and cinnamaldehyde. Many
polysaccharide-based coatings have been developed for fresh-cut
fruits and vegetables. For instance alginate and gellan-based edible
lms have been prepared for increasing shelf-life of fresh-cut Fuji
apples. This coating, signicantly reduced ethylene production and
shelf-life of the coated apples was extended approximately three
times as compared with the control which showed a considerable

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S. Sayanjali et al. / LWT - Food Science and Technology 44 (2011) 1133e1138

loss of quality from the very early days of storage, limiting their
shelf-life to less than 4 days (Rojas-Grau, Tapia, Rodriguez, Carmona,
& Martin-Belloso, 2007). Baldwin, Nisperos, Chen, and Hagenmaier
(1996) studied the performance of carboxymethyl cellulose/cellulose coatings to reduce enzymatic browning of cut apple and potato.
The purpose of this study was to incorporate potassium sorbate
as an antimicrobial agent, into carboxymethyl cellulose based
edible lm and to determine the inhibitory effects of this antimicrobial edible lms against three important species of aatoxin
producer Aspergillus by using agar diffusion method. Fresh pistachios were coated with antimicrobial CMC lms and their effect on
mold growth was investigated. The lms which were targeted as
packaging material for foods require controlled water vapor transition and suitable mechanical characterizations. These properties
also were determined. To evaluate the glass transition temperature
of polymer, differential scanning calorimetry was used.

at 0.4 g/100 mL carboxymethyl cellulose solution. Then potassium

sorbate in four concentrations (1, 2, 3 and 4 g/100 mL) was added to
lm solution. Film solutions were heated until reached to 85  C, and
then they kept at this temperature for 10 min. They were cooled to
room temperature. The lm forming solutions were casted on
20  25 cm polyethylene surface and then dried overnight at 35  C.
Dried lms were peeled and covered with aluminum foil and stored
at room temperature.

2. Materials and methods

2.7. Determination of antimicrobial effects of CMC lms

2.1. Materials

Antimicrobial activity test of edible lms was carried out using

agar diffusion method. Edible lms were cut into a disc form of
20 mm diameter using a sterile scissor in a hood under ultraviolet
light. Film disk was placed on Potato Dextrose agar (Merck) plates
which had been previously inoculated by 0.1 mL of inoculums containing indicator microorganisms in the range of 104e105 spores/mL.
Inoculums were evenly spread on agar using a glass L-rod spreader.
The petri dishes were left at 4  C for 1 h to allow agar surface to dry.
Then plates were incubated at 25  C for 48 h. The diameter of
inhibitory zone surrounding lm discs was then measured with
a caliper with precision of 0.01 mm.

Carboxymethyl cellulose (CMC) with average molecular weight of

41,000 was obtained from Kargam Corporation, Tehran, Iran. Polyethylen glycol (PEG) with average molecular weight of 400, glycerol
80 percent as plasticizers and potassium sorbate (90 percent, food
grade) were obtained from Merck, Darmstadt, Germany.
2.2. Microorganisms and preparation of inocula
Aspergillus avus Persian Type Culture Collection (PTCC-5004),
Aspergillus parasiticus (PTCC-5018) and A. parasiticus (PTCC-5286);
were obtained from the culture collection at Iran Institute of Industrial and Scientic Research. Both of A. avus (PTCC-5004) and A.
parasiticus (PTCC-5018) produce aatoxin B1, whereas A. parasiticus
(PTCC-5286) produces aatoxin B1, B2, G1 and G2. The fungal cultures
were cultivated on potato dextrose agar (PDA; Merck, Mexico) slants
for 10 days at 25  C and the spores harvested with 10 mL of 1 mL/
1000 mL tween 80 (Merck) solutions (Lpez-Malo, Alzamora, & Palou,
2002; Lpez-Malo, Alzamora & Palou, 2005). The spore suspension
was adjusted with the same solution to give a nal spore concentration of 103 spore/mL and was used the same day.
2.3. Sample preparation and coating application
Fresh pistachios were purchased from local market, and
immersed in an aqueous solution of carboxymethyl cellulose lms
with different concentrations of sorbate (0.25, 0.5 and 1 g/100 mL
lm solution). After 2 min, pistachios were dried at 25  C for 24 h
until their coatings became non-tacky to touch.
2.4. Microbiological analysis
The microbiological evaluation of fresh pistachios, were carried
out with 25 g minced pistachios, aseptically weighed and homogenized with 225 mL of peptone water (Merck) by magnetic stirrer
(OGAWA SEIKI, Co, Japan) for 10 min and appropriate decimal
dilutions were done. Counting of molds was carried out on potato
dextrose agar plates which were incubated at 25  C, for 4 days. All
microbiological analyses were done in triplicate. Results corresponded to the average counts and were expressed in Log CFU g1.
2.5. Preparation of antimicrobial lms
Films solutions were produced by dissolving 1 g carboxymethyl
cellulose in 100 mL distilled water. Glycerol was added as plasticizer

2.6. Film thickness

Thickness of lms was measured with a micrometer (Alton), to
nearest 0.01 mm. Measurements were taken at three random
locations of the lm sheets. Average lm thickness was 0.05 mm for
lms with sorbate and 0.02 mm for control lms (without
preservative).

2.8. Tensile strength and elongation at break

Tensile strength and elongation at break of lms were tested
using a Universal testing machine (model 8397, D-89079,
Germany). Five lms were cut into 1  10 cm strips. Films were held
parallel with an initial grip separation of 20 mL and pulled apart at
a head speed of 5 mL/min.
2.9. Differential scanning calorimetry (DSC)
DSC measurements were carried out in DSC instrument
(NETZSCH, Model 200 F3, Maia Germany). Calibration was based on
pure indium. An empty aluminum pan was used as reference.
Samples (1.8 g) were scanned at a rate of 10  C/min between
temperature ranges of 170  Ce600  C. The glass transition
temperatures were determined from the resulting thermo grams as
the midpoint between onset and end temperatures of step changes
in heat ow observed during heating and identied as secondorder transitions.
2.10. Determination of water vapor permeability (WVP)
Water vapor permeability values (WVP) measurements of lms
were conducted at 25  C using the ASTM (D-1653-93, 1999). The
permeation cells contained CaSO4 (0 percent Relative Humidity;
0 Pa water vapor partial pressure). Film was located between the
cells and was placed in RH controlled chamber that maintaining
a temperature of 25  C and an RH of 97 percent (K2SO4, 3074 Pa
water vapor partial pressure). After 24 h a stationary water vapor
transmission rate was attained and, from that moment on, changes
in weight of the cell (to the nearest 0.01 mg) were recorded daily
over a 7 days period. The water vapor permeability value was
expressed in g mm/m2 day kPa.

S. Sayanjali et al. / LWT - Food Science and Technology 44 (2011) 1133e1138

1135

2.11. Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA)
using Excel and SPSS version 13 software. Duncan test was used to
determine the difference at 5 percent signicance level. All tests
were done in three replicates.
3. Results and discussion
3.1. Antimicrobial properties
3.1.1. Antimicrobial activity of potassium sorbate in CMC lms
Selected molds in this study are commonly associated with
aatoxin production. The inhibitory activity was measured based
on clear zone surrounding lm disks.
Previous studies also have shown inhibitory activity of antimicrobial lms against Aspergillus with agar diffusion method
(Appendini & Hotchkiss, 2002; Irkin & Korukluoglu, 2007).
According to results carboxymethyl cellulose based edible lm
without potassium sorbate had no inhibitory zone for three strains
of Aspergillus. The results of the antifungal assessment of edible lm
incorporated with potassium sorbate against three selected fungi
are presented in Table 1. Films containing 1 and 2 g preservative
were not effective against A. parasiticus (PTCC-5018), while lm
contains 1 and 2 g potassium sorbate, showed an inhibitory zone
against A. avus (PTCC-5004) and Aspergillus parasiticus (PTCC5286). Minimal inhibitory concentration of potassium sorbate that
showed inhibition was 1 g for A. avus (PTCC-5004) and A. parasiticus (PTCC-5286). Therefore A. avus (PTCC-5004) and A. parasiticus (PTCC-5286) were observed to be probably more sensitive to
potassium sorbate as compared to A. parasiticus (PTCC-5018).
Among four selected concentrations of sorbate, maximum inhibition zone was observed in 3 and 4 g potassium sorbate for all test
microorganisms. Incorporation of potassium sorbate at higher than
2 g, revealed signicant difference in zone inhibition (p < 0.05).
There was no signicant difference between inhibition zone in 1
and 2 g sorbate concentrations for all strains of Aspergillus.
3.1.2. Microbiological tests of fresh pistachios
Microbiological analyses of pistachios without coating, in Potato
Dextrose Agar media, showed intensive growth of molds compared
to coated samples. In uncoated pistachios, molds count, showed
2.02  106 CFU g1, while coated pistachios with different
concentrations of sorbate (0.25, 0.5 and 1 g/100 mL lm solution),
showed no growth of molds. Carboxymethyl cellulose based edible
lm was also coated on pistachios without potassium sorbate.
Table 1
Antimicrobial activity of carboxymethyl cellulose lms incorporated with potassium
sorbate against test microorganisms.
Microorganism

Sorbate concentration
(g/100 mL)

Inhibition
zone (mm)

Aspergillus parasiticus (PTCC-5286)

1
2
3
4
1
2
3
4
1
2
3
4

1.27
2.16
6.11
6.31
0
0
6.66
6.31
1.11
1.95
5.65
6.94

Aspergillus parasiticus (PTCC-5018)

Aspergillus avus (PTCC-5004)














0.11a
0.13a
0.20b
0.74b
00a
00a
0.11b
0.22b
0.12a
0.10a
0.13b
0.37b

Values (n 3) with different superscript letters in each row are signicantly

different (P < 0.05).

Fig. 1. Fungal growth in uncoated pistachio compared to coated sample (after 2 days
incubation in 35  C).

Results showed that, this type of coating can reduce the growth of
molds. This may be occurred for reduction of respiratory gases
transmission especially oxygen, because molds are aerobic. Visual
observation also indicated fungal growth on uncoated pistachios
after 48 h but in coated pistachios, fungal growth, was observed ten
days later (Fig. 1).
3.2. Barrier and mechanical properties
3.2.1. Water vapor permeability
Water vapor permeability is one of the most important properties in edible lms. Results of Water vapor permeability values
have been shown in Table 2. Addition of potassium sorbate at 2
g/100 mL, signicantly (p < 0.05); increased water vapor permeability from 1.18 g mm/m2 day kPa to 15.5 g mm/m2 day kPa. The
antimicrobial agents contributed to extend intermolecular interaction and furthermore, loosening the compactness of the structure
then moisture passing through the edible lms enhanced and
thereby WVP values of the lms increased (Pranoto, Rakshit, &
Salokhe, 2005). In addition, water vapor transfer generally occurs
through the hydrophilic portions of the lm and depends on the
hydrophilicehydrophobic ratio of the lm components (RojasGrau, Avena-Bustillos et al., 2007). In this study, after addition of
sorbate higher than 2 g, potassium sorbate crystallizes during
drying time. It could be explained that crystallinity of sorbate

Table 2
Water transmission parameters of carboxymethyl cellulose edible lms plasticized
with 0.4 g/100 mL glycerol containing sorbate (1e4 g/100 mL lm solution) and
carboxymethyl cellulose edible lm plasticized with 2 ml/100 mL PEG400.
Sorbate concentration

Thickness
(105 mm)

0
1
2
3
4
0

2.2
5
7.2
8.1
10
8.6

(plasticized with glycerol)

g/100 mL
g/100 mL
g/100 mL
g/100 mL
(plasticized with PEG400)








0.01
0.01
0.01
0.01
0.02
0.02

WVP (g mm/m2 day kPa)

1.18
3.77
15.5
8.74
7.55
12.06








0.15a
0.25a
0.24b
0.22b,c
0.37c
0.16d

Values (n 3) with different superscript letters in each row are signicantly

different (P < 0.05).

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S. Sayanjali et al. / LWT - Food Science and Technology 44 (2011) 1133e1138

Table 3
Mechanical properties of carboxymethyl cellulose edible lms plasticized with 0.4
g/100 mL glycerol containing sorbate (1e4 g/100 mL lm solution) and carboxymethyl cellulose edible lm plasticized with 2 mL/100 mL PEG400.
Sorbate concentration

Ultimate tensile
strength N/mm2

0
1
2
3
4
0

17.62
13.03
10.82
8.65
8.80
20.66

(plasticized with glycerol)

g/100 mL
g/100 mL
g/100 mL
g/100 mL
(plasticized with PEG400)








0.24a,c
0.16a,b
0.34b
0.12b
0.24b
0.36c

Elongation %
6.61
16.31
20.62
28.82
19.0
6.55








0.26a
0.12a,b
0.16b,c
0.22c
0.20b,c
0.26a

Values (n 3) with different superscript letters in each row are signicantly

different (P < 0.05).

blocked the pores of polymer structure, therefore water transmission reduced to 8.74 and 7.55 g mm/m2 day kPa for 3 and 4 g of
sorbate, respectively.
The type of plasticizer also affected the water vapor permeability of edible lms. In present study, PEG400 increased water
vapor permeability from 1.18 for plasticized lm with glycerol to
12.06 g mm/m2 day kPa. On considering poor mechanical characterizations that plasticized lms with PEG400 had produced, this
increase in WVP values, may be attributed to more hydrophilic
property of PEG in comparison with glycerol that caused more easy
transmission of water through the polymer matrix.
3.2.2. Mechanical properties
The tensile strength and percentage of elongation values are
important parameters for edible lms. Tensile strength (TS) is
a measure of lm strength while elongation at break (E value) is
a measure of lm strength ability prior to breakage. The tensile
strength and percentage of elongation values of the edible lm
incorporated with potassium sorbate are summarized in Table 3.
Incorporation of potassium sorbate decreased tensile strength of
lms. A signicant difference (p < 0.05) in tensile strength was
markedly observed after incorporation of 1 g of potassium sorbate.
Tensile strength of lms decreased from 13.03 N/mm2 to
8.65 N/mm2 when sorbate concentration increased from 1 to 3 g.
Addition of potassium sorbate as an antimicrobial component in
polymeric matrix of carboxymethyl cellulose lms, causes reduction in linkage of polymer structure. This result conrms the
outcome of the report by Cagri, Ustunol, and Ryser (2001), who had
concluded that incorporation of additives other than cross linking
agents generally lowers TS value. Similarly, incorporation of
potassium sorbate up to 1 g, in carboxymethyl cellulose lms
plasticized with glycerol, signicantly increased E values (p < 0.05),
while in 4 g sorbate, elongation at break, decreased. This reduction
showed that higher levels of sorbate weakened the polymer
structure and then E value decreased. These results are in a good
agreement with study by others (Pranoto, Rakshit et al, 2005) in
forming chitosan lms containing potassium sorbate and other
preservatives. In both studies there was a peak for E value and after
that, by increasing in sorbate concentration, elongation at break
decreased.
Films which plasticized with polyethylene glycol (400), had greater
tensile strength values compared to plasticized lms with glycerol, but
this increasing was not signicant (p < 0.05). This type of carboxymethyl cellulose lm, indeed, was a brittle lm with high tensile
strength and low exibility that may not be a suitable lm in industrial
application at this concentration (2 mL/100 mL lm solution).
3.3. Thermal properties
The glass transition temperature of the various lms was
determined by differential scanning calorimetry (DSC) and DSC

Fig. 2. Thermograms of carboxymethyl cellulose lms with 0.4 g/100 mL glycerol (a),
2 mL/100 mL polyethylene glycol400 (b) and water (c).

measurements conrmed glass transition phenomena in carboxymethyl cellulose lms. In carboxymethyl cellulose powder thermo
gram, a clear peak at 99.6  C was seen (gure not shown). This peak
probably related to glass transition temperature of carboxymethyl
cellulose. Thermo grams that related to CMC lms with polyethylene glycol and glycerol as plasticizers showed wide peak at
71.8 and 78.1  C respectively (Fig. 2). Glass transition (Tg) of polymer-plasticizer mixture shows plasticizer compatibility with
biopolymer and effectiveness of plasticization process, in fact if
polymer and plasticizer are immiscible, the mixture will exhibit
two Tg corresponding to the two pure phase (Ghanbarzadeh et al.,
2006). These thermo grams indicated that PEG400 and glycerol
caused wideness for curve and they could not reduce glass transition temperature signicantly compared with water in pure carboxymethyl cellulose lm (Fig. 2). Water has signicant role in
decreasing of glass transition temperature. Magoshi, Nakamura,
and Murakamiki (1992) measured the glass transition temperature of zein at various moisture contents and reported it decreased
from 139 to 47  C when water content increased from 0% to 6.6%. In
all of the plasticized carboxymethyl cellulose lms with glycerol
which contain potassium sorbate, a sharp peak at temperature
range of 151e156.9  C, was seen. This sharp peak is probably due to
melting point of potassium sorbate (Fig. 3).

S. Sayanjali et al. / LWT - Food Science and Technology 44 (2011) 1133e1138

1137

temperature of carboxymethyl cellulose and melting point of

potassium sorbate, were determined at 99.6 and 151e156.9  C,
respectively.
The results of this experiment showed that the use of an
antimicrobial coating consisting of potassium sorbate based on
carboxymethyl cellulose is a viable alternative in controlling the
molds growth on pistachios, because the growth of A. parasiticus
(PTCC-5286), A. parasiticus (PTCC-5018) and A. avus (PTCC-5004),
was substantially inhibited by the application of potassium
sorbate. Growth of molds in coated pistachios with carboxymethyl
cellulose edible lms without potassium sorbate was relatively
inhibited due to reduction in oxygen level, and it is a hopeful
concept for edible lms and coatings without preservative to
maintain foodstuff in order to be safer. Therefore, an antimicrobial
carboxymethyl cellulose edible lm incorporated with potassium
sorbate is promising and it seems to have good potential in food
applications.
Acknowledgment
We sincerely thank University of Tabriz for providing nancial
support for this study.
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Fig. 3. Thermograms of plasticized carboxymethyl cellulose lms with 0.4 g/100 mL

glycerol containing potassium sorbate, (a) 1 g/100 mL, (b) 2 g/100 mL, (c) 3 g/100 mL,
(d) 4 g/100 mL.

4. Conclusion
Sorbate presence in edible lms formulated with carboxymethyl
cellulose and glycerol increased exibility of lms. Higher
concentration of sorbate (4 g/100 mL lm solution) approximately
weakened the polymer structure. PEG400 in this study could not
provide suitable mechanical characterizations and lms that were
plasticized with PEG400 (in concentration of about 2 mL/100 mL
lm solution), were brittle compared with plasticized carboxymethyl cellulose lms with glycerol. High water vapor permeability for plasticized carboxymethyl cellulose lms with PEG400
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